Osteoporosis, 2nd Edition (2-Volume Set)

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CHAPTER 1 Chapter Title

Contributors

Numbers in parentheses indicate the volume number and pages on which the authors’ contributions begin.

John P. Bilezikian (2:71) Departments of Medicine and Pharmacology, College of Physicians and Surgeons, Columbia University, New York, New York 10032 Peter V. N. Bodine (1:305) Women’s Health Research Institute, Wyeth-Ayerst Research, Radnor, Pennsylvania 19087 Henry Bone (2:533) Michigan Bone and Mineral Clinic, Detroit, Michigan 48236 Jean-Philippe Bonjour (1:621) Department of Internal Medicine, Division of Bone Diseases (World Health Organization Collaborating Center for Osteoporosis and Bone Diseases), University Hospital, Geneva CH-1211, Switzerland Adele L. Boskey (1:107) Hospital for Special Surgery, Weill Medical College of Cornell University, New York, New York 10021 Mary L. Bouxsein (1:509) Orthopedic Biomechanics Laboratory, Department of Orthopedic Surgery, Beth Israel Deaconess Medical Center, and Harvard Medical School, Boston, Massachusetts 02215 Alan L. Burshell (2:195) Department of Internal Medicine, Alton Ochsner Medical Foundation, Ochsner Clinic, New Orleans, Louisiana 70131 Elizabeth Capezuti (1:795) Nell Hodgson Woodruff School of Nursing, Emory University, Atlanta, Georgia 30322 Dennis R. Carter (1:471) Department of Mechanical Engineering, Biomechanical Engineering Program, Stanford University, Stanford, California 94305; and Rehabilitation Research and Development Center, Veterans Affairs Palo Alto, Palo Alto, California 94304 Jane A. Cauley (1:741) University of Pittsburgh, Pittsburgh, Pennsylvania 15261

M. Arlot (2:501) INSERM Unité 403, Faculté RTH Laennec, Lyon 69372, France Laura K. Bachrach (2:151) Department of Pediatrics, Stanford University School of Medicine, Stanford, California 94305 Daniel Baran (2:229) Departments of Medicine, Orthopedics, and Cell Biology, University of Massachusetts Medical Center, and Merck & Company, Inc., Worcester, Massachusetts 01655 M. Janet Barger-Lux (2:59) Osteoporosis Research Center, Creighton University, Omaha, Nebraska 68131 Elizabeth Barrett-Conner (1:819) Department of Family and Preventive Medicine, University of California, San Diego, La Jolla, California 90293 David J. Baylink (1:405; 2:675) Department of Medicine, Loma Linda University, and the Musculoskeletal Disease Center, Jerry L. Pettis Memorial Veterans Affairs Medical Center, Loma Linda, California 92357 Gary S. Beaupré (1:471) Department of Mechanical Engineering, Biomechanical Engineering Program, Stanford University, Stanford, California 94305; and Rehabilitation Research and Development Center, Veterans Affairs Palo Alto, Palo Alto, California 94304 Belinda R. Beck (1:701) Griffith University, School of Physiotherapy and Exercise Science, Queensland 9726, Australia Daniel D. Bikle (2:237) Department of Medicine, University of California, San Francisco, School of Medicine, and Department of Veterans Affairs, San Francisco Veterans Affairs Medical Center, San Francisco, California 94121

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CONTRIBUTORS

Jacqueline R. Center (2:169) Bone and Mineral Research Program, Garvan Institute of Medical Research, St. Vincent’s Hospital, Sydney, New South Wales 2010, Australia P. Chavassieux (2:501) INSERM Unité 403, Faculté RTH Laennec, Lyon 69372, France Di Chen (1:373) Department of Medicine and Endocrinology, University of Texas Health Science Center, San Antonio, Texas 78284 Michael Chorov (2:769) Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02215 Roberto Civitelli (2:651) Division of Bone and Mineral Diseases, Washington University School of Medicine, and Barnes-Jewish Hospital, St. Louis, Missouri 63110 Cyrus Cooper (1:557) MRC Environmental Epidemiology Unit, University of Southampton, Southampton General Hospital, Southampton SO16 6YD, England Felicia Cosman (2:577) Regional Bone Center, Helen Hayes Hospital, West Haverstraw, New York 10993 Gilbert J. Cote (1:247) Department of Endocrine Neoplasia and Hormonal Disorders, Division of Internal Medicine, University of Texas M.D. Anderson Cancer Center, Houston, Texas 77030 Ann B. Cranney (2:539) Department of Medicine, University of Ottawa, Ottawa, Ontario, Canada K1Y 1J8 Sarah Dallas (1:373) Department of Medicine and Endocrinology, University of Texas Health Science Center, San Antonio, Texas 78284 Bess Dawson-Hughes (2:545) Jean Mayer USDA Human Nutrition Research Center on Aging, Tufts University, Boston, Massachusetts 02111 Chris de Laet (1:585) Institute for Medical Technology Assessment, Erasmus University, Rotterdam 3000 DR, The Netherlands Arthur A. DeCarlo (2:363) Department of Periodontics, University of Alabama School of Dentistry, Birmingham, Alabama 35294 Pierre D. Delmas (2:459) INSERM Research Unit 403, Hôpital E. Herriot, and Claude Bernard University, Lyon 69003, France Marc K. Drezner (2:479) Department of Medicine, University of Wisconsin Medical School, Madison, Wisconsin 53792 Thomas A. Einhorn (1:3) Department of Orthopedic Surgery, Boston University School of Medicine, Boston, Massachusetts 02118

John A. Eisman (2:169) Bone and Mineral Research Program, Garvan Institute of Medical Research, St. Vincent’s Hospital, Sydney, New South Wales 2010, Australia Sol Epstein (2:327) Roche Laboratories, Nutley, New Jersey 07110 Kenneth G. Faulkner (2:433) GE Medical Systems, Madison, Wisconsin 53717 David Feldman (1:257) Department of Medicine, Division of Endocrinology, Stanford University School of Medicine, Stanford, California 94305 Lorraine A. Fitzpatrick (2:259) Division of Endocrinology and Metabolism and Internal Medicine, Mayo Clinic and Foundation, Rochester, Minnesota 55905 H. Fleisch (1:449) University of Berne, CH-3008 Berne, Switzerland Robert F. Gagel (1:247) Department of Endocrine Neoplasia and Hormonal Disorders, Division of Internal Medicine, University of Texas M.D. Anderson Cancer Center, Houston, Texas 77030 Patrick Garnero (2:459) INSERM Research Unit 403, Hôpital E. Herriot, and Synarc, Lyon 69003, France Harry K. Genant (2:411) Department of Radiology, University of California, San Francisco, San Francisco, California 94143 Jayashree A. Gokhale (1:107) Hospital for Special Surgery, Weill Medical College of Cornell University, New York, New York 10021 Deborah T. Gold (2:479) Departments of Psychiatry and Behavioral Sciences, Sociology, Psychology, and Aging Center, Duke University Medical Center, Durham, North Carolina 27710 Steven R. Goldring (2:351) Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, and New England Baptist Bone and Joint Institute, Harvard Institutes of Medicine, Boston, Massachusetts 02215 Gail A. Greendale (1:819) Department of Medicine, Division of Geriatrics, University of California, Los Angeles, School of Medicine, Los Angeles, California 90095 Jeane Ann Grisso (1:795) Center for Clinical Epidemiology and Biostatistics, Division of General Internal Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104 Coleman Gross (1:257) Department of Medicine, Division of Endocrinology, Stanford University School of Medicine, Stanford, California 94305

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CONTRIBUTORS

Gordon H. Guyatt (2:539) Department of Medicine and Clinical Epidemiology and Biostatistics, McMaster University, Hamilton, Ontario, Canada L8N 3Z5 Reinhard Gysin (1:405) Department of Medicine, Loma Linda University, and the Musculoskeletal Disease Center, Jerry L. Pettis Memorial Veterans Affairs Medical Center, Loma Linda, California 92357 Robert P. Heaney (1:669; 2:513) Creighton University, Omaha, Nebraska 68178 Hunter Heath III (2:259) U.S. Medical Division, Eli Lilly and Company, Indianapolis, Indiana 46285 Michael H. Heggeness (2:485) Department of Orthopaedic Surgery, Center for Spinal Disorders, Baylor College of Medicine, Houston, Texas 77030 N. Kathryn Henderson (2:169) Bone and Mineral Research Program, Garvan Institute of Medical Research, St. Vincent’s Hospital, Sydney, New South Wales 2010, Australia Ana O. Hoff (1:247) Department of Endocrine Neoplasia and Hormonal Disorders, Division of Internal Medicine, University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030 Siu L. Hui (1:809) Department of Medicine, Indiana University School of Medicine, and the Regenstrief Institute for Health Care, Indianapolis, Indiana 46202 Marjorie K. Jeffcoat (2:363) Department of Periodontics, University of Alabama School of Dentistry, Birmingham, Alabama 35294 Michael Jergas (2:411) Department of Radiology, University of California, San Francisco, San Francisco, California 94143 C. Conrad Johnston (1:809) Department of Medicine, Indiana University School of Medicine, and the Regenstrief Institute for Health Care, Indianapolis, Indiana 46202 Stefan Judex (1:489) Musculo-Skeletal Research Laboratory, Department of Biomedical Engineering, State University of New York, Stony Brook, New York 11794 Gerard Karsenty (1:213) Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas 77030 Jennifer L. Kelsey (1:535) Department of Health, Research, and Policy, Division of Epidemiology, Stanford University School of Medicine, Stanford, California 94305 Sundeep Khosla (2:49; 2:709) Department of Internal Medicine, Division of Endocrinology and Metabolism, Mayo Clinic and Foundation, Rochester, Minnesota 55905

Donald B. Kimmel (2:29) Osteoporosis Research Center, Creighton University, Omaha, Nebraska 68131 B. Jenny Kiratli (2:207) Spinal Cord Injury Center, Palo Alto Veterans Affairs Health Care System, Palo Alto, California 94304 Michael Kleerekoper (2:403) Department of Internal Medicine, Wayne State University, Detroit, Michigan 48201 Robert F. Klein (2:103) Bone and Mineral Research Unit, Oregon Health Sciences University, and the Portland Veterans Affairs Medical Center, Portland, Oregon 97201 Lynn Kohlmeier (2:341) Spokane Osteoporosis Center, Endocrine Associates of Spokane, Spokane, Washington 99204 Barry S. Komm (1:305) Women’s Health Research Institute, Wyeth-Ayerst Research, Radnor, Pennsylvania 19087 K.-H. William Lau (2:675) Departments of Medicine and Biochemistry, Jerry L. Pettis Veterans Affairs Medical Center, and Loma Linda University, Loma Linda, California 92357 Cassandra A. Lee (1:3) Department of Orthopedic Surgery, Boston University School of Medicine, Boston, Massachusetts 02118 Gary M. Leong (2:169) Bone and Mineral Research Program, Garvan Institute of Medical Research, St. Vincent’s Hospital, Sydney, New South Wales 2010, Australia Jane B. Lian (1:21) Department of Cell Biology, University of Massachusetts Medical School, Worcester, Massachusetts 01655 Robert Lindsay (2:577) Regional Bone Center, Helen Hayes Hospital, West Haverstraw, New York 10993 Kenneth W. Lyles (2:479) Departments of Medicine and Aging Center, Sarah W. Stedman Center for Nutritional Studies, GRECC, Veterans Affairs Medical Center, Duke University Medical Center, Durham, North Carolina 27710 Sharmilla Majumdar (2:3) Department of Radiology, University of California, San Francisco, San Francisco, California 94143 Peter J. Malloy (1:257) Department of Medicine, Division of Endocrinology, Stanford University School of Medicine, Stanford, California 94305 W. J. Maloney (2:385) Department of Orthopedic Surgery, Washington University School of Medicine, St. Louis, Missouri 63110 Robert Marcus (2:3; 2:341) Veterans Affairs Medical Center, Palo Alto, California 94304; and Department of Medicine, Stanford

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CONTRIBUTORS

University School of Medicine, Stanford, California 94305 Thomas J. Martin (1:361) St. Vincent’s Institute of Medical Research, Melbourne 3065, Australia Kenneth B. Mathis (2:485) Department of Orthopaedic Surgery, Center for Spinal Disorders, Baylor College of Medicine, Houston, Texas 77030 Kenneth J. McLeod (1:489) Musculo-Skeletal Research Laboratory, Department of Biomedical Engineering, State University of New York, Stony Brook, New York 11794 L. Joseph Melton III (1:557; 2:49) Department of Internal Medicine, Division of Endocrinology and Metabolism, and Department of Health Sciences Research, Section of Clinical Epidemiology, Mayo Clinic and Foundation, Rochester, Minnesota 55905 P. J. Meunier (2:501) INSERM Unité 403, Faculté RTH Laennec, Lyon 69372, France Subburaman Mohan (1:405) Department of Medicine, Loma Linda University, and the Musculoskeletal Disease Center, Jerry L. Pettis Memorial Veterans Affairs Medical Center, Loma Linda, California 92357 Lis Mosekilde (2:725) Department of Cell Biology, Institute of Anatomy, University of Aarhus, DK-8000, Aarhus C, Denmark Douglas B. Muchmore (2:603) Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46285 Gregory R. Mundy (1:373) Department of Medicine and Endocrinology, University of Texas Health Science Center, San Antonio, Texas 78284 Ran Namgung (1:599) Department of Pediatrics, Yonsei University College of Medicine, Seoul, Korea Dorothy Nelson (1:569) Department of Internal Medicine, Wayne State University, Detroit, Michigan 48201 Lorene Nelson (1:569) Department of Health Research and Policy, Division of Epidemiology, Stanford University School of Medicine, Stanford, California 94305 Robert A. Nissenson (1:221) Endocrine Unit, San Francisco Veterans Affairs Medical Center, and Departments of Medicine and Physiology, University of California, San Francisco, San Francisco, California 94121 Bjorn R. Olsen (1:189) Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115

Eric S. Orwoll (1:339; 2:103) Bone and Mineral Unit, Oregon Health Sciences University, and the Portland Veterans Affairs Medical Center, Portland, Oregon 97201 Babatunde Oyajobi (1:373) Department of Medicine and Endocrinology, University of Texas Health Science Center, San Antonio, Texas 78284 Roberto Pacifici (2:85) Division of Bone and Mineral Diseases, Washington University, St. Louis, Missouri 63110 Charles Y. C. Pak (2:699) Center for Mineral Metabolism and Clinical Research, University of Texas Southwestern Medical Center, Dallas, Texas 75390 Socrates E. Papapoulos (2:631) Department of Endocrinology and Metabolic Diseases, University of Leiden Medical Center, 2333 ZA Leiden, The Netherlands A. Michael Parfitt (1:433) Division of Endocrinology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205 Millan S. Patel (1:213) Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas 77030 Huibert A. P. Pols (1:639) Department of Internal Medicine, Erasmus University Medical School, 3000 DR Rotterdam, The Netherlands Richard Prince (2:621) Department of Medicine, University of Western Australia, Sir Charles Gairdner Hospital, Perth 6009, Western Australia Xuezhong Qin (1:405) Department of Medicine, Loma Linda University, and the Musculoskeletal Disease Center, Jerry L. Pettis Memorial Veterans Affairs Medical Center, Loma Linda, California 92357 Yi-Xian Qin (1:489) Musculo-Skeletal Research Laboratory, Department of Biomedical Engineering, State University of New York, Stony Brook, New York 11794 Lawrence G. Raisz (2:19) Department of Endocrinology and Metabolism, University of Connecticut Health Center, Farmington, Connecticut 06032 Robert R. Recker (2:59) Osteoporosis Research Center, Creighton University, Omaha, Nebraska 68131 Michael S. Reddy (2:363) Department of Periodontics, University of Alabama School of Dentistry, Birmingham, Alabama 35294 Jonathan Reeve (1:585; 2:725) Department of Medicine, Addenbrooke’s Hospital, University of Cambridge, Cambridge CB2 2QQ, United Kingdom

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CONTRIBUTORS

Anthony M. Reginato (1:189) Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115; and Arthritis Unit, Massachusetts General Hospital, Boston, Massachusetts 02114 Ian R. Reid (2:553) Department of Medicine, The University of Auckland, Auckland 1, New Zealand B. Lawrence Riggs (2:49) Department of Internal Medicine, Division of Endocrinology and Metabolism, Mayo Clinic and Foundation, Rochester, Minnesota 55905 Rene Rizzoli (1:621) Department of Internal Medicine, Division of Bone Diseases (World Health Organization Collaborating Center for Osteoporosis and Bone Diseases), University Hospital, Geneva CH-1211, Switzerland Pamela Gehron Robey (1:107) Bone Research Branch, National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland 20892 Gideon A. Rodan (1:361) Department of Bone Biology and Osteoporosis Research, Merck Sharp & Dohme Research Laboratories, West Point, Pennsylvania 19486 Clifford Rosen (2:747) Maine Center for Osteoporosis Research and Education, St. Joseph Hospital, Bangor, Maine 04401 Michael Rosenblatt (2:769) Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02215 F. Patrick Ross (1:73) Department of Pathology, Washington University School of Medicine, St. Louis, Missouri 63110 Clinton T. Rubin (1:489) Musculo-Skeletal Research Laboratory, Department of Biomedical Engineering, and the Center for Biotechnology, State University of New York, Stony Brook, New York 11794 Loran M. Salamone (1:741) University of Pittsburgh, Pittsburgh, Pennsylvania 15261 Adina Schneider (2:303) Mount Sinai Hospital, New York, New York 10029 D. J. Schurman (2:385) Division of Orthopedic Surgery, Stanford University School of Medicine, Stanford, California 94305 Ann V. Schwartz (1:795) Department of Epidemiology and Biostatistics, University of California, San Francisco, School of Medicine, San Francisco, California 94143 Ego Seeman (1:771) Austin and Repatriation Medical Centre, University of Melbourne, Heidelberg, Melbourne 3084, Australia

Elizabeth Shane (2:303; 2:327) College of Physicians and Surgeons of Columbia University, New York, New York 10032 Jay R. Shapiro (2:271) Kennedy Krieger Institute, Baltimore, Maryland 21224; and Uniformed Services University, Bethesda, Maryland 20814 Janet Shaw (1:701) Departments of Exercise and Sport Science, University of Utah, Salt Lake City, Utah 84112 Kathy M. Shipp (2:479) Departments of Physical Therapy and Aging Center, Duke University Medical Center, Durham, North Carolina 27710 Shonni J. Silverberg (2:71) Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, New York 10032 Ethel S. Siris (2:603) Columbia University College of Physicians and Surgeons, and Toni Stabile Center for the Prevention and Treatment of Osteoporosis, Columbia – Presbyterian Medical Center, New York, New York 10032 Charles W. Slemenda† (1:809) Department of Medicine, Indiana University School of Medicine, and the Regenstrief Institute for Health Care, Indianapolis, Indiana 46202 Steven R. Smith (2:195) Pennington Biomedical Research Center, Louisiana State University, Baton Rouge, Louisiana 70808 R. L. Smith (2:385) Orthopedic Research Laboratory, Division of Orthopedic Surgery, Stanford University School of Medicine, Stanford, California 94305; and Veterans Affairs Medical Center, Palo Alto, California 94304 Christine M. Snow (1:701) Bone Research Laboratory, Oregon State University, Corvallis, Oregon 97331 MaryFran Sowers (1:535; 1:721) Department of Epidemiology, University of Michigan School of Public Health, Ann Arbor, Michigan 48109 Bonny L. Specker (1:599) Ethel Austin Martin Program in Human Nutrition, South Dakota State University, Brookings, South Dakota 57007 Gary S. Stein (1:21) Department of Cell Biology, University of Massachusetts Medical School, Worcester, Massachusetts 01655 S. Aubrey Stoch (2:769) Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02215 †

Deceased.

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Steven L. Teitelbaum (1:73) Department of Pathology, Washington University School of Medicine, St. Louis, Missouri 63110 Kathy Traianedes (1:373) Department of Medicine and Endocrinology, University of Texas Health Science Center, San Antonio, Texas 78284 Reginald C. Tsang (1:599) Department of Pediatrics, University of Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio 45229 André G. Uitterlinden (1:639) Department of Internal Medicine, Erasmus University Medical School, 3000 DR Rotterdam, The Netherlands Marjolein C. H. van der Meulen (1:471) Sibley School of Mechanical and Aerospace Engineering, Cornell University, Ithaca, New York 14853; and Biomechanics and Biomaterials, Hospital for Special Surgery, New York, New York 10021

Johannes P. T. M. van Leeuwen (1:639) Department of Internal Medicine, Erasmus University Medical School, 3000 DR Rotterdam, The Netherlands Marie Luz Villa (1:569) Department of Medicine, Division of Gerontology and Geriatrics, University of Washington School of Medicine, Seattle, Washington 98104 WenFang Wang (1:189) Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115 Kristine M. Wiren (1: 339) Bone and Mineral Unit, Oregon Health Sciences University, and the Portland Veterans Affairs Medical Center, Portland, Oregon 97201 Christian Wüster (2:747) Department of Medicine, Novo Nordisk Pharma, 55127 Mainz, Germany

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CHAPTER 1 Chapter Title

Preface

It will come as no surprise to the reader that this second edition of Osteoporosis substantially outweighs its predecessor. The reason for this increase is the astonishing progress in osteoporosis medicine that has taken place during the 5 years since publication of the first edition. Major advances have occurred at both the basic science and clinical levels, and we have sought to incorporate them into the second edition. Some illustrative areas where new insights have emerged are skeletal differentiation and regulation, particularly regarding complementary actions of Hedgehog and PTH-related proteins; the RANK/RANK-ligand osteoprotegerin system as a central feature of osteoclast biology; the skeletal phenotypes of mouse gene knockout models; the discovery of a second estradiol receptor which is relatively enriched in bone; and the molecular basis of bisphosphonate action. These developing areas represent new and important conceptual themes that were unknown in 1995, but which have been accorded detailed consideration in the second edition. Many of the chapters devoted to basic science emphasize new concepts that offer novel molecular targets for future therapeutics. Great progress has also been made in clinical practice. When the first edition was published, therapeutic choices were confined to several antiresorptive drugs. Few controlled clinical trials had been conducted for any agent, and those that had been published relied on the surrogate end point of bone mineral density, rather than on fracture incidence. Large randomized controlled trials using fracture incidence as the primary outcome have now become the industry standard, and results of such trials have led to the registration of new agents for the prevention and treatment of postmenopausal osteoporosis, glucocorticoid-associated osteoporosis, and osteoporosis in men. Perhaps most exciting, the recent demonstration that parathyroid hormone substantially increases bone mineral density and reduces fracture incidence validates the concept of skeletal anabolic

therapy and offers for the first time a potential approach to the eventual cure of osteoporosis. In response to these and other developments, new chapters have been introduced and others have been expanded, updated, or divided. New chapters include contributions on the developmental biology of bone, gene knockout models, major European epidemiological fracture studies, micro-CT assessment of bone architecture, evidence-based analysis of osteoporosis therapy, regulatory considerations for design of osteoporosis trials, skeletal effects of phytoestrogens and selective estradiol receptor modulators, and novel approaches to osteoporosis therapeutics. In the interest of maintaining freshness of ideas, we initiated our own remodeling process by creating a modest degree of authorship turnover. We thank previous contributors for their excellent work and forewarn them that we may call on them again for future editions. We note with sadness the passing of two prominent members of the osteoporosis community who were involved with the first edition of this book, Charles W. Slemenda and Louis V. Avioli. Despite his young age, Charlie provided many valuable insights into multiple aspects of osteoporosis. Lou Avioli elected not to write a chapter, but we benefited greatly from his advice regarding content and authors as well as from his enthusiasm for the project. We are pleased to acknowledge once again the efforts of Dr. Jasna Markovac of Academic Press for her unflagging interest and inspiration for this project. We also greatly appreciate the Academic Press editorial staff, particularly Mica Haley and Jenny Wrenn, for their cheerful hard work and enthusiasm no matter how short the deadline or late the chapter. ROBERT MARCUS DAVID FELDMAN JENNIFER KELSEY Stanford, California

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CHAPTER 1

The Bone Organ System Form and Function CASSANDRA A. LEE AND THOMAS A. EINHORN Department of Orthopedic Surgery, Boston University School of Medicine, Boston, Massachusetts 02118

I. II. III. IV.

Introduction Composition and Organization of Bone Cellular Control of Bone Homeostasis Bone Modeling and Remodeling

V. Bone Biomechanics VI. Summary References

I. INTRODUCTION

its simplest sense, Wolff’s law suggests that “form follows function’’ [3]. To fulfill these structure/function relationships adequately, bone is constantly being broken down and rebuilt in a process called remodeling. The cellular link between bone resorbing cells or osteoclasts, and bone forming cells or osteoblasts, is known as coupling. How bone resorption and bone formation are linked is not entirely understood (see also Chapter 12), but the consequences of accentuating one or the other preferentially leads to disease. Too much bone resorption at the expense of formation results in osteoporosis, a loss of bone strength and integrity, resulting in fractures after minimal trauma. However, under normal states of bone homeostasis, the remodeling activities in bone serve to remove bone mass where the mechanical demands of the skeleton are low and form bone at those sites where mechanical loads are transmitted repeatedly. Hence, bone is a well-designed organ system whose ability to maintain itself depends on the integrated processing of external mechanical input and physiological signals from the systemic environment and the transduction of these demands into cellular and chemical events.

Bone is a vital, dynamic connective tissue that has evolved to reflect a balance between its two major functions, provision of mechanical integrity for locomotion and protection and involvement in the metabolic pathways associated with mineral homeostasis. In addition, bone is the primary site of hemopoiesis, and recent findings support its important role as a component of the immune system [1,2]. Since the observations of Galileo, it has been assumed that the inherent architecture of bone is influenced by the mechanical stresses associated with normal function. A more formal definition of these structure/function relationships was provided by German anatomists and engineers during the late 19th century in what has since been known as Wolff’s law [3]. The tenets of Wolff’s law were based on a recognition of the correlation between the patterns of trabecular alignment in bone and the directions of the principal stresses, which were estimated to occur during normal skeletal function. Under these physiological conditions, the structure/function relationships observed in bone, coupled with its role in maintaining mineral homeostasis, strongly suggest that it is an organ of optimum structural design. In

OSTEOPOROSIS, SECOND EDITION VOLUME 1

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Copyright © 2001 by Academic Press. All rights of reproduction in any form reserved.

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LEE AND EINHORN

II. COMPOSITION AND ORGANIZATION OF BONE Bone is a composite material composed of an organic and an inorganic phase. By weight, approximately 70% of the tissue is mineral or inorganic matter, water comprises 5 to 8%, and the organic or extracellular matrix makes up the remainder. Approximately 95% of the mineral phase is composed of a specific crystalline hydroxyapatite, and this is impregnated with impurities, which make up the remaining 5% of the inorganic phase. Ninety-eight percent of the organic phase is composed of type I collagen and a variety of noncollagenous proteins; cells make up the remaining 2% of this phase [4].

A. Organic Phase The organic phase of bone plays a wide variety of roles, determining the structure and the mechanical and biochemical properties of the tissue. Growth factors and cytokines, and extracellular matrix proteins such as osteonectin, osteopontin, bone sialoprotein, osteocalcin, proteoglycans, and other phosphoproteins and proteolipids, make small contributions to the overall volume of bone and major contributions to its biologic function (see also Chapter 4). Collagen is a ubiquitous protein of extremely low solubility, which consists of three polypeptide chains composed of approximately 1000 amino acids each. It is the major structural component of the bone matrix; constructed in the form of a triple helix of two identical 1(I) chains and one unique 2(I) chain cross-linked by hydrogen bonding between hydroxyproline and other charged residues. This produces a very rigid linear molecule 300 nm in length. Each molecule is aligned with the next in a parallel fashion in a quarter-staggered array to produce a collagen fibril. The collagen fibrils are then grouped in bundles to form the collagen fiber. Within the collagen fibril, gaps, called “hole zones,” exist between the ends of the molecules. In addition, “pores’’ exist between the sides of parallel molecules (Fig. 1). Noncollagenous proteins or mineral deposits can be found within these spaces, and mineralization of the matrix is thought to be initiated in the hole zones. Several noncollagenous proteins have been described in bone. One of the more extensively studied of these in bone is osteocalcin or bone-carboxyglutamic acid-containing protein (bone Gla protein). This is a small (5.8 kDa) protein in which three glutamic acid residues are carboxylated as a result of a vitamin K-dependent post translational modification. The carboxylation of these residues confers on this protein calcium and mineral binding properties. Osteocalcin accounts for 10 to 20% of the noncollagenous protein present in bone and is closely associated with the mineral phase. While the function of this bone-specific protein is

FIGURE 1 Collagen fiber and fibril structure showing putative locations of pores and hole zones. Reprinted with permission from T. A. Einhorn, Bone metabolism and metabolic bone disease, In “Orthopaedic Knowledge Update 4” (J. W. Frymoyer, ed.), pp. 69 – 88. Amer. Acad. Orthop. Surg., Rosemont, IL (1993).

not known, it is thought to play some role in attracting osteoclasts to sites of bone resorption. It may also regulate the rate of mineralization or the final shape assumed by mineral crystals. Other noncollagenous proteins found in bone may also be important to their calcium and mineral-binding properties. In addition, several of the bone matrix proteins, such as osteopontin, bone sialoprotein, bone acidic glycoprotein, thrombospondin, and fibronectin, contain arginine – glycine – aspartic acid (RGD) sequences. Such amino acid sequences, characteristic of cell-binding proteins, are recognized by a family of cell membrane proteins known as integrins. The integrins span the cell membrane and provide a link between the extracellular matrix and the cytoskeleton of the cell. Integrins on osteoblasts, osteoclasts, and fibroblasts provide a means for anchoring these cells to the extracellular matrix. Once anchored, the cells are then enabled to express their phenotype and conduct the type of activities that characterize their funcions [5]. Growth factors and cytokines such as transforming growth factor- (TGF-), insulin-like growth factor (IGF), osteoprotegerin (OPG), the tumor necrosis factors (TNFs), the interleukins, and the bone morphogenetic proteins (BMPs 2 – 10) are present in very small quantities in bone matrix. Such proteins have important effects regulating bone cell differentiation, activation, growth, and turnover (see Chapter 14). It is also likely that these growth factors serve as coupling factors that link the processes of bone formation and bone remodeling (Table 1).

