Physiology of the Gastrointestinal Tract, Volume 1-2, Fourth Edition

PHYSIOLOGY OF THE GASTROINTESTINAL TRACT FOURTH EDITION

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Physiology of the

Gastrointestinal Tract FOURTH EDITION

Volume 1 Editor-in-Chief Leonard R. Johnson Department of Physiology University of Tennessee College of Medicine Memphis, Tennessee

Associate Editors

Kim E. Barrett Department of Medicine University of California, San Diego, School of Medicine University of California San Diego Medical Center San Diego, California

Fayez K. Ghishan Department of Pediatrics Steele Children’s Research Center University of Arizona Health Sciences Center Tucson, Arizona

Juanita L. Merchant Departments of Internal Medicine and Molecular and Integrative Physiology University of Michigan Ann Arbor, Michigan

Hamid M. Said Departments of Medicine and Physiology/Biophysics University of California Irvine, California, and VA Medical Center-151 Long Beach, California

Jackie D. Wood Departments of Physiology and Biology and Internal Medicine The Ohio State University College of Medicine Columbus, Ohio

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Library of Congress Cataloging-in-Publication Data Physiology of the gastrointestinal tract / editor-in-chief, Leonard R. Johnson ; associate editors, Kim Barrett ... [et al.].-- 4th ed. p. ; cm. Includes bibliographical references and index. ISBN 0-12-088394-5 (set : alk. paper) -- ISBN 0-12-088395-3 (v.1 : alk. paper) ISBN 0-12-088396-1 (v.2 : alk. paper) 1. Gastrointestinal system--Physiology. I. Johnson, Leonard R., 1942[DNLM: 1. Gastrointestinal Tract--physiology. WI 102 P578 2006] QP145.P492 2006 612.3′2--dc22

British Library Cataloguing in Publication Data A catalogue record for this book is available from the British Library

Volume 1: ISBN 13: 978-0-120883950 • ISBN 10: 0-12-088395-3 Volume 2: ISBN 13: 978-0-120883967 • ISBN 10: 0-12-088396-1 Two Volume Set: ISBN 13: 978-0-120883943 • ISBN 10: 0-12-088394-5

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Contents Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi Preface to the First Edition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xix Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xxi Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xxii VOLUME 1

Section I. Basic Cell Physiology and Growth of the GI Tract 1. Transcriptional and Epigenetic Regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Juanita L. Merchant and Longchuan Bai

1

2. Translation and Posttranslational Processing of Gastrointestinal Peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cheryl E. Gariepy and Chris J. Dickinson

31

3. Transmembrane Signaling by G Protein–Coupled Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Claire Jacob and Nigel W. Bunnett

63

4. Gastrointestinal Hormones: Gastrin, Cholecystokinin, Somatostatin, and Ghrelin . . . . . . . . . . . . . . . . . . . . . . . . . . Graham J. Dockray

91

5. Postpyloric Gastrointestinal Peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121 Ella W. Englander and George H. Greeley Jr. 6. Gastrointestinal Peptide Hormones Regulating Energy and Glucose Homeostasis . . . . . . . . . . . . . . . . . . . . . . . . . . 161 Daniel J. Drucker 7. Growth Factors in the Gastrointestinal Tract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183 John A. Barnard and Kirk M. McHugh 8. Developmental Signaling Networks Wnt/β-Catenin Signaling in the Gastrointestinal Tract . . . . . . . . . . . . . . . . . . . 247 Guido T. Bommer and Eric R. Fearon 9. Hedgehog Signaling in Gastrointestinal Morphogenesis and Morphostasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271 Gijs R. van den Brink, Maikel P. Peppelenbosch, and Drucilla J. Roberts 10. Developmental Signaling Networks: The Notch Pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287 Guy R. Sander, Hanna Krysinska, and Barry C. Powell 11. Physiology of Gastrointestinal Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 307 Alda Vidrich, Jenny M. Buzan, Sarah A. De La Rue, and Steven M. Cohn 12. Apoptosis in the Gastrointestinal Tract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 345 Leonard R. Johnson 13. Molecular Aspects and Regulation of Gastrointestinal Function during Postnatal Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 375 James F. Collins, Liqun Bai, Hua Xu, and Fayez K. Ghishan 14. Effect of Aging on the Gastrointestinal Tract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 405 Adhip P. N. Majumdar and Marc D. Basson 15. Regulation of Gastrointestinal Normal Cell Growth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 435 Mark R. Hellmich and B. Mark Evers

v

vi / CONTENTS 16. Mucosal Repair and Restitution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 459 Mark R. Frey and D. Brent Polk 17. Mechanisms of Gastrointestinal Malignancies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 477 John Lynch and Anil K. Rustgi

Section II. Neural Gastroenterology and Motility 18. Development of the Enteric Nervous System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 499 Michael D. Gershon and Elyanne M. Ratcliffe 19. Cellular Physiology of Gastrointestinal Smooth Muscle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 523 Gabriel M. Makhlouf and Karnam S. Murthy 20. Organization and Electrophysiology of Interstitial Cells of Cajal and Smooth Muscle Cells in the Gastrointestinal Tract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 533 Kenton M. Sanders, Sang Don Koh, and Sean M. Ward 21. Functional Histoanatomy of the Enteric Nervous System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 577 Simon J. H. Brookes and Marcello Costa 22. Physiology of Prevertebral Sympathetic Ganglia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 603 Joseph H. Szurszewski and Steven M. Miller 23. Cellular Neurophysiology of Enteric Neurons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 629 Jackie D. Wood 24. Integrative Functions of the Enteric Nervous System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 665 Jackie D. Wood 25. Extrinsic Sensory Afferent Nerves Innervating the Gastrointestinal Tract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 685 Michael J. Beyak, David C. E. Bulmer, Wen Jiang, C. Keating, Weifang Rong, and David Grundy 26. Processing of Gastrointestinal Sensory Signals in the Brain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 727 Anthony Hobson and Qasim Aziz 27. Enteric Neural Regulation of Mucosal Secretion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 737 Helen Joan Cooke and Fedias Leontiou Christofi 28. Effect of Stress on Intestinal Mucosal Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 763 Johan D. Söderholm and Mary H. Perdue 29. Effect of Stress on Gastrointestinal Motility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 781 Michèle Gué 30. Central Corticotropin-Releasing Factor and the Hypothalamic-Pituitary-Adrenal Axis in Gastrointestinal Physiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 791 Yvette Taché and Mulugeta Million 31. Neural Regulation of Gastrointestinal Blood Flow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 817 Peter Holzer 32. Neural Control of the Gallbladder and Sphincter of Oddi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 841 Gary M. Mawe, Gino T. P. Saccone, and María J. Pozo 33. Brainstem Control of Gastric Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 851 Richard C. Rogers, Gerlinda E. Hermann, and R. Alberto Travagli 34. Neural and Hormonal Controls of Food Intake and Satiety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 877 Timothy H. Moran

CONTENTS / vii 35. Pharyngeal Motor Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 895 Reza Shaker 36. Motor Function of the Esophagus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 913 Ray E. Clouse and Nicholas E. Diamant 37. Neurophysiologic Mechanisms of Gastric Reservoir Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Jan Tack

927

38. Small Intestinal Motility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . William L. Hasler

935

39. Function and Regulation of Colonic Contractions in Health and Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Sushil K. Sarna and Xuan-Zheng Shi

965

40. Neural Control of Pelvic Floor Muscles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . David B. Vodusek and Paul Enck

995

41. Pathophysiology Underlying the Irritable Bowel Syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1009 Jackie D. Wood VOLUME 2

Section III. Gastrointestinal Immunology and Inflammation 42. Innate Immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1033 Lars Eckmann 43. Biology of Gut Immunoglobulins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1067 Finn-Eirik Johansen, Elizabeth H. Yen, Bonny Dickinson, Masaru Yoshida, Steve Claypool, Richard S. Blumberg, and Wayne I. Lencer 44. Mechanisms of Helicobacter pylori–Induced Gastric Inflammation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1091 Dawn. A. Israel and Richard M. Peek Jr. 45. Mechanisms and Consequences of Intestinal Inflammation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1115 Wallace K. MacNaughton 46. Recruitment of Inflammatory and Immune Cells in the Gut: Physiology and Pathophysiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1137 D. Neil Granger, Christopher G. Kevil, and Matthew B. Grisham 47. Physiology of Host–Pathogen Interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1163 Kim Hodges, V. K. Viswanathan, and Gail Hecht

Section IV. Physiology of Secretion 48. The Cell Biology of Gastric Acid Secretion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1189 Curtis Okamoto, Serhan Karvar, and John G. Forte 49. Regulation of Gastric Acid Secretion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1223 Arthur Shulkes, Graham S. Baldwin, and Andrew S. Giraud 50. Gastroduodenal Mucosal Defense . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1259 Marshall H. Montrose, Yasutada Akiba, Koji Takeuchi, and Jonathan D. Kaunitz 51. Genetically Engineered Mouse Models of Gastric Physiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1293 Linda C. Samuelson 52. Structure–Function Relations in the Pancreatic Acinar Cell . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1313 Fred S. Gorelick and James D. Jamieson

viii / CONTENTS 53. Stimulus-Secretion Coupling in Pancreatic Acinar Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1337 John A. Williams and David I. Yule 54. Cell Physiology of Pancreatic Ducts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1371 Barry E. Argent, Michael A. Gray, Martin C. Steward, and R. Maynard Case 55. Regulation of Pancreatic Secretion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1397 Rodger A. Liddle 56. Bile Formation and the Enterohepatic Circulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1437 Paul A. Dawson, Benjamin L. Shneider, and Alan F. Hofmann 57. Mechanisms of Hepatocyte Organic Anion Transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1463 Allan W. Wolkoff 58. Mechanisms of Hepatocyte Detoxification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1483 Karen F. Murray, Donald J. Messner, and Kris V. Kowdley 59. Physiology of Cholangiocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1505 Anatoliy I. Masyuk, Tatyana V. Masyuk, and Nicholas F. LaRusso 60. Gallbladder Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1535 Sum P. Lee and Rahul Kuver

Section V. Digestion and Absorption 61. Tight Junctions and the Intestinal Barrier . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1559 Thomas Y. Ma and James M. Anderson 62. Protein Sorting in the Exocytic and Endocytic Pathways in Polarized Epithelial Cells . . . . . . . . . . . . . . . . . . . . . . 1595 V. Stephen Hunt and W. James Nelson 63. Physiology of the Circulation of the Small Intestine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1627 Philip T. Nowicki 64. Sugar Absorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1653 Ernest M. Wright, Donald D. F. Loo, Bruce A. Hirayama, and Eric Turk 65. Protein Digestion and Absorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1667 Vadivel Ganapathy, Naren Gupta, and Robert G. Martindale 66. Role of Membrane and Cytosolic Fatty Acid Binding Proteins in Lipid Processing by the Small Intestine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1693 Nada Abumrad and Judith Storch 67. Genetic Regulation of Intestinal Lipid Transport and Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1711 Zhouji Chen and Nicholas O. Davidson 68. Digestion and Intestinal Absorption of Dietary Carotenoids and Vitamin A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1735 Alexandrine During and Earl H. Harrison 69. Vitamin D3: Synthesis, Actions, and Mechanisms in the Intestine and Colon . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1753 J. Wesley Pike, Makoto Watanuki, and Nirupama K. Shevde 70. Vitamin E and Vitamin K Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1773 Ronald J. Sokol, Richard S. Bruno, and Maret G. Traber 71. Intestinal Absorption of Water-Soluble Vitamins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1791 Hamid M. Said and Bellur Seetharam 72. Water Transport in the Gastrointestinal Tract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1827 Jay R. Thiagarajah and A. S. Verkman

CONTENTS / ix 73. Na+-H+ Exchange in Mammalian Digestive Tract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1847 Pawel R. Kiela and Fayez K. Ghishan 74. Intestinal Anion Absorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1881 Pradeep K. Dudeja and K. Ramaswamy 75. Ion Channels of the Epithelia of the Gastrointestinal Tract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1917 John Cuppoletti and Danuta H. Malinowska 76. Integrative Physiology and Pathophysiology of Intestinal Electrolyte Transport . . . . . . . . . . . . . . . . . . . . . . . . . . . 1931 Kim E. Barrett and Stephen J. Keely 77. Molecular Mechanisms of Intestinal Transport of Calcium, Phosphate, and Magnesium . . . . . . . . . . . . . . . . . . . . 1953 James F. Collins and Fayez K. Ghishan 78. Iron Absorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1983 Nancy C. Andrews 79. Trace Element Absorption and Transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1993 Robert J. Cousins

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Contributors Nada Abumrad, Departments of Medicine and Nutritional Sciences, Washington University, Campus Box 8031, 660 South Euclid Avenue, Saint Louis, Missouri 63110

Marc D. Basson, John D. Dingell Veterans Affairs Medical Center, Department of Surgical Service, Wayne State University, Detroit, Michigan 48201 Michael J. Beyak, Gastrointestinal Diseases Research Unit, Queen’s University, Kingston, Ontario, Canada, and Department of Biomedical Science, University of Sheffield, Sheffield S10 2TN, United Kingdom

Yasutada Akiba, Brentwood Biomedical Research Institute, Building 114, Suite 217, West Los Angeles VAMC, Los Angeles, California 90073 James M. Anderson, Department of Cell and Molecular Physiology, University of North Carolina at Chapel Hill, 6312 MBRB, 103 Mason Farm Road, Chapel Hill, North Carolina 27599-7545

Richard S. Blumberg, Department of Medicine, Harvard Medical School, and Division of Gastroenterology, Hepatology, and Endoscopy, Harvard Digestive Diseases Center, Laboratory of Mucosal Immunology, Thorn 1419, Brigham and Women’s Hospital, 75 Francis Street, Boston, Massachusetts 02115

Nancy C. Andrews, Department of Basic Sciences and Graduate Studies, Harvard Medical School, and Department of Pediatrics, Children’s Hospital, Howard Hughes Medical Institute, Gordon Hall, 25 Shattuck Street, Boston, Massachusetts 02115

Guido T. Bommer, Division of Molecular Medicine and Genetics, Department of Internal Medicine, University of Michigan School of Medicine, LSI 5-183A, 210 Washtenaw Avenue, Ann Arbor, Michigan 48109-2216

Barry E. Argent, Institute for Cell and Molecular Biosciences, University Medical School, Newcastle upon Tyne NE2 4HH, United Kingdom

Simon J. H. Brookes, Department of Human Physiology and Centre for Neuroscience, Flinders University, Bedford Park, South Australia 5042

Qasim Aziz, University of Manchester, Clinical Sciences Building, Hope Hospital, Stott Lane, Salford M6 8HD, United Kingdom

Richard S. Bruno, Department of Nutritional Sciences, University of Connecticut, 3624 Horsebarn Road Ext, Unit 4017, Storrs, Connecticut 06269-4017

Liqun Bai, Department of Pediatrics, Steele Children’s Research Center, University of Arizona Health Sciences Center, 1501 North Campbell Avenue, Tucson, Arizona 85724-5073

David C. E. Bulmer, Department of Biomedical Science, University of Sheffield, Sheffield S10 2TN, United Kingdom

Longchuan Bai, Department of Internal Medicine, University of Michigan, 1150 West Medical Center Drive, 3510 MSRB I, Ann Arbor, Michigan 48109

Nigel W. Bunnett, Departments of Surgery and Physiology, University of California, San Francisco, Room C317, 521 Parnassus Avenue, San Francisco, California 94143-0660

Graham S. Baldwin, Department of Surgery, University of Melbourne, Austin Health, Heidelberg, Victoria 3084, Australia

Jenny M. Buzan, Digestive Health Center of Excellence, University of Virginia, Charlottesville, Virginia 22908-0708

Kim E. Barrett, Department of Medicine, University of California, San Diego, School of Medicine, University of California San Diego Medical Center 8414, 200 West Arbor Drive, San Diego, California 92103-8414

R. Maynard Case, Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, United Kingdom Zhouji Chen, Division of Endocrinology, Metabolism, and Lipid Research, Washington University School of Medicine, Campus Box 8046, 660 South Euclid Avenue, Saint Louis, Missouri 63110

John A. Barnard, Department of Pediatrics, Division of Molecular Medicine, The Ohio State University College of Medicine, Columbus Children’s Hospital, 700 Children’s Drive WA2011, Columbus, Ohio 43205

xi

xii / CONTRIBUTORS Fedias Leontiou Christofi, Departments of Anesthesiology and Physiology and Cell Biology, The Ohio State University, 226 Tzagounis Medical Research Facility, Columbus, Ohio 43210

Bonny Dickinson, Department of Pediatrics, Research Institute for Children, Children’s Hospital, Research and Education Building Room 2231, 200 Henry Clay Avenue, New Orleans, Louisiana 70118

Steve Claypool, Laboratory of Mucosal Immunology, Thorn 1419, Brigham and Women’s Hospital, 75 Francis Street, Boston, Massachusetts 02115

Chris J. Dickinson, Division of Pediatric Gastroenterology, University of Michigan, D3252 MPB, 1500 East Medical Center Drive, Ann Arbor, Michigan 48109-0718

Ray E. Clouse, Departments of Medicine and Psychiatry, Washington University, 660 South Euclid Avenue, Campus Box 8124, Saint Louis, Missouri 63110

Graham J. Dockray, Department of Physiology, School of Biomedical Sciences, Crown Street, University of Liverpool, PO Box 147, Liverpool, L69 3BX United Kingdom

Steven M. Cohn, Digestive Health Center of Excellence, University of Virginia, 2 Jefferson Park Ave, Room 2091, Charlottesville, Virginia 22908-0708 James F. Collins, Department of Pediatrics, Steele Children’s Research Center, University of Arizona Health Sciences Center, 1501 North Campbell Avenue, Tucson, Arizona 85724-5073 Helen Joan Cooke, Department of Neuroscience, The Ohio State University, 4066D Graves Hall, 333 West Tenth Avenue, Columbus, Ohio 43210 Marcello Costa, Department of Human Physiology and Centre for Neuroscience, Flinders University, Bedford Park, South Australia 5042 Robert J. Cousins, Boston Family Professor of Nutrition, Center for Nutritional Sciences, University of Florida, PO Box 110370, Gainesville, Florida 32611-0370 John Cuppoletti, Department of Molecular and Cellular Physiology, University of Cincinnati College of Medicine, 231 Albert Sabin Way ML 0576, Cincinnati, Ohio 45267-0576 Nicholas O. Davidson, Division of Gastroenterology, Washington University School of Medicine, Campus Box 8124, 660 South Euclid Avenue, Saint Louis, Missouri 63110 Paul A. Dawson, Department of Internal Medicine, Section of Gastroenterology, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, North Carolina 27157 Sarah A. De La Rue, Digestive Health Center of Excellence, University of Virginia, Charlottesville, Virginia 22908-0708 Nicholas E. Diamant, Departments of Medicine and Physiology, University of Toronto, 6B Fell 6-176, Toronto Western Hospital, 399 Bathurst Street, Toronto, Ontario, Canada M5T 2S8

Daniel J. Drucker, Banting and Best Diabetes Centre, Toronto General Hospital, University of Toronto, 200 Elizabeth Street MBRW 4R-902, Toronto, Ontario, Canada M5G 2C4 Pradeep K. Dudeja, Department of Medicine, University of Illinois at Chicago College of Medicine, Research and Development, Jesse Brown VAMC, MP151, 820 South Damen Avenue, Chicago, Illinois 60612 Alexandrine During, Department of International Health, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland 21205 Lars Eckmann, Department of Medicine, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093-0665 Paul Enck, Department of Internal Medicine VI, University Hospitals Tuebingen, Osianderstr. 5, 72076 Tuebingen, Germany Ella W. Englander, Department of Surgery, The University of Texas Medical Branch, 815 Market Street, Galveston, Texas 77550 B. Mark Evers, Department of Surgery, The University of Texas Medical Branch, 301 University Boulevard, Galveston, Texas 77555-0536 Eric R. Fearon, Division of Molecular Medicine and Genetics, Departments of Internal Medicine, Human Genetics, and Pathology, University of Michigan School of Medicine, LSI 5-183A, 210 Washtenaw Avenue, Ann Arbor, Michigan 48109-2216 John G. Forte, Department of Molecular and Cell Biology, University of California, 241 LSA, MC 3200, Berkeley, California 94720 Mark R. Frey, Department of Pediatric Gastroenterology, Hepatology, and Nutrition, S4322 MCN Vanderbilt University, Nashville, Tennessee 37232-2576

CONTRIBUTORS / xiii Vadivel Ganapathy, Department of Biochemistry and Molecular Biology, Medical College of Georgia, 1459 Laney-Walker Boulevard, Augusta, Georgia 30912-2100 Cheryl E. Gariepy, Division of Pediatric Gastroenterology, University of Michigan, D3252 MPB, 1500 East Medical Center Drive, Ann Arbor, Michigan 48109-0718 Michael D. Gershon, Department of Anatomy and Cell Biology, Columbia University, College of Physicians and Surgeons, 630 West 168 Street, New York, New York 10032 Fayez K. Ghishan, Department of Pediatrics, Steele Children’s Research Center, University of Arizona Health Sciences Center, 1501 North Campbell Avenue, Tucson, Arizona 85724-5073 Andrew S. Giraud, Department of Medicine, University of Melbourne, Western Hospital, Footscray 3011, Australia Fred S. Gorelick, Department of Medicine, VA Healthcare CT, and Yale University School of Medicine, 950 Campbell Avenue, West Haven, Connecticut 06516 D. Neil Granger, Department of Molecular and Cellular Physiology, Louisiana State University Health Sciences Center, 1501 Kings Highway, Shreveport, Louisiana 71130-3932 Michael A. Gray, Institute for Cell and Molecular Biosciences, University Medical School, Newcastle upon Tyne NE2 4HH, United Kingdom George H. Greeley Jr., Department of Surgery, The University of Texas Medical Branch, 301 University Boulevard, Galveston, Texas 77555-0725 Matthew B. Grisham, Department of Molecular and Cellular Physiology, Louisiana State University Health Sciences Center, 1501 Kings Highway, Shreveport, Louisiana 71130-3932 David Grundy, Department of Biomedical Science, University of Sheffield, Sheffield S10 2TN, United Kingdom Michèle Gué, Department of Physiology, Université Paul Sabatier, IFR31, Institut Louis Bugnard, BP 84225, INSERM U388, Laboratoire de Pharmacologie Moléculaire et Physiopathologie Rénale, 31432 Toulouse, Cedex 4, France Naren Gupta, Department of Surgery, University of Virginia Health System, Charlottesville, Virginia 22908

Earl H. Harrison, Phytonutrients Laboratory, United States Department of Agriculture Human Nutrition Research Center, Building 307 C, Room 118 BARC-East, Beltsville, Maryland 20705 William L. Hasler, Department of Internal Medicine, Division of Gastroenterology, University of Michigan Health System, 3912, Taubman Center, Box 0362, Ann Arbor, Michigan 48109 Gail Hecht, Section of Digestive Diseases and Nutrition, University of Illinois, 840 South Wood Street, Room 738A (m/c 716), Chicago, Illinois 60612 Mark R. Hellmich, Department of Surgery, The University of Texas Medical Branch, 301 University Boulevard, Galveston, Texas 77555-0536 Gerlinda E. Hermann, Department of Neuroscience, Pennington Biomedical Research Center, 6400 Perkins Road, Baton Rouge, Louisiana 70808 Bruce A. Hirayama, Department of Physiology, The David Geffen School of Medicine at University of California Los Angeles, Los Angeles, California 90095-1751 Anthony Hobson, University of Manchester, Clinical Sciences Building, Hope Hospital, Stott Lane, Salford M6 8HD, United Kingdom Kim Hodges, Section of Digestive Diseases and Nutrition, University of Illinois, 840 South Wood Street, Room 738A (m/c 716), Chicago, Illinois 60612 Alan F. Hofmann, Department of Medicine MC 0813, Division of Gastroenterology, University of California, San Diego, La Jolla, California 92093-0813 Peter Holzer, Department of Experimental and Clinical Pharmacology, Medical University of Graz, Universitätsplatz 4, A-8010 Graz, Austria V. Stephen Hunt, Department of Molecular and Cellular Physiology, Beckman Center for Molecular and Genetic Medicine, Stanford University School of Medicine, Stanford, California 94305-5435 Dawn A. Israel, Division of Gastroenterology, Department of Medicine, Vanderbilt University Medical Center, Room 1012A MRB IV, 2215 Garland Avenue, Nashville, Tennessee 37232 Claire Jacob, Departments of Surgery and Physiology, University of California, San Francisco, Room C317, 521 Parnassus Avenue, San Francisco, California 94143-0660

xiv / CONTRIBUTORS James D. Jamieson, Department of Cell Biology, Yale University School of Medicine, 333 Cedar Street, New Haven, Connecticut 06510 Wen Jiang, Department of Biomedical Science, University of Sheffield, Sheffield S10 2TN, United Kingdom Finn-Eirik Johansen, Institute of Pathology, University of Oslo, Department of Pathology, Rikshospitalet University Hospital, Sognsvannsveien 20, N-0027 Oslo, Norway Leonard R. Johnson, Department of Physiology, University of Tennessee College of Medicine, 894 Union Avenue, Memphis, Tennessee 38163 Serhan Karvar, Department of Molecular and Cell Biology, University of California, 245 LSA, MC 3200, Berkeley, California 94720 Jonathan D. Kaunitz, Department of Medicine, Division of Digestive Diseases, West Los Angeles VAMC, and UCLA School of Medicine, Building 114, Suite 217, Los Angeles, California 90073 C. Keating, Department of Biomedical Science, University of Sheffield, Sheffield S10 2TN, United Kingdom Stephen J. Keely, Department of Medicine, University of California, San Diego, School of Medicine, University of California San Diego Medical Center 8414, 200 West Arbor Drive, San Diego, California 92103-8414 Christopher G. Kevil, Department of Pathology, Louisiana State University Health Sciences Center, 1501 Kings Highway, Shreveport, Louisiana 71130-3932 Pawel R. Kiela, Department of Pediatrics, Steele Children’s Research Center, University of Arizona Health Sciences Center, 1501 North Campbell Avenue, Tucson, Arizona 85724-5073 Sang Don Koh, Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada 89557 Kris V. Kowdley, University of Washington, Box 356174, 1959 NE Pacific Street, Seattle, Washington 98195 Hanna Krysinska, Child Health Research Institute, North Adelaide, 72 King William Road, South Australia 5006, Australia Rahul Kuver, Department of Medicine, Division of Gastroenterology, University of Washington School of Medicine, Box 356424, 1959 NE Pacific Street, Seattle, Washington 98195

Nicholas F. LaRusso, Departments of Medicine, Biochemistry, and Molecular Biology, Mayo Clinic College of Medicine, 200 First Street Southwest, 1701 Guggenheim Building, Rochester, Minnesota 55905 Sum P. Lee, Department of Medicine, Division of Gastroenterology, University of Washington School of Medicine, Box 356424, 1959 NE Pacific Street, Seattle, Washington 98195 Wayne I. Lencer, Department of Pediatrics, Harvard Medical School, and Division of Pediatric Gastroenterology and Nutrition, Harvard Digestive Diseases Center, Gastrointestinal Cell Biology Laboratories, Enders 720, Children’s Hospital Boston, 300 Longwood Avenue, Boston, Massachusetts 02115 Rodger A. Liddle, Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710 Donald D. F. Loo, Department of Physiology, The David Geffen School of Medicine at University of California Los Angeles, Los Angeles, California 90095-1751 John Lynch, Division of Gastroenterology, University of Pennsylvania School of Medicine, 415 Curie Boulevard, 600 Clinical Research Building, Philadelphia, Pennsylvania 19104 Thomas Y. Ma, Division of Gastroenterology and Hepatology, Departments of Medicine, Cell Biology and Physiology, Inflammatory Bowel Disease Program, University of New Mexico, MSC10 5550, 1 University of New Mexico, Albuquerque, New Mexico 87131-0001 Wallace K. MacNaughton, Department of Physiology and Biophysics, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta T2N 4N1, Canada Adhip P. N. Majumdar, John D. Dingell Veterans Affairs Medical Center, Department of Medicine, Karmanos Cancer Center, Wayne State University, Research Service, Room-B-4238, 4646 John R, Detroit, Michigan 48201 Gabriel M. Makhlouf, Department of Medicine, Virginia Commonwealth University Medical Center, Sanger Hall, Room 12-003, 1101 East Marshall Street, Richmond, Virginia 23298 Danuta H. Malinowska, Department of Molecular and Cellular Physiology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0576

CONTRIBUTORS / xv Robert G. Martindale, Department of Surgery, Oregon Health and Science University, Portland, Oregon 97239 Anatoliy I. Masyuk, Department of Medicine, Mayo Clinic College of Medicine, 200 First Street Southwest, 1701 Guggenheim Building, Rochester, Minnesota 55905 Tatyana V. Masyuk, Department of Medicine, Mayo Clinic College of Medicine, 200 First Street Southwest, 1701 Guggenheim Building, Rochester, Minnesota 55905 Gary M. Mawe, Department of Anatomy and Neurobiology, The University of Vermont, D403A Given Building, 89 Beaumont Avenue, Burlington, Vermont 05405

Karnam S. Murthy, Departments of Medicine and Physiology, Virginia Commonwealth University Medical Center, Sanger Hall, Room 12-003, 1101 East Marshall Street, Richmond, Virginia 23298 W. James Nelson, Department of Molecular and Cellular Physiology, Beckman Center for Molecular and Genetic Medicine, Stanford University School of Medicine, Stanford, California 94305-5435 Philip T. Nowicki, Departments of Pediatrics and Physiology, Center for Cell and Vascular Biology, Columbus Children’s Research Institute, College of Medicine and Public Health, The Ohio State University, Children’s Hospital, 700 Children’s Drive, Columbus, Ohio 43205

Kirk M. McHugh, Department of Pediatrics, Division of Molecular Medicine, The Ohio State University College of Medicine, Columbus Children’s Hospital, 700 Children’s Drive WA2011, Columbus, Ohio 43205

Curtis Okamoto, Department of Pharmaceutical Sciences, University of Southern California, 1985 Zonal Avenue, Los Angeles, California 90033

Juanita L. Merchant, Departments of Internal Medicine and Molecular and Integrative Physiology, University of Michigan, 1150 West Medical Center Drive, 3510 MSRB I, Ann Arbor, Michigan 48109

Richard M. Peek Jr., Division of Gastroenterology, Departments of Medicine and Cancer Biology, Vanderbilt University Medical Center, Room 1012A MRB IV, 2215 Garland Avenue, Nashville, Tennessee 37232

Donald J. Messner, Bastyr University, 14500 Juanita Drive, NE, Kenmore, Washington 98028

Maikel P. Peppelenbosch, Department of Cell Biology, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands

Steven M. Miller, Department of Physiology and Biomedical Engineering, Mayo Clinic College of Medicine, 200 First Street, SW, Rochester, Minnesota 55905

Mary H. Perdue, Intestinal Disease Research Program, HSC-3N5C, McMaster University, 1200 Main Street West, Hamilton, Ontario, L8N3Z5, Canada

Mulugeta Million, CURE/Digestive Diseases Research Center and Center for Neurovisceral Sciences and Women’s Health, Department of Medicine, Division of Digestive Diseases, David Geffen School of Medicine at the University of California, Los Angeles, and VA Greater Los Angeles Healthcare System, CURE Building 115, Room 117, 11301 Wilshire Boulevard, Los Angeles, California 90073

J. Wesley Pike, Department of Biochemistry, University of Wisconsin-Madison, 433 Babcock Drive, Madison, Wisconsin 53706

Marshall H. Montrose, Department of Molecular and Cellular Physiology, University of Cincinnati, Medical Science Building, Room 4253, 231 Albert Sabin Way, Cincinnati, Ohio 45267

Barry C. Powell, Child Health Research Institute, North Adelaide, and School of Pharmacy and Medical Sciences, University of South Australia; Department of Pediatrics, University of Adelaide, 72 King William Road, South Australia 5006, Australia

Timothy H. Moran, Department of Psychiatry and Behavioral Sciences, Johns Hopkins University School of Medicine, Ross 618, 720 Rutland Avenue, Baltimore, Maryland 21205 Karen F. Murray, Children’s Hospital and Regional Medical Center, University of Washington School of Medicine, 4800 Sand Point Way, NE, PO Box 5371/A5950, Seattle, Washington 98105

D. Brent Polk, Departments of Cell and Developmental Biology and Pediatric Gastroenterology, Hepatology, and Nutrition, Digestive Disease Research Center, S4322 MCN Vanderbilt University, Nashville, Tennessee 37232-2576

María J. Pozo, Department of Physiology, Nursing School, University of Extremadura, Avda Universidad s/n, 10071 Cáceres, Spain K. Ramaswamy, Department of Medicine, University of Illinois at Chicago College of Medicine, 840 South Wood Street (m/c 716), Chicago, Illinois 60612

xvi / CONTRIBUTORS Elyanne M. Ratcliffe, Division of Gastroenterology, Hepatology, and Nutrition, Department of Pediatrics, Columbia University, 3959 Broadway, CHN 702, New York, New York 10032

Reza Shaker, Division of Gastroenterology and Hepatology, Digestive Disease Center, Medical College of Wisconsin, 9200 West Wisconsin Avenue, Milwaukee, Wisconsin 53226

Drucilla J. Roberts, Department of Pathology, Massachusetts General Hospital, Boston, Massachusetts 02114

Nirupama K. Shevde, Department of Biochemistry, University of Wisconsin-Madison, 433 Babcock Drive, Madison, Wisconsin 53706

Richard C. Rogers, Department of Neuroscience, Pennington Biomedical Research Center, 6400 Perkins Road, Baton Rouge, Louisiana 70808

Xuan-Zheng Shi, Division of Gastroenterology, Department of Internal Medicine, The University of Texas Medical Branch at Galveston, 9.138E Medical Research Building, 301 University Boulevard, Galveston, Texas 77555-1064

Weifang Rong, Department of Biomedical Science, University of Sheffield, Sheffield S10 2TN, United Kingdom Anil K. Rustgi, Division of Gastroenterology, University of Pennsylvania School of Medicine, 415 Curie Boulevard, 600 Clinical Research Building, Philadelphia, Pennsylvania 19104 Gino T. P. Saccone, Department of General and Digestive Surgery, Flinders Medical Centre, Flinders Drive, Bedford Park, South Australia 5042, Australia Hamid M. Said, Departments of Medicine and Physiology/Biophysics, University of California, Irvine, California, and VA Medical Center-151, Long Beach, California 90822 Linda C. Samuelson, Department of Molecular and Integrative Physiology, The University of Michigan, Ann Arbor, Michigan 48109-0622 Guy R. Sander, Child Health Research Institute, North Adelaide, and Department of Pediatrics, University of Adelaide, 72 King William Road, South Australia 5006, Australia Kenton M. Sanders, Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada 89557 Sushil K. Sarna, Division of Gastroenterology, Department of Internal Medicine, The University of Texas Medical Branch at Galveston, 9.138C Medical Research Building, 301 University Boulevard, Galveston, Texas 77555-1064 Bellur Seetharam, Departments of Medicine and Biochemistry, Medical College of Wisconsin and Clement Zablocki VA Medical Center, Research 151, 5000 National Avenue, Milwaukee, Wisconsin 53295

Benjamin L. Shneider, Department of Pediatrics, Box 1656, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, New York 10029 Arthur Shulkes, Department of Surgery, University of Melbourne, Austin Health, Heidelberg, Victoria 3084, Australia Johan D. Söderholm, Department of Biomedicine and Surgery, Linköping University Hospital, Linköping SE-581 85, Sweden Ronald J. Sokol, Department of Pediatrics, Section of Pediatric Gastroenterology, Hepatology, and Nutrition, Pediatric General Clinical Research Center, University of Colorado School of Medicine, The Children’s Hospital, 1056 East 19th Avenue, Box B290, Denver, Colorado 80218 Martin C. Steward, Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, United Kingdom Judith Storch, Department of Nutritional Sciences, Rutgers University, 96 Lipman Drive, New Brunswick, New Jersey 08901-8525 Joseph H. Szurszewski, Department of Physiology and Biomedical Engineering, Division of Gastroenterology and Hepatology, Mayo Clinic College of Medicine, 200 First Street, SW, Rochester, Minnesota 55905 Yvette Taché, CURE/Digestive Diseases Research Center and Center for Neurovisceral Sciences and Women’s Health, Department of Medicine, Division of Digestive Diseases, David Geffen School of Medicine at the University of California, Los Angeles, and VA Greater Los Angeles Healthcare System, CURE Building 115, Room 117, 11301 Wilshire Boulevard, Los Angeles, California 90073

CONTRIBUTORS / xvii Jan Tack, Department of Gastroenterology, University Hospitals Leuven, Center for Gastroenterological Research, University of Leuven, Herestraat 49, 3000 Leuven, Belgium Koji Takeuchi, Department of Pharmacology and Experimental Therapeutics, Kyoto Pharmaceutical University, Misasagi, Yamashina, Kyoto 607, Japan Jay R. Thiagarajah, Departments of Medicine and Physiology, University of California San Francisco, 1246 Health Sciences East Tower, San Francisco, California 94143-0521 Maret G. Traber, Department of Nutrition and Exercise Sciences, Linus Pauling Institute, Oregon State University, 571 Weniger Hall, Corvallis, Oregon 97331-6512 R. Alberto Travagli, Department of Neuroscience, Pennington Biomedical Research Center, 6400 Perkins Road, Baton Rouge, Louisiana 70808 Eric Turk, Department of Physiology, The David Geffen School of Medicine at University of California Los Angeles, Los Angeles, California 90095-1751 Gijs R. van den Brink, Laboratory for Experimental Internal Medicine, Academic Medical Center, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands A. S. Verkman, Departments of Medicine and Physiology, University of California San Francisco, 1246 Health Sciences East Tower, San Francisco, California 94143-0521 Alda Vidrich, Digestive Health Center of Excellence, University of Virginia, Charlottesville, Virginia 22908-0708 V. K. Viswanathan, Section of Digestive Diseases and Nutrition, University of Illinois, 840 South Wood Street, Room 738A (m/c 716), Chicago, Illinois 60612 David B. Vodusek, Division of Neurology, University Medical Center, Ljubljana 1525, Slovenia Sean M. Ward, Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada 89557

Makoto Watanuki, Department of Biochemistry, University of Wisconsin-Madison, 433 Babcock Drive, Madison, Wisconsin 53706 John A. Williams, Department of Molecular and Integrative Physiology, University of Michigan Medical School, Ann Arbor, Michigan 48109-0522 Allan W. Wolkoff, Marion Bessin Liver Research Center and Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461 Jackie D. Wood, Departments of Physiology and Biology and Internal Medicine, The Ohio State University College of Medicine, 304 Hamilton Hall, 1645 Neil Avenue, Columbus, Ohio 43210-1218 Ernest M. Wright, Department of Physiology, The David Geffen School of Medicine at University of California Los Angeles, 10833 Le Conte Avenue, 53-263 Center for Health Sciences, Los Angeles, California 90095-1751 Hua Xu, Department of Pediatrics, Steele Children’s Research Center, University of Arizona Health Sciences Center, 1501 North Campbell Avenue, Tucson, Arizona 85724-5073 Elizabeth H. Yen, Harvard Medical School Fellowship in Pediatric Gastroenterology and Nutrition, Gastrointestinal Cell Biology, Enders 720, Children’s Hospital Boston, 300 Longwood Avenue, Boston, Massachusetts 02115 Masaru Yoshida, Frontier Medical Science in Gastroenterology, International Center for Medical Research and Treatment, Kobe University School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan David I. Yule, Departments of Pharmacology and Physiology, University of Rochester Medical School, Rochester, New York 14642

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Preface to the First Edition As with any publishing venture and especially one of this magnitude, one must first ask, “Why?” The Associate Editors and I were motivated primarily to collect in one set of volumes the most up-to-date and comprehensive knowledge in our field. Nothing comparable has been attempted in the area of gastrointestinal physiology during the past fourteen years. During this time, there has been a rapid expansion of knowledge and many new areas of investigation have been initiated. More than fifty leading scientists—physiologists, clinical specialists, morphologists, pharmacologists, immunologists, and biochemists—have contributed chapters on their various areas of expertise for these volumes. Our original goal was to review the entire field of gastrointestinal physiology in one work. After examining all of the chapters, however, it was apparent that the final product encompassed more than physiology. The chapters reflect the backgrounds of the authors and the approaches of their different disciplines. As such, these volumes contain information for not only the investigator working in these fields but for the clinician or graduate student interested in the function of the gastrointestinal tract. Anyone involved in teaching gastrointestinal physiology of pathophysiology can readily find the latest and most pertinent information on any area in the discipline. This work is divided into five sections. The first consists of topics such as growth, the enteric nervous system, and gastrointestinal peptides, each of which relates to all areas of the gastrointestinal tract. The second section contains material describing smooth muscle physiology and gastrointestinal motility. The third section presents treatment of the functions of the stomach and pancreas. The fourth series of chapters treats the entire field of digestion and absorption. These chapters vary from basic electrophysiology and membrane transport to reviews of mechanisms leading to clinical conditions of malabsorption. The final section contains chapters on areas peripheral to physiology (such as immunology, parasitology, and prostaglandins) yet necessary for a comprehensive understanding of the subject. No one person can presume to organize and edit a scientific work of this scope. I was fortunate to enlist the aid of four preeminent scientists whose expertises cover the entire field. James Christensen was primarily responsible for the chapters on smooth muscle and motility. Eugene D. Jacobson solicited and edited most of the chapters dealing with secretory mechanisms as well as those covering many of the general topics. Chapters relating to regulation were primarily handled by Morton I. Grossman, and those covering aspects of digestion and absorption were organized and reviewed by Stanley G. Schultz. I am exceedingly grateful to these four men without whom this work would not have been possible. L.R.J.

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Preface This fourth edition of Physiology of the Gastrointestinal Tract follows 12 years after the third edition. The delay was mainly due to buyouts and mergers of the involved publishing houses, certainly not to a lack of new information. On the contrary, the explosion of information at the cellular level, made possible, in part, by the continued emergence of powerful molecular and cellular techniques, has resulted in a greater degree of revision than that of any other edition. Section I, now titled “Basic Cell Physiology and Growth of the Gastrointestinal Tract,” contains numerous new chapters on topics such as transcriptional regulation, signaling networks in development, apoptosis, and mechanisms in malignancies. Most of the chapters in the first section have been edited by Juanita L. Merchant. Section II has been renamed “Neural Gastroenterology and Motility” and has been expanded from 7 chapters with rather classic titles to more than 20 chapters encompassing not only the movement of the various parts of the digestive tract but also cell physiology, neural regulation, stress, and the regulation of food intake. Almost all of the chapters in the second section have been recruited and edited by Jackie D. Wood. Section III is entirely new and contains chapters on “Gastrointestinal Immunology and Inflammation,” which were edited by Kim E. Barrett. Section IV, “Physiology of Secretion,” consists of chapters with familiar titles but with completely updated information to reflect the advances in our understanding of the cellular processes involved in secretion. Section V, “Digestion and Absorption,” contains new chapters on the intestinal barrier, protein sorting, and ion channels, together with those focusing on the uptake of specific nutrients. These chapters have been recruited and edited by Hamid M. Said and Fayez K. Ghishan. The original purpose of the first edition of this textbook—to collect in one set of volumes the most current and comprehensive knowledge in our field—was also the driving force for this edition. As mentioned earlier, this edition includes completely new chapters that cover many new areas. Although the number of chapters has increased by 15, some chapters from the previous edition have been eliminated, some with identical titles have been written by different authors, and a few have been updated by the original authors. The final product again encompasses more than physiology. The information provided is relevant not only to the researcher in the various specialized areas but also to the clinical gastroenterologist, the teacher, and the student. The authors have done an excellent job of presenting their knowledge in a style that is readable and understandable. Much of the effort in organizing and editing these volumes has come from five preeminent scientists whose interest and expertise cover the entire field. Drs. Barrett, Ghishan, Merchant, Said, and Wood met with me to decide on chapter topics, authors, and the overall organization of the material. They were responsible for recruiting authors and for the scientific editing of most chapters. The enthusiasm and abilities of these individuals simplified my task as editor, and without them this work would not have been possible. I also am especially grateful to Philip Carpenter of Elsevier, who contacted authors, tracked submissions, and assisted me in many ways. My Associate Editors and I are all grateful to the contributing authors who were generous enough to make their expert knowledge available. Their efforts have made this work more than a mere review of past contributions to a field. The various chapters synthesize and criticize this accumulated knowledge and identify voids in it, pointing out future directions for research; many of them are superb presentations of information in fields that have been reviewed nowhere else. L.R.J.

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Acknowledgments From the organization stage to actual production the following people provided invaluable assistance, helpful suggestions, and a great deal of support. Their role and efforts have been much more than what is normally provided by a publisher, and I express my thanks to them. Jasna Markovac (Senior Vice President, Global Academic & Customer Relations) Julie Eddy (Publishing Services Manager) Lisa Royse (Production Editor) Mara Conner (Publishing Editor) Judy Meyer (Publishing Editor) Tari Broderick (Senior Publishing Editor) Cate Rickard Barr (Design Manager) Andrea Lutes (Book Designer) Patricia Howard (Senior Marketing Manager) Trevor Daul (Senior Marketing Manager) Philip Carpenter (Developmental Editor)

xxii

CHAPTER

1

Transcriptional and Epigenetic Regulation Juanita L. Merchant and Longchuan Bai Overview of Gene Organization, 1 Gene Composition, 1 Epigenetic Influences, 5 Histone Modifications, 5 DNA Methylation, 8 Chromatin-Binding Proteins, 8 Epigenetics and Development, 9 Epigenetics and Cancer, 9 Anatomy of the Promoter, 9 DNA Elements, 9 DNA-Binding Proteins, 12 Coregulatory Proteins, 15

Methodology, 16 Functional Methods, 16 Structural Methods, 17 Transcriptional Control of Gastrointestinal Peptides, 19 Posttranscriptional Processing, 19 Polyadenylation, 19 RNA Splicing, 20 Transport across the Nuclear Membrane, 21 Conclusion, 22 Acknowledgments, 22 References, 22

With the completion of the human genome sequencing project at the dawn of the third modern millennium, we have come to appreciate that we are only at the start of a new era of genomic enlightenment. Perhaps the most important piece of information that we have learned is that the clues to our genetic destiny are contained in more than just the primary sequence of DNA. Apparently, what distinguishes humans from other life-forms, and most interestingly, other mammals, lies in the complex modifications and function of the 20,000 to 30,000 genes. Not only are these 25,000 or so genes alternatively spliced, but these products are chemically modified to change their function. Therefore, as opposed to our genetic template being composed of a mere 25,000 genetic units, we are actually controlled by 25,000 to the nth power. The latter value has yet to be determined, but likely results in an enormous combination of genetic events.

This chapter reviews what has led us to reformulate our notions of gene expression in the postgenomic era.

OVERVIEW OF GENE ORGANIZATION Gene Composition The molecular definition of a eukaryotic gene is complex, but in the simplest terms, it is a nucleic acid sequence that encodes one polypeptide or messenger ribonucleic acid (mRNA) molecule (1). Genes are composed of two intertwining polymers of DNA that are noncovalently attached to a variety of proteins, including histones and specialized proteins (e.g., polymerases and various accessory proteins). The association of DNA, histones, and specialized nuclear proteins collectively is called chromatin. Chromosomes are composed of continuous strands of chromatin that have been compacted by supercoiling and looping to fit into the nucleus. Most importantly, they are the basic heritable unit in the mammalian cell. In humans, there are 46 chromosomes, or 23 pairs. The smallest unit of the DNA polymer is a nucleotide, a base attached to the first carbon of a five-carbon sugar phosphorylated at its fifth carbon (Fig. 1-1). Nucleosides do not contain phosphates; thus, they differ from nucleotides, which contain one, two, or three

J. L. Merchant: Departments of Internal Medicine and Molecular and Integrative Physiology, University of Michigan, Ann Arbor, Michigan 48109. L. Bai: Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan 48109. Physiology of the Gastrointestinal Tract, Fourth Edition, edited by Leonard R. Johnson. Academic Press, 2006.

1

2 / CHAPTER 1

Base

P

OCH2

Nucleoside Nucleotide

FIG. 1-1. Nucleic acid structure. A nucleoside consists of a purine or pyrimidine base covalently linked to the first carbon of the pentose ring. The addition of one, two, or three phosphate groups yields a nucleotide monophosphate, diphosphate, or triphosphate, respectively. The type of sugar determines the type of nucleic acid: ribose in ribonucleic acid (RNA) and deoxyribose in DNA.

phosphate groups. The four nucleotides are distinguished by the type of base that they contain: adenine (A), thymine (T), cytosine (C), or guanine (G). DNA contains the sugar deoxyribose, whereas RNA contains the sugar ribose and the base uracil (U) instead of thymine. Polymers of nucleotides or nucleic acids (also called nucleoside monophosphates, diphosphates, or triphosphates) are formed when the free phosphate group attached to the fifth carbon of an adjacent nucleotide of the pentose sugar condenses with the hydroxyl group on the third pentose carbon to produce two ester bonds and water (phosphodiester bond). Accordingly, the proximal end of each DNA strand (5′ end) contains a phosphate group in the 5 position of the deoxyribose sugar residue. The terminal nucleic acid at the 3′ end of each DNA strand contains a free hydroxyl group in the 3 position of the deoxyribose ring. By convention, nucleotide sequences are written from 5′ to 3′, reading from left to right, with the sense strand presented as the upper strand. The antisense strand, written on the bottom, is antiparallel and complementary to the sense strand so that the 5′ to 3′ direction proceeds from right to left. Each nucleotide within the polymer is base paired with a particular nucleotide on the opposing strand by hydrogen bonds; adenine pairs with thymine, and guanine pairs with cytosine. The DNA strand containing the same sequence as the mRNA is designated the sense strand, and the strand that it pairs with is designated the antisense strand. The antisense strand becomes the template sequence that will be transcribed by RNA polymerase II (Pol II) into mRNA and subsequently translated into amino acids.

Most studies on transcriptional control focus on genes transcribed by the seven-subunit enzyme Pol II, and thus are designated as class II genes (2). It is Pol II that is responsible for transcribing gene sequences into protein-encoding mRNA. Only 4% of the total RNA in the cell is mRNA. Many of these initial primary transcripts (heterogeneous nuclear RNA [hnRNA]) are further processed as discussed later. Nine percent of cellular RNA is hnRNA, the bulk of which are small nuclear RNA (snRNA; e.g., U2 involved in RNA splicing, 4%) and small nucleolar RNA (e.g., U22 snoRNA comprising 1%). The other 4% of hnRNA is mRNA. An additional 1% of total cell RNA is called guide RNA, which edits mature mRNA transcripts (3). RNA polymerase I (Pol I) transcribes all of the ribosomal genes except for the 5S gene. Ribosomal RNA represents about 75% of the RNA in the cell. RNA polymerase III (Pol III) transcribes the 5S ribosomal gene and the genes encoding transfer RNA. Transfer RNA represents about 15% of the total RNA in the cell. Pol I and III transcribe genes that will not be further translated into peptides, although their primary transcripts are also processed before reaching the cytoplasm. Because Pol II transcribes genes encoding proteins and peptides, Pol II–regulated genes are the primary focus of this chapter. One may conceive of a gene as being analogous to a long sentence read from left to right and composed of letters organized into words separated by spaces and marks of punctuation. Specific DNA sequences “punctuate” the gene with important start and stop signals for transcription and translation. One gene may comprise several hundred to several thousand DNA base pairs. These base pairs (the alphabet) are organized into functional groups (phrases) based on whether a particular sequence is untranscribed, only transcribed, or both transcribed and translated (Fig. 1-2). Exons are DNA sequences that are transcribed into mRNA by Pol II and exit the nucleus. Within the cytoplasm, exons may or may not be translated into peptides. Those exons that are transcribed and translated form the coding sequences (coding exon). In general, the term intron is used to describe the intervening DNA sequence that is transcribed but is removed from the primary transcript by RNA splicing (RNA processing) before it exits the nucleus as a mature transcript (see Posttranscriptional Processing later in this chapter and also Chapter 2). DNA sequences or elements that regulate transcription and are not transcribed into mRNA usually reside in the 5′ portion of a gene upstream (to the left of) of the promoter. The promoter is a group of DNA sequences that binds Pol II in concert with accessory proteins to initiate the synthesis of mRNA. Accessory proteins control the accuracy and rate of polymerase binding. The first nucleotide transcribed into mRNA is assigned the number 1 with subsequent nucleotides (downstream or to the right of the promoter) assigned positive numbers as transcription proceeds toward the 3′ end. Nucleotides preceding the promoter (upstream or 5′) are assigned negative numbers. DNA sequences that encode a polypeptide (open reading frame) begin with the translational start site codon ATG (encoding methionine) and end with one of the three stop codons: TAA, TAG, or TGA.

TRANSCRIPTIONAL AND EPIGENETIC REGULATION / 3 Intron

3′ Exon 2

Exon 3

Exon 2

Exon 3

AATAAA sla ted

lat ed

Exon 1

tra n

tra

un

Transcription

3′

5′

un

Untranscribed

Intron

ns

5′ TTCCAATGACTCAAGTATAAGTCTC −30 +1

Primary Transcript of hnRNA (Nucleus)

m7Gppp

Exon 1

Cap

AAUAAA AAAAAAn Polyadenylation

Posttranscriptional Processing

Processed Transcript (Cytoplasm)

m7Gppp

Exon 1

Exon 2

Exon 3

AAUAAA

AAAAAAn

FIG. 1-2. Gene structure, transcription, and posttranscriptional processing. A gene is composed of several hundred to several thousand base pairs, subdivided into functional elements. The location of 5′ and 3′ untranslated sequences, exons, and introns are shown. The 5′ flanking sequences contain specific DNA elements (e.g., TATA box). Ribonucleic acid (RNA) polymerase II transcribes DNA into heterogeneous nuclear RNA (hnRNA) during transcription. Twenty base pairs after the sequence AATAAA is transcribed to AAUAAA, messenger RNA (mRNA) are cleaved and the polyadenylate tail is added to the 3′ end. A methylated guanylate residue is added to the 5′ end of the mRNA through a triphosphate linkage. Before exiting the nucleus, intron segments are removed by splicing factors during posttranscriptional processing.

(The translational start and stop codons, respectively, are transcribed into mRNA as AUG, UAA, UAG, and UGA.) Because one amino acid is encoded by three nucleotides or a triplet (codon), two or three peptides may be encoded by overlapping codons simply by shifting the reading frame by one or two nucleotides. Regulatory sequences that are transcribed but not translated reside at both the 5′ and 3′ ends of the mature RNA transcript. Both 5′ and 3′ untranslated regulatory sequences, which range from 10 to several thousand nucleotides, are thought to participate in the fidelity of translation and mRNA stabilization or destabilization. RNA molecules that encode proteins (except most histone proteins) are distinguished from ribosomal and transfer RNA by the series of adenosines added to the 3′ end of the molecule (poly(A) RNA; see Fig. 1-2). This feature is a useful means to isolate mRNA from other, more abundant RNA species (transfer and ribosomal RNA) and also designates the functional termination of the protein-encoding portion of the gene. During transcription, the primary RNA transcript is cleaved 20 bp downstream of the AAUAAA site at the 3′ end, and ~150 to 200 adenine nucleotides are added to form the poly(A) tail (4–6). The 5′ end of the mRNA transcript receives a protective “cap” after synthesis of the first 30 nucleotides, which consists of a guanylate residue methylated at the 7 position and linked to the first nucleotide of RNA by three phosphates. The RNA cap is a high-affinity binding site for ribosomes (7,8). Notably, the element

AATAA that signals the site of the poly(A) tail is not necessarily the functional end of the gene. Rather, the 3′ untranslated region (3′UTR) and 3′ untranscribed regions may still contain regulatory elements that can modulate gene expression. Therefore, just as the 5′ end of a gene must be determined empirically, so must the 3′ end of the gene. The 5′ border of a gene is identified by the promoter region (functionally determined) and structurally by the first nucleotide transcribed into mRNA (cap site) as determined by various reverse transcriptase methods—for example, primer extension analysis or anchored polymerase chain reaction (PCR) (9). These techniques use reverse transcriptase to synthesize complementary or copy DNA (cDNA; Fig. 1-3). Radiolabeled primers complementary to the 5′ end of the DNA sequence to be copied are allowed to anneal to mRNA. Reverse transcriptase then adds deoxynucleotides to the primer in the 3′ to 5′ direction. Synthesis of the cDNA will terminate when the 5′ end of the mRNA is reached. Template mRNA molecules are removed by ribonucleases (RNases), and the synthesis of a double-stranded cDNA is completed through the action of DNA polymerase. Because the newly synthesized cDNA is radiolabeled at the 5′ end, the length of the cDNA (and hence the transcriptional start site) is determined by resolving the fragments on a denaturing polyacrylamide gel and comparing the length observed in base pairs to the known cDNA sequence. cDNA is also a useful tool for making probes to detect complementary nucleotide

4 / CHAPTER 1 1

5′

2

3

AATAAA

3′ Gene

Transcription/Processing

5′

1

2

3

AAAAAAAA 3′ mature mRNA

3′

TTTTTTTT 5′ nucleotide primer

Reverse Transcriptase + dNTPs

2

3

AAAAAAAA 3′ mature mRNA

2

1

3′

1

3

5′

TTTTTTTT 5′ Primer Extended Product/single strand copy DNA RNase DNA Polymerase + dNTPs

1

2

5′

3

3′

1

2

3

TTTTTTTT 5′ Copy of Complementary DNA AAAAAAAA 3′ (cDNA)

FIG. 1-3. Complementary DNA (cDNA). Primers complementary to a portion of the messenger ribonucleic acid (mRNA) are allowed to anneal. For unknown sequences, as in the synthesis of cDNA libraries, a primer complementary to the poly(A) tail is used, that is, poly (dT). Reverse transcriptase added together with all four deoxynucleotides (dNTPs) will transcribe mRNA in the 3′ to 5′ direction to make cDNA. The mRNA template is removed by RNases, and double-stranded cDNA is made using DNA polymerase. In primer extension analysis, the 5′ end of mRNA (the cap site) is identified by annealing primers of a known sequence near the 5′ end of mRNA.

sequences and for making cDNA libraries that reflect the spectrum and relative abundance of specific mRNA within a given cell. These cDNA libraries must be contrasted with genomic phage libraries in which the DNA sequences in the phage heads reflect the number of times that a particular gene sequence is represented in the host genome, which is usually once. The 5′ sequences flanking the gene are defined functionally by various methods other than simple structural information. These sequences direct the developmental, tissue-specific, and inducible expression of the gene and can range from a few hundred to several thousand base pairs (10). It is possible to identify the sequences conferring these regulated gene activities by using methods such as DNA transfer into cell lines (11,12) and transgenic mouse models (13,14). For example, the expression of gastrin in the adult occurs in the antrum of the stomach and in the first portion of the duodenum (15–17). However, gastrin is never expressed in skin or kidney. Thus, if 1000 bp of 5′ flanking sequence permits the expression of gastrin in a fibroblast or kidney cell line, but 20,000 bp do not, it may be concluded that the untranscribed sequences between −1000 and −20,000 bp from the promoter are important in shutting off expression of gastrin in skin and kidney, sites where gastrin is never expressed in vivo. Thus, the 5′ regulatory sequences important in normal expression of the gastrin gene may extend as far upstream as −20,000 bp from the start site

of transcription. Alternatively, the 5′ or even 3′ borders may extend even further if functional data indicate that a larger sequence is required for the appropriate tissue and temporal expression to be observed with the native gene. Recently, it has been found that there are specific DNA elements called Insulator elements that mark the boundary of genes (18). These elements, originally identified on the globin gene, bind a transcription factor called CTCF and are capable of preventing the spread of histone acetylation between adjacent genes (19). Specific examples of tissue-specific elements have been reported within the promoters of several gastrointestinal (GI) peptides (e.g., gastrin and secretin), as well as for specific intestinal proteins (e.g., sucrase-isomaltase) (20–26). Similar experiments may also be performed in transgenic mice with constructs containing various lengths of 5′ flanking sequences regulating reporter gene expression. Instead of transferring these reporter constructs into cell lines, they are injected into fertilized eggs and reimplanted into ovulating female mice to be expressed in the mouse germ line (13,14). The expression of these constructs in the offspring is analyzed by cytochemical detection of reporter gene products in various organs or in response to physiologic inducers (27). The transgenic approach to gene expression, like the experiments described earlier, permits anatomic, environmental, and developmental analysis in the whole animal (28,29). This approach is particularly valuable in understanding the

TRANSCRIPTIONAL AND EPIGENETIC REGULATION / 5 regulatory sequences important for the tissue-specific expression of the genes in different cell types of the same organ such as the small intestine (30,31). Given the requirement for larger and larger pieces of DNA to recapitulate native expression in transgenic mouse models, techniques have been developed to clone and manipulate large pieces of DNA (more than 50 kilobases; e.g., yeast artificial chromosomes [YACs] and bacterial artificial chromosomes [BACs]) (32,33). Recombineering is a powerful technique performed in bacteria that permits introduction of foreign DNA or point mutations into these large plasmids that are eventually introduced into transgenic mice (34–37).

EPIGENETIC INFLUENCES Epigenetics literally means “outside of or beyond genetics,” and it refers to the “study of genetic modifications that are mitotically and/or meiotically heritable yet do not change the DNA sequence” (38). Thus, mutations or deletions alter the character or length of the sequence that, in turn, alters the primary sequence of the protein. By contrast, epigenetic influences chemically modify the nucleotide or amino acid structure that, in turn, changes how that particular residue is recognized by nuclear proteins, without changing the sequence itself. Although it is now clear from the completed sequence of the human genome that there are only about 20,000 to 30,000 gene loci, the complexity of the genetic information encoded in human chromosomes must enlist other features of chromatin (39). The epigenetic influences on chromatin appear to be one of the critical features that enhance genomic complexity. Major targets of epigenetic changes are histones, basic proteins coating the naked DNA double helix. The N-terminal tails of histones (H1, H2A, H2B, H3, H4) are positively charged because of the basic amino acid lysine. The positively charged histones attach to DNA because of the negatively charged phosphate backbone of DNA. The ionic interaction is reduced if the positive charge on the lysines is removed. Specific enzymes called histone acetyltransferases (HATs) acetylate the lysine side group, effectively eliminating the positive charge (Fig. 1-4). The loss of the ionic interaction between the histones and phosphate groups on DNA permit greater access to the DNA helix by accessory proteins such as polymerases, transcription factors, and coactivators or repressors. DNA in the form of chromatin becomes open, accessible, and readily transcribed. By contrast, there are enzymes that will “close” chromatin by removing the acetyl groups from the lysines at the N-terminal tails of histone proteins. These enzymes are called histone deacetylases (HDACs). Removal of the acetyl group restores the positive charge to the histones allowing the ionic interaction between histones and DNA to be restored. The nonhistone proteins such as polymerases and transcription factors become excluded from DNA, transcription is silenced, and chromatin is inactive. Collectively, the histones and accessory proteins associated noncovalently with DNA are what forms chromatin.

Closed Chromatin

Open Chromatin

Transcription OFF

Transcription ON Ac

K K K K

K K

HAT p300, CBP

K K

Ac K K

Ac K K K Ac K

K K HDAC

Butyrate

FIG. 1-4. Nucleosome structure. The double-strand DNA helix winds twice around a complex of the four core histones assembled as dimers. Unacetylated histones are positively charged and adhere tightly to the negatively charged DNA, preventing access by transcription regulatory proteins. Histones that are acetylated are less positively charged and do not adhere as tightly to chromatin, allowing access of regulatory proteins to the DNA. The addition or removal of acetyl groups to the ends of histones is regulated by histone acetyltransferase (HATs) and histone deacetylase enzyme complexes (HDACs). The short-chain fatty acid butyrate inhibits the activity of HDACs.

Chromatin exists in two forms: euchromatin and heterochromatin (40). Euchromatin contains the actively transcribed genes and becomes decondensed during DNA replication. Euchromatin is also centrally located in the nucleus. By contrast, heterochromatin contains transcriptionally silent genes that remain condensed at the periphery of the nucleus. The DNA sequences within heterochromatin are repetitive, and only 15% of nuclear chromatin is heterochromatin. The major forms of epigenetic modifications in mammalian cells occur on DNA and histones and include such covalent modifications as acetylation and methylation, but also via the addition of other organic residues. These epigenetic changes affect such events as chromatin folding, gene expression, X-chromosome inactivation, and genomic imprinting (41). Epigenetic events are essential for development and differentiation, during which clusters of genes must be activated or silenced at precisely timed intervals to allow for the organism’s growth and maturation.

Histone Modifications The basic repeating unit of chromatin is the nucleosome. Each nucleosome is composed of 147 bp of DNA wrapped twice around a histone protein octamer consisting of 2 molecules of each of the 4 core histones (H2A, H2B, H3, and H4). The linker histone H1 sits alone between each core nucleosome, facilitating further compaction (42). Each histone contains a structured globular domain with a histone-fold motif important for nucleosome assembly and a highly charged unstructured amino-terminal tail of 25 to 40 residues,

6 / CHAPTER 1 which protrudes from the body of the nucleosome to latch onto the phosphate backbone. The amino termini are the major sites for histone modifications (43). Histones can be modified by acetylation, methylation, phosphorylation, adenosine diphosphate (ADP)-ribosylation, ubiquitination, and sumoylation (44). The mixture of these covalent modifications creates a “code” on the surface of the histone molecule that is subsequently recognized by bromo and chromo domain–containing proteins mediating chromatin compaction, transcription, and DNA repair (45). Acetylation, methylation, ubiquitination, and sumoylation occur on the lysine residues, whereas methylation also occurs on arginine residues. Phosphorylation occurs on serines and threonines, and ADP-ribosylation occurs on glutamic acids. Most of these modifications, particularly acetylation, alter the charge distribution on the amino terminus and also alter nucleosome structure, which may, in turn, regulate chromatin structure (46,47). Some covalent modifications act as molecular switches, enabling or disabling subsequent covalent modifications, which explains the functional complexity of epigenetic modifications (48). Therefore, each modification correlates with a specific physical status of chromatin. Histone Acetylation Acetylation of histones occurs at the ε-amino side group of specific lysines within the N termini of histones. HATs transfer an acetyl group from acetyl-coenzyme A as a donor to the histone terminal lysines (49). In hypoacetylated chromatin, the positive charges on unacetylated lysines are attracted to the negatively charged DNA, producing compact, closed chromatin thereby repressing transcription (50). In contrast, acetylation of the lysines removes their positive charges, resulting in a less compact, open chromatin structure, which facilitates gene transcription. Therefore, HAT activity, and subsequently histone acetylation, is linked mainly to transcriptional activation (51) (see Fig. 1-4). Removal of the acetyl group (deacetylation) by HDACs restores the positive charge on lysines, and chromatin becomes compacted and less accessible to regulatory proteins required for transcription. Thus, HDACs and deacetylation are primarily associated with transcriptional repression (see Fig. 1-4). HATs are divided into five families. These include the p300/CBP (cyclic 3′,5′-adenosine monophosphate [cAMP] response element binding [CREB] protein) HATs (p300 and CBP), Gcn5-related acetyltransferases (GNATs; including Gcn5, p300/CBP-associated factor [PCAF], etc.), MOZ, Ybf2, Sas2, and Tip60 (MYST) (monocytic leukemia zinc finger protein [MOZ], Ybf2/Sas3, Sas2, and Tip60)-related HATs, the general transcription factor (GTF) HATs (TFIID subunit TAF250 and TFIIIC), and the nuclear hormonerelated HATs (SRC1 and ACTR) (52). The most consistent functional characteristic of the HATs is that they are transcriptional coactivators. These proteins are components of large multisubunit complexes that do not bind DNA directly, but instead form protein–protein interactions with DNAbinding transcription factors (53).

The more numerous mammalian HDACs have been grouped into three protein classes (54). Class I includes HDACs 1, 2, 3, and 8. Class II includes HDACs 4, 5, 6, 7, 9, and 10. The class III HDAC family consists of the conserved nicotinamide adenine dinucleotide (NAD)–dependent Sir2 family of deacetylases. Like HATs, HDACs do not bind directly to DNA but rather are recruited by large multisubunit complexes to function primarily as corepressors of transcription (55). The function of HATs and HDACs are of particular relevance in the GI tract because of the effect of butyrate, a by-product of colonic bacterial fermentation, on histone acetylation (see Fig. 1-4). Epidemiologic studies uniformly concur that a diet high in fiber is protective against colon cancer (56). The short-chain fatty acid butyrate is one of several fiber-derived fermentation products capable of maintaining epithelial cell differentiation (57). The differentiation effects were initially demonstrated after treatment of erythroleukemic cells with butyrate (58). Subsequently, it was discovered that the induction of differentiation by butyrate correlated with histone hyperacetylation (59–61) due to suppression of HDACs (62–66). Thus, the HDAC effects of butyrate and resulting histone hyperacetylation may, in fact, be one mechanism by which dietary fiber exerts its anticancer effects (67). Reviews support the viewpoint that butyrate is a potent anticancer agent (68–70). Collectively, early studies emphasized the global effects of butyrate on chromatin remodeling, but the molecular basis for the gene-specific effects of butyrate remains poorly defined. HDAC inhibitors regulate less than 10% of actively transcribed genes. Most of those are up-regulated through GC-rich sites (71,72). In addition to histone acetylation, it is now known that DNA-binding proteins can become acetylated (52). Thus, a possible mechanism by which hyperacetylation induced by butyrate might target specific genes is through acetylation of specific transcription factors. The proposed function of acetylated transcription factors varies and includes increased or decreased DNA binding, as well as protein stability (73). In many instances, the genetic targets of butyrate are GC-rich sequences that bind Sp1 and Sp3. Gamma glutamyl transferase (74), insulin-like growth factor (IGF) binding protein 3 (75), G α(i2) (76), galectin (77), Cox1 (78), and intestinal alkaline phosphatase (79) are all up-regulated by butyrate through Sp1 sites. Sp1 binding sites are also implicated in the butyrate induction of p21WAF1 gene expression (80). HAT p300, recruited to the p21WAF1 promoter, cooperates with Sp1 and Sp3 to mediate the effects of butyrate (81). However, Sp1 does not cooperate directly with p300, but instead binds HDAC1 (82,83). The Sp1-HDAC1 complex, in turn, forms complexes with other corepressors such as Sin3A (84). Thus, Sp1 appears to be the factor that confers p21WAF1 promoter repression by recruiting HDACs and corepressor complexes. HDACs can have opposing functions, especially in cancer. HDACs can prevent the activation of tumor suppressor genes and block the ability of a cancer cell to undergo

TRANSCRIPTIONAL AND EPIGENETIC REGULATION / 7 apoptosis (85). However, HDAC2 silencing can trigger apoptosis (86). Another important feature of HDACs is their interaction with DNA methylation. HDACs cooperate with DNA methyltransferases (DNMTs) by removing the acetyl groups that would otherwise block methylation targets on histones or DNA (87,88). Histone Methylation There are two types of histone methylation, targeting either lysine or arginine residues. Histone methyltransferases perform these modifications using S-adenosyl-methionine as the methyl group donor. Lysine methylation is implicated in changes in chromatin structure and gene regulation, whereas arginine methylation correlates with the active state of transcription, such as acetylation (89). Histone Methylation at Lysines Methylation of lysines residues (K) occurs on histone H3 primarily at K4, K9, and K27 and on H4 at K20 (Fig. 1-5). The lysine residues can be monomethylated, dimethylated, or trimethylated at the ε-amino group. The methylation of H3 is associated with an open chromatin configuration and gene activation (90,91). In contrast, the methylation of H3 at K9 is associated with condensed, repressed chromatin (92). In general, there are at least four families of lysine methyltransferases. All of the lysine methyltransferases are distinguished by the presence of Su(var)3-9, Enhancer of Zeste, and Trithorax (SET) domains. The fourth family of these methyltransferases contains other protein domains aside from the SET domain. SET protein domains are approximately 130 residues homologous to amino acid segments in SET, three Drosophila proteins with intrinsic methyltransferase activity (93,94). The mammalian form of Su(var)3-9 is SUV39H and is involved in stabilizing heterochromatin by trimethylation of histone H3 at lysine K9. The trimethyl

N -S --K --K -- K -K ----K ---- H2A 1 5 9 13 15 36

N --K -- K-S -K --K --K -K ---S --S ---- H2B 5 12 1415 20 23 24 32 36

--R -T - K- K- S -T - K --R - K ----K --R - K --S --- K ---- H3 2 3 4 9 1011 14 1718 23 26 27 28 36

N -S -R -K --K --K -- K --- K ---- H4 1 3 5 8 12 16 20

FIG. 1-5. Histone modifications on histone tails. Shown are the amino-terminal histone residues modified by acetylation (filled circles), methylation (filled squares), and phosphorylation (filled triangles).

group creates an atomic feature or imprint on H3 that, in turn, is recognized by HP1, a chromatin organization modifier (chromo domain proteins) (95). The methylated or acetylated imprints on DNA or histones are recognized by two classes of proteins: those with chromo domains that recognize methyl group imprints and those with bromo domains that recognize acetyl group imprints. Transcriptional coactivators such as CBP, p300, and PCAF are HATs that contain bromo domains. They acetylate histones and other nuclear proteins; thus, not surprisingly, they also recognize an acetyl group imprint. These proteins are discussed in greater detail later in this chapter in Chromatin-Binding Proteins. Histone Methylation at Arginines Methylation at arginines occurs within the tails of histones H3 (R2, R17, and R26) and H4 (R3) and is catalyzed by coactivator-associated arginine methyltransferase 1 (CARM1) and protein arginine N-methyltransferase 1 (PRMT1), respectively, in mammalian cells (see Fig. 1-5). Like lysines, arginines can be either monomethylated or dimethylated (asymmetric or symmetric) on the guanidino nitrogen, and this process is antagonized by human peptidylarginine deiminase 4 (PADI4), which converts methyl-Arg to citrulline (96,97). Less is known about the fate of histones methylated at arginines. However, initial studies indicate that the methylated arginines create an imprint recognized by coregulatory molecules, for example, p300 and switching/sucrose nonfermenting (SWI/SNF) (98,99). Histone Phosphorylation Histone phosphorylation occurs on all four core histones: H2A (S1), H2B (S14), H3 (S10 and S28), and H4 (S1) (see Fig. 1-5). The phosphorylation of S10 in H3 is associated with transcriptional activation (100) and chromosome condensation during mitosis (101). In addition, phosphorylation of S10 in H3 is also associated with the transduction of external signals to chromatin, leading to the transient expression of immediate early genes (102,103). The phosphorylation of H3 is mediated by several specific kinases, activated by distinct pathways. For example, mammalian mitotic H3 phosphorylation is associated with Aurora B kinases (104,105), H3 phosphorylation by IKKα is important for the activation of nuclear factor (NF)-κB (106), and the immediate early gene response is mediated mainly by mitogen and stressactivated kinases MSK1 and MSK2 (107). Histone H2B phosphorylation condenses the chromatin and is involved in apoptosis (108,109). The downstream effects of phosphorylation of H2A and H4 are unknown. Of the histone modifications, acetylation and phosphorylation are reversible. Consequently, if the presence of a modification influences transcription in a particular way, its removal may have the opposing effect. In this way the cell could effectively respond to changes in environmental cues. Different histone modifications may be linked mechanistically. For example, phosphorylation of S10 on H3 enhances

8 / CHAPTER 1 histone acetylation by Gcn5 (110,111), whereas H3 K9 methylation inhibits phosphorylation at H3 S10 (93). Given the number of sites and the variety of possible modifications, the combinatorial possibilities are extremely large. The combinatorial pattern of N-terminal modifications results in a heterogeneous identity for each nucleosome that the cell interprets as a readable code from the genome to the cellular machinery directing various processes to occur. This concept is commonly referred to as the “histone code hypothesis” (45). The precise modification status of a given histone tail on a given gene can also change during the process of transcriptional regulation and each of these different constellations of histone modifications may elicit distinct downstream transcriptional signals (45).

DNA Methylation DNA methylation is a postsynthesis modification that normal DNA goes through after each replication. This modification is catalyzed by DNMTs and occurs on the C-5 position of cytosine residues within CpG dinucleotides located primarily in the promoter of a gene. There are three major DNMTs (DNMT1, DNMT3a, and DNMT3b). Each DNMT plays a distinct and critical role in cells. Murine knockouts of DNMT1 and DNMT3b exhibit embryonic lethality (112). The DNMT3a homozygous knockout mouse appeared normal at birth but died by aged 4 weeks (112, 113). In humans, mutations of DNMT3b are linked to ICF syndrome (immunodeficiency, centromere instability, facial anomalies) (112,114). Sixty percent of human genes contain a CpG island (115). Although methylation can also occur in other parts of the gene, CpG dinucleotides tend to be underrepresented in the genome, and when they are found, they appear in clusters ranging from 0.5 to several kilobases with a GC content greater than 55% (116). These clusters are known as CpG islands (117). Methylation of CpG islands is a late evolutionary development and functions to maintain genome stability by repressing transposons and repetitive DNA elements (118). DNA methylation is an important player in many processes, including transcriptional repression, X-chromosome inactivation, and genomic imprinting. CpG islands located in the promoter region of genes are normally hypomethylated about 40% of the time (116). Their hypermethylation causes stable heritable transcriptional silencing. As observed with HDACs and deacetylation, the methylation status in cancers may seem contradictory. Aberrant de novo hypermethylation of CpG islands is a hallmark of some human cancers and is found early during carcinogenesis (119–121). Tumor suppressor genes are locally hypermethylated in some cancers to silence their expression, whereas oncogenes may be hypomethylated (116). Tumor cells globally demonstrate an overall hypomethylation of DNA, a process that has more recently been linked to nutrition (122). S-adenosylmethionine is the primary methyl donor in the cell and is reduced in conditions predisposed to cancer (123).

Genomic imprinting occurs in gametogenesis and is necessary for development. One of the X chromosomes in female individuals is not expressed because of the heavy methylation of the inactive X chromosome. The epigenetic phenomenon whereby expression of a gene depends on whether it is inherited from the mother or the father is called imprinting, and is caused by differential methylation of specific cytosine bases on the maternal versus the paternal genes.

Chromatin-Binding Proteins The remaining histone methyltransferases also recognize methyl groups on other regulatory proteins; therefore, they are discussed here. The second family of SET domain proteins is related to the Drosophila protein Enhancer of Zeste, with the prototypical mammalian protein named EZH2. EZH2 is part of a complex of proteins called the Polycomb group (PcG). Two variants of these complexes have been designated Polycomb repression complexes 1 (PRC1) and 2 (PRC2). EZH2 belongs to the PRC2 complex that also includes EED and SUZ12; whereas PRC1 includes the proteins RNF2, HPC, EDR, and BMI1. BMI1 has received increased attention because it is an important marker of normal and cancerous hematopoietic stem cells (124–126). The Polycomb group of proteins with their SET domains not only participates in histone lysine methylation, but the complexes that they form (PRC1, PRC2) are also important in recognizing the methylated protein imprint. A human homolog of Drosophila Trithorax is the mixed leukemia gene 1 (MLL1). There are four human MLL homologs. MLL1 has been shown to be a specific methyltransferase for H3 at K4 (127). In turn, it forms protein– protein interactions with coactivators, for example, CBP and corepressors chromatin remodelers (e.g., SWI/SNF) (128,129). Other Trithorax homologs (e.g., Ash1, Trx) form complexes with different coregulatory complexes. Collectively, members of the Trithorax group (TrG) of proteins can either activate or repress transcription depending on the coregulator with which they associate. Retinoblastoma protein-interacting zinc finger protein (RIZ), SMYD3, and MDS-EVI1 form a fourth family of SET domain proteins because they have two isoforms that exhibit opposing functions. The isoform containing the SET domain has tumor suppressor function, whereas the isoform missing the SET domain is cancer promoting. This “yinyang” theory put forth by Huang (123) is especially true for RIZ and MDS-EVI1, in which by an unclear mechanism, the cancer disturbs the normal ratio between the two isoforms. The SMYD3 protein contains another DNA-binding domain called MYND, in addition to a SET domain, and is overexpressed in colorectal and hepatocellular carcinomas (130). Cross talk between DNA methylation and the histone modifications exists. These interactions were shown by the observation that HDAC1 forms a complex with DNMT1 and 5-methyl-cytosine binding protein (MBP) on a methylated promoter to silence gene expression (131). Similar cross talk

TRANSCRIPTIONAL AND EPIGENETIC REGULATION / 9 occurs between the HDACs SUV39 and HP1, the HDACs PRC2 and PRC1, and the HATs MLL1 and BRM (47).

Epigenetics and Development The epigenetic control of gene expression is a fundamental feature of mammalian development, as indicated by the occurrence of developmental arrest or abnormalities in mutants deficient in methylation or acetylation. X-chromosome inactivation is an example of sequence-identical alleles being maintained stably in different functional states. In humans, X-linked inactivation serves to normalize the level of expression of X-linked genes in female (XX) and male (XY) individuals. Mutations in genes that affect global epigenetic profiles can cause human diseases. For example, the Fragile X syndrome results when a CGG repeat in the Fragile X Mental Retardation gene 1 (FMR1) 5′ regulatory region expands and becomes methylated de novo, causing the gene to be silenced and creating a visible “fragile” site on the X chromosome under certain conditions (132). On a more global level, mutations in the DNMT3b gene (which regulates the DNA methylation) lead to ICF syndrome (112,114), and CBP (with acetyltransferases activity) mutations cause Rubinstei–Taybi syndrome (133).

Epigenetics and Cancer Epigenetic changes play an important role in tumorigenesis. The major epigenetic changes that take place during the development of cancer are generally the aberrant DNA methylation of tumor suppressor genes and histones. Chapter 17 covers in greater detail the role of epigenetic influences in cancer, but a few highlights are mentioned here to conclude this section. Genomic methylation patterns are frequently altered in tumor cells, with global hypomethylation accompanying region-specific hypermethylation events. When hypermethylation events occur within the promoter of a tumor suppressor gene, this can silence expression of the associated gene and provide the cell with a growth advantage in a manner similar to deletions or mutations. Although cancer cells are hypomethylated in the genome compared with normal tissues, many tumor-suppressor genes are silenced in tumor cells because of hypermethylation. This aberrant methylation occurs early in tumor development and increases progressively, eventually leading to the malignant phenotype. For example, a high percentage of patients with sporadic colorectal cancers with a microsatellite instability phenotype show methylation and silencing of the gene encoding MutL protein homolog 1 (MLH1) (134). Other methylated tumor suppressors include p16CDKN2A, p14ARF, Rb, E-cadherin, and breast cancer gene-1 (BRCA1). Deregulation of genomic imprinting can also play a role in cancer development, as exemplified by loss of imprinting of the IGF2 gene in Wilms’ tumor (135).

Chromatin remodeling also plays an important role during tumorigenesis. Loss or misdirection of HATs has been linked to embryonic aberrations in mice (136,137) and to human cancers (138,139). Misdirection of HAT activities as a result of chromosomal translocations is associated with multiple human leukemias (140–142). In acute promyelocytic leukemia, the oncogenic fusion protein promyelocytic leukemia-retinoic acid receptor-α (PML-RARα) recruits an HDAC to repress genes essential for the differentiation of hematopoietic cells (143). Similarly, in acute myeloid leukemia (AML), AML1-eight-twenty-one (ETO) fusions recruit the repressive N-CoR-Sin3-HDAC1 complex that, in turn, inhibits normal myeloid development (144). That many human diseases, including cancer, have an epigenetic cause has encouraged the development of a new therapeutic option called “epigenetic therapy” (145). Many agents have been discovered that alter methylation patterns on DNA or the modification of histones, and several of these agents currently are being tested in clinical trials.

ANATOMY OF THE PROMOTER DNA Elements RNA Pol II and its accessory factors bind to a DNA sequence called the promoter, which is located upstream of protein-coding sequences to direct RNA transcription (146). Without the promoter, the genetic sequences that encode the information to make a functional peptide product will not be transcribed. Other 5′ flanking sequences or DNA elements that participate in transcription are sequence-specific binding sites for proteins that regulate the fidelity, rate, and timing of Pol II binding, formation of the preinitiation complex (PIC), and initiation of transcript elongation under basal and regulated conditions (147–149). These sequences are defined as cis-acting elements because they are a part of the same (cis) gene (150–153). DNA elements are categorized according to their ability to regulate transcription as a function of their distance and orientation from the promoter. Sequences that are contained within the first 30 to 100 bp of the promoter and operate in one orientation are considered promoterdependent, cis-acting elements. If they are positive-acting elements and increase the rate of transcription, they are considered activating DNA elements, whereas if they are negative-acting DNA elements and decrease or repress the rate of transcription, they are repressor elements (154–156). The structure of the promoter includes several critical elements that include the TATA element, which lies upstream of the transcription start site, the initiator sequence (Inr) that spans the start site, upstream regulatory elements that bind either transcriptional activators or repressors, and finally downstream poly(dA-dT) elements (157). The TATA element, or “TATA box,” is an element with a DNA sequence that is TATA or variants thereof (151,158–161). This sequence resides at a fixed distance 25 to 30 bp upstream from the transcriptional start site in many Pol II promoters, and its

10 / CHAPTER 1 location relative to the start site is dependent on position and distance (162–164). However, it became apparent that many genes did not have TATA sequences. These “TATA-less promoters” still remain dependent on assembly of the TATAbinding protein (TBP) at the promoter to form the PIC, but the recruitment of TBP is not rate limiting (165). Inr elements, although initially identified at the “TATAless promoters” (166,167), have subsequently been found in both TATA-containing and TATA-less promoters. Their role appears to be in directing the accuracy of Pol II initiation (168). These Inr elements reside within the first 60 bp of the transcriptional start site and directly overlap the start site itself, but they do not have a clearly defined consensus sequence (169). Many of the genes encoding GI peptides (e.g., gastrin, somatostatin, cholecystokinin [CCK], glucagons, and secretin) contain TATA elements (170–174); however, the gene encoding the growth factor, transforming growth factor-alpha (TGF-α), does not (175). Regulatory elements are generally sequence-specific DNA elements that bind transcription factors. In the case of transcriptional activators, there are two variations, upstream activating sequences (UASs) and enhancers. Both elements are orientation and distance independent. However, UAS elements do not function downstream of the TATA box. Thus, their function is restricted by their location relative to the TATA box (176,177). UAS elements, which bind transcription factors, facilitate assembly of the PIC directly by forming protein–protein interactions with GTFs, or indirectly by complexing with coactivators. Upstream repressor sequences (URSs) use several approaches to disrupt formation of the PIC. They can interfere with the activation domain of the activator complex, disrupt interaction with the core promoter factors, or recruit corepressors (e.g., Sin3-Rpd3, HDACs). Homopolymeric dA-dT sequences are required for normal levels of transcription. The repetitive dA-dT sequence has intrinsic structural ability to impair nucleosome assembly or stability (178,179). Models describing the formation of the Pol II initiation complex are constantly evolving and essentially involve the convergence of information gathered from biochemistry, structural biology, and genetics, particularly yeast genetics (148,180,181). Elucidation of the three-dimensional crystal structure of the TBP has advanced our understanding of preinitiation assembly complexes (182,183). Protein folding of TBP into a β-sheet forms a “saddle-shaped” concave surface of sufficient size to contact helical DNA (Fig. 1-6A). On the opposing convex surface are potential binding sites for various regulatory proteins, for example, TBP-associated factors (TAFs), GTFs, and Pol II (see Fig. 1-6A). At least 10 to 14 different human TAFs have been identified from HeLa cells, with their molecular weights ranging from 18 to 250 kDa (167,184,185). TAFs are multiple subunit proteins that associate with TBP to form the essential transcription factor TFIID. The proteins are conserved from yeast to humans with the bulk of our understanding of these factors coming from experiments in yeast and Drosophila. An interesting finding is that TAFs are not universally required for

transcription, but each one is required for only a subset of genes. Thus, for example, one TAF is required for transcription of 8% of genes, whereas three different TAFs are required for 60% of transcribed genes. In addition, TAFs are found in protein complexes other than with TBP. In fact, some TAFs have HAT activity, whereas others are similar to histones. Still other TAFs (e.g., TAFII250) have numerous enzymatic features including ubiquitin-conjugating activity (186). The conclusion from these studies is that TAFs are involved in promoter selection through yet to be defined mechanisms (185). TBP is not specific to Pol II promoters, but also forms PICs at the start site of Pol I and III promoters, as well as Inr promoters that do not contain TATA elements (167,187,188) (see Fig. 1-6B). For example, in Pol I promoters, TBP does not bind DNA directly in a sequence-specific manner, but instead forms protein–protein interactions with the selectivity factor complex (SL1) and the upstream binding factor (UBF) (189). In Pol III promoters, TBP complexes with TFIIIB and TFIIIC (190). In TATA-dependent and -independent Pol II promoters, TBP forms protein–protein interactions with spatially constrained upstream activators that bind DNA; for example, CCAAT-enhancer binding protein (C/EBP) and Sp1. Thus, TBP forms the core of the PIC through both DNA– protein and protein–protein contacts in TATA-dependent promoters but primarily protein–protein interactions in nonTATA promoters (see Fig. 1-6). Apparently, the selection of a promoter by TBP preceding the assembly of the PIC is determined by the type of accessory factors recruited (TAFs, SL1, Sp1, TFIIIC) (188,190,191). Moreover, this recruitment may be regulated by temporal and tissue-specific influences. Inhibition of transcription (repression) may occur simply by preventing one of these general TAFs from participating in the assembly of the PIC (182,187). Like Pol II itself, many TAFs and GTFs are composed of multiple subunits. Thus, there is an enormously complex pattern of assembly of proteins (TBP + TAFs = TFIID, other GTFs, and upstream activators) on specific DNA elements at the promoter (e.g., TATA, INR, UAS) that results in the initiation and elongation of mRNA (182,188,192). Other GTFs besides the TFIID complex include TFIIA, TFIIB, TFIIE, TFIIF, TFIIG, TFIIH, TFIII, TFIIJ, and TFIIK (160,167,193–195). There appears to be a strict requirement for these factors to assemble at the promoter in a specific order (181,182,192). TFIID binds to the TATA elements first, followed by protein–protein interactions of TFIID with TFIIA and TFIIB. The 12-subunit Pol II binds next. TFIIF is then recruited to the TFII-diaminobenzidine complex and facilitates binding of other general (basal) transcription factors E, J, H, and K. Many of these basal factors do not bind DNA directly (e.g., TFIIB, TFIIE, TFIIF), but instead form bridging complexes between the general Pol II transcriptional machinery and TAFs with specific upstream regulators. GTFs are required for the basal activity of the promoter, whereas UAS enhancers are dispensable. Specific functions of some of the GTFs have been elucidated. For example, the larger subunit of TFIIF (Rap74)

TRANSCRIPTIONAL AND EPIGENETIC REGULATION / 11

TBP

TAF

AC

T GF

TBP TAF

GF DNA (TATA)

POL

ACT

TAF

POL

GC-RICH

A

B FIG. 1-6. Schematic diagram of a polymerase (POL) II initiation complex. The saddle-shaped TATA-binding protein (TBP) (A) binds DNA directly at the TATA sequence and (B) is tethered between TBP-associated factors (TAFs) in non-TATA promoters. Thus, TBP forms the core of a complex consisting of TAFs, general transcription factors (GF), upstream activators (ACT), and ribonucleic acid (RNA) POL I, II, and III. (Modified from Comai and colleagues [187], by permission.)

functions as an ATPase-dependent helicase to unwind DNA ahead of the transcription complex (196). TFIIF appears to play a role in promoter stability rather than selectivity. TFIII, a helix-loop-helix (HLH) protein, binds preferentially to Inr promoter with or without TATA elements and cooperates with upstream regulatory factors and the general transcription complex (197). TFIIH is one of several C-terminal domain (CTD) kinases that phosphorylates the CTD of Pol II to signal elongation of the nascent mRNA chain (192,194). Other kinases are now known to phosphorylate CTD (198). Certainly, all genes are not transcribed concurrently; thus, the cell must have various mechanisms for silencing genes either permanently or in response to extracellular cues. The mechanisms for repressing genes may be general (e.g., DNA methylation [199,200]; see also #1375 in Bird [201]) or sequence-specific (202). Alternatively, loss of the ability to inhibit transcription of a gene (derepression) may permit certain cellular functions to proceed unchecked. Examples of the interaction between positive and negative regulators occur during cellular proliferation and differentiation (203). During fetal development, most cells are in the process of rapid proliferation. This period is followed by one of regulated differentiation during which the genes controlling proliferation are repressed. However, proliferative pathways may be derepressed (reactivated) during periods of organ repair or during neoplastic transformation (203,204). Examples include the reexpression of fetal proteins during liver regeneration and neoplasia (e.g., α-fetoprotein) or GI mucosal neoplasia (e.g., carcinoembryonic antigen) (205–207). Negative promoter elements or repressors may serve as the binding sites for proteins that sterically hinder the binding of GTFs (e.g., TFIID) or upstream activators (e.g., Sp1) critical in the formation of the Pol II transcription PICs (DNA–protein interactions). Alternatively, proteins

responsible for gene repression may act by preventing the recruitment of required general or accessory factors (e.g., TFIIB or TAFs) to the bound PIC (protein–protein interactions) (202). The DNA elements CCAAT and GGGCGG, which bind the nuclear proteins C/EBP and Sp1, respectively, are examples of promoter-activating elements that are distinct from the TATA box (151). These upstream promoter elements are distinguished from the TATA element in that mutation or removal of these UASs reduces basal promoter activity without completely eliminating it, whereas mutation or elimination of the TATA sequence completely abolishes transcription. DNA elements that function independently of their position on the gene or their orientation (3′ to 5′ or 5′ to 3′) are called enhancers if they bind nuclear proteins that activate transcription and silencers if they bind nuclear proteins that inhibit transcription (208–210). Many of these enhancer and silencer elements occur far upstream within the 5′ flank, but they may also occur within introns, exons, or 5′ or 3′ untranslated sequences. To identify cis-acting enhancer elements, constructs are made by ligating the regulatory elements to be studied in front of a functional promoter expressing a gene encoding a protein or enzyme that is easily assayed. Typical reporter genes encode proteins that are not normally expressed by the transfected cell. By systematically deleting portions of 5′ flanking sequence, the transcriptional activity of the promoter under various conditions is altered and the regulatory elements of interest are identified. DNA elements responsible for tissue specificity can be identified by transfecting (transferring DNA into eukaryotic cell lines) cell lines derived from different tissues. Transcriptional initiation from a promoter that requires a particular cis-acting sequence for expression in a specific cell type is diminished or abolished if this sequence is eliminated or mutated.

12 / CHAPTER 1 Cis-acting sequences conferring inducible responses are also identified by this method. Alternatively, elements that are only active during development must be identified in eukaryotic systems in which differentiation of a cell line can be controlled, or in transgenic animal models.

DNA-Binding Proteins DNA-binding proteins are also referred to as trans-acting factors by virtue of their ability to bind to the 5′ flanking regions of genes in a sequence-specific manner and regulate transcription (211–213). The term trans was coined to acknowledge that the protein product of one gene regulates the transcription of a different gene. With the genes for several hundred trans-acting factors now cloned, the study of their primary and secondary amino acid structures has demonstrated characteristic protein domains (214,215). In general, these proteins contain specific DNA-binding, transactivation, and oligomerization domains (Fig. 1-7). The amount of a transcription factor binding to a particular sequence initially is considered to be the primary mechanism of control. However, it is now clear that the proteins themselves are regulated by a variety of mechanisms in addition to controlling their levels in the nucleus and include activation or inactivation by proteolysis (e.g., NF-κB), covalent modification (e.g., phosphorylation, acetylation), and ligand binding (e.g., steroid receptors), in addition to regulating translocation

ACTIVATION

to and from the nucleus and transcriptional induction or repression of the trans-acting factor (216). The DNA-binding domain is the portion of the protein that contacts DNA in a sequence-specific manner. However, flanking amino acids may also influence DNA-binding through noncovalent interactions. Examples of four major designs for DNA-binding domains are proteins with a helix-turn-helix domain, “zinc finger” domains, amphipathic helices (e.g., basic-zipper [bZip], HLH), and β-ribbon (prokaryotic proteins) (215) (Fig. 1-8). Most of the proteinDNA contacts occur in the major groove through noncovalent interactions (e.g., hydrogen bonds, hydrophobic interactions, and van der Waals interactions). An α-helical structure appears to be a common motif used in the formation of the DNA-binding domain. The helix-turn-helix motif was initially identified in prokaryotic DNA-binding proteins, but similar motifs have now been identified in the homeodomains of eukaryotic transcription factors (217–219) (see Fig. 1-8). Homeobox factors are a class of DNA-binding proteins that predominantly play a role in the developmental expression of genes. Their discovery arose from the idea that developmental regulation involves control of gene expression by a few regulatory transcription factors called “master switch genes” (220). These DNA regulatory proteins initially were identified in simpler organisms such as the roundworm Caenorhabditis elegans (C. elegans) or Drosophila, in which the genetic development from the single-cell stage to maturity is well defined.

DNA BINDING

OLIGOMERIZATION

VA TI AC

IZA

Methylation

ER OM

Acetylation

TI

Glycosylation

Amphipathic helices e.g., leucine zippers helix-loop-hellix helix-span-helix

IG OL

ON

Phosphorylation

TIO

Sumoylation

N

Glutamine-rich proline-rich acidic domain

DNA BINDING e.g., helix-turn-helix, zinc fingers, basic domains

FIG. 1-7. DNA-binding protein domains. DNA-binding proteins have three functional domains: (1) a surface to bind DNA, (2) a surface to interact with other proteins containing similar oligomerization (dimerization) motifs, and (3) a surface to interact with the signal transduction pathways or the preinitiation complex. Examples of motifs associated with these domains are listed.

TRANSCRIPTIONAL AND EPIGENETIC REGULATION / 13

DNA-binding domain 22 15 8 1

Leucine 5

4

7

Helix-turnHelix

2

3

A

Leucine-zipper domain

6

bZip

B

C

DNA-binding domain

++++

++++

Helical regions

Loop

Activation

Basic

L

F C Helix Span Helix

H

C

H

C

Zn

Dimerization

C

L

F

H Zn H

DNA binding bHSH

bHLH

D

E

Zing finger

F

FIG. 1-8. Example of DNA-binding motifs. (A) Helix-turn-helix. (B) Amphipathic helix formation. An α-helical structure is formed so that all leucines or hydrophobic amino acids line up at one surface. Shown are (C) a leucine zipper adjacent to basic DNA-binding domain (bZip); (D) a helixloop-helix adjacent to a basic DNA-binding domain (bHLH); (E) and a helix-span-helix adjacent to a basic domain (bHSH). (F) A zinc finger motif of the C2H2 type with tetrahedral coordination of a zinc ion between two histidines and two cysteines. (Modified from Berg [241], Falke and Juliano [255], Ellenberger and colleagues [266].)

Through site-directed mutagenesis studies, a specific protein domain required to effect developmental progression of these organisms was identified. This domain shared significant homology with a region within proteins controlling cell lineage in the pituitary (Pit-1) and immune system (B-cell octamer proteins, Oct-1 and -2) (221–225). These proteins also shared significant homology with the C. elegans “homeotic gene,” unc-86. Thus, the ~60- to 75-amino-acid region of shared homology was renamed the “POU” domain after the three proteins Pit-1, Oct-1, and unc-86. Initially, the POU domain was named without knowledge of function or the ability to form specific secondary protein structure. Although some bind similar AT-rich consensus DNA-binding sites (octamer proteins bind an eight-nucleotide sequence ATTTGCAT; Pit-1 [also called GHF-1] binds a nine-nucleotide consensus site TATATATNCAT), others do not (Drosophila eve protein recognizes TCAGCACCG) (217). Mouse homeobox genes have nomenclature based on their similarity to

Drosophila homeobox genes (e.g., caudal, forkhead) and have been associated with control of gut development (226–228). In fact, homeobox genes have emerged as critical regulatory factors in the development of both the luminal GI tract and pancreas (229,230). Homeobox genes in the luminal GI tract are related to the 39-member Hox gene family of transcriptional regulators that control anterior-posterior patterning, and they are related structurally to the Drosophila Antennapaedia gene (229). Hox genes are so strongly conserved in evolution that this cluster of genes has been repeated four times in mammals on different chromosomes (231). Collectively, the replicated genes are called Hox clusters and are expressed primarily in either the mesoderm or ectoderm (e.g., skin, muscle, neural tissue), but not in endodermal tissues. Rather, an evolutionarily related cluster of homeotic genes call the Para-Hox genes appear to play the more important role in endodermal tissue, and therefore gut patterning (232). These genes include Pdx1, which is

14 / CHAPTER 1 essential to the correct development of the pancreas and duodenum (233,234), and the genes related to the Drosophila caudal gene, Cdx1, Cdx2, and Cdx4 (229,235). Cdx2 is not only relevant to development of the luminal gut, but it also is an indicator of neoplastic transformation, especially in the upper GI tract (236,237). The forkhead family of homeotic genes is another group of transcriptional regulators with important implications in the gut because of their role in GI cancers (238,239). There are at least 43 members of the forkhead family spread over three chromosomes. The “winged helix” motif of the forkhead DNA-binding proteins is a variant of the 60-amino-acid homeodomain helix-turn-helix because it has additional peptide domains that have been described as “wings” (240). The forkhead transcription factors are downstream targets of the hedgehog pathway, which is an important developmental signaling cascade originally described in Drosophila (see Chapter 9 for a more detailed discussion). The zinc finger motif is distinguished by the occurrence of cysteine and histidine resides tetrahedrally coordinating a zinc ion (241–243) (see Fig. 1-8F). Two subcategories of zinc finger proteins have been identified: those regulatory proteins in which only cysteine contacts the zinc ion (e.g., the steroid receptor family, GAL4 [244,245]), and those in which both cysteine and histidine residues are involved (e.g., Sp1 and Zif 268 [245,246]). The X-ray crystallographic structures of several zinc finger and helix-turn-helix proteins have now been identified, with identification of more structures still to follow (247–253). Through crystallographic studies and computer modeling, investigators have been able to identify which amino acids within the DNA-binding domain contact particular nucleotides within the DNA element. It is anticipated that most of these interactions will be defined sufficiently well to predict protein–DNA contacts at the molecular level for other trans-acting factors. In the future, this will facilitate the targeting of specific transcription factors (natural or synthetic) to specific promoter sequences (248,254–257). Landschulz and coworkers (258) originally described the bZip/coiled-coiled DNA-binding motif as a dimerization domain (see Fig. 1-8). However, this motif, which consists of 55 to 65 amino acid residues, actually forms two domains: one for dimerization and a second for DNA binding (259). Seven repeating leucine residues forming an α-helical coil compose the dimerization domain (Zip domain) (see Fig. 1-8). Immediately adjacent to the Zip domain, toward the amino terminus, lies the basic/hydrophobic domain (b domain) (215). Thus, the bZip family of proteins, the first of the amphipathic helices to be described, must dimerize to form a complete DNA-binding domain (260,261). Other transcriptional regulatory proteins containing the same heptad repeat are able to dimerize with each other to form a “coiled coil” (262). For stable binding to DNA to occur, some bZip proteins prefer that each dimerization partner be the same (e.g., CREB, C/EBP, or general control of amino acid synthesis 4 [GCN4] homodimers [see #564 in Pu (260); 263,264]), whereas other bZip proteins form more stable complexes as heterodimers (e.g., Fos/Jun), although lower

affinity binding is also possible as homodimers (e.g., Jun/Jun) (265). The first report of a crystal structure for a bZip protein, the yeast transcription factor GCN4, confirmed the predicted model of two α-helical coils, which merge into diverging b domains that straddle and grip the major groove of DNA like “forceps” (266). Other amphipathic helices, which combine a dimerization domain with a basic DNA-binding domain, have been described; however, less is known about their threedimensional structure. The helical domains contain hydrophobic amino acid residues arrayed in an α helix so that they are clustered on one face of the helix, whereas hydrophilic residues reside on the opposing face (see Fig. 1-8). According to thermodynamic principles, the hydrophobic face is sequestered away from the aqueous environment by noncovalent interactions when they dimerize with similar domains on other proteins. In addition to the bZip model described earlier, the HLH and helix-span-helix (HSH) motifs were coined to describe other subclasses of amphipathic helices, albeit with longer linker sequences between the two α helices (267–269) (see Fig. 1-8). In the case of the leucine zipper, the hydrophobic face is formed by a series of leucine residues spaced seven amino acids apart (258). In contrast, the HLH and HSH proteins use a variety of different hydrophobic amino acids in addition to leucine to form two amphipathic α helices separated by a stretch of amino acids (“loop or span”) that do not form a helix. Like the bZip family, HLH and HSH regulatory proteins bind DNA through an adjacent basic domain. Thus, bZip proteins (e.g., CREB, activator protein 1 [AP1], activating transcription factor [ATF], Fos, Jun) are potentially interchangeable partners within homodimeric or heterodimeric complexes with the corresponding ability to recognize a greater repertoire of DNA-binding elements (270–272). For example, the Fos/Jun-binding site differs from the CREB/ ATF-binding site by 1 bp: CREB/ATF binds TGACGTCA, whereas Fos/Jun binds TGAGTCA. Likewise, the bHLH proteins that recognize the CANNTG consensus binding site are also able to complex with each other (273). Currently, there are three family members of the transcription factor AP2, which are the only members of the bHSH family (269, 274,275). An HLH protein without the basic DNA-binding domain called Id was cloned (276). This protein has been shown to combine with three bHLH proteins (MyoD, E12, and E47) and to prevent the formation of normal homodimers or heterodimers, thereby functioning as a dominant negative mutant. Similar types of negative regulatory proteins have been identified for bZip proteins (277,278). Therefore, the combinatorial ability of transcription factors permits flexibility in responding to extracellular signals at the level of DNA–protein and protein–protein interactions. The transactivation domains of regulatory proteins consist of predominantly acidic, basic (glutamine), or proline residues (152,279). These non-DNA-binding surfaces interface with signal transduction pathways and other proteins, but their specific function is not completely understood. Domains with a high degree of acidic charges are thought to represent important contact points for interaction with the Pol II PIC

TRANSCRIPTIONAL AND EPIGENETIC REGULATION / 15 (e.g., Gal 4, VP16). Ptashne (280) coined the phrase acidic blobs to describe such negatively charged trans-activating domains. Glutamine-rich (Sp1) and proline-rich (C/EBP, CTF) domains also presumably cooperate with the transcriptional machinery through protein–protein interactions (160,184,281–284). However, it has more recently been confirmed that transcription factors form protein–protein interactions with other transcription factors not within the same DNA-binding domain family. The most common transcription factor exhibiting this property is Sp1. Sp1 can interact directly with other transcription factors, for example, YY1, Smads, or Jun family members (285,286). A functional interaction between cJun and Sp1 has been shown to mediate epidermal growth factor activation of lipoxygenase gene expression (287). Presumably, the “acidic blob” in the transactivation domain of Sp1 creates a “sticky” surface on which new partnerships are formed at various promoters in response to a variety of extracellular signals. Likewise, Smad proteins, which mediate TGF-β signaling, are also promiscuous in their ability to partner with other transcription factor family members (288,289). Although at one time undetected, protein–protein interactions among transcription factors are now recognized as common occurrences, particularly because there are convenient means to identify the interactions genetically through two-hybrid cloning methods, or biochemically using affinity chromatography, immunoblot assays, and mass spectroscopy. Many of the mechanisms involving transactivation of transcription factors involve protein phosphorylation and dephosphorylation (290). Phosphorylation by protein kinases occurs at serine, threonine, or tyrosine amino acid residues. Several classes of protein kinases exist within the cell; however, the best studied are the protein kinase A (PKA) and C (PKC) pathways. PKA is activated indirectly by the catalytic subunit of adenylate cyclase. Signals that increase intracellular cAMP will activate PKA (291,292). In contrast, PKC is activated by calcium released from intracellular stores and by the phospholipid diacylglycerol (293). Phospholipase Cγ catalyzes the hydrolysis of phosphatidylinositol to diacylglycerol. The tumor promoter 12-0-tetra-decanoyl phorbol-13-acetate (TPA) is a lipid-soluble compound that mimics diacylglycerol and directly activates PKC. Hundreds of additional protein kinases within both the cytoplasm and the nucleus exist that may be implicated in the specific phosphorylation of transcription factors (294). Ligand binding triggers a variety of different activation pathways that appear to result in the direct phosphorylation of transcription factors by protein kinases other than PKC and PKA; for example, casein kinase II (CKII), glycogen synthase kinase III, and several DNA-dependent protein kinases (295–297). Direct phosphorylation of the DNA-binding protein may result in a conformational change that enhances its ability to induce transcriptional activation (e.g., CREB, cJun, C/ EBP-β) or inhibition (e.g., yeast protein A[298]DRI) (290). Alternatively, phosphorylation of an inhibitory subunit may release the transcription factor from an inactive state (e.g., NF–κB) (299–303). Phosphorylation can also regulate the

ability of a protein to dimerize, thereby broadening or narrowing the repertoire of DNA sequences that are recognized (e.g., signal transducer and activator of transcription [STAT] and Fos/Jun family) (304,305). The removal of phosphate groups by sequence-specific phosphatases is an additional mechanism by which the transcriptional activity of DNA-binding proteins may be altered (290,306,307). Interestingly, dephosphorylation appears to be a more common mechanism for regulating transacting factor binding than is kinase-mediated phosphorylation (295). Binding of the Jun family (bZip class), homeodomain proteins, and cMyb to DNA is regulated by dephosphorylation. Phosphorylation of sites within or adjacent to the DNA-binding domain of these proteins inhibits DNA binding, whereas removal of phosphates enhances binding. In contrast, activation of DNA binding by phosphorylation has fewer documented examples. One example is the serum-response factor (SRF) that binds to and activates the cFos promoter (308,309). SRF appears to be activated by phosphorylation at sites adjacent to the DNA-binding domain by CKII. This observation is supported by studies involving both mutational analysis of these phosphorylation sites and increasing cellular CKII kinase activity through microinjection of the enzyme into cells (310,311). Although glycosylated proteins are usually observed on the plasma membrane of cells or in the lumen of intracellular organelles, nuclear proteins have been shown to contain O-linked glycosylated residues as well (312). Sp1 represents the prototypical glycosylated transcription factor, the activity of which is enhanced by the presence of carbohydrate residues (312–315). Other eukaryotic transcription factors such as CTF, AP1, and AP4 are also known to be glycosylated, but the effect of the carbohydrate residues on their transcriptional activity is unknown. Glycosylation may regulate the transcriptional activity of individual transcription factors, perhaps by increasing their resistance to proteolysis, by targeting them to the nucleus, by blocking potential phosphorylation sites, or by facilitating their interaction with coactivators (316).

Coregulatory Proteins By the mid 1990s, it became clear that DNA-binding factors were working in a combinatorial manner, not only with other DNA-binding factors, but with non-DNA-binding proteins that were closely linked to chromatin structure and the PIC. These large molecular weight proteins were initially identified as factors interacting with the steroid hormone receptors, which are DNA-binding proteins that translocate to the nucleus after binding hydrophobic ligands in the cytoplasm (317–319). At about the same time, it was discovered that phosphorylation of the cAMP-activated transcription factor CREB induced its interaction with a 300-kDa coactivator protein called CBP. Subsequent to the discovery of CREB, the homologous transcriptional coactivator designated p300 was also identified (320). Coactivators were

16 / CHAPTER 1 found to facilitate transcriptional activation through intrinsic HAT activity, resulting in an “open” chromatin state at the start site of transcription. There are now several of this class of proteins that include PCAF and GCN5 (321). Conversely, the protein complexes that inhibited transcription were multiprotein complexes that recruited histone deacetylators, which, in turn, deacetylate histones returning chromatin to its closed, inactive state (322). The prototype corepressors were identified because of their ability to suppress activation by the retinoid and thyroid hormones (SMRT/N-CoR) (216). It is now known that there are transcriptional corepressors of a variety of signal transduction pathways, including Sin3A, a corepressor of the cMyc bHLH transcription factor family, and PIAS/SUMO, a corepressor of the STAT signaling pathway (323–328). Collectively, these proteins are considered to be coregulatory factors because they do not contact DNA directly as transcription factors do, but rather form protein bridges between the sequence-specific DNA-binding proteins and the Pol II assembly apparatus, bringing with them enzymatic activity, for example, acetylase and deacetylase activity involved in remodeling chromatin (329–331). Currently, there are three broad categories of coactivators (332). p300 and CBP are the prototypes of the HAT class of coactivators. The TRAP/DRIP/Mediator/ARC complex compose the second class and are proteins that bind transcription factors and recruit RNA Pol II without having intrinsic histone modification capabilities. The third class comprises the yeast SWI/SNF and their mammalian homologues BRG1/BRM. This third class of coactivators contains intrinsic ATPdependent DNA-unwinding activity required for efficient in vivo transcription. Coactivators increase the transcriptional activation of a promoter through its interaction with a sequence-specific DNA-binding protein, but it is not yet clear how the coactivator selects one group of promoters over another. Two concepts have been considered (332). For example, a promoter might need a “threshold level” of positive signals to be activated. Alternatively, some promoters might have a greater requirement for the presence of one coactivator than another. The precise mechanisms of transcriptional activation continue to evolve, and certain themes are emerging. In rare instances, positive or negative enhancer activity is dependent on a single DNA-binding protein that functions as a master switch to activate a family of related genes, for example, the myogenin MyoD family in muscle differentiation (333). However, further scrutiny of this model has indicated a large network of transcription factors that interact with non-DNA-binding complexes involved in chromatin remodeling, for example, histone acetyltransferase proteins p300 and CBP (334–337). Therefore, the more common mechanism implies that most cells respond to their environment by recruiting subsets of ubiquitous and promoter-specific transcription factors that combinatorially produce the desired cellular phenotype (204,338-341). Corepressors SMRT and N-CoR both recruit HDACs, yet they mediate activation downstream of different kinase cascades (342). In addition to the recruitment of classic HDAC-associated corepressors

(e.g., mSin3A and Groucho) (343–345), the runt-related transcription factor (RUNX) proteins exert gene silencing by associating with histone methyltransferases (e.g., SUV39H1) (346). Bifunctional attributes of transcription factors have been attributed to their regulated association with either coactivators or corepressors.

METHODOLOGY This section summarizes some of the molecular techniques used to study transcriptional control of genes. These methods are used to study either genetic structure or function. Three systems have been used to study function: reconstituted cell-free transcription assays, cell culture models, and whole-animal studies. Methods that analyze structural interactions include those techniques that assess DNA– protein interactions and those that assess protein–protein interactions.

Functional Methods Reconstituted Transcription Systems The most basic approach to the functional study of a gene is an in vitro transcription system in which the minimal components required for transcription are isolated and reconstituted to produce the gene product (347,348). mRNA is transcribed from cloned cDNA in the presence of radiolabeled nucleotides, RNA polymerase, and accessory factors isolated from nuclear extracts. The radiolabeled RNA synthesized in vitro is resolved by gel electrophoresis after extraction from the cell. Changes in basal levels of transcription are measured by quantifying the amount of newly synthesized RNA transcripts produced in the presence or absence of cloned or purified gene-specific DNA-binding proteins (349). In this way, differences in gene expression attributable to the activity of a purified transcription factor or enriched nuclear fraction may then be studied under tightly controlled assay conditions. Cell Culture Models The study of transcriptional regulation has been advanced greatly by the use of cell lines derived from the same tissues as the endogenous gene of interest. These cell lines have become the vehicles in which the study of gene expression is performed. Two major advantages of using cell lines are that they are homogeneous populations and they continue to divide in minimal culture conditions. However, in many situations, the cell lines are derived from neoplastic tissues, which may have lost the normal regulatory mechanisms that maintain the differentiated state. In a dedifferentiated state, cells tend to express a variety of genes outside of the repertoire expressed by their normal counterparts. Therefore, studies with cell lines always carry the caveat that they may not reflect activities of native cells.

TRANSCRIPTIONAL AND EPIGENETIC REGULATION / 17 The use of cell lines permits the direct study of regulators of endogenous gene expression, avoiding the confounding effects of contaminating cell types. However, this approach does not permit alteration of the regulatory domains of genes to assess their contribution to transcription. Therefore, techniques have been developed to insert altered genetic material into cells by chemical, electrical, or viral mechanisms. In this way, specific elements controlling transcription can be isolated and studied. To tag the inserted gene, the promoter from which transcription will be initiated is ligated upstream of the coding sequences for a reporter gene, for example, chloramphenicol acetyltransferase, β-galactosidase, growth hormone, green fluorescent protein, or luciferase (9,350–353). The products of the reporter gene are easily measured, and spurious detection of reporter gene activity is kept to a minimum because their products are not normally expressed by most mammalian cells. Regulatory sequences to be analyzed are ligated upstream of a promoter with basal transcriptional activity in the test cell line. Taking advantage of various restriction sites, sequentially shorter 5′ flanking sequences are created, and each resulting construct is then tested by assaying the reporter gene product as an indicator of gene expression. Whole-Animal Models Whole-animal studies have been useful in assessing the contribution of transcriptional control to the regulation of several GI peptides, including gastrin, CCK, and somatostatin (354–356). Brand and Stone (357) showed that gastrin mRNA levels in the antrum increase under conditions of chemical or surgical achlorhydria and coincide with a reciprocal decrease in somatostatin mRNA. These observations are correlated with prior observations that gastrin plasma levels increase under conditions of achlorhydria (354, 358). Furthermore, infusion of the somatostatin analogue octreotide blocks the increase in gastrin mRNA (357). Walsh and coworkers (359,360) found that gastrin mRNA levels are predictably regulated by cycles of fasting and refeeding. Recently, infusion of the proinflammatory cytokine interferon-γ into mice has been used to recapitulate the effect of Helicobacter pylori infection on gastrin and somatostatin (361). Similarly, studies on the dietary control of CCK gene expression have been reported (355). Although such studies permit the linkage of transcriptional regulation to physiologic events, they do not allow dissection of the responsible regulatory elements.

genotypes on the overall phenotype may be amplified or abolished. In many situations, these alterations reproduce clinically relevant pathologic states (362–366). Chapter 53 provides specific details on transgenic technology including the powerful technique of homologous recombination. Cell-Based Knockout Strategies Once a genetic target is identified, whether DNA, RNA, or protein, the next step is to determine the significance of the molecule in a particular signaling, developmental, or neoplastic cascade. This usually is done by blocking, reducing, or removing the gene product at the cellular level before applying the extracellular signal. A change in the expected phenotype would confirm that the gene product makes a significant contribution. At the cellular level, the traditional approach has been to use small molecules, for example, pharmaceutical inhibitors. Once DNA vectors were developed in the early 1980s, antisense and dominant negative approaches to inhibit gene expression came into vogue (367). With the emergence of transgenic technology, it became apparent that one could remove the gene product through genetic manipulation specifically by homologous recombination to disrupt the gene in mice (362,368). With the discovery of snRNA molecules that interfere with either transcriptional initiation or translation, the commercial availability of synthetic “interfering” RNA molecules has emerged (369,370). High-throughput methods using RNA silencing are now being used to complement the gene discovery methods of DNA microarray technologies (371). Nevertheless, RNA interference technology, although relatively easy to use, does not eliminate the gene product as effectively as direct gene targeting. Therefore, genetic methods must be used to generate a complete null cell line. Cell lines are either created from a null mouse model (e.g., embryonic fibroblasts), or somatic cell gene targeting can be performed in the cell line of choice (372,373). The advantage of creating the null cell line from a mouse is that the cells will be from normal tissue and not a tumor cell line. However, unless molecules are introduced to immortalize the cells, the lines are not permanent. Gene targeting in a somatic cell line has not been as widely used because of the difficulty in performing the technique, but it is a powerful approach that permits the study of a null locus without incurring the expense of mice.

Structural Methods Transgenic Animals Through transgenic animals it is possible to introduce genetic information into the mouse genome such that there is permanent alteration of the genetic makeup in both the founder line and successive generations (13,14). Transgenic studies afford the opportunity to study the importance of specific genetic sequences in cell, organ, and whole-animal function. By breeding mice with different transgenic lineages, the interaction between these artificially produced

Once functional regulatory DNA elements have been identified, assays that assess DNA–protein interactions are performed (374). Indeed, in circumstances where a long sequence (>50 bp) must be analyzed, it is simpler to identify DNA–protein interactions first, and then determine whether these DNA elements are involved in transcriptional regulation. DNase I footprinting assays are used to identify DNAbinding elements that interact with crude or purified nuclear proteins by protecting them from chemical or enzymatic

18 / CHAPTER 1 cleavage (375,376). Such assays are particularly well suited for studying cooperative interactions among proteins bound to adjacent DNA elements. The technique can be performed in vivo or in vitro (9). However, in vivo footprinting has been superseded by chromatin immunoprecipitation (ChIP) assays (see the next section). Electrophoretic gel mobility shift assays (EMSAs; gel shift, gel delay, or band-shift assays) permit a more detailed analysis of the following: (1) the type of protein complexes that bind to individual DNA elements, and (2) the specificity of the protein interaction with a specific base pair (377–379) (Fig. 1-9). This assay system is also rapid and easier to use than footprinting assays. Methylation interference assays extend the power of the gel shift assay by identifying specific nucleotide contacts that are required for DNA binding (380). DNA affinity precipitation is a DNA– protein interaction assay that uses the biotinylated DNA binding site to identify the proteins that are recruited to the element (381). The assay uses the DNA element to isolate the protein factors, coupled with immunoblots to identify the proteins that form both the protein–DNA and protein– protein interactions. Southwestern blot analysis takes advantage of specific DNA elements that are used to detect nuclear proteins separated on a denaturing gel and transferred to nitrocellulose or produced by a phage expression library (382–384).

Chromatin Immunoprecipitation Assays ChIP analysis is now the most effective method to document an in vivo interaction at DNA (385–387). First, a fixative, usually formaldehyde, is used to cross-link proteins to DNA. Then antibodies are used to immunoprecipitate the DNA-binding proteins. After a series of extractions to remove the protein from DNA, specific primers are used to PCR amplify the DNA-binding element precipitated with the protein and antibody. Variations of this method are used to identify the in vivo preferred binding sites of known DNA-binding proteins. Alternatively, the immunoprecipitate is resolved on a sodium dodecyl sulfate gel, and mass spectroscopy can be used to identify the proteins that coprecipitate and are likely involved in protein–protein interactions with the DNA-binding proteins. The technique completely depends on the quality of the antibodies, the quantity and quality of genomic DNA precipitated, and primer specificity. ChIP assays complement in vitro DNA–protein interaction assays such as EMSAs or footprinting. Expression vectors or cell-based knockout strategies using dominant negative constructs, antisense technology, or RNA interference may be used to demonstrate functional significance (388). These approaches are rapid and useful to perform before using transgenic mouse approaches.

5 ′ GTCCCTCAGGGGCGGGGATCTAT

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FIG. 1-9. Electrophoretic mobility shift assay (EMSA, gel shift). A DNA element ~30 to 100 bp in length is labeled, and then is incubated with crude nuclear extract or purified protein. A band on the autoradiogram is detected if the radiolabeled probe is retarded and does not migrate to the bottom of the gel. The specificity of binding is determined by competing with unlabeled DNA sequences. Competitor 1 is related to the probe sequence, whereas Competitor 2 is unrelated to the probe sequence.

TRANSCRIPTIONAL AND EPIGENETIC REGULATION / 19 Microarray Technology The latest method to comprehensively analyze gene expression is by microarray technology. At the transcription level, DNA array technology increases by several orders of magnitude the number of genes that can be examined simultaneously under different conditions (389–391). The number of genes that are either stimulated or inhibited under various conditions can be studied simultaneously with the limitations being the number of genomic sequences that are spotted on the glass slide. A glass slide is able to hold the genomic sequences of 25,000 to 30,000 genes, which is the current estimate of the total number of genes in the human genome. Two types of arrays are available: EST/cDNA and oligonucleotide (Affymetrix, [Santa Clara, CA]) based. The EST microarray chips use expressed sequence tags that are fragments of DNA corresponding to segments of the genome that encode mRNA. The Affymetrix gene chips spot commercially designed oligonucleotide sequences. These DNA fragments are subsequently “arrayed” onto glass slides. In most instances, several regions of the genomic sequence unique to that gene are spotted in multiple copies to ensure reproducibility. Different genetic domains are plated because of differences in hybridization affinity. RNA is isolated from cells or tissue after treatment with an extracellular molecule or from cells at different stages of development or transformation. cDNA are then generated and tagged fluorescently, then hybridized under stringent conditions to the DNA arrayed on the glass slide followed by analysis by a special plate reader. Computer-generated algorithms are required to interpret the fluorescent signals and rank the degree of change from baseline fluorescence. The technology is being used to study the gene expression pattern found in various tissues at designated stages, for example, developmental or transformation stages (392–394). The significance of the findings must be confirmed by alternative methods including Northern blot analysis or quantitative PCR. Proteomics Analogous high-throughput approaches have been developed to study protein modifications (395). However, the techniques used to detect protein posttranslational modifications are more complex and use more labor-intensive technology. Protein is extracted from the cell or organelle of interest and resolved by two-dimensional gel electrophoresis, in which proteins are separated by both size and ionic charge (along a pH gradient). The proteins are visualized with a dye either directly on the gel or after transfer to a paper substrate. Both substrates (gel or paper) can be used for further analysis. However, proteins transferred to a paper substrate permit several options for analysis. Resolved proteins that are transferred to paper can be submitted for analysis with an antibody (immunoblot) that might recognize phosphorylated or acetylated peptides. Differences in the size of the spot corresponding to the amount of a particular protein version (phosphorylated, acetylated) can be

quantified by computer. Proteins that cannot be identified by antibody can be analyzed by mass spectroscopy. Therefore, proteomic studies allow the monitoring of regulatory changes that occur because of posttranslational modifications and quantification for large numbers of proteins simultaneously. Taking advantage of the technology used to develop DNA arrays, companies are now developing protein arrays that will be applied to new drug discovery (396).

TRANSCRIPTIONAL CONTROL OF GASTROINTESTINAL PEPTIDES Although knowledge in the transcriptional control of GI peptides has accelerated over the last several years, the field is still hampered by the paucity of gut-derived cell lines that express regulatory peptides. The problem has been circumvented somewhat through the use of neural and endocrinederived hormone-producing cell lines, but application of data obtained with these models to the gut requires assumptions that may not be accurate. Future work in this field will be assisted greatly by the application of high-throughput and transgenic technologies and the development of immortalized and transformed cell lines using in vitro DNA transfer techniques. An overview of what has been accomplished with respect to specific GI peptides can be found primarily in Chapters 4 through 6. Nevertheless, a few peptides deserve brief mention. To date, most studies of the transcriptional control of peptide hormones have focused on somatostatin and vasoactive intestinal peptide because they are expressed in islet or neural-derived cell lines (397–400). The downside of this is that little is known about how somatostatin is regulated in gut-derived tissues; for this reason, the peptide should become a priority for future transcriptional control studies in the GI tract. Studies on the transcriptional control of gastrin have been slow for similar reasons and have been reviewed recently (401). Information on the transcriptional control of secretin and CCK has increased because of the use of transgenic mouse models (25,402–404).

POSTTRANSCRIPTIONAL PROCESSING Polyadenylation Three major events occur at the end of transcription: (1) The poly(A) tail is added, (2) adenine bases are methylated, and (3) hnRNA is processed by removing introns before exiting the nucleus (see Fig. 1-2) (405). All mRNA, except those encoding most histone proteins, have poly(A) tails. The length of the poly(A) tail that is added ranges from 200 to 250 bp and is quite uniform among eukaryotic organisms. Once the transcript reaches the cytoplasm, the length of the poly(A) sequence decreases with the age of the transcript (406). Thus, polyadenylation contributes to mRNA stability and translational activation, processes that also involve a synergistic interaction with the cap site (407–409).

20 / CHAPTER 1 Because there is no poly (dT) sequence within DNA, addition of the poly(A) tail represents a posttranscriptional modification of the newly synthesized mRNA. The AATAAA site in DNA is transcribed as AAUAAA and signals endonuclease cleavage of hnRNA ~20 bp after this RNA element (410). Several factors are required for specific recognition of the AAUAAA element before the addition of adenylate residues by poly(A) polymerase (411,412). Polyadenylation occurs in two phases: (1) an AAUAAA-dependent phase marked by addition of the first 10 residues, and (2) an AAUAAA-independent phase marked by rapid elongation and catalyzed by a poly(A)-binding protein (413). In addition, endonuclease cleavage of polyadenylated histone H1 transcripts have also been shown to require the presence of small nuclear ribonucleoproteins (U7 snRNP, pronounced “snurp”), which are trans-acting factors that participate in RNA splicing reactions (414). Transcription can proceed for up to 2 kb past the polyadenylation site and may terminate prematurely 30% of the time. Adenylate residues within exons are methylated at the sixth nitrogen and are thought to serve a protective role for those sequences that will eventually be translated (415). It is now known that formation of the PIC is linked to the assembly of factors involved in polyadenylation (416).

RNA Splicing The Spliceosome Soon after the termination of transcription, most vertebrate hnRNA (pre-mRNA) will be posttranscriptionally processed after exiting the nucleus into a form that can be translated (see Fig. 1-2). This involves removing intervening sequences that in some transcripts contain transcriptional regulatory signals (cis-acting elements). Splice sites are identified by comparing the genomic sequence with the cDNA prepared from an RNA template. The cis-acting elements within the intron that regulate RNA splicing are GU (GT in the genomic sequence) at the 5′ splice border, AG at the 3′ splice border, and a pyrimidine-rich element that defines the area of the branch point 20 bp upstream from the 3′ splice junction (Fig. 1-10). The branch point lies just upstream of the pyrimidine-rich region (PyPy)n and is a highly conserved sequence in yeast (UACUAAC) but much less so in vertebrates. Five snRNA-U1, U2, U5, U4, and U6-combine with subsets of about 10 different proteins to form small nuclear ribonucleoproteins (snRNPs) (417,418). The snRNA, ranging in size from 56 to 217 nucleotides, are quite abundant in the nucleoplasm and contain a trimethylguanylate cap. Some proteins are components of all five major snRNPs, whereas others are unique to one snRNP. The U7 snRNP, which is present in low concentrations, participates in the 3′ posttranscriptional processing of hnRNA [poly(A)] (419). The five major snRNPs assemble into large multicomponent complexes called spliceosomes to perform the splicing reactions (420). There reactions occur in three steps: cleavage

at the 5′ exon-intron border with formation of a branch point, excision of the branch point as a lariat, and joining of the exons. Splice site selection can be influenced by subtle changes in flanking exon sequences (421–423). The basic steps in RNA processing illustrated in Figure 1-10 are as follows (419): U1 snRNP binds in a sequence-specific manner to the 5′ exon-intron junction of capped pre-mRNA (424). An U2 snRNP accessory factor (U2AF) then binds to the pyrimidine-rich element before sequence-specific recognition of the branch point element by U2 snRNP (425,426). The 5′ exon is released by cleavage of the 5′ exon junction. This allows the freed 5′ guanylate residue to form a phosphodiester bond at the 2′ site of an adenylate residue within the branch point. U4 and U6 snRNPs are paired together by complementary bases and function as a single snRNP complex (427). The recruitment of the U4/U6 snRNPs to the spliceosome is essential to the last excision step and final removal of the intron from the pre-mRNA. U4/U6 snRNP cooperates with the U2 branch point complex without direct contact with RNA (428). U5 snRNP binds just upstream of the 3′ splice junction to initiate cleavage of the 3′ intron border. Finally, the intron is removed as a lariat and the two exons are joined. More recent evidence indicates that small RNA catalyze the splicing reactions without the presence of specific enzymes (429,430). As observed for polyadenylation, the splicing events coincide with transcriptional events (431). It is therefore somewhat surprising that the events involved in splicing are not better understood. Nevertheless, with the understanding that the complexity of the human genome lies beyond the DNA sequence and at the level of epigenetics and alternative splice products, the next decade will likely witness heightened attention to this additional nuclear process (431,432). Alternative Splicing Eukaryotic cells have applied the mechanics of RNA splicing to generate the protein diversity necessary to meet their multiple demands. Thus, in contrast with the original definition of a gene in which only one transcript is produced, complex genes can generate multiple protein isoforms from multiple RNA transcripts through alternative splicing (433). This can be achieved by altering which introns and exons are included in or excluded from the mature mRNA transcript that is used as the template for peptide chain elongation. Accordingly, the definition of introns and exons for each gene is actually a fluid concept because an intron for one gene product may become an exon within another transcript. Alternative splicing is a mechanism used by many protein classes, including muscle-related genes, hormones, and transcription factors (434–438). Regulated Posttranscriptional Mechanisms In addition to cis-acting DNA elements, the cis and trans models of regulation also occur at the posttranscriptional level (439). Ferritin and the transferrin receptor (TfR),

TRANSCRIPTIONAL AND EPIGENETIC REGULATION / 21 Pre-mRNA

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FIG. 1-10. Ribonucleic acid (RNA) splicing reactions. First, small ribonucleoproteins (snRNPs, pronounced “snurps”) and accessory factors (U2 accessory factor [U2AF]) bind in a sequencespecific manner to the branch point and intron-exon borders. Second, the 5′ exon-intron border is cleaved, and a “lariat” is formed by the free end of the intron at the branch point. Third, the 3′ intron-exon border is cleaved, the exons are joined, and the excised intron is removed in the form of a lariat.

which regulate the storage and uptake of iron, are the best known examples of regulated posttranscriptional control (440). Cis-acting RNA elements, responsible for conferring iron regulation on both proteins (iron-response elements [IREs]), reside in the 5′ UTR and 3′ UTR of ferritin and TfR mRNA transcripts, respectively. The same iron-binding protein (IRE-BP) that binds to the IRE in the 5′ UTR of ferritin to block translation can also bind to the 3′ UTR of TfR to block mRNA degradation (439,441,442). Therefore, regulation of iron homeostasis ultimately depends on posttranscriptional mechanisms that either block translation or increase mRNA stability.

TRANSPORT ACROSS THE NUCLEAR MEMBRANE As noted earlier, RNA is synthesized initially as a much larger primary transcript molecule that in many instances undergoes posttranscriptional modification (e.g., splicing,

degradation). However, for any mature RNA transcript to be translated, it must be transported from the nucleus to the cytoplasm. In contrast, nuclear regulatory proteins are translated in the cytoplasm and are eventually returned to the nucleus, either immediately after synthesis or after a dormant state from which they are activated in response to signals (443). This bidirectional shuttling of macromolecules between the cytoplasm and the nucleus occurs through the nuclear pore complex, a specialized compartment of the nuclear membrane regulated by a group of transport receptors called karyopherins. Both import and export processes through the nucleus require energy in the form of the Ras-related GTPase Ran and specific targeting signals on the cargo to be transported (nuclear localization and export signals) (444). The three-dimensional structure of the nuclear pore complex shows a doughnut-shaped structure comprising eight subunits (445). From the eight subunits emanate spokelike structures that radiate inward to form a central plug (446, 447). The cytoplasmic surface of the nuclear pore complex (NPC) is closely associated with ribosomes. Its nuclear

22 / CHAPTER 1 surface is thought to participate in the organization of the genome by binding to specific DNA sequences within transcribed genes with products that may be destined for export from the nucleus (gene-gating hypothesis) (448).

CONCLUSION With the dawn of the postgenomic era on us, our next challenge is to apply the volumes of available genetic, molecular, and cell biological information to tackle questions of GI physiology and development. To accomplish this task and make optimal use of past, ongoing, and future discoveries, physiologists will need to acquire the basic vocabulary of several disciplines including bioinformatics. It is our hope that this chapter has laid the initial foundation necessary to understand those aspects of physiology that pertain to transcriptional control.

ACKNOWLEDGMENTS The work was supported by Public Health Service National Institutes of Health grants DK 55732 and DK 45729 to Dr. Merchant. The authors thank Gail Kelsey and Colleen Hill for assistance with the preparation of both the manuscript and the figures.

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CHAPTER

2

Translation and Posttranslational Processing of Gastrointestinal Peptides Cheryl E. Gariepy and Chris J. Dickinson Translation, 31 Initiation, 32 Regulation of Initiation, 32 Elongation, 34 Termination, 34 Localized Translation Regulation, 34 RNA Silencing, 34 Other Regulators of Messenger RNA Stability, 35 Posttranslational Processing, 35 Transport into the Endoplasmic Reticulum, 35 Signal Peptides, 36 Signal Recognition Particle, 36 Signal Recognition Particle Receptor, 37 Endoplasmic Reticulum Membrane Protein Channel or Translocon, 37 Processing in the Endoplasmic Reticulum, 38 Signal Peptidase, 38 Disulfide Bond Formation, 38 Asparagine-Linked N-glycosylation, 39

Protein N-myristoylation, 39 Protein Folding, 39 Transport from the Endoplasmic Reticulum and through the Golgi, 39 Processing Reactions in the Golgi, 41 Serine- or Threonine-Linked O-glycosylation, 41 O-phosphorylation, 41 Tyrosine Sulfation, 41 Formation of Secretory Vesicles, 42 Processing Reactions in the Secretory Vesicle, 42 Acetylation, 42 Dibasic Cleavage, 42 Monobasic Cleavage, 44 Carboxypeptidases, 45 Aminopeptidases, 46 Glutaminyl Cyclase, 47 Amidation, 47 Posttranslational Processing of Preprogastrin, 48 References, 51

TRANSLATION

as well as energy in the form of guanosine triphosphate (GTP) and adenosine triphosphate (ATP). To be translated into protein, mRNA must contain, in addition to a string of codons, information that specifies nuclear export, translation, and stability. Much of this information is communicated by specific RNA-binding proteins. These proteins first associate with pre-mRNA (primary transcripts of genomic DNA-containing exons and introns) cotranscriptionally and undergo a dynamic series of rearrangements involving the binding and dissociation of numerous proteins throughout the life of mRNA. The mRNA nucleoprotein complex (mRNP) communicates information to the cytoplasm about the structure of the gene from which the mRNA was formed and the processing steps experienced by the mRNA. The mRNP therefore carries significantly greater information than the sequence of the mRNA itself (1).

Translation is the complex process by which a sequence of codons of messenger ribonucleic acid (mRNA) directs the synthesis of a polypeptide chain. Beyond the sequence of codons, the mRNA contains untranslated regions (UTRs) with structural and regulatory sequences that determine its translational fate. Translation involves hundreds of molecules including mRNA, transfer RNA (tRNA), ribosomal RNA, activation enzymes, and many RNA-binding proteins,

C. E. Gariepy and C. J. Dickinson: Division of Pediatric Gastroenterology, University of Michigan, Ann Arbor, Michigan 48109. Physiology of the Gastrointestinal Tract, Fourth Edition, edited by Leonard R. Johnson. Academic Press, 2006.

31

32 / CHAPTER 2 Initiation

Regulation of Initiation

Translation requires the positioning of an elongationcompetent 80S ribosome at the initiation codon (AUG). Binding of the small (40S) ribosomal subunit at the 5′ end of the mRNA is rate limiting and requires energy in the form of ATP. It then forms a 43S preinitiation complex with eukaryotic initiation factors (eIFs) 3, 1, 1A, and 5, and a ternary complex including the methionine-loaded initiator tRNA that will recognize the AUG codon and eIF2 that is coupled to GTP. The preinitiation complex recognizes the mRNA by the binding of eIF3 to the eIF4 protein complex associated with the 5′ cap structure (eIF4F) (2). The eIF4F protein complex contains an enzyme (eIF4A) that unwinds RNA duplexes, allowing the 43S complex to bind and scan the mRNA, and a scaffold protein (eIF4G) that serves as a platform for the assembly of other proteins and interacts with the poly(A)binding protein. This interaction is thought to loop the mRNA and bring the 3′ UTR in close proximity to the 5′ end of the mRNA (3). This provides a means by which sequences in the 3′ UTR can regulate translation initiation. Most known translational regulatory sequences are found within the 3′ UTR. The 43S complex recognizes the initiation codon through the formation of base pairs (bp) between the initiator tRNA and the start codon. Subsequently, eIF2-bound GTP undergoes hydrolysis, a reaction that is necessary for the 60S ribosomal subunit to join the initiation complex. This appears to release most of the initiation factors from the small ribosomal subunit, leaving the initiator tRNA associated with the ribosome (in the P site). Formation of the 80S initiation complex capable of catalyzing the formation of a peptide bond occurs with the hydrolysis of a second molecule of GTP on eIF5B (Fig. 2-1A).

Although the specific translational regulatory mechanisms active in peptide hormone synthesis are not yet clear, translation is generally controlled at the initiation step where regulation may be global or mRNA specific. Global control of mRNA translation generally occurs through changes in the phosphorylation state of initiation factors or regulators that interact with them. Proteolytic cleavage of translation factors can also reduce translation of all mRNA species within the cell. mRNA-specific regulation of translation can be achieved by steric blockage, interference with the eIF4F complex, and cap-independent inhibition of the early initiation steps. Steric blockage refers to the binding of regulatory proteins to message-specific response elements that results in insufficient space for the binding of critical initiation complex proteins (4–7). Interference with the eIF4F complex is achieved by mRNA-specific binding proteins that block eIF4E recognition by eIF4G (8–11). Cap-independent inhibition of translation refers to proteins that bind to specific sites in both the 5′ and 3′ UTRs and recruit corepressors to the 3′ UTR. This affects stable association of the small ribosomal subunit with the mRNA (12–14). Translation can also be controlled later in the initiation process. RNA-binding proteins have been described that prevent the binding of the 60S ribosomal subunit to the 40S subunit at the initiation codon, apparently through interference with initiation factors (15). The existence of more than one open reading frame on an mRNA and the sequence distance between the open reading frames can also play a significant role in determining the likelihood of translation. For example, amino acid deprivation reduces global protein

FIG. 2-1. Translation of messenger RNA (mRNA) into protein, highly simplified. Some translation-initiation factors are omitted (see Preiss and Hentze [526], Ramakrishnan [527], and Hershey [528] for more complete descriptions). (A) Cap-mediated initiation: The methionine-containing ternary complex (methionine-loaded transfer RNA [tRNA], eIF2, and GTP) binds to the 40S ribosomal subunit and other initiation factors (eukaryotic initiation factor-1 [eIF1], 1A, 3, and 5) to form the 43S preinitiation complex. The preinitiation complex recognizes the mRNA through the binding of eIF3 to eIF4 in the cap-binding complex. The cap-binding complex contains eIF4A, an RNA helicase that unwinds the secondary structure of the mRNA during the subsequent scanning step. The cap-binding complex also contains eIF4G, which contacts the poly(A)-binding protein (PABP). This contact is thought to bring the 3′ region of the mRNA in close proximity to the 5′ cap. The 43S preinitiation complex scans the mRNA from 5′ to 3′ until the initiation codon, AUG, is encountered. Stable binding of the preinitiation complex to the AUG codon yields the initiation complex. Subsequent joining of the 60S ribosomal subunit results in the formation of the 80S initiation complex. AUG recognition and the joining of the 60S ribosomal subunit both trigger GTP hydrolysis. The 80S complex contains an aminoacylated initiator tRNA in the P site of the ribosome and an empty A site. It now is competent to catalyze the formation of the first peptide bond. (B) Elongation: A ternary complex containing aminoacylated tRNA and the correct anticodon is brought into the A site of the ribosome. Codon–anticodon recognition leads to guanosine triphosphate (GTP) hydrolysis. This allows for conformational changes within the tRNA and the ribosome. Peptide bond formation (deacylation of the P site tRNA and the transfer of the peptide chain to the A site tRNA) then occurs. Translocation of the tRNA and the mRNA is facilitated by a GTPase, eukaryotic elongation factor-2 (eEF2). The ribosome is then ready for the next round of elongation, with a deacylated tRNA in the E site, peptidyl tRNA in the P site, and an empty A site. (C) Termination: When a stop codon on the mRNA is encountered in the A site, eukaryotic release factor-1 (eRF1) binds to the ribosome A site and triggers the release of the peptide chain from the tRNA in the P site. eRF3 then binds GTP and promotes dissociation of eRF1 from the ribosome. Hydrolysis of GTP is required for subsequent release of eRF3. The ribosome is then left with mRNA and a deacylated tRNA in the P site. The ribosomal releasing factor, together with eEF2 and GTP, is required to disassemble the complex and prepare the ribosome for a new round of protein synthesis. Much of the mechanism of mRNA, translation factor, and subunit release after peptide chain termination remain to be determined. GDP, guanosine diphosphate.

TRANSLATION AND POSTTRANSLATIONAL PROCESSING OF GASTROINTESTINAL PEPTIDES / 33 Met

Met GTP

IF 1,1A,3,5

2

GTP 2

U A C

U A C

40S 43S pre-initiation complex 3′ AAAAAAAAAAAAAAAA ATP

PABP

Met

ADP + Pi GTP

IF4F U A C CAP

5′

AU GC G AU A G

ATP

Scanning

3′ AAAAAAAAAAAAAAAA

2 GDP +2Pi

2 GTP

ADP +Pi PABP

Met

Met

GTP

IF4F U A C CAP

5′

E site P site A site

AUG

U A C C G AU A G

eIF5B + 60S subunit

AUG

C G AU A G

80S initiation complex

A GDP

GTP

Arg

EF-1A

GTP

EF-2

EF-2

GDP +Pi

EF-1A

GCU

Met

Met Arg

Arg

Met Arg GTP

UA C GC U A U G CG AU

AG

EF-2

UA C GC U

UA C GC U

A U G CG A U A G

A U G CG A U AG

B Met Arg

RF-1

RF-1

Met Arg

Met Arg

GTP RF-1 RF-3

RF-1 RF-3 UA C GC U A U G C GA U A G

GC U

GC U

A U G C GA U A G

A U G C GA U A G

RF-3

GTP

RF-3 GDP +

Pi

Factor, tRNA and mRNA release GDP + Pi

GTP RF-3

A U G C GA U A G Ribosomal recycling factor GTP

C

EF-2

+ Pi

34 / CHAPTER 2 synthesis by phosphorylation of eIF2a, which blocks GDPGTP exchange and reconstitution of the functional ternary complex. Paradoxically, the same modification increases the translation of some mRNA that have upstream open reading frames. It appears that the 60S ribosomal subunit dissociates at the stop codon of the first open reading frame and the 40S subunits remain associated with the mRNA and resumes scanning. The 40S subunit must acquire an active ternary complex during scanning to translate downstream open reading frames. The probability of translating the most 3′ open reading frame therefore depends on the distance (scanning time) between the open reading frames and the availability of amino acids within the cell (16). Internal ribosome entry sites (IRESs) mediate translation initiation independent of the cap structure by recruiting the ribosome directly to an internal position of the mRNA (17). Both structural features and short-sequence elements appear to be involved in ribosome recruitment in eukaryotic IRESs. Exactly how these motifs combine to promote internal initiation remains to be determined. The IRES appears to be a complex RNA scaffold that contains multiple sites for interaction with components of the translational apparatus. Structural domains have been identified that interact with the initiation factors eIF4G and eIF4B (18,19), with eIF3 (20,21), or directly with the 40S ribosome subunit at multiple sites. An IRES is also described that can assemble an 80S ribosome at its initiation codon without the aid of any initiation factors or an initiator tRNA (22). A growing body of evidence exists to support the hypothesis that cellular IRESs are involved in the regulation of gene expression under physiologic conditions during which the efficiency of cap-dependent protein synthesis is greatly reduced. IRESs enable cells to respond to these conditions against the background of a general reduction in protein synthesis.

Elongation Each ribosomal subunit has three binding sites for tRNA: designated the A (aminoacyl) site, which accepts the incoming aminoacylated tRNA; P (peptidyl) site, which holds the tRNA with the nascent peptide chain; and E (exit) site, which holds the deacylated tRNA before it leaves the ribosome. The end of the initiation process leaves an aminoacylated initiator tRNA in the P site of the ribosome and an empty A site, which serves to start the elongation process. Aminoacylated tRNA is brought into the A site as a ternary complex with eukaryotic elongation factor-1A (eEF1A) and GTP. Correct codon-anticodon interactions result in conformational changes in the ribosome that stabilize tRNA binding and trigger GTP hydrolysis by eEF1A. This leads to the release of the aminoacyl end of the A site tRNA by eEF1A; the tRNA then swings into the peptidyl transferase site of the large subunit in a process called accommodation. The peptide bond is formed through deacylation of the P site tRNA and the transfer of the peptide chain to the A site tRNA. The ribosome then has a deacylated tRNA in the

P site and peptidyl tRNA in the A site. Translocation of tRNA and mRNA is facilitated by eEF2, which is also a GTPase. The ribosome is then ready for the next round of elongation, with deacylated tRNA in the E site, peptidyl tRNA in the P site, and an empty A site ready to receive the next cognate ternary complex (see Fig. 2-1B).

Termination Termination begins when a stop codon (UAA, UGA, or UAG) is encountered in the A site mRNA. Stop codons are recognized by eukaryotic release factor-1 (eRF1). The GTPase eRF3 then binds the complex of eRF1 bound to the ribosome. Binding of eRF1 to the ribosome at the stop codon A site triggers the hydrolysis and release of the peptide chain from the tRNA in the P site. Hydrolysis of peptidyl tRNA by eRF1 is required for binding of GTP to eRF3 on the ribosome. This, in turn, leads to a conformational change in eRF3 that has high affinity for ribosomes and the dissociation of eRF1 from the ribosome. Hydrolysis of GTP is required for subsequent dissociation of eRF3 from the ribosome (see Fig. 2-1C) (23,24).

Localized Translation Regulation In addition to regulation of the initiation process, mRNAspecific translation regulation also occurs regionally in polarized cells. This is clearly demonstrated in neural tissues where stimulation of synapses induce the polyadenylation and translation of cytoplasmic polyadenylation elementcontaining, but not cytoplasmic polyadenylation elementlacking, mRNA stored in dendrites (25). This allows the generation of protein gradients emanating from particular positions in cells or the restriction of protein expression to a specific region and is a potential mechanism by which a cell may modulate its response to repeated, directional stimuli.

RNA Silencing Small RNA molecules regulate mRNA-specific translation either by translational repression, in the case of microRNA (miRNA) (26–28), or by mediating the degradation of the target mRNA, in the case of small interfering RNA (siRNA) (29,30). The functional difference between miRNA and siRNA (both about ~22 nucleotides in length) depends on the degree of complementation between the small RNA molecule and the mRNA target (31,32). miRNA hybridize by incomplete base paring, usually to several sites in the 3′ UTR of target mRNA. siRNA show perfect complementation to the target mRNA. miRNA and siRNA have distinctly different origins within the nucleus, but they have common RNA-binding proteins (33). It is unclear whether a single type of small RNA-protein complex can mediate both

TRANSLATION AND POSTTRANSLATIONAL PROCESSING OF GASTROINTESTINAL PEPTIDES / 35 target mRNA cleavage and translational inhibition (see review by Sontheimer [34]).

Other Regulators of Messenger RNA Stability Regulation of the rate of decay is an important control point in determining the abundance of an mRNA species, and decay rates of individual mRNA differ widely and can be differentially affected by environmental cues. Several sequence elements can regulate the rate of turnover of a transcript by attracting specific binding proteins that can either destabilize or stabilize the transcript. The strength of the association of these binding proteins can be modified by changes in the cellular environment. The principal mRNAdegradation pathway begins with removal of the 3′ poly(A) tail. Interaction of the cap proteins and the poly(A)-binding proteins with the translational machinery likely protects the 5′ and the 3′ end of the mRNA from attack by deadenylases and decapping enzymes (35,36). This means that translation and mRNA decay are linked. Support for this comes from studies demonstrating that inhibition of translation initiation destabilized mRNA (37) and inhibition of translation elongation (with cycloheximide) promotes mRNA stability (38). The nonsense-mediated decay pathway further links translation to mRNA turnover. This pathway ensures that mRNA with premature stop codons are not translated. To be recognized as premature, a termination codon must lie upstream of the last intron (39–43). Exon-exon junctional complex proteins, which mark the position of exon-exon junctions in the mature mRNA, may play an important role in surveillance for potentially deleterious nonsense mutations (44).

POSTTRANSLATIONAL PROCESSING Although it would appear that the translation of polypeptide hormones is similar to that of other eukaryotic proteins, the posttranslational processing of prohormones is unique. Since the initial discovery of proinsulin (45), it has been evident that the synthesis of polypeptide hormones of the gut involves a series of modification steps after the initial translation of the gene product that are distinct from the biosynthesis of other cellular proteins. These modifications, achieved via a variety of posttranslational processing reactions, may enlarge or diminish the size of the peptide precursor, but, in general, they result in the formation of biologically active and physiologically relevant products. Efforts to determine the nature and mechanisms of peptide hormone posttranslational processing reactions were greatly facilitated by the development of molecular biological techniques that permitted the deduction of peptide precursor sequences. Information on precursor structure has led to the development of molecular probes that can be used to characterize individual processing reactions, as well as patterns of processing reactions for groups of related peptides. Application of these probes to ultrastructural studies has provided

information on the cellular compartments in which processing reactions take place. In vitro reconstitution experiments have led to the elucidation of some of the mechanisms responsible for the transport of peptide precursors between cellular compartments. Development of techniques to isolate and culture functionally intact peptide-secreting cells has permitted physiologists to examine the sequence and dynamics of the complete posttranslational modification and activation process for given peptides. Many of the enzymes responsible for prohormone processing have now been isolated. Coexpression or deletion, or both, of these enzymes within cells has allowed for elucidation of their activities for multiple prohormone substrates. Previously, it was thought that proteins exited from cells via two distinct pathways: the constitutive or the regulated secretory pathways (46,47). As has been the case with other biological systems, more recent evidence suggests that there might be overlap between these pathways (48). Generally, however, the constitutive pathway is reserved for those secreted proteins that are not stored in the cell and usually do not undergo extensive posttranslational processing, as seen with the products of fibroblasts and hepatocytes. Proteins secreted constitutively exit the cell soon after synthesis on the ribosome. Polypeptide hormones, however, enter the regulated pathway of secretion in most neuroendocrine cells. These cells are capable of storing secretory products for hours or days in electron-dense secretory vesicles and releasing them on stimulation. The intracellular pathways and organelles involved in this pathway were first defined in studies (49) in the exocrine pancreas demonstrating that polypeptides are initially synthesized on the rough endoplasmic reticulum (ER), transported to the Golgi apparatus, and finally placed into secretory granules (Fig. 2-2). On cell stimulation, these secretory granules or vesicles fuse in a calcium-dependent manner with the cell membrane to release their contents into the extracellular milieu. This chapter reviews the enormous progress made in recent years in elucidating the mechanisms for posttranslational processing of gastrointestinal peptide hormones, and then presents a detailed analysis of one hormone, gastrin.

TRANSPORT INTO THE ENDOPLASMIC RETICULUM Polypeptide hormones are synthesized ribosomally from the amino-terminal end and enter the secretory pathway via translocation into the ER. This process is of critical importance to both prokaryotes and eukaryotes. Thus, it has been thoroughly examined by several notable scientists including Gunther Blobel, who won a Noble Prize for his work in this area (50). In summary, the first few amino acids of the preprohormone, translated from the leader sequence of the specific mRNA, are called the signal peptide (Fig. 2-3) (51). This peptide (designated as the presequence in preprohormones) is not secreted under normal circumstances but serves as a means of translocating the newly synthesized and

36 / CHAPTER 2 CYTOPLASM Intiation of protein synthesis

ER Completion of precursor synthesis Cleavage of signal peptide Disulfide bond formation Initial glycosylation

GOLGI Further glycosylation Phosphorylation Sulfation

SECRETORY VESICLE Acetylation Dibasic cleavage Monobasic cleavage Carboxypeptidase Aminopeptidase Glutaminyl cyclase Amidation

SECRETION

FIG. 2-2. Intracellular location of posttranslational processing steps. ER, endoplasmic reticulum.

gradually elongating polypeptide chain into the ER (52–54). After emerging from the ribosome, the signal peptide binds to the signal recognition particle (SRP) in the cytoplasm after chain elongation has produced a preprohormone of approximately 50 to 60 amino acids (55–57). This binding results in an arrest of translation, and the SRP initiates the translocation of the nascent polypeptide by binding to the SRP receptor or docking protein located on the cytosolic side of the ER (58–60). The SRP is then released, and the translocation of the peptide continues through a protein channel across the ER membrane (53,59,61–63). The signal peptide is later cleaved by a specific enzyme (signal peptidase) located on the inner membrane of the ER (64). The individual components of these ER translocation events are described in more detail in the next section.

characteristics are shared: (1) a positively charged aminoterminal region of 1 to 10 amino acids, (2) a central hydrophobic region of 7 to 17 amino acids, and (3) a more polar region that often contains an α helix breaking proline or glycine residue, as well as uncharged residues that determine the cleavage site and complex pattern of amino acids adjacent to the site of cleavage between the signal peptide and the prohormone (65,66). The secondary structure of these peptides can assume several different conformations including α helices and β-pleated sheets, depending on the environment (67). Recently, analysis of the new, extensive protein databases has allowed investigators to accurately predict signal peptides (68). The positively charged amino terminus appears to be important in the release of the SRP once docking of the nascent peptide to the ER has occurred. Mutations in this area that result in a net negative charge interfere with both export and synthesis of secretory proteins in prokaryotes (69), although this does not appear to be the case in eukaryotic systems (70). Mutations that substitute polar or charged amino acids for the amino acids present in the hydrophobic region of the signal peptide result in impaired binding of the nascent peptide chain to the SRP (57). Thus, translation is complete, but export of newly synthesized protein is inhibited (71). Initially, it was thought that conservative substitutions of one hydrophobic amino acid for another (e.g., glycine for valine) did not alter the recognition between the SRP and the signal peptide (72). More recently, others have noted that even small changes in the central hydrophobic core can alter SRP binding (73,74). However, in these cases, translocation across the ER membrane still occurs through an unknown mechanism. SRP binding is not dependent on the presence of a net positive charge at the amino terminus or on any identifiable features at the carboxyl terminus (75,76). There do not appear to be any specific structural requirements for the site of signal peptide cleavage, although the carboxyl-terminal amino acid of the signal peptide usually has a small uncharged side chain such as alanine (65,77) (see Signal Peptidase later in this chapter). After translocation through the ER membrane pore, the signal peptide can loop back through the membrane. The signal peptide is then cleaved at its c terminus by signal peptidase. However, recent studies have shown that the signal peptide can be cleaved further by a signal peptide peptidase to release the amino terminal fragment into the cytosol (78). In the case of prolactin, this fragment then binds to calmodulin in a calcium-dependent manner (79). Currently, the full biological implications of this finding are unknown. In addition, it is not known if this applies to multiple other peptide hormones.

Signal Peptides The signal peptide, or presequence, constituted by the amino-terminal 20 to 30 amino acids of a newly synthesized polypeptide chain, directs the translocation of the polypeptide into the ER lumen. There seems to be little primary amino acid homology in the signal peptides of the known gastrointestinal hormone precursors. However, three general

Signal Recognition Particle SRP has three known functions: signal peptide recognition, elongation arrest, and promotion of translocation (55,56, 80–83). This particle consists of six polypeptide components with molecular weights of 72, 68, 54, 19, 14, and 9 kDa,

TRANSLATION AND POSTTRANSLATIONAL PROCESSING OF GASTROINTESTINAL PEPTIDES / 37 A

B

SRP

C SRP receptor

D

E

F

G

H mRNA

ER Cistem

Translocon

BiP

SP

SPP

FIG. 2-3. Signal peptides. (A) Ribosome binds to the messenger RNA (mRNA) and translation begins at the amino terminus. (B) Signal sequence emerges from the ribosome and binds to the signal recognition particle (SRP), which induces an arrest of translation. (C) SRP-ribosome complex binds to the SRP receptor or docking protein located on the endoplasmic reticulum (ER) membrane. (D) The translocon or protein pore binds to the ribosome, releasing the SRP and its receptor. (E) With binding of the ribosome to the cytosolic side of the translocon, BiP is released from the lumenal side of the translocon. (F) Translation resumes at the carboxyl terminus, and the signal peptide can be reinserted into the ER membrane. (G) Signal peptide is cleaved for the prohormone by signal peptidase (SP), and the signal peptide fragment is released into the cytosol via signal peptide peptidase (SPP) cleavage. (H) Translation continues until the entire precursor is located within the ER cistern and properly folded, often in association with chaperones such as BiP.

as well as a 7SL RNA (80,84). Each component is held together in a defined tertiary structure by Mg2+ ions and is essential for the functions of the SRP. The 19- and 54-kDa proteins exist as monomers, but heterodimers of the 9- and 14-kDa proteins and the 68- and 72-kDa proteins are formed (85). The 54-kDa protein contains a series of amphipathic helices with methionine residues, located predominantly on one face, that appear to be important for the binding to the hydrophobic region of the signal peptide (83,86–88). Interestingly, SRP will not bind to signal peptides that are not tethered to a ribosome, although the peptide region responsible for the SRP–ribosome interaction is not known. It appears that the 54-kDa protein binds to the 7SL RNA through the 19-kDa protein that binds directly to the middle of the RNA strand (88). In addition, the 7SL RNA contains 5′ and 3′ Alu-like elements that bind to each other and the 9/14-kDa protein heterodimer (89–91). The 9/14-kDa protein heterodimer mediates elongation arrest of translation, but plays no role in the translocation process (91,92). The 68/72-kDa heterodimer binds to the middle segment of RNA close to the 19-kDa binding site and appears to mediate the binding of SRP to its receptor (93,94). Thus, the 68/72-kDa heterodimer is not involved in elongation arrest but serves to aid in translocation. The 54-kDa protein binds GTP in concert with binding to the signal peptide (80,82,95–97). An additional GTP is required on binding of the SRP to the SRP receptor (96).

When the SRP/SRP receptor complex associates with the ER membrane or translocon there is a subsequent release of GDP (98). The hydrolysis of GTP releases the SRP from the signal peptide and allows translation to proceed.

Signal Recognition Particle Receptor The SRP receptor is located on the cytosolic side of the ER and binds to the SRP–ribosome complex, but not to free SRP as noted earlier. The SRP receptor plays an important role in termination of the elongation arrest and in the translocation of polypeptides into the ER lumen (59). The SRP receptor is a heterodimeric protein consisting of a 30-kDa integral membrane protein (β subunit) and 72-kDa α subunit that possesses domains that are homologous to GTP-binding proteins and the GTP-binding region of the 54-kDa SRP protein (88,95,99–104). The 72-kDa α subunit of the SRP receptor binds to SRP, and GTP is necessary to release SRP from the signal peptide–ribosome complex (95,105).

Endoplasmic Reticulum Membrane Protein Channel or Translocon There have been numerous theories about whether the nascent polypeptide chain is transported directly across the

38 / CHAPTER 2 lipid bilayer or in the aqueous environment of a protein channel. Although the initial thought was that the hydrophobic core of the signal peptide would allow for direct translocation across the membrane, there is a large transmembrane channel that is opened by the presence of signal peptides (106–108). The eukaryotic ER translocon is a heterotrimer (Sec61α, Sec62β, and Sec61γ) estimated to have a diameter of about 30 Å (109–111). The size of the pore is too small for folded proteins, thus ensuring that only nascent, unfolded proteins can enter the ER lumen. The lumenal side of the translocon is sealed by a protein, BiP, that aids in protein folding after passage through the membrane (112). On binding of the ribosome to the translocon, the SRP and its receptor disassociate from the complex, allowing resumption of translation (113). This process also seals the pore on the cytosolic side of the ER membrane, releasing BiP from the lumenal side.

PROCESSING IN THE ENDOPLASMIC RETICULUM Signal Peptidase During translocation, the signal peptide is cleaved from the propeptide by signal peptidase, an integral membrane protein complex on the lumenal surface of the ER. Signal peptidase has been purified from the dog pancreas as a complex of 5 polypeptides with molecular weights of 12, 18, 21, 22/23, and 25 kDa (114,115). The enzyme in the hen oviduct has only 2 subunits of 19 and 22/23kDa (116). The canine and hen 22/23-kDa proteins are glycosylated, and their amino acid sequences are 90% identical (117,118). cDNA encoding the canine 18 (119) and 21 kDa (120) are homologous to 2 yeast SEC11 proteins (121) that are components of the yeast signal peptidase, which contains 4 proteins in total (with molecular weights of 13, 18, 21, and 25 kDa). The 21kDa protein is absolutely required for enzymatic function in yeast (122,123), raising the question of the exact function of the other proteins. As is the case with processing enzymes, there are great similarities between the yeast and mammalian enzymes (124). Although it appears that the structure of eukaryotic signal peptidases are phylogenetically conserved, the Escherichia coli signal peptidase consists of only a single subunit of 323 amino acids (125). Nevertheless, there is some sequence homology between bacterial signal peptidases and subunits of the eukaryotic enzyme. Furthermore, the substrate specificity of the eukaryotic and prokaryotic signal peptidases is similar (126). Eukaryotic signal peptidase has a broad pH optimum and requires phosphatidyl choline as a cofactor (127,128). Determination of the amino acid sequences that define the substrate specificity of signal peptidase has been difficult because of the enormous structural diversity of signal peptides (129,130). However, there is clearly a hierarchy of preferred substrates for amino acids located at the carboxyl terminus of the signal peptide as follows: Ala>Cys>Gly>

Ser>>Thr>Pro>Asn>>Val, Ile, Leu, Tyr, His, Arg, Asp (65,131). Mutations of the signal peptide that increase the number of amino acids between the end of the central hydrophobic domain and the site of cleavage and mutations in the positively charged amino-terminal domain inhibit the cleavage reaction (132–134).

Disulfide Bond Formation After peptide prohormones are translocated into the ER lumen, they can undergo intermolecular or intramolecular disulfide bond formation (e.g., proinsulin) (Fig. 2-4). In the case of proinsulin, disulfide bonds are formed before cleavage of proinsulin into its component A and B fragments by removal of the C peptide. Thus, the disulfide bonds that are intramolecular on the prohormone are subsequently converted to intermolecular linkages that cannot be recreated easily after they are reduced. Although spontaneous formation of disulfide bonds of peptides such as somatostatin can occur in vitro over a few hours under optimal conditions, in vivo, the process occurs either cotranslationally or within seconds after translocation (135). The rapidity of this process suggests that it is catalyzed by an enzyme, the prime candidate being protein disulfide isomerase (PDI) (136–138). In solution, PDI exists as a homodimer (2 × 57 kDa) with a highly acidic isoelectric point (pI) (139). PDI has a broad substrate specificity encompassing relatively small proteins such as insulin, as well as large multidomain proteins such as immunoglobulins (137). PDI also forms the β subunit of a tetrameric enzyme (α2β2) denoted as prolyl-4-hydroxylase, which is responsible for hydroxylation of proline in the formation of procollagen (140). In tissues requiring both PDI and prolyl-4-hydroxylase activities, it appears that the β subunits of prolyl-4-hydroxylase are synthesized in large excess with a fraction being recruited into the prolyl-4-hydroxylase tetramers and the remainder as functional PDI homodimers (140). Previously, it was thought that glutathione provided the oxidizing equivalents for PDI (141,142). More recently, investigators have identified an ER membrane protein S

S

S

A-chain

S

S S

B-chain

Connecting peptide

FIG. 2-4. Structure of proinsulin. A and B peptides are linked by intramolecular disulfide bonds (asterisk), but after the connecting peptide is removed by endoproteolytic cleavage, disulfide bonds are intermolecular. On exocytosis, insulin (A and B peptides) is coreleased with the C peptide.

TRANSLATION AND POSTTRANSLATIONAL PROCESSING OF GASTROINTESTINAL PEPTIDES / 39 (Ero 1p) in yeast (143,144) that serves this function. Indeed, Ero 1p directly oxidizes PDI through disulfide exchange (141,145). The reoxidation of Ero 1 involves flavin adenine dinucleotide (FAD) (145).

N-glycosylated proteins (158). Because few prohormones are glycosylated, it is hypothesized that this pathway is not involved in prohormone processing. An important factor that should not be forgotten is the role that disulfide bond formation plays in maintaining the folded nature of many polypeptides such as proinsulin.

Asparagine-Linked N-glycosylation There are few examples of gastrointestinal peptides with N-linked glycosylation. The primary amino acid sequence of -Asn-X-Thr/Ser, where X can be any amino acid except proline, is obligatory for N-glycosylation of asparagine (146). The anterior pituitary glycoprotein hormone family (thyroid-stimulating hormone, follicle-stimulating hormone, and leuteinizing hormone) is the best example of glycosylated hormones. These are dimeric proteins with a common β subunit and different but homologous β subunits that confer specific biological activities. The glycosylation of both subunits is important for their correct assembly into dimers (147). Proopiomelanocortin (POMC) (148) and proenkephalin A (149) are also glycosylated, although the functional significance of this modification is unknown in these peptides. Secretogranin I (also known as chromogranin B) has a single glycosylation site, but it is uncertain whether it is glycosylated in vivo (150).

Protein N-myristoylation Protein N-myristoylation refers to the cotranslational linkage of myristic acid (C14:0) to the amino-terminal glycine of proteins; protein N-myristoylation is reviewed elsewhere (151). There are no known examples of myristoylated prohormones; however, this modification may play a role in the regulation of a variety of cellular events including posttranslational processing. Examples of N-myristoylated proteins include GTP-binding proteins and the catalytic subunit of cyclic 3′,5′adenosine monophosphate-dependent protein kinase A.

Protein Folding Polypeptides must be folded into a conformation that is compatible with exit from the ER (109,152). Misfolded proteins are tightly but noncovalently bound to a heavy chain binding protein or BiP and retained in the ER (153) until folding is complete and the polypeptide is released on hydrolysis of ATP (154,155). BiP, a member of the heat shock family of proteins (HSP70), binds newly translated and translocated aliphatic single polypeptides and prevents them from folding prematurely (156,157). It is currently unknown whether BiP or other folding proteins (158) are involved in the posttranslational processing of mammalian gastrointestinal prohormones, although BiP is clearly important in the translocation and folding of the yeast prohormone, pro-α factor in the ER (159). Another important folding chaperone is calnexin, but it interacts only with

TRANSPORT FROM THE ENDOPLASMIC RETICULUM AND THROUGH THE GOLGI The mechanisms responsible for protein sorting beyond the ER have been the subject of much investigation. Unlike the well-defined sorting of prohormones to the ER lumen through a signal peptide, there is no single unifying mechanism of prohormone transport from the ER and through the Golgi. Two types of sorting mechanisms have been hypothesized. The first is that prohormones are transported in the nonspecific “bulk flow” of contents from the ER to the Golgi in transport vesicles. An alternative hypothesis is that there is some signal contained in the prohormone structure that specifically directs their sorting through the intracellular compartments. This latter hypothesis is the case for resident soluble ER proteins such as BiP and PDI. Investigators noted in the structures of BiP and PDI a carboxyl-terminal consensus sequence KDEL (LysAspGluLeu) (160). Truncated forms of BiP lacking the KDEL sequence are not retained in the ER, but rather are secreted constitutively. In analogous fashion, prohormones destined for secretion but tagged with KDEL are retained in the ER in an unprocessed form (161). The homologous tetrapeptides DKEL, RDEL, and KNEL are all capable of directing ER retention in mammalian cells, whereas the HDEL sequence is used primarily in yeast (162,163). Although the KDEL-tagged proteins could be retained by a KDEL receptor in the ER membrane, it appears that these proteins initially exit the ER and are then recaptured in a salvage compartment at or near the cis-Golgi and returned to the ER (164). A mutant strain of yeast ERD2 (for ER retention defective) has been shown to have a defect in the KDEL/HDEL receptor (165). The structure of the ERD2 gene was then used to aid in the search for a mammalian homologue (166). This powerful technique of identifying genes of fundamental importance to the sorting of proteins in yeast and then using the yeast model to identify a mammalian homologue has been a fruitful approach in the study of peptide hormone processing. The ERD2 gene encodes a protein of 26 kDa that contains 7 membranespanning regions and is highly homologous to a putative human ERD2-like gene (167). The mammalian KDEL receptor cycles from the ER to the Golgi and back to the ER, thus retaining lumenal ER proteins within that compartment (168). Prohormones proceed from the ER to the Golgi stack where they undergo further posttranslation modification. Prohormone movement through the Golgi stack is by bulk flow (169,170) rather than a process mediated by a sorting signal. Bulk transport of soluble ER proteins to the Golgi and through the various Golgi compartments (cis, stack, and

40 / CHAPTER 2 trans-Golgi network [TGN]) was once thought to occur through transport vesicles (171). In this model, the Golgi was a series of stable, disconnected stacks through which proteins were progressively sorted, modified, or “distilled” toward their final destination. Although this model was attractively simple, it now appears that the Golgi is a much more fluid organelle (171–174). Although the transport vesicle model was indeed attractive, the described vesicles were too small (70 nm in diameter) to transport many secreted proteins. An alternative model is that newly synthesized proteins move from the ER to the cis-Golgi cisternae located near the ER. This newly formed cisternae progresses through the Golgi stack from the ER to the transGolgi (175). The transport vesicles in this case merely shuttle enzymes that characterize the various layers of the Golgi back through the cisternae (176–179). Thus, prohormones are transported from the ER to the cis-Golgi and are not transported out of this compartment, but rather are carried forward to the trans side as newer enzymes and proteins are added to the cis-Golgi. The nature of the ER to Golgi transport has been studied extensively (173). The ER membrane has a fixed number of exit sites from which proteins leave the lumen (180). The ER membranes cause buds that eventually become coated on their outer cytoplasmic surface with dispersed cytoplasmic proteins (coatamers, coat promoter, or COPs) (181–184). The budding ER vesicle is coated with COPII and traps prohormones together with other ER proteins (185–187). The COPII-coated vesicles then uncoat and fuse into a larger vesicular tubular complex (VTC) (185). It appears that the VTC is not continuous with the ER membrane. Eventually, the VTC combines with COPI (188). After fusing with the Golgi membrane (in a GTP-dependent manner) (189–191), the COPI-associated VTCs return ER proteins for recycling. Thus, nonhydrolyzable analogues of GTP such as GTP-γ-s interfere with fusion and block transport through the stack (192). VTCs move from the ER to the Golgi along a microtubular network that is powered by the dynein–dynactin motor (190,193). The fungal metabolite Brefeldin A, which is known to block protein transport through the Golgi stack, blocks the binding of the coatamer complex to budding Golgi membranes (194–197). The finding that Brefeldin A interferes with the posttranslational processing of progastrin suggests that this pathway is involved in the sorting of prohormones, as well as other soluble secretory proteins (198,199). Proteins secreted via either the constitutive or regulated secretory pathways share a common trail from the ER through the Golgi stack, but they diverge in the TGN where proteins are sorted according to their final destination (169,200–202). The sorting signal for enzymes destined for lysosomes involves a glycosylation reaction that occurs in the Golgi stack to attach mannose-6-phosphate residue proteins. A receptor protein in the TGN specifically binds mannose-6-phosphate–modified proteins (203,204) and directs their sorting to lysosomes. To date, searches for a common sequence (KDEL-like) or posttranslational modifications (mannose-6-phosphate–like) in the structure of

prohormones that might direct sorting to secretory vesicles in the TGN have not been successful. Although investigators have long sought to elucidate “the” Golgi sorting signal in neuroendocrine cells, none has been entirely successful. Indeed, it appears that three different mechanisms may be responsible for prohormone sorting to secretory vesicles. These include sorting signal motifs, aggregation, and membrane or lipid raft binding (205). Initially, the search for a sorting signal was pursued vigorously. In a fashion akin to the signal peptide (“pre” region of preprohormones), investigators sought sequences in the “pro” region of prohormones that, although lacking homology in their primary amino acid sequence, still contain sufficient structural information to direct sorting in the TGN. An α-helical motif with three leucine residues occupying one side of the helix was proposed as such a signal, but this hypothesis was not proved (206). In other studies, a chimeric protein containing the “prepro” region of somatostatin at the amino terminus and a constitutively secreted protein such as γ-globulin at the carboxyl terminus were sorted and processed in the secretory pathway (207). Studies with POMC and somatostatin precursors containing deletions in the “pro” region indicate the presence of sorting information at these sites, as well as in other portions of the peptide (208,209). In contrast, deletion of the “pro” sequence from trypsinogen and renin did not disrupt the routing of these proteins into the secretory pathway (210,211). A study expressed neuropeptide Y (NPY) fragments tagged with green fluorescent protein (GFP). GFP, a jellyfish protein not normally secreted, was correctly sorted, stored, and released from neuroendocrine cells when fused to half of the prepro-NPY sequence or only the signal sequence alone of pre-NPY (212). Thus, it appears that some prohormones are likely sorted by a specific signal found in their “pro” regions, but this does not appear to be a universal finding. A second sorting hypothesis is selective aggregation (213–215) of prohormones into acidic clathrin-coated secretory vesicles in the presence of high concentrations of divalent cations such as Zn2+ or Ca2+. Support for this hypothesis comes from observations that specific mutations in the structure of proinsulin that result in inhibition of hexamer formation with zinc also impede processing (48,216). Furthermore, in vitro studies have demonstrated that intravesicular conditions (pH 5.2 and 10 mM Ca2+) can result in selective precipitation of peptides that exit the cell through the regulated pathology of secretion. This applies to secreted proteins such as secretogranin II but not proteins that are constitutively secreted such as immunoglobulins (217). A heterodimeric protein in adrenal chromaffin granules, termed glycoprotein III, can selectively aggregate with two prohormone-processing enzymes carboxypeptidase E (CPE) and a dibasic endoprotease (218). Evidence contradictory to the selective aggregation hypothesis can be found in studies with guinea pig proinsulin, which does not form hexamers with zinc and yet is sorted and processed with high efficiency (219). In addition, in the marine mollusk Aplysia, the egg-laying hormone precursor is processed into two distinct mature hormone products that are sorted into different

TRANSLATION AND POSTTRANSLATIONAL PROCESSING OF GASTROINTESTINAL PEPTIDES / 41 secretory vesicles within a single cell (220). It is difficult to reconcile the selective aggregation model to explain this and other observations. However, aggregation clearly plays a role in the sorting of some prohormones. A third proposed prohormone sorting mechanism is the notion that prohormone-processing enzymes serve as a sorting receptor directing prohormones to secretory vesicles. CPE has been proposed as one putative sorting receptor (221–224). In this model, CPE is associated with Golgi membranes or “lipid rafts” (223,225–229). Others have found little evidence to support these findings with CPE (230–232). Other prohormone-processing enzymes or their activities also have been implicated in sorting. For example, a dibasic prohormone convertase (PC2) is associated with the lipid bilayer during sorting (233). Impairment of dibasic processing for proneurotensin (234), proneuromedin (234), proinsulin (235), and pro-NPY (236) all demonstrate impaired processing and sorting. However, other mutant prohormones are inefficiently cleaved yet sorted correctly (237). Again, it is difficult to reconcile these divergent findings and apply them generally to prohormone sorting. Most of the studies discussed earlier involve expressing a nonnative prohormone in a neuroendocrine cell (e.g., expressing prepro-NPY in insulin-producing β cells), or expressing mutated precursors in neuroendocrine cell lines such as AtT-20 or GH3. Thus, conclusions may be valid only for the studied prohormone or cell line and are not generally applicable. Indeed, prepro-NPY is synthesized, efficiently processed, and stored in cultured neurons (238). However, when these cells are infected with an adenoviral vector expressing POMC, POMC is poorly processed and stored despite that the pro-NPY processing is unaltered. Thus, many investigators now believe that prohormone sorting involves multiple processes that are cell and prohormone specific (48,175,213,239).