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CHAPTER 1 The Bone Organ System

TABLE 1

Noncollagenous Proteins of the Extracellular Matrix

Structural matrix molecules Osteocalcin

Vitamin K dependent. Made by osteoblasts. May inhibit mineral deposition. Regulates activity of osteoclasts and their precursors. May mark turning point between bone formation and bone resorption. Restricted to the osteogenic lineage

Osteopontin

Contains RGD site. Anchors osteoclasts to bone. Supports cell attachments. Possibly inhibits mineralization. May regulate tissue repair and proliferation. Highly expressed in bone and inflammatory tissue

Bone Sialoprotein

Made by osteoblasts and hypertrophic chondrocytes. May initiate mineralization. Supports cell attachment. Binds Ca2 with high affinity. Restricted to skeletal lineage

Matrix Gla protein

Vitamin K dependent. May inhibit mineralization. May function in cartilage metabolism. Expressed in a variety of connective tissues

Chondroiton sulfate proteoglycan I (decorin)

Regulation of collagen fibrillogenesis. Found in extracellular matrix space

Chondroiton sulfate proteoglycan II (biglycan)

May bind to collagen. Regulates mineralization in vitro. Pericellular location

Fibronectin

Contains RGD site. Binds to numerous cell types, fibrin, heparin, gelatin, and collagen. Expressed in variety of connective tissues

Thrombospondin

Contains RGD site. Functions in cell attachment. Binds heparin, platelets, type I and V collagen, thrombin, fibrinogen, laminin, plasminogen, plasminogen activator inhibitor, histidine-rich glycoprotein. Expressed in variety of connective tissues

Osteonectin

Strong affinity for Ca2 and hydroxyapatite. Binds to growth factors. Potential association with osteoblast growth and/or proliferation. May play role in matrix mineralization. Expressed in a variety of connective tissues

Enzymatic matrix modifiers Matrix metalloproteinase (MMP) 9

Regulates growth plate angiogenesis and apoptosis. Degrades components of the ECM. Cleaves type IV, V, and XI collagens, elastin

Lysyl oxidase

Copper-dependent extracellular enzyme that catalyzes oxidative deamination of elastin and collagen precursors, which in turn spontaneously cross-link collagen and elastin, thereby forming a mature and functional ECM

Stromelysin

Is MMP3. Degrades most constituents of the ECM. Activates other MMPs

Latent morphogens and cytokines Bone morphogenetic protein family

Osteoinductive effects, promotes osteogenesis, chondrogenesis, and some induce other tissue types

Tissue growth factor  family

Mitogen for periosteal osteoprogenitors and marrow stromal cells. Direct stimulatory effect on bone collagen synthesis. Decreases bone resorption by inducing apoptosis of osteoclasts. Inhibits osteoblast differentiation in vitro

Fibroblast growth factor

Angiogenic properties. Important with neovascularization and wound healing. Important in healing and bone repair. Promotes bone cell replication

B. Inorganic Phase

C. Organization of Bone

The inorganic component of bone is composed mainly of a calcium phosphate mineral analogous to crystalline calcium hydroxyapatite. This apatite is present as a platelike crystal, which is 20 to 80 nm long and 2 to 5 nm thick. The small amounts of impurities in hydroxyapatite, such as carbonate, which can replace the phosphate groups, or chloride and fluoride, which can replace the hydroxyl groups, may alter certain physical properties of the crystal, such as its solubility [6]. These altered properties may impart important biologic effects that are critical to normal function.

The skeleton is composed of two parts: an axial skeleton, which includes the vertebrae, pelvis, and other flat bones such as the skull and sternum, and an appendicular skeleton, which includes all of the long bones. The long bones are divided into three parts: the epiphysis, metaphysis, and diaphysis. The epiphysis is that portion of the long bone found at either end and develops from a center of ossification that is distinct from the rest of the long bone shaft. It is separated from the rest of the bone by a layer of growth cartilage. This growth cartilage is known as the physis. The metaphysis is the zone between the physis and the

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FIGURE 2

Woven bone. Note arrangement of this bone tissue in which there is active bone formation (top), no particular lamellar organization of the tissue, and a high degree of cellularity.

central portion of the long bone shaft (known as the diaphysis). The metaphysis is the region where remodeling of the bone takes place during growth and development. The diaphysis comprises the majority of the length of a long bone. At the microscopic level, bone appears as an extremely well-organized tissue whereby mineral impregnates the collagen fibril array in such a way as to provide mechanical properties, which, in tension, are nearly as strong as cast iron. There are two types of bone tissue: woven bone and lamellar bone. Woven bone is considered immature or primitive bone and is normally found in the embryo, the newborn, in fracture callus, and in certain metaphyseal regions of the growing skeleton. It is also found in certain bone tumors, in patients with osteogenesis imperfecta, and in patients with Paget disease. Lamellar bone, however, is a more mature bone that results from the remodeling of woven bone or preexisting bone tissue. Woven, or primary bone, is a coarse-fibered tissue that does not show any uniform orientation of the collagen fibers (Fig. 2). It has more cells per unit volume than lamellar bone, and its mineral content and cells are randomly arranged. Newly formed woven bone, which is not as well mineralized as mature lamellar bone, contains particles with a smaller average crystal size. Moreover, the relative disorientation of the collagen fibers gives it isotropic mechanical characteristics; i.e., when tested, the mechanical behavior of woven bone is similar regardless of the orientation of the applied forces. At the time of birth, all bone in the body is woven. However, beginning at approximately 1 month of age, lamellar bone begins to develop. By 1 year

of age, lamellar bone will have effectively replaced much of the woven bone, as the latter is resorbed. By the age of 4, most of the bone in the body will be lamellar. Lamellar bone is a highly organized material with respect to the stress orientation of the collagen fibers (Fig. 3). As a result of this structural organization, lamellar bone exhibits anisotropic properties; i.e., the mechanical behavior of lamellar bone differs depending on the orientation of the applied forces. Moreover, its ability to resist loads is greatest when the forces are directed in a parallel fashion to the longitudinal axis of the collagen fibers. Anatomically, woven and lamellar bone are organized into trabecular (spongy or cancellous) and cortical (dense or compact) bone compartments. Cortical bone has four times the mass of trabecular bone, although the metabolic turnover rate of trabecular bone is much higher than that of cortical bone (bone turnover is a surface event, and trabecular bone has a greater surface area than cortical bone). Trabecular bone is found principally at the ends of long bones and in cuboid bones, such as the vertebrae (Fig. 4). The internal beams or plates of trabecular bone form a three-dimensional branching lattice, which is oriented along lines of stress. Trabecular bone is subject to a complex set of stresses and strains, although it is best designed for resisting of compressive loads. Cortical bone appears as an entirely different structure. It is solid and arranged not as interconnecting plates, but rather as cylinders, which are ellipsoid in cross section (Fig. 5). Cortical bone is usually subject to bending and torsional forces, as well as compressive loads.

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FIGURE 3

Lamellar bone. Note organization of this tissue in which there is a well-delineated orientation of the collagen fibers and a coordinated alignment of the cells.

Haversian bone is the most complex type of cortical bone. It is composed of vascular channels surrounded circumferentially by lamellae of bone (Fig. 6). This complex arrangement of bone around the vascular channel is called

FIGURE 4

the osteon. The osteon is an irregular, branching, and anastomosing cylinder composed of a neurovascular canal surrounded by cell-permeated layers of bone matrix. Osteons are usually oriented in the long axis of the bone and are the

Trabecular bone. Note the perpendicular orientation between the horizontal and the vertical trabeculae.

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FIGURE 5 Cross-section of cortical bone from the middiaphysis of the tibia. Note that cortical bone is a solid structure arranged not as interconnecting plates, but as ellipsoid cylinders. On the inner surface (endosteum), there is a structure that resembles that of trabecular bone.

FIGURE 6

Close-up view of a section of cortical bone. Note the distribution of vascular channels forming the osteons.

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FIGURE 7 Scanning electron micrograph of cortical bone showing individual osteons, surrounded by lamellar bone, which is impregnated with well-aligned cellular lacunae.

major structural units of cortical bone. They are connected to one another by Volkmanns canals, which are oriented perpendicularly to the osteon. Thus, cortical bone is a complex structure composed of many adjacent osteons and their interstitial and circumferential lamellae (Fig. 7). Most vessels in haversian canals have the ultrastructural features of capillaries, although some smaller-sized vessels may resemble lymphatic vessels. When examined histologically, these small vessels contain only precipitated protein; their endothelial walls are not surrounded by a basement membrane. The basement membrane of capillary walls may function as a rate-limiting or selective ion-limiting transport barrier because all material traversing the vessel wall must go through the basement membrane. The presence of this barrier is particularly important in calcium and phosphorous ion transport to and from bone. It is also important in explaining the response of bone to mechanical loads. The capillaries in the central canals are derived from the principal nutrient arteries of the bone: the epiphyseal and metaphyseal arteries [6]. The periosteum lines the outer surface of bone. It is composed of two layers. The outer fibrous layer is in direct contact with muscle and other soft tissue elements and is populated by undifferentiated fibroblast-like cells. The inner layer is known as the cambium layer and it is populated by fibroblast-appearing cells, many of which are committed

progenitors of chondrocytes and osteoblasts (Fig. 8).This layer contributes to appositional bone growth during bone development and is responsible for the expansion of the diameters of the long bones with aging.

III. CELLULAR CONTROL OF BONE HOMEOSTASIS A. Bone Cells Bone metabolism is regulated by bone cells, which respond to various environmental signals, including chemical, mechanical, electrical, and magnetic stimuli. In general, specific responses are governed by cellular receptors found on the membrane of the cell or intracellularly. Cell membrane receptors bind the exogenous signal and transfer the information across the cell’s cytoplasm to the nucleus through a series of interactions that involve a complex set of transduction mechanisms. Intracellular receptors (cytoplasmic or nuclear) bind the stimulus (usually a steroid hormone, which has crossed the cell membrane and entered the cell) and then translocate that effector to the nucleus where the steroid – receptor complex binds to a specific DNA promotor sequence of a gene. Three cell types are found in bone, osteoblasts, osteoclasts, and osteocytes.

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FIGURE 8

Close-up view of the periosteum of a long bone. The darker staining material at the lower portion of the figure is mineralized cortical bone. Above this is the periosteum, which consists of two layers. The outer layer shows elongated fibroblast-like cells embedded in a fibrous-like tissue. The inner layer, known as the cambium layer, is composed of a higher density of cells, which are slightly more plump and are embedded in a loose connective tissue.

Osteoblasts, generally regarded as bone-forming cells, govern bone metabolism (see Chapter 2). Their most obvious function is to synthesize osteoid, the protein component of bone tissue, but they also initiate bone resorption by

FIGURE 9

elaborating various neutral proteases (Fig. 9) [8]. The proteases remove surface osteoid, after which other cells participate in bone resorption. Because osteoblasts contain the receptors for most chemical mediators of bone metabolism,

Low-power view of osteoblasts lining the bone surface.

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CHAPTER 1 The Bone Organ System

FIGURE 10

including bone resorption, they play a critical role in the regulation of bone turnover. Osteocytes are abundant in mineralized bone matrix, but their function is poorly understood. Evidence suggests that they may receive mechanical input signals and transmit these stimuli to other cells in bone (Fig. 10). Osteoblasts and osteocytes are both derived from the same mesenchymal stem cell precursor (found in bone marrow stroma, periosteum, soft tissues, and possibly peripheral blood vessel endothelium). Once an osteoblast has synthesized osteoid and mineralized it, the osteoblast becomes an osteocyte. Evidence suggests that the lineage of the cell type can be identified by the expression of specific cell surface antigens, the expression of alkaline phosphatase when the cell is in its secretory osteoblastic phase, and the loss of alkaline phosphatase activity when the cell evolves to an osteocyte [8]. Osteoclasts, the active agents in bone resorption, are ultimately responsible for the remodeling of bone (see Chapter 3). In cortical bone, they are found at the apex of the classical “cutting cone’’ (Fig. 11, see also color plate). In trabecular bone they create the resorptive cavities, known as Howship’s lacunae (Fig. 12), seen on bone surfaces undergoing active remodeling. Osteoclasts are multinucleated, but their progenitors are hemopoietic mononuclear cells.

Differentiation toward an osteoclastic phenotype occurs early in the development of these cells.

B. Cellular Mechanisms in Bone All bone surfaces are continuous and lined by resting osteoblastic lining cells. On close inspection, it is possible to observe small intercellular gaps between the cells and their cytoplasmic processes. The lining osteoblasts are in communication with osteocytes through cell processes within the canaliculi that form gap junctions. (Fig. 13) Rapid fluxes of bone calcium across these junctions may be involved in the transmission of information between osteoblasts on the bone surface and osteocytes within the structure of bone itself. Although the cellular layer protects the bone from the extracellular fluid space, the osteoblasts on the bone surface are in direct chemical contact with the osteocytes within the mineralized bone by virtue of these cellular processes within the canaliculi. This organizational structure is consistent with the concept that bone cells are in intimate communication with each other and that osteoblasts receive the majority of local and systemic signals and then transmit them to other cells in bone. Conversely, strain-generated signals such as streaming potentials could

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FIGURE 11

A cutting cone in cortical bone. Note that it is composed of an osteoclast, which appears to be tunneling through bone, and this cell is followed closely by a group of endothelial cells that ultimately transition to osteoblasts. (See also color plate.)

be perceived by osteocytes, and their regulatory information can be passed on to the osteoblasts. Osteoclasts at specific bone sites are activated only after disruption of the osteoid layer that covers the bone sur-

FIGURE 12

faces, a bone lining osteoblast-mediated effect. This exposure of the underlying mineralized matrix may be caused by the degradation of surface osteoid by the neutral proteases elaborated by flat, elongated osteoblasts or by the

Low-power view of osteoclasts forming Howship’s lacunae.

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This molecule, related to the TNF family, upregulates osteoclast development and increases the activity of mature osteoclasts [9]. It may also play a role in immunologic tissue by regulating the interaction between T-cells and dendritic cells in vitro [10]. In vivo, RANKL exists in the cell as a ligand, anchored to the surface of an osteoblast or a bone marrow stromal cell and interacts with RANK on a preosteoclast. RANKL also exists in a secreted form that binds directly to the preosteoclast [11]. It is here that it promotes osteoclast differentiation from hemopoietic precursors. Once this reaction occurs, the preosteoclast will differentiate to a mature osteoclast only if the macrophage colony-stimulating factor (M-CSF) is present. Various other factors, such as PTH, interleukin-11, prostaglandin E2 (PGE2), and 1,25(OH)2D, upregulate RANKL on the surface of osteoblasts, hence increasing the development and activity of osteoclasts. The interaction between RANKL and its preosteoclast receptor is controlled by osteoprotegerin (OPG), a secreted protein. OPG is a soluble decoy protein that may modulate communication between osteoblasts and osteoclasts, thus playing a major role in bone homeostasis. Its main function is to halt bone resorption by inhibiting osteoclast formation via interruption of RANKL. In vitro, studies have shown that OPG can induce apoptosis of osteoclast-like cells (see Chapter 3). The proposed mechanism for OPG function is via reduction/disruption of formation of the F-actin ring in isolated osteoclasts. The F-actin ring is a cytoskeletal structure correlated with bone resorption [12].

IV. BONE MODELING AND REMODELING A. Bone Modeling FIGURE 13

Osteocyte (OC) cytoplasmic process in contact with the secreting surface of an osteoblast (OB). TEM  16,125. Reprinted with permission from C. Palumbo, Morphological study of intercellular junctions during osteocyte differentiation, In “Bone” (R. Baron, ed.), pp. 401 – 409. Pergamon Press, Elmsford, NY.

contraction of osteoblasts in response to stimulation by parathyroid hormone (PTH), 1,25-dihydroxyvitamin D, or prostaglandins of the E series. This contraction allows osteoclasts to gain access to the mineralized bone. Three recently discovered molecules that possibly play a key role in the coordinated maintenance of bone homeostasis are osteoprotegerin (OPG), RANK, (receptor activator of NF-B), and its ligand, RANKL, (see Chapter 3). The ligand (RANKL) has proven to be an essential factor in osteoclast differentiation by promoting osteoclast formation.

Bone formation begins in utero and continues throughout adolescence until skeletal maturity (see Chapter 5). Long bones form by two mechanisms. Endochondral ossification occurs in the long bones or the appendicular skeleton. It involves the differentiation of mesenchymal lineage cells to chondroblasts and then chondrocytes with the synthesis of a proteoglycan-rich, type II collagen-based extracellular matrix. This matrix is then modified biochemically by enzymes elaborated by hypertrophic chondrocytes to produce an environment that will permit the deposition of calcium. Once the extracellular matrix is calcified, it becomes a target for blood vessel invasion, and with this angiogenic response comes osteoclasts (which degrade the calcified cartilage) and osteoblast precursors. The calcified cartilage first formed is known as the primary spongiosum, and the bone that is laid down upon this tissue is known as secondary spongiosum. This secondary spongiosum is in the form of woven bone.

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Intramembranous bone formation occurs in the flat bones of the skeleton such as the skull and pelvis. It involves the direct formation of bone tissue by cells of the mesenchymal lineage, which have already undergone a biological commitment to the formation of osteoblastic cells. Bones also grow in length by endochondral ossification and in width by intramembranous or subperiosteal new bone formation.

B. Bone Remodeling Following skeletal maturity, bone continues to remodel throughout life and adapt its material properties to the mechanical demands placed upon it (see Chapters 12 and 15). Remodeling also has the function of maintaining the biomechanical competence of the skeleton by preventing the accumulation of fatigue damage and maintaining a tissue whose components are available for mineral homeostasis. Remodeling is thus a continuous process whereby there is a constant removal and replacement of whole volumes of bone tissue, a process conducted by osteoclasts and osteoblasts on bone surfaces, which, together with their precursor cells, form the bone remodeling system. A knowledge of bone remodeling is essential to the understanding of the pathophysiology of osteoporosis. The bone remodeling process governs the way bone is replaced, gained, or lost at specific sites and the way cumulative effects determine the three-dimensional structure of bone. The rate of turnover also determines the age of the bone tissue, and various physical and chemical properties of the bone are dependent on age and function [13]. According to bone histologists (see Chapter 15), the skeleton is composed of individual structural units or bone metabolic units (BMU) [6]. Cortical bone constitutes approximately 80% of the skeletal mass and trabecular bone approximately 20%. Bone surfaces may be undergoing formation or resorption, or they may be inactive. These processes occur throughout life in both cortical and trabecular bone. Bone remodeling is a surface phenomenon and it occurs on periosteal, endosteal, haversian canal, and trabecular surfaces. The rate of cortical bone remodeling, which may be as high as 50% per year in the midshaft of the femur during the first 2 years of life, eventually declines to a rate of 2 to 5% per year in the elderly. Rates of remodeling in trabecular bone are proportionally higher throughout life and may normally be 5 to 10 times higher than cortical bone remodeling rates in the adult [13]. The BMU of cortical bone is the osteon or haversian system, a cylinder of about 200 to 250 m in diameter running parallel to the long axis of the bone. As described earlier, the canals are connected to each other by transverse Volkmann’s canals and periodically either divide or reunite to form a branching network. Osteons form approximately two-thirds of cortical bone volume, a

proportion that falls with age, with the remainder consisting of interstitial bone representing the previous generations of osteons. There are also subperiosteal and subendosteal circumferential lamellae. In trabecular bone, the BMU is constructed differently. In two-dimensional sections, the trabecular surfaces are shaped like thin crescents about 600 m long and about 60 m in depth. Three-dimensionally, these BMUs are actually larger than they appear in two-dimensional histological sections with prolongations in different directions that interlock with adjacent BMUs [14]. These BMUs follow the same shape as the trabecular surface, most of which are concave toward the marrow. Under normal conditions, the remodeling process of resorption followed by formation is closely coupled and results in no net change in bone mass. As such, the BMU consists of a group of cells that participate in remodeling in a concerted and coordinated fashion. Cortical bone remodeling proceeds via cutting cones (Fig. 11) and is similar to processes in other hard biological tissues. Cuttings cones, or sheets of osteoclasts, bore holes through the hard bone, leaving tunnels, which appear in cross section as cavities. The head of the cutting cone consists of osteoclasts that resorb the bone. Following closely behind the osteoclast is a capillary loop and a population of endothelial cells and perivascular mesenchymal cells that are progenitors for osteoblasts and soon begin to lay down osteoid and refill the resorption cavity. By the end of the process, a new osteon will have been formed. Trabecular bone remodeling proceeds on the surface of bone whereby osteoclasts resorb bone at specific sites (Fig. 14). These areas are then filled in with newly formed osteoid. The mechanisms that control the activity and site specificity of this process are unknown. Cortical bone remodeling occurs in discrete temporal foci that are active for about 4 to 8 months [13]. Mononuclear precursor cells proliferate into a team of new osteoclasts that initiate the cutting cone. The osteoclasts then are removed (or disappear), and there is a quiescent interval or “reversal phase’’ during which time the newly resorbed area of bone is smoothed and a layer of cement substance (osteoid) is deposited. The team of osteoblasts that follow the osteoclast attempts to replace exactly as much bone as has been removed. If it is successful in this venture, bone homeostasis will have been maintained. If not, osteoporosis may result. One of the conditions that affects the ability of the bone-resorbing and bone-forming cells to be coupled and result in no net change in bone mass is aging. During the process of remodeling, changes in the amount of bone are slow and changes in the shape of bone are barely perceptible. According to the model proposed by Parfitt, the normal remodeling sequence in bone follows a scheme of quiescence, activation, resorption, reversal, formation, and return

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FIGURE 14

High-power scanning electron micrograph of an intersection of bone trabeculae showing multiple resorption sites. (Courtesy of S. Goldstein, University of Michigan.)

to quiescence (see Chapter 15). In the adult, approximately 80% of trabecular and approximately 95% of intracortical bone surfaces are inactive with respect to bone remodeling [15,16]. The surface of bone is covered by a layer of thin flattened lining cells approximately 15 m in diameter, which arise by terminal transformation of osteoblasts. Between these lining cells and bone is a layer of unmineralized osteoid. These lining cells have receptors for a variety of substances, which are important for initiating bone resorption (PTH, PGE2), and may respond to such substances by resorbing this surface osteoid, which is covering the bone. In doing so, mineralized bone will be exposed and the activation sequence of bone remodeling may be initiated. The conversion of a small area of bone surface from quiescence to activity is referred to as activation. The cycle of this response begins with the recruitment of osteoclasts followed by the initiation of mechanisms for their attraction (chemotaxis) and attachment to the bone surfaces. Several known growth factors may be active in promoting chemotaxis. In addition, several proteins are known to be attachment factors for osteoclasts, such as those that contain the RGD amino acid sequences as noted earlier. Osteopontin, osteocalcin, and osteonectin

may be important proteins in this process (see Chapters 3 and 4). In the adult skeleton, activation occurs about every 10 s. For intracortical remodeling, osteoclast precursors travel to the site of activation via the circulation, gaining access to the site by either a Volkmann or a haversian canal. In trabecular remodeling, activation occurs at sites that are apposed to bone marrow cells. The osteoclast is a very mobile cell that can resorb bone over an area approximately two to three times the area with which it is in direct contact. In cortical bone, the osteoclast and the cutting cone travel at a speed of about 20 or 40 m per day, roughly parallel to the long axis of the bone and about 5 to 10 m per day perpendicular to the main direction of advance [7]. In trabecular bone, osteoblasts erode to a depth of about two-thirds of the final cavity; the remainder of the cavity is eroded more slowly by mononuclear cells [17]. The reversal phase is a time interval between the completion of resorption and the initiation of bone formation at a particular skeletal site. Under normal conditions, it lasts about 1 to 2 weeks. The appearance of new osteoblasts at the base of the resorption cavity depends on chemotaxis for these osteoblasts and their progenitors, as

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well as conditions that stimulate proliferation. Hence chemotaxis, attachment, proliferation, and differentiation occur in a stepwise and concerted fashion in order for bone formation ultimately to take place. Electron microscope studies of the reversal phase have shown the release of osteocytes and the appearance of mononuclear phagocytes, some of which appear to be smoothing over the ragged surface left by the resorption process [18]. Because the coupling mechanism appears to be important to this bone remodeling process, the release of proteins from the bone matrix may be important in coordinating the activity between osteoclasts and osteoblasts. Unlike bone resorption, bone formation is a two-step process. First, the osteoid is synthesized and laid down at specific sites. Following this, the osteoblast must mineralize this newly formed protein matrix. The new matrix begins to mineralize after about 5 to 10 days from the time of deposition and, as a result, matrix apposition and mineralization are systematically out of step as the osteoid seam first increases and then decreases in thickness. The rate of mineral apposition can be measured directly in vivo after double tetracycline labeling; the mean distance between fluorescent bands divided by the time interval between the midpoints of the tetracycline labels can be used to calculate the mineral apposition rate [15].

V. BONE BIOMECHANICS A. Basic Concepts A basic knowledge of biomechanics must begin with an understanding of the terms stress and strain. These terms are used to describe the phenomenon whereby, when a force is applied to bone, the bone will not only be deformed from its original shape, but an internal resistance will be generated to counter the applied force. This internal resistance is known as stress and it is equal in magnitude but opposite in direction to the applied force. Because stress is distributed over the entire cross-sectional area of a section of bone, it is expressed as units of force per unit area. Strain is a term used to describe the changes in shape that bone experiences when it is subjected to an applied force. Strain is dimensionless and is therefore reported as a fraction or a percentage. It is equivalent to the change in length divided by the original length of the section of bone. Although an applied force can be directed at bone from any angle producing any set of complex stress patterns, all stresses can be resolved into three types: tension, compression, and shear Tension is produced in bone when two forces are directed away from each other along the same straight line. The resistance to a loading situation of this type is produced by intermolecular attractive forces, which

prevent the bone from being pulled apart. An example of a tensile force producing a failure in bone occurs when a tendon or ligament that is inserted into bone undergoes acute loading and, instead of failing within its own substance (tearing), it detaches itself from the bone by actually pulling a piece of bone off with it. Compression results from two forces that are directed toward each other along the same straight line. The common vertebral compression fracture sustained in osteoporotic patients is an example of the failure of bone as a result of this type of loading configuration (Fig. 15). Finally, when two forces are directed parallel to each other but not along the same line, shear stresses are produced. In nature, the three basic stress types can combine as a result of a variety of complex loading configurations and lead to different fracture patterns (Fig. 16). Bending results from a combination of tensile and compressive forces and leads to clinical fractures that show a predominantly transverse fracture pattern. Torsion or twisting produces shear stresses along the entire length of a bone and can result in spiral fractures. Comminuted fractures appear as shattered bones and this occurs because the amount of energy transmitted to the bone is so great that a variety of fracture lines have to be propagated in order for it to be dissipated. Because stress and strain are properties related to the quality of the tissue experiencing the load, the quality of bone can influence the magnitude of the stresses and strains generated. In normal, well-mineralized bone, physiological stresses will generally result in small strains. In poorly mineralized tissue, such as osteomalacic bone, bone will experience larger strains in response to the same stress. Moreover, because in nature, forces are applied to bone not only from perpendicular and horizontal directions but also from oblique angles, conditions will arise in which a variety of complex mechanical relationships will be generated. When bones show different mechanical properties if loads are transmitted from different directions, they express a property known as anisotropy [22]. In general, bone resists loads best when the loads are oriented in the direction of customary loading. For example, the femur is much better adapted to resisting compressive loads than bending loads [20]. Thus, the same stresses generated in the femur if one were to jump from a 4foot wall and land on his feet (compressive stresses) may fracture the femur if they were oriented from a transverse direction(bending stresses). In the proximal femur (hip joint area), loads are best resisted when they are transmitted along lines that are parallel to the trabecular systems [21].

B. Biomechanical Properties The biomechanical properties of bone can be described at two levels. First, the material properties of bone are

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CHAPTER 1 The Bone Organ System

FIGURE 15

Sagittal section of a vertebral compression fracture. Note collapse of the vertebra through the central trabecular

section.

FIGURE 16

Fracture patterns in a cylindrical section of bone subjected to different complex loading configurations. (a) Pure tensile loading produces a purely transverse fracture. (b) Pure compressive loading produces an oblique fracture. (c) Torsional loading produces a spiral fracture. (d) Bending, a combination of compressive and tensile loading, produces an essentially transverse fracture with a small fragment on the concave side. (e) Bending in which compressive forces make a greater contribution to the complex loading configuration, leading to a transverse fracture with a larger fragment on the concave side.

defined by the tissue level qualities of the tissue, which are independent of its structure or geometry. Second, there are structural properties in bone, which are manifest when bone functions as a whole anatomical unit. Classically, the material properties of bone are defined by performing standardized mechanical tests on uniform, machined specimens of bone. Structural properties are determined on whole sections of bone whose normal geometry has been maintained. It is important to recognize that when a patient sustains a fracture, that event most likely represents the failure of bone at either the material or the structural level or both. When a uniform section of bone is tested under controlled laboratory conditions, and the applied forces and deformations are known, four basic mechanical properties of bone can be derived from a plot of the stress – strain relationship. These properties are stress, modulus, energy absorptive capacity, and deformation. By convention, stress is plotted on the ordinate (y axis) and strain on the abscissa (x axis). Figure 17 shows a stress – strain plot of an idealized material. Considering that combinations of the three types of stress can produce different stress patterns as a result of different types of externally applied loads (tension, compression, bending, and torsion), the terms used to define the parameters of the y axis can include any of these loading conditions. Under these circumstances, the stress – strain

18

FIGURE 17 A standard stress/strain curve of bone loaded in bending. The linear portion of the curve represents the elastic region and the slope of this part of the curve is used to derive the stiffness of the bone. Loading in this region will result in nonpermanent deformation, and the energy returned to the bone when the load is removed is known as resilience. The nonlinear portion of the curve represents the plastic region in which the bone will be permanently deformed by the load. The junction of these two regions defines the yield point and the stress here is known as the elastic limit. The maximum stress at the point of failure is known as the ultimate strength of the bone. The maximum strain at this point represents the bone’s ductility. The area under the curve is known as the strain energy, and the total energy stored at the point of fracture defines the toughness of the material. Reprinted with permission from T. A. Einhorn, Calcif. Tissue Int. 51, 333 – 339 (1992).

curve is actually a load versus deformation relationship whereby the y axis could be labeled as torque, compressive load, tensile force, or shear. At low levels of stress there is a linear relationship between the applied load and the resultant deformation. This proportionality is known as the modulus of elasticity or Young’s modulus. It is a measure of the rigidity of the bone tissue and is equivalent to the slope of the linear part of the curve. It is calculated by dividing the stress by the strain at any point along this straight line. This linear part of the curve is also known as the elastic region. The physiological significance of this property relates to the fact that forces applied to bone at any point along this line will only deform the bone temporarily. After the load is removed, it will return to its original shape. At the point where the curve becomes nonlinear, the elastic region ends and the stress at this point is known as the elastic limit. Further loading beyond this point will result in a permanent deformation in the material and this property is known as plasticity. This part of the curve is known as the plastic region. Not all materials (and, for that matter, not all bones) have significant plastic properties. Instead, a material may exhibit elastic deformation but, upon reaching its yield point, will fail. This type of material is considered to be brittle.

LEE AND EINHORN

The maximum height of the curve defines the maximum stress in the bone tissue and is the point at which the material fails. The strain at the point of failure is known as the ductility. The area under the curve is a measure of the energy absorptive capacity or strain energy. This energy is dissipated when the bone fractures and is lost at the point of failure. Note that energy stored by the bone up to the point where it reaches its elastic limit is known as resilience. This energy is recovered if the bone returns to its original shape after the load is removed. When whole bones are subjected to experimental or physiological loading conditions, their mechanical behavior is dependent not only on the mass of the tissue and its material properties, but also on its geometry and architecture. When whole bones are loaded to failure, they, too, produce curves that are configured like the curve shown in Fig. 21. However, under these conditions, the slope of the linear portion defines the stiffness of bone. Strength is determined by extrapolating the height of the curve to the y axis. Depending on the loading conditions, strength can be expressed in terms of compressive, bending, tensile, or torsional strength. The area under the curve now defines the toughness of the bone. Of particular interest in the study of metabolic bone diseases are fractures of the vertebral bodies. Here, the vertebrae fracture as a result of predominantly axially directed compressive loads. The reduced load-bearing capacity of each vertebra is related to the material properties of the bone as well as the way in which the vertebral trabeculation is altered through the processes of postmenopausal and agerelated bone loss. Studies have shown that it is the removal of the horizontal trabeculae or lateral support cross-ties that alter the architectural arrangement and lead to reduced load-bearing capacity [22]. As a result, the vertical trabeculae begin to behave as columns and, as such, are subjected to critical buckling loads [23]. A 50% reduction in the cross-sectional area contributed by these horizontal trabeculae will be associated with a 75% reduction in the loadbearing capacity of the vertebral body [24]. Most fractures occurring in nature result from a combination of axial compression, bending, and torsion. In bending or torsion, the cross-sectional area of a structure is more important in resisting loads than its mass or density [25]. Ideally, in bending or torsion, bone should be distributed as far away from the neutral axis of the load as possible. The geometric parameter used to describe this phenomenon in bending is the “areal moment of inertia’’ [25]. Similarly, in torsion, deformation would be resisted more efficiently if bone were distributed further away from the neutral axis of torsion. This property is known as the “polar moment of inertia’’ [25]. During aging, the outer cortical diameter of bone increases and the cortical wall diameter becomes thinner [26,27]. This results from the combined effects of increased endosteal resorption and periosteal bone formation. Although the net effect may be cortical thinning, the

CHAPTER 1 The Bone Organ System

increased diameter of the bone distributes the material further from the neutral axis and improves its resistance to bending and torsional loads.

VI. SUMMARY Bone is a mechanically optimized organ system whose composition and organization reflect the functional demands made upon it. Far from being an inert substance, it is also a living tissue that serves several important functions in the organism. As a biological entity, bone tissue is a composite material composed of a proteinaceous extracellular matrix or ground substance that has been impregnated by an inorganic calcium phosphate mineral phase. In this sense, it can be likened to a material such as fiberglass with flexibilities, rigidities, and other mechanical properties related to the composite nature of its components. However, what distinguishes a material like bone from other composite tissues and materials is the fact that it is constantly being broken down and rebuilt in the process known as remodeling. The cellular link between bone resorbing cells, osteoclasts, and bone-forming cells, osteoblasts, may be regulated by the release of small molecules from the extracellular matrix during bone resorption. The complexities of the remodeling process require further investigation, but current knowledge suggests that it is composed of several phases, including quiescence, activation, resorption, reversal, and formation. Although the transduction of mechanical signals through bone and the stimulation of bone cells by hormonal agents have been studied extensively, more information is needed before a truly comprehensive understanding of the bone organ system can be developed. Ultimately, it would be important to know how tissue development, cellular function, mechanical input, and the material properties of the tissue are organized in time, space, and function in order to maintain bone homeostasis. The information and concepts described in this chapter form the basis for understanding bone as a tissue as well as an organ system. The ability to maintain homeostasis, prevent bone resorption, and, perhaps more importantly, enhance bone formation will be essential to the management of osteoporosis now and in the future.

References 1. I. A. Bab and T. A. Einhorn, Polypeptide factors regulating osteogenesis and bone marrow repair. J. Cell Biochem. 55, 358 – 365 (1994). 2. M. Horowitz and R. L. Jilka, Colony stimulating factors in bone remodeling, In “Cytokines and Bone Metabolism” (M. Gowen, ed.), pp. 185 – 227. CRC Press, Boca Raton, FL, 1992. 3. J. Wolff, In “Das Gesetz der Transformation der Knochen” (A. Hirchwald, ed.). Berlin, 1892. 4. T. A. Einhorn, Bone metabolism and metabolic bone disease. In “Orthopaedic Knowledge Update 4 Home Study Syllabus” (J. W. Frymoyer, ed.), pp. 69 – 88. Am. Acad. Orthop. Surg., Rosemont, 1994.

19 5. E. Ruoslahti, Integrins. J. Clin. Invest. 87, 1 – 5 (1991). 6. F. S. Kaplan, W. C. Hayes, T. M. Keaveny, A. L. Boskey, T. A. Einhorn, and J. P. Iannotti, Form and function of bone, In “Orthopaedic Basic Science” (S. R. Simon, ed.), pp. 127 – 184. Am. Acad. Orthop. Surg., Rosemont, 1994. 7. T. A. Einhorn and R. J. Majeska, Neutral proteases in regenerating bone. Clin. Orthop. 262, 286 – 297 (1991). 8. S. P. Bruder and A. I. Caplan, Terminal differences of osteogenic cells in the embryonic chick tibia is revealed by a monoclonal antibody against osteocytes. Bone 11, 189 – 198 (1990). 9. T. J. Martin, E. Romas, and M. T. Gillespie, Interleukins in the control of osteoclast differentation. Crit Rev. Eukaryot. Gene Expr. 8, 107 – 23 (1998) 10. Y. Y. Kong, H. Yoshida, I. Sarosi, H. L. Tan, E. Timms, C. Capparelli, S. Morrony, A. J. Oliveirados-Santos, G. Van, A. Itie, W. Khoo, A. Wakeham, C. R. Dunstan, D. L. Lacey, T. W. Mak, W. J. Boyle, and J. M. Penninger, OPGL is a key regulator of osteoclastogenesis, lymphocyte development and lymph-node organogenesis. Nature (England) 397, 315 – 323, (1999). 11. N. Nakagawa, M. Kinosaki, K. Yamaguchi, N. Shimma, H. Yasuda, K. Yano, T. Morinaga, and K. Higasio, RANK is the essential signaling receptor for osteoclast differentiation factor in osteoclastogenesis. Biochem Biophys Res. Commun. 253, 395 – 400 (1998). 12. Y. Hakeda, Y. Kobayashi, K. Yamaguchi, H. Yasuda, E. Tsuda, K. Higashio, T. Miyata, and M. Kumegawa, Osteoclastogenesis inhibitory factor (OCIF) directly inhibits bone-rresorbing activity of isolated mature osteoclasts. Biochem Biophys Res Commun. 251, 796 – 801 (1998). 13. A. M. Parfitt, Bone remodeling: Relationship to the amount and structure of bone, and the pathogenesis and prevention of fractures, In “Osteoporosis: Etiology, Diagnosis, and Management” (B. L. Riggs and L. J. Melton, eds.), pp. 45 – 93. Raven Press, New York, 1988. 14. J. Kragstrup and F. Melsen, Three-dimensional morphology of trabecular bone osteons reconstructed from serial sections. Metab. Bone Dis. Relat. Res. 5, 127 – 130 (1983). 15. A. M. Parfitt, The psychological and clinical significance of bone histomorphometric data. In “Bone Histomorphometry. Techniques and Interpretations” (R. Recker, ed.) pp. 143 – 223. CRC Press, Boca Raton, FL, 1983 16. A. M. Parfitt, The cellular basis of bone remodeling: The quantum concept reexamined in light of recent advances in cell biology of bone. Calcif. Tissue Int. 36, S37 – S45 (1984). 17. E. F. Eriksen, F. Melsen, and L. Mosekilde, Reconstruction of the resorptive site in iliac trabecular bone: A kinetic model for bone resorption in 20 normal individuals. Metab. Bone Dis. Rel. Res. 5, 235 – 242 (1984). 18. A. M. Parfitt, A. R. Villaneuva, M. M. Crouch, C. H. E. Mathews, and H. Duncan, Classification of osteoid seams by combined use of cell morphology and tetracycline labeling: Evidence for intermittency of mineralization. In “Bone Histomorphometry: Second International Workshop” (P. J. Meunier, ed.), pp. 299 – 310. Armour Montagu, Paris, 1977. 19. L. J. Melton, E. Y. S. Chao, and J. M. Lane, Biomechanical aspects of fractures. In “Osteoporosis: Etiology, Diagnosis and Management” (B. L. Riggs and L. J. Melton, eds.), pp. 111 – 131. Raven Press, New York, 1988. 20. A. H. Burstein, D. T. Reilly, and M. J. Martens, Aging of bone tissue: Mechanical properties. J. Bone Jt. Surg. 58A, 82 – 86 (1976). 21. T. D. Brown and A. B. Ferguson, Jr., The development of a computational stress analysis of the femoral head. J. Bone Jt. Surg. 60A, 619 – 629 (1978). 22. Li, Mosekilde, A. Viidik, and L. E. Mosekilde, Correlation between the compressive strength of iliac and vertebral trabecular bone in normal individuals. Bone 6, 291 – 925 (1985).

20 23. P. R. Townsend, Buckling studies of single human trabeculae. J. Biomech. 8, 199 – 201 (1975). 24. H. Yamada, “Strength of Biological Materials” (F. G. Evans, ed.). Williams and Williams, Baltimore, MD, 1970. 25. T. A. Einhorn, Bone strength: The bottom line. Calcif. Tissue Int. 51, 333 – 339 (1992). 26. R. W. Smith and R. Walker, Femoral expansion in aging women: Implications for osteoporosis and fractures. Henry Ford Hosp. Med. J. 28, 168 – 170 (1980).

LEE AND EINHORN 27. C. B. Ruff and W. C. Hayes, Superiosteal expansion and cortical remodeling of the human femur and tibia with aging. Science 217, 945 – 948 (1982). 28. D. B. Phemister, The pathology of ununited fractures of the neck of the femur with special reference to the head. J. Bone Jt. Surg. 21, 681 – 693 (1939). 29. A. M. Pankovich, Primary internal fixation of femoral neck fractures. Arch. Surg. 110, 20 – 26 (1975).

CHAPTER 2

Osteoblast Biology JANE B. LIAN AND GARY S. STEIN Department of Cell Biology, University of Massachusetts Medical School, Worcester, Massachusetts 01655

I. Overview II. Embryonic Origins and Signaling Cascades for Osteogenesis III. In Vivo Tissue Level Organization of Osteoblasts: Phenotypic Features and Function IV. Cellular Cross-Talk of Osteoblast Lineage Cells: Functions in Calcium Homeostasis, Bone Turnover, and Hematopoiesis

V. Osteoblasts in Vitro: Stages in Development of the Osteoblast Phenotype VI. Molecular Mechanisms Mediating Progression of Osteoblast Differentiation VII. Concluding Remarks References

I. OVERVIEW

of genes associated with the biosynthesis, organization, and mineralization of the bone extracellular matrix. This chapter provides a perspective of the signaling pathways and molecular mechanisms mediating osteoblast growth, differentiation, and activity that serve as a basis for understanding the factors regulating bone development and growth, the continued remodeling of bone, and the regeneration of injured tissue. Recent advances in the identification of obligatory factors for development of the skeleton that also contribute to osteoblast growth and differentiation in the adult skeleton will be presented. During the past decade, numerous model systems have been developed to study the cell biology of bone and gain insight into various cell types important for bone formation and function. The advantages and limitations of recently described in vivo and cellular models to address physiological and molecular mechanisms regulating osteoblast growth and differentiation will be discussed. Knowledge of unique properties and definition of the mechanisms that control progression through the osteoblast cell lineage will allow a rational intervention for abnormalities in skeletal development, fracture repair, pathologies of metabolic bone diseases, and implant stability. These are basic biological questions and

Bone formation takes place not only during embryonic development and growth but throughout life to support normal bone remodeling and fracture repair. The requirement for continuous renewal of bone, through the remodeling process involving resorption and formation, necessitates recruitment, proliferation, and differentiation of osteoblast-lineage cells. Subpopulations of osteoblasts are recognizable in vivo morphologically in relation to tissue organization and exhibit subtle phenotypic differences with respect to the expression of genes and responses to physiologic mediators of bone formation. This chapter presents current understanding of the phenotypic definition of the spectrum of bone-forming cells with respect to their functional properties and responses. It is now apparent that growth factor, cytokine, and hormone responsive regulatory signals mediate competency for the expression of genes associated with metabolic responses as a function of the stages of osteoblast growth and differentiation. Osteoblast differentiation is a multistep series of events modulated by an integrated cascade of gene expression that initially supports proliferation and the sequential expression

OSTEOPOROSIS, SECOND EDITION VOLUME 1

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Copyright © 2001 by Academic Press. All rights of reproduction in any form reserved.

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concerns of today’s clinician for the treatment of bonerelated disorders.

II. EMBRYONIC ORIGINS AND SIGNALING CASCADES FOR OSTEOGENESIS The complexities of bone formation are immediately apparent in the embryo where different regions of the skeleton arise from specific primordial structures and skeletogenesis involves two different processes. Intramembranous bone formation, as occurs in the development of the flat bones of the skull, results from the differentiation of mesenchymal cell condensations directly to osteoblasts. The endochondral sequence of bone formation, as occurs for all long bones, involves the differentiation of mesenchymal progenitors first to form a cartilage template of the bone, which is then replaced by bone. Progenitors of the boneforming cells for all osseous tissues derive from the mesodermal germ cell layer. The dorsal paraxial mesoderm gives rise to somites and the sclerotome, which is a source of cells for most of the axial skeleton (vertebrae, skull, ribs, sternum), the lateral plate mesoderm gives rise to the appendicular skeleton (limbs), and the cephalic mesoderm gives rise to the neural crest, which provides progenitor cells for facial skeletal structures. Thus, different regions of

FIGURE 1

the skeleton have distinct embryonic lineages reflecting origins from these specific primordial structures. Considering these different embryonic developmental programs of the mesoderm to form intramembranous bone and subtypes of endochondral bone (e.g., limbs and vertebrae), an early osteoprogenitor may divert from a stem cell at these specific skeletal sites. It has been shown that axial and appendicular-derived osteoblasts exhibit different responses to hormones [e.g., 1,2]. It remains to be determined whether this selective activity reflects the tissue environment or inherent properties of the cells selected at an early stage during osteoblast differentiation. Our understanding of skeletal patterning and limb development has been expanded significantly by characterization of the signaling factors and transcription factors that serve as morphogenic determinants of bone formation [3 – 8]. Well-documented interactions between epithelium and mesenchyme are first necessary for tissue differentiation [9] and are reviewed elsewhere [10]. Signals essential for limb patterning arise from the zone of polarizing activity, which resides in the posterior limb mesoderm, underlying the epithelial apical ectodermal ridge (AER) of the developing limb bud. Gradients of morphogenic factors and integration of the various regulatory cascades reflect the complexity of skeletal development (Fig. 1). Growth factors and their receptors in the transforming growth factor- (TGF-) superfamily, epidermal growth factor (EGF), and the fibroblast

Signaling molecules regulating position and pattern formation and osteoblast differentiation during development of the skeleton. Encircled regulators, the FGF family, BMPs, Hedgehogs, and HOX genes, can be considered major signaling “centers” in that they induce (arrows) the expression of several different  classes of genes essential for skeletal development. Inhibitory controls are indicated ( ). Interrelationships are evident by feedback loops (e.g., FGFs and Hedgehogs), coordinate positive and negative regulation of common genes, and potential interaction of factors from one center with factors regulated by a different center. Relevant references include 21,22,27,28,47,67 – 70,75 – 77,698 – 705, as well as those described in the text.

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CHAPTER 2 Osteoblast Biology

growth factor (FGF) family are implicated in mesenchymal condensations [11,12]. Basic fibroblast growth factor (FGF-2) and retinoic acid have been characterized as soluble mediators of communication between the AER and mesodermal progress zone [10,13 – 17]. FGF-2 activates several signaling pathways [18], including, for example, Wnt genes [19], Notch ligand expression [20,21], Hedgehog factors, and transcriptional regulators, such as helix – loop – helix proteins [22] (Fig. 1). Indian hedgehog (Ihh) is a key factor in the normal development of endochondral bone formation [23]. It is expressed abundantly in the developing growth plate in the mature and hypertrophic chondrocyte zones [24] is a key regulator of chondrocyte maturation. Ihh signaling is mediated through its receptor patch, and this signaling can be antagonized by a cell surface protein [25]. Several studies indicate that the rate of cartilage differentiation by Ihh is mediated by the parathyroid hormone-related protein (PTHrP) and its receptor [24,26 – 28]. PTHrP supports chondrocyte proliferation. Thus together Ihh and PTHrP regulate the proportions of proliferating and hypertrophic chondrocytes. Indeed, the PTHrP receptor will rescue the Ihh null mouse [27]. Knowledge of how mesenchymal condensations are initiated and grow and how their sizes and boundaries are regulated is being accrued through genetic studies in mice and the characterization of molecular defects in skeletal development [7] (Table 1). These studies have identified several specific players. Extracellular matrix molecules, cell surface receptors, and cell adhesion molecules, such as fibronectin, tenascin, syndecan, and N-CAM, initiate condensation and

TABLE 1

set boundaries for the forming mesenchyme, whereas Hox genes modulate the proliferation of cells within condensations [7]. Significant contributions to these earlier stages of skeletal development are provided by the TGF- superfamily of regulatory factors [reviewed in 29 – 32]. Bone morphogenetic proteins (BMPs) are actively involved in determining parameters of size and shape during mesenchymal cell condensation [33,34]. Selective expression of BMPs regulate mesenchymal condensations [35] and contribute to restriction of options for lineages. BMP-2, BMP-4, and BMP-7 (also designated OP-1) are potent inducers of osteogenesis in vivo and cell differentiation in vitro [36 – 41]. Specificity of the activities of TGF-s with target cells is regulated by their activation of distinct factors. TGF- and BMP bind to distinct receptors; each has associated kinase activity that phosphorylates Smad proteins. Smads 2 and 3 mediate TGF- responses, whereas Smads 1, 5, and 8 are activated by BMP receptors and transduce BMP signals. Interactions between receptor-activated Smads and Smad 4, a DNA-binding Smad, result in translocation of the complex to the nucleus for the transcription of target genes [29,42 – 44]. BMPs regulate several classes of genes that are key factors in normal bone development influencing spatial and temporal events (Fig. 1). These include fibroblast growth factors [45], homeobox containing genes [46 – 49], which contribute to position and pattern formation, cell adhesion proteins [50,51], and transcription factors such as helix – loop – helix [22,52], winged helix [53 – 55], SOX9, which is essential for chondrocyte differentiation [56,57], and RUNX2/core binding factor

Representative Mouse Models Involving Factors Related to Skeletal Development and Bone Formation

Homeobox proteins BAPX1 [648,649] (null)

Expressed in prechondrogenic cells, perinatal lethal skeletal dysplasia; malformations and absence of specific bones

Goosecoid [650] (null)

Craniofacial due to condensation defects

DLX-5 [651,652] (null)

Craniofacial and bone defects, die at birth

Msx-2 [653,654] (null)

Skull ossification defects

Msx-2 [655] (gain of function)

Craniosynostasis

TGF- superfamily TGF- receptor type II (transgenic dominant negative) a. Expressed in osteoblasts [656]

a. Decreased remodeling; increased trabecular bone

b. Expressed in skeleton [657]

b. Osteoarthritis

TGF-2 [658] (transgenic)

Development normal; osteoporosis postnatal

BMP-2 [659] (null)

Failure of mesoderm induction

BMP-4 [660,661] (null)

Die early in gastrulation

BMP-5 [34] (mutant)

Short-ear mouse gene (continues)

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TABLE 1 TGF- superfamily GDF-5 [662] (mutant)

(continued)

Brachypodism; bone and joint development problems

BMP-7 [663,664] (null)

Die after birth; polydactly; rib abnormalities

BMP receptor type I [665] (null)

Die 9.5 days post coital

BMP receptor type IB [666] (null)

Appendicular skeletal defects related to reduced chondrocyte proliferation and differentiation

Bone matrix proteins Osteopontin [667] (null)

Development is normal; increased osteoclastogenesis

Osteocalcin [277,278] (null)

Thickened bone increased mineral, but decreased crystal size

Osteonectin [668,669] (null)

Osteopenia; mesenchymal cell proliferation increased

Thrombospondin [670,671] (null)

Increased vascular density; increased marrow osteoprogenitor; increased cortical density

Biglycan [280] (null)

Thin bones, expression in preosteogenic cells

Decorin [672] (null)

Skin fragility

Alkaline phosphatase [673 – 675] (null)

Arrest of chondrocyte differentiation; defective matrix mineralization

Gelatinase [676] (null)

-3 Integrin [677] (null) BMP-5 [34] (mutant)

Transient aberrant growth plate vascularization and ossification Increased bone mass, but osteosclerotic; defective osteoclasts Short-ear mouse

Growth factors and receptors FGF-2 [261] (null)

Trabecular bone mass decreased; decreased mineralization of bone marrow stromal ex vivo cultures

FGF-2 [260] (transgene)

Increased apoptosis in calvaria

FGF-3 receptors [678,679] (null)

Skeletal overgrowth

(mutations) [680]

Achondroplasias dwarfism

FGF-1 receptor [681,682]

Embryonic growth and mesodermal patterning; axial organization and limb development

PTH/PTHrP receptor [80] (transgene)

Delays endochondral bone formation (EBF)

PTH/PTHrP receptor [28] (null)

Accelerates EBF; increase in osteoblast number and matrix; delay in vascular invasion; decrease in trabecular bone

PTHrP [74] (null)

Die at birth; premature chondrocyte differentiation and accelerated EBF

PTHrP x PTH/PTHrPR [78] (double knockout)

Accelerates differentiation of growth plate chondrocytes and EBFs; no delay in vascular invasion

PTHrP [683] (transgene)

In cartilage, delays chondrocyte maturation; mice are born with cartilaginous skeletons

IGF-1 [684] (transgene)

In bone, increases trabecular bone volume without proliferation

Other regulatory factors Indian Hedgehog [23] (null)

Severe dwarfism in the null mouse

Sonic Hedgehog [685]

A segment polarity gene; absence of distal limbs, most of the ribs and spinal column

Noggin [63] (null)

A BMP antagonist; cartilage hyperplasia

C-Abl [686] (null)

A nonreceptor tyrosine kinase; osteoporotic and osteoblast maturation defect

p27kip1 [253,687,688] (null)

A cyclin-dependent kinase inhibitor; larger size animals and bones; increased osteoprogenitors in bone marrow ex vivo cultures

TWRY [689] (mutant)

Nucleotide pyrophosphatase (NPPS) gene mutation; abnormal calcification of cartilage, spine, tendons, limbs

Transcriptional regulators RAR [690] (transgene)

Interferes with chondrogenesis, appendicular skeletal defect

SOX-9 (null)

An HMG domain TF expressed in cartilage lethal malformation syndrome XY reversal

c-Fos [691] (transgene)

Tumors in cartilage and bone

c-Fos [692] (null)

Osteopetrosis

Fra-1 [693] (transgene)

Progressive increase in bone mass; osteosclerotic

Fra-1 [693,694] (null)

Embryonic lethal from a placental defect

MFH-1 [695]

A forkhead or winged helix transcription factor; embryonic lethal and perinatal with skeletal defects

Plzf [696]

Promyelocytic leukemia zinc finger proteins; a growth inhibitory and proapoptotic factor essential for skeletal patterning

ATF-2 [697]

Chondrodysplasia

CBFA1 [58,59] (null)

A runt domain protein, absence of mineralized tissue essential for osteoblast differentiation

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CHAPTER 2 Osteoblast Biology

A1 (CBFA1), which is essential for skeletal formation [58,59]. When growth of condensation ceases, the next stage of skeletal development, cellular differentiation, initiates. Here, Chordin [60], Noggin, and potentially Gremlin [61] play a critical role in skeletal development by inhibiting BMP2 signaling [62 – 65]. Feedback loops and complementary regulation of many of these factors (Fig. 1) ensure the progression of cellular differentiation, cartilage development, endochondral bone formation, and intramembranous osteogenesis [66 – 70]. Systemic factors are also critical for the early stages of limb formation and influence the renewal of osteogenesis throughout adult life. While parathyroid hormone (PTH) stimulates the growth of osteoprogenitor populations [71], PTHrP functions as a cellular cytokine regulating cell growth for differentiation in development [72,73] and is a key regulator of chondrocyte maturation [74]. PTHrP influences Hox gene expression and both peptide and its receptor are upregulated by TGF- [75 – 77]. Ablation of the PTH/PTHrP receptor in mice [28,74,78] and mutations in the receptor in human [79] reveal its central role in the regulation of endochondral bone formation (EBF). The disorganized growth plate of the receptor knockout mouse can be rescued by expression of the receptor; however, endochondral bone formation becomes delayed [80,81] (Table 1). Commitment of stem cells to specific mesenchymal lineages occurs early in development of the limb. Transcription

FIGURE 2

factors which function as “master switches” mediate cell differentiation by induction of a set of phenotypic genes that characterize the muscle, adipocyte, chondrocyte, or osteoblast cells (Fig. 2). Such regulatory proteins have also been implicated in key roles during the progressive differentiation of osteoblasts through specific stages of maturation (Fig. 2, lower panel). Among the most notable are cfos, a protooncogene, the helix – loop – helix proteins (Twist, Id, Scleraxis), leucine zipper proteins (hXBP-1), zinc finger proteins (zif268), homeodomain proteins (Msx-2, Dlx-5), steroid receptors, and runt domain transcription factors (RUNX2/Cbfa1/AML/PEBP2), which are obligatory for osteoblast differentiation. Modifications in the representation of classes of transcription factors at specific sites during embryogenesis and at different stages of osteoblast differentiation (Fig. 2, bottom) reflect linkage to the transcriptional control of osteoblast phenotype development. Helix – loop – helix factors, negative regulators of osteogenesis, illustrate this point. Id (inhibitor of differentiation), twist, and scleraxis are expressed in mesoderm of developing embryo [82,83]. Scleraxis is expressed in cells that form the skeleton and is not detected at the onset of ossification [83]. Id and Twist expression must be downregulated for osteoblast differentiation to proceed [84,85]; overexpression of these factors inhibits osteogenesis in vitro [86]. Msx-2 and Dlx-5 are members of the homeodomain gene family of transcription factors [87]. Both factors are expressed in mesenchymal cells at sites that will undergo skeletogenesis

Transcriptional control of phenotype lineages from mesenchymal stem cells. (Top) Activation of gene transcription for commitment to the muscle cell phenotype by MyoD to adipocytes by PPAR2 and C/EBP, chondrocytes by SOX9, and osteoblasts by BMP-2 and CBFA1 is illustrated. Several in vitro studies demonstrate that phenotype commitment can be blocked or switched by forced expression of the regulatory factor characteristic of a different lineage (designated ) [175,706 – 710]. (Bottom) Developmental expression of several transcription factors influencing osteoblast differentiation. Thick lines represent highest cellular levels.