PROCESSING REACTIONS IN THE GOLGI Serine- or Threonine-Linked O-glycosylation Glycoproteins of the anterior pituitary such as the β subunit of human chorionic gonadotropin are O-glycosylated in the Golgi (240,241). Although there are potential sites for O-glycosylation in some gastrointestinal peptide hormone precursors, there is no evidence to suggest that they are functionally relevant. Confirmation that O-linked glycosylation occurs in the Golgi can be found by inhibiting transport through the Golgi with Brefeldin. Neuroendocrine cells treated with Brefeldin lead to O-linked glycosylation of prohormones not normally glycosylated (242).

O-phosphorylation Serine-linked O-phosphorylation is an uncommon posttranslational modification of peptide hormones that occurs

in the trans area of the Golgi stack just before entry into the TGN (243). Phosphorylation was first reported for POMC (244) and subsequently for proatrionaturetic factor (245,246), proenkephalin (247), and progastrin (248). The physiologic significance of peptide hormone phosphorylation is uncertain, although a hypothesis has been put forth for gastrin (249). At the carboxyl terminus of progastrin is a GlyArgArgSer processing site that eventually causes the amidated and biologically active site of the mature hormone. Varro and colleagues (250) suggest that the formation of amidated gastrin may be correlated with the degree to which the Ser moiety at that site is phosphorylated. Further clarification of the proposed regulatory function of O-phosphorylation on progastrin processing is required. Because there are few known phosphorylated hormone precursors, it has been difficult to identify a consensus sequence to signal phosphorylation. Although there are several phosphorylases capable of catalyzing the reaction, a candidate enzyme has not yet been clearly identified.

Tyrosine Sulfation Sulfation of carbohydrate residues and protein backbones is a common posttranslational reaction that all eukaryotic cells are capable of performing. Sulfation of prohormones at specific tyrosine residues is far less common, but nevertheless it is a critical step in the activation of some hormones. Examples of sulfated gut peptides are gastrin (251), cholecystokinin (CCK) (252,253), the chromogranin/secretogranin family (243), and Leu-enkephalin (254). Whereas sulfation is essential for the biological action of CCK at CCK-A receptors located in the gallbladder and pancreas (255,256), the acid secretagogue effect of gastrin is not influenced by the presence or absence of a sulfate moiety (257). Sulfation results in the inactivation of Leu-enkephalin (254). Some authors have hypothesized that sulfation may play a role in the sorting of prohormones to the secretory pathway; however, the sulfation of chromogranin A, the pancreastatin precursor, does not affect its sorting in the parathyroid (258). From the large number of sulfated secretory proteins it has been possible to identify the structural features that promote tyrosine sulfation (Table 2-1) (259,260). Some authors have examined a larger number of sulfated proteins TABLE 2-1. Structural criteria for tyrosine sulfation 1. Presence of an acidic residue at position −1 (the amino acid directly preceding the sulfated tyrosine). 2. Presence of at least three acidic residues from position −5 to +5. 3. Not more than one basic residue between positions −7 to −2 and +1 to +7. 4. Presence of β-turn-inducing residue (often serine or proline) between positions −7 to −2 and +1 to +7. 5. Not more than three hydrophobic residues between positions −5 and +5. 6. Absence of disulfide bridging or glycosylation sites nearby.

42 / CHAPTER 2 and modified the previous consensus sequence (261). For gastrin, these rules have been confirmed experimentally by mutational analysis (262). Tyrosyl protein sulfotransferase catalyzes the transfer of the sulfate moiety from the sulfate donor, 3′-phosphoadenosine-5′-phosphosulfate, to the protein acceptor (263). The purified enzyme has a pH optimum of 6.5 consistent with its localization in the TGN, where the pH is substantially greater than that of the secretory vesicle (pH 5.5). The enzyme has an absolute requirement for Mg2+ or Mn2+ for biological activity. Two putative tyrosyl protein sulfotransferase genes (TPST-1 and TPST-2) have been identified (262,264–267). The enzymes are highly homologous and are expressed in a wide number of cell types. Homology searches for other family members have been unsuccessful. A TPST-1-deficient mouse has been generated with a reduced body weight phenotype (268). It is unknown if TPST-1 deficiency alters CCK or gastrin sulfation (268). Although other sulfotransferase activities have been identified in other tissues, it is unclear if they can sulfate tyrosine residues. Tyrosine sulfation of CCK and gastrin affects the processing of both peptides. Gastrin sulfation is variable between species and tissues. However, enhanced sulfation of gastrin increases the endoproteolytic maturation of progastrin (269). In contrast, sulfation of pro-CCK is not an absolute requirement for CCK processing, but a lack of sulfation does impair CCK secretion (270).

FORMATION OF SECRETORY VESICLES From early morphologic studies it is known that there is selective condensation of proteins in the TGN to form a dense core that is subsequently enveloped by a membranecontaining clathrin (271,272). Clathrin, which has been reviewed extensively elsewhere, is a 180-kDa polypeptide made up of three light and three heavy chains that coats both endocytic and immature secretory vesicles (273–275). The immature clathrin-containing vesicles contain the prohormones, as well as many of the enzymes responsible for their processing (276). As the secretory vesicles mature, they lose their clathrin coat and contain relatively more processed biologically active peptides and lesser quantities of prohormone precursors. Mutation of proinsulin so that it cannot be processed causes it to remain stored in immature clathrin-coated vesicles, suggesting that prohormone processing may be involved in the uncoating process (277). However, the mechanisms of secretory vesicle formation, clathrin coating, and uncoating remain a mystery. The pH of the secretory vesicle is maintained in the acidic range (pH 5.5) via a proton-transporting Mg2+-dependent ATPase on the membrane (278,279). A low intravesicular pH is believed to be essential for the activation of the processing enzymes contained within the secretory vesicle. Also associated with the acidification and uncoating of the secretory vesicle is an increase in intravesicular Ca2+ that plays a key role in the activation of prohormone dibasic cleavage enzymes (see later).

PROCESSING REACTIONS IN THE SECRETORY VESICLE Acetylation Of the processing reactions that take place in secretory vesicles, the least is known about acetylation because there are only a few examples of acetylated gut peptides. In the rat, acetylation is required to activate a melanocyte-stimulating hormone (280), but it inactivates β-endorphin (281); thus, acetylation is important in the physiologic regulation of these peptides (282,283). The enzyme responsible for acetylation has not been purified, but it has been characterized as a crude enzyme that requires acetyl coenzyme A as an acetyl donor (284,285). Much of the available information on opioid peptide acetylation is derived from experiments in the rat pituitary and brainstem; however, this reaction may be species and organ specific. Because acetylation does not seem to occur in the human pituitary or other nonbrain tissues of the rat, the significance of this reaction as an important step in the posttranslational processing of gut peptides has yet to be determined (286–288). With identification of the five subtypes of melanocortin (MC) receptors (MC1-5), investigators have attempted to determine the potential molecular mechanisms that might mediate the effects of acetylation on MC activity (289). Acetylation of α-MSH results in few, if any, differences in binding or receptor coupling to HEK293 cells expressing MC1 to MC5 receptors. However, native acetylated α-MSH is much more effective than deacetylated α-melanocyte stimulating hormone (MSH) in stimulating rat osteoblast growth (275). Thus, more work clearly is necessary to determine the physiologic implications of MC acetylation.

Dibasic Cleavage Selective endoproteolytic cleavage is perhaps the most important posttranslational processing step for gastrointestinal peptide hormones, because virtually all of them undergo this reaction in one form or another. Furthermore, this step results in the tissue-specific processing of prohormones, resulting in the formation of distinct patterns of mature peptides, often with markedly different biological activities. The most frequent cleavage reactions occur at pairs of basic amino acid residues, although tribasic and tetrabasic sites also serve as cleavage sites. Cleavages usually occur on the carboxyl-terminal side of a dibasic pair (Fig. 2-5). The LysArg pair is the most common dibasic cleavage site (60%), followed by ArgArg (25%), with LysLys and ArgLys sites being the least common (290). Support for the secretory vesicle localization of the cleavage enzymes was initially provided in peptide-release studies demonstrating that processed hormones (e.g., insulin) and prohormone fragments (e.g., C peptide) were cosecreted in equimolar amounts during vesicle exocytosis (291). In more direct immunohistochemical studies, intact proinsulin was shown to be abundantly present in the Golgi stack and in the immature clathrin-coated

TRANSLATION AND POSTTRANSLATIONAL PROCESSING OF GASTROINTESTINAL PEPTIDES / 43

-Lys-ArgTryptic-like cleavage

-Lys-Arg + Carboxypeptidase cleavage

+ Cleaved peptide(s)

FIG. 2-5. Endoproteolysis of prohormones involves tryptic-like cleavage on the carboxyl-terminal side of monobasic or dibasic residues. This is followed by the removal of the basic residues by carboxypeptidase E.

vesicles, whereas processed insulin was present primarily in the mature uncoated vesicles (292). Thus, the dibasic cleavage reaction occurs primarily in the Golgi-derived, clathrincoated, secretory vesicles. There does not appear to be a strict requirement for a specific primary amino acid sequence in the region surrounding the dibasic site for cleavage to occur. Mutation of one of the basic residues to a nonbasic amino acid will result in impaired processing (293). Not all dibasic sites in a given prohormone are cleaved, and different tissues may process the same prohormone at different dibasic sites. These observations may result differential expression of cleavage enzymes in various tissues or from the conformational characteristics of the precursors. Some investigators suggest that the secondary structure (β-turn) near the cleavage site is important for processing. They further hypothesize that dibasic sites buried in an α helix will be cleaved inefficiently (294–296). Although in vitro studies have shown that trypsin can perform dibasic cleavages, the exact nature of the enzymes responsible for cleavage of prohormones in situ had been difficult to ascertain because of their presence in low quantities compared with the relative abundance of proteases associated with cellular organelles (particularly lysosomes) that contaminate many secretory granule preparations (297,298). Although many enzymatic activities were purified and characterized (297,299), it was difficult to determine whether these enzymes were responsible for prohormone processing in vivo. Furthermore, because many enzymes were described from different neuroendocrine tissues, it was unclear whether their actions were specific for a given prohormone substrate in an individual endocrine cell or more generalized and applicable to other prohormones. Gene transfer studies that involve expression of prohormone cDNA in heterologous neuroendocrine cell lines that do not normally express or process a given hormone demonstrated that the posttranslational machinery and enzymes are common to a variety of cell types. For example, the processing of gastrointestinal peptide hormone cDNA encoding progastrin (300,301), pro-NPY (302), pro-CCK (303), pro-SS (304), proglucagon (305), and propancreatic polypeptide (306) has been demonstrated in various neuroendocrine cell lines. The posttranslational processing machinery appears to be specific to neuroendocrine cells because prohormones are processed inefficiently in nonendocrine cells (307). A breakthrough in the field came when Julius and colleagues (308) described a mutation in the kex2 gene of yeast.

This mutation prevented the synthesis of a specific killer toxin and a mating prohormone, both of which require cleavage at dibasic sites for biological activity. The kex2 gene was found to encode a Ca2+-dependent serine protease, not one related to trypsin, as many had expected, but rather one related to the bacterial subtilisins (309–312). In searching for a mammalian homologue for kex2, van den Ouweland and colleagues (313) demonstrated homology between the deduced kex2 protein sequence and the human c-fur gene, which was originally detected in an open reading frame immediately upstream of the fes/fps protooncogene (314). The c-fur gene product, termed furin or PACE (paired basic amino acid residue cleaving enzyme), has a conserved sequence in eukaryotes (315,316). The efforts to identify the mammalian neuroendocrine homologue of kex2 were rewarded with the identification of PC2 (317,318). Similar methods led to the discovery of a second related cDNA encoding a product that was named PC1 (317) or PC3 (319). Like kex2, PC2 and PC1/PC3 are both Ca2+-dependent serine proteases related to the bacterial subtilisins (320,321). The acidic pH optimum (pH 5.5) of both enzymes is consistent with their putative function in secretory vesicles. PC2 and PC1/PC3 transcripts are found only in endocrine and neural tissues (317,322,323). PC4 (324) is expressed only in the testis and not in other neuroendocrine tissues or cell lines. Similar to furin, PC5/PC6, PC7, and PC8 (325–328) are expressed in many tissues including neuroendocrine tissues. These convertases and furin generally are not involved in prohormone processing. Their active forms are localized to the TGN via cytoplasmic tails and in the small vesicles that characterize constitutive secretion (329). These proteases process many secreted protein from organs such as the liver. They also are responsible for the processing of growth factors such as transforming growth factor-β (330) and insulin-like growth factors (331). The cytosolic tail of these enzymes in the constitutive pathway contains motifs that allow for their recycling out of early secretory granules (332,333). The enzymes are not active in the ER inasmuch addition of the ER retention signal, KDEL, to the carboxyl terminus of furin abolishes proteolytic activity (334). Similar to kex2, all members of the PC family including furin are Ca2+dependent proteases. Furin has a pH optimum of 6.5, which is consistent with its localization to the Golgi (335). All members of the PC family are synthesized as a biologically inactive preconvertase. Autoactivation occurs for all

44 / CHAPTER 2 members except PC2 via an intramolecular cleavage (329,336,337). In the case of furin (337), the covalent dibasic bond in the “pro” region is cleaved, allowing exit from the ER. The propeptide remains associated with the protease until the slightly lower pH of the Golgi (pH 6.5) and its calcium-rich milieu remove the propeptide. A second cleavage in the released propeptide prevents its reassociation with the protease. Similar mechanisms have been described for PC1/PC3, PC4, PC5, and PC7 (312). The activation of pro-PC2 requires an accessory protein 7B2. PC2 and 7B2 associate in the ER. It is believed that 7B2 is necessary for pro-PC2 folding and subsequent activation in that PC2 will not autocleave and thus autoactivate in the absence of 7B2 (338). In the more acidic compartment of the TGN, a carboxyl-terminal fragment of 7B2 is cleaved by furin or another similar protease (339,340). This fragment remains near the catalytic site of the enzyme and a LysLys site in the fragment inhibits PC2 activation (341). As the pH in the TGN continues to decrease, the “pro” region of PC2 is removed by autocatalysis (342). Eventually, the partially activated PC2 cleaves the associated carboxyl-terminal fragment of 7B2, allowing for its full activation (340). Although PC1/PC3 is autoactivated, there is another protein that may regulate its activity. Pro-SAAS is a 26-kDa protein produced in neuroendocrine cells and sorted to the secretory pathway (343). Indeed, Pro-SAAS expression overlaps that of PC1/PC3 (344) and can inhibit prohormone processing when expressed in a neuroendocrine cell line (345). The earlier series of events has implications for prohormone processing (346). For example, PC2 is believed to be more efficient than PC1/PC3 at LysLys cleavage sites (347). However, PC2 activation occurs much later than that of PC1/PC3, which can alter the mixture of final peptide products from prohormones with multiple cleavage sites such as POMC and proglucagon (346,348). To examine the physiologic and biochemical effects of individual PCs on prohormone processing, investigators generated several PC-deficient mouse models. Because the motif for the substrate specificity of furin, Arg-X-Lys/Arg-Arg (349), is not present in many prohormone cleavage sites, it has been suggested that PACE/furin may be responsible for processing proteins secreted via the constitutive pathway but not involved in prohormone processing in the secretory pathway (311,349). In addition, the almost ubiquitous expression of furin and its involvement in the processing of many secreted proteins suggests that it is a protein critical to normal development. Indeed, furin-deficient mice all die by E11.5 (350). The mouse embryos fail to undergo axial rotation and heart looping (350,351). PC4 is expressed only in the testis (324). Disruption of PC4 expression results in markedly diminished male fertility without any noted spermatogenic abnormality (352). PC4 deletion also inhibits pituitary adenylate cyclase activating peptide (PACAP) processing in the testis (353). Gene disruption experiments for PC1/PC3 and PC2 are of considerable interest because both enzymes are involved in prohormone processing. Moreover, their substrate specificities

can overlap extensively. PC2-null mice survive but grow more slowly than wild-type mice (354). Proinsulin processing is maintained in these mice, suggesting that PC2 is not necessary for insulin production. However, proglucagon is processed into glucagon but not glucagon-like peptide-1 (GLP-1) or GLP-2 (355,356). Prodynorphin and prosomatostatin processing at its dibasic site are also affected in PC2-null mice (356,357). As anticipated with its role in PC2 activation, 7B2-null mice are phenotypically similar to PC2-null mice. However, 7B2 mice also have a dramatic defect in POMC processing and adrenocorticotropin hormone (ACTH) production, adding to an exaggerated Cushing’s syndrome (358). PC2- and 7B2-null mice also have alterations in pro-CCK and progastrin processing (359,360). PC1/PC3 gene disruption causes dwarfism and multiple peptide processing defects (361). Defects include processing of proinsulin to insulin, POMC to ACTH, proglucagon to GLP-1 and GLP-2, progrowth hormone-releasing hormone. This phenotype and spectrum of prohormone processing defects is similar to a reported human subject with a defect in PC1/PC3 activity (362,363).

Monobasic Cleavage Many gastrointestinal peptide hormones are processed to their biologically active forms by cleavage at single basic amino acids. Examples of such peptides include pancreatic polypeptide (364), gastrin-releasing peptide (365), and CCK (366). The monobasic cleavage reaction must be highly specific because cleavage does not occur at all basic amino acid residues. This specificity no doubt also contributes to the tissue specificity of peptide precursor processing (367). Arginine (81%) is the most commonly cleaved basic amino acid, but lysine cleavage sites also have been identified. Proline, which is thought to induce a β-turn in the prohormone secondary structure, is found in a position adjacent to approximately one third of the known cleavage sites. From the peptide precursors known to be processed, Devi (368) has proposed several principles that define the structural criteria for monobasic processing depicted in Table 2-2 (369). These principles are useful not only in predicting which single basic amino acid sites will be cleaved, but also in predicting which sites will not be cleaved. When the rules are applied to single arginines and lysines that are not cleaved in peptide precursors, approximately 90% are predicted not to be processing sites (368). To determine whether monobasic and dibasic cleavages are distinct phenomena, Schwartz and Tager (370) performed time-course biosynthetic studies involving the pulse-labeling of pancreatic islets producing propancreatic polypeptide, a hormone known to be processed at both monobasic and dibasic sites. In contrast with the dibasic cleavage reaction that usually occurs within 60 to 90 minutes, the monobasic reaction required 120 to 180 minutes for completion. Similar experiments with neurophysin have confirmed these findings (371). Thus, monobasic and dibasic cleavage reactions

TRANSLATION AND POSTTRANSLATIONAL PROCESSING OF GASTROINTESTINAL PEPTIDES / 45 TABLE 2-2. Structural features of monobasic cleavage sites Rules 1. Basic amino acid (Arg, Lys, or His) is always present at either the −3, −5, or −7 position (3, 5, or 7 amino acids preceding the cleavage site). 2. Aliphatic amino acids (Leu, Ile, Val, Met) are never present at the +1 position. 3. Cysteine is never present in the vicinity (−7 to +3) of the cleavage site. 4. Aromatic amino acids (Trp, Tyr, Phe) are never present in the −1 position. Tendencies 1. The monobasic cleavage is usually at a single arginine, occasionally at a single lysine, and never at a single histidine. 2. Short side-chain amino acids, predominantly Ser or Ala, tend to be at the +1 position in approximately 50% of cases. 3. Aliphatic and small side-chain amino acids occur primarily in the −2 position and the +2 to +8 positions. 4. Aromatic amino acids are rarely found at the +1 position.

appear to involve distinct enzymes. Further support for this notion includes the finding that PC2 appears to be unable to cleave peptide substrates containing a single basic amino acid (320). Prosomatostatin also requires processing at a dibasic site to generate SS-14 and is processed at a monobasic site to generate SS-28. Mackin and colleagues (372) have identified two distinct enzymatic activities in tissues that appeared to be responsible for pro-SS processing at monobasic and dibasic sites. The first is a serine protease that cleaved proSS at the dibasic site for SS-14, and a second aspartyl protease cleaved pro-SS and led to the formation of SS-28. There are numerous studies demonstrating the abilities of PC1/PC3 and PC2 to cleave pro-SS at its dibasic site to form S14 (373,374). Finally, others have suggested that SS-14 and SS-28 production occur in different secretory pathways (375), which is consistent with the notion of distinct monobasic and dibasic PCs. To determine whether members of the PC family were capable of monobasic pro-SS cleavage, investigators examined pro-SS processing in PC2-null mice (355). In PC2deficient mice, pro-SS was cleaved at the monobasic but not dibasic site leading to the formation of SS-28 and little SS-14. This suggested that PC2 is not a monobasic pro-SS– processing enzyme, despite that PC2 is capable of monobasic cleavages of other prohormones (357,376). It is not known if PC1/PC3 is the pro-SS monobasic protease in mammals. Although other investigators have identified possible monobasic cleavage activities in various tissues (372,377– 379), the yeast aspartyl protease (YAP3) is perhaps the most promising candidate (380,381). YAP3 has been implicated as an enzyme capable of monobasic processing in anglerfish pro-SS-II to yield S28 (382). Interestingly, both an anglerfish aspartyl protease (372) and a mammalian aspartyl protease not only have similar pH optima and molecular weights to YAP3, but they also share immunologic cross-reactivity

with YAP3 (383). Thus, it is quite possible that a mammalian YAP3 homologue is the monobasic pro-SS–processing enzyme. However, identification of this mammalian homologue has not been as forthcoming as was the identification of the mammalian dibasic PCs after the identification of the yeast kex2 enzyme. Beinfeld and colleagues (378,379) have described two distinct monobasic cleavage activities from brain and gut using a fragment of pro-SS as a substrate. They both have a broad range of pH optima with maximal activity at pH 8.0, but their inhibitor profiles are somewhat different. The activity from the gut was capable of cleavage at both monobasic Arg and dibasic sites. Finally, production of GLP-1 from proglucagon requires processing at a monobasic site. PC1/PC3-null mice cannot generate GLP-1, suggesting that proglucagon monobasic processing can be mediated via PC1/PC3 (384). Although several putative monobasic enzymatic activities have been characterized, final proof of their role in monobasic prohormone cleavage will await their cloning and expression in neuroendocrine cells. Criteria for evaluating candidate processing enzymes initially proposed by Docherty and Steiner (297) for characterization of the dibasic cleavage enzyme(s) are applicable for evaluation of monobasic enzymes (Table 2-3). Dynorphin converting enzyme has been characterized in rat brain extracts and is capable of cleaving the 29-amino-acid peptide leumorphin at a single arginine residue to Dyn B-13 (377). The purified enzyme is unable to cleave substrates at dibasic ArgArg sites (385–387). The enzymatic activity has a pH optimum of 8.0 and appears to be a thiol protease. The amino acid requirements appear to conform to the rules for monobasic enzymatic activity described in Table 2-2. Its tissue distribution and subcellular localization to the secretory vesicle are consistent with a putative role in peptide hormone processing.

Carboxypeptidases A frequently hypothesized mechanism for the formation of biologically active peptides from larger precursors involves TABLE 2-3. Criteria for identification of a prohormone monobasic cleavage enzyme 1. Protease must correctly cleave precursor to generate all known peptide products. 2. Enzyme must be resolved from other contaminating proteases before characterization. 3. Biochemical characterization should include studies of: Cleavage specificity pH characteristics (optimum and stability) Cleavage mechanism (serine, thiol, metalloenzyme) Susceptibility to known protease inhibitors 4. Tissue distribution and subcellular localization must be appropriate for its putative role. 5. Final proof of normal participation in prohormone cleavage requires demonstration that inhibition, inactivation, or mutation of the enzyme prevents cleavage in intact cells.

46 / CHAPTER 2 not only the endoproteolytic cleavages at basic residues, but also the subsequent removal of the remaining basic residues via the action of an aminopeptidase or carboxypeptidase (see Fig. 2-5) (388). This hypothesis arises from the observation that many gut peptides are missing the basic amino acids at their carboxyl termini after undergoing endoproteolytic cleavage. Other experiments demonstrated that addition of carboxypeptidase B to trypsin-induced cleavage of some prohormones in vitro results in the generation of correctly processed peptides (298). The eukaryotic enzyme thought to be responsible for this action in neuroendocrine cells has been extensively characterized and is alternatively known as CPE, carboxypeptidase H, or enkephalin convertase. CPE was partially purified from adrenal medullary secretory granules and found to be maximally active at pH 5.5 (389). CPE has a molecular weight of approximately 50 kDa and exists in both soluble and membrane-bound forms (390). The soluble form of the enzyme appears to be fully active, whereas the membrane-bound form exhibits less than 50% of the activity of the soluble form (391). Enzymatic activity is enhanced by the presence of Co2+ or Ni2+ ions, although it is presumed that the active site metal of the enzyme is Zn2+ (389,390). CPE removes a carboxyl-terminal arginine slightly faster than lysine and much faster than histidine from a variety of peptide substrates (392). Peptides with other amino acids at the carboxyl terminus are not hydrolyzed by CPE. An excess of free arginine and lysine inhibits CPE activity (393). Bovine (394), rat (395), and human (396) CPE share a 93% amino acid homology. The amino acid sequence deduced cloned CPE cDNA contain 41 amino-terminal residues not found in the purified active form of the enzyme. The first 17 to 22 amino acids represent a signal or presequence and the remainder is a prosequence that is endoproteolytically cleaved at five arginine residues that immediately precede the amino terminus of the active enzyme. This cleavage occurs rapidly after synthesis, presumably in the ER or cis-Golgi, or both, and the five consecutive arginine residues conform to the substrate motif necessary for cleavage by the endoprotease PACE/furin active in the cis-Golgi (397). Further analysis of the nucleotide sequences of CPE cDNA indicates that CPE is a member of the metallocarboxypeptidase gene family, although the amino acid homology is only 20 to 50%. Members of this family include carboxypeptidases A and B found in the exocrine pancreas (398), as well as carboxypeptidases M and N, which are thought to function in the processing of serum proteins such as the kinins (399,400). Interestingly, the yeast functional homologue of CPE, the kex1 gene product, has little or no homology to mammalian CPE (401). This is in contrast to the high degree of homology that exists between the yeast kex2 gene product and the mammalian PC1/PC3 and PC2. It is not currently known if a mammalian homologue of the kex1 gene product exists, although kex1 heterologously expressed in mammalian cells is able to process mammalian prohormones (402). The rat CPE gene has been identified and Southern blots indicate only a single copy of the CPE gene (403).

CPE activity and mRNA are widely distributed throughout neuroendocrine tissues of many species, and the tissue levels of CPE mRNA generally reflect the levels of enzymatic activity (404). Stimulation of adrenal chromaffin cells that synthesize and process proenkephalin results in an increase of both CPE activity and mRNA (405). In contrast, stimulation of GH4C1 cells increases CPE activity without altering CPE mRNA levels, suggesting that CPE is regulated differently in various tissues (406). CPE activity has been detected in early embryonic development of the brain, before the appearance of processed neuropeptides, as is expected given its proposed role in the posttranslational processing of prohormones (407). Hyperinsulinemia and obesity are found in mice with a single point CPE mutation that reduces enzymatic activity (408). This exciting discovery led to further examination in these mice of prohormone processing including gastrin (409). Because prohormone cleavages were also affected, investigators examined the role of CPE beyond that of basic amino acid residue removal, including PC activation and activity (225,410,411). Sorting of CPE to the regulated pathway of secretion requires its interaction with lipid rafts via a C-terminal membrane anchor (221,412). It is also hypothesized that CPE might serve as a sorting receptor for prohormones and other processing enzymes, although this hypothesis remains controversial (412).

Aminopeptidases An aminopeptidase capable of removing amino-terminal basic residues may have an important role in prohormone posttranslational processing. Although the endoproteolytic cleavage of most prohormones occurs at the carboxyl side of a dibasic site, there are some examples of gastrointestinal hormones that are characterized by one or more basic amino acids at the amino terminus. Gainer and colleagues (413) have characterized an enzyme from bovine pituitary secretory granules with specificity for amino-terminal arginine residues. The enzyme has a pH optimum of 6.0 and is inhibited by ethylenediaminepentaacetic acid in a fashion reversible with Co2+ and Zn2+, but it cannot be inhibited by serine or cysteinyl protease inhibitors. Although the enzyme can cleave the amino-terminal arginines from β-lipotropin 60-65 (leaving Met-enkephalin) and a variety of synthetic substrates, it does not remove the amino-terminal arginine from fully processed substance P. The enzyme appears to be distinct from lysosomal and degradative aminopeptidases, but its importance in gastrointestinal prohormone processing remains to be clarified. Dipeptidyl aminopeptidase IV sequentially removes two amino acids from the amino terminus of peptides and is clearly involved in the posttranslational processing of prohormones in the honeybee and frog (414,415), although its role in mammals is yet unknown. The STE13 mutant strain of Saccharomyces cerevisiae, which lacks an enzymatic activity present in wild-type yeast capable of hydrolyzing

TRANSLATION AND POSTTRANSLATIONAL PROCESSING OF GASTROINTESTINAL PEPTIDES / 47 mammalian substrates of dipeptidyl aminopeptidase IV, fails to process a mating factor at the amino terminus, thus producing peptides extended by two amino acids (416). Dipeptidyl aminopeptidase IV has been characterized in frog skin and has a molecular weight of 98 kDa with a pH optimum of 8.0 (417). Dipeptidyl aminopeptidase IV is capable of amino-terminal cleavages with an X-Ala, X-Gly, or X-Pro amino terminus wherein X is usually Glu. A variety of gastrointestinal peptides including gastric inhibitory polypeptide (418) and gastrin-releasing peptide (365) are potential substrates for dipeptidyl aminopeptidase IV, because these peptides have been recovered from tissues with the two or four amino acids (e.g., AlaProValPro) missing from their amino termini. The enzyme is expressed in high concentrations of hepatocytes and in nonendocrine epithelial cells of the small intestine (415), thus it may not function in prohormone processing before secretion from neuroendocrine cells, but it may be responsible for cleaving peptides after their release or during the extraction process (419).

Glutaminyl Cyclase N-terminal pyroglutamyl formation is necessary for the full biological activity of thyrotropin- and gonadotropinreleasing hormones (420). The modification also is found in other gastrointestinal peptides such as bombesin and gastrin, although it is not required for their classical functions (421). Other proteins constitutively secreted from nonendocrine cells such as immunoglobulins also contain this modification. The reaction involving the conversion of an amino-terminal glutamine to a pyrole ring structure with the release of ammonia is known to occur spontaneously in the presence of phosphate ions (422). A pituitary enzyme, glutaminyl cyclase (QC; Q is the single letter notation for glutamine), catalyzes this reaction at a rate approximately 70 times faster than the nonenzymatic reaction (423,424). QC has a molecular weight of approximately 40 kDa with a pH optimum of 8.0, despite its localization in the acidic secretory vesicles of neuroendocrine cells (423). The Km is about 100 µM, and the enzyme requires ATP for full activity. A cDNA-encoding QC has been isolated from a bovine (425) and human (426) pituitary. By Northern blot analysis, QC is expressed in many portions of the brain but not in the spleen where large amounts of immunoglobulins containing pyroglutamate residues are produced. It is possible that two forms of the enzyme might exist: one (425) to process prohormone substrates in neuroendocrine cells, and the other to process immunoglobulins and other constitutively secreted proteins. There is some evidence for multiple forms of the enzyme (427).

Amidation Many gastrointestinal peptides are characterized by the presence of a carboxyl-terminal amide moiety that is often essential for biological activity (428). Tatemoto and Mutt

(429) have used this property to identify many gut peptides. The first indication of the source of the amide group was provided by Kreil and coworkers (430,431), who noted that the precursor of melittin, an amidated honeybee venom, contained a glycine residue immediately following the carboxyl-terminal amino acid of the processed peptide. These findings prompted the hypothesis that glycineextended peptides may serve as substrates for the amidation reaction. Since the proposal of this hypothesis, the rapid generation of new prohormone peptide sequences from the cloned cDNA encoding them has confirmed that the carboxyl terminus of an amidated peptide is flanked in the structure of its precursor by a glycine residue followed by one or more basic amino acids. The basic residues are processed by cleavage enzymes, leaving a glycine-extended precursor that, in turn, serves as a substrate for the amidation reaction. Bradbury and colleagues (432) initially identified the amidating enzyme as a 60-kDa protein capable of converting the synthetic substrate D-TyrValGly to D-TyrValNH2. The pituitary form of this enzyme was characterized further by Mains, Eipper, and others (433–435), and initially was referred to as peptidyl-glycine alpha-amidating monooxygenase (PAM). PAM requires Cu2+ and molecular oxygen for function. Enzymatic activity is enhanced by the presence of ascorbic acid and catalase, and there is a biphasic pH optimum (pH 5–7). The requirements for copper, oxygen, and ascorbic acid suggest that PAM is similar to dopamine β-hydroxylase. Amidating activity has been identified in a variety of tissues, as well as in the systemic circulation and cerebrospinal fluid (436–439). The amidating enzyme appears to be specific for a glycine residue in the carboxyl terminus of peptide substrates (440). Peptides terminating in other L-amino acids do not serve as substrates, although D-alanine–containing peptides can serve as a substrate that reacts slowly (441). The enzyme appears to exhibit selectivity for neutral amino acids preceding the glycine residue (442). In vivo amidation enzyme activity can be regulated by the availability of Cu2+ ions as demonstrated by the finding that animals treated with the copper chelator diethyldithiocarbamate have decreased amidated gastrin and increased glycineextended gastrin in the gastric antrum (443,444). Although the importance of copper to peptide amidation was known for some time (444), the mechanisms by which copper reached PAM in cells were unknown. Investigators have identified the Menkes protein as an intracellular copper transport p-type ATPase, ATP7a (445–448). The Menkes protein is related to other copper transport genes such as the Wilson’s disease gene product. However, the expression of the Wilson’s gene is limited to the liver, whereas the Menkes gene is expressed in most tissues except the liver (446). Menkes kinky hair syndrome is an X-linked genetic copper deficiency that is usually fatal early in childhood (449). Children with Menkes syndrome usually do well for the first few months of life, and then develop progressive neurologic degeneration (450). The cause of the neurologic deterioration

48 / CHAPTER 2 is unknown, but it is believed to be a consequence of the low in vivo activities of copper-dependent enzymes such as cytochrome c oxidase, dopamine-β-hydroxylase, and PAM (450–453). The Menkes protein transports copper into the Golgi (454) and has been directly linked to PAM activity (455,456). PAM also requires ascorbate for optimal activity. The monooxygenation performed by PAM requires ascorbate in a reduced state that is subsequently oxidized to dehydroascorbate. The uptake of dehydroascorbate into cells is mediated by the glucose transporters, GLUT3 and GLUT4 (457), but at insufficient levels to account for the biological actions of ascorbate. Two cDNA-encoding ascorbate transporters (SVCT1 and SVCT2) have been isolated (458–461). SVCT1 is expressed at high levels in the kidney and to a lesser extent in the gut. SVCT2 is expressed in levels in neurons but not astrocytes (459,460). To date, there is no direct linkage between SVCT expression and PAM activity. Humans and guinea pigs are two of the more unusual mammals that require ascorbate in their diets. To examine the possible role of ascorbate deficiency (“scurvy”) and peptide amidation, investigators noted that antral extracts of guinea pigs deprived of ascorbic acid contain less amidated gastrin and more glycine-extended gastrin (462). Peptide amidation occurs in a two-step reaction with the initial formation of a hydroxyl intermediate via the action of the monooxygenase domain of PAM (463–468). Another distinct enzyme, with lyase activity, catalyzes the breakdown of hydroxyglycine to form the peptide amide and glyoxylate. The nitrogen of the peptide amide comes from the amino nitrogen of the carboxyl-terminal glycine (432). The oxygen involved in the hydroxylation step originates from molecular oxygen (469). Both enzymes are distributed in same neuroendocrine tissues and are encoded by a single gene. The amino-terminal portion of the mammalian PAM precursor contains a signal peptide followed by a putative “pro” region of unknown physiological significance. The peptidyl-α-hydroxylating monooxygenase (PHM) domain is separated by a linker containing a LysLys endoproteolytic cleavage site from the lyase or peptidyl-α-hyodroxyglycine α-amidating lyase (PAL) domain (470). This is followed by another dibasic endoproteolytic cleavage site and a putative transmembrane domain with a carboxyl-terminal cytoplasmic tail. In the rat, alternative RNA splicing appears to be responsible for the tissue-specific expression of several different mRNAencoding proteins lacking any one of the regions described earlier (470–472). Expression of the full-length PAM cDNA in an endocrine cell line led to increases in monofunctional PHM and PAL activities, although most of the activity was soluble, suggesting that there had been endoproteolytic removal of the carboxyl-terminal transmembrane domain and cleavage between the PHM and PAL domains. Expression of a truncated cDNA without the linker segment resulted in a single bifunctional protein with PHM and PAL activities. Expression of another truncated segment with only the PHM domain resulted in the expression of active soluble monooxygenase activity (471,472).

POSTTRANSLATIONAL PROCESSING OF PREPROGASTRIN Many of the principles of prohormone posttranslational processing can be exemplified in the production of a single gastrointestinal peptide hormone, gastrin. The mechanism of gastrin biosynthesis has long been a focus of interest because of its physiologic importance in the regulation of gastric acid secretion and gastrointestinal cell growth. From the earliest studies, multiple molecular forms of gastrin were characterized in the circulation and antral tissues (257, 473–476). The presence of gastrin heptadecapeptide (G17) in larger molecular forms of gastrin suggested that they shared a precursor-product relation (477). The isolation and sequencing of the gastrin cDNA allowed investigators to deduce many of the mechanisms of gastrin posttranslational processing (478). It has become apparent that preprogastrin undergoes several important modifications necessary for full biological activity, including signal peptide cleavage, tyrosine sulfation, serine O-phosphorylation, dibasic cleavage at two or more sites, carboxypeptidase removal of basic residues, carboxyl-terminal amidation, and amino-terminal pyroglutamate formation. The structure of human preprogastrin, as deduced from the nucleotide sequence of cDNA (479), consists of a 21amino-acid signal peptide, an amino-terminal extension of 35 amino acids, and a carboxyl-terminal extension of 9 amino acids (Fig. 2-6). Analysis of the gastrin precursor yields valuable information and is illustrative of the posttranslational processing steps outlined earlier. Preprogastrin contains a 21-amino-acid signal peptide (479–481) that possesses the three general characteristics shared by signal peptides: (1) a positively charged amino-terminal region of 3 amino acids, (2) a central hydrophobic region of 15 amino acids, and (3) a more polar pattern of amino acids adjacent to the site of cleavage. The alanine residue located at the carboxyl terminus of the signal peptide is one of the preferred amino acids at the signal peptide cleavage site (65,70). The 34-amino-acid “pro” region preceding the Arg57Arg58 site is homologous to progastrins and pro-CKKs in other species, but it does do not have significant homology in primary structure with the “pro” regions of other gastrointestinal hormones. Sorting studies of progastrin have suggested that removal of 30 amino acids within the “pro” does not impair sorting and subsequent processing (482). Indeed, the carboxyl-terminal amino acids including intact processing sites are required for progastrin sorting (482). Sulfation of gastrin at Tyr87 is known to occur in different tissues (483,484). Biosynthetic studies using 35S-sulfate have demonstrated that even large molecular forms of gastrin are sulfated at Tyr87 (251). This is consistent with the presumption that sulfation occurs in the Golgi before endoproteolytic cleavage. If one applies the known structural criteria (259,260) for tyrosine sulfation (see Table 2-1) to the sequence surrounding Tyr87 of gastrin, there is an acidic residue missing at position −1, but many acidic residues (Asp and Glu) are present in positions −5 to +5. Furthermore, there are no basic residues (Arg or Lys) and no more than

TRANSLATION AND POSTTRANSLATIONAL PROCESSING OF GASTROINTESTINAL PEPTIDES / 49 21

1 Signal peptide

Met Gln Arg Leu Cys Val Tyr Val Leu lle Phe Ala Leu Ala Leu Ala Ala Phe Ser Glu Ala

G34

24 26 40 Ser Trp Lys Pro Arg Ser Gln Gln Pro Asp Ala Pro Leu Gly Thr Gly Ala Asn Arg 57 58 Asp LEU Glu LEU Pro Trp Leu Glu Gln Gln Gln Gly Pro Ala Ser His His Arg Arg 59 74 75 Gln Leu Gly Pro Gln Gly Pro Pro His Leu Val Ala Asp Pro Ser Lys Lys

G17

76 87 Gln Gly Pro Trp Leu Glu Glu Glu Glu Glu Ala Tyr Gly Trp Met Asp Phe

Amino-terminal "Pro" fragment

Carboxyl-terminal extension

93 94 95 96 Gly Arg Arg Ser Ala Glu Asp Glu Asn

FIG. 2-6. Deduced amino acid sequence of the human gastrin complementary DNA. The 21-aminoacid signal peptide is shown at the top with the central hydrophobic core underlined. The monobasic (Lys24, Pro25Arg26, and Arg40) and dibasic (Arg57Arg58, Lys74Lys75, and Arg94Arg95) cleavage sites are depicted in boldface. The underlined portion of the amino-terminal “pro” fragment from amino acids 40 to 51 is predicted to form an α helix with three leucine residues shown in capital letters that may be important in prohormone sorting. Gln59 and Gln76 at the amino termini of G34 and G17, respectively, serve as substrates for glutaminyl cyclase. Tyr87 is a site for gastrin sulfation, and Ser96 is a phosphorylation site. After cleavage of Arg94Arg95 and the removal of these residues by carboxypeptidase E, gastrin processing intermediates extended by Gly93 serve as substrates for the amidation enzyme.

three hydrophobic residues from in the adjacent region. On Chou-Fasman analysis there are putative β-turn sites present at −9 and +6. There are no glycosylation or disulfide bridging sites near Tyr87. On the basis of studies using a gastrin peptide fragment as a substrate for the sulfation reaction, it appears that the gastrin sulfation enzyme is similar to the tyrosine sulfotransferase described in other tissues (485). Gastrin conforms to these criteria, and this has been tested experimentally by expressing mutant progastrins in endocrine cell lines and observing the effects of the mutation on gastrin sulfation (262). Interestingly, these studies suggest that sulfation of Tyr87 enhances the subsequent endoproteolytic maturation of progastrin (262). Progastrin has been described to be phosphorylated at Ser96, and this modification can be blocked with Brefeldin in accordance with the localization of the phosphorylation reaction to the distal Golgi stack (198). Because of the proximity of the Ser96 residue to the carboxyl-terminal Gly93Arg94Arg95 amidation site, it has been suggested that phosphorylation of this site may influence the formation of the peptide amide (248,250,486). Indeed, fasting in rats leads to a relative decrease in Ser96 phosphorylation that coincides with decreased amidated gastrin, but with no effect on endoproteolysis at Arg94Arg95 (250). Similar results have been reported in dogs with a surgically excluded antrum (487). After signal peptide cleavage and passage through the Golgi, the 80-amino-acid progastrin molecule is packaged into the secretory vesicle. It appears that there is little endoproteolytic cleavage of gastrin before packaging in the secretory vesicle (488). On the amino terminus, progastrin contains three dibasic cleavage sites—Arg57Arg58, Lys74Lys75, and Arg94Arg95—and three potential monobasic cleavage sites—Lys24, Pro25Arg26, and Arg40 (see Fig. 2-6). Cleavage of

Arg57Arg58 results in the formation G34, whereas cleavage of Lys74Lys75 results in the formation of G17. G17 and G34 are the major molecular forms found in most gastrinproducing tissues, although small amounts of amidated gastrin22-93 or Component I are found in normal antral tissues and in greater concentrations in gastrinoma extracts (489). The initial step in the carboxyl-terminal processing of progastrin is cleavage at the Arg94Arg95 residues. These basic amino acids are subsequently removed by CPE, and the remaining glycine-extended peptide serves as a substrate for PAM, resulting in the formation of amidated gastrin. Sugano and colleagues (490,491) developed region-specific antisera that recognized glycine-extended gastrins (G-Gly), as well as progastrins extended by the Arg94 and Arg95 residues. With these antisera, they demonstrated that progastrin, G-Gly, and amidated gastrin were colocalized within G cells of the antral mucosa (492). Biosynthetic studies with G cells isolated from the canine antrum demonstrated that cells incubated in medium containing 35S-Met incorporated label sequentially into immunoreactive progastrin, G-Gly, and finally amidated gastrin (493–495). In accord with the hypothesis that these posttranslational processing reactions take place in a single enclosed cellular compartment such as the secretory vesicle, they could identify molecular forms of both progastrin and G-Gly that corresponded to heptadecagastrin (G17), tetratriacontagastrin (G34), and Component I (Fig. 2-7; see also later in this chapter). Further confirmation of the notion that G-gly is converted to amidated gastrin in the secretory vesicle is the finding that G-gly and gastrin are cosecreted from isolated G cells (496,497). Endoproteolytic cleavage at the Lys74Lys75 dibasic processing site of progastrin is the major determinant for the relative distribution G17 and G34 in tissues. Thus, we explored

50 / CHAPTER 2 G17 G34 Component I

Signal peptide

GlyArg94 Arg95Ser

Lys74 Lys75

ArgArg “Pro” fragment

Signal peptidase ArgArg

GlyArg94 Arg95Ser

Lys74 Lys75

Sultation and phosphorylation ?SO4

GlyArg94 Arg95Ser

Lys74 Lys75

ArgArg

PO4

Prohormone convertase (PC1/PC3) ?SO4 GlyArg94 Arg95

Lys74 Lys75

Carboxy peptidase E ?SO4 Lys74 Lys75 PAM Lys74 Lys75

Gly PC2

NH2

Gly ?SO4

PC2

NH2

PAM

FIG. 2-7. Posttranslational processing of progastrin.

the ability of two PCs—PC1/PC3 and PC2—to cleave this important site within progastrin. We expressed wild-type human gastrin cDNA in AtT-20 cells. Because AtT-20 cells express PC1/PC3 but not PC2 (319,498), we also coexpressed a cDNA encoding PC2. The Lys74Lys75 progastrin processing site was efficiently cleaved in AtT-20 cells only after coexpression of PC2 (499). These data suggest that PC1/PC3 is not responsible for the cleavage of LysLys sites in wild-type progastrin. Nevertheless, PC1/PC3 is expressed in the duodenum (500) where the predominant form of gastrin is G34. Thus, we sought to explore the possibility that PC1/PC3 may process progastrin at the C-terminal Arg94Arg95 site. We inhibited expression of PC1/PC3 in these cells with two different antisense PC1/PC3 constructs (full and partial length). Coexpression of either antisense construct resulted in a consistent decrease in G34-NH2. We concluded that PC1/PC3 is a progastrin-processing enzyme, suggesting a role for PC1/PC3 progastrin processing in gastric or duodenal G cells (501). Northern blot analysis of enriched canine antral G cells shows PC2 but not PC1/PC3 expression, whereas fundic D cells expressed both enzymes (499,502). Thus, we anticipated that PC2 was responsible for progastrin processing in antral G cells. Inhibition of PC2 expression in

canine antral G cells with antisense oligonucleotide probes resulted in diminished amidated gastrin formation and an accumulation of progastrin in gastric antral G cells. These studies are consistent with our previous studies in endocrine cell lines and confirm that PC2 is a progastrin cleavage enzyme in antral G cells (495). Gastrin biosynthetic studies in the rat (503) demonstrate that progastrin is processed to G34-Gly that undergoes further endoproteolytic cleavage and amidation, resulting in the formation of G17-NH2. Furthermore, it appears that most G17-NH2 arises from cleavage of G34-NH2 rather than amidation of G17-Gly via the action of a PAM, suggesting that G17-Gly is a distinct end-product of progastrin processing. These results suggest that other factors such as cofactor (perhaps ascorbate) availability may account for the presence of G17-Gly in some tissues. Complete posttranslational processing with carboxylamidation of G17 and G34 are required for full biological activity mediated by gastrin/CCKB receptors. Indeed, glycineextended gastrins (G-Gly) are at least four orders of magnitude less potent than amidated G17 in acutely stimulating gastric acid secretion (504,505). Although high concentrations of G-Gly can be found in tumors (506) and during

TRANSLATION AND POSTTRANSLATIONAL PROCESSING OF GASTROINTESTINAL PEPTIDES / 51

Stomach

of other well-characterized monobasic cleavage sites (see Table 2-2). Pro25Arg26 is preceded by a proline and contains a serine in the +1 position, suggesting that it may prove to be a monobasic cleavage site. However, the site lacks a basic amino acid in the −3, −5, or −7 positions, suggesting that it is likely to be a poor site for monobasic cleavage. The glutamine residue at the amino-terminal ends of G17 and G34 serve as substrates for GC after cleavage of Arg57Arg58 or Lys74Lys75, although the activity of this enzyme has not been examined in gastrointestinal tissues or with a gastrin substrate.

REFERENCES G-NH2

jun c-fos and c-jun kinase expression

G-Gly

phosphorylation of c-jun

↑ gene expression and growth

Proliferating gut cell FIG. 2-8. Interaction of amidated gastrin and G-Gly gut growth.

development (507), most investigators believed that the accumulation of G-Gly was of no biological significance. In addition to its well-known regulation of gastric acid secretion, G-NH2 has a proliferative action on normal and malignant gastrointestinal tissues (508–512); therefore, we questioned whether G17-Gly might also act as a trophic agent. To explore this possibility, we noted that both G17NH2 and G17-Gly stimulated DNA synthesis in several cell lines via distinct receptors and signal transduction mechanisms (513-516). Other groups have confirmed the trophic nature of G-Gly on the gut (516–522). In other studies, we noted that the ligand specificity of the G-Gly receptor on parietal cells was similar to that on cell lines (523). Furthermore, although acute administration of G-Gly had no effect on gastric acid secretion (504,505), prolonged treatment (48 hours) of parietal cells with G-Gly does increase histamine-stimulated acid secretion and gene expression of the proton pump, H+,K+-ATPase (523). These chronic effects of G-Gly on acid secretion were confirmed by others in vivo (524) and in gastrin-deficient mice (525). These studies underscore the importance of prohormone-processing mechanisms to gut biology (Fig. 2-8). Cleavage of any of three monobasic sites appears to a relatively rare event, but cleavage of Pro25Arg26 has been observed in gastrinoma tissues. Unlike the other monobasic sites, only the Pro25Arg26 site has any of the structural features

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526. 527. 528.

neutralize amidated and glycine-extended gastrin-17 and inhibit the growth of colon cancer. Cancer Res 1996;56:880–885. Kaise M, Muraoka A, Seva C, Takeda H, Dickinson CJ, Yamada T. Glycine-extended progastrin intermediates induce H+, K+ -ATPase αsubunit gene expression through a nevel receptor. J Biol Chem 1995; 270:11155–11160. Higashide S, Gomez G, Greeley GH Jr, Townsend CM Jr, Thompson JC. Glycine-extended gastrin potentiates gastrin-stimulated gastric acid secretion in rats. Am J Physiol 1996;270:G220–G224. Chen D, Zhao CM, Dockray GJ, Varro A, Van Hoek A, Sinclair NF, Wang TC, Koh TJ. Glycine-extended gastrin synergizes with gastrin 17 to stimulate acid secretion in gastrin-deficient mice. Gastroenterology 2000;119:756–765. Preiss T, Hentze MW. Starting the protein synthesis machine: eukaryotic translation initiation. Bioessays 2003;25:1201–1211. Ramakrishnan V. Ribosome structure and the mechanism of translation. Cell 2002;108:557–572. Calkhoven CF, Muller C, Leut A. Translational control of gene expression and disease. Trends Mol Med 2002;8(12):577–583.

CHAPTER

3

Transmembrane Signaling by G Protein–Coupled Receptors Claire Jacob and Nigel W. Bunnett Structure and Function of G Protein–Coupled Receptors, 64 G Protein–Coupled Receptors Share Structural Motifs, 64 Rhodopsin Is a Model G Protein–Coupled Receptor, 64 Agonists Interact with Different Receptor Domains, 65 G Protein–Coupled Receptors Can Form Dimers, 68 Accessory Proteins Are Required for the Function of Some G Protein–Coupled Receptors, 69 Mechanisms of Signal Transduction, 70 G Protein–Coupled Receptors Interact with Heterotrimeric G Proteins, 70 G Protein–Coupled Receptors and Heterotrimeric G Proteins Exist in Multiple States, 70 Monomeric G Proteins also Mediate G Protein–Coupled Receptor Signaling, 71 Organization of G Protein–Coupled Receptors, G Proteins, and Signaling Proteins into Microdomains Increases the Efficiency and Fidelity of Signal Transduction, 73 Receptor Tyrosine Kinases Are Signaling Partners for G Protein–Coupled Receptors, 74 Receptor Tyrosine Kinases Comprise a Family of SingleTransmembrane Domain Proteins with Intrinsic Kinase Activity, 74

Tyrosine Phosphorylation Recruits and Activates Receptor Tyrosine Kinase Signaling Molecules, 74 G Protein–Coupled Receptors Transactivate Receptor Tyrosine Kinases, 76 Receptor Tyrosine Kinases also Signal through Heterotrimeric G Proteins, 76 Mechanisms That Regulate Signaling by G Protein–Coupled Receptors, 77 G Protein Receptor Kinases and Arrestins Mediate Homologous Desensitization, 77 Other Kinases Regulate G Protein–Coupled Receptor Signaling, 79 Clathrin, β-Arrestins, and Dynamin Mediate Endocytosis of Many G Protein–Coupled Receptors, 81 Receptor Recycling Mediates Resensitization of Signal Transduction, 82 Internalized G Protein–Coupled Receptors Can Continue to Signal, 83 Lysosomal Trafficking Permanently Arrests Signal Transduction, 83 Conclusion, 85 Acknowledgments, 85 References, 85

Cells are required to detect and respond to an extraordinarily diverse array of stimuli from the external environment or from intrinsic sources. Environmental stimuli include signals as diverse as photons, odorant and taste molecules, and both innocuous and noxious physical and chemical agents. Intrinsic stimuli include a range of structurally distinct hormones, neurotransmitters, and paracrine substances.

Receptors in the plasma membrane specifically detect these stimuli and are essential in determining the appropriate physiologic responses to environmental signals and regulatory molecules. Cell-surface receptors can be divided into several broad families based on their structure and mechanisms of action. The superfamily of G protein–coupled receptors (GPCRs) comprises by far the largest and most functionally diverse group of receptors. GPCRs, which include receptors for most hormones and neurotransmitters, are seven-transmembrane domain proteins that interact with heterotrimeric G proteins to initiate signal transduction. The general family of catalytic, enzyme-linked receptors are single-transmembrane domain proteins with cytosolic domains that either have intrinsic enzymatic activity or

C. Jacob and N. W. Bunnett: Departments of Surgery and Physiology, University of California, San Francisco, San Francisco, California 94143. Physiology of the Gastrointestinal Tract, Fourth Edition, edited by Leonard R. Johnson. Academic Press, 2006.

63

64 / CHAPTER 3 associate directly with enzymes. They are subdivided into five families depending on the nature of their enzymatic activities: receptor tyrosine kinases (RTKs), tyrosine kinase– associated receptors, receptor tyrosine phosphatases, receptor serine-threonine kinases, and receptor guanylyl cyclases. This family includes receptors for growth factors, certain hormones, and immune mediators, and the receptors initiate signaling by activation of their intrinsic enzymatic activity or by association with enzymes. Certain ion channels are also directly regulated by extrinsic and intrinsic signals. The ligand-gated ion channels bind directly to neurotransmitters, which regulate their activity. Other channels, such as those of the transient receptor potential vanilloid receptor family, can directly bind regulatory molecules or are indirectly regulated by agonists of other receptors, which control their activity and function. All of these categories of cellsurface receptors play crucial roles in the regulation of the gastrointestinal tract. The purpose of this chapter is to review some of the major recent advances in our understanding of transmembrane signaling by GPCRs. It summarizes current knowledge of the mechanisms of activation, signal transduction, and regulation of these receptors. The RTKs are discussed briefly because of the recent appreciation that signaling by GPCRs and RTKs is inextricably linked: activation of many GPCRs results in stimulation of RTKs, and these receptors share many of the same mechanisms of signal transduction. This chapter does not discuss the roles of specific GPCRs and RTKs in the control of particular aspects of digestion; these topics are covered in other chapters.

disease, and it has been estimated that more than half of all therapeutic drugs target GPCRs, either directly or indirectly (6). The structure and function of GPCRs have been reviewed in detail previously (2,7).

G Protein–Coupled Receptors Share Structural Motifs GPCRs have similar amino acid sequences with some residues that are conserved between family members (2). They have an extracellular amino terminus, an intracellular carboxyl-tail, seven transmembrane domains of hydrophobic amino acids that are organized in α helices, three extracellular loops, and three intracellular loops (see Fig. 3-1A for the structure of rhodopsin, a prototypical GPCR). Consequently, they are also known as heptahelical or serpentine receptors. Sequences of the transmembrane domains are highly conserved among the different GPCRs, and receptors differ more in their loop structures and intracellular and extracellular tails, which vary considerably in length. Based on similarities in their amino acid sequences, the GPCRs have been divided into six families, each with more than 20% amino acid identity in the transmembrane domains (2,8,9). In mammals, the principal families are: A, or rhodopsin-like family (the largest); B, or secretin-like family; and C, or metabotropic family (Table 3-1). Additional families in mammals include the LNB-7TM, frizzled/ smoothened, vomeronasal 1 receptor, and taste 2 receptor; there are also other families of GPCRs that are found only in nematodes, fungi, and plants.

STRUCTURE AND FUNCTION OF G PROTEIN–COUPLED RECEPTORS

Rhodopsin Is a Model G Protein–Coupled Receptor

With at least 1,000 members, the superfamily of GPCRs comprises about 2% of the proteins encoded by the human genome (1). Many GPCRs sense environmental stimuli such as photons, odorants, and tastes. However, there are more than 360 nonsensory GPCRs, 200 of which respond to endogenous agonists including amino acid transmitters, biogenic amines, regulatory peptides, glycoproteins, proteases, nucleotides, nucleosides, prostanoids, phospholipids, and fatty acids (1,2). There are also approximately 160 “orphan receptors” that have been identified in the genome but for which there is no known ligand or function (3). GPCRs as a group participate in all aspects of physiologic regulation, including the sensory perception of light, odors, tastes, and painful stimuli, and they control cell motility, endocrine and exocrine secretion, neurotransmission, metabolism, inflammation, and immune responses (1). GPCRs also play a major role in human disease. Mutations in GPCRs can cause genetic diseases (4), and certain invading organisms, such as viruses, can hijack GPCRs, thereby making use of their intracellular signaling pathways to ensure their replication, which can contribute to their pathogenesis (5). The GPCRs are of great importance in the treatment of

Formidable technical obstacles have hampered attempts to purify GPCRs and study their structure by X-ray crystallography. Obstacles to purifying GPCRs include their low abundance, the instability of these hydrophobic proteins in environments lacking phospholipids, and the tendency of GPCRs to aggregate and precipitate. These obstacles have been overcome for one GPCR, rhodopsin. Rhodopsin is highly concentrated in the retina, allowing purification and investigation of its crystal structure (10) (see Fig. 3-1B). Consequently, much of our understanding about the structure, function, signal transduction, and regulation of GPCRs derives from studies of opsins. Opsins are photoreceptors of the retina. They include rhodopsin, the photoreceptor of retinal rod cells, and blue, green, and red opsins, the photoreceptors of cone cells. They differ from other GPCRs in that they form a covalent link with their ligand, 11-cis-retinal. The link takes the form of a Schiff base between the aldehyde of retinal and the ε amino acid of a lysine residue in transmembrane domain 7. A single photon converts 11-cis-retinal to all-trans-retinal, leading to a conformational change in the receptor and activation of its G protein, transducin, or Gt.

TRANSMEMBRANE SIGNALING BY G PROTEIN–COUPLED RECEPTORS / 65 C C-terminal

C-II Cytoplasmic side

C-III

C-I

E-III Extracellular side

E-I E-II

N-terminal N

A

B FIG. 3-1. The structure of bovine rhodopsin. (A) Two-dimensional model of bovine rhodopsin. Cylinders indicate the transmembrane helices. The disulfide bridge, which is conserved among G protein–coupled receptors, is at residue (110). (B) The three-dimensional model of rhodopsin; parallel view to the plane of the membrane. (Reproduced from [A] Teller and colleagues [223]; and [B] Palczewski and colleagues [10], by permission.)

There are several notable characteristics of the structure of rhodopsin (reviewed by Filipek and colleagues [11]), which has been analyzed with X-ray diffraction at 2.2-Å resolution (12). The transmembrane domains are arranged in an anticlockwise manner when viewed from the extracellular side of the membrane, in the order of 1-2-3-4-5-6-7. The amino acids of the transmembrane domains are in α helices, although there are certain kinks at proline and other residues, and there are also interactions among the helices. For example, helix 6 of rhodopsin interacts with helix 7 by hydrogen bonds and with helices 2, 3, and 5 by van der Waals forces, which together confer mobility to helix 6 that is probably important for receptor activation. The highly conserved motif NPXXY of helix 7 may allow interaction with helix 6, which could hold the receptor in its inactive state. The carboxyl-terminus of helix 3 of rhodopsin is of particular importance because it contains the E(D)RY motif implicated in the regulation of the receptor’s interaction with its G protein. Rhodopsin has an additional short helix in the cytoplasm, which, together with the cytoplasmic loops 2 and 3, constitutes a part of the binding site for Gtα subunit and is involved in the regulation of Gtγ binding (13) (see G Protein–Coupled Receptors Interact with Heterodimeric G Proteins later in this chapter). The cytoplasmic loops are not well defined and must be mobile in solution, and the residues of the carboxyl-tail of the receptor are probably flexible,

lacking a definite conformation (14,15). The amino-terminal extracellular region of rhodopsin consists of 33 residues, which form a glycosylated unit overlaying the extracellular loops. The residues 173 through 198 on the loop between helices 4 and 5 may form a “plug” for binding of retinal arrestin, an important regulator of GPCRs (see G-Protein Receptor Kinases and Arrestins Mediate Homologous Desensitization later in this chapter). Molecular modeling of other class A receptors based on the structure of rhodopsin suggests that they adopt similar conformations. However, information about GPCR structure comes from study of rhodopsin in its resting state, and little is known about how agonist binding alters the receptor conformation to initiate signal transduction.

Agonists Interact with Different Receptor Domains The sites at which agonists and antagonists interact with their receptors have been deduced by a combination of approaches that include molecular modeling, generation of chimeric and mutant receptors, and binding and signaling assays (Fig. 3-2). As may be expected, the sites of interaction between GPCRs and their agonists and antagonists vary, depending on the structures of the receptors and their ligands.

66 / CHAPTER 3 TABLE 3-1. A partial list of G protein–coupled receptors in classes A, B, and C Class

Examples

Class A: rhodopsin-like

Amines: acetylcholine muscarinic receptors, adrenoreceptors, dopamine receptors, histamine receptors, serotonin receptors Peptides: angiotensin, bombesin, bradykinin, chemokine, cholecystokinin, endothelin, galanin, melanocortin, neuropeptide Y, neurotensin, opioid, somatostatin, vasopressin Protease-activated receptors Glycoproteins: follicle-stimulating hormone; luteinizing hormonereleasing hormone; thyrotropin Rhodopsin Olfactory Prostanoids Nucleotides Cannabinoids Platelet-activating factor Lysosphingolipids Calcitonin Calcitonin gene-related peptide Corticotropin-releasing factor Gastric inhibitory peptide Glucagon Growth hormone-releasing hormone Parathyroid hormone Pituitary adenylyl cyclase activating polypeptide Secretin Vasoactive intestinal polypeptide Diuretic hormone Cadherin Metabotropic glutamate Extracellular calcium-sensing Pheromone γ-Aminobutyric acid receptor B Taste receptors (T1R)

Class B: secretin-like

Class C: metabotropic glutamate pheromone

Class A G Protein–Coupled Receptors Small endogenous agonists usually interact in a noncovalent manner within the transmembrane domains of class A receptors. The sites of interaction between the β2-adrenergic receptor (β2-AR) and its agonists have been investigated extensively. For example, the predicted binding site of the agonist epinephrine is deep within the receptor in between helices 3, 4, 5, 6, and 7 (16). Mutagenesis studies have identified some residues of the receptor that are of critical importance for this binding. For example, Asp113 of helix 3 interacts with amine groups of epinephrine. This residue is highly conserved among the receptors for biogenic amines. Ser203, Ser204, and Ser207 of helix 5 form a network of hydrogen bonds with the two catechol hydroxyl groups of epinephrine (17). Asn293 within helix 6 also interacts with the alkyl-hydroxyl group of epinephrine and with Asp113 of

helix 3. Ile169 (helix 4) interacts with the N-methyl group of epinephrine and might be responsible for the stronger affinity of epinephrine for the receptor than norepinephrine, which does not have this N-methyl group. Zn2+ ions act as a positive allosteric ligand for the β2-AR, enhancing agonist affinity for the receptor (18,19). The Zn2+ binding sites responsible for this effect have been identified by mutagenesis as the His269, Cys265, and Glu225 residues, which are localized on the third intracellular loop between helices 5 and 6. Thus, bridging the cytoplasmic extensions of helices 5 and 6 with Zn2+ facilitates agonist binding. Indeed, biophysical studies have demonstrated that agonist binding leads to the movement of helix 6 relative to helix 5 (20). Binding sites within the transmembrane domains have been identified for other small molecular weight agonists of GPCRs, including adenosine (21), nucleotides (22), and lipids such as anandamide, a cannabinoid receptor agonist (23). Receptors for peptide hormones, neurotransmitters, and paracrine agents are the largest subgroup of the A family of GPCRs. Most peptides interact with residues in the amino terminus and the extracellular loops of their receptors, including receptors for angiotensin II (24), bradykinin (25), and cholecystokinin/gastrin (26,27). However, residues in the superficial regions of transmembrane helices 2, 3, 5, 6, and 7 also contribute to binding of certain neuropeptides, such as neuropeptide Y, opioids, and somatostatin (see Gether [7] and references therein). The binding sites for agonists and antagonists of the neurokinin-1 receptor (NK1R) have been studied in considerable detail. Studies of mutant and chimeric neurokinin receptor indicate that natural peptide agonists bind to residues scattered throughout the extracellular regions, whereas nonpeptide antagonists interact with residues in the transmembrane domain (28–33). Cross-linking experiments using photoactivatable substance P (SP) indicate that the peptide interacts with domains in the amino tail and second extracellular loop of the NK1R (34). Different residues are important for interaction with different antagonists (32,33,35,36). For example, His197 in the fifth transmembrane domain of the human NK1R is critical for binding to CP-96,345 but not RP67580 (nonpeptide NK1R antagonists). However, His265 in the sixth transmembrane domain is required for binding of RP67580 but not CP-96,345. Although involving different residues, the binding pocket for epinephrine in the β2-AR is similarly located. Thus, there may be conservation of binding pockets for small molecules in GPCRs. The importance of other residues in the putative binding pocket for nonpeptide antagonists of the NK1R has been further explored by the systematic mutation of residues near to His, to create a metal ion–binding domain (37). Substitution of Glu193 and Tyr272 to His increases the affinity for Zn2+ by 794-fold by creating a pocket of 4 His residues to bind Zn2+. Remarkably, the creation of a high-affinity binding pocket for Zn2+ in this region inhibits binding and signaling of SP. Thus, binding of Zn2+ ions to the mutated receptor may cause an allosteric alteration that mimics that induced by nonpeptide antagonists, which stabilizes the receptor in a conformation that cannot bind SP.

TRANSMEMBRANE SIGNALING BY G PROTEIN–COUPLED RECEPTORS / 67 Competitive antagonist Opsin

Biogenic amine

Glycoprotein (family A)

Non-competitive antagonist

Neuropeptide (family A, B)

Glutamate (family C)

Agonist

Substance P

Protease-activated

Frizzled (F/S family)

FIG. 3-2. Models of ligand binding sites for various G protein–coupled receptors. Small molecules bind to sites within the transmembrane domains. Competitive antagonists and agonists interact with partially overlapping binding sites in biogenic amine receptors, whereas binding sites for allosteric antagonists have been identified in extracellular regions of muscarinic receptors. Peptides bind to extracellular sites in the amino tail and loops. Substance P binds to extracellular domains of the neurokinin-1 receptor. Proteases cleave receptors exposing tethered ligands that bind to sites in the second extracellular loop. Glycoprotein hormones interact with leucine-rich repeat motifs of the large amino tails of their receptors. Family C receptors possess binding sites for agonists and competitive antagonists in a cleft between two extracellular domains. The amino terminus of Frizzled receptors contains the binding site for their native ligands, the Wnt proteins. (Reproduced from Kristiansen [2], by permission.)

The protease-activated receptors (PARs) are a family of four GPCRs that are activated by proteolytic cleavage (reviewed by Ossovskaya and Bunnett [38]). Certain serine proteases (e.g., coagulation factors such as thrombin and factor VIIa and Xa, mast cell tryptase, trypsins) cleave the extracellular amino terminus of these receptors to expose an amino-terminal domain, which acts as a “tethered ligand” by binding to and activating the cleaved receptor. Synthetic peptides that mimic the tethered ligand domain can directly activate the receptors, which provide experimental support for this mechanism of activation. Thus, the PARs could be viewed as specialized peptide receptors–that is, ones in which the peptide is physically part of the receptor molecule and only exposed by proteolysis. Studies of chimeric and mutant receptors indicate that the tethered ligand interacts with the second extracellular domains of these receptors, and mutational analyses have identified residues in the tethered ligand and second extracellular loop that are critical for this interaction (39,40). Receptors for glycoproteins such as luteinizing hormone, follicle-stimulating hormone, and thyroid-stimulating hormone are unusual in that they have large amino-terminal extracellular

tails of up to 500 residues. These domains contain multiple leucine-rich repeat or LRR motifs, which are involved in protein–protein interactions. These regions are essential for interaction of glycoproteins with their receptors (see Kristiansen [2] and references therein). Class B G Protein–Coupled Receptors Members of the class B family of GPCRs have extracellular tails of intermediate length (~120 residues), and the family includes receptors for functionally important peptides such as secretin, vasoactive intestinal polypeptide (VIP), pituitary adenylyl cyclase–activating polypeptide (PACAP), calcitonin gene–related peptide (CGRP), and corticotropin-releasing factor. Agonists of these receptors interact with several sites within the amino-terminal tail and the extracellular loops (41–43). Most B family GPCRs possess a conserved aspartic acid residue and a basic residue in extracellular loop I at the junction of transmembrane domain 2. Mutational analyses indicate that these are essential residues for agonist binding and receptor activation.

68 / CHAPTER 3 Class C G Protein–Coupled Receptors The class C family of GPCRs includes receptors for endogenous ligands such as glutamate (metabotropic glutamate receptors) and γ-aminobutyric acid (GABAB receptors), for Ca2+ ions (Ca-sensing receptors), for pheromones, and for certain tastes. Given the diverse nature of these agonists, it is not surprising that they interact with various regions of their receptors. Class C GPCRs such as the metabotropic glutamate, GABAB, and Ca-sensing receptors have long extracellular tails. This region of the metabotropic glutamate receptor contains a domain known as the “Venus flytrap module.” Mutational analyses indicate that agonists bind to a cleft between two lobes of this domain, resulting in a conformational change that traps the agonist in the cleft (reviewed by Pin and colleagues [44]). This conformational change may then expose amino acid sequences that serve as tethered ligands, which bind to extracellular loops to activate the receptors. Ca2+ ions also appear to interact with extracellular and transmembrane domains of Ca-sensing receptors, allowing the receptors to switch from an inactive to an active state over a narrow range of Ca2+ ion concentration.

G Protein–Coupled Receptors Can Form Dimers GPCRs were thought to function as monomers: a single agonist molecule binds to one receptor, which then associates with a G protein. However, it is now well established that some GPCRs are dimers (see reviews by Terrillon and Bouvier [45] and Milligan [46]). This view is based on a variety of biochemical studies of the patterns of GPCR migration on electrophoresis gels, coimmunoprecipitation of different receptors, and fluorescence or bioluminescence resonance energy transfer that allows observations of protein– protein interactions in living cells. Through these approaches, investigators have shown GPCRs to exist as homodimers and heterodimers in the endoplasmic reticulum, at the cell surface, and in endosomes. This dimerization has been suggested to contribute to the posttranslational processing and plasma membrane trafficking or “ontogeny” of GPCRs, to their interaction with agonists and antagonists or “pharmacologic diversity,” to their mechanisms of signal transduction, and to agonist-induced trafficking of GPCRs (Fig. 3-3A). The correct folding of proteins in the endoplasmic reticulum is essential for their sorting at the plasma membrane. Some proteins form oligomers in the endoplasmic reticulum, which facilitates this sorting by masking retention signals. The formation of GPCR dimers in the endoplasmic reticulum may be required for sorting to the plasma membrane. This is clearly the case for the metabotropic GABAB receptor, which is formed of two receptor subunits: R1 and R2. When expressed separately, GABABR1 is retained in the endoplasmic reticulum because it contains a retention signal, and although GABABR2 traffics to the cell surface, it is unable to signal (47). However, when coexpressed, the subunits form a heterodimer that masks the retention signal of GABABR1 so that both are translocated

to the cell surface where they form a functional receptor. Vasopressin and oxytocin receptors also form dimers in the endoplasmic reticulum (48). Notably, expression of truncated mutants of GPCRs can sometimes impede expression of wild-type receptors at the plasma membrane, possibly by preventing assembly of dimers in the endoplasmic reticulum (49,50). Although many receptors can form dimers at the cell surface, it is unclear whether they dimerize constitutively, or if agonist binding affects dimerization (see Terrillon and Bouvier [45] and references therein). However, it is well established that dimer formation can have a major effect on receptor pharmacology. For example, when expressed together, δ- and κ-opioid receptors form a stable heterodimer that has a low affinity for either δ- or κ-selective ligands alone, but a high affinity for a combination of ligands, suggesting positive cooperativity (51). Both positive and negative cooperativity have been observed for heterodimers of other GPCRs, including δ/µ-opioid (52) and somatostatin SSTR5/dopamine D2 receptors (53). Such diversity could have important implications for drug development, because the efficacy of agonists and antagonists may be influenced by the components of the heterodimers. Heterodimerization may also be important for signal transduction. In the case of the GABAB receptor, the GABABR1 subunit appears to bind GABA, whereas the GABABR2 subunit couples to heterotrimeric G proteins (47). The heterodimerization of taste receptors is also required for signal transduction. In addition, formation of heterodimers has been proposed to explain the potentiation of signal transduction that can occur when GPCRs are coexpressed, including δ/κ opioid receptors (51), δ/µ opioid receptors (52), and somatostatin SSTR5/dopamine D2 receptors (53). Dimerization could also affect receptor endocytosis, as there are reports that activation of one receptor can lead to endocytosis of a second receptor. For example, when the µ-opioid and NK1R are coexpressed, selective activation of either receptor induces endocytosis of the other receptor (54). There are many other examples of cointernalization, including somatostatin SSTR1/SSTR5 (55) and somatostatin SSTR2A/µ-opioid receptors (56). The functional relevance of this trafficking remains to be determined. Despite the aforementioned reports of GPCR dimerization, it remains uncertain whether dimerization plays an important role in physiologic regulation. Most studies of receptor dimerization are in heterologous expression systems, and, to be of physiologic relevance, the receptors should be naturally coexpressed in the same cell. Given the difficulty of specifically localizing GPCRs in tissues, such colocalization studies have been completed for only a few receptors. An obvious caution is that overexpression may promote dimerization and, indeed, certain receptors will only form heterodimers when they are highly expressed (57). However, formation of dimers has been observed in native cells and tissues and in transfected cells expressing physiologically relevant numbers of receptors, where dimerization can have a major influence on receptor pharmacology, signaling, and regulation.

TRANSMEMBRANE SIGNALING BY G PROTEIN–COUPLED RECEPTORS / 69

3 Endocytosis

2 Pharmacological diversity 1

Early endosome

Ontogeny

Endoplasmic reticulum-Golgi apparatus

A

2 Pharmacological diversity Adrenomedullin CGRP

3 RAMP1

RAMP2

RAMP3

Endocytosis

Early endosome

1 Ontogeny

CLR

B

Endoplasmic reticulum-Golgi apparatus

FIG. 3-3. The role of dimerization and of accessory proteins in the ontogeny (1), pharmacologic diversity (2), and trafficking (3) of G protein–coupled receptors (GPCRs). (A) (1) Certain GPCRs dimerize in the endoplasmic reticulum, and dimerization is required for receptor folding and translocation to the plasma membrane. (2) Once at the plasma membrane, dimerization can influence the ability of GPCRs to couple to heterotrimeric G proteins by both positive and negative cooperativity, resulting in pharmacologic diversity. (3) Some GPCRs internalize as dimers, but the function of this process is unknown. (B) (1) Receptor activity-modifying proteins (RAMPs) are chaperones that are required for trafficking of calcitonin receptor-like receptor (CLR) from the endoplasmic reticulum to the plasma membrane. (2) The presence of RAMPs at the plasma membrane leads to pharmacologic diversity as it determines the affinity of CLR for CGRP and adrenomedullin. (3) CLR and RAMP1 cointernalize.

Accessory Proteins Are Required for the Function of Some G Protein–Coupled Receptors The expression and activity of certain GPCRs is critically dependent on the existence of accessory proteins. Perhaps the best example is calcitonin receptor–like receptor (CLR), a receptor for several peptides including CGRP, adrenomedullin, and intermedin (58,59). The function of CLR depends on receptor activity–modifying proteins (RAMPs), a family of three single-transmembrane domain proteins (see Fig. 3-3B). When expressed alone, CLR is retained in the endoplasmic reticulum and does not traffic to the plasma membrane or

signal. RAMPs act as chaperones that mediate CLR trafficking to the plasma membrane and influence the glycosylation state of the receptor. In addition, the presence of RAMPs with CLR at the cell surface determines the affinity of the receptor for agonists. Thus, CLR/RAMP1 shows preference for CGRP over adrenomedullin, whereas CLR/RAMP2 or 3 binds to adrenomedullin with a higher affinity. CLR interacts similarly with intermedin when it is expressed with RAMP1, 2, or 3. RAMPs may also interact with other GPCRs of the B family, including the calcitonin receptor, the VPAC1 VIP/PACAP receptor, the glucagon receptor, and receptors for parathyroid hormone receptors (60).

70 / CHAPTER 3 MECHANISMS OF SIGNAL TRANSDUCTION Activated GPCRs interact with heterotrimeric G proteins. The subunits of these G proteins then regulate an array of enzymes and ion channels, resulting in both transient and sustained effects on cellular function. Many of these pathways are activated by other classes of receptors, such as RTKs, and there is accumulating evidence for cross talk between GPCR and RTK signaling (see G Protein–Coupled Receptors Transactivate Receptor Tyrosine Kinases later in this chapter). However, scaffolding proteins can localize signaling molecules to particular intracellular domains and, in this manner, increase the specificity and fidelity of signal transduction.

G Protein–Coupled Receptors Interact with Heterotrimeric G Proteins Agonist-bound GPCRs adopt a conformation that promotes interaction with heterotrimeric G proteins. These G proteins play a major role in signaling by GPCR (see reviews by Cabrera-Vera and colleagues [61] and Hampoelz and Knoblich [62]). There are three G-protein subunits: α (39– 45 kDa), β (35–39 kDa), and γ (6–8 kDa). Currently, there are 28 α, 5 β, and 12 γ known subunits that can associate in many different combinations to provide great flexibility in the process of signal transduction. The heterotrimeric G proteins follow a common pathway of activation and inactivation after agonist interaction with a GPCR. When bound to guanosine diphosphate (GDP), the Gα subunit associates with Gβγ and the complex is an inactive heterotrimer. Agonist binding induces the change of conformation of a GPCR resulting in an increased affinity for the G protein. The activated GPCR functions as a guanine nucleotide-exchange factor (GEF) for G proteins, allowing dissociation of GDP and almost immediate replacement with guanosine triphosphate (GTP), which is present at greater concentrations than GDP. The GTP-bound Gα subunit dissociates from the Gβγ subunits, and the dissociated Gα and Gβγ regulate downstream effectors. The intrinsic GTPase activity of the Gα subunit terminates the activation, and the resulting GDPbound Gα reassociates with Gβγ to form the heterotrimer of the resting state. Based on structural similarities, there are four main families of Gα proteins: Gs (Gs, Golf), Gi (Gi1-3, Gt, Gg, Go, Gz), Gq (Gq, G11, G14, G15/16), and G12 (G12, G13) (61). The Gα subunits and the Gβγ subunits (Gβγ subunits are tightly associated and can be regarded as a functional unit) associate with a variety of signaling proteins including enzymes and ion channels to regulate multiple signaling pathways (Fig. 3-4). Each of the four main subtypes of heterotrimeric G proteins regulates particular downstream effectors (61). Gαs activates adenylyl cyclases 1 to 9, leading to formation of cyclic 3′, 5′-adenosine monophosphate (cAMP), and also regulates certain Ca2+ channels. Conversely, Gαi inhibits adenylyl cyclase 5 and 6 to suppress generation of cAMP, and also

controls Ca2+ and K+ channels. cAMP regulates cellular functions primarily by activating cAMP-dependent protein kinase A. Protein kinase A is a serine/threonine kinase that is targeted to substrates by A kinase anchoring proteins. cAMP can also act by protein kinase A–independent mechanisms, for example, by controlling the GEF for the GTPase Rap-1 and by activating certain cAMP-gated ion channels. The Gαq family activates phospholipase Cβ1-4 (PLCβ), which hydrolyses phosphatidyl bisphosphate into diacylglycerol and inositol trisphosphate, which subsequently activate protein kinase C and mobilize Ca2+ ions from intracellular stores, respectively. Protein kinase C is a serine/threonine kinase that is targeted to numerous substrates by receptors for activated C kinase or RACKs (63), and regulates multiple events ranging from channel activities to mitogenic signaling. The G12 subfamily regulates the Ras homology (Rho) GEFs. Gβγ subunits contribute to the regulation of these pathways and also have distinct effects of their own (see Fig. 3-4) (64). For example, Gβγ subunits regulate K+ and Ca2+ channels, activate phospholipase A2 leading to release of arachidonic acid, stimulate phospholipase C β, inhibit or activate adenylyl cyclase, control activity of certain G-protein receptor kinases (GRKs), and contribute to Ras-dependent activation of mitogen-activated protein kinases (MAPKs). Receptors vary considerably in their specificity for activating or coupling to distinct G proteins. Some receptors interact specifically with one class of G proteins (Gq/11, Gs, Gi) to activate a limited number of signaling pathways, whereas others couple promiscuously to many G proteins to generate multiple intracellular signals (65). Different agonists that bind to the same receptor can activate different heterotrimeric G proteins (66), and the formation of GPCR homodimers or heterodimers can result in a complex series of signals (45,46).

G Protein–Coupled Receptors and Heterotrimeric G Proteins Exist in Multiple States Several models have been proposed to describe the interactions among GPCRs, their agonists, and heterotrimeric G proteins. In the most simplistic model, receptors are either unoccupied (off) or agonist-bound (on). The first refinement of this model arose from the report that receptor affinity for agonists depends on its interaction with guanine nucleotides (67). Thus, in the presence of GDP, interaction of an agonist with the β-AR induced assembly of a “ternary complex,” comprising agonist, GPCR, and G protein, in which the receptor was GPCR in a high-affinity state. In the absence of G proteins, the receptor was proposed to exist in a lowaffinity state. The second refinement was prompted by the discovery of constitutively active GPCRs, (i.e., GPCRs that are active even in the absence of agonists). The α1b-AR, with a substitution of part of the third intracellular loop of the β2-AR, or even with a mutation of a single amino acid residue, was observed to activate Gq/11 proteins in the absence of agonists (68). These and similar observations of other GPCRs led to the development of the “allosteric ternary

TRANSMEMBRANE SIGNALING BY G PROTEIN–COUPLED RECEPTORS / 71

GPCR

αi

βγ

αs

RTK

Transactivation

α 12/13

βγ

βγ

α q11

βγ

Shc Grb2

AC

AC

PI3K

PLCβ

GEFs

Src

+

−

PLCβ

SRE

Rho

JAK

InsP3

Ras

DAG

PKA

Raf GRK ROK

STAT

Ca2+

PKC MEK

Ki Pyk2 TK

ERK

Morphology proliferation migration secretion adhesion transcription FIG. 3-4. A summary of G protein–coupled receptor (GPCR) signal transduction. GPCRs couples to heterotrimeric G proteins. The Gα subunits Gs, Gi, G12/13, and Gq11 regulate various enzymes. For example, Gs stimulates, whereas Gi inhibits adenylyl cyclase (AC) to increase or decrease cyclic 3′,5′-adenosine monophosphate (cAMP) levels, and cAMP regulates protein kinase A (PKA). Gq11 activates phospholipase Cβ (PLCβ) to generate inositol trisphosphate, which mobilizes Ca2+, and diacylglycerol (DAG), which activates protein kinase C (PKC). G12/13 couple to guanine nucleotide exchange factors (GEF), resulting in activation of Rho, Rho-kinase (ROK), and serum response elements (SRE). GPCRs can activate the mitogen-activated protein kinase (MAPK) cascade by transactivation of the EGF receptor, through activation of PKC, phosphatidyl inositol 3-kinase (PI3K), Pyk2, and other mechanisms. Gβγ subunits couple GPCRs to other pathways, such as activation of G protein receptor kinases (GRKs), potassium channels (Ki), and nonreceptor tyrosine kinases (TK). (Modified from Ossovskaya and Bunnett [38], by permission.)

complex model.” In this model, GPCRs are thought to exist in equilibrium between two allosteric states: R (inactive) and R* (active) (69). Intermolecular interactions between transmembrane helices are thought to maintain unstimulated GPCRs in the inactive R conformation. In the presence of agonists, the equilibrium shifts toward the R* state, where the agonist, activated receptor R*, and G protein exist as a complex. The advantage of the allosteric ternary complex model is that it can explain the actions of various agonists and antagonists. Full agonists stabilize the R* conformation, producing maximal responses. Partial agonists produce a submaximal response, and thus can attenuate responses to full agonists. Neutral antagonists interact similarly with R and R* forms of receptor, having no effect on constitutive activity. Inverse antagonists have a higher affinity for the R state, and thus can suppress constitutive activity. However, the allosteric ternary complex model cannot account for accumulating evidence that suggests GPCRs exist in multiple activated states. Any models that predict that GPCRs exist in

a single active conformation would predict that the response to an agonist should be the same. However, many GPCRs can interact with different heterotrimeric G proteins to activate several different signaling pathways (70–72). Moreover, the order of potencies of different agonists can vary between different signaling pathways, and receptor mutations can affect one pathway but leave others unaffected (73). Finally, the formation of homodimers and heterodimers of GPCRs can have major effects on their pharmacologic properties. Clearly, new and more sophisticated models are required to explain this increased complexity of GPCR activation and signaling.

Monomeric G Proteins also Mediate G Protein–Coupled Receptor Signaling In addition to the well-characterized mechanisms of GPCR signaling that depend on activation of heterotrimeric

72 / CHAPTER 3 G proteins, small G proteins also mediate GPCR signaling (see review by Bhattacharya and colleagues [74]). Small G proteins are 20- to 30-kDa monomeric GTPases, which together form a superfamily with 100 members. There are five subfamilies, depending on their structure: Ras GTPases (e.g., Ras, Rap, and Ral), Rho GTPases (Rho, Rac, and cdc42), Arf GTPases (Arf1-6, Arl1-7, and Sar), Rab GTPases (>60 members), and Ran GTPases. The members of each subfamily have a general task: The Ras family controls gene transcription, the Rho family also regulates transcription and modulates the actin cytoskeleton, the Rab and Arf families control intracellular vesicular trafficking, and Ran family members regulate microtubule organization and the transport of proteins between the cytoplasm and nucleus. Like the heterotrimeric G proteins, the monomeric GTPases are molecular switches that exist in two states: a GDP-bound inactive state and a GTP-bound active state. The interconversion of these states is closely regulated. Thus, interaction of GTPases with GEFs, which, in turn, are controlled by upstream signals such as those arising from the heterotrimeric G proteins, induces the dissociation of GDP to allow association of the more abundant GTP, resulting in activation. GTPase-activating proteins or GAPs bind to the GTP-bound form and enhance the intrinsic GTPase activity, which converts the GTP-bound form back to the GDP-bound inactive form. GPCRs activate monomeric GTPases by a variety of mechanisms. Ras GTPase Ras GTPase plays a major role in mitogenic signaling by growth factors (see G Protein–Coupled Receptors Transactivate Receptor Tyrosine Kinases later in this chapter). Growth factors activate RTKs, leading to the tyrosine phosphorylation and activation of Ras. Ras, in turn, activates the MAPKs, which regulate numerous cytoplasmic and nuclear targets, thereby controlling multiple cellular events. GPCRs also activate the Ras/MAPK pathway by three general mechanisms, depending on the cell type, the GPCR, and the heterotrimeric G-protein involved (Fig. 3-5). The first general mechanism involves established G-protein effectors, such as second messenger kinases and their products. For example, GPCRs that couple to Gαq/11 mobilize intracellular Ca2+ and activate protein kinase C, both of which stimulate Pyk2 (proline-rich tyrosine kinase), a member of the focal adhesion kinase family (75,76). Pyk2 then interacts with Src, resulting in activation of Ras and the MAPK cascade. The second general mechanism involves transactivation of RTKs. Transactivation refers to the process by which GPCRs co-opt RTKs to transduce their signal. One process by which GPCRs transactivate RTKs involves shedding of ligands for RTKs from cells, which can then activate RTKs in an autocrine or paracrine manner (see G Protein–Coupled Receptors Transactivate Receptor Tyrosine Kinases later in this chapter) (77). The third general mechanism involves arrestin-dependent interaction of

GPCRs with upstream components of the MAPK pathways (see G Protein–Coupled Receptors Transactivate Receptor Tyrosine Kinases and also Internalized G Protein–Coupled Receptors Can Continue to Signal later in this chapter). Rho GTPases Agonists of many GPCRs, such as acetylcholine, lysophosphatidic acid, and thrombin, induce reorganization of the cytoskeleton, as well as proliferation (see Sah and colleagues [78] and references therein). The transforming ability of these receptors depends in large part on G12/13 proteins and activation of Rho GTPases. Rho activation involves the interaction of G12/13 with RhoGEF proteins, such as PDZ-RhoGEF and p115-Rho-GEF. Once activated, the RhoGTPases interact with a wide range of effectors to activate multiple signaling pathways controlling the cytoskeleton and the cell cycle. Arf GTPases The Arf GTPases control membrane trafficking that is associated with endocytosis and recycling (see Takai and colleagues [79] and references therein). Two GEFs, ARNO (ADP ribosylation factor nucleotide-binding site opener) and EFA6, regulate Arf6. Both Arf6 and ARNO contribute to the regulation of GPCR signaling and trafficking by direct interaction with receptors or important trafficking proteins such as β-arrestins (see discussion in Mechanisms That Regulate Signaling by G Protein–Coupled Receptors later in this chapter). One consequence of these interactions is enhanced coupling of GPCRs to phospholipase D activation, which is independent of Gq/11 and Gi/o proteins. β-arrestins may serve as scaffolds to promote ARNO-mediated activation of Arf6, thereby stimulating endocytosis of the β2-AR (80). Arf1 and phospholipase D2 also interact with the carboxyltail of the µ-opioid receptor to control endocytosis (81). Many GPCRs, including metabotropic glutamate receptors, can activate phospholipase D2, which is a regulator of vesicular trafficking, cytoskeletal organization, endocytosis, and exocytosis (82). Thus, phospholipase D2 may be a novel adaptor protein inducing GPCR endocytosis, independently or together with β-arrestin. Rab GTPases The Rab GTPases play a major role in vesicular trafficking (see Seabra and colleagues [83] and Rosenfeld and colleagues [84] and references therein; see also Mechanisms That Regulate Signaling by G Protein–Coupled Receptors later in this chapter). Rab1, Rab4, Rab5, Rab7, and Rab11 have been implicated in various steps of GPCR trafficking from the endoplasmic reticulum to the plasma membrane, and from there to early endosomes, sorting endosomes, recycling endosomes, or lysosomes. Rab1 controls the trafficking of β2-AR and angiotensin II type 1a

TRANSMEMBRANE SIGNALING BY G PROTEIN–COUPLED RECEPTORS / 73 EGF-like ligand ADAM GPCR

EGFR 2

Gβγ Gα

AC

1

? PLC

Pyk2 Ca

Shc

2

DAG

cAMP

Grb2

PKC

PKA

Sos

1 Ras

Rap

Raf 3 β-arrestin scaffold

MEK

ERK

FIG. 3-5. A simplified view of some of the mechanisms by which G protein–coupled receptors (GPCRs) activate mitogen-activated protein kinases (MAPKs). Agonist binding to a GPCR activates heterotrimeric G proteins, which dissociate into Gα and Gβγ subunits. In general, GPCRs can activate the MAPK cascade by three mechanisms: (1) Gα-dependent activation of adenylyl cyclase (AC) or phospholipase Cβ (PLC), leading to activation of second messenger kinases such as protein kinases A (PKA) and C (PKC). (2) Activation of ADAMs (a d istintegrin and metalloprotease) by unknown mechanisms, resulting in shedding of ligands for receptor tyrosine kinases (RTKs) such as the epidermal growth factor receptor (EGFR). The EGFR tyrosine kinases, in turn, activate Ras. (3) Formation of MAPK signaling modules, by which β-arrestins recruit and organize components of the MAPK cascade to endosomes containing activated receptors.

(AT1aR) from the endoplasmic reticulum and Golgi apparatus to the plasma membrane (85). Rab5a participates in endocytosis of the β2-AR, AT1aR, NK1R, and PAR2, and in the trafficking of receptors to a perinuclear sorting region (86–90). Both Rab11a and Rab4a contribute to receptor recycling (89,91), whereas Rab7 promotes trafficking of receptors to lysosomes (92). Certain Rabs can associate directly with GPCRs. For example, Rab5a interacts with a domain in the carboxyl-tail of the AT1aR, and agonist binding promotes GDP/GTP exchange, suggesting that the receptor itself can function as a GEF for this GTPase (87). After activation, the AT1aR is directed to early endosomes, which are its final destination. The direct interaction between AT1aR and Rab5 prevents the receptor from being recycled to the plasma membrane or targeted to the lysosomes (87, 92). However, the overexpression of Rab11 promotes AT1aR recycling, whereas Rab7 overexpression leads to AT1aR

degradation, suggesting that the receptor can bind to other Rab proteins and that its trafficking depends on the Rab proteins expressed in a given cell.

Organization of G Protein–Coupled Receptors, G Proteins, and Signaling Proteins into Microdomains Increases the Efficiency and Fidelity of Signal Transduction Although GPCRs, G proteins, and signaling molecules are expressed at low concentrations in most cells, these proteins must physically interact for effective signal transduction to occur. However, many different pathways of signal transduction make use of the same effectors, and these effectors must be spatially confined to ensure specificity. An established theme of GPCR signaling is that signaling molecules are

74 / CHAPTER 3 concentrated into particular microdomains at the plasma membrane or in the cytosol (see reviews by Ostrom and Insel [93] and Luttrell [94]). This compartmentalization enhances the efficiency of signaling by increasing the concentration of proteins in specific regions of the cell, thereby increasing the likelihood of their interaction. It also promotes the fidelity of signaling by confining signaling proteins to particular regions of the cell, thereby increasing the specificity of their effects. Lipid rafts and caveolae are examples of microdomains that are found at the plasma membrane (Fig. 3-6A) (93). Lipid rafts are formed by the coalescence of sphingolipids and cholesterol, and caveolae are pits in the plasma membrane of similar lipid composition, but with caveolin proteins 1 to 3 on the inner leaflet of the membrane. The concentration of GPCRs and their binding partners in lipid rafts and caveolae may be a general mechanism that promotes efficient signal transduction. For example, β1-AR, β2-AR, Gs, and adenylyl cyclase 6 are enriched in lipid rafts and caveolae in cardiac myocytes (95–97). The mechanisms that determine the organization of proteins in lipid rafts are not fully understood, although they may include interactions with caveolin and posttranslational modifications that favor interaction with lipids. For example, coupling of the 5-HT1A receptor to Gi and inhibition of adenylyl cyclase depends on the palmitoylation of two cysteine residues in the carboxyl-tail of the receptor, leading to its retention in lipid rafts (98). The existence of scaffolding proteins is another example of a mechanism that determines the subcellular location and function of activated GPCRs and associated signaling proteins. Scaffolding proteins are of established importance for MAPK signaling, where multiple different stimuli can activate the same MAPK cascade. Scaffolding proteins provide a mechanism by which agonists of particular receptors can activate a specific MAPK, thereby controlling a given cellular response. β-Arrestins are important scaffolds that couple GPCRs to the extracellular signal-regulated kinases 1 and 2 (ERK1/2; see Fig. 3-6B) (99–104). β-Arrestins serve as scaffolds that recruit GPCRs, Src, Raf-1, and MAPKs to clathrin-coated vesicles. In this manner, they can determine the subcellular location and function of activated ERKs. The role of β-arrestins as scaffolds for GPCR signaling is discussed later (see Internalized G Protein–Coupled Receptors Can Continue to Signal).

Receptor Tyrosine Kinases Comprise a Family of Single-Transmembrane Domain Proteins with Intrinsic Kinase Activity Approximately 60 RTKs have been identified in the human genome, and RTKs are classified into 20 subgroups on the basis of sequence similarities (see reviews by Schlessinger [105] and Blume-Jensen and Hunter [106]). All RTKs have certain common structural characteristics: They are singletransmembrane domain proteins with functionally important extracellular and intracellular domains. The agonist-binding domain is extracellular and is usually glycosylated. Various features of the extracellular domain, such as cysteine-rich motifs, immunoglobulin-like repeats, fibronectin type III repeats, and epidermal growth factor (EGF) motifs, determine receptor specificity for different agonists. An intracellular tyrosine kinase domain is highly conserved between different RTKs. The tyrosine kinase domain catalyzes the transfer of γ phosphate of adenosine triphosphate (ATP) to hydroxyl groups of tyrosine residues of the receptor itself, leading to autophosphorylation or to other target proteins. With the exception of members of the insulin receptor family, which exist as preformed dimers that are linked by a disulfide bridge, RTKs exist principally as monomers in the unstimulated state (see Schlessinger [105] and Blume-Jensen and Hunter [106] and references therein). Agonist binding promotes the dimerization of these monomers, and RTKs can homodimerize or heterodimerize with receptors from the same family, which provides increased flexibility of signaling. For example, there are four epidermal growth factor receptors (EGFRs), designated ErbB1 through ErbB4, and EGF can induce heterodimerization of different combinations of the receptors. Receptor–receptor interactions further stabilize the dimers. On agonist binding and dimerization, RTKs adopt a conformation that favors trans-phosphorylation of tyrosine residues in the activation loop by the dimeric receptor partner (see Schlessinger [105] and Blume-Jensen and Hunter [106] and references therein). This phosphorylation results in repositioning of the activation loop away from the active site, which promotes access to target proteins and ATP, thereby increasing phosphorylation of both the receptor and other proteins that mediate signal transduction. Autophosphorylation also occurs outside of the kinase domain and provides docking sites for downstream signaling molecules.

RECEPTOR TYROSINE KINASES ARE SIGNALING PARTNERS FOR G PROTEIN–COUPLED RECEPTORS

Tyrosine Phosphorylation Recruits and Activates Receptor Tyrosine Kinase Signaling Molecules

Once thought to engage distinct signaling pathways, it is now well established that GPCRs and RTKs can activate the same signal transduction pathways, and that GPCRs can signal by activating RTKs, and vice versa, a process known as receptor transactivation. In view of this functional relationship, it is pertinent to review briefly the RTK family, and then to discuss in detail functional interactions between GPCRs and RTKs.

Tyrosine autophosphorylation of noncatalytic domains in the cytoplasmic regions of RTKs allows recruitment and activation of signaling proteins (see Schlessinger [105] and Blume-Jensen and Hunter [106] and references therein). These tyrosine-phosphorylated sites interact with Src homology 2 (SH2) and phosphotyrosine binding (PTB) domains of signaling proteins, which themselves may have enzymatic activity or act as adaptors that recruit other signaling proteins.

TRANSMEMBRANE SIGNALING BY G PROTEIN–COUPLED RECEPTORS / 75 GPCR

Endocytosis Clathrin-coated pit

P

G 1

Gβγ

P

Arrestin

3

GRK

Dynamin

Arrestin

2 Arrestin GRK

Raf-1 MEK1/2

B

Membrane targets

Clathrin 4

Arrestin

Raf-1

ERK1/2 MEK1/2

ERK1/2 Calveolae Early endosome

ERK1/2 signaling modules GPCR Calveolin Gα Cytoskeleton

Gβγ

Cytosolic retention OR Nuclear targets

5

Arrestin

Raf-1 MEK1/2

ERK1/2

A FIG. 3-6. Examples of the spatial organization of signaling by G protein–coupled receptors (GPCRs). (A) Some GPCRs and heterotrimeric G proteins have been identified to associate with lipid rafts of sphingolipid- and cholesterol-rich structures. Caveolae are pit-shaped depressions in the membrane that resemble lipid rafts but which are coated with caveolin proteins. They may associate with the cytoskeleton by poorly understood mechanisms. (B) The role of β-arrestins as molecular scaffolds that mediate activation and targeting of extracellular signal-regulated kinases 1 and 2 (ERK1/2). (1) Agonist-bond GPCRs adopt a conformation that promotes association with heterotrimeric G proteins. (2) G-protein receptor kinases (GRKs) phosphorylate activated GPCRs to increase their affinity for β-arrestins, which translocate to the plasma membrane and interact with GPCRs. (3) β-arrestins are adaptors for clathrin and AP2 to mediate endocytosis. β-Arrestins also recruit Src, Raf-1, MEK1/2 (mitogen-activated protein kinase kinase), and ERK1/2 to GPCRs in clathrin-coated pits (4) and early endosomes (5), thereby forming signaling modules. For some receptors, this recruitment retains activated ERK1/2 in pits or endosomes where they may regulate cytosolic- or membrane-associated targets.

In this manner, the RTKs promote assembly of multiprotein “signaling platforms.” These mechanisms of RTK signaling have been extensively studied (105,106). They include pathways that activate MAPKs, PLCγ, protein kinase C, phosphatidylinositol-3 kinase, and small GTPases such as Rho, Rac, and Cdc42. Activated RTKs induce GTP/GDP exchange on the small GTPase Ras by the following pathway (see Fig. 3-5). RTK autophosphorylation promotes binding of the adaptor proteins SH2-containing (SHC) and growth factor receptor–bound protein 2 (Grb2). Grb2 interacts with the GEF Sos (Son-ofsevenless), which activates Ras. Activated Ras then interacts with effector proteins such as Raf and phosphatidyl inositol 3-kinase (PI3K) kinase, which then regulate multiple targets. Prominent among these targets are the MAPKs. The MAPKs are a family of evolutionarily conserved serine/threonine kinases that play critically important roles in regulating growth, division, differentiation, and apoptosis of cells (see Chang and Karin [107] and references therein). In mammalian cells, there are three major MAPK modules: ERK1/2, the c-Jun N-terminal kinase/stress-activated protein kinases

(JNK/SAPK), and the p38/HOG1 MAPKs. The MAPKs are regulated by a series of parallel kinase cascades, comprising three kinases that successively phosphorylate and activate a downstream target. For example, in the ERK1/2 cascade, Raf-1 (MAPK kinase kinase) phosphorylates and activates MEK1/2 (MAPK kinase), which phosphorylates and activates ERK1/2. Similar cascades activate JNK and p38. Once activated, the MAPKs can translocate to the nucleus to phosphorylate transcription factors, traffic to the plasma membrane to regulate receptors and channels, or remain in the cytosol to phosphorylate a variety of substrates. Activated RTKs also stimulate PLCγ (108,109). PLCγ binds directly to the EGFR through interaction of its SH2 domains with phosphotyrosine residues. The EGFR phosphorylates and activates PLCγ, which then cleaves phosphatidyl inositol bisphosphate, generating inositol trisphosphate, which mobilizes Ca2+, and diacylglycerol. Both Ca2+ and diacylglycerol activate protein kinase C. Most RTKs also activate phosphatidyl inositol 3-kinase (108,109). The phosphatidyl inositol 3-kinases can interact with phosphotyrosine residues of the EGFR or its docking proteins. Activated

76 / CHAPTER 3 phosphatidyl inositol 3-kinase has multiple downstream effects, including protecting against apoptotic cell death and generation of hydrogen peroxide. RTKs can also phosphorylate and activate the STAT (signal transducer and activation of transcription) proteins, which translocate to the nucleus to regulate transcription.

G Protein–Coupled Receptors Transactivate Receptor Tyrosine Kinases The mechanisms by which RTKs activate MAPKs are well established (see “Tyrosine Phosphorylation Recruits and Activates Receptor Tyrosine Kinase Signaling Molecules”). However, GPCRs can also activate MAPKs to induce effects on differentiation and proliferation of cells (see reviews by Luttrell [94] and Waters and colleagues [110]). There are three general mechanisms by which GPCRs can activate MAPKs: by activating effectors of heterotrimeric G proteins, such as protein kinase A, protein kinase C, and Ca2+ ions; by transactivation of RTKs; and by β-arrestin–dependent interactions between GPCRs and MAPKs (see Fig. 3-5; see also Internalized G Protein–Coupled Receptors Can Continue to Signal later in this chapter). In some cell types, such as those of neuronal and hematopoietic origin, Gαs activation results in protein kinase A-mediated activation of the Ras family GTPase Rap-1 and subsequent activation of B-Ras, resulting in ERK1/2 activation (111,112). The Gq/11-coupled GPCRs can activate MAPKs by Ras-dependent and -independent pathways. For example, protein kinase Cα directly phosphorylates Raf-1 (113), and α1B-adrenergic and muscarinic m1 receptors, which couple to Gq/11, activate ERK1/2 in fibroblasts by this Raf-1-dependent but Ras-independent process (114,115). In neuronal cells, Ca2+ and protein kinase C can activate Pyk2, leading to Rasdependent activation of ERK1/2 (75,76,116). GPCRs can transactivate many RTKs, including receptors for EGF, platelet-derived growth factor (PDGF), insulin-like growth factor, vascular endothelial growth factor, brainderived neurotrophic factor, and nerve growth factor (see reviews by Luttrell [94] and Waters and colleagues [110]). The general process entails RTK autophosphorylation and recruitment of RTK signaling molecules and has been described for GPCRs that couple to Gi, Gq, and G13 proteins. The mechanisms of RTK transactivation have been most thoroughly investigated for the EGFR. A prominent mechanism of EGFR transactivation is ectodomain shedding. Most EGFR ligands, such as EGF, transforming growth factor-α heparin-binding (HB)-EGF, amphiregulin, betacellulin, and epiregulin, are produced as transmembrane precursors. Cellsurface metalloproteases cleave these precursors to liberate soluble growth factors, which can signal to RTKs on the same (autocrine signaling) or nearby (paracrine signaling) cells. In ectodomain shedding, agonists of GPCRs activate metalloproteases to induce shedding of EGFR ligands, which then activate the EGFR in an autocrine or paracrine fashion to induce activation of the Ras/MAPK pathway (77,117).

Thus, inhibitors of metalloproteases can suppress GPCRinduced transactivation of RTKs. These metalloproteases are members of the ADAM (a distintegrin and metalloprotease) family of zinc-dependent metalloproteases (118). Some members of this family have been implicated in GPCRmediated transactivation of the RTKs. For example, ADAM10 mediates bombesin-induced shedding of HB-EGF (119), and ADAM12 is required for the shedding of HB-EGF in cardiomyocytes in response to angiotensin II and endothelin-1 (120). ADAM17 (also known as tumor necrosis factor-α– converting enzyme, or TACE) cleaves proamphiregulin in cells treated with carbachol or lysophosphatidic acid, to liberate soluble amphiregulin, which then binds to the EGFR, resulting in its phosphorylation and activation of the MAPKs (121). In tumor cells, ADAM17 also mediates cannabinoidinduced transactivation of the EGFR through release of HBEGF or amphiregulin (122). Transforming growth factor-α also has been implicated in carbachol-induced transactivation of the EGFR in colonocytes (123). However, there remains much to learn about the process of ectodomain shedding. In particular, the signaling mechanism by which activated GPCRs can stimulate cell-surface metalloproteases, many of which have small intracellular domains, is not well understood. Both Gi/o- and Gq/11-coupled receptors can induce ectodomain shedding, and there are various requirements for Gβγ and Gq/11 subunits in this process. The targets of these G proteins that mediate shedding are not known, although phosphatidylinositol 3-kinase and Src have been implicated as intermediates in EGFR transactivation (124). GPCRs can transactivate other RTKs, such as receptors for insulin-like growth factor, vascular endothelial-derived growth factor, and nerve growth factor, but the mechanisms of their transactivation are not completely understood (see review by Waters and colleagues [110]). However, in some cases, transactivation does not entail ectodomain shedding. For example, agonists of 5-HT2A receptors can transactivate EGFR and activate ERK1/2 by mechanisms that are unaffected by metalloprotease inhibitors (125). Lysophosphatidic acid induces transactivation of the PDGF receptor by a phospholipase D–dependent mechanism (126). Transactivation of the PDGF receptor may also involve heterotrimeric G proteins (127).

Receptor Tyrosine Kinases also Signal through Heterotrimeric G Proteins Although heterotrimeric G proteins are viewed principally as signaling partners for GPCRs, there is abundant evidence that they also mediate signaling by other types of receptors, including RTKs (see review by Patel [128]). For example, the EGFR can activate Gs, thereby stimulating adenylyl cyclase and generating cAMP. Indeed, the cytosolic juxtamembrane region of the EGFR is important for association with Gs. The EGFR can also phosphorylate tyrosine residues of Gs, resulting in its activation. There is also evidence that GPCRs and RTKs can directly associate. The EGFR may exist in a preformed complex with the β2-AR, and stimulation with isoproterenol (β2-AR

TRANSMEMBRANE SIGNALING BY G PROTEIN–COUPLED RECEPTORS / 77 agonist) transactivates EGFR and leads to the internalization of the β2-AR-EGFR complex, which is required for the activation of ERK1/2 pathway (101). The internalization of EGFR after transactivation induced by GPCR agonists, such as lysophosphatidic acid, is dependent on G proteins and β-arrestin. However, EGF itself can stimulate the recruitment of β-arrestin-1 to EGFR (129). In addition, EGF ligandinduced EGFR internalization requires Gβγ subunits and β-arrestin-1 (130). It has been shown that EGFR can interact with, and be serine phosphorylated by, GRK2 (131), but the role of this interaction has not yet been elucidated.

MECHANISMS THAT REGULATE SIGNALING BY G PROTEIN–COUPLED RECEPTORS A common feature of GPCR signaling is that G-protein signals are transient even in the continued presence of agonist, and that repeated challenge with agonist results in a diminution of the signal to subsequent challenges, a phenomenon known as receptor desensitization (see reviews by Bohm and colleagues [132] and Gainetdinov and colleagues [133]). The diminution of a signal after repeated challenge with the same agonist is termed homologous desensitization, whereas diminution of a signal by prior activation of a different receptor is termed heterologous desensitization. Receptor desensitization is often transient and reversible, and with time some receptors resensitize. However, other receptors are degraded after activation, resulting in permanent arrest of their capacity to signal, a phenomenon called down-regulation. For some receptors, challenge with an agonist can also magnify or sensitize responses to subsequent stimuli, but the mechanisms of GPCR sensitization are not fully understood.

functions: the amino terminus contributes to receptor recognition, and the carboxyl-terminus is important for membrane targeting, although there are other domains that mediate interactions with many other proteins. The GRK1/7 family is primarily found in the retina, and thus mediates desensitization of opsins, and GRK4 is found principally in the testis. Thus, GRK2/3/5 and 6 account for desensitization of most GPCRs elsewhere in the body. There are four arrestins, two of which (visual arrestin or arrestin-1 and cone arrestin or arrestin-4) are present in the retina, where they regulate opsins (Fig. 3-7). β-arrestin-1 and -2 (arrestin-2 and -3) are widely expressed and mediate desensitization of other GPCRs (138–140). The crystal structure of visual arrestin reveals two major domains, an aminoterminal and a carboxyl-terminal domain, both of which are formed of a seven-stranded β sandwich, with an interdomain

Carboxyl-terminal domain Hinge

Carboxyl-tail

Amino-terminal domain

A N domain

G Protein Receptor Kinases and Arrestins Mediate Homologous Desensitization Homologous desensitization of GPCRs requires the coordinated actions of two families of proteins, the GRKs and arrestins in the following manner (see reviews by Kohout and Lefkowitz [134] and Luttrell and Lefkowitz [135]). On activation, many GPCRs adopt a conformation that favors interaction with GRKs. The GRKs phosphorylate serine and threonine residues in the carboxyl-tail and intracellular loops of GPCRs (136,137). This phosphorylation increases the affinity of the GPCR for arrestins, which interact with the receptor and uncouple its association with heterotrimeric G proteins, resulting in desensitization (138,139). The seven GRKs are serine-threonine kinases that can be divided into three subfamilies based on structural and functional similarities: GRK1/7, GRK2/3, and GRK4/5/6. The GRKs have a common general structure, with an aminoterminal RGS (regulators of G-protein signaling) domain, a middle protein kinase domain, and a variable carboxylterminal domain. These domains have several general

C domain

A

B

β-arrestin1 R1

R2

1 β-arrestin2 R1

R2

418 410

Src-SH3

B

SRC-SH1 Ask 1 (other MAPKKKs?)

IP6

Clathrin

JNK3 NSF (other MAPKs?) Mdm2

AP2 S412

FIG. 3-7. The structure of arrestins. (A) Ribbon diagram of rod arrestin showing the functionally important amino domain, carboxyl domain, and interdomain hinge. The circles highlight positively and negatively charged residues of the polar core. This core is the main phosphate sensor of arrestin. (B) The domain architecture of β-arrestin-1 and 2. The amino domain interacts with activated G protein–coupled receptors and the carboxyl domain is responsible for secondary receptor recognition. The phosphate sensor domain is illustrated by P, between the amino and carboxyl domains. Important binding and regulatory domains are also illustrated. JNK, Jun N-terminal kinase; MAPK, mitogen-activated protein kinase. (Reproduced from [A] Gurevich and Gurevich [224]; and [B] Luttrell and Lefkowitz [135], by permission.)

78 / CHAPTER 3 Membrane targeting of GRK1, 4, 6, and 7 depends on posttranslational modification of the carboxyl-terminus. Thus, GRK 1 is farnesylated, GRK4 and GRK6 are palmitoylated, and GRK7 has a CAAX sequence that predicts a geranylgeranyl modification (see Kohout TA, Lefkowitz [134] and references therein). These lipid groups anchor the kinases to the plasma membrane. GRK5 is also mostly associated with the plasma membrane through interaction between a positively charged domain at its carboxyl-terminus and negatively charged head groups of membrane lipids such as phosphatidylinositol-4,5-bisphosphate. GRK-induced phosphorylation of GPCRs has little effect on G-protein receptor coupling. Rather, GPCR phosphorylation serves to increase the affinity of the receptor for arrestins, and it is arrestin binding that disrupts the interaction of receptors and heterotrimeric G proteins to mediate desensitization. For example, visual arrestin does not bind inactive, unphosphorylated rhodopsin, but it does interacts with phosphorylated rhodopsin with a high affinity. Crystallography and mutagenesis studies suggest that arrestins undergo a major conformational change to expose binding sites for phosphorylated GPCRs, which is made possible by the interdomain hinge. This increased affinity of arrestins for GPCRs promotes the translocation of arrestins

hinge region (141) (see Fig. 3-7). Studies of mutated arrestins indicate the existence of receptor binding domains on the concave surfaces of both the amino and carboxyl domains (142). However, in addition to binding to phosphorylated GPCRs to arrest signal transduction, arrestins interact with a large number of other proteins, and thereby participate in receptor trafficking and signaling (see Clathrin, β-Arrestins, and Dynamin Mediate Endocytosis of Many G Protein– Coupled Receptors and also Internalized G Protein–Coupled Receptors Can Continue to Signal later in this chapter) (135). Therefore, there are many additional protein binding and regulatory domains. Desensitization requires the presence of GRKs at the plasma membrane in close proximity to agonist-occupied receptors (Fig. 3-8). Whereas some GRKs are recruited to the plasma membrane, others are constitutively present at the plasma membrane. GRK2 and GRK3 are cytosolic proteins in unstimulated cells, but they are targeted to agonist-occupied GPCRs by several mechanisms. These mechanisms include interaction with phosphatidylinositol4, 5-bisphosphate through their plekstrin homology domains (143), interaction with free Gβγ subunits (144), and interaction with activated GTP-bound Gαq (145), all of which would target GRK2 and GRK3 to activated GPCRs. Uncoupling and desensitization

Resensitization

Endocytosis

GPCR

Gα 1

Clathrin-coated pit Dynamin 3

P

2

Arrestin

Gβγ GRK

Resensitization

Fast recycling

Gα P

Gβγ

Clathrin

Arrestin

Arrestin AP2

Slow recycling 6

Rab4 4

Rab5 Arrestin

Recycling endosome

H+ Pase

5

Rab11 Recycling endosome

Early endosome

Arrestin

Multivesicular body

Arrestin

P

FIG. 3-8. Mechanisms of agonist-induced desensitization and trafficking of recycling G protein– coupled receptors (GPCRs). (1) Agonist-occupied GPCRs couple to heterotrimeric G proteins, which transduce signals through Gα and Gβγ subunits. (2) G-protein receptor kinases (GRKs) phosphorylate agonist-occupied GPCRs to promote membrane translocation and binding of β-arrestins. (3) β-arrestins are clathrin and AP2 adaptors, and thereby mediate GPCR translocation into clathrincoated pits. The GTPase dynamin mediates the formation of the first formed vesicles. (4) GPCRs that form low-affinity associations with β-arrestin-2 rapidly dissociate from β-arrestins at the cell surface or in endosomes and follow a fast recycling pathway to the plasma membrane that is mediated by Rab4a and Rab11a. (5) GPCRs that form high-affinity interactions with β-arrestin-1 and -2 translocate to perinuclear sorting endosomes and multivesicular bodies. Translocation to sorting endosomes depends on Rab5a. Endosomal acidification promotes agonist dissociation, and endosomal phosphatases remove phosphates and promote dissociation of β-arrestins. (6) These GPCRs then follow a slow recycling pathway to the plasma membrane. Recycling by either pathway can mediate resensitization.

TRANSMEMBRANE SIGNALING BY G PROTEIN–COUPLED RECEPTORS / 79 from the cytosol to the plasma membrane, where they interact with GRK-phosphorylated receptors and hinder their association with heterotrimeric G proteins (see Fig. 3-8). Given that there are 7 GRKs and more than 1,000 GPCRs, it is a formidable task to determine which GRKs can regulate signaling of a particular GPCR. With the exception of GRK1 or rhodopsin kinase, which is confined to the retina and regulates opsins, most other GRKs are widely distributed and could regulate signaling of many different GPCRs. Observations made with heterologous expression systems indicate that, although some GRKs preferentially interact with specific receptors, in many cases, several GRKs can efficiently phosphorylate a single GPCR and induce its desensitization. For example, GRK2, 3, 4, 5, and 6 can phosphorylate and desensitize the β2-AR (136,146–149); GRK2, 3, and 5 phosphorylate and desensitize the secretin receptor (150); and both GRK2 and 3 phosphorylate and desensitize the NK1R (151). However, whereas overexpressed GRK2, 3, 5, or 6 phosphorylate the VIP type 1 receptor, only GRK2, 3, or 5 actually desensitize its signaling (152). Because several GPCRs are capable of phosphorylating particular receptors, at least in transfected cell lines, it is of considerable importance to determine the expression of specific GRKs in tissues and elucidate whether they are colocalized with the receptor of interest. β-Arrestin-1 and -2, like the GRKs, are also widely distributed, and therefore could interact with many different GPCRs. However, GPCRs can be subdivided into two classes on the basis of their affinity and consequent duration of interaction with β-arrestins. The “class A” GPCRs, such as β2- and β1b-AR, µ-opioid receptor, endothelin A receptor, dopamine D1A receptor, and NK3R, interact with low affinity with β-arrestin-2 but do not interact with β-arrestin-1 (153– 155). These receptors interact transiently with β-arrestin-2 at the plasma membrane, and then quickly dissociate from β-arrestin-2 near the plasma membrane; thus, they are largely excluded from endosomes (Fig. 3-9). The “class B” GPCRs, exemplified by AT1aR, neurotensin 1 receptor, vasopressin V2 receptor, thyroid-releasing hormone receptor, and NK1R, interact with both β-arrestin-1 and -2 with high affinity (153–155). These receptors form stable interactions with β-arrestin-1 and -2 at the plasma membrane, and then translocate with β-arrestin-1 and -2 to endosomes, where they remain associated with GPCRs for prolonged periods. (Note that these classes do not correspond to the major GPCR classes described earlier in G Protein–Coupled Receptors Share Structural Motifs). The affinity of interaction between GPCRs and β-arrestins is determined by the phosphorylation of serine and threonine residues in the third intracellular loop and carboxyl-tails of GPCRs, because exchange of these domains can predictably alter the duration of association of certain GPCRs with β-arrestins (155–157). There are no antagonists of GRKs and β-arrestins that can readily be used to study the function of these proteins in animals. However, recent studies of knockout and transgenic mice have provided important new information about the specificity and function of GRKs and β-arrestins under

physiologic circumstances (Table 3-2). In general, animals lacking or overexpressing GRKs and arrestins are phenotypically normal under resting conditions, but they reveal interesting phenotypes when appropriately challenged. Such studies illustrate the physiologic importance of receptor desensitization. For example, mice lacking GRK1 or visual arrestin exhibit exaggerated and sustained responses of retinal rod cells to light because of reduced desensitization of rhodopsin (158,159). Similarly, GRK2+/− mice (GRK−/− animals die in utero) and β-arrestin-1−/− mice show enhanced contractility of the heart to isoproterenol, which can be explained by impaired desensitization of adrenergic receptors (160,161). Although these former results could be predicted from experiments in cell lines, studies of genetically modified mice have identified unexpected new roles for GRKs and β-arrestins, notably in the nervous system (see Gainetdinov and colleagues [133] and references therein). For example, GRK6-deficient mice show remarkable supersensitivity of locomotion in response to psychostimulants such as cocaine and amphetamine (162). These drugs inhibit dopamine transporters, thereby increasing the levels of dopamine in the extracellular fluid, which, in rodents, results in locomotor hyperreactivity. Direct agonists of dopamine receptors also result in exaggerated locomotion in GRK6−/− mice, and there is increased coupling of striatal dopamine D2 receptors to G proteins in these animals (162). These studies indicate a major role for GRK6 in regulating dopamine signaling in the central nervous system, with possible implications for diseases linked to abnormalities in the dopaminergic system. Studies of knockout mice have also shown involvement of β-arrestin-2 in regulating µ-opioid receptors in the brain (163-165). Thus, β-arrestin-2-deficient animals exhibit enhanced and prolonged analgesic responses to acute administration of morphine, which correlates with enhanced coupling of µ-opioid receptors to G proteins. Together, these data suggest diminished desensitization of opioid receptors in β-arrestin-2-deficient animals. Given the capacity of GRKs and β-arrestins to regulate signaling by many GPCRs in heterologous systems, similar studies of genetically modified animals can be expected to reveal important roles for these GRKs and β-arrestins in many aspects of physiologic control. Because experimentally induced alterations in expression of GRKs and β-arrestins can have marked implications for physiologic control, it is also likely that altered expression of these proteins in diseases may contribute to functional abnormalities of many systems.

Other Kinases Regulate G Protein–Coupled Receptor Signaling In addition to GRKs, other kinases such as protein kinase A, protein kinase C, and MAPKs can phosphorylate GPCRs and decrease their capacity to signal, resulting in desensitization. For example, the β2-AR activates protein kinase A, which can phosphorylate β2-ARs and other GPCRs, leading to heterologous desensitization (166–168). Protein kinase C

80 / CHAPTER 3 β-arrestin2-GFP, 10 min

β-arrestin1-GFP, 10 min

[Ca2+]i (% control)

Alexa-MP-NKB β-ARR2

Alexa-SP

Alexa-MP-NKB β-ARR1

NK3R wt

β-ARR2

Alexa-SP

A

B 120

120

100

100

80

80

60

60

40

40

20

20

0

C

NK1R wt

NK3R wt

β-ARR1

NK1R wt

NK1R

0

NK1R/NK3RI3ct

Control 0 min

#

30 min

NK3R

NK3R/NK1Rct

D

FIG. 3-9. Differences in trafficking and resensitization of G protein–coupled receptors (GPCRs) depending on association with β-arrestins. (A, B) Localization of neurokinin-1 receptor (NK1R; detected by binding with the specific agonist Alexa-Sar-Met-substance P [SP]) and the NK3R (detected by binding of the specific agonist Alexa-Me-Phe-neurokinin B [NKB]) with β-arrestin 1 and 2 tagged with green fluorescent protein at 10 minutes after stimulation. Note that both NK1R and β-arrestin-1 (A) and β-arrestin-2 (B) are colocalized in endosomes, indicative of high-affinity, sustained interactions. In contrast, the NK3R is in endosomes, but β-arrestin-1 (A) and -2 (B) have returned to the cytosol. (C, D) Time course of desensitization and resensitization of Ca2+ responses to the NK1R agonist (SM-SP) and NK3R (MP-NKB) in cells expressing NK1R and a chimeric receptor composed of the NK1R with the third intracellular loop and carboxyl tail of the NK3R (C), and the NK3R and a chimera comprising the NK3R with the carboxyl tail of the NK1R (D). Cells were incubated with 10 nM agonist or vehicle for 15 minutes, washed and challenged immediately (0 minute) or after 30 minutes. Results are expressed as the percentage response to the second challenge compared with vehicle-treated controls. Note the slow resensitization of NK1R and the rapid resensitization of NK3R. Replacement of loop 3 and to a lesser extent C tail of NK1R with NK3R accelerates resensitization of NK1R. Conversely, replacement of these domains of the NK3R slows resensitization. *p < 0.05 compared with wild-type NK1R (C) and NK3R (D); #p < 0.05 compared with wild-type NK1R. Together, these results show that receptors that interact with high affinity with β-arrestins slowly resensitize, whereas those that interact with low affinity rapidly resensitize. Domains in the third intracellular loop and carboxyl-tail, which determine these interactions, also influence the rate of resensitization. (Reproduced from Schmidlin and colleagues [155], by permission.)

TRANSMEMBRANE SIGNALING BY G PROTEIN–COUPLED RECEPTORS / 81 TABLE 3-2. Phenotypes of G-protein receptor kinase and arrestin-deficient mice GRK or arrestin

Target GPCR

Phenotype

References

GRK1−/− GRK2−/− GRK2+/− GRK3−/−

Rhodopsin Unknown β1-AR, β2-AR Odorant receptor Muscarinic M2 muscarinic

Prolonged responses of retinal cells to light Embryonic lethal Increased cardiac contractility to isoproterenol Olfactory supersensitivity Enhanced airway response to methacholine Enhanced hypothermia, hypoactivity, cholinergic supersensitivity Dopaminergic supersensitivity of striatal neurons Impaired lymphocyte chemotaxis Prolonged photo response in rods of retina Increased cardiac contractility to isoproterenol Potentiation and prolongation of morphine-induced analgesia Impaired development of morphine intolerance Impaired lymphocyte chemotaxis

Lyubarsky et al. (158) Jaber et al. (225) Rockman et al. (160) Peppel et al. (226) Walker et al. (227) Gainetdinov et al. (228)

GRK5−/− GRK6−/− Visual arrestin−/− β-Arrestin-1−/− β-Arrestin-2−/−

Dopamine D2 Chemokine CXCR4 Rhodopsin β1-AR, β2-AR µ-opioid CXCR4

Gainetdinov et al. (162) Fong et al. (229) Xu et al. (159) Conner et al. (161) Bohn et al. (163–165) Fong et al. (229)

β1-AR, β1-adrenergic receptor; GPCR, G protein–coupled receptor; GRK, G protein receptor kinase.

has also been implicated in desensitization of several receptors including the m1 muscarinic receptor, the vasopressin receptor, and AT1aR (169–171). Clathrin, β-Arrestins, and Dynamin Mediate Endocytosis of Many G Protein–Coupled Receptors On binding agonists, many GPCRs translocate from the plasma membrane to endosomes, a process that is also referred to as receptor internalization or sequestration. Internalized GPCRs can either recycle back to the plasma membrane to mediate resensitization of signaling, or are targeted to lysosomes or proteasomes for degradation, thereby mediating down-regulation, which irrevocably terminates signaling. These processes of agonist-induced trafficking of GPCRs play major roles in the regulation of signal transduction. The molecular mechanisms of GPCR endocytosis have been investigated extensively, and the precise mechanisms depend on the receptor and cell type in question (see Fig. 3-8). GPCRs mostly commonly internalize at sites of clathrincoated pits, but sometimes involve caveolae, depending on the receptor in question and the cell type. The contribution of clathrin to GPCR endocytosis has been studied by treating cells with agents that disrupt clathrin-mediated endocytosis, such as hypertonic sucrose, which causes abnormal clathrin polymerization, and monodansylcadaverine, which blocks invagination of clathrin-coated pits. An alternative approach is to localize GPCRs to clathrin-coated pits by microscopy. By these approaches it is well established that many GPCRs internalize by a clathrin-dependent mechanism, exemplified by the β2-AR (172), NK1R, gastrin-releasing peptide receptor (173), and PAR2 (174,175). By coupling activated GPCRs to clathrin and other endocytic proteins such as AP2, adaptor proteins also contribute to receptor endocytosis. β-Arrestins are prominent adaptors that couple

many GPCRs, such as β2-AR, NK1R, and PAR2 to clathrin and AP2 (176,177). The role of β-arrestins usually is examined by overexpression of wild-type or dominant negative β-arrestin mutants, or by study of cells from β-arrestindeficient mice. For example, overexpression of wild-type β-arrestins promotes endocytosis of GPCRs (176), whereas overexpression of a fragment of β arrestin (residues 319–418), which comprises a clathrin-binding domain and acts as a dominant negative mutant, impairs their endocytosis (176,178,179). Mutagenesis studies have identified the motifs of β-arrestins that interact with clathrin and AP2, and thus are required for function as adaptor proteins. A motif between residues 374 and 377 of β-arrestin interacts with a domain within the amino terminus of the clathrin heavy chain (180), and a RXR sequence between residues 394 and 396 of β-arrestin binds to the β2 adaptin subunit of the heterotetrameric AP2 adaptor complex (181,182). This complex also interacts with dynamin and clathrin, thereby playing an essential role in the formation of clathrin-coated pits. Just as GRK-mediated phosphorylation of GPCRs promotes their interaction with β-arrestins to mediate uncoupling and desensitization, this phosphorylation is also necessary for endocytosis of certain GPCRs, presumably by enhancing interactions of receptors with β-arrestins (183–185). However, β-arrestins do not participate in endocytosis of all receptors because dominant negative β-arrestin mutants do not inhibit agonist-induced endocytosis of the AT1aR or the m1, m3, and m4 muscarinic receptors (178,186). The overexpression of mutants of the GTPase dynamin, such as the GTPase-defective dominantnegative mutant, dynamin K44E, has revealed an important role for dynamin in the formation of endosomes at sites of clathrin-coated pits and caveolae (187,188). Overexpression of dynamin K44E inhibits constitutive endocytosis of the transferrin receptor, a model for receptor trafficking, and also disrupts agonist-induced endocytosis of numerous GPCRs, exemplified by the β2-AR; NK1R; δ-opioid; muscarinic m1,

82 / CHAPTER 3 m3, and m4; and dopamine D1 receptors (178,186,189–193). However, dynamin K44E does not affect agonist-stimulated endocytosis of the AT1aR, muscarinic m2 receptor, dopamine D2 receptor, and α2B-AR, which must internalize by dynaminindependent mechanisms (178,191,192,194). The receptor domains that are required for endocytosis have been determined for some GPCRs by mutagenesis and domain swapping approaches. These domains may be sites of phosphorylation that interact with adaptor proteins such as β-arrestins. For example, truncation of the carboxyl-tail of the NK1R reduces the rate of SP-induced endocytosis by up to 60% (195). Although the explanation of this effect is not firmly established, it is likely that truncated receptors show impaired GRK-mediated phosphorylation, and hence diminished interactions with β-arrestins (102). Thus, whereas SP promotes membrane translocation of β-arrestins in cells expressing wild-type NK1R, there is no β-arrestin translocation in cells expressing a truncated form of the receptor. Some receptors also possess tyrosine-containing endocytic motifs, including β2-AR (196) and NK1R (197). Thus, mutation of Tyr305 within the putative NPX2-3Y endocytic motif of the seventh transmembrane domain of the NK1R impairs SP-induced endocytosis of this receptor. It appears, therefore, that the SP-stimulated endocytosis of the NK1R relies on endocytic motifs in the carboxyl-tail and seventh transmembrane domain. Agonist-induced endocytosis of GPCRs theoretically could contribute to desensitization by removing receptors from the cell surface, and thus making them inaccessible to hydrophilic agonists in the extracellular fluid. However, detectable endocytosis occurs more slowly than desensitization of G-protein signaling. Furthermore, drugs that disrupt endocytosis or expression of mutants of β-arrestins and dynamin have minimal effects on desensitization of G-protein signaling. Thus, endocytosis is not considered to contribute substantively to desensitization. Instead, endocytosis plays a major role in resensitization and signaling of certain GPCRs.

Receptor Recycling Mediates Resensitization of Signal Transduction Once internalized, some GPCRs recycle to the plasma membrane where they can once again interact with agonists and heterotrimeric G proteins, and thereby mediate resensitization (see Fig. 3-8). However, the mechanisms and pathway of receptor recycling, and thus resensitization, vary among receptors and can also depend on the nature of the stimulus. Moreover, compared with our understanding of the molecular mechanisms and function of endocytosis, little is known about the mechanisms of receptor recycling. One factor that determines the rate of recycling is the interaction of GPCRs with β-arrestins. The class B GPCRs, which interact with β-arrestin-1 and -2 with high affinity, cointernalize with both β-arrestins in endosomes and remain colocalized until the receptor recycles and β-arrestins resume their steady-state distribution in the cytosol (153–155). These

receptors slowly recycle and resensitize. In contrast, the class A GPCRs interact only with β-arrestin-2, and do so transiently (153–155). These receptors rapidly dissociate from β-arrestin-2 at the plasma membrane or in endosomes, and they recycle and resensitize more rapidly. For example, the class B neurokinin 1, AT1aR, and vasopressin V2 receptor slowly recycle and resensitize, whereas the class A NK3R and β2-AR rapidly recycle and resensitize (see Fig. 3-9). The differences in the interaction of class A and B GPCRs with β-arrestins are probably attributable to the presence of GRK phosphorylation sites within their intracellular carboxyl-tails and third intracellular loops. For example, exchange of the carboxyl-tail and third intracellular loop of the NK1R with equivalent domains of the NK3R diminishes the duration of association with β-arrestins and promotes recycling and resensitization (155). Exchange of these domains of the NK3R with the same ones from the NK1R has the opposite effect. Another factor that determines the rate of recycling and resensitization is the nature of the stimulus. For example, moderate concentrations of SP (10 nM) induce the “long pathway” of recycling of the NK1R: The receptor traffics from superficial early endosomes to perinuclear sorting endosomes by a mechanism that depends on the GTPase Rab5a, and enters recycling endosomes to translocate to the plasma membrane over a period of hours (89). After stimulation with low concentrations of SP (1 nM), the NK1R follows the “short pathway” of recycling: The receptor is confined to endosomes located immediately beneath the plasma membrane, from which it rapidly recycles to the cell surface by Rab4a- and Rab11amediated mechanisms. The mechanism by which the NK1R enters the long and short recycling pathways remains to be determined. However, SP causes a concentration-dependent phosphorylation of the NK1R, which could affect association with β-arrestins (198). Perhaps low levels of receptor phosphorylation induce a lower affinity interaction with β-arrestins, which could be more rapidly reversed endosomal phosphatases to allow faster recycling. The importance of receptor endocytosis and recycling for resensitization of signal transduction has been investigated by using a variety of genetic and pharmacologic approaches. For example, inhibition of receptor endocytosis with drugs, receptor mutation, or expression of dominant negative mutants of dynamin and β-arrestin, and inhibition of trafficking and recycling by expression of mutants of Rab5a, Rab4a, and Rab11a, impede resensitization of β2-AR and NK1R (88,89,155,199–202) (Fig. 3-10). Recycling and resensitization can also be suppressed by pharmacologic inhibition of phosphatases and of vacuolar H+ ATPase, suggesting a role for endosomal phosphatases and endosomal acidification (203). Together, these findings suggest the following mechanism of recycling and resensitization. Receptors translocate from the plasma membrane to early endosomes, and then to sorting endosomes. Acidification promotes dissociation of agonist, and endosomal phosphatases dephosphorylate the receptors to allow dissociation of β-arrestins and recycling to the cell surface.

TRANSMEMBRANE SIGNALING BY G PROTEIN–COUPLED RECEPTORS / 83

Calcium mobilization (% control)

120 100

NK1R NK1R + DYN

80

NK1R + DYNK44E

60

NK1R + Rab5a NK1R + Rab5aS34N

*

40 *

20 0

control

0 180 Time between SP challenge (min)

FIG. 3-10. Resensitization of substance P (SP)-induced Ca2+ mobilization in cells expressing the neurokinin-1 receptor (NK1R) alone or with dynamin, dominant-negative dynamin K44E, Rab5a, or dominant-negative Rab5a S34N. Cells were exposed to 10 nM SP or vehicle (control) for 10 minutes, washed, and challenged with 10 nM SP 0 to 180 minutes later. Note that expression of dynamin K44E and Rab5a S34N had no effect on desensitization at 0 minute, but strongly inhibited resensitization at 180 minutes. *p < 0.05 compared with wild-type cells. Thus, endocytosis does not affect desensitization but is necessary for resensitization. (Reproduced from Schmidlin and colleagues [88], by permission.)

Internalized G Protein–Coupled Receptors Can Continue to Signal Although β-arrestins uncouple GPCRs from heterotrimeric G proteins, thereby terminating G-protein signaling at the plasma membrane, they also recruit various signaling molecules to activated receptors at the cell surface or in endosomes, thereby permitting receptors to continue to signal through these pathways. Thus, β-arrestins are molecular scaffolds that interact with GRK-phosphorylated receptors, Src family tyrosine kinases, and members of the ERK1/2 and JNK3 MAPK signaling modules (94). Evidence for the existence of this signaling mechanism was first provided by the finding that agonists of the β2-AR promote the assembly of a multiprotein signaling complex in clathrin-coated pits that includes the receptor, β-arrestins, and Src (see Fig. 3-6) (99). In a similar manner, SP promotes interaction of the NK1R with β-arrestin and Src (100,102). These interactions depend on the binding of β-arrestins to both the receptor and to Src. The Src homology (SH) 3 domain of Src interacts with proline-rich PXXP motifs in the aminoterminal domain of β-arrestins, and there are additional interactions between the SH1 domain of Src and β-arrestin epitopes. The assembly of this GPCR/β-arrestin/Src complex contributes to several signaling mechanisms, including tyrosine phosphorylation of dynamin, which may regulate receptor endocytosis, and activation of ERK1/2 (99,204). For example, disruption of assembly of the complex by expression of mutant β-arrestins or truncated receptors impedes

coupling of β2-AR and NK1R mediated to activation of ERK1/2 (99,102). The activation of various MAPK signaling cascades in cells often requires molecular scaffolds that recruit and organize components of the cascade (94,205). These scaffolds have several functions, including increasing efficiency of interaction between the various components of the cascade, minimizing cross talk between different cascades and thereby maintaining the fidelity of the signal, and targeting activated MAPKs to particular regions of the cell to determine their function and specificity. β-arrestins may serve as scaffolds for some signaling pathways. For example, activation of PAR2 promotes the assembly of a complex containing the receptor, β-arrestins, Raf-1 (MAPK kinase kinase), MEK1/2 (MAPK kinase), and ERK1/2 (103). This complex prevents nuclear trafficking of ERK1/2 and retains them in the cytosol where they may regulate cytosolic substrates. Agonists of the AT1AR also induce formation of protein complexes of the receptor, β-arrestin, Raf-1, and activated ERK1/2 (206–208). β-arrestins also interact with JNK, and thus may serve as scaffolds for several MAPK modules (209).

Lysosomal Trafficking Permanently Arrests Signal Transduction Some activated GPCRs, exemplified by PAR2 (174), δ-opioid receptor (210), and chemokine receptor CXCR4 (211), are invariably targeted for lysosomal degradation (Fig. 3-11). Other receptors that generally recycle can, under certain circumstances, traffic to lysosomes. For instance, although the NK1R usually recycles, after prolonged activation with high concentrations of SP (100 nM for several hours), the NK1R is degraded (N. W. Bunnett, unpublished observation). Given that some internalized receptors can continue to signal, this degradation irrevocably arrests signal transduction. It may also serve as an adaptive mechanism to prevent sustained stimulation of cells when there is a continuous release of a receptor agonist. However, the mechanisms that target receptors to degradation have only recently been understood, and many aspects of lysosomal targeting of GPCRs remain to be elucidated. Ubiquitination of GPCRs and associated proteins, such as β-arrestins, contributes to lysosomal trafficking of several GPCRs (see Fig. 3-11). The covalent attachment of the polypeptide ubiquitin to lysine residues alters the location and function of many proteins, including cell-surface receptors (212,213). Three classes of enzyme are responsible for this ubiquitination. First, an ubiquitin-activating enzyme (E1) uses ATP to form ubiquitin C-terminal adenylate, a substrate for formation of an E1-ubiquitin thiol ester. Second, activated ubiquitin is passed to ubiquitin-conjugating enzymes (E2). Third, ubiquitin ligases (E3) attach the C-terminus of ubiquitin to ε-amino lysines of target proteins. Selectivity of this process depends on the selective interactions between target proteins and the many hundreds of E3 ligases. Attachment of single ubiquitin molecules to lysines, or monoubiquitination,

84 / CHAPTER 3 GPCR

Recovery Endocytosis Clatrin-coated pit

Arrestin AP2 Arrestin

ubi

1

6

Clathrin

Clathrin 2

E3

Rab1

Arrestin AP2

E3

Rab5

3

Down-regulation

Golgi apparatus Endoplasmic reticulum

5

4

Rab7 Arrestin

Lysosome

Arrestin

ubi Early endsome Multivesicular body

FIG. 3-11. Down-regulation of G protein–coupled receptors (GPCRs). (1) Activated GPCRs are phosphorylated and interact with β-arrestins, as described for Figure 3-8, and accumulate in clathrincoated pits. (2) E3 ligases such as c-Cbl in the case of protease-activated receptor 2 (PAR2) and AIP4 in the case of CXCR4 attach activated ubiquitin molecules (ubi) to GPCRs at the plasma membrane or perhaps in endosomes. Other E3 ligases such as Mdm2 can ubiquitinate β-arrestins. (3) GPCRs cointernalize with β-arrestins into early endosomes and are then sorted to multivesicular bodies (4) from whence they are sorted to lysosomes for degradation (5). (6) Mobilization of preformed pools of receptors from the Golgi apparatus or synthesis of new receptors is required for recovery from this down-regulation process.

determines the trafficking and subcellular localization of proteins (212). However, chains of ubiquitin molecules can be attached to the three lysine residues of ubiquitin itself. Such polyubiquitinated proteins usually are destined for degradation in the proteasome (213). Agonist binding prompts the ubiquitination of a growing number of GPCRs, including Ste2p and Ste3p in yeast, and β2-AR, chemokine receptor CXCR4, and PAR2 in mammalian cells (211,214–217). These receptors contain several lysine residues in the carboxyl-tails and intracellular loops that could be ubiquitinated, but the precise sites of ubiquitination remain to be defined. Given the large number of E3 ligases that could mediate this ubiquitination, identifying the ligases involved is a formidable challenge. However, the E3 ligase AIP4 mediates ubiquitination of CXCR4 (218), and c-Cbl ubiquitinates PAR2 (217). Although the E3 ligase that ubiquitinates the β2-AR is unknown, GPCR agonists promote interaction between β-arrestin-2 and Mdm2, which ubiquitinates β-arrestin-2 (216,219). Ubiquitination of β-arrestin-2 determines the stability of its interaction with the β2-AR and affects receptor ubiquitination and trafficking (216,220). Ubiquitination can also affect the trafficking

of GPCRs. Although ubiquitination-defective mutants of β2-AR, CXCR4, and PAR2 internalize normally in response to agonists (211,216), ubiquitination contributes to endocytosis of Ste2p, Ste3p, and EGFR (214,215,221). Disruption of ubiquitination of CXCR4 and PAR2 causes their retention in early endosomes, suggesting that ubiquitination is required for the postendocytic sorting of these receptors (217,218). However, some receptors traffic to lysosomes by ubiquitinindependent mechanisms. For example, lysosomal targeting of the δ-opioid receptor is unaffected by mutation of lysine residues, and thus does not require receptor ubiquitination (210). Instead, lysosomal trafficking of the δ-opioid receptor requires its interaction with GPCR-associated sorting protein (GASP). GASP binds to the carboxyl-tail of the δ-opioid receptor but not to the µ-opioid receptor, which efficiently recycles. Therefore, GASP could preferentially interact with receptors that are destined for lysosomal degradation. Domains in the carboxyl-tail of other GPCRs are also required for lysosomal trafficking. PAR1 associates with sorting nexin 1, and disruption of this interaction impedes degradation (222). The interaction is mediated by domains in the carboxyl-tail of PAR1. Clearly, several mechanisms

TRANSMEMBRANE SIGNALING BY G PROTEIN–COUPLED RECEPTORS / 85 can determine the lysosomal trafficking of GPCRs, and the factors that determine which mechanism is used remain to be discovered.

CONCLUSION The superfamily of GPCRs include receptors for an extraordinarily diverse array of stimuli from the external and internal environment. Given the diversity of their agonists— ranging from simple ions, through small molecules such as biogenic amines, to large proteins such as thrombin—it is remarkable that there are such similarities in the mechanisms of activation, signal transduction, and regulation of GPCRs. However, despite the great progress that has been made in understanding the biology of these receptors, there is still much to learn. There are well more than 100 orphan receptors, with no known ligand or biological function. Given that the gastrointestinal tract contains so many hormones and neurotransmitters that signal through GPCRs, and because many of them were first discovered in this organ, it is likely that these orphans, once adopted, will be found to play important roles in the gut. The mechanisms of activation of GPCRs are incompletely understood. To date, rhodopsin is the only GPCR that has been crystallized and examined by X-ray diffraction. Although may GPCRs have been modeled using information about the structure of rhodopsin, structural studies are required for other GPCRs, particularly in their active states. It is unknown how agonist binding alters the conformation of GPCRs to initiate signal transduction, although such information is invaluable for the development of antagonists. Most drugs exert their effects by affecting signaling by GPCRs either directly or indirectly, and GPCRs are thus tractable targets for the pharmaceutical industry. However, many GPCRs that are thought to play important roles in gastrointestinal diseases have been difficult or impossible to antagonize. Clearly, further studies of the mechanisms of activation and signaling of these receptors is required to facilitate the development of therapeutically useful antagonists. The signaling pathways by which GPCRs exert their effects are also incompletely understood. All cells express multiple GPCRs, either for the same or different ligands. Why do cells often express many distinct GPCRs for the same agonist? As certain GPCRs can exist as homodimers or heterodimers in heterologous expression systems, it is possible that they also dimerize when expressed at physiologic levels in cells, with a marked impact on their pharmacologic properties and mechanisms of signal transduction. The complexity of signaling is further amplified by the now established interactions between GPCRs and other cell-surface receptors, most notably the RTKs, where the interactions of these receptors can markedly affect their pharmacology, regulation, and function. The mechanisms that terminate receptor signaling are also incompletely understood. Although the processes of receptor desensitization, down-regulation, and trafficking have been thoroughly studied in heterologous expression system, little is known about

the role of GRKs and arrestins in vivo. A limited number of studies, most notably in the central nervous system, have shown that specific GRKs and arrestins play major roles in controlling responses to agonists of GPCRs in the intact animal, but nothing is known about this type of control in the gastrointestinal tract. Future studies of the basic mechanisms of activation, signal transduction, and inactivation of GPCRs are likely to have a large influence on our understanding of the role of these receptors and their agonists in the control of the digestive system.

ACKNOWLEDGMENTS Research in the authors’ laboratory is funded by National Institutes of Health grants DK39957, DK43207, and DK57480.

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CHAPTER

4

Gastrointestinal Hormones: Gastrin, Cholecystokinin, Somatostatin, and Ghrelin Graham J. Dockray Gastrin, 92 Overview, 92 Peptide Structure, 92 Gene Structure and Expression, 93 Posttranslational Processing, 93 Cellular Origins, 95 Assay, 95 Release Mechanisms, 95 Metabolism, 96 Receptors, Transduction, and Structure–Activity Relations, 96 Biological and Physiologic Actions, 97 Actions of Nonclassical Gastrins, 99 Conditions of Gastrin Excess, 100 Cholecystokinin, 100 Overview, 100 Peptide Structure, 101 Gene Structure and Expression, 101 Posttranslational Processing, 101 Cellular Origins, 101 Assay, 102 Release Mechanisms, 103 Metabolism, 103 Receptors, Transduction, and Structure–Activity Relations, 103 Biological and Physiologic Actions, 104 Conditions of Excess and Deficiency, 106

Somatostatin, 106 Overview, 106 Peptide Structure, 106 Gene Structure and Expression, 106 Posttranslational Processing, 107 Cellular Origins, 107 Assay, 107 Release Mechanisms, 107 Metabolism, 108 Receptors, Transduction, and Structure–Activity Relations, 108 Physiologic Actions, 108 Conditions of Somatostatin Excess or Deficiency, 108 Ghrelin, 109 Overview, 109 Peptide Structure, 109 Gene Structure and Expression, 109 Posttranslational Processing, 109 Cellular Origins, 109 Assay, 110 Release Mechanisms, 110 Metabolism, 110 Receptors, Transduction, and Structure–Activity Relations, 110 Biological and Physiologic Actions, 110 Conditions of Excess and Deficiency, 111 References, 111

Hormones are molecules carried through the bloodstream to transmit information from one cell to another by interacting with specific receptors on the target cell. The first hormone to be discovered was secretin, which is released from the

intestine by acid and stimulates the flow of pancreatic juice (1). The term hormone was then introduced to describe the general properties of substances such as secretin that were carried from their cells of origin (endocrine cells) to their targets through the general circulation. The gastrointestinal hormones are a subset of the hormonal system; they typically are produced in specialized cells of the gastrointestinal epithelium, the gut endocrine, or enteroendocrine cells, and act on other gut or associated cells to regulate the processes of digestion, control energy intake, and regulate

G. J. Dockray: Department of Physiology, School of Biomedical Sciences, University of Liverpool, Liverpool, L69 3BX United Kingdom. Physiology of the Gastrointestinal Tract, Fourth Edition, edited by Leonard R. Johnson. Academic Press, 2006.

91

92 / CHAPTER 4 the secretion of other hormones concerned with nutrient utilization such as insulin. Some of the products of gut endocrine cells act locally rather than via the bloodstream, that is, through paracrine mechanisms. A good example is somatostatin; because somatostatin cells (or D cells) function in a similar way to other gut endocrine cells, and because there are close functional links between somatostatin and gastrin in the control of gastric function, this peptide is considered here. Other locally acting substances include growth factors and cytokines, and these are considered in Chapters 7, 42, and 43. The hormonal peptides produced in gut endocrine cells may also be found elsewhere in the body, most notably in neurons of the central or peripheral nervous systems. The role of neuropeptide transmitters in the enteric nervous system is also considered in other chapters. The term regulatory peptide is sometimes used as a general expression to encompass peptides that function either as hormones, paracrine factors, or neurotransmitters. Most of the currently known gut hormones had been characterized by the early 1980s. However, new hormones continue to be discovered; a good example is the gastric appetite-stimulating peptide ghrelin, which was first described in 1999 (2), although its cell of origin (the gastric A-like or Xcell) had been recognized for many years previously. At the time of the last edition of this book, excellent progress had been made in defining the molecular identities of the then known gut hormones, the receptors at which they acted, their structure–activity relations, and cellular signaling mechanisms. Since then, impressive progress has been made in the use of genetically modified animals to explore the biology of gut hormones. New insights into the physiology of the major gut peptides have come through the study of animals either overexpressing the genes encoding particular hormones or their receptors, or in which these genes have been deleted (3,4). This chapter is concerned with hormones produced in the upper gastrointestinal tract (gastrin, cholecystokinin [CCK], somatostatin, and ghrelin). The main functional links among these hormones are the control of acid secretion, the control of protein and fat digestion, and the regulation of nutrient delivery to the small intestine either through control of food intake or control of gastric emptying. In addition, there are evolutionary links between two of these peptides (gastrin and CCK). In keeping with the previous editions of this book, this chapter is organized around individual peptides. Each is discussed with respect to its structure, cellular origins and mechanisms of release, the relevant receptors, hormonal actions, and physiology, including data from transgenic and gene knockout (KO) models. Integrative actions of the gastrointestinal hormones with neurotransmitters, paracrine, and other humoral mediators are discussed in other chapters throughout this book.

GASTRIN Overview Gastrin is produced by G cells of the pyloric antral part of the stomach. Its main physiologic function is the control

of gastric digestion through the regulation of acid secretion and gastric epithelial cell proliferation. The idea that gastric acid secretion might be controlled by a gastric hormone was first introduced in 1905 by Edkins (5). There was subsequently some doubt whether the putative gastric secretagogue might not be histamine (6), which was eventually resolved by Komarov (7), who produced the first histaminefree extracts of stomach that stimulated acid secretion. The first preparations of pure gastrin were produced by Gregory and Tracy (8) in the early 1960s, and this material was used to elucidate the sequence of the main peptide found in gastric G cells, which is currently known as G17. Gastrin is an important regulator of postprandial acid secretion, parietal cell maturation, and gastric epithelial cell proliferation; it also influences the proportion of different cell types in the gastric epithelium, notably increasing the abundance of the histamine-secreting enterochromaffin-like (ECL) cell. Gastrin is released by food, particularly protein, in the stomach. Its release is inhibited by gastric acid, probably via the release of somatostatin, which then acts as a paracrine mediator in the antrum. Hypergastrinemia may result from gastrin-producing tumors (Zollinger–Ellison syndrome), which cause gastric acid hypersecretion, or from reduced acid inhibition of the G cell, for example, after therapeutic inhibition of acid secretion or autoimmune loss of parietal cells. In both cases, hypergastrinemia is associated with ECL cell hyperplasia. The gastrin gene is also expressed in some gastrointestinal tumors (9). Some of the products of gene expression in these cells lack the peptide structure required for stimulation of acid secretion but are nevertheless able to act as growth factors. These peptides (progastrin and the Gly-gastrins) can be considered “nonclassical gastrins,” in contrast with the “classical gastrins” such as G17, which are acid secretagogues.

Peptide Structure Gastrin is a linear peptide existing in multiple molecular forms. Gregory and Tracy first isolated two peptides consisting of 17 amino acid residues that were identical but for the presence or absence of a sulfated tyrosine residue (8); the unsulfated peptide was named gastrin I and the sulfated peptide was named gastrin II, based on their elution from ion-exchange chromatography columns. Subsequently, larger and less acidic peptides were identified; these were named at the time big gastrin, to distinguish them from the two heptadecapeptides (then called little gastrins I and II) (10). Gregory and Tracy (11) then characterized big gastrin as 2 peptides of 34 amino acid residues corresponding to the 2 heptadecapeptides extended at their N terminus. The heptadecapeptides are now generally called G17, and the big gastrins are called G34; the suffixes -s and -ns are used to define sulfation status if necessary. These peptides are all acid secretagogues. In addition, small amounts of related acid secretagogue peptides have been identified, notably G14 (the C-terminal tetradecapeptide of G17, also called minigastrin)

GASTROINTESTINAL HORMONES: GASTRIN, CHOLECYSTOKININ, SOMATOSTATIN, AND GHRELIN / 93 (12), G6 (the C-terminal hexapeptide of G17) (13), and an N-terminally extended peptide of 71 residues (G71, also called component 1) (14). The physiologic importance of these other gastrins remains uncertain.

Gene Structure and Expression The complementary DNA (cDNA) encoding gastrin was first cloned from pig antrum (15,16), and subsequently from human (17), rat (18), and other common species. The human gene is located on chromosome 17 and contains three exons (19). The sequence in the coding region predicts a precursor of 104 (porcine) or 101 (human) amino acid residues with a characteristic N-terminal signal peptide and a single sequence corresponding to the peptides isolated from tissue (Fig. 4-1). The different forms of gastrin described earlier therefore originate by alternative posttranslational processing from a single precursor generated from a single gene. There is conservation throughout the mammals of primary amino acid sequence in the region corresponding to the C-terminal portion of G17 and of the cleavage sites generating G34 and G17. The C-terminal sequence of gastrin is shared with the intestinal hormone CCK, reflecting a common evolutionary history of the two peptides (Fig. 4-2). Distinct genes encoding peptides more or less similar to mammalian gastrin and CCK have been cloned in chicken (20), turtle (21), bullfrog (22), and an elasmobranch fish (23). Although the C-terminal amide sequence (WMDFamide) is well conserved, there is variation across the major vertebrate groups in the immediately preceding sequence, which seems to be linked to specificity for stimulation of acid secretion. In adult mammals, the gastrin gene is expressed principally in the pyloric antral mucosa. In some species, particularly humans, there is also expression in the duodenal mucosa. In addition, there may be low-level expression in the pituitary. During development, the gastrin gene is expressed in pancreatic islet cells, and in the rat, the shift to an adult pattern of expression occurs around birth (24). Some work indicates that a number of epithelial tumors, including colorectal and lung carcinomas, may express the gastrin gene (25–27); however, the pattern of processing of the precursor peptide in these cells often differs from that in normal endocrine cells (see later). Expression of the gastrin gene in G cells is regulated by the gastric luminal contents. Amino acids that stimulate Preprogastrin gene exon 1

gastrin release also increase gastrin messenger RNA (mRNA), and fasting decreases gastrin mRNA (28). Moreover, intragastric acid decreases gastrin mRNA, and inhibition of acid secretion (e.g., with proton pump inhibitors) increases both mRNA abundance and rates of translation (29,30). The cellular signaling pathways remain uncertain, but activation of the mitogen-activated protein kinase (MAPK) pathway (e.g., by epidermal growth factor) stimulates gastrin gene expression via phosphorylation of Sp1 (31). There is also evidence for control of gene expression via the Wnt pathway and by transforming growth factor-β (32). The gastric pathogenic bacterium Helicobacter pylori is associated with increased plasma gastrin concentrations and increased gastrin mRNA abundance (33). This may be partially attributable to decreased production of somatostatin caused by locally increased concentrations of proinflammatory cytokines. But, in addition, there appears to be stimulation of nuclear factor-κB signaling in G cells of patients infected with Helicobacter pylori, which increases gastrin gene expression (34). In colorectal carcinoma, activation of Ras and the TCF-4/β-catenin signaling pathway has been implicated in gastrin gene expression (35,36).

Posttranslational Processing Studies using antibodies to peptides derived from different regions of progastrin, together with chromatographic separation, first indicated variation in the peptide products of preprogastrin between different cells expressing the gastrin gene. The kinetics of processing have since been studied using pulse-chase labeling methods (37,38). The initial product of translation, preprogastrin, is rapidly converted to progastrin by removal of the N-terminal signal peptide during sequestration into the lumen of the endoplasmic reticulum (Fig. 4-3). Cleavage of the signal peptide occurs at either Ala21 and Ser22, or Arg26 and Ser27 (39,40). Thereafter, the conversion of progastrin to its final secretory products involves endoproteolytic cleavage at pairs of basic residues, carboxypeptidase cleavage of C-terminal basic residues, Serphosphorylation, Tyr-sulfation, and C-terminal amidation. Phosphorylation of Ser-96 and Tyr-87 occurs in the trans-Golgi network (TGN). There is evidence that Serphosphorylation might determine subsequent cleavage at the adjacent pair of Arg residues (41). In endocrine cells, cleavage at pairs of basic residues (Arg-57,58, Lys-74,75, and

exon 2

exon 3

Preprogastrin mRNA RR

KK

RR

Preprogastrin

FIG. 4-1. Schematic representation of the relationship between the preprogastrin gene, preprogastrin messenger RNA (mRNA), and the peptide precursor preprogastrin.

94 / CHAPTER 4 CCK Caerulein Gastrin

SO-3 -Asp-Arg-Asp-Tyr-Met-Gly-Trp-Met-Asp-Phe-NH2 SO-3 pGlu-Gln-Asp-Tyr-Thr-Gly-Trp-Met-Asp-Phe-NH2 SO-3 -Glu-Glu-Glu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2

FIG. 4-2. Amino acid sequence of the C-terminal region of gastrin, cholecystokinin (CCK), and caerulein. Shared sequences are indicated by the box; note also the sulfated tyrosine residue at position 7 (CCK, caerulein) or 6 (gastrin) from the C-terminus; gastrin commonly exists in both Tyr-sulfated and unsulfated forms.

Arg-94,95) is mediated by subtilisin-like prohormone convertases (SPC), followed by carboxypeptidase E removal of C-terminal basic residues. Both SPC1/3 and SPC2 have been identified in G cells (42). Pulse-chase labeling studies indicate that in rat G cells, most progastrin is initially cleaved at the pairs of Arg residues to yield a 35-residue peptide with COOH-terminal glycine (G34-Gly). These cleavages appear to occur after sequestration into vesicles of the

regulated pathway of exocytosis and exhibit a t1/2 of about 12 minutes after exit from the TGN (37). In addition to G34-Gly, there are two other products: an N-terminal progastrin fragment and a COOH-terminal tryptic fragment that includes Ser-96, both of which are thought to be biologically inactive (see Fig. 4-3). Subsequently, G34-Gly is converted to the corresponding C-terminal amidated peptide (G34) via the enzyme peptidyl α-amidating monooxygenase (PAM).

SPC2, SPC3; Carboxypeptidase E Progastrin

G34-Gly

-Gly

G17-Gly

-NH2

G34 G17

A ER:

Preprogastrin

Golgi TGN:

Progastrin

G34-Gly

-NH2

Constitutive secretion G34-GlyOH

G34

Regulated secretion

Secretary vesicles: G17-Gly

B

PAM

-Gly

Non-classical gastrins

G17-GlyOH

G17

Classical gastrins

FIG. 4-3. Biosynthetic relations among different forms of gastrin. (A) Relations among progastrin, the Gly-extended gastrins (G34-Gly, G17Gly), and the amidated gastrins (G34, G17). The relevant posttranslational processing enzymes are shown. The stippled box corresponds to the signal sequence in preprogastrin. (B) Biosynthetic routes by which preprogastrin is converted to the secretory products, the subcellular compartments in which conversion occurs, and the secretory pathway taken by different products of preprogastrin. ER, endoplasmic reticulum; PAM, peptidyl α-amidating monooxygenase; TGN, trans-Golgi network.

GASTROINTESTINAL HORMONES: GASTRIN, CHOLECYSTOKININ, SOMATOSTATIN, AND GHRELIN / 95 This is a two-step reaction involving the generation of a hydroxy-glycine intermediate by the oxygenase domain of PAM, followed by conversion to the C-terminal amide by the lyase domain of the enzyme. The product, G34, may then be converted to G17 by cleavage at Lys-74,75 (38,42–44). There may also be some cleavage of G34-Gly to yield G17-Gly. The pH in secretory vesicles in endocrine cells is approximately 5.5, and prohormone convertases have acidic pH optima. Alkalinization of secretory vesicles has relatively little effect on cleavage at pairs of Arg residues, but it significantly inhibits the cleavage of G34 at Lys-74,75 (45). Some research has suggested that progastrin and the COOH-terminal Gly-gastrins might have biological activities that are distinct from those of the amidated peptides; for this reason, it is useful to refer to these peptides as the “nonclassical gastrins” (see Fig. 4-3). This reflects their new-found status as biologically active peptides, rather than their existence per se, which has been appreciated for many years (46,47). By analogy, the peptides G34 and G17 that were originally isolated and characterized by Gregory and Tracy (8,11,48), and that possess the defining biological property of the hormone, the stimulation of acid secretion, may be called the “classical gastrins” (see Fig. 4-3). There is cell-to-cell variation in posttranslational processing. For example, in human duodenum, G34 predominates over G17 (49), whereas the opposite is true in antral G cells. Moreover, in gastrinoma cells, there is considerable variation between tumors, which probably reflects variable expression of processing enzymes (46). In colorectal carcinoma cells, the main products of gastrin gene expression appear to be progastrin and the Gly-gastrins (25,26,50,51). Because endopeptidase cleavage and COOH-terminal amidation occur in secretory vesicles of the regulated secretory pathway (52) and because in nonendocrine cells these vesicles are scarce or absent, it is thought that the main products of secretion are largely unprocessed peptides that pass directly from the TGN to the cell surface via the constitutive route of secretion.

Cellular Origins In adults of most mammals, G cells identified by immunohistochemistry are found predominantly in the antral mucosa; in humans, there are also significant numbers in the duodenum. G cells are “open”-type endocrine cells; that is, their apical border projects into the gastric lumen and characteristically terminates there in a tuft of microvilli. There is considerable variation in the morphology of secretory vesicles in G cells. Although differences in fixation may account for some of the reported variation seen by electron microscopy, it also seems probable that vesicles recently budded from the TGN are smaller and more electron dense than older vesicles, and that internal pH influences morphology (53–55). The results of [3H]-thymidine incorporation experiments suggest a turnover time for hamster G cells of 10 to 15 days and a cellular derivation from neck precursor cells (56). There is likely to be physiologic control of G-cell numbers

because the labeling index can be increased by administration of bombesin (57), and there is G-cell hyperplasia in achlorhydria (58). Double labeling immunohistochemistry suggests that at an early stage in their differentiation, progenitor endocrine cells in the antrum express both gastrin and somatostatin genes (59). The basic helix-loop-helix (bHLH) transcription factor, neurogenin 3, is required for the development of these cells because both G and D cells are absent in mice lacking the gene, although endocrine cells in the gastric corpus such as ECL and X-cells are present (60,61). In contrast, the bHLH transcriptional repressor, Hes1, is a negative regulator of endocrine cell numbers, and in mice with deletion of the Hes1 gene, there is hyperplasia of pyloric antral and intestinal endocrine cell populations (62). There is also a requirement for the homeobox gene PDX-1 for the final differentiation of G cells, and there is loss of G cells and increased numbers of D cells in mice deficient in PDX-1 (63).

Assay For routine assay purposes, particularly for the determination of plasma gastrin concentrations, radioimmunoassay (RIA) is the method of choice (64). Assays based on determination of acid secretion, or other aspects of parietal cell function, are less sensitive but may be valuable where the distinction between biological and immunological activity is important. Most routine RIAs use antibodies binding at the C-terminus of the amidated gastrins. This sequence is shared with CCK, and thus care is needed to use antibodies with epitopes extending into the unshared sequence to avoid problems of cross-reactivity. Many different antibodies have been described, some showing remarkable capacities to distinguish between peptides differing in individual amino acid residues, in the presence or absence of the C-terminal amide or in Tyr-sulfation status. Even so, many antibodies show approximately similar affinity for the major C-terminally amidated gastrins, and these are usually the most useful for routine assays of plasma hormone concentrations. However, because there are multiple peptides derived from progastrin, the initial validation of a gastrin RIA should include studies of immunoreactive peptides after separation by chromatography. Assays using antibodies reacting at the C-terminus of progastrin (and not with amidated gastrin or Gly-gastrin) or at the C-terminus of Gly-gastrin (and not with progastrin or amidated gastrin) have been used by several groups and are of particular value in the study of nonclassical gastrins (25,26,46,47).

Release Mechanisms Plasma concentrations of the amidated gastrins in fasting humans are in the range 10 to 30 pmol/liter. After a mixed meal, plasma concentrations increase twofold to threefold with a peak at 30 to 60 minutes. The main circulating form

96 / CHAPTER 4 of gastrin in basal conditions is G34, and the main form at the peak of the postprandial response is G17 (65). The secretion of gastrin is regulated by nervous reflexes, by the direct action of luminal chemicals on G cells, and by indirect actions of the luminal contents mediated via other endocrine cells or neurons (Fig. 4-4). The mechanisms regulating gastrin release have been studied extensively in vivo (66), in the isolated perfused rat stomach (67,68), and in isolated G-cell preparations (69). The main luminal nutrients releasing gastrin are aromatic amino acids. The relevant transduction mechanisms remain uncertain. There is, however, evidence that G protein–coupled receptors may play a role as luminal sensors in enteroendocrine cells (70). In other systems, the aromatic amino acids that release gastrin have been shown to bind to and activate the external calcium-sensing receptor (CaR) (71), and there is evidence that CaR is expressed by G cells (72). The release of gastrin is inhibited by luminal acidification less than pH 3, and this provides a feedback-inhibition mechanism autoregulating gastrin release during a meal (73). Acid stimulates antral D cells to release somatostatin that then acts as a paracrine inhibitor of G-cell function (68). A number of neurohumoral factors (e.g., secretin, vasoactive intestinal peptide [VIP]) that inhibit G-cell function probably also work by release of somatostatin (67). Acute neutralization of the gastric contents does not alone stimulate gastrin release. However, prolonged inhibition of acid secretion, which occurs with administration of proton pump inhibitors (74,75) or in conditions where parietal cells are lost (e.g., pernicious anemia) (76), leads to hypergastrinemia. There is also increased gastrin release in conditions of antral inflammation, for example, with H. pylori infection (33,77), which is thought to be caused by direct stimulation of G cells by proinflammatory cytokines and indirect stimulation via cytokine suppression of D-cell function (see Fig. 4-4). In animal models, there is evidence that prolonged inhibition of acid secretion leads to bacterial

H+

H. pylori

Peptides/ aminoacids

+

+ NFκB/ Sp1 G-cell

D-cell −

SOM

TNFα, IL-1β

− +

overgrowth and depression of D-cell function, which could account for increased gastrin release (78). Distension of the stomach has little effect on gastrin release, but sham feeding or vagal stimulation increase gastrin release in humans and dogs. The effects of vagal efferent stimulation on the G cell are, however, complex with evidence for both cholinergic-excitatory and cholinergic-inhibitory pathways regulating gastrin release (66). In addition, there is clear evidence of a noncholinergic excitatory pathway, and the bombesin-like peptide, gastrin-releasing peptide (GRP), has been considered as a putative noncholinergic neurotransmitter controlling gastrin secretion. Thus, there are GRPcontaining nerve fibers in the vicinity of G cells, release of gastric GRP occurs with luminal or vagal stimulation, and administration of GRP antibodies blocks luminal stimulation of gastrin release (79–82). However, studies using a GRP receptor antagonist suggest that in humans, postprandial gastrin release is not mediated by GRP, although there is a role for the latter in control of acid secretion (83).

Metabolism There are important differences in the metabolic clearance rates of G17 and G34. In both humans and dogs, G17 is cleared from the peripheral circulation with a t1/2 that is about fivefold less than that of G34 (84,85). There is no evidence that G34 is converted to G17 in the peripheral circulation. There is, however, evidence that G17 may be cleaved to yield shorter C-terminal fragments such as G14 (86). No single organ has a dominant role in the metabolism of G17, and most capillary beds in the body remove similar proportions of G17 arriving in the arterial blood. Small C-terminal fragments of G17 may be transported by the liver into bile, but this does not apply to G17 or G34; nor do the latter appear in urine. Interestingly, however, N-terminal fragments of G34 (which are biologically inactive) have been described in urine (87). Enzymes responsible for cleavage of G17 include neutral endopeptidase 24.11 (also known as enkephalinase) and angiotensin-converting enzyme. Neutral endopeptidase 24.11 cleaves G17 on the C-terminal side of multiple residues including Trp4, Ala11, Gly13, and Asp16 (88–90).

Receptors, Transduction, and Structure–Activity Relations

Gastrin GRP EGF

FIG. 4-4. Control of G-cell function. G-cell function is stimulated by luminal peptides and amino acids, the neuropeptide transmitter GRP (gastrin-releasing peptide), and the growth factor EGF (epidermal growth factor). It is inhibited by luminal acid, via the paracrine mediator somatostatin from D cells. H. pylori infection depresses D-cell function via proinflammatory cytokines, which also enhance G-cell function. IL-1, interleukin-1; SOM, somatostatin; TNF, tumor necrosis factor.

Carboxy-terminal amidated peptides of the gastrin-CCK family act at the CCK-1 (also known as the CCK-A) and CCK-2 (also known as the CCK-B or gastrin-CCKB) receptors (Table 4-1). The former has low affinity for gastrin, but high affinity for CCK, and the latter has high affinity for both gastrin and CCK (91). The CCK-2 receptor is therefore the primary mediator of the physiologic effects of gastrin. It is normally expressed by parietal cells, ECL cells, some smooth muscle cells, neurons of the central and peripheral

GASTROINTESTINAL HORMONES: GASTRIN, CHOLECYSTOKININ, SOMATOSTATIN, AND GHRELIN / 97 TABLE 4-1. Characteristics of gastrin and cholecystokinin receptors Characteristic

CCK1

CCK2

Alternative names Agonist selectivity Distribution

CCK-A CCK>>>gastrin Pancreatic acinar cells, pylorus, vagus, enteric neurons, CNS neurons L-364,718 (devazepide), loxiglumide

Gastrin/CCK-B CCK = gastrin Parietal cells, ECL cells, CNS neurons

Selective antagonists Receptor coupling Primary GI functions

Gαq/11, Ca2+, PKC Pancreatic enzyme secretion, gall bladder contraction, inhibition of gastric empying, satiety

L-365,260, L-740,093 YM022, CI-988, YF476, spiroglumide, gastrozole Gαq/11, Ca2+, PKC Acid secretion, parietal cell maturation, ECL cell proliferation

CCK, cholecystokinin; CNS, central nervous system; ECL, enterochromaffin-like; GI, gastrointestinal; PKC, protein kinase C.

nervous systems, and (depending on the species) pancreatic acinar cells. The main endogenous ligand for CCK-2 receptors in the brain is likely to be CCK, because this is abundantly produced by central nervous system (CNS) neurons, whereas gastrin is not. In the periphery, however, receptors are exposed to both circulating hormones, and the main endogenous ligand in this case is gastrin because its concentrations in plasma are 5 to 10 times greater than those of CCK. The nonclassical gastrins—that is, progastrin and the Glygastrins—are not endogenous ligands of the CCK-2 receptor. However, these peptides are now known to have their own characteristic pattern of biological activities that are thought to be mediated by non–CCK-1/non–CCK-2 receptors (92,93). The original studies of Tracy and Gregory (94) established that the C-terminal tetrapeptide amide (WMDFamide) is the smallest fragment of G17 with full agonist activity at CCK2 receptors. Thus, the nonclassical gastrins (such as progastrin and the Gly-gastrins), which lack the COOH-terminal amide moiety, have low or no affinity for CCK-2 receptors. Several CCK-2 receptor antagonists have been described, including L-365,260, L-740,093, YM022, YF476, spiroglumide, and gastrozole (91,95,96). Results obtained using these compounds support the idea that gastrin regulates postprandial increases in gastric acid secretion (97). Both CCK-1 and -2 receptors belong to the seventransmembrane domain, G protein–coupled receptor superfamily (91). Homology studies suggest that they are closely related to each other. Signaling mechanisms downstream of the CCK-2 receptor have been studied in isolated parietal and ECL cells (98,99), in cancer cell lines expressing the receptor (e.g., AR4-2J cells) (100), and in transfected cells (101). The receptor is coupled to Gαq/11, and activation leads to an increase in intracellular Ca2+ and protein kinase C (PKC). In addition, there is activation of the MAPK and phosphatidyl inositol kinase (PI3K)/Akt signaling pathways, and these have been linked to a variety of outcomes including proliferation, migration, inhibition of apoptosis, and tubulogenesis (102–105). At least in part, it now seems possible that activation of these pathways may be secondary to autocrine and paracrine activation by growth factors released by gastrin (52). A CCK-2 receptor variant in which intron 4 of the gene is retained (CCK-2Ri4sv) has an extra 69 amino acid residues

in the third intracellular loop. This variant appears to be expressed in colorectal tumors but not adjacent normal colon, in Barrett’s esophagus but not normal esophagus, and in pancreatic cancer (106–108). It has somewhat increased constitutive activity compared with the wild-type receptor and slightly higher affinity for Gly-gastrin, although still relatively low (<0.001) compared with G17 (106). In HEK293 cells, which have low Src activity, expression of CCK2Ri4sv is reported to be associated with an agonist-independent increase in Src, and it has been suggested that expression of this form of the CCK-2 receptor could contribute to the increased Src activity in some tumors (109). Research suggests that induction of CCK-2 receptor expression occurs in some circumstances. Thus, in transgenic mice overexpressing gastrin, there is increased proliferation in the stomach and induction of the CCK-2 receptor on proliferating cells (110). There are also rapid increases in expression of the receptor at the margin of cryoulcers induced in rat stomach (111), and greater expression in the epithelium of Barrett's esophagus compared with esophageal epithelium from unaffected individuals (112). Together, these data indicate that the CCK-2 receptor gene is expressed in response to tissue injury or inflammation. In human colon cancer cells, increased transcription has been reported in response to overexpression of oncogenic Ras via an MEKdependent mechanism (113). Recently, signaling mechanisms activated by the nonclassical gastrins (progastrin and Gly-gastrin) that are independent of CCK-2 receptors have attracted attention (100,114,115). Although a full characterization of the relevant receptors is still required, there is some evidence that progastrin and Glygastrin may act at separate receptors (93,116). Activation of Src has been linked to both progastrin and Gly-gastrin (116,117). The actions of Gly-gastrin may be either MAPKdependent or -independent (114,115) and include stimulation of PI3K (115).

Biological and Physiologic Actions The amidated gastrins have a wide range of biological actions on gastrointestinal tissues. Proof of the physiologic actions of a circulating hormone have, in the past, been

98 / CHAPTER 4 based on experiments showing equivalence in the responses to similar plasma concentrations of hormone released endogenously and administered exogenously. This approach works well for acute effects but is less satisfactory for long-term effects (e.g., cell turnover or control of gene expression). In these cases, the use of receptor antagonists may be valuable, as may be studies of genetically modified animals in which the gene of interest has been deleted. The application of these approaches to gastrin has both confirmed traditional views of the physiology of the hormone in control of acid secretion and produced new insights into roles in the control of cell proliferation and differentiation in the gastric epithelium (3,4,52). Consult the previous edition of this book (118) for a full account of the earlier evidence for the physiologic role of gastrin in control of acid secretion. This edition focuses on more recent work. Parietal Cell Function and Control of Acid Secretion At a given concentration of endogenous plasma gastrin, the increase in gastric acid output after intragastric instillation of an amino acid test meal is comparable with that produced during infusion of exogenous G17 (119). Moreover, the postprandial increase in gastric acid secretion is inhibited by gastrin immunoneutralization (120) and by CCK-2 receptor antagonists (66). Pharmacologic studies using histamine H2 receptor antagonists indicate that gastrin stimulates acid secretion mainly via release of histamine from ECL cells that, in turn, acts as a paracrine mediator in the gastric mucosa–stimulating parietal cells (Fig. 4-5) (52,121,122). In support of this, gastrin releases histamine in isolated perfused rat stomachs (123); increases intracellular Ca2+ in ECL cells (124); and in mice in which the gene encoding histidine decarboxylase (HDC) has been deleted (thereby blocking histamine synthesis), there is little or no acid response to gastrin (125,126). In addition, gastrin regulates ECL cell numbers and the expression of genes in ECL cells that direct histamine synthesis and storage (see later). H+ +

Parietal cell

ECLcell

Histamine Gastrin ECL-cell responses to gastrin ECL-cell proliferation Histamine release Histamine synthesis, and storage, HDC, VMAT2, CgA gene expression

Proliferating cell

HB-EGF TGFα Amphiregulin Parietal-cell responses to gastrin Parietal cell numbers Parietal cell maturation Acid secretion (via histamine) Growth factor synthesis and secretion

FIG. 4-5. Actions of gastrin on histamine-secreting ECL (enterochromaffin-like) and acid-secreting parietal cells. EGF, epidermal growth factor; HDC, histidine decarboxylase; TGF, transforming growth factor; VMAT2, vesicular monoamine transporter type 2.

The question of whether gastrin acts directly on parietal cells has attracted considerable attention. Parietal cells express CCK-2 receptors, and gastrin directly stimulates purified preparations of isolated parietal cells (127,128). One idea is that relatively high plasma concentrations of gastrin are required to directly stimulate acid secretion from parietal cells. More recent data in mice in which the genes encoding either gastrin or the CCK-2 receptor have been deleted suggest there might be other roles for the direct action of gastrin on parietal cells including stimulation of parietal cell maturation and migration (129–131). Thus, in the absence of the gastrin gene (i.e., Gas-KO mice), parietal cells appear morphologically immature, and there is reduced basal acid secretion and insensitivity to stimulation by gastrin, histamine, or carbachol (129,130). However, infusion of gastrin in Gas-KO mice for more than 24 hours induces the capacity for secretagogue stimulation of acid secretion (129,132). With prolonged infusion of G17, acid secretion in Gas-KO mice increased initially, and then faded over a period of 2 weeks; interestingly, when Gas-KO mice were given G17 and G17-Gly, there was maintained secretion over many weeks, suggesting an interaction between the two peptides to maintain parietal cell function (132). Although gastrin is required for the normal regulation of gastric epithelial organization and function, prolonged hypergastrinemia accompanied by inflammation may also be associated with disruption of epithelial organization, at least in experimental animals. For example, in transgenic mice with lifelong increase of plasma amidated gastrin (InsGas mice), there is initially (up to about 3 months of age) increased acid secretion and increased parietal cell numbers, but with age there is a progressive loss of parietal cells and expansion of the mucous neck cell population (133). Infection of these mice with Helicobacter felis accelerates this progression, and by 18 months invasive gastric cancer develops in many (133). Control of Enterochromaffin-like Cell Function As noted earlier, gastrin stimulates histamine release from ECL cells, increases the production and storage of histamine by the ECL cell by stimulating the expression of key genes, and regulates ECL cell number (74,123,124,134–136). Histamine is synthesized from histidine by the cytosolic enzyme HDC (EC 4.1.1.22.), and then is sequestrated into secretory granules by vesicular monoamine transporter type 2 (VMAT2). The abundance of the mRNA encoding HDC is regulated by physiologic concentrations of circulating gastrin and may be increased within the time taken to digest a single meal (137). Hypergastrinemia in both humans and rodents is associated with increased abundance of mRNA encoding HDC and VMAT2, as well as the endocrine cell secretory protein, chromogranin A, which is thought to stabilize amine-containing secretory granules (134,138–142). The transcriptional mechanisms by which gastrin regulates these three genes have been investigated in human gastric epithelial cell lines and commonly involve transcription

GASTROINTESTINAL HORMONES: GASTRIN, CHOLECYSTOKININ, SOMATOSTATIN, AND GHRELIN / 99 factor binding to a variety of GC-rich regions and activation of Cre sites (143–148). In patients and in experimental animals, hypergastrinemia is associated with ECL cell hyperplasia (149). It appears that ECL cells have the capacity for proliferation; thus, this response may reflect a direct mitogenic action of gastrin (150). With prolonged hypergastrinemia there may be a progression to dysplasia and ECL cell carcinoid tumors (9,149,151,152). ECL cell carcinoid tumors are now well recognized to be produced in experimental animals with hypergastrinemia due to long-term administration of proton pump inhibitors (153,154); the ECL cell response is prevented by removal of the pyloric antrum, providing direct evidence for the role of an antral hormone (155). ECL cell carcinoid tumors also develop spontaneously in a sub-Saharan rodent Mastomys, and this has been attributed to amino acid substitutions in the CCK-2 receptor in this species that increase constitutive activity (156,157). In humans, increase of plasma gastrin alone, as occurs, for example, in sporadic gastrinomas, seems to be associated with a low incidence of ECL cell carcinoids (<1 %); the combination of increased plasma gastrin with either mutation of the multiple endocrine neoplasia type 1 (MEN-1) gene or with inflammation (e.g., chronic atrophic gastritis) increases the incidence of ECL cell carcinoids (151,152,158).

H+

TFF-1

Proliferation differentiation migration

HB-EGF, TGFα, amphiregulin, COX-2 products, Regs FGF-1 IL-8 Som

Histamine Gastrin

Control of Gastric Epithelial Cell Proliferation It has been recognized for many years that experimental administration of gastrin stimulates growth of the acidsecreting part of the stomach, leading to increased thickness of the mucosa and increased parietal cell mass (159). Moreover, gastric epithelial cell proliferation rates, determined by BrdU incorporation, increase with feeding partly because of increased gastrin secretion (160). Gastric epithelial progenitor cells are not thought to express CCK-2 receptors; instead, gastrin is considered to act indirectly via release of growth factors (see earlier) (52), presumably originating in parietal or ECL cells (Fig. 4-6). However, in Gas-KO mice, all the major gastric cell types are nevertheless represented, although the mucosa is relatively thin, indicating that development of the gastric mucosa does not exhibit an absolute requirement for gastrin. It appears likely that gastrin plays a role in determining the relative proportions of different cells and their maturation. Thus, in addition to effects on ECL cell number described earlier, there are initially increased numbers of parietal cells in transgenic mice overexpressing gastrin (133), and increased parietal cell mass is found in gastrinoma patients. Parietal cells are terminally differentiated so that gastrindependent increases in cell number may be secondary to either stimulation of progenitor cell proliferation or increased commitment of progenitor cells to the parietal cell lineage. Other Actions of Gastrin Gastrin contracts smooth muscle of the antrum and small intestine (161,162) and the lower esophageal sphincter (163).

FIG. 4-6. Putative paracrine mediators of the action of gastrin in the gastric epithelium relevant to cellular proliferation, differentiation, and cell migration. COX-2, cyclooxygenase-2; EGF, epidermal growth factor; FGF-1, fibroblast growth factor-1; TFF-1, trefoil factor-1; TGF, transforming growth factor.

Depending on the species and tissue, these may be either direct or indirect effects mediated by enteric neurons. Their physiologic significance in normal digestion remains uncertain. Studies using gene arrays and proteomic methods have started to reveal a number of previously unidentified target genes of gastrin. These include plasminogen activator inhibitor-2 (164), matrix metalloproteinase-9 (MMP-9) (165), and trefoil factor-1 (166). Therefore, it appears that in certain circumstances, for example, in cancer or tissue regeneration, gastrin may influence tissue organization, and this involves, at least in part, remodeling of the extracellular matrix (see Fig. 4-6).

Actions of Nonclassical Gastrins Several different roles for Gly-gastrin in the stomach have been described, but the physiologic significance of these effects remains rather uncertain. Synergistic interactions between amidated and Gly-gastrin for stimulation of acid secretion in the rat were reported several years ago (167). More recently, it has been found that administration of the two peptides in combination enhances and maintains

100 / CHAPTER 4 parietal cell function in mice in which the gastrin gene has been deleted (132). Moreover, Gly-gastrin appears to inhibit the loss of parietal cells and gastric atrophy that occur in mice overexpressing amidated gastrin and infected with H. felis (168). Several groups have reported that Gly-gastrin stimulates the proliferation of both transformed and nontransformed cells (169–172). In addition, transgenic mice overexpressing Gly-gastrin exhibit increased proliferation in the colon (173) and are prone to the development of lung adenocarcinomas. Moreover, the expression of Gly-gastrin (but not progastrin or amidated gastrin) in lung adenocarcinomas is associated with decreased patient survival (27). In addition to proliferative effects, the oncogenic properties of Gly-gastrin may also include stimulation of cell migration (115) and cell invasion, at least in part, by induction of MMP-1 and -3 (174,175). Like Gly-gastrin, progastrin stimulates cell proliferation both in vitro and in vivo. In a mouse model, the stimulation of colon proliferation by progastrin was shown to occur with fragments of the precursor that included the C-terminal 26-residues; Gly-gastrin or fragments of the extreme C terminus (e.g., the C-terminal tryptic peptide of progastrin) did not show activity in this assay (176). In transgenic mice overexpressing progastrin, there is increased epithelial cell proliferation in the colon and increased thickness of the colon mucosa (177). These mice also exhibit increased susceptibility to the damaging effects of the carcinogen azoxymethane (177–180). Moreover, susceptibility to the carcinogenic effects of azoxymethane is seen in mice expressing a mutant form of progastrin in which the dibasic amino acid residue cleavage sites have been deleted (180), indicating that the carcinogenic effects of progastrin are not dependent on processing to Gly-gastrin or amidated gastrin. Normally, in mice exposed to 8G γ-irradiation, there is complete inhibition of mitosis in the colon. In contrast, in irradiated mice that overexpress progastrin, colonic cells remain in the cell cycle. Therefore, it appears that progastrin allows cells to stay in the cell cycle even after DNA damage, which presumably contributes to its apparent effects as a cocarcinogen (181). In this irradiation model, there is no difference in rates of colonic epithelial cell apoptosis between control mice and mice overexpressing progastrin, although it has been reported in IEC6 cells that progastrin has inhibitory effects on apoptosis (182).

Conditions of Gastrin Excess Increased plasma gastrin concentrations in humans may be caused by a gastrin-secreting tumor (gastrinoma) or by increased secretion from antral G cells most commonly caused by a failure of acid inhibition. Gastrinomas occur in approximately one patient per million of population per annum and are typically located in either the pancreas or duodenum; they may be sporadic or on a background of MEN-1 (183). Plasma gastrin concentrations in gastrinoma patients are usually in the range 50 to 10,000 pmol/liter and

are accompanied by high basal acid output (>15 mEq/hr). Provocative tests that are useful for diagnosis in those patients with plasma gastrin and acid secretory rates overlapping or just greater than the reference range include rapid intravenous injection of secretin and intravenous calcium (184). As noted earlier, a clear example of the consequences of loss of acid inhibition of the G cell is pernicious anemia, where achlorhydria over many years leads to plasma gastrin concentrations up to 2000 pmol/liter. The long-term use of proton pump inhibitors similarly leads to disinhibition of the G cell and increased plasma gastrin concentration (75, 185,186). Hypergastrinemia also occurs in postvagotomy patients, where again increased gastrin release may be secondary to reduced acid output. In the relatively unusual condition of isolated retained antrum, surgical exclusion of the antrum from the rest of the stomach removes it from acid inhibition, thus increasing gastrin release, which stimulates acid secretion from the rest of the stomach, leading to a clinical picture resembling gastrinoma (183). Antral G-cell hyperfunction (increased gastrin released from antral G cells, hyperpepsinogenemia, but normal G-cell density) and antral G-cell hyperplasia were described many years ago (187,188), but remain poorly defined and may well reflect extreme responses to H. pylori infection.

CHOLECYSTOKININ Overview CCK was first described in 1928 as a stimulant of gall bladder contraction present in intestinal extracts (189). Independently, Harper and Raper (190) later showed that extracts of intestine were capable of stimulating pancreatic enzyme secretion, and that this property was distinct from the capacity of secretin to stimulate the flow of pancreatic juice; they called the active factor pancreozymin (PZ). For many years the relation between CCK and PZ was uncertain. However, the purification of the two activities to homogeneity by Jorpes and Mutt (191) made it clear that they were separate actions of a single hormone. For a while the term cholecystokinin-pancreozymin (CCK-PZ) was in use; the hormone now is commonly referred to as CCK regardless of the biological activity under consideration. CCK is produced by a wide variety of neurons in addition to gut endocrine cells and has multiple biological actions. Its primary gastrointestinal role is the regulation of protein and fat digestion in the upper small intestine (192). This is achieved by matching the delivery of digestive enzymes from the pancreas and bile salts from the gall bladder, with the delivery of nutrients by delaying gastric emptying and inhibiting food intake. Some actions of CCK released from the gut are hormonal and involve direct effects on target cells (e.g., pancreatic acinar cells, gall bladder smooth muscle), but others may be mediated by activation of vagal reflexes (193). In addition, CCK produced in central and peripheral neurons

GASTROINTESTINAL HORMONES: GASTRIN, CHOLECYSTOKININ, SOMATOSTATIN, AND GHRELIN / 101 acts locally as a peptide neurotransmitter. This chapter focuses on the role of CCK as a gut hormone.

Peptide Structure Like the structurally related hormone gastrin, CCK is a linear peptide existing in multiple molecular forms. It was originally isolated from porcine intestine by Mutt and Jorpes (194) as a 33-residue peptide. The C-terminal pentapeptide amide of CCK was shown to be identical to that in gastrin; in addition, it was shown to possess a sulfated tyrosine residue at position 7 counting from the C terminus (whereas in gastrin there is a tyrosine at position 6). At about the same time, the amphibian skin peptide caerulein was isolated and sequenced by Anastasi and colleagues (195); it was shown to possess a closely similar C-terminal sequence and to have CCK-like biological properties (see Fig. 4-2). Multiple molecular forms of CCK have since been isolated and characterized. By analogy with the system for naming the different gastrins, the various forms of CCK that include the C-terminal pentapeptide amide sequence are designated by the number of amino acids in the chain. The peptide first identified by Jorpes and Mutt is therefore CCK-33; subsequently the group identified N-terminal extended forms, namely CCK-39 and CCK-58 (196,197). In addition, both longer (CCK-83) and shorter forms (CCK-8 and CCK-22) have been isolated and sequenced from intestine (198–200). In most cases, these peptides appear to occur predominantly in the tyrosine-sulfated state, although unsulfated CCK-58 is known to exist (201). The stability of different forms of CCK during extraction can be a significant practical problem that requires careful attention in any comparative study of the tissue and species distribution of CCK variants (202).

Gene Structure and Expression A single gene encodes the different forms of CCK and is expressed in both gut endocrine cells and CNS neurons. The cDNA in rat predicts a 345-base mRNA transcript encoding a 115-amino acid-residue precursor with a single copy of the biologically active sequence (Fig. 4-7) (203). The amino acid sequence immediately downstream of the putative -Phe-amide in pro-CCK (i.e., -Gly-Arg-Arg-Ser-Ala-Glu-) is identical to the corresponding sequence in progastrin, indicating conservation of an important site for cleavage and posttranslational processing. Moreover, as in progastrin, species differences in precursor sequence are mainly located in the N-terminal region of the precursor. The CCK gene is located on chromosome 3 (204). The gene structure exhibits similarities in organization with the gastrin gene, including the presence of three exons, the first of which is untranslated (see Fig. 4-7). The similarity in gene organization, as well as in amino acid sequence in biologically important regions, strongly indicates a common evolutionary origin of the two hormones through gene

duplication. Peptides resembling CCK can be found in all the main chordate groups and appear to have been relatively well conserved during chordate evolution (205). Interestingly, in a primitive chordate, Ciona intestinalis, there is an unusual member of this peptide family that has sulfated tyrosine residues at both positions 6 and 7 from the C-terminus, therefore resembling both gastrin and CCK (206). The abundance of CCK mRNA in small intestine is increased by nutrients that also release CCK and is inhibited by fasting (207). The transcriptional control mechanisms have been studied in several cell lines including STC-1 cells. The latter are derived from an intestinal endocrine tumor in a mouse doubly transgenic for the polyoma small T antigen and SV40 early region antigen (208). These cells express CCK and other proximal small intestine hormones. The expression of CCK in STC-1 cells can be increased by peptone by mechanisms that involve increased intracellular cyclic 3′,5′-adenosine monophosphate (cAMP), increased calcium, and stimulation of the MAPK pathway (209,210). Mutational studies have identified a site −72 to −83 upstream of the transcriptional start site that overlaps a putative Cre/AP-1 site and seems to be required for responsiveness to peptone. In addition, CCK gene transcription in STC-1 cells is stimulated by the VIP-like neurotransmitter pituitary adenylyl cyclase-activating polypeptide via activation of the transcription factor cyclic adenosine 3′5′-monophosphate response element binding protein (CREB), which binds the putative AP-1/Cre site (211).

Posttranslational Processing The different forms of CCK described earlier arise through cleavage of pro-CCK in many cases at a single basic residue (Arg) (Fig. 4-8). Some of the N-terminal pro-CCK–derived peptides produced by endopeptidase cleavage have been identified (212). There are apparent differences in the patterns of pro-CCK–derived peptides in intestinal endocrine cells and CNS neurons, which have led to the view that there might be cell-specific patterns of posttranslational processing (212). However, an alternative view is that patterns of posttranslational processing in brain and gut are similar, and that differences in degradation during peptide extraction lead to variation in the identity of the recovered peptide (213). Studies in transfected cell lines suggest that SPC1/3 and SPC5 are capable of cleaving pro-CCK (214,215). There is a requirement for carboxypeptidase E to remove C-terminal basic residues because mice in which the gene encoding this enzyme is deleted are CCK deficient but have increased concentrations of Gly-Arg–extended variants of CCK (216,217).

Cellular Origins CCK in the small intestine is localized to I cells. These are predominantly located in the jejunum, which corresponds

102 / CHAPTER 4 preproCCK gene exon 1

exon 2

exon 3

preproCCK mRNA

preproCCK

FIG. 4-7. Schematic representation of the relationships between the preprocholecystokinin gene, preprocholecystokinin messenger RNA (mRNA), and the peptide precursor preprocholecystokinin. CCK, cholecystokinin.

with the relatively high concentrations of CCK in mucosal extracts of this region of intestine (218). The I cell is an open cell projecting to the intestinal lumen. It has a turnover time of a few days and appears to differentiate from an enteroendocrine cell lineage that includes the secretin cell. The bHLH transcription factor neurogenin 3, is required for the development of I cells and other intestinal enteroendocrine cells (61). Neurogenin 3 increases expression of the bHLH transcription factor, BETA2/NeuroD, which is required for the differentiation of intestinal secretin and CCK cells (219,220). In addition to the CCK produced in I cells, there are also small amounts of CCK elsewhere in the gut that reflect production in enteric neurons, including a subset of cholinergic secretory motor neurons of the submucosal plexus (221). The expression of CCK in CNS neurons is beyond the scope of this chapter; however, it is worth noting that the concentrations of CCK in many regions of the CNS may exceed those in the intestine (222,223). Signal

Neuronal populations expressing the CCK gene include GABAergic cerebral cortex neurons (224) and dopaminergic mesolimbic neurons (225).

Assay Plasma CCK is been measured by RIA (226), although a bioassay based on amylase release from rat pancreatic acinar cells has been useful in some studies (227). In RIAs it is important to take steps to ensure that the shared amino acid sequence between CCK and gastrin does not compromise specificity. Two approaches typically have been used. Antibodies that react equally with all peptides containing the common C-terminal pentapeptide amide may be used, providing steps are taken to separate gastrin and CCK (228). This can be achieved by high-pressure liquid chromatography or gel filtration chromatography, but inevitably such

R

R

K

R

R

preproCCK

RR *

CCK83

NH2

*

NH2 CCK58

*

CCK39

*

CCK33

NH2 NH2

*

NH2 CCK22

*

NH2 CCK8

FIG. 4-8. Relations among the main forms of cholecystokinin (CCK) isolated from small intestine or brain. Cleavage occurs at single Arg (R) or Lys (K) residues except for the sequence causing the C-terminal amide (−NH2).

GASTROINTESTINAL HORMONES: GASTRIN, CHOLECYSTOKININ, SOMATOSTATIN, AND GHRELIN / 103 assays make it difficult to achieve high throughput. Alternatively, antibodies that react selectively with CCK and exhibit little or no cross-reactivity with gastrin can be used (229). Care is needed to determine the relative immunoreactivities of different forms of CCK, and there is some evidence that CCK58 in particular may exhibit lower affinity than other forms of CCK for antibodies binding at the C terminus (230). Because plasma concentrations are relatively low (see later), it is often necessary to include a concentration step before assay.

Release Mechanisms The concentrations of CCK in the peripheral circulation during fasting are about 1 pmol/liter, and they increase up to about fivefold with feeding. In several species, CCK-58 is the predominant circulating form of the hormone (202,231). The main components of the diet responsible for CCK secretion are protein and fatty acids (227). Aromatic amino acids were suggested many years ago to stimulate CCK release based on pancreatic secretory responses in vivo (232); in the same model, intact protein failed to increase pancreatic enzyme secretion, but after digestion with pepsin became a strong stimulus (233). Factors other than CCK may, of course, account for a proportion of the pancreatic response. The question of whether intact protein releases CCK is also complicated by the existence of a number of CCK-releasing factors that are sensitive to protease degradation and are protected by ingestion of dietary protein. Several putative CCK-releasing peptides have been described in the gut lumen (234–236). The so-called luminal CCK-releasing factor has been isolated and sequenced from the luminal contents; it releases CCK when infused intraduodenally, and its effects are blocked by specific antibodies (235). It is produced in pancreas and small intestine (237) and is believed to stimulate CCK release by direct action on I cells to increase intracellular calcium (238). Degradation of CCK-releasing peptides by proteolytic enzymes in the intestinal lumen provides a mechanism to autoregulate CCK release. Consistent with this, it is well established that there is inhibition of CCK release by trypsin in the gut lumen (239). Moreover, prolonged administration of trypsin inhibitors (e.g., soybean trypsin inhibitors) eventually leads to pancreatic hypertrophy, which has been attributed to sustained increase of plasma CCK concentration (240). Stimulation of CCK release by dietary fat requires conversion of triglyceride to fatty acids. In humans, fatty acids with a chain length longer than C12 are effective releasers of CCK in vivo, and fatty acids with a chain length less than 10 are ineffective; similar observations have been made in STC-1 cells (241,242). In the latter, C12 increases intracellular calcium; there appears to be a transient increase in Ca2+ attributable to release from intracellular stores, and a sustained increase caused by Ca2+ entry. Of several cell lines tested (PC12, BON, Caco-2, IIC9), only STC-1 cells responded to C12 in the presence of external calcium, suggesting a cell-specific sensing mechanism for fatty acids

with chain length longer than C12 (243). The primary cellular sensing mechanisms in these two cases remain unclear, but it is notable that a family of at least three G protein– coupled receptors is now known that may function as fatty acid sensors (GRP-40, GPR41, GRP-43); however, whether these play a role in the gut remains uncertain.

Metabolism The half-life of CCK58 in the canine circulation is 4.4 minutes compared with 1.3 minutes for CCK8 (244). There is some evidence that CCK-8 is preferentially cleared by the liver, whereas longer forms are not (245,246). There is degradation of CCK8 by neutral endopeptidase 24.11; however, unlike G-17, where sulfation inhibits degradation by neutral endopeptidase 24.11, in the case of CCK-8, it is enhanced by sulfation (247). A novel CCK-inactivating peptidase has been purified from brain and characterized as a membrane-bound isoform of tripeptidyl peptidase II (EC 3.4.14.10). An inhibitor of this enzyme, butabindide, has been developed that protects endogenous CCK from degradation and enhances the effect of CCK in inducing satiety (248).

Receptors, Transduction, and Structure–Activity Relations CCK acts at both CCK-1 (also known as CCK-A) and CCK-2 (also known as CCK-B or gastrin-CCKB) receptors (see Table 4-1) (91). The latter are likely to be important in mediating the CNS actions of CCK because the alternative ligand, gastrin, is virtually absent from the brain. The CCK-1 receptor is expressed by pancreatic acinar cells, gall bladder and other smooth muscle, some peripheral and CNS neurons (particularly vagal afferent neurons), and some gastrointestinal tumors (149). The CCK-1 receptor was first cloned from rat pancreas, and the cDNA sequence predicted a protein with seven-transmembrane domains belonging to the G protein– coupled family of receptors (249). The receptor is linked to Gαq/11 and consequent stimulation of inositol phosphate turnover, mobilization of intracellular calcium, and activation of PKC (250). There is also activation of the MAPK cascade and tyrosine phosphorylation of p125 focal adhesion kinase and paxillin. The high affinity of naturally occurring peptides for CCK-1 receptors depends on the C-terminal pentapeptide amide shared with gastrin and a sulfated tyrosine residue at position 7 from the C-terminus. Unsulfated peptides, or peptides in which there is a sulfated tyrosine at position 6 from the C-terminus (as in gastrin), have low affinity for CCK-1 receptors. The CCK-1 receptor affinities for CCK-8 and CCK-33 appear to be similar (251). However, studies indicate that the N-terminal sequence of CCK-58 may influence the conformation of the C-terminal octapeptide region so that the biological properties of CCK-58 may differ from that of shorter fragments (252); in particular, CCK-58 appears

104 / CHAPTER 4 relatively more potent as a stimulant of fluid secretion from the pancreas (253,254). A number of useful antagonists of the CCK-1 receptor have been developed. The first to be described (e.g., dibutyryl cyclic guanosine monophosphate, asperlicin, proglumide, and benzotrypt) had relatively low affinity, and in the case of proglumide there was also low specificity. However, some of these compounds have proved to be useful starting points for drug development (255). Thus, glutamic acid derivatives developed from proglumide, such as loxiglumide, have high affinity and are suitable for administration to humans. Moreover, benzodiazepines based on asperlicin, such as L-364,718, have also been widely used in experimental studies. There have been extensive studies of the mechanisms by which CCK increases calcium in pancreatic acinar cells (250,256,257). In response to stimulation by CCK, there is an increase in intracellular calcium in the apical pole of the cell in the region (where secretory granules are located) that triggers exocytosis (256). At physiologic concentrations, CCK produces oscillations of intracellular calcium that are restricted to the apical pole. However, in response to high concentrations, there is a more global increase in intracellular calcium. The latter is also associated with intracellular trypsin activation and vacuolation, which may be relevant in the pathogenesis of acute pancreatitis (258). The spatial restriction of CCK-induced calcium oscillations is thought to be a property of the polarized distribution of mitochondria, which are able to buffer calcium (256). Inositol-1,4, 5-trisphosphate has been recognized as a mediator of CCKinduced increases in intracellular calcium for many years. More recently, cyclic adenosine diphosphate ribose and nicotinic acid adenine dinucleotide phosphate have also been identified as putative mediators of the increase in intracellular calcium induced by CCK, and it has been suggested that the characteristic pattern of intracellular calcium responses evoked by CCK are attributable to interactions between these signaling pathways (257).

Pancreas Postprandial pancreatic enzyme secretion is inhibited by CCK-1 receptor antagonists (263). Pancreatic acinar cells express CCK-1 receptors, and CCK stimulates enzyme secretion in vitro, supporting the idea that CCK acts directly on these cells. However, there is also cholinergic stimulation of pancreatic acinar cells by acetylcholine released from postganglionic vagal efferent fibers. Some evidence indicates that, in addition to direct effects on pancreatic acinar cells, CCK might also activate vagovagal reflexes to stimulate enzyme secretion; these would account, for example, for the rapid pancreatic secretory response to nutrient in the intestine compared with the slower response to injection of CCK (Fig. 4-9) (264,265). In addition to stimulation of secretion, prolonged administration of CCK also stimulates synthesis of pancreatic enzymes and cellular proliferation (266,267). Moreover, there is stimulation of pancreatic growth in rats by diets containing trypsin inhibitors and by protein-rich diets (70% casein); in both cases, the effects are reduced by administration of a CCK-1 receptor antagonist (240,268). However, in mice lacking the CCK gene, the pancreatic hypertrophy that occurs in response to a high-protein diet persists, indicating

+

−

Ach CCK

Biological and Physiologic Actions The main actions of circulating CCK are stimulation of pancreatic enzyme secretion, stimulation of gall bladder contraction, inhibition of gastric emptying, and inhibition of food intake. Mice in which either the CCK or the CCK-1 receptor genes are deleted have, in recent years, provided useful experimental models for studies of these functions (255,259–261). In addition, a strain of rat (the Otsuka Long Evans Tokushima fatty [OLETF] rat) in which the chromosomal locus of CCK-1 receptor is lost has proved useful (262). In addition to gastrointestinal effects of CCK, there are also a number of important CNS actions that may reflect the role of CCK as a transmitter in the brain; these include modulation of opioid analgesia and dopaminergic activity, anxiogenic effects, and inhibition of food intake. This chapter focuses on gastrointestinal effects of CCK.

+

CCK

Pancreatic exocrine secretion Ach

Ach

VIP Ach NO Inhibition of gastric emptying

FIG. 4-9. Activation of vagovagal reflexes by cholecystokinin (CCK). The latter acts on vagal afferent neurons serving the stomach and small intestine; stimulation of these pathways leads to inhibition of food intake, reflex relaxation of the gastric corpus with inhibition of gastric emptying, and reflex stimulation of pancreatic enzyme secretion. There are also direct effects of CCK on smooth muscle in the gastric antrum and on pancreatic acinar cells. Ach, acetylcholine; NO, nitric oxide; VIP, vasoactive intestinal peptide.

GASTROINTESTINAL HORMONES: GASTRIN, CHOLECYSTOKININ, SOMATOSTATIN, AND GHRELIN / 105 that diet-linked pancreatic hypertrophy is not associated with CCK in this case (261). Gall Bladder Contraction The delivery of bile to the duodenum requires contraction of the gall bladder and relaxation of the sphincter of Oddi, and both responses are stimulated by CCK. Gall bladder contraction occurred in response to similar plasma concentrations of endogenous CCK (released by a test meal) and exogenous CCK delivery by intravenous infection (269). Moreover, postprandial gall bladder contraction is blocked by CCK-1 receptor antagonists, which is compatible with the idea that this is a physiologic action of the hormone (270,271). In CCK-1 receptor KO mice, there is evidence of biliary stasis (259). Gall bladder smooth muscle expresses the CCK-1 receptor compatible with direct actions of CCK on the smooth muscle (272). However, in guinea pig gall bladder, there is evidence for a presynaptic site of action to facilitate gall bladder contraction (273,274). In the case of the sphincter of Oddi, the release of inhibitory neurotransmitters (nitric oxide and VIP) appears to be the primary mechanism producing smooth muscle relaxation (275,276). Gastric Emptying and Satiety The control of food delivery to the small intestine is mediated by CCK through inhibition of gastric emptying and inhibition of food intake. The two responses can be considered together because both are likely to involve activation of a vagal afferent pathway by CCK, and because there is evidence for functional links. The gastric emptying of test meals is reduced by administration of CCK-1 receptor antagonists, suggesting this is a physiologic effect of the hormone (263,277). Smooth muscle in the distal stomach may constitute one site of action of CCK in reducing nutrient flow across the pylorus (278), and in humans, ultrasound studies indicate that endogenous CCK plays a role in regulating contractions of the antrum (241). In addition, however, there is considerable evidence that CCK activates a vagovagal reflex leading to relaxation of the proximal stomach (see Fig. 4-9) (277,279,280). Vagal afferent nerve fibers express CCK-1 receptors, and CCK stimulates vagal afferent nerve discharge (281–284). There is evidence to suggest that vagal afferent neurons projecting to both stomach and jejunum are stimulated by CCK (283–285); moreover, brainstem neurons responding to the discharge of vagal afferent neurons activated by gastric mechanoreceptors also respond in the same way to CCK (286). In the case of vagal afferent neurons projecting to the stomach, it would appear that CCK is likely to act after delivery via the circulation; in the case of vagal afferent fibers serving the intestine, it is possible that CCK released by I cells acts locally. The effects of CCK on gastric emptying in the rat appear likely to be mediated by activation of this vagal afferent pathway because these effects are inhibited both by vagal nerve section and by application of the sensory neurotoxin capsaicin (277,279).

The most important behavioral response to peripheral administration of CCK is inhibition of food intake. This was first described in gastric fistula rats and has since been recognized in many different experimental circumstances (287,288). Similar to the effects of CCK on gastric emptying, the effect of peripherally administered CCK on food intake is inhibited by CCK-1 receptor antagonists and by lesion of vagal afferent nerve fibers, either surgically or by perivagal application of the sensory neurotoxin capsaicin. Studies using two CCK-1 receptor antagonists that differ in their capacity to cross the blood–brain barrier (devazepide, which penetrates the CNS, and A-70104, which does not) indicate that endogenous CCK released by nutrient inhibits food intake from a peripheral site, most likely CCK-1 receptors expressed by vagal afferent nerve fibers (289,290). Stimulation of these neurons by CCK increases c-fos expression in brainstem neurons, and research has identified prolactin-releasing peptide (PrRP) as a potential transmitter in these neurons. The latter inhibits food intake on CNS administration, thus the data are compatible with the idea that PrRP is a central peptide transmitter mediating the effects of CCK-vagal stimulation (291). Inhibition of gastric emptying and food intake may well be linked because gastric retention due to inhibition of emptying by endogenous CCK enhances sensations of fullness or satiety in healthy subjects (292). In addition, reduction of food intake in humans is increased by the combination of a low dose of exogenous CCK and moderate gastric distension, suggesting synergistic interactions between CCK and gastric mechanoreceptor stimulation (293). Several different types of interaction between CCK and other regulators of food intake have been identified at the level of the vagus nerve. Thus, potentiating interactions between CCK and leptin have been reported for stimulation of vagal afferent nerve discharge and inhibition of food intake (294–297). In contrast, in both rat and human nodose ganglia, there are neurons expressing CCK-1 and Orexin type-1 receptors, and the orexigenic peptide, Orexin A, inhibits the excitatory effect of CCK on rat vagal afferent nerve discharge (298). In addition, CCK decreases expression of CB1 receptors by vagal afferent neurons. CB1 receptors respond to endogenous cannabinoids such as anandamide that can stimulate appetite via a vagal mechanism (299). Interestingly, the expression of CB1 receptors in vagal afferent neurons is increased in fasted rats and decreased by refeeding through a mechanism sensitive to CCK-1 receptor antagonists (300). Because CB1 receptors are associated with stimulation of food intake, their down-regulation by CCK suggests an additional mechanism by which CCK modulates appetite signaling to the CNS. Gastric Secretory Effects There is expression of CCK-1 receptors by gastric somatostatin-expressing D cells and also by chief cells (301,302). In general, CCK is not a strong stimulant of acid secretion (even though its potency at CCK-2 receptors is comparable

106 / CHAPTER 4 with that of gastrin). However, after administration of CCK-1 receptor antagonists, the acid secretory response to CCK increases, indicating an inhibitory role for these receptors and revealing an acid-secretory response to CCK mediated by stimulation of CCK-2 receptors (303,304). It appears likely that CCK acts as an enterogastrone, that is, an intestinal hormone released by fat that inhibits gastric acid secretion. This effect is attributable to the presence of CCK-1 receptors on D cells and stimulation of somatostatin release by CCK, which, in turn, inhibits acid secretion and gastrin release (302–304). Studies comparing gastric function in mice in which either the gastrin gene or both gastrin and CCK genes have been deleted support the idea that CCK exerts an inhibitory effect via the D cell (305). In addition to CCK-1 receptors expressed by gastric D cells, there may also be expression by pepsinogen-producing chief cells, the physiologic significance of which remains uncertain (306).

Conditions of Excess and Deficiency Increased plasma CCK concentrations occur in vagotomized patients, after administration of CCK-1 receptor antagonists, and in infection of the small intestine (241,307). HyperCCKemia may also occur after prolonged ingestion of protease inhibitors, which accounts for the pancreatic hypertrophy seen in animals fed untreated soy flour (rich in soybean trypsin inhibitor). Conversely, it has been reported that decreased CCK secretion occurs in celiac disease (228). Whether increased plasma CCK concentrations contribute directly to human disease remains to be determined; moreover, the contributions of dysfunctional CCK signaling in the CNS to psychiatric disease remain far from clear.

SOMATOSTATIN Overview Somatostatin was first isolated from sheep hypothalamus on the basis of its ability to inhibit secretion of growth hormone (308). Subsequently, somatostatin-containing cells (D cells) were found to be widely distributed in the gut mucosa and in pancreatic islets (309). Somatostatin is also widely distributed in neurons of the autonomic and central nervous systems. There are two main naturally occurring somatostatins: peptides of 14 (Som-14) and 28 (Som-28) amino acid residues. They act at multiple receptors. Somatostatin released from enteroendocrine and pancreatic endocrine cells is mostly thought to act locally as a paracrine factor (310). This chapter focuses on the role of somatostatin in the gastrointestinal tract.

Peptide Structure The structure of the tetradecapeptide (Som-14) was determined after isolation from ovine hypothalamus; an alternative

name is somatotropin-release inhibiting factor (308,311). The higher molecular weight form (Som-28) was first isolated from porcine intestine and later hypothalamus (312). The sequence of Som-14 is contained within the C-terminal tetradecapeptide of Som-28 and is liberated by cleavage at a pair of basic residues (-Arg-Lys-) (Fig. 4-10). Other peptides of 25 and 20 amino acid residues also have been reported (313,314).

Gene Structure and Expression The human somatostatin gene is located on chromosome 3q38. The predicted precursor, human preprosomatostatin, contains 116 amino acids and has a single copy of the sequence corresponding to Som-28 located at its extreme C terminus (315,316) (see Fig. 4-10). The precursor of rat somatostatin exhibits a similar organization (317), and both rat and human genes contain a single intron (316,318). The somatostatin gene is well conserved in mammals; in some fish (e.g., catfish and anglerfish), there are two somatostatin genes and evidence that, although the sequence of Som-14 is conserved in one of them, there is divergence in the other (319,320). In keeping with the idea that somatostatin acts locally in several different systems, it seems likely that gene expression is differentially regulated in different populations of somatostatin-producing cells (321). For example, there is evidence for differential regulation of somatostatin gene expression in antral versus corpus D cells (322); in the rat gastric antrum, the abundance of Som mRNA is determined by luminal factors, notably gastric acid, whereas in the corpus, it is not. Thus, prolonged increases in gastric pH lead to a decrease in antral somatostatin mRNA, whereas fasting increases mRNA abundance (28,323,324). These changes may be partly because of the action of gastric acid in inhibiting bacterial overgrowth that leads to inflammation in the antral mucosa and suppression of D-cell function (325,326). In contrast, somatostatin gene expression in corpus D cells appears to be under the control of spinal primary afferent neurons and particularly

Preprosomatostatin

Prosomatostatin

RK

63

1

92

Som14

Som28

Som28 1-12

Som14

Som28 1-12

FIG. 4-10. Biosynthetic relations among different forms of somatostatin. Preprosomatostatin (stippled box indicates the signal peptide) is converted to prosomatostatin, which may be cleaved either to generate Som-28 and an N-terminal peptide, or to Som-14 and an N-terminal peptide that includes the 1-12 sequence of Som-28. Som, somatostatin.

GASTROINTESTINAL HORMONES: GASTRIN, CHOLECYSTOKININ, SOMATOSTATIN, AND GHRELIN / 107 calcitonin gene–related peptide released at the peripheral terminals of these nerve fibers (322). This system is thought to be part of the mechanisms by which neurons trigger protective mechanisms in response to mucosal damage by acid or noxious agents (327). The cis-acting mechanisms regulating somatostatin gene expression include stimulation by the cAMP-response element (CRE) and action of its transactivating factor CREB by cAMP or PKC (328–330).

Posttranslational Processing There have been extensive studies of preprosomatostatin posttranslational processing in isolated islets from teleost fish, where the aggregation of islet cells into a relatively compact body is particularly appropriate for biosynthetic studies (331,332). There is a characteristic N-terminal signal peptide in preprosomatostatin that is cleaved in the endoplasmic reticulum, and thereafter prosomatostatin is conveyed through the Golgi complex to secretory granules. Cleavage of the precursor is initiated in the TGN by a decrease in pH (333). The liberation of Som-28 and Som-14 occurs by cleavage at pairs of basic residues mediated by enzymes of the SPC family. In mice in which SPC-2 is deleted, there is impaired processing of prosomatostatin in the islets of Langerhans (and also of proglucagon and proinsulin), whereas in mice in which SPC1/3 is deleted, there is normal production of Som-14 (and normal processing of proglucagon, but impaired cleavage of proinsulin) (334,335). In stomach, duodenum, colon, and pancreas, Som-14 predominates, but in the small intestine, Som-28 accounts for more than 50% of total activity (310). Studies following the incorporation of [35S]-cysteine suggest that in gastric D cells, there is no substantial conversion of Som-28 to Som-14, and that Som-14 is generated by cleavage of prosomatostatin (336). Cleavage of the latter generates prosomatostatin 1-64, which has been isolated from both intestine and pancreas (337).

Cellular Origins Somatostatin-containing D cells are found throughout the gastrointestinal tract and pancreas (309). They are particularly abundant in the corpus and antral regions of the stomach. These cells may extend long cytoplasmic processes that approach nearby target cells (G cells in the antrum, parietal cells in the corpus), thereby facilitating paracrine signaling networks (338). In the gastric corpus, D cells do not project to the lumen, and thus are “closed” endocrine cells; however, in the antrum and throughout the intestine, they do extended into the lumen, and thus are “open” cells. There are also abundant D cells in the pancreatic islets that are often localized around the periphery. In addition, throughout the gastrointestinal tract, somatostatin is produced by some types of enteric neurons (339,340). These may occur in the submucosal plexus and project to the mucosa, or in the myenteric plexus and project distally to other myenteric

plexus neurons (221,341). In submucosal plexus neurons, somatostatin often is colocalized with neuropeptide Y, CCK, and choline acetyltransferase, and in myenteric neurons with choline acetyltransferase (221,341).

Assay RIAs of somatostatin typically make use of antibodies to Som-14 that react with both Som-14 and Som-28. In addition, there are antibodies to the N terminus of Som-14 or to the cleavage site in Som-28 that may provide the capacity for specific assay of different forms (342). Assay of somatostatin in plasma requires an extraction step, for example, by acetone, ethanol, or adsorption to ODS silica (343,344).

Release Mechanisms Concentrations of somatostatin in peripheral plasma are 10 to 30 pM and are thought to represent contributions from many different tissues. After protein- or fat-rich meals, there are increases in circulating peptides corresponding to both Som-14 and Som-28 (345,346). To study secretion from defined populations of somatostatin-producing cells, many authors have made use of assays of peptide in the venous effluent from organs in vivo or from isolated perfused organs, as well as secretion into the media from isolated cells incubated in vitro (67,68,347–351). The control of somatostatin release from gastric D cells has been studied intensively (310,352). Intraluminal acid is a stimulant for antral D-cell somatostatin release (353,354). In vivo, D-cell function also is modulated by a variety of neurohumoral factors and is impaired in infection with H. pylori. A number of hormonal and neuropeptides that inhibit acid secretion, including gastrin-releasing peptide, secretin, glucagon, vasoactive intestinal polypeptide, and CCK, are reported to stimulate gastric somatostatin release, which is consistent with the idea that paracrine release of somatostatin from D cells mediates inhibitory actions on acid secretion (67). In addition, the peptide neuromodulator GRP is reported to stimulate somatostatin release, whereas in the perfused rat stomach, there is cholinergic inhibition of secretion (355,356). The relevant control mechanisms therefore are relatively complex with both excitatory and inhibitory neural and endocrine influences on the D cell (Fig. 4-11). In addition to the hormonal stimuli described earlier, gastrin also is reported to stimulate somatostatin release independently of gastric acid, suggesting a negative feedback relation between G and D cells within the antrum (357). The physiologic mechanisms regulating D-cell function are subverted in antral inflammation. In particular, in H. pylori infection there is depressed D-cell function and depressed D-cell numbers identified by immunohistochemistry (which may simply reflect loss of cellular somatostatin stores). These changes are restored by eradication of the bacterium (358).

108 / CHAPTER 4 Parietal cell

ECL cell

+ Hist

−

Parietal cell

D-cell

− SOM

SOM + D-cell stimulation GRP CCK CGRP/amylin PACAP, VIP, Sec IL-4

− D-cell inhibition Ach GABA Opioids Nociceptin

FIG. 4-11. Control of gastric corpus D-cell function and action of somatostatin (SOM) in the corpus. There are both excitatory and inhibitory neurohormonal inputs to the D cell; paracrine inhibition of enterochromaffin-like (ECL) cell and parietal cell function leads to depression of acid secretion. Ach, acetylcholine; CCK, cholecystokinin; CGRP, calcitonin gene–related peptide; GABA, γ-aminobutyric acid; GRP, gastrin-releasing peptide; IL-4, interleukin-4; PACAP, pituitary adenylyl cyclase–activating polypeptide; VIP, vasoactive intestinal peptide.

Experimental infection of the mouse stomach with other bacteria also inhibits D-cell function (359). The D cell appears to be a target for Th-1 cytokines such as interferon-γ, which depresses function, whereas the Th-2 cytokine IL-4 stimulates D-cell function (360).

Metabolism In the dog, Som-28 has a longer half-life and slower clearance rate than Som-14 (t1/2 2.8 vs 0.6 minutes, respectively) (361,362). Studies of the molecular mechanisms of degradation during perfusion of the rat liver indicate that there are at least three distinct cleavages of Som-28. Tryptic-like cleavage of the Arg-Lys generates Som-14, and both Som-28 and Som14 are subject to endopeptidase cleavage through the cyclized (ring) segment and to aminopeptidase cleavage (342).

Receptors, Transduction, and Structure–Activity Relations There are five different somatostatin receptors, SSTR-1 to SSTR-5, all belonging to the G protein–coupled receptor superfamily (363). The different receptors are differentially expressed, with SSTR-2 and SSTR-5 being important in the gut. In the case of SSTR-2, there are two splice variants: SSTR-2a and SSTR-2b. Immunohistochemical studies indicate that SSTR-2a is expressed primarily by human G cells in the antrum, but also by intestinal G cells (364); interestingly, there was also localization to enteric neurons but not to epithelial cells in the gastric corpus. In contrast, in the rat, there is evidence for localization of SSTR-2 to ECL cells (365). Somatostatin receptors are coupled to pertussis toxin– sensitive G proteins. Multiple signal transduction pathways

have been described, including Gi/o-mediated inhibition of adenylate cyclase, activation of inwardly rectifying K+ channels, inhibition of voltage-dependent Ca2+ channels, and inhibition of intracellular Ca2+ mobilization (361). These effects are likely to account for inhibition of hormone release. In addition, activation of SSTR-2 may depress MAPK activity, and thus inhibit proliferation (361,366). Many somatostatin analogues have been developed (367). The SSTR-2 agonist octreotide is widely used clinically to treat endocrine tumors by suppression of hormone release and inhibition of tumour cell proliferation and metastasis. Other applications include management of intractable diarrhea and bleeding esophageal varices (368). More recently, a number of other agonists showing selectivity for different receptor subtypes have been developed including nonpeptide ligands and a number of antagonists (363). In addition, radioactively labeled somatostatin analogues, for example, [111In-DTPA-D-Phe1]-octreotide, have been used in diagnostic imaging of neuroendocrine and pituitary tumors (368).

Physiologic Actions Somatostatin has a wide spectrum of biological actions in many different parts of the body (363). The first clear role to be identified for somatostatin released by hypothalamic neurons was inhibition of the secretion of pituitary hormones such as growth hormone, prolactin, and thyroid-stimulating hormone. In addition, somatostatin inhibits release of a wide variety of peptide hormones from all regions of the gut and endocrine pancreas; it also has inhibitory effects on gastric acid secretion, pancreatic exocrine secretion, intestinal absorption, and proliferation, and it has complex actions on gastrointestinal motility. It inhibits cytokine secretion from epithelial cells and T lymphocytes, and deactivates macrophages (363,368). Within the gastrointestinal tract, the effects of somatostatin in the gastric epithelium have attracted sustained attention (see Fig. 4-11). In particular, D cells provide a point of convergence for a variety of factors that inhibit G-cell or parietal cell function, and thus represent a crucial link in the negative feedback control of acid secretion. The importance of the system is indicated by evidence that immunoneutralization of somatostatin prevents inhibition of gastric acid secretion caused by upper-intestinal fat, but not ileal or colonic fat (369). Mice in which the SSTR-2 gene has been deleted have high basal acid secretion (but normal plasma gastrin), and this was not changed by administration of Som-14; administration of gastrin antibody reduced basal acid output, indicating a major role for SSTR-2 in the control of acid secretion through inhibition of gastrin release (370).

Conditions of Somatostatin Excess or Deficiency Rare neuroendocrine tumors producing somatostatin have been described, about half of which have been associated with MEN-1. The associated clinical syndrome is

GASTROINTESTINAL HORMONES: GASTRIN, CHOLECYSTOKININ, SOMATOSTATIN, AND GHRELIN / 109 characterized by steatorrhea, cholelithiasis, hyperglycemia, and weight loss (371,372). Within the gastrointestinal tract a clear example of reduced production of somatostatin is provided by patients infected with H. pylori. Infection in the antrum leads to reduced tissue content of somatostatin and reduced D-cell number with a consequent increase in G-cell function. The depression of D-cell function is reversible on eradication of H. pylori infection (373).

GHRELIN Overview Ghrelin is a relatively recently discovered gastric peptide that stimulates food intake. It was identified as a naturally occurring ligand of the growth hormone secretagogue (GHS) receptor (2). It is widely recognized that growth hormone secretion is stimulated by growth hormone– releasing factor and inhibited by somatostatin. In addition, however, some synthetic peptides increase growth hormone secretion through receptors that are distinct from those for growth hormone–releasing factor (374–376). The naturally occurring ligand for these receptors was uncertain until Kojima and colleagues (2) identified a novel 28-residue gastric peptide, ghrelin, as fulfilling this role. Independently, Tomasetto and colleagues (377) identified a novel motilinlike peptide in stomach. It is now clear that ghrelin and motilin are structurally similar (Fig. 4-12), and that ghrelin comes from distinct endocrine cells, X- or A-like cells, of the gastric corpus; in addition to stimulation of growth hormone secretion, ghrelin is a potent stimulant of food intake.

differences at just two positions in the rat and human peptides.

Gene Structure and Expression The human ghrelin gene consists of four exons and three introns and is located on chromosome 3p25-26 (379). The rat ghrelin gene has a similar organization, but the mouse gene has five exons and four introns (380). Studies of the promoter region of the human ghrelin gene have identified an E-box motif that appears to regulate expression in a medullary thyroid carcinoma cell line (381). A number of polymorphisms have been reported. One, Arg51Gln, alters an endopeptidase cleavage site, and thus leads to the production of a proghrelin peptide in place of the 28-residue peptide (382). Another polymorphism, Leu72Met, occurs in the C-terminal cleavage product of proghrelin. There are suggestions that both polymorphisms may be associated with obesity, but their functional significance for human disease remains to be fully explored (383).

Posttranslational Processing Preproghrelin has 117 amino acid residues; the sequence of the biologically active 28-residue peptide follows immediately after the signal peptide and is liberated by cleavage at a pair of basic residues on the C-terminal side (2,377). A C-terminal fragment of the precursor is secreted into plasma (384). The n-octanoylation of Ser-3 is a posttranslational event (see Fig. 4-12); both acetylated and desacyl-ghrelin appear to occur naturally (385). In addition, decanoylated (C10:0) and possibly decanoylated (C10:1) peptides may occur naturally (386).

Peptide Structure Cellular Origins Ghrelin is a 28-amino-acid peptide that is modified by n-octanoylation of Ser in position 3 (2). In addition, alternative mRNA slicing yields a 27-amino-acid residue peptide corresponding to des(Gln-14) ghrelin (378). The structural similarities between human ghrelin and motilin amount to six residues in the N-terminal and midregion of the peptides. Unlike motilin, which is absent in the rat, ghrelin is present in all mammalian species examined so far, and its sequence is relatively well conserved; for example, there are

The X- or A-like cells of the gastric corpus (387) that produce ghrelin account for about 20% of the total endocrine cell population in this region of the stomach. Relatively smaller amounts of ghrelin are also produced in the intestine and colon. Ghrelin-producing cells of the gastric corpus are of the closed type, but in lower regions of the gut, they are the open type, suggesting that there may be different types of regulatory mechanisms in different populations of

CH3 (CH2)6 C=O O

Ghrelin Motilin

G-S-S-F-L-S-P-E-H-Q-R-V-Q-Q-R-K-E-S-K-K-P-P-A-K-L-Q-P-R G-S-S-F-L-S-P-E-H-R-V-Q-Q-R-K-E-S-K-K-P-P-A-K-L-Q-P-R F-V-P-I-F-T-Y-G-E-L-Q-R-M-Q-E-K-E-R-N-K-G-Q

FIG. 4-12. Amino acid sequences of human ghrelin and motilin showing common residues (boxes). Note the presence of an n-octanoyl side chain on Ser-3 of ghrelin.

110 / CHAPTER 4 ghrelin-producing cell (388). Gastrectomy reduces plasma ghrelin concentrations by about 65%, which is compatible with the idea that some circulating ghrelin is of extragastric origin (389). Other extragastrointestinal sites that might contribute to circulating ghrelin include the islets of Langerhans, arcuate nucleus of the hypothalamus, kidney, testis, and immune cells (376). During development, ghrelin appears in the rat stomach at E17; there is also ghrelin expression in the placenta. In pituitary, there are initially high concentrations of ghrelin at birth that decline thereafter (390).

Assay RIAs for ghrelin have made use of antibodies reacting at both the N and C termini (385). Antibodies directed at N terminus require acylation. Importantly, there are greater plasma concentrations of immunoreactive ghrelin measured by C-terminal–specific antibodies, which is consistent with the idea that there are greater circulating concentrations of desacyl ghrelin than the acetylated form (385,391).

Release Mechanisms In humans, circulating concentrations of ghrelin are greatest in the interdigestive period. The mechanisms regulating release of ghrelin currently are attracting considerable attention (376). Plasma concentrations of ghrelin increase before a meal and decrease with ingestion of food (392,393). Oral or intravenous glucose loads also decrease plasma ghrelin (394). There is a relation between ghrelin and body weight: concentrations are high in anorexic subjects (389) and reduced in obese subjects (395). In both cases, plasma concentrations adjust after changes in body weight. Concentrations are markedly reduced in patients after gastric restriction/bypass surgery (396). One study (397) reports eradication of H. pylori to be associated with increased plasma ghrelin, but another study (398) reports similar plasma concentrations of ghrelin in patients infected with H. pylori compared with control subjects. Most human studies of plasma ghrelin to date have not considered H. pylori status, and further work is needed to clarify the significance of infection as a possible confounding variable. In the rat, plasma ghrelin concentrations are greatest at the end of the dark and light periods (399); these periods correspond to maximum and minimum gastric volumes, suggesting that the control of ghrelin is complex. All the major nutrient groups are reported to depress ghrelin secretion when instilled into the rat stomach, and glucose and fat also reduced ghrelin on intravenous administration (400).

Metabolism In human subjects, exogenously administered acylated ghrelin disappeared from plasma more rapidly than total

ghrelin, with half lives of 9 to 13 and 27 to 31 minutes, respectively, suggesting that deacylation occurs after secretion (401). Studies in subjects with renal failure and nephrectomized mice suggest that the kidney is important for the clearance of desacyl ghrelin (391).

Receptors, Transduction, and Structure–Activity Relations Ghrelin acts at the GHS-1 receptor. Alternative mRNA splicing generates two forms of the receptor: type 1a is functional, but a truncated form, type 1b, is not (402). The receptor is coupled to Gαq/11, and activation leads to stimulation of phospholipase C, increased intracellular calcium, increased inositol polyphosphate turnover, and PKC activation (403). Interestingly, the ghrelin receptor has high constitutive, or ligand-independent, activity, which can be revealed using CREB activation as an assay and implying that receptor expression on its own (i.e., in the absence of ligand) may be associated with biological responses (404). GHSs that act via the GHS receptor have been known for many years, including the synthetic hexapeptide, His-D-TrpAla-Trp-D-Phe-Lys-amide (GHRP-6) (374,375). Moreover, a number of other GHS receptor ligands were identified through drug-discovery programs before the discovery of ghrelin as the endogenous ligand (376,405). The characteristic actions of ghrelin on food intake and growth hormone release are properties of the acylated peptide. A report suggests that desacyl ghrelin had actions in the opposite direction to the acylated peptide. These included inhibition of food intake and inhibition of gastric emptying (406); moreover, mice overexpressing desacyl ghrelin appear to be smaller than control animals (407). The mechanism of action of desacyl peptide and the relevant receptor currently are unknown.

Biological and Physiologic Actions The stimulation of growth hormone release by ghrelin occurs in vivo and in vitro and was the defining activity of the peptide (2). In addition, ghrelin exerts a number of other actions including effects on other hormones, the cardiovascular system, sleep, cell proliferation, and carbohydrate metabolism (379,405). Most strikingly, however, ghrelin stimulates food intake and adiposity, and plays a role in the regulation of digestion (408,409). Mice in which the GHS-1 receptor gene is deleted are insensitive to the effects of ghrelin administration, indicating that the orexigenic effects of ghrelin are mediated by GHS-R (410). However, both these mice and mice in which the ghrelin gene has been deleted exhibit normal body weight and food intake (411,412). These observations suggest ghrelin is not an essential regulator of food intake in the mouse. Studies in these mice do, however, suggest that ghrelin plays a role in determining fat versus carbohydrate utilization as energy substrates, particularly during high-fat intake (412).

GASTROINTESTINAL HORMONES: GASTRIN, CHOLECYSTOKININ, SOMATOSTATIN, AND GHRELIN / 111 The idea that ghrelin stimulates food intake by acting directly on the hypothalamus has subsequently been modified by the observation that the effects of circulating ghrelin on appetite require an intact vagus nerve (413). Thus, GHS-1a receptors are localized to vagal afferent nerve fibers that project to the stomach (414), ghrelin inhibits the resting discharge of vagal afferent fibers, and lesion of vagal afferent fibers with capsaicin inhibits the appetite-stimulating actions of ghrelin (413). Ghrelin exerts a number of effects on gastrointestinal function that, taken as a whole, point to a role in stimulating the delivery of nutrients to the upper small intestine. They include stimulation of gastric motility, gastric emptying, and gastric acid secretion (415,416). The stimulation of gastric motility and secretion in the rat after intravenous administration of ghrelin is blocked by vagotomy and atropine, indicating an indirect effect. In conscious rats, intravenous ghrelin and des(Gln-14)ghrelin were equally potent in stimulating gastric emptying; ghrelin also increased intestinal transit times and reversed postoperative delay in gastric emptying (417). On both intracerebroventricular and intravenous administration, ghrelin stimulated fasted-type gastrointestinal motility patterns in the rat (418). Notably, the effects of ghrelin in the rat (which lacks motilin) are reminiscent of those of motilin in other species. Central and peripheral ghrelin administration has potent gastroprotective effects against ethanol-induced ulcers. The mechanism appears to depend on nitric oxide release and primary afferent nerve fibers (419). It is known that activation of spinal afferent neurons releases CGRP, which is gastroprotective (327), and it seems possible that ghrelin activates this pathway.

Conditions of Excess and Deficiency There are increased plasma ghrelin concentrations in anorexia and decreased concentrations in obesity (see earlier). In the Prader-Willi syndrome (characterized by excessive appetite and obesity), there is also increased ghrelin, which it is thought might contribute to hyperphagia (420).

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404. 405. 406. 407. 408. 409.

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CHAPTER

5

Postpyloric Gastrointestinal Peptides Ella W. Englander and George H. Greeley Jr. Neuropeptide Y, 134 Structure, 134 Neuropeptide Y Receptors, 134 Gastrointestinal Localization of Neuropeptide Y, 134 Secretion, 137 Actions, 137 Motilin, 137 Structure, 137 Motilin Receptor, 137 Distribution, 137 Actions on the Gastrointestinal Tract, 138 Mechanism of Action, 139 Release, 139 Pathophysiology, 140 Erythromycin and Motilides, 140 Peptide YY, 140 Structure, 140 Gastrointestinal Localization of Peptide YY and Developmental Appearance of Intestinal Peptide YY, 140 Peptide YY Receptors, 142 Actions on the Gastrointestinal Tract, 143 Regulation of Peptide YY Secretion, 145 Neural Control of Peptide YY Release, 146 Trophic Actions of Peptide YY on the Gastrointestinal Tract, 146 Inhibitory Action of PYY (3-36) on Appetite and Food Intake, 147 Pathophysiology of Peptide YY, 147 References, 148

Secretin, 121 Structure, 122 Gastrointestinal Localization of Secretin, 122 Secretin Receptor, 122 Actions on the Gastrointestinal Tract, 123 Regulation of Secretin Secretion, 125 Pathobiology of Secretin, 126 Intestinal Somatostatin, 126 Distribution, 126 Receptors, 126 Secretion, 126 Actions, 126 Vasoactive Intestinal Polypeptide and Related Peptides, 126 Structure, 127 Gastrointestinal Localization of Vasoactive Intestinal Polypeptide, 127 Vasoactive Intestinal Polypeptide Receptors, 128 Actions on the Gastrointestinal Tract, 129 Regulation of Vasoactive Intestinal Polypeptide Secretion, 129 Vasoactive Intestinal Polypeptide Peptides, 130 Neurotensin, 130 Structure, 130 Gastrointestinal Localization of Neurotensin, 131 Neurotensin Receptors, 131 Actions on the Gastrointestinal Tract, 132 Regulation of Neurotensin Secretion, 133

SECRETIN The current science of endocrinology started with the discovery of intestinal secretin; secretin was the first hormone discovered. The science of endocrinology can be defined as the study of chemical messengers produced within endocrine organs that are secreted into the general circulation and act on specific target tissues some distance away to evoke a physiologic response. In 1902, Bayliss and Starling

E. W. Englander: Department of Surgery, The University of Texas Medical Branch, Galveston, Texas 77550. G. H. Greeley Jr.: Department of Surgery, The University of Texas Medical Branch, Galveston, Texas 77555. Physiology of the Gastrointestinal Tract, Fourth Edition, edited by Leonard R. Johnson. Academic Press, 2006.

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122 / CHAPTER 5 (1) showed that infusion of hydrochloric acid into a denervated loop of the small intestine caused secretion of pancreatic juice in dogs. They reasoned that a chemical substance released by the acidified intestine and transported by the bloodstream to the pancreas triggered the stimulation of pancreatic secretion. They also showed that an acid extract of the intestinal mucosa given intravenously stimulated pancreatic secretion. They then proposed that the exocrine pancreas was stimulated by a chemical factor produced in the intestinal mucosa and released by luminal acid. They called this substance secretin. The endocrine nature of the underlying regulation of pancreatic secretion was also demonstrated by yet another experiment. In 1926, Farrell and Ivy (2) transplanted the tail of the pancreas into the mammary gland of a lactating dog. Feeding the dog resulted in an increase in pancreatic secretion. Because the transplanted pancreas was separated from its normal nidus and nerve supply, these investigators concluded that a bloodborne factor provoked pancreatic secretion after ingestion of a meal. Finally, in 1965, Mutt and coworkers (3) reported the amino acid sequence of secretin. Secretin is a 27-amino-acid peptide hormone produced in enteroendocrine “S” cells of the small intestine. Secretin is released into the general circulation in response to a decrease of luminal pH after a meal; its primary physiologic action is a stimulation of pancreatic bicarbonate secretion, although it also inhibits gastric acid secretion, gastric emptying, colonic contractions, lower esophageal sphincter tone, and motility. Secretin is used clinically in diagnosis of gastrinomas in patients with Zollinger–Ellison syndrome (ZES).

pancreatic exocrine secretion (4). The amidated, carboxy terminal of secretin is also essential for biological activity. Examination of the rat and pig secretin complementary DNA (cDNA) show that the secretin precursor consists of a signal peptide, a short amino-terminal peptide, the secretin sequence, an amidation sequence (glycine-lysine-arginine [GKR]), and a 72-amino-acid carboxyl-terminal peptide (Fig. 5-3) (5). The secretin gene consists of four exons spanning 813 base pairs, and like glucagon, VIP, and PACAP, the secretin sequence is encoded by a single exon (see Fig. 5-3) (6–12). The structure of secretin is highly conserved during mammalian evolution with amino acid substitutions limited to changes in positions 14, 15, and 16 (13–18).

Gastrointestinal Localization of Secretin The greatest tissue concentration of secretin peptide is found in enteroendocrine secretin or “S” cells of the mucosal epithelium, in the villi of the proximal small intestine (19). They are called S cells because of the small size of secretin granules. The density of secretin cells decreases progressively from the duodenum to the ileum. Interestingly, the highest secretin expression (messenger RNA [mRNA]) is seen in the ileum; secretin mRNA is also detected in the colon (5). Developmentally, intestinal secretin cells are first identified on day 17 of gestation in rats (20), and both secretin expression and peptide show their greatest levels 2 days before birth (6). Secretin expression is also detectable in β cells of the fetal rat pancreas (21). The greatest level of secretin expression in the pancreas occurs on day 19 of gestation, and then decreases to undetectable levels during adulthood.

Structure Secretin is a 27-amino-acid peptide (Fig. 5-1) structurally related to other regulatory peptides; together they are called the secretin family of peptides (Fig. 5-2). These peptides are glucagon, vasoactive intestinal polypeptide (VIP), pituitary adenylate cyclase–activating peptide (PACAP), gastric inhibitory polypeptide (GIP), growth hormone–releasing hormone (GHRH), peptide histidine isoleucine (PHI), and peptide histidine methionine (PHM). The entire 27-aminoacid sequence is essential for full activity; although an amino-truncated variant, secretin (5-27) will stimulate

Secretin Receptor The secretin receptor has been cloned (22) and is a G protein–coupled seven-transmembrane receptor linked to adenylate cyclase activation and stimulation of cyclic 3′,5′adenosine monophosphate (cAMP) production (22–24). The secretin receptor has a 427-amino-acid sequence and a molecular weight of 48,696 daltons. The secretin receptor is related structurally to the receptors for VIP, parathyroid hormone, and calcitonin (22).

Human Secretin His-Ser-Asp-Gly-Thr-Phe-Thr-Ser-Glu-Leu-Ser-Arg-Leu-Arg-Glu-Gly-Ala-Arg-Leu-Gln-Arg-Leu-Leu-Gln-Gly-Leu-Val-NH2

Rat Secretin His-Ser-Asp-Gly-Thr-Phe-Thr-Ser-Glu-Leu-Ser-Arg-Leu-Gln-Asp-Ser-Ala-Arg-Leu-Gln-Arg-Leu-Leu-Gln-Gly-Leu-Val-NH2

FIG. 5-1. Comparison of amino acid sequences for human and rat secretin. The sequences differ in only a single residue that is underlined.

POSTPYLORIC GASTROINTESTINAL PEPTIDES / 123 Secretin

THR-PRO-ARG-HIS-SER-ASP-GLY-THR-PHE-THR-SER-GLU-LEU-SER-ARG-LEU-GLN-ASP-

Glucagon

SER-PHE-PRO-ALA-SER-GLN-THR-GLU-PRO-LEU-GLU-ASP-PRO-ASP-GLN-ILE-ASN-GLU-ASP-LYS-ARG-HIS-SER-GLN-GLY-THR-PHE-THR-SER-ASP-TYR-SER-LYS-TYR-LEU-ASP-

SER-ALA-ARG-LEU-GLN-ARG-LEU-LEU-GLN-GLY-LEU-VAL

SER-ARG-ARG-ALA-GLN-ASP-PHE-VAL-GLN-TRP-LEU-MET-ASN-THR-LYS-ARG VIP

SER-SER-ILE-SER-GLU-ASP-PRO-VAL-PRO-VAL-LYS-ARG-HIS-SER-ASP-ALA-VAL-PHE-THR-ASP-ASN-TYR-THR-ARG-LEU-ARG-LYSGLN-MET-ALA-VAL-LYS-LYS-TYR-LEU-ASN-SER-ILE-LEU-ASN-GLY-LYS-ARG

PACAP GIP

HIS-SER-ASP-GLY-ILE-PHE-THR-ASP-SER-TYR-SER-ARG-TYR-ARG-LYS-GLN-MET-ALA-VAL-LYS-LYS-TYR-LEU-ALA-ALA-VAL-LEU-ASP ALA-LEU-PRO-SER-LEU-PRO-VAL-GLY-SER-HIS-ALA-LYS-VAL-SER-SER-PRO-GLN-PRO-ARG-GLY-PRO-ARG-TYR-ALA-GLU-GLY-THR-PHE-ILE-SER-ASP-TYR-SER-ILE-ALA-MET-ASPLYS-ILE-HIS-GLU-GLU-ASP-PHE-VAL-ASN-TRP-LEU-LEU-ALA-GLU-LYS-GLY

GHRH

VAL-ARG-ARG-HIS-ALA-ASP-ALA-ILE-PHE-THR-SER-SER-TYR-ARG-ARG-ILE-LEU-GLY-

PHI

ASN-ALA-ARG-HIS-ALA-ASP-GLY-VAL-PHE-THR-SER-ASP-TYR-SER-ARG-LEU-LEU-GLY-

GLN-LEU-TYR-ALA-ARG-LYS-LEU-LEU-HIS-GLU-ILE-MET-ASN-ARG-GLN-GLN

GLN-ILE-SER-ALA-LYS-LYS-TYR-LEU-GLU-SER-LEU-ILE-

-GLY-LYS-ARG

FIG. 5-2. Alignment of the amino acid sequences for the rat secretin family of peptides. Nonidentity in sequences is indicated by underlining. All sequences shown are for the rat, except for human gastric inhibitory polypeptide (GIP). GHRH, growth hormone–releasing hormone; PACAP, pituitary adenylyl cyclase–activating polypeptide; PHI, peptide histidine isoleucine; VIP, vasoactive intestinal peptide.

Actions on the Gastrointestinal Tract The primary physiologic action of secretin is the stimulation of pancreatic bicarbonate secretion (25–28). Administration of secretin intravenously (0.03 CU/kg/hr [CU = 2.5 pmol]) will increase pancreatic secretion of water and bicarbonate in humans, dogs, and rats (29–33). That secretin is the physiologic effector of pancreatic bicarbonate secretion has been verified in dog experiments where infusion of physiologic amounts of exogenous secretin results in a stimulation of pancreatic bicarbonate secretion. In these experiments, physiologic amounts of secretin means that secretin was given at doses that reproduce circulating secretin levels measured in response to physiologic amounts of acid in the duodenal lumen of dogs. More importantly, the role of secretin is supported by the finding that bicarbonate secretion is reduced by systemic immunoneutralization of secretin in dogs. A significant increase in bicarbonate secretion is measured when plasma secretin levels increase by only 1 to 2 pM, and there is a linear dose–response relation between

plasma secretin levels and bicarbonate secretion up to 40 pM (34). In experimental settings, secretin given alone intravenously is a weak stimulant of pancreatic enzyme secretion. However, small amounts of secretin will potentiate the stimulatory effects of cholecystokinin (CCK) on enzyme and bicarbonate secretion in humans and in dogs (Fig. 5-4) (33). Secretin can also stimulate bile salt-independent bile flow (35–40); however, the physiologic relevance of this finding is unknown. Secretin, administered at physiologic doses, will inhibit pentagastrin-stimulated acid secretion and meal-induced acid secretion in dogs (41–43). Administration of secretin to humans also results in an inhibition of pentagastrin-stimulated acid secretion (44). It has been shown that immunoneutralization of systemic secretin increased plasma gastrin levels and acid secretion in response to a test meal in dogs, implying that secretin has a role as a physiologic inhibitor of acid secretion in the dog (41,43). In dogs, the inhibitory action of secretin on acid secretion has been shown to be mediated in part by prostaglandins (Fig. 5-5) (45). In addition, inhibition

Signal Peptide Rat

N-Terminal Peptide

M E P L L P T P P L L L L L L L L L S S S F V L P A P P R T P R Secretin

H S D G T F T S E L S R L Q D S A R L Q R L L Q G L V G K R S E C-Terminal Extension Peptide

E D T E N I P E N S V A R P K P L E D Q L C L L W S N T Q A L Q

D W L L P R L S L D G S L S L W L P P G P R P A V D H S E W T E

T T R Q P R

FIG. 5-3. Amino acid sequence for rat secretin precursor. The signal peptide, N-terminal peptide, secretin sequence, and C-terminal extension peptide are identified. (Modified from Kopin and colleagues [5], by permission.)

124 / CHAPTER 5 Actual value by Secretin (.03 Cu/kg/hr) and CCK-8 (.03-.25 µg/kg/hr)

∆ HCO3− Output (mEq/15 min)

.35

Sum of values by two individual hormones

.30 .25 .20 .15 .10 .05

SE C C .03 C K S .03 & EC C .0 C 3 K .0 3 SE C C .03 C K S .06 & EC C .0 C 3 K .0 6 SE C C . C 03 K . S 12 & EC 5 C C .0 K 3 .1 25 SE C C .03 C K SE .25 & C C .0 C 3 K .2 5

.00

FIG. 5-4. Mean increased pancreatic bicarbonate outputs greater than basal values in response to intravenous secretin alone (0.08 CU/kg/hr [CU = 2.5 pmol]), cholecystokinin (CCK)-8 alone at four different doses (0.03, 0.06, 0.125, and 0.25 µg/kg/hr), and a combination of secretin and CCK-8 in five dogs with pancreatic fistulas. Bars represent means ± standard error of the mean. (Modified from Chey and colleagues [33], by permission.)

of pentagastrin-stimulated acid secretion and stimulation of pancreatic bicarbonate secretion are not strictly parallel during duodenal acidification in dogs, implying that secretin is not the only factor released by the luminal acid that also exerts an inhibitory action on acid secretion (46). In rats, a low physiologic dose of secretin will inhibit gastric acid secretion that is mediated by prostaglandins (47). In dogs, Pentagastrin

administration of secretin in combination with serotonin (5-HT) results in a greater inhibition of acid secretion when compared with either agent alone (Fig. 5-6) (48). The relevance of the inhibitory action of 5-HT on acid secretion is strengthened by the finding that duodenal acidification causes secretion of intestinal 5-HT (49). Secretin causes a reduction in the serum gastrin response to food in both humans and dogs (50). Administration of secretin will reduce basal gastrin levels in dogs, healthy subjects, duodenal ulcer patients, and patients with pernicious anemia (51–53). The inhibitory actions of secretin on meal-induced gastrin secretion, acid secretion, and gastric emptying are similar in healthy subjects and patients with duodenal ulcer (51,54). In rat studies, secretin has been shown to inhibit gastrin release via its stimulation of stomach somatostatin (SRIF) secretion (55,56). Under a variety of experimental settings, administration of exogenous secretin affects secretion of insulin, glucagon, somatostatin, and pancreatic polypeptide (PP) (55,57–61). Abundant data indicate that secretin has an inhibitory role in regulation of gastric emptying in dogs and humans (41,62–64). Secretin also stimulates pepsin secretion in dogs, cats, and humans (65–68), and secretin stimulates pepsinogen secretion from isolated dog chief cells maintained in culture (69). In humans, the administration of secretin at doses as low as 0.03 U/kg significantly inhibits gastric emptying of a liquid meal (63). This inhibitory action of secretin is not potentiated by CCK. Infusion of secretin will stimulate gastrin secretion in patients with gastrinoma and ZES (53,70,71). In fact, intravenous (IV) infusions of secretin are used in locating gastrinomas during their surgical removal (72). Secretin appears to cause gastrin secretion by a direct action on stomach gastrin “G” cells, because in vitro experiments show that secretin will stimulate gastrin release from cultured gastrinoma cells (73).

Secretin

*

*

0 B1 B2 15

45

75 Minutes

105

135

165

FIG. 5-5. Gastric acid output after infusion of secretin (3.3 CU/kg/hr [CU = 2.5 pmol]) alone or in combination with indomethacin, an inhibitor of prostaglandin synthesis (2 mg/kg bolus + 0.5 mg/kg/hr) in six dogs. Each point represents the means ± standard error of the mean. Note that inhibition of gastric acid secretion was achieved only in the secretin infusion alone group. B, basal value. *p < 0.05 versus values before secretin administration. (Modified from MacLellan and colleagues [45], by permission.)

10

*

PG+5-HT

*

PG+5-HT+Secretin

Secretin * + Indomethacin

1

PG+Secretin

2

PG

3

20 Secretin alone

∆H+ (mEq/30 min)

Gastric acid output (mEq/15 min)

4

*

**

FIG. 5-6. Gastric acid outputs in conscious dogs during infusion of pentagastrin (PG) alone, PG with secretin, PG with serotonin (5-HT), or PG with 5-HT and secretin. *p < 0.05 versus PG alone. **p < 0.05 versus PG + secretin or PG + 5-HT. (Modified from Mate and colleagues [48], by permission.)

POSTPYLORIC GASTROINTESTINAL PEPTIDES / 125

Acidification of the duodenal and jejunal lumen is the primary physiologic stimulus for release of secretin into the bloodstream and the subsequent stimulation of pancreatic exocrine secretion (25–28). In the dog, the pH threshold for stimulation of secretion release is 4.5 (26), and the amount of pancreatic bicarbonate that flows into the intestinal lumen is directly dependent on the length of small intestine that is exposed to the acid and the acid load (82). An increased basal acid secretion will cause an increased secretin release (83). Similar to another intestinal hormone, CCK, systemic levels of secretin are exceptionally low (30,84–86). Plasma secretin levels of approximately 10 pM will stimulate pancreatic bicarbonate secretion, and an increase in plasma secretin levels of only 1 to 2 pM will activate pancreatic bicarbonate secretion significantly (87,88). Measurement of an increase in plasma secretin levels after a normal meal is not agreed on, implying that secretin may not be released after a meal (89,90). However, acidified meals definitely cause secretin release (84). Some studies show that plasma secretin levels do not increase until several hours after a meal (91,92). As explained for the pancreatic bicarbonate response, the increased plasma secretin response is related to the amount of acid entering the duodenal lumen (84,91,93). In a study of humans, a liquid test meal increased plasma secretin levels by 2 to 3 pM when duodenal pH decreased to 4, and it was shown that administration of exogenous secretin at doses that mimicked this plasma concentration increase also stimulated bicarbonate secretion (27). In humans, administration of low amounts of acid into the duodenum, followed by neutralization, caused a short-lived increase in plasma secretin levels of approximately 1 pM (34). Exceptionally small bolus injections of secretin in humans (0.1–0.5 pmol/kg) will result in a stimulation of pancreatic bicarbonate secretion and increases in plasma secretin levels of 2 to 6 pM (34). Direct proof for involvement of secretin in the stimulation of pancreatic bicarbonate secretion has been shown by immunoneutralization of systemic secretin—the pancreatic bicarbonate response was decreased by approximately 80% (41). Together, these findings indicate that secretin acts as a physiologic-hormonal regulator of bicarbonate secretion; however, it is not exactly clear the extent to which this effect is present in humans after a meal (94). Fat in the duodenal lumen is a strong inhibitor of gastrin and gastric acid secretion, but the role of secretin in mediating

30 Plasma secretin (pg/ml)

Regulation of Secretin Secretion

this inhibitory action of fat is not settled (95). Pancreatic bicarbonate and secretin release are stimulated by duodenal infusion of oleate; the small increase in plasma secretin levels implies a role for secretin in the bicarbonate response (Fig. 5-7) (96). Alcohol can stimulate secretin release in humans most likely by a stimulation of gastric acid secretion (97). Intraduodenal administration of bile will also cause secretin release in humans (97). In contrast with other gut hormones, the vagal nerve does not influence secretin release in dogs and humans. In dogs, vagotomy and atropine treatment do not affect secretin release in response to intraduodenal administration of acid in dogs, and atropine treatment does not alter the secretin response to duodenal acidification in humans (95,98). Furthermore, vagotomy does not influence secretin release in response to acid in humans (99). In dogs, vagal stimulation does not affect secretin, and atropine administration did not modify secretin release to acid (98). In contrast with the lack of vagal influence over secretin release, there is a significant interaction between secretin and vagal cholinergic control of the exocrine pancreas. Vagal stimulation will potentiate the stimulatory effects of secretin on pancreatic bicarbonate secretion in dogs and cats (100,101). In addition, vagotomy or anticholinergic drugs will reduce pancreatic secretion of

*

*

20 * 10

0 *

*

0.3 Bicarbonate output (mEq/15 min)

Secretin also stimulates the pylorus or inhibits the lower esophageal sphincter at pharmacologic doses (74,75). Physiologic doses of secretin will relax the sphincter of Oddi in humans (76). Administration of secretin to cats and dogs will increase secretion from intestinal Brunner’s glands (77,78). Although the growth-promoting action of secretin on the pancreas is not completely accepted, exogenous secretin has been shown to exert a trophic action on the exocrine pancreas (79–81).

0.2 * 0.1

0 0

0.25 0.5 1.0 2.0 Sodium oleate (mmol/15 min)

FIG. 5-7. Effects of increasing doses of intraduodenal administration of sodium oleate (0.25–2.0 mmol/15 min) on plasma secretin levels and pancreatic bicarbonate outputs in dogs with modified Herrera cannulas. *p < 0.05 versus 0 minute values. (Modified from Faichney and colleagues [96], by permission.)

126 / CHAPTER 5 water and bicarbonate stimulated by intraduodenal administration of acid or food in humans (29,102–104) and in dogs. Somatostatin will inhibit secretin release (105,106), whereas bombesin has no effect. There is some evidence to suggest that a luminal secretinreleasing peptide mediates secretin release in rats; however, this peptide factor has never been isolated, nor its sequence identified (107).

muscle relaxation. SSTR3 is found in the smooth muscle cells of the rat and can mediate contractions of guinea pig stomach and intestinal smooth muscle cells (125,127,128). SSTR2 may have a role in the regulation of gut motility because SSTR2 is found on nitric oxide synthase-expressing myenteric neurons, which may be inhibitory motor neurons (129). Cortistatin will activate all SRIF receptor subtypes (112,121).

Secretion Pathobiology of Secretin Plasma levels of secretin are altered in various clinical conditions. Hypersecretinemia has been observed in patients with ZES and excessive gastric acid secretion (83). In patients with duodenal ulcers, secretin and pancreatic bicarbonate secretion are unaltered compared with healthy subjects (108). In patients with celiac sprue, the decreased release of secretin may have a role in their reduced pancreatic exocrine secretion (108).

INTESTINAL SOMATOSTATIN Distribution Intestinal somatostatin (SRIF) has been detected by immunoassay in intestinal extracts (Fig. 5-8) (109,110), and immunohistochemical studies show intestinal enteroendocrine somatostatin-containing “D” cells scattered throughout the small and large intestines (111,112). SRIF is also found in intestinal nerves and nerve plexuses; SRIF-containing nerve fibers are observed around ganglion cells of the myenteric plexus of the intestine (113–115). In the intestine, approximately 90% of the SRIF is found in the mucosa and 10% in the muscle layer (116). In the myenteric plexus, SRIF-containing neurons may function as interneurons and are involved in the descending inhibitory reflex of peristalsis (117). In the intestine, the primary molecular form is SRIF-28 (110,118). Interestingly, a novel peptide, called thrittene, was isolated from monkey ileum that is structurally identical to SRIF (119). The amino acid sequence of thrittene is identical to that of the N-terminal 13 amino acids of SRIF-28. Because the gene encoding this peptide has not been isolated, it may represent a processing product of the SRIF precursor and not the product of a novel gene. There is also cDNA of a novel 17-amino-acid SRIF-like peptide called cortistatin (112,120,121).

Receptors Five SRIF receptor subtypes have been identified (SSTR15) (122–124). SSTR1, SSTR2, and SSTR3 are found in the intestine (124–126). In the gastrointestinal (GI) tract, SRIF receptors appear to be involved in regulation of smooth

In humans, SRIF-28 is the primary form of SRIF that is found in the general circulation in response to ingestion of various nutrients (Fig. 5-9). This finding indicates that systemic SRIF is derived from intestinal “D” cells and not the stomach (130).

Actions SRIF exerts a general inhibitory action on release of intestinal hormones, secretion, and absorption. SRIF inhibits the cyclic interdigestive motor and electrical activity of the intestine (131,132). Infusion of physiologic amounts of SRIF will inhibit gastric motility and intestinal phase 2, a period of irregular contractions, and stimulation of phase 3, a period of rhythmic contractions. The interval between activity fronts is decreased by approximately 50% (133). SRIF can inhibit basal and stimulated bicarbonate secretion from rat duodenal Brunner’s glands (134). SRIF inhibits intestinal transport of ions, causing a lower intestinal secretion (135). SRIF inhibits secretion of almost every gut hormone tested including ghrelin, secretin, CCK, GIP, and motilin (126,136,137).

VASOACTIVE INTESTINAL POLYPEPTIDE AND RELATED PEPTIDES VIP is a 28-amino-acid peptide that was first discovered in porcine duodenal tissue extracts. VIP belongs to the secretin family of peptides that includes glucagon, secretin, GIP, PACAP, GHRH, PHI/PHM, and two reptilian peptides, helodermin and helospectin. VIP was named for its strong and persistent vasodilatory effects. VIP relaxes vascular and nonvascular smooth muscle and will stimulate electrolyte secretion from the exocrine pancreas and intestine. VIP and PHI/PHM are processed from the same precursor peptide. Unlike secretin, GIP, and glucagon, VIP is exclusively a neurotransmitter in the GI tract and is not normally secreted into the bloodstream unless it occurs as overflow from nerve endings or VIP-secreting tumors. In the GI tract and pancreas, VIP exerts a broad array of biological activities including regulation of relaxation of gastric and intestinal smooth muscle, stimulation of pancreatic exocrine and intestinal secretion, and an inhibition of gastric acid secretion and blood flow. Humans with a VIP-secreting tumor (VIPoma) will have the watery diarrhea syndrome and high plasma levels of VIP.

POSTPYLORIC GASTROINTESTINAL PEPTIDES / 127 Somatostatin concentration (pmoles/g wet weight) 0 200

1000

3000 Esophagus Cardia Fundus Antrum

Duodenum Jejunum Mid intestine Ileum Cecum

Colon Spleen Pancreas Gall bladder

Muscular layer

Mucosa or parenchyma

Liver

FIG. 5-8. Distribution of somatostatin in the gastrointestinal (GI) tract of the cat. Somatostatin levels are shown in the GI mucosa and parenchyma (right horizontal axis) and muscular layer (left horizontal axis). Maximal levels are seen in antral mucosa and in proximal duodenum. Substantial amounts of somatostatin are measured in the intestinal mucosa. Mean ± standard error of the mean is shown. (From Chayvialle and colleagues [109], by permission.)

Structure Said and Mutt (138–140) discovered VIP when screening extracts of the pig intestine for substances that stimulated vasodilation. VIP is a 28-amino-acid peptide that resembles several other regulatory peptides produced in the GI tract and pancreas including glucagon, GIP, GHRH, secretin, PACAP, PHI/PHM, and the reptilian peptides, helodermin, and helospectin (Fig. 5-10). The sequences for rat, pig, and human VIP are identical, indicating the conserved nature of VIP during evolution. The VIP gene consists of six introns and seven exons. The mRNA for VIP encodes for VIP and a second peptide belonging to the secretin family of peptides called PHM in humans and PHI in rodents. PHM and VIP are encoded by exons 4 and 5, respectively. Prepro-VIP is 170 amino acids; during processing, 5 different peptide fragments are formed, including prepro-VIP (residues 22–79) (also called N-terminal flanking peptide), PHI/PHM (residues 81–107), prepro-VIP (residues 111–122; bridging peptide),

the VIP sequence (residues 125–152), and prepro-VIP (residues 156–170; carboxy-terminal flanking peptide).

Gastrointestinal Localization of Vasoactive Intestinal Polypeptide Neuronal VIP/PHI is found throughout the entire length of the GI tract (109,141–143), the pancreas (144), and in salivary glands (145). In the GI tract, VIP is found in the superior and inferior mesenteric ganglia and in the submucous (Meissner’s) and myenteric (Auerbach’s) plexuses of the intestinal wall (146–149). The greatest concentrations of VIP/PHI are found in the colon and ileum, with lower amounts in the stomach fundus, duodenum, and jejunum (Fig. 5-11) (109,150). During development in rats, intestinal VIP first appears at 3 days of age, and levels increase progressively until 28 days of age, when they achieve adult VIP levels (151). VIP-containing autonomic nerve fibers are detected

128 / CHAPTER 5 Meal 120

*

*

Plasma somatostatin (pg/ml)

*

*

*

Portal

80 * Systemic 40 *

*

* *

0

−15

0

15

30

45

60

90

120

150

Minutes

FIG. 5-9. Somatostatin levels in portal and peripheral plasma after ingestion of a mixed meal in dogs. The test meal caused significant increases in portal and peripheral somatostatin levels lasting for 90 to 120 minutes after ingestion. *p < 0.05 versus basal somatostatin level. (From Chayvialle JA, Miyata M, Rayford PL, Thompson JC. Effects of test meal, intragastric nutrients, and intraduodenal bile on plasma concentrations of immunoreactive somatostatin and vasoactive intestinal peptide in dogs. Gastroenterology 1980;79:844–852, by permission.)

in all layers of the gut and around blood vessels (152). In immunohistochemical studies, VIP-containing fibers are readily evident in the lamina propria, where they are found with blood vessels and reach to the surface epithelium, and in the circular muscle layer. They are absent in the longitudinal muscle layer with the exception of the taenia coli (152). VIP-positive fibers appear to originate from intramural ganglion cells of the submucosal plexus (152,153) because extrinsic denervation will not reduce VIP staining or contents. In diseases that affect autonomic ganglionic cells such as Hirschsprung’s disease and Chagas disease, changes in VIP staining are found (154,155). A characteris-

tic feature of Crohn’s disease is neuronal hyperplasia with an increased VIP content in VIP-containing nerves (156). VIP staining is seen in autonomic nerve fibers of the exocrine and endocrine pancreas (144).

Vasoactive Intestinal Polypeptide Receptors Two VIP, high-affinity, G protein-coupled receptors have been characterized (157,158). They are called VIP receptors 1 and 2 (VPAC1, VPAC2). In the mouse, VPAC1 shows the greatest expression in the small intestine and colon, followed

VIP

HIS-SER-ASP-ALA-VAL-PHE-THR-ASP-ASN-TYR-THR-ARG-LEU-ARG-LYS-GLN-MET-ALA-VAL-LYS-LYS-TYR-LEU-ASN-SER-ILE-LEU-ASP-NH2

Secretin

HIS-SER-ASP-GLY-THR-PHE-THR-SER-GLU-LEU-SER-ARG-LEU-ARG-ASP-SER-ALA-ARG-LEU-GLN-ARG-LEU-LEU-GLN-GLY-LEU-VAL-NH2

PHI

HIS-ALA-ASP-GLY-VAL-PHE-THR-SER-ASP-PHE-SER-ARG-LEU-LEU-GLY-GLN-LEU-SER-ALA-LYS-LYS-TYR-LEU-GLU-SER-LEU-ILE-NH2

PACAP-27

HIS-SER-ASP-GLY-ILE-PHE-THR-ASP-SER-TYR-SER-ARG-TYR-ARG-LYS-GLN-MET-ALA-VAL-LYS-LYS-TYR-LEU-ALA-ALA-VAL-LEU-NH2

GHRH

TYR-ALA-ASP-ALA-ILE-PHE-THR-ASN-SER-TYR-ARG-LYS-VAL-LEU-GLY-GLN-LEU-SER-ALA-ARG-LYS-LEU-LEU-GLN-ASP-ILE-MET-SER ARG-GLN-GLN-GLY-GLU-SER-ASN-GLN-GLU-ARG-GLY-ALA-ARG-ALA-ARG-LEU-NH2

GIP

TYR-ALA-GLU-GLY-THR-PHE-ILE-SER-ASP-TYR-SER-ILE-ALA-MET-ASP-LYS-ILE-ARG-GLN-GLN-ASP-PHE-VAL-ASN-TRP-LEU-LEUALA-GLN-LYS-GLY-LYS-LYS-ASN-ASP-TRP-LYS-HIS-ASN-ILE-THR-GLN

Glucagon Helodermin

HIS-SER-GLN-GLY-THR-PHE-THR-SER-ASP-TYR-SER-LYS-TYR-LEU-ASP-SER-ARG-ARG-ALA-GLN-ASP-PHE-VAL-GLN-TRP-LEU-MET-ASN-THR HIS-SER-ASP-ALA-ILE-PHE-THR-GLU-GLU-TYR-SER-LYS-LEU-LEU-ALA-LYS-LEU-ALA-LEU-GLN-LYS-TYR-LEU-ALA-SER-ILE-LEUGLY-SER-ARG-THR-SER-PRO-PRO-PRO-NH2

Helospectin HIS-SER-ASP-ALA-THR-PHE-THR-ALA-GLU-TYR-SER-LYS-LEU-LEU-ALA-LYS-LEU-ALA-LEU-GLN-LYS-TYR-LEU-GLU-SER-ILE-LEUGLY-SER-SER-THR-SER-PRO-ARG-PRO-PRO-SER-SER

FIG. 5-10. Amino acid sequences of vasoactive intestinal peptide (VIP) and VIP structurally related peptides. Nonidentity in sequences is indicated by underlining. The species for each mammalian peptide are: VIP—human, rat, mouse, pig, sheep; secretin—cow, pig; peptide histidine isoleucine (PHI)—pig; pituitary adenylyl cyclase–activating polypeptide (PACAP)-27—human, rat, sheep; growth hormone–releasing hormone (GHRH)—human; glucagon—human, bovine, porcine; gastric inhibitory polypeptide (GIP)—human.

POSTPYLORIC GASTROINTESTINAL PEPTIDES / 129 by the liver and brain. The VPAC2 receptor is expressed in the stomach, pancreas, and intestine, as well as in the lung and brain. The VIP1 and VIP2 receptors have similar structural binding specificities for VIP, PACAP, and PHI (VIP = PACAP > PHI). VIP receptors are found in the exocrine pancreas, gastric and intestinal epithelial cells, vascular smooth muscle cells, and on pancreatic and biliary ductular epithelial cells. VIP is known to activate multiple intracellular pathways (159) including the activation of cAMP-dependent protein kinases A (PKA) and C (PKC) (160–162). In intestinal epithelial cells, VIP activates IL-8 production by mitogenactivated protein kinase (MAPK)–dependent pathways (163).

Actions on the Gastrointestinal Tract In the GI tract, VIP is considered a neurotransmitter of inhibitory motor neurons, and the primary activity of VIP is its smooth muscle relaxant activity (164–167). VIP mediates relaxation of the lower esophageal and internal anal sphincters and can antagonize the contractile effects of pentagastrin in several species (74,168–170). The role of VIP as a smooth muscle relaxant is supported by the intense VIPergic innervation of gut sphincters. Evidence also indicates that VIP mediates descending relaxation of the peristaltic reflex (171). VIP can inhibit the contractile activity of gastrin (168). The relaxing effect of VIP is most likely secondary to an increased intracellular cAMP level. VIP is released into the gastric venous effluent after either esophageal or fundic distension and electrical stimulation of the vagus (109,172). VIP exerts a dual effect on intestinal smooth muscle: VIP relaxes duodenal and ileal circular muscles in the rat and the colon in the guinea pig (173,174), and at high levels, VIP contracts the longitudinal muscles of the duodenum and ileum in the rat (174) and colon in the guinea pig (173), an effect mediated by intramural cholinergic neurons (174). In guinea pigs, VIP inhibits gall bladder resting tone and CCK-induced contractions (175,176). VIP is a strong stimulant of intestinal secretion, being more potent than secretin and glucagon (177). In fact, VIP is nearly 1000-fold more potent than secretin on intestinal secretion (178). In dog and human studies, VIP can reverse the normal absorptive process of the small intestine to a net secretion of chloride and bicarbonate against an electrochemical gradient (178,179). Patients with increased blood levels of VIP caused by a VIP-secreting tumor (VIPoma) will most likely have watery diarrhea. VIP receptors are found with intestinal enterocytes, suggesting that VIP acts directly to increase intestinal secretion (180). Other activities of VIP include an inhibition of food, histamine, and pentagastrinstimulated gastric acid secretion in dogs (181–183). The inhibitory action of VIP on acid secretion may be mediated by gastric SRIF (182). VIP can simulate pancreatic secretion in numerous species; however, it is less potent than secretin (184–187). VIP may be involved in biliary transport and secretion because VIP is a potent stimulant of fluid and bicarbonate secretion from cholangiocytes via cAMP-independent pathways (188). VIP may also play a role in regulation of

blood flow in the small intestine (189,190), and VIP can stimulate bile secretion in dogs and rats (191,192).

Regulation of Vasoactive Intestinal Polypeptide Secretion Because VIP is a neural peptide in the GI tract, systemic VIP levels do not increase in response to ingestion of a meal. Levels of VIP will increase, however, in the venous outflow of the gut in response to luminal infusion of fat or acid, electrical stimulation of extrinsic nerves, and mechanical distension of the gut mucosa (193,194). Release of VIP by the GI tract is blocked by hexamethonium but not by atropine (172,194). VIP secretion is induced by administration of acetylcholine or neostigmine (a cholinesterase inhibitor) that is blocked by atropine (194,195). These data indicate that

VIP concentration (pmoles/g wet weight) 0 600

400

200

200

400

600 Esophagus Cardia Fundus Antrum

Duodenum Jejunum Mid intestine Ileum Cecum

Colon Spleen Pancreas Gall bladder Liver Muscular layer

Mucosa or parenchyma

FIG. 5-11. Distribution of vasoactive intestinal peptide (VIP) in the gastrointestinal (GI) tract of the cat. VIP levels are shown in the GI mucosa and parenchyma (right horizontal axis) and muscular layer (left horizontal axis). Peaks are seen in both mucosa and muscle layers of duodenum and right colon. Substantial amounts of VIP are also measured in extracts of the gallbladder wall. Mean ± standard error of the mean is shown. (From Chayvialle and colleagues [109], by permission.)

130 / CHAPTER 5 intramural VIP-containing neurons are innervated by preganglionic cholinergic excitatory fibers. Plasma levels of VIP are increased in patients with the watery diarrhea syndrome, also called the Verner and Morrison syndrome that includes the triad of pancreatic islet tumor, severe watery diarrhea, and hypokalemia (196). VIP was shown to be the major effector of diarrhea because tumor removal resulted in the cessation of diarrhea together with the decrease in plasma VIP levels. VIP may also play a role in the increased intestinal fluid secretion induced by cholera toxin and Escherichia coli heat labile (LT) and heat stable (STa) toxins (197). Vasoactive Intestinal Polypeptide Peptides Pituitary Adenylate Cyclase–Activating Peptide PACAP is the most recently discovered peptide of the secretin family of peptides. PACAP is called PACAP because during its isolation from the hypothalamus, it was shown to simulate adenylate cyclase activity in rat anterior pituitary cells in culture (198). There are two PACAP variants, PACAP27 and -38 (160), and both forms are biologically active. The human PACAP gene has been cloned and is composed of five exons (199). The mouse PACAP gene is composed of six exons; the signal peptide, the PACAP-related peptide, and PACAP are encoded within exons 2, 4, and 5, respectively (199). The intron–exon organization and the production of alternate mRNA of the PACAP gene are similar to those of the GHRH gene, which supports the notion that the two genes originated by gene duplication. In fact, the close similarity of the structural organization for the genes of all members of the secretin family of peptides suggests that they originated from an identical ancestral gene by duplication. Although the greatest levels of PACAP peptide are found in the brain, PACAP is also found in nerve fibers and in myenteric and submucous ganglia of the GI tract (200–202). PACAP-27 is found in the intestine, whereas PACAP-38 is primarily nonenteric (203,204). PACAP containing enteric nerve fibers have been found in the stomach and are colocalized with type I PACAP receptors (PAC1) on enterochromaffin-like (ECL) cells. PACAP is localized in the enteric neural plexus of the GI tract, where it is believed to be a mediator of gastric acid secretion and intestinal motility. There are three receptors with a high affinity for PACAP; these are the classical VIP receptor (VPAC1), PAC1, and VPAC2. PAC1 has affinity for only PACAP-38 and PACAP-27, whereas the VPAC receptors show similar affinities for PACAP and VIP (205,206). The PAC1 receptor is found on gastric ECL cells, and the VPAC1 is found on gastric SRIFproducing D cells and chief cells (207). PACAP has been shown to stimulate ECL cell proliferation (208). Centrally administered PACAP also stimulates gastric acid and pepsin secretion in rats (209). Gut smooth muscle also contains VPAC1 and PAC1 receptors. These findings suggest that endogenous PACAP is involved in the neural regulation of gastric acid secretion, relaxation of colonic smooth muscle, and stimulation of pancreatic secretion (210,211). In humans,

PACAP has been shown to stimulate insulin and glucagon secretion (212), and IV infusion of PACAP will stimulate peptide YY (PYY) secretion in dogs (213). Peptide Histidine Isoleucine/Peptide Histidine Methionine PHI and PHM are cosynthesized with VIP from a common precursor, and they are colocalized with VIP in the GI tract (214). There is a carboxy-extended variant of PHI called peptide histidine valine (PHV) (215). The function of PHV is unknown. PHI exerts many of the effects of VIP; however, the potency of PHI can be one to three orders of magnitude less than those of VIP (216). PHI has been considered a weak agonist for VIP. In the GI tract, PHI, like VIP, enhances water and electrolyte transport in the jejunum (216,217), and PHI stimulates pancreatic glucagon secretion (218). A PHI/PHM-preferring receptor has been cloned from the goldfish (219).

NEUROTENSIN During the isolation of substance P from bovine hypothalamic extracts, Carraway and Leeman (220,221) discovered a peptide that potently stimulated vasodilation of exposed cutaneous tissue pieces in rats. This new peptide was sequenced and shown to consist of 13 amino acids. It was named neurotensin because it is found in the brain and exerts hypotensive activity. Neurotensin was later shown in intestinal enteroendocrine “N” cells and neural elements where it has endocrine, paracrine, and neural activity. The neurotensin gene codes for neurotensin and for another smaller peptide structurally related to neurotensin, called neuromedin N. In the GI tract, neurotensin influences gastric acid, pancreatic exocrine and biliary secretion, motility, and intestinal mucosal growth.

Structure Neurotensin is a tridecapeptide—the sequences for human, bovine, canine, porcine, and rat neurotensin are identical (Fig. 5-12) (222). The carboxy-terminal hexapeptide sequence is highly conserved in different species and is the locus of biological activity. Neuromedin N is a hexapeptide structurally related to neurotensin that is derived from the same precursor peptide (Figs. 5-12 and 5-13) (223). The precursor peptide for neurotensin (i.e., preproneurotensin) consists of 170 amino acids and contains both the neurotensin and neuromedin N sequences near the carboxy terminus (see Fig. 5-13). The neuromedin N and neurotensin sequences are flanked by pairs of basic residues that serve as cleavage sites for the endoproteases, prohormone convertases (PCs). In tissues that express pro–neurotensin/neuromedin N, the three carboxy-terminal Lys-Arg sites are differentially processed, whereas the dibasic site upstream of the neuromedin N sequence is not cleaved. In the GI tract,

POSTPYLORIC GASTROINTESTINAL PEPTIDES / 131 Bovine/human/canine/porcine/rat NT

pGlu-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-Leu

Guinea pig NT

pGlu-Leu-Tyr-Glu-Asn-Lys-Ser-Arg-Arg-Pro-Tyr-Ile-Leu

Chicken NT

pGlu-Leu-His-Val-Asn-Lys-Ala-Arg-Arg-Pro-Tyr-Ile-Leu

Opossum NT

pGlu-Leu-His-Val-Asn-Lys-Ala-Arg-Arg-Val-Tyr-Ile-Leu

Amphibian xenopsin

pGlu-Gly-Lys-Arg-Pro-Trp-Ile-Leu

Porcine neuromedin N

Lys-Asn-Pro-Tyr-Ile-Leu

FIG. 5-12. Amino acid sequences of neurotensin (NT) from various species and related peptides. When compared with bovine NI, nonidentity in sequences is indicated by underlining.

Gastrointestinal Localization of Neurotensin In the GI tract, neurotensin is produced primarily in the ileal mucosa with smaller amounts in the jejunum, stomach, duodenum, and colon mucosa (Fig. 5-14) (229,230). Neurotensin is made in open-type enteroendocrine cells called “N” cells (230,231). Neurotensin has also been identified in the

1

20

140

Signal peptide

150 NmN

NmN–125

NmN

160

170

NT

T

NT

T

NT 1-8

myenteric plexus and is released from myenteric neurons in culture (232). Neurotensin found in myenteric neurons most likely functions as a neurotransmitter or neuromodulator. Developmental studies have shown that neurotensin/ neuromedin N expression is low in the fetal ileum, and then increases dramatically on postnatal day 3 in the rat (Fig. 5-15) (233,234). In the human fetal ileum, neurotensin/neuromedin N expression is low and increases dramatically in the adult (233). Interestingly, neurotensin/neuromedin N is transiently expressed in the human fetal colon at midgestation (weeks 16–22) and by 24 weeks of gestation; in newborn and adult specimens, neurotensin/neuromedin N expression is absent (234,235). The significance of these age-related changes in neurotensin/neuromedin N expression is unknown.

Neurotensin Receptors Three receptors for neurotensin have been isolated and characterized (236-239). These are the high-affinity NTS1 and the low-affinity nts2 and nts3. The nts1 and nts2 are G protein–coupled receptors, whereas the nts3 is not a G protein– linked receptor and has a gp95/sortilin structure. The NTS1 4000 Neurotensin concentration (ng/g)

processing of prepro–neurotensin/neuromedin N results primarily in the formation of neurotensin and a large peptide ending with the neuromedin N sequence at its carboxy terminal. It is called large neuromedin N. In the brain, processing of prepro–neurotensin/neuromedin N results in neurotensin and neuromedin N (224,225). Partial sequences of the carboxy terminal of neurotensin that contain five or more amino acids exhibit biological activity. The aminoterminal 10-amino-acid fragment is biologically inactive. There is a neurotensin-like hexapeptide, LANT-6, that is similar to neuromedin N, with an Ile residue substituted by an Asn in chicken LANT (226). Another peptide structurally related to neurotensin is called xenopsin, which is produced in the skin of the frog Xenopus laevis (227). The neurotensin gene has been cloned and is highly conserved among species (228). The rat neurotensin gene consists of 10.2 kb and includes four exons and three introns (228). The neuromedin N and neurotensin coding regions are tandemly arranged in exon 4.

5 dogs 3000

2000

1000 *

NT 9-13

0

FIG. 5-13. Schematic showing processing of the neurotensin/ neuromedin N precursor. Putative cleavage sites and products are shown. Tail peptide is a five-amino-acid peptide found in brain and intestinal extracts. NmN, neuromedin N; NT, neurotensin; T, tail peptide. (Modified from Shulkes and colleagues [252], by permission.)

Esophagus Fundus

Antrum Duodenum Jejunum

Ileum

Colon

Pancreas

Mucosa

FIG. 5-14. Distribution of neurotensin peptide in mucosal extracts of the dog gastrointestinal tract. *p < 0.05 versus esophagus, fundus, antrum, duodenum, ileum, colon, and pancreas. (From Doyle and colleagues [229], by permission.)

132 / CHAPTER 5 Fetal

Postnatal

Day 18 20

1

3

7

10 14 18 21 25 28 60 120

NT/N

Cyclophilin

A

NT/N mRNA/Cyclophilin mRNA (Relative densitometric units)

2 Ileum

1

0 18 20

1 3

60

7 10 14 18 21 25 28

Fetal

B

120

Postnatal Days

FIG. 5-15. Northern blot analysis of neurotensin/neuromedin N (NT/NM) mRNA levels in rat ileum during development. (A) Northern blot containing 10 µg poly (A)+ RNA per lane from the ileum. The Northern blot was probed sequentially with a rat NT/NM and a rat cyclophilin complementary RNA probe. (B) NT/NM mRNA abundance was quantitated in the ileum by scanning densitometric analysis of the autoradiogram. Values are expressed as relative densitometric units after correcting for differences in cyclophilin mRNA abundance in the same RNA preparations. (Modified from Evers and colleagues [233], by permission.)

receptor is expressed in the small and large intestine and in the liver. The nts2 is a low-affinity receptor that is structurally similar to NTS1 and is recognized by levocabastine; therefore, this site corresponds to what was called previously the neurotensin acceptor site. Amino acid substitutions in the second transmembrane domain may underlie the decreased affinity of this receptor when compared with NTS1. nts2 is not expressed in the GI tract. For intracellular signaling pathways, it is well established that neurotensin can stimulate MAPK phosphorylation and activation in different cell types including nontransformed and transformed human colonic epithelial cells (240,241) and pancreatic carcinoma cell lines (240,242) that express NTR1 (243,244). Neurotensin also stimulates the formation of inositol-1,4,5-trisphosphate (IP3), increases intracellular calcium (245,246), and activates extracellular signal-regulated kinase (ERK)1/2 in pancreatic MIA PaCa-2 cells (240), nontransformed human colonic epithelial NCM460 cells (241), and colonic adenocarcinoma HT29 cells (240). In colonic epithelial cells, neurotensin-stimulated MAPK activation depends on activation of small GTPases Ras (241). Binding of neurotensin to NTR1 in human colonic cells also transactivates epidermal

growth factor receptor, resulting in activation of MAPK (247). Studies in PC3 cells show that NTR1 is linked to Gq/11 because neurotensin stimulates phosphatidylinositol-specific phospholipase C (PLC)–mediated formation of inositol phosphate and release of intracellular Ca2+ (248,249).

Actions on the Gastrointestinal Tract Neurotensin exerts a large array of activities in the body (250–252). Systemic administration of neurotensin results in vasodilation, cyanosis, increased vascular permeability, hyperglycemia, hypoinsulinemia, hyperglucagonemia, and stimulation of pituitary hormone release (growth hormone, prolactin, adrenocorticotropic hormone, luteinized hormone, follicle-stimulating hormone) (253). Neurotensin causes a vasoactive stimulation of histamine release; it stimulates pancreatic, gastric, and intestinal secretion, and influences smooth muscle contraction. Administration of neurotensin intravenously to laboratory animals and humans inhibits gastric acid secretion (254,255). One report indicates that the inhibitory action of neurotensin

POSTPYLORIC GASTROINTESTINAL PEPTIDES / 133 (Fig. 5-16) (261–266). Neurotensin alone is unlikely an endogenous effector of pancreatic secretion because intraduodenal administration of oleate increased neurotensin secretion much less than that required to produce an equal pancreatic secretion when neurotensin is given intravenously. As indicated for the inhibitory action of neurotensin in the regulation of acid secretion, neurotensin may act with other enteric factors to regulate pancreatic secretion. Neurotensin also influences esophageal and intestinal motility. In rats and humans, neurotensin can convert a fasting motor pattern to a fed pattern (267,268). Neurotensin appears to exert this effect via a neural mechanism because vagotomy, atropine, or hexamethonium treatment will block the effect of neurotensin on the activity front (269). Neurotensin also stimulates growth of normal of GI tissues including the pancreas, colon, and small intestine (Figs. 5-17, 5-18, and 5-19) (270–275).

on acid secretion is independent of the vagus but requires prostaglandin synthesis (255), whereas other laboratories indicate that neurotensin is ineffective on acid secretion in animals prepared with vagally denervated pouches or in humans with a prior vagotomy (254,256). The inhibitory action of neurotensin on acid secretion, together with the data that dietary fat will cause neurotensin secretion into the bloodstream (257), suggested that neurotensin is an enterogastrone. Although administration of medium doses of neurotensin (2.5–4 pmol/kg/min) will inhibit food-induced acid secretion in humans (258), the plasma neurotensin levels achieved with these infusions (~150 pM) are much greater than the levels measured after a meal. Furthermore, IV administration of neurotensin at doses that reproduced levels seen after a fatty meal or oleic acid failed to inhibit peptoneinduced acid secretion (259). Neurotensin must be given at much greater infusion rates to inhibit acid secretion. Together, these observations indicate that neurotensin is not an enterogastrone by itself, although it may act in concert with other enterogastrone candidates such as PYY, 5-HT, secretin, and GIP. Neurotensin does not inhibit histaminestimulated acid secretion, and neurotensin can also inhibit gastric motor activity (260). Neurotensin given alone has been shown to stimulate pancreatic bicarbonate and protein secretion in humans and dogs (261,262). Neurotensin can also enhance hormonal (i.e., CCK, secretin) and nutrient-induced pancreatic secretion

ID AA

1500

Protein

1000

100 Bicarbonate

500 0

0 ID AA + HCl

ID AA + HCl

10

IV NT (1 µg/kg/hr)

2000

150

1500

100

1000

50 0

Protein

500 0

Integrated protein output (mg/60 min)

Protein (mg/15 min)

Bicarbonate

NT

AA

HCI + HCl NT

AA + AA HCI + + HCl NT

6 4

Secretin

2 0

Bicarbonate (µEq/15 min)

200

8

AA + NT

(2 CU/kg/hr)

150

600

CCK-8

400

(500 ng/kg/hr)

Protein (mg/15 min)

2000

Bicarbonate (µEq/15 min)

IV NT (1 µg/kg/hr) 200

50

Dietary fat is the most potent secretagogue for release of neurotensin from the intestine (257,276). The levels of neurotensin increase from undetectable levels to ~20 to 50 pM when measuring extracted and concentrated plasma in a neurotensin immunoassay. Luminal glucose, amino acids, and saline are weak secretagogues for activation of neurotensin

Integrated bicarbonate output (mEq/60 min)

ID AA

Regulation of Neurotensin Secretion

200 0

FIG. 5-16. (Left) Pancreatic protein and bicarbonate outputs in response to intraduodenal (ID) infusion of amino acids (AA) alone or plus HCl (ID AA + HCl) with and without intravenous neurotensin (NT) in dogs prepared with Herrera-type pancreatic and gastric fistulas. (Right) Comparison of integrated (60 minutes) bicarbonate and protein responses with NT alone; AA versus AA + NT; HCl versus HCl + NT; and AA + HCl versus AA + HCl + NT. *p < 0.05 compared with basal levels. **p < 0.05, significance differences among groups. (Modified from Sakamoto and colleagues [262], by permission.)

134 / CHAPTER 5

Weight, g kg−1

25

*

*

12.5

NEUROPEPTIDE Y

0 150 DNA, mg kg−1

* *

*

75

0

Protein, g kg−1

3

* *

1.5

0

cells (283,284). A role for PKC-α and -δ is suggested because inhibition of PKC-α and -δ can block 4-phorbol12myristate-13-acetate (PMA)-stimulated neurotensin secretion (283). Furthermore, studies show that protein kinase D (PKD) activation plays a role in neurotensin secretion (285).

0

33 100 Neurotensin, µg kg−1

300

FIG. 5-17. Effect of chronic administration of neurotensin on weight, DNA, and protein contents of entire small intestine from same animals shown in Figure 5-18. There are doserelated increases in weight, DNA, and protein contents of entire small intestine after neurotensin treatment. *p < 0.01 compared with control groups. (Modified from Wood and colleagues [273], by permission.)

release (276). In rats, fatty acids and alcohols will cause neurotensin release (277). The minimal fatty acid chain length to cause neurotensin release is four carbons (277). Bombesin, an amphibian skin peptide that is structurally related to the mammalian gastrin-releasing peptide, will stimulate neurotensin secretion in the rat (278). In dogs, resection of the ileum abolishes nutrient-induced neurotensin release (279), indicating that the ileum is the primary source of circulating neurotensin after luminal nutrients. In humans, vagotomy did not affect neurotensin release (256). Release of neurotensin has also been studied in vitro using isolated-perfused organs and enriched intestinal enteroneuroendocrine N-cell populations. In the isolated perfused ileum, bombesin, carbachol, catecholamines, and substance P stimulated neurotensin release (280–282). In vitro cultures of ileal neurotensin cells showed that bombesin- and epinephrine-stimulated neurotensin release can be antagonized by SRIF and carbachol. Intracellular signaling mechanisms underlying regulation of neurotensin secretion have been examined using a human carcinoid cell line called BON that produces neurotensin and chromogranin A, a marker for endocrine/enteroendocrine

Neuropeptide Y (NPY)NPY belongs to the pancreatic polypeptide-peptide YY-neuropeptide Y (PP-PYY-NPY) family of peptides, also called the PP-fold family of peptides. NPY was first characterized from porcine brain extracts using a method that identified amidated peptides at the carboxy terminal (286). NPY is widely distributed in central and peripheral neurons, especially in the GI tract. NPY is one of the most potent orexigenic peptides isolated to date, and brain levels of NPY are changed in conditions where regulation of energy balance is altered including anorexia, bulimia nervosa, and diabetes. In the GI tract, NPY is found within intrinsic neurons of the submucosal and myenteric plexuses and in nerve fibers in the mucosa, submucosa, and muscle layers. NPY exerts primarily inhibitory effects on gut motility, blood flow, and intestinal secretion.

Structure Like PP and PYY, NPY is a carboxy-terminal amidated 36-amino-acid peptide (Fig. 5-20). The sequences for rat, rabbit, guinea pig, and human NPY are identical (287). The NPY gene has been cloned from several species including the rat and human (288–294). The NPY gene consists of four exons with the NPY sequence encoded in the second exon.

Neuropeptide Y Receptors The NPY receptor and the Y receptor subtypes are described later in this chapter (see Peptide YY).

Gastrointestinal Localization of Neuropeptide Y NPY has been localized in neurons of the central, peripheral, and enteric nervous systems of several species, including humans (295–300). The richest peripheral source of NPY is the GI tract (301). NPY and nitric oxide synthase are colocalized in all layers of the human gut wall (Fig. 5-21) (302). Studies on the distribution of NPY in the GI tract of several species show the greatest NPY concentration in the lower esophageal sphincters of the rat and pig and in the pylorus of the guinea pig (297). Immunocytochemical studies show NPY immunoreactivity within nerve cell bodies in the submucosal and myenteric plexuses, thus demonstrating the intrinsic origin of NPY. NPY is also found within nerve fibers in the mucosa, submucosa, and muscle layers of the

+40

First quarter

Second quarter

+40

Weight

**

DNA **

Protein

+20

% Change from control

**

0

+20

**

**

** *

0

−10

−10 Third quarter

* ** **

+20

0 −10

**

**

**

+40

**

**

Fourth quarter

+40

** **

+20

**

*

0 0

33

100

−10 300 0 Neurotensin, µg kg-1

33

100

300

FIG. 5-18. Effect of chronic administration of neurotensin on weight, DNA, and protein contents of proximal to distal quarters of rat small intestine. Saline or neurotensin in gelatin was injected subcutaneously in indicated doses every 8 hours for 5 days in groups of rats. Results are means ± standard error of the mean percentage change compared with control values for each corresponding quarter of small intestine obtained from rats given saline in gelatin. Values for weights of proximal to distal quarters of small intestine from control groups were 5.44 ± 0.16, 5.45 ± 0.15, 5.01 ± 0.09, and 4.83 ± 0.13 g/kg body weight. Values for DNA contents (proximal to distal quarters) from control rats were 27.6 ± 0.6, 24.1 ± 0.5, 22.6 ± 1.2, and 26.6 ± 0.8 mg/kg body weight. Values for protein content (proximal to distal quarters) from control groups were 579 ± 72, 544 ± 25, 499 ± 69, and 476 ± 39 mg/kg body weight. There were dose-related increases in weight, DNA, and protein contents of small intestine after neurotensin treatment. *p < 0.05; **p < 0.01 compared with control groups. (Modified from Wood and colleagues [273], by permission.)

3

*

(9)

4 2

0

(10)

(10)

(10)

(9)

0

25

NT 100

NT 300

NT 600

10

(10)

(10)

(10)

(9)

0

10 8 6 4 2

NT 600

15

12

NT 300

*

*

NT 100

*

*

Control

20

Protein content (mg/kg body weight)

14

Control

RNA content (mg/kg body weight)

6 NT 600

(10)

8

NT 300

NT 600

(10)

10

NT 100

NT 300

(10)

12

Control

NT 100

1

DNA content (mg/kg body weight)

2

Control

Mucosal weight (g/kg body weight)

*

5

*

14 *

(10)

(10)

(10)

(9)

0

FIG. 5-19. (Top) Colonic-mucosal weights and DNA contents, and (bottom) colonic-mucosal RNA and protein contents of rats given neurotensin (NT; 100, 300, 600 µg/kg) administered subcutaneously in gelatin every 8 hours for 10 days, compared with rats given saline (control) for 10 days. *p < 0.05 versus control. (Modified from Evers and colleagues [271], by permission.)

136 / CHAPTER 5 Porcine PYY Tyr-Pro-Ala-Lys-Pro-Glu-Ala-Pro-Gly-Glu-Asp-Ala-Ser-Pro-Glu-Glu-Leu-Ser-Arg-Tyr-Tyr-Ala-Ser-Leu-ArgHis-Tyr-Leu-Asn-Leu-Val-Thr-Arg-Gln-Arg-Tyr-NH2

Porcine PP Ala-Pro-Leu-Glu-Pro-Val-Tyr-Pro-Gly-Asp-Asp-Ala-Thr-Pro-Glu-Gln-Met-Ala-Gln-Tyr-Ala-Ala-Glu-Leu-ArgArg-Tyr-Ile-Asn-Met-Leu-Thr-Arg-Pro-Arg-Tyr-NH2

Porcine NPY Tyr-Pro-Ser-Lys-Pro-Asp-Asn-Pro-Gly-Gly-Asp-Ala-Pro-Ala-Glu-Asp-Leu-Ala-Arg-Tyr-Tyr-Ser-Ala-Leu-ArgHis-Tyr-Ile-Asn-Leu-Ile-Thr-Arg-Gln-Arg-Tyr-NH2

FIG. 5-20. Amino acid sequences for porcine peptide YY (PYY), pancreatic polypeptide (PP), and neuropeptide Y (NPY). When compared with PYY, nonidentity in sequences is indicated by underlining.

Distribution of NPY in rat gastrointestinal tract

pmol/g

60

30

0

us

ag

h op

Es

A

h

S

LE

ac

m

o St

lor

Py

us

pe

Up

um leum un li ina m r Te

ej rj

lon

Co

Distribution of NPY in muscle layers of porcine gastrointestinal tract

pmol/g

140

70

0

us ES dus rum rus m I m II III um um um um um um ing rse ing oid tum L n nt ylo nu u um un un ile ile ile ec nd ve nd m c Fu A P de den en jej r jej per id nal Ca sce ans sce Sig Re o o d id M mi A Tr De r Du Du Duo M owe Up Te L

ag

h op

Es

B FIG. 5-21. (Top) Distribution of neuropeptide Y (NPY) immunoreactivity along the length of the rat and (bottom) pig gastrointestinal tracts. Values shown are in pmol/g wet weight and represent the mean of six rats and four pigs ± standard error of the mean, respectively. LES, lower esophageal sphincter. (From Allen and colleagues [297], by permission.)

POSTPYLORIC GASTROINTESTINAL PEPTIDES / 137 GI tract (301,303-305). Within the submucosa, NPY is also found in cholinergic secretomotor neurons (306,307). A portion of the NPY immunoreactivity is extrinsic in origin because NPY disappears around blood vessels after either surgical or chemical sympathectomy (303,305,308). Colocalization experiments have shown that peripheral NPY is found in extrinsic, postganglionic, sympathetic-catecholaminergic neurons (300,301,305,309–311). In this case, NPY may function as a modulator of sympathetic transmission. In the rat and mouse, NPY is coexpressed with VIP in stomach and intestinal myenteric neurons (311,312). NPY is also produced in the rodent and human endocrine pancreas (313,314).

The mechanisms that regulate the cyclic pattern of motilin secretion and the exact stimulus for the release of motilin are unknown. The physiologic action of motilin is mediated by motilin receptors located on enteric neurons, ultimately leading to activation of muscarinic cholinergic neurons in the gastric antrum. The actions of motilin can be mimicked by the macrolide antibiotic, erythromycin, and a group of motilin agonists known as motilides. The motilides may be useful clinically as prokinetic agents for treatment of patients with delayed gastric emptying and impaired colonic motility.

Structure Secretion Because NPY is a neural peptide, its detection in the systemic circulation most likely will represent measurement of spillover from neural sources (e.g., nerve endings) into the general circulation. For example, stimulation of the vagal nerve in the isolated perfused pig pancreas causes a dramatic release of NPY into the venous effluent, most likely from postganglionic parasympathetic nerves (315).

Actions In the GI tract, NPY affects blood flow, motility, and intestinal secretion (315,316). NPY is a potent inhibitor of intestinal and pancreatic blood flow (317–319). Direct intraarterial administration of NPY to the cat colon causes a dosedependent and persistent vasoconstriction of the splanchnic circulation (317). NPY is approximately 25 times more potent than norepinephrine (317). NPY can also slow jejunal and colonic motility (317). NPY may inhibit colonic motility by an inhibition of excitatory enteric ganglia, as well as by a direct action on colonic smooth muscle in cats (320). NPY, like PYY, has been shown to inhibit fluid and electrolyte secretion from the small intestine (321–323). NPY may also act in the endocrine pancreas to regulate insulin secretion in a paracrine manner (313,314,324).

MOTILIN Motilin is a 22-amino-acid peptide that was characterized from extracts of the pig small intestine based on its ability to stimulate stomach myoelectric activity and pepsin secretion. It is called motilin because it stimulates gut motility. Motilin is produced in enteroendocrine “M” or “Mo” cells of the mucosal epithelium in the upper small intestine. Plasma motilin levels increase during the interdigestive period in a cyclic fashion, simultaneously with phase III contractions of the migrating myoelectric complex (MMC) in the stomach and upper small intestine. Abundant findings indicate that this increase in motilin secretion triggers initiation of phase III contractions of the migrating motor complex.

Motilin is a linear 22-amino-acid peptide (Fig. 5-22) (325,326). The prepromotilin sequence consists of 114 amino acids and includes a 25-amino-acid signal peptide, followed immediately by the motilin sequence, a dibasic cleavage sequence, and a variable (~65–70-amino-acid) carboxyl-terminal extension peptide. Like other GI peptides, prepromotilin is processed to the mature form—that is, the 22-amino-acid sequence, before it is released into the general circulation. Both the amino and carboxyl terminals of motilin are essential for full activity because neither amino- or carboxyl-terminal fragments stimulate gastric motor activity (327). Interestingly, substitution of the first residue in motilin will decrease activity approximately 300-fold (328), whereas substitution of residues 12, 13, 14, or 15 has marginal effects on motilin activity (329,330). The motilin gene consists of five exons separated by four introns and spans approximately 9 kb (331,332). Motilin is produced by exons 2 and 3.

Motilin Receptor The motilin receptor was identified and cloned from human stomach specimens (333). The motilin receptor is a heterotrimeric guanosine triphosphate–binding protein (G protein)– coupled receptor. Its amino acid sequence is 54% identical to the human receptor for the synthetic growth hormone secretagogues and the recently discovered stomach hormone, ghrelin. As expected, erythromycin, the macrolide antibiotic, binds to the motilin receptor. The motilin receptor is also found in enteric neurons of the duodenum, jejunum, and colon.

Distribution Motilin is primarily a duodenal hormone and is localized in enteroendocrine “M” or “Mo” cells. Motilin is also produced at lower levels along the entire length of the GI tract (334–338). In the cat GI tract, motilin mRNA levels are greatest in the duodenum and lowest in the colon; motilin is not detected in the stomach (339).

138 / CHAPTER 5 Human Phe-Val-Pro-Ile-Phe-Thr-Tyr-Gly-Glu-Leu-Gln-Arg-Met-Gln-Glu-Lys-Glu-Arg-Asn-Lys-Gly-Gln

Monkey Phe-Val-Pro-Ile-Phe-Thr-Tyr-Gly-Glu-Leu-Gln-Arg-Met-Gln-Glu-Lys-Glu-Arg-Ser-Lys-Gly-Gln

Porcine Phe-Val-Pro-Ile-Phe-Thr-Tyr-Gly-Glu-Leu-Gln-Arg-Met-Gln-Glu-Lys-Glu-Arg-Asn-Lys-Gly-Gln

Horse Phe-Val-Pro-Ile-Phe-Thr-Tyr-Ser-Glu-Leu-Gln-Arg-Met-Gln-Glu-Lys-Glu-Arg-Asn-Arg-Gly-Gln

Sheep Phe-Val-Pro-Ile-Phe-Thr-Tyr-Gly-Glu-Val-Gln-Arg-Met-Gln-Glu-Lys-Glu-Arg-Tyr-Lys-Gly-Gln

Bovine Phe-Val-Pro-Ile-Phe-Thr-Tyr-Gly-Glu-Val-Arg-Arg-Met-Gln-Glu-Lys-Glu-Arg-Tyr-Lys-Gly-Gln

Canine Phe-Val-Pro-Ile-Phe-Thr-His-Ser-Glu-Leu-Gln-Lys-Ile-Arg-Glu-Lys-Glu-Arg-Asn-Lys-Gly-Gln

Feline Phe-Val-Pro-Ile-Phe-Thr-His-Ser-Glu-Leu-Gln-Arg-Ile-Arg-Glu-Lys-Glu-Arg-Asn-Lys-Gly-Gln

Rabbit Phe-Val-Pro-Ile-Phe-Thr-Tyr-Ser-Glu-Leu-Gln-Arg-Met-Gln-Glu-Arg-Glu-Arg-Asn-Arg-Gly-Gln

Guinea Pig Phe-Val-Pro-Ile-Phe-Thr-Tyr-Ser-Glu-Leu-Arg-Arg-Thr-Gln-Glu-Arg-Glu-Gln-Asn-Arg-Leu-Arg

Chicken Phe-Val-Pro-Phe-Phe-Thr-Gln-Ser-Asp-Ile-Gln-Lys-Met-Gln-Glu-Lys-Glu-Arg-Asn-Lys-Gly-Gln

FIG. 5-22. Motilin amino acid sequences for several species. Nonidentity in sequences is identified by underlining.

Actions on the Gastrointestinal Tract The primary physiologic role of motilin is the stimulation of phase III contractions that originate during the interdigestive period in the antroduodenal region of the gut (340). Motilin exerts marginal effects on postprandial intestinal activity (341). During the interdigestive period, there is a cyclic increase of GI motility that consists of three phases and occurs at intervals of approximately 80 to 100 minutes (Fig. 5-23). Phase I is a period of quiescence lasting about 60 to 70 minutes; it is followed by phase II, which is a period of increased but irregular motor activity lasting approximately 20 to 30 minutes. Phase II is followed by phase III contractions and is characterized by intense and regular contractile activity that lasts approximately 5 to 10 minutes. This contractile activity starts in the stomach/lower esophageal sphincter and moves down the small intestine. As the phase III front reaches the distal small intestine, a new phase III is initiated in the stomach. The function of the MMC is thought to serve a housekeeping role by clearing the stomach and intestine of luminal secretions and contents during the interdigestive period.

Abundant evidence supports the idea that motilin is responsible for the initiation of phase III contractions. The increase in plasma motilin levels coincides with the increase in MMC activity. Other studies show that immunoneutralization of systemic motilin interrupts phase III contractile activity in the proximal gut but has no effect on the motility pattern of the distal small intestine (342,343). These findings support a role for motilin in initiation of phase III contractions in the proximal but not the distal gut. Although there is some argument regarding the role of motilin in humans, findings show that the onset of phase III contractions and motilin secretion are also tightly coordinated (344). A direct relation between motilin secretion and phase III activity, however, is more variable in humans when compared with dog findings (345, 346). IV administration of motilin will induce premature onset of activity fronts (344). Motilin can also stimulate gastric pepsinogen and pancreatic enzyme secretion, gallbladder contraction, and contraction of the lower esophageal sphincter (347–353). These stimulatory effects of motilin resemble those seen during normal phase III activity, suggesting that the observed changes in pancreatic secretion and pepsin secretion during phase III of

Plasma motilin concentration, pg/ml

POSTPYLORIC GASTROINTESTINAL PEPTIDES / 139

500

Feeding 400

300

200

0

Gut motor pattern

Gastric antrum

Upper jejunum Mid intestine

Time intervals, 1 hr

FIG. 5-23. Changes in plasma motilin levels and gastrointestinal motor activity before and after feeding in a dog fasting 17 hours. This dog was prepared with three force transducers that were implanted on the gastric antrum 3 cm proximal to the gastroduodenal junction, on the upper jejunum 20 cm distal to the ligament of Treitz, and on the midintestine just midway between the ligament of Treitz and the terminal ileum. During the interdigestive state, plasma motilin levels increased at constant intervals in close association with the occurrence of episodes of strong contractions in the stomach. However, when bands of strong contractions (arrows) reached the midintestine, plasma motilin levels had decreased. (Modified from Itoh and colleagues [379], by permission.)

the MMC are caused by endogenous motilin. Evidence in dogs and humans shows that motilin can increase the gastric emptying rate (354). Exogenous motilin can be used clinically to treat diabetic gastroparesis or postvagotomy gastroparesis (355,356). Motilin also stimulates interdigestive blood flow in dogs that is apparently mediated by cholinergic and noncholinergic pathways (357). Interestingly, motilin and motilides will stimulate contractions of isolated colonic smooth muscle cells and can enhance propulsive motility of the colon (358–361). Although several studies have shown that motilin and macrolides stimulate gastric and small intestinal motility, there are contradictory findings on the effects of motilin on the colon. In the human, rabbit, and canine colon, motilin shows stimulatory actions (361–368), whereas other laboratories fail to show an effect in the human colon (369,370). The contractile activity of motilin is only partially inhibited by the muscarinic antagonist atropine in the rabbit, suggesting that motilin excites the rabbit colon by mechanisms independent of cholinergic motor nerves, as in the stomach (367). Motilin, at physiologic doses, can also stimulate insulin secretion via a vagal cholinergic muscarinic pathway in the dog (371).

Mechanism of Action The stimulatory action of motilin on phase III contractions is blocked by muscarinic-cholinergic antagonists, indicating that cholinergic neurons mediate motilin-stimulated phase III contractions (372–374). In addition, depletion of 5-hydroxytryptamine in nerve terminals or pharmacologic blockade of the type 3 5-hydroxytryptamine receptors hinders the stimulatory action of motilin on phase III contractions in the dog stomach (375), implying that 5-hydroxytryptamine neurons mediate the phase III effects of motilin.

Release In humans, plasma motilin levels increase after a meal, whereas in dogs, plasma motilin levels increase exclusively during the interdigestive period (376). Plasma motilin levels increase and decrease in a regular, cyclic pattern during the interdigestive period, that is, fasting (377–380). Motilin release occurs at regular intervals of 90 to 120 minutes and coincides with the onset of the phase III contractions of the

140 / CHAPTER 5 interdigestive myoelectric complex in the stomach and duodenum in dogs and humans (see Fig. 5-23) (379–383). Luminal administration of acid or alkali into the duodenum will increase plasma motilin levels in dogs (380,384), and dietary fat is a potent secretagogue for motilin release in humans but not dogs (382,383,385–387). Ingestion of a mixed meal prevents the regular cyclic increase in motilin levels (132, 379,388). The mechanism or the identity of the exact stimulus behind the cessation in the cyclic release of motilin and of the disruption of the MMC is unknown. A cholinergic neural mechanism is suggested for regulation of motilin secretion because the muscarinic antagonist, atropine, and the nicotinic antagonist, hexamethonium, prevent the cyclic increases of plasma motilin, and electrical vagal stimulation causes motilin secretion (372,389,390). Furthermore, carbachol-induced motilin secretion is blocked by atropine but not by hexamethonium, implying that muscarinic receptors mediate motilin secretion. Three to six weeks after truncal vagotomy, plasma motilin levels and alterations seen during the MMC resembled those before surgery, suggesting that enteric cholinergic neurons are not involved in motilin secretion in dogs (391,392). IV administration of secretin and PP inhibit motilin secretion, whereas bombesin stimulates motilin release (386,393,394). Interestingly, exogenous motilin can stimulate secretion of endogenous motilin via muscarinic receptors on motilin cells via preganglionic pathways involving 5-hydroxytryptamine 3 receptors in the dog (395).

Pathophysiology Plasma motilin levels have been measured in patients with a variety of GI conditions. In pregnant subjects or patients with constipation and idiopathic gastroparesis, decreased plasma motilin levels were measured (396–400). In surgical patients with normal gut motility and patients with irritable bowel syndrome or infantile colic, plasma motilin levels are increased (401–405). In addition, plasma motilin levels are increased in some diabetic subjects (406) and patients with ZES (407).

Erythromycin and Motilides The term motilide is derived from the words “motilin” and “macrolide,” indicating that the macrolide antibiotics, including erythromycin, are motilin agonists and bind to the motilin receptor. The macrolide antibiotic erythromycin given to either dogs or humans will cause contractions of the GI tract that resemble phase III contractions (408–410). Motilides exert all of the effects of motilin, causing smooth muscle contraction and activation of cholinergic muscarinic pathways. Erythromycin has been shown to bind to the cloned motilin receptor (333). Because motilides act at subantibiotic doses and are resistant to degradation, they are potentially useful clinically as prokinetic agents for the stomach and colon, especially for patients with diabetic gastroparesis.

PEPTIDE YY PYY is a 36-amino-acid peptide hormone that is structurally similar to two other GI peptides, PP and NPY. This family of peptides is called the PP-PYY-NPY family of peptides, or the PP-fold family of peptides. All 3 peptides are 36 amino acids in length with an amidated carboxy terminus. PYY is produced primarily in enteroendocrine “L” cells in the intestinal epithelium of the ileum-colon and is a candidate enterogastrone because PYY is secreted into the systemic circulation in response to dietary fat and is a potent inhibitor of gastric acid secretion. PYY can also inhibit pancreatic exocrine secretion, gastric emptying, and intestinal transit. Because of its potent antisecretory action, PYY may be an endogenous inhibitor of diarrhea. Based on this array of inhibitory actions in the GI tract, PYY is called an “ileal brake.” The primary role of an ileal brake substance is to enhance intestinal digestion and absorption of ingested nutrients. More recently, a natural variant of PYY, PYY (3-36), has been shown to be a potent inhibitor of food intake in experimental animals and humans.

Structure PYY is a 36-amino-acid peptide that is structurally related to 2 other pancreatic-gut peptides, PP and NPY (see Fig. 5-20) (286,411,412). Together, they compose the PP-fold family of peptides, and with the fish peptide Y (PY), they share a common hairpin-like three-dimensional structure called the PP-fold (412). It is called PYY because it has tyrosine residues at the amino and carboxy terminals. “Y” is the chemists’ shorthand for the tyrosine residue. Another endogenous PYY variant, PYY (3-36), occurs in the intestine and general circulation (413–415). The PYY gene has been cloned from several species including the rat and human (291–294,416–418). The conserved structural organization of the PYY, NPY, and PP genes suggests that each gene is derived from duplication of a common ancestral gene. Prepro-PYY consists of a signal peptide followed by the 36-residue active peptide, a cleavage sequence, Gly-Lys-Arg, and a flanking peptide at the carboxy terminal (290,419). During processing to the mature form, the precursor is cleaved at the carboxy terminal by a PC, and the Lys-Arg sequence is excised by a carboxypeptidase. The carboxy-terminal tyrosine is amidated and is essential for biological activity (420).

Gastrointestinal Localization of Peptide YY and Developmental Appearance of Intestinal Peptide YY PYY is found in “L” cells in the mucosal epithelium of the terminal ileum, colon, and rectum (Figs. 5-24 and 5-25) (421–430). PYY is not found in the muscle layer. Marginal amounts of PYY are found in the stomach antrum, proximal small intestine, and endocrine cells of the pancreas. In the lower intestine, PYY is coproduced with proglucagon (427);

800

Colon

POSTPYLORIC GASTROINTESTINAL PEPTIDES / 141 Rat

†

Distal ileum

Mid-ileum

Mucosa Muscle

*†

Colon

*

Dog

1000

500

*†

Ileum-3

Ileum-2

1500

Jejunum-3

PYY (ng/g tissue)

2000

Fundus

Antrum

200

Jejunum

Duodenum

400

Ileum-1

PYY (ng/g tissue)

600

* †‡

*†

*

FIG. 5-24. Distribution of peptide YY (PYY) immunoreactivity in the digestive tract of the rat (full thickness) and dog (mucosal and muscle layers). In the dog, the duodenum, jejunum, and ileum were divided into three segments of equal length. The colon was divided into two segments. (Top) *p < 0.05 versus antrum, fundus, duodenum, jejunum, and midileum. †p < 0.05 versus distal ileum. (Bottom) *p < 0.05 versus antrum, fundus, duodenum, proximal, and distal jejunum; †p < 0.05 versus muscle layer of respective region; ‡p < 0.05 versus distal jejunum, proximal, and midileal mucosal layers. (Modified from Greeley and colleagues [423], by permission.)

in the pancreas, PYY can coreside with glucagon or PP (421,431,432). PYY is also detected in neuronal elements of the proximal GI digestive tract of the rat, cat, ferret, and pig (430). PYY is found in nerve cell bodies and nerve fibers of the canine stomach and intestinal myenteric plexus, and in the intestinal submucosal plexus (429). PYY is the first major gut peptide to appear during development in the mouse colon (433). On embryonic day 15.5, colonic PYY expression appears, followed by intestinal expression of glucagon, CCK, substance P, 5-HT, secretin, neurotensin, gastrin, and SRIF between embryonic days 16.5 and 18.5. In the rat, expression of PYY occurs on embryonic day 17 in the ileum and colon (417,434). Both PYY mRNA and peptide are detected initially at 17 days of gestation; their levels continue to increase during the postnatal period. The increase in intestinal PYY expression during the first weeks of life may be activated by ingestion of fat contained in the mothers’ milk. Dietary fat is a potent secretagogue for release of PYY (435), and fat is abundant

in rat milk, ranging from 22% in colostrum to 9% in late milk (436). After weaning, there is a coordinated decrease in levels of PYY mRNA and PYY peptide when pups switch from breast milk to an adult diet. Interestingly, the pattern of rat intestinal PYY gene expression agrees with the pattern of plasma PYY levels in human neonates (437). In newborn cord blood, plasma PYY levels are greater when compared with plasma PYY levels in healthy fasting adults, and plasma PYY levels increase during the first 12 days after birth in neonates fed with either breast milk or formula. These observations suggest that intestinal PYY gene expression is controlled by dietary nutrients, and that changes in the intestinal PYY gene expression have a role in the adaptation of the bowel to the changes in dietary intake. PYY has been detected in the fetal mouse pancreas at embryonic day 9.5, and it is detected in each of the four islet cell types (433). In the rat pancreas, PYY expression is first detected on day 15 of gestation and increases to its maximal on day 18 (417). At this time, pancreatic PYY mRNA levels

142 / CHAPTER 5

FIG. 5-25. Flask-shaped peptide YY cell of the open type in the rat colonic mucosa (original magnification × 360). (See Color Plate 1.) (Modified from Jeng and colleagues [495], by permission.)

are sevenfold greater than in the fetal and adult colon and 90-fold greater than those levels in the adult pancreas, in contrast with the intestine. Pancreatic PYY mRNA levels decrease dramatically after birth. Pancreatic PYY peptide levels have not been characterized during development. Although the functional significance of PYY in the pancreas during development is unknown, PYY may participate in the regulation of pancreatic and GI functions by endocrine and paracrine actions. PYY is measurable in the adult canine pancreas with the greatest levels in the right lobe and head of the pancreas, and PYY is released from the canine pancreas by vagal stimulation (438). The early development pattern of intestinal PYY gene expression suggests a role for PYY in the development of the intestine and in the regulation of epithelial cell replication or differentiation. The initiation of PYY gene expression comes before the appearance of mature morphologic characteristics in the colonic mucosa (i.e., formation of the crypts of Lieberkuhn). In addition, cells expressing PYY mRNA in the developing and mature colon are located at the base of the crypts adjacent to populations of immature cells and goblet cells undergoing differentiation (434). PYY may have a role in the regulation of the stomach and pancreas during the developmental period. Although gastric acid and pancreatic secretion are low during the postnatal period and do not achieve adult levels until weaning (439), PYY may have a role in their regulation because it is a potent inhibitor of gastric acid and pancreatic exocrine secretion in adult animals.

Peptide YY Receptors PYY, NPY, and PP bind to a family of G protein–linked receptors, called Y receptors, belonging to the rhodopsinlike superfamily (class 1) of receptors (412,440). There are five different Y receptor subtypes cloned from mammals (Y1, Y2, Y4, Y5, and y6; Table 5-1). The y6 receptor is given the lowercase designation because it encodes for a truncated receptor. All Y receptor subtypes are expressed in either the small or large intestine with specific distributions (440). Y1, Y2, and Y5 bind NPY and PYY preferentially, whereas Y4 binds PP preferentially. The pharmacologic profile of y6 is not agreed on (412,441). The characterization of the responses to PYY, NPY, and PP may reflect the result of the peptide’s activity on local neuronal receptors, as well as direct activity at the cell level on the targeted tissue. Y1 receptors are expressed only in nonepithelial colonic cells, whereas Y2 and Y4 receptors are found in epithelial and nonepithelial cells of either the small or large intestine. Expression of the Y5 receptor is restricted to epithelial crypts of the small intestine and nonepithelial cells of the colon. The Y1, Y2, Y4, Y5, and y6 receptors are coupled to inhibitory G proteins (Gi) and cause inhibition of cAMP synthesis (442–446). Activation of the Y1 receptor is coupled to MAPK in gut epithelial cells (IEC-6 [447]). Y1, Y2, Y4, and Y5 receptors also couple to PLC to cause release of Ca2+ from intracellular stores (442,443,445). The Y1 receptor is found in the gut and mediates an inhibition of fluid secretion in the intestine.

POSTPYLORIC GASTROINTESTINAL PEPTIDES / 143 TABLE 5-1. Cloned neuropeptide Y receptors with ligand-binding profiles, example locations, and example functions Receptor type Y1 Y2

Ligand-binding profile NPY ≈ PYY ≈ [Leu , Pro ] NPY > NPY2-36 > NPY3-36 ≥ PP > NPY13-36 NPY ≥ NPY2-36 ≈ NPY3-36 ≈ NPY13-36 >> [Leu31, Pro34] NPY 31

34

Y4

PP > PYY ≥ NPY > NPY2-36

Y5

NPY ≈ PYY ≈ NPY2-36 > hPP > [D-Trp32] NPY > NPY13-36 > rPP 1. NPY ≈ PYY ≈ [Leu31, Pro34] NPY >> PP 2. PP > [Leu31, Pro34] NPY > NPY ≈ PYY

y6

Example of locations

Example of functions

Gut, brain, heart, kidney

Vasoconstriction, anxiolysis; antisecretory in intestine

Brain, low levels in peripheral tissues

Presynaptic inhibition of neurotransmitter release, regulation of GI motility Inhibition of pancreatic secretion, GI motility Increased food intake

Small intestine, colon, prostate, and CNS Brain areas involved in food intake Small intestine and adrenal (mouse)

Unknown

CNS, central nervous system; GI, gastrointestinal tract; NPY, neuropeptide Y; PP, pancreatic polypeptide; PYY, peptide YY. Modified from Adrian TE. Peptide YY. In: Encyclopedia of hormones, Vol. 3. San Diego: Academic Press, 2003;161–170.

The Y4 receptor is found in the pancreas and mediates an inhibition of pancreatic exocrine secretion. On rabbit gastric circular smooth muscle cells (441), Y1, Y2, and Y4 receptors were shown to be negatively coupled to adenylyl cyclase by activation of Gi2, and Y2 and Y4 receptors were shown to be coupled to IP3-dependent (Ca2+)i release and activation of PLC-β by activation of Gq.

Actions on the Gastrointestinal Tract Studies in rats, dogs, and humans demonstrate that PYY is a general inhibitor of GI function. Based on its inhibitory actions on gastric acid secretion, gastric emptying, pancreatic secretion, and intestinal transit, PYY has been called an “ileal brake.” Infusion of PYY IV has been shown to inhibit gastric acid secretion in the dog, rat, rabbit, cat, and in humans (Fig. 5-26) (448–451). PYY can inhibit pentagastrin-stimulated, but not histamine and cholinergic-stimulated (i.e., bethanechol), gastric acid secretion in the dog (452). There is also evidence that PYY can decrease gastric acid secretion by blocking the cephalic phase of gastric acid secretion in the dog (453). In humans, PYY is a potent inhibitor of the cephalic phase of gastric acid secretion, indicating that part of the enterogastrone action of PYY is mediated via neural pathways (454). Furthermore, the inhibitory actions of PYY on acid secretion in humans are independent of SRIF and gastrin secretion (454). In the cat, PYY inhibits pentagastrin-stimulated acid secretion by stimulation of SRIF secretion. In the dog, PYY (1-36) is more potent than PYY (336) and PYY (4-36), indicating that the complete sequence of PYY is essential for full expression of its inhibitory action on acid secretion (420). A study using isolated rat gastric ECL cells showed that PYY may inhibit gastric acid secretion by blocking gastrin-induced histamine release and Ca2+ entry via activation of the Y1 receptor (455). In addition, there is evidence that PYY acts centrally in the brain to regulate gastric acid secretion (456). PYY, given peripherally, will inhibit thyrotropin-releasing hormone–activated vagal efferent

cholinergic projections to the stomach and acid secretion. Furthermore, PYY injected into the cisterna magna and dorsal vagal complex (DVC) results in an inhibition of gastric transit and antral motility (457,458). Interestingly, administration of PYY into the medullary brainstem area of the vagus (dorsal motor nucleus [DMN]) causes a dose- and vagal-dependent stimulation of gastric acid secretion in rats (459). The DMN is referred to as the DVC and includes the area postrema, the nucleus of the solitary tract, as well as the DMN. The medullary region contains vagovagal circuits that communicate between the periphery—that is, the GI tract and central nervous system (CNS). PYY receptors have been identified autoradiographically in the DVC of rats, and systemic radiolabeled PYY can penetrate into the DVC (460). These neural PYY receptors may be activated by PYY released from the GI tract or the brain because PYY is expressed in the brainstem (425,426) and GI tract (423). This centrally PYY-activated inhibition of gastric acid secretion may be part of the ileal brake. IV administration of PYY also inhibits pancreatic exocrine secretion. PYY can decrease the stimulatory effects of CCK, secretin, and neurotensin on the pancreas (Fig. 5-27) (461). PYY can also inhibit the stimulatory effects of endogenous secretagogues that are released in response to ingestion or luminal instillation of nutrients (461–463). PYY inhibits pancreatic secretion of juice, bicarbonate, and pancreatic enzymes. In addition, physiologic levels of PYY can inhibit meal-induced pancreatic secretion, and treatment with antiPYY serum enhances pancreatic secretion in the rat (464). PYY (1-36), as well as PYY (3-36) and PYY (4-36), exerts inhibitory actions with similar potencies on pancreatic secretion in the dog (420). With guinea pig acini, PYY cannot inhibit CCK bombesin-stimulated amylase secretion; however, PYY can inhibit VIP- and forskolin-induced amylase release (465). PYY may exert its inhibitory actions on pancreatic secretion by multiple pathways. PYY may act via adrenergic pathways to inhibit pancreatic secretion because phentolamine plus proprandolol administration will prevent the inhibitory effects of PYY on pancreatic secretion (466). The delay in

144 / CHAPTER 5 Pentagastrin (0.5 µg/kg-h) 6 4 2 0 PYY (100 pmol/kg-h)

Gastric acid output (mEq/15 min)

6 *

4

*

2 0 PYY (200 pmol/kg-h) 6 4

*

*†

* *

2 0 PYY (400 pmol/kg-h) 6 *

4

*†

2

* *†

*†

0 B1 B2

15 30 45 60 75 90 105 120 135 150 165 180 Minutes

FIG. 5-26. Inhibition of pentagastrin-stimulated gastric acid secretion by peptide YY (PYY) given intravenously in dogs prepared with gastric fistulas. *p < 0.05 versus pentagastrin alone; †p < 0.05 versus 100 pmol/kg/hr dose of PYY. (From Jeng and colleagues [495], by permission.)

intestinal transit of luminal nutrients by PYY is dependent on a β-adrenergic pathway that acts via serotonergic and opioid pathways (467). Evidence also supports the idea that PYY acts via intrapancreatic neural pathways (468) and centrally to inhibit neurally mediated pancreatic secretion (469). In the rat, PYY has been shown to exert its inhibitory action on the exocrine pancreas through the DVC of the CNS (469). The area postrema is the major site of PYYmediated inhibition of CCK and secretin-stimulated pancreatic secretion (470). PYY may also regulate pancreatic exocrine secretion by its inhibition of CCK secretion because IV administration of PYY blocks release of CCK in response to intraduodenal infusion of a fatty acid in dogs (463). Administration of PYY will also block CCK release in humans (471). PYY inhibits gastric emptying and intestinal transit. Unabsorbed nutrients, especially fats in the lumen of the distal small intestine (ileum), will inhibit upper gut motility (472–476). Ileal perfusion of carbohydrates alone or with an amylase inhibitor delays gastric emptying (476). This negative-feedback mechanism of the lower intestine represents a major and fundamental control mechanism of upper

gut motility and is part of the ileal brake. Because PYY is localized in the lower intestine and because it can inhibit gastric emptying and intestinal transit (319,477,478), PYY has been proposed as an ileal brake factor because it has all of the properties of the ileal brake. There is additional evidence to indicate that PYY is the ileal brake (479–481). In dogs, immunoneutralization of PYY increased intestinal transit of a test meal (479), and ileal perfusion of fat in dogs will increase the migrating motor (myoelectric) complex (MMC) cycle length, decrease the number of MMCs, and increase circulating levels of PYY (480). The fasting motor patterns of the upper gut, also called the interdigestive MMC, are characteristic patterns of intestinal electrical activity and are disrupted by ingested nutrients (480). The interdigestive MMC, cycle length, and blood levels of PYY are also parallel (481). Because of its potent antisecretory action, PYY may be an endogenous inhibitor of diarrhea (482). PYY or synthetic PYY analogues, given directly into the intestinal lumen are proabsorptive (483). PYY has been shown to enhance intestinal absorption of fluid and electrolytes in the jejunum and ileum (484,485). In agreement with the antisecretory action

POSTPYLORIC GASTROINTESTINAL PEPTIDES / 145 IV Secretin (100 ng/kg-h) PYY 12.5 25

50

Secretin

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FIG. 5-27. (Top) Pancreatic bicarbonate output in response to a submaximal dose of secretin (100 ng/kg/hr, intravenously [IV]) alone (control) or in combination with graded doses of peptide YY (PYY; 12.5, 25, 50, 100, 200, 400 pmol/kg/hr) given each for 30 minutes in conscious dogs. PYY at 200 and 400 pmol/kg/hr inhibited secretin-stimulated pancreatic bicarbonate output significantly. (Bottom) Pancreatic protein output in response to a submaximal dose of cholecystokinin (CCK)-8 (50 ng/kg/hr, IV) alone or in combination with PYY (400 pmol/kg/hr, IV). PYY inhibited CCK-stimulated pancreatic protein secretion significantly. *p < 0.05 versus control; n = 6 dogs. (From Jeng and colleagues [495], by permission.)

of PYY, PYY receptors are found in the intestinal crypt cells where chloride secretion occurs (486). Occupation of the PYY receptor by PYY inhibits small bowel chloride ion secretion by decreasing basal and VIP or prostaglandinstimulated levels of cyclic AMP (321,442,444). PYY (3-36) has been shown to reduce appetite and food intake, both in normal and obese subjects (487). Interestingly, PYY was shown to reduce plasma ghrelin levels (ghrelin stimulates food intake), and endogenous PYY levels were reduced in obese subjects (488), suggesting that the PYY deficiency has a role in the cause of obesity. PYY may cause satiety by inhibition of gut motility and give a sense of fullness that is mediated by vagal afferents that originate in the GI tract and project to the hindbrain, terminating in the hypothalamus. In animal studies, PYY has been shown to inhibit hypothalamic NPY neurons and agouti-related protein neurons through inhibitory NPY-Y2 receptors that disinhibit adjacent proopiomelacortin-expressing neurons and decrease food intake. Systemic PYY can cross the blood–brain barrier, indicating that intestinal-derived PYY can influence central ingestive regulatory pathways (489).

Regulation of Peptide YY Secretion In humans, dietary fat is a potent stimulus for release of PYY from the intestine (490). In humans, PYY is released from the intestine in proportion to the calories ingested (490). In dogs, the ability of different nutrients given either into the duodenum or colon to stimulate PYY secretion was tested (435). Fatty acids (oleic acid) and glucose (2 g/kg) were shown to be the strongest PYY secretagogues when administered into the duodenum, whereas fatty acids and glucose are moderate stimulants of PYY release when given into the colon lumen. Amino acids and intact protein do not cause PYY release when given intraduodenally. However, a mixture of tryptophan and phenylalanine or intact protein administered into the colon of dogs will cause PYY secretion (435,438). Short-chain fatty acids (acetate, propionate, butyrate) given into the isolated perfused rabbit colon stimulate PYY release (491). Short-chain fatty acids are normal constituents of feces (492). In dogs, bile salts stimulate a dose-related release of PYY (493). Interestingly, plasma levels of PYY increase significantly within 15 to 30 minutes after ingestion of nutrients or in

146 / CHAPTER 5

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Neural pathways may have a role in the regulation of PYY secretion. In dogs, either atropine, hexamethonium, or atropine plus hexamethonium administration decreased food-induced secretion of PYY (499). Furthermore, β-adrenergic blockade with proprandolol or depletion of nerve terminal stores of catecholamines with reserpine did not influence nutrientinduced PYY secretion. IV administration of terbutaline, a β2-adrenergic agonist, causes PYY secretion (500). Truncal vagotomy increased resting-fasted and food-stimulated release of PYY. IV administration of bethanechol to dogs, a cholinergic agonist, also stimulated release of PYY and electrical stimulation of the vagus-stimulated PYY release. 350 Food Plasma PYY (pg/ml)

‡

14

PYY (pg/ml)

response to luminal administration of a fatty acid in humans, dogs (Fig. 5-28), and rats (464,490,494). PYY secretion in response to intraduodenal administration of fat causes PYY release even when the flow of chyme is prevented from reaching the distal intestine, and removal of the ileum-colon abolishes the PYY response to fat. This rapid release of PYY resulted in the speculation that another gut hormone that is released from the upper gut in response to a luminal nutrient triggered PYY release. It was also shown that IV administration of CCK caused a dose-dependent release of PYY (Fig. 5-29), and further experiments showed that a CCK-Atype receptor antagonist prevented the PYY response to a fatty meal or to exogenous CCK, supporting the role of intestinal CCK in causing PYY secretion (494,495). The influence of growth factors on PYY homeostasis has been studied (496,497). Both growth hormone and insulinlike growth factor-1 (IGF-1) stimulate intestinal PYY expression and peptide levels in rodents. Growth hormone and IGF-1 also stimulate PPY secretion. In terms of a molecular mechanism, the increase in intestinal PYY expression by IGF-1 involves Sp1 binding sites in the proximal promoter region of PYY (498).

300 250 200 150

30

60 90 Minutes

120

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FIG. 5-29. (Top) Integrated release of peptide YY (PYY) in response to graded doses of cholecystokinin (CCK)-8 given intravenously in six dogs. *p < 0.05 versus lower doses of CCK-8; †p < 0.05 versus 50 and 100 pmol/kg/hr CCK-8; ‡p < 0.05 versus lower CCK-8 doses; N = 5–7 dogs/dose). (Bottom) Plasma PYY levels in response to intravenous administration of CCK-8 (600 pmol/kg/hr) in conscious dogs. (From Jeng and colleagues [495], by permission.)

Together, these findings in the dog suggest that stimulated PYY levels are dependent on ganglionic transmission and an atropine-blockable postganglionic parasympathetic pathway. A vagal cholinergic mechanism may act in a tonic fashion to inhibit PYY secretion. Adrenergic mechanisms do not participate in nutrient-stimulated PYY release; however, electrical stimulation of the splanchnic nerves will increase PYY secretion, implying that the sympathetic nervous system has a role in PYY secretion. Cutting of intestinal intramural neural pathways does not interfere with food-induced release of PYY, suggesting that intramural neural mechanisms also do not participate in food-induced release of PYY (494). In the isolated, perfused rat ileum, bethanechol stimulated PYY release and the β-adrenergic agonist isoproterenol stimulated transient release of PYY (501,502).

0 B1

B2 15

30 45 60 Minutes

90

120

150

FIG. 5-28. Plasma peptide YY (PYY) levels in response to an oral mixed meal in conscious dogs. Notice that circulating PYY levels are increased 15 minutes after feeding the meal. B1 and B2 refer to basal plasma PYY levels. (From Jeng and colleagues [495], by permission.)

Trophic Actions of Peptide YY on the Gastrointestinal Tract The early developmental expression of PYY in the intestine suggests a regulatory role in the development of the alimentary

POSTPYLORIC GASTROINTESTINAL PEPTIDES / 147 canal, as well as in mucosal cell replication or differentiation. PYY has been shown to stimulate intestinal growth in mice and rats (434). In adult female mice, administration of PYY increases intestinal growth in a dose-related manner (434). The stimulatory effects of PYY are greater on intestinal protein contents than on intestinal DNA contents. High doses of PYY (200 nmol/kg) increase DNA contents by 14% in the proximal small intestine, 18% in the distal small intestine, 20% in the proximal colon, and 28% in the distal colon. The same PYY dose increases protein contents by 47% in the proximal small intestine, 77% in the distal small intestine, 66% in the proximal colon, and 52% in the distal colon. Smaller doses (30 and 100 nmol/kg) of PYY did not affect bowel DNA content; however, they significantly increased the protein contents of the small intestine and colon to values that were significantly greater than those of control animals. High doses of PYY also increased the RNA contents of the various intestinal segments by 15% to 39%. In nursing-unweaned rats, the trophic effects of PYY are seen only in the proximal small intestine (434). PYY treatment increased DNA, RNA, and protein levels. In nursing rats, PYY increased the weight of the proximal small intestine by 12% to 15%, the total DNA contents by 43% to 50%, and the total protein contents by 22% to 20%. There were no stimulatory effects of PYY on the distal small intestine or colon in nursing rats. The absence of a trophic effect of PYY on the distal small intestine and colon in rats may be attributed to the low density of PYY receptors in the distal intestine (444). In rats, there are abundant high-affinity PYY/NPY binding sites in the duodenum and jejunum, a lower level in the ileum, and a complete absence in the colon (444). However, the intestinal pattern of PYY receptors cannot entirely explain the stimulatory actions of PYY. For example, PYY did not exert a stimulatory effect on the distal small intestine in rats, which has PYY binding sites (444), and PYY increased growth of the colon in mice, a place where PYY receptors are not expected. PYY may also act indirectly to stimulate intestinal growth through receptors located in stroma cells of the lamina propria or submucosa. In situ hybridization studies show the presence of PYY mRNA at several levels of the intestinal wall, including the lamina propria, submucosa, and muscle layer of the colon. PYY may act on neighboring cell targets that, in turn, influence gut cell proliferation. Other evidence supports an indirect mechanism of action for in vivo stimulation of intestinal cell growth by PYY. PYY cannot stimulate growth of a rat intestinal epithelial cell line (IEC-6, IEC-18 cells) (G. Gomez and G. H. Greeley Jr., unpublished data). PYY treatment, over a broad dose range and under different tissue culture conditions (e.g., serum-free or with serum), fails to increase cell numbers that are normally responsive to mitogens such as epidermal growth factor, platelet-derived growth factor, and insulin (503). Other studies have examined the trophic actions of PYY on the GI tract and reported that PYY administration failed to stimulate growth of the duodenum in rats (503). In this study, PYY was administered for 4 days only, and at a daily

dose that was approximately 15 to 60 times smaller than the dose range tested in rats or mice described earlier (434). Another earlier study also failed to show a trophic effect of a long-term PYY infusion on the intestine (504). This latter study showed that intestinal resection resulted in a dramatic increase in circulating PYY levels, as well as in the expected mucosal adaptive response. However, because exogenous PYY failed to stimulate bowel growth, the report concluded that PYY does not have a role in the trophic response after intestinal resection in the rat. Differences in experimental design may explain discrepancies among these various studies. Further evidence suggesting that PYY may be involved in the trophic adaptation of the intestine is provided by the finding that PYY content increases in the ileum that undergoes hyperplastic adaptation after subtotal colectomy in rats (G. Gomez and G. H. Greeley Jr., unpublished data). The study described earlier (434) did not address whether PYY exerts a trophic action on the intestine at physiologic doses. Evidence that the Y1 receptor is involved in intestinal cell growth is shown by a study where activation of the Y1 receptor in IEC-6 cells stably expressing this receptor caused cell growth and increased MAPK phosphorylation (505).

Inhibitory Action of PYY (3-36) on Appetite and Food Intake PYY (3-36) infusions in subjects of normal weight (Fig. 5-30) and in rodents will reduce appetite and food intake, and repeated administration of PYY will reduce weight gain in rodents (506). In mice, the inhibitory action of PYY (3-36) on food intake is not influenced by circulating ghrelin levels (507).

Pathophysiology of Peptide YY Plasma levels of PYY are altered in various clinical conditions (508–511). Plasma PYY levels are increased in patients with diarrhea associated with malabsorption of fat (510). In patients with gastric resection–induced dumping syndrome, due to rapid gastric emptying, plasma PYY levels are also increased (509). Systemic basal and postprandial levels of PYY are increased in patients with celiac and tropical sprue and in patients with massive small-bowel resection. The increase in PYY homeostasis is an expected adaptation because there is an increased load of unabsorbed nutrients in the distal intestine that stimulate PYY secretion via a luminal contact of PYY cells (485). In morbidly obese patients, both basal plasma PYY and postprandial PYY levels are reduced despite increased food intake (512,513). These observations are consistent with the idea that a decrease in PYY secretion is involved in the pathogenesis of obesity. Interestingly, in obese subjects, IV administration of PYY will reduce appetite and food intake with magnitudes similar to those seen in healthy subjects,

148 / CHAPTER 5 Obese subjects

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FIG. 5-30. Caloric intake by obese and lean subjects after infusion of peptide YY (PYY) (3-36) or saline. (A) Caloric intake by individual obese subjects. (B) Caloric intake by individual lean subjects during a buffet lunch 2 hours after the infusion of PYY or saline. (C) Mean ± standard error (SE) caloric intake by obese subjects. (D) Caloric intake by lean subjects during a buffet lunch 2 hours after infusion of saline or PYY. (E) Mean ± SE cumulative 24-hour caloric intake by obese subjects. (F) Caloric intake by lean subjects after infusion of saline or PYY. In all panels, the lean and obese groups each consisted of 12 subjects: 6 women and 6 men. (From Batterham and colleagues [513], by permission.)

indicating that obesity is not associated with an impaired response to PYY (see Fig. 5-30) (513).

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478. Savage AP, Adrian TE, Carolan G, Chatterjee VK, Bloom SR. Effects of peptide YY (PYY) on mouth to caecum intestinal transit time and on the rate of gastric emptying in healthy volunteers. Gut 1987;28:166–170. 479. Lin HC, Zhao XT, Wang L, Wong H. Fat-induced ileal brake in the dog depends on peptide YY. Gastroenterology 1996;110:1491–1495. 480. Dreznik Z, Meininger TA, Barteau JA, Brocksmith D, Soper NJ. Effect of ileal oleate on interdigestive intestinal motility of the dog. Dig Dis Sci 1994;39:1511–1518. 481. Wen J, Phillips SF, Sarr MG, Kost LJ, Holst JJ. PYY and GLP-1 contribute to feedback inhibition from the canine ileum and colon. Am J Physiol 1995;269:G945–G952. 482. Playford RJ, Domin J, Beacham J, Parmar KB, Tatemoto K, Bloom SR, Calam J. Preliminary report: role of peptide YY in defence against diarrhoea. Lancet 1990;335:1555–1557. 483. Liu CD, Hines OJ, Whang EE, Balasubramaniam A, Newton TR, Zinner MJ, Ashley SW, McFadden DW. A novel synthetic analog of peptide YY, BIM-43004, given intraluminally, is proabsorptive. J Surg Res 1995;59:80–84. 484. Bilchik AJ, Hines OJ, Zinner MJ, Adrian TE, Berger JJ, Ashley SW, McFadden DW. Peptide YY augments postprandial small intestinal absorption in the conscious dog. Am J Surg 1994;167:570–574. 485. Bilchik AJ, Hines OJ, Adrian TE, McFadden DW, Berger JJ, Zinner MJ, Ashley SW. Peptide YY is a physiological regulator of water and electrolyte absorption in the canine small bowel in vivo. Gastroenterology 1993;105:1441–1448. 486. Voisin T, Rouyer-Fessard C, Laburthe M. Distribution of common peptide YY-neuropeptide Y receptor along rat intestinal villus-crypt axis. Am J Physiol 1990;258:G753–G759. 487. Batterham RL, Bloom SR. The gut hormone peptide YY regulates appetite. Ann N Y Acad Sci 2003;994:162–168. 488. Korner J, Leibel RL. To eat or not to eat-how the gut talks to the brain. N Engl J Med 2003;349:926–928. 489. Nonaka N, Shioda S, Niehoff ML, Banks WA. Characterization of blood-brain barrier permeability to PYY3-36 in the mouse. J Pharmacol Exp Ther 2003;306:948–953. 490. Adrian TE, Ferri GL, Bacarese-Hamilton AJ, Fuessl HS, Polak JM, Bloom SR. Human distribution and release of a putative new gut hormone, peptide YY. Gastroenterology 1985;89:1070–1077. 491. Longo WE, Ballantyne GH, Savoca PE, Adrian TE, Bilchik AJ, Modlin IM. Short-chain fatty acid release of peptide YY in the isolated rabbit distal colon. Scand J Gastroenterol 1991;26:442–448. 492. Rubinstein R, Howard AV, Wrong OM. In vivo dialysis of faeces as a method of stool analysis. IV. The organic anion component. Clin Sci 1969;37:549–564. 493. Izukura M, Hashimoto T, Gomez G, Uchida T, Greeley GH Jr, Thompson JC. Intracolonic infusion of bile salt stimulates release of peptide YY and inhibits cholecystokinin-stimulated pancreatic exocrine secretion in conscious dogs. Pancreas 1991;6:427–432. 494. Greeley GH Jr, Jeng YJ, Gomez G, Hashimoto T, Hill FL, Kern K, Kurosky T, Chuo HF, Thompson JC. Evidence for regulation of peptide-YY release by the proximal gut. Endocrinology 1989;124: 1438–1443. 495. Jeng Y-J, Hill FLC, Lluis F, Gomez G, Izukura M, Kern K, Chuo S, Ferrar S, Greeley GH Jr. Peptide YY release and actions. In: Thompson JC, Cooper CW, Greeley GH Jr, Rayford PL, Singh P, Townsend CM Jr, eds. Gastrointestinal endocrinology: receptors and post-receptor mechanisms. San Diego: Academic Press, 1990;371–386. 496. Gomez G, Udupi V, Qi X, Lluis F, Rajaraman S, Thompson JC, Greeley GH Jr. Growth hormone upregulates gastrin and peptide YY gene expression. Am J Physiol 1996;271:E582–E586. 497. Lee HM, Udupi V, Englander EW, Rajaraman S, Coffey RJ Jr, Greeley GH Jr. Stimulatory actions of insulin-like growth factor-I and transforming growth factor-alpha on intestinal neurotensin and peptide YY. Endocrinology 1999;140:4065–4069. 498. Wang G, Leiter AB, Englander EW, Greeley GH Jr. Insulin-like growth factor I increases rat peptide YY promoter activity through Sp1 binding sites. Endocrinology 2004;145:659–666. 499. Zhang T, Uchida T, Gomez G, Lluis F, Thompson JC, Greeley GH Jr. Neural regulation of peptide YY secretion. Regul Pept 1993;48:321–328. 500. Kogire M, Izukura M, Gomez G, Uchida T, Greeley GH Jr, Thompson JC. Terbutaline, a beta 2-adrenoreceptor agonist, inhibits gastric acid secretion and stimulates release of peptide YY and gastric inhibitory polypeptide in dogs. Dig Dis Sci 1990;35:453–457. 501. Plaisancie P, Bernard C, Chayvialle JA, Cuber JC. Release of peptide YY by neurotransmitters and gut hormones in the isolated, vascularly perfused rat colon. Scand J Gastroenterol 1995;30:568–574.