26 [88 – 90]. Their importance for normal bone formation is realized by skeletal abnormalities that result from mutations or misexpression of these factors [91; and see Table 1]. Msx-2 must be down regulated for progression of osteoblast differentiation [92,57,8]. The runt homology domain-related core-binding factor RUNX2 has been shown to play an essential role in bone formation in the embryo, demonstrated by the inhibition of mineralized tissue formation in the CBFA1/ RUNX2 null mutation mouse model [58,59]. The family of RUNX/CBFA transcription factors comprises three related genes that each support tissue specification and organogenesis [93– 95]. RUNX1 (CBFA2/AML-1B/PEBP2B) and its partner proteins CBF are critical for hematopoietic cell differentiation [96 – 98]; RUNX3 (CBFA3/AML-2/PEBP2C) is required for gut development (Dr. Y. Ito, Kyoto University, Japan, personal communication); RUNX2 (CBFA1/ AML-3/ PEBP2A) is essential for the differentiation of osteoblastic cells for formation of the mineralized skeleton. In human, these factors were first designated as AML because they were identified as the protein encoded by a gene locus rearranged in acute myelogenous leukemia (AML). Other names include core-binding factor  (CBFA) and polyoma enhancer-binding protein (PEBP2). The Human Genome Nomenclature Committee has now referred to this family as RUNX. CBFA1 was initially identified in bone as an osteoblastspecific DNA-binding protein that activated transcription of the tissue-specific osteocalcin gene [99 – 103] (also see Chapter 6). The obligatory role of CBFA1 for the formation of mature bone in the developing skeleton has been shown by the absence of a calcified skeleton and bone formation in CBFA1 null mutant mice [58,59]. The heterozygous mice exhibited phenotypic features akin to mouse models [104] and human cleidocranial dysplasia (CCD) abnormalities. Various mutations in CBFA1 were then identified in patients with CCD [105 – 108]. CBFA1 is expressed not only in abundance in osteoblasts and hypertrophic chondrocytes, but in cartilage [109 – 111], thymus [112], and testis [113], as well as early stage osseous and chondroprogenitor cells Marrow mesenchymal stromal cells lacking overt expression of chondrocyte or osteoblast markers and several nonosseous mesenchymal cell lines also have significant CBFA1 expression [114 – 116]. The transient expression of CBFA1 in early embryogenesis, followed by an upregulation in late stages of bone development [58,102,103], suggests that CBFA1 may be important in both early specification of the mesenchymal stromal phenotype and in supporting the final stages of osteoblast differentiation [103,117]. Evidence is also provided by the observations that RUNX antisense blocks in vitro differentiation of osteoblasts to the final mineralization stage [103]. Furthermore, CBFA1 can induce the expression of bone-related genes in nonosseous

LIAN AND STEIN

cells [102,103,118]. Finally, a dominant-negative CBFA1 mutant protein, expressed only in mature osteoblasts by the osteocalcin promoter, resulted in osteopenic bone due to decreased osteoblast activity [117]. The activities of growth regulators of skeletal development, FGFs, TGF-1, or BMP and CBFA1, appear to be closely linked. Activating mutations in FGF receptors cause premature fusion of the calvarial sutures in human disorders [119] and in a recently described mouse model [120]. FGF expression in this disorder is linked to expression of CBFA1. FGFR1 and ligands FGF2 and FGF8 induce CBFA1 in vivo and in C3H10T1/2 cells. Several reports have shown that CBFA1 is a downstream target of BMP-2 in osteoblastic and nonosseous cell lines [114,118,121,122] and BMP-7 [102]. Evidence has been presented demonstrating a protein – protein interaction of the CBFA2 factor with Smad1 or Smad 5 for functional cooperativity of TGF-mediated gene transcription [123]. Furthermore, Smad2 and CBFA1 cooperativity in osteoblasts has been shown [124]. These studies reflect cross-talk between two classes of signaling factors essential for osteogenesis, the Smad proteins, which mediate TGF-1 and BMP activities, and CBFA1 factors. Other studies suggest that CBFA1 induction of osteogenesis may require a BMP responsive factor. For example, while both TGF and BMP-2 induce CBFA1 expression [121] in the premyoblastic C2C12 cell line, only BMP-2 leads to expression of the osteoblast phenotype. Thus, BMP-2, but not TGF-1 signaling, leads to a factor that, together with CBFA1, may be required to mediate osteogenesis. This concept is further supported by the ability of BMP-2 to activate osteocalcin in calvarial cells from CBFA1 null mice [58]. In addition, subclones of the osteoblastic MC3T3-E1 cell line, which expressed CBFA1, were found incompetent for the synthesis of a mineralized bone ECM [125]. Thus, while CBFA1 gene ablation studies in mice revealed that CBFA1 is necessary for bone formation and can activate some osteogenic genes in nonosseous cells, CBFA1 may not be sufficient for de novo skeletal formation. We still do not understand if CBFA1 can provide a cell with the same cascade of signals induced by the bone morphogenetic proteins necessary for bone formation. In summary, our present knowledge of signaling factors and transcriptional regulators of bone development is growing. The significance of many of these growth factors and morphogens in regulating the progression of the pluripotent stem cell and multipotential mesenchymal cell to the committed osteoprogenitor and finally to recognizable osteoblasts is appreciated from mouse models in which these genes or receptors for these proteins have been expressed in transgenic mouse models or ablated (null mutations) with consequences on formation of the skeleton (summarized in Table 1). However, identification of the sequelae of events and integration of their activities can only be postulated at the present time. It is apparent that the convergence of multiple pathways and the

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CHAPTER 2 Osteoblast Biology

coordination of activities are operative through complex feedback loops as illustrated above (Fig. 1), not only in development, but in renewal of osteogenesis in the adult [126,127].

III. IN VIVO TISSUE LEVEL ORGANIZATION OF OSTEOBLASTS: PHENOTYPIC FEATURES AND FUNCTION Based on morphological and histological studies, osteoblastic cells are categorized in a presumed linear sequence progressing from osteoprogenitor cells to preosteoblasts, which mature to osteoblasts and then to lining cells or osteocytes [128 – 134] (Fig. 3). There is a gradient of differentiation that can be observed morphologically either in the periosteum or in the marrow as the osteoprogenitor cell reaches the bone surface and the osteoblast phenotype becomes fully expressed. Osteoblasts that are derived from proliferating osteoprogenitors can be observed in clusters at the bone surface (Fig. 4, see also color plate). These cells synthesize the bone extracellular matrix, designated osteoid (Fig. 5, see also color plate). In metabolic bone disorders leading to decreased calcium or phosphate deposition in bone, as in vitamin D deficiency, wide osteoid seams are evident (Fig. 5B). Mineralization leads to the

FIGURE 3

final stage of osteoblast differentiation. When the boneforming osteoblast becomes encased in its own mineralized matrix, they are osteocytes. On a quiescent bone surface, the osteoblast flattens to a lining cell, forming an endosteum. Four forms of the osteoblast cell lineage are thus recognized in vivo. They are the committed progenitors (preosteoblasts), mature osteoblasts, osteocytes, and the bone-lining cell (Figs. 4 and 5). Particularly important in defining phenotypic differences is an understanding of gene expression and protein localization at the single cell level in relation to the development of bone tissue organization. During the past, high-resolution in situ hybridization methods have been developed for bone sections that effectively support the assessment of mRNA levels in specific cells and with respect to intracellular localization [135 – 139]. The application of immunological detection methods [140 – 142] to sections of intact bone has supported the establishment of linkage between patterns of gene expression observed in culture with those that occur developmentally in tissue. Distinct gradients of particular markers are evident, a phenomenon that may be related to a coupling of cell polarity with physiological function [143]. Polymerase chain reaction (PCR) methods have been applied successfully to determinations of cellular RNAs in single cells [144], and in situ PCR protocols offer the potential for extending this approach [145,146]. Variable levels of expressed genes are observed in neighboring cells.

Growth and differentiation of osteoblast lineage cells. Progression through the osteoblast lineage from a pluripotent mesenchymal stem cell to a mature osteocyte is regulated by numerous physiologic mediators, including, but not limited to, transforming growth factor- (TGF-), superfamily members, steroid hormones, growth regulators, and transcription factors. Each of these subpopulations of osteoblasts have many common features, but exhibit distinct different properties/functions and express osteoblast phenotypic genes to varying extents (see text for details).

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FIGURE 4 Organization of osteoblasts in bone tissue. (A) Intramembranous bone formation (40). (B) Bone-forming surface (200), Goldner and von Kossa stain shows (A) osteoblast clusters and (B) surface osteoblasts, preosteocytes below the osteoid (blue), and mineralized tissue (black) with an osteocyte in its lacunae. Osteoprogenitor cells are evident at higher magnification behind the large surface cuboidal osteoblasts. Osteocyte cellular processes that extend through canaliculi cannot be seen with light level microscopy. (See also color plate.)

Viable approaches are thereby provided for investigating protein functions within multiple contexts that range from transactivation in the nucleus to support of extracellular matrix mineralization. The following summarizes the morphologic and phenotypic functional features of each osteoblast population. For a more detailed description of bone cell ultrastructure, the reader is referred to Refs. 130,132, 134, and 147.

A. Osteoprogenitors and Preosteoblasts: More Cells Build Bigger Bones Progression of the most primitive pluripotent cell to the undifferentiated multipotential mesenchymal cell and presumed osteoprogenitor is not understood. The expression and subsequent differentiation of the early osteoprogenitor

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FIGURE 5 Mineralization defects due to vitamin D deficiency and low serum calcium results in osteomalacia. Cortical bone sections from a 6week control rat (A) and a rat maintained on a vitamin D-deficient diet from weanling to 6 weeks. (B) Osteoblast differentiation from the periosteal and endosteal surfaces is seen, as well as a wide osteoid due to a much greater decrease in the rate of mineral deposition. Toluidine blue/von Kossa stain (200). (See also color plate.)

cells should be considered within the context of embryologic development, bone formation during growth, and bone tissue remodeling in the adult skeleton. Under each of these circumstances, progenitor cells must be responsive to a broad spectrum of regulatory signals that mediate their proliferation, commitment, and progression of phenotype development, as well as sustaining their structural and functional properties. In fully developed bone, there is a requirement for utilization of the same factors that can mediate the growth and differentiation of osteoprogenitor cells during skeletal development, as well as for osteoblast differentiation during bone remodeling and fracture healing in the adult [127,148]. From a developmental perspective, mesenchymalderived osteoprogenitor cells arise/reside in the periosteal tissue or the bone marrow stroma. The marrow and its stromal “bedding” give rise to multipotential cells of both

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hematopoietic lineage (origin of osteoclasts) and nonhematopoietic lineage cells from which many tissuespecific cells derive, such as chondrocytes, myoblasts, and adipocytes. When suspensions of marrow cells are plated in vitro, clonal colonies of adherent fibroblasts are formed; each derived from the single cell that has been designated as the colony-forming fibroblastic unit or CFU/F [149,150]. Formation of CFUs requires the presence of hematopoietic cells [151]. A proportion of these cells have high proliferative and differentiation capacity and exhibit characteristics of stem cells when transplanted in the closed environment of a diffusion chamber [152] or transplanted into the circulation [153]. Studies have demonstrated that marrow stromal cells can be transfected with reporter genes and then transplanted either into specific tissue sites (Subcutaneous, bone tumors, fracture sites) or systemically (e.g., by tail vein infusion) and they maintain competency for differentiation in vivo [154]. For clinical applications of genetically engineered cells for the local reconstruction of bone tissue [155 – 159] or to treat systemic bone diseases [153,160 – 163], in vitro expansion and modification of the cells forming adherent marrow colonies are important considerations. However, mechanisms related to the retention of in vivo properties, homing, engraftment, and differentiation after transplantation must be addressed [159,164 – 166]. Many groups have shown the adherent marrow population differentiates in vivo and in vitro to several mesenchymal lineage cells: adipocytes, chondrocytes, osteoblasts, and myoblasts [reviewed in 167,116,168 – 174]. The plasticity of these lineages is indicated by several lines of evidence. Forced expression of the transcription factors that function as “master switches” (Fig. 2) in phenotype commitment can transdifferentiate a cell to a different phenotype. The reciprocal relationship between adipocyte and osteoblast differentiation is suggested by such studies [115,175]. Forced expression of PPAR2 in marrow stromal cell lines results in the inhibition of terminal osteoblast differentiation with concomitant downregulation of CBFA1 [115,175]. The bipotential property of the late stage osteoprogenitor or preadipocyte [176 – 183] is markedly sensitive to biological regulatory signals influencing “master switch” transcription factor expression. Regulatory signals influencing osteogenesis in preference to adipogenesis can include BMP-2 and the BMP receptor [115,179,182, 184 – 187] and TGF-1 [188]. Retinoid signaling pathways [189] and leptin signaling (through the central nervous system) [181,190] favor adipogenesis. In contrast, 1,25(OH)2D3 inhibits adipogenesis [191]. One molecular mechanism for commitment to the osteogenic or adipogenic lineage which has been identified involves the role of mitogen-activated protein kinase family members (ERK, JNK, and p38). ERK functions as positive regulators of osteogenic differentiation. Inhibiting ERK activation blocked

29 the bone phenotype and resulted in the adipogenic differentiation of human mesenchymal stem cells [192]. Implications for the plasticity of osteoprogenitor and adipocyte cells in relation to osteoporosis and the aging skeleton have been reviewed in detail [180,193]. Presently, a key obstacle in understanding the origin of osteoblast lineage cells is the inability to identify an osteoprogenitor cell prior to the expression of bone phenotypic properties. Using characterization of hematopoietic stem cells as a paradigm, several groups have developed antibodies to cell surface proteins using presumptive marrow stromal cell populations. These reagents have the potential for both recognition and purification of skeletal stem cells [172,194 – 202]. STRO-1 positive cells [203] are well documented with respect to their pluripotential and osteoprogenitor properties [196,204 – 206]. Antigens for other antibodies have been characterized. Interestingly, an antigen to a cell surface marker antibody (SB-10) produced in response to mesenchymal stem cells is the activated leukocyte cell adhesion molecule ALCAM [195]. Expression of ALCAM becomes downregulated in concert with changes in morphology and detection of alkaline phosphatase activity of the periosteal osteoprogenitors as they migrate and develop into osteoblasts. There still remains considerable debate as to whether pluripotential osteoprogenitors (marked, for example, as STRO-1 or ALCAM positive cells) have the capacity to function as a “stem cell.” Proliferation and differentiation of the osteoprogenitor pool is influenced by several regulatory factors. Plateletderived growth factor and epidermal growth factor [207] have been identified as important in stimulating expansion of the CFU/F [151]. The leukemia inhibitory factor (LIF) maintains stem cell populations and osteoprogenitors and inhibits their differentiation in vitro [208,209], but has also been reported to have osteogenic activity in vivo [210]. The fibroblast growth factor-2 [211] and TGF-1 are potent mitogens for periosteal osteoprogenitors and marrow stromal cells [212 – 214]. The osteoinductive effects of bone morphogenetic proteins are complex, modulating growth, osteoinduction, and even apoptosis, depending on the specific BMP, concentration dependency, and the progenitor cell phenotype [36,185,215,216]. BMP-2 rapidly induces osteoblast differentiation in marrow stromal cells [37,183, 217,218], but the effect is slower in a number of pluripotent cell lines [219,220] and in the mouse myogenic C2C12 cell line [221]. These growth factors are expressed and produced by osteoblast lineage cells and are stored in the bone extracellular matrix. A local mechanism for stimulating the proliferation of progenitors in the bone microenvironment is thereby provided [222,223]. The osteoprogenitor appears to have limited selfrenewal capacity compared to the stem cell [224]. In contrast, a key feature of the osteoprogenitor/preosteoblast population is its capacity to divide and increase the size of

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bone. Labeling studies ([3H]thymidine and autoradiography) indicate that the proliferating cells are principally confined to progenitor cells and preosteoblasts with very few osteoblasts labeled [225 – 227]. The determined osteoprogenitor is recognizable in bone as a preosteoblast. Preosteoblasts are usually observed as one or two layers of cells behind the osteoblast near bone-forming surfaces [130 – 134,147,228]; i.e., they are usually present where active mature osteoblasts are laying down a bone matrix. These cells appear elongated, fibroblastic, or spindle shaped with an oval or elongated nucleus and with notable glycogen content. However, some of the cells in the layer found directly behind the active osteoblasts may appear morphologically closer to the cuboidal osteoblast and can clearly be defined as preosteoblasts (Fig. 4). Preosteoblasts may express a few phenotypic markers of the osteoblast, e.g., alkaline phosphatase activity, but less than mature osteoblasts [147,229]. The preosteoblast, however, has not yet acquired many of the differentiated characteristics of mature osteoblasts; for example, there is no evidence of a developed rough endoplasmic reticulum [130].

B. Cell Cycle Regulatory Parameters: Control Mechanisms Supporting Osteoblast Growth With recognition of decreased osteoblast surfaces in osteoporotic bone [230] and reports of decreased marrow

osteoprogenitors with age [231 – 235], defining mechanisms contributing to the regulation of proliferative activity in osteoblast lineage cells is increasing in importance. To understand regulatory parameters of proliferation, one must consider mechanisms that support the requisite responsiveness to growth factors through signaling pathways and the consequent induction of proliferation. To explain the induction, synthesis, activation, and suppression of the complex and interrelated regulatory factors associated with the growth control of osteoprogenitor cell proliferation in vivo, an understanding of mechanisms that control cell proliferation is required. Proliferation is controlled through the cell cycle by the activity of regulatory proteins which support progression of cells that have responded to a mitogenic stimulus through DNA replication and cell division. The cell cycle is a stringent growth-regulated series of sequential biochemical and molecular events that support genome replication and mitotic division [reviewed in 236]. The stages of the cell cycle and checkpoints that monitor competency of cells to progress through DNA replication and mitotic division are illustrated in Fig. 6. Suppression of certain cell cycle regulated genes is requisite for the cessation of proliferation and upregulation of phenotypic genes. When quiescent cells (G0) are stimulated to proliferate and divide, they enter G1, the first phase of the cell cycle where the enzymes required for DNA replication are synthesized. Before a cell can progress through G1 and begin DNA synthesis (S phase), it must pass through a checkpoint

FIGURE 6 Control of cell cycle progression in bone cells. The cell cycle is regulated by several critical cell cycle checkpoints (indicated by check marks), at which competency for cell cycle progression is monitored. Entry into an exit from the cell cycle is controlled by growth-regulatory factors (e.g., cytokines, growth factors, cell adhesion, and/or cell – cell contact) that determine the self-renewal of stem cells and expansion of precommitted progenitor cells. The biochemical parameters associated with each cell cycle checkpoint are indicated. Options for defaulting to apoptosis during G1 and G2 are evaluated by surveillance mechanisms that assess the fidelity of structural and regulatory parameters of cell cycle control. Apoptosis also occurs in mature differentiated bone cells.

CHAPTER 2 Osteoblast Biology

in late G1, which is known as the restriction point [237]. At this cell cycle restriction point, both positive and negative external growth signals are integrated. If conditions are appropriate, the cell proceeds through the remainder of G1 and enters the S phase. Once the cell passes the restriction point, it is refractory to withdrawal of mitogens or to growth inhibitory signals and is committed to progressing through the remainder of the cell cycle unless it is subjected to DNA damage or metabolic disturbance [237]. In mammalian cells, progression through the cell cycle is regulated by a cascade of cyclin-containing growth regulatory factors that transduce growth factor-mediated signals into discrete phosphorylation events, controlling the expression of genes responsible for both initiation of proliferation and competency for cell cycle progression. Cyclin activity is modulated by the formation of complexes with a family of threonine/serine kinases designated cyclin-dependent kinases (cdk) [238,239]. Cdks are regulated by both positive and negative phosphorylation, as well as by their reversible association with specific cyclins during defined phases of the cell cycle [240]. In general, the levels of cdk proteins remain relatively constant during the cell cycle, whereas the expression of specific cyclins is confined to distinct phases of the cell cycle where they are degraded quickly after having completed their function. An emerging concept is that the cyclins and cdks are responsive to regulation by the phosphorylation-dependent signaling pathways associated with activities of the early response genes, which are upregulated following the mitogen stimulation of proliferation [reviewed in 240 – 243]. Cyclindependent phosphorylation activity is functionally linked to activation and suppression of both p53 and RB-related tumor suppressor genes [244,245]. p53 accumulates in response to stress, inducing arrest at G1 or G2. The retinoblastoma protein (Rb), a tumor suppressor, is a member of a family of related proteins that include p105, p107, and p130. Rb has been shown to have a critical role in the regulation of cell proliferation, particularly in progression through G1 [reviewed in 244]. Rb functions as a signal transducer, receiving both growth-promoting and -inhibitory signals and linking them to the transcriptional machinery required for cell cycle progression or cell cycle arrest. In quiescent cells or cells reentering G1 from mitosis, Rb exists in an underphosphorylated or dephosphorylated state. Phosphorylation of Rb occurs late in G1 and modifies the activities of regulatory complexes that are required for gene expression linked to the onset of S phase [246]. The activities of the cdk are downregulated by a series of inhibitors (designated CDIs) and mediators of ubiquitination, which signal destabilization and/or destruction of these regulatory complexes in a cell cycle-dependent manner [247]. The cyclin inhibitory protein (CIP) class of CDI includes the proteins p21, p27, and p57. Growth arrest is, in part, due to induction of the cyclin-dependent

31 kinase inhibitor protein p21, which can interact with multiple cyclin – cdk complexes. The INK class is represented by proteins p15, p16, p18, and p19, which are linked to apoptosis control mechanisms. Expression of cell cycle regulatory proteins, cyclins, and cyclin-dependent kinases appears not to be solely confined to control of proliferation but, for example, associated differentiation in bone osteoblasts and nonosseous cells [248 – 250]. Cell cycle regulatory factors, particularly cyclin E, have been noted in several systems involved in the regulation of differentiation, in myoblasts [251], in osteoblasts [249], and in promyeloid cell differentiation into macrophages [252]. During osteoblast differentiation, cyclin-dependent kinase inhibitors (cdki) are also developmentally expressed. The cdki p21 (CIP/WAF1) is expressed in the growth period. In contrast, p27 (KIP-1) is expressed in the immediate postproliferative period and is upregulated again during differentiation [253]. Studies characterizing bone abnormalities associated with null mutations of cell cycle and cell growth regulatory factors have revealed their significance in providing signals for the control of both the number and the differentiation of bone-related cells. For example, marrow harvested from p27-/- mice shows a three- to fourfold increase in osteogenic nodule formation compared to wild type. Thus absence of this cdk inhibitor allows the marrow population to extend their growth phase, increasing the cell numbers. This expansion of the osteoprogenitor population is consistent with the larger size of the animals and the proportionally increased cortical width of the long bones [253]. Investigations of the effects of growth factors and osteogenic hormones on cell cycle target genes are increasing our understanding of their precise molecular mechanisms in the regulation of growth, differentiation, and apoptosis of osteoprogenitor cells and osteoblasts (Fig. 6). Several studies have reported BMP-2 and BMP-4 induction of cell cycle arrest in the G1 phase that is mediated by enhanced expression of the p21 cyclin inhibitor [254] and rapid induction of cyclin G, a cyclin that is increased after the induction of p53 by DNA damage [255]. Both of these events are linked to the induction of apoptosis, and in the developing tooth, p21 and BMP-4 are coexpressed in cells destined to undergo apoptosis in a transitional epithelial structure known as the enamel knot [256]. The apoptotic-promoting effects of BMP-2 have been reported to oppose the estradiol-induced growth of human breast cancer cells. Where estradiol stimulates cyclins and cyclin-dependent kinases, the BMP induction of the cyclin kinase inhibitor p21 leads to the inactivation of cyclin D1 [257]. The abundance of TGF- and BMPs in the early stages of osteoblast maturation and the targeting of BMP action to p21 may provide a mechanism not only for promoting osteogenic differentiation, but for apoptosis of proliferating cells that are recruited to the bone surface and may not progress to the mature osteocyte.

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Other cytokines and growth factors that target the proliferation phase have their effects coupled through p21. IL-6 promotes differentiation and exhibits antiapoptotic effects on human osteoblasts [258]. The effects of IL-6 on the p21 promoter are mediated by STAT-binding proteins and a STAT response element in the p21 promoter. Fibroblast growth factors are classic mitogens of the osteoprogenitor pool, as well as modulators of osteoblast differentiation [259 – 263]. FGF signaling also activates STAT1 and p21 [264], a mechanism that accounts for the ability of FGF-2 to induce both mitogenic responses and growth arrest in cancer cells [264,265]. TGF- also inhibits cell cycle progression in part through the upregulation of p21 gene expression [253,266]. Regulation of the p21 promoter is mediated by the TGF- induction of Smad3 and Smad4 [266,267]. The steroid hormone 1,25(OH)2D3 exerts antiproliferative effects in undifferentiated cells also mediated by the enhanced expression of p21 [268] and p27 [253]. This finding is consistent with the high levels of p27 in mature osteoblasts and 1,25(OH)2D3 induction of markers of the mature osteoblast phenotype. It is becoming increasingly evident that each step in the regulatory cycles (cell cycle, cyclin/cdk cycle, cdki cycle) governing proliferation is responsive to multiple signaling pathways and has multiple regulatory options. The diversity in cyclin – cyclin-dependent kinase complexes accommodates the control of proliferation under multiple biological circumstances and provides functional redundancy as a compensatory mechanism. Similarly, the inhibitors of cyclin – cdk complexes bind to and regulate multiple cyclin – cdk-containing complexes at several checkpoints [240,269,270]. The regulatory events associated with these proliferation-related cycles support control within the contexts of (a) responsiveness to a broad spectrum of positive and negative mitogenic factors, (b) cell – cell and cell – extracellular matrix interactions, (c) monitoring genome integrity and invoking DNA repair and/or apoptotic mechanisms if required, and (d) competency for differentiation. Perturbation of any of these cell cycle regulatory mechanisms can result in unregulated or neoplastic growth.

C. Osteoblasts: Producer of the Bone Extracellular Matrix When the preosteoblast ceases to proliferate, a key signaling event occurs for development of the mature osteoblast from the spindle-shaped osteoprogenitor. The osteoblast expresses all of the differentiated functions required to synthesize bone. Osteoblasts are defined in vivo by their appearance along the bone surface as large cuboidal cells actively producing matrix (Figs. 4 and 5), which is not yet calcified (osteoid tissue). Several structural features characterize this osteoblast, including its size and

cuboidal morphology, a round distinguishing nucleus at the base of the cell (opposite to the bone surface), a strongly basophilic cytoplasm, and a prominent Golgi complex located between the nucleus and the apex of the cell [271]. At the ultrastructural level, one observes an extremely welldeveloped rough endoplasmic reticulum with dilated cisternae and a dense granular content, and a large circular Golgi complex consisting of multiple Golgi stacks. These are typical characteristics of a secretory cell. The osteoblast synthesizes and vectorially secretes most of the bone ECM protein; others are accumulated. Fetal bone is enriched in type III collagen, whereas type I collagen accounts for 80 – 90% of the osteoid in the adult with minor collagens type V. Specialized noncollagenous proteins for cell adhesion and with calcium and phosphatebinding properties are found in variable amounts changing with age. The most abundant noncollagenous proteins include osteonectin, osteocalcin, bone sialoprotein, and osteopontin; and based on many in vitro studies, it is assumed that they participate in specialized functions necessary for both the structural integrity of bone tissue and bone turnover. The protein properties and gene regulation of bone extracellular matrix constituents are detailed elsewhere in this volume (see Chapter 4). The primary functional activity of the active surface osteoblast is production of an extracellular matrix with competency for mineralization. In this regard, the high level of the tissue-nonspecific alkaline phosphatase (TNAP) (bone, kidney, liver isoform) and the ability to synthesize a number of noncollagenous proteins that are in either representative or restricted abundance in mineralized tissues are important features. For example, hypertrophic chondrocytes and odontoblasts share some phenotypic features with osteoblasts, as osteocalcin expression [272,273] and high alkaline phosphatase activity, sufficient to allow for histochemical detection in cells associated with the active formation of mineralized matrix. Alkaline phosphatase activity, a hallmark of the osteoblast phenotype, is a widely accepted marker of new bone formation and early osteoblast activity. Gradations of enzyme intensity and mRNA expression are found in bone with lowest levels (or absence) in osteocytes and osteoprogenitors and maximal levels in surface osteoblasts and hypertrophic chondrocytes at the mineralization front [134,229]. Alkaline phosphatase activity is still considered critical to the initiation of mineralization, a concept supported by characterization of the genetic defect in hypophosphatasia [274]. Several of the noncollagenous bone-related proteins are implicated in initiating or regulating the mineral phase of bone from studies of the biochemical, molecular, and functional properties [275,276] (Table 1). However, ablation of the genes encoding some of the more abundant and bone restricted noncollagenous proteins (osteocalcin [277,278], osteopontin [279], biglycan [280]) have resulted in subtle changes in

CHAPTER 2 Osteoblast Biology

the mineral phase of bone. Future directions in which double mutants are derived may reveal severe mineralization defects. The anabolic activities of osteoblasts are regulated in large part by the insulin-like growth factor (IGF)/IGFbinding protein (IGFBP) system [281; reviewed in 282]. IGF-I and IGF-II are synthesized in may tissues and both are highly expressed in active osteoblasts [283]. IGFs stimulate cell proliferation and collagen synthesis [284 – 286] and, at the same time, inhibit matrix collagen degradation by decreasing collagenase 3 transcription [287]. The synthesis of IGF-I is regulated by physiologic mediators of bone formation, PTH stimulates [288], whereas glucocorticoids [289] and growth factors (e.g., FGF-2, PDGF-2) are inhibitory [290]. The activities of IGF-I and IGF-II are regulated by a family IGFBPs, designated IGFBP-1 through IGFBP-6 [291]. These binding proteins have either stimulatory effects (e.g. IGFBP-5) [286,292 – 294] or inhibitory activity (e.g., IGFBP4) [294] and appear to be expressed at different levels in subpopulations of osteoblasts [295]. Several clinical studies are revealing associations of IGF-1 and IGFBPs with metabolic bone diseases [296 – 298, and reviewed by 299]. Regulation of bone development and cellular differentiation by the extracellular matrix (ECM) is well established [300 – 302] (see Chapter 4). Osteoblast differentiation and functional activity are supported by cell – matrix interactions [303,304]. Functional studies establishing requirement of the type I collagen and other ECM components in promoting osteoblast/osteocyte differentiation have been carried out by modifying the production of osteoblastsecreted products or culturing osteoblasts on various matrices [305 – 308]. The molecular mechanisms mediating osteoprogenitor/osteoblast – matrix interactions are being identified. A spectrum of integrins have been shown to be expressed by osteoblasts [309]. Interactions between integrin receptors and fibronectin are required for both osteoblast differentiation [310,311] and cell survival [312]. Osteoblasts appear to use 1 integrins to adhere to the full range of RGD-containing bone matrix proteins. Disruption of the collagen – 21 interaction suppresses expression of the osteoblast phenotype [313,314]. The noncollagenous RGD containing bone matrix protein, bone sialoprotein (BSP), was also shown to mediate the collagen-induced osteoblast differentiation of bone marrow cells [315]. This activity of BSP is consistent with the reported coordinated expression of 3 integrin and BSP during osteoblast differentiation in vitro [316,317]. Based on early findings of Vukicevic et al. [306], who reported laminin-1 stimulates osteoblast differentiation, and recent observations that osteoprogenitors selectively attach to laminin-1 [318], it will be instructive to address the specificity of cell – matrix interactions with respect to subpopulations of osteoblast lineage cells. Studies are identifying integrin receptors in the

33 regulation of BMP-2-mediated differentiation of mesenchymal cells [319]. In addition to cell – matrix interactions, cell – cell communication is important for the differentiation and maturation of osteoblasts. Cytoplasmic processes on the secreting side of the surface osteoblast extend deep into the osteoid matrix and are in contact with the extended cellular processes of osteocytes. Junctional complexes (gap junctions) are often found between the osteoblasts on the surface as well as between cellular processes. In this manner, surface osteoblasts establish cell – cell communication with neighboring cells in the mineralized matrix. Gap junctions are a structure of six multiple protein units (connexins) that couple with an identical unit in a neighboring cell to form a channel connecting the two cytoplasms. Studies in osteoblasts [320,321] suggest that the selective utilization of connexin proteins contributes to the modulation of molecular permeability. Several members of the cadherin family of cell – cell adhesion proteins are expressed in osteoblasts, including cadherin-11, cadherin-4, N-cadherin, and OB-cadherin [50,51]. N-cadherin is present on proliferative preosteoblastic cells and may support osteoblast differentiation [322], but is lost as they become osteocytic [323]. In contrast, OB-cadherin is barely detected in osteoprogenitor cells and is upregulated in alkaline phosphatase-expressing cells [324]. Indeed, the relative abundance of cadherin defines the differentiation pathway of mesenchymal precursors to specific lineages [51,325]. Expression of R-cadherin, N-cadherin, and cadherin-11, present in progenitor cell lines, is modified in response to differentiation, e.g., R-cadherin is downregulated and cadherin-11 upregulated in response to BMP2-induced osteogenesis. In addition, ICAM-1 and VCAM-1 have been reported on the osteoblast surface, thereby providing a potential mechanism for T-cell interactions that contribute to the regulation of bone turnover [326]. Signaling pathways from the extracellular matrix through the cytoskeleton and finally to the nucleus, which allow expression and upregulation of bone-specific and bone-related genes, need to be investigated. For example, -catenin, which colocalizes and coprecipitates with cadherins [50], is a potential candidate. CD44, the hyaluronate receptor, is a nonintegrin adhesion receptor that is linked to the cytoskeleton. CD44 has been identified as a useful marker for osteocyte differentiation [200,201] and is also expressed in osteoclasts [327].