POSTPYLORIC GASTROINTESTINAL PEPTIDES / 159 502. Dumoulin V, Dakka T, Plaisancie P, Chayvialle JA, Cuber JC. Regulation of glucagon-like peptide-1-(7-36) amide, peptide YY, and neurotensin secretion by neurotransmitters and gut hormones in the isolated vascularly perfused rat ileum. Endocrinology 1995;136: 5182–5188. 503. Kurokowa M, Lynch K, Podolsky DK. Effects of growth factors on an intestinal epithelial cell line: transforming growth factor beta inhibits proliferation and stimulates differentiation. Biochem Biophys Res Commun 1987;142:775–782. 504. Savage AP, Gornacz GE, Adrian TE, Ghatei MA, Goodlad RA, Wright NA, Bloom SR. Is raised plasma peptide YY after intestinal resection in the rat responsible for the trophic response? Gut 1985; 26:1353–1358. 505. Mannon PJ, Mele JM. Peptide YY Y1 receptor activates mitogenactivated protein kinase and proliferation in gut epithelial cells via the epidermal growth factor receptor. Biochem J 2000;350(pt 3): 655–661. 506. Batterham RL, Cowley MA, Small CJ, Herzog H, Cohen MA, Dakin CL, Wren AM, Brynes AE, Low MJ, Ghatei MA, Cone RD, Bloom SR. Gut hormone PYY(3-36) physiologically inhibits food intake. Nature 2002;418:650–654.

507. Adams SH, Won WB, Schonhoff SE, Leiter AB, Paterniti JR Jr. Effects of PYY[3-36] on short-term food intake in mice are not affected by prevailing plasma ghrelin levels. Endocrinology 2004; 145:4967–4975. 508. Wahab PJ, Hopman WP, Jansen JB. Basal and fat-stimulated plasma peptide YY levels in celiac disease. Dig Dis Sci 2001;46:2504–2509. 509. Adrian TE, Long RG, Fuessl HS, Bloom SR. Plasma peptide YY (PYY) in dumping syndrome. Dig Dis Sci 1985;30:1145–1148. 510. Adrian TE, Savage AP, Bacarese-Hamilton AJ, Wolfe K, Besterman HS, Bloom SR. Peptide YY abnormalities in gastrointestinal diseases. Gastroenterology 1986;90:379–384. 511. Adrian TE, Savage AP, Fuessl HS, Wolfe K, Besterman HS, Bloom SR. Release of peptide YY (PYY) after resection of small bowel, colon, or pancreas in man. Surgery 1987;101:715–719. 512. Alvarez Bartolome M, Borque M, Martinez-Sarmiento J, Aparicio E, Hernandez C, Cabrerizo L, Fernandez-Represa JA. Peptide YY secretion in morbidly obese patients before and after vertical banded gastroplasty. Obes Surg 2002;12:324–327. 513. Batterham RL, Cohen MA, Ellis SM, Le Roux CW, Withers DJ, Frost GS, Ghatei MA, Bloom SR. Inhibition of food intake in obese subjects by peptide YY3-36. N Engl J Med 2003;349:941–948.

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CHAPTER

6

Gastrointestinal Peptide Hormones Regulating Energy and Glucose Homeostasis Daniel J. Drucker Proglucagon Gene Structure and the ProglucagonDerived Peptides, 161 Regulation of Proglucagon Gene Expression, 162 Control of Proglucagon-Derived Peptide Secretion, 164 Proglucagon-Derived Peptide Metabolism and Clearance, 164 Glucagon Receptor Family, 165 Glucagon Receptor, 165 Glucagon-Like Peptide-1 Receptor, 165 Glucagon-Like Peptide-2 Receptor, 166 Glucose-Dependent Insulinotropic Polypeptide Receptor, 166 Biological Actions of Glucagon, 166 Glucagon Administration in Human Subjects, 167 Biological Actions of Glicentin, 167 Oxyntomodulin, 167 Biological Actions of Glucagon-Like Peptide-1, 167

Glucagon-Like Peptide-1 Receptor Agonists and the Treatment of Type 2 Diabetes, 169 Enhancing Incretin Action via Inhibition of Dipeptidyl Peptidase-IV, 170 Biological Actions of Glucagon-Like Peptide-2, 170 Glucagon-Like Peptide-2 Administration to Human Subjects, 171 Glucose-Dependent Insulinotropic Polypeptide, 171 Synthesis and Secretion, 171 Biological Actions of Glucose-Dependent Insulinotropic Polypeptide, 171 Glucose-Dependent Insulinotropic Polypeptide Administration in Human Subjects, 172 Pancreatic Polypeptide, 172 Orexins, 172 References, 173

Gastrointestinal hormones synthesized in functionally distinct populations of enteroendocrine cells and neurons play diverse roles in regulation of energy intake, nutrient absorption, and nutrient disposal. Most gut hormones are secreted at low basal levels in the fasting state, and plasma levels of most gut peptides increase rapidly but transiently after nutrient ingestion. The effects of gut hormones are increasingly complex and include regulation of food intake, exocrine secretion, gut motility, mucosal growth, nutrient absorption, and pancreatic endocrine function. Gut hormones also communicate with regulatory centers in the central nervous system (CNS) via afferent ascending nerves. This chapter

focuses on the biology of the proglucagon-derived peptides (PGDPs) and gut hormones with related actions on control of insulin secretion and energy balance.

PROGLUCAGON GENE STRUCTURE AND THE PROGLUCAGON-DERIVED PEPTIDES Before the molecular biology era, the best characterized member of the proglucagon peptide family was 29-aminoacid glucagon produced in pancreatic α cells. Antisera directed against pancreatic glucagon were shown to crossreact with immunoreactive peptides in gut extracts that were also detected in the circulation (1). These peptides, originally known collectively as “enteroglucagons,” were subsequently shown to consist of 2 principal forms: a larger protein of 69 amino acids designated glicentin, and a smaller 37-amino-acid peptide named oxyntomodulin (2). After cloning of complementary DNA (cDNA) and genes encoding mammalian

D. J. Drucker: Banting and Best Diabetes Centre, Toronto General Hospital, Toronto, Ontario, Canada M5G 2C4.

Physiology of the Gastrointestinal Tract, Fourth Edition, edited by Leonard R. Johnson. Academic Press, 2006.

161

162 / CHAPTER 6 Proglucagon GRPP

Glucagon

IP-1

Glicentin Oxyntomodulin Pancreas Glucagon MPGF

GLP-1

IP-2

GLP-2

MPGF Intestine & Brain Glicentin Oxyntomodulin GLP-1 GLP-2 IP-2

FIG. 6-1. Structural organization of proglucagon gene and the proglucagon-derived peptides (PGDPs). Proglucagon undergoes tissue-specific posttranslational processing in the pancreas, intestine, and brain to liberate the indicated PGDPs. GRPP, glicentin-related pancreatic polypeptide; GLP-1 and GLP-2, glucagon-like peptide-1 and -2; IP-1 and IP-2, intervening peptides 1 and 2; MPGF, major proglucagon fragment.

preproglucagon, the structural relation among various PGDPs was clearly elucidated (Fig. 6-1). Two PGDPs related in sequence to glucagon, designated glucagon-like peptide-1 (GLP-1) and -2 (GLP-2) were found to be coencoded together with glucagon in a single proglucagon precursor (3–5). The human proglucagon gene is located on the long arm of chromosome 2 (6) and consists of six exons, with the sequences of glucagon and the GLPs encoded within separate exons (see Fig. 6-1). To date, no variants of the human proglucagon gene have been linked to heritability of specific human diseases. The mammalian genome encodes a single proglucagon gene that is transcribed to yield identical proglucagon messenger RNA (mRNA) transcripts predominantly in three cell types: pancreatic α cells, gut enteroendocrine L cells, and neurons in the caudal brainstem. The proglucagon mRNA transcript is structurally identical in all three tissues (7–9); hence, diversity in the generation of tissue-specific profiles of PGDPs is accomplished through tissue- and cell-specific expression of the prohormone convertases, proteases that differentially process proglucagon to yield either 29-aminoacid glucagon in the islet α cell or glicentin, oxyntomodulin, and both GLP-1 and GLP-2 in the gut L cell (see Figs. 6-1 and 6-2). Experimental studies have provided evidence supporting an essential role for PC2 in the cleavage of proglucagon to yield glucagon in pancreatic α cells, together with a larger, incompletely processed, secreted polypeptide designated major proglucagon fragment (10–13). The importance of PC2 for the generation of mature proglucagon is exemplified by the phenotype of mice with a targeted disruption of the PC2 gene. These mice exhibit mild hypoglycemia and markedly increased levels of incompletely processed proglucagon, together with deficiency of mature 29-amino-acid glucagon (14). In contrast with the importance of PC2 for generation of glucagon in islet α cells, PC1/3 appears to be required for processing of proglucagon in intestinal L cells (11,15). PC1 knockout mice exhibit multiple defects in prohormone processing including failure to generate significant amounts

of GLP-1 and GLP-2 (16). Although levels of mature GLP-1 are reduced in human subjects with an inactivating mutation of PC1, small amounts of bioactive GLP-1 can be generated in the absence of a functional PC1 enzyme (17). Intriguingly, PC1/3 has been localized to α cells in the embryonic pancreas, raising the possibility that GLP-1 may be liberated during development of the endocrine pancreas (18). PC1/3 expression, together with increased production of bioactive GLP-1, has also been localized to α cells in the setting of experimental diabetes; however, the biological implications of this finding remain uncertain (18,19).

Regulation of Proglucagon Gene Expression In the pancreas, proglucagon gene expression is stimulated by fasting and hypoglycemia, but is inhibited by insulin (20, 21), whereas in the intestine, proglucagon gene expression is up-regulated by nutrients (22). Gastrin-releasing peptide (GRP) and glucose-dependent insulinotropic polypeptide (GIP) (23) have been shown to increase levels of intestinal proglucagon mRNA transcripts in rodents. Fasting reduces and refeeding stimulates intestinal proglucagon gene expression (24), and a high-fiber diet (25) and short-chain fatty acids are potent inducers of proglucagon mRNA transcripts in enteroendocrine cells (26,27). Intestinal resection is associated with increased levels of proglucagon mRNA transcripts in the remnant intestine (28,29); however, the signals and mechanisms underlying this up-regulation remain unknown. Agents that activate the cAMP and protein kinase A (PKA) signaling pathways also increase proglucagon gene expression in the pancreas and intestine (8,30,31). Protein hydrolysates directly stimulate intestinal proglucagon gene expression through induction of gene transcription in part via DNA-promoter elements that mediate the response to cyclic 3′,5′-adenosine monophosphate (cAMP) (32). Intestinal proglucagon gene expression has been studied using primary cultures of intestinal cells, enteroendocrine cell lines, and transgenic mice. A primary determinant of intestinal proglucagon gene expression in studies of nontransformed intestinal cells appears to be the level of intracellular cAMP (8). Although agents such as phorbol esters, cholinergic agonists, calcium ionophores, and both GIP and GRP stimulate PGDP secretion in these cultures (33), only activators of cAMP generation enhanced proglucagon biosynthesis. Similarly, agents acting through the cAMP-dependent pathway appear to be the principal factors regulating proglucagon gene expression in mouse enteroendocrine glucagon SV40T antigen (GLUTag) (31,33) and secretin tumor cell line (STC-1) cells (34). Although GRP increases proglucagon gene transcription in STC-1 cells via the cAMP-response element (CRE) (23), GRP has not yet been shown to stimulate proglucagon gene expression in primary cell cultures or in the rodent intestine in vivo. Much less is known about the factors regulating the human proglucagon gene, largely because of the paucity of suitable models for studying human proglucagon gene expression.

GASTROINTESTINAL PEPTIDE HORMONES REGULATING ENERGY AND GLUCOSE HOMEOSTASIS / 163 RSLQDTEEKSRSFSASQADPLSDPDQMNEDKRHSQGTFTSDYSKYLDSRRQDFVQWLMNTKRNRNNIA Glicentin HSQGTFTSDYSKYLDSRRQDFVQWLMNTKRNRNNIA Oxyntomodulin Glucagon HSQGTFTSDYSKYLDSRRAQDFVQWLMNT HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG GLP-1(7-37) HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR GLP-1(7-36)amide HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPS Exendin-4 GLP-2 HADGSFSDEMNTILDNLAARDFINWLIQTKITD RNRNNIA IP-1 DFPEEVAIVEELG IP-2 YAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ GIP APLEPVYPGDNATPEQMAQYAADLRRYINMLTRPRY

PP

FIG. 6-2. Amino acid sequences of the proglucagon-derived peptides (PGDPs), exendin-4, glucosedependent insulinotropic polypeptide (GIP), and pancreatic polypeptide (PP). The lizard-derived exendin-4 shares 53% amino acid sequence identity with mammalian glucagon-like peptide-1 (GLP-1). Peptides that contain an alanine or proline at position 2 (indicated by underlining) are substrates for cleavage by the enzyme dipeptidyl peptidase-IV. IP-1 and IP-2, intervening peptides 1 and 2.

Transgenic mice expressing a human proglucagon gene promoter growth hormone transgene in the gut provide a potential model for studies of the human proglucagon gene promoter (35). Although nutrients up-regulated expression of the endogenous murine proglucagon gene, feeding had no effect on the human growth hormone (hGH) transgene, suggesting that important control elements for nutrientregulated control of human proglucagon promoter activity do not reside within the 1.6 kb of transgene human proglucagon gene promoter sequences (36). The control of human proglucagon gene expression has also been examined in NCIH716 cells, a cell line derived from a human adenocarcinoma exhibiting features of endocrine differentiation. Multiple factors, including palmitic acid, oleic acid, meat hydrolysate, carbachol, and GRP stimulate PGDP secretion from NCIH716 cells. In contrast with results of studies in rodent cells, cAMP enhanced GLP-1 secretion but failed to increase levels of proglucagon mRNA transcripts in NCI-H716 cells (37). Similarly, insulin, phorbol myristate acetate, or forskolin, all known regulators of rodent proglucagon gene expression, had no effect on proglucagon gene expression in NCI-H716 cells, and transfection studies using either the human or rodent proglucagon gene promoters demonstrate that NCIH716 cells do not support transcriptional activation of the isolated proglucagon gene promoter sequences (38). Hence, the available data suggest that NCI-H716 cells may express the human proglucagon in a constitutive, nonregulated manner. Cis-acting sequences within the rat proglucagon gene promoter have been identified as important control regions for islet cell–specific gene expression. Transcription factors important for islet cell–specific proglucagon gene expression include Brn4, Pax-6, Cdx-2/3, Isl-1, and members of the hepatocyte nuclear factor 3 (HNF-3 or Foxa) family (39–46). Pax6 and Cdx-2/3, in association with a coactivator protein, p300, interact synergistically to regulate proglucagon gene expression in islet cells (47). Pax-2 binds elements within the proglucagon gene promoter (48), but whether Pax-2 plays a role in the control of proglucagon gene expression remains uncertain (49). Similarly, although Brn4 is a potent activator of islet proglucagon gene expression, targeted disruption

of the Brn4 gene in mice does not produce abnormalities in α-cell development or changes in the levels of pancreatic glucagon mRNA transcripts (50). The proglucagon gene 5′-flanking sequences contain a CRE that confers cAMP responsivity to proglucagon gene transcription in pancreas and intestine (8,30,31,51). In contrast with our understanding of proglucagon expression in pancreatic islets, less is known about the factors that specify proglucagon gene expression in the intestine, partly because of the limitations of models for analysis of enteroendocrine gene transcription. Cell transfection studies using immortalized STC-1 or GLUTag cell lines indicate that DNA sequences between −1,252 and −2,292 in the rat proglucagon promoter are essential for specifying intestinal proglucagon expression (52). Several transcription factors thought to be important for control of islet proglucagon gene transcription may not be essential for enteroendocrine gene transcription. Genetic inactivation of the murine Foxa1 (HNF-3α) gene results in mice with mild hypoglycemia and inappropriately low levels of pancreatic proglucagon mRNA transcripts; however, levels of intestinal proglucagon mRNA transcripts are normal in Foxa1 mutant mice (46,53). Similarly, although members of the Foxa3 (HNF-3β ) family have been implicated in the control of proglucagon gene transcription in transfection studies, levels of pancreatic and intestinal proglucagon mRNA transcripts are normal in Foxa3−/− mice (54). The Pax-6 gene is important for cell lineage development in the gut and pancreas, as well as for control of pancreatic and intestinal proglucagon gene expression. Global inactivation of the murine Pax-6 gene results in major defects in formation of islet cell lineages (55), whereas mice homozygous for a dominant negative form of Pax-6 (SEYNeu) have significantly reduced levels of proglucagon mRNA transcripts in the pancreas (56) and both the small and large bowel, indicating that this transcription factor is essential for proglucagon gene expression in both islet and enteroendocrine cells (57). Conversely, increased expression of Pax-6 in primary intestinal cell cultures or in the rodent intestinal epithelium is associated with up-regulation of the levels of proglucagon gene expression (58).

164 / CHAPTER 6 Control of Proglucagon-Derived Peptide Secretion Glucagon, the principal secretory product from islet α cells, is secreted in response to hypoglycemia; however, the mechanism for sensing hypoglycemia and stimulating α-cell secretion likely involves the central and peripheral nervous system. In contrast, insulin and other factors secreted from the β cell, such as γ-aminobutyric acid (GABA) and zinc, appear to be the predominant factors inhibiting islet α-cell secretion. Glicentin, oxyntomodulin, GLP-1, and GLP-2 (Fig. 6-2) are cosecreted from enteroendocrine L cells, which are predominantly localized to the distal ileum and colon (59,60). GLP-1– immunopositive cells may also be located in more proximal regions of the small bowel, in cells that produce both GLP-1 and GIP (61). Multiple forms of GLP-1 are released in vivo, including GLP-1(1-37) and GLP-1(1-36)NH2, which appear to be biologically inactive (62–64), and GLP-1(7-37) and GLP-1(7-36)NH2, which are biologically active. A major proportion of GLP-1 is amidated at the C-terminal glycine residue [GLP-1(1-36)NH2 and GLP-1(7-36)NH2], likely via the activity of peptidylglycine α-amidating monooxygenase (65). C-terminal amidation may enhance the survival of GLP-1 in plasma (66). Both GLP-1(7-37) and GLP-1(7-36) NH2 appear equipotent (66–68); however, most circulating GLP-1 in humans is GLP-1(7-36)NH2 (69). In pigs and rats, approximately half of the GLP-1 is glycine extended, whereas in dogs, the glycine-extended forms of GLP-1 predominate. GLP-1 secretion from intestinal endocrine cells is stimulated by neural signals, endocrine factors, and direct nutrient contact with gut L cells (70). Intestinal PGDP secretion is regulated by several intracellular signals, including PKA, PKC, and calcium (33,71,72). A combination of studies using fetal rat intestinal cultures, the perfused rat ileum, and experiments performed in humans, rodents, and dogs have demonstrated that GLP-1 secretion is regulated directly by nutrients such as fatty acids, butyrate, peptones, or amino acids such as glutamine. Nevertheless, the rapid increase in plasma levels of the PGDPs within minutes of nutrient ingestion invokes a role for both neural and endocrine factors in control of L-cell secretion (70,73–75). Several neurotransmitters, including muscarinic agonists (76,77), regulate L-cell secretion both in vitro and in vivo. In humans, basal plasma levels of intact GLP-1 typically are within the 5- to 10-pM range in the fasting state, and increase to approximately 50 pM after meal ingestion (69,78). A small but detectable defect in meal-stimulated GLP-1 secretion has been observed in some subjects with obesity and type 2 diabetes (78). Mixed meals appear to be the most potent stimulus for GLP-1 secretion; however, individual nutrients including glucose, fatty acids, and dietary fiber also stimulate PGDP secretion (74,79–81). As most GLP-1–producing L cells, and hence stored GLP-1, is localized to the distal portion of the small intestine, a role for neural, endocrine mediators, or both in the rapid, nutrient-stimulated increase in plasma GLP-1 seems likely (70,82). Candidate mediators for indirect stimulators of GLP-1 include the more proximally located duodenal hormone GIP and the neurotransmitter acetylcholine (77,82–84). Nevertheless, species-specific

differences have been identified in studies of GLP-1 secretion because GIP does not stimulate GLP-1 secretion in humans (85,86), whereas the neuropeptide GRP stimulates GLP-1 secretion in both humans and rodents (87). Further evidence for the importance of GRP derives from studies of mice with inactivation of the GRP receptor that exhibit a reduced plasma GLP-1 response to gastric glucose (88). The neuropeptide calcitonin gene–related peptide may also regulate GLP-1 release (89). The vagus nerve plays a major role in controlling GLP-1 release from the distal L cells in response to ingested nutrients (90). Intestinal GLP-1 secretion appears to be inhibited by insulin and somatostatin-28 (33,91), as well as by the neuropeptide galanin (92).

PROGLUCAGON-DERIVED PEPTIDE METABOLISM AND CLEARANCE Little is known about the metabolism of glucagon, glicentin, and oxyntomodulin; however, the kidney plays a major role in the catabolism and clearance of all three peptides (93,94). Glucagon action is terminated via extracellular and intracellular degradation pathways. Glucagon-degrading activity within hepatic endosomes has been attributed to cathepsins B and D (95), and glucagon and GLP-1 are substrates for the widely expressed membrane-bound neutral ectopeptidase (NEP) 24.11 (96). The clearance and degradation of GLP-1 has received considerable attention because of the therapeutic potential of the peptide. The half-life of circulating native GLP-1 is less than 2 minutes (97,98), principally because of the protease activity of dipeptidyl peptidase-IV (DPP-IV), an aminopeptidase that specifically cleaves dipeptides from the amino terminus of proteins containing an alanine or proline at position 2. Oxyntomodulin and GLP-2 are also substrates for DPP-IV (99,100). DPP-IV is widely expressed in a large number of cell types in many tissues including the vascular endothelium of the small intestine, directly adjacent to the sites of GLP-1 release (97,101,102). Furthermore, in addition to a cell-associated membrane-bound form, a soluble DPP-IV molecule is also found in the circulation. DPP-IV catalyzes the cleavage of GLP-1 at the position 2 Ala residue to yield GLP-1(9-37) or GLP-1 (9-36)NH2. More than 50% of GLP-1 is metabolized to its N-terminal truncated form by DPP-IV within 2 minutes of peptide administration (103). Complementary evidence for the importance of DPP-IV in GLP-1 metabolism is derived from studies of rodents with mutations or inactivation of the DPP-IV gene. DPP-IV– deficient rats exhibit a prolonged half-life of GLP-1 (104,105), and mice with a targeted inactivation of DPP-IV exhibit increased levels of plasma GLP-1 (106). Moreover, rats and mice with mutant or inactivated DPP-IV genes exhibit improved glucose tolerance and increased levels of circulating intact GLP-1. DPP-IV is also critical for GLP-1 inactivation in humans, because intravenous or subcutaneous GLP-1 is rapidly degraded (within 30 minutes) to GLP-1(9-36)NH2 after administration of GLP-1 to healthy or diabetic human subjects, and this N-terminally shortened peptide accounts

GASTROINTESTINAL PEPTIDE HORMONES REGULATING ENERGY AND GLUCOSE HOMEOSTASIS / 165 for more than 75% of the immunodetectable circulating GLP-1 (98). Although GLP-1 (9-36)NH2 functions as a weak competitive pharmacologic antagonist of the GLP-1 receptor at the β cell and in the gastrointestinal system (107), experiments in anesthetized pigs treated with DPP-IV inhibitors revealed that GLP-1 (9-36)NH2 could paradoxically elicit modest insulin-independent antihyperglycemic effects (108). In contrast, infusion of GLP-1 (9-36)NH2 in healthy human subjects had no effects on glucose tolerance, insulin secretion or sensitivity, or GLP-1 action (109). Thus, the biological importance of N-terminally cleaved GLP-1 (9-36)NH2 remains uncertain. NEP 24.11 also exhibits endoproteolytic activity on GLP-1 and may also contribute to the metabolism of GLP-1 (96,110). Because GLP-2 is cosecreted with GLP-1, factors identified as important for GLP-1 secretion are similarly important for GLP-2 secretion, predominantly nutrients (111–113). GLP-2 also contains an alanine at position 2 and is degraded by DPP-IV (100). However, the half-life of exogenously administered GLP-2 is comparatively longer than that measured for GLP-1, at ~7 minutes (114). Although limited information is available about perturbations in levels of GLP-2 in human disease, patients with extensive resection of the small and large bowel exhibit significant reductions in circulating levels of GLP-2 after meal stimulation, whereas preservation of the colon appears important for maintaining levels of GLP-2 in adult human subjects (115,116). In contrast, the colon may be less important for preservation of plasma levels of GLP-2 in infants with nutrient malabsorption consequent to intestinal surgery (117). Patients with inflammatory bowel disease often exhibit increased circulating levels of GLP-2, in association with reductions in plasma levels of DPP-IV activity (118). The primary route of clearance for GLP-1 and GLP-2 appears to be through the kidney via mechanisms that include glomerular filtration and tubular catabolism (119–122). Patients with uremia have increased levels of circulating, immunoreactive GLP-1 (123), and bilateral nephrectomy or ureteral ligation in rats is associated with increases in the circulating half-life of GLP-1 (119). A role for tissues other than the kidney, such as the liver and lung, in GLP-1 clearance has not been clearly established (119).

intracellular cAMP and intracellular calcium (125). Oxyntomodulin is capable of binding to and activating both the glucagon and GLP-1 receptors; however, the anorectic actions of oxyntomodulin appear to require a functional GLP-1 receptor (126). The human Gcgr gene is localized to chromosome 17q25, and several reports have described an association between a Gly40Ser Gcgr mutation and an increased incidence of type 2 diabetes; however, this finding has not been confirmed in different populations with type 2 diabetes (127,128). Furthermore, cells expressing a transfected Gcgr containing the Gly40Ser mutation exhibit decreased affinity for glucagon in vitro, and human subjects with the Gly40Ser mutation exhibit a paradoxically decreased glycemic response to glucagon infusion in vivo (128,129). Hence, the biological significance of the Gly40Ser substitution remains uncertain. Gcgr mRNA transcripts have been detected in liver, islet β cells, brain, adipocytes, heart, and kidney. Rat Gcgr mRNA transcripts have also been detected in tissues such as spleen, thymus, adrenal gland, intestine, ovary, and testis where glucagon action remains poorly defined (130). Glucagon binding sites have been identified in multiple brain regions, and Gcgr transcripts have been detected in cortex, cerebellum, hypothalamus, and brainstem; however, the biological actions of glucagon in specific CNS regions remain unclear (131). Loss of glucagon action has been studied using antisense oligonucleotides directed against the Gcgr in diabetic mice (132,133), or in Gcgr−/− mice. Remarkably, overlapping phenotypes were seen in these experiments, including increased circulating levels of glucagon, mild fasting hypoglycemia together with improved glucose tolerance in Gcgr−/− mice, decreased body weight, and increased pancreatic mass associated with considerable hyperplasia of islet α cells (134,135). Furthermore, reduction of Gcgr expression in db/db mice was associated with lower levels of blood glucose, triglycerides, and free fatty acids (132,133). The improvement in glycemic control after loss of Gcgr function in these rodent studies appears to be attributable in part to increased circulating levels of bioactive GLP-1 derived from the pancreas. Gcgr expression is regulated in a tissue-specific manner with glucose and cAMP, important for Gcgr expression in hepatocytes (136,137), whereas glucose increased but cAMP and the glucocorticoid dexamethasone decreased levels of Gcgr mRNA transcripts in rat islets (138).

GLUCAGON RECEPTOR FAMILY GLUCAGON-LIKE PEPTIDE-1 RECEPTOR Separate receptors have been identified for glucagon, GLP-1, GLP-2, and GIP (124); however, the mechanisms underlying actions of PGDPs such as glicentin and oxyntomodulin remain poorly understood.

GLUCAGON RECEPTOR The glucagon receptor (Gcgr) is a member of the seventransmembrane–spanning G protein–coupled receptor superfamily, and it responds to glucagon with increases in both

The glucagon-like peptide-1 receptor (GLP-1R) was cloned from a rat pancreatic islet cDNA library (139). The human GLP-1R gene has been mapped to chromosome 6, band p21.1 (140); however, human GLP-1R mutations or polymorphisms have not been linked to the development of type 2 diabetes. The GLP-1R is expressed in lung; kidney; stomach; heart; intestine; α, β, and δ cells of the pancreatic islets; and multiple regions of the CNS (131,141–143). The GLP-1R couples to multiple G proteins, including Gαs, Gαq/11, and Gαi1,2, leading to activation of several intracellular

166 / CHAPTER 6 signaling pathways (144), increased adenylate cyclase and phospholipase C, and activation of PKA and PKC, respectively (139,145,146). GLP-1R activation also increases intracellular calcium (147–149), phosphatidylinositol-3 kinase (PI3K), and mitogen-activated protein kinase (MAPK) signaling pathways (144,150). Little is known about the factors that regulate levels of GLP-1R mRNA. GLP-1R expression is down-regulated in response to GLP-1, activation of PKC, high glucose, or dexamethasone, whereas treatment of diabetic rodents with DPP-IV inhibitors is associated with up-regulation of pancreatic GLP-1R expression (151). The GLP-1R undergoes homologous and heterologous desensitization and internalization in islet cell lines, in association with receptor phosphorylation (152–154). However, desensitization of the GLP-1R has not been observed after long-term administration of GLP-1R agonists in vivo. Exendin (9-39), an N-terminally truncated peptide derived from the lizard GLP-1R agonist exendin-4, functions as a relatively specific GLP-1 receptor antagonist (155,156) and is commonly used to examine the consequences of transient loss of GLP-1R action.

GLUCAGON-LIKE PEPTIDE-2 RECEPTOR The GLP-2 receptor was cloned from hypothalamic and intestinal cDNA libraries and is also coupled to cAMP generation when expressed in heterologous cell lines (157). GLP-2R expression is highly restricted, predominantly to the stomach, small and large bowels, and the CNS (158–160). Immunocytochemistry localized the GLP-2R to human enteroendocrine cells (158) and specific regions of the murine and rat CNS (159,160), whereas in situ hybridization has localized the receptor to neurons in the CNS and the murine enteric nervous system (161,162). The GLP-2R is highly specific for GLP-2 and is not activated by physiologically relevant concentrations of related members of the glucagon peptide superfamily (157,163). GLP-2 dose-dependently activates adenylyl cyclase, cAMP formation, and PKA in cells expressing a heterologous rat or human GLP-2R, as well as in primary cell cultures from the CNS and the intestinal mucosa (157,160,164,165). Furthermore, GLP-2R activation activates c-fos in cells transfected with the GLP-2R (164) and in the murine gastrointestinal tract (161). GLP-2 also activates constitutive nitric oxide synthase (NOS) activity and endothelial NOS protein abundance in the gut, which appears to regulate GLP-2– induced intestinal blood flow and glucose uptake (166). Activation of the GLP-2 receptor is also associated with enhanced cell survival. GLP-2R activation inhibits cycloheximide-induced apoptosis in a PKA-independent manner (167); in contrast, PKA regulates the antiapoptotic properties of GLP-2R signaling after inhibition of PI3K (168) in transfected fibroblasts or exposure to glutamate in hippocampal neurons (160). Although GLP-2 administration promotes rapid growth of the intestinal epithelium after administration in vivo (169), whether GLP-2R activation can

directly stimulate cellular proliferation requires further study. Incubation of colonic intestinal cells or rat astrocytes with GLP-2 results in increased cell proliferation (170–172); however, baby hamster kidney (BHK) fibroblasts transfected with the GLP-2 receptor did not exhibit a significant mitogenic effect in response to GLP-2 (164). Furthermore, GLP-2 treatment paradoxically inhibits cell proliferation in epithelial cells derived from the small intestine, yet stimulates cell proliferation in cell lines derived from the colon in a GLP-2R–independent manner (173). Hence, the molecular basis for GLP-2R activation promoting mitogenesis appears complex, likely indirect, and highly cell-type or tissue specific.

GLUCOSE-DEPENDENT INSULINOTROPIC POLYPEPTIDE RECEPTOR Genetic mapping and linkage studies have localized the human glucose-dependent insulinotropic polypeptide receptor (GIPR) gene to chromosome 19 band q13.3 (174). The GIPR is a member of the seven-transmembrane domain, heterotrimeric, G protein–coupled Gcgr superfamily (124, 175), and it is expressed in the pancreas, stomach, small intestine, adipose tissue, adrenal cortex, lung, pituitary, heart, testis, vascular endothelium, bone, and brain (175,176). Activation of GIPR signaling leads to generation of cAMP and increases in intracellular calcium in pancreatic islet cells, adipocytes, osteoblasts, endothelial cells, and heterologous cell lines transfected with GIPR (176–179). The GIPR also signals via PI3K, MAPK, and phospholipase A2 pathways (180–182). The GIPR undergoes rapid and reversible homologous desensitization (73) in vitro. Studies using the βTC3 islet cell line implicate a role for regulator of G-protein signaling 2 (RGS-2), G-protein receptor kinase 2 (GRK-2), and β-arrestin-1 in GIP-induced receptor desensitization (183). GIP action is diminished in human subjects with type 2 diabetes (86), and defective GIP action in Zucker diabetic fatty rats has been correlated with reduced levels of GIPR mRNA transcripts in pancreatic islets (184). The absence of GIP action has been studied in mice with genetic disruption of the GIPR (185). GIPR knockout mice exhibit a mild defect in glucose tolerance (185), but they are resistant to the development of obesity and insulin resistance after high-fat feeding (186). Despite the roles of GIP and GLP-1 as the two dominant incretins regulating insulin secretion, combined disruption of both the GLP-1 and GIP receptors produces only modest impairment of glucose-dependent insulin secretion (187,188).

BIOLOGICAL ACTIONS OF GLUCAGON Glucagon exerts a number of important physiologic actions in tissues such as the liver, the endocrine pancreas, the vascular bed, and the gastrointestinal tract. Glucagon regulates hepatic glucose production via activation of glycogenolysis

GASTROINTESTINAL PEPTIDE HORMONES REGULATING ENERGY AND GLUCOSE HOMEOSTASIS / 167 and gluconeogenesis and by inhibition of glycolysis. Glucagon modifies the activity of enzymes important for glucose production and modulates expression of genes encoding enzymes in the glycolytic or gluconeogenic pathways (189). Glucagon also regulates fatty acid metabolism via reduction of malonyl coenzyme A (CoA) and stimulation of fatty acid oxidation. The cAMP-dependent transcription factor CREB is a downstream mediator for glucagon action. CREB, together with activation of the nuclear receptor coactivator peroxisome proliferator-activated receptor (PPAR) gamma coactivator-1 (PGC-1) and suppression of PPARγ activity, ultimately results in activation of hepatic gluconeogenesis (190). Glucagon increases cAMP and stimulates lipolysis in adipocytes, thereby providing free fatty acids as substrate for fat-burning tissues. Glucagon also inhibits insulin-stimulated glucose transport in adipocytes (191). In the peripheral vascular system, glucagon acts as a vasodilator via effects on local vascular tone, and glucagon increases cardiac output and heart rate, possibly via direct effects on the heart. Pharmacologic doses of glucagon increase renal blood flow, glomerular filtration rate (GFR), and urinary electrolyte excretion; however, lower concentrations of glucagon do not affect renal blood flow, GFR, or solute excretion (192). Although the kidney exhibits significant gluconeogenic capacity, the available evidence does not support a role for endogenous glucagon in the control of renal glucose output (193). The β cell expresses receptors for glucagon coupled to cAMP and stimulation of insulin secretion. The threshold for glucagon-stimulated cAMP accumulation in isolated β cells is ~1 nM glucagon, which is greater than the concentrations required for cAMP stimulation by GLP-1 or GIP (194). The physiologic importance of intraislet or circulating glucagon for β-cell physiology remains unclear, given the high concentrations of glucagon required to stimulate the β cell in vitro.

GLUCAGON ADMINISTRATION IN HUMAN SUBJECTS The most common use of glucagon therapeutically is in the acute management of severe hypoglycemia. Diabetic patients with hypoglycemia generally respond quickly, with a rapid increase in blood glucose, after glucagon administration (195,196). Glucagon is also used to inhibit gastrointestinal motility during radiologic or endoscopic investigations, and glucagon administration has been shown to benefit selected patients with refractory bronchospasm or symptomatic bradycardia (197,198).

BIOLOGICAL ACTIONS OF GLICENTIN Glicentin is a 69-amino-acid PGDP that contains the sequence of 29-amino-acid glucagon flanked by peptide extensions at both the amino and carboxy termini (see Fig. 6-2). Although cosecreted with GLP-1 and GLP-2 from

gut L cells, the specific biological actions of glicentin remain elusive. Glicentin has been shown to activate signal transduction pathways leading to stimulation of intracellular calcium and reduction of cAMP formation in smooth muscle cells derived from rabbit antrum (199). Furthermore, administration of glicentin to rodents induces small-bowel growth in some (169,200) but not all studies (201). To date, a distinct receptor responsible for transducing the biological actions of glicentin has not yet been identified.

OXYNTOMODULIN Oxyntomodulin contains the sequence of 29-amino-acid pancreatic glucagon together with an 8-amino-acid carboxyterminal extension (see Fig. 6-1). The original biological action described for oxyntomodulin was stimulation of acid secretion from the oxyntic glands in the stomach (202). Additional actions ascribed to oxyntomodulin include stimulation of intestinal glucose uptake (203), regulation of insulin secretion (204), reduction of gastric emptying (205), and inhibition of meal-stimulated gastric acid secretion (205–207). More recent studies have demonstrated an inhibitory effect of oxyntomodulin on food intake after both intracerebroventricular and peripheral administration in rats (208,209) and after intracerebroventricular administration in mice (126). Oxyntomodulin infusion also produces satiety and inhibits food intake after short-term intravenous infusion in human subjects (210). Although oxyntomodulin is a weak agonist at both the GLP-1 and glucagon receptors (211–214), the anorectic actions of oxyntomodulin are blocked by the GLP-1R antagonist exendin (9-39) (208) and are eliminated in the absence of a functional GLP-1R (126). Given the overlapping actions of oxyntomodulin with glucagon and GLP-1 and the lack of evidence for a distinct oxyntomodulin receptor, the available evidence suggests that many of the pharmacologic actions of oxyntomodulin represent heterologous activation of related PGDP receptor systems.

BIOLOGICAL ACTIONS OF GLUCAGONLIKE PEPTIDE-1 GLP-1 exerts multiple physiological actions leading to control of energy intake and nutrient assimilation (Table 6-1) (215). The original physiologic role described for GLP-1 was that of an incretin hormone. GLP-1 is secreted after nutrient ingestion and stimulates insulin secretion in a glucosedependent manner (85,216,217). GLP-1 also increases insulin gene transcription, mRNA stability, and biosynthesis by mechanisms that involve both cAMP/PKA-dependent and -independent pathways, as well as increases in the levels of intracellular Ca2+ (145,218,219). GLP-1 increases expression of the sulfonylurea receptor (SUR1) and inwardly rectifying potassium channel (Kir 6.2) in β cells, and it prevents glucose-dependent inhibition of KATP-channel activity (220).

168 / CHAPTER 6 TABLE 6-1. Summary of glucagon-like peptide-1 action Pancreas Stimulates glucose-dependent insulin secretion Increases insulin gene transcription, messenger RNA stability, and biosynthesis Inhibits glucagon secretion Stimulates somatostatin secretion Enhances β-cell responsivity and glucose competence Induces β-cell neogenesis and proliferation Inhibits β-cell apoptosis Gastrointestinal tract Inhibits gastric emptying Inhibits gastric acid secretion Cardiovascular system Increases heart rate and blood pressure in rodents Improves myocardial contractility Reduces cardiomyocyte apoptosis Central nervous system Inhibits food and water intake Stimulates luteinizing hormone–releasing hormone secretion Increases thyroid-stimulating hormone, luteinizing hormone, adrenocorticotropic hormone, and corticosterone/cortisol secretion in vivo Thyroid, lung, and kidney Stimulates calcitonin secretion from C cells in the thyroid gland Enhances mucous secretion, pulmonary muscle relaxation, and surfactant secretion in lung Promotes diuresis and natriuresis in kidney

The physiologic importance of GLP-1 has been demonstrated using GLP-1R antagonists, immunoneutralizing antisera, and GLP-1R knockout mice. Elimination of GLP-1 activity with GLP-1 immunoneutralizing antisera or the GLP-1R antagonist exendin (9-39) results in impaired glucose tolerance and diminished glucose-stimulated insulin levels in animals and humans (221–224). Similarly, mice with a targeted inactivation of the GLP-1R gene (GLP-1R−/−) are glucose intolerant and exhibit defective glucose-stimulated insulin secretion (225). GLP-1 confers glucose sensitivity to β cells, thereby improving the ability of the endocrine pancreas to sense and respond to glucose (226,227); however, GLP-1R signaling is not required for preservation of β-cell glucose sensitivity in the mouse (228). The demonstration that GLP-1 up-regulates the expression of components of the β-cell glucose sensing system (i.e., glucose transporters and glucokinase) may provide a partial mechanism for the effects of GLP-1 on β-cell glucose responsivity (229,230). GLP-1 also inhibits glucagon and stimulates somatostatin secretion (217). The increase in somatostatin secretion appears to be direct, via GLP-1Rs on somatostatin-secreting pancreatic δ cells (231), whereas the inhibitory effect of GLP-1 on glucagon secretion may be indirect, perhaps through stimulation of insulin and somatostatin, both of which inhibit glucagon secretion. However, GLP-1 may also inhibit glucagon secretion directly, via interaction with GLP-1Rs on α cells (142). Basal GLP-1 signaling in the fasting state is important for glucoregulation because administration of the antagonist exendin (9-39) to humans increases fasting glucose

and glucagon, suggesting that even the low basal levels of GLP-1 exert a tonic inhibitory effect on glucagon-secreting α cells (156). The insulinotropic and glucagonostatic effects of GLP-1 are glucose-dependent (232,233). Thus, when blood glucose levels decrease, GLP-1 no longer stimulates insulin secretion or inhibits glucagon secretion, thereby reducing the possibility of hypoglycemia. GLP-1 administration also leads to a number of changes in β-cell differentiation and function, including induction of proliferation and neogenesis of pancreatic β cells, reduced apoptosis, and increased differentiation of exocrine-like cells toward a more differentiated β-cell phenotype (234). GLP-1 activates the expression of immediate early genes which encode for transcription factors that regulate islet cell proliferation and differentiation. Furthermore, the differentiation of pancreatic exocrine cells toward a differentiated endocrine and β-cell–like phenotype is associated with induction of genes required for glucose-sensing and insulin gene expression (235–237). The molecular mechanisms mediating the GLP-1R–dependent activation of the endocrine differentiation pathway are poorly understood but may involve synergy with transforming growth factor-β and coordinate changes in Smad transcription factor activity (238). GLP-1R agonists also stimulate β-cell neogenesis and proliferation, and increase β-cell mass in both normal and diabetic rodents (235,239–242). Conversely, elimination of GLP-1R signaling in the mouse is associated with reduced numbers of large β-cell clusters and alterations in islet α-cell topography (243), and GLP-1R−/− mice exhibit a reduced adaptive response and greater hyperglycemia after partial pancreatectomy (244). The intracellular signal transduction pathways whereby GLP-1 mediates its proliferative effects have not been clearly defined, but they likely involve the induction of PI3K, epidermal growth factor receptor transactivation, and activation of p38 MAPK and PKCξ (245,246). GLP-1 may also regulate β-cell mass via increasing the resistance to apoptosis (246–250). GLP-1 inhibits apoptosis in β-cell lines and in nontransformed rodent β cells. Similarly, treatment of diabetic mice or rats with GLP-1R agonists reduces β-cell apoptosis (247,251,252). Furthermore, GLP-1R signaling appears to be essential for β-cell survival because GLP-1R−/− mice exhibit more severe hyperglycemia and enhanced sensitivity to the cytotoxic effects of streptozotocin on β cells in vivo (247). Importantly, the antiapoptotic actions of GLP-1 have been demonstrated in experiments using human islets (249,250). Incubation of freshly isolated human islets with GLP-1 for 72 hours reduced the expression of proapoptotic genes, improved cell viability, and enhanced glucose-stimulated insulin secretion (249). Complementary studies demonstrated that GLP-1 prevented apoptosis induced by high glucose or palmitate in human islets in vitro through mechanisms involving PKB and Akt (250). In the gastrointestinal tract, GLP-1 inhibits pentagastrinand meal-induced gastric acid secretion and gastric emptying (253,254). The reduction of gastric emptying attenuates meal-associated excursions in blood glucose, appears

GASTROINTESTINAL PEPTIDE HORMONES REGULATING ENERGY AND GLUCOSE HOMEOSTASIS / 169 dependent on the vagus nerve, and likely involves both central and peripheral GLP-1 actions (255,256). Vagal afferent denervation or GLP-1R antagonism with exendin (9-39) eliminates the effects of central and peripheral GLP-1 on gastric emptying. Furthermore, a recombinant GLP-1-albumin protein (Albugon) that does not appear to cross the blood– brain barrier retains the ability to inhibit gastric emptying (256), which is consistent with the importance of ascending GLP-1R–dependent pathways for control of gut motility. GLP-1 increases systolic, diastolic, and mean arterial blood pressure and heart rate in rats (257) and increases heart rate in calves (258). The effects of GLP-1 on the cardiovascular system are mediated through central and peripheral mechanisms (257,259,260). In experimental models of cardiovascular dysfunction such as pacing-induced ventricular failure or coronary occlusion and reperfusion in dogs, GLP-1 administration significantly improved ventricular function (261,262). GLP-1 may also exert a direct effect on cardiovascular parameters through interaction with GLP-1Rs in the heart (263). GLP-1 improved myocardial glucose uptake and cardiac contractility in dogs with pacing-induced heart failure (262). Furthermore, GLP-1 improved regional wall motion recovery after transient coronary artery occlusion and a 24-hour reperfusion period in dogs (261). Moreover, a 72-hour infusion of GLP-1 in patients after acute myocardial infarction and angioplasty resulted in markedly improved ventricular function (264). Conversely, GLP-1R signaling appears essential for normal cardiac structure and function because GLP-1R−/− mice exhibit ventricular hypertrophy and an impaired cardiovascular response to external stress (265). Intracerebroventricular administration of GLP-1 inhibits short-term food and water intake in rodents (225,266,267), and peripheral administration promotes satiety and suppresses energy intake in healthy, diabetic, and obese humans (268–270). Pharmacologic administration of GLP-1 may modify feeding behavior through direct interaction with satiety centers, or through mechanisms involving induction of interoceptive stress and visceral illness (271,272). The recombinant GLP-1-albumin protein Albugon rapidly inhibits food intake and activates neuronal c-fos expression without directly penetrating the CNS (256). Administration of either GLP-1 or noxious substances such as lithium chloride results in a similar pattern of neuronal c-fos activation in rats and elicits comparable aversive responses consistent with induction of visceral illness (272–275). Thus, the observation that excess GLP-1 induces nausea and reduction of food intake in human subjects may reflect activation of central aversive signaling pathways. Consistent with the cytoprotective actions of GLP-1 on β cells, activation of GLP-1R signaling promotes neuronal survival in diverse modes of cytotoxic injury (276,277). Furthermore, GLP-1R−/− mice exhibit a learning deficit that is improved after localized restoration of CNS GLP-1R signaling and display enhanced sensitivity to neuronal injury and increased seizure activity after kainate administration (278). These findings have engendered interest in the potential use of GLP-1R agonists as neuroprotective agents (279).

GLP-1 also modulates components of the hypothalamicpituitary axis. GLP-1 stimulates cAMP formation and thyroidstimulating hormone (TSH) release from cultured mouse pituitary thyrotrophs and isolated rat anterior pituitary cells (280) and enhances luteinizing hormone-releasing hormone secretion from rodent hypothalamic neuronal cell lines. Furthermore central GLP-1 administration stimulates TSH, luteinizing hormone, corticosterone, and vasopressin secretion in rats (281,282); however, GLP-1R−/− mice do not exhibit a major impairment of hypothalamic-pituitary function (283). Nevertheless, short-term infusions of GLP-1 transiently increase plasma levels of adrenocorticotropic hormone and cortisol in healthy human subjects (284). Notably, experimental evidence in rodents clearly indicates that systemic administration of GLP-1 or exendin-4 results in rapid activation of brainstem proglucagon neurons, indicating that peripheral GLP-1 is capable of activating the CNS circuits that, in turn, produce GLP-1 in the brain (285).

GLUCAGON-LIKE PEPTIDE-1 RECEPTOR AGONISTS AND THE TREATMENT OF TYPE 2 DIABETES GLP-1 reduces blood glucose levels through stimulatory effects on insulin secretion, reduction of glucagon secretion and gastric emptying, and indirectly through promotion of satiety leading to weight loss and improved insulin sensitivity. Short-term administration of native GLP-1 rapidly decreases plasma glucose in patients with type 2 diabetes (286–288). Furthermore, postprandial administration of native GLP-1 significantly attenuated meal-related glycemic excursion in diabetic patients, and a 3-week trial of preprandial subcutaneous GLP-1 injections improved postprandial glycemic control and reduced plasma glucagon in subjects with type 2 diabetes (289). The efficacy of native GLP-1 for the treatment of type 2 diabetes was illustrated in studies of continuous subcutaneous GLP-1 infusion for 6 weeks. Subjects treated with GLP-1 exhibited improved β-cell function, decreased fasting and postprandial glucose levels, a significant reduction in HbA1c, in association with modest weight loss and improved insulin sensitivity (290). Hence, there are considerable data from human clinical studies that administration of native GLP-1 exhibits therapeutic utility in the treatment of diabetic patients (291,292). Nevertheless, the therapeutic use of native GLP-1 would require multiple daily subcutaneous injections or continuous administration via subcutaneous infusion, because of the short circulating half-life of the native peptide in vivo (98,103,293,294). Accordingly, considerable effort has been devoted to development and characterization of GLP-1R agonists that are resistant to DPP-IV–mediated degradation, and that exhibit more prolonged durations of action in vivo. Exendin-4 is a naturally occurring GLP-1–related peptide isolated from the venom of the Heloderma suspectum lizard (295). Exendin-4 exhibits 53% identity to mammalian GLP-1, is a potent agonist at the GLP-1 receptor (155), and is

170 / CHAPTER 6 encoded by a separate gene distinct from lizard GLP-1 (296). Analysis of exendin-4 action in preclinical studies demonstrates that exendin-4 is a long-acting GLP-1R agonist that exhibits a full spectrum of GLP-1R–dependent actions in normal and diabetic rodents (297–299). Exendin-4, subsequently renamed Exenatide for clinical use, also has been shown to potently reduce blood glucose in human subjects with type 2 diabetes. Acute intravenous infusion of exendin-4 to healthy volunteers reduced fasting and postprandial glucose in association with inhibition of gastric emptying and reduced food intake (300). Repeated subcutaneous administration of exendin-4 for 4 weeks improved blood glucose and significantly decreased HbA1c (301). The clinical efficacy of Exenatide administration was examined in a series of phase 2 and 3 trials in patients with type 2 diabetes. Addition of Exenatide twice daily to treatment regimens previously encompassing metformin, a sulfonylurea, or both agents produced a significant reduction in HbA1c with prevention of weight gain in a 4-week study (302,303). Exenatide has completed phase 3 clinical trials as add-on therapy in diabetic patients with inadequate glycemic control who were treated previously with metformin or a sulfonylurea agent, or both, and is the first GLP-1R agonist approved for the treatment of type 2 diabetes. Additional GLP-1R–based agonists in clinical development include Liraglutide, a human DPP-IV–resistant analogue that exhibits noncovalent binding to albumin and a prolonged pharmacokinetic profile after once daily administration (304,305). Liraglutide has completed phase 2 clinical trials and significantly reduced HbA1c with no associated weight gain in a 12-week monotherapy study (306). CJC1131 is a human GLP-1 analogue that forms a covalent bond to human serum albumin and exhibits GLP-1R–dependent actions in preclinical studies (242). CJC-1131 also has been shown to effectively reduce glycemia in 12-week studies of subjects with type 2 diabetes. Taken together, the available evidence strongly suggests that one or more injectable GLP-1R agonists may be used for the treatment of type 2 diabetes.

stimulate insulin secretion, and increase the levels of intact GLP-1 and GIP in preclinical models of diabetes (307–309). Moreover, administration of DPP-IV inhibitors to human subjects with type 2 diabetes results in significant reduction of glycemia, together with decreased levels of circulating glucagon and an improvement in the insulin/glucose ratio (310,311). Several DPP-IV inhibitors currently are in latestage clinical development for the treatment of type 2 diabetes (291,292,312).

BIOLOGICAL ACTIONS OF GLUCAGONLIKE PEPTIDE-2 GLP-2 is a 33-amino-acid peptide cosecreted with GLP-1 from gut endocrine cells in a nutrient-dependent manner (112,113). The principal biological consequence of exogenous GLP-2 administration is the expansion of the mucosal epithelium in the small bowel (169), caused by enhanced crypt cell proliferation and a reduction in enterocyte apoptosis (313,314). The intestinotrophic actions of GLP-2, which are summarized in Figure 6-3, have been demonstrated in mice (169,315), rats (100,316,317), pigs (318,319), and human subjects (320). GLP-2 also acutely enhances hexose transport (321,322), reduces epithelial permeability (323), enhances gut barrier function (324), and reduces gastric motility and acid secretion (325,326) after exogenous administration in vivo (see Fig. 6-3). Whether GLP-2 action is essential for the growth and survival of the developing or adult mucosal epithelium remains uncertain. Although GLP-2(3-33) modestly attenuates exogenous GLP-2 action, it exhibits properties of both a partial antagonist and a weak agonist (327); hence, optimal tools for analysis of loss of GLP-2 action have not yet been identified.

Gut endocrine L cells Blood flow

ENHANCING INCRETIN ACTION VIA INHIBITION OF DIPEPTIDYL PEPTIDASE-IV The rapid degradation of native GLP-1 has fostered efforts directed at preventing GLP-1 degradation for the treatment of diabetes. DPP-IV, also known as CD26, is a widely expressed cell surface–associated peptidase that also circulates as a soluble form in the plasma. DPP-IV cleaves peptides at the position 2 alanine and appears to be essential for normal glucose homeostasis, because mice with genetic inactivation of CD26 exhibit increased levels of GLP-1 and reduced glycemic excursion after glucose challenge (106). Because DPP-IV is the dominant enzyme regulating GLP-1 degradation (98,103,106), considerable efforts have focused on the development of DPP-IV inhibitors for the treatment of type 2 diabetes. DPP-IV inhibitors reduce blood glucose,

Hexose transport

GLP-2R+ gut endocrine cells

GLP-2

Proliferation Apoptosis

Secondary mediators

Permeability Motility GLP-2R+ Enteric neurons

FIG. 6-3. The actions of glucagon-like peptide-2 (GLP-2) in the gastrointestinal epithelium. GLP-2 secreted from gut endocrine cells acts via gut endocrine cells, myofibroblasts, or enteric neurons to promote diverse actions in the gut epithelium. GLP-2R, glucagon-like peptide-2 receptor.

GASTROINTESTINAL PEPTIDE HORMONES REGULATING ENERGY AND GLUCOSE HOMEOSTASIS / 171 The trophic and cytoprotective actions of GLP-2 also have been examined in the setting of experimental intestinal injury. GLP-2 facilitates mucosal adaptation in rats after major small-bowel resection (317), and intravenous GLP-2 prevents mucosal hypoplasia in rats fed parenterally (328). The extent of mucosal damage is markedly attenuated by exogenous GLP-2 in mice with dextran-sulphate–induced colitis (329) or after indomethacin-induced enteritis (330). Similarly, GLP-2 administration improved intestinal disease activity scores and survival in mice with chemotherapyinduced enteritis (331), in rats with autoimmune enteritis (332), and in rats after occlusion of the superior mesenteric artery (333). Hence, pharmacologic GLP-2 administration promotes intestinal healing and prevents mucosal injury in a diverse number of experimental models of gut injury (234,334).

GLUCAGON-LIKE PEPTIDE-2 ADMINISTRATION TO HUMAN SUBJECTS Because exogenous GLP-2 administration significantly increases the surface area and absorptive capacity of the small-bowel epithelium, GLP-2 or structurally related GLP-2 analogues may exhibit therapeutic potential for the treatment of human subjects with short bowel syndrome (335). Administration of native GLP-2, 400 µg subcutaneously twice daily for 35 days, to human subjects without a terminal ileum and colon significantly improved energy absorption and body weight, and increased lean body mass. GLP-2– treated subjects exhibited reduced enteral fluid loss and reduced gastric emptying but no change in small-bowel transit time (320). The potential therapeutic efficacy of a more potent DPP-IV–resistant GLP-2 analogue currently is being examined in separate clinical trials of patients with Crohn’s disease or short bowel syndrome. GLP-2 also appears to regulate bone mass through direct effects on bone resorption. Administration of GLP-2 twice daily to human subjects with short bowel syndrome reduced markers of bone turnover in a 5-week study (336). Because food ingestion is known to regulate gut hormone release and bone resorption, the relation between gut peptides and bone resorption was studied after single-dose administration of various peptides in healthy postmenopausal women. Although administration of GIP and GLP-1 had no significant effect on bone turnover, GLP-2 reduced circulating levels of the C-terminal telopeptide region of type I collagen and decreased urinary excretion of dihydropyrimidine dehydrogenase (DPD)/ creatinine, both markers of bone resorption (337).

GLUCOSE-DEPENDENT INSULINOTROPIC POLYPEPTIDE Synthesis and Secretion GIP, a 42-amino-acid peptide and the first incretin hormone to be identified (338,339), is synthesized in and released

from intestinal K cells in response to nutrient ingestion. GIP was originally identified on the basis of its ability to inhibit gastric acid secretion in dogs, and subsequently was shown to potentiate glucose-stimulated insulin secretion (338,339). The sequence of GIP is highly conserved across species, with more than 90% identity at the amino acid level for human, rat, mouse, porcine, and bovine GIP. The peptides encoded within the N- and C-terminal segments of pro-GIP have no known biological function. GIP is expressed predominantly in the stomach and in the K cells of the proximal small intestine. GIP mRNA also has been detected in the rat submandibular gland (73). There is little information about the factors that regulate GIP gene expression; however, nutrients up-regulate levels of GIP mRNA in the rat duodenum and submandibular salivary gland, whereas fasting significantly reduces levels of GIP mRNA (340,341). GIP secretion reflects the rate of nutrient absorption, rather than the simple presence of nutrients in the small intestine. Fat is a potent stimulus for GIP secretion in humans, whereas in rodents and pigs, carbohydrates are more effective secretagogues (342). GIP contains an alanine at position 2 and is a substrate for enzymatic inactivation by DPP-IV (293). The half-life of biologically active intact GIP(1-42) is estimated to be less than 2 minutes in rats (103, 343) and approximately 7 and 5 minutes in healthy subjects and patients with type 2 diabetes, respectively (343). After intravenous infusion of GIP in humans, intact bioactive GIP accounted for approximately 40% of the total detectable amount of immunoreactive GIP, whereas only about 20% of total immunoreactive GLP-1 remained intact in the same studies (343). GIP is cleared through the kidney, and levels of GIP are increased in patients with uremia or chronic renal failure.

Biological Actions of Glucose-Dependent Insulinotropic Polypeptide The increasingly diverse actions of GIP on various tissues are summarized in Table 6-2. The dominant actions of GIP on the islet β cell are the enhancement of glucose-dependent insulin secretion via increase of intracellular cAMP, inhibition of adenosine triphosphate–sensitive K+ channels, increase in intracellular Ca2+, and engagement of the β-cell secretory machinery (344). GIP also enhances insulin gene transcription and up-regulates the expression of glucose transporters and glucokinase, which are components of cellular glucose sensors (345). GIP functions as a β-cell growth factor for islet β cells in vitro via activation of cAMP/PKA, MAPK, and PI3K-dependent pathways (346,347). Whether GIP is also important for growth and survival of islet β cells in vivo remains uncertain. GIP also regulates adipocyte lipid metabolism (342) including stimulation of glucose uptake and increases the sensitivity of insulin-stimulated glucose transport. GIP enhances fatty acid production and insulin-stimulated incorporation of fatty acids into triglyceride, augments lipoprotein lipase

172 / CHAPTER 6 TABLE 6-2. Summary of glucose-dependent insulinotropic polypeptide action Pancreas Enhances glucose-stimulated insulin secretion Stimulates insulin gene expression Promotes β-cell proliferation and reduces β-cell apoptosis Adipose tissue Stimulates insulin-dependent glucose uptake Increases insulin receptor affinity Enhances fatty acid synthesis and incorporation into triglyceride Augments lipoprotein lipase synthesis and activity Reduces glucagon-stimulated lipolysis Bone Stimulates alkaline phosphatase activity and collagen type I messenger RNA Increases bone mineral density in rodents Other tissues Up-regulates intestinal hexose transport Stimulates glucocorticoid secretion in rodents Modulates vascular bed-type–dependent endothelial tone

synthesis and activity, and reduces glucagon-stimulated lipolysis in adipose tissue. GIP may also have lipolytic effects in adipocytes (348). The signaling mechanism(s) activated by GIP in adipocytes have not been fully elucidated (342). Intriguingly, GIPR null mice are resistant to diet-induced obesity and exhibit relative reductions in adipose tissue mass after high fat feeding, together with a reduction in expression of the key enzyme Acyl CoA:diacylglycerol transferase 1 (Dgat1) in adipose tissue (186). GIP may also regulate bone formation in osteoblast cells including increases in alkaline phosphatase activity and levels of collagen type I mRNA (178). GIP treatment increases bone mineral density in the ovariectomized rat (349), although acute administration of GIP to human subjects was not associated with changes in markers of bone turnover (337). GIP stimulates glucocorticoid secretion in rats via a cAMP/PKAdependent signaling pathway (350). Although GIP does not appear to regulate cortisol secretion in healthy human subjects (351), aberrant expression of the GIPR in adrenocortical adenomas is associated with the pathogenesis of meal-induced Cushing syndrome (351–353).

Glucose-Dependent Insulinotropic Polypeptide Administration in Human Subjects Because GIP stimulates glucose-dependent insulin secretion, several studies have examined the therapeutic potential of GIP for the treatment of type 2 diabetes. Remarkably, although GIP is a potent insulinotropic agent in healthy humans, GIP action is significantly diminished in human subjects with type 2 diabetes (86,354). Unlike GLP-1, GIP does not significantly inhibit glucagon secretion or gastric emptying in humans (355,356). Although GIP analogs engineered for resistance to DPP-IV action exhibit enhanced insulinotropic properties in preclinical studies (357), the

potential of these analogs to reduce blood glucose in human subjects with type 2 diabetes has not been carefully examined. Hence, the available evidence suggests that GIP is unlikely to be a therapeutic candidate for the treatment of diabetic human subjects (358).

Pancreatic Polypeptide Pancreatic polypeptide (PP) is a structurally related member of the peptide YY/neuropeptide Y (PYY/NPY) family. PP is a 36-amino-acid peptide predominantly expressed in the endocrine pancreas; however, rare PP-immunopositive enteroendocrine cells have been described in some but not all species (359). PP appears to preferentially recognize the NPY 4 receptor (360). PP secretion is stimulated by food ingestion and exercise, and vagal tone is an important determinant regulating PP secretion in rodents and human subjects. Studies in rodents have also demonstrated an anorectic role for either centrally or peripherally administered PP (361). Intriguingly, PP not only reduces food intake but also increases energy expenditure after intraperitoneal administration in genetically obese mice (362). Similarly, transgenic mice with PP overexpression in pancreatic islets exhibit reduced milk intake during the neonatal period, with decreased food intake together with reduced body weight a feature of older PP transgenic mice (362). Although the available evidence clearly implicates PP as a regulator of food intake, whether PP is essential for body weight homeostasis has not yet been determined. The biological actions of PP in human subjects remain somewhat obscure; however, PP, like PYY(3-36), exerts anorectic actions in vivo. Administration of PP reduces food intake in healthy human subjects (363) and in patients with Prader–Willi syndrome (364).

Orexins The orexin peptide families, also referred to as the hypocretins, are produced predominantly in the CNS. Original studies of orexin biology linked orexin action to the control of feeding behavior (365), whereas more recent data have implicated a role for orexins in the regulation of arousal and sleep physiology (366,367). Orexin-like immunoreactivity and orexin receptor mRNA transcripts have also been localized to the enteric nervous system in both the submucosal and myenteric ganglia (368). Intriguingly, fasting activates orexin plus neurons in the gut, and circulating levels of plasma orexin-A are increased after fasting in both rodents and humans (369). Intriguingly, orexin is also secreted from islet α and β cells in a glucose-dependent manner, and administration of orexin-A increases plasma levels of glucagon and glucose in fasted rats (370). Both orexin and the OX2 receptor have been localized to enteroendocrine cells; however, the precise function of the orexin system in gut endocrine cells remains poorly understood.

GASTROINTESTINAL PEPTIDE HORMONES REGULATING ENERGY AND GLUCOSE HOMEOSTASIS / 173 In summary, enteroendocrine peptides exert increasingly complex actions on the control of gut motility, epithelial integrity, cytoprotection, satiety, and pancreatic endocrine function. The actions of many of these peptides, as revealed through studies using antagonists and genetic loss of function mutants, are essential for control of glucose and energy homeostasis. Moreover, GLP-1 and GLP-2 agonists are being evaluated in clinical trials for the treatment of diabetes and short bowel syndrome. Hence, understanding the pleiotropic actions of these peptides has relevance for understanding the biology of gut hormone action and the potential treatment of specific human diseases.

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CHAPTER

7

Growth Factors in the Gastrointestinal Tract John A. Barnard and Kirk M. McHugh Introduction, 184 Growth Factors Defined, 184 Compartmentalization of Growth Factor Function in the Gastrointestinal Tract, 185 “Redundancy” in Growth Factor Families, 185 β Family of Peptides and Transforming Growth Factor-β Receptors, 186 Overview and Discovery of Transforming Growth Factor-β, 186 Transforming Growth Factor-β Genes, 186 Transforming Growth Factor-β Protein and Activation, 186 Transforming Growth Factor-β Receptors and Receptor Interacting Proteins, 187 Smad Signaling Is Induced by Transforming Growth Factor-β, 188 Biological Actions of Transforming Growth Factor-β in the Gastrointestinal Tract, 189 Lessons from Gene-Targeted and Transgenic Mice, 192 Epidermal Growth Factor Family of Peptides and Receptors, 193 Overview and Discovery of the Epidermal Growth Factor Family, 193 Epidermal Growth Factor, 194 Transforming Growth Factor-α, 195 Amphiregulin, 198 Heparin-Binding Epidermal Growth Factor–Like Growth Factor, 198 Betacellulin, 199 Epiregulin, 199 Epigen, 199 Regulated Shedding of Epidermal Growth Factor–Related Peptides by Metalloproteases, 200 Epidermal Growth Factor Receptor Family, 200 Insulin-Like Growth Factors, 205 Overview and Discovery of Insulin-Like Growth Factors, 205

Insulin-Like Growth Factor Genes and Proteins, 206 Insulin-Like Growth Factor Receptor Genes and Proteins, 206 Insulin-Like Growth Factor Binding Protein Gene and Protein Family, 206 Insulin-Like Growth Factor Family Distribution in the Gastrointestinal Tract, 207 Biology of the Insulin-Like Growth Factor Family in the Gastrointestinal Tract, 208 Elimination of Insulin-Like Growth Factor Function by Gene Targeting, 209 Trefoil Factor Family of Peptides, 209 Overview and Discovery of Trefoil Factor Family, 209 Trefoil Factor Family Genes, 210 Trefoil Factor Family Proteins, 210 Trefoil Factor Family Receptors and Binding Proteins, 211 Intracellular Signaling by Trefoil Factor Family, 211 Biological Actions of Trefoil Factor Family Proteins, 211 Hepatocyte Growth Factor, 213 Overview and Discovery of Hepatocyte Growth Factor, 213 Hepatocyte Growth Factor Gene, 213 Hepatocyte Growth Factor Protein, 213 Hepatocyte Growth Factor Receptors, 214 Intracellular Signaling by Hepatocyte Growth Factor, 214 Biological Actions of Hepatocyte Growth Factor, 214 Fibroblast Growth Factor Family, 216 Overview and Discovery of Fibroblast Growth Factor Family, 216 Fibroblast Growth Factor Genes, 216 Fibroblast Growth Factor Proteins, 216 Fibroblast Growth Factor Receptors, 217 Intracellular Signaling by Fibroblast Growth Factor, 218 Biological Actions of Fibroblast Growth Factor, 219 References, 221

J. A. Barnard and K. M. McHugh: Department of Pediatrics, Division of Molecular Medicine, The Ohio State University College of Medicine, Columbus Children’s Hospital, Columbus, Ohio 43205. Physiology of the Gastrointestinal Tract, Fourth Edition, edited by Leonard R. Johnson. Acedemic Press, 2006.

183

184 / CHAPTER 7 INTRODUCTION

Historically, growth factors have been named based on their most prominent biological activity or the activity that led to their discovery. Subsequent work has, in most cases, resulted in the description of activities that are misrepresented by the original name. For example, EGF was discovered and so named because it stimulated epithelial cell growth in the newborn mouse eyelid, but further studies showed that EGF is also a powerful mitogen for fibroblasts. TGF-β was discovered and so named because it induced morphologic transformation of fibroblasts in culture, although, ironically, it ultimately proved to be one of the most important tumor suppressors ever discovered. Some growth factors also exhibit activities that are quite unrelated to cell growth; for example, EGF inhibits gastric acid secretion and hepatocyte growth factor (HGF) stimulates cell motility. Notably, the distinction between the term growth factor and related terms such as cytokine, gut peptide, and hormone is not distinct and other chapters in this volume cover peptides that may, in some classifications, be considered “growth factors.” Polypeptide growth factors modulate cellular functions by several potential pathways. Most do not regulate function by a classical endocrine mechanism, but rather by autocrine, paracrine, or both pathways (Fig. 7-1). A growth factor that regulates cellular function by an autocrine pathway is synthesized and secreted by a cell type that also has functional cellsurface membrane receptors and intact intracellular signaling pathways for that same factor. If adjacent but histogenetically

Growth Factors Defined Growth factors are polypeptides that regulate cell growth and modulate other critical cellular functions by binding to specific homodimeric or heterodimeric, high-affinity, cellsurface membrane receptors. Interaction between ligand and receptor activates intracellular signaling pathways, typically by phosphorylation of cytoplasmic proteins that translocate into the nucleus and specifically modify gene expression. Growth factors usually are secreted, but may also be found in transmembrane, cytosolic, and extracellular matrix (ECM)– associated compartments. Biological effects usually are apparent at extremely low concentrations, usually in the nanogram per milliliter range. Structurally and biologically related growth factors are classified into “families,” although this is not necessarily reflected in the confusing nomenclature of individual growth factors. For example, two of the most relevant and extensively studied growth factor families in gastrointestinal biology are the transforming growth factor-β (TGF-β) and the epidermal growth factor (EGF) families. TGF-β1, a member of the TGF-β family, is not related structurally or biologically to transforming growth factor-α (TGF-α), which is a member of the EGF-related peptide family, and is closely related to EGF structurally and functionally.

2

1

3

Epithelial cells

Mesenchymal cells

Splanchnic vasculature

FIG. 7-1. Paradigms for growth factor signaling in the gastrointestinal tract. Polarized epithelial cells are shown in relation to mesenchymal elements in the lamina propria and the splanchnic vasculature. Endocrine (1), paracrine (2), and autocrine (3) signaling pathways are shown; the pathways for each growth factor family are described in detail in the text.

GROWTH FACTORS IN THE GASTROINTESTINAL TRACT / 185 different cell types also possess functional receptors and signaling pathways, these cells are regulated by a paracrine route. Juxtacrine growth regulation occurs when a growth factor anchored in the cell-surface membrane stimulates a biological activity in an adjacent cell (1). Intracrine growth regulation occurs when a polypeptide growth factor binds to an intracellular receptor and is biologically active without exiting the cell (2). It is likely that most growth factors contribute to cellular and tissue homeostasis by simultaneous use of multiple pathways. The biological significance of autocrine growth factor pathways is considerable. Autocrine regulation of normal cellular homeostasis is an intricate servomechanism for fine regulation of biological function and cellular homeostasis. However, disruption of autocrine control may have devastating pathophysiologic consequences. This is best exemplified by the autocrine hypothesis that emerged in the early 1980s to explain the unchecked growth characteristic of malignant cells in human neoplastic disorders; that is, malignant cells secrete stimulatory polypeptide factors for which the cells themselves have functional receptors (3). Numerous examples now exist in support of amplified autocrine growth stimulatory pathways or interrupted autocrine growth inhibitory pathways leading to human cancers. This chapter discusses many examples of normal physiologic responses and pathophysiologic states that may result from a disturbance in this intricate balance of regulatory control.

Compartmentalization of Growth Factor Function in the Gastrointestinal Tract Perhaps more so than other organ systems, study of the biological activity of growth factors in the gastrointestinal tract requires consideration of multiple complex spatial variables. The apical domain of the gastrointestinal epithelium contacts a highly heterogeneous external environment, including dietary constituents and endogenous luminal factors such as proteases, extremes of pH, bacteria, and growth factor– laden secretions from more proximal organs. The basolateral domain of the epithelium is largely protected from these intraluminal influences by the tight junction, but this cell surface contacts extracellular fluids and is subject to physiologic and pathophysiologic alterations therein. A healthy tight junction maintains this spatial compartmentalization. However, in the context of epithelial injury and disruption of tight junctions, growth factors and their receptors that are ordinarily spatially segregated may contact and exert biological activity in cell or tissue compartments to which they are not usually exposed. Other compartmental variables to be considered are the horizontal (proximal-to-distal) and vertical (crypt-to-surface epithelium) axes in the gastrointestinal tract. Thus, a large number of complex variables contribute to growth factor biology in the gastrointestinal tract. With the exception of the EGF-related peptide family, little progress has occurred in the understanding of these spatial relations. Later, this chapter makes an effort to synthesize what is

known about these spatial relations into three dimensions for the EGF-related peptide family.

“Redundancy” in Growth Factor Families Virtually every growth factor family is composed of multiple structurally and biologically related proteins, implying a potential for redundancy of biological function. The concept of redundancy should be viewed with caution. True, in the artificial “one-dimensional” setting of cultured cells, studies clearly support functional redundancy in most growth factor families. However, the development of growth factor knockout mice in the early to mid-1990s emphasized that complex spatial, compartmental, and kinetic considerations must be invoked in the analysis of growth factor function. Under these terms, redundancy is much less commonly observed. Analysis of EGF and TGF-α, two of the best-studied members of the EGF-related peptide family, is illustrative of a potentially redundant system that, in reality, is much less redundant than might be predicted. In cell culture, these growth factors are usually equipotent in stimulation of cell proliferation and migration. However, differential activity potentially exists at the cell, tissue, and organ levels, and it may explain differences in biological activity between TGF-α and EGF in more complex models. At the cellular level, differences in receptor–ligand interaction are observed. For example, although EGF and TGF-α bind the mammalian EGF receptor (EGFR) with similar affinity, Winkler and colleagues (4) developed an anti-EGFR antibody that markedly inhibits TGF-α when compared with EGF binding, suggesting that the two peptides bind differently to the same receptor. This line of evidence is further supported by differential binding of mammalian TGF-α and EGF to NIH3T3 cells transfected with a chicken EGFR (5). Moreover, there are convincing data that intracellular routing and degradation of TGF-α receptor and EGFR complexes are different, partly because of differences in the isoelectric point of the two proteins (6). An integrated quantitative model of endocytic trafficking of EGF and TGF-α predicts whether signaling begins at the cell membrane in the case of TGF-α, or the endosomal compartment in the case of EGF (7). Compartmentalization of the polarized epithelial cell membrane into brush border and basolateral domains raises interesting questions regarding targeting of the transmembrane precursors of TGF-α and other EGF-related peptides to the cell surface, as well as the localization of EGFRs on these two domains. TGF-α is sorted to the basolateral membrane with approximately 95% efficiency in Madin– Darby canine kidney (MDCK) cells (8), an observation with important implications regarding biological activity in vivo. This observation is even more intriguing in light of subsequent observations that EGF is targeted to the apical domain of MDCK cells (9). Additional considerations occur at the tissue and organ levels. For example, that EGF is not synthesized by the normal small intestinal epithelium, whereas TGF-α is synthesized

186 / CHAPTER 7 by cells in the differentiated compartment of the epithelium (10,11) suggests that cell and tissue compartmentalization of EGF and TGF-α synthesis may result in major differences in activity. Thus, TGF-α is a more physiologic ligand for the small intestinal epithelial EGFR than is EGF. Similar paradigms of differential growth factor distribution have been observed in other organ systems (12). In pathophysiologic situations, alternate patterns of growth factor expression may be observed. For example, Wright and coworkers (13) described emergence of a novel EGF-producing cell lineage in chronically injured gastrointestinal mucosa. EGF is not normally produced in the small bowel, with the possible exception of Brunner’s glands of the duodenum, (14); thus, expression may depend, in part, on the coexistence of cellular injury and other pathophysiologic processes. Other examples of EGF-related peptides demonstrating differential biological activity despite structural similarity and affinity for the same receptor are observed in the complex milieu of the gastrointestinal luminal contents. Fasting human pancreatic juice destroys both TGF-α and EGF, whereas feeding the milk protein casein preserves EGF but not TGF-α bioactivity (15). EGF and TGF-α are also partially degraded by gastric juice, resulting in attenuation of bioactivity (16). These studies highlight the potential contribution of luminal and extracellular events on differential bioavailability of EGF-related peptides.

β TRANSFORMING GROWTH FACTOR-β FAMILY OF PEPTIDES AND RECEPTORS Overview and Discovery of Transforming β Growth Factor-β TGFs were first identified in 1978 as a reversible, transforming activity in cell culture medium conditioned by murine sarcoma virus–transformed mouse fibroblasts (17). Treatment of normal fibroblastic cells suspended in soft agar with a partially purified protein preparation from transformed mouse fibroblasts stimulated growth of colonies (anchorage-independent growth) and morphologic transformation; hence, the term transforming growth factors. Purification of the transforming activity identified two structurally unrelated peptides: TGF-α and TGF-β (18,19). Thereafter, the history of these two important growth regulatory proteins diverges rather sharply and has left in its wake a confusing nomenclature. Currently, three mammalian TGF-β “isoforms” are designated TGF-β1, TGF-β2, and TGF-β3. TGF-β is the prototype for a superfamily of structurally related, evolutionarily conserved peptides that share a cluster of conserved, disulfide-linked cysteine residues and exert a multitude of biological activities related to cell growth and differentiation, morphogenesis, and cell fate determination. The TGF-β superfamily includes activins and inhibins (20), bone morphogenetic peptides (21,22), nodal (23), growth and differentiation factor, Mullerian inhibiting substance (24),

Drosophila decapentaplegic peptide (25), and TGF-β, among others. TGF-β–related peptides are synthesized by a wide variety of cell types as large precursor molecules undergo regulated activation to release the biologically active protein. In some instances, activation occurs intracellularly, and mature peptide is secreted bound to a peptide antagonist. Other members of the family undergo regulated activation after secretion into the extracellular space. Cell-surface receptors and their primary intracellular signaling proteins also are related structurally and are conserved within the TGF-β superfamily. By far, the most extensively studied TGF-β superfamily member in gastrointestinal biology is TGF-β, which is the focus of this section. β Genes Transforming Growth Factor-β The three mammalian TGF-β growth factor family members have nearly identical biological activity in cell culture systems, but complex and distinctive phenotypes in gene-targeted and transgenic mouse models. The proteins are designated TGF-β1, -β2, and -β3 and are often referred to as TGF-β isoforms (26). The genes have been cloned in humans and many other species. In humans, TGF-β1, -β2, and -β3 are localized to chromosomes 19, 1, and 14, respectively (27,28). Major structural differences exist in the 5′-flanking regions of each gene, suggesting that observed differences in tissue distribution and differential regulation of TGF-β expression may be mediated by gene transcription. Transactivating and cis-acting factors in the promoters of all three isoforms have been extensively mapped and characterized (29–33). β Protein and Activation Transforming Growth Factor-β The three mammalian TGF-β isoforms share approximately 70% amino acid homology. All are first synthesized and secreted in biologically inactive, latent precursor complexes that are not structurally competent to bind to cell-surface TGF-β receptors. “Activation” of TGF-β occurs when the mature, biologically active form is liberated from the precursor complex by a process described later. Mature, activated TGF-β is a 25-kDa homodimer that functionally interacts with the receptor. The carboxy terminal 112 amino acids exhibit 70% to 75% conservation of sequence between isoforms and 100% conservation of the 9 cysteine residues. The amino-terminal precursor sequences are strikingly dissimilar (34). The overall amino acid sequence for mature isoforms is highly conserved in mammals. Expression of individual isoforms in cells and tissues in vivo is highly variable, especially during embryogenesis (35), perhaps accounting for the variable phenotype observed in gene-targeted mice. Activation of TGF-β under physiologic and pathophysiologic situations has been a relatively neglected area of TGF-β research, despite its critical importance in regulation of TGF-β activity. Latent TGF-β consists primarily of the N-terminal TGF-β precursor sequence, termed the

GROWTH FACTORS IN THE GASTROINTESTINAL TRACT / 187 latency-associated peptide (LAP), noncovalently associated with the mature, C-terminal TGF-β sequence (36,37). The latent TGF-β complex also includes a second gene product, the latent TGF-β binding protein (LTBP). Four LTBPs have been cloned and fully sequenced (38). These molecules are structurally closely related to fibrillins, large extracellular glycoproteins with EGF-like and cysteine repeats. LTBPs are structurally unrelated to TGF-β and associate with the latent molecule by disulfide bridging. Mature, biologically active TGF-β is released from the latent complex by physicochemical phenomena such as extremes of pH, but the physiologic mechanism operative in vivo is less well understood. Proteolytic activation by proteases such as plasmin has been extensively characterized in vitro (39). Thrombospondin has been proposed as an attractive physiologic activator in vivo, based on the observation that plasminogen null animals do not replicate the abnormalities observed in TGF-β1–deficient mice, whereas thrombospondin null mice do (40). It has been hypothesized that thrombospondin confers a conformational change in the TGF-β latent complex by specifically binding to sequences in the LAP, releasing mature TGF-β for binding to TGF-β receptors. Other factors contributing to the activation of latent TGF-β include the presence of mannose-6 phosphate residues on the LAP (41a), transglutaminase-dependent cross-linking of latent TGF-β to ECM proteins (41b), and interaction with αvβ6 integrin (42). Finally, other extracellular proteins may bind TGF-β after activation, further modifying its activity. Some of these include decorin, biglycan, and α2-macroglobulin (43,44). β Receptors Transforming Growth Factor-β and Receptor Interacting Proteins The TGF-β family of receptors also has a complex nomenclature (45). Herein, this chapter focuses on receptors that bind the three mammalian TGF-β isoforms, fully recognizing that other members of the TGF-β superfamily such as the activins and bone morphogenetic proteins (BMPs) bind to structurally related TGF-β receptors and activate closely related signaling pathways that result in closely related biological activities. TGF-β receptors originally were described based on crosslinking patterns to 125I-TGF-β and designated receptor types I (~53 kDa), II (~70 kDa), and III (~300 kDa). Each receptor, now named TGF-βRI (or Alk5 in some systems), TGF-βRII, and TGF-βRIII, was cloned and extensively characterized in the early 1990s. The TGF-β receptors have an extracellular ligand-binding domain, a transmembrane sequence, and an intracellular sequence. Intracellular serine/threonine kinase domains are present in type I and II receptors, whereas the intracellular domain of the type III receptor has no known catalytic activity (46–48). All three isoforms of TGF-β bind to receptor types I, II, and III, but exceptions exist (see later in this chapter). Once the ligand is appropriately engaged, TGF-βRI and TGF-βRII heterodimerize to form the active

signaling complex. TGF-βRIII (betaglycan) is thought to function by binding TGF-β and increasing its affinity for TGF-βRII. TGF-β signaling is initiated from a heterotetrameric receptor complex involving TGF-βRI and TGF-βRII (49) (Fig. 7-2). In the basal state, TGF-βRII is constitutively homodimerized and constitutively autophosphorylated in a ligand-independent manner by its intrinsic serine/threonine kinase activity (50). Signaling from TGF-βRII does not occur in the absence of TGF-βRI, even when a ligand is engaged (51–53). TGF-βRI also forms ligand-independent homodimers, but it is unable to bind TGF-β in the absence of TGF-βRII. Ligand binding to TGF-βRII recruits TGF-βRI into a heterotetrameric receptor complex resulting in transphosphorylation and activation of TGF-βRI. Multiple TGF-βRII– mediated serine and threonine phosphorylation sites are present in the Gly/Ser (GS)-rich domain of TGF-βRI, which is located just C terminal to the juxtamembrane domain. A T204D (aspartic acid substitution for threonine) mutation in the GS region of TGF-βRI results in a constitutively active receptor, and this mutant receptor has become a valuable reagent for the study of TGF-β signaling in vitro (54). TGF-βRIII, or betaglycan, is an abundant, heavily glycosylated, high-molecular-weight proteoglycan that migrates diffusely in the range of 280 to 330 kDa in polyacrylamide gels (48). Endoglin, another TGF-βRIII, is found predominantly in endothelial cells (55). Mutations in an activin receptor-like kinase (Alk-1) and endoglin lead to altered TGF-β signaling and vascular arteriovenous malformations comprising the clinical entity termed hereditary hemorrhagic telangiectasia (56). The TGF-βRIII receptors are believed to function as “reservoirs,” acting as a sump for TGF-β and a presenting receptor for the active signaling TGF-βRI/ TGF-βRII complex. Like TGF-βRI and TGF-βRII, TGF-βRIII is constitutively homodimerized. Multiple lines of evidence support the hypothesis that betaglycan functions to facilitate high-affinity binding of all three TGF-β isoforms to TGF-βRII. This is especially true for TGF-β2, which has a low affinity for TGF-βRII in the absence of TGF-βRIII, but a 10-fold enhancement of activity when betaglycan is coexpressed (57). Data suggest that endoglin binds only TGF-β1 and TGF-β3 in the presence of TGF-βRII (58). Both endoglin and betaglycan can be coimmunoprecipitated with TGF-βRII (59,60). Many TGF-β receptor interacting proteins have been identified in cell culture experiments, but the exact importance of most of these in human biology needs further study. Examples of TGF-β receptor interacting proteins include the FK-506 binding protein FKBP12 (61), the α subunit of farnesyl transferase (62), serine threonine receptor-associated protein (STRAP) (63), SARA (Smad anchor for receptor activation) (64), Bα (a subunit of phospholipase A2) (65), Daxx (66), Disabled-2 (67), transforming growth factor-β receptor interacting protein (TRIP)-1 (68), tumor necrosis factor receptor-associated protein-1 (TRAP)-1 (69), TLP (TRAP-1 like protein) (70), SMIF (71), and mammalian light chain 7 (mLC7)-1 (72). All bind to cytoplasmic sequences in TGF-βRI or TGF-βRII and modulate TGF-β signaling.

188 / CHAPTER 7 Smad2

Smad4

Smad6/7 P P

P

P P

Smad3

P

P P Smad complex TGFβRI + TGFβRII P

P

P

P

Target gene -growth inhibition -differentiation

TGF TGFβ

FIG. 7-2. Transforming growth factor-β (TGF-β) signaling in the gastrointestinal epithelium. TGF-β is secreted from the basolateral surface of polarized intestinal epithelial cells and activates heterotetrameric TGF-β receptor (TGF-βR) complexes, also on the basolateral membrane domain. The primary growth inhibitory signaling pathway activated by TGF-β is the Smad pathway. Negative feedback from inhibitory Smad6 and Smad7 are also shown. The primary outcome of TGF-β signaling in the intestinal epithelium is growth inhibition.

Individual discussion of these proteins is beyond the scope of this chapter, but the role of FKBP12 on TGF-β signaling is illustrative of how these proteins potentially modulate TGF-β signaling. FKBP12 binds to the GS region of TGFβRI and is believed to help stabilize its inactive conformation. Phosphorylation of the GS region by TGF-βRII is predicted to dissociate FKBP12, creating a conformation for signaling. Thus, FKBP12 is a brake on TGF-β signaling.

Smad Signaling Is Induced by Transforming β Growth Factor-β TGF-β signaling occurs primarily by activation of Smad proteins, although a relatively substantial body of literature describes Smad-independent signaling by TGF-β (26,73). Herein, this chapter focuses on Smad signaling, a signaling cascade unique to the TGF-β pathway. Nine distinct Smad proteins have been identified in mammals. These are highly conserved signaling molecules related to “Mad” proteins in Drosophila and “Sma” proteins in Caenorhabditis elegans, hence the designation “Smads.” Smads are categorized as receptor-regulated R-Smads (Smad1, Smad2, Smad3, Smad5, Smad8, and Smad9), commonpartner Co-Smads (Smad4), and inhibitory Smads (Smad6 and Smad7) (74). For mammalian TGF-β signaling, Smad2 and Smad3 are the R-Smads, whereas activins and BMPs activate alternate R-Smads. Phosphorylation of Smad2 and Smad3 by TGF-βRI is necessary for TGF-β signaling. Phosphorylation by the TGF-βRI kinase occurs on serine residues in a highly

conserved SSXS motif in the MH-2 domain (75). Smad4, which does not contain this motif, is not phosphorylated. SARA facilitates interaction among Smad2, Smad3, and TGF-βRI (64). Phosphorylated Smad2 and Smad3 form heteromeric complexes with Smad4 (76). This configuration of Smads is believed to be required for translocation of the TGF-β signal into to the nucleus. However, variations on this “traditional” model of TGF-β signaling are increasingly being invoked to explain differences in the genomic and biological responses to TGF-β in models of diminished or deficient Smad2 or Smad3 function (see review by Flanders [77]). TGF-β signaling is negatively regulated at multiple points in the ligand/receptor/signaling cascade. Key proteins involved in negative regulation of mammalian Smad signaling are Smad6 and Smad7 (78,79). Although structurally related, Smad6 and Smad7 lack the SSXS phosphorylation site required for activation of Smad2 and Smad3. Both are induced by TGF-β treatment and exert negative feedback on TGF-β signaling (80). Smad7 binds to activated TGF-βRI and inhibits phosphorylation of Smad2, assembly of Smad complexes, and nuclear translocation of Smads (79). Smad6 and Smad7 also recruit E3-ubiquitin ligase activities known as Smurfs to the activated TGF-βRI, resulting in ubiquitination and degradation of the receptor (81). Other potent negative regulators of Smad function are the c-SnoN and c-Ski nuclear oncoproteins. C-SnoN and c-Ski interact with Smad2, Smad3, and Smad4 in the nucleus to repress transcription of TGF-β–responsive genes (82). After TGF-β exposure, c-SnoN is rapidly targeted for ubiquitin-dependent degradation by the proteasome (83), releasing TGF-β target genes from

GROWTH FACTORS IN THE GASTROINTESTINAL TRACT / 189 transcriptional repression. Later, c-SnoN is markedly up-regulated, a presumed mechanism for negative feedback of TGF-β signaling (84). As the Smad complex accumulates in the nucleus in response to TGF-β signaling, transcription is modulated, both positively and negatively, by direct binding to DNA and interaction with a large number of potential DNA-binding proteins. Smad3 and Smad4 bind to the Smad-binding element (SBE) with low affinity, whereas Smad2 does not bind at all (85). The SBE consists of the 4-base-pair sequence 5′-AGAC-3′ that contacts a specific domain in the MH-1 region of Smad3 and Smad4. Because the SBE is short, interactions with Smad3 and Smad4 occur with low affinity. The SBE also occurs often in the mammalian genome. Thus, it is interesting that TGF-β does not signal promiscuously and nonspecifically. TGF-β also leads to both gene activation and gene repression in a given cell, depending on the target gene. These complexities are explained by the extensive repertoire of cell type–specific transcriptional corepressors and coactivators available to form a transcriptional complex with Smad proteins. In their review, Shi and Massague (26) point out “much work is needed for the delineation of cell type specific gene responses that mediate the action of the TGF-β family.” Although a great deal is known about the multifaceted biological response to TGF-β, the specific transcriptional programs induced by a growth inhibitory versus an angiogenic response, for example, is likely a function of the specific expression and stoichiometry of Smad coactivators and repressors within a given cell type. That is, the TGF-β response is read in different ways depending on the cellular context and the portfolio of available transcription factors. Consideration of all the potential DNA-binding partners is beyond the scope of the present application; however, many of these are catalogued in Table 7-1 and include well-characterized transcriptional modulators such as histone deacetylases and intermediate binding partners such as mSin3A and NcoR (86). Several merging concepts in TGF-β signaling deserve comment. These relate to progress in the understanding of how Smad proteins are partitioned between the cytoplasm and nucleus in the quiescent state and in response to receptor activation. First, Smad distribution within the cell is actively TABLE 7-1. Smad-associated transcriptional regulators Sequence-specific transcription factors Jun/Fos Sp1 Basic helix-loophelix (bHLH) CREBP E2F ATF-2 β-catenin Others

Coactivators

regulated by nucleocytoplasmic shuttling. It is clear that Smad2 and Smad3 are predominantly retained in the cytoplasmic compartment in the absence of TGF-β, but Smad4 is distributed in both the nuclear and cytoplasmic compartments. Evolving theories and pathways are being explored to more clearly understand how nuclear pore complexes and cytoplasmic and nuclear retention factors contribute to Smad localization and function (87). Second, it has become clear that TGF-β receptor signaling and Smad activation occur in distinct endocytic compartments, including caveolin-1 lipid raft–dependent and clathrin-dependent pathways. Segregation of receptor and Smads in these compartments may contribute to the intensity and duration of Smaddependent TGF-β signaling (88). Finally, it has been determined that Smad translocation into the nucleus is critically dependent on the scaffolding protein ELF, because ELF β-spectrin–deficient mice have a phenotype similar to Smad mutant mice (89). A variety of additional signaling pathways are activated in response to TGF-β. In some instances, these pathways directly influence Smad signaling, whereas in others, signaling may occur in parallel with or independent of Smad activation. A critical challenge in the field will be to integrate all TGF-β signaling into a clear comprehension of how the multitude of TGF-β biological responses occurs. In some cell types, TGF-β induces mitogen-activated protein kinase (MAPK) activity (90), whereas it does not in others (91). It has been suggested that increased MAPK activity in oncogenic Ras-transformed cells phosphorylates Smad2 and Smad3 in the linker domain, resulting in defective translocation of the Smad complex into the nucleus and TGF-β resistance (92). Conversely, others have found that EGF and HGF both appear to phosphorylate Smad2 and induce Smad2dependent transcriptional responses (93). These interactions clearly deserve further study. c-Jun N-terminal kinases (JNKs) phosphorylate Smad3 outside its -SSXS motif and facilitate TGF-β signaling (94). A TGF-β–stimulated MAPK kinase kinase (TGF-β activated kinase [TAK-1], TGF-β activated kinase 1) and downstream cascade has been identified (95), together with activating, interacting proteins TAK-1 binding protein (TAB)1 and TAB2 (96). TAK-1 activity simulates nuclear factor (NF)-κB activation (97). TGF-β signaling via protein kinase C (PKC) (98), Rho-like GTPases (99,100), p38 (101), and JNKs (94,99,102) have been reported and are reviewed extensively by Yue and Mulder (73).

Corepressors

p300/CBP ARC105 Smif

c-ski c-SnoN TGIF

Others

Histone deacetylase complex (HDAC) mSin3 N-CoR Others

CBP, CREB-binding protein, CREBP, cyclic AMP response element binding protein.

β Biological Actions of Transforming Growth Factor-β in the Gastrointestinal Tract TGF-β is described as a “multifunctional” or “pleiotrophic” peptide because of its diverse biological activities, many of which have important implications for gastrointestinal homeostasis. Alterations in TGF-β signaling have been extensively studied in the context of gastrointestinal neoplasia. These and other diverse activities of TGF-β are reviewed later.

190 / CHAPTER 7 Differentiation and Inhibition of Cell Growth The three mammalian TGF-β isoforms are potent and reversible growth inhibitors in epithelial cells, including those of the gastrointestinal tract (103,104). Although TGF-β is a differentiating factor in certain cellular contexts (105), a complete program of enterocyte differentiation is not induced by TGF-β. Under precise experimental conditions, TGF-β may induce limited characteristics of the differentiated phenotype in vitro (103,106). TGF-β arrests cells growth in the mid and late G1 phase of the cell cycle by inhibition of critical cellular events required for completion of G1 traverse and S-phase entry. Important early G1 events include inhibition of the growthpromoting transcription factor c-myc (107) and stimulation of c-jun expression (108). Late G1 events include inhibition of expression or activity of cyclin-dependent kinase activities (e.g., cyclin D, cyclin E, cyclin A, and their respective catalytic partners, the cyclin-dependent kinases: cdk2, cdk4, and cdk6) (26,109–114). TGF-β may also stimulate cyclin-dependent kinase inhibitor activities such as p21/waf1/cip1 (115), p27/kip (116), and p15/ink (117). To a large extent, the specific mechanism appears to be dependent on the cell type being studied. In cultured intestinal epithelial cells, down-regulation of cyclin D1 expression is a key step in G1 arrest induced by TGF-β (118). A result of depressed cyclin-dependent kinase activity is inhibition of phosphorylation of the retinoblastoma gene product, leading to a late G1 cell cycle arrest (111). Apoptosis, or programmed cell death, is critical for maintenance of the gastrointestinal epithelium. Although TGF-β induces or suppresses apoptosis depending on the cell type being studied, in the intestinal epithelium, TGF-β induces apoptosis in vitro (119,120) and in vivo (121). The mechanisms regulating TGF-β induction of apoptosis in the intestine are not well understood, but they appear to be Smad3 dependent (120) and mediated in part by down-regulation of Bcl-2 expression (122). In summary, TGF-β is a critical autocrine and paracrine growth inhibitor in epithelial cells, including those of the gastrointestinal tract. Consequently, it is not surprising that TGF-β, its receptors, and its signaling transduces all have tumor suppressor functions; this concept is explored later in this chapter. Although TGF-β is widely appreciated as a potent growth inhibitor in epithelia, it stimulates growth of fibroblastic cells. The kinetics of TGF-β–stimulated growth are delayed, suggesting involvement of a secondary mitogen. This is believed to occur by induction of the profibrotic cytokine, plateletderived growth factor (PDGF) (123). One reported study indicates that Smad-independent activation of c-abl kinase activity may be an important mediator of the proliferative effect of TGF-β on fibroblasts (124). Induction of Extracellular Matrix Formation and Fibrosis A major biological effect of TGF-β is modulation of cells and genes that contribute to ECM deposition. The result is a

prominent increase in ECM formation, recruitment of fibroblasts, and fibrosis. As a consequence of this potent activity, TGF-β has been implicated in fibrotic diseases of virtually every organ system, including the intestine, pancreas, and liver. TGF-β induction of fibrosis occurs by multiple pathways, including increased synthesis of ECM proteins (collagen, fibronectin) and proteinase inhibitors (plasminogen activator inhibitor), decreased synthesis of matrix metalloproteinases (MMPs; plasminogen activator, collagenase, elastase), and modulation of matrix receptors and binding proteins (integrins). Several comprehensive reviews highlight the effect of TGF-β on ECM (125–127). It has been of long-standing interest to determine whether signaling pathways responsible for the fibrogenic response to TGF-β are separate and distinct from the growth responses. This distinction remains relatively unclear but is an area of intense ongoing investigation, because it may ultimately have important therapeutic consequences. It appears increasingly clear that most of the fibrogenic responses to TGF-β are dependent on Smad3, because Smad3-deficient mice are resistant to fibrosis in several models (77). Although these studies are convincing, it remains difficult to reconcile these observations with other published work. For example, in a widely referenced study, Hocevar and coworkers (128) described fibronectin synthesis in a Smad4-deficient fibrosarcoma cell line, indicating independence from Smad4, and presumably Smad3, signaling. The effect of TGF-β on ECM synthesis appears to have clinical significance because a large number of fibrotic diseases are associated with increased TGF-β activity, and TGF-β increases wound healing in several models, including incisional wounds in the gastrointestinal tract (129). This may occur as a result of increased synthesis of collagen by human intestinal smooth muscle cells (130). Notably, however, unexpected observations have been made in models of wound healing in Smad3 gene–targeted mice. In these mice, wound healing is accelerated in the absence of Smad3. More analysis is needed to ascertain the role of TGF-β in the complicated milieu of a wound, which includes inflammatory cells, epithelial cells, fibroblasts, and myofibroblasts that respond in well-characterized stages of wound healing such as reepithelialization and wound contraction. Each of these stages may be differentially affected by TGF-β. Effect on Immune Function TGF-β is produced by many immune system cells, including T cells, B cells, macrophages, neurokinin cells, and others. The effects of TGF-β on the immune system are complex because both proinflammatory and immunosuppressive activities are modulated by TGF-β (131). The marked inflammation observed in the gastrointestinal mucosa of TGF-β1 gene–targeted mice (see later in this section) underscores the importance of TGF-β1 as an immune suppressor in the intestine (131–133). In mice, TGF-β promotes the expansion and suppressive function of CD4+/CD25+ T-regulatory cells (134), perhaps by induction of the transcription factor Foxp3.

GROWTH FACTORS IN THE GASTROINTESTINAL TRACT / 191 Transgenic mice with impaired TGF-β signaling in T cells are more susceptible to dextran sodium sulfate–induced colitis (134). Similarly, transgenic mice overexpressing a dominant negative TGF-βRII directed to the intestinal epithelium by an intestinal trefoil factor promoter or a liver fatty acid binding protein promoter are also markedly more sensitive to induction of colitis by dextran sodium sulfate (135,136). Interestingly, TGF-β levels are increased in the colonic mucosa of patients with inflammatory bowel disease, a disorder characterized by excessive immune response to mucosal antigens (137). This apparent paradoxical observation has been potentially explained by increased mucosal levels of inhibitory Smad7 and impaired TGF-β signaling in active inflammatory bowel disease (138). One of the most prominent effects of TGF-β on immune cell function is its activity as an IgA switch-inducing factor in naive B cells. TGF-β induces IgA− B cells to initiate IgA synthesis and secretion, which is a key observation because IgA is the dominant isotype in the gastrointestinal tract (139,140). The immunosuppressive effects of TGF-β may also have important implications for colorectal tumorigenesis. Early studies in fibrosarcoma cells suggested that TGF-β suppresses tumor immune surveillance, perhaps favoring establishment of metastasis (141). More recent studies show that TGF-β production by tumor-infiltrating T lymphocytes suppresses tumor growth (142). Stimulation of Cell Migration and Angiogenesis TGF-β promotes cell migration and chemotaxis in a broad variety of cell types, including fibroblasts, neutrophils, glial cells, and intestinal epithelial cells (126,143,144). The concentration of TGF-β required to stimulate migration is far less than that required for modulation of cell growth. In a model of intestinal epithelial wounding, TGF-β expression is upregulated, and restitution of the epithelial surface is accelerated (145). Conversely, loss of TGF-β signaling diminishes wound healing and renders mouse models more susceptible to chemically induced colitis (136). TGF-β appears to be much less potent than growth factors such as EGF or HGF in models of chemotaxis (directed migration) in intestinal epithelial cells (144). Some studies suggest migration in epithelial cells is stimulated by activation of Smad-dependent pathways, as well as p38 and JNK pathways (146). As a result of its pleiotrophic effects on cellular migration, cell proliferation, and ECM deposition, TGF-β is a major contributor to blood vessel growth and angiogenesis (147). Defects in vasculogenesis occur in TGF-β1 (148), TGF-βRI (147), and TGF-βRII gene−targeted mice (149). TGF-β stimulates expression of genes such as vascular endothelial growth factor (VEGF) (150,151), basic fibroblast growth factor (bFGF), and cyclooxygenase-2 that contribute to angiogenesis, as well as a large number of genes expressed in endothelial cells related to matrix deposition, a necessary precursor for angiogenesis (150,152). VEGF- and bFGFstimulated invasion of matrices in vitro is further supported

by low concentrations of TGF-β (pg/ml range) (153). The proangiogenic properties of TGF-β are believed to contribute to its tumor-promoting effects late in oncogenesis (154). Transforming Growth Factor-β Expression in the Normal Gastrointestinal Tract In the mouse intestinal epithelium, immunoreactivity for all three TGF-β isoforms is seen beginning midgestation (155). All three TGF-β isoforms are found in the adult normal gastrointestinal mucosa (10,104,156), but the distribution along the vertical axis of the crypt-villus unit and the epithelial and nonepithelial compartments of the intestine has been reported in various forms. The preponderance of data suggest TGF-β1 immunostaining in the colon is greatest in differentiated epithelial cells, although expression in lamina propria cells is also quite prominent (104,121,137,156–162). Expression of TGF-βRII protein in the murine and human small intestine and colonic epithelium occurs in a distribution nearly identical to ligand; that is, increased expression in the differentiated compartment and little or no expression in the proliferative compartment (156,163,164). Expression of TGF-βRI and TGF-βRIII in normal intestine has not been examined extensively, but it appears to mirror, in part, the distribution of ligand (165). Studies show that TGF-βRII is localized and activation of Smad phosphorylation occurs from the basolateral domain of polarized intestinal epithelial cells (166). Collectively, data on the distribution of TGF-β and its receptors in the gastrointestinal epithelium support the view that TGF-β is a major autocrine or paracrine inhibitory axis, or both, in the intestinal epithelium. A Complex Role for Transforming Growth Factor-β in Gastrointestinal Cancer Resistance to the tumor-suppressive actions of TGF-β is common and occurs by genetic and epigenetic mechanisms. Progress has delineated well-described genetic defects in the TGF-β tumor suppression pathway. Examples of genetic defects include: (1) inactivating mutations in noncoding polyadenine repeats of the TGF-βRII gene in individuals with microsatellite instability, including hereditary nonpolyposis colon cancer syndrome (167,168); (2) sporadic genomic mutations in TGF-βRII (169,170); (3) mutations in Smad proteins including sporadic, inactivating, missense mutations in Smad2 and Smad4 (171–173) and germline Smad4 mutations, primarily in exon 9, in juvenile polyposis (174). Epigenetic defects in TGF-β signaling are also increasingly recognized. Epigenetic defects in TGF-β signaling also occur in a significant fraction of colon cancer cells, such that in total, more than 90% of colorectal cancers are resistant to growth inhibition by TGF-β. An important epigenetic mechanism for resistance to growth suppression by TGF-β is oncogenic activation of Ras, a guanine nucleotide binding protein that plays a central role in organizing and transmitting cellular growth signals. Activating Ras mutations occur in more than 50% of

192 / CHAPTER 7 colorectal cancers (175), and Ras-transformed cells in culture are resistant to growth inhibition by TGF-β (92,176–182). Proposed mechanisms, among others, include decreased expression of TGF-βRII (177,182), ineffective Smad complex assembly and nuclear translocation (92), and degradation of Smad4 before nuclear translocation (183). For example, Smad2 and Smad3 do not accumulate in the nucleus of TGF-β–resistant mammary and lung epithelial cells overexpressing oncogenic Ras (92). This observation is explained by Ras-mediated phosphorylation of extracellular signal– regulated kinases 1 and 2 (ERK1/2) consensus sites in the linker region of Smad2 and Smad3, a site distinct from the TGF-β phosphorylation site in the SSXS domain. The result is inhibition of Smad accumulation in the nucleus. An analogous Ras-mediated phosphorylation event interferes with translocation of Smad1 in BMP-treated cells (184). Using an inducible Ras model, investigators have observed Smad4 degradation in the cytoplasm, prohibiting accumulation of a functional transcriptional complex in the nucleus (183). In contrast, studies in TGF-β–resistant, Ras/ErbB2-transformed, mammary epithelial cells show that Smad complexes are able to translocate into the nucleus and activate transcription from the Smad7 promoter (185). Others have also found that Smad complexes accumulate in the nucleus without hindrance in Ras-transformed intestinal cells, but their function is inhibited by interference with the transcriptional repressor TIEG (186). EGF and HGF both appear to induce Smad2dependent transcriptional responses, presumably by stimulation of ERK activity (93). In aggregate, these studies indicate that the effect of Ras on Smad signaling is dependent on the cell line being studied, specific technical aspects of the assay being used, or the stoichiometry of Smad complex formation in individual cell types. It has been established that tumor-suppressive TGF-β activity occurs by growth inhibitory Smad-dependent signaling. Until recently, however, the “double-edge” role of TGF-β as both a tumor suppressor and a tumor promoter could only be indirectly inferred (128,147,187). For example, it has been noted often that TGF-β levels are increased in metastatic tumors, including those of the gastrointestinal tract, potentially further driving prooncogenic signaling (188,189). In fact, increased expression of TGF-β is a risk factor for recurrence and reduced survival of colorectal cancer (190). Metastatic lesions express relatively more TGF-β1 than primary colorectal cancers, and the serum of patients with colorectal cancers has approximately fourfold more TGF-β than that of control subjects (191). Some colon carcinoma cell lines are growth stimulated by TGF-β (192). In colorectal cancers with mutant, inactivated TGF-βRII in which there is presumably no potential for prooncogenic signaling, the prognosis is better than in individuals with retention of one allele (193). Conversely, restoration of TGF-βRII in cells that carry a mutation in this gene increases cell invasiveness in vitro (187). Thus, once tumors escape growth regulation by TGF-β, if the potential remains for residual TGF-β signaling, the result is an enhanced potential for tumorigenesis.

More definitive evidence has emerged in support of tumor promotion by TGF-β using bitransgenic mice that overexpress the ErbB2 oncogene and a mutant-activated TGF-β receptor, each under the direction of a mouse mammary tumor virus (MMTV) mammary epithelial cell promoter (194). In these mice, induction of tumor formation by ErbB2 is delayed because of tumor-suppressive actions of TGF-β. However, an increased percentage of metastatic foci developed in the lungs, which is consistent with a prooncogenic effect for TGF-β later in the stages of tumor formation. Similar tumor-promoting actions of TGF-β were reported in cyclin D1/TGF-β1 bitransgenic mice (195). It is clear that events such as Ras transformation may have an additive adverse interaction with TGF-β signaling. In Ras-transformed cells, loss of autocrine growth inhibitory TGF-β signaling occurs coincident with a synergistic stimulation of angiogenic molecules (196), matrix molecules (197), and epithelial-to-mesenchymal transition (187), which is believed to be a key factor in tumor growth and metastasis in vivo. Ras overexpression also transcriptionally activates the TGF-β1 gene (198). Increased levels of TGF-β in this context further contribute to “undesirable” tumor-promoting signaling in growth-resistant cells.

Lessons from Gene-Targeted and Transgenic Mice Use of gene-targeted and transgenic mice has become a valuable methodology for study of TGF-β biology, especially relating to carcinogenesis. Illustrative examples have been cited throughout this chapter, and this section describes additional mouse models. Several investigators nearly simultaneously accomplished disruption of TGF-β1 by homologous recombination in the early 1990s (132,133,148). Embryonic lethality caused by defective yolk sac vasculogenesis is observed in approximately half of TGF-β1 null mice (148). In the remainder, no developmental defect is observed at birth. At 2 to 3 weeks of age, the deficient animals experience development of a rapidly fatal wasting syndrome characterized by massive infiltration of multiple organs with mixed inflammatory cells. Inflammatory lesions are most prominent in the heart, lungs, and gastric mucosa. The small bowel and colon are less involved. Peyer’s patches are small. These studies suggest an important role for TGF-β1 in immune homeostasis. Housing mice in a germ-free environment does not ameliorate inflammation, indicating independence of the inflammatory process from microorganisms (199). The predominant phenotype in TGF-β2 knockout mice is perinatal mortality caused by multisystem developmental abnormalities (200). TGF-β3 – deficient mice have defective palate formation (201). No overt abnormalities in gastrointestinal development are observed in TGF-β2 and TGF-β3 knockout mice. The rapidly lethal phenotype in TGF-β1 gene–targeted mice was cleverly circumvented by breeding TGF-β1–deficient mice with Rag2-/- mice that lack both B and T cells (202). Survival was prolonged, and carcinoma of the cecum and

GROWTH FACTORS IN THE GASTROINTESTINAL TRACT / 193 the three knockout approaches, a lethal wasting syndrome develops early in life and is associated with immunodeficiency (208,209). In the remaining model, metastatic colon adenocarcinoma was found at 4 to 6 months of age in Smad3-deficient mice (210). Compound mutant Smad4 and adenomatous polyposis coli (APC) mice experience development of more aggressive tumors than APC mice alone, indicating cooperativity in these two tumor-suppressive pathways (211).

colon developed as early as 3 months of age, indicating loss of tumor suppression by TGF-β. Overexpression of TGF-β in the liver of transgenic mice has been reported to cause hepatic fibrosis and atrophy of the pancreas (203). Overexpression in the pancreas causes chronic pancreatitis and fibrosis (204). An attractive alternate approach for determination of the roles for TGF-β in development has been study of mice with a dominant-negative TGF-βRII transgene, thereby circumventing the “problem” of ligand redundancy. A dominant-negative TGF-βRII under control of a metallothionein (MT) promoter results in fibrosis of the pancreas together with macrophage inflammation, neoangiogenesis, and defective acinar cell differentiation (205). No discernible effects on the liver or other gastrointestinal structures were observed. Disruption of the gene encoding LTBP results in reduced deposition of TGF-β in ECM, decreased phosphorylation of Smad2, and development of colorectal cancer (206). This observation highlights the biological importance of the latent form of TGF-β. Targeted mutagenesis of various Smad genes has been accomplished by a number of investigators, and the characteristics of these mice have been summarized in an excellent review (207). Deletion of Smad2 and Smad4 results in profound defects in embryologic cellular differentiation and is lethal in the perigastrulation interval. Heterozygotes show no abnormalities. Targeted deletion of Smad3 has been accomplished by three groups of investigators. Gross defects in embryogenesis are not observed, although in two of

EGF TGFα AR EPIGEN

EPIDERMAL GROWTH FACTOR FAMILY OF PEPTIDES AND RECEPTORS Overview and Discovery of the Epidermal Growth Factor Family The EGF-related family of regulatory peptides shares sequence homology, affinity for the same receptors, and a profile of similar biological activities (212,213). All EGFrelated peptides are expressed in the gastrointestinal tract, although there are important differences in the distribution and circumstances of expression (214). These proteins are defined by high-affinity binding to the EGF receptor (Fig. 7-3) and conserved spacing of six cysteine residues and intervening amino acids in the mature peptide sequence according to the formula XnCX7CX4CX10CXCX5GX2CXn (212,213). Using this definition, the mammalian EGF family now includes the following seven members: EGF, TGF-α, amphiregulin (AR),

ErbB2: No ligand, preferred heterodimerization partner for all other ErbB receptors, ErbB2/ErbB3 is most active

BTC HB-EGF EPR

Heregulins Neuregulins

ErbB-1: Traditional EGF receptor, commonly overexpressed in GI neoplasia

Extracellular domain

Cysteine-rich domain TM domain

Transmembrane domain

Kinase domain

Cytoplasmic domain ErbB-1 HER1 EGFR

ErbB-2 HER2 Neu

ErbB-3 HER3

ErbB-4 HER4

ErbB4: Stimulates cellular migration when expressed alone

ErbB3: Lacks kinase activity; contains 6 putative PI3K binding sites

FIG. 7-3. Epidermal growth factor (EGF) receptor family. The four mammalian EGF receptors (EGFRs) are shown, together with their ligand specificities. The text boxes illustrate some of the distinctive features of each receptor. Note that ErbB2 and ErbB3 do not have specific high-affinity binding for members of the closely related EGF peptide family, but signaling may occur as a result of heterodimerization with ErbB1 or ErbB4. AR, amphiregulin; BTC, betacellulin; EPR, epiregulin; GI, gastrointestinal; HB-EGF, heparin-binding epidermal growth factor–like growth factor; HER1, human epidermal growth factor receptor 1; PI3K, phosphatidyl inositol kinase; TGF-α, transforming growth factor-α; TM, transmembrane.

194 / CHAPTER 7

TGFα

AR

HB-EGF

BTC

EPR

EPIGEN

EGF Ectodomain cleavage sites EGF-like domain Heparin-binding domain

FIG. 7-4. Epidermal growth factor (EGF)–related peptide family. Diagram showing each transmembrane precursor of the seven EGF-related peptides discussed in this chapter. Note that amphiregulin (AR) and heparin-binding epidermal growth factor–like growth factor (HB-EGF) are unique in the occurrence of heparin binding domains. All are processed proteolytically to yield the “mature” peptide sequence. BTC, betacellulin; EPR, epiregulin; HB-EGF, heparin-binding epidermal growth factor-like growth factor; PI3K, phosphatidyl inositol kinase; TGF-α, transforming growth factor-α.

heparin-binding epidermal growth factor–like growth factor (HB-EGF), betacellulin (BTC), epiregulin (EPR), and epigen; these are shown in Figure 7-4. Each is synthesized as a glycosylated integral membrane propeptide with functionally distinct cytoplasmic, transmembrane, and extracellular domains. The extracellular domain includes a mature peptide sequence that is processed by regulated proteolytic cleavage by ADAM (a disintegrin and metalloprotease) sheddases (215). There are a large number of more distantly related EGF-like molecules and molecules that contain EGF-like repeats. These include trefoil peptides, cripto and various viral proteins, among others. Glial growth factor, acetylcholine receptor–inducing activity, heregulin, and neu differentiation factor (now collectively termed the neuregulins) are distantly related to EGF and bind to EGFR-like molecules, but they are not further referenced in this chapter. Many key regulatory proteins have single or multiple EGF-like repeats that uniquely contribute to their biological function.

Epidermal Growth Factor Epidermal Growth Factor Gene and Protein During the past half century, the study of EGF, its receptors, and signaling pathways has defined many important paradigms for understanding modern-day molecular and cell biology. As a consequence of career-long work in this field, Stanley Cohen and Rita Levi-Montalcini were awarded the Nobel Prize in 1987 for their seminal studies of EGF. As discussed throughout this chapter, more key emerging therapeutic modalities are relevant to EGF biology than to any other growth factor. The human EGF gene, located on chromosome 4q25-27, is 120 kb in length and contains 24 exons, 23 introns, and 9 EGF-like repeats (216–218). The 1217-amino-acid

transmembrane precursor is encoded by a 4.7- to 4.9-kb mature RNA species. Promoter sequences that regulate expression of EGF are not well understood, and factors that regulate EGF expression largely remain unknown (219). The mature, soluble peptide is 53 amino acids in length. The major sites of EGF synthesis under basal physiologic circumstances are the salivary glands and the kidney (220). Extremely low levels of EGF mRNA are detectable in the lactating mammary gland, the pancreas, and the duodenum. EGF immunoreactivity has been reported in a variety of gastrointestinal cells and tissues, including the mouse and human pancreas, the liver, Brunner’s glands of the duodenum, the normal stomach, and the normal esophagus. In aggregate, these observations of low-level EGF expression should be viewed with caution, because RNA levels are low or undetectable, and immunohistochemical findings have not been confirmed in other studies (10,11,221–232). These discrepant findings may be due to antibody cross-reactivity with other EGF-related peptides (especially in early years of study), developmental regulation of EGF expression, species variability, and other technical differences. A study using a semiquantitative polymerase chain reaction (PCR) assay detected EGF, TGF-α, AR, HB-EGF, BTC, and EPR transcripts throughout the gastrointestinal tract (233). Notwithstanding these discrepant observations, it is clear that gastrointestinal secretions have sufficient EGF concentrations, at least from the salivary glands, to exert a biological effect on the gastrointestinal mucosa (234,235). EGF expression may be induced in certain pathophysiologic states. For example, although undetectable in the normal liver, EGF expression is induced in the liver after partial hepatectomy and may be up-regulated in cirrhosis (224,225). In the context of chronic intestinal injury and ulceration, a novel EGF-producing epithelial cell lineage emerges in the intestine, perhaps contributing to epithelial restitution and

GROWTH FACTORS IN THE GASTROINTESTINAL TRACT / 195 repair (13). EGF secretion by the salivary gland and pancreas can be stimulated by drugs and gastrointestinal hormones such as secretin, pentagastrin, theophylline, and sildenafil (234,236). Although aberrant EGF expression occasionally is detected in gastrointestinal neoplasia, including pancreatic (223,237,238), esophageal (239), gastric (230,240–242), hepatocellular (243), and colorectal carcinoma (244), far more attention has been given to overexpression of other EGFrelated peptides in these cancers (see later). The potential for EGF to contribute to gastrointestinal neoplasia certainly exists as suggested by the occurrence of hepatocellular carcinoma in transgenic mice overexpressing a secretable form of EGF (243). Biological Effects of Epidermal Growth Factor in the Gastrointestinal Tract Hundreds of studies have been published on EGF and gastrointestinal function. Multiple examples are given in the subsequent section on TGF-α, the rationale being that EGF and TGF-α bind the same receptor, and a nearly identical spectrum of biological activity has been reported. The basis for focusing on TGF-α is that it appears to be the more physiologic ligand, at least in the healthy adult gastrointestinal tract. Substantive differences in the mitogenic activities of EGF and TGF-α in gastrointestinal cell cultures have not been found (214,245). The nonmitogenic effects of EGF and TGF-α on the gastrointestinal tract, such as inhibition of gastric acid secretion, are also similar. Any proposed differences can potentially be related to varying loci of growth factor production, subtleties in receptor binding, targeting to domains of polarized cells, intracellular routing, and differential stability in gastrointestinal fluids. The result is, at most, a modest difference in bioactivity (246).

α Transforming Growth Factor-α Transforming Growth Factor-α Gene and Protein TGF-α was discovered as a secreted, EGF-like transforming activity in virally transformed cells. The human TGF-α gene is located on chromosome 2p11-13, contains 6 exons, and spans 70 to 100 kb of DNA (247,248). Its 4.5- to 4.8-kb TGF-α mRNA transcript encodes a 160-amino-acid integral transmembrane precursor protein. A signal peptide in the amino terminus is cleaved before insertion of the TGF-α precursor into the cell membrane. The extracellular domain includes a “mature” 50-amino-acid sequence that is 35% identical to EGF. The 23-amino-acid hydrophobic transmembrane domain is followed by a 39-amino-acid intracellular “cytoplasmic tail” that contains 7 cysteine residues, some of which are covalently linked to palmitate (249). The reported sizes of the mature TGF-α protein range from 5 to 30 kDa, a variation that may reflect differential glycosylation and proteolytic cleavage.

Mature TGF-α is cleaved from its membrane-bound precursor by a zinc-dependent metalloprotease disintegrin tumor necrosis factor–converting enzyme (TACE), also known as ADAM17 (250). This process is termed ectodomain shedding. ADAM-mediated proteolysis has emerged as a critical step in regulating the bioactivity of TGF-α and other EGF-related peptides. The ADAM proteases and shedding of EGF-related peptides are discussed separately later. Early studies performed before the discovery of ADAMs showed that the initial step in release of mature peptide is rapid cleavage at an extracellular N-terminal alanine/valine site (215,249). Release of mature peptide results from a slower proteolytic cleavage step at alanine/valine residues that flank the C terminus of the mature sequence (251). Factors that regulate cleavage include a carboxy-terminal valine in the cytoplasmic tail, adenosine triphosphate, PKC, and G-protein signaling pathways (249,251–253). TGF-α mRNA and protein are widely expressed in normal cells and tissues, including those of the gastrointestinal tract (254,255). The TGF-α promoter has been extensively characterized, is GC rich, does not have a TATA box, and is highly dependent on the Sp1 transcription factor (256). Additional promoter elements include binding sites for AP-2 (257), p53 (258), and TEF1 (259); sequences responsive to tumor necrosis factor-α (TNF-α) (260); estrogen (261); and a site responsive to EGF and TGF-α (262). The latter element is of interest because autoinduction and cross-induction between EGF-related peptide family members has been described in vivo and in vitro (245,263,264). This phenomenon underscores the complexity of interactions within the EGF-related peptide family. Transforming Growth Factor-α Expression and Regulation of Proliferation in the Liver, Pancreas, and Intestine Hepatic TGF-α mRNA appears early in gestation in the rat, peaks during embryogenesis, and subsequently declines approximately 90% from late gestation to adulthood (265,266). The normal adult liver is a quiescent organ, and TGF-α expression is low. However, expression increases in scenarios marked by robust hepatocyte proliferation, a finding that is compatible with TGF-α and EGF being potent autocrine mitogens for hepatocytes (267). For example, in the partial hepatectomy model, 70% resection of liver mass initiates a burst of hepatic DNA synthesis within 12 to 15 hours, resulting in a complete recovery of liver mass in 96 hours (268). Hepatic TGF-α mRNA begins to increase within 6 to 8 hours of a partial hepatectomy and peaks at about twice baseline levels at 24 hours, the time of peak DNA synthesis (269,270). TGF-α protein levels increase approximately 13 hours after partial hepatectomy, coincident with the onset of DNA synthesis (271), and peak about twofold greater than baseline levels (272). TGF-α levels also increase the blood of rats subjected to partial hepatectomy (273). Notwithstanding, TGF-α is not the primary stimulus for hepatocyte proliferation after partial resection, because a normal

196 / CHAPTER 7 proliferative response to hepatectomy is observed in TGF-α null mice, suggesting other EGF-related peptides may compensate for TGF-α deficiency (274). Overexpression of TGF-α in transgenic mice increases liver weight and DNA content twofold to threefold, and the fraction of cells engaged in DNA synthesis fivefold (275,276). Multifocal, well-differentiated hepatocellular carcinomas (277) develop in some strains of TGF-α transgenic mice, and coexpression of c-myc accelerates this process (278). In humans with hepatoblastoma and hepatocellular carcinoma, TGF-α is expressed at increased levels both in serum and tumors (279–281). A key role for EGF-related peptide signaling in a rat model of hepatocellular carcinoma is suggested by the prevention of tumor formation by administration of gefitinib, an EGFR tyrosine kinase inhibitor (282). In the pancreas, TGF-α mRNA and immunoreactivity are detected in fetal pancreatic acini, ductular epithelium, and in islet cells, whereas expression in adult pancreas is predominantly in the ductular epithelium (223,254,283,284). No abnormalities in pancreatic morphology or function are noted in TGF-α knockout mice (285,286), indicating the growth factor is dispensable for normal pancreatic development. Studies in a variety of in vivo and in vitro models indicate that both EGF and TGF-α stimulate cellular proliferation in the pancreas, predominantly in ductal cells (287–291). TGF-α is overexpressed in pancreatic cancers and is increased in the serum of patients with pancreatic carcinoma (237,238,292). Coexpression of the receptor and either EGF or TGF-α occurs in 38% of tumors and correlates with advanced disease. Overexpression of TGF-α in the pancreas of transgenic mice (275,276) results in interstitial pancreatic fibrosis, multifocal pseudoductular acinar metaplasia, and sometimes pancreatic carcinoma (293). Development of pancreatic carcinoma is accelerated in TGF-α /c-myc (294) and TGF-α /p53−/− bitransgenic mice (295). Several interesting observations suggest that TGF-α and other EGF-related peptides are important in regulation of progenitor cell populations in the pancreas. For example, the homeobox gene Pdx1, a marker for primitive pancreatic epithelium, is increased in MT-TGF-α transgenic mice (296a). Newly formed β-cell populations in a ligated pancreatic duct model are associated with increased expression of HB-EGF and BTC, as well as TGF-α (296b). Finally, evidence suggests that TGF-α activates expression of Notch signaling, which is important in the regulation of cell fate decisions, and together these proteins contribute to pancreatic carcinogenesis (297). Overexpression of EGF in pancreatic islets causes disorganization and enlargement of islets of Langerhans. Glucose homeostasis is not disturbed (298). TGF-α is readily detectable in gastrointestinal tissues in fetal and adult life (228,299–302). In humans, expression is found as early as the 10th week of gestation and persists throughout the gastrointestinal tract into adulthood. The preponderance of evidence suggests that TGF-α expression is greatest in differentiated epithelial cell compartments of the gastrointestinal tract, implying that the nonmitogenic actions of TGF-α may predominate under

normal physiologic circumstances. This assumes that physical access of this autocrine factor to the proliferative cell compartment may be limited; this is a speculation that remains to be proved. Because TGF-α is sorted, shed, and released from the basolateral surface of the intestinal epithelial cell, it is feasible that autocrine proliferative activity is able to access the proliferative compartment via the lamina propria (303). In the human esophagus, TGF-α mRNA and protein are found primarily in the differentiated epithelial cell population (226,304,305). In the human and murine stomach, expression is also found in the differentiated, nonproliferative cell population of the epithelium, especially in parietal cells (229,304,306–309). In most reported studies, TGF-α in the small intestine and colon is also principally found in differentiated, surface epithelial cells (10,11,303,304,310). Gastrointestinal biologists and physiologists have long been intrigued by the compartmentalization of the gastrointestinal epithelial functions into apical (brush border) and basolateral domains. This spatial compartmentalization is especially important for understanding how autocrine and juxtacrine growth factors function. The sorting of TGF-α and other EGF-related peptides therefore has been of intense interest. It is now clear that TGF-α is sorted to the basolateral domain of polarized epithelial cells with greater than 90% efficiency (8). Redundant motifs in the cytoplasmic tail, including a dileucine motif and eight amino acids in the juxtamembrane region of pro–TGF-α, are responsible for sorting (311). Myristoylation-dependent binding of Naked-2 to the cytoplasmic tail is believed to contribute to the escort of pro–TGF-α to the basolateral membrane (312), and evidence suggests that binding of membrane associated guanylate kinase inverted-3 (MAGI-3) to the PDZ domain also facilitates sorting (313). Once targeted to the basolateral membrane, the protein is rapidly cleaved by ADAM17 and quickly bound to EGF receptors, which are also located on the basolateral domain. EGF and TGF-α stimulate epithelial proliferation in normal gastrointestinal epithelium in vitro and in vivo. For purposes of this discussion, it is assumed that the mitogenic effects of TGF-α and EGF are equivalent, as this is the case in most assays. Studies are collated for this discussion. Neutralizing antibodies to EGF and TGF-α reduce the growth of cultured cells, including epithelial cells, exposed to human milk (314–316). TGF-α is a mitogen for gastric mucous cells, fundic epithelial cells, small intestinal epithelial cells, and colonic epithelial cells cultured in vitro (103,245,317– 320). TGF-α may also be an “indirect mitogen.” For example, some work shows that treatment of intestinal epithelial cells with substance P activates EGFR signaling by rapidly promoting metalloproteinase-induced cleavage of membraneanchored TGF-α (321). A number of studies have examined a role for EGF-related peptides on gastrointestinal epithelial cell proliferation in vivo. The methods used to deliver exogenous EGF-related peptide in these studies are of interest in light of the basolateral (serosal) distribution of EGFR in the gastrointestinal tract (see later in this section for a description of this observation). Parenteral administration of EGF or TGF-α stimulates

GROWTH FACTORS IN THE GASTROINTESTINAL TRACT / 197 small intestinal and colonic epithelial growth (322–325). Overexpression of TGF-α in the small intestine of MT-TGF-α transgenic mice increases crypt cell proliferation, resulting in increased villus height (326). In contradistinction, transgenic mice overexpressing EGF under the direction of an β-actin promoter are growth retarded, but they show no substantive intestinal, pancreatic, or hepatic phenotypes (327). Limited evidence also suggests EGF stimulates proliferation in humans (328). In most reported studies enteral administration of EGF or TGF-α does not stimulate proliferation of normal adult gastrointestinal mucosa. These findings are congruous with the observation that the EGFR is localized to the basolateral domain of the intestinal epithelial cell. It should be recognized, however, that enteral administration of EGF might permit access to the basolateral receptor and stimulate growth under circumstances of developmental immaturity or pathologic injury to the epithelial barrier. It is well known that early in development, the intestinal epithelial barrier is more permeable to macromolecules. A significant body of evidence suggests luminal growth factors may actually gain access to and activate EGFRs on the basolateral membrane in immature animal models, although cellular proliferation is not necessarily the observed biological end point in these studies (329–331). EGF may also traverse the mucosa intact by transcytosis in the newborn interval (332). In experimental models of intestinal atrophy, parenteral administration of EGF or TGF-α promotes epithelial growth and reverses atrophy (333–337). In most, but not all, partial small intestinal resection models, the adaptive proliferative response observed in residual intestine is enhanced by administration of EGF, perhaps, in part, by increased expression of EGFR (338–340). Overexpression of EGF in the intestinal epithelium of transgenic mice also enhances the proliferative response to resection (341). However, postresection proliferation occurs in TGF-α–deficient transgenic mice, suggesting that sufficient overlap exists with other EGF-related peptides (342). The gastric mucosa of TGF-α overexpressing mice is also hyperplastic and shows lineage disturbances reminiscent of Ménétrièr’s disease (343,344). Clearly, the preponderance of experimental evidence cited in this chapter suggests that TGF-α and EGF stimulate proliferation in the intestinal mucosa. However, these observations do not establish an absolute requirement for EGF or TGF-α in normal intestinal epithelial proliferation. Genetargeting experiments that specifically address this issue clearly conclude that EGF and TGF-α are not required for normal intestinal proliferation (285,286). Although TGF-α gene–targeted mice have hair follicle abnormalities leading to wavy whiskers and occasional ocular abnormalities, no gastrointestinal phenotype is observed (285). A gastrointestinal phenotype is also not observed in EGF gene–targeted mice (345). In the context of disruption of the epithelial barrier, luminal EGF may also gain access to the basolateral domain, leading some experts to call EGF a “luminal surveillance factor” poised to contribute to proliferation, migration, and

repair of injured mucosa (346). In support of this, enteral EGF administration ameliorates mucosal injury in a rat model of neonatal enterocolitis (331), and improved outcomes were reported in a clinical trial of EGF enemas in patients with ulcerative colitis (347a). Interestingly, neither transgenic overexpression of EGF in the intestinal epithelium nor intravenous EGF ameliorates chemotherapeutic agent–induced mucosal injury induced by fluorouracil (347b). A different study found that EGF administered intraperitoneally protects the small intestinal mucosa against methotrexate-induced injury (348). EGF null mice are more sensitive to cysteamineinduced duodenal lesions, but they are no more sensitive to dextran sulfate sodium–induced colitis (233). Interestingly, TGF-α–deficient mice have increased susceptibility to dextran sulfate sodium–induced colitis (349). Triple knockout EGF, TGF-α, and AR mice are growth retarded, have reduced levels of goblet cells, develop spontaneous duodenal erosions, and show blunted ileal villi (233). Whether these findings are the result of reduced proliferation or a loss nonmitogenic activity contributing to cytoprotection is unclear. A number of factors may contribute to the variable results in experiments testing in vivo activities of EGF-related peptides. These include the route and dose of EGF-related peptide, the presence or absence of mucosal injury, the developmental stage of the model being studied, and the model of injury or read-out of EGF-related peptide biological activity. Even the feeding status of the animal may play a factor, because inhibition of luminal proteases by food may increase bioactivity of EGF in the intestinal lumen that is otherwise degraded soon after entry into the proximal bowel (350). Wong and Wright (351) provide an excellent review of the effects of EGF-related peptides on intestinal growth. Additional evidence for the mitogenic properties of TGF-α in the gastrointestinal epithelium is the substantial body of data showing it is overexpressed in gastrointestinal cancers. TGF-α may be overexpressed in neoplasms of the esophagus (352,353), stomach (230,240,241,354–356), and colon (357–362). Often, the level of overexpression correlates with tumor aggressiveness. Serum levels of TGF-α are also increased in patients with gastrointestinal tumors. Activation of the EGFR by excessive ligand production or other key mechanisms leads to an amplified autocrine axis that is amenable to therapeutic intervention. As discussed later in this chapter, this field has evolved to the development of inhibitors of EGFR as a therapeutic strategy for the treatment of gastrointestinal neoplasia. Nonmitogenic Activities of Epidermal Growth Factor and Transforming Growth Factor-α EGF-related peptides have key biological activities that are unrelated to cellular proliferation. These so-called nonmitogenic activities are outlined in a superb review by Uribe and Barrett (363). Nonmitogenic activities of EGF-related peptides include inhibition of gastric acid secretion; modulation of sodium, chloride, and glucose transport; stimulation of amylase and mucin secretion; stimulation of mesenteric

198 / CHAPTER 7 and mucosal blood flow; stimulation of smooth muscle contraction; and stimulation of cell motility, migration, and chemotaxis, among others. In aggregate, these activities work to promote adaptation to gastric, small intestinal, and colonic mucosal injury and ulceration (364), as discussed earlier.

Amphiregulin AR was first identified in MCF-7 breast carcinoma cells treated with the tumor promoter phorbol myristate acetate. The name amphiregulin comes from the ability of AR to either stimulate or inhibit cell growth, depending on the cell line being studied (365). The proteins originally described as colorectum-derived growth factor and Schwannoma cell– derived growth factor are now known to be AR (366,367). The AR gene is located on human chromosome 4q13-4q21 (368). Many proteins and compounds that stimulate cellular proliferation also induce AR gene expression. Examples include other members of the EGF-related peptide family (245,369), oncogenic Ras (370,371), phorbol esters (368), and prostaglandin E2 (372), likely through a cyclic 3′,5′adenosine monophosphate response element in the AR promoter. Structurally, AR is distinguished from EGF and TGF-α by an N-terminal heparin-binding domain in the mature peptide sequence. The N-terminal sequence of AR is extremely hydrophilic and contains a nuclear localization signal that is required for mitogenic activity of AR (366). Because of differential processing and variable glycosylation, multiple molecular weights of AR are noted on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The membranebound form has a molecular weight of 50 kDa, whereas the mature peptide ranges from 22 to 46 kDa (373,374). Shedding of AR occurs by the TACE/ADAM family of metalloproteinases. The metalloproteinase inhibitor batimastat inhibits shedding of AR, as it does other EGF-related peptides (374). Inhibition of tyrosine phosphatase activity promotes metalloproteinase-dependent cleavage of AR (375). AR binds exclusively to ErbB1. AR mRNA is expressed in normal and neoplastic cells of the gastrointestinal tract (368,376–378). It is detectable in the surface epithelium of the normal colon, whereas little or no expression is found in the proliferative zone (377,378). Most colon cancers express AR at a greater level than adjacent normal colon tissue (376–379). Polarizing Caco-2 and HCA-7 colon carcinoma target AR to the basolateral membrane, and secretion occurs into the basolateral cell culture medium (380). Cytoplasmic sequences in pro-AR are required for sorting (381). AR is a potent mitogen for most epithelial cells, and all the requisite elements of a growth stimulatory autocrine loop exist in colon carcinoma cells (378–380). In vitro, inclusion of heparin-like glycosaminoglycans in growth assays enhances proliferation induced by AR (382), whereas heparin antagonists block proliferation (383). Treatment of the GEO colon cancer cell line with antisense AR oligomers reduces cell growth (384).

AR is also an important mitogen in the liver and pancreas. In the normal human liver, AR gene expression is barely detectable; however, expression is increased in the context of cirrhosis (385). In the partial hepatectomy model, AR expression is induced to a maximum level 6 hours after resection, and the onset of DNA synthesis is delayed in AR gene–targeted mice (385). As expected, AR is mitogenic in isolated hepatocyte cultures. Transgenic overexpression of AR in the exocrine pancreas results in proliferation in small intralobular ducts (386), but the excessive fibrosis observed in TGF-α transgenic mice is not observed (275,276). AR-deficient mice are normal and have no obvious defects in the normal gastrointestinal system, although the proliferative response to partial hepatectomy is delayed and less robust (345,385).

Heparin-Binding Epidermal Growth Factor–Like Growth Factor HB-EGF was first identified in conditioned medium of a cultured macrophage cell line using heparin-affinity chromatography (387). The human gene locus for HB-EGF is 5q21 (388). Like AR, a host of proliferative stimuli and other perturbations rapidly induce HB-EGF gene expression, including TNF-α, oxidative stress, serum, other EGF-related peptides, insulin-like growth factor-1 (IGF-1), gastrin, v-jun, oncogenic Ras, phorbol esters, angiotensin II, and TGF-β (389–400). The mRNA is super induced by the protein synthesis inhibitor cycloheximide (245,391), thus defining HB-EGF as an “immediate-early gene.” This suggests HB-EGF plays a key role in mediating the earliest responses to proliferative stimuli and other cellular perturbations. Like other EGF-related peptides, HB-EGF is synthesized as a transmembrane propeptide that is processed by the ADAM family of metalloproteinases to release soluble mature peptide (401). The HB-EGF transmembrane precursor is apparently unique among EGF-related peptides in that it is capable of functioning as a receptor molecule by binding and internalizing the diphtheria toxin (402). The molecular weight of mature HB-EGF is 20 to 22 kDa, although multiple higher molecular weight forms have been noted (390,403). Mature HB-EGF contains an N-terminal hydrophilic heparinbinding domain similar to that found in AR and BTC. Interestingly, the C-terminal product of HB-EGF shedding is retained intracellularly, translocates to the nucleus, and triggers nuclear export of the promyelocytic leukemia zinc finger transcriptional repressor (404). Thus, one of the most prominent activities of HB-EGF, stimulation of cellular proliferation, may occur by two distinct mechanisms: activation of the EGFR by soluble HB-EGF and release of cells from transcriptional repression by intracellular C-terminal HB-EGF. HB-EGF is found in many human tissues and body fluids in the picogram per milliliter concentration range, including human amniotic fluid and breast milk (405,406). However, with the exception of prominent expression of HB-EGF mRNA and protein expression in normal gastric parietal cells (407), expression is low or undetectable in the normal

GROWTH FACTORS IN THE GASTROINTESTINAL TRACT / 199 gastrointestinal epithelium and in the liver (408,409). In vitro, the Cdx-2 homeobox gene induces expression of HB-EGF, which contains a Cdx-2 binding element (410), suggesting it may play a role in intestinal development. HB-EGF is found in the developing pancreas and the homeodomaincontaining transcription factor PDX-1 binds to the HB-EGF promoter (411). The rapid induction of HB-EGF in response to a variety of experimental perturbations in vitro is also observed in vivo. For example, HB-EGF mRNA is rapidly induced after acute injury to the kidney (397), brain (412), and intestine (413). In the intestine, HB-EGF protects against hypoxic/ ischemic injury in vitro (413) and in vivo (414), perhaps by suppression of NF-κB activation (415). Substantial data support a role for HB-EGF in wound healing (390). HB-EGF binds to ErbB1 and ErbB4 and induces tyrosine phosphorylation of each (416–418). In cells expressing ErbB4 but no EGFR, HB-EGF signals a chemotactic, but not a proliferative, response, suggesting these two receptors mediate different biological activities (417). Receptor-binding affinities and biological activities of HB-EGF are accentuated by cell-surface heparan sulfate proteoglycans (HSPGs) (419–421). There are interesting, but limited, data suggesting that HB-EGF also specifically binds to N-arginine dibasic convertase to modulate cellular migration in an EGFRdependent manner (422). The dominant activities of HB-EGF are stimulation of cellular proliferation, cell survival, and cell motility (390). Most cell types proliferate in response to HB-EGF, including gastric cell, pancreatic cell, intestinal epithelial cells, and hepatocytes (245,420,421,423). Although liver-specific overexpression of HB-EGF in transgenic mice has no effect on basal measures of proliferation in the liver, in the context of partial hepatectomy, labeling indices are fivefold greater than in nontransgenic control animals (424). In renal epithelial cells, intestinal epithelial cells, and hepatocytes, enforced expression of a membrane-anchored form of HB-EGF or exogenous administration of HB-EGF causes resistance to apoptotic stimuli such as TNF-α (425–427). In this capacity, HB-EGF serves as a “survival” factor. Although only a limited number of studies have addressed HB-EGF expression in gastrointestinal neoplasms (428), overexpression has been observed in pancreatic, liver, and gastric neoplasia (429–432). Transgenic overexpression of HB-EGF in the pancreatic islet cells leads to fibrosis and metaplasia of ductular epithelium (433).

Betacellulin BTC was first isolated from a mouse insulinoma cell line (434). The gene is located on human chromosome 4q13-q21. The molecular weight of unprocessed BTC is 40 kDa (435). The mature peptide is released by an ectodomain-shedding mechanism that appears to be uniquely regulated in comparison with other EGF-related peptides (see later) (436). Mature, soluble BTC is approximately 32 kDa and consists of 80 amino acids. Like AR and HB-EGF, BTC contains

consensus glycosylation sequences and a heparin-binding domain (434). The pattern of BTC binding to ErbB receptors mirrors that of HB-EGF and EPR (437–439); however, BTC is unique in its ability to bind ErbB2 and ErbB3 heterodimers in the absence of ErbB1 and ErbB4 (437,438). Studies suggest that BTC induces distinctive patterns of EGFR tyrosine phosphorylation, leading to alterations in the profile of adapter protein recruitment and differences in signaling pathway activation (440,441). Again, these findings emphasize that redundancy in growth factor families should be viewed with some skepticism, because detailed scrutiny of the complexity of regulation of these proteins typically reveals unanticipated differences. BTC is found in bovine milk (442). The 3-kb BTC mRNA transcript is widely expressed in normal tissues, including the small intestine, liver, colon, and pancreas (434,443,444). BTC is increased in intestinal epithelial cells and in epidermal cells transformed by oncogenic Ras (371,445). Gastric cancer cell lines also express BTC mRNA, although the transcript is detectable in primary tumors only by PCR (429). In the pancreas, BTC is detectable in this normal fetus, in the adult by immunohistochemistry (446), and as early as embryonic day 13 by reverse transcriptase PCR (447). Studies of BTC function in the gastrointestinal tract are limited. Treatment of fetal pancreas explants with BTC favors development of a β-cell lineage (447). BTC is mitogenic for a number of fibroblastic and epithelial cell lines (434,448). BTC gene-targeted and transgenic mice have not yet been generated.

Epiregulin EPR was relatively recently discovered (449,450). The gene is on human chromosome 4q13.3. The molecular weight of the purified protein is 5.4 kDa, and the mature peptide is 46 amino acids in length. N-terminal sequences seen in AR, HB-EGF, and BTC are not present in EPR, and binding to heparin does not occur. The 4.8-kb EPR RNA transcript has been detected in a wide variety of cell lines and in normal stomach, small intestine, and colon (450,451). Similar to AR, EPR inhibits growth of some cell types and stimulates growth of others (449,452). It stimulates growth of gastric epithelial cells (453), and its expression is increased in models of partial hepatectomy (454) and in tumors of the colon and pancreas (455,456). Although the affinity of EPR for the ErbB1 is less than EGF, EPR is more potent in mitogenic assays (449,457–459). EPR gene-targeted mice show no overt developmental defects, a normal proliferative response to partial hepatectomy, but increased sensitivity to dextran sulfate sodium-induced colitis (451).

Epigen Epigen, the newest and presumably the last EGF-related peptide, has been identified as an expressed sequence tag in a mouse keratinocyte library (460). It is located on human

200 / CHAPTER 7 chromosome 4q13.3. The mRNA is found in the liver. Expression in other gastrointestinal organs has not been investigated. The protein is composed of 152 amino acids, and the biological function appears to be similar to EGF, although preliminary evidence suggests it may be far more potent (461).

Regulated Shedding of Epidermal Growth Factor–Related Peptides by Metalloproteases All EGF-related peptides are first synthesized as anchored transmembrane precursor proteins that are subsequently cleaved to release biologically active autocrine or paracrine peptide growth factor. This process is termed shedding. One of the most exciting areas of EGF-related peptide biology in recent years has been elucidation of the cellular molecular mechanisms by which shedding occurs. It has been learned that this is a complex, highly regulated process subject to therapeutic manipulation in diseases such as cancer in which EGF-related peptide signaling is often hyperactive. The significance of shedding as a key regulatory step in EGF-related peptide signaling was first highlighted by the observation that TACE gene–targeted mice develop a phenotype similar to TGF-α and EGFR null mice (250,285,462). TACE null fibroblasts isolated from these mice were dramatically impaired in their ability to shed mature TGF-α. This mouse model implied that cleavage of EGF-related peptides by TACE is critical for growth factor activity. Subsequent studies support this assertion. For example, it is clear that TACE is required for activation of the EGFR by TGF-α in tumors, a finding that also calls into question the importance of juxtacrine signaling by membrane-anchored growth factor (463). TACE belongs to a large family of zinc-dependent disintegrin-metalloproteinases designated ADAMs (464–466). These proteins are also variably referred to as sheddases or convertases. TACE, or ADAM17, is the major metalloproteinase involved in EGF-related peptide shedding, although multiple other members of this large family of 39 proteins may also participate. Adding to the complexity is a great deal of cell-type specificity of ADAM function. Notwithstanding, currently, it appears that ADAM17 is the major protease involved in TGF-α, AR, and HB-EGF shedding (467–469), whereas ADAM10 is the major protease involved in shedding of EGF and BTC (436,468). Cleavage of HB-EGF by other metalloproteinases such as MMP-3 may also contribute to shedding (470). Shedding can be activated by a variety of stimuli, including phorbol esters, calcium ionophores, Ras activation, calmodulin antagonists, tyrosine phosphatases inhibitors, GTP-γs, and EGFR autocrine signaling (374,471–477). Emerging evidence indicates that the well-known phenomenon of EGFR transactivation by ligands of G protein-coupled receptors (GPCRs) occurs by activation of ADAM activity, release of mature EGF-related peptide, and activation of the EGFR (478).

A potentially important dividend realized by expanding our understanding of ADAM and MMP-induced shedding is the potential for modulating EGFR activation using metalloproteinase inhibitors. The broad-spectrum metalloprotease inhibitor batimastat blocks shedding of EGF-related peptides in a manner that suggests an integral role for metalloproteases in the regulation of biological activity of these proteins (375,479). However, to date, clinical trials of metalloproteinase inhibitors have not been successful, perhaps because of a lack of specificity of currently available inhibitors (480).

Epidermal Growth Factor Receptor Family Overview The mammalian EGFR family is composed of four membrane-anchored receptor tyrosine kinases. Each is characterized by a single extracellular binding domain, a transmembrane domain, and a single intrinsic tyrosine kinase domain extending into the cytoplasm (481–484). The classical EGF receptor (ErbB) is also known as HER1 (human epidermal growth factor receptor 1), or ErbB1, because of homology with the v-erbB oncogene encoded by the avian erythroblastosis virus (485). The remaining three receptors are designated HER2/ErbB2, Her3/ErbB3, and Her4/ErbB4. The overall homology within the ErbB family is 40% to 50%, and all cysteine positions are conserved in the extracellular domain. The phenomenon of receptor dimerization is a key principle in understanding ligand-induced signaling by EGFRs (486), and it is a recurrent theme in the biology of most growth factor families. Within the EGFR family, ligand binding is able to transduce signals by two distinct pathways, receptor homodimerization and autophosphorylation and receptor heterodimerization and transphosphorylation. When one collectively considers the seven EGF-related peptides discussed in this chapter in the context of the extraordinary number of potential dimerizing partners in the ErbB family, the rich complexity and diversity of signal transduction within the EGF family quickly becomes apparent. In most contexts in this chapter, the classic EGF receptor, ErbB1, is referred to as the EGFR. When it is desirable to facilitate understanding of the pattern of homodimerization and heterodimerization with other members of the mammalian family, the designation ErbB1 will be used. The remaining three EGFR family members are designated by ErbB2, ErbB3, and ErbB4, respectively. Epidermal Growth Factor Receptor (or ErbB1) The EGFR gene is found on human chromosome 7p11 (487,488) and contains 110 kb DNA, including 26 exons and 25 introns (488). The molecular weight of the protein is 170 kDa. It is no doubt the most extensively characterized receptor protein in the field of growth factor biology. Both extracellular and intracellular domains have been extensively

GROWTH FACTORS IN THE GASTROINTESTINAL TRACT / 201 characterized by point mutants, deletion mutants, insertional mutants, and chimeric receptors. The extracellular domain is characterized by an extensively N-glycosylated N-terminal domain and a cysteine content of about 10%. The hydrophobic transmembrane sequence traverses the cell membrane only once. Located in the cytoplasmic domain are a C-terminal tyrosine kinase and five tyrosine autophosphorylation residues. When phosphorylated, the latter serve as docking sites for intracellular signaling proteins that bind Src homology-2 (SH2) domains in intracellular adaptor molecules that link directly to signaling cascades. Studies suggest that EGFRs may exist in a ligandindependent, “predimerized” configuration (489), the existence of which is regulated by sequences in the cytoplasmic domain. When ligand is engaged, the EGFR homodimerizes or heterodimerizes with ErbB2, ErbB3, or ErbB4, depending on the ligand and cell type. Biophysical studies and X-ray crystallography indicate that receptor dimerization requires interaction between two molecules of ligand and two molecules of receptor (490–492). Engagement of a single ligand with a monomeric receptor results in a conformational change in the receptor that favors receptor-receptor interaction. Dimerization of EGFRs results in intermolecular autophosphorylation of key tyrosine kinases in the cytoplasmic domain. Congruous with this model is the dominant-negative effect observed by transfection of cells with a mutant EGFR containing an inactive tyrosine kinase moiety resulting in a reduction of high-affinity binding sites for EGF, a reduced rate of receptor endocytosis, and diminished receptor signaling (493). After binding any of its ligands, EGFRs are rapidly internalized by clathrin-dependent and clathrin-independent endocytic pathways. The extent to which signaling continues after endocytosis remains unclear, although it is generally believed that signaling continues and endocytic mechanisms are able to modulate the magnitude, specificity, and duration of signaling (494,495). Interestingly, endocytosis is inefficient for all other ErbB receptors, and signaling from the cell membrane is sustained (496). Once internalized, most EGFR is sorted to lysosomes for degradation, whereas approximately 25% is recycled to the cell membrane. The result is receptor down-regulation and attenuated signaling. Precisely how the various ErbB heterodimers undergo trafficking is not entirely clear. Data suggest that the EGFR is ubiquitinated by the ubiquitin protein ligase, c-Cbl, thus targeting the receptor/ligand complex for endocytosis (497,498). An old but intriguing observation that must be accommodated in models of EGFR signaling is that continuous exposure of EGFR to ligand for at least 8 hours is required for S-phase entry and DNA synthesis (499,500). Epidermal Growth Factor Receptor Signaling When ligand is engaged, specific residues in the cytoplasmic domain of dimerized or heterodimerized EGFR are tyrosine autophosphorylated. These phosphotyrosine sites create docking motifs for signaling proteins or adapter proteins with

SH2 or phosphotyrosine binding domains. SH2 domains are found in a large number of proteins and consist of approximately 100 amino acids that recognize phosphopeptide motifs (501). Recruitment of SH2 proteins to phosphotyrosine motifs in the activated EGFR may result in their direct phosphorylation by EGFR tyrosine kinase, activation of their intrinsic catalytic activities, or establishment of platforms for recruitment of additional signaling or adaptor proteins, as shown in Figure 7-5. Examples of catalytic activities recruited to the activated EGFR include Src kinases, Abl, protein tyrosine phosphatase (PTP), phospholipase Cγ (PLCγ), and phosphoinositol-3 kinase (PI3K) (502). Examples of adapter proteins include Shc, Grb2, Crk, Grb7, and Nck, among many others (503,504). Thus, the complexity of EGFR signaling is multiplied many fold by the diversity of potential catalytic activities and adaptor molecules recruited to the EGFR. A full discussion of all adaptor proteins, catalytic activities, and downstream signaling cascades activated by the EGFR is beyond the scope of this chapter, but there are several excellent reviews of this topic (502,503,505). A few classical and illustrative examples are provided below. The most extensively studied signaling pathway activated by the EGFR tyrosine kinase is the Ras/MAPK pathway (501,506). Central to activation of the Ras pathway is Grb2, a cytoplasmic adaptor protein with two SH2 motifs. Grb2 is constitutively bound to the Ras exchange factor Sos. Binding of Grb2 to the activated EGFR relocates Sos to the EGFR and the cell membrane, facilitating interaction and activation of Ras by guanine nucleotide exchange. Activation of Ras is a central event in a multiplicity of physiologic and pathophysiologic cellular events, the most prominent of which is activation of cellular proliferation and survival. This variety of cellular response results from Ras activation of a complex downstream network of interacting and intersecting Ras effectors (507–510). In addition to activation of the classical Raf/MEK/ERK signaling pathway, other Ras-effector cascades activated by Ras include PI3K, Ral GEF, and Rho-kinase pathways, among others (510). Mutational activation of the K-Ras gene occurs in about 50% of colorectal cancers (175) and more than 75% of pancreatic cancers (511). Activated Ras increases levels of EGF-related peptides and phosphorylation of the EGFR (512). Interestingly, this creates amplifying loops of autocrine EGF-related peptide production and activation of EGFR, as detailed in Figure 7-6. Termination of this autocrine loop by EGFR tyrosine kinase inhibitors reduces cell growth and indices of cellular transformation in Rastransformed cells (513). This in vitro phenomenon is also observed in vivo. The hypomorphic EGF tyrosine kinase activity characteristic of wav-2 mice decreases formation of epithelial tumors when these mice are mated with mice overexpressing the adapter protein Sos, a Ras activator (514). Study of cells isolated from these mice attributed the decreased number of tumors to loss of an EGFR-dependent “survival function,” which is a well-recognized consequence of EGFR activation (515).

202 / CHAPTER 7

PIP2

DAG IP3 JAK1 STATs

PI-3K PLCγ

EGFR/ErbB2

P P

Ca++

PKC

GPCR

AKT

Shc Grb2

Ras

Sos TGFα

MKK6

MEKK-1

Raf

p38

MKK -7

MEK

MAPKAP2

JNK

ERK

TACE/ADAM17

TGFα (pg/ml)

FIG. 7-5. Epidermal growth factor receptor (EGFR) signaling. In the gastrointestinal epithelium, EGFR activation typically occurs by ligand that has been targeted to and proteolytically cleaved from the basolateral domain of the polarized enterocyte. EGFRs, also located on the basolateral domain, are activated by ligand engagement, resulting in activation of intrinsic tyrosine kinase activity. Scaffolding proteins dock to the activated receptor and link to a complex variety of signaling cascades. ADAM, a disintegrin and metalloprotease; DAG, diacylglycerol; ERK, extracellular signalregulated kinases; GPCR, G protein–coupled receptor; IP3, inositol-1,4,5-triphosphate; MAPK, mitogen-activated protein kinase; PKC, protein kinase C; PLCγ, phospholipase Cγ; STAT, signal transducer and activator of transcription; TACE, tumor necrosis factor–converting enzyme; TGF-α, transforming growth factor-α.

60 50 40 30 20 10 0

PGE2 production

Ras activation

Pai, et al. Nature Med 8:289 Shao, et al. J Biol Chem 279:14287

RIE Ras Du et al. Mol Cancer Res 2:233

24 h

8h 12 h

4h

1h 2h

45′

0

30′

15′

Ligand auto- and cross-induction

COX-2 induction

EGFR family activation

EGFR ligand

TGFα AR HB-EGF

Bernard et al. J Biol Chem 269:22817

BTC 1B15

FIG. 7-6. Autoamplifying loops of epidermal growth factor (EGF)–related peptide activity that potentially contribute to unregulated growth in the intestinal epithelium. A large body of data now supports the “autoamplifying” nature of EGF-related peptide signaling. In the context of neoplastic disorders, multiple autoamplifying loops contribute to ongoing, unregulated cellular proliferation. Contributors to this loop are increased production of EGF-related peptides and induction of cyclooxygenase-2 (COX-2) in cells transformed by oncogenic Ras, extensive induction of other EGF-related peptides, induction of COX-2 expression, and increased prostaglandin production by EGF-related peptides. Recent evidence suggests that prostaglandin E2 (PGE2) transactivates the EGF receptor.

GROWTH FACTORS IN THE GASTROINTESTINAL TRACT / 203 PLCγ is also a key signaling molecule for receptor tyrosine kinases, including members of the EGF family (502,516). PLCγ hydrolyses phosphatidylinositol 4,5-biphosphate, releasing the second messenger molecules inositol-1,4, 5-triphosphate (IP3) and diacylglycerol (DAG). It may also function as a guanine nucleotide exchange factor (517a). PLCγ is distinguished among the PLC isoenzymes by two SH2 and one SH3 motifs that associate with the activated EGFR. Activated EGFR directly phosphorylates and activates PLCγ (517b). The result is stimulation of cellular proliferation and migration, including cells isolated from the gastrointestinal tract (518). PLCγ also appears to be an essential signaling molecule for maintenance of intestinal epithelial integrity by EGF (519). Involvement of members of the Src family of cytosolic, nonreceptor, tyrosine kinases in EGFR signaling has long been recognized, especially with regard to oncogenesis (520,521). Both SH2 and SH3 domains are found in Src family members. Downstream targets include PI3K and PLCγ, among others. Evidence suggests that the EGFR itself is phosphorylated by Src, leading to increased tyrosine kinase activity (521). Overexpression of Src proteins enhances EGF-mediated cell proliferation and transformation, whereas inhibition of Src activity blocks EGF-induced cellular proliferation. Thus, there is a bidirectional synergy between Src family members and the EGFR family (521). The phospholipid kinase PI3K phosphorylates the membrane lipid phosphoinositol to generate phosphoinositides (522). Phosphoinositides are second messenger molecules that recruit cytoplasmic proteins to the cell membrane. PI3K is activated by association with virtually all receptor tyrosine kinases, including the EGFR, or their associated adaptor proteins. PI3K contains two SH2 and one SH3 domain and is a critical downstream effector of Ras. The serine threonine kinase Akt, also known as protein kinase B (PKB), accumulates as sites of PI3K activation by binding directly to phosphoinositides. Akt catalytic activity results in phosphorylation of a host of other cellular proteins that are critical for cell proliferation and survival. Signal transducer and activator of transcription (STAT) proteins are a family of seven latent cytoplasmic transcription factors that are well-known signaling molecules for cytokines such as interferons (523). Less well studied is the phosphorylation and activation of STAT proteins by the activated EGFR. Activated STAT proteins dimerize and translocate to the nucleus where they bind to specific sequences of target genes. The activation of STAT proteins in response to EGFR activation appears to be Src-dependent in many cell types (524). Some work suggests that EGF treatment of primary hepatocytes results in activation of STAT5b in a Src-dependent manner, although activation of STAT5b is not required for stimulation of cellular proliferation (525). Migration of esophageal epithelial cells appears to be dependent on EGF activation of STAT proteins (526). The outcome of STAT activation in normal and transformed cells of gastrointestinal origin deserves further investigation.

Epidermal Growth Factor Receptor Expression in Normal Gastrointestinal Tissues and Gastrointestinal Neoplasia The EGFR is expressed in all cells at a level ranging from 20,000 to 200,000 receptors per cell. An exception is hematopoietic cells, which do not express receptor. Receptor levels can be much greater in neoplastic cells. Expression in the normal gastrointestinal tract begins during embryonic development and persists throughout adulthood. In the liver, multiple studies have shown that EGFR levels are quite high in the fetus, and then decrease after birth (527–529). Notwithstanding the high fetal levels of EGFR, the normal hepatic architecture observed in EGFR gene-targeted mice indicates hepatic organogenesis is not dependent on EGFR signaling (462,530). EGFR mRNA is detectable in normal human hepatocytes, most prominently at the periphery of the hepatic lobule (225). EGF is found in significant concentrations in bile, and it has been suggested that hepatic EGFR serves as a means to clear plasma of EGFR ligands by biliary excretion (531,532). After partial hepatectomy, the EGFR is initially up-regulated, and then down-regulated (529,533), perhaps because of increased ubiquitination and internalization of receptor (534). EGFR is also expressed in normal human pancreas, primarily in ductal and acinar epithelial cells (232). The EGFR is expressed in normal fetal and adult gastrointestinal tract. In the human fetus, EGFRs are present in the human gastric glands as early as the 12th week of gestation (535) and are detectable in fetal jejunum and colon between week 15 and 19 of gestation (536). The ontogeny of intestinal EGFR expression is reviewed comprehensively in an excellent article by Chailler and Menard (537). In the adult esophagus, EGFRs are localized to the basal and suprabasal layers, and little or no receptor is found in the surface epithelium (226,346,538–540). Expression in increased in the chronically inflamed esophagus (305). In the stomach and intestine, EGFR is found in parietal cells and has been variously reported in the proliferative regions of the crypt/villus axis or all along the horizontal axis of the epithelium in the small intestine and colon (346,541). An extensive body of literature concludes that the EGFR is located primarily on the basolateral membrane of polarized gastrointestinal epithelial cells and polarized epithelial cells in other tissues (329,380,542–548). A 22-amino-acid sequence in the EGFR juxtamembrane sequence contains information necessary for targeting the receptor to a basolateral localization (549). In polarized Caco-2 human colon carcinoma cells, levels of basolateral EGFR are 15-fold greater than apical receptor, and no bioactivity is detected when the apical compartment is treated with ligand (550). Similar observations have been made using polarized MDCK cells, rat proximal tubular cells, and other colon cancer lines. Despite these findings, it has been proposed that low levels of apical EGFR in the gastric epithelium protect against apical gastric acid (551) and a functional role for apical receptor should not be dismissed.

204 / CHAPTER 7 The EGFR is overexpressed in many types of cancers. In many instances, overexpression or amplification correlates with a poor prognosis. Overexpression of the EGFR and coexpression of its ligands are common in pancreatic carcinoma and also appear to portend a poor prognosis (238,287,291,552–555). In human hepatocellular carcinoma, overexpression of EGFR and its ligands may play a less important pathogenetic role (556–559). EGFR is overexpressed in Barrett’s epithelium and in adenocarcinoma of the esophagus and may correlate with a poor prognosis (353,558,560,561). In gastric cancer, a correlation has been established between overexpression of the EGFR and a poor prognosis, but overexpression is not particularly common (562–564) and not all studies corroborate this correlation (565–567). Expression of the EGFR and its ligands have been detected in human colon adenoma and carcinoma cell lines, in animal models of colon carcinoma, and in human disease (242,244,359–361,562,568,569). Metastatic colorectal carcinomas express more receptor than the primary tumors (570,571). Collectively, overexpression of EGFR and its ligands in gastrointestinal neoplastic disorders has led to a number of exciting and intensively studied therapeutic approaches that target the EGFR system.

distantly related neuregulin family are ligands for ErbB3 (585). When ErbB3 is expressed alone, binding with the EGF-related peptide family does not occur, but binding and signaling may occur when ErbB3 heterodimerizes with remaining EGFR family members. ErbB3 is considered a “kinase-impaired” receptor because its tyrosine kinase activity is significantly less than other ErbB receptors (586). ErbB3 is also unique among ErbB receptors in that multiple (six) YXXM consensus sites in the cytoplasmic tail permit binding of and signaling by PI3K (587). Distinct physiologic roles for ErbB3 in the gastrointestinal tract have not been identified. ErbB3 protein expression is detectable in normal human esophagus, stomach, small intestine, and colon (588). The predominant expression is in the differentiated, surface epithelium. Expression is also detectable in fetal and adult pancreas and liver (529,589). ErbB3 expression is upregulated by insulin and partial hepatectomy in the rat (529, 590). ErbB3 immunoreactivity is low or undetectable in normal human colonic epithelium, whereas expression is often detectable in most colon carcinomas (376,578). Increased expression is also found in gastric, hepatic, and pancreatic cancers (591–593).

ErbB2

ErbB4

c-ErbB2 is located on human chromosome 17q21, and the protein product is a 185-kDa glycoprotein with an intrinsic, cytoplasmic tyrosine kinase domain (572). ErbB2 is also referred to as the neu protooncogene, or HER2, and is the only member of the EGFR family with no identified ligand. ErbB2 appears to be the preferred heterodimeric partner with the other ErbBs, and heterodimer formation with ErbB2 increases affinity of ligand for the heterodimeric receptor complex (439,573). Formation of heterodimers with ErbB1, ErbB3, and ErbB4 induces tyrosine transphosphorylation of ErbB2. Overexpression of ErbB2 is associated with uncontrolled cell proliferation in animal models, as well as human neoplastic disorders (574,575). ErbB2 amplification and overexpression have been most extensively studied in mammary carcinoma, where it is overexpressed in about 20% of cases and is associated with a poor prognosis (575). Expression of immunoreactive ErbB2 in normal colonic epithelium ranges from undetectable to low and is found from the base of the crypt to the surface epithelium (576–579). ErbB2 overexpression in human colon cancer is infrequent (507–510,579,580). Increased expression of ErbB2 occurs in a small subset of tumors of the stomach, pancreas, and esophagus, and in tumors of the neuroendocrine system, but not the liver (488,511,512,567,581–583).

The ErbB4 gene is found on human chromosome 2q33 (594). The gene product is a 180-kb glycosylated, transmembrane protein with a tyrosine kinase domain in the cytoplasmic tail. Several distinctive features of ErbB4 have been described are reviewed in detail by Carpenter (595). Alternative spicing of the RNA occurs as evidenced by the absence of a PI3K domain in the cytoplasmic tail of one variant (596) and variation in the juxtamembrane domain of another (596). Interestingly, ErbB4 is the only EGFR that is susceptible to proteolytic cleavage by TACE (375,597,598). After cleavage, the intracellular domain translocates to the nucleus (599), but its function in the nucleus is uncertain. Cells that exclusively express ErbB4 undergo chemotaxis, but fail to proliferate (417). This differential activity of ErB4 may be the result of differential expression alternatively spliced isoforms with and without PI3K domains (600). The dominant ligands for ErbB4 are BTC, HB-EFG, EPR, and neuregulins. Expression has been identified in the fetal pancreas and the upper gastrointestinal tract, but not the liver (589,601). Expression of ErbB4 in gastrointestinal neoplasms has not been reported extensively, but misexpression of ErbB4 in pancreatic, gastric, and colon cancer has been observed (601–603).

ErbB3

Epidermal Growth Factor Receptor and ErbB Receptor Knockout Mice

The c-ErbB3 gene is located on human chromosome 12q13 and encodes a glycosylated, transmembrane protein with a molecular weight of 180 kDa (584). Members of the more

Multiple EGFR mutant and gene-targeted mice have been described and offer an interesting insight into the importance of this ligand in gastrointestinal physiology. A naturally

GROWTH FACTORS IN THE GASTROINTESTINAL TRACT / 205 occurring recessive mouse mutation termed waved-2 has striking phenotypic similarities with the TGF-α knockout mouse described earlier in this chapter (285). Waved-2 mice have a single amino acid substitution in the tyrosine kinase domain of EGFR, the result of which is 80% to 90% reduction of EGFR tyrosine kinase activity, but normal levels of receptor mRNA and protein (604). Thus, the waved-2 mouse is an EGFR hypomorph or “partial” EGFR knockout. No gastrointestinal phenotype is apparent in normal waved-2 mice; however, under pathophysiologic circumstances, a phenotype emerges. For example, the normal proliferative response is blunted, and the accelerated enterocyte apoptosis is increased after partial small-bowel resection in waved-2 mice (605,606). The waved-2 allele reduces the number of small intestinal polyps in the APCmin mouse model by 90% (607). Waved-2 mice have an increased susceptibility to dextran sulfate sodium-induced colitis (608). These observations emphasize that EGFR signals critically important proliferative, survival, and cytoprotective functions in the gastrointestinal epithelium. The waved-5 mutation is an EGFR tyrosine kinase “dead” mutant with dominant negative activity (609). Three groups of investigators accomplished EGFR knockout nearly simultaneously in the mid-1990s (462, 530,604). The phenotype is quite variable, ranging from peri-implantation lethality to normal newborn pups that develop wasting and death in the first and second postnatal weeks. The predominant defects in EGFR null pups are observed in epithelial tissues, although a great deal of phenotypic heterogeneity is seen, likely because of the genetic background of the deficient mice. Null mice demonstrate delayed eyelid development, thin skin, disorganized hair follicles and limited hair growth, impaired central nervous system development, impaired pulmonary alveolar development, and renal structural and functional abnormalities. Miettinen and coworkers (462,610) described short, attenuated intestinal villi, reduced jejunal epithelial DNA synthesis, altered pancreatic islet morphology, and reduced pancreatic duct branching. Terminally, the intestine of EGFR null mice was hemorrhagic and dilated with complete loss of villus architecture. Threadgill and colleagues (530) found reduced esophageal bromodeoxyuridine incorporation, but normal intestinal morphology, differentiation, and proliferation in a CD1 background. Sibilia and Wagner (611) found no obvious gastrointestinal abnormalities. ErbB2 and ErbB4 null mice die on embryonic day 10.5 because of defective cardiac myocyte development and central nervous system development (612–614). The effect of deficiency of these receptors on gastrointestinal, liver, and pancreas development is uncertain, although a cell-specific knock out of ErbB2 in the enteric nervous system and colonic epithelial cells has been reported (615). The mutant mice were growth retarded and had distended colon, reminiscent of Hirschsprung’s disease. This phenotype was dependent on the loss of ErbB2 expression in the colonic epithelium, because mice with absent ErbB2 expression in

the enteric nervous system were normal. ErbB3 null mice survive until approximately embryonic day 13.5, but they have marked abnormalities in the central nervous system and the heart and show attenuation of the gastric epithelium and mesenchymal layers, decreased numbers of enteric ganglia, and abnormal pancreatic development (612,616). Transactivation of ErbB Signaling A major development in the understanding of EGFR biology in recent years has been the elucidation of various mechanisms of receptor transactivation. Transactivation is the term used to describe activation of the EGFR directly or indirectly by heterologous ligands. Transactivation has been reported extensively for the ErbB1 receptor tyrosine kinase and is now considered a well-established pathway for receptor activation. GPCR agonists, cytokine receptors, prostaglandins, growth hormone (GH) receptor, and various adhesion receptors such as integrins all cause tyrosine phosphorylation of ErbB1 (372,617–620). In many cases, GPCR-mediated transactivation of the EGFR occurs by activation of ADAMs that induce ectodomain shedding to produce EGF-related peptides (478,621). In addition, physical, chemical, and electrical stimuli also increase the level of tyrosine phosphate on ErbB1. Blockade of Epidermal Growth Factor Signaling: A Therapeutic Option in Clinical Medicine Inhibition of ErbB receptors and their signaling constituents has become a viable option for treatment of human tumors that overexpress EGF-related peptides, EGFR-related receptors, or both. In fact, the first approved cancer therapeutic agent based on growth factor biology was the anti-ErbB2 monoclonal antibody trastuzumab. Multiple potential therapeutic options to block EGFR signaling currently are in the drug development pipeline, ranging from antibody-mediated receptor blockade, antibodies that serve as carriers of cytotoxic compounds, highly specific EGFR tyrosine kinase small molecule inhibitors, ADAM inhibitors, specific signaling pathway inhibitors, and antisense oligonucleotide or ribozyme inhibitors. All these strategies are at various stages of testing in animal models and in human trials (465,622–624).

INSULIN-LIKE GROWTH FACTORS Overview and Discovery of Insulin-Like Growth Factors The IGF ligand/receptor system includes three structurally related ligands (insulin, IGF type I [IGF-I], and IGF-II) and two high-affinity cell-surface receptors (IGF-I and IGF-II receptors). An important physiologic factor regulating the interaction between the IGFs and their receptors is the IGF binding protein family (IGFBP1-6). GH is structurally unrelated to IGFs, but its actions are primarily mediated by regulation of IGF-I synthesis and secretion in hepatocytes;

206 / CHAPTER 7 hence, GH and its biology are referenced occasionally in this chapter. The IGF system functions as a leading endocrine, paracrine, and autocrine regulatory axis for cellular proliferation, survival, and apoptosis in many organ systems, including the gastrointestinal tract. In addition, it has general activities relating to energy metabolism, body size, carcinogenesis, and various organ specific functions. A number of excellent, comprehensive reviews are available for further study (625–633).

Insulin-Like Growth Factor Genes and Proteins IGFs are two closely related members of the insulin superfamily of peptide hormones. IGF-I and IGF-II are 67% identical, single-polypeptide chains that share ≈40% amino acid identity with insulin. Historically, IGF-I and IGF-II were referred to as somatomedin-C and somatomedin-A, respectively. Current nomenclature, and this chapter, uses the terms IGF-I and IGF-II. Unlike insulin, IGFs are not produced and stored in pancreatic islets, but they are synthesized and secreted by most cells in the body in a highly regulated manner. The IGF-I gene is located on human chromosome 12q22q23. The genomic sequence is large, spanning more than 80 kB DNA, including 6 exons (634). At least 4 transcriptional start sites have been identified, and IGF-I mRNA species range from about 1.0 to 8.0 kB. Complex, tissuespecific alternate slicing patterns have been observed, but the biological significance of these variants has not been clearly delineated for the gastrointestinal tissues (635). For human pro-IGF-I, two primary translation products exist, IGF-IA and IGF-IB. The most commonly recognized mature IGF-I sequence is a 70-amino-acid, secreted protein. Most IGF-I in the peripheral circulation is synthesized in the liver in response GH. In this context, IGF-I is an endocrine growth factor and it exerts feedback to the pituitary to down-regulate GH synthesis. Most growth-stimulating activities of GH can be mimicked by IGF-I. It is clear that GH regulates IGF production in other tissues as well, including the gastrointestinal tract (see later). In malnutrition, the stimulatory effect of GH on IGF-I is markedly reduced. IGF-I binds IGF receptor 1 with high affinity and IGF receptor 2 with low affinity. The human IGF-II gene is located on chromosome 11p15.5. Like the IGF-I precursor, the IGF-II precursor is large, and multiple splice variants have been described, many of which are developmentally regulated and contain gutspecific promoters (636). A pro-IGF-II–containing carboxyterminal precursor sequence is secreted from cells before processing to the mature 67-amino-acid protein (629). The IGF-II gene is highly unique in that it is “imprinted” in 90% of humans, meaning that normally one allele is silenced on the basis of parental origin. When IGF-II genomic imprinting is lost, IGF-II levels are increased and a substantially increased risk for colorectal cancer exists (637). IGF-II binds IGF receptors 1 and 2 with high affinity.

Insulin-Like Growth Factor Receptor Genes and Proteins The IGF-I receptor (IGF-1R) gene is located on human chromosome 15q26.3 and is 70% identical to the insulin receptor gene (638). The protein is synthesized as a single polypeptide chain that is cleaved by proteolysis at a tetrabasic amino acid site resulting in α and β subunits. These subunits associate by disulfide bridging into α and β heterodimers that further associate by disulfide bridging to the mature heterotetrameric α2β2 receptors. Structurally, this overall configuration is identical to the insulin receptor; in fact, hybrid IGF-1R and insulin receptors are well recognized (639). The α subunits form the extracellular ligand-binding domain, whereas the β subunits are transmembrane and intracellular sequences that carry the tyrosine kinase signaling domain (626). IGF-1Rs bind IGF-I and IGF-II with high affinity (KD is ≈ 1 × 10−8 to 1 × 10−9) and insulin with a 100-fold reduced affinity. Most biological activities of IGF-I and IGF-II are mediated by the IGF-IR. IGF-I binding results in tyrosine autophosphorylation of the receptor, activation of its intrinsic tyrosine kinase activity, and phosphorylation of intracellular substrates (Fig. 7-7). Foremost is the insulin receptor substrate 1 (IRS-1), a 185-kDa intracellular signaling protein with multiple phosphorylation sites and SH domains permitting docking of multiple intracellular signaling molecules (640). The result is activation of a variety of signaling cascades, the most prominent of which are the Ras and phosphoinosotide-3 kinase pathways. As expected from previous discussion on EGF signaling, activation of these pathways results in cellular proliferation and survival, the predominant activities of IGFs. The IGF-IIR is identical to the cation-independent mannose-6 phosphate receptor and has no structural homology with the insulin receptor or the IGF-IR (633). It is located on human chromosome 6q26. The IGF-IIR receptor is a single, transmembrane polypeptide that binds IGF-II with a 100-fold greater affinity than IGF-I. It has 15 cysteine-laden, contiguous, extracellular repeats and a short intracellular sequence with no recognizable signaling motifs (628). Its role in IGF-II signaling is controversial, and most experts believe it down-modulates IGF-II activity by regulating its endocytosis and intracellular degradation by targeting the proteins to the lysosome. This is consistent with a growing body of data suggesting that the IGF-IIR is a tumor-suppressor gene, acting as a “sink” or reservoir for IGF-II (630,641).

Insulin-Like Growth Factor Binding Protein Gene and Protein Family IGFBPs are a well-characterized family of six secreted proteins, designated IGFBP1-6, that bind IGFs with high affinity and display a broad spectrum of biological activity. Extensive information about the gene organization, protein structure, and molecular biology and physiology of IGFBPs can be found in the reviews referenced at the beginning of this section.

GROWTH FACTORS IN THE GASTROINTESTINAL TRACT / 207

PI-3K

IRS-1 β-chain

IGFIR α-chain

AKT

α-chain β-chain

IGF-1

IRS-1

Shc Grb2 Sos

Ras

Raf

MEK

Target gene

ERK

-growth stimulation -survival

FIG. 7-7. Insulin-like growth factor (IGF) signaling. Signaling is initiated from the basolateral membrane domain of the polarized enterocyte. Intrinsic tyrosine kinase activity in the cytoplasmic domain of the IGF-I type I receptor (IGF-IR) is activated by IGF-I or IGF-II. Insulin receptor substrate 1 (IRS-1) is a key adaptor protein in IGF receptor signaling, linking activated receptor to the canonical Ras signaling pathway, as well as activation of phosphatidylinositol-3 kinase (PI3K) signaling. The result is stimulation of cellular proliferation and cell survival.

The IGFBPs are potent modulators of IGF activity, in part because the affinity of IGF-I and IGF-II for their binding proteins is 10- to 100-fold greater than affinity for the IGF receptors. Both IGF-I and IGF-II bind to all six IGFBPs, albeit with some differences in affinity. The structure of all six IGFBPs can be divided into three domains, highly conserved C- and N-terminal domains and a central domain that is unique among IGFBP family members. The C- and N-terminal domains bind IGFs. It is the central domain that is modified by posttranslational modifications and confers functional diversity among IGFBPs (631). Depending on the experimental setting and IGF function being assessed, IGFBPs may potentiate or inhibit IGF activity. In most contexts, IGFBPs reduce IGF activity by reserving it in a biologically inaccessible pool. Regulated proteolysis of IGFBPs is a key mechanism for release of IGF, thus regulating its bioavailability and bioactivity (631). For example, addition of IGFBP-3 to colon cancer cell culture medium decreases bioactivity of IGF-I. Addition of MMP-7 cleaves IGFBP-3 into four fragments, releases IGF-I, and potentiates IGF-I biological activity (631). Other IGFBP functions include transport of IGFs in extracellular fluids. IGFBPs are detectable in plasma and extracellular fluids and are expressed during development, as well as during adult life. The primary circulating IGFBP is IGFBP-3, which carries approximately 75% of circulating IGF-I and IGF-II (631). Activities of IGFBPs that take place independent of IGF binding are recognized; for example, IGFBP-3 activates TGF-β receptor signaling in several cell culture models,

including intestinal smooth muscle cells (642). This may be an additional mechanism by which IGFBPs inhibit cellular proliferation.

Insulin-Like Growth Factor Family Distribution in the Gastrointestinal Tract A large number of studies have described the presence and distribution of IGFs and IGF receptors in the gastrointestinal tract (635,636,643–653). IGF-I, IGF-II, and IGFBPs are also found in human breast milk and gastrointestinal tract secretions (654–656). At least a portion of enterally administered 125I-IGF-I and -II can be recovered intact from gastrointestinal tissues of suckling rats, indicating that the peptide is stable in the milieu of the neonatal stomach and small intestine (657,658). Collectively, these studies unambiguously document expression of this ligand receptor system in the gastrointestinal tract; however, a consistent picture of distribution along the crypt villus axis and in the epithelial versus mesenchymal compartments has not emerged. This lack of clarity is a result of the variety of species studied and the variety of technical and molecular approaches used to study the distribution of IGF family members. Several general conclusions can be drawn, however. First, it is clear that both IGF-I and IGF-II bind to intestinal epithelial cells in vitro and in vivo (648,650,652,659,660). Second, similar to the EGF and TGF-β receptor systems, both IGF-IR and IGF-IIR are targeted to the basolateral membrane domain of the

208 / CHAPTER 7 enterocyte and other epithelial cells (661,662). Finally, multiple components of the IGF system, including IGFBPs, are expressed in the lamina propria of this intestine, implying an important role in regulation of epithelial-mesenchymal interactions (663,664). In support of this, transgenic mice in which IGF-I is overexpressed in the intestinal lamina propria under the direction of an α-smooth muscle actin promoter shows increased proliferation of the ileal epithelium (665). As pointed out earlier, most circulating IGF-I is synthesized in the hepatocytes in response to GH. Consequently, hepatocyte levels of IGF-I are very high and IGFBPs are also synthesized in the liver. Interestingly, however, the normal hepatocyte is not considered a major target for IGF action, because the receptor is expressed at extremely low levels (666). Hepatic stellate cells and myofibroblasts also express IGFBPs and IGF-I (667), and it is these cells that are believed to play a role in fibrogenic response to IGFs in the liver (668). IGF-II and IGF-IIR are highly developmentally regulated. Intestinal IGF-II RNA transcripts are readily detectable during late fetal gestation, but are much less apparent in adult rat samples (635,645). Similar observations have been made in human stomach and intestine (653). IGF-IIR levels also decrease during rat and human development, suggesting the IGF-II/IGF-IIR axis may play an important role in intestinal development (653,669). A clear pattern of developmental expression of IGF-I/IGFIR has not emerged. Fluctuations in expression are observed and the degree of change is less apparent than that described for IGF-II (635,652,653,670).

Biology of the Insulin-Like Growth Factor Family in the Gastrointestinal Tract The gastrointestinal tract is a major target organ of IGF action (671). One of the most prominent effects is stimulation of intestinal epithelial proliferation and maintenance of cell survival by reduction of apoptosis. Other activities relate to the diverse effects of this ligand/receptor family on somatic growth, energy balance, and metabolism of glucose, carbohydrate, and proteins. Insulin-like Growth Factor Stimulates Cellular Proliferation Multiple lines of evidence indicate that IGFs are mitogenic in intestinal epithelial and smooth muscle cells and on hepatic stellate cells in vitro and in vivo. In vitro, IGFs stimulate intestinal epithelial proliferation, but, in most instances, significantly less so than other growth factors such as EGF (103,660,672–675). When EGF is provided with IGF-I or insulin, a synergistic effect on intestinal epithelial proliferation often is observed. Isolated intestinal smooth muscle cells and isolated hepatic stellate cells in vitro also proliferate in response to IGF (676,677). The in vivo evidence for a proliferative effect of IGF on the intestine is dramatic and has been observed in multiple

experimental models using both enteral and parenteral IGF. Several illustrative examples are cited here. Oral feeding of IGF-I to neonatal pigs increases indices of small intestinal weight, DNA content, protein content, and villus height in the small intestine (678). These effects are most prominent in the proximal small intestine and are not seen in the pancreas or stomach. In a fetal sheep model, in utero ligation of the esophagus deprives the distal intestine from growth regulatory peptides in amniotic fluid. A 10-day infusion of IGF-I distal to the ligation results in increased small intestinal growth (679). Potten and coworkers (680) found a modest stimulation of small intestinal crypt labeling index after brief treatment of mice with an intraperitoneal injection of IGF-I. Similarly, a 14-day parenteral treatment of adult rats with IGF-I increased crypt depth and villus height 30% over control values (681). In this study and others, the proximal small intestine is the most responsive region. In multiple models, infusion of LR3IGF-I, an N-terminal–extended analogue of IGF-I that has a reduced affinity for IGFBPs, better stimulates proliferation in the epithelium and muscularis of the small intestine than the parent peptide (682,683). IGF-I also has prominent effects on intestinal growth under various pathophysiologic conditions. For example, IGF-I augments adaptive mucosal proliferation in the small intestine of rats that have undergone a partial small intestinal resection (684–686). The mucosal atrophy observed in the jejunal mucosa in parenterally fed rats is blunted by administration of inclusion of IGF-I in the parenteral nutrition solution (687). Similarly, the small intestinal atrophy that occurs in the context of chronic liver disease or sepsis is also reduced by administration of IGF-I (688,689). Studies in transgenic mice also support a proliferative effect of GH or IGFs on the intestinal mucosa. Transgenic mice overexpressing GH have increased bowel length and mass, but a normal intestinal crypt cell proliferation rate, suggesting that GH increases survival of intestinal epithelial cells (690). As expected, plasma and intestinal mucosal IGF-I concentrations were increased, suggesting that IGFs are “survival factors.” In transgenic mice overexpressing IGF-I under a MT-I promoter, circulating GH levels are undetectable because of negative feedback from IGF-I, thus permitting dissection of the specific effects of GH and IGF-I on the intestine. MT-IGF-I mice exhibit a significantly greater small intestinal length and mass, an increased villus height, greater crypt depth, and a higher crypt cell mitotic index compared with control littermates (691). Indices of differentiation are not altered. Overexpression of IGF-II in a transgenic mouse either does (692) or does not (693) increase the mass of the gastrointestinal tract, but detailed effects on the gastrointestinal tract were not reported in either case. Thus far, detailed attention to the development and function of the intestine in IGFBP overexpressing mice has not been reported. Collectively, the studies outlined previously suggest that IGF-I and GH, the latter of which is already used extensively in the clinical arena for other indications, may be useful therapeutic agents in patients with short bowel syndrome.

GROWTH FACTORS IN THE GASTROINTESTINAL TRACT / 209 Indeed, an increasing body of data suggests GH improves intestinal function in patients with short bowel syndrome (694), although not all studies and authors support this view (695). In animal models of short bowel syndrome or intestinal atrophy, it is clear that IGF-I is more potent in stimulating intestinal growth than GH. It has been hypothesized that this is because of induction of suppressor of cytokine signaling-2 (SOCS-2) by GH, but not IGF-I (696). In cell culture systems, SOCS-2 is growth inhibitory, apparently blunting the proliferative response to GH in the intestine. New clinical trials using IGF-I may offer more encouraging results in subjects with decreased gastrointestinal mucosal function. Insulin-like Growth Factor Is a “Survival Factor” Studies referenced earlier suggest that IGFs increase intestinal growth by inhibition of apoptosis. Indeed, MT-IGF-I transgenic mice exhibit a lower background level of spontaneous apoptosis in small intestine crypts and also a lower level of apoptosis in response to irradiation (697). This observation is congruent with reports that IGF-I inhibits apoptosis in cultured cell lines (698). Insulin-like Growth Factor Is Profibrogenic Several lines of evidence support the profibrogenic actions of IGFs in the intestinal tract. IGF-I stimulates proliferation and inhibits apoptosis of cultured fibroblasts, myofibroblasts, and smooth muscle cells and increases collagen synthesis in intestinal smooth muscle cells (699,700). Transgenic overexpression of IGF-I in the lamina propria of SMP8-IGF-I mice increases the mass of the muscularis propria and length of the intestine (665). The adaptive response to surgical resection of the small intestine in this same SMP8-IGF-I mouse is characterized by a marked lengthening of the residual bowel, suggesting IGF-I autocrine activity increases the mesenchymal elements in the intestine (701). In Crohn’s disease, a human disorder characterized by fibrosis and stricture formation, mucosal IGF-I and IGFIR levels are increased relative to normal intestine (702). Despite the weight of this evidence, it should be noted that not all models support a profibrogenic response to IGF-I. For example, carbon tetrachloride injury of the SMP8-IGF-I mouse liver results in reduced collagen synthesis and amelioration of the extent of liver injury (703). Insulin-like Growth Factor in Gastrointestinal Cancer The prominent effects of IGF ligands and receptors on cellular proliferation and survival suggest this growth factor axis may play an important role in neoplastic disorders. Multiple lines of evidence now support this view (630,632). Circulating IGF-I levels vary considerably among healthy individuals, and population studies suggest an overall trend toward increasing cancer risk, including cancer of the gastrointestinal tract, in persons at the high end of the normal range of IGF-I blood levels. Loss of IGF-II imprinting, resulting in

a modest increase in IGF-II levels, is now widely accepted as a marker for colorectal cancer risk (637). Increased expression of IGF-I, IGF-II, and IGF-IR occur in colorectal cancers (704).

Elimination of Insulin-Like Growth Factor Function by Gene Targeting Gene targeting has been used extensively to study the function of the IGF system (this topic has been reviewed by Dupont and Holzenberger [633], Wood [705], and Butler and LeRoith [706]). A prominent effect is fetal growth retardation, implying that the IGF system is a major regulator of in utero growth. For example, IGF-IR–deficient mice weigh approximately 45% of their normal littermates and die of respiratory failure within minutes of birth (707). Significant abnormalities occur in several organ systems and include hypoplasia of skeletal muscle, central nervous system anomalies, skin and hair follicle abnormalities, and delayed bone development. IGF-I–deficient mice are about 60% of normal birth weight. Depending on the genetic background, some mice die in the newborn period, whereas others survive into adulthood. Placental development is normal (708). No significant intestinal, liver, or pancreatic phenotype has been observed in IGF, IGF receptor, or IGFBP gene–targeted mice.

TREFOIL FACTOR FAMILY OF PEPTIDES Overview and Discovery of Trefoil Factor Family Trefoil factor family (TFF) represents a relatively new class of biologically active peptides that appear critical in maintaining gastrointestinal epithelial integrity under normal and pathologic conditions (709–713). TTF peptides are differentially synthesized wherever mucin secretion occurs, being prominently expressed within the gastrointestinal mucosa. TFF proteins are constituents of mucous gels and have been shown to rapidly modulate mucosal restitution through a combination of motogenic, cell-scattering, antiapoptotic, and anti-inflammatory activities. Several members of the trefoil family have also been implicated as gastrointestinal-specific tumor-suppressor genes, whereas the coordinate expression of TFF peptides within neural tissues suggests a potential neuromodulatory role for these proteins both within the brain and peripheral targets including the gastrointestinal tract. The trefoil, or P domain, is a conserved 38- to 39-aminoacid motif containing 6 cysteine residues held together by 3 pairs of disulfide bonds configured 1-5, 2-4, and 3-6 (714). This domain produces a compact “three-leafed clover” protein structure that has marked thermal and enzymatic stability, a property that is most likely critical to the function of trefoil proteins within the harsh environment of the gastrointestinal tract (715–719). The trefoil domain has been highly conserved through evolution and is found in numerous species from

210 / CHAPTER 7 Xenopus to humans (709). Three distinct trefoil peptides have been identified in mammals. TFF1, originally designated pS2, was isolated from an estrogen-sensitive breast cancer cell line (720). TFF2, or spasmolytic polypeptide, was identified in the early 1980s during purification of porcine insulin (715), whereas intestinal trefoil factor, or TFF3, was isolated from rat intestine in 1991 (721). TFF1 and TFF3 each contain a single-trefoil domain, whereas TFF2 contains two-trefoil motifs as a result of genomic duplication (721–723).

Trefoil Factor Family Genes The TFF proteins are the product of a multigene family organized in a 50-kb head-to-tail linear arrangement on human chromosome 21q22.3 (724–726). A similar clustered organization for the TFF genes is observed on a 40-kb syntenic region on mouse chromosome 17q (721,728). Genetic studies indicate that TFF genes share several common regulatory motifs including those binding hepatocyte nuclear factor 3 (HNF-3)/forkhead, GATA6, AP-1, upstream stimulatory factor (USF), and the Myc/Max/Mad network of proteins (729–734). In addition, the individual TFF genes each contain a subset of specific regulatory elements that modulate cell-specific and physiologic expression (729). The combinatorial use of these regulatory motifs therefore provides the ability for coordinate as well as independent expression of each TFF member. The 4.3-kb human TFF1 gene is composed of three exons with its single-trefoil domain encoded by exon 2 (735). The proximal promoter domain of TFF1 contains a series of standard regulatory elements that includes a TATA-box, a CAAT element, and a GC-rich element. Additional specific regulatory elements including an AP-1 binding site, estradiol responsive element (ERE), heat shock factor 2 (HSF-2) motif, GATA consensus sequence motif, and a tissue-specific enhancer that potentially binds EGF, TPA, c-Ha-ras, and c-jun have also been characterized in association with the TFF1 gene promoter (729,733–737). TFF1 gene expression has been shown to be responsive to insulin, IGF-1, bFGF, and retinoic acid (738,739). The presence of an ERE within the TFF1 promoter provides rationale for the original identification of this TFF member in estrogen-sensitive breast cancer cell lines. Interestingly, the ERE is missing from the mouse TFF1-promoter domain (740). The human TFF2 gene is 4.6 kb is length and is composed of 4 exons with its duplicated trefoil domains encoded by a single exon (734). TFF2 expression appears to be modulated by AP-1, USF, PEA3, Ets-like factor, HSF-2, and GATA6 and has been shown to be responsive to asparin (730,731,734,741,742). No ERE is observed within the TFF2 promoter, which explains its lack of responsiveness to estradiol. Stomach-specific expression of both TFF2 and TFF1 appears to be principally modulated by the HSF-2 and GATA consensus motifs found within their promoters (731). The 3.5-kb human TFF3 gene is composed of 3 exons with the trefoil domain encoded by exon 2 (743). The TFF3 gene contains potential binding sites for SP-1, AP-1, CF1,

and HRE (743,744). Unlike its gastric counterparts, TFF3 does not contain HSF-2 and GATA consensus motifs and is unresponsive to GATA6 (729,731). Goblet cell–specific transcription of TFF3 appears to be modulated by the presence of a 9-bp goblet cell response element (GCRE), the expression of which can be modified by adjacent positive and negative regulatory elements (745). In addition, a second goblet cell silencer inhibitor (GCSI) that interacts with a specific GCSI binding protein has been shown to modulate TFF3 expression in certain cancer cell lines (746–748). Interestingly, the mucin-2 (MUC2) promoter also contains two GCREs providing a common regulatory pathway for coordinate regulation of these genes (749). TFF3 expression has been shown to be responsive to neuropeptides, acetylcholine, hypoxia, and short-chain fatty acids (744,750–752). A variety of studies suggest that the epigenetic regulation of TFF transcription may play an important role in modulating their expression (751,752). Variations in histone acetylation have been postulated to modulate TFF gene transcription in cancer cell lines, although hard evidence for this mode of regulation remains to be identified (751). In contrast, good evidence exists to support the observation that CpG methylation of TFF genes plays an important role in their expression. Mouse studies indicate that all three TFF genes appear demethylated in gastrointestinal tissues and methylated in nongastrointestinal tissues, whereas TFF1 and TFF3 possess reciprocal methylation states in the stomach and intestine specifically limiting their expression patterns to these tissues (752).

Trefoil Factor Family Proteins TFF proteins are small 7- to 12-kDa, highly stable proteins that are produced by mucin-secreting epithelial cells found throughout the body (709–713,753–756). Synthesized as precursors containing an N-terminal signal sequence, TFF proteins are copackaged into mucous granules within the Golgi apparatus and eventually secreted onto the epithelial cell surface (713). Although trefoil peptides are principally expressed within the gastrointestinal tract, their expression has also been observed in a variety of other tissues including conjunctiva, uterus/endocervix, salivary glands, respiratory tract, nonlactating breast, gallbladder, pituitary, hypothalamus, hippocampus, frontal cortex, and cerebellum (757–769). Human serum contains approximately 150 pg/ml TFF1, whereas human urine has a TFF1 concentration of 14 ng/mg creatinine (770–772). The concentration of TFF2 in gastric juice has been reported to be between 1 and 20 µg/ml with dramatic diurnal variation (773). Within the gastrointestinal tract, trefoil proteins display cell-specific patterns of expression that are both temporally and spatially regulated (753). TFF1 and TFF2 are considered “gastric” peptides showing complementary expression within the pit mucous cells and mucous neck cells of the stomach, respectively (753,758,764,774–777). TFF2 expression is also observed within Brunner’s glands of the duodenum

GROWTH FACTORS IN THE GASTROINTESTINAL TRACT / 211 (776,778,779). In contrast, expression of TFF3 appears “intestinal,” being primarily limited to goblet cells of the small intestine and colon (740,780–782). These cell-specific patterns of TFF expression result in a regional colocalization of specific TFF proteins and secretory mucins, thereby providing a potential molecular basis for the functional mucosa specificity. TFF expression occurs early in embryonic development, with all three TFF proteins coordinately expressed within the gastrointestinal tract (753,783,784). As development proceeds, the expression of the individual TFF peptides becomes progressively more restricted, resulting in the cell-specific patterns observed in the adult. Similar developmental regulation of TFF expression has also been observed in the brain (767). It is important to note that there is a large degree of species variation in the expression patterns of TFF proteins.

Trefoil Factor Family Receptors and Binding Proteins The direct receptors or binding proteins, or both, for the various TFF peptides have not yet been identified. TFF peptides are expressed at high levels at the apical surface of the normal gastrointestinal tract, suggesting that their association with high-affinity receptors may be problematic (709–710). It has been proposed that TFF–mucin interactions may mediate many of the biological properties of trefoil factors; however, TFF3 elicits a functional response only when applied to the basal surface of monolayers (785). In addition, findings that TFF proteins rapidly activate several intracellular signaling pathways strongly suggest that specific receptors/binding proteins exist. Although two TFF2-binding proteins have been identified, neither has been shown to mediate the cellular responses observed for TFF2 (786–789).

Intracellular Signaling by Trefoil Factor Family A variety of mechanisms have been postulated as modulating the intracellular responses of TFF proteins. These include activation of the EGFR and ras-dependent MAPK pathway (790–792). TFF3 induces phosphorylation of β-catenin and the EGFR, resulting in the intracellular redistribution of E-cadherin, which complexes with catenins (619,793,794). In addition, TFF proteins directly decrease the expression of catenins and the APC gene product, which completes with E-cadherin (794). The subsequent convergence of these two pathways therefore has a direct negative effect on the adhesive properties of cells, thereby promoting cell migration. TFF proteins inactivate apoptosis by a similar convergence of pathways including the phosphorylation of Akt, as well as the blockage of p53-independent and -dependent apoptosis through the EGFR and PI3K pathways (711). These pathways promote cell survivability in the absence of proliferation, which when combined with the motogenic pathways above result in a coordinated cellular response

supporting migration in the absence of programmed cell death and proliferation.

Biological Actions of Trefoil Factor Family Proteins In vitro studies, as well as recent transgenic knockout models, have helped to clarify the specific roles of TFF peptides within the gastrointestinal tract (795–800). From this work, it seems clear that the primary function of TFF proteins is the establishment and maintenance of mucous epithelial surface integrity (Fig. 7-8). TFF proteins function both in an autocrine and paracrine manner within the gastrointestinal tract, with optimal biological activity involving the interaction of two distinct trefoil domains (712,801). TFF2, which contains two trefoil domains, is eight times more efficient at promoting cell migration that monomeric TFF1 (799–800). The monomeric TFF proteins, TFF1 and TFF3, have been shown to form homodimers and heterodimers through disulfide linkage at a seventh conserved cysteine residue found within the C-terminal dimerization domain (775,802–806). Even so, monomeric TFF3 is capable of promoting cell migration, suggesting that dimerization may not be essential for TFF function (717,800,807). That TFF peptides are compact, highly stable proteins that are cytoprotective and promote healing when administered orally, subcutaneously, or intravenously makes them prime therapeutic targets for the prevention and resolution of gastrointestinal mucosal injury (808–810). Mucosal Defense TFF proteins have been shown to be coexpressed with mucins and enhance the protective characteristics of the mucosal defense barrier within the gastrointestinal tract (753,811,812). The addition of TFF2 to mucin preparations results in increased elasticity and viscosity of the mixture (755,780,813–816). It is postulated that TFF proteins are capable of cross-linking with mucin glycoproteins to form a physical barrier designed to protect the epithelial lining of the gastrointestinal tract (775,817). In addition, TFF peptides may directly affect the packaging of mucin glycoproteins within goblet cells, thereby regulating their secretion and subsequent function (711,712). Tissue Repair One of the fundamental actions of TFF proteins in the mature gastrointestinal tract is to promote epithelial cell restitution after mucosal injury (709–713,818). Loss of mucosal continuity within the gastrointestinal tract results in a coordinate increase in TFF expression in the regions immediately juxtaposed to the insult (712,819). This response is rapid, occurring as quickly as 30 minutes after injury, and is evident during inflammatory bowel disease, as well as a variety of ulcerative conditions (810). The coordinate expression of all three TFF members at the site of injury is similar to the pattern observed during early gastrointestinal

212 / CHAPTER 7 Mucosal defense

Cell proliferation

Cell-cell adhesion

Cell migration/ tissue repair

Differentiation Inflammatory response

Apoptosis

= Mucins = TFF

Transcriptional effects Enterocyte

Enterocyte Goblet cell Cell-substrate adhesion

Tumor suppression Potential neuropeptide

FIG. 7-8. Schematic representation of the biological action of trefoil factor family (TFF). TFFs are cosecreted with mucins from goblet cells and deposited on the apical surface of the gastrointestinal epithelium where they interact to increase mucosal defense. In addition, TFFs promote restitution by decreasing cell proliferation, cell–cell adhesion, cell–substrate adhesion, and apoptosis, thereby promoting cell migration in the absence of cell death. TFFs have also been proposed to be circulating neuropeptides, as well as play a role in tumor suppression.

development and may reflect a return to a more permissive microenvironment that is necessary for normal wound healing. It has also been suggested that the TFF expression supports the appearance of the ulcer-associated cell lineage, thereby directly stimulating gland regeneration in areas of ulceration (779,780,820,821). During restitution, TFF peptides act as motogens, supporting cell migration in the absence of cellular proliferation and apoptosis (709–713,717,754,755,783,790,799–800, 811,813,822–824). TFF proteins are believed to promote cell migration by the disruption of cell–cell and cell–substrate adhesion while promoting cell survivability. TFF-induced disruption of cell–cell adhesion appears to occur via rapid phosphorylation of β-catenin, causing the down-regulation of adhesion molecules associated with adherens junctions such as E-cadherin (619,712,793,794,825). In a similar manner, TFF treatment has been shown to induce focal adhesion kinase (FAK) phosphorylation, resulting in the downregulation of focal adhesion complexes and disruption of cell–substrate adhesion (712,822). Although the precise mechanism remains ambiguous, TFF proteins are believed to prevent cell death through the convergence of multiple pathways causing ERK/MAPK activation (712,717,790–791). TFF function is linked to the expression of other growth factors and signal transduction pathways that work in synergy to promote healing in the gastrointestinal tract. For example, although TFF proteins appear to work independent of TGF-β, they function synergistically with EGFR ligands such as TGF-α and erbB family members (619,709–713, 778,790,798,807,813,826–829). These complex interactions

generate a coordinate and sustained response to mucosal injury that is most likely critical to its eventual resolution. Although TFF3 knockout mice appear to develop normal gastrointestinal tissues, they show significantly impaired mucosal healing after injury and eventually die of extensive colitis (797). In a similar manner, TFF2 knockout mice display an increased susceptibility to injury by nonsteroidal antiinflammatory drugs (796). These observations highlight the important role that TFF plays in the mucosal defense and repair of the gastrointestinal tract. Development The coordinate expression of all three TFF isotypes in the developing gastrointestinal tract before the appearance of mature mucin-secreting cells suggests that TFF peptides may play a role in the differentiation and maturation of gastrointestinal epithelium. TFF1 promotes gastrointestinal differentiation by decreasing apoptosis and delaying the G1-S-phase transition in cells (830). This hypothesis is supported by several knockout studies, which demonstrate that TFF1 is essential for the normal differentiation of antral and pyloric mucosa, whereas TFF3 is critical in modulating intestinal cell proliferation rates (795,797). Inflammation Both TFF1 and TFF2 are expressed at high levels in rat lymphoid tissues and have been shown to participate in the immune response (831,832). In addition, the TFF proteins

GROWTH FACTORS IN THE GASTROINTESTINAL TRACT / 213 have been shown to induce directed monocyte migration, block complement deposition in vitro, and increase interleukin-6 (IL-6) secretion (829,831–835). Transgenic studies suggest that the SHP2-ERK/MAPK pathways are capable of inducing TFF1 expression, whereas IL-6 appears capable of regulating TFF3 expression (836). These observations suggest that TFF proteins may have direct effects on the inflammatory response providing an additional mechanistic synergy designed to promote wound healing within the gastrointestinal tract. Tumor-Suppressor Genes TFF1 was originally isolated from an estrogen-responsive human breast carcinoma cell line. Changes in TFF gene and protein expression have been documented in a wide range of epithelial cancers including those of the gastrointestinal tract, breast, pancreas, lung, endometrium, ovary, prostate, bladder, and cervix (754,837–845). Both TFF1 and TFF3 knockout mice show increased rates of gastrointestinal tumor development, whereas loss of TFF1 expression has been reported in human gastric cancers as a result of somatic mutation (795,797,846). In addition, overexpression of TFF1 and TFF3 has an antiproliferative effect on a variety of cell lines (837–845). These observations support the contention that at least TFF1 and TFF3 may represent tumorsuppressor genes within the gastrointestinal tract. It is postulated that TFF peptides promote the metastatic capacity of tumor cells by promoting cell migration in the absence of apoptosis. Neuropeptide TFF proteins are coordinately expressed in the developing and mature brain independent of mucous production, suggesting a potential role for these factors in normal neural development (767). Expressed of TFF1 has been reported in the rodent hippocampus, frontal cortex, and cerebellum with staining principally occurring within astrocytes (767). TFF3 is expressed in the hypothalamus of both rats and humans, with TFF3 and oxytocin being stored in the same secretory vesicles (767–769,847). These observations suggest that TFF3 may be playing a neuromodulatory role within the brain, as well as in peripheral targets including the gastrointestinal tract.

HEPATOCYTE GROWTH FACTOR Overview and Discovery of Hepatocyte Growth Factor HGF, or scatter factor, was originally identified in the early 1980s as a component of serum that was highly mitogenic for hepatocytes (848–850). More recent studies indicate that HGF plays a key role in gastrointestinal development and repair (851–857). HGF is principally synthesized in cells of mesenchymal origin and represents a pleiotrophic cytokine that induces cell proliferation, motility, differentiation, and

morphogenesis via its high-affinity receptor c-met (857–861). HGF can stimulate the growth of gastrointestinal epithelial cells in vitro and enhance intestinal mucosal repair in vivo, and it plays a critical role in gastrointestinal morphogenesis during development (851–857).

Hepatocyte Growth Factor Gene The single-copy human HGF gene is composed of 18 exons and is located on chromosome 7q21.1 (862–864). IL-1, TNF-α, and TGF-β and a variety of growth factors have all been shown to regulate HGF expression at the level of gene transcription (865–868). A novel regulatory element that binds inducible transcription factors in response to extracellular stimuli has been identified within the basal promoter of the HGF gene (869). Both HGF mRNA and protein expression are up-regulated by EGF, TGF-α, and PDGF, whereas TGF-β down-regulates expression of HGF (870,871). Heparin is a strong inducer of HGF expression, whereas estrogen stimulation through the EGFR induces HGF transcription (872,873). HGF has been shown to regulate met transcription providing a direct regulatory feedback loop between ligand and receptor (874,875).

Hepatocyte Growth Factor Protein Principally a product of mesenchymal-derived cells, HGF is a heparin-binding glycoprotein that is synthesized as a 728-amino-acid precursor that contains a 29-amino-acid signal sequence and 25-amino-acid prosequence (876,877). HGF is synthesized and secreted as an inactive single-chain pro-HGF that is processed into a mature, biologically active heterodimer by extracellular cleavage (876–882). Mature HGF heterodimer is composed of a 60-kDa α-chain and a 30-kDa β-chain that is proteolytically cleaved from pro-HGF at 494 arg-495 val by the serine proteinase HGF activator (HGFA). The α-subunit of HGF possesses four kringle domains that are believed to mediate receptor-ligand binding and have been shown to be necessary for the correct biological activity of HGF, whereas the β-subunit of HGF has an inactive serine protease domain (876,883–886). HGFA is related to coagulation factor XII and has been shown to activate pro-HGF in response to liver, renal, and gastrointestinal injury, as well as during gastrointestinal development (870,887–891). Although HGFA is produced and secreted mainly in the liver and circulates in the plasma as an inactive precursor, HGFA is also expressed at high levels throughout the gastrointestinal tract (887,891,892). The developmental and physiologic expression patterns of HGFA provide a secondary mode of regulation for HGF biological activity by spatially and temporally delimiting the production of active HGF heterodimer formation. HGFA is synthesized as an inactive precursor that is activated by thrombin (890,893). HGFA activity has also been shown to be regulated by HGFA inhibitor type 1 (HAI-1) and HAI type 2 (HAI-2), both of which are Kunitz-type serine

214 / CHAPTER 7 proteinase inhibitors (894–896). In vitro studies indicate that HAI-1 is also a specific cell-surface binding protein for active HGFA (896).

Hepatocyte Growth Factor Receptors The high-affinity receptor for HGF is the transmembrane tyrosine kinase c-met (880). Originally identified as an activated oncogene in an N-methyl-N′-nitrosoguanidine-treated human osteogenic sarcoma cell line, c-met is a member of the receptor tyrosine kinase family that possesses significant homology to Ron and c-sea (897–901). The c-met receptor is expressed by a variety of epithelial/endothelial cells including those of the gastrointestinal tract, as well as gastrointestinal smooth muscle cells (902). The c-met receptor is a heterodimer composed of a 50-kDa α-chain that is disulfide linked to a 145-kDa β-chain forming a 190-kDa complex (903). The c-met receptor α-chain is exposed to the cell surface, whereas the β-chain spans the cellular membrane and contains an intracellular tyrosine kinase motif (880, 904–906). As with many growth factor receptors, c-met is found primarily on the basolateral domain of polarized epithelial cells supporting the proposed paracrine stimulation via HGF secreted from closely associated mesenchymal cells (903,907). However, studies indicate a second paracrine mode of action for HGF involving luminal expression of HGF in body fluids that is bound by a c-met/src-related gastrointestinal tyrosine kinase (gtk) complex found in the brush border membranes of enterocytes (907). Autocrine regulation of HGF action has also been described in some cancer cell lines (908,909). c-Met expression is regulated by a number of factors including HGF, PMA, ConA, EGF, IL-1, IL-6, TNF, and the steroidal hormones estrogen, progesterone, tamoxifen, and dexamethasone (874,903,910,911). HGF binding has been shown to result in c-met polyubiquitination leading to its degradation, thereby providing a potential regulatory feedback loop between HGF activation and receptor turnover (903). HGF also directly promotes expression of the met gene (875).

Intracellular Signaling by Hepatocyte Growth Factor Binding of HGF to the c-met receptor has been shown to initially induce tyrosine phosphorylation of the β-chain (912). The differential recruitment of substrate molecules to the phosphotyrosylated segments on the C-terminal cytoplasmic domain of the c-met receptor is proposed to activate distinct signaling pathways (901,913–915). For example, STAT has been shown to be critical in mediating tubular morphogenesis, whereas cell scattering appears to be regulated through the Ras-MAPK signaling pathway. In a similar manner, PLCγ activation appears critical for HGF-induced cell motility, with phosphorylation of PLCγ resulting in the

formation of DAG and IP3, thereby stimulating the PKC and Ca2+ mobilization signaling pathways, respectively (903,910). HGF-mediated ERK activation appears critical in signaling cell proliferation, whereas the nuclear translocation of ERK is believed to regulate transcription factors (903). HGF binding to c-met has also been shown to activate the guanine-nucleotide binding protein, ras, thereby inducing alterations in cell movement and cell shape by cytoskeletal remodeling (903).

Biological Actions of Hepatocyte Growth Factor HGF has been shown to be a mitogen, motogen, and morphogen (915a). HGF activity represents a classic example of epithelial/mesenchymal induction, with the most common mode of action for HGF being activation of the c-met receptor via paracrine mechanisms. It is generally proposed that cells of mesenchymal origin produce pro-HGF, releasing it into the local microenvironment where it is processed to biologically active mature HGF heterodimers (Fig. 7-9). As discussed earlier, active HGF associates with heparin and is bound to epithelial cells via the high-affinity c-met receptor, resulting in receptor phosphorylation and activation of multiple intracellular signaling pathways that ultimately lead to a variety cellular responses. The biological activity HGF is directly regulated within the local microenvironment by the complex interaction of both activator and inhibitor molecules. The precise interplay between these molecules appears essential in determining whether active HGF heterodimers are present in sufficient quantities to bind to the c-met receptor and elicit a biological response. Development The morphogenic properties ascribed to HGF are believed to occur by a unique combination of actions including alterations in cytoskeleton, focal contacts, and cellular junctions (903). HGF stimulates the growth of gastrointestinal epithelial cells in a dose-dependent and region-specific manner, with distinct gastrointestinal epithelial subpopulations displaying different mitogenic responses (851,854,857). In vitro studies indicate that subepithelial intestinal fibroblasts regulate intestinal epithelial cell proliferation through the paracrine action of HGF (916). Studies indicate that HGF is capable of cross-linking with gtk, a src-related kinase that is highly expressed in the brush border of the small intestine, thereby inducing gtk kinase activity and promoting modulation of the growth and differentiation of gastrointestinal epithelial cells (907). Developmental studies indicate both HGF and c-met are expressed in human fetal tissue by 6 weeks of development with expression observed in the placenta, lung, liver, pancreas, and throughout the developing gastrointestinal tract (917,918). During development, HGF has been shown to induce branching tuburogenesis in the kidney, lung, and mammary gland, whereas in the adult, HGF appears critical

GROWTH FACTORS IN THE GASTROINTESTINAL TRACT / 215 HGFA

Thrombin

HGFA

IL1β HAI-1

Mucosal Injury

HGFA Liver & GI Tract

HS HS proHGF

Activation

proHGFA

HGF

Met

HGF Promotes proliferation migration differentiation

Epithelial cell

Epithelial cell

Epithelial cell

Promotes angiogenesis HS HGF Mesenchymal cell t Me

Endothelial cells

FIG. 7-9. Proposed model of hepatocyte growth factor (HGF) wound healing in the gastrointestinal (GI) tract. Circulating pro-HGF activator (proHGFA) is synthesized in the liver and gastrointestinal tract and becomes activated by thrombin on mucosal injury. In the presence of cytokines such as IL-1β, sequestered HGFA inhibitor type 1 (HAI-1)/HFGA complexes are released from the site of injury, thereby activating pro-HGF into active HGF, which interacts with heparin sulfate and binds to the c-met receptor promoting proliferation, differentiation, and migration in gastrointestinal epithelial cells and angiogenesis in vascular endothelial cells. HS, heparan sulfate.

in the regeneration of the same organs (859,919–926). Genetargeting studies indicate that HGF-met signaling is required for normal development of liver, skeletal muscle, placenta, and specific neural crest lineages (917,927). Consequently, HGF knockout mice are embryonic lethal as a result of impaired placental and liver development. Overexpression of HGF in transgenic mice results in an increase in renal failure and tumorigenesis (928,929). Interestingly, transgenic mice that overexpress HGF throughout the digestive tract experience development of ulcerative proctitis, rectal prolapse, and intestinal pseudoobstruction (857). These studies provide strong evidence that the HGF-met signaling is critical in the development and pathogenesis of numerous organ systems including the digestive tract (930). Tissue Repair HGF has been shown to induce the proliferation and migration of gastrointestinal epithelial cells in vitro, and gastrointestinal expression of HGF and its receptor c-met is greatly increased after acute injury and in regions of ulceration (856,896,931). In addition, HAI-1 expression has been shown to be up-regulated in regenerative epithelium (896). During epithelial injury, inflammatory cytokines such as IL-1β have been shown to promote the release of active HGFA/HAI-1 complexes from the cell surface (896). These studies suggest that HAI-1 may function as a reservoir of active HGFA in

regions of epithelial repair, thereby providing efficient localization of active HGF at the site of mucosal injury. Studies suggest that HGF-mediated repair occurs through a TGF-β–dependent pathway that involves the integration of both motogenic and mitogenic pathways (932). For example, HGF has been shown to inhibit the assembly of adherens junctions by directly modifying the stability and phosphorylation pattern of E-cadherin (933). In addition, HGF appears capable of directly regulating cell adhesion by decreasing expression of E- and P-cadherin at the protein level (934). HGF has also been shown to enhance the phosphorylation status of β-catenin, resulting in the dissociation of β-catenin and E-cadherin, thereby promoting the dismantling of cell– cell adhesion complexes (935,936). HGF is capable of modulating cell-matrix adhesion by altering the pattern of integrin expression, as well as modulating the recruitment of integrins, cytoskeletal proteins, pp125FAK, and paxillin into focal adhesion complexes (930,937). HGF stimulates matrix degradation by several mechanisms including increasing the production of plasminogen activator type 1, tissue factor, MMPs, collagenase-1, and stromelysin-1 (938,939). In addition, HGF binds directly to several matrix molecules such as fibronectin, heparin sulfate proteoglycan, and thrombospondin-1 (903). HGF has been shown to regulate cell motility by the selective up-regulation of motor-related proteins including α-tubulin, myosin II, actin, kinesin, and myosin 1α (940).

216 / CHAPTER 7 Tumorigenesis HGF and c-met are expressed in a wide range of tumors and have been implicated in regulating tumor cell motility, growth, proliferation, invasion, progression, and neovascularization (903,941–944). A variety of studies indicate that both HGF and c-met are highly expressed or overexpressed in gastrointestinal tumors including those of the esophagus, stomach, colon, and rectum (903,936,945–953). Distinct mutations and isoforms of c-met have been reported in both hereditary and sporadic forms of cancer (903,954). Both c-met and HGF have been shown to be valuable prognostic indicators, and HGF represents a viable target for cancer treatment, with inhibition of its function reducing many of its effects in cancer cells (947,951,955–963).

FIBROBLAST GROWTH FACTOR FAMILY Overview and Discovery of Fibroblast Growth Factor Family Fibroblast growth factors (FGFs) are a large family of structurally related proteins that are involved in diverse cellular processes including proliferation, differentiation, migration, and apoptosis (964). Originally identified in the early 1970s, FGF was characterized and named based on its ability to stimulate the proliferation of 3T3 fibroblasts (965). Since then, an additional 21 members of the FGF family have been identified in vertebrates (Table 7-2) (964,966). FGFs elicit their biological responses by binding to and activating FGF receptors (FGFRs) (967–969). Studies suggest that FGFs and their receptors are differentially expressed throughout the developing and mature gastrointestinal tract, where they

appear to play an important role in modulating gut development and epithelial repair mechanisms after injury (964, 970–973).

Fibroblast Growth Factor Genes Each FGF family member is the product of a single, unlinked gene ranging in size from 5 to 100 kb (966,974). Although most FGF genes are found scattered throughout the genome, some clusters have been observed in both humans and mice (966). Human FGF3, FGF4, and FGF19 are clustered on chromosome 11q13; FGF6 and FGF13 map to chromosome 12p13; and FGF17 and FGF20 are clustered on 8p21-p22. The most common genomic organization of the prototypic FGF gene is three coding exons, with exon 1 containing the initiation start site (966). Even so, alternative splicing, initiation sites, and amino termini have been reported for distinct FGF family members, making the precise organization and regulation of these genes best examined on an individual basis.

Fibroblast Growth Factor Proteins The FGF family currently consists of 22 members that have been subdivided into 7 distinct subclasses based on a combination of differential criteria including structure, biochemical properties, receptor affinities, and expression patterns (see Table 7-2) (966,975). Human FGF15 and mouse FGF19 have not been identified. However, evolutionary studies indicate that these two FGFs are most closely related to one another, suggesting that FGF19 may be the human ortholog

TABLE 7-2. Fibroblast growth factor family members and receptor affinity Ligand

Subfamily

Synonym

FGF-1

FGF1

Acidic FGF, aFGF

FGF-2 FGF-3 FGF-4 FGF-5 FGF-6 FGF-7 FGF-8 FGF-9 FGF-10 FGF-11 to -14 FGF-16 FGF-17 FGF-18 FGF-19 FGF-20 FGF-21 FGF-22 FGF-23

FGF1 FGF7 FGF4 FGF4 FGF4 FGF7 FGF8 FGF9 FGF7 FGF11 FGF9 FGF8 FGF8 FGF19 FGF9 FGF19 FGF7 FGF19

Basic FGF, bFGF Int-2 kFGF, Kaposi FGF, hst-1 hst-2 KGF AIGF GAF KGF-2

High-affinity receptors FGFR1, IIIb and IIIc; FGFR2, IIIb and IIIc; FGFR3, IIIb and IIIc; FGFR4 FGFR1, IIIb and IIIc; FGFR2, IIIc; FGFR3, IIIc; FGFR4 FGFR1, IIIb; FGFR2, IIIb FGFR1, IIIc; FGFR2, IIIc; FGFR3, IIIc; FGFR4 FGFR1, IIIc; FGFR2, IIIc FGFR1, IIIc; FGFR2, IIIc, FGFR4 FGFR2, IIIb FGFR1, FGFR2, IIIc; FGFR3, IIIc; FGFR4 FGFR2, IIIc; FGFR3, IIIb and IIIc, FGFR4 FGFR1, IIIb; FGFR2, IIIb Function intracellularly independent of FGFRs FGFR4 FGFR1, IIIc; FGFR2, IIIc FGFR2, IIIc, FGFR3, IIIc FGFR4 FGFR1, IIIc unknown FGFR2, IIIb FGFR3, IIIc

AIGF, androgen-induced growth factor; GAF, glia-activating factor; FGF, fibroblast growth factor; FGFR, fibroblast growth factor receptor; KGF, keratinocyte growth factor.

GROWTH FACTORS IN THE GASTROINTESTINAL TRACT / 217 of mouse FGF15 (966). Human FGFs range in size from 17 to 34 kDa and share several common features including a high affinity for heparin and the presence of a homologous 120-amino-acid central core domain that has been shown to be critical in FGFR binding (966). Structural studies have identified 12 antiparallel β strands within the central core domain including a β trefoil structure composed of four β sheets organized in a triangular array (967,968,976). The primary heparin-binding site is composed of several basic amino acids conserved on two β sheets and appears structurally distinct from the FGFR-binding site (969,976). Most FGFs contain an amino-terminus signal sequence and are consequently readily secreted from cells in which they are produced (966). However, a significant subset of FGFs including FGF1, 2, 9, 11 to 14, 16, and 20 lack an obvious signal peptide (977–986). Of this group, FGF9, 16, and 20 are nevertheless secreted from cells, and FGF9 has been shown to contain a noncleaved amino-terminal hydrophobic domain that is required for secretion. Although FGF1 and FGF2 are not secreted from cells, they can be found on the cell surface and within the ECM, suggesting that they are released from cells by a mechanism that is independent of the endoplasmic reticulum–Golgi pathway. In contrast, FGF11 to 14 do not appear to be secreted from cells, and it remains to be determined whether these FGFs function by interacting with FGFRs or in a receptor-independent manner. Interestingly, FGF2 and FGF3 have alternative high-molecular weight isoforms possessing an additional nuclear localization amino-terminus sequence that results in their transport to the nucleus. The precise biological function of these nuclear-localized FGFs remains unclear. Mammalian FGFs are differentially expressed in most tissues examined in both a developmental and tissue-specific manner (964). Although FGF subfamilies tend to display similar patterns of expression, individual FGFs are clearly expressed in unique sites as well (966). FGF3, 4, 8, 15, 17, and 19 appear to be expressed almost exclusively in the developing embryo, whereas the remaining FGFs are expressed both during embryonic development and in the adult (966). These patterns of expression strongly suggest that FGFs play an important role in embryonic development, as well as a potential physiologic role in adult tissues.

of FGFR and FGF ligand expression provide one mechanism whereby their biological functionality is delimited during development and in the adult. Structurally, FGFRs consist of an extracellular ligandbinding domain, a single-transmembrane domain, and a split intracellular kinase domain (Fig. 7-10) (964,992). The extracellular region contains two to three immunoglobulin (Ig)-like domains that are necessary for FGF binding. These Ig-like domains are capable of regulating both ligand affinity and specificity. The sites for heparin binding and ECM and cell adhesion association are located between Ig domains I and II. The intracellular region contains the functional domain responsible for FGFR tyrosine kinase activity, as well as additional sites that are necessary for protein binding and phosphorylation or autophosphorylation of the receptor molecule. Alternative splicing of FGFR transcripts is capable of generating FGFR isotypes that possess unique functionalities (988,991). For example, alternative splicing of the C-terminal portion of the third Ig domain generates distinct FGFR isotypes designated IIIb or IIIc that have been shown to possess unique binding specificities for FGFs (993–995). In addition, FGFR isotypes that lack the transmembrane domain, acid box, or signal peptide have also been reported (988,996–1000). Clearly, these changes in functional domain inclusion would have profound effects on the biological activity associated with receptor-ligand binding. Despite the complexity of receptors and receptor isoforms, most FGFRs Signal peptide targeted secretion I

Ig-like domains affects FGF binding affinity and selectivity II

III

Heparin-binding domain/ CAM homology domain proper interaction with extracellular components

Alternatively spliced Ig III domain

Transmembrane domain maintains receptor conformation for ligand activation

Fibroblast Growth Factor Receptors FGFRs are a gene family of receptor tyrosine kinases with at least four signaling members identified in vertebrates and designated FGFR1, FGFR2, FGFR3, and FGFR4 (987). FGFR genes generally contain 18 exons, possess similar exon-intron organization, and are randomly dispersed throughout the genome with no apparent linkages to FGF gene locations (987). FGFRs are differentially expressed in a tissue-specific manner throughout development and into adulthood, and they are capable of producing multiple receptor isoforms through alternative splicing of primary transcripts (964,966,974,987–991). The overlapping patterns

Acidic sequence optimal HSPG interaction

Juxtamembrane domain adaptor molecule binding

Kinase domains catalytic activity & adaptor molecule binding

FIG. 7-10. Schematic representation of a prototypic fibroblast growth factor receptor (FGFR) with functional domains identified. CAM, cell adhesion molecule; HSPG, heparan sulfate proteoglycan.

218 / CHAPTER 7 associate with similar adapter molecules to transmit intracellular signaling, and consequently have conservation of their intracellular adapter protein-binding sites (987).

Intracellular Signaling by Fibroblast Growth Factor FGFs bind to FGFRs, inducing their dimerization resulting in the autophosphorylation of specific cytoplasmic tyrosine residues and activating intracellular signal transduction pathways by controlling the protein tyrosine kinase activity and recruitment of signaling complexes (Fig. 7-11) (964, 1001). The binding of SH2 and phosphotyrosine binding signaling proteins to the phosphorylated FGFR provides a platform for the intrinsic catalytic activity of these molecules, as well as the binding of additional adapter proteins (964,1002,1003). In this manner, cell-specific signaling pathways can be modulated by ligand binding to the FGFR. Three primary signaling pathways have been associated with FGF binding to the FGFR including the Ras/MAPK, PLCγ/Ca2+, and PI3K/Akt pathways (964). Activation of the Ras/MAPK pathways involves the interaction of the lipidanchoring docking protein FRS2, which is bound by the adapter protein Grb2 and the PTP Shp2, resulting in complex formation with Son of sevenless and activation of ras by GTP exchange (1004–1009). This signal transduction

FGFR FGFR

pathway results in the phosphorylation of downstream transcription factors including c-myc, AP-1, and ETS family members through the MAPK signaling cascade (1010). Binding of PLCγ to FGFR results in the formation of two second messengers, IP3 and DAG, activating PKC and stimulating the release of Ca2+, respectively (964). This signaling cascade appears important in FGF-stimulated neural development including neurite outgrowth (1011–1013). FGFR is also capable of activating the PI3K/Akt pathway via three overlapping mechanisms involving Ras, Grb2/Gab1, and the FGFR alone (964,1014). Activation of this FGFR pathway has been shown to be critical in mesoderm induction (1014). Studies indicate that the activity of FGF signaling is regulated both within the cell, as well as within the extracellular space (964). Within the cell, many of the FGF signaling cofactors are regulated by FGF signaling directly, thereby creating regulatory feedback loops designed to modulate FGF activity (1015–1018). In addition, specific intracellular regulators of FGF have also been identified in several species. These include PTPs, which act as positive and negative regulators of FGF signaling, and Sprouty proteins, which interfere with FGF signaling via multiple mechanisms (1019–1028). A variety of transmembrane modifiers of FGF activity have also been identified including Sef, FLRT3, N-CAM, N-cadherin, and L1 (964,1011,1017,1029–1036). These interactions result in the formation of multiprotein

FLRT3

FGF HS FGF

P PLC

Ca2+

S2 FR P

Shp-2 DAG

FGF

FGFR

HS FGF

FGFR N-CAM

IP3

P13K

Gab1

Akt PKC

SOS

2. Differentiation

3. Proliferation & differentiation

Grb2

Raf

Ras

MEK

Transcriptional effects Nucleus

P

ERK

MKP3

1. Proliferation & differentiation

FIG. 7-11. Some of the intracellular signaling pathways activated by fibroblast growth factor (FGF) binding to the FGF receptor (FGFR). Fibroblast growth factor-heparan sulfate (FGF-HS) binding to the dimerized FGFR causes autophosphorylation of the receptor and activation of multiple intracellular signaling pathways including the Ras/mitogen-activated protein kinase (MAPK) pathway (1), phospholipase Cγ (PLCγ)/Ca2+ pathway (2), and the phosphatidylinositol-3 kinase (PI3K)/Akt pathway (3). DAG, diacylglycerol; ERK, extracellular signal–regulated kinase; FRS, FGF receptor substrate 2; MKP3, MAP kinase phosphatase 3; N-CAM, neural-cell adhesion molecule; PKC, protein kinase C; SOS, Son of sevenless.

GROWTH FACTORS IN THE GASTROINTESTINAL TRACT / 219 TABLE 7-3. Developmental phenotypes observed in fibroblast growth factor knockout mice Subfamily

Ligand

FGF1

FGF-1 FGF-2 FGF-4 FGF-5 FGF-6 FGF-3 FGF-7 FGF-10 FGF-22 FGF-9 FGF-16 FGF-20 FGF-8 FGF-17 FGF-18 FGF-11 FGF-12 FGF-13 FGF-14 FGF-19 FGF-21 FGF-23

FGF4 FGF7

FGF9 FGF8 FGF11

FGF19

FGF−/− phenotype Viable Viable Lethal, Viable Viable Viable Viable Lethal, ND Lethal, ND ND Lethal, Viable Lethal, ND Viable ND Viable Lethal, ND Viable

E4-5

P0 P0 E8 P0

E12.5

Description None identified Mild cardiovascular, skeletal, neural defects Inner-cell mass proliferation Long hair, angora mutation Subtle, muscle regeneration Mild inner ear, skeletal (tail), CNS defects Hair follicle growth, ureteric bud growth Multiple organ development, duodenal and colonic atresia ND Lung, heart, GI, skeletal, testes development ND ND Gastrulation defect; CNS, limb development Cerebellar development Skeletal and lung development ND Neuromuscular phenotype ND Neurologic phenotype Not clear ND Growth retardation, bone abnormalities

CNS, central nervous system; FGF, fibroblast growth factor; GI, gastrointestinal; ND, not determined.

complexes that appear critical in modulating FGFR interactions and its resultant signaling. Within the extracellular compartment, the interaction of FGF with heparin/HSPG has been shown to be an important feature of FGF functionality (990,1037–1040). HSPGs are critical in stabilizing FGF proteins, as well as modulating their binding and specificity FGFRs (990,1041–1044). In addition, these interactions appear important in modulating FGF release and diffusion within the extracellular compartment, thereby helping to delimit FGF activity within the local microenvironment (1045,1046).

Biological Actions of Fibroblast Growth Factor FGFs are a diverse family of growth factors that effect a wide range of biological functions including tissue morphogenesis, tissue repair, angiogenesis, and tumorigenesis via paracrine activation of FGFs (964). The precise temporal and spatial distribution of FGF ligands, FGFR isotypes, and cell-specific adaptor molecules provides a basis for functional diversity that is capable of establishing unique developmental and tissue-specific responses based on individual expression patterns alone. The critical, and yet

diverse, roles that FGFs and their receptors play in embryonic development are borne out by the wide range of anomalies observed in FGF and FGFR knockout mice (Tables 7-3 and 7-4) (964,966). In these studies, FGFs have been shown to be critical in implantation, mesoderm formation, gastrulation, chemotaxis, morphogenetic movements, neural induction, anterior-posterior axis formation, and endoderm formation, subsequently affecting a wide range of target tissues including those of the gastrointestinal tract. The physiologic recapitulation of these developmental programs in adult tissues, such as the gastrointestinal tract, appears critical in maintaining tissue homeostasis and promoting repair after injury. Finally, the role of FGFs in mediating cell growth and differentiation makes them and their receptors potential targets for gastrointestinal tumorigenesis. Development During embryonic development, FGFs can act as classic morphogens being both diffusible and capable of eliciting a biological response in a graded, dose-dependent manner (964). FGFs have been shown to be mitogenic for a wide range of cell types including fibroblasts, endothelial cells, and epithelial cells (964). A variety FGFs and FGFR subtypes are

TABLE 7-4. Developmental phenotypes observed in fibroblast growth factor receptor knockout mice Receptor FGFR1 FGFR2 FGFR3 FGFR4

FGFR−/− phenotype

Description

Embryonic lethal Embryonic lethal Viable Viable

Die around gastrulation with many defects Die either just after implantation or E10-11 Skeletal defects, long bone overgrowth, deafness No overt defects (lung defects/dwarfism with FGFR3−/−)

FGFR, fibroblast growth factor receptor.

220 / CHAPTER 7 differentially expressed within the gastrointestinal tract during development, suggesting that they play a key role in gut morphogenesis (964,1047). In general, FGF is expressed by mesenchymal cells and their derivatives found within the submucosal tissue, whereas expression of FGFR isotypes is observed in the epithelial cells of the overlying mucosa. Within the gastrointestinal tract, FGF-7, or keratinocyte growth factor (KGF) and its corresponding receptor, FGFR2-IIIb, have been extensively studied (1048–1051). KGF was originally isolated from the conditioned medium of human lung fibroblasts and characterized as a potent mitogen for keratinocytes (1052). Since then, KGF has been shown stimulate the proliferation of a variety of epithelial cell types (1053–1056). KGF and its receptor are expressed throughout the developing and mature gastrointestinal tract where they have been shown to enhance the proliferation, migration, and differentiation of gastrointestinal epithelial cells (1048,1054,1057,1058). KGF is principally produced by mesenchymal cells and acts in a paracrine manner by binding to the FGFR2-IIIb splice variant found in adjacent epithelial cells (1048). Recombinant KGF stimulates growth of the gastrointestinal tract, and unlike many other FGFs, its activity is principally limited to epithelial cells (1050, 1051,1054). KGF is believed to promote goblet cell differentiation through interactions with the GCSI binding protein, and KGF has also been shown to differentially regulate digestive function by specifically modulating enzyme activities within the gastrointestinal tract (747,1051,1059). Involvement of the FGF signaling pathway in early mesoderm and endoderm formation places the members of this growth factor family in a position to dramatically affect the early stages of embryonic development. Consequently, ablation of FGF or FGFR expression, or both, in mice results in a significant degree of embryonic lethality, making the observation of specific gastrointestinal defects difficult. Even so, alterations in gastrointestinal development have been observed in FGF and FGFR knockout mice. For example, FGF-10 (KGF-2) and FGFR2-IIIb knockout mice show a wide range of gastrointestinal defects with incomplete penetrance and variable phenotype (1060–1064). These defects include duodenal and colonic atresia, as well as defects in cecal and anorectal development. The duodenal and colonic atresia observed in FGF-10−/− and FGFR2-IIIb−/− mice occurs independent of mesenteric vascular occlusion and appears to represent a fundamental defect in the development of these gastrointestinal segments (1062–1064). The defects in cecal development observed in these animals occurs as a result of improper epithelial proliferation and invasion into the developing cecal bud permitting maintenance of this immature gastrointestinal segment throughout development (1060). The anorectal defects observed in FGF-10−/− mice represent a developmental failure of anorectal communication that becomes evident as early as embryonic day 13.5 (1061). Additional studies suggest that misexpression of FGF-1, -7, and -10 within the developing foregut may result in esophageal atresia and tracheoesophageal fistula formation (1065). Taken together, these observations highlight that

important role that the FGF signaling plays in mediating distinct epithelial–mesenchymal interactions within the gastrointestinal tract, thereby controlling normal gut development, maturation, and function. Tissue Repair and Angiogenesis A variety of studies indicate the FGFs play an important role in wound healing and tissue repair (970,971,973,1050, 1066,1067). Within the gastrointestinal tract, FGFs have been shown to restore connective tissue in injured mucosa by promoting epithelial cell migration and proliferation, fibroblast proliferation, and angiogenesis (1067,1068). FGF expression appears critical in the formation of granulation tissue, which is composed of fibroblasts, macrophages, and endothelial cells and represents the primary source for regeneration of the lamina propria and microvessels within the healing ulcer (1067,1068). Within this context, it has been suggested that the chronic overexpression of FGF may promote fibrosis (1069). Expression of FGF2 and its receptor increases along ulcer edges after acute gastrointestinal injury, and KGF, or FGF-7, overexpression has been observed in inflammatory bowel disease (1070,1071). FGF2 expression increases VEGF mRNA synthesis and protein secretion in a dosedependent manner promoting reconstruction of the microvasculature within the mucosal scar, thereby providing critical oxygen and nutrients to the region under repair (1067, 1068,1072). The administration of recombinant FGF has been shown to decrease the severity of gastrointestinal ulceration, reduce the susceptibility to experimental ulceration, and promote epithelial restitution (970,1073–1076). For example, KGF pretreatment ameliorates chemotherapy and radiationinduced mucositis by significantly reducing atrophy, accelerating regrowth, decreasing ulcer formation, and improving crypt and villus survival (1074,1077–1080). KGF has also been shown to prevent graft-versus-host disease by maintaining gastrointestinal integrity and preventing subsequent proinflammatory cytokine generation (1081). KGF appears to mediate some of these cytoprotective responses by up-regulating the expression of genes involved in mucosal protection such as antioxidant enzymes and intestinal trefoil protein (TFF3) (1080). As with most other growth factors, the biological actions of FGF during ulcer healing are tightly associated with the expression and activity of additional growth factors found within the gastrointestinal tract. These growth factors appear to cooperatively interact in the process of ulcer healing by differentially affecting nearly all the cell types required for epithelial restitution. These include epithelial cell proliferation and migration by EGF>FGF>PDGF, fibroblast proliferation by FGF>PDGF, and angiogenesis by VEGF>FGF>>PDGF>>EGF (973). The overlapping functions associated with these growth factors are most likely designed to guarantee a high success rate in wound healing after acute gastrointestinal injury.

GROWTH FACTORS IN THE GASTROINTESTINAL TRACT / 221 Tumorigenesis That FGFs have been characterized as potent mitogens for a variety of cell types makes them potential targets for tumor development within the gastrointestinal tract, as well as other tissue types. FGF expression has been reported in a variety of gastrointestinal tumors, with FGF1 overexpression observed in a high percentage of colorectal adenomas, colorectal cancers, and gastric cancers (1082–1084). In contrast, expression of FGF2 was reduced in many of the same tumor types, and this reduction in FGF2 expression was strongly correlated with gastric tumor grade. Expression of FGF2 and FGFR1 has been reported in human esophageal cancers, and expression of KGF and FGFR2-IIIb has been reported in neoplastic colonic mucosa, with overexpression of FGFR2-IIIb observed in more differentiated tumor samples (1047,1084). In addition, FGF-induced signaling through the pp57 mitogen-activated kinase has been shown to promote cell growth in undifferentiated colon carcinoma cell lines, whereas FGF1 expression has been correlated with the proliferation and activity of stromal fibroblasts found within endocrine tumors of the gut (1085,1086). FGFs may also play a secondary role in promoting tumor growth by mediating localized angiogenesis within the developing tumors (1087). These studies indicate that aberrant FGF and FGFR expression may be important in the development and progression of gastrointestinal tumors.

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246 / CHAPTER 7 1063. Fairbanks TJ, Kanard RC, Del Moral PM, Sala FG, De Langhe SP, Lopez CA, Veltmaat JM, Warburton D, Anderson KD, Bellusci S, Burns RC. Colonic atresia without mesenteric vascular occlusion. The role of the fibroblast growth factor 10 signaling pathway. J Pediatr Surg 2005;40:390–396. 1064. Kanard RC, Fairbanks TJ, De Langhe SP, Sala FG, Del Moral PM, Lopez CA, Warburton D, Anderson KD, Bellusci S, Burns RC. Fibroblast growth factor-10 serves a regulatory role in duodenal development. J Pediatr Surg 2005;40:313–316. 1065. Crisera CA, Maldonado TS, Longaker MT, Gittes GK. Defective fibroblast growth factor signaling allows for nonbranching growth of the respiratory-derived fistula tract in esophageal atresia with tracheoesophageal fistula. J Pediatr Surg 2000;35:1421–1425. 1066. Szabo S, Sandor Z. Basic fibroblast growth factor and PDGF in GI diseases. Baillieres Clin Gastroenterol 1996;10:97–112. 1067. Jones MK, Tomikawa M, Mohajer B, Tarnawski AS. Gastrointestinal mucosal regeneration: role of growth factors. Front Biosci 1999; 4:D303–D309. 1068. Tarnawski A, Szabo IL, Husain SS, Soreghan B. Regeneration of gastric mucosa during ulcer healing is triggered by growth factors and signal transduction pathways. J Physiol Paris 2001;95:337–344. 1069. Okunieff P, Barrett AJ, Phang SE, Li A, Constine LS, Williams JP, Rubin P, Wang X, Wu T, Chen Y, Ding I. Circulating basic fibroblast growth factor declines during Cy/TBI bone marrow transplantation. Bone Marrow Transplant 1999;23:1117–1121. 1070. Bajaj-Elliott M, Breese E, Poulsom R, Fairclough PD, MacDonald TT. Keratinocyte growth factor in inflammatory bowel disease. Increased mRNA transcripts in ulcerative colitis compared with Crohn’s disease in biopsies and isolated mucosal myofibroblasts. Am J Pathol 1997;151:1469–1476. 1071. Finch PW, Cheng AL. Analysis of the cellular basis of keratinocyte growth factor overexpression in inflammatory bowel disease. Gut 1999;45:848–855. 1072. Sako A, Kitayama J, Yamaguchi H, Kaisaki S, Suzuki H, Fukatsu K, Fujii S, Nagawa H. Vascular endothelial growth factor synthesis by human omental mesothelial cells is augmented by fibroblast growth factor-2: possible role of mesothelial cell on the development of peritoneal metastasis. J Surg Res 2003;115:113–120. 1073. Zeeh JM, Procaccino F, Hoffmann P, Aukerman SL, McRoberts JA, Soltani S, Pierce GF, Lakshmanan J, Lacey D, Eysselein VE. Keratinocyte growth factor ameliorates mucosal injury in an experimental model of colitis in rats. Gastroenterology 1996;110: 1077–1083. 1074. Danilenko DM. Preclinical and early clinical development of keratinocyte growth factor, an epithelial-specific tissue growth factor. Toxicol Pathol 1999;27:64–71. 1075. Han DS, Li F, Holt L, Connolly K, Hubert M, Miceli R, Okoye Z, Santiago G, Windle K, Wong E, Sartor RB. Keratinocyte growth

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CHAPTER

8

Developmental Signaling Networks Wnt/β-Catenin Signaling in the Gastrointestinal Tract Guido T. Bommer and Eric R. Fearon History, 248 Discovery of the Wnt Signaling Pathway, 248 Adenomatous Polyposis Coli Tumor Suppressor Gene at Chromosome Region 5q21, 248 β-Catenin Pathway, 249 Wnt/β Overview, 249 Wnt/β-Catenin Pathway Ligands, Receptors, and Extracellular Inhibitors, 249 Wnt/β-Catenin Signaling from the Cell Surface to the Nucleus, 252 Nuclear Access of β-Catenin: Subcellular Distribution, 258 Nuclear Action of β-Catenin, 258 Wnt/β-Catenin/T-Cell Factor Target Genes, 259

Noncanonical Wnt Signaling Pathways, 260 β-Catenin Pathway in Gastrointestinal Physiology, 260 Wnt/β Wnt/β-Catenin in Gastrointestinal Development, 260 Wnt/β-Catenin Pathway in Gastrointestinal Homeostasis and Architecture, 260 β-Catenin Pathway Defects in Gastrointestinal Wnt/β Tumors, 261 Colon and Rectum, 261 Esophagus, 263 Stomach, 263 Liver, 264 Pancreas, 264 References, 264

A major objective for the gastrointestinal biology field is to obtain in-depth and comprehensive knowledge of specific signaling pathways, factors, and mechanisms that regulate crucial cell fate decisions in epithelial cells in the developing and adult gastrointestinal tract. Substantial progress toward a more complete understanding of pathways and proteins with key roles in regulating cell proliferation, survival, differentiation, and motility, among other processes, has been achieved, in part through the recognition that cancers in the

gastrointestinal tract often co-opt critical developmental pathways, with essentially constitutive (unregulated) activity of the pathway in cancer cells. As such, understanding how the deregulated pathways contribute to the altered phenotype of the cancer cells can provide important clues into the function of the pathway in normal cells, complementing other types of research investigations that might be performed to explore the function of the pathway in normal cell physiology. The Wnt proteins represent a conserved family of secreted proteins with pleiotropic and context-specific activities in embryogenesis and adult tissues, including the gastrointestinal tract. Defects in Wnt signaling play a major role in the pathogenesis of colorectal and subsets of various other gastrointestinal cancers. This chapter highlights how findings from a broad array of different models and approaches have revealed the significance of Wnt pathway function in development and normal adult tissues and how derangements in Wnt signaling underlie the genesis of tumors of the gastrointestinal tract.

G. T. Bommer: Division of Molecular Medicine and Genetics, Department of Internal Medicine, University of Michigan School of Medicine, Ann Arbor, Michigan 48109. E. R. Fearon: Division of Molecular Medicine and Genetics, Departments of Internal Medicine, Human Genetics, and Pathology, University of Michigan School of Medicine, Ann Arbor, Michigan 48019. Physiology of the Gastrointestinal Tract, Fourth Edition, edited by Leonard R. Johnson. Academic Press, 2006.

247

248 / CHAPTER 8 HISTORY Discovery of the Wnt Signaling Pathway In the early 1980s, Nusse and Varmus (1) determined that the proviral integrations of the mouse mammary tumor virus (MMTV) that were present in the genomic DNA of many independent MMTV-induced carcinomas were often associated with the same chromosomal region, and the cellular gene that was inappropriately activated by these integrations was hence termed Int-1, an abbreviation for “integration site 1.” After the introduction of the Int-1 gene into certain nontumorigenic epithelial cells, the cells acquired abnormal phenotypic traits associated with cancer cells, implicating Int-1 as a presumptive oncogene (2). Independent studies found that mutations of a Drosophila homologue of the Int-1 gene can lead to flies without wings, termed wingless (wg) mutants (3,4). After recognition of the relationship between Int-1 and Wg and further efforts revealing that the proteins encoded by Int-1 and Wg are members of a much larger collection of related signaling molecules, the family of genes and proteins was collectively termed WNTs (pronounced “wints”), for the combination of wingless and integration site 1 (5). A considerable fraction of current knowledge of key components in Wnt signaling has been obtained from genetic analysis of mutant phenotypes in Drosophila. As noted earlier, Wg mutants in Drosophila initially caught the attention of investigators because of the major defects seen in wing morphogenesis. However, it was subsequently found that embryos and larvae carrying Wg mutations also show other phenotypes, such as rather subtle changes in the distribution of specific mechanosensory hairs (so-called cuticular denticles) within body segments, as well as changes in the development of the eye. Of some interest is that a similar denticle phenotype akin to that seen in Wg mutants subsequently was also described for other Drosophila mutant flies, including those with mutations in the Dishevelled (dsh), Armadillo (arm), and Pangolin genes. In flies carrying inactivating mutations in a gene termed Zeste-white-3 (zw-3), essentially the opposite phenotype was observed, namely a naked cuticle phenotype. By examining flies for more pronounced phenotypes, or conversely more subtle phenotypes, in progeny generated by crossing a particular mutant fly with a defined phenotype to another mutant fly carrying a defect in some other gene, it is possible to order signaling pathways genetically, even in the absence of in-depth knowledge of the molecular changes leading to the observed phenotypes. These types of studies are referred to as epistasis experiments. Using this type of epistasis approach, it was possible to define the relationship and order of major components of the Wg pathway in Drosophila by the mid1990s (6–9). Wnt genes in certain vertebrate species, such as Xenopus laevis, were studied quite intensively in the late 1980s and early 1990s. In Xenopus, ectopic expression of certain Wnts was found to lead to a duplicated body axis (10,11).

However, it was not until the mid-1990s that a confluence of events led to major breakthroughs in understanding of the mechanisms through which Wnt proteins mediate signaling activity in invertebrate and mammalian cells. Arguably, one of the more significant events in elucidating Wnt pathway function in mammals was the discovery of the means by which the adenomatous polyposis coli (APC) protein functions to regulate β-catenin levels and localization.

Adenomatous Polyposis Coli Tumor Suppressor Gene at Chromosome Region 5q21 Familial adenomatous polyposis (FAP) is an autosomal dominant syndrome affecting about 1 in 7000 to 1 in 10,000 people worldwide (12). In general, hundreds to thousands of benign adenomatous polyps (adenomas) arise in the colon and rectum of individuals with FAP by late in the second decade of life. Because of the large number of adenomas that arise and the risk that one or more lesions will progress to cancer, the lifetime risk for colorectal cancer approaches 100% if affected individuals are not screened and definitively managed by surgical intervention. Overall, FAP accounts for less than 1% of all colorectal cancer cases. The relative rarity of FAP germline mutations in colorectal cancer belies the importance of insights into Wnt pathway function gleaned from work on FAP. The tumor suppressor gene at chromosomal region 5q21, which when mutant underlies FAP, was first identified by linkage analysis and positional cloning, and subsequently was termed APC (13–15). Notwithstanding the central role of the APC gene in the development of adenomas and carcinomas in patients with FAP, interest in the APC gene was considerably heightened because of its prominent role in the development of sporadic colorectal tumors. Roughly 80% of sporadic colorectal adenomas and carcinomas have somatic mutations inactivating APC (16). Consistent with the “two-hit” hypothesis for the role of tumor suppressor gene mutations in cancer originally proposed by Knudson to explain the origins of sporadic and familial forms of retinoblastoma, somatic mutation or chromosomal loss of the remaining wild-type APC allele occurs in colorectal neoplasia arising in patients with FAP, as well as in sporadic lesions (17). Although the primary amino acid sequence of the 2843 amino acid APC protein was found to have some repeated sequence motifs when the APC gene was first identified, definitive insights into APC protein function were initially slow to emerge. The identification of the β-catenin protein as a major binding partner for APC was perhaps the major catalyst for uncovering what appears to be the most significant function of APC in normal and cancer cells. β-catenin is an abundant cellular protein that was first identified because of its role in linking the cytoplasmic domain of the E-cadherin cell–cell adhesion molecule to the cortical actin cytoskeleton, in part via binding of β-catenin to α-catenin, a vinculinlike molecule. However, the role of APC in binding to β-catenin appears to have little direct relation to the function

DEVELOPMENTAL SIGNALING NETWORKS / 249 of β-catenin in E-cadherin–mediated cell adhesion. Rather, APC appears to be a major regulator of the “free” pools of β-catenin in the cytosol and nucleus. By combining insights gained from Drosophila genetics, it was soon possible to identify glycogen synthase kinase-3β (GSK-3β), the mammalian homologue of the Drosophila zw-3 protein described earlier, and an apparent scaffolding protein termed Axin as key players that work together in a “destruction complex” targeting the free pool of β-catenin for proteasomal degradation. By mutational inactivation of APC, stabilizing β-catenin mutations, or in the presence of Wnt ligands, this destruction machinery is inhibited and allows β-catenin to translocate to the nucleus and act as a transcriptional coactivator for a family of HMG box proteins termed T-cell factor (TCF) or lymphoid enhancer factor (LEF) transcription factors (18–20). As described further later in this chapter, the role of β-catenin/TCF–mediated transcription in normal and neoplastic gastrointestinal cells has continued to be a major focus of attention for researchers in the Wnt and gastrointestinal biology fields.

β-CATENIN PATHWAY Wnt/β Overview The pathway initiated by the binding of a number of different Wnt proteins to their cell-surface receptors and which leads ultimately to the accumulation of β-catenin (the vertebrate homolog of Armadillo) in the nucleus has been termed the canonical pathway by many authors to distinguish it from other distinct intracellular signaling pathways, so-called noncanonical pathways, that appear to be activated in parallel by Wnt proteins binding to surface receptors (Fig. 8-1). These noncanonical Wnt pathways include activation of c-Jun N-terminal kinase (JNK) signaling or increases of intracellular calcium ion levels that lead to activation of protein kinase C (PKC). For simplicity, this chapter uses Wnt/β-catenin pathway to refer to the canonical Wnt pathway. In addition, this chapter emphasizes the terminology for the mammalian proteins, pointing out the relevant findings from other species where appropriate. In the absence of Wnt proteins, a “destruction complex” consisting of proteins such as APC, Axin, Casein kinase 1α (CK1α), and GSK-3β subjects β-catenin to a cascade of phosphorylation at several serine and threonine residues in the amino (N)-terminal sequences of β-catenin. These phosphorylation events cause β-catenin to be recognized and ubiquitinated by an E3 ubiquitin ligase complex containing the protein β-TrCP. After polyubiquitination, β-catenin is degraded by the proteasome. Binding of Wnt proteins to their receptors inhibits the phosphorylation cascade, allowing free β-catenin to accumulate in the cytoplasm and translocate to the nucleus where it can activate the transcription of target genes via interaction with TCF transcription factor proteins. The transcriptional program activated by β-catenin/TCF likely varies to some degree from one cell type or context to

another, reflecting that β-catenin/TCF may act with other transcription factors to modulate the relative levels of expression of target genes, rather than mediate all-or-none effects on the expression of target genes. Many β-catenin/TCF target genes likely play a role in physiologic cell fate decisions in embryonic development, adult tissue homeostasis, and the development of cancer, and the identities and functional activities of a number of presumptive β-catenin/TCF target genes are discussed later. β-Catenin Pathway Ligands, Receptors, Wnt/β and Extracellular Inhibitors Wnt proteins are small cysteine-rich glycoproteins. Currently, 19 human Wnt genes have been identified. Although secreted, Wnt proteins are not easily recovered from the supernatant of Wnt-expressing cells, which has been attributed to posttranslational modifications, particularly palmitoylation of the proteins, rendering them more hydrophobic than expected (21). Despite their apparent tight association with cells, Wnt ligands still achieve a significant spread from their site of production in vivo, allowing for the formation of a morphogen gradient. This observation has led to the concept that Wnt proteins might be transported by an extracellular vesicle-based transport system, termed the argosomes (22). Similar to other morphogens, the development of a morphogen gradient for Wnts seems to be regulated by the local composition of high-molecular-weight proteoglycans (23–26) and possibly by the presence of extracellular inhibitors such as secreted frizzled-related protein 1 (SFRP1) or Wnt inhibitory factor 1 (WIF1; see later; and Fig. 8-1). Wnt proteins interact with a heterooligomeric receptor complex consisting of a member of the so-called Frizzled (Fz) family and a low-density lipoprotein receptor–related (LRP) protein (27) (Figs. 8-2 and 8-3). Given the existence of 19 human Wnt genes, 12 human Frizzled genes, and 2 human LRP genes involved in Wnt signaling, there is a considerable potential for combinatorial diversity and functional redundancy in this signaling pathway, making it difficult to identify the physiologic role of specific receptor–ligand pairs by standard genetic approaches. Expression of Wnt ligands and receptors is tightly regulated spatially and temporally during development. This tight regulation of expression would seem to imply that, even though the activity of Wnt proteins might be redundant in certain model systems, the differing Wnt expression patterns seen in vivo lead to unique functional consequences. Importantly, Wnt proteins are defined by sequence similarity, rather than functional activity. As such, Wnt proteins can be subsetted according to their ability to induce β-catenin stabilization, a property that correlates well with the ability of the Wnt proteins to induce significant morphologic transformation of mammary epithelial cells (2,28,29). The Wnt proteins that stabilize β-catenin and induce morphologic transformation include Wnt-1 and Wnt-3, among others.

250 / CHAPTER 8

β–cat. β–cat.

AXIN

α–cat. β–cat.

β–cat. β–cat.

A FIG. 8-1. Two-state model of Wnt/β-catenin signaling pathway. (A) Absence of Wnt stimulation. In the absence of Wnt ligands, free β-catenin is continuously phosphorylated in its N-terminal region. This phosphorylation cascade takes place in a destruction complex consisting of adenomatous polyposis coli (APC) and Axin, as well as the enzymatic activities glycogen synthase kinase-3β (GSK-3β) and casein kinase 1α (CK1α). Phosphorylation of serine 45 by CK1α allows for the subsequent phosphorylation of threonine 41, serine 37, and serine 33. Phosphorylated serine 33 is then recognized by β-TrCP, which initiates ubiquitination and proteasomal destruction of β-catenin. This degradation is supported by the nuclear export of β-catenin by APC and Axin. In the absence of nuclear β-catenin, target gene promoters are silenced by recruitment of transcriptional repressors of the transducin-like enhancer of split/groucho-related gene (TLE/Grg) family via T-cell factor/lymphoid enhancer factor (TCF/LEF) DNA-binding factors. Several extracellular antagonists can prevent the effective stimulation of Wnt signaling: Secreted frizzled-related protein (SFRP) and Wnt inhibitory factor 1 (WIF1) directly bind to Wnts, thereby preventing binding to their receptor. Dickkopf (Dkk) binds to low-density lipoprotein receptor–related proteins 5 and 6 (LRP5/6) via the third and fourth β-propeller domain. Although binding sites to not overlap with Wnt binding sites on these proteins, establishment of LRP5/6-Wnt-Frizzled receptor complexes is inhibited. Recruitment of Kremen proteins via Dickkopf leads to internalization of Wnt receptor complexes.

This group of Wnt ligands has sometimes been referred to as canonical Wnt proteins, whereas others have called them activating Wnts. Both terms essentially highlight the effects on β-catenin. Interestingly, although the Wnt-1 and Wnt-3 genes were initially discovered because they were activated by proviral insertions in a murine mammary carcinoma model, as yet, gene amplification, marked overexpression of the Wnt-1 or Wnt-3 genes, or both have not been revealed as common mechanisms for Wnt pathway dysregulation in human cancer. This issue is discussed further later.

Frizzled (Fz) genes encode proteins with seventransmembrane domains, and they have a high affinity for Wnt ligands, with a binding affinity (Kd) less than 10−8 M (30,31) (see Fig. 8-2). Binding of Wnt ligands to Fz proteins is assumed to occur via Wnt interactions with a cysteine-rich domain (CRD) in Fz, because this domain is conserved among all Fz proteins and also in some extracellular inhibitors of Wnts that bear considerable similarity to Fz proteins, namely the SFRP family. Different affinities of the CRDs of Fz proteins for different Wnt ligands have been proposed as an explanation

AXIN

β–cat. α–cat.

β–cat.

β–cat.

β–cat.

β–cat.

B FIG. 8-1. cont’d (B) Presence of Wnt stimulation. Wnt ligands bind to a receptor complex consisting of members of the Frizzled and the low-density lipoprotein receptor related protein (LRP5 and LRP6) families. Intracellular phosphorylation of LRP5/6 and phosphorylation of Dishevelled allow for the recruitment of Axin to the cell membrane, thereby disrupting the destruction complex that normally targets β-catenin for proteasomal degradation. Efficient action of the destruction complex is also inhibited by the recruitment of FRAT1/GBP (frequently rearranged in T-cell lymphoma/GSK-3β binding protein) that displaces GSK-3β from its binding to Axin. Stabilized β-catenin translocates to the nucleus and activates target gene transcription via recruitment of chromatin remodeling proteins (Brg1, CBP/p300), basal transcription apparatus (TIP49), and coactivators (Legless/BCL9 and Pygopus). HDAC, histone deacetylase complex; RYK, related to tyrosine kinase.

ii iii

Wnt

Wnt norrin

Dickkopf

EGF LDL-A LDL-A LDL-A

Propeller

EGF

Propeller

EGF

Propeller

EGF

Propeller

SXV

TM

TM

TM

TM TM

TM TM

CRD

PPP(S/T)P

LRP5/6

Axin

Dishevelled 6 JNK

FIG. 8-2. Schematic drawing of Frizzled protein structure. Frizzled receptors contain seven-transmembrane domains (TM). The N-terminal extracellular cysteine-rich domain (CRD) mediates binding to ligands such as Wnt of Norrin (30,36). Interaction with Dishevelled occurs immediately intracellular of the transmembrane domain (109). Several proteins involved in noncanonical signaling interact with the C terminus (265, 266). JNK, c-Jun N-terminal kinase. Binding regions for interacting proteins are shown later.

FIG. 8-3. Schematic drawing of low-density lipoprotein (LDL) receptor-related proteins 5 and 6 (LRP5/6). LRP5/6 have a single-transmembrane region. The extracellular N-termini contain four YWTD β-propellers (propeller), each of which is followed by an epidermal growth factor (EGF) repeat. β-Propellers 1 and 2 seem to mediate ligand binding, whereas propellers 3 and 4 mediate interaction with antagonists of the Dickkopf family (32,33,47,48). Three LDL receptor type A domains (LDL-A) allow for oligomerization. In the intracellular domain, PPPSP/PPPTP motifs (P) mediate the phosphorylation-dependent interaction with Axin (75,95). Binding regions for interacting proteins are shown later.

252 / CHAPTER 8 why certain Fz receptors may preferentially signal via the Wnt/β-catenin pathway, whereas other Wnts signal predominantly or exclusively through noncanonical pathways (31). As noted earlier, the LRP5 and LRP6 proteins are singletransmembrane domain proteins with significant similarity to the low-density lipoprotein receptor (32,33) (see Fig. 8-3). Although the presence of both classes of cell-surface receptors, Fz and LRP5/6, seems to be required for canonical signaling to occur in cells, there is evidence indicating that, in some instances, the two parts of the receptor complex may independently transmit Wnt signals (34,35). Evidence is also accumulating that LRP5/6 generally are not involved in β-catenin–independent Wnt signaling (i.e., noncanonical signaling), which seems mainly to rely on Fz receptors. An additional complexity is that additional interacting proteins (apart from the Wnts and LRP5/6) may exist for certain Fz receptors (36). For instance, it has been suggested that, in some settings, the protein encoded by the gene known as RYK (for “related to tyrosine kinase,” the mammalian homologue of the Drosophila derailed gene) might be an essential component of the canonical signaling pathway, functioning as a Wnt coreceptor in conjunction with Fz proteins (37). Several inhibitors of Wnt ligand–receptor interactions have been identified (see Fig. 8-1A). Whereas SFRPs and WIF1 seem to inhibit ligand–receptor interactions by binding to Wnt proteins directly, Dickkopf (Dkk) proteins do so by interacting with the LRP receptors. There are five human SFRP proteins (SFRP1-5), and they constitute a family of secreted proteins containing a CRD similar to that present in Fz receptors. Binding of SFRPs to Wnts via the CRD domain prevents their interaction with membrane-bound Fz and LRP5/6 receptors, and antagonizes Wnt signaling. In addition, as is discussed later, there seems to be evidence that SFRPs, via unknown mechanisms, may even be able to antagonize β-catenin signaling that has been activated by mutations in cancer cells downstream of the receptor–ligand interaction. The observation that epigenetic inactivation of several of the SFRP genes may be an early event in the pathogenesis of several cancer types (particularly colorectal cancer) is an intriguing one, given this apparent antagonistic activity of the SFRPs on Wnt/β-catenin signaling (38–40). Despite the apparent inhibitory effects of the SFRPs on Wnt/ β-catenin signaling, it has been suggested that in some settings SFRPs could function to increase Wnt β-catenin signaling, possibly by serving as a carrier for the highly hydrophobic acylated Wnt proteins that are not readily water soluble (41). These apparently conflicting activities for SFRPs might be reconciled, in part, because different reagents were used in the various studies (wingless vs mammalian Wnts). Nonetheless, currently, it seems reasonable to suggest that the physiologic role of SFRPs in vivo has not been fully established. WIF1, a secreted protein, was uncovered in a functional screen for inhibitors of vertebrate Wnt signaling (42). WIF1 interacts with Wnts via a poorly understood domain termed the WIF domain (42). In Drosophila, the transmembrane Wnt-interacting protein Derailed, which likely functions in

noncanonical Wnt signaling, appears to contain a WIF domain. Evidence suggests that, in contrast to the situation for the Drosophila Derailed protein, the human homologue of Derailed, termed RYK, might actually have an essential role in Wnt/β-catenin signaling (37). Notably, Shifted recently has been identified as a putative Drosophila homolog of WIF1. Whereas no modulation of Wnt signaling by Shifted could be demonstrated in the fly, it was observed that Shifted supports another important developmental pathway, namely hedgehog signaling by increasing the ligand’s extracellular half-life and diffusion (43,44). It is currently unclear whether a similar cross talk also exists in mammals. Dkk proteins were originally identified because of their ability to inhibit dorsalization of Xenopus embryos, a process that is promoted by activating Wnt ligands (45,46). Dkk proteins are secreted proteins that bind to the extracellular domain of LRP5/6 with high affinity (47–49). The interaction of Dkk1 with LRP5 or LRP6 can inhibit the formation of an Fz-LRP5/6 receptor complex, thereby inhibiting Wnt signaling (48). Based on sequence similarity, the Dkk family in vertebrates comprises four members, known as Dkk1-4. Whereas Dkk1 and Dkk4 have been shown to act as antagonists of Wnt signaling, Dkk3 does not interact with LRP5/6, and Dkk2, in some experimental settings, might actually play a positive role in Wnt/β-catenin Wnt signaling. Mechanistic insights into these differential functional activities of the Dkk family members are lacking. In addition to the simple disruption of the formation of Fz-LRP5/6 receptor complexes (48), another model for the action of Dkk proteins has been suggested. Specifically, Dkk1 has been shown to interact with the transmembrane proteins Kremen1 and Kremen2. The Kremen proteins, upon Dkk binding, appear to promote internalization of the LRP5/6 proteins (50,51). The net consequence of decreased cell-surface levels of LRP5/6 is reduced responsiveness of a cell toward Wnt ligands.

Wnt/β-Catenin Signaling from the Cell Surface to the Nucleus As noted earlier, stabilization of the free pool of β-catenin is believed to be a pivotal event in the cellular response to activating Wnt ligands. In the absence of activating Wnt ligands, the half-life of the “free” pool of β-catenin is tightly regulated by phosphorylation at its N-terminus, ubiquitination, and proteasomal degradation. To understand how the interaction of Wnt proteins with their respective receptors leads to the inhibition of the destruction machinery regulating β-catenin, it is important to understand first how the destruction of β-catenin takes place in the absence of activating Wnts. Thus, this section first focuses on the physiologic life cycle of β-catenin molecules. β-Catenin and the Destruction Complex β-Catenin, a protein of 781 amino acids encoded by the CTNNB1 gene, serves a dual role as a cell adhesion molecule

DEVELOPMENTAL SIGNALING NETWORKS / 253 and transcriptional regulator (Fig. 8-4). In normal epithelial cells, most β-catenin seems to be bound in a cytoskeletal complex that on one side (via E-cadherin) mediates contact to neighboring cells and on the other side (via binding to α-catenin) is tethered to the intracellular actin cytoskeleton. β-Catenin is therefore a major constituent of specialized cell–cell contacts termed adherens junctions. β-Catenin bound in this compartment seems to be long-lived and not subject to regulation via Wnt signaling. In contrast to the membraneassociated β-catenin pool, in cells not exposed to activating Wnt ligands, only a small fraction of the cellular β-catenin seems to be present in the cytoplasm and the nucleus; this is the so-called free β-catenin pool. This free pool is subject to rapid degradation via the proteasome, and consequently has a short half-life. Stabilization and nuclear translocation of β-catenin from this pool is essential for the ability of β-catenin to modulate transcription via interaction with TCFs and potentially other transcription factors. Given the large capacity of the cytoskeletal pool, it is not surprising that ectopic expression of E-cadherin can decrease the amount of the free β-catenin and inhibit neoplastic growth (52,53). In its N-terminus, β-catenin contains a stretch of evolutionary conserved serine and threonine residues that are each localized four amino acids apart (i.e., serine 33, serine 37, threonine 41, and serine 45). CK1α appears to phosphorylate serine 45 constitutively (54). This so-called priming phosphorylation event allows for the sequential phosphorylation of positions 41, 37, 33, and 29 mediated by GSK-3β (55–58). The phosphorylation of serines 33 and 37 ultimately

33

37

41

creates an epitope that is recognized by the F-box protein β-TrCP, which as a part of an SCF E3-ubiquitin ligase complex triggers the transfer of ubiquitin to β-catenin (59). Ubiquitinated β-catenin is then rapidly degraded via the proteasome (60,61). The sequential phosphorylation of β-catenin, as described earlier, takes place in a multiprotein complex that has been termed destruction complex. It assembles on two essential scaffolding proteins: APC and Axin. Both seem to be necessary for full activation, but both seem to play different roles (62–65). In addition to the catalytic components CK1α and GSK-3β, this complex also contains various regulatory components that are discussed later in this chapter. A series of phosphorylations of APC mediated in part by GSK-3β seems to be important for the full binding capacity of APC to β-catenin (66) and might support the release of phosphorylated β-catenin from the destruction complex, allowing unphosphorylated β-catenin to bind to Axin (67,68), thereby recharging the destruction complex. Axin binds not only to β-catenin, but also to APC (63,69,70). By this scaffolding function, it is believed to increase the effective concentration of enzyme (GSK-3β and CK1α) and substrate (β-catenin) of the destruction complex (71). Although conflicting evidence has been published, interaction of Axin with β-catenin, as well as the stability of Axin, might also be regulated by GSK-3β–dependent phosphorylations (72–75). Inhibition of the destruction complex leads to the accumulation of N-terminally unphosphorylated forms of β-catenin in the cytoplasm and the nucleus (76,77). Similarly, mutation

45

βTRCP1, Ebi GSK3β, CKIα, α-catenin

APC, E-Cadherin, Axin, TCF/LEF Legless, TIP49

TAdomain

ARM ARM ARM

ARM ARM ARM ARM ARM ARM ARM ARM ARM

SYLDSGIHSGATTTAPSLSGK

CBP/p300

Brg1

FIG. 8-4. Schematic drawing of β-catenin protein structure. β-Catenin plays a dual role as a transcription factor and cell adhesion molecule. Its stability is regulated by the sequential phosphorylation of several N-terminal serine and threonine residues (indicated by boldface) mediated by glycogen synthase kinase-3β (GSK-3β) and casein kinase 1α (CK1α), which allows for recognition by the F-Box protein β-TrCP and consecutive ubiquitin- dependent proteasomal degradation (55,59). Phosphorylation-independent recognition of the N terminus by the F-Box Ebi leads to SIAH-dependent proteasomal degradation (84). Twelve armadillo repeats (ARM) mediate the interaction with adenomatous polyposis coli (APC), E-cadherin, α-catenin, Axin, T-cell factor/lymphoid enhancer factor (TCF/LEF), Legless (267), and TIP49/Pontin52 (164). The C-terminal transactivation domain interacts with histone acetyltransferase CBP/p300 (160) and chromatin remodeling factor Brg1 (163). Interaction sites with other proteins are indicated below the schematic representation.

254 / CHAPTER 8 of any of the potential GSK-3β and CK1α phosphorylation sites, such as is often seen in certain cancer types, leads to the accumulation of β-catenin and subsequent activation of the downstream transcriptional program. Given its dual role in cytoskeletal organization and transcriptional activation, β-catenin is capable of a variety of protein–protein interactions. The central portion of βcatenin contains 12 repeats that are homologous to the Drosophila armadillo protein, and are therefore termed armadillo repeats. These repeats form a superhelix of α-helices that shows a prominent groove on one side (78). This groove mediates several of the most important protein–protein interactions for β-catenin, including those with APC, E-cadherin, Axin, and TCFs. In the cytoplasm, APC and E-cadherin compete for the same binding site on β-catenin (79), which is consistent with the notion that cytoskeletal pool is not amenable to the destruction complex. Assembly of the adherens junctions additionally requires the binding of α-catenin to the N-terminus. Interestingly, this interaction, as well as the interaction with E-cadherin, is regulated by phosphorylation of specific tyrosine residues of β-catenin (Y142 and Y654, respectively), suggesting that this could represent a major determinant of the distribution into the cytoskeletal versus the signalingcompetent pool (80–82). Whereas the fruit fly genome encodes only a single homolog of β-catenin called armadillo, vertebrate genomes also encode the protein γ-catenin/plakoglobin, which also serves a dual role in cytoskeletal and nuclear processes. Both proteins seem to mediate similar protein–protein interactions with their partners APC, Axin, and GSK-3β. It also has been shown that γ-catenin is capable of transforming cells. In addition to the classical destruction complex, there are other pathways of β-catenin destruction. One of these alternative pathways also seems to be dependent on APC function. Through an interaction with the C terminus of APC, a protein complex involving the SIAH1 and Ebi proteins can trigger degradation of β-catenin in a p53-dependent manner, independent of any requirement for GSK-3β (84–86). Most tumors with biallelic inactivation of APC express C-terminally truncated APC proteins that are unable to support the degradation of β-catenin via the classical destruction complex and the SIAH1-dependent pathway. Consistent with the view that SIAH1-mediated degradation of β-catenin is independent of GSK-3β, cancer-derived mutants of β-catenin with Nterminal mutations in the phosphorylation motif can still be regulated by SIAH1. Mutations of the SIAH1 gene in gastric cancers have been shown to be associated with increased nuclear localization of β-catenin (87). Given that mutations of SIAH1 should leave the activity of the classical destruction complex intact, this suggests that SIAH1-dependent β-catenin degradation might be the major determinant of β-catenin stabilization in certain tissues. In addition to the classical GSK-3β–dependent destruction mechanism for β-catenin and the SIAH1-dependent destruction, β-catenin degradation can be induced by retinoid X receptor (RXR). This has been shown to be independent of APC (88).

Regulation of the Destruction Complex in Wnt/ β-Catenin Signaling The end point of the Wnt/β-catenin pathway is the stabilization of the free β-catenin pool and its translocation to the nucleus where it can activate transcription of target genes. The function of the destruction complex has been discussed earlier, so this chapter now discusses the mechanisms by which binding of Wnt proteins to their receptors leads to the inhibition of the destruction complex and subsequent nuclear accumulation of β-catenin. In general, the Wnt receptor complexes seem to consist of both LRP5/6 and Fz proteins (27,89). Although these two signaling components seem to work together in a physiologic setting, the mechanism of cooperation is not entirely clear. There are also instances when only one of the two receptor components may be required for β-catenin stabilization. For example, genetic data from Drosophila suggest that Arrow, the Drosophila counterpart of LRP5/6, does not function in noncanonical Wnt signaling, though Fz proteins are clearly required for noncanonical Wnt signaling. Another example is that Fz proteins do not seem to be required for the stabilization of β-catenin resulting from ectopic overexpression of certain mutant “activated” forms of LRP5/6, such as membrane-anchored forms of LRP5/6 lacking the extracellular domain (32,34). However, the physiologic significance of these latter observations is unclear, and the larger body of data appears to support the view that Wnt-mediated stabilization of β-catenin requires both a Fz and an LRP5/6 component. To make the receptor functions easier to understand, this section describes the main effects of LRP5/6 and Fz separately, trying to point out the possibilities for cooperation as the discussion progresses. This section also introduces the function of Dishevelled, an additional signaling protein that genetically acts downstream of Fz and LRP5/6 receptors and upstream of β-catenin stabilization. The Role of Axin and LRP5/6 in Wnt/ β-Catenin Signaling Axin acts as a scaffold for both substrates and kinases of the degradation complex (Fig. 8-5). It can bind to APC (90), β-catenin, GSK-3β (91), and CK1α (55). The N-terminal RGS (regulator of G-protein signaling) domain of Axin seems to be required for its interaction with APC (90). Binding to β-catenin, GSK-3β, and CK1α takes place in the central portion of Axin. The C-terminal DIX (Dishevelled Axin) domain can interact with a corresponding DIX domain on the adaptor protein Dishevelled (92) and mediate homodimerization of Axin. By bringing both substrate (β-catenin) and enzymatic activities (GSK-3β and CK1α) in close vicinity, Axin can increase the rate of phosphorylation of β-catenin enormously, by some estimates more than 24,000-fold (63,71). In addition to this scaffolding function, Axin also seems toplay a role in supporting the nuclear export of β-catenin (see later). Experimental data and mathematical models have suggested that Axin protein seems to be the limiting factor in the

APC

DIX

RGS

DEVELOPMENTAL SIGNALING NETWORKS / 255

GSK3β

βcatenin

Dishevelled

LRP5/6

FIG. 8-5. Schematic drawing of Axin protein structure. Axin acts as a scaffold protein for the destruction complex. Binding to adenomatous polyposis coli (APC) is mediated by a N-terminal RGS domain (regulator of G-protein signaling domain) (70,90), whereas interaction with glycogen synthase kinase-3β (GSK-3β) and β-catenin occurs in the central domain (69,91). The C-terminal DIX (Dishevelled Axin) domain mediates homodimerization and binds to Dishevelled (268). On Wnt signaling, Axin is recruited to the phosphorylated intracellular domain of LRP5/6 (75,95), thereby disrupting the destruction complex for β-catenin. Binding regions for interacting proteins are shown below.

β-catenin destruction complex, being present in the cell at levels estimated 5000-fold less than those of APC (93). Thus, regulation of the levels and localization of the Axin protein would seem to be a highly effective way of regulating the overall activity of the destruction complex. In the absence of activating Wnt proteins, Axin is bound to APC, GSK-3β, and CK1α, markedly increasing the rate of β-catenin phosphorylation (71). Also, in the absence of extracellular Wnts, LRP5/6 proteins seem to form inactive receptor complexes, perhaps via homodimerization mediated by their extracellular epidermal growth factor repeats (94). On binding of Wnt proteins to the receptor complex, LRP5/6 is rapidly phosphorylated within one or more of five highly conserved PPP(S/T)P motifs that are present in the intracellular domain (95) (see Fig. 8-3). Once phosphorylated at the serine/threonine residue in the motif, one or more of the motifs can bind Axin, resulting in sequestration of Axin by LRP5/6 at the plasma membrane, thereby reducing Axin’s availability to the destruction complex. Interestingly, these phosphorylated PPP(S/T)P motifs in LRP5/6 seem not only to be essential, but also sufficient for Axin binding, as demonstrated through studies showing that transfer of a single PPP(S/T)P motif to the C terminus of the low-density lipoprotein receptor (which lacks any such motifs normally) will allow the chimeric mutant low-density lipoprotein receptor to stabilize β-catenin. Membrane-tethered mutant forms of LRP5/6, harboring a deletion of the extracellular domain, actually mediate Wnt ligand-independent stabilization of β-catenin, suggesting that the binding of Wnts must abolish a negative regulatory effect of the extracellular domain of LRP5/6 on the PPP(S/T)P motifs in the cytoplasmic domain (34,94). In addition to the ligand-induced change of subcellular distribution of Axin, it has also been reported that this process is accompanied by increased phosphorylationdependent proteasomal degradation of Axin. It is currently unclear which kinase triggers this process, although GSK3β has initially been implicated (72–75). It has been

suggested that recruitment of Axin to the plasma membrane also requires Dishevelled, another key player of intracellular Wnt signal transduction (34,106) that genetically acts downstream of LRP5/6 and Fz receptors. Both Axin and Dishevelled contain DIX domains that mediate their interaction. Dishevelled itself seems to be recruited to activated receptor complexes by direct binding to Fz receptors. The complex of Dishevelled and Axin could therefore mediate an indirect interaction of Fz and LRP5/6 receptors, and might be responsible for the cooperation of both receptor components in Wnt signal transduction. LRP5/6-mediated relocation of Axin most likely not only inhibits the assembly of the classical destruction complex, but also inhibits the role of Axin in the nuclear export of β-catenin (96). This might explain the observation that Wnts can regulate the activity of β-catenin to some degree even in the complete absence of GSK-3β activity, which is considered to be essential for the classical destruction complex (74). Obviously, in the case of cells lacking GSK-3β, Wnt proteins cannot regulate β-catenin levels via inhibition of a destruction complex that is already dysfunctional. In this situation, alternative constitutive degradation pathways (e.g., SIAH1) and Axin dependent nuclear export of β-catenin could possibly keep the concentration of nuclear β-catenin low in the absence of Wnt ligands. Wnt ligand binding could then abrogate the Axin dependent nuclear export of β-catenin and allow a Wnt signal transduction even in the absence of GSK-3β function (74, 96–98). Notably, mammalian genomes contain a second Axin-like gene called Axin2 (initially termed Axil in the rat and conductin in the mouse). The Axin and Axin2 proteins are roughly 45% identical in sequence. Similar to Axin, Axin2 can increase the degradation of β-catenin (91). In contrast to the apparently ubiquitous expression of Axin, Axin2 expression seems to be much more tightly regulated (91,99). Interestingly, the Axin2 gene has been identified as a target gene for Wnt signaling, presumably mediating a negative feedback function by targeting β-catenin for destruction (100–103). Mutations in the genes for Axin and Axin2 that prevent their function in the destruction complex have been found in hepatocellular carcinoma, colorectal carcinoma, and gastric cancers at varying frequencies (104). To date, germline mutations associated with colorectal cancer and congenital developmental defects of the teeth (oligodontia) have only been described for Axin2 and only in a single kindred from Finland (105). Information is lacking on whether the second allele is missing in the tumors arising in individuals carrying a germline mutation, though both Axin and Axin2 might be expected to function as a classical “Knudson-type” tumor suppressor genes with biallelic inactivation in tumor cells. The Role of Fz and Dishevelled Proteins in Wnt/ β-Catenin Signaling As described earlier, Fz proteins are high-affinity receptors for Wnts (see Fig. 8-2). Whereas LRP5/6 proteins seem to be mainly involved in the pathway leading to β-catenin

256 / CHAPTER 8

Axin, actin & vesicles

DEP

PDZ

DIX

stabilization, Fz receptors also play a role in the signaling network that leads to the correct orientation of cells within a plane, so-called planar cell polarity signaling, as well as Ca2+/PKC and JNK signaling (see review by Veeman and colleagues [107]). Although the intracellular domain of Fz receptors is the least conserved region of this protein family, it seems to be necessary for the Fz-dependent β-catenin stabilization. The precise mechanism of action of Fz in stabilizing β-catenin, however, is still mysterious but seems to involve the action of Dishevelled proteins. Based on genetic epistasis analyses, Dishevelled proteins act downstream of both Fz and LRP receptors and upstream of the stabilization of β-catenin (Fig. 8-6). On binding of Wnts to receptors, Dishevelled rapidly gets phosphorylated. Notably, this phosphorylation seems to occur independent of the subsequent downstream signaling events (35,108). Some part of the total cellular Dishevelled then becomes redistributed to the plasma membrane, possibly by interactions between the N-terminal PDZ domain of Dishevelled and the intracellular domain of Fz (109). As described earlier, Axin also gets redistributed to the plasma membrane when the intracellular domain of LRP5/6 becomes phosphorylated. Dishevelled seems to be required for the effective redistribution of Axin to the plasma membrane (106), possibly by interaction of DIX domains that are present in both proteins (92). The importance of this interaction is underlined by the observation that the disruption of the Dishevelled–Axin interaction by the protein IDAX (inhibitor of Dishevelled Axin complex) can inhibit Wnt signaling (110). In addition, Dishevelled can bind to the GSK-3β inhibitor FRAT1/GBP (i.e., frequently rearranged in T-cell lymphoma/ GSK-3β binding protein). This protein is thought to compete with Axin for the binding of GSK-3β. By this mechanism, Axin molecules that are bound to Dishevelled might be

Frizzled FRAT1/GBP, Naked, IDAX




JNK, Rho

FIG. 8-6. Schematic drawing of Dishevelled protein structure. The N-terminal DIX (Dishevelled Axin) domain mediates the binding to Axin (268) and to the actin cytoskeleton (269). Interaction with Frizzled seems to require the PDZ (PSD, Disc-large, and ZO1) and the DEP (Dishevelled, Egl-10, and Pleckstrin) domains (109,270). Several inhibitory proteins also bind to the central PDZ domain such as GBP/FRAT1 (GSK3β-binding protein/frequently rearranged in T-leukemia-1) (112), IDAX (inhibitor of Dishevelled-Axin complex) (110), and naked (271). The DEP domain seems to mediate noncanonical signaling such as activation of c-Jun N-terminal kinase (JNK) and Rho (272,273). Binding regions for interacting proteins are shown later.

prevented from binding to GSK-3β and are therefore unable to act in the classical degradation complex (111–113). Because Fz receptors contain seven-transmembrane domains similar to G protein–coupled receptors, it has been suggested that Fz action might also depend on G proteins. Although there is some evidence that β-catenin stabilization upon activation of rat Fz 1 can be mediated by G proteins (114), the physiologic contribution of G proteins to most downstream effects of Wnt signaling is not yet clear (see review by Malbon and colleagues [115]). The Role of Adenomatous Polyposis Coli in Wnt/ β-Catenin Signaling Activation of the Wnt/β-catenin signaling pathway occurs in most colorectal cancers. In about 80% of these cases, this is attributable to biallelic inactivation of the APC tumor suppressor gene. Stabilizing β-catenin mutations accounts for about 10% of the cases (116,117). The biallelic inactivation of APC has some curious aspects that suggest that APC fulfills move functions than simply its role in the destruction complex for β-catenin. First, biallelic inactivation requires two distinct genetics events leading to either two distinct mutations in the APC alleles, or a mutation in one allele and chromosomal loss involving the other allele. This seems rather inefficient in light of the fact that Wnt/β-catenin signaling can be efficiently activated by a single point mutation in one of the two alleles of the gene encoding for β-catenin. Indeed, most noncolonic tumors with activation of the Wnt/β-catenin signaling pathway carry stabilizing β-catenin mutations. It is unclear what factors are responsible for the preference to activate Wnt/β-catenin signaling by biallelic inactivation of APC in the colon. Second, in most cases at least one APC allele is retained that leads to the expression of a truncated protein. Complete loss of APC expression is exceedingly rare. The situation is especially interesting in the case of patients with familial adenomatous polyposis. These patients carry a germline mutation in the APC gene. The localization of the germline mutation (i.e., the first hit) seems to predetermine the way of inactivation of the second allele in the tumors, suggesting that some truncated APC proteins are not sufficient to support the transformation of colonic epithelium. Third, the amount of APC protein does not seem to be the rate limiting factor in the assembly of the classical destruction complex. In fact, Axin is present at a 5000× lower concentration in the cells (93). The vast majority of cellular APC protein is therefore localized outside the classical destruction complex. Taken together, those observations imply that APC might have other tumor-suppressor functions in colon epithelial cells in addition to its role in the regulation of β-catenin stability. They also suggest that mutant APC proteins might either retain some functions that are essential for colorectal carcinogenesis or might acquire new “gain-of-function” activity.

DEVELOPMENTAL SIGNALING NETWORKS / 257

APC

Asef



Rac

15aa 15aa

15aa

ARM ARM ARM ARM ARM ARM ARM

MCR

Kap3



microtubules

β-catenin (15 and 20aa repeats) GSK3β (20aa repeats)

SIAH,

 EB1  micro- tubules

Axin (SAMP)

FIG. 8-7. Schematic drawing of adenomatous polyposis coli (APC) protein structure. APC binds to β-catenin via four 15-amino-acid (aa) repeats and seven 20-aa repeats. The latter also mediate binding of glycogen synthase kinase-3β (GSK-3β). Axin binds to SAMP repeats (containing the sequence serine-alanine-methionine-proline), which are interspersed in the last five 20-aa repeats. Most inactivating somatic mutations occurs in the mutation cluster region leading to truncated APC proteins that cannot bind to Axin anymore. Effects of APC on the actin cytoskeleton seem to be mediated by binding of the guanosine triphosphate exchange factor Asef to the armadillo repeats (142). Modulation of microtubule function depends on direct interaction of the C terminus, as well as indirect interactions via EB1 and Kap3 (134,274,275). Binding regions for interacting proteins shown later. MCR, mutation cluster region.

Human APC is a large protein of roughly 300 kDa (Fig. 8-7). Multiple interactions between APC and other proteins have been described (see review by Nathke [118]). The N-terminus of APC is predicted to promote dimerization with another APC molecule via a coiled-coil structure (119), and the N-terminal region might be involved in intramolecular interaction with the C terminus of APC. The main interactions of APC with Wnt-signaling pathway components take place in its central region. Four 15-aminoacid repeats in the central region mediate constitutive binding to β-catenin. Seven additional 20-amino-acid repeats in the central region also mediate binding to β-catenin (120) and are regulated in their affinity for β-catenin binding by phosphorylation of APC: The affinity of APC for β-catenin increases greatly when APC is modified by a cascade of phosphorylation events involving GSK-3β, a scenario somewhat reminiscent of the phosphorylation of β-catenin and the resultant effect on the affinity of β-catenin for β-TrCP binding (66). Interspersed between the last five of the seven 20-aminoacid repeats are 3 serine-alanine-methionine-proline (SAMP) repeats. These repeats are characterized by the presence of the SAMP sequence and mediate the interaction of APC with Axin (70,121). Mutations in colorectal cancers tend to occur in a central 650-amino-acid region of APC, which is commonly referred to as the somatic mutation cluster region (MCR). Most mutations lead to the expression of a C-terminally truncated APC protein that lacks the Axin-interacting SAMP repeats and several of the β-catenin–interacting 20amino-acid repeats (122). These proteins are therefore not capable of participating in the destruction of β-catenin. As explained earlier, the nature of the first hit, that is, the localization of the first mutation, seems to dictate the localization

of the second mutation, suggesting a selective advantage in the cancer cells for combinations of certain truncated APC proteins (123,124). Mammalian genomes encode for another homologue of Drosophilia armadillo, termed γ-catenin or junctional plakoglobin. γ-Catenin binds to APC, and its levels and localization in cells appear to be regulated by APC function (83). When overexpressed, wild-type γ-catenin seems to be capable of transforming cells. Although it had been controversial for some time whether γ-catenin does indeed have a transactivating function of its own or whether its function is mainly exerted by freeing β-catenin from its cytoskeletal location (125,126), recent data confirm that γ-catenin can act as a transcriptional activator independent of β-catenin perhaps in a cell-context manner (127–129). In addition to the classical destruction complex, APC seems to be involved in an alternative pathway of destruction for β-catenin. This pathway is dependent on the E3-ubiquitin ligase complex containing SIAH1 and Ebi, and requires interactions with the C terminus of APC, which usually is lost in colorectal carcinoma (84,85). Interestingly, the SIAH1 gene seems to be a target for regulation by p53. Notably, p53 is activated by accumulation of excessive amounts of βcatenin, suggesting a negative feedback regulation mechanism (130,131). In addition to its role in the degradation of β-catenin, APC seems to play important roles in intracellular shuttling of proteins (e.g., β-catenin) and cytoskeletal organization, thereby possibly influencing the chromosomal segregation in mitosis (see review by Nathke [118]). Several nuclear export and localization sequences are localized throughout the sequence of APC. The role of the nucleocytoplasmic

258 / CHAPTER 8 shuttling of β-catenin in the regulation of the transcriptional activity of β-catenin is discussed later. The C terminus of APC can interact directly with microtubules (132,133). Via interaction with the protein Kap3, APC associates with microtubule-associated motor proteins of the kinesin family, allowing its directional transport to the microtubule’s “plus” end (134). This leads to the formation of APC clusters at the microtubule’s end and seems to be able to stabilize them (135). APC’s action on microtubule function seems to be important in the correct segregation of chromosomes during mitosis. Consequently, it has been observed that cells lacking functional APC show more chromosomal segregation defects, and that tumor-derived C-terminally deleted fragments of APC seem to have a dominant effect on spindle function (136–140). Taken together, these data suggest that defects in APC function might lead to a certain degree of chromosomal instability that could foster the further development of gastrointestinal cancers. The significance of this effect in vivo has not yet been clearly established (141). Via interaction with the GTP exchange factor Asef, which modulates the activity of small G proteins such as Rac, APC may also function in modulating the actin cytoskeleton. Tumor-derived mutations might thereby regulate cell migration (142,143). Nuclear Access of β-Catenin: Subcellular Distribution The dual role of β-catenin in cell adhesion and nuclear transcriptional regulation implies that effective ways must exist not only to regulate the cytoplasmic levels of free βcatenin, but also to regulate the subcellular localization of β-catenin. Even though most colorectal carcinoma contain mutations activating the APC/β-catenin pathway, β-catenin is only localized in the nucleus of a minority of the cells (144,145), preferentially in cells with mesenchymal phenotype and localized at the invasion front. This has been attributed to differences in the microenvironment and also to differences in differentiation status. Given the necessity for nuclear accumulation of β-catenin to perform its transcriptional activity, understanding the pathways that regulate the distribution of β-catenin is of obvious importance in the understanding of the molecular pathogenesis of colorectal carcinoma development and progression. Interestingly, in tissue culture systems, dramatic changes of the subcellular distribution of β-catenin within the cells can be observed when cells are grown at different cell densities. It has been postulated that, in addition to the membranebound pool, different free pools of β-catenin should exist that can preferentially bind to TCFs or α-catenin (146). Whether and what kind of structural differences account for these different pools currently is not well understood. To a certain degree, phosphorylation of specific tyrosine residues in β-catenin seems to govern the decision whether β-catenin acts as a cytoskeletal protein (via interaction with α-catenin and E-cadherin) or whether it is capable of fulfilling its transcriptional function (via interaction with BCL9-2) (80).

Much more is known about factors that regulate the nuclear access of β-catenin. Initially, it was suggested that because of the similarity of the armadillo repeats with the repeats of Importin-β, β-catenin could directly interact with the nuclear pore complex. Despite its size, β-catenin was therefore postulated to freely shuttle between nucleus and cytoplasm. In this model, the subcellular distribution of β-catenin would be mainly determined by the distribution of its interaction partners between the nucleus and the cytosol (97). However, evidence has been presented that nuclear export of β-catenin might actually be supported by APC (147,148) and Axin (96). Although nuclear export of β-catenin mediated by APC seems to play a role in terminating the transcriptional activity of nuclear β-catenin, it has been noted that overexpression of APC constructs that are targeted to the nucleus leads to a nuclear accumulation of β-catenin without an increase in target gene transcription (149). The molecular basis for this apparent paradox seems to lie in the capacity of APC to reduce the amount of functional β-catenin-TCF complexes by its interaction with the protein CtBP (C-terminal binding protein) (150). Taken together, in addition to its contribution to the destruction complex, APC seems to reduce the nuclear activity of β-catenin not only by increasing its nuclear export, but also by sequestering it away from TCF. It has been suggested that the nuclear export functions of APC and Axin are being antagonized by the nuclear import function of the proteins Legless/BCL9 and Pygopus, which also serve as essential coactivators of β-catenin’s transcriptional function. However, the physiological relevance of this effect is still subject to debate (151–154). Nuclear Action of β-Catenin Stabilization of β-catenin and nuclear accumulation allows for an increased interaction with proteins of the TCF/LEF DNA-binding protein family. These proteins bind to the consensus site CWTTGWW. Mammalian genomes have four distinct genes encoding for TCF-LEF transcription factors. The proteins contain a highly conserved high mobility group box (HMG box) that mediates sequence-specific DNA binding (155; see also review by van Noort and Clevers [156]) (Fig. 8-8). Binding to its recognition sequence induces a bend in the DNA that potentially can alter the binding and access of other DNA-binding proteins (157). β-Catenin interacts with the N terminus of TCF/LEF transcription factors (18). In the absence of high levels of β-catenin, they repress the transcription of target genes by virtue of their interaction with transcriptional repressor proteins of the transducin-like enhancer of split/groucho-related gene (TLE/Grg) family (related to the Drosophila groucho protein) and possibly the repressor CtBP (14,158,159). These repressors recruit histone deacetylases, and induce changes in the chromatin structure, making the promoter inaccessible for transcriptional activation. In addition, CtBP might cooperate with nuclear APC to prevent the formation of functional β-catenin-TCF complexes (150). β-Catenin,

DEVELOPMENTAL SIGNALING NETWORKS / 259

HMG

β-catenin

Grg/TLE

DNA

FIG. 8-8. Schematic drawing of Lymphoid enhancer factor (LEF)/T-cell factor (TCF) protein. β-Catenin binds to the N terminus of TCF/LEF proteins. Recruitment of transcriptional repressor complexes is mediated by the interaction with TLE/Grg proteins (transducin-like enhancer of split/grouchorelated genes). The high mobility group (HMG) domain mediates interaction with specific DNA target sequences. Binding regions for interacting proteins are shown later.

when present in the nucleus, appears to displace the transcriptional repressors from the TCF proteins. Association with histone acetyl transferases (such as CBP/p300), (160,161) ATPases (such as TIP49) (162) and components of the SWI/SNF chromatin remodeling complexes (such as Brg1) (163) leads to a modification of the chromatin in the promoter region, making the DNA more accessible for transcription factors. The basal transcriptional machinery gets recruited to responsive promoters (164), and target gene transcription can begin. The proteins Legless/BCL9 (i.e., B-cell lymphoma 9) and Pygopus seem to play a major role as essential coactivators of β-catenin–dependent transcriptional activation (152–154). Previously, BCL9 has been implicated in the development of non–Hodgkin’s lymphoma in the mouse. Legless/BCL9 has been shown to directly interact with β-catenin and recruit Pygopus to this complex. In addition to the transcriptional coactivator function of BCL9/Legless and Pygopus, a role of these proteins in nuclear import of β-catenin has been suggested (151). However, it is still subject to debate how much this contributes to the essential function of Legless/ BCL9 and Pygopus in the Wnt/β-catenin signaling pathway. The importance of the nuclear import function is supported by that expression of armadillo mutants that are artificially targeted to the nucleus are transcriptionally active even in the absence of either BCL9 or Pygopus (151). Mammalian genomes encode for two BCL9/Legless proteins, BCL9 and BCL9-2. BCL9 seems to exert its nuclear transport function of β-catenin mainly due to nuclear localization sequences on its interaction partner, Pygopus. In contrast, the other homolog, BCL9-2, does not seem to rely on Pygopus for nuclear localization (159) and can specifically interact with β-catenin only when β-catenin is tyrosine phosphorylated at residue 142. This modification in parallel prevents the interaction of β-catenin with α-catenin, thereby representing an intriguing model of a switch between the cytoskeletal and transcriptional functions of β-catenin (80).

Wnt/β-Catenin/T-Cell Factor Target Genes Accumulation of β-catenin leads to fundamental changes of gene expression of a large number of genes. Several

microarray-based studies have formed our current knowledge of this transcriptional program in different tissue culture systems, animal models, and tumor entities (165–169). Interestingly, only a few genes seem to be regulated consistently in all experimental systems, suggesting that this transcriptional is highly dependent on the context in which its activation occurs. Several target genes are discussed briefly here and later in this chapter (see Wnt/β-Catenin Pathway in Gastrointestinal Physiology). For further information on this rapidly expanding field, refer to several reviews (170–172) and the Wnt Home Page (access online at: http://www. stanford.edu/~rnusse/wntwindow.html). The protooncogene c-myc was among the first target genes of β-catenin to be identified, and it plays an important role in cell proliferation and growth (173). By interaction with MIZ1, it is capable of down-regulating the cell cycle inhibitor p21CIP1, thereby releasing a major brake from the cell cycle (165). The progression through the cell cycle is also supported by induction cyclin D1, which is also directly up-regulated by β-catenin (174). Proinvasive factors also seem to be subject to β-catenin– dependent transcriptional regulation. So far, good evidence suggests that matrix metalloproteinase-7 (MMP-7)/Matrilysin (175,176), membrane-type 1 MMP (MT1-MMP) (177,178), Laminin-5 γ2 chain (178), and CD44 (179) are target genes of this pathway. When analyzing the transcriptional program induced by stabilized β-catenin, a marked resemblance to the expression pattern of intestinal cell progenitor cells was noted (165). Inhibition of this pathway conversely led to a pattern resembling differentiated epithelial cells. Several of the previously identified target genes subsequently have been found to be up-regulated in the presumptive intestinal cell progenitor population in vivo. In addition, it was noted that β-catenin coordinates the mutually exclusive expression pattern of EphB2/EphB3 receptors and their ligand Ephrin B1, which are capable of mediating repulsive signaling during cell migration. It also was shown that this coordinated expression is responsible for the correct positioning of proliferative and differentiating intestinal cells along the crypt axis (180). Interestingly, several well-conserved feedback mechanisms have been described. TCF/LEF proteins seem to play a significant role in two of those feedback loops: Stabilized β-catenin seems to specifically activate the transcription of an isoform of TCF1 that does not contain the binding site for β-catenin and can therefore block the transcriptional activation of TCFresponsive promoters. Consistent with this notion, TCF1 knockout mice spontaneously form intestinal polyps consistent with a tumor-suppressor function of this protein in the intestine (181). In contrast, β-catenin specifically increases the transcription of the LEF1 transcripts that can interact with β-catenin, and therefore harbor the potential to increase the transcription of target genes in a positive feedback loop (182). Among the most robust transcriptional targets are the Axin2/Conductin gene and the NKD1 and NKD2 genes (related to the Drosophila naked cuticle gene). Similar to Axin, Axin2/Conductin can mediate a negative feedback

260 / CHAPTER 8 by supporting the degradation of β-catenin. Nkd1 and Nkd2 seem to mediate negative feedback inhibition at the level of Dishevelled (102,103). Both proteins could play an important role in the termination of signaling after physiologic stimulation of this pathway.

NONCANONICAL WNT SIGNALING PATHWAYS Early studies have led to the notion that the transforming activity of Wnts correlates with their ability to stabilize β-catenin (29). It also was realized that Wnt proteins play a role in several signaling pathways without the involvement of β-catenin. These pathways collectively are referred to as “noncanonical Wnt signaling pathways” in the literature (see review by Veeman and colleagues [107]). One of the classic examples of noncanonical Wnt signaling is the establishment of the polarity of cells within a plane in vivo, that is, the organization perpendicular to the apicobasal axis of the cells. Signaling pathways involved in the specification of these decisions involve the JNK pathway and intracellular calcium-dependent signaling pathways. Disruption of components of this so-called planar cell polarity pathway in mammals has been shown to lead to an inhibition of the development of the inner ear (183) by disturbing the orientation of the cilia. Interestingly, disruption of Fz 6 leads to a lack of orientation of the hair fur (184). So far, there is no evidence that planar cell polarity pathways are involved in gastrointestinal physiology or pathology. There is evidence that the Wnt proteins that preferentially serve in planar cell polarity and other noncanonical pathways, such as Wnt5a, might act antagonistic to activating Wnt proteins and induce degradation of β-catenin (185). Thus, they seem to be able to antagonize the function of β-catenin in the transformation of cell lines (186–188). Consistent with this possibility, data implicate Wnt5a as a tumor suppressor in the lymphoid system (186). Part of the Wnt signaling pathway is mediated by inhibition of GSK-3β, which is a central player in multiple signaling pathways (189). In addition to the stabilization of β-catenin, numerous other important pathways are therefore possibly modulated by Wnt signaling, for example, Snail (190) and Notch (191), which are both involved in epitheliomesenchymal transformation (192–194). Future studies need to address the relevance of the cross talk between these pathways to developmental processes in vivo.

WNT/β-CATENIN PATHWAY IN GASTROINTESTINAL PHYSIOLOGY Wnt/β-Catenin in Gastrointestinal Development The gastrointestinal tract develops as a derivative of the endoderm (see reviews by Gregorieff and Clevers [170] and Wells and Melton [195]). Using tissue-specific knockout

approaches, it was demonstrated that β-catenin is essential for endoderm formation around embryonic day 6 of mouse development (196). Experiments in Xenopus suggest that this may be, at least in part, mediated by binding to Sox17, a DNA-binding protein containing an HMG box similar to TCF/LEF proteins (197,198). The source, as well as the identity, of the activating Wnt protein is currently unknown. Given the lack of formation of a primitive streak (a prerequisite for endoderm formation) in the respective knockout embryos, Wnt3 could possibly serve this function (199). In the further development of the gut tube from the endoderm, Tcf1 and Tcf4 seem to be essential mediators as demonstrated by severe developmental defects of Tcf4−/−/ Tcf1−/− embryos. In addition to the truncation of the hindgut, these embryos show an expansion of anterior gut structures such as the stomach (200). Interestingly, it has been shown that overexpression of stabilized β-catenin mutants in the lung endoderm of transgenic animals leads to the expression of genes typically specific for the intestine (201). Taken together, these two observations suggest that Wnt/β-catenin signaling is not only essential for endoderm development, but also for the development of an intestinal fate.

Wnt/β-Catenin Pathway in Gastrointestinal Homeostasis and Architecture The intestinal mucosa is exposed to one of the most toxic, aggressive, and changing milieus of the whole body. In contrast with the outer surface of the body, the intestinal barrier consists only of a single layer of fragile epithelial cells. To maintain an active barrier and also fulfill the main tasks of digestion and absorption, the intestine relies on the high turnover of several specialized cell types that continuously form from intestinal stem cells (171,202). Mature cells can be mainly subdivided into a secretory and an absorptive lineage. The secretory lineage consists of goblet cells producing the intestinal mucus and a small number of enteroendocrine cells that are further subdivided based on the type of hormone that they produce. In the small intestine, a third secretory cell type, the so-called Paneth cells, localizes to the bottom of the crypts, producing a variety of antibacterial substances. The absorptive lineage is also called enterocyte and fulfills the task of absorbing the nutrients from the intestinal contents (170). All intestinal cell types derive from intestinal stem cells. These stem cells localize to the base of the crypt in the colon and above the Paneth cells in the lower part of the crypts in the small intestine (203). With the exception of the Paneth cells, the differentiating daughter cells migrate actively up the crypt in a coordinated way and populate the intestinal surface and villi. After a lifetime of less than a week, these cells undergo apoptosis and are shed in the intestinal lumen. Wnt/β-catenin signaling seems to play a major role in maintenance of a progenitor cell compartment, in the maintenance of the intestinal architecture, and in the differentiation of the secretory lineage.

DEVELOPMENTAL SIGNALING NETWORKS / 261 Several lines of evidence demonstrate an essential role for Wnt/β-catenin signaling in the maintenance of a functioning intestinal progenitor compartment. Knocking out the DNAbinding adaptor TCF4 leads to severe developmental defects with a total absence of the intestinal progenitor cell compartment (204). A similar phenotype is observed in transgenic animals overexpressing the Wnt antagonist Dkk1 in the gut and on induced deletion of β-catenin in the small intestine (205–207). This is consistent with the previous observation that inhibition of Wnt/β-catenin signaling in colorectal carcinoma cell lines leads to a shift in gene expression from a progenitor type to a more differentiated absorptive cell type (165). As expected, overexpression of stabilized β-catenin mutants or induced inactivation of APC in the gut leads to epithelial hyperproliferation and expansion of the proliferative compartment (169). Evidence for a role of Wnt/β-catenin signaling in maintaining the architecture of the intestinal crypts originated from the observation that the tyrosine kinase receptors EphB2 and EphB3 are direct target genes of this pathway (180). These receptors play a well-characterized function in the guidance of axonal outgrowth and cell migration during the development of the central nervous system (208). As expected for a Wnt/β-catenin target gene, a high expression in the proliferative compartment of the intestinal crypts was noted. Analysis of double-knockout mice for EphB2 and EphB3 showed that this expression pattern is essential for a coordinated movement of differentiating cells and the localized maintenance of a stem cell compartment. Consequently, in these mice, proliferative and differentiating cells intermingled, and Paneth cells no longer homed for the base of the crypt (180). A role of Wnt/β-catenin signaling in the commitment to a secretory fate was initially suggested by the observation that induced deletion of β-catenin in the intestine or overexpression of Wnt antagonists leads to a striking reduction of goblet cells, Paneth cells, and enteroendocrine cells (204,205,207). In reverse experiments, it was demonstrated that induced activation of the Wnt/β-catenin signaling leads to an increased production of Paneth cells, suggesting that Wnt signaling might play a specific role in mediating the differentiation of this specific cell type. In this context, several Paneth cell– specific proteins have been shown to be direct Wnt/β-catenin target genes (209,210). Interestingly, a phenotype of increased goblet cell differentiation also has been described in animals that were treated with γ-secretase inhibitors thereby preventing efficient Notch signal transduction (211). Similarly, an increase of secretory lineage cells has been observed in mice with a targeted disruption of the Notch target gene HES1 (212), suggesting that the balance between Notch signaling and Wnt/β-catenin signaling could govern the decision between absorptive and secretory lineage differentiation. Although much has been learned about the role of Wnt/βcatenin signaling in cellular differentiation and the maintenance of a progenitor cell compartment, very little is known about which specific Wnt proteins and receptor complexes

are mediating these effects in vivo. Given the vast potential for redundancy and combinatorial diversity, this poses a major challenge to the understanding of the physiologic role of this signaling pathway. An additional layer of complexity is added by the extensive cross talk of Wnt/β-catenin with other signaling pathways such as Notch signaling and the bone morphogenic protein pathway (202).

WNT/β-CATENIN PATHWAY DEFECTS IN GASTROINTESTINAL TUMORS This section summarizes some of the data that are available on the involvement of the Wnt/β-catenin signaling pathway in gastrointestinal malignancy. See Table 8-1 for more detailed information.

Colon and Rectum Sporadic Colorectal Cancer Activation of the Wnt signaling pathway seems to be one of the earliest changes in the development of colorectal neoplasia, occurring as early as in aberrant crypt foci (213). Inactivating mutations of APC seem to account for up to 80% of the cases (214,215). APC is a large gene coding for 2843 amino acids and spanning more than 90 kBp. Although a plethora of mutations have been described, they do not seem to be distributed evenly over the coding sequence, and 60% seem to be clustered in a small region of exon 15 between codon 1250 and 1450, which therefore has been termed mutation cluster region (MCR) (16,216). The capacity to destabilize β-catenin is highly dependent on the presence of the SAMP repeats, which, via binding to Axin, seem to recruit APC to the destruction complex. Biallelic inactivation of APC almost invariably leads to the expression of truncated APC proteins that do not contain any of the Axin-interacting SAMP repeats and are therefore not capable of efficiently stimulating degradation of β-catenin. Interestingly, complete deletion of both APC alleles with complete loss of APC protein expression is rarely observed, suggesting that the selective advantage of APC mutations not only relies on the inactivation of APC functions, but also possibly on the retention of certain APC functions or even the generation of new functions of the truncated proteins (123,124). Different explanations to account for this selection advantage such as increased genomic instability (139,140) and increased migratory potential (143) induced by truncated APC proteins have been proposed. In aggregate, less than 10% of colorectal carcinomas have been reported to carry stabilizing β-catenin mutations, and most colorectal carcinomas with functionally significant β-catenin mutations are mismatch repair deficient (215,217). In mismatch repair–deficient colorectal carcinomas, mutations in a microsatellite repeat tract in the coding region of the Axin2 gene also seem to be present in a

262 / CHAPTER 8 TABLE 8-1. Percentage of activation of Wnt/adenomatous polyposis coli/β-catenin pathway in selected tumor entities Tumor entity

APC

β-catenin

Colon Carcinoma

80%

10%

Stomach Fundic gland polyps (236) Gastric adenoma (240) Carcinoma (242,243)

43–64% 8–27%

Esophagus Barrett’s esophagus Adenocarcinoma Squamous carcinoma

Axin

Axin2

0%

<10%

4%

2%

10%

3%

64–94%

1%

Lung Adenocarcinoma

3%

Ovary Endometrioid carcinoma

2%

Rare, but frequent nuclear localization <2% Rare

14–50%

Uterus Endometrial carcinoma Cervical carcinoma

11–45% Rare

Thyroid Carcinoma

46%

Liver Hepatocellular carcinoma

Rare

Hepatoblastoma

≤69%

Pancreas Ductal carcinoma Acinar cell carcinoma Solid pseudopapillary tumor Pancreatoblastoma

6%

Skin Melanoma Pilomatricoma

3–7%

10–43% (correlated with hepatitis C virus) ≤89%

7%

Rare 18% 90% 55% 2–22% 26–100%

For references see corresponding chapters and Gilles and colleagues (172). APC, adenomatous polyposis coli.

considerable fraction of cases, though such mutations are not seen in colorectal cancers where mismatch repair activity is intact (218). In contrast with the situation in hepatocellular carcinoma, the pathogenetic relevance of the rarely observed Axin mutations in colorectal cancers is unclear (219,220). Familial Adenomatous Polyposis Coli FAP is an autosomal dominant condition caused by the germline mutation of one allele of the APC gene that leads to the development of thousands of colorectal polyps in the second decade of life. The disease almost invariably progresses to the development of colorectal cancer unless the colon is surgically removed before its occurrence. It is observed in 1 in 7000 to 1 in 10,000 people and is responsible for less than 1% of colorectal cancers (12). Most mutations are nonsense or frameshift mutations leading to the production of truncated APC proteins lacking APC protein synthesis lacking Axin-interacting regions but still retaining some capacity to interact with β-catenin. The majority is localized in the N-terminal half, and almost 35%

of the mutations occur in two mutational hotspots (codon 1061 and 1309) (116,123). Only a small subset of mutations are large deletions or mutations that completely abolish APC expression. Some analyses also suggest that the localization of the germline mutation predetermines the mechanism by which the second allele is lost. This is consistent with the notion that truncated APC proteins serve important functions in colorectal carcinogenesis and complete loss of APC expression is selected against. Greater severity of polyposis is associated with mutations between codons 1250 and 1464 (221). However, clinical presentation is not consistent even within kindreds carrying the same germline mutation. The same is also true for the presentation of extracolonic manifestations. The most common extracolonic symptom is a benign alteration of the eye’s pigment epithelium, called congenital hypertrophy of the retinal pigment epithelium (CHRPE). It occurs in up to 60% of patients and is associated with mutations between codons 457 and 1444 (222). In addition, other tumors occur in greater frequency in patients with FAP such as desmoid tumors (associated with mutations between codon

DEVELOPMENTAL SIGNALING NETWORKS / 263 1445 and 1578 [223]), osteoma, gastric adenocarcinoma, thyroid carcinoma, central nervous system tumors, hepatoblastoma, as well as duodenal ampullary carcinoma. Depending on the constellation of extracolonic manifestations, several distinct syndromes have been described. Among these, Gardner syndrome refers to FAP cases with extensive polyposis, epidermoid cysts, desmoid tumors, and osteomas. Turcot syndrome comprises cases with polyposis and brain tumors, such as medulloblastoma (12). Attenuated forms of FAP (termed AAPC for attenuated adenomatous polyposis coli or AFAP for attenuated FAP) have also been described. In AAPC kindreds, some affected individuals in the family have as little as 10 to 20 colonic adenomas and no cancers by 50 years old, though other affected AAPC family members can have colonic adenoma numbers essentially indistinguishable from those with classical FAP (224). Although the likelihood of development of colorectal cancer is much smaller for an individual with AAPC, in some populations, a high frequency of a predisposing allele might lead to a significant contribution to the incidence of colorectal carcinoma (225). For example, 6% to 10% of Ashkenazi Jews carry the APC variant I1307K, which increases the colorectal carcinoma risk only about twofold without leading to overt polyposis (226–228). However, because of its high prevalence in this population, this mutation can significantly influence colorectal carcinoma incidence in this population.

Esophagus Malignancies of the esophagus can be subdivided into squamous cell carcinoma and adenocarcinoma. The latter seems to develop via a premalignant glandular metaplasia termed Barrett’s esophagus. Squamous Cell Carcinoma Human Fz 2 was first cloned from an esophageal squamous cell carcinoma (229). The percentage of carcinomas overexpressing Fzd2 was reported to increase from 23% of differentiated carcinomas to 86% of undifferentiated carcinomas. However, nuclear localization of β-catenin was reported in only 18% of all cases (230). The significance of these findings is still unclear. Barrett’s Esophagus and Esophageal Adenocarcinoma Activating mutations of β-catenin have been described in less that 2% of the cases of esophageal adenocarcinoma (233–235). Although loss of heterozygosity (LOH) for APC was reported to occur in up to 40% of the cases, inactivating mutations in the remaining allele were only found in 1 of 38 tumors with LOH (235). Given the fact that only the mutation cluster region was screened in these studies, mutations in the remaining APC gene cannot be excluded. The role of genetic events leading to the activation of the Wnt/β-catenin signaling pathway is therefore unclear.

However, reduced membranous and increased nuclear localization of β-catenin seem to be common and early events in the pathogenesis of Barrett’s esophagus, and are correlated with the development of dysplasia (231). The cause of these changes and their relevance to the progression to cancer are unclear because they have been reported to be associated with both better and worse clinical outcomes in esophageal adenocarcinoma (231,232).

Stomach Fundic Gland Polyps Fundic gland polyps are benign nondysplastic hamartomatous or hyperplastic lesions that constitute most gastric polyps. Sporadic fundic gland polyps rarely, if at all, progress to adenocarcinoma. This is in contrast with the gastric fundic gland polyps that have been observed in patients with familial APC mutations. These usually display biallelic inactivation of the APC gene product and occasionally progress to adenocarcinoma. Surprisingly, up to 94% of the polyps predominantly benign sporadic fundic gland polyps show stabilizing mutations of the β-catenin gene (236,237). In the rare instances of fundic gland polyposis, polyps from single individuals often do not all show the same mutation, suggesting that they are of polyclonal origin (238). That gastric fundic gland polyps almost never progress to gastric adenocarcinoma (or even only to dysplasia) and the activation of the Wnt/βcatenin signaling pathway in most cases seems difficult to reconcile. However, Hassan and colleagues (239) reported that syndromic and sporadic cases with stabilizing β-catenin mutations rarely show abnormal β-catenin staining. Although these findings require further confirmation, they could suggest that so far unknown mechanisms are able to control the activation of the Wnt/β-catenin pathway in gastric epithelium. Gastric Adenoma Unlike benign gastric fundic gland polyps, about 25% of the much rarer gastric adenomas progress to gastric adenocarcinoma, marking these polyps as premalignant lesions. Depending on the histologic subtype, biallelic inactivation of APC has been described in 64% (intestinal type) and 43% (foveolar type) of cases (240). Interestingly, the presence of an APC inactivation was reported to be inversely correlated with the tendency to progress to adenocarcinoma (241). The small number of studies and rare occurrence of these lesions currently limits our understanding of these observations. Gastric Adenocarcinoma Although nuclear localization occurs in approximately one third of gastric adenocarcinomas, less than 25% of these

264 / CHAPTER 8 cases show activating mutations of β-catenin (242,243). It is unclear what causes the nuclear localization of β-catenin in the remaining carcinomas without mutations. In rare instances, biallelic inactivation of SIAH1 gene, encoding a component of a E3 ubiquitin ligase complex, have been reported to be associated with nuclear β-catenin accumulation (87). Given the fact that the regulation of β-catenin via the classical destruction complex is still functional in these cases, the exact molecular mechanism leading to the stabilization of β-catenin awaits further elucidation. Germline mutations in the gene encoding E-Cadherin are associated with familial gastric adenocarcinoma. Epigenetic inactivation of ECadherin also seems to occur in a high percentage of sporadic diffuse-type gastric adenocarcinoma. However, although these changes would be expected to free a large part of the cytoskeleton-bound β-catenin, they do not seem to lead to an increase in TCF/LEF-dependent transcription (243,244), suggesting that effects outside of the Wnt/β-catenin signaling pathway might be more relevant.

Liver Nuclear localization of β-catenin is observed in a significant proportion of hepatocellular carcinoma (HCC). The pathogenesis of this tumor shows marked differences between different geographic regions which can be attributed to the different prevalences of hepatitis B, hepatitis C, and the exposure to aflatoxin. The occurrence of nuclear β-catenin seems to be associated only with some of these causative agents, which might explain the marked differences of its frequency in the literature ranging from 12% to 41%. Stabilizing β-catenin mutations seem to occur in a distinct subset of these tumors that are characterized by their good prognosis and lack of involvement of the hepatitis B virus. This subset seems to be associated rather with hepatitis C virus and is devoid of high chromosomal instability (245–249). Distinct from this group is a rare childhood tumor called hepatoblastoma that shows activating β-catenin mutations in a high percentage (250–252). Axin and Axin2 mutations have been observed in HCC in 8% to 10% and 3%, respectively (104,245,253). APC mutations seem to be extremely rare in HCC. It has been reported that the prolyl-isomerase PIN1 can inhibit the interaction of β-catenin and APC. Overexpression of PIN1 has therefore been suggested to account for the stabilization of β-catenin in the fraction of tumors that do not show other genetic defects in the Wnt/β-catenin pathway (254). Overall, the importance of the Wnt/β-catenin signaling pathway in hepatic carcinogenesis has also been demonstrated in animal models. Biallelic inactivation of APC in the liver leads to the development of HCCs. Overexpression of stabilized β-catenin mutants only leads to a hypertrophy of the liver, and coexpression with H-Ras leads to the development of HCCs (255–257). In addition, β-catenin seems to play an important role during hepatic development (258).

Pancreas Ductal adenocarcinoma of the pancreas, the most common form of pancreatic cancer, usually does not show abnormal localization of β-catenin. Except for two mutations in pancreatic cancer cell lines, no mutations in β-catenin or APC currently have been described (243,259,260). However, several rare tumor entities display a high percentage of abnormalities in the Wnt/APC/β-catenin signaling pathway. Up to 90% of solid pseudopapillary tumors (261,262) and twothirds of pancreatoblastomas harbor activating β-catenin mutations (263). In contrast, activation of Wnt/APC/β-catenin signaling pathway in 25% of the acinar cell carcinoma is mediated by biallelic inactivation of APC in most cases (264).

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270 / CHAPTER 8 252. Wirths O, Waha A, Weggen S, Schirmacher P, Kuhne T, Goodyer CG, et al. Overexpression of human Dickkopf-1, an antagonist of wingless/WNT signaling, in human hepatoblastomas and Wilms’ tumors. Lab Invest 2003;83:429–434. 253. Ishizaki Y, Ikeda S, Fujimori M, Shimizu Y, Kurihara T, Itamoto T, et al. Immunohistochemical analysis and mutational analyses of betacatenin, Axin family and APC genes in hepatocellular carcinomas. Int J Oncol 2004;24:1077–1083. 254. Pang R, Yuen J, Yuen MF, Lai CL, Lee TK, Man K, et al. PIN1 overexpression and beta-catenin gene mutations are distinct oncogenic events in human hepatocellular carcinoma. Oncogene 2004;23: 4182–4186. 255. Colnot S, Decaens T, Niwa-Kawakita M, Godard C, Hamard G, Kahn A, et al. Liver-targeted disruption of Apc in mice activates beta-catenin signaling and leads to hepatocellular carcinomas. Proc Natl Acad Sci U S A 2004;101:17216–17221. 256. Harada N, Miyoshi H, Murai N, Oshima H, Tamai Y, Oshima M, et al. Lack of tumorigenesis in the mouse liver after adenovirus-mediated expression of a dominant stable mutant of beta-catenin. Cancer Res 2002;62:1971–1977. 257. Harada N, Oshima H, Katoh M, Tamai Y, Oshima M, Taketo MM. Hepatocarcinogenesis in mice with beta-catenin and Ha-ras gene mutations. Cancer Res 2004;64:48–54. 258. Micsenyi A, Tan X, Sneddon T, Luo JH, Michalopoulos GK, Monga SP. Beta-catenin is temporally regulated during normal liver development. Gastroenterology 2004;126:1134–1146. 259. Gerdes B, Ramaswamy A, Simon B, Pietsch T, Bastian D, Kersting M, et al. Analysis of beta-catenin gene mutations in pancreatic tumors. Digestion 1999;60:544–548. 260. Kubozoe T, Tsujiuchi T, Murata N, Sasaki Y, Tsunoda T, Konishi Y, et al. Absence of beta-catenin gene mutations in pancreatic duct lesions induced by N-nitrosobis(2-oxopropyl)amine in hamsters. Cancer Lett 2001;168:1–6. 261. Abraham SC, Klimstra DS, Wilentz RE, Yeo CJ, Conlon K, Brennan M, et al. Solid-pseudopapillary tumors of the pancreas are genetically distinct from pancreatic ductal adenocarcinomas and almost always harbor beta-catenin mutations. Am J Pathol 2002;160:1361–1369. 262. Tanaka Y, Kato K, Notohara K, Hojo H, Ijiri R, Miyake T, et al. Frequent beta-catenin mutation and cytoplasmic/nuclear accumulation in pancreatic solid-pseudopapillary neoplasm. Cancer Res 2001;61: 8401–8404. 263. Abraham SC, Wu TT, Klimstra DS, Finn LS, Lee JH, Yeo CJ, et al. Distinctive molecular genetic alterations in sporadic and familial adenomatous polyposis-associated pancreatoblastomas: frequent alterations in the APC/beta-catenin pathway and chromosome 11p. Am J Pathol 2001;159:1619–1627.

264. Abraham SC, Wu TT, Hruban RH, Lee JH, Yeo CJ, Conlon K, et al. Genetic and immunohistochemical analysis of pancreatic acinar cell carcinoma: frequent allelic loss on chromosome 11p and alterations in the APC/beta-catenin pathway. Am J Pathol 2002;160:953–962. 265. Yao R, Natsume Y, Noda T. MAGI-3 is involved in the regulation of the JNK signaling pathway as a scaffold protein for frizzled and Ltap. Oncogene 2004;23:6023–6030. 266. Yao R, Maeda T, Takada S, Noda T. Identification of a PDZ domain containing Golgi protein, GOPC, as an interaction partner of frizzled. Biochem Biophys Res Commun 2001;286:771–778. 267. Kramps T, Peter O, Brunner E, Nellen D, Froesch B, Chatterjee S, et al. Wnt/wingless signaling requires BCL9/legless-mediated recruitment of pygopus to the nuclear beta-catenin-TCF complex. Cell 2002; 109:47–60. 268. Kishida S, Yamamoto H, Hino S, Ikeda S, Kishida M, Kikuchi A. DIX domains of Dvl and axin are necessary for protein interactions and their ability to regulate beta-catenin stability. Mol Cell Biol 1999;19: 4414–4422. 269. Capelluto DG, Kutateladze TG, Habas R, Finkielstein CV, He X, Overduin M. The DIX domain targets dishevelled to actin stress fibres and vesicular membranes. Nature 2002;419:726–729. 270. Axelrod JD, Miller JR, Shulman JM, Moon RT, Perrimon N. Differential recruitment of Dishevelled provides signaling specificity in the planar cell polarity and Wingless signaling pathways. Genes Dev 1998;12:2610–2622. 271. Rousset R, Mack JA, Wharton KA Jr, Axelrod JD, Cadigan KM, Fish MP, et al. Naked cuticle targets dishevelled to antagonize Wnt signal transduction. Genes Dev 2001;15:658–671. 272. Li L, Yuan H, Xie W, Mao J, Caruso AM, McMahon A, et al. Dishevelled proteins lead to two signaling pathways. Regulation of LEF-1 and c-Jun N-terminal kinase in mammalian cells. J Biol Chem 1999;274:129–134. 273. Moriguchi T, Kawachi K, Kamakura S, Masuyama N, Yamanaka H, Matsumoto K, et al. Distinct domains of mouse dishevelled are responsible for the c-Jun N-terminal kinase/stress-activated protein kinase activation and the axis formation in vertebrates. J Biol Chem 1999; 274:30957–30962. 274. Berrueta L, Kraeft SK, Tirnauer JS, Schuyler SC, Chen LB, Hill DE, et al. The adenomatous polyposis coli-binding protein EB1 is associated with cytoplasmic and spindle microtubules. Proc Natl Acad Sci U S A 1998;95:10596–10601. 275. Su LK, Burrell M, Hill DE, Gyuris J, Brent R, Wiltshire R, et al. APC binds to the novel protein EB1. Cancer Res 1995;55:2972–2977.

CHAPTER

9

Hedgehog Signaling in Gastrointestinal Morphogenesis and Morphostasis Gijs R. van den Brink, Maikel P. Peppelenbosch, and Drucilla J. Roberts Patterning, 271 The Hedgehog Pathway, 272 Hedgehog Genes, 272 The Unusual Posttranscriptional Fate of Hedgehog Proteins, 273 The Receptor Complex, 274 Signal Transduction and Target Genes, 274 Role of Hedgehog Signaling in the Development of the Gut, 275 Development of the Gastrointestinal Tube, 275 Hedgehog Signaling and Foregut Development, 275 Hedgehog Signaling and Development of the Midgut, 278 Hedgehog Signaling and Development of the Hindgut, 278

Hedgehog Signaling and the VACTERL Association in Humans, 279 Hedgehog Signaling in Homeostasis of the Adult Gastrointestinal Tract, 279 Morphostasis, 279 Role of Ihh and Shh in Adult Gut, 280 Shh and Gastric Epithelial Homeostasis, 281 Shh and the Small Intestinal Crypt, 281 Ihh and Colonic Epithelial Differentiation, 281 Hedgehog Signaling and Carcinogenesis of the Gastrointestinal Tract, 282 Future Developments, 283 References, 283

The mechanisms that orchestrate the development of a multicellular organism from a single common ancestor cell have fascinated people for centuries. We are still a long way from truly understanding the robust programs that allow building of our specialized organs that regulate digestion, respiration, and hemodynamics and that try to guard us from invasion by other unsolicited organisms. However, in the past three decades, many of the genes that play a role in patterning our bodies have been identified, and much has been learned about the mechanisms involved in development by characterizing the nature of these patterning genes and the way they work together. This chapter discusses

patterning mechanisms, introduces the Hedgehog pathway, and then summarizes our current understanding of the role of Hedgehog signaling in development and maintenance of the gastrointestinal (GI) tract.

PATTERNING The human body is built from hundreds of specialized cell types that make up its organs. The development and postnatal maintenance of these cell lineages is tightly controlled and incredibly stable in time. Cells are capable of going through major shifts in protein expression in response to physiologic and environmental cues without changing their cellular phenotype. Each cell type is defined by the expression of a unique combination of proteins transcribed and posttranscriptionally modified from the templates of the 20,000 to 25,000 genes in the body. One of the central questions in biology is how the information contained in these genes can translate into a spatially complex multicellular organism. The idea that such complexity is possible because cellular phenotype is coupled to cellular position goes back

G. R. van den Brink: Laboratory for Experimental Internal Medicine, Academic Medical Center, Amsterdam, The Netherlands. M. P. Peppelenbosch: Department of Cell Biology, University of Groningen, Groningen, The Netherlands. D. J. Roberts: Department of Pathology, Massachusetts General Hospital, Boston, Massachusetts 02114.

Physiology of the Gastrointestinal Tract, Fourth Edition, edited by Leonard R. Johnson. Academic Press, 2006.

271

272 / CHAPTER 9 at least a century (1). Much has been learned about the proteins involved in the specification of cellular position from developmental biologists who have used models such as the regulation of segmentation of the Drosophila larval epidermis (2), development of the vertebrate limb bud (3), and neurogenesis (4). Most of the evolutionary conserved signaling pathways that act to couple cellular position to cellular phenotype have been identified in genetic screens in Drosophila, most notably the systematic screens performed by Nusslein-Volhard and Wieschaus (5). The type of molecules that have emerged from these genetic screens can be classified in two broad categories: intrinsic and extrinsic factors. Intrinsic signals such as kinases, phosphatases, and transcription factors function within the cell, and thus act in a cell-autonomous manner. Extrinsic signals are soluble or transmembrane factors that act among cells and regulate cell-nonautonomous processes. Extrinsic information is critical for a complex tissue composed of multiple different cell types to organize appropriately. The idea that tissues might contain gradients of soluble factors that act in a concentration-dependent manner to control cell fate was already postulated at the beginning of the twentieth century (1). Such factors have been termed morphogens. Signaling molecules from the Hedgehog, Wnt, and transforming growth factor-β have been shown to act as morphogens in several models of development and are used in the regulation of cell fate throughout evolution and in most developing tissues. This chapter summarizes our current understanding of the Hedgehog pathway and its role in the development and postnatal maintenance of the GI tract.

THE HEDGEHOG PATHWAY Hedgehog Genes The Hedgehog gene was identified in the first systematic screen for patterning genes in Drosophila. In larvae of wildtype Drosophila, a band of bristles called denticles is present across the anterior half of each segment, whereas the posterior half is smooth (the so-called naked cuticle). In screening for mutations that affect the segmental pattern of the Drosophila larva, Nüsslein-Volhard and Wieschaus (5) identified a group of mutants that affected the patterning within the segments, but at the same time left the number of segments unaltered. In one of these segment polarity mutants, the posterior half of each segment failed to develop, resulting in a larva that was entirely covered by denticles (Fig. 9-1). This phenotype gave the larva the aspect of a Hedgehog (Hemiechinus), hence the name. After much effort, the nucleotide sequence of the Hedgehog gene was finally obtained (6), and the mammalian Hedgehog homologues were identified. Echelard and colleagues (7) identified three Hedgehog genes in the mouse. They were named Sonic Hedgehog (Shh), after a popular Sega computer game character, Desert Hedgehog (Dhh), after an Egyptian species of Hedgehog (Hemiechinus auritus), and Indian Hedgehog (Ihh), which is a Hedgehog species endemic in Pakistan (Hemiechinus micropus). Riddle and coworkers (8) found that Shh protein mediates the action of the zone of polarizing activity (ZPA) in the chick limb bud (Fig. 9-2). The ZPA is a region at the posterior margin of the chick limb

A

B FIG. 9-1. The hedgehog mutant. An artistic impression of the Drosophila Hedgehog mutant. Each segment of the wild-type Drosophila larva (A) has bristles or denticles at the anterior end and a smooth posterior end, the so-called naked cuticle. Hedgehog signaling specifies the posterior end of each segment; as a result, the mutant lacks the naked cuticles (B).

HEDGEHOG SIGNALING IN GASTROINTESTINAL MORPHOGENESIS AND MORPHOSTASIS / 273 Anterior

S

O

Posterior

Cys 258

Gly 257

Shh ZPA

Hh-C

N

Hh-N

A N

O

A Hh-N

Hh-C

S

Shh soaked bead

Cholesterol

B S O

Shh Hh-N

B FIG. 9-2. Shh and the zone of polarizing activity (ZPA). (A) A small group of cells in the ZPA at the posterior end of the developing limb produces Shh and specifies digit identity along the anteroposterior axis. (B) Transplantation of an extra ZPA at the anterior end of the limb bud leads to a mirrorimage duplication of the digit pattern. The same can be achieved by transplantation of a bead coated in Shh protein.

bud that is responsible for normal anteroposterior patterning. Shh was shown to colocalize with this previously described region and to alter limb patterning in a way similar to ZPA grafting. At the same time, a Hedgehog homologue was identified in zebra fish (Danio rerio) and shown to be expressed in tissues with organizing activity (9). In 1995, a new Hedgehog homolog was found in zebra fish and was named TiggyWinkle Hedgehog, after a character in a children’s book (10). Most phyla of the eukaryotic kingdom have now been shown to contain Hedgehog homologues. The expression of vertebrate Hedgehog genes has been detected in a variety of tissues during development. Sonic hedgehog is expressed in the limb buds and midline tissues such as the gut, notochord, floor plate, and ventral forebrain. The Indian hedgehog gene is expressed in the gut, kidney, liver, and developing bones (11–13). The mouse Desert hedgehog gene is expressed in the developing gonad, endothelium, and Schwann cells (12). Amphioxus, the closest living relative of vertebrates, has a single Hedgehog gene (14). Amphioxus Hedgehog is expressed in the notochord, the floor plate, and the endoderm (14); this pattern of expression is maintained by vertebrate Sonic hedgehog, suggesting that Indian and Desert hedgehogs have been able to diverge in their expression patterns and their role in embryonic development.

The Unusual Posttranscriptional Fate of Hedgehog Proteins For the Hedgehog protein to act as a potent morphogen, it needs to be processed in different ways. After removal of the signal sequence, the approximately 45-kDa Hedgehog

Signaling peptide

N

Hh-C Inactive processed peptide

C FIG. 9-3. SHH protein processing. (A) In the first step of SHH precursor protein processing the 257Gly-258Cys peptide bond is converted to an internal thioester via an N to S shift. (B) This processing intermediate is cleaved by cholesterol transferase activity of the C-terminal end of the precursor protein that mediates a nucleophilic attack by cholesterol on the internal thioester. (C) Processing results in a 19-kDa biologically active N-terminal fragment that is covalently attached to cholesterol and a biologically inactive C-terminal fragment.

proteins are cleaved autocatalytically, yielding an 19-kDa N-terminal fragment that contains all the signaling functions and a 26-kDa C-terminal fragment that has no other function than to catalyze the cleavage (Fig. 9-3) (15,16). The unusual posttranslational processing of Hedgehog proteins is not limited to this autocatalytic processing of the primary translated product. In the course of the cleavage, the SHH protein is covalently palmitoylated (17). This is atypical because palmitoylation usually is restricted to the cytoplasm, and thus affects only proteins without a signal peptide and not proteins entering the secretory vesicular pathway. Palmitoylated recombinant SHH has approximately 30-fold greater biological activity than unpalmitoylable mutants, suggesting that this acylation is important for enhancing biological activity. In accordance, in a Drosophila mutant in the acyl transferase mediating the Hedgehog palmitoylation (the so-called skinny hedgehog mutant [ski]), the morphogen is functionally inactive (18–20). The main function of the palmitoylation seems to be to increase the hydrophobicity of the molecule, which may serve to target the molecule to lipid rafts, which could, for example, facilitate efficient interaction with the Hedgehog receptor Patched (21,22). Even more atypical than the palmitoylation is the covalent attachment of a cholesterol moiety to the C-terminal part of Hh-N (23), making Hedgehog proteins the only established example of protein sterolation. A role for the cholesterol moiety in regulating diffusion or release of Hedgehog has been suggested (24–26): the sterol anchors hedgehog to

274 / CHAPTER 9 biological membranes, thus hampering diffusion. In limbs of embryos containing sterolation-deficient hedgehog, digits close to the ZPA (the source of hedgehog in limb bud) are normal, but the more distant digits are absent, suggesting that sterolation determines the spatial distribution of Hedgehog protein but is not required for biological activity.

The Receptor Complex Hedgehog signal transduction is highly unusual, containing many features unique to this signaling system (27). The Hedgehog binding receptor is a 12-transmembrane protein patched (Ptc). However, unlike a conventional receptor, Ptc does not convey the Hedgehog signal to the intracellular components of the pathway itself. Rather, binding of Hedgehog to Ptc alleviates the inhibitory effect of Ptc on another membrane receptor, the seven-transmembrane protein Smoothened (Smo). In the absence of Ptc, Smo will constitutively activate Hedgehog target gene transcription (28). The mechanism of Smo inhibition by Ptc is unknown, but it is presumably a catalytic effect, because Ptc and Smo do not need to bind or colocalize, and Ptc also inhibits Smo when present substoichiometrically (29). Some homology exists between Ptc and some prokaryotic transporter molecules, but also the Niemann–Pick C1 (NPC1) protein, a transport protein involved in cholesterol homeostasis (30). This homology gave rise to the conception that, in the absence of Hedgehog, Ptc might act as a pump that controls the containment of a yet unidentified compound that binds to and inhibits Smo (Fig. 9-4) (29).

PTCH

SMO

Signaling downstream of Smo is still only partially understood but involves a microtubule-bound complex of proteins that regulates the activity of the Hedgehog pathway. This complex consists of the proteins Costal2 (Cos2), Fused (Fu), Suppressor of Fused (Su(fu)), and a Hedgehog target gene– activating transcription factor. In Drosophila, the Hedgehog pathway acts through a single transcription factor, Cubitus interruptus (Ci). In vertebrates, the Hedgehog pathway regulates three Ci homologues that have been termed Gli-1 to Gli-3 (31–34). Because Hedgehog signal transduction is even more poorly understood in vertebrates than in Drosophila, this section focuses on Hedgehog signal transduction in Drosophila. Transcriptional regulation by the Ci protein has been hard to elucidate because of the presence of both an activating and a repressor form of the protein. In Drosophila, all Hedgehog-responsive cells contain a 75-kDa repressor form of Ci that is generated from the 155-kDa full-length protein by a proteolytic event that splices off the transcriptional activation domain and nuclear export signal (see Figs. 9-4 and 9-5). The resulting truncated protein retains a DNA-binding domain and an N-terminal transcriptional repression domain (Fig. 9-6) (35). Hedgehog signaling inhibits Ci proteolysis and results in nuclear accumulation of full-length Ci and activation of target genes. Ci proteolysis is regulated by Shaggy, an orthologue of the vertebrate glycogen synthase kinase-3β (GSK-3β) that can hyperphosphorylate Ci after a priming phosphorylation by protein kinase A (PKA) (36,37). This aspect of Hedgehog signaling is reminiscent of the mechanism of action of the Wnt pathway, where GSK-3β negatively regulates Wnt signaling through its control of the breakdown of the effector of the Wnt pathway, β-catenin (38). Interestingly, Smo is genetically closest related to the Wnt receptor Frizzled, and both receptors seem to work by inhibition of GSK-3β with resulting inhibition of the proteolytic breakdown of the effector protein of the pathway. Several studies have shown that the kinesin-related protein Cos2 provides a physical link between Smo and Ci processing. On activation of Smo, the stability of Smo is increased and Ci processing is inhibited potentially through

Hh-N

A

Signal Transduction and Target Genes

PTCH

SMO

B FIG. 9-4. The Hedgehog receptor complex. A model of patched in human (PTCH)-Smoothened (SMO) interaction. (A) In the absence of Hedgehog, PTCH functions as a transmembrane pump that pumps out an SMO inhibitory compound that is yet to be identified. (B) Binding of Hedgehog to PTCH shuts down its pump function; as a result, the inhibition of SMO is released.

FIG. 9-5. The Cubitus interruptus (Ci) protein. Ci is a 155-kDa protein that is proteolytically processed in the absence of Smoothened (SMO) signaling. Splicing generates a 75-kDa N-terminal protein that contains that DNA-binding zinc finger (ZnF) and the nuclear localization signal (NLS); because this protein lacks the transactivating C-terminal end of the protein, it acts as a repressor of gene transcription. Activity of the fulllength protein (that also contains a nuclear export sequence [NES]) can be inhibited by binding of Suppressor of Fused (Su(fu)) to the N-terminal Su(fu) binding site.

HEDGEHOG SIGNALING IN GASTROINTESTINAL MORPHOGENESIS AND MORPHOSTASIS / 275

SMO

Su(fu) Fu Cos2 Ci Proteasome P

Ci-R

P

PKA, GSK3β, CK1

FIG. 9-6. Cubitus interruptus (Ci) processing. A schematic representation of the factors that influence Ci processing. Ci processing occurs after phosphorylation by protein kinase A (PKA), glycogen synthase kinase-3β (GSK-3β), and casein kinase 1 (CK1), and it requires the activity of the proteasome. Inhibition of Ci processing via activation of Smoothened (SMO) requires functional Cos2 and Fu kinase activity. The full-length Ci protein can be inhibited by Su(fu).

release of Ci from the Cos2 complex (39–42). Cos2 forms a close association with Fu, a kinase that phosphorylates Cos2 and is required for Hedgehog signaling, potentially influencing the interaction of the Ci/Cos2/Fu complex and microtubules (31,43). Su(fu) was identified in genetic screens in Drosophila as a suppressor of mutants in Fu (44). Su(fu) is a negative regulator of Hedgehog signaling and acts either by sequestration of Ci in the cytoplasm or by direct interaction with Gli proteins on DNA (43). After the release of transcriptional repression, Hedgehog target genes are transcribed. Hedgehog target genes include Gli-1, HNF3β, Hox genes, bone morphogenetic proteins 2 and 4 (BMP2 and BMP4), and Patched- and Hedgehoginteracting protein (HIP, an extracellular hedgehog antagonist). Of these genes, the up-regulation of Patched and HIP seem to be involved in a negative feedback response to Hedgehog signaling, whereas transcription factors such as HNF3β and Hox genes are implicated in translating the extracellular morphogenetic signal to intracellular phenotypic interpretation. Studies that used microarray techniques have now identified a large number of possible Hedgehog target genes (45–48). Hedgehog target genes found in microarrays include molecules involved in angiogenesis, cell cycle, cell adhesion, signal transduction, apoptosis, and nerve formation; transcriptional regulators; Wnt signaling antagonists; protease inhibitors; and a metal-binding protein. The actual in vivo relevance of these genes remains largely unclear and awaits functional studies for further validation.

Cells from the endoderm will form the epithelial layer; mesodermal cells differentiate into mesenchymal cell types such as myocytes and fibroblasts, whereas neural cells originate from the ectoderm. The gut tube acquires regional specialization along the longitudinal axis of the developing embryo. The fate of cells that form this tube is restricted to become part of one of the roughly four specialized tissue units of the esophagus, stomach, small intestine, and colon. Other small groups of cells bud from the original tube to form the thymus, lungs, liver, and pancreas. A second axis of specialization along which the different issue layers of muscle, submucosa, and epithelium develop is the crosssectional or vertical axis. The constant renewal of epithelial cells in the adult occurs along this vertical axis. Hedgehog signaling plays a role in the specification of cellular fate along both the longitudinal and the vertical axes, and its regulation is involved in budding of gut tube–derived organs. The endodermal cell layer expresses hedgehog family members SHH and IHH. This section discusses our current understanding of the role of Hedgehog signaling in GI development in three subsections that cover the three segments of the gut and its derivatives: foregut (esophagus, trachea, lungs, thymus, stomach, liver, and pancreas), midgut (small intestine), and hindgut (colon and anus).

Hedgehog Signaling and Foregut Development Esophagus and Trachea

ROLE OF HEDGEHOG SIGNALING IN THE DEVELOPMENT OF THE GUT Development of the Gastrointestinal Tube The GI tract develops from a simple tubular structure composed of cells derived from all three germ layers.

After formation of the foregut primordium, the trachea buds from the ventral wall of the foregut (49,50). In the mouse foregut, Shh mRNA expression is initiated at 9.5 days postcoitum (dpc) in the endoderm of the foregut primordium and will extend along the ventral wall of the developing foregut toward the midgut (7,51). Expression of Shh in the murine esophagus has been described until at least 15.5 dpc (12,51).