D. Osteocytes and Bone-Lining Cells: Gatekeepers of the Structural Integrity of Bone As the active matrix-forming osteoblast becomes encased in the mineralized matrix, the cell differentiates further into osteocytes. Labeling studies suggest that the

34 transition from an osteoblast to an osteocyte is approximately 3 – 5 days [169,328]. The osteocyte is considered the most mature or terminally differentiated cell of the osteoblast lineage. Osteocytes are embedded in bone matrix occupying spaces (lacunae) in the interior of bone and are connected to adjacent cells by long cytoplasmic projections, enriched in microfilaments, and lie within channels (canaliculi) through the mineralized matrix. These cell processes maintain contact with other osteocytes or with processes from the cells lining the bone surface [134,329]. It is estimated that 25,000/mm3 osteocytes are found embedded within the bone, contributing to the metabolic functions of bone [330]. Some, but not all, of the biochemical features of the osteoblast are expressed in the osteocyte. The morphologic properties of osteocytes change as the surrounding osteoid mineralizes and reflects their functional activity (Fig. 3). Being derived from osteoblasts, a young osteocyte has many of the ultrastructural characteristics of a cell involved in protein synthesis (rough endoplasmic reticulum, large Golgi), except that there is a decrease in the volume of the cell. An older osteocyte, located deeper within the calcified bone, shows less of these features; and in addition, glycogen stores become evident in its cytoplasm. Osteocytes have been shown to synthesize new bone matrix at the surface of the lacunae, and there is some evidence for their ability to resorb calcified bone from the same surface [331]. The osteocyte is a terminally differentiated cell, not capable of cell division even when separated from its matrix. In isolated cultures, they retain their cellular projections [332]. Osteocytes will be phagocytized and digested together with the other components of bone during osteoclastic bone resorption. On quiescent bone surfaces, the osteoblast develops into a flattened bone-lining cell of a single layer forming the endosteum against the marrow and underlying the periosteum directly on the mineralized surfaces. These osteoblasts are in direct communication with the osteocytes within the mineralized matrix through cellular processes that lie within the canaliculi. In the adult, the majority of bone surfaces are occupied by another osteoblast subtype with distinct phenotypic features. The bone-lining cell displays a flat and highly elongated cell shape with a spindle-shaped nucleus and fewer organelles than active osteoblasts [130, 271,329,333]. They are considered to provide a selective barrier between bone and other extracellular fluid compartments and contribute to mineral homeostasis by regulating the fluxes of calcium and phosphate in and out of bone fluids [333]. The organization of osteocytes in bone reflects their function and their capacity to respond, allowing these cells to communicate and transmit regulatory signals. The osteocytes and surface-lining cells form a continuum, or syncytium, by connection of their cytoplasmic projections through gap junctions that facilitate the exchange of both mechanical and metabolic signals [334,335] for

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responsiveness to physiologic demands on the skeleton. Osteoblasts and osteocytes are coupled metabolically and electrically through different gap junction proteins called connexins, described earlier [321,336,337]. Rapid fluxes of bone calcium across these junctions are thought to facilitate the transmission of information between osteoblasts on the bone surface and osteocytes within the structure of bone [338]. This structural organization and the direct contact of the active osteoblast or surface lining cells with the osteocytes is consistent with the concept that bone cells, responding to varying physiological signals, can communicate their responses. Bone-lining cells receive the majority of systemic and local signals and can transmit these to osteocytes. Reciprocally, mechanical forces on the bone produce stress-generated signals that are perceived by osteocytes, which then transmit the regulatory information to surface osteoblasts. Stress-generated electric potentials experienced by bone are either produced by strain in the organic components (piezo electric potential) or result from electrolyte fluid flow produced by deformation of the bone (streaming potential) [339 and reviewed in 340]. Thus, the ability of bone to act as a tissue responding to physiological homeostatic demands and functioning as a structural connective tissue organ to meet physical demands depends on communication among its resident cells. Studies in bone tissue and isolated cells following applied stress have advanced our understanding of osteocyte functions and responses [341]. Osteocytes produce IGF-1 and release prostaglandins in response to stress [342]. Direct evidence that osteocytes sense mechanical loading [343] has been demonstrated by rapid changes in metabolic activity by [3H]-uridine uptake [344], increased metabolic activity (e.g., glucose-6-phosphate dehydrogenase) [345], increased gene expression [346 – 348], and activation of a volume-sensitive Ca2 influx pathway potentiated by PTH [349]. Signals induced by fluid flow that have been reported include prostaglandin PGE-2, cAMP, and nitrous oxide [350 – 352]. In addition, extracellular matrix receptors, such as the integrins and CD44 receptors, are thought to mediate cellular sensing of mechanical loads [353]. The disruption of cell – matrix interactions by loading could induce a mechanical twisting of the integrins [354]. Integrins are tightly coupled to the cytoskeleton [355] and together the integrin – cytoskeleton complex facilitates the transduction of mechanical signals that may ultimately lead to modifications in gene expression [356]. Thickening of actin stress fibers and increased synthesis of cytoskeleton components in osteoblasts in response to mechanical strain have been documented [357]. The majority of the evidence to date suggests that mechanical tension can trigger bone remodeling and may favor bone formation. Increased osteopontin expression and synthesis may facilitate bone remodeling by osteoclasts [348,358,359]. However, it has been reported that mechanical strain inhibits expression of the osteoclast

CHAPTER 2 Osteoblast Biology

differentiation factor TRANCE [360]. Related to bone formation, several studies have demonstrated significant increases in PGE-2, which stimulates IRF-1 in osteocytes in response to mechanical strain [361 and reviewed in 282]. BMP-2 and BMP-4 (but not other BMPs) were induced in a model of distraction osteogenesis [362]. Thus, mechanical strain induces factors for the proliferation, differentiation, and anabolic activities of osteoblasts [363]. The life span of osteoblast lineage cells is dependent on several factors. Because more osteoblasts are recruited to bone remodeling sites than can be organized on the bone surface for further differentiation by mineralizing osteoid, a high percentage of surface preosteoblasts will die [364]. Apoptosis of preosteoblast clusters may be triggered by the lack of an adequate ECM and appropriate cell – matrix interactions for survival [312]. In vitro, osteoblast apoptosis is particularly evident in cells on the surface of multilayered bone nodules formed in primary calvarial cell cultures [365]. Apoptosis is a general mechanism for limiting organ size in embryonic development [366] and in the adult when there is a need to regenerate tissue. Here growth factor and cytokine effects on apoptosis of specific cell subpopulations in bone are likely to be contributing to tissue turnover [258,364,367]. In contrast to osteoblasts, osteocytes are very long lived in their lacunae, but will undergo apoptosis when their structural integrity is compromised. Microfracture in bones [368] and disruption of cell – cell contacts with the consequent inability to receive stimulatory signals and cell nutrients will lead to apoptosis. Increased empty lacunae and apoptotic cells (detected by DNA fragmentation using the TUNEL assay) are observed during bone turnover in aged human bone [369,370], in glucocorticoidtreated mouse models [371], and following estrogen withdrawal [372]. Parathyroid hormone [373], bisphosphonates [374], and estrogen [375,376] have been reported to prevent apoptosis. Of significance, calbindin-D (28k), which is expressed in osteoblasts, suppresses apoptosis by reducing caspase-3 activity through protein – protein interactions [377]. More studies are required to address intracellular apoptotic control mechanisms and pathways operative in the environment of the osteoblast [365].

IV. CELLULAR CROSS-TALK OF OSTEOBLAST LINEAGE CELLS: FUNCTIONS IN CALCIUM HOMEOSTASIS, BONE TURNOVER, AND HEMATOPOIESIS The biological significance and unanswered questions of the interrelationship of bone tissue cells with the hematopoietic and immune systems were highlighted at a National Institute of Health conference [378]. Osteoblasts

35 control osteoclast differentiation from hematopoietic precursors. They also support long-term bone marrow cultures and regulate hematopoiesis by the production of stimulatory factors (e.g., GM-CSF) [379,380], as well as by cell – cell interactions between early hematopoietic cells and osteoblasts via 1 integrins on CD34 cell and various cell adhesion on bone marrow stromal cells (e.g., VCAM1) [381] [reviewed in 382]. The immune and bone organ systems are linked by the production of multiple cytokines from T lymphocytes regulating bone turnover by the modulation of both osteoblast and osteoclast activities. Cross-talk between osteoblasts and other cellular systems is beginning to be investigated. Endothelial cell (EC) and osteoblast cross-talk is likely to be important for vascular invasion into the bone matrix. Osteoblasts secrete paracrine factors that regulate endothelial cell function [383], including vascular endothelial growth factor (VEGF) and its receptors [384]. VEGF secreted by ECs has been reported to enhance the anabolic effects of vitamin D3 on osteoblasts [385] and to be necessary for angiogenesis during endochondral bone formation in vivo [386]. Of note, bone sialoprotein, which is upregulated in osteogenic tumors and mediates cell attachment via V3 integrins, can promote adhesion of endothelial cells [387]. An important function of osteoblast lineage cells is their response to endocrine factors and the production of paracrine and autocrine factors for the sequelae of events mediating bone turnover. At all stages, from the initial activation of bone resorption to formation of new bone at the resorption site in the adult (bone remodeling unit), crosstalk between osteoblast lineage cells and other cell phenotypes is necessitated for the regulation of bone remodeling. The coupling of osteoblast and osteoclast activities through cross-talk is mediated by several mechanisms, which are detailed later. Direct interactions between a ligand on osteoblast lineage cells and its receptor on mononuclear preosteoclasts activate signaling cascades for osteoclast differentiation. Numerous indirect pathways are operative in which calciotrophic hormones and cytokines stimulate the secretion of factors from one cell type that activate or suppress activities of the other cell phenotype. Osteoprogenitors in the marrow, surface osteoblasts, and osteocytes have receptors for cytokines [reviewed in 388,389], parathyroid hormone, 1,25(OH)2D3 [390,391], and estrogen [392], which are key regulators of osteoclast activities. In addition to cytokine and hormone feedback loops, the transcriptional control of genes involved in the activation or suppression of osteoclast and osteoblast functions are coordinated. Two observations suggest a potential interrelationship between osteoblasts and osteoclasts by an unknown mechanism. Annexin II was identified as a vitamin D3 receptor [393] mediating the nongenomic rapid effects of 1,25(OH)2D3, which increase intracellular calcium in osteoblasts [394 – 396]. Annexin II was also reported to be

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secreted by osteoclasts and stimulate osteoclast differentiation and GM-CSF [397], an autocrine and paracrine product of osteoblasts [398]. These features provide mechanisms for regulating osteoblast and osteoclast activities to support calcium homeostasis and bone turnover. Osteoblasts and stromal osteoprogenitors primarily support osteoclastogenesis by the secretion of soluble cytokines and through cell – cell interactions with osteoclast precursors. Stromal osteoblastic cells are the major source of the macrophage colony-stimulating factor (M-CSF/CSF1) and the IL-6 family of cytokines, which are potent stimulators of bone resorption and participate in osteoclastogenesis at early and later stages. Other cytokines, such as TNF- and IL-1, which are predominantly derived from monocytes and have mitogenic effects on the mononuclear osteoclast precursor, feed back on stromal cells to regulate their production of M-CSF, as well as IL-11, another key regulator of osteoclast formation [reviewed in 399; 400,401]. IL-6, IL-11, and other bone-resorbing factors, such as LIF and oncostatin M, transduce their regulatory signals through the gp130 signal transduction pathway. Cytokine production by human bone marrow stromal cells can be effected by age and estrogen status [402]. PTH and vitamin D stimulate osteoblast production of IL-6, as do IL-1,

TNF- and TGF-. The mechanisms by which these factors affect the generation of early osteoclast precursors from CFU-GM colonies, osteoclast differentiation, and activity have been well documented (in several reviews [403 – 406]) (see Chapter 3). Two discoveries provide insight into mechanisms for the essential role of osteoblasts in mediating osteoclastogenesis directly through cell – cell interactions (Fig. 7). One is the characterization of osteoprotegerin (OPG), also designated osteoclastogenesis inhibitory factor (OCIF), a secreted protein with strong homology to the TNF receptor family. OPG is expressed in several tissues, including bone, cartilage, kidney, and blood vessels [407,408]. Several experimental approaches established OPG as a soluble factor competent to inhibit osteoclast differentiation [409 – 411]. Expression of the gene in osteoblast lineage cells is upregulated by calcium and is downregulated by the glucocorticoid, dexamethasone [411]. These findings are consistent with the conditions required for osteoclast differentiation in cocultures of bone marrow stromal cells and spleen cells [412]. Overexpression of OPG in transgenic mice resulted in severe osteopetrosis [407]. The presence of F4/80 positive osteoclast precursors in these mice suggested that OPG inhibits terminal stages of osteoclast differentiation.

FIGURE 7 Osteoblast – osteoclast cross-talk in the regulation of bone turnover. M-CSF and OPGL/RANKL are osteoblast factors that promote osteoclast differentiation: M-CSF binds to the c-fos receptor on the mononuclear preosteoclast, whereas RANKL mediates cell – cell interaction with the RANK receptor to promote fusion of the preosteoclast to the differentiated multinucleated resorbing cell. Other regulatory hormones, cytokines, and bone matrix proteins are shown. A soluble “decoy” OPG receptor is also produced by osteoblasts that can block osteoclast precursor interactions with stromal cells.

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In search of the specific ligand for OPG, several groups cloned a novel member of the transmembrane TNF ligand family, leading to the discovery of an osteoclast differentiation factor [411,415,416]. The ligand is identical to a TNF cytokine family member designated TRANCE (TNF-related activation induced cytokine) [417], a cytokine that regulates T-cell-dependent immune responses and to RANK (receptor activation of NF-,) a TNF receptor family member cloned from T cells [418]. A standard nomenclature has been proposed for the TNF factors related to bone resorption, designating the ligand which is expressed at high levels in osteoblasts as RANKL [419]. Importantly, RANKL was demonstrated to have competency for inducing osteoclast formation from hematopoietic cells in the absence of stromal cells [415,416]. The studies define RANKL as acting directly with RANK on osteoclast precursors. The soluble decoy receptor which blocks this interaction remains designated OPG (Fig. 7). Mice with a disrupted RANKL gene completely lack osteoclasts because of the inability of osteoblasts to support their differentiation [420]. Of interest, activating mutations in RANK have been identified as the cause of the bone disorder familial expansile osteolysis [421]. Thus, RANKL is essential for the differentiation of osteoclasts from the early stages of mononuclear cell fusion, activation, and survival. Together RANK, RANKL, and M-CSF represent essential factors required for coupling stromal/osteoblastic cells to the formation of osteoclasts and are approximately controlled by cytokine and hormonal mediators of bone resorption for regulated bone turnover.

It is instructive to consider regulated expression of RANK and RANKL in osteoblasts in an environment poised for bone resorption or bone formation. Expression of the RANKL ligand in osteoblasts is upregulated by stimulator of bone resorption while OPG is downregulated ([411]; and reviewed in Hofbauer et al. [422]). Notably, regulatory elements for the osteoblast differentiation transcription factor CBFA1 occur in both the OPF and OPG-L gene promoters [423,424]. CBFA1 upregulates OPG in osteoblasts [423], thereby suppressing osteoclastogenesis when bone formation is needed. Regulation of the RANKL promoter by CBFA1 was not examined; however, CBFA1 regulates RANKL mRNA, while vitamin D decreases CBFA1 cellular levels [425,426] and increases RANKL expression. These changes are consistent with vitamin D mediating osteoclast differentiation. Thus, through transcriptional control of the CBFA1 promoter by the same steroid hormone that regulates osteoclastogenesis, as well as transcriptional regulation of OPG and OPG-L by CBFA1, feedback loops can contribute to a balance between bone resorption and bone formation (illustrated in Fig. 8). The OPG system is discussed extensively in Chapters 3, 6, 12, and 14. Crosstalk between osteoclast activity and osteoblast recruitment maintains the fidelity of bone tissue organization. Following the activation and resorption phases of the bone remodeling sequence, the recruitment, proliferation, and differentiation of osteoprogenitors and osteoblasts on the resorbed surface is accomplished in part by the resorbed bone microenvironment. Stored growth factors in the bone matrix provide a local concentration to initiate the formation phase by recruitment of osteoprogenitors to the

FIGURE 8 CBFA and vitamin D3 regulate bone turnover by coordination of gene transcription. Vitamin D promotes osteoclast differentiation in part by increasing RANKL. Modest downregulation of the mouse CBFA1 promoter by 1,25(OH)2D3 [425] may ensure physiologic levels of this factor. CBFA1 is a positive regulator of OPG expressing bone resorption while promoting bone formation. Thus, OPG (RANK) /RANKL ratios may be regulated through the combined activities of vitamin D and CBFA.

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resorbed bone surface. Here the role of TGF-1 is central to increasing osteoblast differentiation at sites of bone resorption [427].

V. OSTEOBLASTS IN VITRO: STAGES IN DEVELOPMENT OF THE OSTEOBLAST PHENOTYPE A. Cell Culture Models 1. PRIMARY CELL CULTURES Valuable insight into the biology and pathology of bone has been obtained over the past several years from cultured mammalian and avian osteoblasts that undergo developmental expression of genes and establishment of bone tissue-like organization, analogous to osteoblast differentiation in vivo. The clinical relevance of culture models should not be overlooked. Primary cell cultures offer

TABLE 2 Cell Culture Models for Studying Commitment, Growth, Differentiation, and Physiological Responses of Osteoblastic Cells Primary cell cultures for osteoblast differentiation Periosteum [441] Calvariae [432] Trabeculae [434] Cortical bone [435] Marrow stroma [437,439] Tumor-derived cell lines [477] ROS 17/2.8; ROS 24/1 UMR106 SaOS-2 U2-OS MG-63 Clonal and transformed cell lines i. Pluripotential/bipotential C3H10T1/2 [462] ROB-C26 [459] 2T3 [182] MLB13 Myc [183] ii. Monopotential MC3T3-E1 (preosteoblast) [125] C2C12 (premyoblast) [461] MLO-Y4 (osteocytic) [473] iii. Marrow stromal origin ST-2 [463] W20-17 [37]

several advantages, particularly for studying cell growth control mechanisms and differentiation in the context of a mineralizing matrix. Primary cultures often reflect the in vivo status of the osteoblast and phenotypic properties of a mouse model [253,261] (Table 2). Cultured calvarial osteoblasts from osteopetrotic rats that exhibit precocious and intensified mineralization within the intact animal during development retain this defect in vitro [428,429]. The pathology is reflected by parallel in vitro and in vivo modification in the temporal sequence of cell growth and tissue-specific gene expression as well as by features of cell organization and extracellular matrix mineral deposition that are revealed at the histochemical and ultrastructural levels. In several mouse models, marrow cultures supporting osteoblast differentiation and osteoclastogenesis have revealed cell type-specific defects and provided insight into molecular mechanisms [430]. The method of releasing populations of cells from a bone extracellular matrix by outgrowth cultures of bone fragments or by enzymatic digestion with trypsin and collagenase was developed several decades ago [431 – 435]. Calvarial or trabeculae-derived osteoblasts support the exploration of mechanisms associated with the differentiation of cells committed to the osteoblast phenotype. Marrow cell cultures permit the evaluation of gene expression related to the commitment of stem cells to the osteoblast lineage, initial stages of differentiation, and the limits of plasticity that characterize pluripotent osteoprogenitor cells [133,171,202, 436 – 440]. Osteoprogenitor cells that differentiate have also been isolated from the periosteum [441,442]. Primary cultures of normal diploid fetal rat calvarialderived osteoblasts form multilayered cellular nodules having bone tissue-like properties [143,443 – 447], whereas adherent marrow-derived cells give rise to osteoblastic colonies. Osteoblasts isolated from more mature bone as described for chick [446], bovine, and human [447] form a uniform layer of mineralized matrix. The isolated cells from bone tissue are not a heterogeneous mixture, but will become heterogeneous in long-term culture as the bone matrix is synthesized and mineralized osteoblasts result from outgrowth or released cells, particularly from fetal tissues. Although primary cell cultures are a mixed population of cells, ultrastructural, histochemical, single cell gene analyses of the cell layers and bone nodule colonies revealed that preosteoblastic, osteoblast, and osteocyte-like cells can be identified in these different models. Primary cell cultures may not always be appropriate or practical for some lines of experimentation. They are limited in their ability to maintain phenotypic properties with passaging. The cumulative observations from primary cultures of fetal and adult bone, marrow, and periosteum reported over the last decade reveal considerations for the following in interpretation of these studies. The age of isolation influences the growth properties and representations of subpopulations of bone-forming cells. The expression of

CHAPTER 2 Osteoblast Biology

osteogenic and other phenotypic responses appears to also be related to bone sites and cell passages. Thus, studies of osteoblast activities must be controlled carefully. Cells closer to the progenitor/preosteoblast stage are differentiated more readily in vitro. It should additionally be noted that protocols have been developed for the culture of human bone cells from multiple sites offering viable options for the application of culture techniques to the evaluation of skeletal diseases or for the evaluation of selective responses of osteoblasts that have been observed in vivo [435,448,449]. Although limitations must be acknowledged, the effective use of cultured human osteoblasts in assessing functional activity related to disease has been validated, e.g., with cells from patients with Paget’s disease [450], osteogenesis imperfecta [451], and other skeletal disorders [452]. Several studies have indicated that osteoblasts from osteoporotic patients and control subjects exhibit few differences when compared in vitro [453,454]. Caution must be exercised in the interpretation of osteoblast responses to agents in vitro relative to in vivo effects. For example, osteoblasts harvested from bone biopsies of osteoporotic patients treated with fluoride exhibit increased proliferative activity [455]. In contrast, fluoride is not mitogenic to osteoblasts in culture [456]. 2. TUMOR-DERIVED, TRANSFORMED, AND IMMORTALIZED CELL LINES Several classes of nonmalignant, clonal murine and rat cell lines have facilitated the investigation of biochemical and molecular parameters of osteoblast differentiation (Table 2). Calvarial-derived cells that undergo spontaneous immortalization during passage in cultures [433,457] exhibit selective, and often unstable [458], expression of bone cell phenotypic properties and responses to steroid hormones and growth factors. Pluripotent clonal cell lines are being used frequently to examine molecular mechanisms of cell phenotype commitment. The ROB-C26 [459], isolated from neonatal rat calvaria, has tripotential (osteogenic, myogenic, and adipogenic) properties. A myogenic cell line (C2C12 cells) has been well characterized with respect to its differentiation potential in response to TGF-1 and BMP-2/4 [221]. TGF-1 blocks myotube formation only, whereas BMP-2/4 induces the osteoblast phenotype. The expression of BMPs and their receptors [460] and Smad signaling factors, as well as responses to different BMPs [461], have been studied [43,408]. The pluripotential C3H10T/2 cell line, which differentiates into adipocytes, chondrocytes, and myotubes when treated with azacytidine, will also differentiate to osteoblasts in response to BMP-2 [219,462]. The stromal cell line W20-17 [37], which was subcloned (by limiting dilution from bone marrow), and other similar mouse stromal lines (ST-2 cells) [463] have bipotential properties. These cells, which are widely used to support osteoclast formation, can

39 be differentiated to mature osteoblasts by BMP-2. The MC3T3-E1 monopotential preosteoblastic cell line [464] that was obtained by subjecting calvarial-derived osteoblasts (from C57B1/6 mice) to scheduled passaging retained the ability to undergo a development sequence of gene expression [465]. These cells establish a mineralized bone extracellular matrix [466] and, like primary rat calvarial cells, differentiation can be modulated by various stimuli. In recent years, subclones from these cell lines have been isolated with distinct properties, likely reflecting stable stages of osteoblast maturation [125]. Viral transformation of calvarial-derived rodent [467], marrow stromal cells, or human osteoblasts has also provided model systems for addressing regulatory mechanisms operative in bone cells [183,467 – 469]. The introduction of a temperature-sensitive viral gene, which at permissive temperatures selectively supports proliferation or postproliferative phenotypic gene expression, offers new options in the pursuit of skeletal regulatory mechanisms in both murine [182,183] and human [470,471] cells that are involved in development and bone disease. Characterized properties reveal that viral transformation appears to restrict their properties to mono- or bipotential lineages at an early stage of commitment, e.g., mouse limb bud-derived cell lines (MLB13 Myc clones 14 and 17) [183] and human marrow stromal cells [472]. The 2T3 cell line from mouse calvaria [182] and the MLO-Y4 osteocyte-like cell line from long bone [473] have been established from transgenic mice harboring the SV40 large T antigen. Stable cell lines permit conditional and reversible expression of osteoprogenitor or osteoblast phenotypic properties that are developmentally regulated. The limitation is that these cell lines may not reproducibly support formation of a mineralized matrix. Osteosarcoma cell lines, which typically express a limited gene characteristic of bone cells in vivo, have been utilized by many investigators to support studies directed to the control of genes expressed during osteoblast proliferation and differentiation. A series of rat osteosarcomaderived cell lines (ROS) exhibit a wide range of phenotypic responses. The ROS 17/2.8 [474] exhibits most properties of mature osteoblasts, including high levels of osteocalcin [475]; the ROS 24/1 cell line lacks the vitamin D receptors [396]. Several human osteosarcoma cell lines have been widely used for the characterization of hormonal responses. These include the rat UMR-106 and human MG-63, SAOS-2, U2-OS, and TE-85 cell lines. The large quantity of cells available permits isolation and characterization of gene regulatory molecules that control transcription at the level of the gene, as well as the signaling pathways that mediate responsiveness to growth and phenotypic cues [476]. However, caution must be exercised in assuming that identical regulatory parameters are operative in normal diploid osteoblasts and osteosarcoma cells

40 [477 – 482]. The abrogation of key components of growth control and proliferation – differentiation interrelationships in transformed and tumor cells results in a consequential coexpression of cell growth and tissue-specific genes that are, in normal diploid cells, sequentially expressed in response to stringently regulated physiological signaling mechanisms.