276 / CHAPTER 9 At some later point in development, expression of Shh is down-regulated in the esophagus, because no Shh expression is found at 18.5 dpc (52) or in the adult esophagus (53). In the elongating trachea, Shh expression initially is lost at 11.5 dpc, but then it reappears around 15.5 dpc (51). In contrast, Shh is not expressed by the cells of the thyroid, which form as a primordium at 9 dpc, is completely dissociated from the gut tube by 11.5 dpc, and descends to its final position until 15 dpc (54). Ihh is not expressed at any point in foregut development; this is confirmed by that Shh and Gli mutant mice show similar foregut phenotypes (see later). The role of Shh signaling in foregut development is revealed in Hedgehog pathway mutant mice. In Shh−/− mice, the formation of the tracheal bud is delayed and the lungs become hypoplastic. The normal elongation of the trachea fits well with that no Shh expression is found during this phase. The hypoplastic lungs suggest a role for Shh signaling in branching morphogenesis. The esophagus fails to develop normally; only a small esophageal remnant is found in Shh−/− mice at later stages of development. Because Shh has been shown to signal mainly to the mesenchyme during development and seems to act as a growth factor for mesenchymal cells (55), it is possible that the esophagus degenerates because of a lack of mesenchymal cell growth to support its epithelium. The stomach seems to connect to the respiratory tract in Shh null mice (51). The importance of Hedgehog signaling in foregut development was confirmed in Gli mutant mice (56). Although Gli3−/− mice have a normal trachea and esophagus, in the Gli2−/− mutant mouse, the esophagus has a small lumen, has little mesenchymal cells, and fails to develop a small muscle layer. The lungs in the Gli2−/− mutant mouse are hypoplastic and have only one right lobe instead of the four lobes that are normally found. Although the trachea elongates normally in the Gli2 mutant mouse, it does appear hypoplastic at 18.5 dpc, suggesting that the induction of Shh expression in the trachea at 15.5 dpc (51) is involved in its growth along the vertical axis. The Gli2−/−Gli3−/− mouse has a phenotype that is more severe than the Gli2−/− mouse and seems identical to the Shh null mutant. Therefore, although Gli3 is not necessary for normal foregut development, it can rescue part of the phenotype in Gli2−/− mice. Interestingly, the Gli2−/−Gli3−/− phenotype is more severe than the Shh null phenotype, demonstrating no development of lung, trachea, or esophagus at all. This suggests that Gli signaling in foregut development is not entirely Shh dependent (56). This discrepancy between the Shh and Gli2−/−Gli3−/− null phenotype can be explained by the finding that Fgf signaling may be capable of activating Gli transcription factors (57). Some effectors of Hedgehog signaling have been found by their similarity of their null phenotype to the Hedgehog pathway mutant phenotypes. The forkhead transcription factor Foxf1 seems to play a role as a mesenchymal effector downstream of Gli signaling. Foxf1 is expressed in the splanchnic mesoderm. Foxf1+/− mutant mice display a phenotype comparable to Shh mutant mice with esophageal atresia

and tracheoesophageal fistula. It was further shown that ectopic Shh expression activates Foxf1 expression whereas Foxf1 expression is lost in Shh−/− mice (58). Together these data imply that Foxf1 is a crucial mesodermal mediator of the endodermal Hedgehog signal in the normal tracheoesophageal bifurcation and elongation (59). The homeodomain transcription factor Nkx2.1 (thyroidspecific enhancer-binding protein [TTF1] or thyroid transcription factor-1 [T/EBP]) also plays a role in tracheoesophageal bifurcation. In the mouse, Nkx2.1 is expressed in the endoderm of the tracheal diverticulum and endoderm of the thyroid, trachea, and lung at later stages of development, whereas no Nkx2.1 is detected in the esophagus or the rest of the GI tract (59). In this mouse, however, the Nkx2.1−/− tracheoesophageal pathology seems different from that observed in the mice defective in Hedgehog signaling. The Nkx2.1−/− mouse has a single tube that connects the pharynx to both the lungs and the stomach. The pathology in this mouse seems to arise from a failure of the tracheal diverticulum to elongate (see Fig. 1B of Minoo and colleagues [59]). This is consistent with the selective expression of Nkx2.1 in the trachea and lung, but not with the formation of a fistula between airway structures and the esophagus or stomach as in the Shh−/−, Gli2−/−Gli3+/−, and Foxf1−/− mice. It is therefore unclear whether this mouse is truly a model of tracheoesophageal fistula as Minoo and colleagues suggested (59). In conclusion, Hedgehog signaling seems to play an essential role in the normal separation of trachea and esophagus, the maintenance and growth of the esophagus, and the growth of the lungs. It seems to play this role through regulation of mesenchymal growth through induction of Foxf1. Thyroid Although the thyroid primordium is correctly specified at 9 dpc in the absence of Hedgehog signaling (56), careful examination of the thyroid phenotype in the Shh null mouse does reveal a role for Shh signaling at later stages of thyroid development (54). The thyroid primordium buds in the foregut endoderm and becomes completely separated at 11.5 dpc. It descends between 10 and 15 dpc to its final position after which it separates into two lobes and organizes into a follicular structure, then genes involved in thyroid hormone production are induced (60). In Shh−/− mice, the thyroid primordium forms, but separation from the foregut is delayed, because it remains connected through a narrow stalk initially. The thyroid does not separate into two distinct lobes but becomes a single unilateral mass, the size of a single thyroid lobe in control mice (54). The single thyroid gland of Shh null mice formed normal follicles that contained apparently normally differentiating thyrocytes. The observed thyroid phenotype may be indirect and secondary to Hedgehog signaling in an adjacent tissue because no Shh or Ptc expression was detected in the thyroid at any point of development. Interestingly, small foci of

HEDGEHOG SIGNALING IN GASTROINTESTINAL MORPHOGENESIS AND MORPHOSTASIS / 277 ectopic thyroid tissue were found in the tracheal tube at 15.5 to 17.5 dpc in the Shh−/− mouse (54), suggesting that the induction of Shh in the trachea not only plays a role in tracheal growth along the vertical axis (see later) but also acts to suppress thyroid cell fate in the trachea. Stomach During development of the stomach, the gastric epithelium forms tubular structures that invaginate into the underlying mesoderm. These tubules contain two cell populations. The superficial or pit cells are similar throughout the stomach and produce soluble mucins that contain a protein backbone encoded by the MUC5AC gene. The cells that form the bottom of the tubules form the glandular region and have both exocrine and endocrine functions. In the adult, both cell populations originate from a common stem cell compartment between the pit and the gland region from where daughter cells migrate in two directions. The gastric glands of the proximal (fundus) and distal (antrum) stomach are functionally distinct. Both Shh and Ihh are expressed in the developing stomach in mice (12). At 12.5 dpc, Shh is expressed in the proximal but not the distal stomach (61). High expression of both Shh and Ihh is found in glandular epithelium of the stomach at 18.5 dpc (52). The epithelium of Shh−/− mice (but not that of the Ihh mutant) is hyperplastic, and patches of gastric epithelium are positive for intestinal brush border markers alkaline phosphatase and wisteria floribunda agglutinin (WFA) lectin (52). These data suggest that Shh signaling is necessary to induce a gastric phenotype in endodermal cells in the stomach. The chick has a proximal glandular stomach (proventriculus) and adjacent muscular stomach (gizzard). In the chick, proventriculus Shh is initially expressed uniformly throughout the endoderm (62). During proventricular unit formation, the gastric units start to form a superficial pit region and a glandular region. No Shh is expressed in the glandular region at this point. When Shh is ectopically expressed in developing gastric units in a proventricular explant model, gastric units fail to develop (62). These data may suggest that gastric unit formation has some similarities with the development of endodermal appendages such as the thymus and pancreas. It is unclear, however, if this downregulation of Shh during gastric unit formation also occurs in the mouse or human. Both Shh and Ihh are uniformly expressed throughout the developing gastric units in mice at 18.5 dpc (52). However, maturation of gastric glands in mice occurs after birth, and restriction of Hedgehog signaling may therefore occur at a later point in gastric unit maturation. In the fully developed stomach, Shh is expressed exclusively in gastric glands in both mice and humans (see later) (53). In other experiments using a chick stomach explant model, it was shown that treatment inhibition of Hedgehog signaling with cyclopamine disturbed endodermal differentiation in the gizzard (63). In the absence of hedgehog signaling, the differentiating epithelium failed to stratify and contained

nonpolarized cells. The mesenchyme of cyclopamine-treated animals contained multiple vascular structures (something that was also observed in the thyroid of Shh−/− mice [54]). The endoderm of both in vitro and cyclopamine-treated chick stomachs expressed both glucagon and insulin, suggesting that the epithelium differentiates toward a pancreatic cell fate. Chick embryos treated with cyclopamine in ovo also displayed pancreatic differentiation of the gizzard endoderm (63). These data suggest that Shh is necessary to suppress a pancreatic cell fate in chick gizzard endoderm. In conclusion, Shh seems to act to suppress more distal cell fates in gastric endoderm (small intestinal in the mouse and pancreatic in the chick), and data in the chick suggest that a temporary loss of Shh expression in the developing gastric unit may play a role in budding of units from the endodermal layer. Pancreas The pancreas is an endodermal-derived gland with both endocrine and exocrine functions. The bulk of pancreatic tissue (approximately 95–99%) is composed of exocrine acinar cells that produce digestive enzymes and pancreatic ducts that are lined by duct cells. The rest of the pancreas consists of small clusters of endocrine cells or Islets of Langerhans. Each cluster of endocrine cells has a core of insulin-producing β cells surrounded by glucagon-producing a cells, somatostatin-producing D cells, and pancreaticpolypeptide cells. The development of the pancreas is probably the best studied aspect of endodermal differentiation, and many of the intrinsic and extrinsic determinants have been described (64,65). The pancreas develops from two primordia that originate at the ventral and dorsal side of the gut tube. In the mouse, the pancreatic anlage is specified around 8.5 dpc. Pancreatic morphogenesis initiates at 9.5 dpc with the formation of the dorsal pancreatic bud. At this time, the first endocrine cells are also detectable (64). At 10.5 dpc, Shh is expressed uniformly throughout the endoderm of the gut tube with the exception of pancreatic precursor cells that are distinguished by their Pdx-1 positivity (66). Although no Shh is expressed in the developing or adult pancreas, low levels of Ihh and Ptc mRNA are found with reverse transcriptase-polymerase chain reaction in the murine pancreas throughout development and in the adult (61). When a Pdx-1 promoter was used to drive ectopic Shh expression in the pancreatic buds, development of the pancreatic mesoderm was strongly disturbed. The mesenchyme contained α-actin–positive smooth muscle cells and c-kit–positive cells of Cajal. Both cell types are normally found in intestinal mesenchyme. The pancreatic buds in transgenic animals did contain endocrine and acinar cells, but these failed to cluster into islets and acinar structures and produced mucins that are typical for intestinal epithelial cells (66). These data suggest that during early pancreatic development, suppression of Shh signaling is especially important for normal development of the pancreatic mesoderm,

278 / CHAPTER 9 and that although the pancreas contains both endocrine and acinar cells, their differentiation is not entirely normal. Data from Xenopus further support this concept (67). Xenopus injected with a constitutive active form of the Hh receptor Smo completely lack a pancreas, a phenotype more severe than that of the Pdx-1-Shh mouse. This may be because their transgenic mouse expressed Shh behind a pancreas-specific promoter and the ectopic Shh transcription therefore starts after the initial specification of a pancreatic domain has already been completed. It has been suggested that Hedgehog signaling may act to restrict pancreas growth (61). Shh−/− mice have a body weight of approximately 30% of wild-type embryos at 18.5 dpc, but their pancreas weight is similar to that in wild-type mice. The Ihh−/− mouse has a body weight of approximately 65% of wild-type mice at this stage of development and its pancreas is similarly 65% of the weight of the wild-type pancreas. It is difficult to infer from these observations if, for example, Shh affects body size but not pancreas size or if Shh restricts pancreas growth as suggested (61). Almost half of Ihh−/− mice develop an annular pancreas in which the pancreas encircles the duodenum. Histologically, the pancreas appears intact and contains normal islets. Cellular differentiation seems to be more or less normal in both Shh−/− and Ihh−/− mice. Only if the number of endocrine cells is expressed relative to body weight would there appear to be a relative increase in the number of endocrine cells in the pancreas, especially in the Shh−/− mice (61). The number of endocrine cells/pancreas weight seems a more logical measure, however, and this seems only modestly increased in Shh−/− mice (22% increase), whereas the number of endocrine cells/pancreas weight is increased by 75% in Ihh−/− mice (61). Because Ihh and not Shh is expressed in the developing pancreas, it seems that Ihh may act as a negative regulator of endocrine cell fate in the developing pancreas.

Hedgehog Signaling and Development of the Midgut The midgut will form most of the intestine distal from the common bile duct that derives its blood supply from the superior mesenteric artery: the distal duodenum, the jejunum, ileum, cecum, and the ascending and proximal transverse colon. The intestinal endoderm initially is pseudostratified and is converted into an epithelial monolayer in a proximalto-distal morphologic wave that starts at 15 dpc. In contrast with human development, morphogenesis of the murine intestinal epithelium is not complete until 3 weeks after birth when crypts have developed from intervillous epithelial cells (68). Ihh and Shh are both expressed in the developing midgut, and therefore have partially overlapping functions. They regulate endodermal–mesodermal interactions in the developing small intestine because both are produced by endodermal cells and the Hedgehog target gene and receptor Ptc is strongly expressed in the surrounding mesoderm (52,69). Experiments in the chick gut have shown that during early gut development, Hedgehog signaling acts to recruit mesoderm.

This induction of mesodermal proliferation may be limited by that Shh induces BMP4 expression in the mesoderm (70,71). Ihh is expressed throughout the midgut at 11.5 dpc (12). Ihh expression is maintained along the length of the midgut until at least at 18.5 dpc when Ihh is expressed at the base of the villi in the small intestine (52). Initially, Shh is expressed uniformly throughout the endoderm; however, similar to the expression of Ihh, this expression is restricted to the base of the villi of the proximal midgut around 14.5 dpc (12,52). What underlies the down-regulation of Shh in the intestine at 14.5 dpc remains to be determined. It seems to follow a restrictive influence exerted by the mesoderm around 13.5 dpc with down-regulation of Foxa2 (HNF3β) and GATA factors in the endodermal layer (72). Interestingly, the intestinal endoderm is converted to a monolayer at 15 dpc, and this event seems to follow these molecular changes that are orchestrated by the underlying mesenchyme. Indeed, experiments in Xenopus with constitutively active Smo (SmoM2) suggest that down-regulation of Shh expression may not only be essential for the appropriate differentiation of pancreatic mesenchyme, but also important in the midgut. After injection of SmoM2 mRNA into one to eight cell-stage Xenopus embryos, intestinal epithelial differentiation was completely disrupted specifically in the midgut (67). The interpretation of these experiments is difficult, however, because they involved injection of SmoM2 mRNA that is not regulated by a cell type–specific promoter; this will also induce Hedgehog signaling in the endoderm where it does not normally occur in intestinal development. Shh and Ihh mutant mice displayed different phenotypes. Whereas the duodenum of Shh−/− mice was overgrown with villi, villus size was reduced in the Ihh−/− mouse and intestinal precursor cell proliferation also was reduced (52). Both Ihh and Shh null mice show a reduction in the thickness of the smooth muscle layer, indicating that Hedgehog signaling stimulates smooth muscle cell differentiation or proliferation (52).

Hedgehog Signaling and Development of the Hindgut The hindgut forms the distal transverse colon, descending colon, sigmoid, and anorectum that develop around the inferior mesentery artery. Low levels of Shh are expressed at the base of the crypts in the colon at 18.5 dpc (52), whereas Ihh mRNA is expressed throughout the epithelial layer from 11.5 dpc until birth (12,52). In Ihh−/− mice, the epithelial cells fail to organize into a polarized monolayer and the epithelium does not organize into cryptlike structures (73). The colon is partially dilated in Ihh−/− mice with an abnormally thin wall and lack of neurons in the dilated areas, a phenotype reminiscent of Hirschsprung’s disease (52). These data suggest that signaling by Ihh plays a role in the conversion of the pseudostratified endodermal layer to an epithelial monolayer in the hindgut and regulates the survival of neural crest cells that have migrated into the intestinal mesoderm.

HEDGEHOG SIGNALING IN GASTROINTESTINAL MORPHOGENESIS AND MORPHOSTASIS / 279 Signaling by Shh is important in the distal hindgut because Shh null mice were shown to have anorectal malformations (52,74). The anorectum develops with a complex interplay of all three germ layers as it forms at the junction of the endoderm and ectoderm (71,75). Ramalho-Santos and colleagues (52) found an imperforate anus in their Shh−/− mice: the colon ends in a blind sack and does not form a connection with the perineum. Shh−/− mice maintained by Mo and colleagues (74) display a failure to separate the anorectum from the lower urinary tract, which drain in a common cavity or outlet. Gli2−/−, Gli3+/−, and Gli2+/− Gli3−/− compound mutant mice also have a persistent cloaca and a common outlet for the digestive and urogenital tract but a phenotype that is less severe than Shh−/− mice. Gli2−/− mice have an imperforate anus with a rectovesical fistula (between the distal intestine and the bladder) and a single urethral opening in the perineum (see also the study by Kim and coworkers [76]). The phenotype of Gli3−/− mice is mild with slight anal stenosis (74). These phenotypes indicate that, although Gli3 does play a role in the transduction of the Shh signal to the anorectal mesenchyme, the contribution of Gli2 is much more important and is similar to the role of Gli signaling in foregut development.

Hedgehog Signaling and the VACTERL Association in Humans All of the developmental abnormalities observed in the Hedgehog pathway mutant mice also are found in humans. In humans, the nonrandom association of these abnormalities is denoted by the acronym VACTERL. VACTERL stands for vertebral defects (V), anal atresia (A), cardiac defects (C), tracheoesophageal fistula, renal anomalies (R), and limb defects (L) (77–80). This association is not recognized as a specific syndrome, and its different components are found together in varying constellations (see Rittler and colleagues [80]) and have been assigned various acronyms (such as VATER, VATERL, among others). One study that examined 2,493,999 births reported only a single case with combined recognized deformities in all 6 organs (80). However, all of these defects have been found by many observers in one or more of the several known human and murine mutants of the Hedgehog pathway (51,52,56,64,74,81–83; see also the review by Kim and colleagues [76]). The atresia or stenosis of the esophagus with associated tracheoesophageal fistula that was found in hedgehog mutant mice is observed in approximately 1 in 2000 to 1 in 5000 live human births (77). Just like in the Hedgehog mutants, this anomaly can be found in association with other GI malformations in humans such as anal atresia, midgut malrotation, duodenal atresia, and annular pancreas (84), and with malformations of other organs as part of the VACTERL association mentioned above. Although the similarity between malformations found in the VACTERL association and Hedgehog mutant mice suggests a role for defective Hedgehog signaling, in patients

with the VACTERL association, such a role has not yet been established.

HEDGEHOG SIGNALING IN HOMEOSTASIS OF THE ADULT GASTROINTESTINAL TRACT Morphostasis The molecules that are involved in the regulation of position-dependent cell fate have been found in genetic screens that used developmental models. However, positional information is something that is equally important to many cell types in the adult organism. Although adult tissues seem to be static and stable, many tissues are not, because they have a high cellular turnover rate and their microarchitecture can be disturbed, for example, in chronic inflammation. The GI tract is a good example of the importance of positional information in cellular renewal in the adult. At each level of the gut a common precursor cell produces a module of specialized cell types appropriate for its position along the anterior-posterior axis. These modules display elaborate patterns of differentiation along the radial axis. At some parts of the GI tract precursor cells generate two functionally different compartments of cells that migrate in opposite directions (e.g., the stomach). Even among cells that migrate in the same direction, opposite gradients of maturation can be observed for different cell types. Although stability of the resulting complex epithelial microarchitecture is the norm, instability often is observed in humans. This instability leads, for example, to the formation of metaplasia, atrophy, or hyperplasia such as in polyposis. Often, these unstable epithelial states are at a high risk to progress to GI cancer. GI epithelial cells undergo a well-controlled maturation process. Precursor cells generate descendants that withdraw from the cell cycle and induce transcription of cell lineage– specific proteins. When the cell is fully differentiated it is shed into the gut lumen or undergoes apoptosis to maintain cellular census. This is a rapid process that is dependent on the cell’s position along the axis of renewal. It was shown in an important experiment by Hermiston and colleagues (85) that such processes are regulated in a cell nonautonomous manner, and thus are controlled by extrinsic regulators of cell fate. The authors performed an elegant experiment in chimeric mice in which some of the intestinal epithelial cells overexpressed the cell–cell adhesion protein E-cadherin (85). In these mice, the descendants of the transgenic precursor cells migrated considerably slower along the axis of renewal than the progeny of normal precursor cells present in the same chimeric animal. Despite this difference in the speed of migration, differentiation and cell death of the epithelial cells occurred at the appropriate moment along the vertical axis and homeostasis was maintained. This experiment demonstrated that differentiation of the epithelial cell is completely dependent on its position along the vertical axis of renewal, and therefore is extrinsically regulated.

280 / CHAPTER 9

Villus

Pit Precursor cell

Crypt

Shh

Parietal cell Gland

Precursor cell

Shh

FIG. 9-7. SHH signaling in the adult stomach and small intestine. (A) SHH is expressed in the fundic glands of the stomach where it may act as a polarizing factor. Gastric epithelial cells migrate in two directions from the precursor cell compartment. Cells that migrate up form the gastric pit, whereas those cells that migrate down form the gastric gland. In humans, Shh is produced by parietal cells in the gastric gland. Gland but not pit cells express PTCH, suggesting that Shh signaling acts on gland cells only. (B) SHH is produced by a few cells at the base of the small intestinal crypt and may be involved in the regulation of precursor cell proliferation and triglyceride absorption.

Evidence also exists that the proliferative compartment of precursor cells receives negative feedback information from cells in the differentiated compartment (86). Through such negative regulation the amount of differentiated cells can regulate the pace of their own replacement. If differentiated cells in the superficial epithelium are lost by damage, such a mechanism allows for increased proliferation to guard epithelial integrity. The mediator(s) of this negative feedback loop have not been identified to date but are clearly soluble molecules capable of long-range signaling. The identification of the extrinsic controls of cell fate is fundamental to the understanding of the regulation epithelial homeostasis and its deregulation. Work from several laboratories has shown that morphogens seem to play an important role as extrinsic regulators of cell fate in the adult, and this section reviews what is known about the role of Hedgehog signaling in the adult GI tract.

zymogenic cells in both mice and rats (53,87). IHH is expressed in the colonic epithelium where it is produced by terminally differentiated enterocytes (73). During development of the GI tract Hedgehog signaling occurs mostly from the endodermal layer to the mesodermal layer, with the exception of the stomach where Hedgehog receptor and target gene Ptc is also expressed in the differentiating epithelium at later stages of development in the mouse (12,52,69). In contrast, in the adult epithelium of both humans and rodents, Hedgehog signaling seems to be mostly autocrine within the epithelium because the greatest levels of PTCH/Ptc are found in the epithelium (73,87). Intercrypt table

Ihh

Role of Ihh and Shh in Adult Gut Crypt

The expression patterns of IHH, SHH, and Hedgehog receptor patched (PTCH) in humans suggest that Hedgehog signaling occurs throughout the human adult GI tract, with the exception of the squamous epithelium of the esophagus (53,87,88). Both SHH and IHH are expressed in the adult GI tract. The expression pattern of SHH has been examined most carefully; SHH mRNA is expressed at high levels in the glands of the fundic stomach. Low levels of SHH mRNA are found in epithelial cells at the base of the crypts of the small intestine and colon (Figs. 9-7 and 9-8). SHH protein can be detected in the fundic glands where it is expressed by parietal cells in humans and by mucous neck cells and

Precursor cell

Shh

FIG. 9-8. Hedgehog signaling and the colonic crypt. Similar to the crypts of the small intestine, low levels of Shh are expressed around the precursor cells at the base of the colonic crypt. High levels of Ihh, however, are produced by differentiated colonic enterocytes at the luminal end of the crypt and at the intercrypt tables. Hedgehog receptor and target gene PTCH is also expressed by the differentiated enterocytes, indicating that these cells are the major target for hedgehog signaling in the colon.

HEDGEHOG SIGNALING IN GASTROINTESTINAL MORPHOGENESIS AND MORPHOSTASIS / 281 Shh and Gastric Epithelial Homeostasis Renewal of gastric epithelial cells is a bidirectional process. The gastric mucosa is a flat surface that contains multiple invaginations or gastric units. Gastric precursor cells are located somewhere approximately halfway up these units. Cells that migrate toward the luminal surface secrete the protective soluble mucin MUC5AC and form the gastric pit (the upper part of the unit) (89). Cells that migrate down form the gastric gland and produce a variety of exocrine and endocrine factors involved in defense against bacterial invasion, digestion of food, and regulation of metabolism. In this homeostatic system, factors have to control asymmetric cellular differentiation where the appropriate amount of cells adopts either a pit or gland cell fate. The presence of such factors that act as polarizing signals in the gastric units is clear from histopathologic observations in patients with hypertrophic gastropathies. These gastropathies include Ménétrier disease and hyperplastic hypersecretory gastropathy (HHG) (90). In Ménétrier disease, transforming growth factor-α, a ligand for the epidermal growth factor receptor, is overexpressed, leading to excess proliferation and differentiation of pit cells and loss of glandular epithelial cells (91–93). In contrast, the hypertrophy in patients with HHG is characterized by an increased mass of gland cells. We have shown that Shh expression is restricted to one side of the gastric precursor cell region. In humans, Shh expression forms a gradient in gastric glands as it is highest in parietal cells close to the precursor cell compartment and gradually diminishes to low levels as parietal cells migrate to the bottom of the fundic gland (87). We have therefore proposed that SHH may act as a polarizing factor in the adult gastric unit, in analogy with its role as a polarizing factor in the developing limb bud. We have, indeed, been able to show that Hedgehog signaling controls proliferating cell nuclear antigen (PCNA) expression in gland cells and not pit cells, and it regulates the expression of gland cell–specific transcription factors (87). The gastric epithelium of Shh null mice shows intestinal transformation (52), and loss of gastric gland cell differentiation in patients with intestinal metaplasia of the fundic epithelium is characterized by loss of SHH expression, indicating that SHH seems to be essential in the induction and maintenance of a fundic gland cell phenotype (53,94). In vitro experiments in a gastric cancer cell line that expresses SHH suggest that SHH expression may be pH dependent and is stimulated by low pH (94). Interestingly, treatment of newborn rabbits with retinol increases the number of parietal cells (95). Retinoids have been shown to act through their control of Shh expression in the developing limb bud (96), thus the control of parietal cell differentiation by retinoids may well be Shh dependent. A role for Shh as a fundic gland cell type–promoting factor is further supported by mice deficient for α-Inhibin (97). α-Inhibin−/− mice acquire gonadal sex-cord-stromal tumors producing large quantities of Activin A and B. The fundic glands of these mice show increased amounts of pit cells, loss of parietal cells, and absence of zymogenic cells. It has been

shown in developmental models that Activin acts to inhibit Shh signaling (98), and that loss of activin signaling in the foregut leads to expansion of both Shh expression and gastric epithelial differentiation (99). Together, these data suggest that SHH may act as a central regulator of gastric gland cell fate that is stimulated by retinoids and inhibited by Activins.

Shh and the Small Intestinal Crypt In the adult small intestine, SHH is expressed by a few cells at the base of the small intestinal crypt (53). Levels of Shh protein diminish in a gradient from the duodenum to the ileum in 2-week-old mice (100). As judged by the expression of Ptc, Hedgehog signaling targets both epithelial cells at the base of the small intestinal crypt and mesenchymal cells (88). A small reduction of precursor cell proliferation was observed in small intestinal crypts of cyclopaminetreated adult mice, suggesting that Shh stimulates precursor cell proliferation (87). Wang and colleagues (100) observed increased proliferation of small intestinal precursor cells in anti-Hh–treated postnatal mice. In their experiment, mice were treated from postnatal day 1 to 15, when morphogenesis of the small intestinal crypts occurs, polyclonal crypts are “purified” to monoclonality, and crypts multiply by a high rate of crypt fission (68). Hedgehog signaling thus seems to play different role during crypt morphogenesis and in the fully mature intestinal crypt. These authors also treated adult mice with an anti-Hh antibody and observed disturbed triglyceride absorption in Hedgehog-inhibited mice (101). In conclusion, the limited data available about the role of Shh signaling in the small intestinal crypt suggest a role in precursor cell proliferation and the regulation of triglyceride absorption.

Ihh and Colonic Epithelial Differentiation Epithelial renewal in the colon is a unidirectional process from a precursor cell compartment at the base of the crypts toward the lumen where cells reach the flat surface (intercrypt tables) and undergo a death program. The colonic crypt contains three different functional cell types. A gradient of differentiating absorptive enterocytes is found from the crypt base toward the lumen of the gut, whereas most mucusproducing goblet cells are found in the middle of the crypt and the endocrine cells form a gradient toward the base of the crypt (102). Both IHH mRNA and protein are expressed by terminally differentiated enterocytes in the adult colon (73). Low levels of SHH mRNA can be detected at the base of colonic crypts, and SHH protein is difficult to detect but is probably expressed by a few cells at the base of the colonic crypt (53,88). Most Hedgehog signaling, however, occurs in the differentiated enterocytes as they express the highest level of PTCH protein and mRNA (73,88). Oniscu and colleagues (103) found SHH

282 / CHAPTER 9 expression in differentiated enterocytes using immunohistochemistry; however, because these cells do not express SHH mRNA but do express IHH mRNA, these results are most likely explained by cross-reactivity of the SHH antibody with IHH protein. The role of Hedgehog signaling in colonic enterocytes was addressed by treating rats with the Hedgehog inhibitor cyclopamine, a veratrum alkaloid that binds and inhibits Smo (104–106). Histologic examination of the colonic epithelium of these rats showed a remarkable change in the appearance of the colonic enterocytes, with altered distribution of the cytoskeletal protein Villin that is specific for microvilli and loss of expression of the brush border enzyme carbonic anhydrase IV. Interestingly, the enterocytes gained the expression of intestinal trefoil factor, a marker of the goblet cell lineage. In vitro experiments in the HT-29 cell differentiation model showed that butyrate-dependent HT-29 cell differentiation is dependent on induction of IHH expression. These data suggest that Hedgehog signaling is essential to the appropriate specification of the enterocyte cell lineage and acts to suppress a goblet cell phenotype in these cells (73). A possible mode of action for Ihh was suggested by that the expression of Wnt target genes Engrailed-1 and BMP4 was increased in the colon of cyclopamine-treated rats, and that the expression of both proteins that is normally restricted to the base of the colonic crypt extended up toward the differentiated enterocytes. These results suggested that IHH might act to suppress WNT signaling as a differentiating enterocyte moves from the precursor cell compartment in the crypt toward the differentiated compartment at the luminal end of the axis of renewal. Additional evidence for a role for IHH as a suppressor of WNT signaling was obtained in DLD1 cells in vitro (73). The importance of inhibition of WNT signaling in enterocyte differentiation lies in that WNT signaling seems to act to specify a precursor cell phenotype in colonic enterocytes (107). We found that expression of IHH is in its turn suppressed by WNT signaling, and because WNT signaling is aberrantly activated in most colon cancer cells, these cells have lost their expression of IHH, and therefore have lost their autocrine Hedgehog signaling. These data correlate well with findings by Berman and colleagues (108), who showed that growth of most carcinoma cells derived from the esophageal, gastric, biliary tract, and pancreas was dependent on Hedgehog signaling, but that this was not the case for colon carcinoma cells. Colon carcinoma cells did not display any endogenous activity of the Hedgehog pathway, nor was their viability affected by cyclopamine, an observation that correlates well with our own unpublished observations. Qualtrough and colleagues (109) have found that cyclopamine treatment resulted in detachment of cultured colon cancer cells; this detachment was not observed in our own studies or those of Berman and colleagues (108), and further experiments are necessary to show if this is caused by methodologic differences. Oniscu and coworkers (103) found that recombinant Hedgehog protein stimulates the proliferation of isolated primary murine colonic enterocytes. Because differentiating enterocytes do not proliferate, this

study examined the effect of Hedgehog signaling on intestinal precursor cells. Because low levels of SHH are found in the precursor cell area, SHH may act to stimulate precursor cell proliferation. In conclusion, low levels of SHH in the precursor cell area of the colonic crypt may act to control precursor cell phenotype or proliferation. The major target for Hedgehog signaling, however, is the differentiating colonic enterocyte. These cells produce IHH that seems to act in an autocrine fashion to suppress WNT signaling and stimulate differentiation.

HEDGEHOG SIGNALING AND CARCINOGENESIS OF THE GASTROINTESTINAL TRACT It is becoming more and more evident that disturbance of cellular interpretation of extrinsic information is a major step in the initiation of GI malignancies. Several morphogenetic pathways have been implicated, for example, in the development of colorectal cancer. First, mutations in the WNT pathway were identified as the cause of the familial adenomatous polyposis (FAP) syndrome and most sporadic colonic adenomas (38). Then hamartomatous polyposis syndromes such as juvenile polyposis were linked to mutations in the BMP receptor and the related intracellular signal transducer SMAD4 (110). Disturbed Hedgehog signaling has already been shown to lead to basal cell carcinoma, medulloblastoma, and rhabdomyosarcoma (111). Two independent groups have found a role for Hedgehog signaling in GI tract– derived malignancies. One showed that most cell lines derived from esophageal, gastric, biliary, and pancreatic cancers express both SHH and IHH mRNA and PTCH mRNA, indicating that autocrine Hedgehog signaling occurs in those cells. This was indeed confirmed by transfection of Hedgehog reporter constructs, which showed endogenous Hedgehog signaling that was inhibited by cyclopamine. Treatment with cyclopamine also greatly reduced the viability of these cell types, suggesting that Hedgehog signaling maintains a malignant phenotype in most GI cancer types (108). Another group made similar observations in the development of pancreatic cancer; the group showed that SHH is not normally expressed in ductal epithelium in the pancreas, but that expression of SHH is induced in pancreatic intraepithelial neoplasia in humans and seems to increase with the severity of the lesions (112). Mice with a Shh transgene behind a pancreas-specific promoter not only developed mesenchymal transformation to an intestinal phenotype, but also displayed an epithelial histology that resembled pancreatic intraepithelial neoplasia in human patients. The viability of 6 of 12 SMO-positive pancreatic cancer cell lines was shown to be responsive to cyclopamine, suggesting that cyclopamine nonresponsive cells either have Hedgehog activating mutations downstream of SMO or alternative growth in a Hedgehog-independent manner (112). No such role for Hedgehog signaling was found in the colon (108). Our own data suggest that this may be caused

HEDGEHOG SIGNALING IN GASTROINTESTINAL MORPHOGENESIS AND MORPHOSTASIS / 283 by the fact that the initiating mutation in most colonic adenomas is a mutation in the WNT pathway that would suppress the expression of IHH, and thus the activity of the Hedgehog pathway (73). Indeed, we found that expression of IHH is lost in HT-29 and DLD1 colon carcinoma cells in vitro and in sporadic adenomas and those in patients with FAP in vivo (73). Loss of IHH expression is an early event in patients with FAP and was found in histologically normal epithelium with an aberrant β-catenin expression pattern that is typical for APC mutant epithelial cells (73). Loss of IHH expression could be restored in DLD1 cells by blocking WNT signaling with an inducible dominant negative TCF4. The WNTdependent loss of IHH expression may play an important role in the loss of differentiation that is one of the defining hallmarks in the earliest stages of adenoma formation. Indeed, many of the phenotypic abnormalities that we observed in the colonic enterocytes of cyclopamine-treated rats such as redistribution of villin expression, loss of brush border enzyme expression, and induction of intestinal trefoil factor (ITF) expression are also observed during colorectal carcinogenesis (113,114). We have not examined what happens to Hedgehog signaling in the later stages of colorectal carcinogenesis when colon cancer cells progressively accumulate novel gene mutations and assume a more poorly differentiated phenotype. Data from Oniscu and colleagues (103) suggest that with the expansion of the precursor cell compartment in the later stages of colorectal cancer development, the zone of SHH-expressing cells increases as well. Thus, Hedgehog signaling seems to maintain a malignant phenotype in cancers that originate from foregutderived epithelial tissues such as the esophagus, stomach, bile duct, and pancreas. In the adenoma to carcinoma sequence that leads to colorectal cancer, secondary loss of IHH signaling caused by WNT pathway activating mutations seems to lead to loss of differentiation in maturing colonic enterocytes.

FUTURE DEVELOPMENTS The similarities between morphogenesis and morphostasis of GI epithelium will receive much more attention in the coming years. We hope that the further application of insights derived from a variety of developmental models will increase our understanding of the molecular mechanisms of homeostasis of GI epithelial tissues. In most cases, signaling modules that have been studied in developmental models will be found to be extremely similar in the adult GI tract. They have the same ligands, the same receptors, the same intracellular signal transduction molecules, and importantly, the same target genes. Therefore, a large body of experimental data from a variety of experimental developmental systems awaits application in the adult system. For Hedgehog signaling, the first steps have now been made. The finding that Hedgehog signaling may influence the survival of proximal GI tract–derived cancers and the availability of a variety of Hedgehog pathway inhibitors suggest

that the first experimental clinical data in oncologic patients will soon appear.

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CHAPTER

10

Developmental Signaling Networks: The Notch Pathway Guy R. Sander, Hanna Krysinska, and Barry C. Powell Overview of Notch Signaling, 287 Structure of the Notch Receptors, 288 Structure of the Notch Ligands, 289 The Delta Subfamily, 289 The Jagged Subfamily, 289 The Notch Signaling Pathway, 290 Functions of Different Notch Receptors and Ligands, 292 Modifiers of Notch Signaling, 293 Interactions of Notch with Other Signaling Networks, 294

Notch Signaling in the Gastrointestinal System, 295 Overview of Gastrointestinal Structure and Development, 295 Notch in the Developing Gut, 297 Notch in the Enteroendocrine System, 298 Notch in the Mucosal Immune System, 301 Notch in the Enteric Nervous System, 302 Future Directions, 302 References, 302

The Notch signaling pathway is fundamentally important in biology. In broad terms, it enables the formation of different cell types in a tissue. Notch is a receptor, and interaction of Notch with its ligands on adjacent cells initiates a cascade of biochemical actions, causing Notch to move from the membrane to the nucleus to alter the activity of gene expression networks through transcriptional regulation. Notch is expressed in all manner of cells in the gut, the epithelium, the enteric nervous system, the mucosal immune system, and the vascular network, yet with few exceptions, we know little about the specifics of Notch function in the gut. Much of what we know about Notch comes from its action in other tissues. This chapter describes the Notch signaling pathway and its role in development. Then what is known of Notch signaling in the gastrointestinal tract is discussed, focusing

on recent research that implicates a vital role for Notch in the development of the enteroendocrine system.

OVERVIEW OF NOTCH SIGNALING The name Notch originates from an observation made by Mohr (1), who noted the formation of notches at the wing margin of the fruit fly, Drosophila melanogaster, caused by partial loss of function of what was later named the Notch gene. The Notch locus was cloned in 1983 by Spyros Artavanis-Tsakonas (2), who has continued to pioneer the field. One of the distinguishing features of the Notch signaling pathway is that it is activated by cell–cell contact. The Notch receptors are large, cell-surface proteins that are activated by a group of cell-surface ligands often collectively referred to as the DSL ligands, after the founding members: Delta, Serrate, and Lag2. Both receptors and ligands are singlepass, transmembrane (TM) proteins. Activation of the receptor by ligand results in a proteolytic cleavage of the intracellular portion of the receptor, which is then transported to the nucleus, where it induces specific gene transcription. Notch signaling is important in a multitude of processes; that is, in growth, differentiation, proliferation, and patterning in a wide variety of tissues through its control of cell fate (3–6). From the earliest stage of development of an organism

G. R. Sander: Child Health Research Institute, North Adelaide, and Department of Pediatrics, University of Adelaide, South Australia 5006, Australia. H. Krysinska: Child Health Research Institute, North Adelaide, South Australia 5006, Australia. B. C. Powell: Child Health Research Institute, North Adelaide, and School of Pharmacy and Medical Sciences, University of South Australia; Department of Pediatrics, University of Adelaide, South Australia 5006, Australia. Physiology of the Gastrointestinal Tract, Fourth Edition, edited by Leonard R. Johnson. Academic Press, 2006.

287

288 / CHAPTER 10 there is a recurring need to regulate the production of different cell types and at the same time maintain the pluripotential capacity of the progenitor cell. Notch signaling is important in the specification of cell fate as groups of undifferentiated cells segregate into different cell lineages. As such, Notch signaling is not deterministic of a particular fate, but is instead permissive, enabling the choice of different fates. Depending on the cellular context, Notch signaling may result in diametrically different outcomes—inhibition or promotion of differentiation, proliferation or apoptosis.

STRUCTURE OF THE NOTCH RECEPTORS Notch proteins have been identified in a number of species. In general, invertebrates possess one or two Notch genes, whereas vertebrates have four. Much of the pioneering work has been performed in the fruit fly, D. melanogaster, which has only one Notch receptor and two ligands, Delta and Serrate (4). The sea urchin, Lytechinus variegatus (7), the invertebrate chordate, Amphioxus (8), and the frog, Xenopus, also have only one Notch gene (9). The worm, Caenorhabditis elegans (10,11), and the chicken have two Notch receptors (12), whereas, intriguingly, zebra fish, Danio rerio, have four receptors (13). In humans and mice there are four Notch receptors (4). The Notch genes are highly conserved and are thought to have arisen by duplication of a single ancestral gene. Notch receptors are heterodimeric TM proteins of about 300 kDa (Fig. 10-1). Both the extracellular ligand binding domain and the Notch intracellular domain (NICD) contain a number of conserved regions. Unlike other receptors, Notch receptors have no enzymatic activity of their own, but instead rely on a ligand-induced proteolytic cleavage to induce activity through release of the NICD. The NICD is translocated to the nucleus where it interacts with the CSL protein (CBF1 or RBP-Jκ in vertebrates, Su(H) in Drosophila, and Lag1 in C. elegans). This interaction converts CSL from a transcriptional repressor to an activator, leading to the activation of other transcription factors and gene networks (3,4). All four Notch receptors signal through CSL, the one nonredundant member of the Notch signaling pathway. The Notch extracellular domain contains multiple, tandemly arranged epidermal growth factor-like repeats (EGFR). The number of EGFRs varies between different Notch receptors; Notch1 and Notch2 have 36 repeats, Notch3 has 34, and Notch4 has 29 (14,15). Comparison of the amino acid sequences of the individual EGFRs shows that the homology between similarly positioned repeats in Notch receptors in different species is greater than the homology between EGFRs within the same Notch molecule (16). This linear conservation suggests that the tertiary structure of the EGFRs might be important for function of the receptor, perhaps conferring some degree of ligand specificity, enhancing or suppressing the interaction with ligands, or affecting the stability of the Notch protein. In Drosophila, EGFRs 11 and 12 have been shown to be necessary for ligand binding (17), but the

functions of the remaining EGFRs are not well understood. Modification of adjacent EGFRs, referred to as the Abruptex domain in Drosophila Notch (18,19), suggest that they mediate the interaction of adjacent receptors and ligands in the same cell membrane, an interaction that results in a reduced ability of the Notch receptor to be stimulated by a ligand on adjacent cells (20). Adjacent to the EGFRs are three repeats of a cysteine-rich region termed LNR (after the Notch-related region in the C. elegans LIN-12 gene). This region is implicated in dampening receptor activity, because deletion or missense mutations in the LNR region result in a constitutively active receptor (15,16). Proteolytic cleavages of the Notch receptor occur during maturation of the receptor and in its activation (21). The first site of proteolytic cleavage (S1) is located between the LNR and TM domains, and cleavage there is required for formation of the mature Notch heterodimer (22). The receptors are synthesized as single-polypeptide precursors of approximately 2500 amino acids. The precursor contains a leader peptide that is removed during maturation. In the trans-Golgi apparatus, a furin-like convertase cleaves the precursor protein, and it is reassembled as a heterodimer of the extracellular and TM domains. The two subunits of the mature Notch receptor interact by a noncovalent calcium-dependent bond. Processed receptor is transported from the Golgi to the cell membrane. There are two other proteolytic cleavage sites in Notch. The second site (S2) is located near the TM region and is the site at which Notch is initially cleaved on ligand binding (23–26). The TM region contains the S3 and S4 sites at which cleavage occurs after ligand binding (27). The cytoplasmic domain of the Notch receptor contains several conserved regions that regulate its function. The subTM region known as RAM and the six adjacent ankyrin (ANK) repeats are involved in binding the CSL transcription factor (28). Ankyrin repeats may mediate homotypic interaction between Notch molecules expressed by a cell (29). The ANK region is also known to interact with Deltex, a widely expressed ubiquitin ligase (30,31) that can act either as a positive or negative regulator of Notch signaling (see Deltex section later in this chapter). Directly following the ANK repeats is a region identified in Notch1 and Notch2 as a Notch cytokine response region (32). In myeloid cells, this region regulates differentiation by granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor. Human Notch1 and Notch2 also have a transcriptional activation domain (TAD) located downstream of the ANK region that has been shown to interact with general transcriptional coactivators such as p300/CBP associated factor (PCAF) and p300 (33). Farther toward the C terminus is a polyglutamine tract (OPA) of unknown function (34). Glutamine-rich regions are found in some transcriptional regulators where they appear to mediate protein–protein interaction, but a direct role for the OPA repeats in Notch function currently is lacking. A PEST sequence (proline, glutamic acid, serine, and threonine-rich region), a characteristic of short-lived proteins and frequent site of ubiquitination (35), is located at the C terminus of the receptor (36).

DEVELOPMENTAL SIGNALING NETWORKS: THE NOTCH PATHWAY / 289 A C terminus Intracellular

N terminus Extracellular

TM S2

NLS

NLS ANK

Ligand-binding site

TAD

c c EGFR

LNR

PEST OPA

S4 S3

B Drosophila

TM

Notch

Vertebrate Notch1 Notch2 Notch3 Notch4

FIG. 10-1. Structure of Notch receptors. (A) The mature Notch receptor is a heterodimer, formed after proteolytic cleavage and noncovalent reassembly of the newly synthesized protein. The receptors contain a variety of domains that are often tandemly repeated. The extracellular domain is characterized by between 29 and 36 tandem epidermal growth factor-like repeats (EGFRs). EGFRs 11 and 12 are important in mediating receptor–ligand interaction. These are followed by three cysteine-rich LNR repeats, the transmembrane region (TM), the RAM domain, and six ankyrin (ANK) repeats that mediate protein–protein interaction and bind the CSL (CBF1 or RBP-Jκ in vertebrates, Su(H) in Drosophila, and Lag1 in C. elegans) transcription factor through which all Notch receptors signal. The downstream Notch cytokine response region (NCR) and transcriptional activation domain (TAD) appear to be specific for Notch1 and Notch2. At the carboxy terminus lie a polyglutamine tract (OPA) of unknown function and the PEST region (proline, glutamic acid [amino acid symbol, E ], serine, threonine rich) that may mediate receptor stability. (B) Notch receptor structure (but not number) is highly conserved among species. Whereas the invertebrate Drosophila has one receptor, most vertebrates have four. Primary differences between vertebrate receptors are in the variation in the number of EGFRs and the length of the region between the ANK repeats and the carboxy terminus. (Modified from Weinmaster [15], by permission from Elsevier and the author.)

STRUCTURE OF THE NOTCH LIGANDS The Notch ligands are also known as the DSL family (after Drosophila Delta and Serrate and C. elegans Lag2), and, like Notch, they are single-pass, TM proteins (Fig. 10-2). The DSL family also has tandem, multiple EGFRs in its extracellular domain. However, the most N-terminal EGFR differs from the others and is referred to as the DSL repeat. It is essential for ligand function (37). As for the Notch receptors, invertebrates have a more simplified repertoire of Notch ligands. Drosophila possesses two ligands, Delta and Serrate, C. elegans has three (each containing remarkably few EGFRs), and vertebrates possess several ligands. Higher vertebrates appear to have three Delta genes and two Jagged genes (15), Jagged being the vertebrate equivalent of Drosophila and chicken Serrate. Zebra fish exhibit more variability, with four Delta genes and three Jagged genes (38,39). The distinctive structural difference between the Delta subfamily and the Jagged/Serrate subfamily of ligands is the presence of a cysteine-rich region in the extracellular domain of the latter. The intracellular domains of all the

ligands are quite short, and although they do not share any substantial homology, they are essential for normal function.

The Delta Subfamily Delta ligands contain an N-terminal domain and DSL and EGFR motifs, but they lack the cysteine-rich region of the Jagged subfamily (see Fig. 10-2). The three vertebrate Delta proteins have a variable number of EGF repeats, with Delta1 and Delta4 having eight repeats and Delta3 having six repeats (40–42). Delta3 appears to be the most divergent member of this family. Drosophila has one ligand, and vertebrates have three.

The Jagged Subfamily The proteins of the Jagged subfamily of Notch ligands, referred to as Serrate in Drosophila and chicken but Jagged in other vertebrates, are much larger than the Delta family

290 / CHAPTER 10 A Equipotent cells

Difference in receptor/ligand expression initiated

Feedback amplification

B

1° fate

2° fate

FIG. 10-3. Schematic of classical, lateral inhibition model of Notch function. A population of equipotent cells expressing Notch receptor and ligand equally receives a stimulus that initiates a change in the receptors and ligands. A fluctuation in Notch receptor expression is amplified through positive and negative feedback mechanisms such that adjacent cells eventually express receptors or ligands. This results in adjacent cells that adopt distinct fates driven by their receptor–ligand profile. In the schematic, receptors are identified as cup-shaped molecules, and ligands as hammer-like molecules.

FIG. 10-2. Structure of Notch ligands. (A) The notch ligands are single-pass transmembrane proteins that also contain a variety of conserved tandem repeats. There are two families of ligands, Delta and Jagged (also known as Serrate in some species), but the overall structure of their extracellular domains is remarkably similar to that of the Notch receptors. A variable N-terminal domain (NT) and a DSL motif (Delta/ Serrate/Lag) are followed by tandem epidermal growth factorlike repeats (EGFRs), typically from 6 to 16. The two families of ligands then differ substantially. The Jagged family (upper) is distinguished by a conserved cysteine-rich domain (CR) preceding the transmembrane (TM) region. All ligands have short intracellular regions lacking any conserved structure that typifies the Notch receptors. (B) Notch ligand complexity, similar to that of the receptors, increases from invertebrates to vertebrates. Drosophila has one Delta and one Serrate ligand, whereas humans have three Delta ligands and two Jagged ligands. Within each family the ligands are similar. (Modified from Weinmaster [15], by permission from Elsevier and the author.)

with, on average, twice the number of EGFRs. Drosophila has one ligand, and vertebrates have two. The extracellular portion of the Jagged ligands contain an N-terminal domain of 100 to 165 amino acids, followed by the highly conserved DSL motif (see Fig. 10-2). C terminal to the DSL motif is 16 EGF repeats and a cysteine-rich region that distinguishes

Jagged ligands from Delta. Following the TM region is an intracellular domain of variable length.

THE NOTCH SIGNALING PATHWAY Most of our knowledge about the function of Notch signaling comes from studies of hematopoiesis, neurogenesis, and embryonic development. The amount of information regarding Notch signaling of these events is extensive. The essential requirement for Notch signaling in early embryonic development has made it difficult to tease out the functions of Notch by traditional gene knockout studies because, generally, the genome-wide knockouts are embryonic lethal. However, sophisticated studies using conditional transgenic and gene knockout mice are beginning to reveal more about Notch function in the adult (43–45). Notch signaling can be regulated in three ways: by lateral inhibition, by inductive signaling, or in a cell autonomous manner. Notch is best known for its role in lateral inhibition, the process whereby positive and negative feedback loops that amplify or down-regulate Notch signaling are established to prevent neighboring cells from adopting the same fate at the same time (Fig. 10-3). Initially, during the process of lateral inhibition, there is an equipotent population of precursor cells that express both Notch receptors and ligands on their surface. By as yet unknown means, Notch expression decreases in some cells, and they up-regulate ligand expression.

DEVELOPMENTAL SIGNALING NETWORKS: THE NOTCH PATHWAY / 291 Increased ligand expression stimulates Notch signaling on previously unaffected neighboring cells, which then reduce ligand expression by mechanisms such as those described later. The resulting distinct cell populations (receptor-low/ ligand-high and receptor-high/ligand-low) adopt different fates and become different cell types (5,46,47). An example of lateral inhibition is provided by the formation of neurons and glial cells during vertebrate neurogenesis (48). When neural stem cells encounter a neurogenic signal in their environment, only some of them respond to it by up-regulating the Notch ligand, Delta, on their cell surface. This, in turn, activates the Notch receptor expressed on the neighboring cells and stops them from responding to neurogenic signals in the environment. The outcome is that the Delta-high cells differentiate into neurons, whereas the cells that were initially prevented from differentiating by Notch signaling adopt an alternative fate and become glial cells. When Notch receptors and ligands are present on different cells, inductive signaling can occur. During this signaling, cells expressing the ligand activate Notch signaling in the cell expressing the receptor, thus influencing their developmental fates (5,46,47). When the Notch receptor and ligand are expressed in the same cell, there is a possibility of a cis interaction between them that could drive them into differentiation of one lineage. Coexpression can also result in an inhibition of Notch signaling (49), a process that is mediated, in part, by glycosylation (50). Notch receptors and ligands that are expressed by adjacent cells interact via their extracellular domains. Epidermal growth factor repeats 11 and 12 of the receptor and the DSL motif of the ligand mediate this interaction. Attachment of fucose to serine or threonine residues within the EGF domains by the enzyme O-fucosyltransferase is essential for interaction (51), but how this mediates interaction and initiates downstream events is unknown. Interaction may induce conformational changes in the receptor that expose proteasesensitive sites. After ligand interaction, the receptor is cleaved, initially at the extracellular S2 site, followed by concurrent cleavages at sites S3 and S4 in the TM region (see Fig. 10-1). On ligand binding to the extracellular domain, Notch receptors undergo several proteolytic cleavages, resulting in a release of the NICD (Fig. 10-4). The first ligand-induced cleavage occurs at the S2 site and is mediated by an ADAMtype proteinase (23,25). Cleavage at S2 results in shedding of the extracellular domain and is followed by intramembrane, presenilin-dependent γ-secretase cleavages at S3 and S4 (52,53). The S3 cleavage site is close to the cytosolic membrane border, and S4 is approximately 12 amino acids upstream from S3, near the middle of the TM. The presenilin-dependent cleavages at sites S3 and S4 result in the liberation of NICD, which translocates to the nucleus, where it interacts with the CSL repressor complex to convert it to an activation complex, thereby altering gene expression. CSL is the major effector of Notch signaling and is the one nonredundant member of the Notch signaling pathway through which all four Notch receptors signal. In the absence of Notch signaling, CSL represses the transcription of target

genes via a repressor complex that includes histone deacetylases and various corepressors, SMRT (silencing mediator of retinoid and thyroid hormone receptors), SAP30, CIR, and SHARP (54–56). Ligand activation and nuclear transport of the NICD enables it to interact with CSL via the Notch RAM and ANK regions. This interaction displaces SMRT, histone deacetylases, and the corepressors and in their place recruits histone acetyltransferases and other coactivators, creating a transcriptional activation complex. In association with coactivators, histone acetyltransferase mediates chromatin remodeling to activate expression of Notch target genes (57,58). The level of nuclear NICD is controlled by various modifications including phosphorylation and ubiquitination. Degradation of NICD down-regulates signaling and resets the cell for future instructive Notch signaling. Most research has focused on the fate of Notch after ligand interaction, but there is some evidence for a role for the ligands after Notch interaction (59,60). First, Drosophila Delta has an intrinsic nuclear localization capability; second, the C terminus of Jagged1 is capable of interacting with PDZ-containing proteins. These features may reflect the existence of signaling pathways initiated in the ligand-expressing cell in response to Notch–ligand interaction, invoking bidirectional signaling mechanisms. The primary Notch target genes include transcription factors of the basic helix-loop-helix (bHLH) superfamily (61,62). Original target genes identified in Drosophila, Hairy and Enhancer of Split, have given the name to the vertebrate homologs, Hes (63). Since discovery of the Hes family, another family of Hes-related bHLH genes has also been shown to respond to Notch signaling (64–66). This is now referred to as the Herp (Hes-related bHLH protein) family (61). These two families include approximately 10 Notch bHLH target genes. Basic HLH transcription factors function as dimers, with two activator bHLH proteins required to bind DNA and activate transcription. With one exception (67), the primary Notch target genes are all bHLH transcriptional repressors that inhibit transcriptional activity by forming nonproductive dimers. Because there are more than 200 bHLH genes with diverse biological roles, there is incredible potential for Notch signaling to influence many cellular processes through this family. What genes are regulated by the bHLH Notch target genes? Because bHLH proteins function as dimers, other families of bHLH genes are often immediate targets. Two well-studied families are involved in neural and endocrine development. Originally identified in Drosophila as the Achaete-Scute complex and the Atonal proneural genes, they encode bHLH proteins involved in neuronal and epidermal lineage formation (62,68,69). In neuronal formation in Drosophila, the AchaeteScute genes, in turn, regulate Delta expression (70,71). Because Notch signaling is also thought to up-regulate itself through Hes (72), the outcome of these activities is to create a feedback loop to polarize receptor and ligand expression and reinforce Notch signaling via different bHLH proteins. Vertebrate homologs of the Drosophila Atonal genes include BETA2, neurogenin3, and Math1. These genes are also

292 / CHAPTER 10 Notch

Delta jagged

HDAC CoR

NICD

CSL

HAT

X

Hes

CoA

CSL

P

Hes

Ub

Hes

X

Math1

FIG. 10-4. Mechanism of Notch signaling. Interaction of the Delta or Jagged ligand on one cell with the extracellular domain of a Notch receptor on an adjacent cell leads to proteolytic cleavage that releases the Notch intracellular domain (NICD). The NICD then translocates to the nucleus, where it binds to the CSL (CBF1 or RBP-Jκ in vertebrates, Su(H) in Drosophila, and Lag1 in C. elegans) transcription factor, displacing a complex of corepressors (corepressor R [CoR] and histone deacetylase [HDAC]) that have hitherto blocked transcription of CSL target genes. Binding of NICD to CSL occurs via the Notch ankyrin domains and leads to recruitment of transcriptional coactivators (CoA and histone acetyltransferase (HAT)) and transcription of basic helix-loop-helix (bHLH) target genes from the Hes and Herp families. These proteins typically act as transcriptional repressors and turn off downstream genes such as Math1 that regulate the selection of a secretory or absorptive cell fate in the gastrointestinal tract (see Notch in the Enteroendocrine System). Posttranslational modification of the NICD, involving phosphorylation and ubiquitination mechanisms, leads to its degradation and a downturn in Notch signaling, potentially resetting the Notch switch.

important in vertebrate neuronal differentiation; however, exciting research has revealed their involvement in the differentiation of enteroendocrine cell lineages in the gut (73–79) (see Notch in the Enteroendocrine System later in this chapter). The cell cycle regulators cyclin D1 and p21 may also be primary targets of Notch signaling (80,81). In keratinocytes, activated Notch induces p21 and promotes differentiation, whereas in other cells, Notch increases cyclin D1 expression to promote progression of cells through the G1 phase of the cell cycle. Both pathways are CSL dependent. CSL and the bHLH genes are the main effectors of Notch signaling, but there is accumulating evidence that Notch can signal through CSL independently of the bHLH genes and is also able to signal independently of CSL itself (82,83). Further identification of specific target genes in different cell types is clearly important to understand the functions of Notch signaling in proliferation, differentiation, and apoptosis. Notch signaling is tightly regulated at a number of levels. The receptor, ligand, and other components of the Notch signaling pathway all appear to be targets for ubiquitination and degradation via the proteasome. Ubiquitination of Notch and Delta is mediated by E3 ubiquitin ligases. The C terminus of Notch, including the PEST sequence, appears to be essential for ubiquitination by the E3 ligase mSel-10 (84),

and phosphorylation of specific Notch residues appears to be the signal for mSel-10 action (85). This rapid downregulation of Notch, coupled with its intrinsically low level of expression, provides an explanation for difficulty in detecting nuclear Notch. Delta has also been shown to be a target for another ubiquitin ligase (86).

Functions of Different Notch Receptors and Ligands In general, vertebrates have four Notch receptor genes and five ligand genes, and these are usually expressed in different patterns throughout development, potentially providing a diverse array of receptor–ligand combinations. In addition, different ligands can signal through the same receptor and produce different outcomes. In Drosophila, in which there is only one Notch receptor, mutations in Delta and Serrate produce different phenotypes, even though they act through the same receptor (87). In this model system, cell interactions mediated between Delta and Notch appear to be more stable than interactions between Serrate and Notch (17,88), and this may lead to stronger or more sustained Notch signaling. The distinctive cysteine-rich region of Serrate may mediate this effect. However, the glycosyltransferase Fringe

DEVELOPMENTAL SIGNALING NETWORKS: THE NOTCH PATHWAY / 293 may also modify the responses to these two ligands (see Glycosylation section later in this chapter). Both Delta and Serrate are expressed during wing formation in Drosophila, where, at the wing margin, Notch activation is modified by Fringe (89). Fringe enhances Delta-activated Notch signaling and inhibits Serrate-induced signaling to establish a dorsal ventral boundary at the wing margin that is necessary for development of the wing. Given the large size of the Notch receptors and the presence of various conserved domains, it is likely that they will exhibit different signaling capacities. However, the data on this aspect of Notch signaling are only beginning to emerge. The primary bHLH Hes target genes appear to be differentially activated by Notch, but the precise effects are not clear. Whereas Notch1 is a potent activator of Hes1 and Hes5, an ability that appears to be mediated by the ANK repeat region and another adjacent region of the intracellular domain (90), the action of Notch3 is controversial (91,92). There is evidence that Notch3 is a much weaker activator of the Hes1 promoter than Notch1 (91), but there is also evidence to the contrary (92). Interestingly, Shimizu and colleagues (92) show that the level of coexpressed CSL affects the ability of Notch to transactivate Hes promoters, a finding that may explain the controversy regarding Notch3 transactivation. They also report that the NICD of Notch1 and Notch3 differentially transactivates Hes1 and Hes5, and that Notch2 is also a weak activator. Interestingly, Shimizu and colleagues (92) show that coexpression of Notch2 inhibited activity of Notch1 and Notch3, indicating that each receptor can not only activate different downstream genes, but is also able to competitively influence each other. An explanation for this may stem from the fact that Notch2 appears to have a weaker TAD compared with Notch1 (47).

the mammalian glycosyltransferases of the Fringe family: manic Fringe, radical Fringe, and lunatic Fringe (95,96). There are a number of O-fucose sites in the extracellular domain of the Notch receptor, particularly in EGFRs 11 and 12, that mediate receptor–ligand interaction (50,51). Modification of Notch by Fringe results in a different responsiveness of the receptor to ligand binding, because elongation of glycans by Fringe increases the ability of the receptor to bind Delta (97) but inhibits binding of Jagged (95). Glycosylation by Fringe has been shown to play an important role in the boundary formation in Drosophila wing, eye, and leg, in formation of the chick limb, and in vertebrate somitogenesis (98). Phosphorylation

A range of interactions with other molecules and posttranslational modifications of the receptor and ligand act to regulate Notch activity (93). Posttranslational modifications include glycosylation, phosphorylation and ubiquitination, and they affect each step in the signaling pathway, from ligand binding outside the cell to gene activation in the nucleus. This section briefly discusses these three processes and the function of three specific modifiers of Notch signaling: Numb and Deltex, which are involved in ubiquitination of Notch, and Mastermind, a nuclear coactivator that may also facilitate Notch phosphorylation.

Phosphorylation is a well-known and widespread mechanism for regulating protein function, and a role for phosphorylation in regulating the Notch signaling pathway is starting to emerge. After ligand activation, the NICD appears to be phosphorylated at several sites downstream of the ANK repeats by glycogen synthase kinase-3β (99–102). Glycogen synthase kinase-3β has been shown to bind to the Notch2 NICD near the ANK region and phosphorylate several serine and threonine residues, a process that inhibits activation of the Hes1 target gene and leads to down-regulation of Notch signaling (102). Notch1 has also been shown to be phosphorylated by glycogen synthase kinase-3β, but, in contrast with the effect on Notch2, this appeared to stabilize Notch1 signaling (101). The sites of phosphorylation need to be clarified in these studies, and whether they represent genuine distinctions in the response of different receptors to phosphorylation or inherent differences in the cells studied is awaited with interest. Notch signaling is integral to myeloid cell differentiation in response to cytokines (32). Research now shows that phosphorylation of specific amino acids in a 30-residue segment of the Notch cytokine response region has a key role in this process (103). Granulocyte colony-stimulating factor induces phosphorylation of the Notch2 NICD, including a critical site at Ser2078 (different to that phosphorylated by glycogen synthase kinase-3β), that inactivates the NICD and promotes differentiation. In contrast, when cells are stimulated with granulocyte-macrophage colony-stimulating factor, the Notch2 NICD is not phosphorylated at these residues, the NICD is able to initiate the Notch signaling cascade in the cells, and differentiation is inhibited. The direct effects of phosphorylation of the NICD in myeloid differentiation remain to be determined but may well signal ubiquitination and protein degradation (104).

Glycosylation

Ubiquitination

Glycosylation is the process of attaching carbohydrates to the side chains of amino acids. The EGFRs of Notch receptors are heavily glycosylated (94), a process that can potentiate or inhibit Notch signaling. The addition of O-fucose is particularly important, because it becomes the site of a further elongation by addition of N-acetylglucosamine by

Ubiquitination, the attachment of ubiquitin molecules by ubiquitin ligases, marks proteins for internalization from the plasma membrane and for proteasome- or lysosome-mediated degradation. Because activation of Notch occurs via an extracellular signal, and the signaling portion of the receptor is cleaved and released intracellularly and is therefore

Modifiers of Notch Signaling

294 / CHAPTER 10 dissociated from further extracellular events, internal mechanisms must operate to ensure tight control over Notch signaling. At least four ubiquitin ligases appear to regulate Notch signaling via ubiquitination (105). Two regulate rapid turnover of the NICD, a third regulates the Delta ligand, and a fourth regulates the Notch antagonist, Numb. Ubiquitination of the NICD is regulated by the ubiquitin ligases Sel-10 and Itch in a process coupled with Notch phosphorylation. The NICD is ubiquitinated by these ligases at sites located C terminal to the ANK region, leading to degradation of the NICD by the proteasome, thereby down-regulating the Notch signal (84,104,106). Although both Notch1 and Notch4 have been shown to interact with the Sel-10 ubiquitin ligase, they respond differently (104). Whereas Notch1 NICD appears to be rapidly degraded, the Notch4 NICD is not. How Notch4 evades proteasome degradation and what the consequences are for Notch4-mediated signaling are unclear. Sel10 also interacts with presenilin (107), the protease that cleaves Notch at the membrane in response to ligand interaction and may therefore regulate Notch signaling at a number of steps. Both the membrane-bound and the activated nuclear Notch can be ubiquitinated by the E3 ubiquitin ligase Itch and degraded by the proteasome (108,109). However, downregulation of the membrane-bound Notch in differentiating myoblasts is achieved by ubiquitination of tyrosine residues by cCbl ubiquitin ligase and degradation by the lysosomal pathway, rather than the proteasomal pathway (110). Thus, several mechanisms for ubiquitinating Notch appear to operate to maintain tight regulation of Notch signaling. Numb Numb was discovered as a key protein in the regulation of neuronal development. Knockout of Numb causes embryonic lethality before embryonic day (E) 11.5 with defects in many neuronal lineages (111). In the Drosophila peripheral nervous system, a sensory organ precursor cell undergoes rounds of asymmetric divisions in development of the sensory organ. Numb is a membrane-associated protein that segregates into one daughter cell during precursor cell division and acts as an inherited determinant of cell fate (112). Notch acts as a binary switch in this system to specify cell fate, and Numb acts to negatively regulate Notch in the process. Numb is a cytoplasmic, membrane-associated protein that binds to the RAM and C-terminal sequences of the NICD to suppress Notch activity (93). It does this by recruiting the E3 ubiquitin ligase, Itch, enhancing ubiquitination of membrane-tethered and nuclear Notch (109). Deltex Deltex was identified in Drosophila in a genetic screen for molecules that interact with Notch to specify cell fate (113). Deltex binds to the ANK region of the NICD and can modulate Notch signaling either positively or negatively (30,114,115). There are several vertebrate Deltex homologs (115) that are widely expressed with Notch (116). Deltex has

been identified as an E3 ubiquitin ligase that transfers ubiquitin to itself and to heterologous substrates (117). This finding, together with the elucidation of Numb’s involvement in Notch ubiquitination, further emphasizes the importance of ubiquitination in regulating Notch signaling. Mastermind Like Numb and Deltex, Mastermind was identified in a screen for Notch modifiers in Drosophila. Mastermind is a nuclear protein that binds to the ANK region to coactivate Notch signaling, amplifying Notch induction of the target gene Hes1 (58). Mastermind has a key role, recruiting the general coactivators CBP/p300 to Notch target gene enhancers to promote nucleosome acetylation and gene activation in response to Notch signaling (118). Mastermind also appears to enhance phosphorylation and proteolytic turnover of the NICD. Fryer and colleagues (118) speculate that Mastermind may function as a timer, coupling transcriptional activation with disassembly of the Notch enhancer complex, in effect coordinating the turning on and off of transcription. The “off switch” may be completed by Notch ubiquitination via the ubiquitin ligase Sel-10, followed by degradation of the NICD by the proteasome (84,104,106).

INTERACTIONS OF NOTCH WITH OTHER SIGNALING NETWORKS The Notch signaling pathway is important in the specification of cell fate in a wide variety of cell types and is perhaps instructive for the generation of all cells in invertebrates and vertebrates (4). Depending on the context, Notch signaling may result in diametrically different outcomes, from inhibition or promotion of differentiation to proliferation or apoptosis. Not surprisingly therefore, the Notch pathway appears to interact with a variety of other important signaling networks. Key pathways where interactions with Notch have emerged are the fibroblast growth factor pathway, the transforming growth factor-β and bone morphogenetic protein (BMP) pathway, and the nuclear factor-κΒ, Wnt, Ras, and signal transducer and activation of transcription (STAT) signaling pathways (119–130). All these pathways are involved at some stage in gut function and, with the widespread expression of Notch in the gut (see Notch in the Developing Gut later in this chapter), potentially herald its extensive involvement in diverse cellular processes (76). In foregut development in Drosophila, STAT signaling is required for expression of Notch target genes that regulate epithelial morphogenesis (124). Development of the proventriculus, an organ derived from the foregut, depends on Notch signaling in two rows of boundary cells in the proventriculus primordium. This results in invagination of the foregut epithelium into the endodermal midgut layer, a process driven by a cytoskeletal protein of the spectraplakin family that responds to Notch signaling (131). Drosophila Notch and Janus-activated kinase (JAK) STAT mutants exhibit similar

DEVELOPMENTAL SIGNALING NETWORKS: THE NOTCH PATHWAY / 295 defects in cell movement and proventriculus development. Although the exact nature of the link between STAT and Notch remains to be determined in this system, Kamakura and colleagues (130) have shown that in the central nervous system, the Notch target gene Hes interacts with JAK and STAT to promote STAT phosphorylation and activation. Other signaling pathways appear to modulate Notch signaling via the Hes and Herp Notch target genes (120,122,128), although interaction with NICD and CSL can also occur (121,127). In contrast, the Wnt pathway, which operates in diverse cell types during development and acts through the lymphoid enhancer factor 1/T-cell factor (LEF1/TCF) transcription factors (132), appears to regulate Notch signaling through direct control of Delta transcription (126). There are several LEF1 binding sites in the Delta1 promoter, and mutagenesis studies have shown that these are necessary for Delta1 expression, at least in the presomitic mesoderm. Wnt signaling is involved in the proliferation of intestinal epithelial progenitor cells, and mutation of TCF4 has dramatic consequences for intestinal morphogenesis (133). TCF4 knockout mice die soon after birth. At a time when the gut epithelium is normally in a highly proliferative phase, these mice lacked any proliferative epithelial compartments, and the neonatal epithelium was composed exclusively of differentiated, nondividing villus-type cells. TCF4 thus appears to have a critical role in maintaining intestinal crypt stem cells. Notch1, Notch2, and Delta1 are expressed in the intervillus epithelium at E18.5, where the stem cells and proliferative cells are found (134), and it is possible that Wnt acts through TCF4 to up-regulate Delta1 expression in gut epithelial stem cells, in turn activating Notch signaling in adjacent cells. In this scenario, in normal gut epithelium, the TCF4/Delta cells would retain their stemlike capability, and the Notch expressing cells would have a high proliferative capacity that fuels gut growth. This work offers tantalizing glimpses of how diverse signaling pathways may interact with Notch, and it appears certain that such interactions will prove relevant to gut development and function.

NOTCH SIGNALING IN THE GASTROINTESTINAL SYSTEM Overview of Gastrointestinal Structure and Development Much about the adult gastrointestinal tract is large. In humans, it is several meters in length with a surface area of more than 200 square meters! Furthermore, the gut epithelium is in a state of continual renewal, replenishing its epithelial surface every few days. In the small intestine, this amounts to replacement of a staggering 109 cells in mice and more than 1011 cells in humans every 2 to 4 days (135). Endocrine cells in the epithelium of the gastrointestinal tract represent the largest population of hormone-secreting cells in the body (136). The mucosal immune system and enteric nervous system, which are intimately linked with gut function, form

substantial and complex networks in their own right. The mucosal immune system, which is involved in the development of oral tolerance and defense against intestinal pathogens and toxins, is estimated to contain about half the total number of T cells in the body (137) and about 80% of all B cells (138). The enteric nervous system, which contains more than 14 different neuronal cell types, has a key role in intestinal motility, absorption, and secretion (139,140). This section discusses the emerging role for Notch signaling in the development and maintenance of these tissues and networks that underpin gastrointestinal function. The adult gut derives from all three germ layers in the embryo, namely the ectoderm, mesoderm, and endoderm. The gut epithelium and its associated glands derive from embryonic endoderm, whereas the nervous system derives from embryonic ectoderm. The mucosal immune system derives from the endoderm and mesoderm. The early stages of gut development—that is, the formation of the primitive gut tube—are similar between Drosophila and vertebrates (141,142), and much of our knowledge of Notch signaling in gut development is informed by studies in Drosophila. Essentially, in Drosophila and in mice, the endodermal layer invaginates in the anterior and posterior positions, forming foregut and hindgut primordial tubes, which then extend toward each other, fuse, and give rise to the primitive gut tube (142). The alimentary canal is divided into foregut, midgut and hindgut sections, each with distinct physiologic functions. In mice, the foregut gives rise to the proximal part of the duodenum, the midgut gives rise to the remaining parts of the small intestine, distal duodenum, jejunum and ileum, and the hindgut gives rise to the colon, caecum, and rectum (143). Development from the primitive gut tube to the established organ sees many changes. In mice, formation of the primitive gut tube commences at E8; by E13.5, the intestinal epithelium is beginning to undergo morphogenesis and cytodifferentiation, and at E15.5 appears as a pseudostratified epithelium. The epithelium then starts to fold, forming an undulating layer of epithelium folded upward into villi separated by proliferating intervillus epithelium. After birth, the intervillus epithelium is folded downward into pocket-like crypts. The crypts increase in number through fission in the next few weeks such that each villus is “fed” by cells emanating from several crypts. Looking down onto the epithelium from the lumen of the gut, a regular array of villi and crypts with several crypts surrounding each villus and a number of villi about each crypt can be seen. Apoptosis of cells at the apex of the villi occurs at birth, but the villi continue to lengthen for a few weeks as cell production exceeds the rate of apoptosis and cell loss. In humans, formation of mature crypts and villi occur earlier in gestation, and both are evident well before birth (144). The epithelium of the small intestine produces five types of differentiated cells: absorptive enterocytes, mucinproducing goblet cells, antimicrobial peptide-secreting Paneth cells, peptide-secreting enteroendocrine cells and antigen sampling M cells; the latter are specifically found in the

296 / CHAPTER 10 follicle-associated epithelium that overlies Peyer’s patches. All cell types are formed in crypts, which are bottle-shaped invaginations of the small intestine interspersed between finger-like villi (Fig. 10-5). The stem cells reside near the base of the crypt; four to six are predicted for small intestinal crypts (145–147). As the gut epithelium is replenished every few days, the cycle of production of new cells continues throughout life. The overwhelming majority of these cells become absorptive enterocytes, which, together with secretory goblet cells and enteroendocrine cells, move upward as they differentiate. A small number of cells, about 10 per crypt, behave differently. Known as Paneth cells, they migrate downward from the stem cells and complete their differentiation at the bottom of the crypt. Paneth cells are designed for antimicrobial defense, containing many secretory granules, and are relatively long lived with a lifespan of 20 days. The upwardly mobile cells are shed from the top of the villus in a continuous

stream, taking between 2 and 4 days to make the trip from newly minted cell to an apoptotic body. How this prolific and carefully orchestrated production of four different cell lineages is maintained is now coming to light (see Notch in the Enteroendocrine System later in this chapter). In the formation of the colon, the epithelium also becomes folded and forms villi and crypts, although this a transitory arrangement. In humans, the villi have flattened by birth, and the colonic epithelium has matured into an array of regularly spaced crypt pockets (Fig. 10-6). The stem cells are thought to reside at different crypt locations in the ascending and descending colon (145–147). In the ascending colon, they are thought to be located in the midcrypt, whereas in the descending colon, they are believed to be located at the crypt base. As for the small intestine, there are several stem cells per crypt, and they produce enterocyte, goblet, and enteroendocrine lineages. There are no Paneth cells in the adult intestine, perhaps because the stem cells have different potential or because an initiating signal is lacking.

Shedding cells

Villus

Epithelium Enterocytes Goblet cells Enteroendocrine cells

Epithelium Differentiation

Enterocytes Goblet cells Enteroendocrine cells

Differentiation

Proliferation

Proliferation Crypt Stem cells

Migration Crypts Migration

Paneth cells Stem cells

FIG. 10-5. Schematic of the crypt-villus structure of the small intestine. The epithelium is massively folded into finger-like villi and pocket-like crypts. The epithelial stem cells reside near the base of each crypt (white cells). Proliferative daughter cells variously migrate down to the crypt base and differentiate into Paneth cells or up toward the villus tip and differentiate into enterocytes, goblet cells, and enteroendocrine cells. Each crypt feeds a number of surrounding villi. (Modified from Sancho and colleagues [76], with permission, from the Annual Review of Cell and Developmental Biology, Volume 20 © 2004 by Annual Reviews www.annualreviews.org and from the author.)

FIG. 10-6. Schematic structure of the large intestine. The epithelial surface is packed with crypts but otherwise lacks the villus structure of the small intestine. The epithelial stem cells reside at or near the crypt base and give rise to enterocytes, goblet cells, and enteroendocrine cells. Paneth cells are not found in the large intestine. (Modified from Sancho and colleagues [76], with permission, from the Annual Review of Cell and Developmental Biology, Volume 20 © 2004 by Annual Reviews www.annualreviews.org and from the author.)

DEVELOPMENTAL SIGNALING NETWORKS: THE NOTCH PATHWAY / 297 In the stomach, the epithelium forms deep tubular glands in the stomach wall, each gland with several different zones: the foveolus, isthmus, neck, and base (146). The cells lining the epithelium comprise different types of secretory cells; gastric foveolar cells of the mucosal surface and foveolae secret mucous, whereas the chief or peptic cells of the neck and isthmus secrete pepsinogen. Acid-secreting or parietal cells are found in the body compartment of the stomach at the base of the glands, endocrine cells are located in the fundus and body, and gastrin-producing cells are predominantly found in the antral mucosa. The stem cells are believed to reside in the middle of the glands in the vicinity of the neck and isthmus. Thus, the type of differentiation is connected with the direction of migration from the progenitor pool.

Notch in the Developing Gut The development of the primitive gut tube along the anteriorposterior axis is known to be regulated by Hedgehog and BMP signals (141,148), and the involvement of Notch at this stage has not been studied. Until recently, the mechanisms that regulate the development of the gut tube in the dorsoventral axis have not been well defined. It is now known through studies of cell fate specification in the Drosophila hindgut that Notch signaling plays an important instructive role in this patterning (142). A single row of boundary cells separates the dorsal and ventral cells in the hindgut. Notch is exclusively expressed in presumptive boundary cells (which lack Delta), and Notch signaling is activated on interaction with Delta expressed by adjacent ventral cells. This results in expression of steroid receptor genes and Crumbs, an apical polarity organizer, in the boundary cells that determines their fate. How the Delta expression domain is established in the ventral cells is unknown. A number of survey studies show that genes involved in Notch signaling are widely expressed throughout embryonic and adult gut, with varying spatial and temporal patterns, which is indicative of a variety of roles in intestinal function (77,134,149,150). The earliest stage in mouse embryonic gut development investigated for the expression of Notch receptors, ligands, and downstream signaling molecules is E13.5 (134). Schroder and Gossler (134) undertook a comprehensive survey of expression for Notch1-4; the ligands Jagged1, Jagged2, and Delta1-3; the three modifying glycosyltransferases of the Fringe family; and the downstream targets Hes1, Hes5, Hes6, and Hes7 at E13.5, E18.5, and postnatal day 25 by nonradioactive in situ hybridization. These experiments were the first to provide important spatial information on the expression of Notch genes in the gut, although the low sensitivity of the technique compromised the landscape view and is probably an oversimplification. The genes were variously expressed in the gut epithelium and in the mesenchyme, sometimes throughout each compartment and sometimes in discrete cells, pointing to complex and perhaps redundant interactions between the various receptors and ligands. At E13.5, when the gut is a relatively simple tube, most Notch pathway genes appeared to be expressed more

abundantly in the mesenchyme compared with the epithelium. Delta1 was expressed in mesenchymal cells adjacent to the epithelium and in scattered cells around the periphery of the mesenchyme. A knock in of LacZ into the Delta1 locus confirmed high Delta1 expression in the circular and longitudinal muscle layers in the mesenchyme and revealed a similar pattern in the fetal stomach (151). Delta3 and Delta4 were expressed more discretely in the E13.5 gut tube; Delta3 was weakly expressed in rare, scattered cells at the periphery of the mesenchyme, whereas Delta4 appeared to be more abundant in cells that overlapped the Delta1 and Jagged expression domains. Jagged1 and Jagged2 were primarily expressed in a mesenchymal layer beneath Delta1. All Notch receptors appeared to be expressed in low amounts; Notch1-3 were expressed in both the epithelium and mesenchyme, whereas Notch4 was restricted to the mesenchyme. Notch1 and Notch4 were expressed in a similar pattern to Delta4. The expression domains of the Fringe modifier genes and the Hes target genes generally overlapped with receptor or ligand expression. These expression domains indicated involvement of Notch signaling in diverse developmental processes in the epithelium and mesenchyme from an early age. At E13.5, the gut tube mesenchyme contains a variety of different cell types that are developing into muscle, angiogenic, and neuronal structures. Although the spatial distribution of the Notch components was consistent with a role for Delta1 in muscle development, and a role for Delta4 in angiogenesis was inferred based on expression in other tissues (42), it is important to test these assumptions by examining coexpression with diagnostic markers. At E18.5, the gut tube is undergoing dramatic changes in organization as the epithelium folds into villi-like extensions that project into the lumen. The intervillus epithelium, which is reshaped over the next few days into the crypts characteristic of the adult intestine, contains proliferating cells and in all probability the intestinal epithelial stem cells. There is a clear demarcation in Notch receptor and ligand expression at this time. Notch1 and Notch2 are expressed in a few cells in the intervillus epithelium, where they are partnered by Delta1 (134). That they are involved in the process of cell renewal seems certain, but at what stage this occurs is unclear. The other Notch receptors and ligands are expressed in the lamina propria or submucosa. Expression of the Notch target gene, Hes1, is largely confined to the intervillus epithelium, whereas Hes5 and Hes6 are mainly observed in the villus epithelium. Immunohistochemical detection of Hes1 protein shows that is restricted to the villi at E18 (73,77) and is not in the proliferative intervillus cells where Hes1 messenger RNA (mRNA) has been detected (134). These studies suggest the Hes genes and proteins may mediate the effects of Notch signaling that commenced in the intervillus region. In their study of Hes1 knockout mice, Jensen and colleagues (77) recorded the expression of several Notch signaling genes in the developing intestine of E18 mice. Where genes were expressed, these authors saw little change in their expression in intestinal compartments ranging from the stomach to the colon. Hes2 and Hes3 were absent from embryonic gut, and in contrast with the in situ localization studies of Schroder

298 / CHAPTER 10 and Gossler (134), Jensen and colleagues (77) detected Delta3 but not Hes5. Different findings in these studies may be attributable to the relative strengths of the different detection techniques used. Jensen and colleagues (77) used reverse transcriptase-polymerase chain reaction to detect mRNA expression in segments of gut, whereas Schroder and Gossler (134) used in situ hybridization. The Notch signaling genes and proteins are invariably expressed at low levels, and the detection techniques often need to succeed at their highest level of sensitivity. Thus, probe optimization, “macroscopic” versus “microscopic” tissue analysis, and regional variation can all contribute to apparent discrepancies in expression data. The use of knockin mice to express a readily detectable and long-lived reporter protein from the endogenous promoter can be particularly effective, as demonstrated for studies of Delta1, Math1, and neurogenin3 expression (73,74,151). At postnatal day 25, the gut resembles its adult form, with typical villus and crypt architecture. At this time, Notch receptor expression in the epithelium is restricted to the crypts where Notch1 appears to be the major active receptor. Notch2 is weakly and diffusely expressed in the crypts, whereas Notch3 and Notch4 are not expressed there. However, Notch3 and Notch4 are expressed in the vasculature of the mesenchyme, a pattern that has continued throughout gut development. Of the ligands, Delta1, Delta4, and Jagged1 are expressed in individual cells in the crypt epithelium, and Delta4 and Jagged1, together with Jagged2, are also expressed in the vasculature beneath the crypts. Hes1, Hes5, Hes6, and Hes7 are all expressed in the crypt epithelium, and as at E18.5, Hes5 expression extends up the villi. Hes5 is also expressed in the mesenchyme. Hes1 protein expression is also confined to the crypts in adult mice and is in direct contrast with what is seen in late embryonic (E17) gut, where it is present in the differentiating villus cells (73,77). This transition may occur just before birth, as Kayahara and colleagues (152) report that Hes1 protein is present in the intervillus region at postnatal day 1. Hes1 expression in nonproliferating villus cells at E17 suggests that it does not control the choice between proliferation and differentiation at this stage, whereas its expression in adult crypts implies it might have a role in what is considered to be the more classical aspect of Notch function. Work in our laboratory confirmed the presence of transcripts for Notch1-3, Jagged1, Jagged2, and Delta1 in adult rat gut from the esophagus through all the distinctive compartments of the stomach and small intestine to the large intestine (149). In situ hybridization techniques located Notch1 (Fig. 10-7), Notch2, Jagged1, and Jagged2 in a variety of cells in these tissue compartments. In the small intestine, they were abundantly expressed in the lower parts of the crypts, with the exception of Notch2, which was present in only a few crypt cells. The nature of these cells was not clear, and in the colon, Notch2 was not detected at all. The expression of Notch receptors and ligands in cells of the lamina propria and enteric nervous system (150) point to the much wider involvement of Notch signaling in gut function, extending beyond its role in epithelial function.

Notch in the Enteroendocrine System Endocrine cells in the gut epithelium are the largest population of hormone-producing cells in the body (136), and unlike other organs in which endocrine cells are found, enteroendocrine cells are scattered among other differentiating cell types. This necessitates an additional degree of control over differentiation of enteroendocrine cells from progenitor cells. It has been known for many years that enteroendocrine cells derive from common progenitor cells in the intestinal crypts, yet the mechanisms have remained elusive. Exciting research using several different strains of knockout mice now implicate the Notch signaling pathway as a critical player in the differentiation of enteroendocrine cell lineages in the gut (73–79). More than 10 different enteroendocrine cell types are found in the gastrointestinal tract of mammals based on their hormone profile and their ultrastructural appearance (153). Their distribution can vary markedly. Some occur throughout the gastrointestinal tract, some are found mainly in the stomach and small intestine, and yet other enteroendocrine cell types preferentially occur in the large intestine. Early hints of a role for Notch signaling in forming these lineages came from mice in which a bHLH transcription factor, BETA2, had been ablated (78). BETA2, also known as NeuroD, is a member of the proneural class of bHLH genes related to the Achaete-Scute genes of Drosophila, which are regulated by Notch (62,154). The BETA2-deficient mice acquired severe diabetes and died perinatally. Development of pancreatic islets appeared to be arrested between E14.5 and E17.5, a stage characterized by expansion of the insulinproducing β cell population. They lacked enteroendocrine cells expressing secretin or cholecystokinin, although they did make endocrine cells expressing other hormones. BETA2 interacts with the p300 transcriptional coactivator to regulate transcription of the secretin gene and the gene encoding the cyclin-dependent kinase inhibitor, p21 (79). By examining the effect of overexpression of BETA2 on secretin production and apoptosis in cell lines, Mutoh and coworkers (79) proposed that BETA2 coordinates transcriptional activation of the secretin gene and cell cycle arrest in the intestinal epithelium. In a different approach, Rindi and colleagues (75) used other sophisticated gene technology that specifically ablated three types of enteroendocrine cells in the mouse gut. Using the promoter of the secretin gene that is expressed by a subset of enteroendocrine cells, they made transgenic mice in which secretin-producing cells could be ablated by addition of the antiviral agent gangciclovir. Identification of endocrine cells in these mice showed loss of secretin and cholecystokinin- and peptide YY/glucagon-expressing cells. Reduced numbers of enteroendocrine cells producing gastric inhibitory peptide, substance P, somatostatin, and serotonin were also observed. They showed that BETA2 was expressed in enteroendocrine cell but not nonendocrine cells. Thus, BETA2 is a marker for enteroendocrine cells and is essential for secretin and cholecystokinin expression, but it is not

DEVELOPMENTAL SIGNALING NETWORKS: THE NOTCH PATHWAY / 299

A

B

C

FIG. 10-7. Expression of Notch1 in adult duodenum. Specific radioactive riboprobes for Notch1 were hybridized to adult rat duodenal sections and developed; sections were counterstained with hematoxylin. (A) Bright field images; (B, C) dark field images. Notch expression is indicated by white grains in the dark field images. (C) This panel has been manipulated in Photoshop to remove the gray background cast by the stained tissue in B, which makes it difficult to distinguish the expression. Notch is expressed in the crypt region (bracketed) and in the lamina propria (arrow) and epithelium of the villus (arrowhead).

required for development of all enteroendocrine cells, indicating that there are at least two types of multipotent enteroendocrine progenitor cells. Direct involvement of Notch signaling in the development of endocrine cells has been provided by knockout of Delta1 and CSL, the transcription factor that mediates NICD function in the nucleus (155). Knockout of either of these genes resulted in accelerated differentiation of pancreatic endocrine cells. Knockout of Hes1, one of the immediate Notch target genes, provides further evidence for the role of Notch signaling in enteroendocrine development in the gut (77). A deficiency in Hes1 produces abnormal endocrine differentiation in the gastrointestinal tract and pancreatic hypoplasia. Normal enteroendocrine cell differentiation begins in the antral region of the stomach at E14.5, followed in the intestine at E16.5. Hes1 deficiency produced an increase in the number of enteroendocrine cells producing glucagon, cholecystokinin, gastrin, and somatostatin. The proportion of goblet cells was increased, whereas there were reduced numbers of enterocytes; these effects ultimately distorted villus morphology. Interestingly, although mitotic activity of E17 epithelium appeared unchanged, there was increased apoptosis at E19, which, surprisingly, was apparent in the crypts. In the knockout mice, there was an increase in expression of two Notch ligands, Delta1 and Delta3, and several bHLH transcriptional activators, principally Mash1, NeuroD/BETA2, and neurogenin3, in the gastrointestinal tract. These are consistent

with the notion that Hes1 inhibits Notch signaling via two mechanisms, feedback inhibition of the activating ligands and direct inhibition of Notch bHLH transcriptional activators, a hallmark of the lateral inhibition hypothesis for Notch signaling. Thus, Hes1 may act as a general repressor of bHLHcontrolled enteroendocrine development. Work by Lee and colleagues (74) extends our knowledge of enteroendocrine cell development, shedding light on their development in the stomach and the potential involvement of Notch in that process via the bHLH transcriptional activator, neurogenin3. Neurogenin3 lies downstream of Notch and is a target for repression by the Notch target gene, Hes1. Hes1 negatively regulates neurogenin3 through repression of its promoter (156). Knockout of neurogenin3 causes severe diabetes and death at postnatal day 3 (157). Neurogenin3 mice lack glucagon-secreting cells, somatostatin-secreting cells and gastrin-secreting cells in the glandular stomach. Serotonin-expressing enterochromaffin cells are not absent but are dramatically decreased. Similar effects occur in the pancreas. In addition to enteroendocrine cells, two other types of secretory cells are found in the gut epithelium, mucinproducing goblet cells and antimicrobial, peptide-secreting Paneth cells. Paneth cells differ in two notable respects from all other differentiated cells of the gut epithelium in that they migrate to the base of the crypt as they mature, where they reside for approximately 20 days before being shed.

300 / CHAPTER 10 They are thus much longer lived and migrate in a different direction to all other differentiating gut epithelial cells. A molecular explanation for the origin of these lineages has remained elusive. Knockout of another bHLH gene, Math1, a downstream target for Notch signaling, now provides insight into how differentiation of secretory lineages in the gut is coordinated (73). Math1 was previously known to be involved in cell fate determination in the nervous system (158,159). In using a LacZ knockin at the Math1 locus, Yang and colleagues (73) were able to carefully delineate where Math1 was expressed. In the gastrointestinal tract, Math1 expression is turned on specifically in the epithelium of the small and large intestine from E16.5 (73). Interestingly, it is not expressed in the stomach, which also has a number of resident secretory cell types. Math1 is abundantly expressed in the proliferative compartment of the gut epithelium of embryonic and adult mice, with scattered positive cells in the villi. Because LacZ staining is cytoplasmic, they were able to readily double stain for a nuclear proliferative marker, Ki-67, to precisely correlate Math1 expression with proliferation. This revealed that Math1 was expressed in most but not all proliferative cells, from the 4th to the 13th cell position in the crypts of the small intestine and from the 2nd to 4th position in the colon. Using a battery of morphologic, histologic, and immunohistochemical tests, Yang and colleagues (73) confirmed that Math1 is expressed in goblet, Paneth, and enteroendocrine cells, and that all these cells are absent in the knockout mice. Because enterocyte differentiation appears unaffected, Math1expressing progenitor cells appear to give rise to secretory cell lineages, and Math1-negative proliferative cells give rise to enterocytes. As with previous knockout studies of this family of bHLH genes, loss of Math1 decreased expression of one of the Notch ligand genes, Delta3, and another bHLH gene, NeuroD/BETA2. In contrast, expression of Notch1, Notch2, Notch3, Notch4, Delta1, or Hes1 was not affected. These effects are consistent with a model in which Math1 is upstream of NeuroD/BETA2 and provides positive feedback to down-regulate Delta3 and Notch signaling. Figure 10-8 outlines a simple model for specification of the four major types of differentiated cells in the intestine, enterocytes, goblet cells, Paneth cells, and enteroendocrine cells. It is suggested that they arise through a series of binary decisions mediated by the Notch signaling pathway. Simply, a stem cell produces two cells, one of which retains its “stemness” and a daughter cell that adopts a transit-amplifying phenotype and gives rise to all other lineages. There is circumstantial evidence for Notch involvement in the earliest binary decision. An RNA-binding protein known as Musashi1 has been found to have a central role in the maintenance of neural stem cells (160–163) and perhaps in breast epithelial stem cells (164). Musashi1 is expressed in a few select cells in the intestinal crypt at positions defined by labeling studies as likely sites for intestinal epithelial stem cells (152,165,166). Notch and Hes1 are abundantly expressed in the crypt (134,149), and both expression domains overlap with Musashi1. Musashi1 has also been shown to down-regulate

Secretory lineages Paneth

Goblet

Absorptive lineage

Enteroendocrine (10)

Notch?

Enterocyte

NeuroD/BETA2

Notch? Ngn-3

Notch? Math-1+ Math-1−

Math-1− Math-1+ Stem Notch? Stem

FIG. 10-8. Potential role of Notch signaling in intestinal epithelial differentiation. This model proposes that the four major cell lineages of the intestine arise from common pluripotent stem cells through binary cell decisions potentially mediated by Notch signaling. It is likely that Notch signaling activates the basic helix-loop-helix (bHLH) transcription factor Hes1, which antagonizes Math1, another member of the same superfamily. Expression of Math1 commits progenitor cells to a secretory cell fate, whereas Math1−ve cells become enterocytes. Subsequent expression of neuorogenin3 and neuroD/BETA2 in Math1+ve cells is necessary for specification of various enteroendocrine cell types. (Modified from Sancho and colleagues [76], with permission from Elsevier and the author.)

Numb, a Notch antagonist, thereby promoting Notch signaling (93,167). In the context of the intestine, the stem cells are predicted to express Notch, a Notch ligand, Musashi1, and Hes1, promoting autonomous Notch activity and inhibiting downstream transcriptional activators, such as Math1 and NeuroD/BETA2. The transit-amplifying cells are predicted to express Notch and Hes but not Musashi. The net result of this is that Hes continues to inhibit Math1 and NeuroD/BETA2 but results in negative feedback regulation of the Notch ligand. This stops autonomous Notch signaling and instead promotes polarization of Notch receptor and ligand expression on adjacent cells, effectively establishing lateral Notch signaling between adjacent cells. Cells expressing Notch and Hes continue to block Math1 and NeuroD/BETA2 induction and adopt an enterocyte fate. Adjacent cells expressing ligand down-regulate Notch and Hes, Math1 is derepressed, and its expression commits these cells to the secretory lineage. Subsequent activation of NeuroD/BETA2 in this lineage provides further specification in the development of enteroendocrine cell fates, principally the secretin, cholecystokinin, and L-cell lineage. Conceivably, other transcriptional activators from the large bHLH family may be regulated by Notch signaling to dictate the individual enteroendocrine cell types.

DEVELOPMENTAL SIGNALING NETWORKS: THE NOTCH PATHWAY / 301 Notch in the Mucosal Immune System The intestine is a major T- and B-cell organ, estimated to contain about half the T cells and about 80% of all B cells in the body (137,138). These cells reside in the gut epithelium, the underlying lamina propria, and specific gut-associated lymphoid structures, such as Peyer’s patches, cryptopatches, and lymphocyte-filled villi. Much work has shown that Notch signaling is essential in the development of T and B cells (168), but its involvement in the mucosal immune system is relatively unexplored. In their in situ hybridization study of Notch receptors and ligands in the adult rat gastrointestinal tract, Sander and Powell (149) noted their expression in the lamina propria and Peyer’s patches. Notch1 was expressed at a low level in Peyer’s patches, in the coronal zone just beneath the follicle-associated epithelium, which typically contains T cells. Notch2 was more abundant and expansive in the corona and germinal center, the latter predominantly containing B cells. Neither of the two ligands investigated, Jagged1 or Jagged2 appeared to be expressed in Peyer’s patches; however, Jagged1 was expressed in scattered cells in associated high endothelial venules. Notch1 is also expressed in the lamina propria of the small and large intestines, whereas Notch2 is present only in the lamina propria of the large intestine. A downstream Notch regulator, Deltex (see Deltex section earlier in this chapter), has also been discovered in sheep Peyer’s patch B cells (169). These studies provide tantalizing evidence for a role for Notch in the adult mucosal immune system and warrant further investigation. In lymphocyte development, the decision to adopt a T- or B-cell fate involves Notch signaling. This requirement has been shown in a series of elegant experiments based on conditional inactivation of Notch signaling in mice expressing Cre recombinase under the control of the interferon-αregulated Mx promoter. Inducible inactivation of the Notch1 receptor or the CSL gene that mediates signaling by all Notch receptors results in a block in the development of T cells and ectopic development of immature B cells in the thymus (168,170–173). Wilson and colleagues (170) specifically investigated the effect of the inducible inactivation of Notch1 on the development of extrathymic T cells in the gut using a bone marrow reconstitution assay. Because inactivation of Notch1 was nearly completely effective in the bone marrow but only about 60% in the mice, they used a competitive, mixed bone marrow chimera assay to investigate the effect on T-cell production. They found a 10- to 15-fold decrease in T-cell receptor αβ and γδ T cells in the gut as a result of Notch1 knockout, confirming that Notch1 is necessary for development of extrathymically derived gut T cells. Because CD117+ lineage–negative T cells located in gut cryptopatches have been described as potential intraepithelial T-cell precursors (174), they then examined their relative abundance in the gut and thymus of reconstituted chimeras. In the gut of Notch1-deficient mice, they were reduced twofold, compared with ninefold in the thymus. Thus, Notch signaling is essential for the development of T cells in the thymus and extrathymic T cells in the gut. One model put

forward proposes that bipotential T-/B-cell progenitors migrate from the bone marrow to the thymus or the gut, at which locales Notch signaling is initiated and T-cell development continues. In addition to the classic functions of Notch in regulating development and cell fate selection, new insights into an ongoing role for Notch in lymphocyte function are emerging that are relevant to the gut. These studies reveal that Notch signaling can regulate T-cell receptor–mediated T-cell proliferation and apoptosis, development of Th1/Th2 immune responses, and the decision between immunity and tolerance (175–180). Jehn and colleagues (175) showed that Notch1 has an antiapoptotic capacity in T cells, where it is able to interact with a member of the orphan nuclear receptor family that normally orchestrates T-cell receptor–mediated apoptosis. In other work, the same group showed that Notch is required for T-cell receptor–mediated activation of peripheral CD4 and CD8 T cells (176). Engagement of the T-cell receptor resulted in Notch synthesis and activation and appeared to have a direct effect on interferon-γ production, specifically in CD8 T cells. Hoyne and colleagues (177) provide data that Notch signaling regulates the decision between immunity and tolerance. They showed that Notch1 and Jagged1 were expressed on peripheral CD4 and CD8 T cells and dendritic cells in the spleen of naive mice. Using retroviral gene transfer to constitutively express Jagged1 on antigen-presenting cells, they then showed that peripheral naive CD4 T cells differentiated into regulatory T cells, which were able to transfer tolerance to naive recipient mice. If blockade of Notch signaling in the receiving T cells could be shown to prevent development of regulatory T cells, that would add further weight to these provocative findings. Current work now also highlights a role for Notch signaling in mediating Th1 and Th2 responses to infection (179,180). Th1 and Th2 T cells are derived from CD4 T cells. Th1 cells mediate immunity to bacteria and viruses, characteristically secreting interferon-γ, whereas Th2 cells mediate immunity to multicellular pathogens, characteristically via secretion of interleukin-4 (IL-4), a key cytokine that is also involved in their differentiation (181,182). T cells do not directly recognize pathogens, but instead depend on dendritic cells to pass on this “information,” in the process initiating development of a Th1 or Th2 response. Amsen and coworkers (179) showed that dendritic cells engineered to express Delta induced a Th1 response by interacting T cells, whereas Jagged induced a Th2 response. They further showed that the IL-4 promoter contains several binding sites for the Notch effector, CSL, and thus provide a mechanism through which Notch signaling could initiate Th2 differentiation. Taken together, these studies indicate that Notch signaling can have profound effects on lymphocyte function, controlling the balance of differentiation of CD4 T cells into Th1 or Th2 lineages and regulating their proliferation and survival. Although these capabilities are yet to be specifically linked to functioning of the mucosal immune system, they have obvious implications for this major lymphocyte reservoir in its response to enteric infection.

302 / CHAPTER 10 Notch in the Enteric Nervous System Notch signaling has been well studied in the central nervous system, where it is known to have key roles in the maintenance and differentiation of neural precursor cells (183), whereas its role in the enteric nervous system is largely unexplored. The enteric nervous system is an independent and complex nervous system that controls gastrointestinal motility reflex and has a major role in regulating water and electrolyte transport and intestinal blood supply. The enteric nervous system is derived from the neural crest, and its precursors migrate along defined pathways to the gut (184). These precursors remain pluripotent even after colonizing the gut (185). There are more than 14 different neuronal cell types in the enteric nervous system (139,140) that appear to be derived from at least 2 precursor lineages: a sympathoenteric lineage that expresses the bHLH transcription factor and Notch target gene, Mash1, and a sympathoadrenal lineage that does not express Mash1 (186,187). Mash1 is the mammalian homolog of the Achaete-Scute complex of Drosophila, which participates in neurogenesis in the central and peripheral nervous systems. In Drosophila, Mash1 and Notch have been shown to interact. The vertebrate homologs show similar expression patterns in the developing brain and retina, suggesting that both genes participate in the development of vertebrate neural lineages (188). Mash1 knockout mice lack sympathetic and enteric serotenergic neurons and enteric neurons in the esophagus and die soon after birth (186). Enteric neurons of the stomach and small intestine are only partly affected. During development in Mash1 knockout mice, neural crest cells migrate to their correct positions but do not differentiate into neurons (189). These studies implicate Notch signaling in the development of enteric neurons. Notch signaling is traditionally associated with development and developmental decisions that lead to diversification of cell fates; however, it also has a role in postmitotic cells. Berezovska and colleagues (190) were the first to show that Notch is expressed in mouse and human adult brain, and subsequently, Sestan and coworkers (191) showed that Notch is involved in regulating the capability of neurons to elaborate and extend neurites. They found that formation of neuronal contacts activated Notch signaling, leading to restriction of neuronal growth and an arrest in maturation. Increased Notch activity was concomitant with an increase in the number of interneuronal contacts and cessation of neurite growth, whereas inhibition of Notch signaling promoted neurite extension. With the report by Sander and colleagues (150), we now know that Notch is involved in functioning of the adult enteric nervous system. They documented differential expression of Notch receptors and ligands in adult rat gut and used immunohistochemical markers to correlate their expression with specific neuronal subtypes. Notch1 and Jagged2 but not Notch2 or Jagged1 were detected in ganglia of the submucosal and myenteric plexus. Notch1 was expressed by cholinergic neurons lacking calretinin or nitric oxide synthase, and

therefore may be restricted to neurons that elaborate polarized projections to the intestinal muscle layers (192,193). In contrast, Jagged2 was more widely expressed than Notch1, prompting speculation that other Notch receptors may have roles in the enteric nervous system.

FUTURE DIRECTIONS In the years since the first Notch gene was cloned, we have come to appreciate how important the Notch signaling pathway is in biology. Our increasing knowledge of its interaction with other signaling networks heralds a better understanding not only for how cell fate is determined by this pathway, but also for the impact of Notch signaling on other cellular functions. There is still much to learn, and that is no more evident than when one considers the complexity of the gastrointestinal tract and the diversity of cells that express Notch. We are really only at the beginning of understanding Notch function in the gastrointestinal tract, but the few studies that have been done reveal the broad scope of Notch involvement in the gut. Current evidence implicates Notch in the development of intestinal epithelial cell lineages, but its role remains to be proven formally by direct manipulation of receptor or ligand activity. To better understand the extent of Notch signaling in the gut, we need to better define the cells in which the Notch receptors and its ligands are expressed. Given the low level of endogenous Notch expression, expanded application of knockin technology to monitor expression of Notch gene promoters with sensitive and stable reporters is expected to provide more precise data on expression. Is Notch expressed in intestinal epithelial stem cells? What are the cells in the lamina propria, in the Peyer’s patch, or in the enteric nervous system that express Notch, and what role does Notch play in them? With this knowledge in hand, studies of Notch function in gut cell lines and targeting of inducible gene knockouts to specific gut cells are needed to help map the mechanisms and consequences of Notch action in the gut.

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306 / CHAPTER 10 154. Sriuranpong V, Borges MW, Strock CL, Nakakura EK, Watkins DN, Blaumueller CM, Nelkin BD, Ball DW. Notch signaling induces rapid degradation of achaete-scute homolog 1. Mol Cell Biol 2002;22: 3129–3139. 155. Apelqvist A, Li H, Sommer L, Beatus P, Anderson DJ, Honjo T, Hrabe de Angelis M, Lendahl U, Edlund H. Notch signalling controls pancreatic cell differentiation. Nature 1999;400:877–881. 156. Lee JC, Smith SB, Watada H, Lin J, Scheel D, Wang J, Mirmira RG, German MS. Regulation of the pancreatic pro-endocrine gene neurogenin3. Diabetes 2001;50:928–936. 157. Gradwohl G, Dierich A, LeMeur M, Guillemot F. Neurogenin3 is required for the development of the four endocrine cell lineages of the pancreas. Proc Natl Acad Sci U S A 2000;97:1607–1611. 158. Bermingham NA, Hassan BA, Wang VY, Fernandez M, Banfi S, Bellen HJ, Fritzsch B, Zoghbi HY. Proprioceptor pathway development is dependent on Math1. Neuron 2001;30:411–422. 159. Ben-Arie N, Hassan BA, Bermingham NA, Malicki DM, Armstrong D, Matzuk M, Bellen HJ, Zoghbi HY. Functional conservation of atonal and Math1 in the CNS and PNS. Development 2000;127:1039–1048. 160. Okano H, Imai T, Okabe M. Musashi: a translational regulator of cell fate. J Cell Sci 2002;115:1355–1359. 161. Sakakibara S, Okano H. Expression of neural RNA-binding proteins in the postnatal CNS: implications of their roles in neuronal and glial cell development. J Neurosci 1997;17:8300–8312. 162. Kaneko Y, Sakakibara S, Imai T, Suzuki A, Nakamura Y, Sawamoto K, Ogawa Y, Toyama Y, Miyata T, Okano H. Musashi1: an evolutionally conserved marker for CNS progenitor cells including neural stem cells. Dev Neurosci 2000;22:139–153. 163. Sakakibara S, Nakamura Y, Yoshida T, Shibata S, Koike M, Takano H, Ueda S, Uchiyama Y, Noda T, Okano H. RNA-binding protein Musashi family: roles for CNS stem cells and a subpopulation of ependymal cells revealed by targeted disruption and antisense ablation. Proc Natl Acad Sci U S A 2002;99:15194–15199. 164. Clarke RB, Anderson E, Howell A, Potten CS. Regulation of human breast epithelial stem cells. Cell Prolif 2003;36(suppl 1):45–58. 165. Nishimura S, Wakabayashi N, Toyoda K, Kashima K, Mitsufuji S. Expression of Musashi-1 in human normal colon crypt cells: a possible stem cell marker of human colon epithelium. Dig Dis Sci 2003;48: 1523–1529. 166. Potten CS, Booth C, Tudor GL, Booth D, Brady G, Hurley P, Ashton G, Clarke R, Sakakibara S, Okano H. Identification of a putative intestinal stem cell and early lineage marker; musashi-1. Differentiation 2003;71:28–41. 167. Imai T, Tokunaga A, Yoshida T, Hashimoto M, Mikoshiba K, Weinmaster G, Nakafuku M, Okano H. The neural RNA-binding protein Musashi1 translationally regulates mammalian numb gene expression by interacting with its mRNA. Mol Cell Biol 2001;21: 3888–3900. 168. Radtke F, Wilson A, MacDonald HR. Notch signaling in T- and B-cell development. Curr Opin Immunol 2004;16:174–179. 169. Gupta-Rossi N, Storck S, Griebel PJ, Reynaud CA, Weill JC, Dahan A. Specific over-expression of deltex and a new Kelch-like protein in human germinal center B cells. Mol Immunol 2003;39:791–799. 170. Wilson A, Ferrero I, MacDonald HR, Radtke F. Cutting edge: an essential role for Notch-1 in the development of both thymusindependent and -dependent T cells in the gut. J Immunol 2000;165: 5397–5400. 171. Wilson A, MacDonald HR, Radtke F. Notch 1-deficient common lymphoid precursors adopt a B cell fate in the thymus. J Exp Med 2001;194:1003–1012. 172. Radtke F, Wilson A, Stark G, Bauer M, van Meerwijk J, MacDonald HR, Aguet M. Deficient T cell fate specification in mice with an induced inactivation of Notch1. Immunity 1999;10:547–558.

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CHAPTER

11

Physiology of Gastrointestinal Stem Cells Alda Vidrich, Jenny M. Buzan, Sarah A. De La Rue, and Steven M. Cohn What Is a Stem Cell? 308 Organization of Structural/Proliferative Units in the Gastrointestinal Epithelium, 308 Small Intestine, 308 Colon, 308 Gastric Epithelium, 308 Morphogenesis of the Gastrointestinal Proliferative Units, 309 Clonality of the Structural/Proliferative Unit, 310 Studies of Chimeric Organisms, 310 X-Chromosome Inactivation, 312 In Vivo Stem Cell Mutagenesis Assays, 312 Stem Cell Number and Hierarchy, 313

Stem Cell Number, 313 Stem Cell Hierarchy, 314 Regulation of Stem Cell Number, 314 Putative Stem Cell Markers, 315 The Stem Cell Niche, 315 Regulation of Stem Cell Function, 316 Signaling Pathways, 316 Growth Factors and Peptides, 327 Inflammation: Cytokines and Other Mediators, 332 Stem Cell Plasticity, 335 Conclusion, 336 Acknowledgments, 336 References, 336

Epithelia, such as those lining the gastrointestinal tract, are able to undergo continuous self-renewal and repair because they contain a population of undifferentiated stem cells and long-lived progenitor cells that have a large capacity for selfrenewal and can give rise to daughter cell lineages that differentiate into the mature epithelial cell types needed for normal function of the epithelial layer. The “Unitarian hypothesis,” which advanced the concept that all of the differentiated cell types found in the small intestinal and colonic epithelia arise from a common multipotent progenitor or “stem” cell located near the base of each intestinal crypt of Lieberkühn was first proposed by Cheng and Leblond in 1974 (1–5). In the ensuing decades since this seminal work, investigators have further refined our knowledge of the role that epithelial stem cells play in normal gut development, in epithelial regeneration associated with mucosal injury or inflammation, and in intestinal neoplasia. A number of functional and kinetic properties of intestinal stem cells have also been defined from studies of the topographic arrangement of differentiated stem cell descendents in aggregation

chimeras (tetraparental mice), from in vivo mutagenesis studies, and from the radiobiologic response of intestinal crypts after γ-irradiation (6–16). However, the most dramatic progress in deciphering many of the molecular pathways involved in regulating these processes has been made through advances in our ability to experimentally manipulate the expression of genes in transgenic and knockout mice. The goal of using stem cells as a therapeutic modality for the restoration and repair of diseased organs has captured the imagination of both scientists and the lay public. However, the possibility of isolating and manipulating embryonic stem cells first became a reality more than 20 years ago, with the successful growth of murine embryonic stem cells in culture (17,18). The derivation of embryonic stem cell lines from human blastocysts was realized more recently (19,20). The potential therapeutic uses of human embryonic stem cells have met with significant opposition based on ethical considerations. Nevertheless, a number of human embryonic stem cell lines have been produced. Opposition to the generation of human embryonic stem cells has fueled the investigation of stem cells from adult tissues. Although obviously promising, these studies are hampered by the lack of identifiable stem cell markers and limited knowledge of the requirements for growth and maintenance of these cells in culture. Nevertheless, in vivo studies using knockout mice and mice expressing a variety of

A. Vidrich, J. M. Buzan, S. A. De La Rue, and S. M. Cohn: Digestive Health Center of Excellence, University of Virginia, Charlottesville, Virginia 22908. Physiology of the Gastrointestinal Tract, Fourth Edition, edited by Leonard R. Johnson. Academic Press, 2006.

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308 / CHAPTER 11 mutated genes have begun the process of identifying and characterizing the genes, signals, and pathways that regulate epithelial stem cells in their native tissue environment or niche. These have been supplemented by studies of human diseases with underlying gene mutations that alter stem cell fate or function. Understanding the biology of stem cells, both embryonic and adult, is of fundamental importance to the studies of tissue and organ development, the homeostatic maintenance of renewing tissues, tissue repair, and intestinal neoplasia, as well as advancing developments in tissue engineering. This chapter reviews the current knowledge pertaining to the regulation of epithelial stem cells of the gastrointestinal tract during both gut morphogenesis and maintenance of intestinal homeostasis.

WHAT IS A STEM CELL? Certain tissues of the body display continual turnover because cells lost through injury or terminal differentiation must be replaced to maintain the functionality of the tissue. The rate of cell replacement changes to match the rate of cell loss so that the tissue can maintain homeostasis under either normal or perturbed conditions. The gastrointestinal tract is a prime example of a continuously and rapidly renewing tissue. Terminally differentiated cells of the intestine are shed from the villus tip into the gut lumen. These cells are replaced by enterocytes arising from the proliferation of rapidly amplifying transit cells, which are the progeny of stem cells residing in the bottom half of the crypts. Currently, there are no specific morphologic or molecular criteria for identifying epithelial stem cells in the gut. Although different characteristics have been attributed to the stem cell population in the literature, the definition of a stem cell advanced by Potten and Loeffler (16) is used for purposes of discussion in this chapter. They proposed that a number of characteristics should be used to identify stem cells, or the properties of “stemness” that specify the cells’ functional capability under a variety of circumstances (16). The properties defining intestinal epithelial stem cells include: (1) an undifferentiated cell with a relatively unlimited potential for proliferation, (2) the capacity for self-maintenance, (3) the ability to give rise to a variety of cell lineages (pluripotency), and (4) the ability to regenerate the entire epithelium in response to an injurious event.

ORGANIZATION OF STRUCTURAL/ PROLIFERATIVE UNITS IN THE GASTROINTESTINAL EPITHELIUM

and colonic epithelia arise from a common multipotent progenitor cell located in the lower region of the crypt or replicative unit of the intestine as first proposed by Cheng and Leblond (1–5). In this view of the organization of the crypt, stem cells are thought to reside in a restricted zone just above the Paneth cells at the crypt base. These stem cells have a high potential for self-renewal and a large potential for cell division. A small fraction of epithelial cells at this location (cell positions 4–5 from the crypt base) appear to replicate slowly (Tc > 30 hours) and are thought to represent the stem cell and long-lived progenitor cell populations (Fig. 11-1) (14,21,22). Transit-amplifying (TA) cells are more rapidly proliferating (Tc ~12–13 hours in the mouse) daughters of stem cells that undergo 4 to 6 divisions within a zone of proliferation located in the mid-crypt. Differentiation occurs as the progeny of the TA cell population migrate in vertically coherent and topologically constrained bands either onto the villus epithelium or toward the base of the crypt (1,16,23). Goblet cells and enterocytes undergo terminal differentiation as they are rapidly translocated onto the villus. Paneth cells arise during downward migration to the crypt base, while enteroendocrine cells differentiate during migration from the zone of proliferation in either direction. Despite the high turnover of cells in the adult small intestine, the sizes of the crypts and the villi remain constant. Thus, epithelial cell differentiation appears to be tightly coupled to cell migration, and the rates of cell renewal, migration, and cell loss must be tightly regulated and interrelated processes.

Colon The organization of the adult colonic epithelium is similar to the small intestine except that there are no villi in the colon. A pleuripotent stem cell located at or near the base of each colonic crypt gives rise to each of the three principal epithelial cell lineages present in the colon (Paneth cells are not found in the mouse colon under normal circumstances) (6,10). Rapidly replicating TA cells are found in the lower half of the crypt, and cell-cycle times in this region of the crypt have been estimated to be approximately 30 to 33 hours (24–27). As in the small intestine, the pleuripotent epithelial stem cell is thought to have a much longer cell-cycle time than the TA cell population (24). Cells differentiate during migration from the proliferative zone, which lies in the lower third of the colonic crypt, to a polygonal-shaped surface epithelial cuff that surrounds the orifice of each gland (9).

Gastric Epithelium Small Intestine In the small intestine, the progeny of replicating undifferentiated epithelial cells located in the crypts rapidly and continuously replace the differentiated epithelial cell population. All of the differentiated cell types found in the small intestinal

Patterns of gastric epithelial cell renewal in the mucous and zymogenic units of adult CD1 mice have also been defined by Lee and Leblond (28,29). Their studies suggest that granule-free stem cells located in the isthmus region give rise to daughter cells. These daughter cells differentiate

PHYSIOLOGY OF GASTROINTESTINAL STEM CELLS / 309

3 days Differentiated cells– enterocytes, goblet cells, enteroendocrine cells

Villus Crypt

Transitamplifying cells Tc~12_13 hrs

Pericryptal fibroblast

~150 cells

"Anchored" stem cell Tc > 30 hrs

Paneth cell

FIG. 11-1. Epithelial renewal and differentiation in the adult intestine. Each crypt contains one or more slowly cycling “anchored” clonogenic stem cells located at approximately cell positions 4 to 5 from the crypt base. One of the daughters of this stem cell eventually gives rise to long-lived progenitor cells and subsequently to the rapidly proliferating, undifferentiated transit-amplifying cell population. Enterocytes, goblet cells, and enteroendocrine cells differentiate during upward migration of epithelial cells onto the villi, whereas Paneth cells differentiate as they migrate to the crypt base.

as they migrate away from the isthmus in a bipolar fashion. In the peripyloric mucous gland, two types of exocrine cell precursors are produced. One type contains homogeneous dense mucous granules. These cells replicate several times, move upward into the pit region, and then migrate to the surface. The lifespan of these surface mucous cells is approximately 3 days. The other type of daughter cell possesses apical granules with dense cores. These latter cells migrate downward and subsequently differentiate into relatively longlived glandular mucous cells (28,29). Rapidly proliferating undifferentiated progenitor cells of zymogenic glands are also found in the isthmus. These undifferentiated cells are thought to give rise to immature forms of several cell types that subsequently undergo a complex bipolar migration. Surface mucous cells migrate upward to the luminal surface as their name implies. Parietal (acidsecreting) cells migrate downward to the neck and base of

the glands. Mucous neck cells also migrate downward and are believed to undergo successive transformation into mucozymogenic and then long-lived zymogenic cells. Enteroendocrine cells of the gastric epithelium arise from stem cells in the isthmus and join the downward migration into the glands (30).

MORPHOGENESIS OF THE GASTROINTESTINAL PROLIFERATIVE UNITS The molecular mechanisms that regulate intestinal development have been difficult to study in humans because many of these maturational and morphogenic events occur during the first trimester of prenatal development and are not accessible to easy experimental manipulation. In contrast to humans, rodents are altricial animals, thus many of

310 / CHAPTER 11 these same developmental events occur shortly before birth or during the suckling period and the suckling/weaning transition. This difference in the timing of developmental events has made the rodent an attractive model system for studying intestinal ontogeny. The rodent gastrointestinal epithelium undergoes a remarkable morphologic reorganization beginning in late gestation, changing from a poorly differentiated pseudostratified cuboidal epithelium to a simple stratified columnar epithelium (31–33). Nascent villi form by the upward growth of mesenchymal tissue into this monolayer of columnar epithelial cells. At birth, the proliferative unit of the mature intestinal epithelium, the crypt, has not yet formed (Fig. 11-2). The fetal and neonatal homolog of the crypt, the intervillus epithelium, is populated by the descendants of several pluripotent stem cells (7,10). Formation of the small intestinal crypts begins during early postnatal life by invagination of the intervillus epithelium into the underlying mesenchyme. This poorly understood process is completed by the suckling/weaning transition (postnatal day 14 in the mouse). Evidence suggests that, during the process of nascent crypt formation, the newly forming polyclonal crypt epithelium is converted to monoclonality (i.e., each crypt contains an epithelial cell population derived from a single multipotent stem cell; see Clonality of the Structural/Proliferative Unit later) (7). Morphogenesis of crypts in the colon resembles that of the small intestine but begins in late gestation (34–36). The number of glands in the stomach, small intestine, and colon expands greatly by a process of gland or crypt division (fission) beginning during the suckling period and extending through early (post-weaning) adulthood to reach the number of crypts present in the adult small and large intestine or glands present in the stomach (37). Since each crypt or gland is ultimately derived from at least one pluripotent stem cell, it Late suckling intestine

Newborn intestine

is likely that a marked expansion of the number of resident clonogenic stem cells within the gastrointestinal epithelium also occurs during this developmental period. During late fetal and early postnatal life, regional gradients of differentiation are also established along both the duodenal-colonic and the crypt-villus axes of the gut (6,38–43). Several studies have suggested that pericryptal fibroblasts in the mesenchymal tissue underlying the epithelium and secreted extracellular matrix components are important in regulating the establishment of region-specific differentiation programs (44–48).

CLONALITY OF THE STRUCTURAL/ PROLIFERATIVE UNIT The direct identification of stem cells in the gastrointestinal epithelium has been difficult due to the lack of specific morphologic or immunohistochemical markers that identify this unique and rare cell population within each intestinal crypt or gastric gland. Rather, the existence and functional properties of the parental stem or progenitor cell have been inferred by experimental characterization of the daughter lineages they produce. A number of studies have suggested that each adult crypt is monoclonal, that is, genetically derived from a single stem cell. These data come from a variety of experimental systems in which histochemical markers were used to identify the genetic origin(s) or tag lineages of cells within the crypt epithelium. The model systems used include allophenic tetraparental mice (7–9), use of X chromosome– linked enzyme markers (10,49–51), in vivo mutagenesis in mice heterozygous for cell-surface lectin markers (11,12), and studies tracing age-dependent extinction of transgene expression in the colon (6). In each of these systems (see later), the crypt behaves as if all of its cells are derived from a single multipotent stem cell. Pulse-chase labeling studies to investigate cell migration suggest that these stem cells reside at about the fourth cell position from the base of the crypt (14).

Studies of Chimeric Organisms Villi

Inter-villus epithelium

Replicating cells

Crypt

Stem cell

FIG. 11-2. Morphologic remodeling of the intestinal epithelium occurs in the suckling mouse. Arrows indicate cell migration pathways. Enterocytes, goblet cells, and enteroendocrine cells differentiate during migration onto the villus epithelium from the zone of replicating cells located midcrypt. Paneth cells differentiate during migration of progenitors to the crypt base.

Mouse embryo aggregation chimeras are formed from the fusion of two zygotes that differ in the expression of a marker for the tissue of interest. Adult mice derived from these embryonic chimeras are composed of tissues that are a mosaic of cells derived from two parental genotypes (7–9,52,53). This technique has provided many important insights into the clonal origins of epithelial cells in various tissues including the intestine. C57BL/6J Lac (B6) mice express binding sites for the lectin Dolichos biflorus agglutinin (DBA), whereas SWR mice do not. The intestines of C57BL/6J Lac (B6),SWR aggregation chimeras are mosaics of cells derived from the two parent strains. However, when binding of labeled DBA to crypt epithelial cells was examined histologically in these chimeric mice, crypts were composed of cells that were wholly positive or wholly negative for DBA staining, with no mixed

PHYSIOLOGY OF GASTROINTESTINAL STEM CELLS / 311 A

B

Villi

Crypt Pericryptal fibroblast Goblet cells Enterocytes

Paneth cells Enteroendocrine cells

CO

MO

−?

GLP-2

Stem cell

GLP-2R

Sign

al

Endothelial cell

Enteric neuron

Crypts

FIG. 11-3. The intestinal stem cell niche. (A) Illustration of the stem cell hierarchy of the crypt. (See Color Plate 2.) Chimeric mice can be generated by injecting pluripotent embryonic stem cells from one genetic background into blastocysts representing another genetic background. The small intestine of the resulting mouse contains cells representing both genetic backgrounds. The image on the left is a section prepared from the proximal small intestine of an adult chimeric mouse. The finger-shaped villus is supplied with epithelial cells from two adjacent crypts. One crypt is populated entirely by blue epithelial cells, representing one genetic background. The other crypt is populated entirely by nonblue cells, representing the other genetic background. Crypts do not contain a mixture of cells from both genetic backgrounds. Thus, they are monoclonal, that is, composed of epithelial cells ultimately derived from a single progenitor. This progenitor occupies the highest position in the stem cell hierarchy. The precise number of progenitor-derived multipotent stem cells that are active in each crypt is uncertain. Note that cells from each crypt migrate up the villus in an orderly fashion, forming distinct columns with discrete borders. Migration is completed in 3 to 5 days. The highly organized migration is illustrated further in the inset that shows a whole-mount preparation of intestine from a chimeric mouse. Striped villi contain differentiating epithelial cells derived from monoclonal blue crypts and nonblue (white) crypts. (B) Diagrammatic representation of the stem cell niche. An active multipotent stem cell (SC) gives rise to a C0 daughter that produces the enterocytic lineage and to descendants that generate secretory lineages (goblet, Paneth, and enteroendocrine cells; note that it is uncertain whether all are derived from M0). Glucagon-like peptide-2 (GLP-2) produced by a subset of enteroendocrine cells is able to stimulate proliferation of the Co daughter via interaction with enteric nervous system neurons that express the GLP-2 receptor (GLP-2R). The nature of the neuronal signal that affects Co is unknown. (Reproduced from Mills JC, Gordon JI. The intestinal stem cell niche: there grows the neighborhood. Proc Natl Acad Sci U S A 2001;98:12334–12336, by permission.)

crypts being observed (Fig. 11-3A). This strongly implies that each crypt is composed of a single clonal population of epithelial cells (7,8). The data from these mice were informative for the Paneth, mucous, and columnar cells, but a result could not be determined for the endocrine cells (8). Interestingly, nascent crypts were polyclonal during the first 2 weeks after birth—that is, composed of a mixture of cells from C57BL/6 and SWR parents (7). However, after this point, crypts were composed of cells derived from either C57BL/6 parents or the SWR parents, but not a mixture of cells from both parents. This subsequent “cleansing,” or purification, of the crypt is proposed to occur either by overgrowth and extrusion of

one of the stem cells over the other(s), or by fission of the crypts, with individual stem cells segregating into separate crypts. Another approach for identifying the clonal origins of cells within a crypt is by detecting the presence of the Y chromosome in crypt epithelial cells of mouse aggregation chimeras produced from fusion of a female and a male blastocyst. These chimeras were constructed in a similar manner to the C57BL/6J Lac (B6),SWR mice aggregation chimeras described earlier (30). Experiments conducted in the resulting XX/XY mice confirmed the observation that crypts (glands) were monoclonal (i.e., crypts containing a mixture of female cells and

312 / CHAPTER 11 male cells were not observed) from 2 weeks after birth. In addition, these experiments were extended to confirm the origin of the neuroendocrine cells within the crypt epithelium. A combination of in situ hybridization for the Y chromosome and immunohistochemistry for the enteroendocrine cell-specific marker gastrin was used. This technique demonstrated that enteroendocrine cells in the female areas of the chimeric stomach were Y chromosome negative, while those in the male areas were almost exclusively Y chromosome positive (30). Human chimerism can also result spontaneously from the occurrence of nondisjunction within a cell of the early developing embryo. An example of this is loss of the Y chromosome, which results in an XO/XY chimeric individual (54). Tissue was obtained from a single XO/XY individual during a prophylactic colectomy for familial adenomatous polyposis (FAP). The tissue was then probed using Y-chromosome– specific probes in a non-isotopic in situ hybridization assay. These probes revealed that the intestinal crypts in normal areas of the tissue were almost entirely composed of either Y chromosome positive or negative cells, with only 4 of the 12,614 crypts scored containing a combination of both. This observation is consistent with the Unitarian hypothesis, as proposed from the observations made during the aforementioned murine studies. Interestingly, in regions of the colonic tissue that contained tubular adenomas, approximately 76% of the crypts were polyclonal. This observation suggests that crypt monoclonality is actively maintained in the normal mucosa.

X-Chromosome Inactivation X-chromosome inactivation has also been used as a tag to trace the origins of epithelial cell lineages in the mouse intestinal tract. In gut epithelium, X-chromosome inactivation is completed by embryonic day 11.5 (E11.5), well before the commencement of stomach gland morphogenesis (49). A transgenic mouse line that contains a tandem insertion of copies of the lacZ reporter gene on the X chromosome (H253 mice) was used to examine the origins of cell lineages in the gastric epithelium (50). Hemizygous female H253 mice show mosaic expression of the transgene as a result of X-chromosome inactivation that normally occurs during development. The clonality of the gastric glands was examined in the fundic and pyloric regions of female H253 hemizygotes at different developmental time points. Most of the glands were found to be polyclonal at early developmental time points, with an estimated three or four stem cells per gland. However, by 6 weeks of age, the glands had become monoclonal, being either entirely positive or negative for β-galactosidase activity. Two alternative mechanisms were proposed as possible explanations for the appearance of this monoclonality: either emergence of a dominant stem cell clone, or purification through segregation of stem cells to daughter glands as gastric glands undergo fission (50).

In another study, the colonic crypts of H253 mice were also shown to be monoclonal (51). The additional observation was made that 5% to 10% of the glands retain their polyclonal nature into adulthood. The authors proposed that a possible explanation for this could be that these glands have not undergone fission, are doing so at a reduced rate, or had a greater original number of stem cells. None of these alternative explanations has been confirmed. The clonality of the fundic and pyloric glands in the human stomach has been studied by investigating polymorphisms in two X chromosome–linked genes: the phosphoglycerate kinase (PGK) gene and the androgen receptor (HUMARA) gene (50). These were carried out in a similar manner to the X-inactivation studies in mice; however, a somewhat different result was obtained. Although the pyloric glands appeared to be homotypic for both the PGK and HUMARA loci (i.e., they were monoclonal), approximately half of the fundic glands were observed to be heterotypic for both loci. This indicates that approximately half of the fundic glands are polyclonal and demonstrates that the situation in humans is more complex than in mice, with regional differences apparent in the gastric mucosa (50).

In Vivo Stem Cell Mutagenesis Assays Characterization of animals with induced somatic mutations in the intestine has provided further information regarding cell lineage relationships, and properties of stem cells and progenitor cells within the crypt epithelium. For example, mutations at the Dlb-1 locus, which encodes the DBA lectin-binding site, have been studied in the intestine in situ after in vivo mutagenesis with ethylnitrosourea. The F1 progeny of C57BL/6J (DBA receptor–positive) crossed with SWR (DBA receptor–negative) mice are heterozygous for expression of the DBA receptor. When this locus is abolished, either spontaneously or using ethylnitrosourea, cells that are negative for DBA binding occur. Examination of the crypts in the small intestine of these mice showed a subset of wholly DBA-negative mutated crypts that persisted indefinitely. This observation is consistent with a Dlb-1 gene mutation in a single stem cell that is able to give rise to the entire epithelial cell population in a given crypt (11,12). In addition to wholly DBA-negative crypts, partially unstained crypts were also seen. However, the partially negative crypts were extinguished over time, suggesting that they resulted from a mutation in a progenitor or TA cell rather than an anchored stem cell within the crypt. More recently, Bjerknes and Cheng (13) found that the mutagen, N-nitroso-N-ethylurea, can induce clones of cells in SWR mice (Dlb-1−/−) that are able to bind DBA. This results in a clearer signal, where clones of lectin-positive cells can be observed on an otherwise unstained background. Various progenitor cell populations were characterized within the crypt by the DBA-positive staining of differentiated progeny that they produce. Bjerknes and Cheng have used this specific

PHYSIOLOGY OF GASTROINTESTINAL STEM CELLS / 313 progenitor assay by somatic mutation (SPASM) technique to demonstrate the presence of long-lived progenitor cells committed to columnar cell differentiation (C0), or committed to mucous/secretory cell differentiation (M0). A third mixed progenitor cell type was also identified that could give rise to either mucous or columnar cell lineages. Data obtained in these studies allowed the researchers to estimate that mucous cell clones (Mo) persist for an average of 100 days. Such long-lived progenitors appear to be intermediate in the hierarchy between the stem cell and the rapidly replicating transit cell population (13). Short-lived committed progenitor cells for both columnar (C1) and mucous (M1) cells were also identified, in addition to pluripotent stem cells that were able to give rise to all epithelial cell types. Somatic mutagenesis of genes on the X chromosome has also been useful in studying clonal relations within the crypt. As one allele of an X-linked gene is inactivated in the adult intestine due to X-chromosome inactivation, a single mutation in the remaining active allele is sufficient to induce a mutant phenotype that can be topologically examined by immunohistochemistry or histochemical staining for enzyme activity. For example, Griffiths and colleagues (10) studied intestinal mutations induced by dimethyl hydrazine (DMH) in the X-linked gene, glucose-6-phosphate dehydrogenase (G6PD). When female mice were administered a single dose of DMH, either partially or wholly G6PD-negative crypts resulted. The presence of partially negative crypts was found to be transient, with a decrease in frequency occurring relative to the time after administration of DMH. In parallel to this decrease in the number of partially negative crypts, there was an increase in the frequency of wholly negative crypts. The initial explanation proposed for this phenomenon was based on the presence of multiple stem cells in a crypt, with random loss occurring after time. However, the possibility that “cleansing” could occur by crypt fission, with a concomitant increase in wholly negative crypts, has gained credence more recently. This would involve the segregation of stem cells from partially negative crypts into separate, monoclonal crypts that were wholly negative or wholly positive (55). Insights into the clonality of human colonic crypts have been gained from studies of spontaneous mutations within the gene locus encoding the enzyme O-acetyl transferase (OAT). The mucus secreted from the goblet cells in the colon of most of the human population contains O-acetylated sialic acid residues. However, 9% of the population are homozygous for a mutation in the gene that encodes the enzyme OAT, and instead secrete mucus containing non–O-acetylated sialic acid (56,57). O-acetylated mucus is not stained by the mild periodic acid-Schiff (mPAS) technique, whereas the non–Oacetylated mucus is. An individual who is heterozygous expresses active OAT, and therefore stains negative for the mPAS technique. However, when this individual loses the remaining active gene, the genotype is converted to OAT −/−, which can result in occasional positively mPAS-stained crypts. When this occurs, the goblet cells are observed to stain uniformly PAS positive, from the base to the luminal surface

of the crypt (58). This could be caused either by a spontaneous somatic mutation within a crypt stem cell or by loss of a functional OAT allele through mitotic nondisjunction within an individual crypt stem cell, which subsequently colonizes the crypt.

STEM CELL NUMBER AND HIERARCHY Stem Cell Number Although the many genetic studies cited in the previous section suggest that the adult crypt is ultimately derived from a single genetically determined stem cell, other systems for estimating stem cell numbers have yielded differing results. For example, estimates of stem cell number based on mathematical modeling from cell proliferation data obtained by either 3H-thymidine labeling or by quantifying mitoses have suggested that there are approximately four to six ultimate clonogenic stem cells per crypt in the small intestine (23,24). Although the rates of epithelial renewal in the colon can be accounted for by the presence of only a single stem cell per colonic crypt, the models are also consistent with a greater number of stem cells per colonic crypt. Another powerful experimental approach that has been used to assess the number and functional potential of progenitor cells is based on the capacity of these cells to regenerate individual crypts after injury. Originally described by Withers and Elkind, this approach has been used by a number of investigators to estimate the stem cell number in the intestinal and colonic epithelium after γ-irradiation or cytotoxic therapy (14,15,24,59–63). The microcolony formation assay is based on the capacity of surviving stem cells (clonogens) to regenerate crypt-like foci of replicating epithelial cells, which can be scored histologically 3 to 4 days after radiation injury (15,62). Within 3 to 6 hours after irradiation, many of the cells within the lower third of the crypt undergo p53-mediated apoptosis. The remaining transit cells within the crypt undergo cell-cycle arrest and cease replication, although they continue to migrate out of the crypt and onto the villus. Thus, the crypt is rapidly depleted of cells and disappears as a recognizable structure 24 to 36 hours after irradiation. If one or more clonogenic stem cells within a crypt survive irradiation, they will proliferate, ultimately giving rise to regenerative crypts that first appear in the small intestine approximately 72 to 96 hours after irradiation. These crypts, or “microcolonies,” can easily be counted, and the fractional crypt survival in response to varying radiation doses can be analyzed. An estimate of the number of stem cells (clonogens) in each crypt that retain the capacity to regenerate the epithelium can be derived from experiments measuring fractional survival of crypts after one or two dose fractions of irradiation (63–66). When extrapolated to zero dose, data from these experiments suggest that there may be four to six stem cells per crypt under unperturbed conditions in the small intestine.

314 / CHAPTER 11 Stem Cell Hierarchy The functional studies detailed earlier which indicate that crypts contain several stem cells with clonogenic potential is, on first appearance, at odds with the previously cited genetic analyses of crypt clonality that imply that crypts are monoclonal (i.e., derived from a single ultimate stem cell) in the adult intestine. However, the concept of crypt hierarchy proposed by Potten and colleagues (24,63,66) provides a potential resolution for this paradox. This concept arises from the observation that the number of clonogenic stem cells determined using the split-dose radiation injury model described earlier is highly dependent on the radiation dose used (63). When low doses of γ-irradiation are used to make this determination, there are approximately four to six clonogenic stem cells per crypt. However, as the dose of radiation is increased, the estimate of the number of clonogenic stem cells increases, reaching as high as 36 stem cells per crypt (24,63,66). Observations such as these coupled with studies examining the distribution of apoptosis as a function of cell position in the crypt, have led to the proposition of a threetiered hierarchy of stem cell potential within the lower crypt. In this model, the ancestral stem cell occupies the first tier, while a second tier of potential stem cells (the asymmetric daughters of the ultimate stem cell) is located above this level. This second tier of undifferentiated cells normally enter the TA population; however, they retain the ability to function as stem cells if the ancestral stem cells are lost or rendered nonfunctional. At even greater doses of irradiation, the immediate daughters of the second tier stem cells may also be recruited to function as clonogens to reconstitute crypts after injury. Thus, estimates of stem cell number obtained from functional assays such as radiation injury may depend on the nature of the experimental perturbations produced in the stem cell microenvironment.

Regulation of Stem Cell Number At steady state in the normal intestine, there is a continuous flow of cells from the crypt stem cell/TA cell compartment onto the villus tips. The number of stem cells in the crypt is tightly regulated with production of stem cells being balanced by cell loss. It is widely believed that, under unperturbed conditions, stem cell proliferation is asymmetric; that is, division of the parental stem cell gives rise to a daughter cell in addition to a new stem cell (self-renewal), thus maintaining a balance between crypt stem cell number and the production of differentiating cells. The precise mechanisms that regulate the proliferation of stem cells and whether they divide symmetrically or asymmetrically are unknown. In early studies, Potten and colleagues (67) postulated that selection of stem cells may occur through the expression of specific genes after division that enable autonomous stem cell function of one daughter cell. Alternatively, the expression of certain genes during mitosis results in a polarized cell division committing one of the

daughter cells to the differentiation pathway and the other to functioning as an autonomous stem cell (67). A third alternative is that local environmental signals may dictate stem cell behavior of progeny within the stem cell niche (68,69). Programmed cell death may also be an important mechanism for regulating stem cell number in the intestinal epithelium. In the adult small intestine, under unperturbed conditions, a persistent low frequency of apoptosis is seen in the crypts of animals and humans (14,70,71). This process, referred to as “spontaneous apoptosis,” occurs predominately in the lower regions of the crypt and appears to be restricted to the stem cell region, averaging four to five cell positions above the base of the crypt. Tight control of stem cell number is required to maintain a steady level of cellular output from the crypt onto the villus, thereby maintaining a stable crypt size. Thus, spontaneously occurring apoptosis is likely an inherent part of the regulatory mechanism that determines stem cell numbers and the size of the replicating transit cell population in the normal adult tissue (14,71). Various gene families have been found to be important in the regulation of apoptosis including p53, which is involved in the response of cells to DNA damage, and the Bcl-2 family of genes, whose products are capable of both suppressing and promoting apoptosis. In contrast with p53, Bcl-2 family members may be involved in regulating both spontaneous apoptosis and apoptosis that occurs in response to cellular injury. There are regional differences in the expression of both p53 and Bcl-2 family proteins between the large and small intestine. These differences directly relate to the frequency of spontaneous apoptosis and to tumor incidence in these regions of the gastrointestinal tract (72). Forced alterations in β-catenin/lef1 signaling can also induce apoptosis in the intervillus epithelium and alter the process of crypt stem cell purification to monoclonality described earlier, reinforcing the suggestion that apoptosis is a critical part of this process (73). The regional expression of the Bcl-2 gene family along the crypt-villus axis of the normal adult intestine has also been addressed. The family can be split into two groups: those proteins that are involved in suppression of apoptosis, which include Bcl-2 and Bcl-XL, and those that promote apoptosis, which includes Bax, Bad, Bcl-Xs, and Bak. Bak and Bad are expressed in the crypt, while Bcl-2 and Bcl-XL are expressed along the length of the villus (74). The proapoptotic protein Bax is strongly expressed in the stem cell region of the small intestine, that is, the lower region of the crypt. However, the distribution of Bax is quite different in the colon with little or no Bax found at the base of the crypts and moderate to high levels in cells of the surface epithelium (70). The precise cell-specific and regional expression of these proteins during normal intestinal development has not been fully characterized. Overall, these observations fit with the hypothesis that the ratio of deathpromoting and death-suppressing members of the Bcl-2 family in a given cell plays an important role in controlling its sensitivity to apoptosis.

PHYSIOLOGY OF GASTROINTESTINAL STEM CELLS / 315 Bcl-2–related proteins and p53 also play a role in preserving the genomic integrity of the stem cell population in response to genotoxic damage. Low doses of radiation or genotoxic compounds can induce p53-dependent apoptosis in stem cells in the lower crypt (14,75–77). These studies suggest that altruistic cell death may be one mechanism for ensuring the genetic integrity of crypt stem cells faced with an onslaught of environmental agents with genotoxic or mutagenic potential. Stem cells in the small intestine are more prone to initiate this process of altruistic apoptosis than stem cells in the colon. These regional differences in damage-induced apoptosis may partially explain the propensity of stem cells within the colonic epithelium to accumulate mutations, leading to a greater incidence of carcinoma in this tissue. Induction of cell cycle arrest is another potential mechanism for enhancing the genomic integrity of the stem cell population. Together with the expression of p53 that occurs rapidly after radiation injury, the cell cycle regulatory gene p21waf1/cip1 is also induced (24,72,78). Cell-cycle arrest induced by p21waf1/cip1, p53, or other regulatory proteins at G1/S or G2 checkpoints likely plays a role in providing sufficient time for repair of DNA damage before S phase or mitosis. A third mechanism has been proposed by Potten and colleagues (79) for protecting the genomic integrity of intestinal stem cells. In this model, the stem cell retains the template DNA, while newly synthesized DNA strands are selectively segregated to the committed daughter cells after the second round of stem cell division. Although this proposal remains controversial, it offers another potential mechanism for protecting stem cells from mutations that inevitably occur during DNA replication.

Putative Stem Cell Markers Although no definitive markers of intestinal stem cells have as yet been established, Potten and colleagues have identified Musashi-1 (Msi-1) as a potential candidate marker for the intestinal epithelial stem cell (80). Msi-1 encodes a neural RNA-binding protein originally identified in Drosophila that appears to be required for the asymmetric division of both Drosophila and mammalian sensory neural precursor cells (81–83). The expression of the Msi-1 gene was examined in murine intestine after irradiation, as well as at several different developmental stages (80). In the neonatal mouse intestine, Msi-1 is expressed in the cytoplasm of undifferentiated epithelial cells in nascent crypts, while in the adult intestine, it is expressed by a small number of cells just above the Paneth cell zone and in cells intercalated between Paneth cells. In addition, not all proliferating cells (i.e., Ki67-positive cells) express Msi-1. The distribution of Msi-1 immunoreactivity is consistent with the location of the stem cell population within the crypt epithelium. Radiation injury increases the expression of Msi-1, which is localized in the lower half to one third of the crypt, although it is only present in a subset of proliferating cells.

The wave of Msi-1–expressing cells correlates with the restoration of the clonogenic stem cell compartment after injury. These data suggest that Msi-1 is a marker of stem cells and early lineage specific progenitor cells. Further examination of both Msi-1 expression in the developing and adult intestine (84) and Hes-1, a transcription factor under the control of Msi-1, showed that both Msi-1 and Hes-1 are expressed in the intervillus epithelium. However, Msi-1 is found in only a few cells located above Paneth cells, whereas Hes-1 is more broadly distributed in epithelial cells in the lower and mid crypt. This finding that Hes-1–expressing cells have a broader distribution supports the argument that they may represent rapidly replicating TA cells of the crypt. This is consistent with the demonstrated need for Hes-1 expression for the differentiation of the absorptive enterocyte (see later).

THE STEM CELL NICHE The existence of a specialized physical and structural microenvironment, or niche, in which stem cells reside was first proposed by Schofield (85) and is now widely accepted. The stem cell niche is understood to encompass both cellular and molecular components that surround, support, and interact with stem cells. It provides a stable, protective environment in which the function of stem cells and their daughters, the TA cells, is maintained and regulated by factors and signals produced by the cells forming the niche. Stem cells also interact with the niche components as the needs of the tissue, organism, or both change during development or in response to injury or other physiologic changes. In the intestine, the crypt is the niche that houses epithelial stem cells and their TA daughters (see Fig. 11-3). This structure is formed early in intestinal development by the invagination of the intervillus epithelium into the surrounding mesenchyme. A number of cell types comprise the mesenchymal component of the intestinal stem cell niche including intraepithelial lymphocytes, blood vessels, enteric neurons, and pericryptal fibroblasts. Subepithelial myofibroblasts surround the intestinal crypts, while a basement membrane lies between the epithelial cells lining the crypt and the myofibroblasts. The intestinal subepithelial myofibroblasts (ISEMFs) form a syncytium that merges with the pericytes of the blood vessels, as well as extending throughout the entire lamina propria. These cells form a fenestrated sheath that is tightly apposed to the epithelial layer, and they have been indicated to play an essential role in interactions between the epithelium and the mesenchyme (86). In addition, secretion of growth factors such as hepatocyte growth factor (HGF), transforming growth factor-β (TGF-β), various fibroblast growth factors (FGFs), and perhaps other growth factors by the ISEMFs plays an essential role in the differentiation of the epithelial cells that express receptors for these factors (86). The interstitial cells of Cajal are another type of myofibroblast found in the intestine. These cells localize proximal to mural neurons, where they play

316 / CHAPTER 11 various roles, including as pacemakers for smooth muscle in the intestines, propagating electrical events, and modulating neurotransmission (87). Although both types of fibroblasts exist as a syncytium, it is unclear whether the two networks connect (86). Bjerknes and Cheng suggest that enteric neurons may promote proliferation of stem cell clones and long-lived columnar progenitor cell clones that differentiate into absorptive cells when the neuronal circuit is activated by glucagon-like peptide-2 (GLP-2), an enteroendocrine cell product (88). The existence of a niche is demonstrated by its independent existence in the absence of stem cells. In the gut, proof of the existence of a niche came from targeted disruption of the T-cell factor 4 (Tcf-4) gene (89). In the absence of Tcf-4 intestinal crypts were found to be devoid of a proliferating cell population, while the pericryptal mesenchymal components of the niche remained. Similarly, a dose of lethal radiation will deplete the epithelial component of the intestinal niche. If a single stem cell survives, the remaining mesenchyme is sufficient to support restoration of the stem cell compartment within the regenerating crypt.

REGULATION OF STEM CELL FUNCTION Signaling Pathways TCF-4 and the Wnt Signaling Pathway Wnt proteins are a family of secreted, small signaling molecules (90) that are known to regulate embryonic development, tissue morphogenesis, and maintenance of adult tissue homeostasis, as well as to play a key role in cancer development. Furthermore, studies have demonstrated the central role of Wnt signaling in regulating crypt epithelial stem cell proliferation during normal development and in tissue homeostasis. This is illustrated by the observation that mice lacking Tcf-4, a downstream component of Wnt signaling, do not have replicative cells, and presumably stem cells, in the intervillus epithelium from E16.5 (89). These mice are unable to form nascent crypts and die shortly after birth. The intracellular signaling cascade that links Wnt signaling to activation of β-catenin/Tcf-4 has been largely defined and is summarized in Figure 11-4 (90). The primary receptors for Wnts are the Frizzled family of proteins (91,92). However, Wnt signaling also requires a transmembrane molecule of the low-density lipoprotein (LDL) receptor–related protein (LRP) family, with which it forms a trimeric receptor complex that transduces the Wnt signal to intracellular proteins (91,92). Signaling via Wnt receptors is complex and thought to involve Wnt binding to the Fz/LRP receptor, which activates the complex and causes Axin to bind to LRP with subsequent stabilization of β-catenin (a transcriptional regulator) (90,93–97). Stabilized, cytoplasmic β-catenin translocates to the nucleus, where it complexes with the TCF/LEF DNA-binding

proteins, converting the TCF complex from a transcriptional repressor to an activator of gene transcription (98–102). In the absence of Wnt signaling, cytoplasmic β-catenin complexes with Axin and adenomatous polyposis coli (APC), at which point it is phosphorylated by the serine/ threonine kinases glycogen synthase kinase-3 (GSK-3) and casein kinase 1α (103–106). Axin and APC are thought to provide scaffolding for the interaction between the kinases and β-catenin (107,108). Once phosphorylated, β-catenin is ubiquitinated and targeted for degradation by the proteosome (109–111). Support for the role of the β-catenin/Tcf family in the regulation of epithelial morphogenesis comes from a report by van Noort and colleagues (112). They demonstrated the presence of unphosphorylated nuclear β-catenin in the undifferentiated intervillus epithelium, confirming that activation of the β-catenin/Tcf signaling pathway occurs as early as E16.5 in the fetal homolog of the adult crypt. Studies by Wong and colleagues (73,113) suggest that members of the β-catenin/Tcf family also regulate the selection of a single, genetically dominant pleuripotent stem cell in each crypt. This selection occurs during postnatal intestinal development in the mouse as described earlier (see Morphogenesis of Structural/Proliferative Units and Clonality of the Structural/ Proliferative Unit). This group engineered the in vivo expression of a stabilized β-catenin mutant only in the 129/Sv cells of B6-ROSA26,129/Sv chimeric mice (73,113). In these mice, artificially sustained expression of a Lef-1/β-catenin fusion protein during the time when signaling by this complex would normally be declining resulted in a dramatically greater frequency of apoptosis in the population of cells expressing the transgene. The 129/Sv stem cells, which expressed the stabilized β-catenin mutant, were selectively eliminated from the chimeric intestine after weaning, by which time crypt morphogenesis was completed. These experiments suggest that early extinction of Lef-1/β-catenin signaling in a pleuripotent crypt stem cell results in its selection as the genetically dominant stem cell within the crypt. However, other regulatory mechanisms affecting this selection process cannot be excluded. It has been proposed that the Wnt family of ligands provides the extracellular signals that induce activation of β-catenin/Tcf-4 intermediate signaling pathways, although the precise identity of these extracellular signals remains to be elucidated. Thus, other extracellular signals may contribute to the activation of the β-catenin/Tcf-4 signaling cascade. For example, FGF2 can regulate Tcf/β-catenin– mediated transcriptional activity in endothelial cells (114). This may occur in intestinal epithelial cells, as has been indicated by our studies of FGF receptor-3 (FGFR-3)–mediated signaling events in intestinal mucosal development. During normal morphogenesis of the small intestine, robust expression of FGFR-3 was noted in the intervillus epithelium and the stem cell region of newly forming crypts (115). A marked reduction in both the number of crypts and the size of the epithelial stem cell population was observed in the intestine of suckling FGFR-3 null mice.

PHYSIOLOGY OF GASTROINTESTINAL STEM CELLS / 317

FIG. 11-4. Diagrammatic representation of Wnt signaling. Cytoplasmic components of the Wnt signaling pathway. In naive cells (left), β-catenin forms a complex with Axin and adenomatous polyposis coli (APC). Axin acts as a scaffold for the protein kinases casein kinase 1α (CK1α) and glycogen synthase kinase-3 (GSK-3), and the PP2A protein phosphatase. PP2A may act on Axin, as well as other substrates in the Axin/APC complex. After phosphorylation by GSK-3 and CK1α, β-catenin is degraded by ubiquitination involving interactions with Slimb/-TrCP. After binding of the Wnt ligand (right), the Fz and low-density lipoprotein receptor–related protein (LRP) receptors recruit Dsh and Axin to the membrane, where they may interact with each other (dashed lines). Wnt signaling leads to inhibition of β-catenin degradation and its accumulation. As a result, β-catenin is stabilized in the cytoplasm and is no longer degraded. Negative regulators are outlined in black. Positively acting components are outlined in color. (Reproduced from Logan and Nusse [90], by permission.)

Furthermore, stimulation of the CaCo2 intestinal cell line by FGFR-3 ligands or transfection with a constitutively active form of FGFR-3 induced up-regulation of Tcf-4–mediated transcriptional activity in these cells (116). Conversely, inhibition of Tcf-4–mediated signaling in preconfluent Caco-2 cells by transfection of a dominant negative Tcf-4 mutant triggers precocious differentiation of these cells (117). Decreased activation of Tcf-4/β-catenin signaling is therefore a possible mechanism for the decreased size of the stem cell pool in FGFR-3 null mice, suggesting that regulation of intestinal stem cell dynamics by the Tcf-4/β-catenin family involves diverse extracellular signaling events that may be context dependent. Genes Downstream of β -Catenin/Tcf-4 Activation A number of target genes that respond to β-catenin/ Tcf-4 transcriptional regulation have been identified as mediators of stem cell proliferation that maintain the topologic organization of stem cells and progenitors within the crypt.

Eph Receptors and Ephrin Ligands The organization and hierarchy of cells within the intestinal crypt are highly ordered and topologically constrained, as discussed earlier. Studies have provided insights into possible mechanisms that may be involved in maintaining the relative positions of cells, including stem and progenitor cells, along the crypt–villus unit. Not surprisingly, the β-catenin/ Tcf transcription complex is involved in regulating this aspect of cellular organization along the crypt-villus axis. van de Wetering and colleagues (118) have identified a number of Tcf-4–regulated genes in colon cancer cells by expression of a dominant negative TCF (dnTCF) mutant. Among the genes down-regulated by expression of the dnTCF mutant were the EphB2 and EphB3 receptors. Conversely, ephrin ligands were up-regulated in these cells (118). Eph receptors and ephrin ligands play essential roles in cell–cell interactions, providing repulsive signaling in a wide range of developmental processes (119). In addition, the observation that the Tcf4–deficient E18 mouse embryos lacked intestinal EphB2 and EphB3 expression, while proliferating cells of normal intervillus epithelium express both Tcf-4 and EphB receptors,

318 / CHAPTER 11 Villus Crypt

Ephrin B1 Ephrin B2 EphB2

Stem cell

EphB3 Paneth cells

FIG. 11-5. Schematic representation of the expression gradients of EphB2, EphB3, and their ephrin ligands in crypts of the adult small intestine. Arrows show the direction of the gradient of Eph receptor and the respective ephrin ligand expression.

corroborated the regulation of EphB/ephrinB expression by signaling through Tcf-4 (118). The receptors for ephrins constitute a large subfamily of receptor tyrosine kinases. These receptors are grouped into EphA receptors that bind type A ephrin ligands and EphB receptors that bind type B ephrins (119). Both Eph receptors and ephrin ligands are membrane-bound proteins; thus, the interaction between the receptor and ligand requires direct cell–cell contacts. The most common type of interaction is generally one of repulsion of a cell neighbor, although examples of increased adhesion/attraction have been documented (119). Studies of EphB2 and EphB3 null mice demonstrate that expression of EphB receptors is necessary for the correct positioning of epithelial cells along the crypt-villus axis (120). EphB2 is expressed in a gradient along the crypt-villus axis with the greatest levels in cells at the bottom of the crypt (Fig. 11-5). Conversely, ephrinB ligands are expressed in an opposing gradient, with the highest levels observed in cells at the crypt-villus junction in adult intestine and at the periphery of the intervillus pockets in the neonatal gut, whereas no expression is observed at the crypt base. Only EphB3 is necessary for the downward migration of Paneth cells to the crypt base as Paneth cells distribute randomly throughout the crypt-villus axis in EphB3 null mice. Paneth cells do not express any of the ephrinB ligands, and therefore differ from the surrounding epithelial cells at the base of the crypt. Thus, the lack of ligand expression coupled with high expression of the EphB3 receptor provides Paneth cells with a positional address, or “zip code,” that directs their migration downward to the crypt base. Presumably, the lack of ephrinB ligands eliminates the “repulsive” response such expression would elicit in an area of EphB receptor–positive (crypt base) cells (120).

In the neonatal intestine, the interaction of EphB receptors on proliferating cells with their ephrin B1 ligand on adjacent differentiating cells gives rise to an organized topologic localization of these cells. This restricted topologic positioning is lost in the EphB2/EphB3 double-mutant intestine, where proliferating and differentiated cells intermingle. In addition, some intervillus regions of the double-mutant mice contain differentiated cells without an alteration in the position of proliferating cells in this region. This result suggests that in wild-type mice, expression of EphB receptors on proliferating cells actively restricts the migration of the ephrinBexpressing differentiated cells (120). Expression of the EphB receptor and the ephrinB ligand is more complex in the adult intestinal crypt, with different levels of expression along the length of the crypt (120). Nonetheless, EphB receptors still restrict the positions occupied by cells with different levels of ephrinB expression, suggesting that expression of EphB defines a location in the crypt that is restricted to stem cells and their rapidly proliferating daughters. Thus, in EphB2/EphB3 double-mutant mice, proliferating cells that normally localize toward the bottom of the crypt can be found at the crypt-villus junction, while differentiated cells with high levels of ephrinB that are generally found at the crypt-villus junction may relocate to the bottom of the crypt, where they are intermixed with islands of undifferentiated replicating cells. C-Myc The myelocytomatosis oncogene (c-Myc) is a member of the MYC family of transcription factors that are characterized by a basic helix-loop-helix leucine zipper (bHLH-LZ) motif. It binds E-box sequences as a heterodimer with MAX, another (bHLH-LZ) protein (121,122). The activated β-catenin/Tcf-4 complex has opposing effects on the expression of c-Myc and p21cip1/waf1, a key cell-cycle inhibitor and marker of differentiated cells (118). Transfection of colorectal carcinoma cell lines with dnTCF mutants showed that activation of c-Myc through this signaling pathway inhibits differentiation and promotes proliferation of these cells. More importantly, the stable transfection of only c-Myc into the dnTCF-expressing colorectal cell lines abrogated the inhibition of cell growth, and the induction of p21cip1/waf1 imposed by the expression of dnTCF. Finally, c-Myc regulation of p21cip1/waf1 expression is suggested by the demonstration that c-Myc binds to the p21cip1/waf1 promoter (118). These findings strongly suggest that c-Myc mediated the proliferative effects of the β-catenin/Tcf-4 complex in colorectal carcinoma cells through transcriptional inhibition of the cell cycle inhibitor p21cip1/waf1. This regulatory pathway also appears to be operative during mucosal morphogenesis that occurs in the normal fetal small intestine. Genes that are transcriptionally upregulated by β-catenin/Tcf-4, such as c-Myc, Enc1, and CD44, were either not expressed or expression was decreased in the intestinal intervillus epithelium of late fetal Tcf-4 null mice, which lack stem cells in the intervillus epithelium. In contrast, gene products that are normally down-regulated

PHYSIOLOGY OF GASTROINTESTINAL STEM CELLS / 319 by activation of Wnt signaling, such as p21cip1/waf1 and liver fatty acid–binding protein, were abundantly expressed in this region (118). Cohen and colleagues (123) transiently decreased c-Myc expression in mid-to-late gestation embryos by in utero adenoviral-directed introduction of either antisense c-Myc messenger RNA (mRNA) or the engineered F-TrCP-MAX ubiquitin-protein ligase, which targets the degradation of c-Myc. Both means of diminishing c-Myc expression resulted in highly decreased complexity of the intestinal villus structure and the lung alveoli, suggesting a failure of adequate expansion of the stem cells found in these epithelial structures at this developmental time point. Finally, gene expression analysis of mRNA expressed by epithelial cells at the crypt base, isolated by laser capture microdissection from Paneth cell–ablated CR2-tox176 mice, lends further (circumstantial) support for the hypothesis that c-Myc expression is necessary for maintaining a proliferative stem cell population (124). Many of the genes expressed by the epithelial cells occupying the lower crypt were identified as those involved in regulating c-Myc transcription and c-Myc protein stability, as well as transactivation of c-Myc target genes such as the recombinant RNA–binding proteins nucleolin and nucleophosmin. Caudal-Related Homeobox Genes As the name implies, the caudal gene of the fruit fly Drosophila melanogaster determines anterior-posterior patterning during development (125–127). Cdx1 and Cdx2 are members of the caudal-related homeobox gene family sharing sequence homology to the caudal gene and encode transcription factors that regulate key aspects of normal mammalian development (128,129). These transcription factors have important roles in the early morphogenesis of the intestine when the visceral endoderm is converted to a columnar epithelium with nascent villi (125,126,130). At this developmental stage, Cdx1 expression is restricted to the intervillus epithelium, which contains stem progenitor cells, suggesting a role for this transcription factor in crypt morphogenesis (125). Cdx1 and Cdx2 proteins are expressed in gradients along both the duodenal-colonic axis and the crypt-villus axis of murine intestine from early embryonic development through adulthood (131). Expression of both Cdx1 and Cdx2 is greatest in the distal small intestine with expression decreasing proximally. Cdx1 is expressed throughout the entire colon, while Cdx2 is only present in the proximal colon. Finally, there is a crypt-villus gradient of expression for both Cdx1 and Cdx2. In the adult intestine, Cdx1 is localized primarily in the nuclei of crypt epithelial cells, while Cdx2 is found mostly in the villi with some crypt expression (131). A number of studies suggest that Cdx1 and Cdx2 regulate region-specific morphogenic programs in the adult intestine (127,131–137). Thus, heterozygous Cdx2 null mice develop colonic polyp-like lesions that do not express Cdx2, presumably resulting from mutations that result in biallelic Cdx2 inactivation but do not include loss

of heterozygosity (138,139). These polyps display a mucosal heterogeneity with areas of keratinized stratified squamous epithelium, as well as areas of gastric and small intestinal– type mucosa (138–140). In contrast, Cdx1 expression is seen in gastric or esophageal mucosa that has undergone intestinal metaplasia (141). In addition, a role for Cdx1 and Cdx2 in intestinal differentiation is suggested by the observation that colorectal carcinoma has low or no expression of these transcription factors (141–143). Both Cdx1 and Cdx2 are involved in the regulation of intestinal epithelial cell differentiation and expression of intestine-specific gene products. For example, IEC6 cells, an intestinal epithelial cell line that does not differentiate in culture, can acquire a differentiated phenotype when induced to express transfected Cdx1 and Cdx2 (132,135). Cdx1 and Cdx2 also regulate the transcription of intestine-specific enzymes such as colonic carbonic anhydrase 1, sucrase isomaltase, and intestinal phospholipase A/lysophospholipase (IPAL) (127,133,144). In the case of Cdx2, this transcriptional regulation is achieved through the binding of Cdx2 to cis elements present in the promoters of sucrase isomaltase and carbonic anhydrase 1 genes, as well as several other intestine-specific genes (127,145–149). More recently, Yamamoto and colleagues (136) identified CDX consensusbinding sites in the MUC2 promoter. They determined that CDX2, but not CDX1, can bind to these sites and up-regulate the transcription of MUC2 mRNA, suggesting that CDX2 plays a role in the differentiation of intestinal goblet cells. Expression of Cdx1 by epithelial cells residing in the lower portion of crypts in the adult murine intestine raises the possibility that it plays a role in regulating some aspect of epithelial stem or progenitor cell fate, or both. Consistent with this hypothesis, the Cdx1 promoter contains four Tcf/ Lef1 binding motifs; Cdx1 expression is up-regulated by activation of Wnt/β-catenin signaling, which occurs in the intestinal stem and progenitor cell population; and Tcf-4 null mice at E17 specifically lack Cdx1 expression in the small intestine. These findings suggest a role for Cdx1 in mediating the β-catenin/Tcf-4 regulation of crypt morphogenesis, and possibly proliferation of some as yet unidentified epithelial cells in the stem cell niche (150). In vitro studies have yielded conflicting data regarding the role of Cdx1 in regulating intestinal epithelial cell proliferation. Some studies led to the conclusion that Cdx1 stimulated proliferation of cells in culture, while other studies reported inhibition of their proliferation (135,151,152). Similar conflicting data were obtained in studies examining the effect of Cdx1 expression in colon cancer cell lines (137,142). In contrast, Cdx2 inhibited cellular proliferation when expressed in both a cell line derived from normal intestine (IEC6 cells) and in a colon carcinoma–derived cell line (Caco-2 and HT29 cells) displaying tumor-suppressor activity (132,137,142,153,154). More recent investigations have clarified the role of Cdx1 in regulating intestinal epithelial cell proliferation, especially with respect to colon cancer. In addition, these studies point to differences in in vitro methodologies as the source of some of the prior conflicting results (155,156). The colon cancer

320 / CHAPTER 11 cell lines, DLD1 and HCT116, which transiently express CDX1, were found to undergo cell-cycle arrest in G0/G1 with an accompanying reduction in G1 kinase activity, cdk6 and cdk2 [156]. In addition, cyclin D1 mRNA and protein were diminished due to a reduction in the transcriptional activity of the cyclin D1 promoter. Lynch and colleagues (156) therefore postulated that Cdx1 may play a role in coordinating the exit of a crypt epithelial (TA) cell from the cell cycle, along with the induction of differentiation as it migrates out of the crypt. The observed loss of CDX1 in human colon cancer may be a contributing factor to the attainment of uncontrolled proliferation due to the inability to exit the cell cycle. Guo and colleagues (155) presented compelling evidence that both Cdx1 and Cdx2 can inhibit β-catenin/Tcf transcriptional activity. These data raise the possibility that expression of Cdx may mediate the transition of crypt epithelial cells from the progenitor compartment to the TA cell compartment. However, the current data do not allow for a definitive identification of the Cdx-responsive crypt epithelial cell population. Cell lines displayed variable sensitivity to the Cdx1/Cdx2-mediated inhibition of β-catenin/Tcf transcriptional activity and subsequent inhibition of cellular proliferation. The authors speculate that a gradient of sensitivity to the inhibition of proliferation by Cdx factors may exist among cycling crypt epithelial cells (155). Indeed, it could be envisioned that stem and progenitor cells might be relatively unresponsive to the antiproliferative effects of Cdx, while TA cells may be a more sensitive population. A model is proposed that reconciles the presence of both Cdx1 and Cdx2 in proliferating epithelial cells of the crypt with the findings of an antiproliferative role for these transcription factors (Fig. 11-6) (155). In epithelial stem and cycling progenitor cells of the crypt, the activated β-catenin/ Tcf signaling pathway promotes the expression of key target

FIG. 11-6. Model of Cdx1 and Cdx2 interaction with the Wnt/ β-catenin signaling pathway. TCF, T-cell factor. (Reproduced from Guo and colleagues [155], by permission.)

genes, such as cyclin D1, c-Myc, and Cdx1. Cdx1 enhances the expression of Cdx2, and together they function to slow cellular proliferation. However, this activity is dampened or inhibited in Cdx-expressing epithelial cells of the crypt by as yet unknown mechanisms. As cells migrate out of the crypt base, inhibition of Cdx1 and Cdx2 antiproliferative activities ceases and expression of Cdx gene targets, such as p21cip1/waf1 and KLF4, is induced. This regulates the withdrawal of differentiation-committed cells from the cell cycle. Active β-catenin/Tcf-4 signaling inhibits the expression of both p21cip1/waf1 and KLF4. Thus, the demonstrated ability of Cdx to inhibit β-catenin/Tcf-4 may be necessary for the subsequent induction of p21cip1/waf1 and KLF4 (118,155,157). SOX9 SOX proteins are a family of transcription factors that share a common high-mobility group DNA-binding domain with the TCF/LEF transcription factors. While some Sox proteins play a role in inhibiting cellular differentiation and maintaining stem cell multipotency, others are involved in the regulation of cell fate decisions and cellular differentiation (158–163). SOX9 expression is found throughout the developing and adult intestine (164). It localizes to the nuclei of epithelial cells located in the lower portion of crypts and also colocalizes with Ki-67, a marker of proliferating cells. Studies of LS174T cells (human colon carcinoma cells) transfected with an inducible dominant negative TCF4 construct demonstrated that the Wnt signaling pathway regulates SOX9 expression. Direct interference with the formation of the β-catenin/Tcf-4 complex in these cells also resulted in the immediate loss of SOX9 expression. The in vivo link between SOX9 expression and Wnt signaling is illustrated in Tcf-4 null mice that have decreased numbers of replicating cells within the intervillus region of the developing small intestine, do not form crypts, and also fail to express SOX9 in the intervillus epithelium (89,164). These findings suggest that SOX9 may be a downstream effector of Wnt/β-catenin/Tcf-4 signaling. More direct proof for this hypothesis comes from the identification of Wnt/β-catenin/ Tcf-4 target genes that are directly regulated by SOX9 (164). Thus, the expression of Cdx2 and Muc2, genes that are normally expressed in differentiated villus epithelial cells, was repressed when SOX9 was overexpressed in LS174T cells. Therefore, SOX9 likely mediates some aspects of the maintenance of the undifferentiated, proliferating stem cell compartment in the intestine by the Wnt/β-catenin/Tcf-4 pathway. Interestingly, other important regulators of this compartment (i.e., Cdx1 and c-Myc) that are direct downstream target genes of the Wnt/β-catenin/Tcf-4 pathway were not affected by SOX9 expression (164). It has therefore been speculated that SOX9 may represent a branching point of the Wnt signaling pathway (164). Finally, Sox9 is also found in the nucleus of Paneth cells in the small intestine, that is, in differentiated cells that do not cycle. This observation led Blache and colleagues (164) to suggest that SOX9 expression is not so much cell specific,

PHYSIOLOGY OF GASTROINTESTINAL STEM CELLS / 321 as specific to a cellular location within the tissue, in this case the intestinal stem cell niche (164). CD44 In rapidly renewing tissues such as the intestine, the rate of proliferation of epithelial cells in the intestinal crypts must be tightly matched to the rate of the cell loss. CD44 had been indicated to play a role in the maintenance of intestinal homeostasis under normal conditions. This family of cell-surface–expressed glycoproteins mediates both cell– cell and cell–matrix interactions (165,166). A single gene encodes all of the family members, while alternative splicing and differential glycosylation generate different isoforms (167–169). CD44 is thought to function as a cell-adhesion receptor linking extracellular hyaluronate to the cell and cytoskeleton in tumor progression and metastasis, as well as other processes (167,168,170–173). In addition, CD44 variants carrying the v3 exon contain heparan sulfate side chains that bind growth factors and facilitate growth factor receptor–mediated signaling (174–176). This finding is of significance for intestinal stem cell maintenance and function since heparan-sulfated CD44 binds to mesenchymal FGF2, heparin-binding epidermal growth factor, and HGF (174,176). This ability to bind growth factors is especially important for cells occupying the intestinal stem cell niche since the surrounding mesenchyme is known to secrete FGF2, which is produced during epithelial repair after injury and increases stem cell survival in radiation injury (60). In the intestinal epithelium, CD44 expression is confined to the basolateral membrane of a subset of proliferating epithelial (proliferating cell nuclear antigen–positive) cells that reside in the lower portion of the crypt and include the stem and progenitor cells (177). Tcf-4 null mice do not develop small intestinal crypts and show a loss of cycling cells in the intervillus epithelium, and also lack expression of CD44 (177). However, cells of the small bowel lamina propria, which are not affected by the disruption of Tcf-4 expression, expressed CD44 (177). These data suggest that the β-catenin/Tcf-4 signaling pathway controls the expression of CD44 by small intestinal epithelial cells, but that other regulatory pathways exist in nonepithelial cell types. Interestingly, the epithelia of colon and stomach that were not affected by Tcf-4 disruption also expressed CD44. Such epithelial compartments may be spared the effects of Tcf-4 disruption due to the expression of Tcf-3, which can also complex with β-catenin (89,178). Several investigators have raised the possibility that CD44 may regulate stem cell or progenitor cell fate as opposed to proliferation. Currently, there is no direct evidence that CD44 plays a role in regulating the proliferation of crypt epithelial cells, while its presence on only a subset of PCNA-positive epithelial cells would suggest that it does not (179). Nevertheless, CD44 expression is up-regulated in colorectal cancer as early as the adenoma stage, suggesting that it may play a role in adenoma formation through effects on nonproliferative mechanisms (173,180,181).

Indeed, studies by Lakshman and colleagues (182) demonstrate that CD44 is a negative regulator of apoptosis. Ultrastructural changes in the colons of CD44 null mice were consistent with events in the mitochondria associated with apoptosis including enlargement of the organelles and disrupted cristae. In addition, these mice displayed an altered balance in the expression of antiapoptotic and proapoptotic members of the Bcl-2 family in favor of increased apoptosis (183). Thus, expression of antiapoptotic Bcl-xl was decreased relative to wild-type mice, while the expression of Bax was unchanged (182). In addition, activation of both caspase-9 and -3 was evident in CD44 null mouse colon. Levels of the cell-cycle proteins cyclin A, p21, and pRb were found to be low, or the proteins were absent in the colon of CD44 null mice. These key regulators of the cell cycle have also been implicated in apoptosis in a variety of tissues (184–189). Although the small intestine of CD44 null mice was not examined, these data suggest that crypt epithelial cell expression of CD44 may act to limit apoptosis of these cells. Indeed, the frequency of apoptosis is low in small intestinal crypts of healthy animals, suggesting that this process is highly regulated and probably plays an important role in maintaining crypt stem cell numbers (70).

Genes That Modify Wnt Signals Dickkopf1 Dickkopf (Dkk) is a vertebrate mesenchymal multigene family that encodes a group of secreted proteins that inhibit Wnt signaling (190,191). Dkk1 is the prototype family member and is known to be critically important in head formation during vertebrate development, a process that requires the inhibition of Wnt signaling (192–194). Dkk1 binds to the Wnt coreceptors LRP5/6, and to the transmembrane molecule Kremen. The resulting complex affects the internalization of LRP, thereby abrogating Wnt signaling by eliminating the formation of the Wnt/Frizzled/LRP complex (195–197). Dkk1 is believed to be a pan inhibitor of the canonical Wnt signaling pathway (198,199). Although it is currently unknown whether Dkk1 is operative in the adult intestine under normal conditions, experimental expression of this inhibitor in mice has provided compelling and direct evidence that Wnt signaling is critical for regulation of the function of intestinal stem cells. Two studies have examined the impact that expressing Dkk1 has on intestinal physiology, either in plasma through an adenovirus shuttle mechanism or by transgenic expression in the intestine using villin to target Dkk1 to that tissue (198,199). Both studies demonstrated that expression of Dkk1 resulted in disorganization of the intestinal epithelium and distortion of crypt and villus architecture. Kuhnert and colleagues (198) report down-regulation of the Wnt target genes CD44 and EphB2 in small intestine and colon together with inhibition of epithelial proliferation. Pinto and colleagues (199) report a reduction in crypt size and number in villinDkk1 transgenic mice, together with decreased proliferation

322 / CHAPTER 11 and an absence of nuclear β-catenin expression. In addition, the epithelium that replaces the crypts in Dkk1 transgenic mice displays a differentiated enterocyte phenotype, indicating that inhibition of the canonical Wnt signaling pathway in the intestine leads to terminal differentiation. Of significance was the observation that secretory cells were absent in the Dkk1-expressing intestine, which suggests that Wnt signaling is also necessary for secretory cell lineage allocation, possibly through the regulation of the proliferation of a secretory lineage progenitor (199). Prior studies have demonstrated that secretory lineage progenitors express Math1 (see Notch Signaling Pathway later in this chapter). Consistent with this observation, Dkk1 transgenic intestines were devoid of Math 1 expression, indicating that Wnt/ β-catenin/Tcf-4 signaling may be necessary for the maintenance of epithelial cell progenitors committed to the secretory cell lineage (199,200). As detailed earlier, maintenance of the intestinal crypt stem and TA cell population is dependent on activation of the canonical Wnt signaling pathway, via the expression of c-Myc and repression of p21cip1/waf1 (118,199). As expected, Dkk1 transgenic intestines did not express c-Myc. Rather, p21cip1/waf1 was expressed by the epithelium replacing the crypts. These data further support opposing roles of c-Myc and p21cip1/waf1 in regulating proliferation and differentiation of the intestinal epithelium (118). Lastly, although there is no question that Dkk1 is a potent inhibitor of Wnt signaling, it is currently unknown whether Dkk1 is operative in the adult intestine under normal conditions. However, a study by Niida and colleagues (201) demonstrated that the Dkk1 promoter contains four TCF-binding elements, which suggests that Dkk1 is a downstream target of the Wnt signaling pathway that may participate in a negative feedback loop in Wnt signaling. There are two possible ways in which Dkk1 may act. First, it may have a localized “braking action” of Wnt signaling, thereby ensuring that crypt epithelial cell proliferation does not outstrip the ability of cells to transit out of the crypt and onto the villus, thus maintaining homeostasis in the crypt-villus axis. Second, it may also act as a facilitator of epithelial differentiation by blocking proliferation through the inhibition of Wnt signaling and its downstream proproliferation genes (c-Myc), with subsequent induction of p21cip1/waf1. Foxl1 (Fkh6) Foxl1 is a winged helix transcription factor that is expressed by mesenchymal cells (202–204). In the small intestine, Foxl1 is expressed by fibroblasts surrounding the crypts (203,205). Both the cellular localization of Foxl1 and the finding that mice with a targeted inactivation of Foxl1 display abnormal intestinal architecture suggest that this transcription factor mediates cross talk between the intestinal epithelium and the surrounding mesenchyme (206). Some research has provided a glimpse of the mechanism through which Foxl1 might affect intestinal architecture (207). Foxl1 null mice were found to have thickened gastric mucosa with branching or fission of the

gastric glands after weaning, while small intestinal villi were lengthened and there was a significant expansion of the crypt compartment compared with wild-type mice. At 4 and 12 weeks of age, Foxl1−/− mice had 40% and 22% more crypts than their wild-type littermates, suggesting an expansion of the crypt stem cell population (205). Increased epithelial cell proliferation was seen in Foxl1 null mice, which correlated with an increase in the nuclear levels of β-catenin in these cells, suggesting an activation of the Wnt/β-catenin pathway (207). Additional experiments revealed increased expression of the heparan sulfate proteoglycans (HSPGs) Syndecan-1 and Perlecan. Extracellular proteoglycans can modify Wnt signaling by acting as coreceptors to facilitate/enhance the interaction of Wnts with their high-affinity receptors, the frizzled proteins (207). Thus, it is postulated that Foxl1 modulates Wnt signaling indirectly by limiting the expression of HSPGs. Indeed, a mutation in the mammalian HSPG Glypican-3 results in a tumor-susceptibility syndrome (208). Notch Signaling Pathway A number of studies emphasize the role of the Notch signaling pathway in directing cell lineage decisions of epithelial cell progenitors in the intestinal crypts (200,209,210). Notch genes encode transmembrane receptors that interact with membrane-bound ligands on adjacent cells (211). This signaling pathway is operative in a variety of diverse tissues and cells including T and B cells (reviewed by Sander and Powell [211]). In the vertebrate intestine, four receptors (Notch1-4) and five ligands have been identified (Delta1, Delta3, Delta4, Jagged1, and Jagged2) (212). Activation of the Notch receptor results in cleavage of the intracellular domain of this protein and translocation of the fragment to the nucleus. Once it reaches the nucleus, truncated Notch activates the transcription factor Su(H) (Suppressor of Hairless), which then binds to regulatory sequences in downstream target genes such as hairy/enhancer of split (Hes). Hes protein inhibits the activity of a variety of bHLH transcriptional activators (213). The downstream effects of Notch signaling are context specific and can result in modulation of proliferation, either inhibition or induction of differentiation, or inhibition of apoptosis (213). Math1, a bHLH transcription factor, is a downstream effector of Notch signaling that is required for secretory cell fate specification (200). Normal architecture of the crypt-villus unit was present in Math1 null mice; however, the villi were populated only by enterocytes, and all secretory cell lineages were absent. In contrast, expression of Hes-1 likely supports the differentiation of the absorptive enterocyte since Hes1 null mice display increased expression of enteroendocrine cell markers and increased numbers of goblet cells (210). It is postulated that Hes1 exerts a suppressive influence on Math1 to ensure that the correct number of secretory cells and enterocytes is generated. In parallel with Math1 expression, Hes-1 is found in the embryonic intestinal precursor and persists in the adult intestine. Interestingly, Hes-1 is localized

PHYSIOLOGY OF GASTROINTESTINAL STEM CELLS / 323 to the nonproliferative compartment in the developing (E17) proximal intestine, while in the adult intestine, it is found only in crypts, suggesting multiple roles for Hes1 in enterocyte differentiation. As noted earlier, in Drosophila development, Notch signaling induces Drosophila E(spl) (enhancer of split, the Drosophila homolog of mammalian Hes 1) and regulates cell fate decisions by lateral inhibition, whereby a cell that is beginning to differentiate prevents its neighbor from differentiating (209,210). The reciprocal interaction of Notch and Delta on adjacent cells results in a cascade of intracellular signals in the Notch-bearing cell that suppresses the expression of differentiation-specific genes (Fig. 11-7). The observation that Delta1 and Delta3 are up-regulated within

the intervillus epithelium of the Hes1 null mouse supports the concept that Notch–Delta interactions regulate whether progenitor cells become committed to enterocyte-specific or secretory cell lineages. Other genes known to regulate differentiation of various secretory cell types, including Math1, NeuroD, and Neurogenin3 are prematurely expressed in the developing gut of Hes1−/− mice and provide further evidence that Hes1 functions as a suppressor of secretory cell differentiation (210). Other bHLH transcription factors are involved in the commitment of secretory cell lineage progenitors to differentiate into various secretory cell subtypes. For example, a study by Jenny and colleagues (214) demonstrates that

FIG. 11-7. Notch signaling pathway. (A) Low-power 4-mm section of adult murine small intestine. Precursor cells (brown) are stained for cyclin proliferating cell nuclear antigen (PCNA); enterocytes (red) express intestinal alkaline phosphatase (IAP); goblet cells (blue) secrete mucins. Inset shows high-power image of small intestine enterocytes and goblet cells. (B) Math1, a component of the Notch signaling pathway, influences intestinal epithelial cell fate decisions. In crypt progenitor stem cells that express high levels of Notch, the Hes1 transcription factor is switched on, and the expression of Math1 and other “prosecretory” genes is blocked. The result is that the precursor cells become enterocytes. In cells expressing low amounts of Notch, levels of Delta are high, production of Hes1 is blocked, and Math1 expression is induced. Production of the Math1 helix-loop-helix transcription factor allows precursor cells to make a choice whether to become goblet cells, Paneth cells, or enteroendocrine cells. Cr, crypt; Vi, villus. (See Color Plate 3.) (Reproduced from van den Brink GR, de Santa Barbara P, Roberts DJ. Development. Epithelial cell differentiation—a Mather of choice. Science 2001;294:2115–2116, by permission.)

324 / CHAPTER 11 neurogenin3 (ngn3) functions downstream from Math1 to direct enteroendocrine cell fate specification during development, as well as in the adult intestine. Neurogenin3 was expressed in a decreasing gradient along the cephalocaudal axis of the developing mouse intestine and in cells located in the proliferative compartment of the adult crypt. Expression of Ngn3 protein was also restricted to the crypt in adult mice. The role of ngn3 in regulating differentiation of the enteroendocrine cell populations was underscored by the observation that ngn3 null mice did not express any of the markers characteristic of enteroendocrine differentiation beginning at E15.5, a time when such markers are first detectable in the wild-type mouse. Other aspects of intestinal development appeared normal, suggesting that the effects of ngn3 disruption were specific to the development of the enteroendocrine cell lineages. NeuroD regulates the final stage of enteroendocrine cell differentiation by inducing cell-cycle arrest and up-regulating expression of genes involved in the synthesis of the neuroendocrine mediators made by these cells. Although Math1 was expressed in the intestine of ngn3 null mice, NeuroD was not expressed in these mice, suggesting that ngn3 functions downstream of Math1 but upstream of NeuroD, regulating the commitment of secretory cell progenitors to enteroendocrine cell lineage development before their exit from the cell cycle. Klf4, a zinc-finger transcription factor, is required for differentiation of the mature goblet cell phenotype in the colon (215). A marked decrease in the number of goblet cells in the colon was observed in Klf4−/− mice on postnatal day 1. However, all other epithelial subtypes differentiated normally, and colonic cellular proliferation was not altered compared to wild-type mice. Staining for acidic and neutral mucins confirmed the presence of some immature cell or goblet cell precursors, raising the possibility that Klf4 may not be involved in the initiation of the goblet cell lineage, but rather plays a role in its terminal differentiation. The small bowel did not show similar goblet cell alterations in response to Klf4 ablation. Therefore, it is likely that the differentiation programs for goblet cell specification in the colon are different from that of the small bowel. The Klf4 promoter contains binding sites for the caudalrelated homeobox protein Cdx2 (216). Transfected Cdx2 has been shown to effect the in vitro differentiation of rat small intestinal cells (IEC6) into goblet cells (132). These observations raise the possibility that Cdx2 functions via activation of Klf4 to effect terminal colonic goblet cell differentiation. However, as pointed out earlier, goblet cell differentiation in the colon may proceed by a different mechanism, since Cdx2 is primarily expressed in the proximal gut during embryonic development, as well as in the adult tissue (131). Whether Klf4 is involved in goblet cell lineage progenitor commitment or is operative in the terminal differentiation of the goblet cell phenotype remains to be determined. Hedgehog Signaling Pathway The hedgehog (Hh) pathway is involved in the developmental patterning of a variety of tissues including the

gastrointestinal tract (217,218). In the intestine, the Hh pathway regulates anterior-posterior and axial patterning during development. More recent work shows that Hh regulates intestinal stem cell proliferation and commitment of epithelial progenitors to differentiate (219–222). The vertebrate Hh family is composed of three members: Sonic Hedgehog (Shh), Indian Hedgehog (Ihh) and Desert Hedgehog (Dhh), each of which encodes secreted protein products (223). Two of these, Shh and Ihh, are expressed in the intestine. The receptor for Shh is composed of two proteins: Patched (Ptch), a tumor suppressor and repressor of Hh target genes, and Smoothened (Smo), a seven-transmembrane domain protein that is the signaling component of the receptor complex (Fig. 11-8) (224). In the absence of Shh, Ptch represses Smo, while Shh binding of Ptch abrogates Smo inhibition. This leads to the activation of the transcription factors Gli1 and HRK4 (human Kruppel-related gene 4), which affects downstream gene expression in response to Shh binding (224,225). The induction of Ptch by Shh may be Hh

Ptch

Smo

Gli complex − Hh

Gli repressor

+ Hh

Gli activator Ptch Gli Bmp4

FIG. 11-8. Hedgehog (Hh) signaling pathway. The cellular response to Hh is controlled by two transmembrane proteins, Patched (Ptch) and Smoothened (Smo). The secreted ligand Hh binds to Ptch and relieves the repression of Smo signaling. Smo undergoes a conformational change, leading to the activation of its intracellular target(s), and a cascade of intracellular events that result in the translocation of the active form of the transcription factor Gli to the nucleus. Nuclear Gli activates target gene expression, including Ptch and Gli itself, as well as genes involved in controlling cell proliferation. In the absence of Hh signaling, Ptch represses Smo signaling and Gli becomes a transcriptional repressor of Hh target genes.

PHYSIOLOGY OF GASTROINTESTINAL STEM CELLS / 325 a way to limit the response of Hh signaling in a given tissue (223,224,226). The importance of limiting Hh pathway signaling is underlined by data from human studies indicating that unrestrained Hh signaling in adults results in the development of various cancers (225,227). Shh and Ihh are expressed in the gut epithelium at sites of epithelial–mesenchymal interactions. Secretion of Hh induces the mesenchymal production of Ptch and of bone morphogenic protein 4 (BMP-4), a member of the TGF-β superfamily (218). Shh and Ihh display overlapping expression in the mouse gut endoderm; but at later developmental ages, more regionalized intestinal expression is established. At E14.5, Shh is expressed primarily in the fore stomach and duodenum, while Ihh is found from the hind stomach to the anus (218). Expression of mesenchymal Bmp in the small intestine parallels the epithelial expression of Hhs (218,219). For example, in the small intestine of E18.5 mice, both Shh and Ihh are found in epithelial cells at the base of villi where crypts will eventually form, while expression of Bmp4, Ptch, and Gli1 parallels this in the underlying mesenchyme (219). Similarly, Bmp4, Ptch, and Gli1 expression is found throughout the mesenchyme of the colon at E18.5. Whereas Ihh is expressed throughout the colonic epithelium at this developmental time point, expression of Shh is restricted to colonic crypt epithelial cells (219). The expression of Hh ligands and downstream target genes in late intestinal development and in the postnatal gut suggests a role for this gene family in regulating the size and location of the proliferative compartment, the patterning of the crypt-villus unit, or the maintenance of adult intestinal homeostasis (219,220,222). The potential role of Hh signaling in regulating small intestinal epithelial (stem) cell proliferation has been suggested by a number of studies. In one study, Ihh−/− mice displayed intestinal villi of reduced size compared with those of wildtype mice; this was accompanied by a 54% reduction in intervillus epithelial cell proliferation (219). The intestinal phenotype of Ihh−/− mice resembled that of Tcf-4 null mice, which lack a stem cell compartment. However, the intestinal localization and expression of Tcf-4 in Ihh−/− mice was not abnormal. These results suggest that normal Ihh pathway signaling may either induce additional Wnts or stimulate other pathway intermediates leading to the stabilization of β-catenin with subsequent activation of β-catenin/Tcf-4 signaling. This activation of β-catenin/Tcf-4 signaling is needed to maintain epithelial stem or progenitor cell proliferation in the intestinal crypts (118,199). However, when both Shh and Ihh signaling is inhibited, a different effect on stem or progenitor cell proliferation is obtained. Blockade of total Hh signaling in the intestine through the expression of an intestine-specific pan-Hh inhibitor, the hedgehog-interacting protein (Hhip), resulted in increased epithelial stem or progenitor cell proliferation, or both (220). This effect was mediated by mesenchymal cells that expressed: (1) Ptch1 and Ptch2, Hh receptors; (2) Gli1, a downstream transcription factor; and (3) BMP4, a target gene of the Hh signaling pathway (220). The strictly mesenchymal expression of Ptch1 and Gli1 suggests that Hh signaling in the small intestine is paracrine. Mice expressing

a villin-Hhip transgene in intestinal epithelial cells exhibited varied levels of transgene expression that correlated with the severity of the observed phenotype. The highest transgene expression resulted in dramatic histologic alterations in the architecture of the neonatal intestinal crypt-villus unit. This was exemplified by a flattened epithelium with regions that were pseudostratified, and the epithelium was hyperproliferative as assessed by Ki67 staining. These histologic alterations suggest that inhibition of Hh signaling interferes with the normal process of epithelial remodeling and villus formation. Lower transgene expression resulted in milder intestinal phenotypes showing disorganized villus architecture with crypt-like structures seen on the sides of thickened villi and villus branching (220). Again, proliferating epithelial cells were evident along the villi. Proteins expressed by differentiated villus epithelial cells were absent or greatly reduced from large areas of the intestine of villin-Hhip mice, underscoring the perturbation of epithelial differentiation along the crypt-villus axis. Hyperproliferation of crypt epithelial cells and disorganization of the villi was also reported in a study that inhibited Hh pathway signaling by the administration of a pan-Hh–specific antibody to mice late in gestation and after birth (228). The above studies with villin-Hhip mice indicate an interrelation between Wnt signaling and the Hh pathway. Gene targets of the β-catenin/Tcf-4 signaling pathway, Cdx1, Cd44, EphB2, and c-myc, were up-regulated and found in the ectopic cryptlike structures that formed in villin-Hhip mice (220). In addition, there was increased cytoplasmic and nuclear β-catenin localization in epithelial cells of the villus. Finally, both ISEMFs (which express Hh receptors) and the muscularis propria displayed abnormalities in the intestines of villin-Hhip mice. ISEMFs were mislocalized to the villus tips in close association with the ectopic proliferating epithelial cells. Taken together, these data suggest that epithelial Hh signaling to the surrounding mesenchyme defines the location of the proliferative compartment through reciprocal interaction between mesenchymal cells and the overlying epithelium. Hh signaling also establishes the polarity of the villus axis before crypt formation. Hh signaling might restrict epithelial stem or progenitor cell proliferation, or both, by interacting with the Wnt/β-catenin/Tcf-4 pathway, possibly through the (down) regulation of Wnt expression that is provided by the ISEMFs (86,118,229). Hh signaling seems to play a different role in the colon from that of the small intestine. Both in vivo and in vitro studies suggest a role for Hh signaling in regulating colonic crypt morphogenesis and commitment of progenitor cells to colonocyte specific differentiation. Differentiated colonic epithelial cells express Ihh; the colonic epithelium of wildtype mice at E16.5 consists of a monolayer of polarized epithelial cells with organized crypts and proliferating cells located at the crypt base. In addition, cells that have ceased to proliferate are located in the cuff region (221). By contrast, the colon of Ihh−/− mice is a multilayered epithelium composed of proliferating cells. However, no cryptlike structures were observed (221). The absence of cryptlike structures in Ihh null mice suggests that Ihh provides the requisite signals

326 / CHAPTER 11 for initiating crypt morphogenesis and subsequent epithelial cell differentiation in the fetal colon. Confirmation of this hypothesis was obtained by examining the consequence of inhibiting Hh signaling in vivo (221). The colon of adult rats treated with cyclopamine, an inhibitor of Hh signaling that binds the Smo receptor, displayed marked histologic changes, as well as loss of markers of mature differentiated colonocytes such as villin (specific for microvilli) and carbonic anhydrase IV (a brush border enzyme). In the colon, Ihh signaling also negatively regulates the Wnt pathway and restricts expression of downstream Wnt target genes to the crypt compartment. Cyclopamine-treated animals showed up-regulation of the Wnt pathway gene targets cyclin D1, BMP4, and Engrailed-1, with a concomitant increase in crypt epithelial cell proliferation (221). In the normal adult colon, two Wnt-regulated genes, Engrailed-1 and BMP4, display a gradient of expression along the crypt length, with greatest expression seen at the crypt base. Cyclopamine treatment abolished this gradient of expression, suggesting that normal Ihh signaling acts to restrict the effects of Wnt pathway activation, that is, expression of downstream target genes, to cells localized to the crypt base. Studies have confirmed that Ihh can regulate β-catenin/ Tcf-4 signaling in vitro (221). Finally, colonic adenomas from patients with FAP, where β-catenin/Tcf-4 signaling is increased as a result of mutations in the APC gene, do not express Ihh, suggesting that constitutive activation of the canonical Wnt pathway abrogates Ihh signaling (221). These data point to reciprocal regulation between the Wnt and Ihh signaling pathways that results in the restriction of epithelial cell proliferation to the crypt compartment. Bone Morphogenic Protein Signaling Pathway BMPs comprise a family of mesenchymal-derived, secreted, signaling molecules that are members of the TGF-β superfamily (230). Previous investigations show that the BMP pathway is involved in gut development and in the maintenance of adult intestinal homeostasis (230–232). More recent work has also begun to elucidate some of the mechanistic details of the role that BMP signaling plays in shaping the intestinal stem cell niche. BMPs bind to receptors (BMPR) with serine-threonine kinase activity to transduce a signal to the nucleus via (multiple) nuclear binding proteins, the Shads (230). BMP activity is antagonized by two soluble inhibitors, Noggin (gene product of Nog) and Gremlin (product of mouse gene Cktsf1b1) (233–235). Compelling evidence that Bmp4 plays a direct role in shaping the stem cell niche by restricting the topographic location of newly forming crypts comes from the in vivo inhibition of Bmp function (231,236). Intestinal expression of the Bmp inhibitor Noggin, under control of the mouse villin promoter resulted in ectopic formation of crypts perpendicular to the villus axis in the mouse small intestine (231). These crypts appeared normal in that there was a proliferative compartment at the base. Expression of crypt-specific genes was observed, most notably c-myc and

EphB3, genes regulated by the activated β-catenin/Tcf-4 signaling pathway. Finally, there was proper positioning of Paneth cells to the crypt base, and all the epithelial lineages appeared to be expressed. Gastric and duodenal juvenile polyps also develop in Smad4 null mice due to their inability to respond to a BMP signal (237,238). Smad4 forms part of the intracellular complex that transduces the Bmp signal from the cell surface to the nucleus to effect gene transcription in response to Bmp signaling (239). Nonsense mutations in human BMPR1A, a receptor for BMP4, underlie the formation of intestinal polyps in a subset of juvenile polyposis patients, further implicating BMP4 in the proper architectural arrangement of cells to form the crypt-villus unit (240). The BMP signaling pathway serves as an interface between two important signaling pathways: the canonical Wnt signaling pathway and the Hh pathway. Wnt/β-catenin/Tcf-4 signaling is known to be critical in maintaining the stem cell compartment and is a regulator of stem and progenitor cell proliferation in the crypt (118). BMP4 is a well known downstream target gene of this pathway. Hh proteins secreted by intestinal epithelial cells, known to be regulators of intestinal patterning during embryogenesis, are also emerging as key regulators of epithelial differentiation in the postnatal intestine through the repression of Wnt signaling (220,221). BMPs are also gene targets of the Hh pathway, especially BMP4 (220). BMP4, as well as other mediators responsive to Hh signaling, are expressed by mesenchymal cells surrounding Hh-secreting epithelial cells and constitute part of the communication network between the epithelium and the mesenchyme in the stem cell niche. Current data support the hypothesis that the spatial restriction of crypt formation to the intervillus epithelium at the base of villi is achieved by the cooperative interaction of the Bmp and Hh pathways during late intestinal development. Furthermore, this cooperation also serves to limit Wnt/β-catenin/Tcf-4 signaling to the stem cell niche (Fig. 11-9) (220,221,236). More direct evidence that BMP4 may restrict crypt epithelial stem and progenitor cell replication through the direct perturbation of downstream components of the Wnt signaling pathway has been reported (236). These authors show that mice in which the BMP receptor Bmpr1a had been conditionally inactivated develop small intestinal polyps. These polyps display an increase in the number of crypts in polypoid regions compared with the number of crypts found in normal intestinal segments of control mice (236). Wnt signaling, as measured by phosphorylation of LRP6, a component of the Wnt receptor complex, was active in both wild-type and Bmpr1a mutant mice (236). Thus, the observed expansion of the crypt proliferative compartment was not due to activation of the Wnt receptor in the Bmpr1a mutant mice. Rather, BMP regulates a downstream component of Wnt receptor interaction, the nuclear levels and activity of β-catenin (236). Based on their observations, He and colleagues (236) propose that activation of stem cell self-renewal depends on two signals, the Wnt signal as previously proposed by other groups and a

PHYSIOLOGY OF GASTROINTESTINAL STEM CELLS / 327 and epithelial cells to regulate a number of differing functions, including mucosal morphogenesis, normal cellular proliferation and epithelial turnover, differentiation, mucosal regeneration after injury, and tumorigenesis. Many different growth factor families have been found to affect mucosal function in the gastrointestinal tract including FGFs, the epidermal growth factor family, TGF-β, growth hormone and insulinlike growth factors (IGFs), hepatocyte growth factors, platelet-derived growth factors, and GLP-2 (241–251). In the following sections we discuss several growth factor families for which substantial evidence exists to imply a direct effect in regulating epithelial stem and/or progenitor cell fate or proliferation in the gastrointestinal tract.

Ihh

BMP4

Ihh BMP4

Fibroblast Growth Factors Notch

Stimulatory

Wn

t

Inhibitory

W

nt

nt

W

Wnt

Wnt

FIG. 11-9. Model of the signaling network that defines the intestinal stem cell niche and regulates the gradient of proliferation to differentiation. The stem cell niche is defined by epithelial-mesenchymal signaling, which restricts Wntexpressing cells to the base of the crypt. Mesenchymal expression of BMP4 is maintained by two signals, Wnt-expressing cells that surround the crypt epithelium and Indian hedgehog (Ihh)–expressing villus epithelium. Bone morphogenic protein 4 (BMP4), in turn, has an inhibitory effect on Wnt expression, restricting Wnt-expressing mesenchymal cells to around the crypt base. The Ihh expression domain is also regulated by an unknown Wnt-responsive factor, restricting the domain to differentiated villus epithelium. A gradient of factors sets up a microenvironment within the stem cell niche to promote adhesiveness of the stem cell, proliferation of the transit-amplifying population, and differentiation of epithelium. Wnt signaling is a likely candidate for creating morphogendependent differential responses within the microenvironment. Low levels of Wnt signaling near the mid and upper regions of the crypt may permit Notch signaling to affect cellular differentiation of the intestinal lineages. Notch, in turn, acts to suppress the Wnt signal. (Reproduced from Rizvi AZ, Wong MH. Epithelial stem cells and their niche: there’s no place like home. Stem Cells 2005;23:150–165, by permission.)

transient suppression of BMP signaling, possibly through the expression of the BMP inhibitor Noggin.

Growth Factors and Peptides Growth factors are a large heterogeneous group of peptides that mediate interactions between cells in the mesenchyme

The FGF family consists of a series of heparin-binding proteins encoded by at least 22 distinct genes that have been identified in many organisms, including human and mouse (252). FGFs regulate pleiotropic responses, during normal development, tissue homeostasis, and in injury repair. FGFs are biologically active for a wide variety of cell types, including cells of epithelial, mesodermal, and neuroectodermal origin. FGF1 and FGF2, the prototypical members of this family, lack a consensus signal sequence required for the classical secretory pathway. The lack of a signal sequence suggests that a major mode of release of FGF1, FGF2, or both may be through cell death or injury, and that these growth factors may have a prominent role in wound repair. However, the mechanism by which these two prototypical FGFs are released from cells has yet to be fully elucidated. FGF3-8, 10, 17-19, and 21-23 are readily secreted from cells due to the presence of appropriate amino-terminal signal peptides. FGF9, FGF16, and FGF20 lack a signal peptide but are nevertheless secreted (252). FGF11-14 also lack signal sequences and are thought to remain intracellular (253). Expression of a number of FGFs has been described in the gastrointestinal tract (60,115,243,244,254–260). However, the levels and patterns of individual FGF genes being expressed depend on the particular developmental stage or physiologic context. For instance, FGF2 is expressed during embryogenesis in the developing foregut and regulates patterning of the foregut endoderm for the formation of hepatic and pancreatic buds (241). Similarly, FGF10 can be detected in the developing mouse gut as early as E10.5 and is expressed in the mesenchyme surrounding the cecal bud at E11.5 (243,244). FGF10 regulates proliferation and infiltration of the colonic endoderm into the surrounding mesenchyme. Mice with a targeted knockout of FGF10 or its receptor, FGFR-2, have decreased endodermal replicative activity and do not develop a cecum (244,261). In the suckling mouse intestine when crypt morphogenesis is occurring, expression of FGF1, FGF9, and to a lesser extent FGF2 predominates (260). Expression of FGF10 is difficult to detect at this developmental stage but is induced in the adult intestine after experimental injury (262).

328 / CHAPTER 11 In contrast to the patterns of FGF ligand expression associated with early development of the gut and with nascent crypt formation in the suckling mouse intestine, basal expression of FGFs in the adult gastrointestinal tract is low. However, expression of several of the FGF genes can be induced in response to injury or inflammation. Levels of FGF7 (keratinocyte growth factor) are increased in the intestinal mucosa of patients with inflammatory bowel disease (IBD). Vidrich and colleagues (260) found that increased expression of FGF2, FGF7, and FGF10 in the ileum of SAMP1/YitFc mice, a mouse model that develops chronic ileal inflammation, paralleled the increase in the number of potential clonogenic stem cells per crypt that occurs in this model. The response to radiation injury is also characterized by marked increases in the expression of FGF2, FGF4, and FGF10. This coincides temporally with the appearance of regenerative crypts derived from surviving clonogenic stem cells and the subsequent expansion of crypt numbers through branching or fission (60,259). Proliferation of the surviving crypt stem cell population must occur to support the expansion in crypt numbers that replenishes the complement of proliferative units in the epithelium. Several FGFs have been shown to regulate proliferation, differentiation, or migration of intestinal epithelial cells in culture (259,263,264). Dignass and Podolsky (263) have demonstrated that FGF1, FGF2, and FGF7 have mitogenic activity for the intestinal epithelial cell lines IEC6, Caco-2, and HT-29 in culture. In an in vitro wound-healing model, these researchers also demonstrated that FGF1 and FGF2 promote cell migration and epithelial restitution through a TGF-β-dependent mechanism. More recently, the mitogenic activity of FGF4 and FGF10 on cultured intestinal epithelial cells has been described (259,264). FGFs may also play a role in regulating lineage commitment of undifferentiated progenitors. For example, a study by Iwakiri and colleagues (265) demonstrated that FGF7 could mediate goblet cell differentiation in the H2 subclone of the human colonic epithelial cell line HT-29 through regulation of the goblet cell silencer inhibitor element. The ability of FGFs to regulate the proliferation or fate of intestinal stem cells and progenitor cells in vivo has also been described (60,254–256,264,266,267). Pharmacologic studies of FGF2, FGF7, and FGF10 have demonstrated that these ligands can each enhance clonogenic crypt stem cell survival in the microcolony assay when they are administered before irradiation. Interestingly, these growth factors must be administered for several days before irradiation to achieve a dramatic effect on crypt stem cell survival after irradiation. Potten and colleagues (268) have suggested that a possible explanation for this effect is that FGF7 (and potentially other FGFs) increases stem cell number or induces changes in the cell cycle dynamics of the clonogenic crypt stem cell before radiation injury. The biological effects of FGFs on target cells are mediated by binding to high-affinity, cell-surface receptor tyrosine kinases encoded by at least four separate genes (269–277). Alternative splicing of the primary RNA transcripts results

in two distinct isoforms of each receptor (IIIb and IIIc) that differ in their ligand-binding affinities for various members of the FGF family. Tissue-specific and cell-specific differences in the pattern of receptor RNA splicing and expression of various isoforms have been found (252). The IIIb isoforms of FGFR-1, -2, and -3 are expressed predominantly in epithelial cells and have a more restricted ligand-binding profile than the corresponding IIIc isoforms. Our understanding of the role of FGFRs in regulating the fate or proliferation of stem and progenitor cells in the gastrointestinal tract is complicated by the existence of multiple isoforms of each receptor. In addition, individual FGFR isoforms may have significant binding affinity for multiple FGF ligands that overlap with the ligand-binding characteristics of the other FGFRs (252,278). Thus, multiple FGFRs may play overlapping or distinct roles in regulating the physiology of intestinal stem cells and progenitor cells under a variety of physiologic or developmental circumstances. For example, studies using laser capture microdissection and immunohistochemistry have shown that both FGFR-2 and -3 are expressed in intestinal epithelial cells. FGFR-2 mRNA was detected throughout the gastrointestinal epithelium during early gut development (243,244,261) and in both crypt and villus epithelial cells in the adult murine small intestine. FGFR-2 IIIb has been implicated in the regulation of epithelial regeneration after intestinal injury or associated with intestinal inflammation (254–256,264,266,267). The FGFR-2 IIIb ligands, FGF7 and FGF10, are both able to increase the number of surviving crypt epithelial stem cells when they are administered before irradiation (267,268,279). FGF7 can induce a human colonic epithelial cell line, HT29 subclone H2, to differentiate into goblet cells in vitro (265). Given the specificity of this ligand for FGFR-2 IIIb, these findings suggest that signaling via FGFR-2 may mediate aspects of lineage allocation decisions within the progenitor cell population, in addition to its effects on the clonogenic stem cell population. Expression of FGFR-3 in the small intestine and colon is developmentally regulated. Increased expression of FGFR-3 was observed as early as E15 and E16. FGFR-3 is also expressed at high levels in the suckling mouse intestine (postnatal days 4–21), a developmental time period characterized by formation of small intestinal crypts and expansion of crypt numbers through crypt fission (115). In contrast to the relatively uniform distribution of FGFR-2 along the crypt-villus axis, we found that cell-surface FGFR-3 was expressed only in crypt epithelial cells (Fig. 11-10) (115). Expression of FGFR-3 was not observed in the entire TA cell population, but rather was restricted to a smaller subset of replicating cells in the lower half of the crypt, where epithelial stem cells and undifferentiated progenitor cells are found (Fig. 11-11) (115). FGFR-3 IIIb was the dominant isoform expressed in both murine small intestinal and colonic crypts. In humans, FGFR-3 IIIb is expressed in the normal colon and in multiple intestinal epithelial cell lines (280). FGFR-3 is also highly expressed within regenerating crypts after radiation injury (115). The developmental stage-specific

PHYSIOLOGY OF GASTROINTESTINAL STEM CELLS / 329 A

B

C

D

BG

FIG. 11-10. Fibroblast growth factor receptor-3 (FGFR-3) expression in the suckling mouse intestine. Sections were incubated with rabbit anti–FGFR-3. (A) Immunoreactive FGFR-3 in the proximal jejunal crypts from a 14-day-old mouse (original magnification ×100). Open arrowheads point to Paneth cells, while the black arrowheads indicate FGFR-3–positive ganglion cells. The inset shows a higher magnification view of a crypt from the section in A (original magnification ×400). Staining is localized to the basolateral membranes of undifferentiated epithelial cells in the lower half of the crypt. Black arrow indicates the crypt-villus junction. Arrowheads denote Paneth cells showing barely detectable staining. (B) The specificity of the anti–FGFR-3 immunohistochemistry is demonstrated in a section from the proximal jejunum incubated with normal rabbit IgG in place of the primary anti–FGFR-3 antibody. (C) In the duodenum (original magnification ×100), staining is confined to the crypt epithelium with no staining in the adjacent Brunner’s glands (BG). (D) FGFR-3 immunoreactivity also is detectable in the lower third portion of colonic crypt epithelium (original magnification ×200). (See Color Plate 4.) (Reproduced from Vidrich and colleagues [115], by permission.)

changes in FGFR-3 expression, coupled with the observation that FGFR-3 is highly expressed within adult regenerating crypts after radiation injury, strongly suggest that one major function of this receptor is regulation of stem cell fate during crypt morphogenesis (115). Activation of FGFR-3 IIIb can induce proliferation of Caco-2 intestinal epithelial cells in vitro (280). Expression of FGFR-3 IIIb was down-regulated as these cells differentiated and correlated with the loss of proliferative responses to FGFR-3 IIIb–specific ligands. Induction of FGFR-3 IIIb expression in differentiated cells restored the proliferative response only to those FGF ligands that bind to FGFR-3 IIIb.

These data, as well as the observation that cell-surface expression of this receptor is restricted to undifferentiated cells in the lower crypt, imply that FGFR-3 has a restricted role in crypt morphogenesis. This likely involves mediating the proliferation or fate of stem cells or their immediate descendents, rather than regulating the proliferative response of the entire replicating crypt cell pool. The downstream signaling pathways through which FGFRs mediate their effects on epithelial stem and progenitor cells have only been partially defined. FGFRs can activate a number of downstream signaling pathways, including signal transducer and activator of transcription 1 and 3 (STAT1/3),

330 / CHAPTER 11

A

B

C

FIG. 11-11. Cell-specific patterns of fibroblast growth factor receptor-3 (FGFR-3) expression in mouse proximal jejunum. Proximal jejunum from 21-day-old FVB/n mice (A–C) received BrdUrd 2 hours before tissue harvest. (A) S-phase cells incorporating BrdUrd (red fluorescence) are found primarily in the lower two-thirds crypt. Solid white arrows indicate the location of the crypt-villus junction. (B) FGFR-3 is expressed along the basolateral surfaces of epithelial cells in the lower crypt (green fluorescence). Open arrows indicate the basilar surface of a crypt. (C) Colocalization of BrdUrd and FGFR-3. The S-phase cells in the upper portions of the crypt epithelium do not stain with anti–FGFR-3 (e.g., white arrowhead ). (See Color Plate 5.) (Modified from Vidrich and colleagues [115], by permission.)

and the phosphatidylinositol-3 kinase-dependent, phospholipase Cγ–dependent, and Ras/Raf/mitogen-activated protein kinase–dependent signaling cascades (280–282). An intriguing report demonstrates that FGF2, a ligand of FGFR-2 and -3, can stimulate Tcf-4/β-catenin signaling in some cell types (114). Activation of Tcf-4/β-catenin–mediated signaling is required for proliferation of the crypt stem cell population and normal morphogenesis of the intestinal crypt epithelium (see earlier in this section) (89,178). Although much attention has been focused on Wnt(s) as the candidate upstream mediator(s) of Tcf/β-catenin signaling pathway, the finding that other growth factors such as FGF2 can also regulate Tcf/β-catenin activity suggests the possibility that other upstream signaling pathways may be involved (114). The cellspecific pattern of FGFR-3 expression reported by Vidrich and colleagues (115) encompasses the stem cell and progenitor cell populations, which are regulated via Tcf-4 (89). In addition, the timing of FGFR-3 expression in the intestinal epithelium relative to Tcf-4 mRNA expression would support such a speculation (115,178). Insulin-like Growth Factors The IGFs, IGF-I and IGF-II, are small polypeptide growth factors (~7 kDa) that are expressed in many different tissues, including the gastrointestinal tract. A number of reports have emphasized the potential role of the IGF family in enhancing intestinal growth (245–247). IGFs also promote epithelial regeneration in a number of injury models including methotrexate-induced enteropathy (283), dextran sulfate–induced colitis (284,285), radiation injury (286–288), and intestinal adaptation after small-bowel resection (289–291). IGF-I also suppresses apoptosis of colon cancer cells (292,293) and promotes restitution of epithelial cell monolayers in culture after experimental wounding (294). IGF-I and -II bind to both type I and II IGF receptors in the gastrointestinal tract and may mediate muscular hypertrophy and fibrosis

in Crohn’s disease (295,296). Several IGF-binding proteins modulate the biological activity of IGFs. Evidence that IGFs may regulate intestinal stem cell or progenitor cell function, in addition to their effects on mesenchymally derived cells in the intestine, has emerged from recent studies. Several groups have reported that administration of IGF or overexpression of IGF-I in the intestine is protective against radiation enteritis (286–288,293). In IGF transgenic mice, this effect is mediated in part by suppression of radiation-induced apoptosis in the lower crypt (286). Maximal suppression of apoptosis was noted in the stem cell zone, at cell positions 3 to 5 above the crypt base. In the IGF transgenic mice, induction of the proapoptotic gene Bax by irradiation was also suppressed in the lower third of the crypt epithelium. The authors concluded that IGF-I enhanced mucosal regeneration in this model through direct effects on crypt cell replication or apoptosis within the stem cell compartment. Studies of intestinal development in transgenic mice that overexpress IGF-II in the intestine and in IGF-II knockout mice point to a role for this growth factor in regulating epithelial stem cell proliferation or crypt morphogenesis (51). The surface area and the number of crypts (structural/proliferative units) in the colon of IGF-II–deficient mice were reduced during the suckling–weaning transition and early postweaning period. Conversely, mice overexpressing an IGF-II transgene under the control of a keratin promoter (K:IGF-II transgenic mice) manifested a significant increase in the surface area of the colonic mucosa. This increase in luminal surface area was attributable to increased numbers of colonic crypts, while changes in the size of the crypts or the rates of spontaneous crypt epithelial apoptosis were not observed. Rather, the authors conclude that the increase in the number of crypts in the K:IGF-II transgenic mice was due to an accelerated rate of crypt fission. Since crypt fission results in the segregation of clonogenic crypt stem cells to daughter crypts, an increased rate of symmetric clonogenic stem cell division must accompany this process.

PHYSIOLOGY OF GASTROINTESTINAL STEM CELLS / 331

Transforming Growth Factor-β Members of the TGF-β family are thought to be important regulators of epithelial homeostasis in the normal gastrointestinal tract. TGF-β mediates its

KGF

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Glucagon-like peptide-2 is a 33-amino-acid peptide produced by enteroendocrine cells (L cells) in the distal small intestine and colon (297). Studies have identified GLP-2 as a potent intestinal growth factor in rodents that acts by expanding the mucosa through stimulation of crypt epithelial cell replication (248,298,299). The trophic effects of GLP-2 are greater in the small intestine than in the colon (297,298). In addition to its trophic effects in the uninjured intestine, GLP-2 promotes epithelial regeneration in a number of models of epithelial injury and inflammation (300–303). GLP-2 may also regulate crypt epithelial apoptosis in response to experimental injury. Administration of GLP-2 in the setting of nonsteroidal anti-inflammatory drugs or chemotherapy-associated injury to the intestinal mucosa significantly reduced crypt epithelial apoptosis induced by these agents (301,304). The elegant studies reported by Bjerknes and Cheng (88) have provided important new insights into the mechanism of the effects of GLP-2 on epithelial stem cell and progenitor cells in the gut. An in vivo assay was developed that could distinguish effects of growth factors and other mediators on specific stem cell or progenitor cell subsets defined by the cell lineage(s) tagged by induced mutations in the Dlb-1 locus. Dlb-1 encodes a cell-surface binding domain for the lectin, Dolichos bisflorus, and the progeny of Dlb-1+ stem cells and progenitor cells can be identified histologically by their ability to bind this lectin. Thus, the lineages represented in the Dlb-1+ clone of cells along a crypt-villus unit define the functional capacity of the specific mutant progenitor or stem cell that gave rise to the clone (Fig. 11-12). Stem cell clones and columnar-specific progenitor cells specifically responded to GLP-2 administered to mice and resulted in expansion of columnar cell lineages in these mutant clones. Mucous cell progenitors were unaffected. These data point to a rather specific effect of GLP-2 on stem cells and only a subset of long-lived lineage committed progenitors within the lower crypt epithelium. The biological activity of GLP-2 is mediated by binding to a G protein–coupled receptor, GLP2R. However, expression of this receptor was found only in enteric neurons. GLP2R was not detected in crypt epithelial cells in the rodent gut in this study (88). The GLP2R+ neurons were shown to respond to GLP-2 stimulation, and the direct effect of these neurons on the crypt progenitor cell population was demonstrated by local inhibition of neuronal signaling. This intriguing finding implies that GLP-2 mediates its effects on epithelial stem cells and progenitor cells through an intervening local neural circuit that impinges directly on the function of the crypt epithelium and emphasizes the potential importance of the enteric nervous system in integrating the response of the stem cell population to physiologic stimuli.

GLP-2

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1 0 Stem Clones Epithelium

FIG. 11-12. Progenitor behavior detected by specific progenitor assay by somatic mutation (SPASM) after glucagon-like peptide-2 (GLP-2) or keratinocyte growth factor (KGF) administration. (A) Photomicrographs of typical NEU-induced Dlb-1+ clones derived from mucous progenitors, columnar progenitors, or stem cells. Dlb-1+ cells, labeled with horseradish peroxidase–conjugated lectin are stained brown. The tissue is counterstained with Alcian blue to aid identification of mucous cells (which are blue [arrow], unless Dlb-1+ [arrowhead] ). Columnar cells are pale blue (arrow), unless Dlb-1+ (arrowhead). Dlb-1+ mucous (arrowhead) and columnar (arrow) cells are both seen in stem cell clones. Original magnification ×320. (B) After GLP-2 treatment, columnar cell and stem cell clones are larger in comparison with those in control samples. Mucous cell clones were larger after KGF treatment. Control clones had 129.2 ± 28.3, 3.4 ± 0.3, 12.0 ± 3.5 (mean ± standard error of the mean) cells for stem cell, mucous cell, and columnar cell clones, respectively. (C) GLP-2 treatment decreases, but KGF treatment increases mucous cell density in the epithelium and stem cell clones. Control mucous cell density was 0.039 ± 0.001 in the epithelium and 0.032 ± 0.003 in stem cell clones. (See Color Plate 6.) (Reproduced from Bjerknes and Cheng [88], by permission.)

332 / CHAPTER 11 effects through binding to and forming a multimeric complex with type 1 and 2 TGF-β receptors on the cell surface (305). Binding of ligand results in phosphorylation of Smad2 and Smad3, which form a heterotrimeric complex with cytoplasmic Smad4. On translocation to the nucleus, this complex modulates the transcriptional activity of TGF-β target genes. Expression of several TGF-β isoforms (TGF-β1, TGF-β2, TGF-β3) has been reported in the adult intestine. TGF-βs are expressed in multiple epithelial and nonepithelial cell types within the intestinal tract (306). However, the epithelium is thought to be the predominant source of TGF-β synthesis under basal conditions (306,307). A number of observations suggest that TGF-βs may have a direct role in regulating epithelial homeostasis along the crypt-villus axis of the intestinal epithelium. Administration of exogenous TGF-β3 resulted in suppression of proliferation at cell positions 3 to 4 and at cell positions 13 to 27 from the base of the crypt, suggesting that TGF-β can regulate proliferation of both the stem cell and the TA cell populations in the small intestine and colon (242). A direct effect on the stem cell population is also suggested by the observation that prolonged administration of TGF-β results in significant protective effect on crypt stem cells using the microcolony formation assay after radiation injury (242,308). Booth and colleagues (242) suggest that this protective effect is mediated by cell-cycle inhibition of the stem cell population, rendering fewer stem cells sensitive to the cytotoxic effects of radiation injury during the sensitive phase of the cell cycle. A similar protective effect of TGF-β2 was observed using a methotrexate intestinal injury model and was attributed to alterations in the cell-cycle dynamics of the epithelial stem cell population (309). TGF-β also has a role in regulating intestinal inflammation, injury repair, and tumorigenesis. Mice homozygous for a targeted disruption of the TGF-β1 gene develop a multifocal inflammatory disease and die of organ failure at about 3 weeks old. Increased expression of TGF-β has also been noted in experimental models of intestinal injury in vivo and in IBD and may provide a mechanism for cross talk between the epithelial stem cell niche and the mucosal immune system (310,311). The inflammatory phenotype and early mortality observed in Tgf β1 −/− mice can be largely abrogated by introducing this targeted mutation into a Rag2−/− background. The Tgf β1−/− Rag2−/− mice develop multiple carcinomas in the cecum and colon by 5 months old, suggesting that TGF-β1 acts as a potent tumor suppressor gene altering some aspect of stem cell physiology in these regions of the gut. Surprisingly, the epithelial cell proliferation was not grossly altered in the hyperplastic epithelium, suggesting that other compensatory mechanisms may regulate the response of the TA cell population in the absence of TGF-β1. Mice heterozygous for targeted disruption of Smad4, a signaling molecule downstream of the TGF-β receptor, and an Apc gene mutation develop increased numbers of polyps due to somatic loss of the wild-type Apc and Smad4 alleles (237). The lesions formed in these compound mutant mice progress to form malignant

adenocarcinomas to a far greater degree than mice that are heterozygous for the mutant Apc allele alone (237). However, polyps found in compound heterozygotes for a target mutation of Smad2 and the same Apc mutation were not different from mice heterozygous for the Apc mutation alone, suggesting that there may be a much greater degree of complexity to this signaling pathway than originally thought (312).

Inflammation: Cytokines and Other Mediators The adult gastrointestinal tract is characterized not only by the rapid and continuous replacement of differentiated epithelial cells, but also by a remarkable capacity to quickly repair damage that disrupts mucosal integrity. Mucosal injury and ulceration are common features found in a variety of gastrointestinal diseases that can be induced by numerous causative agents including infectious agents, toxins, acute or chronic radiation injury, ischemia, and immune activation as seen in IBD. For example, IBD is characterized by cycles of clinically apparent disease activity marked by the presence of mucosal injury and inflammation, followed by periods of disease quiescence during which the injured mucosa is repaired and normal gut function is restored. Loss of mucosal integrity from any cause leads to a common set of histopathologic features that constitute the host response, which include inflammation and mucosal regeneration (313). Mucosal regeneration in the intestine after intestinal injury or inflammation is a multistep process that must ultimately involve changes in the dynamics of the proliferating crypt epithelial cells and the epithelial stem cell population (22, 229,314,315). Initially, a continuous simple epithelial cell monolayer has to be restored through migration of adjacent epithelial cells over the wound. Subsequently, intestinal crypts (the morphologic unit of cell renewal) must form within the reconstituted epithelium. A single surviving clonogenic crypt stem cell is sufficient to give rise to a regenerating crypt. Crypts may also undergo branching or fission as part of the regenerative process to replenish the epithelium with sufficient numbers of proliferative units (crypts) in areas where mucosal disruption is extensive and severe. Since the epithelial stem cells within a crypt segregate to daughter crypts when crypt fission occurs, an expansion of the stem cell number must accompany this reparative response. Finally, the normal morphologic relationship of the epithelium to the underlying mesenchymal tissue and vasculature must be reestablished, and normal region-specific epithelial differentiation and function must be reconstituted. Inflammation may have both localized and systemic effects on the epithelial stem cell and progenitor cell populations. An illustration of this phenomenon is provided by studies of the SAMP1/YitFc mouse. These mice are a recombinant inbred strain in which many features of human Crohn’s disease develop including segmental small intestinal inflammation that is confined to the ileum (316). These mice develop localized perturbations in lineage-specific epithelial differentiation in the ileum at an early age, when

PHYSIOLOGY OF GASTROINTESTINAL STEM CELLS / 333 histologic evidence of acute or chronic inflammation is first beginning to occur (260). Marked expansion of multiple secretory cell lineages, including increased numbers of Paneth, goblet, and intermediate cells, occur in regions of ileal inflammation (Fig. 11-13). Accompanying this localized expansion of secretory cell lineages is a loss of absorptive enterocytes. Changes in epithelial differentiation within the ileal mucosa persist into adulthood and are further amplified with the development of a local inflammatory response, reciprocal interactions between inflammation and epithelial differentiation. These observations favor a model in which the local inflammatory response in the SAMP1/YitFc ileum is associated with an increase in the number of the common secretory cell lineage progenitors at the expense of the columnar (absorptive) progenitor cell population. The number of stem cells per crypt also increases markedly at the onset of inflammation in the SAMP1/YitFc mouse (Fig. 11-14). However, the increase in crypt stem cell number occurs globally, both in inflamed regions of the

small intestine (ileum) and in the proximal jejunum, a region of the small intestine where inflammation does not develop in this model. The global increase in crypt stem cell number follows a time course that parallels the severity of inflammation. Thus, the mechanisms that regulate the global effects of inflammation on the crypt stem cell population are distinct from those that mediate local perturbations in progenitor cell replication, differentiation, or both, suggesting that different components of the inflammatory response are involved. A number of cytokines and other mediators associated with intestinal inflammation or injury can induce alterations in the crypt stem cell or progenitor cell populations when exogenously administered. These include tumor necrosis factor (TNF)-α, TGF-β3, trefoil family factor 3 (TFF3), interferon-γ (IFN-γ), interleukin-1 (IL-1), IL-11, prostaglandins, cyclooxygenase-1 (COX-1), COX-2, FGF2, FGF7, and FGF10 (59,60,242,264,308,317–325). Expression of FGF2, FGF7, FGF10, and IL-11 were each increased

A

B

C

D

FIG. 11-13. Altered lineage allocation and crypt morphology accompany ileitis in SAMP1/YitFc mice. Ileitis in SAMP1/YitFc mice is characterized by the presence of a marked transmural acute and chronic inflammatory infiltrate and crypt abscesses. Loss of villi, marked crypt elongation, and increased numbers of Paneth cells and intermediate cells containing eosinophilic granules (A, open arrows ) are evident throughout the crypt of SAMP1/YitFc mice at 20 weeks of age. Note the eosinophilic granules in numerous cells located from the base of the crypt to the crypt-villus junction. In control AKR or C57BL/6 mice, Paneth cells are normally restricted to cell positions 1 to 4 at the crypt base. Periodic acid-Schiff-Alcian blue staining demonstrates increased numbers of goblet cells in areas of inflammation (B). Electron microscopic analysis of crypt epithelium of 10-week-old SAMP1/YitFc mice shows cells in the mid and upper crypt containing immature Paneth cell granules characteristic of intermediate cells (C). Electron micrograph of Paneth cells with mature granules from 10-week-old AKR mice is shown for comparison (D). (See Color Plate 7.) (Reproduced from Vidrich and colleagues [260], by permission.)

334 / CHAPTER 11 A

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FIG. 11-14. Regeneration of crypts in SAMP1/YitFc mice after radiation injury. The appearance of regenerating crypts is shown in hematoxylin and eosin–stained sections of ileum from 40-week-old SAMP1/YitFc mice (A) and age-matched AKR (B) mice 3.5 days after 14 Gy γ-irradiation. In adjacent sections, S-phase cells were detected by immunohistochemistry with anti-BrdUrd (C, D). A regenerative crypt (microcolony) is observed if one or more clonogenic stem cells within the crypt survive radiation injury and are able to subsequently replicate. Note that the number of regenerating crypts is markedly increased in the SAMP1/YitFc (C) mice compared with the age-matched AKR control mice (D). Crypt stem cell survival was globally increased in both inflamed (ileum, E) and noninflamed (proximal jejunum, F) regions of the small intestine in SAMP1/YitFc mice compared with AKR or C57BL/6 control mice (*, p < 0.01). Crypt stem cell survival after 14 Gy γ-irradiation (G) increases in parallel to the severity of the inflammatory response (H). (See Color Plate 8.) (Reproduced from Vidrich and colleagues [260], by permission.)

significantly coincident with the onset of inflammation and the increase in stem cell number in SAMP1/YitFc mice (260). Prior studies have suggested that FGF2, FGF7, and FGF10 may each play a role in regulating repair of injury to the intestinal epithelium (60,254–256,258,264,266,267, 326). Serum levels of FGF2 are significantly increased in patients with active IBD (258,326) and in the mouse small intestine after radiation injury. As discussed in the preceding sections, FGF2 enhances crypt stem cell survival after irradiation (60). FGF7 and its receptor are also expressed at greater levels in the colonic mucosa of patients with IBD (254–256). Given the effects of FGF7 or FGF10, or both, on crypt epithelial progenitors and mucous cell differentiation reported in other models of injury repair (264,279), it is tempting to speculate that the increase in FGF7 or FGF10 that occurs with inflammation in the SAMP1/YitFc ileum is responsible for the local perturbations in epithelial differentiation observed in this model, although this speculation has yet to be confirmed.

IL-11 is produced by intestinal myofibroblasts and possibly by other intestinal cells in response to various cytokines that regulate the inflammatory response (327). Some studies have shown that administration of IL-11 reduced the severity of colitis in HLA-B27 rats with established colonic disease (328,329). In other studies, IL-11 increased crypt stem cell survival in a model of severe cytoablative chemotherapy (324,328) or after γ-irradiation (325). This occurred in conjunction with increased villus length, preserved crypt/villus ratio, and reduced hepatic bacterial foci, as well as decreased apoptosis of intestinal epithelial crypt cells (324). Increased IL-11 levels are also associated with the development of inflammation in the ileum of SAMP1/Yit mice and may mediate the global increase in crypt epithelial stem cell number observed in this model (260). These data have led to the hypothesis that IL-11 may exert its beneficial effects by simultaneously affecting stem cell production or survival and immune system modulation including reduction of proinflammatory cytokines (324,325,329–332).

PHYSIOLOGY OF GASTROINTESTINAL STEM CELLS / 335 The effect of a particular cytokine or inflammatory mediator may also depend on the particular physiologic context present in the stem cell/progenitor cell niche. For instance, in mice without inflammation, blocking IFN-γ has little effect on proliferation of stem cells or progenitors in the lower crypt (323). However, in mice infected with the parasite, Trichuris muris increased proliferation was observed at cell positions 1 to 4, the region of the crypt containing the stem cell population, and at cell positions 11 to 15, corresponding to the TA cell zone. The increased proliferation in both the stem cell zone and the TA cell zone was abrogated by treatment with neutralizing anti–IFN-γ antibody but not by isotype-matched control immunoglobulin, suggesting that IFN-γ mediates stem cell proliferation in this physiologic context. The source of a particular mediator that regulates stem cell function may also depend on which cellular or enzymatic pathway(s) are activated in a particular inflammatory context. For example, in the absence of inflammation, prostaglandins produced by Cox-1 but not by Cox-2 were found to enhance crypt stem cell survival in the microcolony assay and to suppress radiation-induced apoptosis in the lower crypt of normal mice (59,61). However, when mice were exposed to a proinflammatory stimulus such as bacterial lipopolysaccharide before irradiation, crypt stem cell survival and radiation-induced apoptosis became highly dependent on prostaglandins produced by COX-2 (319). Protection of the stem cell population and inhibition of apoptosis in the lower crypt by lipopolysaccharide are dependent on TNF receptor I, suggesting that complex signaling networks are involved in mediating this effect. Distinct subsets of cells within the adaptive mucosal immune system may mediate the response of the stem cell/ progenitor niche to different immunologic or inflammatory stimuli. Kamal and colleagues (333) show that a distinct epithelial response occurs after infection with another parasitic worm, Trichinella spiralis. The predominant response in the small intestinal crypt epithelium to infection with T. spiralis was an increase in Paneth and intermediate cell numbers. The increase in Paneth and intermediate cells occurred in infected euthymic and nude mice but not in infected TCR(β/δ)−/− mice. Adoptive transfer of normal mesenteric lymph node cells to infected TCR(β/δ)−/− mice restored the increase in Paneth/intermediate cells observed after T. spiralis infection, suggesting a unique T-cell population mediates this effect on epithelial lineage allocation as part of the immune response to this parasite. This phenotype is reminiscent of the local perturbation in secretory cell lineage allocation observed in SAMP1/YitFc mice as described earlier (260). In contrast to T. spiralis infection, increases in crypt progenitor cell replication in colitis induced by dextran sodium sulfate (DSS) depended on the presence of luminal flora and activated macrophages (334). Intact Toll-like receptor signaling was also required since the increased proliferation of colonic epithelial progenitors did not occur after DSS treatment in Myd88−/− mice, which are defective in signaling via Toll-like receptors that recognize various components of the microbiota. Experiments using Csf1op/op

(lacking macrophages) Rag1−/− mice (depleted of lymphocytes) and mice depleted of neutrophils by treatment with a neutrophil-specific monoclonal antibody indicated that the increase in crypt progenitor cell proliferation in DSS colitis required macrophages but not T cells or neutrophils. Pericryptal macrophages isolated by laser capture microdissection from DSS-treated animals displayed an activated phenotype. These studies illustrate that a complex network of signals generated by both the luminal environment and the mucosal immune system are integrated within the stem cell niche to regulate the response of epithelial stem cells and progenitors to a broad range of differing physiologic or environmental circumstances.

STEM CELL PLASTICITY The traditional view that organ-specific stem cells have irreversibly lost the capacity to differentiate into cell types of other tissues has been long standing. However, this dogma has been challenged in the past few years by studies demonstrating that adult stem cells isolated from one organ or tissue may be able to differentiate into cells of other tissues under certain circumstances (reviewed by Anderson and colleagues [335]). An example of this phenomenon is illustrated by studies showing that transplantation of bone marrow cells into humans or lethally irradiated animals can give rise to the expected hematopoietic cell lineages, as well as to nonhematopoietic cells such as cardiomyocytes, hepatocytes, skeletal muscle fibers, and others (315,336). These findings raise the possibility of using bone marrow–derived cells for repopulating a failing organ with autologous functional cells. One demonstration of this concept is the rescue from liver failure of mice that are deficient in fumarylacetoacetate hydrolase (FAH) enzyme by the transplantation of purified hematopoietic stem cells (337). The transplanted cells differentiated into functionally normal, FAH-expressing hepatocytes and restored organ function. Several studies have begun to address the question of whether hematopoietic stem cells can also repopulate the gastrointestinal tract and give rise to the relevant intestinal cell lineages. Bone marrow cells from male donors transplanted into syngeneic female mice engrafted into the intestine and differentiated into ISEMF cells. The bone marrow origin of the ISEMF cells was verified by the presence of the Y chromosome. The expression of smooth muscle α-actin and absence of desmin, the macrophage marker F4/80, and the hematopoietic precursor marker CD34 was consistent with a myofibroblast phenotype (338). The persistence of these cells in the lamina propria for a period of at least 6 weeks suggested that the donor stem cells at this site were capable of sustained turnover and differentiation. Donorderived ISEMFs have also been observed in intestinal biopsies from human female patients with graft-versus-host disease who received bone marrow from a male donor (338). Another study reported the long-term persistence of bone marrow–derived epithelial cells in lung, intestine, and skin from the transplantation of a single purified hematopoietic

336 / CHAPTER 11 stem cell (339). However, these investigators did not find any evidence of mesenchymal ISEMF being derived from the single hematopoietic stem cell transplantation. This result suggests that the prior studies demonstrating the differentiation of bone marrow cells into intestinal mesenchymal ISEMF may have been due to the inclusion of fibroblast precursors in the transplanted bone marrow cell preparation. However, stromal cells do not generally survive transplantation, thus the discrepancy in some of the results of the two studies cited remains to be resolved (315). Nevertheless, both studies demonstrate that the transplanted bone marrow– derived cells were able to populate and persist in the recipient intestine, a heterotypic tissue. This demonstration is considered a prerequisite to proving that tissue-specific stem cells transplanted into another tissue have functionally crossed lineage boundaries and differentiated into cells of the recipient tissue (335). Before it can be accepted that organ-specific adult stem cells can differentiate into a cell of another organ, more rigorous criteria must be applied to the isolation of pure stem cells, as well as to the identification of the resulting lineages, which must include morphologic, molecular, and functional parameters (335).

CONCLUSION Significant progress has been made in elucidating the mechanisms and pathways involved in the establishment and maintenance of the intestinal stem cell compartment and crypt-villus unit. It is becoming increasingly clear that crypt morphogenesis, maintenance of the proliferative epithelial stem cell compartment, and crypt-villus homeostasis require the cooperation and convergence of multiple signaling pathways, some of which were hitherto believed to be active primarily during intestinal specification and development. The orchestration of these processes is achieved by sustained cross talk between the epithelial compartment and those cells that comprise the stem cell niche. Studies examining the signaling pathways involved in maintaining intestinal homeostasis have also yielded important insights into the mechanisms that underlie some gastrointestinal cancers, as well as clues to important control points for these pathways that may be targeted for therapeutic interventions. Although current research has addressed many questions, many more have been raised. What are the extracellular signals involved in the establishment of the crypt stem cell compartment during normal gut development, as well as in the maintenance of crypt stem cell homeostasis in the adult, and how are they integrated to determine the fate of individual stem cells or progenitor cells? What is the nature of the molecular signals that regulate stem cell selection within the crypt compartment that occurs during epithelial morphogenesis? The issue of whether crypts become genetically monoclonal by an active selection process involving apoptosis of unwanted cells or through a process of stem cell “competition” is open to debate and research. Indeed, the molecular definition of the intestinal stem cell is only beginning to emerge.

What are the molecular mechanisms for tightly regulating stem cell number within a narrow range in the normal adult crypt under steady-state conditions, and what is the nature of signals that regulate changes in the size of the stem cell population that occur in pathologic processes, such as mucosal injury repair, inflammation, and neoplasia? Finally, although a type of molecular addressing system has been identified that specifies cell positioning along the cryptvillus axis, characterization of the molecular signals controlling its expression has only just begun. It is clear that the many recent advances described earlier have added a significant new molecular dimension to the elegant and insightful studies of Cheng and Leblond that first outlined more than a quarter of a century ago the hierarchical organization of epithelial cells within the intestine. Although many questions remain, the ongoing lively research in intestinal stem cell biology is certain to provide intriguing and perhaps unanticipated answers and, in the process, to raise many new questions as well.

ACKNOWLEDGMENTS This work was supported by grants from the National Institute of Diabetes and Digestive and Kidney Diseases (R01 DK06475, to S.M.C.) and through the University of Virginia Digestive Diseases Research Center (NIH P30 DK56703).

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331. Trepicchio WL, Bozza M, Pedneault G, Dorner AJ. Recombinant human IL-11 attenuates the inflammatory response through down-regulation of proinflammatory cytokine release and nitric oxide production. J Immunol 1996;157:3627–3634. 332. Trepicchio WL, Wang L, Bozza M, Dorner AJ. IL-11 regulates macrophage effector function through the inhibition of nuclear factorkappaB. J Immunol 1997;159:5661–5670. 333. Kamal M, Wakelin D, Ouellette AJ, et al. Mucosal T cells regulate Paneth and intermediate cell numbers in the small intestine of T. spiralis-infected mice. Clin Exp Immunol 2001;126:117–125. 334. Pull SL, Doherty JM, Mills JC, et al. Activated macrophages are an adaptive element of the colonic epithelial progenitor niche necessary for regenerative responses to injury. Proc Natl Acad Sci U S A 2005; 102:99–104. 335. Anderson DJ, Gage FH, Weissman IL. Can stem cells cross lineage boundaries? Nat Med 2001;7:393–395. 336. Bhatia M, Wang JC, Kapp U, et al. Purification of primitive human hematopoietic cells capable of repopulating immune-deficient mice. Proc Natl Acad Sci U S A 1997;94:5320–5325. 337. Lagasse E, Connors H, Al-Dhalimy M, et al. Purified hematopoietic stem cells can differentiate into hepatocytes in vivo. Nat Med 2000; 6:1229–1234. 338. Brittan M, Hunt T, Jeffery R, et al. Bone marrow derivation of pericryptal myofibroblasts in the mouse and human small intestine and colon. Gut 2002;50:752–757. 339. Krause DS, Theise ND, Collector MI, et al. Multi-organ, multi-lineage engraftment by a single bone marrow-derived stem cell. Cell 2001; 105:369–377.

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CHAPTER

12

Apoptosis in the Gastrointestinal Tract Leonard R. Johnson Cell Death, 345 Apoptosis and Necrosis, 345 Proteins Involved in Apoptosis, 346 Apoptotic Pathways, 347 Intestinal Epithelia: In Vivo Studies, 349 Growth and Differentiation, 349 Spontaneous Apoptosis, 350 Induced Apoptosis, 351 Intestinal Cells: In Vitro Studies, 358 Apoptotic Pathway in Intestinal Cells, 358

Polyamines and Apoptosis, 359 Additional Studies, 362 Anoikis, 363 Gastric Mucosa, 364 Helicobacter Pylori, 364 Additional Studies, 365 Conclusion, 366 References, 366

CELL DEATH

Apoptosis and Necrosis

Cell death is a common event in multicellular organisms and is responsible for tissue modeling during embryonic development, as well as homeostasis of cell number in various proliferative adult tissues. Examples of the former include the deletion of the tail in human embryos, the resorption of the tadpole tail as it turns into a frog, and the loss of tissue between developing digits. An example of the latter is the death of extra stem cells in gut crypts so that the number of proliferating cells is kept in balance with those being lost as they are shed into the lumen. Another example of cell death in the maintenance of tissues is the killing of targeted or infected cells by elements of the immune system. In addition to these types of cell death, which are obviously regulated, cells can be killed by outright injury. This often results in an inflammatory response and tissue damage. This type of cell death is usually considered to be accidental, whereas the former is usually referred to as programmed.

Programmed cell death occurs through a process known as apoptosis and is the result of triggering a signal cascade that activates specific enzymes that dismantle the cell. The word apoptosis originates from Greek and literally means “falling off ” as leaves fall from a tree. Although apoptosis had been recognized as a form of cell death for many years, and was described as pyknosis, the term itself was proposed by Kerr and colleagues in 1972 (1). The apoptotic process can be initiated by a variety of stimuli. These include cell damage, such as caused by radiation, chemotherapy, or reactive oxygen species. Withdrawal of growth factors or trophic substances also results in apoptosis of some cells. The binding of ligands to cell membrane receptors of the tumor necrosis factor (TNF) family is responsible for cytokine-induced apoptosis. The full course of apoptosis, from its initiation to the final fragmenting of the cell, usually requires several hours. At the start of apoptosis, dense chromosome condensation occurs at the periphery of the nucleus. The chromatin is compacted and segregated. At this time, the cell also begins to shrink as the cytoplasm condenses. The nucleus becomes fragmented, and the cell breaks up into membrane-bound apoptotic bodies, which are phagocytosed by surrounding cells. Morphologically, the defining feature of apoptosis is that the contents of the cell are never released but remain membrane bound.

L. R. Johnson: Department of Physiology, University of Tennessee College of Medicine, Memphis, Tennessee 38163.

Physiology of the Gastrointestinal Tract, Fourth Edition, edited by Leonard R. Johnson. Academic Press, 2006.

345

346 / CHAPTER 12 Regulator

Adapter

Effector

C. elegans

Ced-9

Ced-4

Ced-3

Apoptosis

Vertebrates

Bcl-2

APAF-1

Caspase-9

Caspase-3

Apoptosis

FIG. 12-1. Three types of proteins involved in apoptosis and first identified in Caenorhabditis elegans (C. elegans) are shown with their vertebrate counterparts. (Modified from Vaux and Korsmeyer [5], by permission.)

In contrast, in cells undergoing necrosis in response to tissue damage, the plasma membrane becomes permeable early in the process. The cells typically swell and burst, releasing their contents and often triggering an inflammatory response. Detection of cells undergoing necrosis is based on the early increase in membrane permeability. Small charged molecules, such as propidium iodide and various nucleic acid dyes, which would normally be excluded from the cell, enter and can be detected by their fluorescence.

Proteins Involved in Apoptosis The general steps involved in apoptosis are remarkably conserved during the evolutionary process. This suggests that the proteins involved are also similar. Identification of these proteins and the molecular mechanisms involved was largely the result of genetic studies in the roundworm Caenorhabditis elegans (2,3). Three C. elegans gene products (CEDs) are involved in apoptosis. CED-3 and CED-4 promote apoptosis, whereas CED-9 prevents it. CED-3 is an effector, a cysteine protease that cleaves its target proteins following aspartic acid residues. It exists as a zymogen and is activated by self-cleavage. CED-4 can bind to CED-3 and activate it. CED-9 can bind to CED-4 and prevent its activation of CED-3. Under normal conditions, apoptosis is prevented because CED-4 is bound to CED-9. Thus, CED-9 may be considered a regulator and CED-4 an adaptor protein. Mammals have evolved similar groups of proteins that fill the roles of effector, adaptor, and regulator (4). These are shown in Figure 12-1, together with their C. elegans counterparts (5). Effectors In vertebrates, apoptosis results from a complex set of biochemical events catalyzed by a family of cysteine proteases called caspases (6,7). These effectors exist in the cytoplasm as presynthesized proenzymes and are activated in response to physiologic, pathogenic, or cytotoxic stimuli (8). There are two basic types of caspases (Fig. 12-2): executioner caspases are responsible for the actual dismantling of the cell, and initiator caspases are capable of activating executioner caspases. The morphologic hallmarks of apoptosis in the nucleus, chromatin condensation and nuclear fragmentation, are initiated by caspase cleavage of specific proteins. Caspase-3 and -6 cleave lamin and various nuclear proteins responsible for the structural integrity of the

nucleus (9). The fragmentation of DNA is the result of the activation of a number of caspase substrates, such as caspase-activated DNAase (CAD) and DNA fragmentation factor (DFF). Both of these enzymes are constitutively present bound to inhibitory proteins and are released by the action of the caspases (10). Additional proteins cleaved and inactivated during apoptosis are responsible for the repair of DNA damage. These include DNA-dependent protein kinase and poly adenosine diphosphate ribose polymerase (11). DNA fragmentation results in a “laddering” that can be detected by enzyme-linked immunosorbent assay (ELISA) after staining with appropriate DNA antibodies linked to peroxidase and various dyes. Various membrane and cytoplasmic proteins are also targets of caspases. Many of these are involved in the maintenance of the cytoskeleton and include actin, β-catenin, gelsolin, fodrin, and spectrin. Actin itself is the major cytoskeletal protein responsible for the shape of the cell and cell motility. Early during the apoptotic process, phosphatidyl serine is externalized to the plasma membrane, providing a phagocytotic signal to other cells. Annexin V binds to phosphatidyl serine and, conjugated to fluorochromes, provides another means to detect apoptotic cells via fluorescent microscopy. The activation of caspase-3 itself also serves as an excellent assay of apoptosis. Colorimetric substrates are available for all the caspases, or Western blotting can be used to determine the shift in the amount of procaspase to the active form (12). Accessory Molecules Adaptor or accessory molecules are involved in the activation or inhibition of caspases (Fig. 12-3). Perhaps the most familiar of these is apoptotic protease activating factor-1 (APAF-1), which is analogous to CED-4 (see Fig. 12-1). Another such molecule is cytochrome c, which is released from mitochondria during the apoptotic process. Cytochrome c binds to APAF-1 monomers in the cytosol, resulting in the formation of the heptameric apoptosome. Caspases Initiator Caspases

Executioner Caspases

Caspase-8

Caspase-3

Caspase-9

Caspase-6 Caspase-7

FIG. 12-2. The major vertebrate caspases.

APOPTOSIS IN THE GASTROINTESTINAL TRACT / 347 Adapter molecules APAF-1

cIAP1

Cytochrome c

cIAP2

Smac / Diablo

BIRC3

Htr A2

Survivin

XIAP FIG. 12-3. Accessory or adaptor molecules that regulate caspase activity. Htr A2, high-temperature requirement service protease A2; XIAP, X-linked inhibitor of apoptosis protein.

The apoptosome then recruits and binds procaspase-9, which is proteolytically cleaved to form active caspase-9 (8,13). Caspase-9 then activates the executioner caspase-3, -6, and -7 (8,13). Inhibitors of apoptosis proteins (IAPs) are capable of directly inhibiting activated caspases and probably ensure that cells are not killed by spontaneous activation of caspases. Synthesis of IAPs is also stimulated by transcription factors such as nuclear factor-kappa B (NF-κB) and the extracellular signal–regulated kinases (ERKs) (14,15). Strong anticaspase activity has been demonstrated for a subset of IAPs, which includes XIAP (X-linked IAP), cIAP1, cIAP2, and ML-IAP (16,17). The most potent of these is probably XIAP, which has been targeted for anticancer therapy (18,19). IAP activity can be inhibited by additional regulators released from mitochondria. These include SMAC (second mitochondrial activation of caspase) and high-temperature requirement service protease A2. These will bind to XIAP in the cytoplasm, interfering with the inhibition of caspases (19,20). Regulators The final group of molecules involved in apoptosis is composed of the members of the Bcl-2 family of proteins

that regulate the release of cytochrome c from mitochondria and alter the ability of APAF-1 to activate caspases. Bcl-2 was originally discovered as an oncogene in B-cell lymphoma (21). Subsequent studies showed that Bcl-2 is capable of inhibiting apoptosis induced by a variety of signals (22,23). A large number of Bc1-2 proteins have been identified using a variety of techniques. All of these possess at least one of four conserved motifs known as Bcl-2 homology (BH) domains. Based on their structures and function, the Bcl-2 proteins can be divided into three groups (Fig. 12-4). The first consists of the antiapoptotic channel-forming proteins having four BH domains and a transmembrane anchor sequence. The second group consists of proapoptotic channelforming proteins having domains BH1, BH2, and BH3 plus the transmembrane region. The third group contains proapoptotic proteins having only the BH3 domain. They are often referred to as BH only proteins and dimerize with the membrane-anchored, channel-forming Bcl-2 receptors (24). Proapoptotic and antiapoptotic members can form homodimers and heterodimers. Whether a cell lives or dies may depend on the relative amounts of the two forms (25). Thus, a preponderance of proapoptotic Bcl-2 proteins will result in heterodimers with all available antiapoptotic members and an excess number of proapoptotic proteins to form homodimers. An excess of prosurvival proteins prevents the release of cytochrome c from mitochondria and may also prevent the activation of procaspase-9 by APAF-1 (26).

Apoptotic Pathways Activation of two major pathways initiates the process of apoptosis in most cells. An extrinsic pathway is initiated by ligands such as TNF-α, a pleiotypic cytokine that, depending on cell type and surrounding circumstances, can result in growth arrest, apoptosis, or proliferation (27). The second major pathway is the intrinsic or mitochondrial pathway,

Antiapoptotic channel-forming BH4

BH3

BH1

BH2

TM

Bcl-2, Bcl-w Bcl-XL, Mcl-1 Bcl-w

BH2

TM

Bax, Bok Bak

TM

Bik, Blk

Proapoptotic channel-forming BH3

BH1

Proapoptotic ligands BH3

BH3

Bad, Bid, Bim

FIG. 12-4. Regulators of apoptosis. Subgroups of the Bcl-2 family of proteins, showing their Bcl-2 homology (BH) domains and presence or absence of a transmembrane (TM) region, which is involved in mitochondrial anchoring. Examples of each are listed.

348 / CHAPTER 12 which is activated by DNA damage and often depends on p53 (28). Extrinsic Pathway: Death Receptors Death receptors of the TNF receptor (TNFR) family, such as Fas, TNF-α–related apoptosis-inducing ligand (TRAIL), and TNFR1, recruit, on binding of ligand, molecules that make up a death-inducing complex that includes procaspase-8 (13) (Fig. 12-5). TNF trimerizes TNFR1 on binding, inducing the association of the receptor’s death domains, which, in turn, bind to adapter proteins that recruit and activate a number of other molecules to the receptor complex (4). This results not only in the activation of caspase-8, which, in turn, activates caspase-3, but also the activation of pathways mediated by NF-κB, mitogen-activated protein kinases (MAPKs), and c-Jun NH2-terminal kinases (JNKs), which regulate apoptosis (29–31). The activation of these additional pathways explains why TNF-α usually does not cause apoptosis unless an inhibitor of protein synthesis is present. Nuclear factor-κB protects most cells from apoptosis, whereas its inhibition enhances cell death (29,32). Normally, NF-κB is bound in the cytoplasm to inhibitory κB (IκB) and remains inactive. Activation results from phosphorylation of IκB on serine residues, which involves phosphatidylinositol-3 kinase (PI3K) and the Akt serine-threonine kinase (33). Phosphorylation of IκB releases NF-κB to go to the nucleus and regulate gene transcription. Among its protein products are

Bcl-2, Bcl-XL, and various IAPs (34). Sustained activation of JNK induces apoptosis in a variety of cell lines (35,36). In some cell lines including intestinal epithelial cell-6 (IEC-6), JNK is inhibited by the sustained activation of MAP kinase kinase (MEK1), ERK1/2, or both (37,38). Intrinsic Pathway: The Mitochondrion The second major pathway is the intrinsic or mitochondrial pathway, which results in the release of cytochrome c from mitochondria (Fig. 12-6). As mentioned earlier, cytochrome c binds to APAF-1 in the cytoplasm and in the presence of adenosine triphosphate forms the apoptosome, which recruits and activates procaspase-9. Additional proteins such as SMAC are also released from the mitochondria. SMAC (or Diablo) binds to and inhibits cytosolic IAPs. The mitochondrial pathway is regulated by the Bcl-2 family proteins. DNA damage is the usual trigger for the release of cytochrome c. Agents such as ultraviolet irradiation, γ-irradiation, chemotherapeutic drugs, and some instances of cellular damage, such as hypoxia, often activate p53 (39). Activated p53 promotes the expression of a number of apoptotic genes including members of the Bcl-2 family such as Bax (39). Much of the tumor-suppressive activity of p53 depends on its ability to induce apoptosis, and many cancers

DNA damage

p53 TNF-α

FasL Bax, BH3-only proteins Fas

TNFR1

Bcl-XL, Bcl-2

Extrinsic pathway F ADD

TRADD

t-Bid

Procaspase 8

NF-κB

Caspase-8

Bcl2, Bcl-XL

Bid

Bad

ERK1/2 Cyto C IAPs

Procaspase-3

t-Bid SMAC

Nucleus

Caspase-3

APAF 1

Mitochondrial pathway

Procaspase 9

Caspase 9 IAPs Procaspase 3

Bcl-2

Apoptosis

FIG. 12-5. Extrinsic or death receptor pathway. ERK, extracellular signal–regulated kinase; FADD, Fas-associated death domain; IAP, inhibitor of apoptosis protein; NF-κB, nuclear factor-κB; TNF, tumor necrosis factor; TNFR, tumor necrosis factor receptor; TRADD, TNFR-associated death domain.

Caspase 3

Apoptosis

FIG. 12-6. Intrinsic or mitochondrial pathway, also sometimes referred to as the damage pathway. BH, Bcl-2 homology; IAP, inhibitor of apoptosis protein; SMAC, second mitochondrial activation of caspase.

APOPTOSIS IN THE GASTROINTESTINAL TRACT / 349 contain deletions or mutations that inactivate p53. Although most of the effects of p53 are believed to be due to its actions as a transcription factor, Chipuk and colleagues (40) show that p53 directly activated Bax to permeabilize mitochondria and initiate apoptosis. Depolarization and permeabilization of the mitochondrial membrane and the subsequent release of cytochrome c are determined by the relative amounts of antiapoptotic channelforming Bcl-2 family members such as Bcl-2 and Bcl-XL and proapoptotic channel-forming members such as Bax and Bak (see Fig. 12-4). However, BH3 only family members play an important role in this process by inactivating Bcl-2 in the mitochondrial membrane. Some BH3 only proteins such as Noxa and PUMA are transcriptional targets of p53 (41,42). Other members of this group are inactivated by posttranslational modifications that prevent them from interacting with prosurvival Bcl-2 family members (43). In some cells, the withdrawal of a growth factor initiates apoptosis. In these cells, Bad is sequestered by a cytosolic protein known as protein 14-3-3. That sequestration (binding) depends on the serine phosphorylation of Bad, which is dependent on Akt activation by growth factors (44). Removal of growth factor frees Bad to move to the mitochondria and bind Bcl-2 or Bcl-XL, resulting in their inactivation and apoptosis. The BH3 only protein Bid is cleaved by caspase-8 in response to the activation of the intrinsic pathway through TNF, Fas, or TRAIL receptors. Cleavage converts Bid to the active, truncated form, t-Bid, which is capable of binding to Bcl-2 and causing apoptosis (45). This process results in cross talk between the extrinsic pathway and the mitochondrial pathway (43). p53 has also been shown to release proapoptotic multidomain proteins and BH3 only proteins that were sequestered by Bcl-XL (40).

INTESTINAL EPITHELIA: IN VIVO STUDIES This section covers studies done in both the small and large intestines. Although there are certainly differences involving apoptosis, the primary one being the high incidence of colon cancer compared with cancer of the small bowel, there are also many similarities. The comparison and contrast between the two tissues avoid redundancy and often provide obvious explanations of differences in physiology and pathology. There is an abundance of studies examining the place of apoptosis in the development of colon cancer and other gastrointestinal tract malignancies. This is a subject in itself and is covered in Chapter 17. The intestinal epithelium provides an interesting yet complicated model in which to study apoptosis. This tissue has one of the most rapid turnover rates known, as cells are lost into the lumen and replaced by the progeny of stem cells. Apoptosis plays a role both in the loss of cells and in regulating the number of stem cells. Both processes, proliferation and apoptosis, are affected by hormones, growth factors, and influences that regulate the growth of all tissues in the body. In addition, however, the lining of the gut is

subjected to luminal contents in the form of nutrients, digestion products, bacteria, and growth factors. All of these have been shown to affect the growth of the tissue (46). The intestinal epithelium of the mouse has served as the primary model for studying gut apoptosis. For the most part, these studies have focused on describing spontaneous apoptosis and damage- or stress-induced apoptosis. Many of these have identified the roles of members of the Bcl-2 family of proteins and p53. However, few studies have examined the overall regulatory process of apoptosis in the intestine. There has been little progress in developing appropriate in vitro models of normal intestinal cells. The most widely used intestinal cell lines have been developed from tumors and are abnormal. Some such as the popular CaCo-2 line contain a mutated p53 gene. Although all cell lines by definition have altered rates of proliferation and death, the IEC-6 line has proved useful to elucidate the mechanisms regulating apoptosis in untransformed crypt cells that have not differentiated.

Growth and Differentiation The small intestinal epithelium of the adult mouse, like most vertebrates, is composed of protruding villi interspaced with numerous cavities called crypts (Fig. 12-7). The crypt-to-villus ratio varies from approximately 14 crypts per villus in the duodenum to about 6 crypts per ileal villus (47). This difference is the crypt-to-villus ratio probably accounts for the difference in the sizes of villi in the two locations. The longer duodenal villi each contain approximately 7800 cells, whereas those in the ileum contain about 2100 (47). The approximately 1.1 million crypts of Lieberkühn of the adult mouse (48) are flask-shaped structures and are the proliferative units of the intestine. Numerous studies in normal, transgenic, and chimeric mice have shown that crypts are monoclonal and are populated by the descendents of a single stem cell anchored near the base of the crypt (48–53; see review by Hermiston and colleagues [54]). After division of the stem cell, one daughter cell remains anchored as the new stem cell, whereas the other undergoes several divisions in the lower and middle thirds of the crypt. The total number of dividing and nondividing cells in a crypt varies according to its location just as the number of cells per villus varies. Duodenal crypts contain approximately 530 cells, whereas the average number of cells per ileal crypt is 360 (47). Each crypt produces 13 to 16 cells per hour (47). Most of these cells migrate out of the crypt and onto a villus, where they continue to migrate toward the villous tip. During migration the cells mature into three of the four terminally differentiated cell types of the adult small intestinal epithelium: the absorptive enterocyte, the enteroendocrine cell, and the mucus-secreting goblet cell. Some cells migrate to the base of the crypt where they differentiate into Paneth cells that produce lysozyme. Thus, each of the four main cell types arise from progenitors of a single multipotent stem cell (51,54). The cells that migrate onto a villus are subsequently shed

350 / CHAPTER 12

3 days

Villus Enterocytes Goblet cells Enteroendocrine cells

As in the small intestine, proliferation takes place in crypts. Cells migrate from the colonic crypts to a group of surface epithelial cells surrounding the crypt opening. Together with cells from additional crypts, the surface cells make up the so-called intracryptal tables, the flat areas between the crypts. Colonic crypts, like the crypts in the small intestine, are monoclonal, that is, populated by the progeny of a single stem cell (52,54,59). Crypts in the proximal colon produce approximately 6 cells per hour, whereas those in the distal colon have greater proliferative rates of about 21 cells per hour. The average cell turnover rate in the colon is increased to approximately 8 days compared with the 2 to 3 days found in the small intestine (60).

Spontaneous Apoptosis

Crypt

Proliferative transit cells

Stem cell(s)

In both animals and humans, a low rate of apoptosis occurs in the intestinal crypts unrelated to a known stimulus. The greatest frequency of apoptosis coincides with positions 4 and 5 from the base of the crypt, which is the presumed location of the stem cells (61). Apoptosis may therefore be responsible for eliminating extra stem cells. Apoptosis may also be responsible for removing excess cells from the villous tip. Although few apoptotic cells have been observed in sections from the lower and mid regions of the villi, this incidence increases with increasing distance from the crypt. This process of cell removal is certainly regulated to maintain the number of villous cells. However, it is uncertain whether apoptosis itself is regulated, or whether apoptosis occurs in response to detachment from the basement membrane.

Paneth cells

Crypt FIG. 12-7. Schematic of the crypt-villus unit of the small intestine, showing progress of an enterocyte from stem cell to transit cell to mature cell located on the villus to its exfoliation. Migration from the crypt-villus junction to the zone of extrusion at or near the villus tip takes approximately 3 days.

into the intestinal lumen by a process that is incompletely understood (55,56). Shedding involves apoptosis, and the question is whether the apoptosis occurs on the villus, or whether it is triggered by detachment from the substrate, a process known as anoikis. The whole process from proliferation to differentiation to shedding occurs in a few days. It takes only 2 to 3 days for a cell to move from the crypt to the apex of the villus where it is exfoliated (47,57). Cells are shed from the villous tip at the rate of 1400 cells per villus during a 24-hour period (58), making the epithelium of the small intestine one of the most rapid turning over tissues in the body. From the previous discussion, it is obvious that cell death, apoptosis, plays a significant role in maintaining the normal cell population of the small bowel epithelium. Despite the absence of villi, proliferation and migration are also involved in cellular differentiation in the colon.

Potten (62,63) has estimated that one apoptotic cell is found in every fifth longitudinal histologic section through the crypt of the mouse small intestine. These cells, for the most part, occupy positions 4 and 5 from the base of the crypt (the stem cell positions). Spontaneous apoptosis in the small intestinal crypt occurs at the same rate in p53-deficient mice as in wild-type animals (64). Thus, p53 does not appear to play a role in regulating the number of stem cells in the small intestine. Bcl-2 is not expressed in small-bowel epithelial cells (65), and as one would surmise, Bcl-2−/− and normal mice have the same rate of apoptosis of small intestinal epithelial cells (66). The apoptotic protein Bax, however, is strongly expressed in the stem cell region of the small intestine and in enterocytes at the villous tips (61). Thus, Bax may stimulate the removal of extra stem cells and play a role in the shedding of effete villous cells. Little or no Bax is found in the crypts of the large intestine (61). Thus, the localizations of the antiapoptotic and proapoptotic Bcl-2 family members may provide the explanation for the relative resistance of the small intestine to the development of cancer compared with the colon (61,67). These findings indicate that apoptosis plays an important role in regulating the number

APOPTOSIS IN THE GASTROINTESTINAL TRACT / 351 of stem cells in the epithelium of the small intestine, and therefore the number of cells leaving the crypts and migrating onto intestinal villi. Potten and colleagues (61) have estimated that an extra stem cell will cause 60 to 120 additional cells per crypt. Thus, by virtue of the several divisions undertaken by daughter cells before they exit the crypt, an extra stem cell would seriously alter the equilibrium of cell number normally maintained in the crypt-villus unit. Apoptosis in the colonic epithelium occurs at a much lower rate than it does in the small intestine and is not confined to the crypts. Instead, cells are spontaneously eliminated at low rates regardless of their positioning. Knockout studies have shown that, as in the case of the small intestinal epithelium, spontaneous apoptosis in the colonic epithelium occurs independently of both p53 and Bax (65,68). In contrast, Bcl-2 is expressed in colonic epithelium, and knocking out this gene results in an increased rate of spontaneous apoptosis, which occurs primarily in the crypt at positions 1 and 2, the location of the stem cells (66). Bcl-2 is believed to play an important role in the regulation of cell number in the colonic epithelium, and its presence is an explanation for why the colonic mucosa is susceptible to cancer. Villus There is no doubt that detachment from the underlying matrix or substrate induces epithelial cell apoptosis. Detachment-induced cell death, or anoikis, has been shown to occur in isolated human intestinal cells (69) and in mammary, renal, and bronchial epithelial cells (70–72). In normal human IECs, activation of caspase-6 occurred within 15 minutes of detachment (73). The major questions being debated are whether apoptosis begins while cells are still attached to the villi, and, if so, how it is initiated. The first question arose out of the relatively infrequent observations of obvious apoptotic cells on the villus (61). Hall and colleagues (74) used sensitive microscopic and histologic techniques to determine that, on average, one apoptotic body was present per section of villous epithelium. Each section was 3 µm in thickness, and because the villi of the mice were 120 to 150 µm, these authors calculated that at any time 40 to 50 apoptotic cells were present on each villus. The distribution of apoptotic cells was not uniform, and they increased in number with position toward the villus tip. Apoptotic bodies are rapidly cleared, usually in less than 1 hour. Thus, during a 24-hour period, each villus may shed 960 to 1200 apoptotic cells (74). This calculation fits with that of Potten and Hendry (75) who report that 1200 cells migrate onto a villus each day. The finding that apoptosis occurred while cells were still attached to the villus was confirmed by Grossman and colleagues (76), who used an antibody directed against activated caspase-3 to detect dying cells. These authors report that epithelial cells of the small bowel activated caspase-3 as they approached the area of shedding and before they displayed the morphologic features of apoptosis. Because caspase-3 is the major executioner caspase, and its activation

is the culmination of the signaling pathway leading to apoptosis, we can conclude that the apoptotic process is well under way before effete cells appear apoptotic. In conclusion, these studies (74,76) provide strong evidence that intestinal cell number is regulated by apoptosis, as well as by proliferation, and that the apoptotic rate is linked to the proliferative rate. Do cells detach from the extracellular matrix (ECM) before being shed into the lumen? Using scanning electron microscopy, Shibahara and colleagues (77) observed protruding enterocytes at the tips of villi. These cells showed evidence of apoptosis such as vacuoles and chromatin condensation. Frequently, the intercellular spaces beneath these cells were occupied by large lymphocytes. Taken together, these data suggest, although they certainly do not prove, that mature enterocytes may detach from the ECM and undergo apoptosis before being shed into the lumen. The mechanisms involved in this process and anoikis are discussed later in this chapter in the Intestinal Cells section. Regardless of the mechanisms involved, the processes of cell proliferation and loss are linked. Additional support for this relation comes from a study in elderly adults. Ciccocioppo and colleagues (78) compared the rates of enterocyte apoptosis and proliferation in the duodenal mucosa of 12 humans with a mean age of 77.6 years with 12 others with a mean age of 37.7 years. The TUNEL technique was used to detect apoptosis, whereas proliferation was measured immunohistochemically by M1B-1 detection in endoscopically obtained biopsy specimens. Apoptosis had increased in the elderly adults to 15.3% of the cells examined from 2.1% in the younger age group. This was matched by an increased proliferative rate to 37.7% versus 15.8%. What is not known is whether the increased proliferation compensates for increased cell loss or whether the opposite situation exists.

Induced Apoptosis Many damaging agents have been shown to induce apoptosis of intestinal epithelia. These include ionizing radiation, chemical mutagens, and chemotherapeutic drugs and food products. In addition, activation of the extrinsic pathway via the so-called death receptors results in a more physiologic apoptotic response. Apoptosis is part of the result of procedures such as small-bowel resection and ischemia–reperfusion and has been investigated to determine its contribution to the overall response. A variety of agents has been tested for their protective effect on the gut mucosa and their ability to prevent apoptosis during exposure to radiation or chemotherapeutic agents. The goal of these studies has been to discover a means to protect the lining of the intestine during cancer therapy. A variety of cytotoxic drugs, mutagens, and ionizing radiation result in apoptosis of intestinal epithelia (79,80). In the whole animal, the most widely studied of these and the one most used as a model to study the effects of other agents on

352 / CHAPTER 12 apoptosis is radiation. Dose-dependent increases in apoptosis occur within the intestinal crypt after 3 to 6 hours of exposure to γ-irradiation (81,82). The cells most susceptible to radiation-induced apoptosis were at position 4 of the crypt—again, the stem cell location. These were the cells that died within the first 3 to 6 hours. Ijiri and Potten (80) found that all cells in the crypt were capable of apoptosis, but their susceptibility was dependent on the type of damaging agent. The early- and low-dose apoptotic response associated with stem cells of the small intestine is dependent on p53. Studies using p53−/− mice showed the absence of apoptosis that normally occurred 3 to 6 hours after γ-irradiation of wild-type animals (64,84). Knockout studies using Bcl-2−/− mice showed similar levels of apoptosis in small intestinal crypts 3 to 4 hours after γ-irradiation regardless of the presence of Bcl-2 protein (84). This result is entirely predictable, because the small intestinal epithelium does not express Bcl-2. As would also be expected from the distribution of the protein, increased numbers of apoptotic cells were found in colonic crypts of the Bcl-2−/− animals. Results using the chemotherapeutic agent 5-fluorouracil are similar to those with γ-irradiation, because p53 null mice are resistant to apoptosis after administration of this drug compared with wild-type animals (85). Apoptosis initiated by 5-fluorouracil could be attenuated by the administration of uridine, but not thymidine. The authors suggest that the p53-dependent cell death initiated by 5-fluorouracil was caused by the ability of the drug to damage RNA rather than DNA (85). In Bcl-2 null mice, 5-fluorouracil also had effects similar to those of γ-irradiation, because there was a significant increase in apoptosis at the base of colonic crypts 4.5 hours after administration of the agent (86). This same group has also examined the role of the antiapoptotic protein Bcl-w using the identical experimental design (87). Immunoblotting showed that Bcl-w was expressed in both the small intestine and the colon. Interestingly, spontaneous apoptosis was not altered by the absence of Bcl-w in crypts from either tissue. Both γ-irradiation and 5-fluorouracil, however, caused significantly greater levels of apoptosis in Bcl-w−/− mice than in their wild-type counterparts. Bcl-w, therefore, seems to play a significant role in damage-induced apoptosis in both tissues, whereas Bcl-2 regulates apoptosis only in the colon. Whereas p53, Bcl-2, and Bcl-w have important roles in the regulation of damage-induced apoptosis in the gut epithelia, Bax expression was without effect (86). Neither γ-irradiation nor 5-fluorouracil altered the incidence or location of apoptosis in intestinal epithelia of Bax null mice compared with their normal counterparts. In all of the studies discussed previously, apoptosis was quantitated by identifying apoptotic cells. Marshman and colleagues (88) characterized the activation of caspase-3 along the crypt-villus axis, comparing it with the existing histologic techniques for detecting apoptosis. In both large and small intestines, caspase activation was rare in control,

untreated mice. Exposure to 8 Gy radiation increased caspase-3 activity in the crypts of both tissues, where it was confined to apoptotic bodies. Thus, the determination of caspase-3 activity produced results identical to histologic techniques and provides an additional method for examining cell death in vivo. It is obvious from the experiments discussed earlier that knockout studies are extremely useful in determining whether a specific protein is involved in apoptosis. In fact, these data could not have been obtained in any other way. The primary disadvantages to these studies are: (1) the gene is absent from the entire animal, and it is unknown what compensatory changes have occurred; (2) the development of null mice is expensive and time consuming; and (3) most importantly, these studies tell nothing about the regulatory pathways that actually influence the levels of the proteins being studied. A study by Egan and colleagues (89) uses selective gene ablation to provide important data on the regulation of apoptosis in vivo. These authors examined the role of the transcription factor NF-κB (see Fig. 12-5) in radiation-induced apoptosis in small intestinal epithelium, in which the gene coding IκB kinase had been selectively ablated (thereby preventing the activation of NF-κB) using the Cre/loxP system of somatic DNA recombination. Using morphologic techniques and caspase-3 activation, the authors report the number of apoptotic cells to be approximately twofold greater in the mice in which IκB kinase had been ablated compared with control mice after treatment with 8 Gy γ-radiation. There were no significant effects of gene ablation on spontaneous apoptosis. Whole-cell extracts of isolated IECs from the conditional IκB kinase knockout mice contained greater levels of p53 than the control mice. The conditional knockouts also expressed significantly lower levels of the antiapoptotic proteins Bcl-2 and Bcl-XL. The authors concluded that IκB kinase–dependent NF-κB activation is a key signaling event leading to protection against radiation-induced apoptosis in intestinal epithelia (89). It is likely that one of the mechanisms involved is the stimulation of the transcription of Bcl-2 and Bcl-XL by NF-κB. To this point in the chapter, nothing has been stated about the role of the extrinsic, or “death receptor,” pathway in damage-induced apoptosis. Few experiments have been conducted in vivo regarding the TNFR family, although many have been done using cell lines (see discussion later in this chapter). However, the role of TNFR1 has been examined during the response to radiation-induced damage of the intestinal epithelium. These studies were prompted by observations indicating that p53 mediates apoptosis partly through its ability to transactivate members of the TNFR family (90). Mice in which TNFR1 had been knocked out (TNFR1−/−) and those injected with TNFR1-fusion chimeric (a competitive inhibitor of TNFR1) were partially (30–40%) protected from p53-dependent apoptosis induced by γ-irradiation. The authors also found that radiation induced a p53-dependent increase in the intestinal level of TNF, and that injection of a neutralizing anti-TNF antibody

APOPTOSIS IN THE GASTROINTESTINAL TRACT / 353 decreased p53-dependent intestinal cell apoptosis by 60%. These data indicate that p53-dependent, radiation-induced apoptosis of IECs is mediated, in part, by the upregulation of TNF by p53 and its ability to subsequently induce cell death through TNFR1 (90). A large body of literature exists that deals with the death receptor and ligand system known as the Fas/Fas ligand (FasL) system (see Fig. 12-5). Pinkoski and colleagues (91) have reviewed this topic as it pertains to the liver and the gastrointestinal tract. The best characterized system of apoptosis involving the Fas/FasL system is that found in lymphoid cells. A major role in these cells of the Fas/FasL system is controlling the immune response by regulating the number of lymphocytes. Fas is also expressed in nonlymphoid tissues at sites of immune privilege (ovaries, uterus, testes) and at sites commonly infiltrated by lymphocytes (small and large intestines, liver, and lung). The role of the Fas/FasL system in controlling lymphocytes in these tissues as it pertains to immune privilege, graft-versus-host disease, inflammatory bowel disease, hepatitis B and C, alcohol liver disease, and Wilson’s disease is covered in Pinkoski and colleagues’ review (91) and is not discussed here. Small-Bowel Resection The most widely used model to study intestinal adaptation and mucosal proliferation is partial intestinal resection (92,93). After resection of a significant portion of the small bowel in many species, including humans, the remaining intestinal mucosa undergoes both structural and functional changes including small-bowel dilation, villus enlargement, increased crypt depth, epithelial cell hyperplasia, increased cell migration rate, and increased absorptive capacity per length of intestine (92–95). There have been a plethora of studies attempting to elucidate the mechanisms involved in the growth of the mucosa, but until fairly recently these have all targeted proliferation. Growth results from the equilibrium between cell renewal and cell loss. Cell loss is especially important in tissues that proliferate as rapidly as the intestinal epithelium. Interestingly, Helmrath and colleagues (96) found increased numbers of apoptotic bodies in both crypts and villi of mice after a 50% resection of the small bowel. Several groups have explored the significance of apoptosis in the response to small-bowel resection by examining the effects of various gene products involved in apoptosis on the adaptive response. Shin and colleagues (97) performed 50% intestinal resections on wild-type and homozygous p53-null mice to examine the role of p53 during increased enterocyte apoptosis and the overall adaptive response of the gut mucosa. In general, these investigators found approximately equal values for DNA content, protein content, villus height, crypt depth, and the proliferative index in the transected control and p53 null animals. Seven days after resection, there were significant increases in all these parameters in both groups and no significant differences in the increases themselves; that is, p53 had no significant effect on the

adaptive response. The apoptotic index was determined by counting apoptotic cells in the crypts, and the results indicated that apoptosis was not altered in the p53 null mice after small-bowel resection. In both groups, apoptosis increased approximately 25%, which matched the 23% increase in proliferation. Although this correlation supports a role for apoptosis in maintaining the new equilibrium in cell growth, its significance has not been established. The major conclusion from this study was that the increase in apoptosis after small-bowel resection occurs via a p53-independent mechanism (97). The Bcl-2 family of proteins also has been targeted to explain the increased rates of apoptosis and proliferation after resection. Welters and colleagues (98) examined the effects of 75% small-bowel resection in wild-type control mice and those overexpressing Bcl-2 in the small intestinal epithelium. They concluded that intestinal hyperplasia after resection was significantly increased in the mice overexpressing Bcl-2. Small-bowel resection produced the expected increases in jejunal mucosal weight and protein and DNA content compared with the sham-operated animals. These increases were significantly greater in the mice overexpressing Bcl-2 than in the wild-type resected group. The investigators did not quantitate apoptosis, thus the actual role of cell death in the results can only be surmised. Studies involving the genetic manipulation of the Bax gene, however, have shed considerable light on the role of apoptosis in the adaptive response to small-bowel resection. Stern and colleagues (99) compare adaptation to resection in Bax null mice with that of wild-type counterparts. They observed the usual increases in ileal wet weight, crypt depth, and proliferative rates in both groups. Resection significantly increased the rate of apoptosis in the control group; however, the apoptotic index in the Bax null mice was unchanged. These data support the conclusion that increased apoptosis after small-bowel resection is Bax dependent, and that enterocyte proliferation and apoptosis are regulated via different mechanisms during intestinal adaptation (99). Tang and colleagues (100) have approached the same question but have also examined the pathways involved. One week after resection, they observed significant increases in messenger RNA (mRNA) for caspase-8, Fas, FADD (Fas-associated death domain), and TRADD (TNFR-associated death domain) in Bax+/+ mice, but not in Bax−/− animals. They also found significant increased levels of caspase-8 and Fas protein after resection of Bax+/+ mice. Fas protein was increased in the control Bax+/+ animals as well. In response to resection, apoptosis occurred only in the Bax+/+ mice, prompting the conclusion that adaptation-induced apoptosis is mediated through the extrinsic or FAS pathway but requires the mitochondrial pathway as evidenced by the requirement for Bax. In the adapting Bax+/+ intestine, increased apoptosis was confined to the crypts and was accompanied by increased cell proliferation. Bax−/− mice exhibited an adaptive response similar in magnitude to the Bax+/+ animals; however, it occurred without an increase in cell proliferation and with

354 / CHAPTER 12 an actual decrease in apoptosis. The authors concluded from these data that adaptation-induced, Bax-dependent apoptosis may be the limiting factor determining the magnitude of the adaptive response (100). Knott and colleagues (101) examined the involvement of the extrinsic pathway directly by comparing the adaptive responses to resection in TNFR1 null mice and Fas null mice with wild-type control animals. They found that apoptotic and adaptive responses occurred normally in both null models and concluded that increased apoptosis in response to smallbowel resection does not appear to involve the extrinsic or death receptor pathway. Thus, these data and conclusions disagree with those described by Tang and colleagues (100). In support of their data, Knott and colleagues (101) also examined the expression of caspase-8, TNFR1, FasL, Fas, and TNF in the gut remnant at various postoperative times. They reported only modest changes in these parameters that did not correlate with the histologic appearance of apoptosis. Based on observations that inhibition of the epidermal growth factor receptor (EGFR) decreases adaptation and increases apoptosis and that intestinal levels of Bax correlated with EGF signaling, Jarboe and colleagues (102) examined whether Bax was required for the exaggerated apoptosis after resection in mice with deficient EGFR signaling. They crossed waved-2 mice (impaired EGFR signaling) with Bax null animals. After small bowel resection, enterocyte apoptosis increased in the EGFR-impaired mice and was prevented in the Bax−/− mice. Crossing the animals prevented the increased apoptosis observed in the waved-2 mice. The authors conclude that Bax is required for the increased apoptosis observed after resection, and that it is also necessary for the enhanced apoptotic response to defective EGFR signaling. To summarize the studies, apoptosis after massive smallbowel resection appears to be independent of p53 and dependent on Bax. There are differing results as to the involvement of the extrinsic or death receptor pathway, although the study in which this pathway was inactivated genetically (101) found no involvement of either TNFR1 or Fas. Ischemia–Reperfusion Ischemia–reperfusion induces an apoptotic response in the adult rat intestinal epithelium (103). These experiments demonstrate that apoptosis was induced by ischemia and exacerbated by reperfusion. Coopersmith and colleagues (104) examined this response in mice by quantitating the effects of ischemia–reperfusion on both immature crypt cells and differentiated villous cells. After transient occlusion of the superior mesenteric artery, apoptosis occurred in the undifferentiated epithelial cells of the proliferative compartment, as well as in the differentiated cells of the villus. The response in the villous cells was sevenfold over baseline compared with twofold in the crypt cells. The greatest increase in apoptosis occurred at the villous tip, perhaps reflecting the possibility that hypoxia was greatest in this region. Comparisons of p53+/+ and p53−/− mice also indicated that apoptosis after

ischemia–reperfusion is p53 independent (104). These investigators used a fatty acid–binding protein promoter to produce transgenic mice that overexposed Bcl-2 fivefold in both crypt and villous cells. Forced expression of Bcl-2 reduced apoptosis by approximately 50% in both crypts and villi compared with normal littermates. The authors suggest that these data indicate that both mature and immature cells undergo a commitment phase that is sensitive to Bcl-2. They also found that forced expression of Bcl-2 caused a similar reduction of p53-dependent apoptosis of crypt epithelial cells in response to γ-irradiation. Thus, the ability of Bcl-2 to inhibit apoptosis occurs independently of whether p53 is involved in the response. Factors affecting apoptosis of the intestinal epithelium after ischemia–reperfusion have been studied in detail by Fujimoto and his group of investigators (103,105–107). These studies demonstrate that injury and apoptosis were mediated by reactive oxygen species, inflammatory leukocytes, mitochondrial disfunction, and the release of cytochrome c. Another study from this group examined whether reactive oxygen species induced preoxidative mitochondrial damage by decreasing the mitochondrial membrane potential after ischemia–reperfusion, and whether ischemic preconditioning attenuated apoptosis by affecting the mitochondrial pathway (108). Ischemic preconditioning refers to the fact that a tissue can be made resistant to the effects of long-term ischemia–reperfusion by a prior brief period of occlusion. In this study, 60 minutes of ischemia followed by 60 minutes of reperfusion was used to generate damage. In some instances, the 60-minute occlusion of the superior mesenteric artery was preceded by a process of preconditioning, which consisted of 20 minutes of occlusion followed by 5 minutes of reperfusion. These experiments were conducted in rats, and the major stimulus caused an approximate sevenfold increase in apoptosis, which was decreased 30% by preconditioning. The investigators found that ischemia–reperfusion by itself caused mitochondrial respiratory disfunction, which resulted in a significant increase in the production of reactive oxygen species. Increased production of reactive oxygen species enhanced mitochondrial glutathione oxidation and lipid peroxidation. Peroxidation of lipids reduced the mitochondrial membrane potential, which led to the release of cytochrome c, caspase-9 activation, and finally, the activation of caspase-6-in the small-bowel mucosa. Each step in this scheme was inhibited significantly by ischemia preconditioning, which resulted in the reduction of apoptosis (108). This study therefore shed light on the mechanism by which ischemia–reperfusion damages the intestinal mucosa and the role of the mitochondria in that damage. Prevention of Apoptosis Radiation and chemotherapy for human tumors target the rapidly dividing cancer cells but also affect normal cells with high rates of proliferation. Foremost among these are the cells of the bone marrow and intestinal epithelia.

APOPTOSIS IN THE GASTROINTESTINAL TRACT / 355 Acute injury to the intestinal mucosa is the major complication of this therapy. The toxicity derived from the loss of mucosal cells consists of diarrhea, dehydration, and bacterial infection, which can lead to sepsis and shock. This sequelae often limits the dose of therapeutic agent, and hence the effectiveness of the therapy itself. In addition, the quality of the lives of patients can be compromised severely. For these reasons, considerable time and effort have been invested in attempting to discover an agent or procedure that would decrease the severity of the side effects on the mucosa of the gastrointestinal tract. There are threshold levels of radiation above which the bone marrow and intestinal mucosa are unable to repopulate themselves. In mice, exposure to 7 to 15 Gy total body radiation kills most proliferating cells of the bone marrow, but sufficient stem cells survive to repopulate the tissue (109). Greater doses result in an insufficient number of surviving stem cells, and the animal dies. After 8 to 14 Gy radiation, many proliferating IECs are killed, but a sufficient number of stem cells survive to repopulate the mucosa (62,81,110). The greater the dose of radiation, the smaller the number of surviving stem cells and the fewer the number of regenerating crypts. Three to four days after the radiation insult, the number of active crypts can be counted histologically as an assay of stem cell survival and the overall damage (110,111). This assay is quite useful for evaluating “protection” and has been termed a clonogenic assay, because a single surviving stem cell can generate a new crypt. Numerous studies have identified several different classes of compounds with radioprotective effects on the small-bowel mucosa. Growth Factors Although several growth factors have been shown to protect enterocytes from apoptosis, there are few systematic studies or series of studies involving these compounds. Epidermal growth factor (EGF) was shown to protect against mucositis induced by 5-fluorouracil (112). However, there have also been conflicting results, and a more recent report examined the role of EGF using both transgenic and classical approaches. Huang and colleagues (113) found that mice overexpressing EGF specifically in the small intestine showed no differences in weight, apoptosis, crypt cell proliferation, and mucosal architecture when compared with control animals after a course of fluorouracil. Treatment of normal mice with exogenous EGF also did not protect, despite a variety of doses, schedules, and routes of administration. The investigators also induced mucositis in mutant mice with specific defects in EGF signaling. Compared with control animals, clinical and histologic parameters after fluorouracil were not different in mice with functionally defective EGFRs or those lacking transforming growth factor-α (TGF-α), a major ligand for the EGFR (113). Granulocyte-macrophage colony-stimulating factor has been shown to reduce chemotherapy-induced intestinal damage in a rat model by decreasing glutathione generation and lipid peroxidation (114). Glucagon-like

peptide-2 also decreases injury of the gut caused by chemotherapeutical agents by preventing some of the apoptotic response (115,116). A similar claim has been made for keratinocyte growth factor in mice subjected to both radiation- and chemotherapy-induced injury. The authors found increased stem cell survival, and they suggest that it acts by stimulating cell proliferation (117). Interestingly, the protective effects of TGF-β3 have been attributed to its ability to induce cycle arrest before exposure to either radiation or chemical agents (118–120). Dunker and colleagues (121) found decreased rates of apoptosis in mice heterozygous for TGF-β2 and TGF-β3 with concomitant increases in Bcl-2 and Bcl-XL. Because it is unlikely that one peptide, keratinocyte growth factor, prevents apoptosis by stimulating cell proliferation, whereas another, TGF-β3 protects stem cells by inducing cell cycle arrest, it is safe to conclude that the mechanism(s) of action of these compounds remains to be elucidated. Podolsky and coworkers have undertaken a series of studies involving intestinal trefoil factor (ITF). Intestinal trefoil factor is produced and secreted by goblet cells of both the small and large bowels, enhances cell migration, and protects enterocytes from damage (122). Intestinal trefoil factor has been shown to decrease apoptosis of colonic epithelial cells in response to ceramide, serum starvation, and etoposide (123,124). Because apoptosis of goblet cells is characteristic of the intestinal response to radiation and chemotherapy, Beck and colleagues (125) hypothesize that these procedures decrease ITF expression, and that the loss of its effects on migration and cellular function impair healing. Three and a half days after exposure to 14 Gy total-body radiation, there was a marked reduction in goblet cell number and ITF mRNA. Intestinal trefoil factor enhanced migration of IEC-6 cells and wound repair in vitro after methotrexate. intestinal trefoil factor–negative (ITF−/−) mice were significantly more susceptible to 5-fluorouracil–induced weight loss, diarrhea, and increased intestinal permeability compared with wild-type control animals. Survival of the ITF−/− mice was also decreased after chemotherapy. Exogenous ITF given orally was able to improve crypt survival significantly after radiation and chemotherapy. Interestingly, ITF did not alter the expression of a host of molecules involved in apoptosis, including Fas, FasL, TNF, TNFR1, TNFADD, and numerous members of the Bcl-2 family of proteins, as assessed by microarray analysis. The authors suggest that ITF is involved in the protection against, and recovery from, radiation and chemotherapy (125). Several early studies demonstrated that basic fibroblast growth factor (bFGF) could protect endothelial cells from radiation-induced injury (126–128). Dignass and colleagues (129) found that bFGF is expressed in intestinal mucosa, and that it may regulate epithelial cell growth and migration. Houchen and colleagues (130) examined the effect of radiation injury on the expression of bFGF and the survival of crypt stem cells in the mouse small intestine. Fibroblast growth factor mRNA increased significantly 48 hours after exposure to 13 Gy and reached a peak at 96 hours.

356 / CHAPTER 12 COX-2) and by a specific COX-2 blocker. Lipopolysaccharide was protective in COX-1−/− mice, but its effects were eliminated in COX-2 knockout animals. Thus, the radioprotective effect of LPS on the mouse intestine is mediated through prostaglandin synthesized by COX-2 (137). Interestingly, the same group had previously obtained evidence that crypt cell survival in the mouse small intestine after irradiation was dependent on prostaglandin synthesized via COX-1 (138). This conclusion was based on the findings that COX-1 localized to the crypt cells, and 3 days after irradiation, prostaglandin E2 (PGE2), COX-1 mRNA, and COX-1 protein levels were increased. Although these two studies implicate PGE2 in an important role in the response to radiation damage, they also imply different mechanisms. A follow-up study identified the EP2 receptor as being responsible for mediating at least some of the survival effects of PGE2 on the murine intestinal mucosa (139). Riehl and colleagues (140) have obtained evidence that TNFR1 mediates the radioprotective effects of LPS. Crypt cell apoptosis and the number of surviving crypts were measured in wild-type, TNFR1−/−, and TNFR2−/− mice after 12 Gy radiation with and without pretreatment with LPS. Lipopolysaccharide reduced radiation-induced apoptosis in wild-type and TNFR2−/− mice but had no effect in the TNFR1 knockouts. Exogenous PGE2 protected the TNFR1−/− animals. TNFR1 and COX-2 expression were found in subepithelial fibroblasts and villous cells, but not crypt cells. The authors suggest that the subepithelial fibroblasts could be the source of the protective prostaglandins in vivo. Thus, as in the case of bFGF, the protective actions of LPS on crypt stem cells may be mediated by a second cell type.

The expression of FGF protein in the small intestine followed a similar time course. Treatment with FGF before, but not after, irradiation enhanced crypt cell survival threefold. Additional studies involving bFGF provided evidence that intestinal injury after irradiation in mice is secondary to apoptosis of endothelial cells (131). Receptors of bFGF were found in intestinal endothelial cells, but not in crypt or villous cells, with the exception of Paneth cells. Intravenous administration of bFGF prevented radiation-induced endothelial apoptosis and crypt damage, organ failure, and death from the gastrointestinal syndrome. To investigate whether endothelial apoptosis was causally responsible for the damaged intestinal epithelium, these investigators examined the responses of mice in which the acid sphingomyelinase (asmase−/−) gene had been knocked out. These animals did not synthesize the proapoptotic lipid, ceramide, in endothelia after irradiation. Apoptosis of the intestinal epithelia, damage to the gut, and death were prevented after irradiation in the asmase−/− mice. The authors conclude that endothelial apoptosis is the primary lesion initiating intestinal radiation damage in mice (131). They suggest that these data provide a new approach to prevent radiation damage to the bowel. Prostaglandins The E-type prostaglandins protect both the intestine and bone marrow from radiation damage (132–134). Lipopolysaccharide (LPS) protects the bone marrow from radiation injury (135) and stimulates prostaglandin synthesis by inducing cyclooxygenase-2 (COX-2) (136). Stenson’s group demonstrated that LPS administered at least 2 hours before irradiation led to a threefold increase in the number of surviving crypts 3.5 days later (137). Parenteral administration of LPS stimulated prostaglandin production in the intestine and induced COX-2 expression in subepithelial cells and villous but not crypt cells. The protective action of LPS was prevented by indomethacin (inhibits COX-1 and

Polyamines The polyamines, spermidine and spermine, and their precursor, putrescine, are found in virtually all cells of higher eukaryotes (141); they are intimately involved in, and

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FIG. 12-8. Apoptotic cells in mice subjected to 15 Gy γ-irradiation and killed 4 hours later. Mice received vehicle (control) or 2% DL-α-difluoromethylornithine (DFMO) in their drinking water for 4 days before irradiation. (A) Apoptotic cells per crypt-villus unit. (B) Position of apoptotic cells along crypt-villus axis. (Reproduced from Deng and Johnson [148], by permission.)

APOPTOSIS IN THE GASTROINTESTINAL TRACT / 357

Lysophosphatidic Acid The phospholipid, lysophosphatidic acid (LPA), a normal constituent of serum, mimics the actions of polypeptide growth factors. Lysophosphatidic acid acts on a G-protein– coupled family of receptors to influence cell-cycle progression, the actin cytoskeleton, cellular differentiation, Ca+2 homeostasis, and cell survival (149–151). Lysophosphatidic acid has been shown to enhance the survival of Schwann cells and lymphocytes (152,153) and to suppress apoptosis induced by ischemia–reperfusion in cardiomyocytes (154) and by serum withdrawal in fibroblasts and renal epithelial cells (155,156). Deng and colleagues (157) examined the ability of LPA to prevent apoptosis in IEC-6 cells and the intestinal epithelia of mice. Injection of LPA 2 hours before exposure to 15 or 18.75 Gy γ-radiation significantly reduced the number of apoptotic bodies per crypt-villus unit compared with intestines from mice injected with vehicle alone. In IEC-6 cells, LPA rescued cells from apoptosis when applied up to 1 hour after camptothecin treatment or 2 hours after exposure to radiation. Lysophosphatidic acid also inhibited the activation of caspase-3. The ability of LPA to prevent apoptosis

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1 Control 2 Irradiation

1.00 Caspase-3 activity pmol/mg protein/min

required for, cell growth and proliferation (142,143). Intracellular polyamine levels are highly regulated and are dependent primarily on the activity of ornithine decarboxylase (ODC), which catalyzes the first rate-limiting step in polyamine biosynthesis, the decarboxylation of ornithine to form the diamine putrescine (144,145). DL-αdifluoromethylornithine (DFMO) blocks ODC and depletes cells of polyamines. DL-α-difluoromethylornithine is a specific and irreversible inhibitor of ODC, having no effects except those caused by the inhibition of ODC and the subsequent depletion of polyamines (145,146). Polyamines are involved in apoptosis, and whether they increase or inhibit it depends on the cell type or tissue involved and the stimulus for apoptosis (147). We have found, however, that DFMO and subsequent polyamine depletion prevent apoptosis in IEC-6 cells. (These studies are discussed in detail in the next section.) We have also shown that polyamine depletion inhibits irradiation-induced apoptosis in the epithelium of the mouse small intestine (148). Four hours after exposure to 15 Gy radiation, apoptotic cells increased from 0.6 to 2.7 per cryptvillus unit. In polyamine-depleted animals, the increase was reduced to 1.6 cells (Fig. 12-8A). Most apoptotic cells occurred at positions 4 through 7 within the crypts (see Fig. 12-8B). Polyamine depletion also prevented most of the increases in caspase-3 activity (Figs. 12-9A and B) and the proapoptotic protein Bax (see Fig. 12-9C) after irradiation. Four days after irradiation, the crypt survival rate was increased more than twofold in the mice treated with DFMO compared with control animals. Because DFMO is already approved for use in patients to retard tumor growth and because it can be administered orally, polyamine depletion may be useful therapeutically to counteract gastrointestinal toxicity caused by radiation and chemotherapy (148).

3 Irradiation DFMO

0.75

*

0.50

0.25

0

1

A

3

2

kDa Pro-caspase-3

−32

Active Caspase-3 unit

−19

Beta-actin

−42

B Irradiation

1

2

3

−

+

+

1

2

3

Bax

Beta-actin

C

FIG. 12-9. Polyamine depletion inhibits caspase-3 activation and Bas after irradiation of the mouse small intestine. Animals were treated as in Figure 12-8. (A) Caspase-3 activity determined by enzyme-linked immunosorbent assay. (B) Pro-caspase-3 and caspase-3 Western blots. (C) Bax levels assayed by Western blotting. DFMO, DL-α-difluoromethylornithine. (Reproduced from Deng and Johnson [148], by permission.)

even when administered after the apoptotic stimulus is interesting. It remains to be seen whether this effect can be reproduced in whole animals. Others Several cytokines have been reported to protect intestinal epithelia from programmed cell death. Interleukin-11 (IL-11) protects clonogenic stem cells in the intestinal crypts of mice after radiation (158), stimulates the recovery of small-bowel epithelial cells after cytoablative therapy (159), and reduces

358 / CHAPTER 12 increased both caspase-3 activity and DNA fragmentation. Thus, NO appears to exert an antiapoptotic effect on the rat gastrointestinal tract.

the severity of chemotherapy-induced mucositis in hamsters (160). Interleukin-15 has been shown to protect the intestinal mucosa from several chemotherapeutic agents including 5-fluorouracil (161). Leukotriene D4 increases cell survival by preventing apoptosis in IECs (162). The mechanism of this effect has been explored Wikström and colleagues (163). These investigators found that leukotriene D4 blocked the release of cytochrome c caused by the inhibition of COX-2. They found that COX-2 inhibition resulted in the activation of caspase-8 and the subsequent production of t-Bid, indicating that this pathway to apoptosis is blocked by prostaglandins. Leukotriene D4 also reduced the basal level of caspase-8 activation and the t-Bid generation. The effect of nitric oxide (NO) on intestinal apoptosis has been examined by Bersimbaev and colleagues (164) in the rat gastrointestinal tract. They exposed mucosal cells to NO delivered from the NO donor, S-nitroso-N-acetylpenicillamine, and examined the effect of intravenous LPS in the presence and absence of the inhibition of NO synthesis. Nitric oxide inhibited caspase-3 activity and apoptosis as measured by DNA fragmentation. Lipopolysaccharide increased both caspase-3 activity and apoptosis in gastric, ileal, and colonic mucosa 5 and 24 hours after LPS. Administration of an inhibitor of inducible nitric oxide synthase (iNOS) immediately after LPS injection significantly

INTESTINAL CELLS: IN VITRO STUDIES This section covers in vitro studies involving cultured cell lines and isolated enterocytes. Cell lines have been used to elucidate apoptotic pathways and to identify points at which these pathways can be controlled. Isolated enterocytes have been used primarily to examine the onset and events involved in apoptosis induced by detachment from substrate or ECM, the process referred to as anoikis.

Apoptotic Pathway in Intestinal Cells The extrinsic and intrinsic apoptotic pathways shown in Figures 12-5 and 12-6 are the consensus of many experiments conducted with a variety of different cell types. Figure 12-10 illustrates the responses to activation of the extrinsic pathway by TNF-α and cycloheximide (CHX), which is given to block the synthesis of antiapoptotic proteins (165), and activation of the intrinsic or mitochondrial pathway by either the

TNF-α

(167)

(167) IκBa

PI3K Akt

p53

Procaspase-8 IκBa = NF-κB (167)

ERK1/2 (38)

(166) Bax Caspase-8

NF-κB (167)

MEKK

Damage (12,148)

(167)

Bcl-XL Bcl-2 (12) MEK4/7

Bid (12)

IAPs

Procaspase-3 (12,38,120)

(38)

t-Bid

JNK (166)

MEK1 (38)

Nucleus Bcl-XL (12)

Caspase-3

Bad

Erk1/2

Bcl-2 CHX (166)

(12,38,166) Cyto c

APAF-1

Caspase-9 Apoptosis (12,38,165,166,167,168)

Procaspase-9 (12,166)

FIG. 12-10. Apoptotic pathways activated in intestinal epithelial cell-6 (IEC-6) cells by tumor necrosis factor-α (TNF-α) and damage induced by γ-irradiation or camptothecin. CHX, cycloheximide; Cyto c, cytochrome c; ERK, extracellular signal–regulated kinase; IAP, inhibitor of apoptosis protein; IκBα, inhibitory κBα; JNK, c-Jun NH2-terminal kinase; NF-κB, nuclear factor-κB; PI3K, phosphatidylinositol-3 kinase; TNF, tumor necrosis factor.

APOPTOSIS IN THE GASTROINTESTINAL TRACT / 359 topoisomerase I inhibitor, camptothecin (12), or γ-irradiation (148) in the IEC-6 cell line. This line was originally described by Quaroni and colleagues (166) and was derived from adult rat intestinal crypt cells. It remains a nontransformed, undifferentiated cell line. The pathways shown in Figure 12-10 are the first comprehensive portrayal of events after an apoptotic stimulus in IECs. As shown, the primary apoptotic effect of TNF-α is the activation of procaspase-8 to caspase-8 to initiate the extrinsic pathway. The mitochondrial pathway is also activated in response to TNF-α because caspase-8 cleaves Bid to produce t-bid (12). Tumor necrosis factor-α also initiates two survival pathways by activating PI3K, which leads to activation of NF-κB (167) and MEK, which leads to the activation of ERK1/2 (38). These transcription factors produce IAPs and Bcl-2, which would block the apoptotic response in the absence of CHX (165). Damage to IEC-6 cells causes the activation of Bax and the mitochondrial pathway with the subsequent release of cytochrome c (12).

Polyamines and Apoptosis Depletion of polyamines by the inhibition of ODC with DFMO leads to cell-cycle arrest without apoptosis in IEC-6 cells (168). Cell-cycle arrest was the result of the induction of p53 and the cell cycle inhibitors p21 and p27. The absence of apoptosis under these conditions prompted Ray and colleagues (169) to examine whether DFMO and polyamine depletion altered the response of IEC-6 cells to apoptotic stimuli. Both camptothecin and TNF-α/CHX significantly increased apoptosis as measured by DNA fragmentation and caspase-3 activity. Polyamine depletion prevented most of both responses. Cells that were cultured with exogenous putrescine together with DFMO had apoptotic responses indistinguishable from control samples, indicating that the protective effect was caused by polyamine depletion and not DFMO itself. Increasing the levels of putrescine by blocking its conversion to spermidine induced apoptosis in the absence of other stimuli (169). This result was in keeping with data from other cell types indicating that excess polyamines could stimulate apoptosis (170,171). An additional interesting report by Li and colleagues (172) indicates that polyamine depletion can either sensitize IEC-6 cells to apoptosis or prevent it, depending on the stimulus. These authors found that DFMO increased apoptosis in response to staurosporine and decreased it after TNF-α/CHX. Both responses appeared to depend on NF-κB, because they were prevented by the inhibition of NF-κB binding activity. To my knowledge, this is the only report where polyamine depletion sensitized IECs to an apoptotic stimulus. This observation has not been pursued, thus the mechanism remains unknown. Effects of Polyamine Depletion Yuan and colleagues (12) have shown that polyamine depletion prevented the release of cytochrome c from

mitochondria after camptothecin. This was due, in part, to increased levels of Bcl-2 and Bcl-XL and decreased levels of Bax and t-Bid in the cells incubated with DFMO. In addition to inhibiting the activation of caspase-3, the depletion of polyamines significantly reduces the activation of caspase-6, -8, and -9 in response to TNF/CHX (165). The remaining proapoptotic step shown in Figure 12-10 involves JNK, which has been shown to be activated by TNF-α/CHX through TNFR1 and TNF receptor-associated factor (TRAF2) (31,173). Tumor necrosis factor-α/CHX also activates JNK in IEC-6 cells, and that activation is dependent on the presence of polyamines (165). Polyamine depletion prevented JNK activation, caspase-9 activation, DNA fragmentation, and caspase-3 activation. Inhibition of JNK with the specific inhibitor SP-600125 prevented apoptosis, caspase-9 activation, and the release of cytochrome c. Thus, part of the antiapoptotic effect of polyamine depletion appears to be caused by the inhibition of JNK activity (165). The mechanism by which JNK activates apoptosis is not clear; however, these results indicate that JNK regulates a step(s) upstream of caspase-9 and modulates the mitochondrial pathway by influencing cytochrome c release or Bcl-2. Two other pathways shown in Figure 12-10 involve ERK and NF-κB. Both of these are activated by TNF/CHX and enhanced by polyamine depletion. A large number of reports point to the role of ERKs in mediating a strong antiapoptotic response (37,174,175). Polyamine depletion causes the sustained activation of ERK in response to TNF-α/CHX (38). Pretreatment of polyamine-depleted cells with a membranepermeable MEK1/2 inhibitor, U-0126, significantly inhibited TNF-α/CHX-induced ERK phosphorylation and increased DNA fragmentation, JNK activity, and caspase-3 activity in response to TNF-α/CHX. IEC-6 cells expressing constitutively active MEK1 had decreased TNF-α–induced JNK activation and were significantly protected from apoptosis. Conversely, a dominant negative MEK1 resulted in high basal activation of JNK, cytochrome c release, and spontaneous apoptosis. Polyamine depletion of the dominant negative MEK-1 cells did not prevent JNK activation or cytochrome c release and failed to protect cells from both TNF-α/CHXand camptothecin-induced apoptosis (38). The stable expression of a dominant negative mutant of JNK significantly protected IEC-6 cells from apoptosis. Thus, polyamine depletion in IEC-6 cells activates ERK, which inhibits JNK activation, leading to the protection of cells from apoptosis (38). Polyamine depletion rapidly activates NF-κB (176). Within 1 hour after administering DFMO, p65 had redistributed from the cytoplasm to the nucleus of IEC-6 cells. The degradation of IκBα also began within 1 hour and was complete by 5 hours. These times correlated with a 50% decrease in putrescine levels at 1 hour and a total absence of putrescine by 3 hours. The activation of NF-κB was prevented by the addition of exogenous putrescine to the DFMOtreated cultures (176). Li and colleagues (172) also reported that polyamine depletion led to activation of NF-κB from 4 to 8 days after constant exposure to DFMO. They found that inhibition of NF-κB binding activity by sulfasalazine or

360 / CHAPTER 12 MG-132 prevented the increased apoptotic response to staurosporine and the decreased apoptotic response to TNF/ CHX normally found in polyamine-depleted cells. Wang and his coworkers (177) have followed this study by showing that activation of NF-κB by polyamine depletion led to the synthesis of cIAP2 and XIAP, members of the inhibitors of apoptosis family, and the inhibition of caspase-3 activity. They did not try to reconcile these findings with their previous data indicating that NF-κB activation enhanced staurosporine-induced apoptosis (172). TNF-α–induced activation of NF-κB has been shown to require Akt (178,179). Akt, also known as protein kinase B1, is a serine/threonine protein kinase, originally identified as the oncogene transduced by the acute transforming retrovirus Akt (180,181). In quiescent cells, Akt resides in the cytosol in an inactive state. After cytokine stimulation, it translocates to the inner surface of the plasma membrane where PI3K-generated 3′-phosphoinositides reside, and Akt is phosphorylated at threonine 308 and serine 473 by pyruvate dehydrogenase kinase-1 (PDK1) for its full activation (see Fig. 12-10) (182). Akt has been shown to inhibit apoptosis by directly phosphorylating and inactivating Bad (183). Akt protects cells from apoptosis by inactivating caspase-9 and by activating NF-κB (184,185). Although Akt inhibits apoptosis by regulating the release of mitochondrial cytochrome c, the mechanism is not clear (186,187). Glycogen synthase kinase-3β (GSK-3β), an important downstream target of Akt, is inhibited by Akt phosphorylation (188). Increased activity of GSK-3β has been shown to induce apoptosis of several cell types (189). Zhang and colleagues (190) found that inhibition of Akt increases apoptosis in polyamine-depleted IEC-6 cells, and they suggest that Akt activation is responsible for suppression of caspase-3 activity through a process involving the phosphorylation of GSK-3β. Bhattacharya and colleagues (167) found that inhibition of GSK-3β had no effect on apoptosis induced by TNF-α in IEC-6 cells. They went on to show that inhibition of PI3K and overexpression of dominant-negative Akt (DN-Akt) significantly increased apoptosis in polyaminedepleted cells. Constitutive activation of Akt in response to polyamine depletion was prevented in DN-Akt cells, reversing the protective effect of polyamine depletion. Expression of DN-Akt, as well as PI3K inhibitors, prevented Akt activation and subsequent translocation of NF-κB to the nucleus. Constitutively active Akt (CA-Akt) expression increased the resistance to TNF-α–induced apoptosis and increased the activation of NF-κB. Polyamine depletion of cells transfected with DN-Akt prevented basal and TNF-α–induced IκBα phosphorylation. Prevention of NF-κB activation in cells transfected with DN-IκBα increased apoptosis basally and restored it in polyamine-depleted cells. Thus, protection from apoptosis by activated Akt in polyamine-deficient cells is dependent on the nuclear translocation and activation of the transcription factor NF-κB and is independent of GSK-3β activity. Akt regulates the mitochondrial pathway, preventing activation of caspase-9, and thereby caspase-3, via the actions of NF-κB (167).

Signal transducers and activators of transcription (STATs) proteins are a family of latent transcription factors that undergo ligand-dependent tyrosine phosphorylation and activation (191). STAT3 is the transcription factor responsible for activation of the acute phase-response genes and binds to the sis-inducible element (SIE) found in the c-fos promoter regulated by platelet-derived growth factor (PDGF) (192). Pfeffer and colleagues (193) found that inhibition of ODC with DFMO rapidly induced STAT3 activation as determined by its tyrosine phosphorylation, translocation from the cytoplasm to the nucleus, and presence in SIE-dependent DNA-protein complexes. Activation of STAT3 was accompanied by the activation of a STAT3-dependent reporter construct. Additional phosphorylation of STAT3 at serine 727 is thought to increase its transcriptional activity and is mediated by the MAPK pathway (194,195). Constitutive activation of STAT3 has been described in a wide variety of primary tumors and has been reported to induce cell survival in association with surviving expression in gastric cancer cells (196,197). STAT3 signaling appears to mediate the survival of oncogenic Ras-transfected IECs (198). Bhattacharya and colleagues (199) found that polyamine depletion induced STAT3 phosphorylation at both Tyr-705 and Ser-727, resulting in localization to the cell periphery and nucleus, respectively. Sustained phosphorylation of STAT3 at both residues occurred after polyamine-depleted cells were exposed to TNF-α. Inhibition of STAT3 increased the sensitivity of polyamine-depleted cells to apoptosis, and the protective effect of polyamine depletion was totally eliminated in cells expressing DN-STAT3. DFMO increased mRNA and protein levels of Bcl-2, Mcl-1, and BIRC3. These levels were significantly decreased in DN-STAT3– expressing cells. Inhibition of STAT3 in polyamine-depleted cells induced the activation of caspase-3 and apoptosis. Thus, STAT3 appears to play a prominent role in protecting polyamine-depleted cells from apoptosis. This protection may be mediated by the expression of antiapoptotic proteins, Bcl-2, Mcl-1, and BIRC3 (199). Regulation of Apoptotic Pathways It is naive to believe that polyamines affect each of the steps or even several steps in Figure 12-10. In eukaryotic cells, reversible protein phosphorylation by protein kinases and phosphatases regulates numerous processes, including apoptosis (200). The kinases themselves are dephosphorylated and inactivated by a type 2A protein phosphatase (PP2A). Previous studies have implicated serine/threonine PP2A in a wide variety of cellular functions including metabolism, transcription, translation, ion transport development, proliferation, differentiation, and apoptosis (200–202). Figure 12-10 depicts two major regulatory pathways—the ERK/JNK pathway and the PI3K/Akt/NF-κB pathway— that are altered by polyamine depletion. In addition, each of these pathways controls the levels or activities of Bcl-2 family proteins that are responsible for determining whether cytochrome c is released from mitochondria. The activities

APOPTOSIS IN THE GASTROINTESTINAL TRACT / 361 Protein

Activity of Protein

Apoptosis

ERKs Akt IκBα NF-κB Bcl-2 Bad FIG. 12-11. The effects of serine-threonine phosphorylation on the activities of proteins regulating apoptosis. The consequences of the change in activity on apoptosis also is shown. ERK, extracellular signal–regulated kinase; IκBα, inhibitory κBα; NF-κB, nuclear factor-κB.

of many components of these pathways and the Bcl-2 proteins themselves are regulated by serine/threonine phosphorylation. Bcl-2, for example, is inactivated by PP2A, whereas Bad is activated by PP2A (201). An analysis of the effects of serine/threonine phosphorylation on the activities of proteins in these signaling pathways is shown in Figure 12-11. In each case, this effect is in the same direction as the change in activity reported for polyamine depletion. Also in each case, the change in activity, whether an increase or a decrease, acts to prevent apoptosis. In addition, Akt and the ERKs are activated within minutes of exposing IEC-6 cells to TNF-α. These activities peak around 3 to 4 hours, and then decrease to basal levels by 9 hours (38, 167). In polyamine-depleted cells, however, the activities of

300 DFMO 250

Activity

200 150 100 Control

50 0 0

3

6

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Time (h) TNF/CHX

FIG. 12-12. Generalized time course of the activities of Akt and extracellular signal–regulated kinase (ERK) after exposure of control and polyamine-depleted DL-α-difluoromethylornithine (DFMO) cells to tumor necrosis factor-α/ cycloheximide (TNF-α/CHX).

these enzymes increase to greater levels, and then plateau. Figure 12-12 is a generalized representation of these findings. Thus, there appears to be no inactivation of Akt and ERK in DFMO-treated cells so that polyamine depletion renders these enzymes constitutively active (38,167). The findings depicted in Figures 12-11 and 12-12 could be explained if PP2A were inactive in polyamine-depleted cells and its inactivation protected those cells from apoptosis. In support of this explanation are earlier reports indicating that polyamines can activate PP2A specifically compared with PP1 (203,204). Ray and colleagues (205) have shown that the inhibition of PP2A with either okadaic acid or fostriecin protected IEC-6 cells from apoptosis induced by TNF-α/CHX. Cells transfected with the siRNA for PP2A were also protected from apoptosis. Polyamine-depleted cells had only about one-third the PP2A activity of control cells or those incubated with putrescine in addition to DFMO. Inactivation of Bad involves the phosphorylation of serine residues 112 and 136, which decreases its affinity for Bcl-XL at the mitochondrial membrane (200,206). This frees Bcl-XL to prevent apoptosis. PP1 activates Bad by serine dephosphorylation. Current data suggest that PP1 itself is dephosphorylated and activated by PP2A, which is also responsible for dephosphorylating and inactivating Bcl-2 (200). Thus, decreased PP2A activity in polyamine-depleted cells may not only prevent the inactivation of Bc1-2, but also cause Bad to remain phosphorylated and inactive. Polyamine-depleted cells have been shown to have significantly greater levels of pSer112-Bad than control cells (205). Furthermore, the level of active pSer Bcl-2 decreased after TNF-α/CHX in control cells but increased significantly in cells treated with DFMO. In addition, okadaic acid increased the phosphorylation of these proteins similar to the effects of polyamine depletion. The inhibition of PP2A with okadaic acid also mimicked other effects of polyamine depletion, namely the prevention of cytochrome c release, inhibition of JNK activity, and the inhibition of caspase-9 and -3 in response to TNF-α/CHX (205). Type 2A protein phosphatase, therefore, appears to be a crucial point for controlling apoptotic pathways in IECs. Type 2A protein phosphatase itself is regulated by tyrosine kinases such as Src kinase and the EGFR, which phosphorylate it, inhibiting the enzyme (207,208). Signals transduced through a single receptor, such as the TNF-α receptor, can promote either cell proliferation or apoptosis, depending on how the cell is programmed to respond to receptor proximal events. Expression of v-Src rescues several cell types from apoptosis induced by cytokine withdrawal, irradiation, chemotherapeutic drugs, or detachment from the ECM (209–213). These findings suggest that constitutively activated Src can mimic the effects of cytokine receptors, integrins, and other receptor pathways that protect cells from apoptosis. The mechanism by which Src inhibits apoptosis is not well defined because of the divergent nature of Src signaling. However, current studies suggest the Src may regulate apoptosis by altering the strength and balance

362 / CHAPTER 12 of signals activating the various MAPK and PI3K/Akt pathways (214–217). We have shown that Src is constitutively activated in polyamine-depleted IEC-6 cells (218). Phosphorylation of Tyr 416 in the kinase domain completely activates Src and provides a binding site for SH2 domains of other proteins (219). Incubation of IEC-6 cells with DFMO for 4 days significantly increased the amount of pY 416-Src without affecting the level of Src protein. Pharmacologic inhibition of Src increased basal apoptosis and apoptosis in response to TNF/CHX in control cells. In polyamine-depleted cells, the inhibition of Src restored apoptosis to control levels. The expression of DN-Src in IEC-6 cells increased apoptosis significantly compared with vector-transfected cells and eliminated all protection by DFMO. Conversely, the expression of CA-Src produced cells resistant to apoptosis (218). Figure 12-13 depicts a model showing how the activation of Src might account for the effect of polyamine depletion on apoptosis. As noted earlier, the activation of STAT3 contributes to the resistance of polyamine-depleted cells to apoptosis. Interestingly, Src activation may also account for the activation of STAT3. The activation of STAT3 has been associated with Src activity in cell lines transformed with v-Src (220,221). Src is known to activate STAT3 in PDGF-stimulated fibroblasts (222). Zhang and colleagues (223) have proposed a mechanism for STAT3 activation in which Src, as part of a membrane-anchored scaffold, phosphorylates and activates Janus kinase (JAK), which, in turn, phosphorylates the cytoplasmic tail of a membrane receptor, which acts as the docking site for STAT3, which is then phosphorylated by Src. In conclusion, polyamine depletion has not only allowed investigators to define the effects of polyamines on apoptosis, but it has contributed to our understanding of the apoptotic process and how it is regulated in IECs. In DFMO cells:

+Src

+ Bcl-2-pSer

− PP2A-PTyr

+ Akt-pSer

+ ERKs-pTyr/Thr

− IKB-α-pSer

+ NFκB

− JNK

− Bad-pSer112

FIG. 12-13. Summary of the changes (positive [+] and negative [−]) in activity occurring as a result of polyamine depletion and how they can be accounted for by a Src-induced inhibition of type 2A protein phosphatase (PP2A). DFMO, DL-αdifluoromethylornithine; ERK, extracellular signal–regulated kinase; IκB-α, inhibitory κB-α; JNK, c-Jun NH2-terminal kinase; NF-κB, nuclear factor-κB.

Additional Studies Numerous studies using cultured or isolated intestinal cells have examined the regulation of various steps in the apoptotic pathway. For the most part these experiments agree with those involving polyamine depletion in cultured cells and those examining the same compounds in vivo. The importance of the Ras/MAPK/ERK pathway was emphasized in a study that showed dying enterocytes downregulate pathways converging on Ras (224). Ras-convergent proteins such as the EGFR, Erb-B2, and the Son of sevenless guanine nucleotide exchangers were rapidly down-regulated. Caspase inhibitors, however, prevented both down-regulation and apoptosis. Yan and colleagues (225) found that expression of the DN kinase inactive kinase suppressor of Ras (KSR), which regulates the TNF activation of the Raf/MAPK/ERK pathway, decreased survival and increased apoptosis of TNFα–treated IECs. Antiapoptotic pathways including NF-κB, cIAP2 gene expression, and ERK/MAPK activation were all inhibited in the DN-KSR cells. In the T84 cell line, Rudolph and colleagues (226) found that cyclic adenosine monophosphate (cAMP) inhibits apoptosis by up-regulating ERK1/2 through a protein kinase A (PKA)–independent mechanism. It is obvious from these studies and those discussed previously that a number of upstream regulators converge on the MAPK signaling pathway to prevent cell death in IECs. Denning and colleagues (227) obtained evidence that in response to oxidative stress, enterocytes are primed for apoptosis via Fas-mediated pathways. After exposure to H2O2, mRNA and protein levels increased for both Fas and FasL, and IECs underwent apoptosis. Apoptosis was associated with the activation of caspase-8. The susceptibility of normal enterocytes to Fas-induced apoptosis depended on levels of cIAP1 and cIAP2 (228). Thus, these results are basically identical to those showing that TNF-α will not cause apoptosis without concurrently inhibiting protein synthesis. The antral hormone gastrin is an important trophic agent for gastric and colonic mucosa. These trophic effects, as opposed to the acid secretion stimulatory action of the hormone, are primarily produced by progastrin (see Chapter 15). Although it is well established that gastrin increases growth by stimulating DNA synthesis and increasing cell proliferation, it has also been shown to inhibit apoptosis (229,230). Gastrin has been shown to prevent apoptosis in a pancreatic acinar cell line by activating the Akt (229) and MAPK (231) pathways. In IECs, pentagastrin decreased the release of cytochrome c from mitochondria and inhibited the activation of caspase-9 and -3 (230). Most studies involving effects of gastrin on apoptosis have dealt with cancer progression and are covered in Chapter 17. As frequently pointed out, COX-2 is overexpressed in colorectal adenomas and adenocarcinomas, and its inhibition by nonsteroidal anti-inflammatory drugs (NSAIDs) protects against colorectal cancer (232–234). One study produced some insight into the mechanisms involved in the prostaglandinmediated protection of cells from apoptosis. Nishihara and colleagues (235) show that cAMP agonists, such as PGE2,

APOPTOSIS IN THE GASTROINTESTINAL TRACT / 363 cholera-toxin, and a cAMP analog that was membrane permeable, protected both normal and transformed intestinal cells (RIE-1 and T84) from apoptosis induced by anti-Fas antibody and staurosporine. Protection was associated with the rapid induction of cIAP2, but not six other members of the IAP family. Inhibition of COX-2 in cells overexpressing the enzyme decreased cIAP2 expression and increased apoptosis. Both of these were reversed by exogenous PGE2 and its subsequent cAMP-dependent induction of cIAP2. These results suggest an explanation for the anticancer effects of NSAIDs by identifying a mechanism whereby cAMP promotes cancerogenesis by inhibiting the normal apoptotic process (235). Two recent studies have examined the cytokines, plateletactivating factor (PAF) and IL-11, which may be involved in mediating the pathogenesis of ulcerative colitis and protecting against it, respectively, for their effects on the apoptotic pathway (236,237). Lu and colleagues (236) stably transfected IEC-6 cells to overproduce Bcl-2 under the control of a lactose-inducible promoter. In the absence of Bcl-2 overexpression, PAF caused Bax to translocate to the mitochondria, resulting in the collapse of the mitochondrial membrane potential and activation of caspase-3 and apoptosis. This was the first demonstration that PAF can activate apoptosis in enterocytes by a Bax-dependent mechanism, and that the entire process could be controlled by Bcl-2 expression. An earlier study had shown that Bax is down-regulated in the inflamed mucosa involved in ulcerative colitis compared with normal colon and inactive mucosa from a patient with ulcerative colitis (238). Even though IL-11 is being evaluated in patients for its effects on ulcerative colitis, its mechanism of protecting colonic cells has not been determined. Kiessling and colleagues (237) found that IL-11 induced signaling in HT-29 cells and colonic epithelial cells by activating the JAK/STAT pathway. IL-11 resulted in a dosedependent phosphorylation of Akt and decreased activation of caspase-9 and the inhibition of apoptosis.

Anoikis In 1994, Frisch and Francis (239) showed that detachment of epithelial cells from the ECM induced apoptosis, which they termed anoikis for the Greek word meaning “homelessness.” Cells undergoing anoikis have all the characteristics of apoptosis that have been discussed earlier in this chapter. The ECM controls cells through signals via a family of cellsurface receptors called integrins (240,241). Integrins are composed of α- and β-subunits. At least 15 α- and 8 βsubunits have been described, and the combination of particular α- and β-subunits accounts for binding and signaling specificity (241). As integrins bind to the ECM, they aggregate and direct the formation of a cytoskeletal and signaling complex that results in the assembly of actin filaments. The reorganization of actin filaments into stress fibers produces a positive feedback, resulting in additional integrin clustering. This causes the formation of a transmembrane complex

of ECM proteins, integrins, and cytoskeletal proteins such as talin, paxillin, vinculin, and actin. These aggregates are known as focal adhesion complexes. Focal adhesion kinase (FAK) is one of a variety of protein tyrosine kinases activated by integrins and targeted to the focal adhesion complex (242). Focal adhesion kinase is further phosphorylated within the focal adhesion complex, which then provides binding sites for other proteins such as paxillin. The formation of focal adhesions is necessary for cell migration, spreading, growth, and differentiation (239,243–245). Epithelial cells that have lost attachment undergo anoikis, and are thus unable to move and attach elsewhere. Anoikis is often lost in cancer cells because of the mutation and inactivation of genes necessary for apoptosis. These malignant cells are able to proliferate free of attachment to the ECM and to metastasize (246). Focal adhesion kinase is the primary enzyme linking detachment from the ECM to apoptosis. Focal adhesion kinase is overexpressed in tumor cells, and the inhibition of FAK results in apoptosis in breast cancer cells by activating a caspase-8–dependent mechanism (247). In IECs, activation of caspase-6 occurs within 15 minutes of detachment and 30 to 45 minutes before the activation of caspase-3 (248). In a second report, the same group of investigators found that FAK is cleaved in an orderly fashion by two different caspases. The first cleavage was mediated by caspase-3, resulting in a 94- to 92-kDa fragment, which was further cleaved by caspase-6 to an 84-kDa fragment (249). Thus, the targeting of FAK during anoikis not only dismantles the cytoskeleton, but also alters the cell’s signaling mechanisms. During the first 15 minutes after detachment, two initiator caspases, caspase-2 and -9, were activated followed by the activation of caspase-6 and -3. The release of cytochrome c was not detected before 60 minutes (250). These investigators concluded, therefore, that cytochrome c release did not play a role in the initiation of apoptosis. These results imply that caspase-9 activation proceeded by a mechanism different from the formation of the apoptosome with APAF and cytochrome c. The activation of EGFR signaling inhibits anoikis (251), and one well-established mediator of EGFR signaling is the c-Src kinase. Src is recruited to the focal adhesion complex, and the ability of FAK to transduce downstream signals is dependent on this protein tyrosine kinase (252). Rosen and colleagues (251) used TGF-α to activate the EGFR in IEC-18 and rat intestinal epithelial (RIE) cells. Transforming growth factor-α strongly inhibited anoikis by preventing the inhibition of Src activity that normally follows detachment. Detachment caused the down-regulation of Bcl-XL, which was necessary for anoikis and was prevented by Src activation. The authors conclude that anoikis was, at least in part, mediated by the loss of Src activity and the subsequent decreased expression of Bcl-XL (251). A follow-up article from the same group showed that v-Src induced resistance to anoikis was mediated by increased Bcl-XL (253). Transfection of IEC-18 cells with v-Src made them resistant to anoikis and up-regulated Bcl-XL. Both of these findings depended on the induction of the MEK/MAPK pathway.

364 / CHAPTER 12 From these studies it appears that detachment disrupts Src signaling from the focal adhesion complex, leading to the down-regulation of the MAPK pathway and decreased expression of Bcl-XL. Although Filmus and coworkers (251,253) have documented the importance of Bcl-XL in attenuating anoikis, they have also shown that exogenous Bcl-XL, no matter how highly expressed, cannot completely prevent it (254). A second mechanism was identified, in which anoikis was stimulated by detachment-induced up-regulation of FasL. They also demonstrated that the increase in the expression of FasL was dependent on a detachment-induced increase in stress-activated protein kinase 38. Vachon and colleagues (255) induced anoikis in enterocytes by pharmacologically inhibiting FAK or by expressing DN-FAK. They found that p38 activation increased transiently after the induction of anoikis in both differentiated and undifferentiated enterocytes. The activation of p38 was associated with the downregulation of the FAK/PI3/Akt pathway. Only p38β and p38δ were required for anoikis, specifically in undifferentiated and differentiated cells, respectively. The studies discussed previously have established that Bcl-XL levels play an important role in anoikis, and that Src can regulate Bcl-XL expression through a MAPK-dependent mechanism. Additional studies have examined the role of Ras during anoikis and found that it regulates Bcl-XL through a mechanism that is independent of both the MAPK and the PI3/Akt pathways (256). Using nonmalignant rat and human IECs, Rosen and coworkers (256) found that activated H- or K-ras oncogenes completely prevented the down-regulation of Bcl-XL triggered by detachment from the ECM. Inhibition of Bcl-XL in the Ras-transformed cells stimulated anoikis and inhibited tumorigenicity. Ras also induced the downregulation of the proapoptotic Bak protein, which could be reversed by pharmacologic inhibition of PI3K. Thus, the authors concluded that Ras-induced resistance to anoikis was mediated by at least two separate mechanisms: one that results in the down-regulation of Bak, and one that stabilizes the expression of Bcl-XL in the absence of ECM (256). These findings were supported by recent data showing that FTS (farnesylthiosalicylic acid), a direct inhibitor of Ras, restored the sensitivity to anoikis in Ras-transformed RIE cells (257). Ras inhibition of anoikis did not depend on Akt or MAPK. Several pathways have therefore been identified that appear to mediate anoikis in IECs. One involves Src, another the FasL, and apparently two separate pathways mediated by Ras.

GASTRIC MUCOSA The stem cells for the gastric mucosa are located in the neck area between the gastric pits and glands. Cells migrate toward the surface, differentiating into surface mucous cells and downward into the pits differentiating into parietal, chief, and endocrine cells. The surface cells are shed continuously with a 3- to 5-day rate of renewal under normal

conditions (258). Cell loss is enhanced by the physical presence of food in the lumen of the stomach and the friction between it and the surface cells produced by gastric motility. The turnover rates of the cells present in the gastric glands are considerably longer (259).

Helicobacter Pylori Apoptosis is involved in gastric ulceration, and the mediators are basically identical to those regulating apoptosis in IECs. Slomiany and colleagues (260) induced chronic gastric ulcers in rats with acetic acid and observed a 33-fold increase in caspase-3 activity at the onset of ulceration 2 days after damage. Caspase-3 activity then decreased as the ulcers healed. Konturek and colleagues (261) used water immersion and restraint for 3.5 hours to induce acute gastric stress ulcers in rats. Apoptosis was maximal 4 hours after the period of stress and declined to near basal levels 24 hours after injury. Bax levels and mRNA expression were high immediately after damage and peaked at 4 hours. They then decreased approximately 50% by 24 hours but were still significantly increased. Bcl-2 expression, in contrast, was low after injury, and then increased fourfold between 4 and 6 hours. It remained near this level throughout the remainder of the 24-hour period. Gastric infection by Helicobacter pylori is associated with gastritis, ulceration, and carcinoma (262). Additional studies have shown that incubation of cultured gastric epithelial cells with H. pylori inhibits cell proliferation (263,264). In addition to the inhibition of proliferation, the induction of apoptosis contributes to the decreased growth rate after H. pylori infection. Moss and colleagues (265) found low rates (2.9% of epithelial cells) of apoptosis in gastric biopsies from uninfected patients. The apoptotic cells were confined to the most superficial regions of the gastric glands. In H. pylori infection they were more numerous, comprising 16.8% of the epithelial cells, and were located throughout the depth of the glands. These investigators suggested that increased apoptosis could provide the stimulus for a compensatory increase in proliferation, a response associated with chronic H. pylori infection (266,267). It is unknown whether the rate of apoptosis in individuals infected with H. pylori depends on the presence of metaplasia. Eradication of H. pylori did not alter cell proliferation in patients with chronic gastritis, and H. pylori increased apoptosis only in the absence of metaplasia (268). A second study with similar conclusions found that after eradication of H. pylori a significant decrease in apoptosis occurred in nonmetaplastic but not in metaplastic tissue from the same individual (269). In general, H. pylori induces apoptosis in gastric epithelial cells in cell culture experiments (263,264,270–272). In addition, in gastric biopsy samples, H. pylori has been associated with twofold to fivefold increases in epithelial cell apoptosis that return to normal levels after eradication of the bacteria (265–267,270,273–275). Epidemiologic studies have implicated the cag island in the pathogenesis of

APOPTOSIS IN THE GASTROINTESTINAL TRACT / 365 H. pylori (276), possibly resulting from the insertion of the CagA protein into host epithelial cells after bacterial attachment (277). Some studies have shown that cag-positive strains increase proliferation but not apoptosis (274,275), whereas others have found that apoptosis is increased significantly in patients with duodenal ulcer disease who, for the most part, are infected with cag-positive bacteria (278–280). Moss and colleagues (281) examined the effect of H. pylori on gastric epithelial proliferation and apoptosis in a large prospective study of 60 patients with nonulcer dyspepsia. They found that proliferation was increased twofold in both antral and corpus epithelial cells and was not related to cagA status but was positively correlated with the presence of gastritis. Apoptosis was increased in the antrum and body only in those with cagA-positive strains of H. pylori. Thus, these results do not support the hypothesis that a deficiency of apoptosis in the presence of cagA strains may lead to neoplasia. Additional evidence implicates another strain-specific H. pylori locus, vacA, in apoptosis. VacA encodes a secreted bacterial toxin, VacA, which induces apoptosis of gastric epithelial cells (282,283). Although a vacA gene is present in virtually all strains of H. pylori, the strains vary greatly in the production of vacuolating cytotoxin activity (284). The variation is due to a difference in the vacA gene structure and the resulting toxins produced. Cover and colleagues (284) have shown that VacA purified from a potent strain induced apoptosis in a dose-dependent manner that required acid activation of the toxin and the presence of ammonium. A mutant inactive toxin neutralized the effect of the toxin from the potent strain. The authors conclude that differences in levels of gastric epithelial cell apoptosis among individuals infected with H. pylori may depend on strain variations in VacA structure. A number of other mechanisms related to the bacteria have been suggested to account for the apoptotic activity of H. pylori (259). Of particular relevance is that H. pylori infection stimulates the release of cytokines such as IL-1, -2, -6, and -8, TNF-α, and interferon-γ (285–287). Their effects may be mediated by the TNF receptor family, and H. pylori has been shown to induce the expression of Fas and FasL (288,289). The induction of apoptosis by H. pylori in vitro is related to the ability of the bacteria to induce and activate the Fas/FasL pathway (288–290). Wu and colleagues (291) found that H. pylori sensitized human gastric epithelial cells and enhanced their susceptibility to TRAIL-mediated apoptosis. Urease has also been shown to contribute to apoptosis. Fan and colleagues (292) found that H. pylori urease binds to class II major histocompatibility complex molecules and induces apoptosis in N87 and Kato III gastric epithelial cells. There is additional evidence that LPS contributes to apoptosis (293). H. pylori can activate NF-κB via the IKKα signaling pathway (294). Suppression of NF-κB activation has been shown to sensitize cells to H. pylori–induced apoptosis, suggesting that NF-κB inhibits apoptosis after infection by the bacteria (295,296). In contrast, other studies indicated that NF-κB

activation enhanced H. pylori–induced apoptosis (297,298). Gupta and colleagues (297), for example, showed that direct inhibition of NF-κB abolished H. pylori–induced apoptosis in gastric epithelial cells. Kim and colleagues (299) found that suppression of NF-κB significantly increased caspase-3 activity and apoptosis in H. pylori–infected MKN-45 and Hs 746T gastric epithelial cell lines, as well as in primary gastric epithelial cells. Inhibition was accompanied by a reduction in iNOS mRNA and protein levels. These investigators conclude that NF-κB activation by H. pylori protects cells from apoptosis by up-regulating iNOS. As far as NF-κB is concerned, all results seem possible because Chu and colleagues (300) found that apoptosis in H. pylori–infected cells was not correlated with NF-κB but was related to a reduction in Bcl-2. The variable results in these studies may well be due to the use of different cell types, the measurement of protein levels at different times after infection, and the use of different strains of H. pylori. Nagasako and colleagues (301) found that Smad5, a protein that is involved in differentiation and known to inhibit growth, is induced by H. pylori infection, but only by strains positive for the CagA pathogenicity island. Suppression of Smad5 by interference RNA totally inhibited the induction of apoptosis by H. pylori. Thus, from this study it appears that the up-regulation of Smad5 may be responsible for the induction of apoptosis by H. pylori under some circumstances.

Additional Studies Unlike the case for the small intestine, there have been relatively few studies of apoptosis of normal gastric epithelial cells, other than those involving H. pylori. The only real exception involves NSAIDs. Because of the interest in gastric ulcer and the effects of aspirin, indomethacin, and other NSAIDs on the gastric mucosal barrier, there are a plethora of studies involving damage to the gastric mucosa. Beginning in the late 1990s, investigators started to examine the effect of these substances on apoptosis of gastric epithelial cells. Nonsteroidal anti-inflammatory drugs induce apoptosis in isolated rat gastric mucosal cells, as well as in gastric epithelial cell lines (302–305). Kusuhara and colleagues (302) induced apoptosis with both indomethacin and sodium diclofenac (nonselective inhibitors of COX). A selective COX-2 inhibitor had a weak effect. Apoptosis caused by the NSAIDs could be inhibited by caspase inhibitors, but not by exogenous PGE2. The authors suggest that the induction of apoptosis did not involve the inhibition of prostaglandin synthesis. This conclusion is in disagreement with a host of studies showing that gastric damage caused by a variety of agents can be prevented by exogenous prostaglandin. Zhu and colleagues (303) found evidence that indomethacin-induced apoptosis involved the up-regulation of the c-myc oncogene. Further investigation by the same group focused on protein kinase C (PKC). Indomethacin decreased the abundance of PKC-β1, and overexpression of

366 / CHAPTER 12 this isoform attenuated the apoptotic response of AGS cells (306). They obtained additional evidence that PKC-β1 protected against indomethacin-induced apoptosis through the up-regulation of the cell-cycle inhibitor p21 (waf1/ cip1). Fujii and colleagues (305) examined the effect of indomethacin on the mitochondrial pathway. Indomethacin induced apoptosis and caspase-3 activity in a time- and dosedependent manner. Cytochrome c release was also related to the dose of indomethacin and occurred immediately before and at the same time as caspase-3 activation. Z-VAD-FMK, a caspase inhibitor, inhibited both the increase in caspase-3 activity and apoptosis without altering cytochrome c release. This is one of the few studies that have examined the steps involved in the apoptotic pathway in gastric epithelial cells. A study by Hoshino and colleagues (307) examined the effects of PGE2 on apoptosis caused by the gastric irritant ethanol. This study followed a series of reports by Mizushima and coworkers (308) showing, in 48-hour primary cultures of guinea pig gastric epithelial cells, that gastric irritants induced apoptosis. They also determined that these irritants— ethanol, acid, NSAIDs, and hydrogen peroxide—induced apoptosis through a common pathway that involved mitochondrial disfunction (309). In each case, exogenous PEG2 inhibited irritant-induced apoptosis (310). In the Hoshino study (307), subtype-specific agonists were used to show that EP2 and EP4 receptors were involved in the PGE2-mediated protection from ethanol-induced apoptosis. Activation of these receptors was coupled to an increase in cAMP, and a cAMP analogue inhibited the ethanol-induced apoptosis. The mechanism of inhibition was shown to involve the activation of PKA, but not PI3K. This article also reported experiments showing that PGE2 blocked cytochrome c release and the activation of caspase-9, -8, and -3, which were stimulated by ethanol exposure. This thorough study provides the best description of the apoptotic process in gastric epithelial cells to date. In addition, it is relevant to the large volume of studies documenting gastric mucosal damage after exposure to various irritants. These data are also in agreement with all of the experiments indicating that prostaglandins can prevent gastric mucosal damage.

CONCLUSION The field of apoptosis is relatively new. Although the process of apoptosis or programmed cell death has been known for many years, its importance was only realized beginning in the early 1990s. Since that time the number of manuscripts being published in this field has increased at a logarithmic rate. To my knowledge, there are at least two journals devoted entirely to this field. Many of the published reports involve cancers or the development of neoplasia. I have not included these because they are dealt with elsewhere in these volumes as separate chapters. I have tried to review and summarize the literature pertaining to normal apoptotic processes in gastrointestinal epithelial cells. A great deal has been learned about programmed

cell death in the small and large intestines including the identification of susceptible cell types, the role of apoptosis in maintaining the equilibrium of cell numbers, apoptotic pathways, and the roles of Bcl-2 family proteins and IAPs. For the most part, similar information is sparse or lacking for the gastric mucosa. Studies are underway to identify agents that might prove useful in blocking unwanted intestinal cell apoptosis that occurs during radiation and chemotherapeutic treatment of cancer. Although we have reasonable evidence indicating how apoptosis is induced through the TNF receptor family or by DNA damage, information is lacking indicating the mechanism of protection from apoptosis initiated by other compounds. Additional studies of various signaling pathways that regulate apoptosis such as the PI3K, MAPK, and STAT cascades are necessary to determine their utility in clinical medicine and whether they can provide therapeutic regimens for conditions such as inflammatory bowel disease and cancer. The influence of phosphatases, which often determine the activity of many additional enzymes, remains largely unidentified in apoptotic pathways. Given the general interest in apoptosis, research will continue to expand at a rapid pace. Hopefully, a significant proportion of those studies will be directed at the gastrointestinal tract.

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Molecular Aspects and Regulation of Gastrointestinal Function during Postnatal Development James F. Collins, Liqun Bai, Hua Xu, and Fayez K. Ghishan Ontogeny of Secretory Function, 376 Ontogeny of Digestive Function, 377 Ontogeny of Intestinal Transport, 379 Transport of Macromolecules during the Neonatal and Suckling Periods, 379 Intestinal Transport of Hormones and Growth Factors during the Neonatal Period, 380 Postnatal Development of Transport Function, 381

Ontogeny of Transport Function along the Vertical and Horizontal Gut Axes, 391 Developmental Regulation of Gastrointestinal Function, 392 Acknowledgments, 394 References, 394

The mammalian intestinal tract undergoes dramatic maturational changes during the first few weeks of postnatal life. These changes in digestive and transport function occur under the influence of genetic and neurohormonal regulators that mediate development of the gut. In precocial species such as rats and mice, gut maturation occurs predominantly after birth during the suckling and weaning periods. Conversely, in altricial species such as humans and pigs, significant gut maturation occurs during the late fetal period with limited absorptive and secretory function being present at birth. In all mammals, dramatic genetic and functional changes occurring around the suckling/weaning transition. The neonatal intestine differs from that of adults in that digestive and secretory function is not fully developed, and thus nutrient absorption occurs throughout the small and large intestines and along the entire length of the cryptvillus axis. Many milk-derived factors are now known to have effects on intestinal function and to regulate the expression of genes involved in nutrient transport and immune function. These factors include immunoglobulins, growth

factors and peptide hormones, and antigens such as bacteria. On weaning, the gut epithelium is under the influence of a different set of physiologic regulators and dietary factors; thus, alterations in function rapidly ensue. This chapter reviews the key discoveries that have led to current knowledge and understanding of gastrointestinal function during ontogeny. This chapter discusses secretory, digestive, absorptive, and regulatory aspects of gut maturation and, whenever possible, provides insight into the molecular physiology of experimental findings. Ontogeny is development of a single individual, or a system within the individual, from the fertilized egg to death (1). As observed in several mammalian species, ontogenetic development of the intestine is an organized process driven by genetic and environmental factors that results in an adaptive, functional intestinal tract. Advances in cellular and molecular biology have allowed a better understanding of the mechanisms responsible for the ontogenetic changes of the mammalian intestine. The development of the gastrointestinal tract in the early stage of life is characterized by morphologic and functional changes of the intestinal epithelium. However, there are significant variations in the timing and rate of maturation that depend on the gestational period of mammals. For example, the mouse and rat, which have short gestational periods, show maturation of their gastrointestinal function at weaning. In contrast, intestinal development

J. F. Collins, L. Bai, H. Xu, and F. K. Ghishan: Department of Pediatrics, Steele Children’s Research Center, University of Arizona Health Sciences Center, Tucson, Arizona 85724. Physiology of the Gastrointestinal Tract, Fourth Edition, edited by Leonard R. Johnson. Academic Press, 2006.

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376 / CHAPTER 13 occurs earlier in utero in species with long gestational periods, such as the sheep, pig, and human (2,3). The complete development of gastrointestinal function includes fully developed secretive, digestive, and absorptive functions as guided by genetic factors and influenced by neurohormonal regulation. The maturation of digestive and absorptive functions of carbohydrates, proteins, fats, minerals, and vitamins in the newborn have been determined in relation to the availability of hydrolytic enzymes, such as amylase, glycosidases, lactase, protease, and lipase. The anatomic differentiation of the human fetal gastrointestinal tract occurs by 20 weeks of gestation and resembles that of a newborn. However, secretive and absorptive functions develop at different stages; the gastric and pancreatic secretive processes are only minimal and can only be partially stimulated in the full-term newborn, whereas the intestinal absorptive function is partially developed by 26 weeks gestation. The gastrointestinal epithelium consists of multiple functional cell types, including enterocytes, enteroendocrine cells, goblet cells, and Paneth cells in the intestine, as well as parietal cells, chief cells, and mucous neck cells in the stomach. All four epithelial cell types of the intestine differentiate from common undifferentiated stem cells in the crypt compartment (4). These crypts contain only a small group of proliferating, undifferentiated stem cells that give rise to several cell phenotypes that migrate onto several adjacent villi (5). Enterocytes absorb water, solutes, vitamins, and other nutrients. Enteroendocrine cells secrete peptide hormones. Goblet cells produce a variety of mucins. Paneth cells secrete digestive enzymes, growth factors, and antimicrobial cryptdins. The enterocytes, goblet cells, and enteroendocrine cells differentiate as they migrate up the crypt-villus axis turning over every 5 days, whereas Paneth cells migrate downward turning over more slowly (4). As mature enteroendocrine cells and enterocytes migrate to the tips of the villi, they presumably undergo apoptosis and are extruded into the lumen (6). How stem cells are allocated to differentiate into different cell types at different stages of development is not fully understood. It has been proposed that the development of the gastrointestinal function is dependent on “hardwired” genetic programming coupled with dietary influences as triggered by neurohormonal factors (7). In the past decade, molecular biology techniques have been widely applied to investigate the development of gastrointestinal function. This chapter summarizes the most recent updates in the ontogeny of gastrointestinal function, especially in the developmental regulation of overall gastrointestinal and absorptive function.

ONTOGENY OF SECRETORY FUNCTION Secretion occurs in different segments of the gastrointestinal tract and is an important process of normal gastrointestinal function. Gastrointestinal secretion occurs throughout the gut from salivary glands, stomach, pancreas, small intestine, and colon. Acid secretion, through gastric H+,

K+-ATPase activity, results in acidification of the gastric lumen and creates an environment that promotes protein and bacterial degradation. Bicarbonate secreted by the pancreas neutralizes gastric acid, ensuring proper pH conditions for digestive enzyme function in the intestine. Chloride secretion via the cystic fibrosis transmembrane conductance regulator (CFTR) provides active intestinal secretion. Secretory processes thus are critical for normal gastrointestinal functions, including digestion and absorption. Developmental changes occur in secretory function of the various components of the gastrointestinal tract and are determined by genetic, environmental, and dietary factors. Furthermore, alterations in secretory function, as seen in patients with cystic fibrosis, result in chronic diarrhea and malnutrition, which is commonly observed in children. Normal growth of the human infant is dependent on a well-adapted gastrointestinal system that ensures adequate secretion, digestion, and absorption, as well as protection from infection. The development of gastrointestinal secretory function, particularly in response to dietary and hormonal stimulation, has been studied extensively over the last decade. Gastric acid secretion is a critical step for food digestion. Alterations in gastric acid secretion are seen in neonates in intensive care units (8,9). Parietal cells of the gastric mucosa contain a secretory membrane protein, H+, K+-ATPase, that is primarily responsible for gastric acid production (10). Gastric acid is produced in premature and term infants (11–15). Parietal cells and gastric H+, K+-ATPase are reported to be present at 13 weeks gestation (16) and increased significantly with gestational age (17). Using H+, K+-ATPase β subunit–deficient mice, researchers showed that gastric H+, K+-ATPase is required not only for the normal digestive function, but also for normal development of the gastric mucosa and attainment of the normal structure and composition of secretory membranes (18). Regulation of gastric H+, K+-ATPase during development has been studied predominantly with respect to gastrin. Human newborns have high circulating levels of gastrin, yet they have low acid secretion. Furthermore, exogenous gastrin is ineffective in stimulating acid secretion, despite the presence of anatomically developed parietal cells in newborns. Thus, it is unclear the precise role that gastrin plays in regulation of acid secretion during early postnatal life. Secretion of water and electrolytes by the gastrointestinal tract is central to its physiologic functions. Secretion of bicarbonate and potassium occurs along the entire length of the intestine, with the predominant electrolyte driving fluid secretion being chloride. Chloride is secreted into intestinal lumen via the coordinated action of the basolateral Na+-2Cl−-K+ cotransporter (NKCC1) and the apical CFTR (19). Chloride secretion is accompanied by paracellular and transcellular movement of sodium, and the resulting luminal accumulation of sodium chloride provides the osmotic basis for water movement. Potassium is secreted through apical K+ channels following the electrochemical gradient (20,21). A portion of bicarbonate secretion is also secondary to active secretion of chloride, which recycles into the cell through

MOLECULAR ASPECTS AND REGULATION OF GASTROINTESTINAL FUNCTION / 377 apical anion exchangers in exchange for bicarbonate (22). The secretion of HCO3− by ductal epithelium of the pancreas has several functions. First, it neutralizes the gastric acid and protects the intestinal mucosa; second, it protects the pancreatic-biliary tract from ascending infection. Overall, the mechanism of bicarbonate secretion has been implicated as a complex regulatory process including neural, hormonal, and intracellular signal transduction in ductules and acinar pancreatic cells (23). Developmental change of intestinal secretion of electrolytes has been observed, especially for K+, Cl−, and HCO3−. Suckling rats have high colonic K+ secretion, and this has been shown to be a result of regulation by aldosterone (21). Intestinal Cl− secretion was also found in suckling animals from different mammalian species (24–28). The activity of cyclic adenosine monophosphate (cAMP)– dependent chloride channels and the CFTR transcript has been detected in fetal small and large intestines, with similar expression levels seen throughout prenatal and early postnatal development (29–32). Several studies have suggested that adults are more resistant to bacterial enterotoxin-induced secretory diarrhea. This phenomenon reflects that the development of secretive function is age dependent. In animal studies, the immature intestine is more susceptible than the mature intestine to bacterial enterotoxins (24,33,34). The jejunum of 2- and 3-week-old rats have higher sensitivity for stable toxin (ST) of Escherichia coli than that of the adult (34). Furthermore, the small intestine of 14-day-old pigs was more sensitive to cholera toxin (CT) challenge than that of 14-week-old animals (24). This reduced response to CT with age was found to be partially due to the presence of an antisecretory factor, which naturally inhibits fluid losses in enterotoxigenic diarrhea. CT and ST are members of a larger family of A-B toxins of bacterial (and plant) origin that are composed of structurally and functionally distinct, enzymatically active A and receptor-binding B subunits or domains. The mechanism of CT- and ST-induced diarrhea involves B pentamer–mediated receptor binding of the toxins, entry into a vesicular pathway, and translocation of the enzymatic A1 domain of the A subunit into the target cell cytosol, where covalent modification of intracellular targets leads to activation of adenylate cyclase (cAMP). The activation of cAMP opens Cl− channels and inhibits Na+/H+ and Cl−/HCO3− exchangers, subsequently causing a sequence of events culminating in life-threatening diarrhea (35). It has been suggested that differential expression of G proteins and ion transporters (e.g., Na+, K+-ATPase and Cl− channels) during development may also alter the neonatal host’s responsiveness. Therefore, the developmental control of microvillous membrane receptors, signal transduction mechanisms, and ion transport systems in the gastrointestinal tract may, in part, contribute to the altered host sensitivity to toxigenic diarrhea of infancy (35). The developmental changes in responsiveness of intestinal secretory function by enterotoxins may include affinity or number of receptors on enterocyte membranes, receptor–effector transduction pathways, and neurohormonal

regulation (36). It has been shown that the number of ST receptors was increased in the brush-border membranes of porcine, murine, rat, and human intestine during development (34,37–39). In a pig model, the increase of the susceptibility during the first week of life and at weaning to ST-mediated diarrheal disease is partially caused by an increase of ST receptor numbers (38). In addition, ST receptor messenger RNA (mRNA) expression was found to be greatest in the perinatal period in the jejunum and ileum, suggesting that the age-dependent changes in ST receptors are transcriptionally or posttranscriptionally controlled, or both (40). However, the immature gut exhibited increased host sensitivity to CT stimulation that was not correlated with initial receptor binding, but was related to decreased Na+-K+-ATPase activity, suggesting that an underexpressed sodium pump may be partially responsible for the high incidence of secretory diarrhea in neonates (33). The signal transduction pathways of receptor–effector also show developmental changes. One study found that the interaction of adenylate cyclase and it receptors on stimulation of CT is significantly greater in preweaned rats than that in weaned rats (41). In addition, the CT-induced fluid secretion was closely correlated with increased adenylate cyclase activities. Thus, increases in adenylate cyclase responsiveness to CT are, at least in part, responsible for the increased incidence of toxigenic diarrhea in neonates. Furthermore, high levels of antisecretory proteins were found in 28-weekold pigs compared with 35-week-old pigs that are capable of inhibiting enterotoxin-induced secretion in older animals (42). These observations suggest that multiple factors are involved in the developmental regulation of intestinal secretion. Moreover, the enteric nervous system appears to modulate intestinal secretion. There are at least two major, final, common neuronal pathways influencing the secretory epithelium of the crypts, cholinergic and noncholinergic. The direct stimulatory effect of CT binding to enterocytes is estimated to cover only one third of the secretory response, and the remainder is induced indirectly through activation of the enteric nervous system. Cholera toxin–induced secretion in the small intestine is accompanied by the release of mucin from goblet cells (43). However, exposing cultured goblet cells in vitro to CT does not evoke mucous secretion (44). These observations indirectly suggest that the CT-induced mucous secretion is mediated by the enteric nervous system. Furthermore, CT-induced secretion can be attenuated by 5-HT, or serotonin (45–47). These findings suggest that the release of toxins activates the enteric nervous system and inflammatory mediators, thus evoking fluid secretion or influencing enterocytes directly, or both (48,49).