B. Developmental Sequence of Gene Expression Characterizes Stages in the Osteoblast Differentiation Pathway Primary cell cultures and cultures of established lines that produce an organized bone-like matrix provided a basis for studies that have mapped the temporal expression of cell growth and tissue-specific genes during the progressive establishment of the osteoblast phenotype [446,477, 483 – 486]. Profiles of gene expression defined developmental stages of osteoblast maturation and allowed for investigating regulatory mechanisms that support the progression of osteoblast growth and differentiation and developmental stage-specific responses to physiological mediators of bone formation and remodeling that include growth factors and steroid hormones [486 – 497]. These characterizations have facilitated the investigation of gene regulatory signaling pathways and control mechanisms associated with development and maintenance of bone tissuelike organization. The sequential expression of cell growth and tissue-specific genes presented in Fig. 9 (see also color plate) has been mapped during progressive development of the bone cell phenotype in cultured osteoblasts and marrow stromal cells from several species and several sites [128,167,320, 483,486,498,499] by the combined application of Northern blot analysis, in situ hybridization, nuclear run-on transcription and histochemistry. Four principal developmental periods can be defined [499] by expression of the major functional bone matrix proteins, often designated “phenotypic markers”. Initially, proliferation supports expansion of the osteoblast cell population to form a multilayered cellular nodule and biosynthesis of the type I collagen bone extracellular matrix (ECM). At this time, genes requisite for the activation of proliferation (e.g., c-myc, c-fos, c-jun) and cell cycle progression (e.g., histones, cyclins) are expressed together with the expression of genes encoding growth factors (e.g., FGF, IGF-I), cell adhesion proteins (e.g., fibronectin), and others associated with the regulation of ECM biosynthesis (e.g., TGF, type I collagen). However, several of the BMPs reach peak expression in immediate postproliferative osteoblasts [500] and may function in the regulation of related BMPs supporting osteoblast growth and differentiation [501,502]. For example, BMP-2 is not only autoregulated but additionally downregulated by

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BMP-4 and BMP-6 in osteoblasts [503]. However, BMP-2 enhances BMP-3 and BMP-4 expression during the mineralization stage [504]. Following the initial proliferation period, a second stage of gene expression is associated with the maturation and organization of the bone ECM. Genes that contribute to rendering the extracellular matrix competent for mineralization (e.g., alkaline phosphatase) are upregulated. Collagen synthesis continues and undergoes cross-link maturation [505]. Two principal transition points are key elements of this temporal expression of genes that support the progression of differentiation (Fig. 9B, bottom). These transitions have been established experimentally and defined functionally as restriction points during osteoblast differentiation to which developmental expression of genes can proceed but cannot pass without additional cellular signaling [483]. The first transition point is at the completion of the proliferation period when genes for cell cycle and cell growth control are downregulated, and expression of genes encoding proteins for extracellular matrix maturation and organization is initiated. The absence during the proliferation period of gene expression observed in postproliferative mature osteoblasts is called “phenotype suppression” [506]. The model is supported by the binding of regulatory factors abundant in proliferating osteoblasts (e.g., oncogene-encoded [507] or helix – loop – helix proteins [84,85,508,509]) to regulatory elements in postproliferative expressed genes, which results in the suppression of transcription of the genes until later in development. The second transition is at the onset of extracellular matrix mineralization. Signals for the third developmental period involve gene expression related to the accumulation of hydroxyapatite in the ECM. Genes encoding several proteins with mineral binding proteins (e.g. osteopontin, osteocalcin and bone sialoprotein) exhibit maximal expression at this time when mineralization of the bone tissue-like organized matrix is ongoing. This profile suggests functional roles for these proteins in regulation of the ordered deposition of hydroxyapatite. A fourth developmental period follows in mature mineralized cultures during which time collagenase is elevated, apoptotic activity occurs, and compensatory proliferative activity is evident [510,511]. Although the biological significance of gene expression during the fourth developmental stage remains to be formally established, it appears to serve an editing/remodeling function for modifications in the bone extracellular matrix that sustain the structural and functional properties of the tissue. This working model of osteoblast differentiation has been supported by studies that demonstrate the stage-specific expression of family members of numerous classes of proteins that regulate development of the bone cell phenotype. These include, for example, the IGF family of binding proteins [512], cyclin and cyclin-dependent kinases [249], bone morphogenic proteins [492,500], oncogene-encoded proteins

CHAPTER 2 Osteoblast Biology

41

FIGURE 9 Stages of maturation in primary cultures of bone-derived cells. (A) Morphology and histochemical staining of fetal rat calvaria-derived osteoblasts at three stages: proliferation (P) with toluidine blue, matrix maturation (MM) with alkaline phosphatase staining, and mineralization (M) detection by the von Kossa stain. Multilayers of osteoblasts form a typical bone nodule having a mineralized extracellular matrix. (See also color plate.) (B) Northern blot analyses of marker genes expressed maximally at each stage. (See also color plate.) (C) Schematic illustration of several genes temporally expressed during 35 days of culture. Increases in mRNA transcripts of OC and OP during the culture period parallel calcium (Ca2 ) deposition. The increase and peak levels of collagenase are related to remodeling or editing of the extracellular matrix. Induction of genes reflecting the apoptosis of cells associated with the mineralized nodules is shown [365,483,494]. (D) The reciprocal and functionally coupled relationship between cell growth and differentiation-related gene expression is illustrated by arrows. Broken vertical lines between the three principal periods indicate experimentally established transition or restriction points requiring the downregulation of cell growth and ECM signaling events for the progression of differentiation.

42 [507], heat shock proteins [513], homeodomain proteins [514], and TGF- receptors [515]. Notably, several genes associated with skeletal cells, osteonectin [305,485] and matrix Gla protein [516], are expressed constitutively. The sequential and stringently regulated expression of genes that defines periods of osteoblast phenotype development is illustrated schematically as a reciprocal and functionally coupled relationship between proliferation and differentiation (Fig. 9B, bottom). The extent to which developmental expression of genes is sequential and mutually exclusive rather than concomitant is in part dependent on the position in the osteoblast lineage. For example, while in calvarial-derived osteoblasts, alkaline phosphatase expression is only observed in postproliferative cells, and alkaline phosphatase is compatible with proliferation in earlier stage bone marrow-derived osteoprogenitor cells. The interrelationship between proliferation and differentiation often times provides explanations for discordance between in vivo and in vitro effects. FGF, for example, is a potent mitogen for mesenchymal cells, chondrocytes, and osteoblasts and stimulates endosteal bone formation [212]. FGF-2 is useful for fracture repair in vivo [517]. However, FGF effects in vitro are stage specific. If added to a proliferation stage, mineralization and subsequent maturation of osteoblasts are inhibited [260,263,518]. In part, the results are mediated by continued proliferation modifying the differentiated phenotype and induced levels of collagenase modifying the ECM in a manner incompatible with ECM mineralization. In postproliferative cultures, FGF-2 promotes differentiation [263].

C. Differential Responsiveness of Hormones and Growth Factors as a Function of Stage of Osteoblast Maturation Based on the in vitro models of osteoblast differentiation, we can better understand the properties and physiologic responses of the cells at their individual stages of differentiation. This is best exemplified by selective responses in bone marrow stromal cell cultures and calvarial-derived osteoblasts to growth factors [263,491,518] and hormones [487,519 – 525]. The parathyroid hormone will promote the differentiation of preosteoblasts but suppress late stages of maturation [526,527]. Although caution should be exercised in translation from in vitro to in vivo effects of PTH on bone formation, these studies indicate that even pulsed PTH administration may increase bone formation by stimulating the proliferation of progenitors, and not by anabolic effects of differentiated osteoblasts [528]. It is established that TGF- stimulates the replication of progenitor cells and directly stimulates collagen synthesis. When proliferating calvarial osteoblasts are exposed to TGF-, a block in differentiation is observed

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[490 – 492,529]. The mitogenic effects of TGF- are not apparent on mature postproliferative osteoblasts. The insulin-like growth factors [284] and fibroblast growth factors (FGF-1, FGF-2) [530], which have distinct roles in development [11], also have selective effects on osteoblast subpopulation in vitro. The various steroid hormones, including glucocorticoids [519,531], 1,25(OH)2D3 [487,489,520], and estrogen [521], also have selective effects, either promoting differentiation of the cells at early stages of maturation or inhibiting anabolic activities and promoting resorptive properties of the osteoblast at later stages. In general, growth factors and steroid hormones have the most robust responses in immature osteoblasts and can radically modify their program of differentiation when added to proliferating cells. Glucocorticoids regulate numerous osteoblast-expressed genes, which contribute to bone formation, including cytokines, growth factors, and bone matrix proteins [reviewed in 193,531,532] (see Chapter 44). The molecular mechanisms by which glucocorticoids exert selective effects on a particular gene are complex, but numerous examples have been documented. Increases in alkaline phosphatase, osteocalcin, and collagen are observed at early maturation stages, but inhibition of these genes occurs in differentiated osteoblasts [519,533,534]. Both transcriptional and posttranscriptional mRNA stability contribute to positive and negative regulation of a gene, as shown for osteocalcin [519] and collagen [535]. Glucocorticoids promote osteoblast colony formation in human and rat marrow-derived cells and accelerate osteoblast differentiation in proliferating calvarial derived cells, reflected by both increased numbers and size of the bone nodules and early mineralization [439,493,494]. Because postproliferative cultures cannot be stimulated to produce more mineralizing nodules, the mature osteoblast is refractory to growth stimulation by dexamethasone [129,493,494,536]. This may be a consequence of glucocorticoids pushing cells to terminal differentiation or apoptosis, thereby depleting the pool of preosteoblasts capable of nodule formation [129,371,493]. It is noteworthy that dexamethasone exerts an antiproliferative effect on mouse osteoblasts [525,537] and blocks their maturation. These findings, together with glucocorticoid effects on osteoclast activity (reviewed in [193], contribute to glucocorticoid-induced osteopenia observed in vivo when pharmacologic doses of glucocorticoids are required [290,531, 538 – 541] (see Chapter 44). The active metabolite of vitamin D, 1,25(OH)2D3, has complex effects on the skeletal system related to targeting of many cell types, dose, and timing [542 – 544] (see Chapter 9). Vitamin D is a biphasic regulator of osteoblast activity for bone formation and bone resorption. Vitamin D regulates the expression of genes in osteoblasts that form the bone ECM or provide signals for osteoclast differentiation. The up-

CHAPTER 2 Osteoblast Biology

regulation by 1,25(OH)2D3 of numerous osteoblast parameters related to bone matrix formation and mineralization (e.g., collagen, alkaline phosphatase, osteopontin, osteocalcin, matrix Gla protein), and bone resorption by cytokines (e.g., osteoprotegerin [545]), reflects influences of the hormone on osteoblast function and regulation of bone turnover. However, pharmacological doses and long-term exposure of this hormone to rats can result in abnormalities of bone formation [544,546,547]. In vitro analysis of 1,25(OH)2D3 in primary cultures of rat osteoblasts show stage-dependent effects. The steroid is antiproliferative in the growth period and can block formation of the mineralized nodule when introduced during the growth period [487 –489,524,548] or stimulate differentiation-related gene expression in mature osteoblasts. Because of these properties, acute versus continuous exposure of cells to 1,25(OH)2D3 can lead to opposing results [487,489]. The selective effects based on the stage of osteoblast maturation are evident by in situ hybridization studies, in vivo and in vitro, and accompanying changes in cell morphology in vitro [136,137,143,536,549]. These changes in morphology and gene expression may relate to bone-resorbing effects of 1,25(OH)2D3 on surface-lining osteoblasts, which must (a) retract to allow for osteoclast interaction with the bone mineral and (b) provide local factors for the induction of osteoclast activity. Studies from many groups using different osteoblast models have reinforced two important concepts: (1) that the stage of osteoblast maturation influences the selective responsiveness of specific genes to hormones or growth factors and (2) that there is a window of responsiveness of a cell during which the factor can alter development and maintenance of the bone cell phenotype. These analyses have provided clinically relevant information toward an understanding of the consequences incurred by the osteoblast when exposed to therapeutic agents that may stimulate or inhibit cell proliferation or differentiation.

VI. MOLECULAR MECHANISMS MEDIATING PROGRESSION OF OSTEOBLAST DIFFERENTIATION A. Stage-Specific Expression of Transcription Factors and Their Contribution to OsteoblastSpecific Gene Expression The progression of osteoblast differentiation requires the sequential activation and suppression of genes that encode phenotypic and regulatory proteins. Key molecular events initiate from the extracellular matrix and signaling molecules, such as BMPs and TGF-s, that can indirectly result in a cascade of gene expression (see earlier discussion). In addition, regulatory factors that directly engage in protein – DNA, as well as protein – protein interactions, are

43 important mechanisms for both the activation and the suppression of genes reflecting stages of osteoblast maturation [550]. Transcription factors described in Section I, which contribute to position and pattern formation in the developing embryo, provide mechanisms for regulating the progression of osteoblast differentiation in the adult. The selective representation of these factors during osteoblast differentiation and family members within a class of transcription factors (Fig. 2), as well as evidence for their functional consequences (e.g., by forced expression, antisense or antibody blocking studies) on osteoblasts, provides compelling evidence for their regulatory effects in driving osteoblast maturation. The focus on transcription factors that regulated the developmental expression of osteocalcin [reviewed in 551,552] has been further supported by the characterization of mice carrying null mutations of several transcription factors (Table 1). Homeodomain proteins, Fos/Jun family members, helix – loop – helix factors, and RUNX2/CBFA1 proteins are among the well-characterized transcription factors (see Fig. 2). The homeodomain protein-binding sites contribute to bone-specific expression of several genes, collagen type I [553,554], osteocalcin [514,555], and, most recently, regulated expression of bone sialoprotein by Dlx5 [556]. During osteoblast differentiation in vitro, Msx-2 is expressed maximally in the preosteoblast and is subsequently downregulated [514] with the onset of osteocalcin expression. In situ hybridization studies confirm the reciprocal expression of osteocalcin and Msx-2 in cells of developing bone [557]. In a reciprocal fashion, Dlx-5 appears in the postproliferative osteoblast and increases during mineralization [558]. It appears that there is selectivity for the expression of homeodomain factors during osteoblast differentiation as well as developmental variations in activities [557]. Consistent with the developmental expression of Msx-2 in early osteoprogenitors and Dlx-5 in mature osteoblasts, Msx-2 downregulates OC, whereas Dlx-5 can positively regulate OC and collagen as well. Protein – protein interactions between Msx-2 and Dlx-5 are also determinants for developmental activities of these factors, as demonstrated by Zhang et al. [559]. The molecular mechanisms of this heterodimer interaction in alleviating suppression of the osteocalcin gene were demonstrated [555]. These activities may in part reflect the sequence content of homeodomain responsive promoter elements in genes that are expressed developmentally in osteoblasts [514,553,558,560,561]. Helix – loop – helix transcription factors are expressed at high levels in proliferating osteoblasts, thereby facilitating downregulation of the gene in these immature cells [85,509]. This complexity ensures developmental, tissuespecific regulated expression of the postproliferative bonespecific genes in osteoblasts. The fos and jun family members also exhibit developmental stage-specific expression and activities during osteoblast

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differentiation in vivo [562] and in vitro [507]. Studies of c-fos expression in transgenic mouse models reveal the importance of c-fos in establishing the osteoblast phenotype [563]. In vivo immunohistochemical staining reveals that c-fos is expressed in osteoprogenitor cells, in the perichondrium and periosteal tissues, but not in mature osteoblasts [562]. During osteoblast differentiation in vitro, c-fos and jun expression is expressed maximally in proliferating preosteoblasts. However, retention and upregulation of fra2 and jun-D protein levels and mRNA expression were observed postproliferatively in differentiated osteoblasts [564]. Confirmation of the functional activities of these factors in regulating progressive maturation of the osteoblast phenotype has been demonstrated by several experimental approaches, including specific repressor and enhancer activities of c-fos/ c-jun and fra2/jun-D, respectively, on the osteocalcin promoter, as well as inhibition of osteoblast differentiation resulting from antisense inhibition of fra2 expression [564]. RUNX2/CBFA1, described in Section I as an essential transcription factor for osteogenesis, regulates osteoblast differentiation. It is noteworthy that CBFA1 is present in the osteoprogenitors where osteoblast-specific genes are not expressed. CBFA1 mRNA, cellular protein levels, and DNA-binding activity are increased during osteoblast differentiation [103] and may contribute to specific CBFA1 activities in cells at distinct stages of osteoblast differentiation. In consideration of the precise functional activities of CBFA1 in the regulation of osteoblastic genes, multiple molecular mechanisms are operative. Oftentimes expression of a gene can be regulated during development (embryonic versus postnatal) or in relation to cell-type specificity by the production of protein isoforms resulting from either alternative usage of ATG initiation sites or splicing variant. Several examples of alternatively spliced products imparting tissue specificity have been recorded in genes. The Fibronectin gene gives rise to multiple splice variants, and the splicing process is cell type, developmental stage, and age regulated [565,566]. In 2(XI) collagen, alternative

FIGURE 10

splicing gives rise to proteins that are expressed in a tissuespecific manner. Isoforms lacking exons 6 – 8 of the 2(XI) collagen gene code for a protein that is found in cartilaginous tissue of developing limbs and axial skeleton, whereas the transcript containing exons 6 – 8 is found in nonchondrogenic tissue such as calvaria and periosteum [567]. These mechanisms may contribute to the timing of expression or the protein isoforms may exhibit specialized functions. Several NH2 and COOH-terminal CBFA1 isoforms have been reported (Fig. 10) [93,568,569]. Both the aminoterminal isoforms, type I (MRIPV) and type II (MASNS) isoforms, have nearly equivalent transactivation activity on different promoters and in various cell lines [118, 570 – 572]. To date, no studies have systematically addressed the relative expression of these CBFA1 isoforms and other isoforms resulting from C-terminal splicing events. Noteworthy in the regulation of CBFA1 activities are posttranslational modifications, including phosphorylation by MAPK [573]. In addition, CBFA1 interacts with several coregulatory proteins, both activators and suppressors of CBFA1 activity [95,96,574,575,575,576], which also appear to be developmentally expressed during osteoblast differentiation [574]. CBFA1 also has properties that can mediate modifications in chromatin, an important component of tissue-specific transcriptional control that is described in the following section.

B. Contributions of Nuclear Architecture to Transcriptional Control during Osteoblast Differentiation It is becoming increasingly apparent that nuclear architecture provides a basis for support of the stringently regulated modulation of cell growth and tissue-specific transcription necessary for the onset and progression of osteoblast differentiation. Here, multiple lines of evidence point to contributions by three levels of nuclear organization to in

CBFA1 NH2 terminal isoforms present in osteoblast lineage cells. Schematic illustration of the origin of CBFA1 regulated by an upstream (PU) promoter is transcribed at MET-1 in exon 1 (MASNS isoform or type II designation by Harada et al. [118]). The type I isoform (MRIPV), regulated by the downstream promoter (PD), was the first CBFA1 protein to be cloned from T cells [94], which is also expressed in mesenchymal lineage cell lines, chondrocytes, and osteoblasts [114 – 118]. The MASNS isoform was initially identified by Stewart et al. [96a] and originates at MET 69 regulated by the same downstream promoter (PU) from which OSF-2 was first characterized [102]. OSF-2 is poorly transcribed from the MET-1, which does not reside in a Kozak sequence [96b].

CHAPTER 2 Osteoblast Biology

vivo transcriptional control where structural parameters of the genome are functionally coupled to regulatory events. The primary level of gene organization establishes a linear ordering of promoter regulatory elements. This representation of regulatory sequences reflects competency for the responsiveness to physiological regulatory signals as discussed earlier. The osteocalcin gene provides a paradigm for the involvement of nuclear organization in transcriptional control that is linked to bone formation, homeostatic regulation, and bone remodeling. The regulatory elements of the bone-specific osteocalcin gene are organized in a manner that supports responsiveness to physiologic mediators and developmental expression in relation to bone cell differentiation. Characterized regulatory elements and cognate transcription factors can support osteocalcin suppression in nonosseous cells, immature osteoblasts, and growthstimulated osteoprogenitors/osteoblasts, transcriptional activation in postproliferative cells, and steroid hormone enhancement (Fig. 11). For example, the OC box (99 to 76 bp) is a multipartite element that binds several classes of transcription factors. The OC box binds homeodomain proteins (e.g., Msx-2, Dlx-5 [514,577]) and a bone tissuespecific complex of unknown origin, designated the OC box-binding protein (OCBP), which contributes to the activation of gene transcription [578,579]. The minimal OC promoter containing the OC box is sufficient to support osteoblast-specific expression in vitro. A bipartite element in the proximal promoter confers FGF-2 and cAMP responsiveness [580]. This element regulates the suppression of osteocalcin synthesis in response to numerous modulators of cell growth, including IGF-1, bFGF, cAMP, and PTH [581]. A series of AP-1 sites occur in the OC gene promoter [506,582,583], including one that is the TGRE [584]. Fos (c-fos, fral, fra2) and jun (c-jun, jun-D, jun-B) oncogeneencoded families of transcription factors form homo or heterodimeric complexes that regulate gene expression at AP-1 motifs. These AP-1 sites provide another example of regulatory sequences that contribute to positive and negative regulation dependent on the biological circumstances. The c-fos/c-jun heterodimer represses, whereas the fra2/jun-D heterodimer activates transcription. The latter is more abundant in postproliferative osteoblasts. The vitamin D responsive element (VDRE) functions as an enhancer [585 – 588], binding the transcriptionally active VDR/RXR heterodimer complex (see Chapter 9). The core motif, two steroid half elements with a three nucleotide space, is highly conserved [589 – 591]. However, subtle variations, both within the core domain and in the flanking sequences, render VDRE promoter elements of various genes selectively ligand responsive in a developmental and tissue-specific manner. Specificity of VDRE utilization is further conferred by protein – DNA and/or protein – protein interactions in addition to the VDR/RXR complexes

45 [479,591 – 595]. The osteocalcin VDRE transcription factor complex appears to be a target for modifications in vitamin D-mediated transcription by other physiologic factors, including glucocorticoids [592], TNF-a [596], and retinoic acid [597 – 601]. It is evident from available findings that the linear organization of gene regulatory sequences is necessary but insufficient to accommodate the requirements for physiological responsiveness to homeostatic, developmental, and tissue-related regulatory signals. Parameters of chromatin structure and nucleosome organization are a second level of genome architecture. There is a requirement to render promoter regulatory elements competent for protein – DNA and protein – protein interactions that mediate positive and negative controls. Additionally, activities of regulatory complexes at the proximal and distal promoter must be integrated. Modifications in chromatin reduce the distance between promoter elements, thereby supporting interactions between the modular components of transcriptional control. Each nucleosome (approximately 140 nucleotide base pairs wound around a core complex of two each of H3, H4, H2, and H2B histone proteins) contracts linear spacing by sevenfold. Folding of nucleosome arrays into solenoid-type structures provides a potential for interactions that support synergism between promoter elements and responsiveness to multiple signaling pathways. The molecular mechanisms that mediate chromatin remodeling are being defined [reviewed in 602 – 605]. A family of proteins comprising multimeric protein complexes has been described in yeast (SWI/SNF complex) and in mammalian cells that promote transcription by altering chromatin structure [604 – 608]. These alterations render DNA sequences containing regulatory elements accessible for binding cognate transcription factors and mediate protein – protein interactions that influence the structural and functional properties of chromatin. Multiple lines of evidence suggest that the remodeling of nucleosomal structure involves alterations in histone – DNA and/or histone – histone interactions. Histone acetylation and phosphorylation posttranslational modifications have been functionally linked with changes in nucleosomal structure that alter the accessibility to specific regulatory elements [605]. Core histone hyperacetylation enhances the binding of most transcription factors to nucleosomes [609 – 611]. Alterations in the chromatin organization of the osteocalcin (OC) gene promoter during osteoblast differentiation provide a paradigm for remodeling chromatin structure and nucleosome organization that is linked to a long-term commitment to phenotype-specific gene expression [612 – 615]. In nonosseous cells, the packing of chromatin contributes to the extent that promoter elements are accessible to transcriptional activation complexes. An array of nucleosomes on the OC promoter in nonosseous cells contributes to maintaining the suppression of gene transcription. Figure 11 schematically depicts modifications in chromatin structure and

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FIGURE 11

Developmental remodeling of nucleosome organization in the osteocalcin gene promoter correlates with transcriptional activity during the differentiation of normal diploid osteoblasts. The positioning of nucleosomes in the osteocalcin gene promoter was determined by combining DNase I, micrococcal nuclease, and restriction endonuclease digestions with indirect end labeling [272]. Filled circles represent the placement of nucleosomes with the shadows indicating movement within nuclease-protected segments. Note the differences in nucleosome organization of the gene between nonosseous cells when the OC gene is not transcribed (line 1) and in osteoblasts expressing osteocalcin (lines 2 and 3). Vertical arrows correspond to the limits of the distal (600 to 400) and proximal (170 to 70) sites of DNase I hypersensitivity (DHS), which are observed in osteoblasts when the OC is expressed. DHS is increased in response to vitamin D (represented by larger arrows). Positions of CBFA1 elements (sites A, B, C), the VDRE (465 to 437), the osteocalcin box (OC box 99 to 77), and TATA element (31 to 28) are designated.

nucleosome organization that parallel competency for transcription and the extent to which the osteocalcin gene is activated and transcribed in bone cells. Basal expression and enhancement of osteocalcin gene transcription are accompanied by two changes in the structural properties of chromatin when the gene is activated in a bone cell. When the gene is activated in osteoblasts, there is a rearrangement in nucleosome placement [613]. A single nucleosome becomes positioned between proximal regulatory elements, and distal steroid hormone-dependent regulatory sequences provide a basis for accessibility of transactivation factors to cognate sequences (Fig. 11). This model is derived from experimen-

tal evidence. DNase I hypersensitivity is detected in two regulatory domains of the promoter encompassing the VDRE and CBFA sequences in the proximal and distal promoter [612 – 614]. Vitamin D treatment enhances DNase I hypersensitivity. These domains contribute to tissue-specific and vitamin D enhancer activity. A third level of nuclear architecture that contributes to transcriptional control is provided by the nuclear matrix [616]. The anastomosing network of fibers and filaments that constitute the nuclear matrix supports the structural properties of the nucleus as a cellular organelle and accommodates structural modifications associated with

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proliferation, differentiation, and changes necessary to sustain phenotypic requirements of specialized cells. As the intricacies of gene organization and regulation are elucidated, the implications of a fundamental biological paradox become strikingly evident. With a limited representation of gene-specific regulatory elements and a low abundance of cognate transactivation factors, how can sequence-specific interactions occur to support a threshold for the initiation of transcription within nuclei of intact cells? Viewed from a quantitative perspective, the in vivo regulatory challenge is to account for the formation of functional transcription initiation complexes with a nuclear concentration of regulatory sequences that is approximately 20 nucleotides per 2.5 yards of DNA and a similarly restricted level of DNAbinding proteins. Regulatory functions of the nuclear matrix include, but are by no means restricted to, DNA replication [617], gene location [618,619], imposition of physical constraints on the chromatin structure that support the formation of loop domains, concentration, and targeting of transcription factors [620 – 623], RNA processing and transport of gene transcripts [624 – 628], and posttranslational modifications of chromosomal proteins, as well as imprinting and modifications of chromatin structure [629]. Chromatin loop domains (10 – 100 kb) are tethered to components of the nuclear matrix through MAR (matrix attachment regions) sequences.

The initial indication of linkage between the nuclear matrix and control of osteoblast differentiation was first provided by the observation of a parallel representation of nuclear matrix proteins and developmental stage-specific gene expression [630]. Direct involvement of the nuclear matrix in the control of bone-specific gene transcription is provided by several lines of evidence. Two nuclear matrix DNA-binding proteins regulate the activation of osteocalcin gene transcription, identified as NMP1. YY1 modulates vitamin D enhancer activity and NMP2 characterized as RUNX/CBFA1 is a transcriptional activator protein and contributes to promoter organization [99]. The mechanism by which transcription factors contribute to overall promoter architecture is becoming clear. Transcription factors and chromatin-remodeling factors associated with the nuclear matrix, such as CBFA factors [631] and the YY1 transcription factor [632], which regulate osteocalcin gene activity, can contribute to conformational modifications in the promoter structure. Through protein – DNA interactions of the CBFA element with nuclear matrix-associated CBFA factors, local chromatin changes are induced by coactivator/corepressor proteins associated with the DNAbinding complex, and the OC promoter becomes poised for transcription. Figure 12 extends the model presented in Fig. 11 by illustrating the three-dimensional organization of the promoter that is permissive for the binding of

FIGURE 12 Role of the nuclear matrix-associated CBFA1 factor in organizing promoter architecture. A three-dimensional representation of the osteocalcin promoter is shown. The interaction of OC CBFA recognition motifs with CBFA1 provides a conformation stability mediated by the tight association of CBFA1 with the nuclear matrix scaffold. This conformation, together with the positioned nucleosome, facilitates the integration of regulatory signals between the proximal (TATA) and the distal (VDRE) elements necessary for basal and vitamin D enhancer activity. Mutations of three CBFA elements in the rat OC gene resulting in loss of DNase I hypersensitivity and vitamin D responsiveness support this model [647].

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FIGURE 13

CBFA-interacting proteins regulating CBFA transactivation functions. Coregulator proteins interact directly with the indicated domains of CBFA transcription factors. Structurally and functionally homologous segments include the conserved DNA-binding runt homology domain, transcriptional activation, and suppression domains [643,711,712], as well as subcellular targeting signals [631,713]. The promoter organizing functions of CBFA factors may involve interacting proteins, including CBF [714], necessary for DNA binding of CBFA to regulatory elements, coactivators ALY [633] and p300, the latter having histone acetylase (HAT) activity [634], and Groucho/TLE [574,575,639], a negative regulator of CBFA enhancer activity. In addition, other transcription factors [635], Smad signal transduction proteins [123], and steroid receptors [637] contribute to synergistic gene expression with CBFA1 (shown in Fig. 14) [reviewed in 94,95].

transactivation factors and protein – protein interactions between distal and proximal elements (e.g., TFIIB TATA box-binding protein and the VDR/RXR). We postulate that this promoter architecture is mediated and stabilized by transcription factors that are targeted to subnuclear domains, such as CBFA1. Among the numerous CBFA-interacting proteins that have been identified (Fig. 13), several have enzyme activities for chromatin remodeling (Fig. 14). Coactivators include ALY [633] and p300, which has histone acetylase (HAT) activity [634], as well as interacting transcriptional regulators [635], ear-2 [636], signal transduction proteins [123], and steroid receptors [637,638]. Corepressor modulators include HES [575] and the negative regulatory factor Groucho/TLE [576,639], which is also associated with the nuclear matrix [640]. This protein has histone deacetylase activity and a histone H3-interacting domain [641,642]. We have shown that the highest levels of Groucho at the mRNA and cellular protein levels are observed in early stage osteoblasts [574]. Groucho/TLE is also abundant in muscle tissue and may contribute to the suppression of osteocalcin in nonosseous cells and proliferating osteoblasts. As indicated in Fig. 13 (bottom), cooperative interactions between CBFA and other transcription factors, through as yet undefined mechanisms, have been reported. The synergistic transactivation of gene transcription between CBFA

and the Ets factors is well documented [95,96], as well as CBFA with C/EBP factors [643] and Smad factors. These interactions provide options for positive and negative regulation of a spectrum of CBFA-regulated genes within the cell or related to a cellular phenotype. Of interest, osteoblast-specific synergistic interactions between an estrogen response element and CBFA elements have been reported [638]. These respective activities contribute to the enhancer and suppressor transcriptional properties of CBFA factors as illustrated in Fig. 14 [574,575,644]. Several reports have indicted that not all osteoblast-expressed genes containing CBFA regulatory sequences are activated by Cbfa1, some are repressed; e.g., BSP [645] and the CBFA1 gene are autoregulated by CBFA1 [646]. The significance of the localization of these transcription factors in specific subnuclear domains, as well as their ability to interact with chromatin modifying proteins, is related to tissue-specific, as well as steroid hormone-dependent gene control of transcription. Colocalization of these regulatory components that are functionally linked to activation and suppression have been established in situ [574]. Validation of these models is further provided by studies in which the CBFA sites in the rat OC promoter were mutated. Three CBFA motifs are strategically positioned in the bone-specific rat osteocalcin promoter. Sites A and B flank the vitamin D response element in the distal

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FIGURE 14

Model of CBFA transcriptional regulation of gene activation and suppression mediated by modifications in chromatin architecture. CBFA factors interact with histone acetylases (HATs) (e.g., p300) to open chromatin domains for the binding of regulatory factors that lead to transcriptional activation. CBFA interactions with coregulators having associated histone deacetylase activity (HDAC) result in the suppression of transcription.

promoter, and sites B and C define the orders of a positioned nucleosome in the proximal promoter. The functional significance of multiple CBFA elements in contributing to promoter structure was addressed by mutating individual or multiple AML sites within the context of the native rat ( 1.1 kb) osteocalcin promoterCAT [647]. All three sites are required for maximal basal expression of the rOC promoter. Strikingly, mutation of the three CBFA1 sites leads to abrogation of responsiveness to vitamin D, glucocorticoids, and TGF-. The mechanism of this loss of promoter responsiveness was related to an absence of DNase I hypersensitive sites at the vitamin D response element and over the proximal tissue-specific basal promoter. These findings strongly support a multifunctional role for AML factors in regulating gene expression, not only as a simple transactivator, but also by facilitating modification in promoter architecture and chromatin organization. It is already apparent that local nuclear environments generated by the multiple aspects of nuclear structure are intimately tied to the developmental expression of cell growth and tissue-specific genes. Membrane-mediated initiation of signaling pathways that ultimately influence transcription has been recognized for some time. Here, the mechanisms that sense, amplify, dampen, and/or integrate regulatory signals involve structural as well as functional

components of cellular membranes. Extending the structure – regulation paradigm to nuclear architecture expands the cellular context in which cell structure – gene expression interrelationships are operative. The nuclear structure is a primary determinant of transcriptional control. Thus, the power of addressing gene expression within the threedimensional context of nuclear structure would be difficult to overestimate.

VII. CONCLUDING REMARKS This chapter has presented the cell biology of osteoblasts within the content of our current understanding of the regulatory controls operative in promoting osteoblast differentiation. We have attempted to address how physiologic parameters of gene expression are integrated to support the requirements of bone development and functional integrity of the tissue. During osteoblast phenotype development and bone formation, stages of maturation are defined by levels of expression of subsets of osteoblast genes. A cohort of tissue-specific, developmental, steroid hormone and growth factor-related transcription factor complexes impinge on gene transcription, providing a complex and integrated series of regulatory signals for the selective activation and repression of genes related to osteoblast activity. Additionally,

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the effects of a hormone or growth factor on expression of a specific gene are related to the phenotype as well as the stage of cellular maturation because of the different representation of proteins that contribute to gene regulation. Thus clinical consideration for treatment and therapeutic regimens can be approached with greater knowledge of the consequential effects at the level of gene-regulating responses. We have presented a growing body of evidence for cross-talk at all levels of bone biology, including among the subpopulations of bone cells, between the extracellular matrix and intracellular signaling factors, and finally at the DNA level between transcription factor complexes at multiple elements. Such interactions and complexities need to be considered in future applications of therapeutic strategies.

Acknowledgments The authors gratefully appreciate preparation of the manuscript by Judy Rask and thank colleagues Janet Stein and André van Wijnen for helpful discussions and members of our research group: Chaitali Banerjee, Amjad Javed, Hicham Drissi, Kaleem Zaidi, and Eva Balint. The National Institutes of Health grants supporting the research program related to this chapter include AR45688, AR45689, AR39588, and DE12528. The contents of this chapter are solely the responsibility of the authors and do not necessarily represent the official views of the National Institutes of Health.

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CHAPTER 3

Osteoclast Biology F. PATRICK ROSS AND STEVEN L. TEITELBAUM Department of Pathology, Washington University School of Medicine, St. Louis, Missouri 63110

I. II. III. IV. V.

VI. Mechanisms of Bone Resorption VII. Humoral Regulation of Osteoclastic Bone Resorption VIII. Diseases of the Osteoclast References

Introduction Osteoclast Morphology Origin of Osteoclasts Models of Osteoclast Function Osteoclast Attachment and Polarization

I. INTRODUCTION

polykaryons generally juxtaposed to bony surfaces and often in resorption pits (Howship’s lacunae) (Fig. 1). These excavations are the products of the capacity of the osteoclast to degrade both organic and inorganic matrices of bone, an event in which mineral removal precedes collagenolysis [1]. Consequently, resorption pits are lined by demineralized collagen, which has not yet been degraded, a phenomenon best appreciated by scanning electron microscopy [2] (Fig. 2). All forms of adult osteoporosis reflect enhanced bone resorption relative to formation and should be viewed in the context of the remodeling cycle. Bone remodeling, a process characterized by the coupling of osteoclast and osteoblast recruitment, occurs throughout life in thousands of sites within the human skeleton. While the fundamental purpose of bone remodeling is unknown, it probably serves to replace effete, aged bone with that which is newly synthesized. Remodeling is initiated by osteoclasts, or their precursors, attaching to trabecular or endosteal bone surfaces. The mechanism by which the osteoclast binds to bone has been a focus of intense investigation and its recent unraveling promises to yield novel approaches to osteoporosis prevention. In this regard, it has been hypothesized that a thin layer of unmineralized type I collagen covering the surface of bone must be removed prior to osteoclast attachment [3]. Degradation of this surface collagen is proposed to be the

Osteoporosis, regardless of etiology, always represents enhanced bone resorption relative to formation. Thus, insights into the pathogenesis of this disease, and progress in its prevention and/or cure, depend on understanding the mechanisms by which bone is degraded. The osteoclast is the principal, if not exclusive, resorptive cell of bone. It is a member of the monocyte/ macrophage family, and most successful treatments of osteoporosis, to date, target osteoclastic bone resorption. Major advances have been witnessed in delineating the mechanisms by which osteoclasts are generated from their precursors and stimulated to degrade bone once they are fully differentiated. In fact, the essential osteoclastogenic molecules are now in hand and, most importantly, are the targets of rational antiosteoporosis drug design. These potential therapeutic agents reflect advances made in understanding osteoclast biology.

II. OSTEOCLAST MORPHOLOGY The osteoclast is a multinucleated cell whose capacity to degrade hard tissues depends on cell/matrix contact. Thus, when viewed in histological sections, osteoclasts appear as

OSTEOPOROSIS, SECOND EDITION VOLUME 1

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FIGURE 1 Osteoclasts (arrows) in resorption bay (Howship’s lacuna). Resorptive cells are juxtaposed to bone, and their nuclei are polarized toward the antiresorptive plasma membrane (nondecalcified, Goldner stain). consequence of secretion of neutral collagenase by osteoblasts [4]. While provocative, this hypothesis is unproven and, in fact, the putative layer of lining collagen may be an artifact of tissue fixation.

FIGURE 2

Upon attaching to a nascent remodeling site, osteoclasts polarize their resorptive machinery (infra vide) toward the cell/bone interface and generate a Howship’s lacuna approximately 50 m deep. Once the degradative phase of

Scanning electron micrograph of resorption pit formed on bone slice by isolated avian osteoclast. Bundles in the bottom of the pit represent bone collagen that the cell has demineralized in preparation for degradation.

CHAPTER 3 Osteoclast Biology

the remodeling cycle is complete, osteoclasts are replaced in the resorptive bay by mononuclear cells of unknown origin, which in turn are replaced by cells of osteoblastic lineage. Histological sections of normal human osteoclasts contain varying numbers of nuclei generally maximizing at 10. Nuclear number may mirror osteoclast activity. For example, osteoclasts in Paget’s disease, a disorder of accelerated resorption, are enormous and often contain as many as 100 nuclei [5]. Alternatively, hypernucleated osteoclasts are also encountered in circumstances of resorptive dysfunction, such as various forms of osteopetrosis. Whether the abundant osteoclast nuclei seen in some osteopetrotic patients reflect increased levels of osteoclastogenic agents, such as parathyroid hormone, or an abnormality intrinsic to the osteoclast per se is not known. Osteoclast nuclei are distinct from one another, a feature useful in distinguishing the cell from the megakaryocyte. Moreover, osteoclast nuclei are typically polarized away from the plasma membrane juxtaposed to bone, residing closest to the antiresorptive surface of the cell. Formation of the osteoclast polykaryon probably depends on matrix attachment. Furthermore, osteoclast multinucleation is not the product of endomitosis, as mitotic figures are rarely encountered. However, when placed in culture with mononuclear phagocytes, osteoclasts incorporate new nuclei and expel others [6]. Thus, the most compelling hypothesis holds that once an osteoclast, or its progenitor, attaches to a putative resorptive site, additional mononuclear precursors that fuse with the immobilized cell are recruited. While the life span of osteoclasts is unknown, they clearly undergo programmed cell death. Increased numbers of apoptotic osteoclasts appear when exposed to specific families of bisphosphonates, particularly those that modulate the mevalonate pathway [7]. Alternatively, agents such as interleukin-1 (IL-1), M-CSF, and RANK-ligand (RANKL) extend the cell’s survival (vide infra). Osteoclasts are rich in lysosomal enzymes, with the most studied being tartrate-resistant acid phosphatase (TRAP). While presence of this enzyme in histological sections serves to identify osteoclasts, the bone isoform of TRAP is also expressed by macrophages derived from other organs, most notably lung and spleen [8,9]. At first glance these data would suggest that TRAP is of little use in identifying osteoclasts formed in culture. Alternatively, Suda and colleagues generated bona fide osteoclasts in vitro from alveolar and splenic macrophages, and the same is true regarding circulating human monocytes [10], suggesting that TRAP-expressing cells resident in these tissues are, in fact, capable of osteoclastogenesis [11]. Similarly, the antiosteoclastogenic cytokine interleukin-4 (IL-4) prompts appearance of TRAP-negative polykaryons in culture, thereby associating resorptive activity with expression of

75 the enzyme [12]. Thus, while osteoclasts generated in vitro from macrophages invariably express TRAP, confirmation of their identity requires demonstration of other phenotypic features, preferably the capacity to resorb bone. Because osteoclasts are enormous, often larger than 100 m in diameter, histological sections, typically 5 to 10 m thick, accommodate only a small proportion of each cell. Reflecting the size and complexity of the osteoclast, a given section may contain apparently separate fragments of the same cell, generally considered distinct osteoclasts by bone histomorphometrists. Thus, so-called mononuclear osteoclasts may represent a portion of a multinucleated cell. The likelihood of multiple fragments of the same cell in a single slide underscores the limitations of attempting to distinguish altered numbers of osteoclasts from changes in size or plasma membrane complexity. Being a member of the monocyte/macrophage family, osteoclasts share a number of morphological features with foreign body giant cells, such as abundant lysosomes. However, the unique capacity to resorb bone endows the osteoclast with morphological features distinct from those of other related macrophage-like cells. For example, unlike foreign body giant cells, osteoclasts are rich in mitochondria and, in fact, probably represent the cell containing the greatest density of these organelles [13]. Clearly, however, the morphological feature of the osteoclast that most dramatically distinguishes it from related cells is its ruffled membrane (Fig. 3). This complex infolding of plasma membrane, polarized to the cell/bone interface, is best appreciated by transmission electron microscopy. The ruffled membrane is unique to the osteoclast and matrix specific, appearing only when the cell is in contact with bone. Because of the enormity of an osteoclast, its plasma membrane may touch bone in more than one location and thus separate sites of ruffling will develop within the same cell. Moreover, the extent of ruffled membrane formation appears to parallel the degradative activity of the cell and its exposure to resorptive agonists or antagonists [14]. In fact, as osteoclasts alternate between their resorptive/adherent and mobile/detached states, they respectively express and lose their ruffled membranes. While yet to be visualized in situ, ruffled membrane formation probably represents the insertion of proton pumpbearing vesicles into the plasma membrane, a process similar to exocytosis (Fig. 4). In fact, the small GTPase, Rab-3, which modulates the fusion of exocytic vesicles to the plasma membrane, may also regulate ruffled membrane formation [15]. The ruffled membrane is structurally distinct from the nonresorptive plasma membrane in that it contains abundant, “spike-like” structures shown by light [16] (Fig. 5, see also color plate) and ultrastructural immunocytochemistry to represent vesicular proton pumps. These same projections are present in acidifying vesicles within the cytoplasm. This

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FIGURE 3

Transmission electron micrograph of osteoclast attached to bone (B). The convoluted infolding of plasmalemma is the ruffled membrane (R). The sealing or “clear” zone (C) is rich in actin filaments oriented perpendicular to the bone surface. Note the abudance of mitochondria (arrows).

observation supports the hypothesis that these proton pumpbearing vacuoles are precursors of the ruffled membrane. While its role, if any, in the resorptive process is not clear, TRAP activity also polarizes to the ruffled membrane and, like other lysosomal enzymes, is secreted into the resorptive microenvironment [17], where it dephosphorylates osteopontin, thereby altering the adhesive properties of this important bone matrix protein [18]. Osteoclasts contain a prominent cytoskeleton, components of which are critical to resorptive function. Microtubules extend from organizing centers to the periphery of the cell. These structures polymerize on cell/substrate adherence and, in so doing, acquire the capacity to transport vesicles [19]. Thus, microtubules are likely to play a central role in osteoclast polarization, delivering proton pump-containing vacuoles for insertion into the resorptive membrane [20] (Fig. 6). The intimacy between osteoclasts and bone required for resorption is reflected, in the cell, by the matrix attachment or “sealing” zone (Fig. 3). This distinct morphological entity, organized as a ring, completely surrounds the ruffled membrane. While organelle free, and thus also referred to as the “clear zone,” this attachment area is, in actuality, rich in microfilaments polarized perpendicularly to the bone surface [21]. The sealing zone is a well-demarcated structure, not unique to the osteoclast, and is also formed

when other members of the macrophage/monocyte family attach to the matrix. Like microtubules, microfilaments organize upon adherence to bone, and the filamentous distribution of F-actin directly correlates with the resorptive activity of osteoclasts [22] (Fig. 7, see also color plate). In fact, when osteoclasts are in the process of degrading bone, Factin localizes in a ring-like structure of punctate plasma membrane protrusions known as focal adhesions or podosomes [23] (Fig. 8). In addition to F-actin, these structures contain matrix-recognizing integrins and proteins such as vinculin and talin, which link these attachment heterodimers to the cytoskeleton [24]. Thus, actin filaments in osteoclasts probably anchor the plasma membrane to the cytoskeleton and are central to the means by which osteoclasts recognize bone. Following formation of a resorptive lacuna, the cell becomes motile, an event attended by dissolution of the actin ring. Evidence in hand indicates that the cyclical mobilization of the actin cytoskeleton in osteoclasts is under the aegis of the Rho family of GTPases [25]. While the magnitude of podosome expression appears to mirror the degradative activity of the cell [26], the punctate appearance of these attachment structures suggests that they do not serve as the osteoclast’s “tight seal.” Unraveling the molecular mechanisms serving to isolate the resorptive microenvironment

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FIGURE 4

Model of ruffled membrane formation. In the nonattached state the osteoclast is unpolarized, with acidifying vesicles distributed throughout its cytoplasm. Once in contact with bone, matrix-derived signals, probably mediated via integrins such as v3, prompt targeting of acidifying vesicles to the apical (resorptive) surface of the cell. Insertion of these vesicles into the bone-adjacent plasma membrane yields the characteristic, highly ruffled resorptive surface of the cell. The “spikes” within the vesicles represent the H-ATPase (proton pump).

of the osteoclast is a challenge likely to yield important insights into regulating its activity.

III. ORIGIN OF OSTEOCLASTS Avian osteoclasts (OC) immunostained for vacuolar HATPase (proton pump). (A) Cells are juxtaposed to bone, and the pump (brown-staining reaction product, arrows) is polarized toward the cell – bone interface. (B) Osteoclasts are unattached to bone, and HATPase is distributed diffusely throughout the cytoplasm. Reprinted with permission from Blair et al., Science 245, 855 – 856. Copyright 1989 American Association for the Advancement of Science. (See also color plates).

FIGURE 5

The hematopoietic origin of osteoclasts is now in hand. Its recognition, however, follows a contentious history with initial debate focusing on whether osteoclasts and osteoblasts derive from a common precursor [27]. Early attempts to resolve this controversy involved experiments in which the circulations of two rats were joined. Using this parabiotic approach, Gothlin and Ericsson (reviewed in Ref. 28) established that osteoclasts migrating to a fracture are derived from the blood of its partner. In contrast, osteoblasts are not donor derived, suggesting that their ontogeny differs from that of bone-resorbing cells.

The hematopoietic origin of osteoclasts is also suggested by experiments in which quail cartilaginous limb buds were transplanted onto chick chorioallantoic membranes [29]. The host circulation vascularized the rudiments, which

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FIGURE 6 Model of osteoclast polarization. In the nonattached state the osteoclast is unpolarized, with acidifying vesicles distributed throughout its cytoplasm. Once in contact with bone, matrix-derived signals, probably mediated via integrins such as v3, prompt targeting by trafficking along microtubules of acidifying vesicles to the apical (resorptive) surface of the cell. Insertion of these vesicles into the bone-adjacent plasma membrane yields the characteristic, highly ruffled resorptive surface of the cell.

FIGURE 8 Isolated murine osteoclast stained with rhodamine phalloidin to delineate F-actin. The fluorescent rings are well-organized sealing zones containing several layers of punctate, focal adhesions, or podosomes.

FIGURE 7 Varying organization of the actin cytoskeleton during the different stages of osteoclast activity. Prior to resorptive phase (stages 1 – 3), actin (red) and vinculin (green) are found in punctate, podosomal structures. During resorption (stage 4), podosomes are lost and actin forms a dense continuous band surrounded by a double ring of vinculin and talin (not shown). During the postresorptive period (stage 5), the system reverts toward its original state. (See also color plate.)

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ultimately developed into bone. Most of the osteoclasts present in the neovascularized graft were of chick origin, indicating that they were derived hematopoietically. The hematopoietic origin of osteoclasts, and its distinction from that of osteoblasts, is also established by studies of murine and human osteopetrosis, a disease discussed in detail later (Section VIII). Given the hematopoietic derivation of the osteoclast, efforts turned to identifying its precursor. Because of phenotypic similarities, the cell was suspected of membership in the monocyte/macrophage family. Initial experiments aimed at determining if such is the case involved the injection of thorium dioxide-labeled macrophages into sibling rats who immediately thereafter had a femur fractured. Initially, only labeled monocytes and macrophages were present in the fracture but, ultimately, thorium dioxide-bearing osteoclasts appeared. These observations prompted the search for functional similarities between osteoclasts and macrophages [28]. Early in vitro studies suggested that peripheral blood monocytes and elicited rodent peritoneal macrophages are capable of degrading bone [30 – 32], but it is unlikely that the models used in these experiments are reflective of bona fide resorption. Moreover, despite formation of TRAP-expressing polykaryons by monocytic leukemic lines placed in osteoclastogenic conditions [33], these cells are not authentic osteoclasts. The first successful attempt at generating osteoclasts in vitro utilized long-bone primordia [34]. Culture of these rudiments with embryonic liver and marrow mononuclear cells, but not peripheral blood monocytes or peritoneal macrophages, propagates osteoclasts. Colony-forming unitgranulocyte – macrophage (CFU-GM) or more primitive stem cells are also capable of differentiating into bone resorptive polykaryons. The suggestion that osteoclasts are derived from pluripotent hematopoietic stem cells [35] is supported by experiments in which spleen cells taken from 5-fluorouracil-treated mice were cultured with IL-3 [36]. Blast cell colonies so formed mature into TRAP-expressing polykaryons responsive to calcitonin and are capable of resorbing bone. In contrast, mature macrophages are unable, in this system, to differentiate into osteoclasts. The experiments discussed thus far indicate that isolated, primitive hematopoietic precursors, but not more differentiated macrophages, are capable of osteoclast differentiation in vitro. The studies of Takahashi et al. [37] provided insight into the apparent osteoclastogenic capacity of immature and mature macrophages. In these experiments, mouse marrow cells in the presence of 1,25(OH)2D3 or parathyroid hormone (PTH) formed osteoclasts [38]. Interestingly, most TRAPexpressing polykaryons appeared near alkaline phosphataseexpressing cells, suggesting that osteoblasts play a role in osteoclast differentiation. This group next demonstrated that osteoclasts are induced by coculture of mouse spleen cells and osteoblasts [39]. Furthermore, marrow-derived stromal

cell lines may substitute for osteoblasts in inducing osteoclastogenesis [38,40]. Most importantly, in this coculture system, mature monocytes and macrophages differentiate into osteoclasts [11]. As will be discussed, the means by which stromal cells and osteoblasts promote osteoclastogenesis are in hand. Colony-forming assays, along with osteoclast and macrophage markers, serve to define further the lineage of human osteoclasts. CD34 hematopoietic stem cells, maintained with granulocyte – macrophage colony-stimulating factor (GM-CSF), form CFU-GM colonies [41]. Treatment of these colonies with 1,25(OH)2D3 generates granulocyte, macrophage, and mixed colonies, as well as those consisting of polygonal cells. In defined conditions, only CFUGM and polygonal cell colonies yield osteoclast-like cells, a few of which are capable of forming small resorptive pits. More recent studies, discussed in Section VII, provide further evidence that cells in the myeloid lineage contain osteoclast precursors. Thus, the hematopoietic origin of osteoclasts is established as is the cells’ membership in the monocyte/ macrophage family. Moreover, it is likely that macrophages at various maturational states may differentiate into osteoclasts.

IV. MODELS OF OSTEOCLAST FUNCTION The majority of resorptive experiments in whole animals involve the administration of drugs, systemic hormones, and cytokines (reviewed in Ref. 42). Additional studies include those exploring the effects of weightlessness [43,44], a relevant question in view of the possibility of long-term space travel. Interpretation of these experiments has depended on the quantitation of osteoclast number or net resorptive activity. The advent of densitometric techniques applicable to small animals [45] and of assays for products of rodent bone degradation [46] has increased the usefulness of these models. This approach has yielded important phamacological information, but the possibility that the target for the intervention is not the osteoclast itself, but rather an intermediary cell which modulates the bone resorptive polykaryon, is a major problem confounding whole animal experiments. However, gene deletion techniques have led to invaluable information regarding the molecular mechanisms of osteoclastogenesis in vivo. Fetal mouse [34] and rat bone cultures [47] utilizing intact explanted rudiments facilitate the exploration of paracrine, as opposed to endocrine factors in the resorptive process. The presence of an infinite variety of other cells confounds the direct assessment of osteoclast function. None the less, these systems continue to yield important data regarding specific aspects of osteoclast biology. Bone biopsy has been used as a means of estimating osteoclast activity in humans [48]. In addition to its invasive

80 nature, this approach is limited in that it does not actually measure resorptive activity but provides an estimate of osteoclast number. Noninvasive determinants of human osteoclast function include urinary markers such as hydroxyproline [49] and the dehydropyridinoline moiety [50]. This last approach utilizes a sensitive enzyme-linked immunosorbent assay (ELISA) to detect fragments arising directly from the degradation of mature bone collagen and is probably the current method of choice. Highly enriched or pure populations of osteoclasts would obviate many of the difficulties attending whole animal or organ culture systems. Early attempts utilized enzymatic digestion of bone and/or bone marrow suspensions, coupled with rapid sedimentation yielding small numbers of both mammalian osteoclasts [51 – 54]. The discovery that hens fed a low-calcium diet mobilize abundant osteoclasts [55] permits isolation of these cells in numbers sufficient for biochemical studies, including characterization of an acidic, cathepsin B-like collagenolytic enzyme [56] and a reconstitutible proton pump complex [57]. The same cells, following extensive purification, were used to confirm the presence of pp60c-src in osteoclasts [58], a protein essential to the cells’ polarization [59]. Because avian osteoclasts probably lack the calcitonin receptor, their relationship to mammalian osteoclasts is tenuous. Large numbers of rabbit osteoclasts have been obtained by simple adhesion techniques [60]. Sequencing of the cDNA library generated from these cells confirmed the presence of mRNA for osteopontin and cathepsin K [60,61]. Finally, freshly isolated rat osteoclasts lend themselves to the exploration of intracellular signaling [62,63]. A related strategy, applied to both human and avian systems, is based on immunoisolation of osteoclasts using monoclonal antibodies to suitable surface markers [64 – 66]. Such antibodies were raised to human osteoclastomas and screened for their ability to inhibit bone resorption in vitro. A single clone, 23C6, recognizes the integrin receptor, v3 [67]. This reagent has been used as a blocking antibody and to obtain highly purified polykaryons from which cDNA libraries have been generated [68]. Expression cloning of such a library led to the discovery that annexin II, a putative plasma membrane calcium channel, is secreted by osteoclasts and stimulates their activity [69]. In analogous studies, monoclonal antibodies were raised to purified avian osteoclasts [64]. Such an antibody, 121F, identifies a cell surface protein related structurally to superoxide dismutase [70]. The potential importance of this finding is underscored by the fact that oxygen-free radicals inhibit osteoclastic bone resorption [71]. While the ability to isolate and study mature osteoclasts is essential to understanding the mechanisms by which they degrade bone, the use of differentiated resorptive polykaryons precludes the identification of osteoclast precursors and the biochemical signals regulating osteoclastogenesis. Thus,

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efforts have focused on the generation of osteoclasts in vitro. The first successful attempts utilized mononuclear cells derived from calcium-deficient hens known to contain many mature osteoclasts [55]. When these TRAP-negative monocytic cells are cultured, at high density, they fuse within 5 – 6 days, yielding an almost homogeneous population of bone resorptive polykaryons with many, but not all, components of the osteoclast phenotype [2]. The large numbers of both precursors and fused cells allow for performance of a range of biochemical and cell biological studies. Thus, mature cells were characterized with respect to the integrins present and, most importantly, the ability of an antibody against the integrin v3 to block bone resorption [72]. Given that 1,25(OH)2D3 and retinoic acid stimulate osteoclastogenesis in vivo, the precursors served as a means to examine regulation, by these hormones, of this functionally important integrin [73,74]. In fact, the promoter of the 3 gene contains both vitamin D and retinoic acid responsive elements [75,76]. The same generated avian polykaryons have been used to demonstrate a decrease in intracellular calcium concentrations following liganding of the integrin v3 with soluble peptides [77]. Attempts to generate mammalian osteoclasts in vitro have also been rewarding. In early studies, unfractionated marrow cultures, treated with 1,25(OH)2D3, led to the production of small numbers of rabbit, primate, and human osteoclast-like cells [78 – 80]. The cells, most notably those of human origin, exhibit a limited ability to excavate characteristic resorption pits. In contrast, primary murine osteoblasts [39] and several clonal stromal cells lines [11,40], when cocultured with purified murine monocytic precursors, engender greater numbers of multinucleated cells, which resorb bone avidly and express the complete osteoclast phenotype. This system, which depends on contact between stromal cells/osteoblasts and osteoclast precursors, has been used to demonstrate that prostaglandin E2 and IL-4 have opposite effects on osteoclastogenesis [81]. Using the same assay and a function-blocking antibody to macrophage colony-stimulating factor (M-CSF), it was possible to reproduce, in vitro [82], in vivo data concerning the essential nature of this cytokine (M-CSF) for osteoclast production [83,84]. Studies have elucidated the minimal requirements for the generation of murine and human osteoclasts. In short, three proteins are sufficient to induce the differentiation of macrophages into osteoclasts. These osteoclastogenic proteins are M-CSF, receptor for activation of nuclear factor-kB (RANK), and RANK ligand (RANK-L).1 Interestingly, the soluble protein, osteoprotegerin (OPG), competes with RANK as a decoy receptor for RANK-L and thus attenuates osteoclast differentiation. Human bone marrow-derived CD34 cells [85,86] and murine 1 RANK-L is synonymous with osteoprotegerin ligand (OPGL), TRANCE, and osteoclast differentiation factor.

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spleen- or bone marrow-derived macrophages serve as osteoclast precursors, targeted by RANK-L and M-CSF [87,88]. RANK-L exists as soluble and membrane-bound forms and is produced by vascular endothelium [89], activated T cells [90], and, probably of most significance to osteoclast biology, those of the osteoblast/stromal lineage. This observation explains the mechanism by which osteoblasts and marrow stromal cells prompt macrophage differentiation into osteoclasts. OPG is also a product of osteoblastic/stromal cells [91], whereas RANK is found on cells of the osteoclast lineage [92,93]. With these proteins in hand, it is now feasible to generate large numbers of highly purified osteoclasts, using as targets, cells of the macrophage/monocyte lineage [88], now known to be osteoclast precursors. This approach has facilitated the execution of a wide range of biochemical and molecular experiments, adding substantially to our understanding of bone biology, a subject discussed further in Section VII.