ONTOGENY OF DIGESTIVE FUNCTION Expression levels of digestive enzymes in the small intestinal brush-border membrane change dramatically from the neonatal period into adulthood because of dietary adaptation.

378 / CHAPTER 13 This topic has been investigated intensively since the 1970s. Disaccharidases are located in the brush-border membrane of mature enterocytes of the small intestine. The major disaccharidases found in humans are β-galactosidase (lactase) and α-glucosidases (sucrase, isomaltase, and glucoamylase). Lactase hydrolyzes lactose to glucose and galactose. Sucrase hydrolyzes sucrose to fructose and glucose. Isomaltase hydrolyzes the α-1, six bonds of α-limit dextrins to two glucose units. Maltase hydrolyzes maltose to two glucose units. Disaccharidase activities are greatest in the proximal and midjejunum. Glucoamylase activity is distributed evenly throughout the small intestine. Lactase levels continue to increase for 30 cm beyond the ligament of Treitz, and then decrease. This distribution is established in fetal life and continues into adulthood. Milk is enriched with lipids, but it contains relatively low carbohydrate levels. Lactose is the major carbohydrate in the human milk. Lactase activity, which peaks soon after birth, is low before the 12th fetal week and increases to only 30% of perinatal expression levels by the 26th to 34th week of gestation (50). The rapid ontogenic change in lactase activity in the intestine during the early stage of human life clearly demonstrates physiologically relevant dietary adaptation after birth. The lactase levels remain relatively high through 6 months of age, and then they decline at 5 years of age (51). In adults, two phenotypes exist in regard to lactose digestion. Most of the world population is characterized by a loss of lactase activity before adulthood, resulting in adult-type hypolactasia. Therefore, the ingestion of lactosecontaining products will result in osmotic-type diarrhea. The lactase-persistent phenotype is characterized by continued high lactase activity throughout adulthood. The adult-type hypolactasia is inherited in a recessive manner, whereas the persistence of lactase activity is inherited in a dominant manner. Several nucleotide polymorphisms have been described in the promoter region of the lactase gene. Two particular single-nucleotide polymorphisms (SNPs) are tightly associated with adult-type hypolactasia. A C at position− 13910 upstream of the lactase gene is 100% associated, and a G at position −22018 is more than 95% associated with lactase nonpersistence in the Finnish population (52). The functional role of these SNPs has been confirmed in two articles (53,54) demonstrating that the genomic region containing these two polymorphisms possesses enhancer activity. Currently, it is believed that lactase expression in both humans and animals is regulated via a complex spatial and development pattern in the small intestine. A conserved 150-base pair (bp) proximal promoter is important for regulation of lactase expression. This promoter region binds transcription factors such as CDX-2, hepatocyte nuclear factor 1α (HNF-1α), and GATA that are important for regulation of the lactase expression. In humans, all glucosidases are present by the third intrauterine month. An increase in maltase, glucoamylase, and sucrase-isomaltase occurs at the 12th week of gestation, and 70% of adult levels are reached between 26th and 34th

weeks gestation (50,55). Although these developmental changes have been well documented over the past several decades, the molecular mechanisms of how those genes are precisely regulated remain largely unknown, but transcriptional mechanisms have been suggested (56). Indeed, in the mouse and the rat intestine, sucrase-isomaltase is increased by weaning, and a combinatory role of HNF-1α, Cdx-2, and GATA-4 was shown to be involved in the timing and position of the sucrase-isomaltase expression along the small intestine (56). Similar to our advanced understanding of the molecular physiology of digestive enzymes on the intestinal brushborder membrane, there has been a remarkable advance in our knowledge of the events of the ontogeny of islets of Langerhans in the mammalian pancreas. In particular, there has been a dramatic shift from the previously held concept of one secreted hormone per cell type during ontogeny to a wider acceptance of a precursor cell for each of the four types of principal endocrine cell types, which ultimately produce insulin, glucagon, somatostatin, and pancreatic polypeptide (57). The current understanding of ontogenic change of the endocrine pancreas is that there is a series of stages in the differentiation of islet cells, and in one of these stages, they often can secrete more than one hormone (58). Early in development, glucagon and insulin secretion are usually mediated by the same cell (59). Somatostatin and pancreatic polypeptide are secreted together by cells much later in development, but at one point in mouse development, polypeptide YY is present in all four principal islet cell types (60). More recently, developmental biologists have demonstrated that pancreatic development requires complex combinations of transcription factors to control organogenesis and cell differentiation (61–63). In particular, the Pax family of transcription factors is involved in the formation of the pancreas and many other organs. Two of its members, Pax4 and Pax6, play important roles in islet differentiation (64,65). Pax genes encode key regulators, which are involved in the embryonic development of many organs including the eyes, brain, kidney, thyroid gland, immune system, and pancreas. A number of murine and human genetic disorders are linked to mutations in specific Pax genes. Pax genes 4 and 6 are required for development of the mouse pancreas with Pax6 directing the differentiation of A cells (65). Pax4 null mice lack serotonin and somatostatin-secreting cells in the gastric antrum, as well as most endocrine cell types in the proximal small intestine (66). Deletion of Pax6 eliminates glucagon-like peptide-1 (GLP-1)– and GLP-2–expressing cells in the distal intestine (67), gastrin and somatostatin cells in the antrum, and gastric-inhibitory-peptide (GIP) cells in the duodenum (66). In addition, reduced numbers of all four pancreatic islet cell types was seen in Pax6 null mice (62), and pancreatic endocrine cells were absent in Pax4 and Pax6 double knockout mice (65). A new member of the protein tyrosine phosphatase (PTP) family, called PTP-NP (for neural and pancreatic), is a receptor-type transmembrane molecule and an early marker of development (68). Overall, the absence of these endocrine

MOLECULAR ASPECTS AND REGULATION OF GASTROINTESTINAL FUNCTION / 379 cell types in Pax4 and Pax6 null mice indicates that Pax4 and Pax6 play crucial roles in normal pancreatic development. Another regulatory peptide, adrenomedullin, has been found to modulate insulin secretion (69). Adrenomedullin is an almost ubiquitously expressed peptide that is involved in growth, development, and endocrine signaling. It is expressed by islet cells during development (70). This hormone appears first within cells containing glucagon, then eventually within all principal islet cell types, and ultimately only within those cells expressing pancreatic polypeptide (71). Thus, early expression of adrenomedullin is critical for normal islet development. The pancreatic duodenal homeobox gene-1 (Pdx-1) is a master regulator of both pancreatic development and the differentiation of progenitor cells into the β-cell phenotype (72,73). Pdx1 regulates insulin and somatostatin gene expression in the endocrine pancreas (74). The stable expression of Pdx-1 in a glucagon-secreting α cell line is sufficient to induce expression of the β-cell–specific genes insulin, glucokinase, and islet amyloid polypeptide (75,76). The patterns of expression of Pdx-1 in the developing pancreas are maintained throughout development and provide both spatial and temporal contributions to the commitment of endoderm to a pancreatic phenotype (77). Targeted disruption of the Pdx-1 gene in mice results in agenesis of the pancreas (78,79). Notably, a child born without a pancreas was shown to be homozygous for an inactivating mutation in Pdx-1 (80), underscoring the importance of the Pdx-1 transcription factor in pancreatic development.

ONTOGENY OF INTESTINAL TRANSPORT Two important developmental periods must be taken into account when one considers developmental aspects of intestinal transport function: the neonatal/suckling and the weaning/postweaning periods (36). These developmental stages are important because milk-derived factors, hormones, and macromolecules affect physiology of the gut, and there are distinct hormonal changes associated with weaning, when new dietary factors influence gut physiology. The degree of intestinal maturation also plays a major role in differences in absorptive function during ontogeny. Thus, mechanisms and levels of intestinal transport of nutrients and electrolytes vary throughout postnatal development. Furthermore, nutrient transport in the neonatal intestine occurs along the entire crypt-villus axis, whereas only enterocytes along the upper villus have an absorptive phenotype after weaning (81). Changes in transport function during postnatal development can be related to alterations in transporter turnover rates, density of transporters in the plasma membrane of intestinal epithelial cells, or changes in affinity constants and/or expression levels of different transporter isoforms. Changes in transport activity can also be related to several nonspecific factors, such as alterations in mucosal surface area, proliferation and differentiation of epithelial cells, membrane fluidity, and paracellular permeability (36). Plasma membrane fluidity

of intestinal cells changes during ontogeny due to altered cholesterol-to-phospholipid and protein-to-phospholipid ratios and also to changes in the lipid composition (82–85). Furthermore, these changes in the biophysical properties of the membrane can modify the activity of various transporters (86,87). Also during intestinal development in the postnatal period, there is a transient appearance of villus-like structures in the proximal colon. Enterocyte amplification also occurs, in which the absorptive surface area of microvilli increases (88,89), a phenomenon that may be caused by decreased cell turnover rates.

Transport of Macromolecules during the Neonatal and Suckling Periods In general, the neonatal intestinal epithelium is much more permeable than the adult intestine. During early postnatal life, growth factors, immunoglobulins, food antigens, other macromolecules, and bacteria may adhere to the gut epithelium and be absorbed from colostrum and milk. This transport occurs through enterocytes and specialized “M” cells (90), which appear early in fetal development (91,92). This process may be mediated by interaction of milk-derived factors with specific receptors on epithelial cells. Whole proteins derived from dietary sources may also be transported by M cells. The high permeability of the neonatal intestine for macromolecules decreases after birth, but the precise timing of this event is species dependent. In some rodent species and in pigs, transport capacity decreases a few days after birth, whereas in rats and rabbits, transport ceases completely around weaning (93–96). The process of “gut closure,” the dramatic decrease in epithelial permeability that occurs around the suckling/weaning transition, is dependent on epithelial-derived factors and increased pancreatic secretions into the gut lumen (97–99). Transport of macromolecules during the neonatal period may also occur by a nonspecific process, whereby adherent or soluble molecules are moved across the epithelium by vesicular transport. Only molecules adhered to the microvillus membrane are transported across the cells, whereas soluble molecules in the fluid phase are mostly degraded (100–102). This ability of the neonatal gut is due to an apical, tubular system and numerous supranuclear vacuoles, of which distribution and developmental pattern vary dramatically between mammalian species (36). That macromolecules are more likely to adhere to intestinal epithelial cells during the neonatal period (103) cannot be fully explained, although differences in membrane composition are one possible factor. Furthermore, the relative contribution of enterocytes versus M cells in nonspecific transport of macromolecules is unknown, although selective adherence of milk IgGs to M cells has been reported (104). Immunoglobulin transport in the neonatal period is enhanced by protease inhibitors found in colostrum and milk (105,106), which ensure that IgGs escape degradation and bind to their cognate receptors in the intestinal epithelium.

380 / CHAPTER 13 This process is important for the development of passive immunity, a process that is known to be involved in regulating the expression of development-specific genes in the developing intestine (107). Rodents express a specific IgG receptor that selectively binds to the Fc portion of immunoglobulin (the Fc receptor of the neonate [FcRN]), which mediates endocytosis and transport across epithelial cells (Fig. 13-1) (108,109). Fc receptor of the neonate is maximally expressed in the proximal small bowel, with decreasing expression levels in more distal gut segments (97). The FcRN/IgG interaction occurs at the low pH of the duodenum and uptake is mediated by endocytosis (109,110). Transported IgGs are then trafficked within vesicles that are protected from fusion with lysosomes to the basolateral membrane for extrusion into the interstitial space (111). Humans and other precocious mammals are born in a hypoglobulinemic state, and thus absorption of IgGs from the maternal milk is critical for development of passive immunity (112). Studies suggest that IgG may also be transferred by the placenta during late fetal development in humans. However, the intestinal epithelium also expresses IgG receptors in the fetal and neonatal periods, and macromolecular transport continues until after birth, when it gradually diminishes (113–115). Studies suggest that FcRN expression may be transcriptionally mediated by Sp family member transcription factors (116), which are known to regulate many genes during postnatal development (117,118). Furthermore, the neonatal intestine also actively A. Receptor-mediated absorption

secretes IgAs via the activity of the polymeric IgA receptor (PIgR) (119,120) (see Fig. 13-1), and interestingly, PIgR gene expression may be modulated at the transcriptional level by glucocorticoid hormones (121).

Intestinal Transport of Hormones and Growth Factors during the Neonatal Period Several lines of experimental evidence suggest that milkbased hormones and growth factors can survive digestion and are absorbed by epithelial cells of the neonatal, mammalian intestine (Table 13-1). Because gastric acid secretion and pancreatic secretion of proteases is decreased during the neonatal and suckling periods, peptide hormones and other proteins are able to reach the absorptive enterocytes of the proximal small bowel. The principal target of these milk-borne substances seems to be the intestinal epithelium, where they influence proliferation and differentiation of enterocytes (122–126). A small fraction of these absorbed molecules may survive intracellular proteolytic processes and become absorbed into the neonatal circulation; however, the physiologic importance of this observation is currently unknown. In suckling animals, degradation of proteins is low, but it increases dramatically coincident with weaning (126–129). This low degradative capacity facilitates the transport of biologically active substances by the neonatal intestine, as intestinal epithelial cells express B. Transcytotic absorption

C. Secretory component

IgG FcRN IgA PIgR

FIG. 13-1. Immunoglobulin absorption and secretion during neonatal life. (A, B) Two different mechanisms used during the suckling period to transport IgGs across the intestinal epithelium. The receptormediated pathway involves the Fc receptor of the neonate (FcRN) and endocytosis, followed by vesicular trafficking and release across the basolateral membrane. The transcytotic pathway can be observed in some mammals, and it involves nonselective endocytosis, where endocytosed molecules are either partially destroyed or partially transported into the interstitial fluids. (C) IgA secretion is shown, which also occurs at this developmental stage and involves the polymeric IgA receptor (PIgR). IgA dimers bind to the receptor on the basolateral membranes and are endocytosed and transported across the cellular cytoplasm, where the complexes are secreted into the gut lumen. (Modified from Pacha [36], by permission.)

MOLECULAR ASPECTS AND REGULATION OF GASTROINTESTINAL FUNCTION / 381 TABLE 13-1. Transport of milk-derived hormones and growth factors across the gastrointestinal epithelium Milk-derived factor

Physiologic effects

Corticosterone Epidermal growth factor (EGF) Insulin

Hormone shown to be absorbed from formula-fed, glucocorticoid-deficient rats (435). Transport occurs in suckling and weanling rats. Some EGF is degraded during transport, whereas only a small amount is actually transported into the circulation (133,443,444). Orally ingested insulin decreases blood glucose levels in suckling rats (445). Neonatal calves receiving greater amounts of dietary insulin have higher circulating levels of insulin (colostrum vs milk) (446). Insulin-like growth factor may be absorbed in an undegraded form in pigs. Plasma concentration of IGF is greater in pigs fed a diet containing higher IGF levels (colostrum vs milk) (122,447). Orally ingested IGF-I modulates intestinal growth in rats (125) and is absorbed intact (448). Insulin-like growth factor type I is absorbed by a nonsaturable, nonreceptor-dependent mechanism (449). Prolactin is transferred from mother to rat pups and can be detected in the serum of the offspring; it can also be detected in the serum of suckling rats given an oral dose of prolactin (450). Some prostaglandins are absorbed intact and can be detected in the liver of neonatal rats (451,452). Oral administration to rats results in a small amount being absorbed in an undegraded form (453,454). Thyroid-releasing hormone treatment of maternal rats leads to an increase of serum levels in the pups (455), and TSH was shown to be absorbed in a biologically active form (456). Injection (subcutaneous) of neonatal rats with TSH increased serum T4 in both sucklings and weanlings, whereas oral administration had the same effect only in sucklings (457). Mouse pups born to TGF-β knockout mouse heterozygotes have detectable tissue levels of TGF-β, whereas those born to null females do not. Transforming growth factor-β given orally can cross the intestinal epithelium and can be detected in various tissues (458).

Insulin-like growth factor (IGF)

Prolactin Prostaglandins Somatostatin Thyroid-releasing hormone Thyroid-stimulating hormone (TSH) Transforming growth factor-β (TGF-β)

Modified from Pacha (36), by permission.

receptors that mediate transepithelial transport of these molecules across intestinal mucosa (Table 13-2) (130–132). Furthermore, in rat ileum, it has been demonstrated that epidermal growth factor (EGF) and nerve growth factor may be transported by fluid-phase and receptor-mediated mechanisms, but that only small fraction of both peptides actually reach the basolateral membrane for extrusion into the circulation (133,134).

Postnatal Development of Transport Function Most nutrient transporters are first expressed during gestational development and are particularly important during the third trimester when the fetus actively absorbs nutrients from the amniotic fluid. The postnatal ontogeny of nutrient transporters reflects the need to absorb increasing quantities of nutrients that are required for rapid growth and high metabolic activity. The developmental changes in transport capacities are due to several factors: (1) changes in intestinal mass, (2) physicochemical properties of the cell membranes, (3) and types of transporters being expressed and their kinetic properties. Furthermore, colonic absorption of some nutrients occurs in sucklings, a process that may compensate for decreased transport capacity in the small bowel and less developed colonic bacterial fermentation. Total intestinal transport capacity increases with age, predominantly because of increased intestinal mass, but surprisingly, the absorptive capacity for many carbohydrates and amino acids decreases in proportion to intestinal mass (135–138). In some mammalian species, this decline may

be up to 50% of initial values (137,139) and is not attributable to decrease of mucosal surface area (135). The phenomenon is not, however, fully understood, but three possible mechanisms could be hypothesized: (1) changes in transporter expression levels, (2) induction of other isoforms or related transport systems, and (3) alterations in transporter turnover rates. Decreased absorptive capacity could also be related to the smaller proportion of enterocytes that express various active transporter processes during early ontogeny. Ontogenic Regulation of Intestinal Monosaccharide Absorption Sugars are a major source of caloric intake at all life stages. The dietary sources of sugars vary from lactose in milk to complex carbohydrates found in other foods. All these carbohydrates are digested as mainly glucose, galactose, and fructose, before being absorbed in the small intestine (140). The existence of multiple intestinal carbohydrate transporters has been proposed on the basis of kinetic analysis (141,142), and two families of monosaccharide transporters have been identified: the facilitated-diffusion glucose transporter (GLUT) family and the Na+-dependent glucose transporter (SGLT) family. These transporters play a pivotal role in the transfer of monosaccharide across epithelial cell layers in the gut; and overall, several transporters contribute to the absorption of glucose, galactose, and fructose. Lactose in milk is hydrolyzed by lactase into glucose and galactose, and these sugars are absorbed by a Na+-dependent mechanism (143–146). This pathway is mediated by the sodium-dependent glucose transporter 1 (SGLT1), a member of SLC5 gene

382 / CHAPTER 13 TABLE 13-2. Developmental regulation of mucosal receptors of the small intestine Receptor

Developmental stage

Experimental observations

Epidermal growth factor (EGF)

F, S, W, Ad

Glucocorticoid

N, S, W, PW

Insulin

S, PW

Insulin-like growth factor type I (IGF-I)

N, S, W

Epidermal growth factor binding capacity decreases in the perinatal period, and then increases to adult levels by postnatal day 14. A factor present in rat milk decreases EGF binding (459). Epidermal growth factor receptor expression is greater during the gestational period than in suckling and adult rats (425). A higher binding capacity and lower receptor affinity is present after weaning in newborn pigs. Virtually no receptors were observed in suckling pigs (131). Binding capacity increases from fetal life through the suckling period, and then decreases until adulthood in rats (412). Binding capacity reaches a maximum during gestation, followed by a decrease to adult levels within 3 days of parturition of rabbits (460). Decreased binding capacity occurs during development in high- and low-affinity receptors, with no changes in binding affinity. Binding capacity is much greater in crypt cells than in villus cells in rats (130). Binding capacity rapidly declines after birth and remains low during the suckling period, and then increases by postnatal day 21 in pigs. Receptor affinity remains unchanged during ontogenic development (461). Age-dependent increase of binding capacity during suckling and weaning periods in rats, with decreased capacity noted thereafter (462). Receptor messenger RNA expression is significantly higher during fetal development of the rat intestine (463). Age-dependent decrease of binding capacity from early suckling period until adulthood in rats (462). An age-dependent increase in binding capacity has been noted in rats (464). Binding capacity decreases immediately after birth, and then increases within 2 weeks to adult levels, in the absence of alterations in receptor affinity. In the perinatal period, only high-affinity receptors are expressed, whereas later in postnatal development, high- and low-affinity receptors are expressed in rats (465). Receptor messenger RNA expression is not altered during postnatal development in rats (466). An age-dependent increase in binding capacity and expression of both highand low-affinity receptors has been noted in the absence of changes in affinity in rats (467). High- and low-affinity receptors have been described; the binding capacity of both receptors has been noted during ontogeny (468). Receptor binding capacity dramatically increases on weaning in rats, although receptor affinity does not change during postnatal development (469).

S, W, PW, Ad IGF-II

F, W S, W, PW, Ad

Prostaglandin E1 Somatostatin

W, PW, Ad N, S, W, PW, Ad

Thyroid hormone

N, S, W, PW

Vasoactive intestinal polypeptide

S, W, PW F, N, W

Vitamin D

S, W, PW, Ad

Ad, adult; F, fetal; N, neonatal; PW, postweaning; S, suckling; W, weanling. Modified from Pacha (36), by permission.

Sodium-Dependent Glucose Transporter 1 family (147), whose mRNA expression varies during ontogeny (148–151). Furthermore, SGLT1-mediated sugar absorption shows a gradient of activity along the horizontal axis of the small intestine, with greater activity in more proximal sections and decreasing activity distally. SGLT1 expression is high during neonatal life, and it decreases on weaning (138,152); also, because SGLT1 also transports galactose, the developmental pattern of galactose transport is thus identical to that of glucose (135,138). In contrast, fructose absorption is mediated by a Na+-independent, facilitated diffusion pathway involving the glucose transporter GLUT5. The monosaccharide exits enterocytes by facilitated diffusion via the glucose transporter GLUT2. The absorption process mediated by these transporters is regulated by many developmental and dietary factors. The following sections focus only on development-related regulation of these transporters.

Intestinal SGLT1 was cloned and characterized many years ago from rabbits and humans (153,154). SGLT1 is located at the brush-border membrane of the intestinal epithelial cells and is responsible for transporting glucose and galactose across the intestinal brush border (155). Mutations in this transporter gene in humans results in glucose-galactose malabsorption (GGM) syndrome (140,156), a disease characterized by neonatal onset of severe, osmotic diarrhea. Na+dependent glucose uptake in the intestine is modulated during postnatal development. A high glucose absorption rate is observed in the perinatal period, but absorption decreases thereafter (157–159). A similar pattern is also observed in intestinal SGLT1 transporter expression. In human intestine, SGLT1 mRNA expression first occurs in midgestation (~17 weeks) and the expression level increases in adults (148). In rat intestine, SGLT1 expression is higher in both

MOLECULAR ASPECTS AND REGULATION OF GASTROINTESTINAL FUNCTION / 383 weanlings and adults compared with sucklings (138,159). These observations suggest that the functional regulation of glucose absorption during ontogeny may involve transcriptional regulation of SGLT1 gene expression. Some evidence suggests that another Na+-dependent glucose transporter may also be present in the intestinal epithelium. Kinetic studies of sugar absorption in intact animals, and in in vitro preparations and brush-border membrane vesicles, indicate that a low-affinity glucose transporter is also present in the apical membrane of the epithelial cells (160). The presence of a second glucose transporter in the brushborder membrane of enterocytes may explain why less severe symptoms are observed in some GGM patients with deletion mutations in the SGLT1 gene, and why symptoms may be observed in some GGM patients with no mutations in the SGLT1 gene (140). Facilitated Glucose Transporter 5 Fructose absorption is mediated by GLUT5 (140,161,162), which is an apically expressed, transmembrane protein that facilitates fructose movement down a chemical gradient. GLUT5 transporter complementary DNA (cDNA) has been cloned from the human, rabbit, rat, and mouse intestine (148,163–168). Fructose absorption increases dramatically in the small intestine during weaning in rats and rabbits, and to a lesser extent in pigs (135,137,138), and a similar pattern is also observed in intestinal GLUT5 transporter expression. In human and rat intestines, GLUT5 gene expression is very low before weaning and sharply increased at weaning (148,167,169,170). This stimulation in rats and in mouse (163,168,171–174) is linked to an increase of GLUT5 mRNA abundance. These observations then suggest that the functional regulation of fructose absorption during development involves transcriptional regulation of GLUT5 gene expression (175). Furthermore, GLUT5 expression may be precociously induced just before weaning by exogenous fructose administration (171). Fructose uptake in the intestine is also modulated by dietary factors including fructose content of the diet. Facilitated Glucose Transporter 2 As discussed earlier, glucose, galactose, and fructose are transported across the apical membranes of enterocytes by two different transport systems; however, they all share a common transport pathway for extrusion across the basolateral membrane, mediated by GLUT2. This transporter is involved in transporting these sugars out of enterocytes along the existing electrochemical gradients (161,174). GLUT2 is thus a facilitated diffusion glucose transporter expressed at the basolateral membrane of intestinal epithelial cells (174, 176). GLUT2 cDNA has been cloned from human, rat, and mouse (177–179). Similar to GLUT5, the expression of GLUT2 protein is also modulated during ontogeny and regulated by dietary factors. GLUT2 expression decreases during the late suckling period. The expression of GLUT2

protein increases with age; in the perinatal period, expression level of GLUT2 is low, and it reaches plateau after weaning (148,162,180–183). Diets containing high fructose or glucose levels can enhance intestinal GLUT2 expression in neonatal (suckling) rats through increasing GLUT2 gene transcription (184). Ontogenic Regulation of Intestinal Amino Acid Transport The adult mammalian intestine transports amino acids via Na+-dependent and -independent mechanisms, and single amino acids may be transported by multiple transport systems. In the neonatal intestine, Na+-independent lysine and alanine absorption, and Na+-dependent alanine, taurine, and glutamine absorption have been demonstrated (143,145, 185,186).The intestinal transport capacity for various amino acids varies throughout postnatal development and, in general, is greater in the neonatal period than later in life (135,138). These developmental changes may be due to changes in expression levels of individual amino acid transport systems expressed in the intestinal epithelium; this has been directly demonstrated in primary enterocytes and brushborder membrane vesicles (186,187). Na+-dependent amino acid and carbohydrate transport systems also are expressed in the colon during the neonatal period but rapidly decline toward weaning (188–191). The unusual transport ability of the colon for sugars and amino acids may compensate for lower transport capacity in the small intestine of neonates. In support of this supposition, lactose absorption has been detected in the proximal colon of newborns (192,193). However, despite advances in the understanding of intestinal amino acid transport during postnatal development, information about the relative contribution of individual amino acid transporter systems is lacking. Ontogenic Regulation of Intestinal Lipid Absorption Lipid absorption is particularly important in the neonatal period, because lipids constitute a major portion of consumed calories. In addition to absorbing lipids by pinocytosis (194), the neonatal intestine can also absorb fatty acids and cholesterol from dietary sources (195,196). Triglycerides are digested by gastric and lingual lipases (197) in the absence of bile acids, which are low in the neonatal period. Resulting catabolic products, fatty acids and 2-monoacylglycerols, are transported at greater rates in the neonatal intestine compared with adults (195,196,198). The brush-border fatty acid binding protein may also enhance lipid absorption by both diffusional and specific carrier-mediated processes (199), although a passive mechanism(s), independent of fatty acid binding protein, likely also exists (195). Furthermore, intestinal absorption of carnitine, which is an important player in lipid metabolism, is enhanced in the neonatal small intestine (200). It is then a logical assumption that absorbed fatty acids and monoacylglycerides are efficiently resynthesized into triglycerides, phospholipids, and cholesteryl esters, because the activity of reesterification enzymes in the neonatal gut is

384 / CHAPTER 13 comparable with adult levels (201). Expression of apolipoproteins, which are involved in the formation of macromolecular complexes of lipoproteins, is also regulated during ontogeny, with moderate expression levels during gestation (202,203). Postnatal Development of Water Transport Water is absorbed across the small intestinal epithelium in the absence of external driving forces and is secondary to active sodium-chloride transport. In proximal small bowel, sodium and water absorption are glucose dependent. In vivo experiments in suckling rats demonstrated a fourfold decrease in water transport in the small intestine compared with the colon (204), and a decrease in colonic water absorption during early postnatal development (205). Greater water absorption detected in these perfusion experiments may be mediated by paracellular pathways, as supported by the observation that an artificially created osmotic gradient results in water secretion. Furthermore, the connection between active sodium transport and glucose is sodium-glucose cotransport across the apical membrane of enterocytes by the Na+-glucose symporter, SGLT1. Na+ that enters cells with glucose is pumped out across the basolateral membrane by the Na+, K+-ATPase pumps, and glucose passes out across the basolateral membrane passively via GLUT2, with the net result being that glucose and sodium are transported across the epithelium. However, the coupling among Na+, glucose, and water transport is less well understood. It is commonly thought that Na+ transport increases the local osmotic pressure in the lateral intercellular spaces, and that this, in turn, generates osmotic water flow across the epithelium. Some work, however, suggests that water is cotransported together with Na+ and sugar through SGLT1 (155), but the physiologic importance of this observation is unknown. The gastrointestinal tract, like many other cells and tissues, also expresses water channels called aquaporins, with at least six aquaporin isoforms being expressed in the digestive system (206). Aquaporin-1 (AQP1) is widely distributed in endothelial cells of capillaries and small blood vessels, as well as in the central lacteals in the small intestine. Aquaporin-1 is also present in the duct system in the pancreas, liver, and bile duct and in colonic crypts (207). Aquaporin-3 is mainly expressed in the epithelia of the upper digestive tract, from the oral cavity to the stomach, and in the lower digestive tract, from the distal colon to the anus. Aquaporin-4 has been shown to be expressed in the basal membrane crypt cells and in gastric parietal cells (206), but its function in these tissues is currently unknown. Aquaporin-5 is expressed in acinar cells of the salivary, pyloric, and duodenal glands. In addition, the recently cloned AQP8 has been found to be strongly expressed in enterocytes (208,209), although evidence demonstrates that AQP8 null mice do not have an intestinal water transport defect (210). Aquaporin-8 is also expressed in salivary glands, pancreas, and liver, whereas AQP9 has been detected in the liver and intestinal goblet cells. Aquaporins have

important roles in the digestive system, such as the participation of AQP5 in saliva secretion, as demonstrated by the studies on AQP5 null mice (206). Currently, little is known regarding the developmental pattern of AQP expression in the intestine, even though remarkable changes in AQP expression have been reported in the developing kidney (211). In addition, water transfer across the digestive epithelia seems to occur not only via aquaporins, but also via other transporter or channel systems. Ontogenic Development of Sodium Chloride Absorption Ion and fluid transport processes undergo ontogenic changes in both the small and large intestines. In neonatal mammals, the colon appears to have a significant role in absorption of water and ions and in maintenance of normal fluid and electrolyte homeostasis. This role may be extremely important during early postnatal growth, when the small intestinal transport functions are not fully developed. Overall, the gastrointestinal tract processes about 9 liters water and 800 mEq Na+ daily in adulthood. Thus, a major function of the gastrointestinal tract is to absorb virtually all water and Na+ to maintain overall body water and Na+ homeostasis. Increased water and NaCl transport occurs early in postnatal development; high Na+ absorption occurs in the neonatal period and decreases later in postnatal life in the rat, rabbit, and human colon (205,212,213) and in the rat small intestine (36). The Na+ absorptive pathway that predominates in the colon is mediated by amiloride-sensitive Na+ channels (213–215), which gradually increase in abundance from fetal life through the suckling period (95,216,217) after which time they decline in abundance. Colonic Na+ transport persists until adulthood in rabbits (213), but in rats, it ceases after weaning (214,215) and is replaced by Na+-H+ exchange (36). In pigs, Na+ absorption changes from an electroneutral mechanism in the perinatal period to an electrogenic process later in postnatal development (218). Na+-H+ exchange activity increases during development and reaches maximal values after weaning (219–221). Three Na+-H+ exchanger (NHE) isoforms, NHE2, NHE3, and NHE8, are expressed in the mammalian small intestine, and the expression NHE2 and NHE3 is developmentally regulated (219,220,222). A functional coupling exists between Na+-H+ and Cl-HCO3 exchangers in the small intestinal epithelium, but the ontogenic expression pattern of the two transport systems are opposite to one another (223). As has been hypothesized for NHE activity in the developing gut, ontogenic changes in Cl-HCO3 exchanger expression may be transcriptionally mediated (224). Another colonic Cl transport system, which is unresponsive to luminal Na+, also has been reported (189,213). Chloride may also be transported by Cl channels and the NKCC1 in the neonatal intestine (32). In addition, the mucosal electrical gradient generated by active Na+ ion transport promotes diffusion of Cl across the epithelium via alternative mechanisms that may include transcellular- and paracellular-mediated pathways.

MOLECULAR ASPECTS AND REGULATION OF GASTROINTESTINAL FUNCTION / 385 Sodium-Hydrogen Exchangers The sodium absorptive process in the small intestine involves carrier-mediated pathways. The NHEs play an integral role in electroneutral sodium absorption in the mammalian intestine. To date, nine NHE isoforms (NHE1-9) have been identified, and NHE isoforms 1, 2, 3, and 8 are known to be expressed in the mammalian intestinal epithelium. These NHEs have different tissue distribution, membrane localization, inhibitor sensitivity, and physiologic regulation (225), and they show 25% to 70% amino acid sequence identity between isoforms (226). NHE1 is expressed on the basolateral membrane of intestinal epithelial cells throughout the gastrointestinal tract and plays an important role in cell volume and intracellular pH (pHi) regulation (225). Loss of NHE1 function in knockout mice results in growth retardation, ataxia, seizures, and altered intracellular pH in pancreatic acinar cells (227,228). Because NHE1 does not contribute to the intestinal Na+ absorption process, it is not discussed here in detail. NHE2 protein is expressed on the apical membrane of enterocytes and is involved in transepithelial Na+ absorption in the mammalian gastrointestinal tract (225). Knockout of NHE2 in mice leads to abnormal parietal cell viability and enhanced, compensatory NHE3 expression in the colon (229,230). Intestinal NHE2 expression is modulated during postnatal development. NHE2-mediated Na+ uptake in rat jejunum is lowest in suckling rats, higher in weanling and adult rats, and still higher in adolescent rats (220) (Fig. 13-2). A similar pattern is also seen in the NHE2 protein and mRNA expression; NHE2 protein levels are lowest in sucklings and higher in weanling and adult rats (fourfold to sixfold increase), whereas NHE2 mRNA is lowest in suckling rats and higher after weaning (threefold to fivefold increase) (219). The mechanism of this ontogenic regulation on NHE2 function is thought to be at the transcriptional level; an approximately twofold greater transcription rate was observed in adolescent rats compared with suckling rats by nuclear run-on analyses (219).

Epidermal growth factor has shown to enhance Na+ absorption in gastrointestinal tract. In isolated rat hepatocytes, EGF treatment also significantly increases the pHi recovery rate (recovery from an intracellular acid load; a measurement of NHE activity) by 2.4-fold (231). In isolated rat enterocytes, administrating EGF (200 ng/ml) stimulates Na+-H+ exchange activity by 1.8-fold (232). In in vivo studies, EGF treatment increases jejunal NHE2 activity by almost twofold in suckling rats. The increase of NHE2 activity correlates with the increase of NHE2 mRNA abundance. Epidermal growth factor administration has no effect on NHE3 gene expression in these young rats. In vitro studies show that EGF stimulates pHi recovery in cultured rat intestinal epithelial cells by twofold, and a similar induction is observed in NHE2 gene promoter activity (233). These observations suggested that the effect of EGF on NHE gene expression in suckling rat jejuna is isoform specific, only for NHE2 but not NHE3, and this regulation is mediated by transcriptional activation of NHE2 gene expression. The NHE3 protein is also apically expressed NHE in intestinal epithelial cells. It is involved in transepithelial Na+ absorption in the mammalian gastrointestinal tract (225). Knockout NHE3 mice exhibit a moderate to severe absorptive defect in the intestine and mild diarrhea (229,234,235). As a key transporter involved in electroneutral Na+ absorption in the small intestine, NHE3 gene expression is regulated by many factors, and like intestinal NHE2, NHE3 function, and gene expression, it is also subject to developmental regulation. At the functional level, NHE3-mediated Na+ uptake in rat jejuna changes as follows: suckling = weanling < adult << adolescent rats (220) (see Fig. 13-2). At the protein level, NHE3 expression is lowest in suckling rats and increased up to sevenfold after weaning, whereas NHE3 mRNA expression is also lowest in suckling rats and increased approximately twofold after weaning (219). The ontogenic regulation of NHE3 expression likely involves both transcriptional and posttranscriptional mechanisms. The increase in rat small intestinal NHE3 expression after weaning coincides with a surge in levels of endogenous

2w

A

NHE activity in rat ileum Na+ nMol/mg protein/10 sec.

Na+ nMol/mg protein/10 sec.

NHE activity in rat jejunum 2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 3w

6w

0.40 0.35 0.30 0.25 0.20 0.15 0.10 0.05 0.00 2w

3-4 mo.

3w

6w

B

FIG. 13-2. Sodium-hydrogen exchange (NHE) activity in rat small intestine. Brush-border membrane vesicles were purified by the Mg++ precipitation method from rats and pH-dependent uptake of radiolabeled sodium was assayed. (A) Sodium-hydrogen exchange activity in jejunum increases with maturation, and then declines into adulthood. (B) Sodium-hydrogen exchange (NHE) activity in ileum increases with maturation. The lowest activity is seen in suckling rats, and the highest activity is seen in adolescent rats. 3-4 mo., 3 to 4 months old; 2w, 2 weeks old; 3w, 3 weeks old; 6w, 6 weeks old. (A: Modified from Collins and colleagues [220], by permission. B: Modified from Kikuchi and colleagues [221], by permission.)

386 / CHAPTER 13 glucocorticoid hormones (236). In fact, studies have demonstrated glucocorticoid regulation of intestinal NHE3 gene expression in an age- and region-specific manner. In suckling rats, the responsiveness to methylprednisone treatment is observed only in the proximal, but not in the distal, small intestine. NHE3-mediated Na+ absorption is also increased by threefold, together with a twofold to threefold increase in NHE3 protein and a twofold increase in NHE3 mRNA expression (220,237). Conversely, in adult rats, the stimulatory effect of methylprednisone is observed only in the distal, but not in the proximal, small intestine, where NHE3mediated Na+ absorption is increased together with NHE3 protein and mRNA levels (237,238). These observations suggest that the effect of glucocorticoids on NHE3 expression is most likely due to genomic effects mediated by the glucocorticoid receptor. NHE8 protein has also been shown to be expressed on the brush-border membrane of intestinal epithelial cells. The function of NHE8 has not been fully defined, although it may contribute to intestinal Na+ absorption early in life, based on its protein localization and gene expression pattern (239). In the proximal small intestine, NHE8 expression is also regulated by development, but opposite to the expression pattern of NHE2 and NHE3. NHE8 protein expression is highest in weanling rats and ~56% lower in adults, whereas NHE8 mRNA abundance is similar in suckling and weanling rats, but twofold less in adult rats (239). Potassium Absorption K+ homeostasis is an important physiologic function, and it is maintained by K+ transport across the renal and intestinal epithelia and via regulation by hormonal factors and movement between extracellular and intracellular compartments. Na+ is extruded and potassium transported into enterocytes across the basolateral membrane of intestinal epithelial cells by the Na+-K+-ATPase, which is more strongly expressed in sucklings and weanlings than in adults (215, 240–242). The developmental increase in Na+-K+-ATPase activity is paralleled by mRNA expression for α- and β subunits of this protein, with expression levels decreasing toward adult levels around weaning (242,243). Although Na+-K+-ATPase activity and the number of ATPase molecules increase during development, total functional protein activity surprisingly is not altered, suggesting that additional regulatory mechanisms are involved (244). This ATPase plays a major role in intestinal potassium homeostasis as described later. The intestinal epithelium actively absorbs potassium from the body fluids, followed by secretion into the gut lumen; thus, the intestine plays a major role in overall body potassium homeostasis. K+ secretion has been documented in rat jejunum and ileum (36) and in pig and human colon (212,245), whereas absorption was detected in other experimental situations (246,247). These contradictory results are most likely related to methodologic differences used in the different studies, but it is likely that both processes do occur and are of physiologic importance. Additional studies

demonstrated active K+ absorption via H+-K+-ATPase in both the small (248) and large intestines of neonates (249,250). Furthermore, it was demonstrated that active K+ absorption and H+-K+-ATPase activity are higher during the neonatal period (249,250). Calcium Absorption Intestinal Ca2+ absorption in mammals involves two parallel processes: paracellular, nonsaturable movement through tight junctions between enterocytes, and transcellular transport, which is saturable and 1,25(OH)2 vitamin D3 dependent. In adult mammals, the paracellular pathway predominates under conditions of high calcium intake, whereas the transcellular process is more important under conditions of low calcium intake. In newborn rats, calcium absorption seems to be dependent on both pathways, but a paracellular, 1,25(OH)2 vitamin D3–independent pathway is predominant (251,252). The nonsaturable diffusional pathway also functions as the only absorptive pathway for Ca2+ in preterm, low-birth-weight infants (253). The appearance of saturable active Ca2+ absorption is associated with a substantial increase in the capacity for other steps involved in 1,25(OH)2 vitamin D3–dependent, transcellular Ca2+ absorption, including movement across the apical membrane of enterocytes along a steep electrochemical Ca2+ gradient (mediated by transient receptor potential channel isoform 6 [TRPV6]) (254–256), translocation through the cell (mediated by the intracellular calcium binding protein calbindin-D9k) (257–259), and extrusion at the basolateral membrane against an electrochemical gradient by plasma membrane Ca2+-ATPase (259–262). Many of the proteins responsible for intestinal calcium transport have been identified (as described earlier) (257); however, to date, expression patterns of these genes during postnatal development have not been determined. Phosphate Absorption Phosphate is one of the most important minerals in the body; it is a constituent of bone and teeth, and it plays an important physiologic role in the regulation of acid–base balance. Phosphate is found primarily in bone matrix (85%), whereas the remaining phosphate is divided between intracellular (14.5%) and extracellular fluids (<0.5%). In intracellular fluids, phosphate is a component of nucleotides (DNA and RNA), high-energy molecules (e.g., ATP), and metabolic intermediates. In extracellular fluids, phosphate is present in its inorganic form and serves as a buffer for H+. About 90% of the phosphate in plasma is free, whereas the remaining 10% is bound to serum proteins. The phosphorus level in blood in humans is between 3.0 and 4.5 mg/dl. Phosphate absorption in the small intestine involves transcellular and paracellular pathways. The paracellular pathway appears to be dependent on the magnitude of the electrochemical gradients across the intestinal epithelium (263) and on dietary inorganic phosphate (Pi) intake levels; it predominates under conditions of high Pi intake. Meanwhile, the transcellular pathway involves a carrier-mediated process and

MOLECULAR ASPECTS AND REGULATION OF GASTROINTESTINAL FUNCTION / 387 is most important when dietary Pi intake levels are low. Early studies showed that active transport of Pi through the apical membrane of small intestinal epithelial cells is coupled with sodium in human, rat, and rabbit (263–268). Other studies showed that Pi is absorbed predominantly by secondary active Na+-dependent transport in a stoichiometry of 2Na+:1Pi, and it is now known that this process is mediated by the type IIb sodium-phosphate (NaPi-IIb) cotransporter (as discussed in detail later) (269–271). In agreement with the increased requirements of neonatal mammals for phosphate, the capacity of intestinal phosphate transport decreases during development (Fig. 13-3). Furthermore, sodiumphosphate cotransport activity is restricted to the duodenum, jejunum, and proximal ileum in suckling rats, and only to the duodenum and proximal jejunum in adult animals (271). For decades, the molecular identity of proteins involved in intestinal phosphate absorption was not known. However, beginning in 1998, the intestinal sodium-coupled phosphate transporter (NaPi-IIb) gene was cloned from mouse and human (270,272,273). The NaPi-IIb protein has moderate homology with the renal sodium-phosphate cotransporter (NaPi-IIa). The NaPi-IIb cotransporter is expressed at the apical membrane of intestinal epithelial cells (as well as in numerous other tissues) and is responsible for phosphate absorption in the intestine. Pi absorption across the epithelial cells (transcellular pathway) in the small intestine is modulated by many factors including age, dietary Pi content, hormones (vitamin D, estrogen, glucocorticoids, EGF). Here, the focus is on developmental regulation of this key intestinal transporter. Phosphate absorption in the small intestine decreases with age. The highest Pi uptake rate is observed in the suckling period, with the lowest uptake rate observed in the adult. This pattern has been reported in several species including rat, mouse, pig, and rabbit. In pigs, sodium-dependent phosphate uptake in intestinal brush-border membrane vesicles is greater in newborn piglets and decreased ~66% after weaning (274). In mouse, Na-dependent Pi uptake is greatest

in sucklings, then decreased ~85% at weaning, and further reduced ~94% at 8 weeks and 8 to 9 months of age (275). In rat, Na-dependent Pi uptake in suckling rats is significantly greater (threefold) than in adult rats (276). And finally, in rabbits, sodium-phosphate cotransport is greatest in sucklings and decreased 87% in 3-month-old adults (271). The functional expression pattern of Pi absorption correlates well with the NaPi-IIb expression pattern in the small intestine. In mouse and rat, the expression levels of NaPi-IIb protein and mRNA are at the highest levels at the suckling stage, with NaPi-IIb protein and mRNA expression being threefold to fivefold higher than in adults (275,276). Furthermore, it is a well-known fact that a low-phosphate diet (LPD) stimulates intestinal sodium-coupled phosphate absorption. An LPD increases intestinal Na+-dependent Pi transport by ~3-fold in adult rats and 2.6-fold in adult mice (277,278). The molecular mechanism of LPD on intestinal Pi absorption involves regulation of NaPi-IIb expression at transcriptional or posttranscriptional levels, or both. An LPD stimulates intestinal Pi absorption in mice by enhancing NaPi-IIb mRNA and protein synthesis (279–282). An LPD stimulates intestinal Pi absorption in rats by stimulating apical NaPi-IIb protein expression, but not NaPi-IIb mRNA expression (280). The effect of LPD on Pi absorption in mice is independent of the vitamin D receptor, and may thus involve a nongenomic action of 1,25 (OH)2 vitamin D3 (279). In metabolic acidosis, the body accumulates excessive protons and acids, and thus the body requires more phosphate for buffering of the body fluids. In a rat model of metabolic acidosis, a decrease in renal NaPi-IIa expression and renal phosphate reabsorption were observed (283). A similar result was also observed in a mouse model of metabolic acidosis, where urinary fractional Pi excretion was increased twofold, whereas serum Pi levels remained normal. Interestingly, intestinal Na+-dependent Pi transport activity in these mice is increased 20-fold. Together with functional changes, the abundance of NaPi-IIb protein is induced approximately fivefold, but no change is detected in NaPi-IIb mRNA expression (284).

Pi absorption in mouse jejunum

Pi absorption in rat jejunum

2.0 1.5 1.0 0.5 0.0

A

120 Pi nMol/mg protein/10 sec.

Pi nMol/mg protein/10 sec.

2.5

2w

3w

8w

100 80 60 40 20 0

8-9 mo.

2w

3-4 mo.

B

FIG. 13-3. Sodium-dependent phosphate absorption in mouse and rat jejunum. (A) Sodiumdependent phosphate cotransport (NaPi) activity in mouse jejunum decreases with maturation: the greatest activity is seen in suckling rats, and then significantly decreases after weaning. (B) Sodiumdependent phosphate cotransport activity in rat jejunum decreases with maturation: the greatest activity is seen in suckling rats, and then significantly reduces in adult rats. (A: Modified from Arima and colleagues [275], by permission. B: Modified from Xu and colleagues [276], by permission.)

388 / CHAPTER 13 1,25(OH)2 vitamin D3 is a potent regulator of calcium and phosphate homeostasis, and it stimulates intestinal phosphate absorption via its effect on the sodium-coupled phosphate absorption process. Administration of 1,25(OH)2 vitamin D3 increases Na+-dependent Pi uptake by twofold in rabbit duodenum (285) and in suckling and adolescent rats (286). Similar results were observed in 1,25(OH)2 vitamin D3–treated adult rat jejuna (287). The mechanisms of 1,25(OH)2 vitamin D3 on intestinal Pi absorption involve transcriptional and posttranscriptional regulation of NaPiIIb expression, depending on the developmental stage of the animals. In adult rats and mice, 1,25(OH)2 vitamin D3 enhances intestinal sodium-dependent phosphate absorption by increasing the apical expression of NaPi-IIb transporter protein (276,280,282). In suckling rats, however, 1,25(OH)2 vitamin D3 stimulates intestinal sodium-dependent phosphate absorption by increasing NaPi-IIb mRNA and, concomitantly, protein expression (276). Endogenous glucocorticoid levels are low during early life and increase dramatically on weaning (288,289). Glucocorticoids are well known to reduce inhibit intestinal Pi absorption. In suckling rabbits, Na+-dependent Pi uptake is reduced 50% by methylprednisolone administration (290). Similar results were also seen in rat studies (291). The effect of glucocorticoids on intestinal Pi absorption is more significant during the neonatal period, with methylprednisolone administration decreasing intestinal Na-dependent Pi uptake by 70% to 83% in suckling and weanling mice, whereas no changes were observed in older animals. At the protein expression level, glucocorticoid treatment decreases apical NaPi-IIb abundance by fourfold in suckling mice, whereas NaPi-IIb mRNA abundance decreases more than threefold in suckling and weanling mice (275). Thus, the mechanism of glucocorticoid action on intestinal Pi uptake in suckling age involves inhibition of NaPi-IIb cotransporter expression in neonatal mice. Epidermal growth factor is a 53-amino-acid polypeptide that is secreted predominantly by salivary glands, with lower levels excreted by kidney and many other tissues. Epidermal growth factor receptors are present along the intestinal tract (292). As a growth factor, EFG has broad effects on cell division, DNA synthesis, tissue proliferation, cellular differentiation, and electrocyte and nutrient absorption (293,294). Epidermal growth factor reduces renal Pi uptake by inhibiting NaPi-IIa cotransporter mRNA and protein expression (295). Furthermore, in patients with intestinal ischemia injury, serum Pi levels (296) and EGF production and utilization are increased (297). These observations suggest that EGF may play an important role in Pi homeostasis by regulating renal and possibly intestinal Pi absorption. Studies on NaPi-IIb transporter expression show that NaPi-IIb mRNA abundance is decreased 50% by EGF administration in suckling rats and in human intestinal epithelial cells (Caco-2). This reduction may be due to inhibition of human NaPi-IIb promoter activity (298). The EGF response element has been identified as -AACTGG-, which is located between −739 and −734 bp of the human NaPi-IIb gene promoter. The trans-acting

factor that interacts with the EGF response element at the human NaPi-IIb gene promoter has been identified as the c-myb protein, a nuclear protein involved in regulation of cell growth, differentiation, and apoptosis (299). Estrogen is another important physiologic regulator of calcium and possibly phosphate homeostasis. Estrogen plays important roles not only in the regulation of intestinal Ca2+ absorption, but also in the maintenance of bone mineralization (300–302) and 1,25(OH)2 vitamin D3 activation (301, 303–306). Studies have shown that estrogen also plays a role in modulating intestinal Na+-dependent Pi uptake. In female adult rats, estrogen treatment increased Na+-dependent Pi absorption by ~45%, NaPi-IIb protein expression by ~75%, and NaPi-IIb mRNA abundance by ~50%. The activity of the human NaPi-IIb gene promoter also increased by ~36% after estrogen treatment (307). Absorption of Trace Mineral Elements Mammals have a greater absorptive capacity for many trace minerals during the neonatal period, which is likely because of the need for sufficient amounts of these key ions during peak growth and development. Trace minerals are easily absorbed from colostrums and milk (308). Kinetic studies have demonstrated that both a passive, linear and carrier-mediated processes are involved in the absorption of trace minerals in the neonatal intestine. Specific carriers have been identified over the past several years for iron (309), copper (310), zinc (311), and magnesium (312), but not for manganese (313). Interestingly, many of these metal ions can also be transported by a recently discovered iron transporter, divalent metal transporter 1 (Dmt1) (314), but whether this occurs in vivo is not known. Copper absorption is enhanced in the neonatal period, likely mediated by passive, nonsaturable transport localized predominantly in the ileum (310,315), which is replaced by a saturable pathway in more proximal gut segments later in life. Intestinal copper transport may be mediated by a recently identified copper transporter 1 (Ctr1) (316–318), but its developmental pattern of expression has not been described. The onset of saturable copper transport correlates with increased expression of metallothionein, which is an intracellular regulator of copper and zinc homeostasis (310,319). Similarly, iron absorption is also increased early in life, even in the setting of high body iron levels (309,320,321). Increased iron absorption is due, in part, to enhanced transport in the developing jejunum and ileum, whereas iron transport then occurs predominantly in the duodenum and proximal jejunum in adulthood. Furthermore, intestinal zinc transport has been unraveled at the molecular level with many intestinal zinc transporters recently identified (322), but their expression levels during ontogeny and their relative contribution to total intestinal zinc transport have not been determined. Intestinal iron absorptive transport systems are known to be induced by iron deprivation. This process plays a pivotal role in overall body iron homeostasis, and most known physiologic regulators of iron homeostasis alter gut

MOLECULAR ASPECTS AND REGULATION OF GASTROINTESTINAL FUNCTION / 389 absorption (323). Knowledge of the molecular mechanisms of transepithelial iron transport has increased dramatically with the discovery of several of the key players (324–326). These include the brush-border–associated proteins duodenal cytochrome b (Dcytb) and divalent metal transporter 1 (Dmt1), which allow iron transport into intestinal epithelial cells. Other proteins are involved in iron extrusion into the interstitial space, namely hephaestin and iron-regulated gene 1 (Ireg1). Ferritins are iron-sequestering proteins found in the cytoplasm of enterocytes, and developmental changes in their expression have been reported (327). Although the developmental patterns of expression of most of these genes have not been studied extensively, their inducibility by iron deficiency across several postnatal developmental stages has been investigated. Collins and colleagues (328) used an oligonucleotide microarry approach to characterize gene expression patterns in the duodenal mucosa of iron-deprived rats over the course of rat ontogeny. These authors found strong induction of Dmt1, Dcytb, and transferrin receptor 1 (Tfr1) in suckling, weanling, adolescent, and adult rats. Other known iron transport protein-encoding genes were not as strongly or consistently regulated. Furthermore, induction of other novel genes potentially associated with intestinal metal iron homeostasis was reported, including the Menkes copper ATPase (Atp7a), metallothionein (Mt1), lipocalininteracting membrane receptor (Limr), and the sodiumdependent vitamin C transporter 2 (Svct2) (Table 13-3) (328). Interestingly, many of these known and novel genes were also similarly regulated in the jejunal gut segment. These data overall suggest that some aspects of intestinal

iron transport are similar across several developmental stages, including the neonatal period. Iron in milk is bound to lactoferrin, transferrin, or both, depending on which mammalian species is being considered (308), and it has been suggested that these interactions enhance iron transport in the neonatal period (329,330). Putative lactoferrin receptors have been identified in the fetal and neonatal human small intestine (331), whereas transferrin receptors are present in the apical membranes of enterocytes in the rat small intestinal epithelium (332). Putative brush-border membrane lactoferrin receptors are expressed at the highest levels in sucklings (333). Even though iron bound to lactoferrin is transported into intestinal epithelial cells (334), it is unclear whether this process plays an important role in developmental changes seen in iron uptake capacity (320). Ontogenic Regulation of Intestinal Transport of Water-Soluble Vitamins Water-soluble vitamins are essential micronutrients for the body, and they play critical roles in maintaining normal metabolism, energy, differentiation, and growth. These vitamins can not be synthesized by humans and mammals; therefore, they have to be obtained from exogenous sources via the intestinal absorptive processes. Exogenous sources of these vitamins are dietary sources and bacterial sources, whereby vitamins are produced by the normal microflora of the small and large intestines. Vitamins are transported in the intestine via a saturable, nonlinear process (when dietary

TABLE 13-3. Genes induced in rat duodenum at five different postnatal ages by dietary iron deprivation Gene name

Gene symbol

Aliases/Biological function

Divalent metal ion transporter 1, +/− IRE Duodenal cytochrome B (highly similar to mouse) Duodenal cytochrome B (same chromosomal region) Transferrin receptor Metallothionein 1 and 2 Tripartite motif protein 27 (similar to mouse) ATPase, Cu++-transporting, α polypeptide (Menkes) Divalent metal ion transporter 1, + IRE Metallothionein Glycerol-3-phosphate acyltransferase (similar to mouse) Factor-responsive smooth muscle protein Pyruvate carboxylase Phosphoglucomutase-related protein (same chromosomal region) Calponin 3, acidic Glutathione peroxidase 2 (gastrointestinal) Lipocalin-interacting membrane receptor (similar to mouse and human) Gap junction protein, β2 Retinoblastoma binding protein 7

DMT1 CYBRD1 CYBRD1 TFRC MT1/2 TRIM27 ATP7A DMT1 MT1 GPAM SM20 PC PGM-RP

Divalent metal ion transport Rat sproutin; ferric-reductase Ferric-reductase Iron ion homeostasis; transferrin-iron endocytosis Metal ion binding Oncogenesis; formation of cellular compartments Copper ion efflux from enterocytes Divalent metal ion transport Metal ion binding Glycerolipid synthesis Apoptosis; protein metabolism; oxidoreductase Gluconeogenesis; metabolism; lipid biosynthesis Acidulin; cell adhesion; carbohydrate metabolism Actin/Calmodulin binding Defense against oxidative stress Lipocalin receptor; possible cytoplasmic iron delivery Connexin 26; intercellular communication Interaction with histone deacetylases; corepressor complex Regulation of tissue calcification Cell adhesion; cell-surface–mediated signaling Ascorbic acid (vitamin C) transport

Progressive ankylosis (highly similar to mouse) Integrin, α6 Sodium-dependent vitamin C transporter 2

CNN3 GPX2 LIMR GJB2 RBBP7 ANK ITGA6 SVCT2

Ages studied include 8 days (suckling), 21 days (weanlings), 6 weeks (adolescent), and 12 and 36 weeks (adults). Modified from Collins and colleagues (328), by permission.

390 / CHAPTER 13 intake levels are low or normal) and by passive, linear diffusion (with high-intake levels). Saturable transport occurs via the action of specific transporters by facilitated diffusion or ATP-driven active transport (335). The ontogenic patterns of vitamin absorption have been investigated for only a few vitamins, but it is known that the developmental patterns are not identical for all vitamins. The intestinal transport of folate and riboflavin are carrier-mediated, Na+-dependent processes, the capacity of which decreases after the neonatal period (336,337). A large portion of folate in milk is bound to a high-affinity folate-binding protein that enhances folate uptake probably through a specific receptor on the membranes of enterocytes (337). Unbound folate is absorbed at much lower rates, by an endocytotic process that has greater capacity in the ileum than jejunum (338). Similarly, riboflavin absorption is also enhanced by milk factors (339). Biotin absorption has a different developmental pattern; adult rats absorb biotin preferentially in the jejunum, whereas suckling rats absorb biotin predominantly in the ileum. Furthermore, the nonsaturable pathway decreases in jejunum and ileum during postnatal development, and the capacity of saturable, carrier-mediated transport increases in the jejunum and decreases in the ileum (340,341). Milk-derived factors also strongly promote the transport of cobalamin (342). Cobalamin absorption occurs in all small intestinal gut segments and exhibits saturation kinetics. The intrinsic factor (IF), which promotes cobalamin absorption in the adult intestine, is distinct from milk factors that affect cobalamin absorption. The IF enhances transport only in the ileum and is more effective in weanling animals. In contrast, milk-derived factor(s) are more effective in promoting transport in suckling rats where IF/cobalamin receptor activity is low (342,343). Ontogenic development of IF and IF/cobalamin receptor activity is maximal in rats around weaning (343), and this timing correlates with enhanced production of IF in the stomach (344). In contrast, in human intestine, IF/cobalamin receptors are expressed in early gestation (345) and fetal parietal cells produce IF (346). Vitamin C is present in its reduced (ascorbic acid) and oxidized (dehydroascorbic acid [DHAA]) forms in the diet. Ascorbic acid is a cofactor for a number of enzymes involved in maintaining transition metal ions in their reduced forms, in the biosynthesis of extracellular matrix proteins and neurotransmitters, and in the regulation of iron intake. Deficiency of vitamin C leads to various clinic symptoms including scurvy, poor wound healing, and bone and connective tissue disorders. In the guinea pig and human ileum, the uptake for ascorbic acid is saturable, indicating a carrier mechanism, and the brush-border membrane uptake of ascorbic acid appears to involve Na+ movement (347–349). The Na+-dependent ascorbic acid uptake was confirmed later by using brush-border membrane vehicles isolated from guinea pig intestine (350–352). The carrier protein involving in the intestinal ascorbic acid absorption is SVCT1. Sodiumdependent vitamin C transporter 1 is highly expressed in the small intestine in human, mice, and rats. Sodium-dependent vitamin C transporter 1 mediates the transport of ascorbate,

the reduced form of vitamin C, in a Na+-dependent manner (353–355). Furthermore, DHAA uptake may occur through the GLUT proteins, which are facilitated diffusion glucose transporters (356,357). Vitamin C uptake decreases with age; there is a general decline of plasma ascorbic acid levels in elderly populations as a function of dietary intake compared with the total adult population. Over the entire range of ascorbic acid consumption, plasma vitamin C levels in the elderly are lower than those in young to middle-aged adults (358). These studies strongly suggest that dietary ascorbic acid uptake is impaired with age. A similar result is also reported in rats, where isolated hepatocytes from old rats showed a lower vitamin C uptake rate compared with cells isolated from young rats. The decline in uptake levels is correlated with the reduced SVCT1 mRNA levels (359). Biotin (vitamin H) is necessary for the metabolism of carbohydrates and fatty acids. Biotin cannot be synthesized by humans and other mammals, and therefore must be obtained from exogenous, dietary sources. Deficiency in biotin results in neurologic disorders, growth retardation, and dermal abnormalities (360). Studies have shown that the absorption mechanism of biotin in the small intestine involves a Na+-dependent carrier-mediated process (361). The carrier protein is localized at the apical membrane of the enterocytes of human, rat, and rabbit (362–364). The protein responsible for biotin transport in the intestine is identified as the sodium-dependent multivitamin transporter (SMVT). The intestinal SMVT cDNA has been cloned and characterized from rat, rabbit, and human (365–367). The SMVT protein transports not only biotin, but also pantothenate and lipoate (365,367,368). The activity of Na+-dependent biotin absorption is regulated during ontogeny and maturation of intestinal tract. Biotin uptake rates in the ileum are greater in suckling rats and not different between jejunum and ileum in weanling rats. In contrast, biotin transport is twofold greater in the jejunum than in ileum in adult rats (340,341). Considering the developmental regulation of the biotin absorptive process in the same region of the intestine, Na+-dependent biotin absorption shows an increase during postnatal development. In rat jejunum, the uptake rate in adult rats is about twofold greater than in suckling rats (369). These functional changes are correlated with the expression pattern of SMVT protein and mRNA. Sodium-dependent multivitamin transporter mRNA transcript in the jejunum of adults is 2.6-fold greater than in suckling rats, and nuclear run-on assays using nuclei isolated from suckling and adult rat jejunum indicated an approximately twofold higher nascent transcription rate for SMVT in adult compared with suckling rats (369). Folate is important for the formation of normal red blood cells and in the production of nucleic acids. Folate deficiency results in a serious anemia. Folate absorption occurs mainly in the proximal intestine; involves a pH-dependent, carrier-mediated process (360); and has been observed in isolated intestinal brush-border membrane vehicles from humans and rats (370–372). The molecular mechanism of

MOLECULAR ASPECTS AND REGULATION OF GASTROINTESTINAL FUNCTION / 391 this absorption has been delineated; the protein now known to be responsible for folate uptake in the intestine is the reduced folate carrier (RFC). The cDNA-encoding RFC protein has been cloned from mouse and human intestine (373–375). Studies in animal models have shown that folate absorption in the intestine changes with age. Brush-border membrane vesicle (BBMV) uptake of folate decreases with age in rat jejunum, with the highest uptake being seen in sucklings (7-fold greater than adult), decreased uptake in weanlings (2.4-fold greater than adult), and the lowest uptake in adults (376). The decline in folate uptake with age is correlated with the RFC protein and mRNA expression, with protein abundance being 2.3-fold greater in suckling rats and 1.4-fold greater in weanling rats compared with adults. Reduced folate carrier mRNA abundance is also greater in suckling and lower in adult rats (376). Nuclear run-on assays confirmed a parallel decline in the RFC transcriptional initiation rate in jejunal epithelia with maturation; the transcriptional rate was 2-fold greater in sucklings and 1.6-fold greater in weanling rats compared with adults (376). Intestinal Transport of Lipid-Soluble Vitamins during Postnatal Development Lipid-soluble vitamin absorption is dependent on bile acids secreted from the liver, a process that is decreased in the neonatal period. Variations in bile acid secretion during the neonatal period thus likely explain some ontogenic differences reported for the absorption of lipid-soluble vitamins (377). Transport of retinol has been widely investigated and is discussed briefly here. Retinol is transported into intestinal epithelial cells by a carrier-mediated mechanism that decreases during ontogeny, and retinol absorption along the gut is higher in the jejunum and lower in the ileum in sucklings and in adults (378,379). Milk-derived factors have also been reported to increase retinol absorption (380). Retinol may also be complexed with retinol-binding protein, and the complex is transported via a specific, saturable, receptor-mediated process, a mechanism that is distinct from the transport of unbound retinol (381). Ontogenic patterns for the absorption of other lipid-soluble vitamins have not been reported to date. Intestinal Absorption of Bile Acids during Ontogeny Bile acids are an essential component of the process of solubilization and digestion of dietary lipids. The critical micellar concentration of bile acids in the duodenum determines, in part, the efficacy of lipid absorption through the gastrointestinal tract. Initial studies using brush-border membrane vesicles have shown that bile acid transport during the suckling period in the rat is markedly decreased, with a significant increase during the weanling period. Moreover, bile acid transport in the ileum has been shown to be enhanced by the administration of glucocorticoids. Advances in molecular cloning have allowed the identification of an apical

sodium-dependent bile acid cotransporter (ASBT clone). The ASBT is a 48-kDa protein, which is primarily expressed in the brush-border membranes of the ileum (382). Western and Northern blot analysis have confirmed the original observation that the ASBT expression is markedly increased during the weanling period (383,384). These findings explain the decrease in the bile acid pool size in the neonatal period and the relative decrease in fat absorption during early life. The second step in bile acid transport involves the cytocylic movement of bile acid. An ileal bile acid binding protein (IBABP), which is a 14-kDa protein, was identified as the major cytocylic bile acid binding protein, and again, this binding protein was shown to be markedly increased during the third postnatal week of rat development (385). Both ASBT and IBABP mRNA are precociously increased by early weaning and delayed by weaning prevention. Moreover, glucocorticoids administration induces both ASBT and IBABP mRNA. These findings suggest that a hardwired (preprogrammed) system together with dietary factors is involved in maintaining the enterohepatic circulation of bile acids.

ONTOGENY OF TRANSPORT FUNCTION ALONG THE VERTICAL AND HORIZONTAL GUT AXES Studies of gastrointestinal morphology and function have demonstrated that horizontal (proximal-to-distal) and vertical (crypt-to-villus) axes, where functional properties can vary greatly, exist in the mammalian intestine. These axes are first established during fetal development in precocial species, but in species with longer gestational periods such as pigs and humans, they are not fully established until several weeks after parturition (386–388). The physiologic factors that direct ontogenic development of these intestinal gradients have not been fully defined, but some studies suggest that predetermined genetic factors regulate development of intestinal gradients. However, other influences such as dietary factors may also be involved in this important physiologic process (149,389). The differences in gene expression patterns seen along these gut axes are regulated by a variety of trans-acting factors, which are expressed differentially in intestinal segments during postnatal development (390). The fetal and neonatal colon, where the most prominent developmental differences are noted, have distinct morphologic and functional features that are absent in adults. For example, the colons of neonates have villus-like structures (391,392) and active transport of some dietary components, which does not occur in the colon of adults. Furthermore, colonic Na+ absorption occurs by an electrogenic process during early postnatal development, whereas an electroneutral process predominates in adults, presumably mediated by NHE. In addition, other transport functions also vary along the length of the small intestine during postnatal development. Other notable differences are the distribution and

392 / CHAPTER 13 presence of vacuolated enterocytes and saturable absorption of bile acids and some vitamins, all of which are present only in early neonatal development. There are also distinct changes in transport function along the crypt-villus axis noted during development (81,149, 393). Some of these observations may be explained by the differential expression of genes encoding various transporters during early life, or by changes in rate of enterocyte migration and turnover. This latter possibility seems particularly relevant as cell replacement and mitotic activity is slower in the perinatal period, and it increases around the suckling/weaning transition (36). These observations may also be explained by the transcriptional repression of some transporter-encoding genes during neonatal life (394).

DEVELOPMENTAL REGULATION OF GASTROINTESTINAL FUNCTION The development of normal gastrointestinal function requires differentiation of four types of principal epithelial cells that include enterocytes, goblet cells, enteroendocrine cells, and M cells. All of these cells originate from common

M-cell

pluripotent stem cells in the intestine. The developmental regulation of gastrointestinal function involves complex interactions among diet, hormones, growth factors, and neurotransmitters (Fig. 13-4). The development of the multicellular gut depends on morphogenetic signals between cells that direct changes in gene expression. The signals that trigger age-dependent changes are originated from genetic and external factors, with both acting in concert to mediate intestinal development. An example of this type of regulation is that hormones or neurotransmitters deliver extracellular signals to nuclei by binding their cognate receptors on the cell surface, and transducer signals by binding to specific regulatory sequences in the DNA, thus inducing changes in expression of specific genes (395). Genetic hardwired signals predominantly determine organogenesis during early development (396). Studies of suckling and weanling rodents have shown that shifts in the activities of different intestinal enzymes and transporters are genetically programmed and relatively less influenced by dietary factors or hormones (397–400). One study showed that rats prevented from normal weaning by being maintained on powdered milk were generally similar to normal rats weaned onto chow. Notably, their fructose uptake still

Enteroendocrine cell

Goblet cell

Enterocytes

Basal lamina

Fibroblasts Sensory neurons

Immune cells Capillary Enteric neurons

Lamina propria

Myenteric ganglion

Luminal stimuli Paracrine effects Nervous stimuli Endocrine stimuli

FIG. 13-4. Signaling interactions that regulate nutrient absorption and secretion during ontogenic development of the intestine. Several physiologic factors that influence intestinal transport function are shown. Luminal effectors, paracrine effectors, nervous stimuli, and hormonal secretions all play a role in regulating transport function during postnatal development. Dietary (e.g., luminal) stimuli interact with different epithelial cells as shown. M cells have a modified basolateral surface that forms an invagination into which immune cells can migrate. Enteroendocrine cells and lymphocytes and other immune cells respond to stimuli by the release of signaling mediators that influence other cell types of the lamina propria through paracrine action. The enteric nervous system receives its input from paracrine effectors, from sensory neurons, and from the central nervous system. This information is processed in the enteric nervous plexus, which results in the activation of motor neurons that may control various mucosal functions. Lastly, hormonal or endocrine stimuli influence the cells of lamina propria, as well as cells of the epithelial layer. (Modified from Pacha [36], by permission.)

MOLECULAR ASPECTS AND REGULATION OF GASTROINTESTINAL FUNCTION / 393 increased steeply on weaning, despite low dietary fructose levels, demonstrating hardwired programming of intestinal transporter development (138). Although internal hardwired programming mechanisms remain in control of development of gastrointestinal function during early developmental stages, external factors, including diet, hormones, growth factors, and neurotransmitters, absolutely play a role in the development of normal gastrointestinal function in the following stages. Many of these factors control the development of gastrointestinal function through transcriptional pathways. The transcriptional control of intestinal gene expression is regulated by multiple positive and negative cis-elements in gene promoters or enhancers (394). Cdx1 and Cdx2, caudal-related intestine-specific homeodomain transcription factors, are selectively localized in the mucosal epithelial cell nuclei in the small intestine and colon of the fetus and adults in both humans and mice (401,402). Whereas normal gastric mucosa does not express transcription factors Cdx1 or Cdx2, strong nuclear immunoreactivity for Cdx1 was detected in human gastric intestinal metaplastic mucosa (403). Furthermore, Cdx1 and Cdx2 are also expressed in human intestinal metaplastic mucosa (404,405). These findings suggest that Cdx1 and Cdx2 may play a pivotal role in the development of intestinal metaplasia in the human stomach. Hormonal factors also play an important role in the ontogeny of the gastrointestinal tract function (406). Much attention has been given to the role of glucocorticoid hormones on development of the intestinal function. Glucocorticoid administration of thyroxine to suckling animals has broad effects on many digestive functions, including sucrase, alkaline phosphatase, enteropeptidase, diamine oxidase, pepsin, and amylase expression (407–409). The cellular mechanism of the action has now become clear. These hormones bind to specific intracellular receptors (glucocorticoids receptors). The hormone/receptor complex undergoes conformational change, and then activates a transcriptional complex to bind to hormone-specific cis-elements on gene promoters or enhancers, thus inducing expression of genes that control intestinal development (410). Glucocorticoids receptor expression has been found in both suckling and adult animals, with decreased expression by age (411,412), which suggests a regulatory role of glucocorticoids on the developmental regulation of intestinal function at weaning (413). Both stimulatory and inhibitory effects of glucocorticoids on intestinal functions have been demonstrated for many genes. Glucocorticoids stimulate Na+-dependent glucose transport, bile acid transport, and Na+-K+-ATPase expression (185, 247,414–417), whereas inhibition is observed with Na+dependent phosphate transport and Ca2+ absorption (290,418). Although glucocorticoids have been demonstrated to have a primary role in the developmental regulation of gastrointestinal functions, many other regulatory peptides and hormones are an essential part of this regulatory machinery, including calcitriol, cholecystokinin, gastrin, secretin, insulin, and several growth factors (419–423). Two important growth factors are transforming growth factors (TGF) and EGFs.

Both TGF and EGF receptors have been found in fetal, neonatal, and adult intestine; however, age-dependent differential responses to intraluminal administration of EGF has been demonstrated, in which mucosal EGF-stimulated EGF receptor phosphorylation occurs in suckling jejunum, but not in weanling or adult jejunum (131,424,425). Although EGF receptor abundance was similar in 22-day-old fetal, 8-day-old suckling, and adult jejunum (425), adult animals exhibited a higher expression of EGF mRNA in comparison with suckling animals (426). It has been shown that milkborne growth factors provide important regulatory signals to the neonatal intestine under normal states (427,428). In contrast with EGF, strong expression of TGF mRNA was found in the small intestine of both adult and suckling rats, whereas rat milk is free of TGF. The small intestine is directly exposed to environmental factors as the result of daily dietary intake and is prepared to adapt its structure and function in response to variations in the diet. Food processed in the gastrointestinal tract induces a series of physiologic responses, including the release of trophic hormones, the stimulation of the enteric nervous system, and the activation of secretary, digestive, and absorptive functions. The normal diet also contains growth factors that directly stimulate the growth of intestinal mucosal cells independently of their nutritive role (429). Significant changes of diet occur during early developmental stages, such as during birth and the weaning period. The developmental patterns of the gastrointestinal tract at these stages are controlled by both genetic and dietary factors (430). Interactions between diet and intestinal maturation occur immediately after birth, by means of feeding in humans and in animals. In some species, the neonatal intestine undergoes rapid growth and reorganization, possibly as a result of programmed gene expression associated with the immediate response to milk (431). The stomach and intestines of newborn rats, rabbits, and dogs increase in mass within hours after the initiation of enteral nutrition (432). Milk-feeding in animals results in a greater DNA, RNA, and protein content in the stomach and intestine than in those with no milk consumption (433). This suggests that rapid growth of the small intestine in naturally fed animals is the result of the specific composition of natural milk, probably caused by stimulation of gene expression by milk-borne factors responsible for intestinal growth. The milk of mammals contains a range of trophic factors, hormones, and other biologically active substances that may cause growth-enhancing effects on the gastrointestinal mucosa (434,435). Differences in pancreatic and gastrointestinal hormone release between breast-fed and bottle-fed infants have also been described (436). Epidermal growth factor, insulin and insulin-like growth factors, polyamines, and glucocorticosteroids are important factors in milk necessary for proper intestinal development (94, 437,438). Luminal administration of EGF stimulates the proliferation and differentiation of enterocytes (439,440). The direct effect of nutrients on the postnatal shift in gene expression and processing of gene products has been implicated in the postnatal disappearance of glucose absorption

394 / CHAPTER 13 in the small intestine of lambs. As a consequence of the rumen maturation, which coincides with weaning, dietary carbohydrates are fermented into short-chain fatty acids by rumen microflora, the amount of hexoses entering the small intestine gradually decreases (441), and the capacity of Na+glucose cotransport declines (146). An example of diet-induced developmental regulation of gastrointestinal function is the dietary effect on lactase expression. Under normal circumstance, lactase activity decreases by age. However, the ontogenic decline in lactase activity can be promoted or delayed by changing the time when rats are switched from milk to the adult-type diet (442). Preweaned animals force-fed an artificial diet without lactose retained a high amount of lactase mRNA in the jejunum, whereas this transcript precociously decreased in the distal ileum. Conversely, prolonged nursing delayed the specific decrease of lactase mRNA levels in the latter segment. These results suggest that switching over from milk to the adulttype diet at weaning contributes to the modification of the longitudinal distribution of the lactase mRNA that normally occurs at this stage. Thus, although the ontogenic decline in intestinal lactase is intrinsically programmed, luminal factors modulate this process. The transition to the adult diet is required for the normal progression of events.

ACKNOWLEDGMENTS Our efforts in writing this chapter were supported by National Institutes of Health grants 1-R21-DK-06834 (J.F.C.), R01-DK-412174 (F.K.G.), R01-DK-33209 (F.K.G.), and R01-DK-063142 (L.B.).

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CHAPTER

14

Effect of Aging on the Gastrointestinal Tract Adhip P. N. Majumdar and Marc D. Basson The Aging Esophagus, 406 Structural and Functional Properties, 406 Barrett’s Esophagus, 407 Key Areas for Future Research, 407 The Aging Stomach, 407 Structural and Functional Properties, 407 Mucosal Injury and Repair, 408 Gastric Secretion and Digestion, 410 Key Areas for Future Research, 411 The Aging Intestine, 411 Structural and Functional Properties, 411 Immunologic Effects, 412 Gut Peptides and Other Key Molecules, 412 Contractility and Motility, 412 Mechanisms of Fecal Incontinence, 413 Key Areas for Future Research, 414 Aging and Gastrointestinal Cancers, 414 Epidemiologic Observations, 414 Epithelial Growth and Renewal as Potential Premalignant Events, 415

Inhibition of Apoptosis as a Potential Premalignant Event, 416 Changes in Susceptibility to Carcinogens, 417 Mutational Events, 418 Key Areas for Future Research, 419 Regulation of Mucosal Growth during Aging, 420 Intracellular Mechanisms, 420 Nutritional and Hormonal Factors, 420 Growth Factors and Protein Kinases, 421 Gene Expression Patterns, 423 Key Areas for Future Research, 423 Aging and Surgery of the Gastrointestinal Tract, 423 Adaptation to Fasting and Sepsis, 423 Adaptation to Bowel Resection, 423 Surgical Diseases of the Gut, 424 Angiodysplasia, 425 Key Areas for Future Research, 425 Acknowledgments, 425 References, 425

Census and other data suggest that persons older than 65 years constitute about 12.3% of the U.S. population, or 1 in 8 Americans. By 2030, the fraction of older Americans has been predicted to increase to 20%, with a total number of 71.5 million, more than twice as many as in 2000. The number of Americans older than 85 years has been predicted to increase from 4.2 million (based on the 2000 census) to

8.9 million in 2030. The predicted life expectancy of an American child born in 2000 is 76.9 years. Aging is defined as the progressive accumulation of changes over time that is associated with or is responsible for increasing susceptibility to disease and death, the final event of advancing age. A primary conceptual challenge in the study of mammalian aging is the occurrence of disease and the intricate relation of disease processes to aging. Aging is associated with marked changes in the structural and functional properties of the gastrointestinal (GI) organ system. Thus, consideration of the effects of aging on the GI tract is likely to become increasingly relevant as the population ages. This chapter therefore reviews the structural and functional changes in different parts of the GI tract with aging. In addition, how such changes disturb homeostatic mechanisms and predispose the gut to certain diseases is discussed. This latter discussion in particular focuses on malignant transformation.

A. P. N. Majumdar: John D. Dingell Veterans Affairs Medical Center, Department of Medicine, Karmanos Cancer Center, Wayne State University, Detroit, Michigan 48201. M. D. Basson: John D. Dingell Veterans Affairs Medical Center, Department of Surgical Service, Wayne State University, Detroit, Michigan 48201. Physiology of the Gastrointestinal Tract, Fourth Edition, edited by Leonard R. Johnson. Academic Press, 2006.

405

406 / CHAPTER 14 THE AGING ESOPHAGUS Structural and Functional Properties Aging has been associated with a diversity of alterations in the oropharyngeal components of swallowing. In general, aging has been found to be associated with increased oropharyngeal segmental transit times (1,2), as well as increased pharyngeal peristaltic pressure wave activity and amplitude (1–4). Peak pharyngeal pressures were found to be greater in older subjects (5), whereas the anteroposterior diameter of the upper esophageal sphincter (UES) and the anterior hyoid bone were both shown to be significantly less in elderly subjects than in their younger counterparts, as is laryngeal excursion (2). Although the mean UES resting pressure tends to decrease with advancing age (5), possibly as the result of striated muscle degeneration, the UES residual pressure after deglutition is increased (6). Rapid anterior movements of the hyoid, normally observed in younger individuals, are found to be delayed in elderly subjects, as is the subsequent anticipated relaxation of the UES. Consequently, smooth passage of a luminal bolus from the oropharynx to the esophagus is likely to be compromised. During the esophageal phase of swallowing, the ingested bolus is propelled through the esophagus by primary peristaltic waves and is delivered into the stomach with a coordinated relaxation of the lower esophageal sphincter (LES). The normal esophageal motor activity consists of an orderly peristaltic contraction that propagates itself from the UES through the striated and smooth muscle sections and terminates in closure of the lower sphincter (7). There have been numerous studies examining the effects of aging on esophageal motility in patients with dysphagia, as well as in healthy asymptomatic control subjects. A variety of abnormalities have been described in both subgroups; however, the consistency of these aberrant findings has been the subject of debate. Earlier, Soergel and colleagues (8) evaluated the esophageal motor function of 15 nonagenarians by cineradiography, intraluminal manometry, and balloon kymography. In this group of subjects, disturbed esophageal motility consisted of disorganization of the response to deglutition, as a result of a defect in initiating relaxation of the LES, as well as primary peristalsis, whereas the incidence of secondary peristalsis was found to decrease less frequently. Numerous nonpropulsive, often repetitive, esophageal contractions were also seen. A similar observation was also made by Zboralske and coworkers (9), who studied 41 subjects, including 15 subjects aged between 90 and 98 years. The pathogenesis of this disorganization, however, is currently unknown. Ueda and colleagues (10) have studied the motor and electrical activities of feline esophagus by intraluminal pressure detectors and by chronically implanted platinum electrodes, before and after vagotomy. The prominent features after vagotomy were persistent weakness, incoordination, and repetitive responses in the lower esophagus preventing normal emptying of the organ. The significance of possible degenerative changes of vagal

innervation in the aged remains unknown. Grande and coworkers (11) studied 79 healthy subjects and noted an inverse correlation of age with LES pressure, as well as peristaltic wave amplitude and velocity. Alternatively, Richter and colleagues (12) did not detect any differences in LES pressure between young and elderly subjects using either the rapid or stationary pull-through methods. This finding has been confirmed by others (13) who further noted that the total length of the LES high-pressure zone was similar between young and elderly subjects. Several investigators have found an age-related decrease in the amplitude of peristaltic wave pressure (11,14). These studies also noted a decrease in peristaltic velocity and an increase in wave duration, whereas Hollis and Castell (15) found no age-related differences in these parameters. Secondary peristalsis, which is essentially a peristaltic wave that is not induced by a swallow, occurs less often in elderly subjects and may contribute to reduced clearance of residual, as well as refluxed, esophageal contents (14). Finally, several investigators have shown an increased frequency of tertiary or spontaneous nonperistaltic contractions in elderly subjects (10,11,16). The antireflux barrier at the gastroesophageal junction is generally attributed to the interaction of intrinsic LES tone, the crural diaphragm that exerts extrinsic pressure on the LES, and the length and thoracoabdominal distribution of the LES (17,18). Xie and coworkers (19) found that elderly subjects had increased gastroesophageal reflux events after pharyngeal water swallows significantly more often than their younger counterparts. This increase in reflux events was further associated with a decreased intraabdominal segment of the LES in the elderly. Other investigators (20–22) have noted an increased duration of reflux episodes in elderly patients. Interestingly, however, elderly patients seem to perceive reflux episodes less often than younger patients (15,22,23). The prevalence of Helicobacter pylori infection increases during aging (24). It has been estimated that 40% to 60% of elderly subjects may be infected with H. pylori (24). Because aging is also associated with an increased incidence of gastroesophageal reflux disease (GERD), it has been suggested that H. pylori infection may be a contributory factor for GERD. However, this relation has not been thoroughly investigated, particularly with respect to aging. Earlier studies by Liston and colleagues (25) failed to show a significant association between H. pylori and reflux esophagitis during aging. In a more recent study, Haruma and coworkers (26) observed a lower prevalence of H. pylori in subjects older than 60 years with reflux esophagitis. However, in a double-blind, placebocontrolled study, Schwizer and colleagues (27) observed in patients with GERD that H. pylori–positive patients relapsed earlier than those in whom H. pylori was eradicated. In contrast, Pilotto (28) reports that eradication of H. pylori infection in elderly patients did not influence the short- and long-term healing rates of esophagitis between young and elderly patients. Limited information is available with respect to agerelated changes of the esophageal mucosa. Furthermore, the significance of the development of Barrett’s esophagus with

EFFECT OF AGING ON THE GASTROINTESTINAL TRACT / 407 advancing age remains unknown. Barrett’s esophagus, a premalignant lesion, requires the replacement of normal stratified squamous epithelium with intestinal metaplastic epithelium at the distal esophagus (29). The prevalence of Barrett’s esophagus increases with age and plateaus in the seventh decade of life (30). Although this increasing prevalence with aging certainly reflects the cumulative effect of protracted esophageal exposure to acid, aging may independently contribute to the evolution and progression of Barrett’s epithelium through age-related reduction in perception of acid reflux (22,23,31). Similarly, elderly patients would also be more likely to require medications that potentially diminish LES pressure, such as calcium channel blockers, β-adrenergic agonists, and nitrates, thereby enhancing the propensity for acid reflux disease (32).

Barrett’s Esophagus It is well recognized that Barrett’s esophagus is a premalignant lesion with the potential to transform into adenocarcinoma of the esophagus. The presence of Barrett’s esophagus is associated with a 30- to 40-fold increased risk for esophageal adenocarcinoma (33). The development of adenocarcinoma in Barrett’s esophagus is a multistep process during which the metaplastic epithelium ultimately transforms into low-grade and subsequently high-grade dysplasia. There is evidence that continuous acid exposure can induce differentiation and reduce proliferation, whereas short bursts of acid exposure increase proliferation (34). More importantly, normalization of intraesophageal pH can reduce esophageal mucosal cell proliferation. Interestingly, complete symptom eradication does not guarantee normalization of intraesophageal pH (35). Therefore, it is certainly conceivable that elderly individuals with intrinsically enhanced reflux but compromised sensitivity to reflux is particularly susceptible to induction of mucosal cell proliferation. Cyclooxygenase-2 (COX-2) or prostaglandin synthase 2, a membrane-bound protein involved in the generation of prostaglandins from arachidonic acid, mediates prostanoid release involved in inflammatory and tumorigenic responses (36). Cyclooxygenase-2 expression is restricted in certain tissues, but it can be induced in response to various stimuli (37,38). Increased expression of COX-2 has been observed in both animal models of inflammation and human inflammatory diseases (39–42). There is increasing evidence to implicate COX-2 in colorectal, gastric, and esophageal carcinomas (43–45). Moreover, it has been demonstrated that COX-2 protein expression is significantly higher in Barrett’s metaplastic and dysplastic epithelium, as well as adenocarcinoma, and is correlated with progressive morphologic changes (46). Certainly, nonselective COX inhibitors (nonsteroidal anti-inflammatory agents), as well as newer selective COX-2 inhibitors, may have a role in cancer chemoprevention for patients with Barrett’s esophagus. Finally, mutations in the tumor-suppressor gene p53, one of the most common genetic alterations in human cancer,

have been described in most Barrett’s esophagus cases with high-grade dysplasia or adenocarcinoma (47). Similarly, immunohistochemical staining of p53 has been demonstrated in low-grade dysplasia associated with Barrett’s esophagus and may be predictive of future evolution to high-grade dysplasia. Elderly patients with comorbid conditions precluding definitive surgical intervention for Barrett’s dysplasia are often considered for a variety of endoscopic ablative techniques. Despite the ability of many of these techniques to destroy Barrett’s mucosa, there is still evidence that the genetic aberrations associated with Barrett’s esophagus may persist (48).

Key Areas for Future Research Although the impact of aging on esophageal motility is clear, the mechanisms of these effects require further study, particularly with reference to the relative contributions of neural and hormonal influences. This is particularly important because the development of targeted modalities for the reconstitution of esophageal motility in the elderly could substantially reduce morbidity and mortality from aspiration pneumonia. The triggers for the development of Barrett’s esophagus and subsequent malignant degermation also require further study. Although repeated exposure to gastric refluxate is assumed to be pathogenic in this regard, the possibility that aging esophageal mucosa may be more susceptible to such refluxate at a cellular level is a fertile ground for investigation. Finally, the potential role of H. pylori in the pathogenesis of GERD, Barrett’s esophagus, and esophageal carcinoma requires further investigation.

THE AGING STOMACH Structural and Functional Properties Like other parts of the GI tract, the gastric mucosa is composed of numerous exocrine and endocrine cells (49), which renew at different rates. In the oxyntic gland area of the stomach, most of the newly produced cells migrate rapidly to the surface while differentiating into mucous cells. The migration time represents the time for replacement of the total cell population above the dividing zone. This migration time is about 3 days in adult rats (50) and 4 to 6 days in the adult dog or human (51). Most studies have indicated that parietal cells are unable to divide, and that some newly formed cells migrate slowly down the gland to differentiate into acid-producing cells (51). In the adult mouse, zymogen (chief) cells are replaced by mitosis (52), but after injury, they have been found to originate from undifferentiated cells. Aging is associated with marked changes in the structural and functional properties of the gastric mucosa. In rats, aging is found to be associated with gastric atrophy, as evidenced

408 / CHAPTER 14 by a significant reduction in mucosal glandular height, gland density (number of glands per square centimeter), and total mucosal DNA content (53,54), and it may lead to a reduction in the number of glandular epithelial cells of all types. In aged rats, gastric epithelial cells also show evidence of decreased secretory activity, with decreased number of intracytoplasmic secretory granules and decreased cell size (53). In contrast, the connective tissues of lamina propria of aged rats, especially between the glands and the muscularis, are found to be thickened due to massive collagen deposition (53). Electron microscopy studies in rats have further demonstrated ultrastructural degenerative changes in both parietal and chief cells (53,55). Although earlier studies have suggested that the gastric mucosa remains normal in older human subjects, several reports have documented a marked increase in the incidence of atrophic gastritis among the elderly (56,57). Earlier studies by Andrews and colleagues (56) have demonstrated some degree of chronic atrophic gastritis in 23 of 24 subjects older than 60 years. Bird and coworkers (57), who analyzed 201 gastric biopsy specimens from individuals between 65 and 90 years old, observed signs of atrophy in most of the specimens. Hradsky and colleagues (58) also found an increased incidence of gastritis with aging by comparing the gastric biopsy results in groups of patients 44 years or younger, between 45 and 59 years old, and between 60 and 84 years old. Although the underlying regulatory mechanism(s) for the development of gastric atrophy is unknown, one plausible explanation could be that the ability of the gastric mucosa to replenish the lost surface cells decreases with aging. Indeed, it has been demonstrated that in aged rats, reepithelialization of the gastric mucosa after hypertonic saline-induced mucosal erosion is markedly delayed compared with young animals (59).

Mucosal Injury and Repair Aging is also associated with increased susceptibility of the gastric mucosa to damaging agents (60). This, together with impediment for the repair process, may contribute to the increased incidence of gastric and duodenal ulcers observed among the elderly. Because intake of aspirin and other nonsteroidal anti-inflammatory drugs (NSAIDs) is generally greater among the elderly, studies have been performed to determine the frequency of NSAID-induced injury in elderly subjects. Epidemiologic data suggest that aging is associated with increased NSAID-induced gastric mucosal injury (61). It is estimated that in the United States, approximately 16,000 deaths could be related to adverse events associated with NSAID use (62). Approximately 34% to 46% of patients receiving NSAID therapy are expected to have some manifestation of adverse GI events, and the overall risk for development of a serious GI complication is three times greater in NSAID users than nonusers (63). The risk tends to increase linearly with aging and becomes particularly high in the presence of disability (64), comorbidity

(congestive heart failure, hypertension, renal or hepatic insufficiency) (62), and comedication and concomitant intake of corticosteroids (65) and anticoagulants (66). Moreover, it has been demonstrated that both H. pylori infection and NSAID use independently and significantly increase the risk for peptic ulcer and bleeding (67). H. pylori infection increased the risk for peptic ulcer in NSAID users by about 3.5-fold in addition to the risk associated with NSAIDs (67–69). These observations are in agreement with pathophysiologic studies that demonstrated that aging, as well as H. pylori infection, may decrease gastric mucosal hydrophobicity (70) and gastric mucus gel formation (71), which is known to provide cytoprotection. Again, eradication of H. pylori infection before NSAID use has been shown to reduce the occurrence of NSAID-related peptic ulcer (72). Clearly, further studies are needed to evaluate the roles of various endogenous and exogenous factors in the age-related increase in the frequency of gastric mucosal injury. To determine the age-related susceptibility of the gastric mucosa to damaging agents and the subsequent reparative processes, we used a hypertonic saline-induced gastric mucosal injury model in Fischer-344 rats. Our investigation with hypertonic saline, which virtually eliminates the surface epithelium, showed a 67% higher lesion index (percentage of damaged area) in the gastric mucosa of 24-month-old Fischer-344 rats compared with their 4-month-old counterparts (73). Electron microscopic examination also showed that, although administration of hypertonic saline virtually eliminated the surface epithelium in both young and aged rats, the extent of injury in older animals extended beyond the surface epithelium (74). In aged rats, epithelial cells in deeper parts of the gastric glands demonstrated severe swelling with vacuolization and disintegration of the cell organelles, with dying and dead cells (74). Aspirin and ethanol have also been shown to produce a greater number of lesions in the gastric mucosa of aged than in young rats (75). Taken together, these observations suggest that aging is associated with reduction in mucosal protective mechanisms that may predispose aged animals to mucosal injury. Although the underlying biochemical mechanisms for this increased susceptibility are poorly understood, several investigators have demonstrated that gastric mucosal prostaglandin content, which provides cytoprotection to the surface epithelium, decreases with aging in humans (76,77). In addition, Gronbech and Lacy (78) have suggested that impaired mucosal defense and reduced restitution in aged rats is partly due to decreased density of calcitonin gene–related peptide in nerve fibers and the biosynthetic capacity of the mucosa in prostaglandins. A decline in mucosal blood flow in aged rats has also been suggested as another causative factor for the age-related decline of mucosal repair (79). In addition, the H. pylori infection, which increases among diabetic patients and the elderly, is thought to be an important causative factor for duodenal and gastric ulcers (80). Type II diabetes, the incidence of which increases with advancing age, has also been shown to affect the structural and functional integrity of many organ systems, including

EFFECT OF AGING ON THE GASTROINTESTINAL TRACT / 409 the GI tract. Diabetes has been linked to “premature aging.” Although gastroparesis diabeticorum is the most common complication of diabetes in the digestive tract (81), clinical studies have demonstrated that diabetes ketoacidosis is often associated with abdominal pain, nausea, and vomiting (82–85). This could partly be the result of upper GI hemorrhage, the prevalence of which is increased in diabetic ketoacidosis (83,84). In addition, results from experimental models of diabetes have shown that diabetes is associated with spontaneous gastric ulceration and enhanced susceptibility of the gastric mucosa to exogenous damaging agents, as well as with stress-induced gastric ulcer (86–88). Food deprivation in streptozotocin-induced diabetic rats has been shown to produce severe gastric lesions (87,88). Although the underlying mechanisms remain to be elucidated, this could be partly due to the loss of surface mucosal cells resulting from spontaneous mucosal injury. Induction of diabetes mellitus by streptozotocin in rats has been shown to result in gastric mucosal atrophy with reduction in mucosal thickness (89). Whether these changes are worsened with aging, however, remains to be determined. However, the gastric mucosa of healthy adult animals possesses a remarkable capacity to repair promptly after an acute injury. Aging and diabetes are associated with decreased healing of the gastric mucosa (59,60,89). The regulatory mechanisms that direct this repair, as well as the potential modulating effects of aging or diabetes, are poorly understood. The process of mucosal reconstitution, which is accomplished by cell migration, begins within minutes after injury and is completed within a few hours. Earlier in vitro studies using frog mucosa in the Ussing chamber have demonstrated that the surface epithelium is restored within a few hours of exposure to 1 M NaCl (90,91). A similar phenomenon was also observed in rats (92). Although similar studies have not been performed during aging, we have demonstrated that, whereas the gastric mucosa of young rats shows signs of reepithelialization as early as 6 hours after hypertonic saline (2 M NaCl)-induced injury, which is essentially complete by 24 hours, in aged rats, only intermittent surface cells can be seen 24 hours after injury (59,60). The partial reepithelialization of the gastric mucosa of young rats that occurs within 6 hours after injury is thought to be the result of increased cell migration during this period. Aging appears to attenuate this process. A number of growth factors, including basic fibroblast growth factor (90,93), intestinal trefoil peptide (94–96), as well as epidermal growth factor (EGF) and transforming growth factor-α (TGF-α) (97–101), all of which stimulate cell migration, are thought to play a role in this process in healthy young animals. However, their role in mucosal cell migration during aging remains to be evaluated. Similarly, polyamines, which are known to be essential for cell proliferation (102) and for repair of gastric and duodenal ulcers, have also been shown to stimulate cell migration in vitro (103). Therefore, it is tempting to speculate that they may also be involved in regulating healing of the gastric mucosa after injury during aging. To the best of our knowledge, no information is available about the role

of either growth factors or polyamines in regulating the reparative processes of the gastric mucosa with advancing age. However, in assessing the role of growth factors in mucosal restitutional process, Polk and colleagues (99) have demonstrated a marked increase in TGF-α, but not EGF, in the gastric mucosa of young rats within 30 minutes after an acute injury. They suggest that local production of TGF-α may have an important role in the early reparative phase of the gastric mucosa. In aged rats, we have also observed a marked increase in gastric mucosal TGF-α levels 30 minutes after hypertonic saline-induced injury (104). Yet, mucosal restitution, as determined by reepithelialization of the gastric mucosa, was attenuated (59). Because TGF-α exerts its physiologic action by activating the intrinsic tyrosine kinase of its receptor, the epidermal growth factor receptor (EGFR) (105), we postulated that decreased activation of EGFR tyrosine kinase might, in part, be responsible for the diminished mucosal repair in aging animals. The role of EGFR tyrosine kinase in gastric mucosal was initially evaluated by examining the effect of tyrphostin, an inhibitor of tyrosine kinases with a greater specificity for EGFR than other protein kinases (106,107), on gastric mucosal regeneration in 3- to 4-month-old rats at 24 hours after hypertonic salineinduced injury (108). In the absence of tyrphostin, the gastric mucosa showed signs of extensive regeneration, whereas in its presence, regeneration was greatly attenuated (108). These changes were accompanied by parallel alterations in the number of proliferating cell nuclear antigen (PCNA)– immunoreactive cells and EGFR tyrosine kinase activity in the gastric mucosa (108). The observation of increased PCNA immunoreactivity in the gastric mucosa of control rats (which were not treated with tyrphostin) at 24 hours after injury suggests that induction of cell proliferation is a necessary step for regeneration of the mucosa after injury. To further evaluate the role of EGFR tyrosine kinase in the early reparative phase of the gastric mucosa, we examined time course changes in total and EGFR tyrosine kinase activity in young adult rats over a period of 60 minutes after hypertonic saline-induced injury (109). Injury caused an immediate increase in both total and EGFR tyrosine kinase activity in the gastric mucosa, with the maximal stimulation occurring at 30 minutes after injury (109). When gastric mucosal EGFR tyrosine kinase activity was examined in young and aged rats at 30 minutes after injury, we observed that the enzyme activity and the expression of the receptor protein in the gastric mucosa of both age groups were significantly increased 30 minutes after injury; however, the magnitude of stimulation was lower in the gastric mucosa of aged than in young rats, when compared with the corresponding basal levels (109). A similar phenomenon was also noted for phospholipase C (PLC) and tyrosine phosphorylation of PLCγ (109). Taken together, the results suggest that TGF-α is less effective in enhancing activation of EGFR tyrosine kinase in aged rats. This could be attributed, in part, to increased sensitivity of the aged gastric mucosa to EGF family of peptides such that low doses are stimulatory, whereas high doses of these peptides may inhibit the physiologic

410 / CHAPTER 14 actions of these peptides in aged rats. The basis for this hypothesis comes from the observation that administration of a relatively high dose of EGF, which caused a significant stimulation in mucosal proliferative activity and tyrosine kinase activity in the gastric mucosa of young rats, produced a marked inhibition in aged rats (110,111). This was further substantiated by the observation that in isolated gastric mucosal membranes from aged rats, the concentration of TGF-α required to achieve the maximal stimulation of EGFR tyrosine kinase was several fold less than that required to attain the same stimulation in young animals (111). Because injury causes significant increase in TGF-α production in the gastric mucosa of both age groups (109), it is likely that increased levels of endogenous TGF-α or other EGFR ligand(s) may prevent the injury-induced stimulation of EGFR tyrosine kinase and the subsequent signaling events in aged rats through an autocrine/paracrine mechanism. Another possibility could be that the maximal stimulation in mucosal EGFR tyrosine kinase in aged rats after injury occurs at a different time than in young animals, and that it takes longer for complete regeneration of the mucosa in aged animals. Undoubtedly, further studies are needed to evaluate the participation of EGFR and other signaling pathways in early and late reparative phases of the gastric mucosa during aging.

Gastric Secretion and Digestion The primary function of the gastric mucosa is to continue the process of digestion initiated in the mouth through the secretion of acid and pepsin. Although the major constituents of the gastric juice are hydrogen ion, pepsin, mucus, and intrinsic factor, the only indispensable ingredient is intrinsic factor, which is required for absorption of vitamin B12 by the ileal mucosa. Pernicious anemia resulting from vitamin B12 deficiency has been observed in the aging population (112). A decreased mucosal cell mass and, in turn, a diminished production of intrinsic factor by the parietal cells could, in part, be responsible for the development of pernicious anemia observed in the aging population. However, an association of atrophic gastritis with pernicious anemia has been disputed. Considering that a severe reduction in intrinsic factor is required before vitamin B12 absorption is impaired by ileal mucosa, evidence has been presented to show that in atrophic gastritis appreciable quantities of intrinsic factor may still be produced when acid production is greatly diminished (112). However, pernicious anemia may occur in some patients with severe atrophic gastritis, and it is characterized by the presence of a megaloblastic anemia caused by vitamin B12 deficiency (113). At times, atrophic gastritis is also associated with intestinal metaplasia, and both conditions are age related and considered to be precancerous lesions (114). Thus, aged individuals with gastritis are statistically at risk for development of gastric cancer, but precise measurement of this risk has not been determined. Although chronic atrophic gastritis in the

aged often is found to be associated with H. pylori infection, the precise role of H. pylori in the initiation and progression of gastritis during aging is still controversial. Studies by Feldman and colleagues (115) on healthy volunteers suggest that aging was independently related to chronic gastritis, and several others have found that, although atrophic changes of the gastric mucosa were associated with H. pylori infection, these changes could not be related to aging. However, it has been demonstrated that in subjects infected with H. pylori, the intensity of mononuclear infiltration of chronic gastritis and the increase in intestinal metaplasia were higher in older subjects when compared with their younger counterparts, and that H. pylori eradication was beneficial in preventing the progression of atrophy and intestinal metaplasia regardless of age differences (116). In a study with 132 adult volunteers with gastric atrophy, a significant improvement of the ailment was observed after treatment for H. pylori infection (117). Moreover, in a study carried out in 22 elderly subjects from Finland who had H. pylori infection and moderate to severe atrophic gastritis, a significant improvement in histologic scores of inflammation, atrophy, and intestinal metaplasia in the corpus mucosa was observed 2.5 years after H. pylori eradication (118). Taken together, the results suggest that the structural and functional changes that occur in older subjects after H. pylori infection are no different from those observed in young patients, and that all age groups show similar beneficial effects after eradication of H. pylori. Because one of the main functions of the stomach is to secrete acid, considerable work has been done to examine the age-related changes in acid output by the gastric mucosa. Earlier studies have demonstrated a decline in both basaland histamine-stimulated acid output in aging humans (119), and this has been related to the degree of gastric mucosal atrophy and the parietal cell number. In Fischer-344 rats, we have also observed an age-related decline in basal and gastrin-induced acid secretion (54), which could be partly due to the reduced number of acid secretory cells (53,55). In contrast, some studies on healthy humans indicated no significant change in either basal or stimulated gastric acid output with aging (115,120). Similarly, the pentagastrininduced stimulation of acid output was found to be only slightly decreased among the elderly (121). Thus, the earlier studies, which reported a decline in both basal and maximal gastric acid output in aging human populations, could be the result of inclusion of subjects with atrophic gastritis, a condition that is recognized to be common among the elderly, affecting 25% of those older than 60 years (57,121). Another factor that may affect gastric acid secretion in humans is H. pylori infection (120,122), the incidence of which is also increased with aging (80,123). H. pylori has been recognized as a causative factor in the development of chronic antral gastritis (124). In rats, basal pepsin secretion and mucosal pepsinogen concentration were also found to be lower in aged than in young rats (54). The regulatory mechanism(s) for the agerelated decline in mucosal pepsinogen content in rats is

EFFECT OF AGING ON THE GASTROINTESTINAL TRACT / 411 unknown. We have reported a decline in steady-state mRNA levels of several gastric proteases including pepsinogen C with aging (125). Whether this could result in decreased synthesis of the enzyme remains to be determined. In humans, aging has also been shown to decrease both basal and stimulated gastric pepsin output, and it was found to be independent of atrophic gastritis, H. pylori infection, or smoking (126,127). In contrast, serum pepsinogen I levels in normal female individuals, but not in male individuals, were shown to increase with age (128). A similar observation was also made in patients aged 50 or older with duodenal ulcer (129).

Key Areas for Future Research The phenomenon of gastric hyposecretion and hypochlorhydria in the aging stomach is well known, but its fundamental mechanisms await elucidation. The relative contributions of decreased expression of mucosal receptors for secretory hormones, abnormalities in intracellular signal transduction, and abnormalities in physiologic levels of the secretory hormones themselves should be studied further. The reduction in gastrin-induced acid secretion in the aging animal or human may be paradigmatic in this regard. This is particularly important because the achlorhydric stomach is associated with increased pernicious anemia and may also be at more risk for malignant degeneration, although the precise mechanism of this latter relation also awaits study. In addition, the impact of aging on mucosal healing also represents an opportunity for future research. It is becoming increasingly clear that growth factors and matrix proteins activate a complex cascade of intracellular signals during restitution that regulate the velocity and quality of mucosal healing. Signals at the focal adhesion complex such as focal adhesion kinase (FAK) and Src are likely to be of particular relevance in this regard. Surprisingly, there are little data available on the impact of aging on such signals or about changes in integrin expression or organization that may occur in the aging mucosa.

THE AGING INTESTINE Structural and Functional Properties Small intestinal mucosal permeability does not appear to change in aging humans, measured by lactulose/mannitol absorption (130), and the histology of the small mucosa is relatively well preserved in aging humans and animals. However, several investigators have described an increase in enterocyte proliferation and apoptosis with aging (131,132). Proliferating cell nuclear antigen immunoreactivity increases with advancing age in both the crypts and the villi, raising the possibility that abnormal absorptive function in the villi could coexist with normal proliferative biology in the crypts (133). The significance of this observation awaits study, but one

could question whether this hyperproliferative state might contribute to the increased likelihood of malignant degeneration or affect responses to various pathophysiologic stimuli, or both. Dietary restriction seems to potentiate small-bowel mucosal apoptosis in rats during aging (134). The density and distribution of mucosal neuroendocrine cells also seems to alter with aging in a complex fashion that seems to vary with the site in the GI tract and also the type of neuroendocrine cell being analyzed (135). Colonic mucosal proliferation is similarly enhanced in aging rats, with accelerated G1- to S-phase transition and progression through the S-phase, together with parallel alterations in several of the conventional mediators of the cell cycle, although colonocyte apoptosis may actually be decreased (136,137). An interesting question for further study is whether the disparity between increased apoptosis with aging in the small bowel and decreased apoptosis in the colon might contribute to the difference between the incidence of adenocarcinoma in the small and large bowels. In addition to differences in the histology of the mucosa itself, changes in the mucosal microenvironment may also occur with aging. The pH of the rat jejunal epithelial surface is significantly greater in senescent rats than in young adult rats, independent of the effects of sodium removal or amiloride. This may reflect the thinner mucous layer in these aged rats (138). Duodenal enterocytes from older rats exhibit decreased responsiveness to parathyroid hormone, possibly, at least in part, because of decreased expression of parathyroid hormone receptors (139). This correlates with age-related impairments in activation of PLC (140) and mitogen-activated protein kinase phosphorylation (141) in response to parathyroid hormone. Such signal loss may impair the regulation of intracellular calcium and proliferation in duodenal enterocytes (140,142). An age-dependent decrease in intestinal inorganic phosphate absorption has also been described and appears to correlate with a decrease in 1,25-dihydroxy vitamin D–dependent stimulation of expression of the type IIb sodiuminorganic phosphate cotransporter (143). 1,25-Dihydroxy vitamin D–dependent signals are similarly impaired in aging duodenal mucosal enterocytes, including protein kinase C activation and isozyme translocation, and the generation of inositol-1,4,5-trisphosphate and diacylglycerol (144–147). The role of steroids in supporting the small-bowel mucosa may also differ between younger and older rats. Adrenal resection results in mucosal atrophy, decreased crypt cell proliferation, and histologic disorganization in the proximal small intestinal epithelium of both young and old rats, together with increased numbers of Paneth cells. However, adrenal insufficiency in older rats also diminishes the activity of a broad spectrum of brush-border enzymes in the mucosa, including the disaccharidases, in contrast with the more specific patterns of loss observed in younger animals (148). In contrast, although ion transport in the kidney changes substantially with aging, it appears that mineralocorticoidsensitive, electrogenic sodium absorption in the rectosigmoid colon does not change substantially as humans age (149).

412 / CHAPTER 14 Intestinal mucosal L-DOPA and dopamine levels decrease in aging rats because of increased aromatic L-amino acid decarboxylase activity, but these changes do not correlate with inhibition of the sodium-potassium ATPase in the gut mucosa as clearly as they do in the aging kidney (150,151).

Immunologic Effects A substantial clinically significant effect of aging on small-bowel mucosal immunity has not been demonstrated, although two interesting articles have described an impairment of immunity to intraduodenal cholera toxin in older rats, and that orally administered kefir fermented milk enhances the intraduodenal immune response to cholera toxin in young but not senescent rats (152,153). Decreased specific antibody titers in intestinal lavage fluid from aging animals may correlate with compromised IgA immunoblast homing in the aging rat or monkey intestinal mucosa (152,154). Several more subtle deficiencies have also been described in animal models and humans. For instance, the repertoire of the mucosal gamma delta T cells, which constitute an important aspect of mucosal defense mechanisms, becomes increasingly restricted in aging humans and pigs, and it displays a striking variation along the length of the GI tract (155). Serum IgA decreases in humans with age (156). One investigator has suggested that lamina propria lymphocytes display an age-related loss of immune function (assessed by proliferation and interleukin-2 [IL-2] production) similar to peripheral blood lymphocytes, but intestinal epithelial lymphocytes appear to be spared from this decline with aging (156). Additional research is needed to elucidate the potential significance of such changes in immune function in aging humans. These alterations could also prove of greater significance in aging humans with clinical conditions directly or indirectly impairing other aspects of immune function.

of these cells also changes with aging in a complex and cell type-specific pattern (163). In general, proliferation of these cells does not seem to change, but apoptosis increases as the mice age (135). The density of murine colonic neuroendocrine cells immunoreactive for peptide YY (PYY), enteroglucagon, and serotonin displays a biphasic pattern, high in 1-month-old mice, decreased in 3-month-old mice, and then increasing again in 12- to 24-month-old mice (164). Although murine enteroendocrine cells resemble those of humans in many important ways (165), it should be noted that potentially relevant differences in the patterns of gut peptide appearance and change during development and aging are found when different species are compared. For instance, PYY cells increase with age in rodents, but this has not been demonstrated in humans (166). Human duodenal neuroendocrine cells have also been reported to vary with age in a complex fashion. For instance, gastrin/CCKimmunoreactive cells were found to be more numerous in 1- to 2-year-old and 60- to 69-year-old healthy humans than in 20- to 29-year-old humans, whereas somatostatinimmunoreactive cells were less common in 20- to 29-yearold humans than 40- to 49-year-old humans. The numbers of immunoreactive secretory granules within many of these cells was somewhat greater in 1- to 2-year-old than 20- to 29-year-old humans (167). Interestingly, aging also increased glutathione-S-transferase, but not glutathione peroxidase, superoxide dismutase, or lipid peroxidation within the small-bowel mucosa of aging Wistar rats. This pattern differs from that observed within the liver of these animals (168) and also seems to vary somewhat with the strain of rat studied (169), but its overall significance is not yet clear. Aging also alters the patterns in which brush-border enzyme expression alter in response to germ-free conditions (170), whereas small intestinal mucosal lipid esterification and absorption have been reported to appear altered in aging rats (171). In humans, most evidence suggests that lipid digestion and absorption are relatively preserved during aging (172). Thus, the significance of these interesting observations awaits further study.

Gut Peptides and Other Key Molecules Aging is correlated with complex changes in gut peptide levels (157). These have been best studied in the plasma, but they have also been characterized within bowel mucosa. Antral gastrin decreases in the aging rat mucosa (158,159), but neurotensin levels increase with age in the plasma, as well as in the ileal mucosa, of aging rats (160). Conversely, cholecystokinin (CCK) levels within the duodenal or jejunal mucosa increase with age in guinea pigs (161) and humans (162). Several investigators have characterized the density, morphology, proliferation, and apoptosis of neuroendocrine cells within the aging mouse duodenum, demonstrating a complex pattern in which some cells increase, whereas others decrease (135,163). For instance, cells immunoreactive for somatostatin decrease with age, whereas serotoninimmunoreactive cells may increase, and gastrin/CCKimmunoreactive cell density does not change. The morphology

Contractility and Motility Although numerous animal studies have addressed the effects of aging on GI tract motility, their relevance to human physiology and pathophysiology is not always clear. It seems a clinical truism that aging patients may complain more about bowel function. However, it is often difficult to distinguish the effects of aging on the gut per se from influences outside the gut, including altered states of health, diet, activity, and medication. In general, the effects of aging appear most pronounced at both ends, altering swallowing, esophageal contractility, and the mechanisms of fecal continence, whereas the effects of aging on midgut motility are more subtle and of less clear clinical significance. This section moves from the swallowing mechanism distally, considering each segment of the gut in turn.

EFFECT OF AGING ON THE GASTROINTESTINAL TRACT / 413 Constipation seems common in elderly patients in clinical practice (173). However, the mouth-to-anus transit time does not appear significantly prolonged in older individuals, despite slower gastric emptying (174). Indeed, one study comparing young with middle-aged subjects actually suggested that both gastric and small intestinal transit are accelerated in middle-aged women compared with transit in their younger counterparts (175). The issue is complex, and differences in sex, dietary habits, ethnicity, and other factors may all contribute to subtle differences among studies. In general, the effects of healthy aging on intestinal motility appear relatively slight (176), compared with other factors that may influence this issue and may become more prevalent with age. For instance, pelvic radiation to treat prostate cancer may affect anorectal function (177), and this certainly occurs more commonly among the elderly. A recent metaanalysis (178) suggests a disparity between subjective reports of constipation and laxative use in the elderly and objective clinical assessments of constipation. It seems likely that a subgroup of deconditioned elderly patients with other medical issues may indeed have increasing constipation, together with slow rectosigmoid transit (178), but it is unclear whether this reflects aging itself or stems from alterations in diet, fluid intake, exercise, medication use, or surgical procedures such as cholecystectomy that may occur in many elderly individuals. Cigarette smoking, perhaps more common among older patients in some societies, is also associated with slowing of GI transit (179) and should also be considered in such evaluations. At least one study has demonstrated an increase in mouthto-cecum transit in the elderly independent of the effect of cholecystectomy on transit time (180). Studies of small intestinal motility and transit suggest an increased frequency of propagated clustered contractions and a slowing in the propagation velocity of phase III of the migrating motor complex, but the clinical significance of such findings is unclear (181,182). In general, small-bowel transit appears clinically nonproblematic in most elderly individuals without complicating factors. Conversely, age does not predict significant differences in the small-bowel motility of patients with unexplained bowel symptoms (183). However, it should be acknowledged that mouth-to-anus transit time has been noted to be prolonged in elderly patients with small intestinal bacterial overgrowth (184). In this subgroup of patients, such bacterial overgrowth may be associated with clinical consequences such as malabsorption and malnutrition. The overgrowth is effectively managed by antibiotic therapy rather than intervention directed at small intestinal motility. Interestingly, the development of disabilities that substantially decrease physical activity may be associated with an increased risk for small intestinal bacterial overgrowth (185), although the precise causal linkage here awaits elucidation. In the colon, studies in rats suggest a decrease in colonic transit time in senescent (22- to 28-month-old) rats, which is associated with decreased contractility in response to cholinergic stimulation, possibly related to decreased calcium influx

via membrane calcium channels in the rat colon myenteric plexus (186). Another study of rat colonic histology suggests that the density of myenteric neurons decreases in the sigmoid colon as rats age. Interestingly, this decrease seems to have been aborted by serosal application of the neurotoxic agent benzalkonium chloride (187). Such animal observations have not yet been confirmed in healthy elderly humans, although an age-related decline in inhibitory junction potentials has been described in the descending human colon independent of any age-related variation in immunoreactivity for vasoactive intestinal peptide, peptide histidine-methionine, met5enkaphalin, neuropeptide Y, or somatostatin (188). One recent study found no difference in small-bowel transit time between young and elderly women, but it found a decrease in colonic fermentation in the elderly (189). Many factors could alter colonic fermentation, including differences in dietary fiber intake, colonic bacteriology, luminal mixing or transit, and medication use. The key regulatory factors that influence colonic fermentation are a subject of continuing study. However, although the mechanism of age-related differences in colonic fermentation awaits clarification, such differences in colonic fermentation could well alter subjective perceptions of colonic function in aging patients even without a direct effect on colonic transit per se. Constipation in the elderly may also be associated with outlet obstruction, particularly in women. Elderly women more commonly display abnormal perineal descent, perhaps because of the cumulative effects of pregnancy, as well as general aging and failure of the anorectal angle to open with defecation (190). Either may cause constipation. Constipation in the elderly has also been attributed to decreased rectal sensation. The threshold for sensing rectal distension appears increased in aged but healthy humans, independently of rectal compliance or tone (191). One interesting study, although not specifically directed at aging patients, compared electrical stimulation therapy with biofeedback in patients with impaired rectal sensation and otherwise idiopathic chronic constipation. (This represented about one fifth of all patients seen with functional constipation by this group.) The investigators responsible for this study found that either electrical stimulation or biofeedback therapy improved overall symptoms. However, biofeedback appeared to specifically improve anal residual pressures, whereas electrical stimulation specifically improved rectal sensory thresholds and the maximum tolerated volume in the rectum (192).

Mechanisms of Fecal Incontinence Although many elderly patients report symptoms of constipation, other older patients describe fecal incontinence. Fecal incontinence is often underreported because patients are embarrassed to mention this symptom (193), and yet it represents an important factor that limits activity and reduces quality of life in the elderly (194). Although fecal incontinence is more prevalent among younger women than younger men, this sex gap may be reduced with aging,

414 / CHAPTER 14 as other factors associated with incontinence become more prevalent (195). A sonographic study suggests that the internal anal sphincter thickens with age, but becomes less elastic, because of increased fibrosis, whereas the external sphincter becomes thinner, and anal canal resting and squeeze pressures and rectal reservoir function decrease (196). However, most elderly individuals who display such findings remain continent, and the magnitude of these changes does not necessarily predict incontinence (197). Thus, as for constipation, incontinence of stool is a complex problem, often as much associated with comorbidities or dietary changes as with the direct effects of aging on sphincter function or anorectal sensation. For instance, rectal hyposensitivity (198), possibly related to occult pelvic or spinal nerve injury, has been implicated as possibly causative in some patients with fecal incontinence, chronic constipation, or functional bowel disorders (199). Continence is generally linked to the external sphincter, and fatty atrophy of this sphincter may play a role in incontinence after external sphincter repair procedures (200). However, abnormalities of the internal anal sphincter have also been linked to incontinence in some patients, with higher thresholds required to initiate the rectoanal inhibitory reflex and progressive increases in internal anal sphincter relaxation (201). Despite the details of anorectal physiology that may appear in the literature, it should be emphasized clinically that decreased mental status, chronic constipation with fecal overflow, diabetic neuropathy, and altered ability to reach the toilet in a timely fashion may all predispose to fecal incontinence in the elderly without the incontinence being the direct result of aging. Thus, although sacral nerve stimulation (201–203), biofeedback, radiofrequency energy application (193), and various surgical procedures including anterior levatorplasty and anal sphincteroplasty (204,205) have been advocated for the management of fecal incontinence, primary therapy should first be directed at thorough geriatric assessment of the patient’s diet, stool habits, and interaction with the environment, with manipulation of fecal consistency as indicated.

Key Areas for Future Research Although the aging intestinal mucosa exhibits complex variations in absorptive and secretory capacity, this seems not to have substantial clinical consequences in the healthy aging human because of the large surface area available and corresponding excess capacity. An important subject for future study, however, may be the impact of such changes in disease states in which this excess capacity has been reduced. At the most simplistic level, for instance, the question of how such changes impact small or large intestinal absorption and secretion after major bowel resection may merit additional investigation. In addition, the Scylla and Charybdis of constipation and fecal incontinence in the aging represents a major clinical problem. Improved understanding of the molecular

mechanisms of colonic absorption and motility may ultimately permit the development of targeted therapeutic interventions. In addition, more basic research is required at the physiologic level to understand the muscular mechanisms of continence and incontinence in younger and aging humans.

AGING AND GASTROINTESTINAL CANCERS Epidemiologic Observations Although the incidence of most human malignancies including cancer of the GI tract increases dramatically with advancing age, the precise role of aging in that increase remains a matter of continued controversy and raises a number of questions. For example, is the aging process itself inherently carcinogenic? Does aging enhance carcinogenic susceptibility? Or, is an increased cancer incidence with time merely reflective of a protracted exposure to carcinogenic agents (206)? Numerous endogenous processes inherent to both aging and carcinogenesis implicate potentially shared derangements in the pathway to tumor formation. These include, but are not limited to, alterations in DNA methylation, protooncogene expression, chromosomal fragile sites, DNA mismatch repair, immune system dysfunction, and free radical reaction damage (207). This section discusses these issues with respect to aging and its relation to carcinogenesis of the GI tract, with particular reference to gastric and colorectal cancers, the two most common GI malignancies, which have incidences that increase with aging. Aging has been shown to be associated with increased incidence of premalignant lesions in the stomach and colon. Yamaji and colleagues (208) have studied the effect of age on the malignant potential of colorectal polyps. They have observed that the occurrence of nonmalignant and malignant colorectal neoplasms increases with advancing age, and that the polyps of older persons show greater malignant potential than younger subjects regardless of the size of the lesion. With respect to gastric cancer, atrophic gastritis and its associated intestinal metaplasia are age-related lesions and are considered to be precancerous (209). Aged individuals with atrophic gastritis and intestinal metaplasia are statistically at risk for development of gastric cancer, but precise surveillance recommendations have not been determined. In addition, it has been reported that patients who had partial gastrectomy 15 to 25 years earlier for peptic ulcer disease are at increased risk for gastric cancer (210). Jaszewski and coworkers (211) have analyzed time-dependent changes in ornithine decarboxylase (ODC) activity, an indicator of mucosal proliferation, in the gastric mucosa of subjects who underwent Billroth I or II gastrectomy. They report that ODC activity was significantly higher in Billroth II patients who underwent gastrectomy more than 15 years earlier compared with those in whom gastrectomy had been performed less than 15 years earlier or in normal control subjects (211). They subsequently examined the frequency of H. pylori in postgastrectomy remnants and noted a 58%

EFFECT OF AGING ON THE GASTROINTESTINAL TRACT / 415 frequency in Billroth II patients, which was associated with a more severe histologic gastritis (212). Atrophic gastritis and its associated intestinal metaplasia are age-related lesions and are considered to be precancerous (213). Baldini and colleagues (214) examined gastric biopsies from antrum and corpus of 117 healthy volunteers to determine a relation between age and H. pylori infection and risk for gastric cancer. They report that in the area with high gastric cancer rate, H. pylori infection was associated with atrophic gastritis and hyperproliferative state of the gastric mucosa, which were observed in both young and aged subjects, indicating a potential risk for development of gastric cancer (214). Ohkuma and colleagues (215) evaluated the effect of H. pylori infection and aging on atrophy and intestinal metaplasia of the gastric mucosa by examining 163 patients. Their results suggest that atrophic gastritis is not a normal aging process, but instead is likely to be the result of H. pylori infection, whereas intestinal metaplasia, a precancerous lesion, is associated with aging and H. pylori infection (215).

Epithelial Growth and Renewal as Potential Premalignant Events Many probable explanations for the age-related increase in cancer incidence have been offered including altered carcinogen metabolism and the cumulative effects of protracted exposure to cancer-causing agents. Neoplasia of the GI tract, including that of the stomach and colon, is a multistage process with hyperproliferation being central to the initiation of carcinogenesis. In view of this, this chapter discusses the growth-related processes of the mucosa of different parts of the GI tract during aging and the regulation of these processes. Cells of the GI tract mucosa are subject to a constant process of renewal, which in healthy adults reflects a balance

between proliferation of precursor cells and exfoliation of surface cells (216–218). The mucosa of the GI tract actually has one of the most rapid cell turnover rates of any tissue in the body. The continuous cell renewal is maintained by the sustained proliferative activity of a small number of mucosal stem cells (216,217,219). The proliferative zone is composed of actively dividing cells distinct from the stem cell, whereas in a larger nonproliferative zone, cells are undergoing progressive differentiation before being extruded into the lumen. Cell production not only compensates for cellular shedding from the surface epithelium, but also assures the renewal of certain specialized cells in the glandular tubes. Any dysfunction in this process may accelerate or diminish the growth rate, resulting in either hyperplasia/hypertrophy or atrophy of the organ, respectively. Knowledge of normal cell proliferation kinetics and regulation of GI mucosal growth during aging is, therefore, essential for the understanding of the pathogenesis of many GI mucosal diseases and disorders linked to aging including cancer. Although earlier observations in the mouse suggest that proliferative activity of the small intestine either decreases (220,221) or remains unchanged (222) with aging, some morphologic and biochemical studies from our and other laboratories have demonstrated that, in barrier-reared Fischer-344 rats, aging is associated with increased mucosal proliferative activity in the stomach and small and large intestines (53,132, 136,223–228). Representative figures (Figs. 14-1 and 14-2) show the age-related increase in proliferative activity in the mucosa of the stomach and different regions of the colon of Fischer-344 rats, as evidenced by increased immunoreactive PCNA. The age-related increase in mucosal proliferative activity, at least in the colon, is also found to be accompanied by an increase in the proliferative zone, a phenomenon that has been observed in patients with precancerous conditions, as well as in subjects with cancer of the colon, rectum, or both. For example, we have reported a marked increase in the proliferative zone of the disease-free colonic mucosa

Gastric mucosa 4 Mo

24 Mo

FIG. 14-1. Photomicrograph showing changes in gastric mucosal proliferative activity, as assessed by bromodeoxyuridine immunoreactivity, in 4- and 24-month-old Fischer-344 rats. (Reproduced from Majumdar and Jaszewski [235], by permission.)

416 / CHAPTER 14 COLON Proximal

Middle

Distal

4 mo

13 mo

22 mo

FIG. 14-2. Photomicrograph showing changes in mucosal proliferative activity in the proximal, middle, and distal colon of 4-, 13- and 22-month-old rats. (Reproduced from Xiao and colleagues [136], by permission.)

from subjects with adenomatous polyps, ulcerative colitis, and colorectal cancer, compared with control subjects (229). In experimental animals, administration of azoxymethane, a colonic carcinogen, has also been shown to cause widening of the proliferative zone of the colonic crypts (230,231). Morphologic studies of the colonic mucosa of human volunteers have further shown that, whereas cell proliferation in young subjects is confined to the lower two thirds of the crypt, with aging there is a major shift from the base to the middle and upper third of the gland (232–234). The observations of the age-related increase in mucosal proliferative activity, together with expansion of the proliferative zone in the colon, both of which are common occurrences in precancerous conditions and in cancer, have led us and others to postulate that aging may predispose the GI tract to neoplasia (132,224,228,235,236).

Inhibition of Apoptosis as a Potential Premalignant Event In addition to increasing proliferation of the mucosa of much of the GI tract, alterations in mucosal apoptosis (programmed cell death) may also contribute to age-related increases in predisposition of the tissues to carcinogenesis. Alterations in apoptosis are now recognized to be an important event in development, in normal cell turnover, in hormone-induced tissue atrophy, and in many pathologic

conditions including cancer (237–239). It has been demonstrated that cells derived from a variety of cancers, including colorectal cancer, show decreased apoptosis in response to physiologic stimuli (237,240,241). Additional evidence supporting the involvement of apoptosis in the development of colorectal neoplasia comes from the observations that transformation of colorectal epithelium to carcinoma is associated with progressive inhibition of apoptosis (240). Little information is available on age-related changes in apoptosis in tissues of the GI tract. To determine whether changes in apoptosis could contribute to the age-related increase in the incidence of gastric or colorectal carcinomas, or both, we examined the number of apoptotic cells in the gastric and colonic mucosa by TUNEL assay. We have observed that the number of apoptotic cells in the mucosa of the stomach and all regions of the colon of 22- to 24-month-old (aged) Fischer-344 rats that fasted overnight is lower than in the corresponding tissues from 4-month-old (young) counterparts (Table 14-1). Further support for the age-related decrease in apoptosis in GI mucosa comes from the observations that cleavage of 115-kDa full-length poly(ADP-ribose) polymerase, a substrate for caspase-3 and probably for other caspases, to the 85-kDa product was decreased by colonic mucosal lysates from 13- and 22-month-old Fischer-344 rats compared with their 4-month-old counterparts (136). These changes were accompanied by a concomitant reduction in the levels of 20-kDa active caspase-3 and -8, as well as 10-kDa active caspase-9 (136). To further determine the intracellular

EFFECT OF AGING ON THE GASTROINTESTINAL TRACT / 417 TABLE 14-1. Changes in the number of apoptotic cells, as assessed by TUNEL assay, in the mucosa of the stomach and different regions of the colon of overnight-fasted 4- to 5-, 12- to 14-, and 22- to 24-month-old Fischer-344 rats Number of apoptotic cells per mm2 Tissue Stomach Colon Proximal Middle Distal

4–5 mo

12–14 mo

22–24 mo

15.2 ± 1.2

11.2 ± 1.5

10.6 ± 1.2a

8.0 ± 0.6 10.2 ± 0.8 8.2 ± 1.3

7.4 ± 1.1 7.3 ± 1.8 5.3 ± 0.6

6.7 ± 0.5a 5.3 ± 0.7a 4.9 ± 1.0a

Values represent mean ± standard error of the mean. ap < 0.01, compared with the corresponding values in 4- to 5-month-old rats.

events regulating the apoptotic processes in the colonic mucosa during aging, the levels of Bak, the induction of which promotes apoptosis, and BclxL, which promotes cell survival, were also examined in the colonic mucosa of 4- to 12- and 22-month-old Fischer-344 rats that fasted overnight. Whereas the levels of Bak were found to be about 50% lower, Bcl-xL levels were increased by about 50% in 22-month-old rats compared with 4- or 12-month-old animals (136). Lee and colleagues (242) have examined age-related changes in colonic mucosal apoptosis in Fischer-344 rats. In contrast with what we reported, immunohistochemical studies conducted by Lee and colleagues (242) show only a few apoptotic bodies in the colonic with no significant difference between young and aged rats. Although the discrepancy between the two studies is not fully understood, one possibility could be the nutritional status of the animals. Whereas Lee and colleagues (242) killed the animals in fed conditions, in our studies, the animals fasted for 24 hours to minimize the effect of food-induced stimulation of apoptosis. In fact, Lee and colleagues (242) report that the expression of Bax protein, which promotes apoptosis, is higher in the upper part of the colonic crypt of aged rats than in their younger counterparts, which they suggest increases the sensitivity of the colonic mucosa to apoptotic stimuli. However, in view of our observation of age-related decrease in apoptosis in the colonic mucosa, it is tempting to speculate that aberrant survival of a small group of cells or even a single cell in the colonic mucosa during aging could promote accumulation of secondary genetic changes, and thereby enhance susceptibility of the tissue to certain carcinogen(s) or tumor promoter(s). Although reduction of apoptosis was more modest in the gastric mucosa, that the marked increase in proliferative activity in the gastric mucosa in aged rats was not accompanied by a parallel increase in apoptosis suggests that aging may also promote accumulation of cells with secondary genetic changes in the gastric mucosa.

Changes in Susceptibility to Carcinogens Although many probable reasons have been suggested for the age-related increase in malignancies, including altered

carcinogen metabolism and cumulative effects of long-term exposure to cancer-causing agents (243–245), the possibility that aging may render target tissue(s) more susceptible to carcinogen(s) or tumor promoter(s), or both, has not been thoroughly investigated, and the results remain inconclusive in the studies that have been performed (246–248). Magnuson and colleagues (249) compared the susceptibility of the colon to azoxymethane, a colonic carcinogen, in 1- (young) and 12-month-old (old) Sprague–Dawley rats by examining the formation of aberrant crypt foci (ACF). Aberrant crypt foci, which were initially described by Bird (250) as lesions consisting of large, thick crypt in methylene blue–stained specimens of azoxymethane-treated mice, have since been detected in humans with colon cancer, as well as in benign adenomas, which are believed to be the precursors of colorectal cancer (251–254). Several investigators have demonstrated that patients with colon adenomas or cancer have significantly more ACF than control subjects (253). In addition, ACF showed 60% to 70% K-ras mutations (254–257). These and other observations have led to the postulation that ACF are neoplastic and likely are precursors of adenomas and carcinomas (258,259). In the azoxymethane model of colorectal cancer in mice, azoxymethane has been shown to produce a single to multiple aggregates of ACF, leading to the development of colonic adenocarcinomas (252,257). Magnuson and colleagues describe results that show a significantly greater number of ACF in older rats both at 6 and 14 weeks after azoxymethane treatment when compared with their corresponding younger counterparts (249). Moreover, they report that an increase in the number of ACF in older animals occurred in each size category and was not restricted to the small ACF only (249). A similar observation was also made by us in Fischer-344 rats, when 4- (young) and 24-month-old (aged) rats were treated with azoxymethane for 4 weeks to induce ACF formation in the colon (260). We observed that the number of ACF formed in aged Fischer-344 rats in response to azoxymethane treatment was markedly greater than in their 4-month-old counterparts (260). To further evaluate the age-related increase in susceptibility of the GI tract to cancer-causing agent(s), we examined the early events of colorectal neoplasia by measuring changes in proliferative activity in the colonic mucosa of young (4-month-old) and aged (24-month-old) Fischer-344 rats 5 days after a single injection of azoxymethane or vehicle (control animals). Morphometric analysis revealed the number of bromodeoxyuridine (BrdU)-immunoreactive cells (a measure of proliferative activity) per colonic crypt of control aged rats to be about 40% greater than in their younger counterparts (8.63 ± 2.3 in young control animals vs 12.3 ± 1.9 in aged rats; p < 0.025), indicating a higher basal proliferative activity in the colonic mucosa of aged rats (Fig. 14-3). Although azoxymethane markedly stimulated the number of BrdU-positive cells in both age groups, the magnitude of stimulation was found to be greater in aged than in young rats when compared with the corresponding control animals (see Fig. 14-3). In young rats, the number of BrdU-positive cells were increased by about 220% to an average of 28.2 ± 3.5 cells/crypt, whereas in aged rats, it was

418 / CHAPTER 14

A

B 4-mo Cont.

C 4-mo AOM

D 24-mo Cont.

24-mo AOM

FIG. 14-3. Photomicrograph showing bromodeoxyuridine-immunoreactive cells in the colonic mucosa of young (4-month-old) and aged (24-month-old) Fischer-344 rats 5 days after a single subcutaneous injection of the colonic carcinogen azoxymethane (AOM; 20 mg/kg).

increased by about 300% to an average of 49.8 ± 2.3 cells/ crypt when compared with the corresponding control animals. In addition, azoxymethane produced a change in distribution of BrdU-positive cells in the colonic mucosa. Whereas in young rats, most of the BrdU-positive cells were located at the bottom half of the crypt, in aged rats, they were distributed throughout the crypt with the exception of 10% to 20% of the upper region (see Fig. 14-3). The latter is similar to what we noted earlier in the colonic mucosa of patients with adenomatous polyps and ulcerative colitis (229), conditions that are considered precancerous (218). To further explore the age-related susceptibility of the colonic mucosa to carcinogens, we examined the in vitro changes in ODC and tyrosine kinase activities in the colonic mucosa in response to methylazoxymethanol, the active metabolite of the colonic carcinogen azoxymethane. We observed a greater degree of stimulation of ODC and tyrosine kinase activities in aged than in young colonic mucosa in response to methylazoxymethanol, the active component of azoxymethane (261), indicating an increased susceptibility of the colonic mucosa of aged rats to this carcinogen. A similar phenomenon was observed with TGF-α (262), a potent mitogen for mucosa of various parts of the GI tract including the colon (263,264). A growing body of evidence also suggests that TGF-α may play a critical role in the development of colorectal neoplasia through autocrine/ paracrine mechanisms. This is supported by the observation that cell lines derived from adenocarcinomas of various tissues, including the colon, show increased expression of TGF-α and its receptor, EGFR (263–269). Because TGF-α exerts its mitogenic action by activating the intrinsic tyrosine kinase activity of EGFR, we compared the changes in TGF-α-mediated activation of EGFR-associated tyrosine kinase between young and aged rats (262). Despite a higher basal EGFR tyrosine kinase activity level in the colonic mucosa of aged rats, TGF-α produced a significantly greater stimulation of EGFR tyrosine kinase activity and tyrosine phosphorylation of EGFR and several other mucosal proteins.

Because this occurred despite a relatively high basal EGFR tyrosine kinase in aged rats, we suggest that aging is associated with increased responsiveness of the colonic mucosa to TGF-α (262). A similar observation was also made with isolated colonocytes from azoxymethane-treated rats. Both EGF and TGF-α caused a comparatively greater degree of stimulation of EGFR tyrosine kinase in colonocytes isolated from rats 5 days after a single injection of azoxymethane than those from control animals (231). Taken together, these alterations in GI mucosal proliferation and differentiation strongly suggest that aging is associated with increased susceptibility of the GI tract not only to carcinogen, but also to certain growth factors, particularly the EGF family of peptides. These factors may play a role in malignant transformation of the GI tissues during aging.

Mutational Events Carcinogenesis, which is a multistep process, results from accumulation of mutations during progression from normal epithelium to carcinoma (270). Genetic changes that occur at different stages of epithelial cell carcinoma have been extensively studied by Fearon and Vogelstein in human colon cancer (270) and have been reviewed by others. However, at least for colon cancer, it has been suggested that the loss or inactivation of the tumor-suppressor gene in adenomatous polyposis coli (APC) initiates genomic instability that may produce the phenotypic appearance of an adenoma. Advanced tumors, however, possess mutation or deletion, or both, of a number of oncogenes and tumor-suppressor genes not seen early adenoma (270). Although such detailed analysis of genetic alterations has not been performed for gastric cancer, inactivation of several tumor-suppressor genes, including APC, p53, and deleted in colorectal cancer (DCC), has been observed in gastric cancer (271). However, little information is available as to whether aging, which is thought to predispose the GI tract to carcinogenesis, is associated with

EFFECT OF AGING ON THE GASTROINTESTINAL TRACT / 419 increased inactivation of tumor-suppressor genes. If aging predisposes the GI tract to carcinogenesis, we postulate that aging may be associated with altered expression or mutations of genes involved in cell growth, or both. To test this hypothesis, we investigated the age-associated changes in mutations of APC, DCC, p53, and K-ras genes in the gastric mucosa of 19 healthy volunteers of varying ages (25–91 years) (272). Specifically, we studied the loss of heterozygosity (LOH) of these genes in the gastric mucosa. We observed that 3 of 19 subjects (16%) older than 60 years showed LOH of at least one of the tumor-suppressor genes. Among the subjects older than 60 years, the incidence of LOH was 38%, and two thirds of the subjects had mutations in more than one tumorsuppressor genes (272). In addition, we noted that in all three affected subjects, mutations in APC, DCC, or p53 were located in the body of the stomach, suggesting increasing susceptibility of this region to neoplastic changes (272). To further determine a link between genetic alterations and GI cancers during aging, Hastle and coworkers (273) examined telomere length in relation to age and colon adenomas. Telomeres are believed to be critical in the maintenance of chromosomal integrity and are composed of a sequence of six nucleotides (TTAGGG) that is repeated from several hundred to a thousand times (273,274). These sequences are synthesized by telomerase, a ribonucleoprotein enzyme composed of both protein and RNA. Telomerase makes a DNA copy of its own RNA sequence by reverse transcription. The extension of telomeres by telomerase is necessary to offset the normal shrinkage of chromosomes that occurs after each DNA replication. However, the telomeres shorten progressively with aging in somatic cells. In contrast, immortalized and some carcinoma cells show maintenance of telomere length despite repeated cell division due to the activity of telomerase (275). Hastle and colleagues (273) note a reduction in telomere length in normal colonic tissue of older compared with their younger individuals. Length reductions were also seen in colon adenomas and carcinomas. These authors hypothesize that telomere loss may render chromosomes less stable and impair viability. Alternatively, they may make chromosomes more prone to fusion bridge breakage cycles, leading to daughter cells that receive partly deleted and duplicated chromosomes. These alterations may have a role in generating loss of alleles of restriction fragment length polymorphisms that often occur in colorectal cancers (274). In a similar study, Furugori and colleagues (276) examined the telomere length in nonneoplastic gastric mucosal specimens from 38 human subjects aged between 0 and 99 years. They report an average annual loss rate of 46 base pairs. Telomere lengths in gastric carcinomas were also shorter than those for intestinal metaplasia (276). Issa and colleagues (277) studied the estrogen receptor (ER) in relation to aging and colorectal cancer. The ER may actually serve as a tumor-suppressor gene in that it is expressed in normal tissue, is altered in tumors, and suppresses growth in tumor cell lines and tissues. CpG islands located in the promoter region of genes are normally free of methylation, but methylation is common in islands of the ER in aging,

in colonic mucosa, and in colon tumors (277). Moreover, methylation of the ER in older healthy subjects or those with colon cancer is associated with inactivation of the gene. Folic acid is the primary methyl donor for DNA methylation and for producing the purines and pyrimidines required for DNA synthesis. Lack of folate or methyl group nutrients in the diet has been shown to cause DNA hypomethylation in both rats and humans, whereas an opposite phenomenon is noted with excess folic acid (278–280). Crott and coworkers (281) have studied the effects of dietary folate and aging on gene expression in the colonic mucosa of rats, and its implications in colon cancer. Gene expression was measured using oligonucleotide arrays. The authors observed that in folate-deficient rats, aging induced the down-regulation of immune-related genes, urokinase, p53, insulin-like growth factor binding protein-3 (IGF-BP3), and vav-1 oncogene, whereas folate supplementation caused down-regulation of vascular endothelial growth factor and caspase-2. They suggest that folate depletion with aging may increase the risk for development of colon cancer, and that folate supplementation may reduce the risk for age-associated cancers by suppressing deleterious changes in the expression of certain genes. Hypermethylation has been shown to cause functional inactivation of a number of tumor-suppressor genes (282). In some cases, CpG islands in the 5′ regions of tumorsuppressor genes are methylated, and their expressions are switched off. For example, in the breast cancer cell line MDA-MB-435, methylation of the promoter of connexin 26 has been shown to be associated with a concomitant loss of its expression (283). Hypermethylation has also been shown to cause functional inactivation of p16INK4A (284). In Fischer-344 rats, aging has been shown to be associated with increased expression and activation of EGFR in the GI mucosa (285,286), a phenomenon that has also been observed in preneoplastic and neoplastic lesions (229,230, 263,269). Results from several laboratories, including our own, have demonstrated that folic acid could be chemopreventive (287–290), and that this could be attributable, in part, to inhibition of EGFR activation (291). Indeed, we observed that supplemental folic acid and its metabolite 5-methyltetrahydrofolate greatly attenuated serum-induced activation of EGFR promoter activity in colon cancer cells in vitro (292). Supplemental 5-methyltetrahydrofolate also attenuated the basal EGFR promoter activity in colon cancer cells in vitro (292). However, whether the EGFR promoter function in the GI tissues during aging would also be similarly affected by supplemental folic acid or its metabolite(s) remains to be determined.

Key Areas for Future Research It is obvious that aging is associated with increased incidence of both premalignant and malignant gastric and colonic lesions. Interestingly, there is little evidence that the small intestine is similarly affected. Investigation of the disparate mechanisms of small intestinal malignant transformation

420 / CHAPTER 14 and colonic malignant transformation may therefore provide some clues as to why gastric and colonic cancers increase in the aging population. In addition, further research into the general biology of such tumors obviously is required. Increasing evidence suggests that the aging colonic mucosa is more susceptible to chemical carcinogens, but the mechanism of this effect is unknown. The changes in bacterial flora within the colonic lumen during aging are poorly characterized and may also contribute to the development of colonic cancer in the elderly. Finally, although somewhat outside the scope of basic physiology, it has become increasingly obvious that the real key to curing colon cancer is prevention. Thus, research into chemoprevention and improved screening modalities in high-risk elderly patients clearly is warranted.

REGULATION OF MUCOSAL GROWTH DURING AGING Intracellular Mechanisms Although aging, as described in the previous section, is associated with increased proliferation and decreased apoptosis in much of the GI tract, the intracellular events responsible for regulating these processes are poorly understood. With respect to the age-related increase in GI mucosal proliferative activity, however, it has been demonstrated that in Fischer-344 rats, aging is associated with a progressive increase in activity and protein levels of cyclin-dependent kinase (cdk2) in the gastric and colonic mucosa (137,293). Cyclin-dependent kinases are a highly conserved family of serine/threonine protein kinases that phosphorylates several molecules involved in cell cycle progression. Binding of cyclins to Cdks activates their intrinsic kinase activity, triggering events that induce progression of cells through different phases of the cell cycle (294,295). Moreover, activation of Cdks depends on the binding of a specific cyclin (295). For example, whereas Cdk4 and Cdk6 require D-type cyclins for their activation (296), Cdk2, which is involved in G1/S transition, is activated by cyclin E (297,298). In addition, Cdks are also regulated by a class of Cdk inhibitors. Of these, p21Waf1/Cip1 and p27kip1, which have been studied extensively, are considered universal inhibitors of different cyclin-Cdk complexes (299,300). The age-related increase in gastric and colonic mucosa was accompanied by a concomitant increase in cyclin D1 levels and reduction of p21Waf1/Cip1 (137,293). In addition, it was observed that the levels of phosphorylated retinoblastoma, a form that is involved in regulating progression of cells through the S-phase, are greater in both gastric and colonic mucosa of aged compared with young rats (137,293). Taken together, these results suggest that aging is associated with increased transition of G1- to S-phase, as well as progression through the S-phase of the cell cycle in both gastric and colonic mucosa.

Nutritional and Hormonal Factors Gastrointestinal mucosal cell proliferation is known to be under the regulation of a number of nutritional and hormonal factors. In general, deprivation of food results in decreased cell proliferation, and refeeding reverses the situation (301). Holt and Yeh (224,226) have compared the effect of fasting and refeeding on small and large intestinal proliferative activity in young (3- to 4-month-old) and aged (24- to 28month-old) Fischer-344 rats. In the mucosa of both small and large intestines, a 3-day fasting resulted in 40% to 60% reduction in proliferative activity in young rats, whereas it was decreased by only 10% to 20% in old rats when compared with their corresponding fed control animals (224,226). Although in both age groups refeeding increased small intestinal proliferative activity, in aged rats, this increase was associated with broadening of the proliferative zone (224,226). Moreover, in fasted aged rats, proliferative responsiveness of the large intestine to food was blunted (223–225). The latter observation suggests that nutritional modulation of mucosal cell proliferation is affected by aging. Age-associated changes in GI mucosal cell proliferation could be secondary to alterations in hormonal influences. Over the past few decades considerable evidence has appeared to show that a number of GI hormones/growth factors, specifically gastrin, bombesin (an amphibian peptide that is structurally and functionally analogous to gastrin-releasing peptide), and the EGF family of peptides regulate mucosal proliferation in much of the GI tract, including the stomach (235,302). However, responsiveness of the gastric mucosa to these peptides changes at different stages of life. We have reported earlier that in rats, the gastric mucosa becomes responsive to the growth-promoting action of gastrin around the third postnatal week of life (303). At this time, the parietal cells also become sensitive to gastrin and secrete acid in response to the hormone, when gastrin receptors also appear (304). In contrast, the functional properties are either decreased or remain unchanged during advancing age. For example, basal gastric acid and pepsin output decline during aging, as does gastrin secretion (54,158). In contrast, antral gastrin levels increase during this period, as does mucosal histidine decarboxylase activity (54,158). The age-related decline in gastrin secretion could be attributed partly to a higher ratio of somatostatin (D) to gastrin (G) cells in the antral mucosa (158). With aging there is also a progressive loss of gastric mucosal responsiveness to both acid secretory (54) and growth-promoting (158) actions of gastrin. Although the underlying mechanisms are unknown, one possibility could be the loss of functional receptors of gastrin. Singh and colleagues (305) report that the number of gastrin binding sites in the gastric mucosa of 24-month-old rats was lower than in their 3- and 6-month-old counterparts. Taken together, the results suggest a relation between gastrin receptor populations and gastric mucosal responsiveness to gastrin. Like gastrin, bombesin has also been shown to stimulate gastric mucosal cell proliferation in young animals (306,307).

EFFECT OF AGING ON THE GASTROINTESTINAL TRACT / 421 To evaluate its role in GI mucosal cell proliferation during aging, we examined changes in gastric mucosal DNA synthesis and ODC activity in young (4-month-old) and aged (24-month-old) rats after continuous infusion of bombesin (300 ng/kg/h) for 2 weeks. We observed that whereas in 4-month-old rats bombesin infusion resulted in a significant twofold increase in gastric mucosal DNA synthesis and ODC activity, in aged rats, the peptide had no effect (308). Whether the lack of response of aged gastric mucosa to the growth-promoting effect of bombesin could be partly the result of loss of receptors of the peptide is unknown.

Growth Factors and Protein Kinases Epidermal growth factor family of peptides, specifically EGF and TGF-α, are known to regulate GI mucosal cell proliferation (235,263,264). Feldman and Aures (309) were the first to demonstrate that administration of EGF to suckling mice stimulates gastric and duodenal mucosal ODC activity. Subsequently, Malo and Menard (310) reported that EGF not only stimulates growth of the proximal, but also the distal small intestine. Similar observations were also made by us and other investigators (311–314). Using an organ culture system, we have demonstrated that EGF stimulates protein and DNA synthesis in both prenatal and neonatal rats (311) and ODC activity in the colonic mucosa of young mature rats (312). This observation not only indicates a direct growth-promoting effect of EGF on both small and large intestinal mucosa, but also shows that EGF may have a role in regulating mucosal growth from before birth to adulthood. Gastric mucosa also responds to the growthpromoting action of EGF. Dembinski and Johnson (314) report that in 10-day-old suckling rats, repeated injections of EGF for 5 days result in a significant increase in gastric mucosal DNA, RNA, and protein content. We have further demonstrated that in unweaned rats, daily injections of EGF at a dose of 20 µg/kg for 7 days significantly stimulate both gastric and small intestinal protein and nucleic acid content (110). Repeated injections of EGF for 2 days have also been shown to stimulate gastric mucosal DNA synthesis in young mature rats (111,315). More recently, we examined the responsiveness of gastric mucosa of 4- and 24-monthold Fischer-344 rats to TGF-α, the structural and functional analogue of EGF (315). We observed that pharmacologic doses of TGF-α, which stimulated gastric mucosal proliferative activity in 4-month-old rats, caused a marked inhibition of the same in their 24-month-old counterparts (315). We postulated that this inhibition is, in part, due to increased sensitivity of aged gastric mucosa to these peptides such that low doses of these peptides are stimulatory, whereas high doses inhibit proliferative processes (111). In support of this postulation, we have observed that the concentration of TGF-α needed to induce maximal stimulation in EGFR tyrosine kinase activity in gastric mucosal membrane preparations from aged rats is considerably less than that required

for the same induction in young rats (111). Likewise, Evers and colleagues (316) observed that whereas administration of neurotensin, a peptide present in the GI mucosa, increased the crypt cell density in the intestine of both young and aged rats, in aged rats, the peptide enhanced an increase in crypt depth and villous height. This observation is similar to observations after refeeding of rats that had been fasted for 72 hours (224,225,317) and after administration of the carcinogen dimethylhydrazine (318). In evaluating the age-related increase in GI mucosal proliferative activity, we have focused on the role of tyrosine kinases, which catalyze phosphorylation of tyrosine residues in proteins and play a crucial role in regulating proliferation, differentiation, and transformation of cells (319,320). The notion that tyrosine kinases may play a role in regulating GI mucosal cell proliferation comes from our earlier observation that the age-related increase in gastric mucosal proliferative activity is also accompanied by a parallel increase in overall mucosal tyrosine kinase activity and tyrosine phosphorylation of several membrane proteins (53,227). However, tyrosine kinases are associated with the products of many protooncogenes, as well as with receptors of several growth factors, including the EGFR, the common receptor for EGF and TGF-α (319,320), which is a 170-kDa transmembrane glycoprotein that spans the plasma membrane. It contains an amino-terminal extracellular ligand-binding domain, a single-transmembrane–anchoring region, and a carboxyl-terminal intracellular domain that has tyrosine kinase activity (105). High-affinity binding sites for the EGF family of peptides, specifically EGF and TGF-α, have been detected in much of the GI tract, with the greatest concentrations in the esophagus, stomach, and colon (321–324). Moreover, mRNA for TGF-α, as well as its receptor, has been detected in gastric and colonic mucosal cells (324–327). Overexpression of EGFR with increased tyrosine kinase activity has been associated with many human malignancies, including gastric and colonic cancers (264,328,329). Results from our laboratory have demonstrated that activation of EGFR tyrosine kinase is associated with increased GI mucosal proliferative activity. We have reported that increased mucosal proliferative activity in the colon in response to azoxymethane (a colonic carcinogen) and in the gastric mucosa after injury is accompanied by a concomitant increase in EGFR tyrosine kinase activity, and that these increases can be greatly attenuated by prior administration of tyrphostin, an inhibitor of EGFR tyrosine kinase (230,330). These and other relevant observations suggest a role for EGFR tyrosine kinase in GI mucosal cell proliferation. We have reported that an age-related increase in gastric and colonic mucosal cell proliferation is accompanied by a marked increase in expression and activation of several tyrosine kinases, including EGFR and its family member ErbB-2/HER-2 (262,285,331). Aging is also found to be associated with activation of EGFR in the gastric and colonic mucosa, as evidenced by increased tyrosine phosphorylation of EGFR (286). Ligand binding is one of the primary causes

422 / CHAPTER 14 for activation of intrinsic tyrosine kinase activity of EGFR, triggering a complex array of enzymatic and biological events leading to stimulation in cell proliferation. There is considerable evidence that shows that TGF-α, which is synthesized in mucosa of much of the GI tract including the stomach and colon, may play a key role in regulating EGFR tyrosine kinase activity in different pathophysiologic conditions. For example, cell lines derived from adenocarcinomas of the colon express both TGF-α and EGFR (264,328,329). Our observation that the azoxymethane-induced colonic mucosal EGFR tyrosine kinase activity is accompanied by a parallel increase in synthesis and secretion of TGF-α (230) suggests that TGF-α may be partly responsible for regulating this process through an autocrine/paracrine mechanism. However, results from cell-surface and biochemical studies have demonstrated that the presence of the transmembrane TGF-α is a normal consequence of TGF-α synthesis, and in most cases, the peptide is present on the cell surface in its precursor form, which can also activate EGFR (332–334). We have observed that the age-related increase in EGFR tyrosine kinase in the gastric mucosa is accompanied by a marked increase in 16- to 20-kDa precursor forms of TGF-α in the mucosal membrane fraction, but not in the cytosol (285). The colonic mucosa also shows a similar phenomenon (286). In addition, our recent in vitro studies show that the membrane-bound precursor form(s) of TGF-α from the gastric and colonic mucosa of aged rats induces a greater stimulation in EGFR tyrosine kinase activity than those from young rats (286). This could be attributed partly to a greater amount of TGF-α bound to its receptor, the EGFR (286). However, because TGF-α, but not EGF, is synthesized in much of the GI mucosa of normal adult rats (230,332,335), we postulate that TGF-α plays a key role in regulating gastric and colonic mucosa proliferation during aging, probably through an autocrine/ paracrine mechanism. Results from our laboratory also suggest that aging enhances sensitivity of the GI mucosa to both EGF and TGF-α such that low doses of the peptide stimulate the EGFR signaling cascades leading to stimulation in proliferation, whereas high doses inhibit these processes. This inhibition is found to be caused by enhanced internalization of the ligand-receptor complex (118). Likewise, responsiveness of the colonic mucosa to both EGF and TGF-α is also augmented after azoxymethane treatment (230,231). Activation of EGFR triggers a complex array of enzymatic events through activation of Ras, converging on mitogenactivated protein kinases, which after translocation to the nucleus activate transcription factors and induce activation of genes that promote growth (336–338). It has been demonstrated that with aging there is a marked activation of extracellular-signal regulated kinases and c-Jun NH2-terminal/ stress-activated kinases in the gastric mucosa of Fischer-344 rats, and that these changes are accompanied by a concomitant stimulation of the gastric mucosal DNA-binding activity of activator protein-1 (AP-1) and nuclear factor-κB (NF-κB) (339). In addition, it has been demonstrated that aging is associated with a marked induction of TGF-α-induced activation of AP-1 in the gastric mucosa (340). Because activation of

AP-1 and NF-κB is thought to result in induction of proliferation and inhibition of apoptosis (341,342), the age-related induction of transcriptional activity of AP-1 and NF-κB in the gastric mucosa suggests that they might be involved in regulating GI mucosal growth during aging. Although the precise regulatory mechanisms for the age-related increase in EGFR activation in the mucosa of the GI tract is unknown, our data suggest this could be partly the result of loss of ERRP (EGFR-related peptide), a novel “negative regulator,” which we have identified and characterized (343). Epidermal growth factor receptor–related peptide, a ~55-kDa secretory protein, possesses 85% to 90% homology to the external ligand binding domain of EGFR and 50% to 60% homology to the external domain of other members of the EGFR family (343). Expression of ERRP is high in benign human colon, stomach, liver, and pancreas but low in the respective invasive adenocarcinomas (343–348). We report that ERRP inhibits cellular growth by attenuating EGFR signaling processes (343,344). Overexpression of ERRP in colon cancer cell lines, HCT-116 and Caco2, or exposure of the cell lines to recombinant ERRP inhibits EGFR activation, as well as proliferation (343,344). In addition, intratumoral or subcutaneous injections of recombinant ERRP cause regression of palpable tumors in SCID mice xenograft of colon cancer cells (343). Because aging and carcinogenesis of GI tissues are associated with increased expression and activation of EGFR, we hypothesize that these increases could be partly the result of loss of ERRP, which we believe to be a suppressor of EGFR function. To test this hypothesis, we examined the relation between age-related changes in EGFR activation and expression of ERRP in the gastric mucosa of rats. Indeed, we observed that in the gastric mucosa of Fischer-344 rats, the age-related increase in EGFR activation was associated with marked reduction in ERRP levels (345). The same phenomenon was also noted in the colon, where the levels of ERRP in both the proximal and distal colon of the Fischer-344 rats were found to be lower in aged (21-month-old) than in young (6-month-old) animals (260). The age-related decrease in ERRP expression in the colonic mucosa was further decreased by administration of the colonic carcinogen azoxymethane (260). Because ERRP possesses a substantial homology to the ligand-binding extracellular domain of EGFR, we hypothesized that ERRP may compete with EGFR for the ligands. To test this hypothesis, we analyzed the amount of TGF-α bound to EGFR and ERRP in the gastric mucosa of 6- and 24-month-old Fischer-344 rats. We observed the amount of TGF-α bound to ERRP was about 50% less in the gastric mucosa of aged than in young rats (345). In contrast, the amount of TGF-α bound to EGFR in the gastric mucosa of aged rats was found to be about 70% greater than in young rats (345). These changes were accompanied by parallel alterations in EGFR and ERRP levels (345). We suggested that the higher amount of TGF-α bound to EGFR in the gastric mucosa of aged rats could be the consequence of a greater availability of TGF-α resulting from decreased amount of TGF-α bound to ERRP. The age-related decline in

EFFECT OF AGING ON THE GASTROINTESTINAL TRACT / 423 ERRP levels, together with a concomitant reduction in the amount of precursor forms of TGF-α bound to ERRP, suggests that increased activation of EGFR, observed in the GI mucosa of aged rats, could be partly due to a greater availability of TGF-α and possibly other ligands of EGFR resulting from the loss of ERRP.

Gene Expression Patterns Advances in genomic techniques have allowed assessment of global changes in gene expression patterns. Using this technology, investigators have demonstrated that aging results in marked changes in the expression of IGF-I and IGFBPs in the colonic mucosa of rats (349). Because IGF-I plays a major role in the regulation of mucosal proliferation in the intestine (349), the age-related increase in IGF-I expression in the colon, it is tempting to speculate a role for IGF-I in modulating intestinal mucosal growth during aging.

Key Areas for Future Research Some research suggests that abnormalities in growth factors, growth factor receptors, and their regulators may play a major role in the regulation of these events. This is an important area for future basic science research because changes in mucosal growth affect the structural and functional properties of the mucosa and may also contribute to growth-related diseases such as cancer.

AGING AND SURGERY OF THE GASTROINTESTINAL TRACT As human life expectancy is prolonged and medical science advances, elderly patients increasingly have intraabdominal septic pathology or are increasingly subjected to the stresses of elective or urgent surgical procedures, or both (350,351). It therefore becomes important to begin to understand how aging affects the adaptation of the intestine to fasting and refeeding, sepsis, and bowel resection.

differences in mucosal composition and surface area, particularly if these studies include preadult humans or animals (354). Although the mechanisms of these various effects are therefore not all clear, they represent an important target for future study, because elucidation of the responsible factors may identify targets by which nutrition may be improved in elderly patients consuming marginal diets. Indeed, animal studies suggest that aging appears to alter intestinal responses to fasting and feeding in complex ways, often decreasing the response or inhibiting biological pathways. For instance, basal small intestinal ODC activity and polyamine synthesis appear higher in aging rats than young rats, and they are more dysregulated by fasting and refeeding in the older animals (355). Similarly, jejunal epithelial cells from aged mice demonstrate decreases in oxygen uptake in the absence and presence of exogenous substrate, glucose oxidation, entry of glucose and glutamine into the TCA cycle, and lower contribution of glucose and glutamine carbon to anaplerosis and subsequent synthetic compounds. These differences are greater in fed mice than fasted mice. These changes are all consistent with suppression of the role of the TCA cycle in biosynthesis and oxidative metabolism (356). Such results should be interpreted with caution, however, because another study found that age-related differences in oxidative metabolism in rats varied with the strain of rats studied (357). The induction of apolipoproteins A-1 and A-IV in the intestine and liver by fasting and refeeding has also been reported to be markedly blunted in 25- to 34-monthold mice compared with 6- to 9-month-old mice (358). Although the metabolic response to moderate surgical stress such as an elective colon resection may not differ between healthy younger humans and healthy older humans (359), it has long been a clinical observation that aging humans may be more susceptible to the severe stress of intraabdominal sepsis than younger humans (360). Murine studies seem to confirm this suspicion, even in the absence of human comorbidities. Although the mechanisms of the effect are not entirely clear, senescent mice die more rapidly and at lower bacterial thresholds in cecal ligation and puncture models, possibly related to an age-related defect in glucocorticoid inhibition of TNF-α (361,362).

Adaptation to Bowel Resection Adaptation to Fasting and Sepsis Intestinal absorption may be decreased in aging humans. For instance, ileal reabsorption of conjugated bile acids is decreased in older adults (352). Indeed, older patients may exhibit malabsorption of carbohydrates, lipids, amino acids, minerals, or vitamins (353). It remains unclear to what extent these defects represent aging per se and to what extent they reflect the development of comorbidities. For instance, although substantial abnormalities are found in lipid absorption in many elderly patients, these often correlate with intercurrent disease (172). The interpretation of studies of transport capacity may also be obscured by age-dependent

The adaptation to bowel resection poses particular challenges in postsurgical patients, because these patients must not only deal with the stress of surgery and a subsequent period of fasting due to ileus, but may also be required to adapt nutrient absorption to the demands of a short bowel syndrome thereafter. Although most clinical bowel resections are relatively well tolerated, some are more major. Such major bowel resections and the short gut syndrome that follows represent important animal models in which to study the stress of adaptation to bowel resection. Because many sick elderly patients undergoing surgery are already malnourished, the adaptation of the small bowel

424 / CHAPTER 14 to small-bowel resection is important for subsequent restoration of the normal nutritional state. At least one study suggests that the morphologic and proliferative adaptive response of the remnant small bowel to massive (70%) small-bowel resection is similar between young (2-month-old) and old (24-month-old) rats (363). Some studies suggest that the early adaptive response may be delayed in older animals, although adaptation is equivalent in the long term (364,365). Another investigator determined that the residual small bowel retained sufficient absorptive capacity even after 90% proximal small-bowel resection, although a delay in early response correlated with a decrease in ODC mRNA expression (364). In contrast, pancreatic hyperplasia after 90% proximal small-bowel resection appears inhibited in old rats, possibly because of an inadequate postresection increase in plasma enteroglucagon (366). Ileal-jejunal transposition, which creates a physiologic short gut syndrome without actually removing bowel, is associated with increases in mucosal mass, villus height, and crypt depth throughout the small bowel, as well as increased colonic and pancreatic weight and increased plasma neurotensin levels; but none of these parameters were observed to be affected by the age of the rats studied (367). It should also be pointed out that although many studies of intestinal adaptation focus on morphologic parameters, these may not necessarily correlate with gut mucosal function. For instance, duodenal adaptation to 70% distal small-bowel resection has been reported to induce equivalent morphologic adaptation in young and old rats, but sucrase-, maltase-, and lactase-specific activities in the duodenal mucosa were only found to increase in young rats (368). In addition, although the final morphologic results of adaptation may be similar between young and older animals, it is possible that the mechanisms by which these effects are achieved may differ. For instance, neurotensin has been hypothesized to induce colonic mucosal thickening in young rats by hyperplasia, but in older rats by hypertrophy (317). This may not be true for the small bowel (369). Delayed intestinal adaptation may nevertheless be more clinically important in the elderly because delayed adaptation may make the elderly less able to recover from malnutrition or combat other comorbidities. Neurotensin (363) and growth hormone, together with a high-protein diet (370), have each been reported to promote intestinal adaptation after massive bowel resection in aging rats, but neither has been demonstrated to be more effective in older animals than in younger animals. Furthermore, such agents may have systemic effects. One study of the effects of growth hormone on colonic anastomotic strength in old diabetic rats found no effect of the agent on anastomotic healing, but growth hormone administration doubled the overall mortality of the procedure (371). An interesting parallel case to the adaptation to smallbowel resection is the adaptation of a jejunal pouch to distal proctectomy and anastomosis between the new jejunal reservoir and the anus. Functional outcomes after distal proctectomy and coloanal reconstruction with a jejunal pouch seem reasonably equivalent between younger patients and selected

elderly patients, although reported constipation may be more common in the older patients (372). Interestingly, aging but otherwise healthy rats exhibit impaired healing of their abdominal wounds after colonic transection and anastomosis, but aging does not appear to impair colonic healing (373). However, although colonic anastomotic healing in rats is not generally affected by aging under optimal conditions, anastomotic bursting strength may be more substantially impaired by a low dose of the chemotherapeutic and antimitogenic agent 5-fluorouracil (374). In general, intestinal repair after injury by systemic alkylating agent toxicity is faster in younger animals than in older animals (375). Such observations raise the more general question of whether studies of responses to surgery and stress in otherwise healthy aging animals may adequately represent the pathophysiology of the responses to surgery and stress in aging humans whose status may be further compromised by other comorbidities.

Surgical Diseases of the Gut Many GI diseases of surgical significance increase in prevalence with age. The most obvious is malignancy. However, peptic ulcer disease is also more common among the elderly because of increasing NSAID use and increasing H. pylori colonization (376). Gastroesophageal reflux problems also become more prevalent, although it is difficult to distinguish the contribution of changes in GE junction physiology from the effects of longer exposure to refluxate, NSAID, aspirin or other barrier breakers, and obesity. Certain colonic diseases also become more prevalent with age. Aside from colon cancer, among the most obvious of these diseases are diverticular disease and arteriovenous malformations that cause lower GI bleeding. Either of these may cause patients to require surgery. In the Western world, aging and fiber-poor diets clearly predispose to the development of diverticulosis, defined as the presence of “false” diverticula of the colon as opposed to the rarer true colonic diverticulum that contains all layers of the colonic wall (377–379). Aging is associated with increased collagen deposition in the colonic wall (380), and a small electron microscopic study suggested that collagen fibrils in the left colon become smaller and more tightly packed than those in the right colon with age and especially in patients with diverticular disease (381). Indeed, tensile strength in the left colon also declines with age (382). Although a causal link has not been established, it seems plausible that these alterations in collagen support and mechanical properties could contribute to the development of diverticulosis as patients age. Interestingly, high-fiber diets appear to protect against age-related changes in collagen cross-linking, as well as the development of diverticulosis in a rat model (383), thus it is possible that either chemical or mechanical properties of the luminal contents may influence submucosal collagen metabolism. A high-fiber diet is expected to alter concentrations of luminal nutrients

EFFECT OF AGING ON THE GASTROINTESTINAL TRACT / 425 and bioactive agents, including short-chain fatty acids (384,385) and bile acids (386), as well as pressure and strain within the colon (382,387). Either might exert direct effects on the colonic submucosal layers or indirect effects by influencing colonic epithelial cells to release paracrine factors (388,389). By comparison with Western white individuals, right-sided true diverticula may be more common among Asians (390). However, studies suggest that such diverticula are also increasing in incidence over time and with age, and may reflect environmental factors, as well as genetic background (391,392).

Angiodysplasia Colonic bleeding from angiodysplasia is also substantially more common among the elderly. Bleeding from such arteriovenous malformations may be more common in the right colon and has been attributed to degenerative changes in the colonic mucosa and vasculature (393).

Key Areas for Future Research The impact of aging on patients undergoing or adapting to GI surgery is an extremely important clinical problem that merits substantial additional basic and clinical investigation. It is clear from some of the work that has already been done that adaptation is slow, growth is less, and the regulation of these processes is abnormal. However, better elucidation of the mechanisms responsible for these events is required so that interventions may be designed to correct these abnormalities. As the population ages and perioperative management improves, more aging patients will undergo more complex GI surgery. Improved understanding of the impact of aging on the gut will facilitate their management and recovery.

ACKNOWLEDGMENTS This work was supported by grants from the National Institutes of Health (5 R01 AG14343) and the Department of Veterans Affairs.

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