V. OSTEOCLAST ATTACHMENT AND POLARIZATION As a prerequisite to initiating resorption, the osteoclast must attach to bone and create a functional, polarized plasma membrane. This binding event involves interactions between membrane-bound receptors on the cell surface and matrix proteins in bone. A number of surface markers have been identified on osteoclasts and/or their precursors [94], but, with the exception of integrins, few have been characterized functionally with respect to attachment. Antibody-blocking experiments suggest that members of the 1 family of integrins may participate in the resorptive process, presumably by binding to type I collagen [95]. However, the most compelling data indicate that v3 is the integrin most essential to osteoclast activity. Initial studies implicating this heterodimer in bone degradation were performed in vitro using antibodies to v3 [72] or peptides that block its function [96]. These experiments indicate that the integrin, on osteoclasts, binds one or more bone matrix proteins containing the motif Arg-Gly-Asp (RGD) [72,97 – 100]. The most convincing evidence that v3 is essential to osteoclast function comes, however, from the 3 knockout mouse [101]. This animal has dysfunctional osteoclasts as evidenced by their failure to form actin rings, a normal ruffled membrane or to resorb bone in vitro. Most importantly, these mice, which fail to express v3, are hypocalcemic and become progressively osteosclerotic with age. These studies suggest that v3 is a promising therapeutic target to inhibit accelerated bone resorption as occurs in postmenopausal osteoporosis. In fact, an organic molecule mimicking RGD prevents the profound bone loss rapidly following oophorectomy in the rat [102].

While the precise bone matrix protein(s) recognized by v3 the integrin in vivo is not known, osteopontin is a reasonable candidate. For example, immunoelectron microscopy suggests that v3 and OPN colocalize in bone [103], and an immunopurified antibody to OPN inhibits osteoclast/bone interaction [72]. Perhaps related more directly to polarization, the avian osteoclast cytoskeleton reorganizes upon v3 occupancy by soluble osteopontin [104]. In this instance, the mechanism involves activation of the important intracellular enzyme phosphoinositol-3 kinase, inhibition of which arrests bone resorption in vitro [105]. Activated phosphoinositol-3 kinase generates phosphotidylinositol-3,4 bisphosphate. This phospholipid, by binding to the actin-capping protein gelsolin, causes actin depolymerization and subsequent reelongation [106]. The most compelling evidence that osteopontin is pivotal to osteoclast function is the fact that the osteopontin knockout mouse is protected from oophorectomy-induced osteoporosis [107]. While the v3 integrin is pivotal to the resorptive process, its most important function is probably not formation of the tight seal, but transmission of bone-derived, intracellular signals. Thus, plating of mononuclear cells committed to osteoclast differentiation on a matrix recognized by v3 activates c-src [108], a plasma membrane protooncogene essential for osteoclast polarization and bone resorptive activity [109]. While it has been hypothesized that activated c-src associates with the tyrosine kinase pyk-2, which prompts a cascade of events probably responsible for organizing the osteoclast cytoskeleton during bone resorption [108], this remains to be proven. The dramatic polarization of the osteoclast, a phenomenon probably mediated by v3, distinguishes the cell from other members of the monocyte/macrophage family. It is clear that osteoclast polarization requires bone matrix recognition. The means by which the most dramatic manifestation of such polarization, namely the ruffled membrane, is generated are beginning to emerge. The information in hand suggests that the process involves migration, via microtubules, of acidifying vesicles from the trans-Golgi network to the bone-apposed plasma membrane into which they insert under the aegis of a Rab GTPase [15]. Thus, ruffled membrane formation is a process akin to regulated exocytosis, eventuating in focal complexity of the plasma membrane due to vesicular incorporation. A provocative alternative hypothesis holds that it is the endosomal, rather than exocytic, pathway that contributes to the ruffled membrane [110].

VI. MECHANISMS OF BONE RESORPTION The functional role of the osteoclast is to resorb bone, a composite matrix consisting of both inorganic and organic elements. The inorganic component is largely substituted

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FIGURE 9

Essential components of osteoclast-mediated bone degradation. See text for details.

hydroxyapatite, whereas the organic phase contains 20 or more proteins, with type 1 collagen the single major species (90% of total protein by weight) [111]. The initial step in bone resorption is attachment of the cell to the matrix, followed by the creation of an isolated, extracellular, resorptive microenvironment bordered by the ruffled membrane. Events leading to attachment and formation of the resorptive space have been discussed earlier. Studies with isolated osteoclasts reveal that dissolution of the inorganic phase of bone precedes that of protein [1]. Demineralization involves acidification of the extracellular microenvironment [112] (Fig. 9), a process mediated by a vacuolar H-ATPase in the ruffled membrane of the polarized cell [113] (Fig. 5). The structure and functional activity of this multienzyme complex are very similar, if not identical, to that of the analogous proton pump in the intercalated cell of the kidney [114,115]. This pump is a multimer, in which only some of the units are intrinsic membrane proteins. Others, including the 70-kDa protein containing ATPase activity, are attached, noncovalently, to subunits buried in the membrane. It is possible that one or more subunits may be present as an isotype, producing an osteoclast-specific form of the pump [116]. In support of this complex being the critical moiety in osteoclast acidification, the fungal metabolite bafilomycin A, a potent and specific inhibitor of all vacuolar proton

pumps, blocks bone resorption [117]. The intact proton pump has been isolated from avian osteoclasts, and the identity of several subunits to those present in other vacuolar pumps was established by Western analysis [57]. Importantly, the activity of the isolated complex is restored by the incorporation into lipid vesicles [57]. As with all members of the vacuolar pump family, extrusion of a proton through the plasma membrane is accompanied by the hydrolysis of one molecule of ATP. Given the fact the osteoclast transports protons extracellularly by an electrogenic mechanism raises the issue of maintenance of intracellular pH. Turning to the paradigm of the renal intercalated cell, Teti et al. [118] found that osteoclasts express, on their antiresorptive border, an energy-independent Cl/HCO3 exchanger similar to band 3 of the erythrocyte. Finally, electroneutrality is preserved by a plasma membrane Cl channel, charge coupled to the HATPase, resulting in the secretion of HCl into the resorptive microenvironment [119,120]. Because blockade of the Cl channel arrests H secretion, it is likely that impaired anion transport also impedes bone resorption. Acidification of the isolated resorptive environment prompts mineral mobilization as well as subsequent solubilization of the organic phase of bone [1], the products of which are endocytosed by the osteoclast, and transported to, and released at its antiresorptive surface [121,122].

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Given the low pH at the osteoclast/bone interface, the lysosomal family of acidic hydrolases, delivered to the resorptive microenvironment via the mannose-6-phosphate receptor [123,124], presented themselves as candidate molecules to degrade bone collagen and noncollagenous proteins. In fact, there is compelling evidence that, in the mammalian osteoclast, cathepsin K fulfills this role. Cathepsins have the capacity to fragment bone organic matrix [56] and, most importantly, mice deleted of the cathepsin K gene have increased skeletal mass [125]. Thus, cathepsin K is a potential antiosteoporosis target. Several studies reveal a possible role for neutral collagenases in the early phase of bone resorption. Bone is believed to be covered by a thin layer of osteoid (unmineralized matrix), which, some posit, needs be removed prior to osteoclastic degradation [4]. This hypothesis is, however, controversial, and the osteoblast is proposed to be the source of the putative neutral collagenase in this circumstance [126 – 129]. The most compelling evidence that these enzymes participate in the resorptive process comes from the demonstration that mutation in vivo of the site in type 1 collagen targeted by neutral collagenases attenuates bone resorption [130]. In addition to degrading bone, the osteoclast also resorbs mineralized cartilage in the hypertrophic zone of the growth plate. There is little evidence that the so-called “chondroclast” is distinct from the osteoclast; in fact, cartilage resorption by the cell is mediated via matrix metalloproteinase-9 [131]. While the means by which osteoclasts degrade bone are reasonably well defined, less is known regarding the mechanisms terminating their activity. The most provocative argument holds that sensing a high ambient Ca2 within the resorptive space, by a plasma membrane cation receptor, prompts withdrawal of the osteoclast from the bone surface and terminates resorption [132].

osteoclast recruitment. Injection of RANK-L into mice results in a rapid increase in serum calcium, indicating that the molecule also stimulates the resorptive activity of mature osteoclasts [134]. Targeted deletion of the RANK-L, OPG, and RANK genes prompts phenotypes consistent with the role of each protein as described in the current model. Thus, mice lacking either RANK-L [135] or RANK [136] are severely osteopetrotic secondary to lack osteoclasts, while the absence of OPG results in profound osteoporosis [137]. Binding of M-CSF and RANK-L to their respective receptors, c-fms and RANK, is the necessary and sufficient event to initiate osteoclastogenesis. Discovery of these molecules has yielded insights into the interplay between regulatory humoral and local factors and the intracellular signals controlling formation of the osteoclast (Fig. 10, see also color plate). In brief, a range of hormones, cytokines, and growth factors, targeting primarily to mesenchymederived cells in the bone microenvironment, control the expression of M-CSF, RANK-L, and OPG, with the overall impact on osteoclast recruitment dependent mainly on the ratio of OPG to RANK-L. Finally, the macrophage products, IL-1, TNF, and IL-6, regulate the capacity of stromal cells to promote osteoclast precursor differentiation (reviewed in Ref. 138). These proinflammatory cytokines also directly target myeloid precursors and mature osteoclasts, initiating signals prompting cell fusion and altered function and survival. Treatment of osteoclast precursors with IL-1 accelerates fusion and generates bone-resorbing polykaryons [139]. The cytokine also increases the life span of mature osteoclasts by a mechanism involving the activation of NF-B [140]. A more controversial

VII. HUMORAL REGULATION OF OSTEOCLASTIC BONE RESORPTION As discussed earlier, four molecules, M-CSF, RANK, RANK-L, and OPG, acting in concert, are major regulators of osteoclast formation and function. OPG, a member of the TNF receptor family, inhibits osteoclast formation. RANK-L, a novel member of the TNF superfamily, is the OPG ligand. Whereas RANK-L is primarily membrane bound, it also exists as a soluble form. Not surprisingly, RANK, the receptor for RANK-L, expressed on osteoclasts and their precursors, is a TNF receptor-related protein. The fact that purified macrophages give rise to numerous functional osteoclasts, when treated only with M-CSF and RANK-L [88] and that osteoclastogenesis is blocked by OPG [133], establishes these molecules as central to

FIGURE 10

Regulation of osteoclast formation. A range of hormones and cytokines, targeting to stromal cells, enhance expression of M-CSF and RANK-L, the pivotal osteoclastogenic molecules, and the proinflammatory cytokines IL-1, IL-6, and TNF. These proteins act to stimulate both formation and activity of mature polykaryons. HSC, hematopoietic stem cell. (See also color plate.)

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FIGURE 11

Key elements of RANK signaling. Receptor ligation is followed by the recruitment of adaptor molecules, including TRAF6, which, interacting with c-src, stimulates the PI-3 K/Akt pathway. Additionally, the NF-B and AP-1 families of transcription factors, key elements in osteoclast formation and function, are activated. Akt plays a role in phosphorylation of the IKK complex [367]. (See also color plate.)

hypothesis holds that TNF induces osteoclast formation in a manner not requiring RANK-L [141]. While the extracellular domain of RANK identifies it as a member of the TNF receptor superfamily, the intracellular amino acid sequence is unlike that of all orthologs. Despite these differences, the known proximal and distal signaling events arising from ligation of RANK and other members of the family are similar (Fig. 11, see also color plate). Two critical distal events in RANK signaling are the activation of JNK and the NF-B transcription complex. Mice lacking both the p50 and p52 NF-B subunits fail to generate osteoclasts [142]. Similarly, the AP-1 family proteins, c-fos and c-jun, are targets of JNK and play a role in osteoclast formation. Thus, animals lacking c-fos generate no multinucleated, bone-resorbing cells [143], nor do precursors which overexpress a form of c-jun containing a mutation that renders the transcription factor nonactivatable by JNK [144]. The proximal RANK-initiated signals are more complex and may reflect those following ligation of other members of the TNF receptor superfamily. For example, activation of the type 1 TNF receptor, which promotes osteoclast formation [145], is followed by binding of TRAFs, adaptor proteins capable of driving a variety of downstream signals [146]. Similarly, overexpressed RANK in embryonic human kidney cells recruits TRAFs 1, 2, 3, and 5 [147]. Because mice lacking TRAF 2, 3, or 5 have normal bones [148 – 150], these molecules may not be necessary for RANK-induced osteoclastogenesis. However, these three TRAFs bind to overlapping sites in the RANK cytoplasmic tail [146] and thus it may be necessary to generate mice lacking combinations of TRAFs 2, 3, and 5 to establish the role of these adaptor proteins in osteoclast function.

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Absence of TRAF6 results in osteopetrosis. While this observation raises the possibility that TRAF6 links RANK-L to osteoclastogenesis, TRAF6-/- osteopetrotic mice have numerous osteoclasts, which, however, are unable to attach to bone, form a ruffled membrane, or degrade the matrix [151]. These animals bear a striking resemblance to those lacking c-src [59], a fact explained by the constitutive binding of the protooncogene to RANK and its interaction with TRAF6 following receptor occupancy [152]. The subsequent downstream events (Fig. 11) include stimulation of phosphatidylinositol-3 kinase (which by phosphorylating Akt, initiates cytoskeletal reorganization), antiapoptotic signals, and activation of NF-B [153], all key components of osteoclast function. In murine osteoclasts, RANK-derived signals stimulate JNK1 (but not JNK2) via the MEKMEKK axis (Srivastava et al., unpublished data). In short, the link between RANK activation and the AP-1 and NF-B families of transcription factors suggests these signals represent key components of RANK-induced osteoclast formation. However, the presence of numerous, albeit nonfunctional, osteoclasts in TRAF6 null mice indicates that generation of these cells does not require TRAF6-initiated signals. As will become evident, the majority of the osteoclasttargeting cytokines/hormones/steroids exert their influence, wholly or in part, by their ability to increase or decrease M-CSF, OPG, and RANK-L expression. Thus, if M-CSF production is enhanced, the number of precursors, and their ability to differentiate, is augmented. Similarly, a net excess of RANK-L activity (as a result of decreased OPG secretion, increased RANK-L synthesis, or both) stimulates osteoclast recruitment and function. A net deficiency of RANK-L activity (arising from the reciprocal set of circumstances) will arrests osteoclast differentiation and activity. Finally, a number of cytokines, in addition to controlling formation of osteoclasts, through their effects on OPG and RANK-L expression, also target the mature polykaryon, altering its resorptive capacity. Table 1, which summarizes the role of the various cytokines regulating production of M-CSF, OPG, and RANKL, reveals an apparent conundrum, namely that a given osteoclast agonist can increase both OPG and RANK-L. The net effect for any single molecule is explained by assuming greater potency for the induction of either the osteoclaststimulating or inhibiting moiety. It must be recognized that in vivo, the processes of osteoclast formation and subsequent function are controlled in a continuous and rapid manner, reflecting the net effect of the numerous regulators discussed next.

A. Parathyroid Hormone Parathyroid hormone is a major accelerator of bone remodeling. Because the remodeling process is initiated by

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TABLE 1 Summary of Factors that Modulate Expression of RANK-L, OPG, and M-CSFa RANK-L

OPG

M-CSF

q

q

q

q

p

Hormones 1,25(OH)2vitamin D3 Estrogen q

p

TNF

q

q

IL-1

q

q

IL-6

q

IL-11

q

PTH Cytokines

q

modulating cells and osteoclasts is necessary [11,82]. The capacity of PTH to increase OPGL and decrease OPG [167] explains the osteoclastogenic properties of the hormone. Intact PTH and parathyroid hormone-related protein (PTHrP) target the same receptor [168] and exert similar biological effects on bone cells [169]. In contrast, the carboxyl-terminal portion of PTHrP may uniquely dampen osteoclastic bone resorption [170]. It should be pointed out that the biological implications of this phenomenon are controversial, as it occurs only in selected systems [171]. Whether carboxyl-terminal PTHrP targets osteoclasts or exerts its inhibitory effects via intermediary cells is not clear [172].

Growth factors TGF-

p

q q

BMP-2 Others Prostaglandin E2

B. Cytokines and Chemokines

q

p

a Adapted, with additions, from Hofbauer et al. [138]. Data on the role of PTH are found in Lee and Lorenzo [167].

osteoclast recruitment, states of PTH excess are typically associated with morphological and biochemical evidence of increased resorptive activity. Importantly, PTH sensitizes bone to 1,25(OH)2D3 [154]. Thus, it is not surprising that 1,25(OH)2D3, in the parathyroprivic state, does not mobilize osteoclasts [155]. Osteoclasts, in vivo or in organ culture, undergo profound changes when exposed to PTH. The population of these cells grows proportional to PTH concentration and the hormone augments the ruffled membrane area and the number of cells exhibiting this resorptive organelle [21,156]. Clear zone and overall cell size are also enhanced [157]. The fact that PTH administration induces c-fos mRNA expression in osteoclasts [158] indicates that the hormone prompts the differentiation of precursors along an osteoclastic pathway [143]. Taken together, these observations suggest that PTH stimulates osteoclast recruitment and activates differentiated polykaryons. While it had been proposed that the hormone targets osteoclasts directly, high-affinity PTH receptors have not been demonstrated in these cells [159,160], nor is there is convincing evidence that the hormone exerts a direct biological effect on isolated osteoclasts [161,162]. However, osteoblasts [158] and marrow stromal cells [163] from which they are derived are sensitive to PTH. The fact that isolated osteoclasts, cocultured with these cells, respond to PTH indicates that osteoblasts and marrow stromal cells serve as intermediaries in PTH-stimulated, osteoclastic bone resorption [164]. PTH-treated osteoblasts or stromal cells secrete resorptive activity [165,166], but contact between

Cytokines regulating osteoclast biology can be divided broadly into three groups: those that facilitate differentiation, survival, and proliferation of precursors; those that promote osteoclast precursor differentiation, and those that alter the resorptive capacity of the mature polykaryon. As discussed later, a number of cytokines exert more than one function. Some act at both proliferative and differentiation stages, whereas others also target mature osteoclasts, modulating bone degradation. Osteoclast formation and activity are controlled by both systemic hormones and locally produced cytokines/chemokines. The latter molecules are themselves regulated in a complex manner in which paracrine and/or autocrine events play a central role. Proteins such as stem cell factor, IL-1, IL-3, IL-6, GMCSF, M-CSF, erythropoietin, epidermal growth factor, and basic fibroblast growth factor are required for the optimal differentiation of stem cells to CFU-GM [85,173], an early osteoclast precursor [41]. As discussed elsewhere in this chapter, GM-CSF and M-CSF play overlapping/redundant roles as regarding the formation of precursors capable of being induced to form bone-resorbing polykarons. Members of the hematopoietic family of cytokines target osteoclast precursors, stimulating both proliferation and differentiation. Other molecules, produced in the bone microenvironment by marrow stromal cells, immature monocytes, and osteoblasts and their precursors, as well as T lymphocytes [88,174,175], are also capable of modulating osteoclastogenesis by acting in a paracrine and/or autocrine manner to regulate the production of M-CSF, RANK-L, and OPG. Whereas most cytokines promote osteoclast formation by stimulating proliferation and/or differentiation of osteoclast precursors, several, including M-CSF, RANK-L, IL-1, IL-6, and TNF, also directly modulate mature osteoclasts. When binding to early osteoclast precursors, M-CSF provides a signal required for survival, proliferation, and maturation [176,177]. In contrast, the same protein

86 increases the motility of mature osteoclasts, with consequent diminution in bone resorption [178,179]. The cytokines IL-6, IL-11, oncostatin M, and leukocyte inhibitory factor share a common signaling pathway. The heterodimeric signaling complex comprises a common subunit, gp130, linked to a second protein, specific for each cytokine [180]. Whereas IL-6 and IL-11 [181,182] are important regulators of osteoclast function, the roles of oncostatin M and leukocyte inhibitory factor are less clear [183,184]. Mice unable to transduce signals from any member of the gp130 family as a result of targeted deletion of the common subunit have normal bones [185]. This result is not surprising given the multiplicity of pathways whereby control of the critical regulator of osteoclast formation and function, namely RANK-L, can be exercised (Fig. 10). Ligation of IL-6 to its receptor, in the mature osteoclast, promotes bone resorption [68], a process accentuated in Paget’s disease [186], in which the cytokine appears to play an important pathogenetic role [187]. The resorptive properties of IL-6 have been documented with osteoclastomaderived, osteoclast-like cells treated with antisense IL-6 oligonucleotides [188]. An antibody that blocks IL-6 function arrests bone loss in vivo [189,190] and blunts oophorectomy-induced osteoclastogenesis [190,191]. Mice in which the IL-6 gene has been removed by targeted deletion do not lose bone following oophorectomy [192]. Finally, IL-6 has been shown to play a role in PTH-induced bone resorption in vivo [193]. Taken together, these results provide strong support for the hypothesis that IL-6 appears to modulate, in part, the effects of estrogen deprivation on bone resorption (see Chapter 41). A molecular explanation for this is provided by the observation that IL-6, targeting to mature osteoclasts, blunts the inhibitory role of high extracellular calcium by a pathway that involves induction, on the surface of the cell, of ADP-ribosyl cyclase [194,195]. In contrast, Kitazawa et al. [189] found decreased numbers of osteoclasts in both oophorectomized and sham-operated mice administered the anti-IL-6 antibody. Furthermore, this antibody, administered in vivo, decreases the histological evidence of bone resorption but not urinary excretion of pyrodinoline cross-links. A naturally occurring inhibitor of IL-1 action, the IL-1 receptor antagonist is produced by monocytes [196] and dampens osteoclast formation and bone resorption in oophorectomized rats and mice [189,197]. Similarly, TNFbinding protein, a soluble form of the TNF receptor that blunts TNF activity, decreases the number of osteoclasts produced by murine cells in vivo and in vitro [189,198]. Furthermore, mice transgenic for the TNF-binding protein, a soluble TNF receptor, are resistant to oophorectomy-induced bone loss, indicating a central role for this cytokine in the most significant form of osteoporosis [199]. Regulation of cytokine expression as relating to osteoclastogenesis is incompletely understood. As regarding

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inductive proteins, IL-1 and TNF stimulate the synthesis of M-CSF and IL-11 by marrow stromal cells [200,201]. The same agonists, as well as IL-6, regulate their own production by monocytes via a complex set of paracrine and autocrine signals [202]. The release of IL-6 by stromal cells is stimulated by PTH [203]. As discussed later, estrogen controls the production of several cytokines central to different aspects of osteoclast biology. There are relatively few reports examining the role of chemokines in bone biology. Macrophage inflammatory protein-1, a product of osteoblasts, stimulates motility but decreases the resorptive activity of isolated osteoclasts [204]. Similarly, IL-8, secreted by osteoclasts [205], acts via an autocrine loop to inhibit bone resorption [206]. The latter finding is in conflict with an earlier report that osteoclast recruitment and matrix degradation increase in parallel with IL-8-induced neoangiogenesis [207].

C. Estrogen Estradiol is critical to skeletal preservation in both women [208] and men [209 – 211], as its denial prompts rapid loss, especially of cancellous bone. The estrogendeprived skeleton, particularly as seen shortly after menopause, exhibits histological features of accelerated remodeling, including abundant osteoclasts and resorption bays [3]. Biochemical evidence of enhanced osteoclastic activity in estrogen-deficient patients includes increased hydroxyproline excretion [212]. Interestingly, tamoxifen, which antagonizes estrogen in other organs, is an estrogen agonist in bone, wherein the effects of both steroids are additive [213,214]. Given the identification of molecular targets of estrogenic activity, the means by which the steroid dampens osteoclast function are now more clearly understood. While the role of estrogen is discussed in greater detail in Chapters 10 and 41, it is pertinent that the sex steroid not only modulates M-CSF, but, by controlling monocytic secretion of IL-1 and TNF, the ratio of OPG to RANK-L as well. Likewise, there are reports that the steroid either does [190] or does not [189,215,216] block the secretion of IL-6 from the same cells. There is, however, agreement that estrogen inhibits the production of IL-6 by monocytes [217 – 220]. Additionally, while the sex steroid dampens the secretion of GM-CSF and IL-1 receptor antagonist from cultured human monocytes [220,221], it does not alter the production of IL-11 by stromal cells [181]. Estrogen also diminishes expression of the type I IL-1 receptor on osteoclast and their precursors, thereby limiting the capacity of the cytokine to target these cells [222]. Finally, in addition to modulating the expression of factors that stimulate osteoclast formation and function, estrogen also induces osteoclast apoptosis, a subject discussed in Section VIII.

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D. Vitamin D 1,25(OH)2D3, the biologically active form of vitamin D, is a potent inducer of resorption whether administered in vivo [223,224] or added to bone organ culture [22,225]. The molecular basis of this activity is thought to lie largely in the ability of this secosteroid to stimulate, by osteoblastic/stromal cells, a net increase in RANK-L as opposed to OPG. The molecular basis for this result has been defined by the work of Kitazawa and colleagues [226], who demonstrated a functional vitamin D response element in the murine RANK-L promoter. A secondary component of the activity of 1,25(OH)2D3 is that it increases the production of M-CSF, the only cytokine, besides RANK-L, necessary to produce osteoclasts in vitro from both murine and human precursors [88]. A number of structural modifications of the vitamin D molecule have yielded analogues of 1,25(OH)2D3 with distinct biological properties of potential therapeutic importance. For example, 22-oxacalcitriol, while being an effective inducer of cell maturation, is only 1% as effective as 1,25(OH)2D3 in stimulating osteoclastic bone resorption [227]. The molecular basis of this observation is not clear at this time. Vitamin D analogues are discussed in detail in Chapter 9. The fact that vitamin D receptor-ablated mice in whom normal calcium and phosphate levels had been preserved by dietary means exhibited no differences from their wild-type littermates in bone histomorphometry suggests that the major role of the steroid in bone function is to maintain mineral homeostasis [228]. Finally, by promoting expression of type 1 IL-1 and E2 receptors in marrow-derived stromal cells [229,230], the secosteroid primes them to respond to these agonists.

E. Glucocorticoids Gluocorticoid-induced osteoporosis is among the most devastating forms of osteopenia. It predominantly affects trabecular bone [231,232], being manifest most profoundly in the axial skeleton. The subject is discussed extensively in Chapter 44. While glucocorticoids appear to increase osteoclastic activity in vivo, their effect on resorbing cells is complex. Patients chronically treated with glucocorticoids have increased numbers of osteoclasts [233] and resorption bays [234]. There is also biochemical evidence of accelerated skeletal degradation such as enhanced hydroxyproline excretion [235]. Given their osteoclastogenic properties, it is surprising that the resorptive activity and survival of isolated osteoclasts exposed to these agents are attenuated [236]. This conundrum prompted a hypothesis that glucocorticoids, in low dose, permit osteoclast precursor differentiation, whereas higher concentrations blunt resorption [237]. A more likely explanation

of the apparent discrepancy between in vivo and in vitro effects of glucocorticoids on osteoclasts relates to the steroid’s actions on mesenchymal marrow cells. By simultaneously increasing RANK-L, and decreasing OPG, the net effect is an increase in osteoclast number and resorptive capacity [138]. In addition to these direct effects, glucocorticoids suppress intestinal absorption of calcium [238]. This event, in conjunction with augmented renal calcium loss [239], provokes secondary hyperparathyroidism [233,240]. Furthermore, the resorptive impact of such relatively mild hyperparathyroidism is amplified as glucocorticoids sensitize bone cells to PTH [237] and 1,25(OH)2D3 [241], agents known to stimulate osteoblasts to accelerate osteoclast activity. Given these data, it is not surprising that osteoclasts do not proliferate in parathyroidectomized animals receiving glucocorticoids [242]. Finally, while their effects on PTH responsivity are in keeping with stimulated osteoclastic bone degradation, glucocorticoids also inhibit the production of other molecules known to enhance resorption. For example, the steroid suppresses the expression of IL-1 and IL-6 [243] and blunts the synthesis of prostaglandins [244].

F. Thyroid Hormone Thyroid hormone excess, whether endogenous [232,245] or iatrogenic [246-248], prompts bone loss in humans and experimental animals. The skeletal changes of hyperthyroidism are those of accelerated remodeling with an abundance of osteoclasts, osteoblasts, and osteoid [246,247]. In some instances, one encounters peritrabecular marrow fibrosis (osteitis fibrosa) [246,249]. The effects of thyroid on bone are discussed in Chapter 47. Thyroid hormone stimulates bone resorption in vivo [250,251], in organ culture [252,253], and in isolated cell systems consisting of osteoclasts and osteoblasts [254]. The urine of hyperthyroid patients contains increased amounts of the products of bone degradation, including hydroxyproline [250,255] and pyrodinoline cross-links [256,257]. However, the effect of thyroid hormone on osteoclasts is indirect and, like other stimulatory agents, is mediated by osteoblasts [254]. At this time there are no reports as to the impact of thyroid hormone treatment of osteoblastic/stromal cells on the OPG/RANK-L ratio.

G. Prostaglandins Prostaglandins exert dramatic and diverse effects on osteoclast function. While most is known about the resorptive impact of prostaglandins of the E series (PGE), thromboxane [258] and products of the lipooxygenase pathway [259,260] also modulate the osteoclast (see Chapter 13).

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The predominant effect of a relatively prolonged exposure of the osteoclast to PGE is enhanced resorption. Interestingly, the resorptive effects of intense mechanical force on osteoclast function may also be prostaglandin mediated [261]. While administration of prostaglandins in vivo has not been shown to accelerate resorption, cyclooxygenase inhibitors dampen bone remodeling [262]. Moreover, PGE potently induces osteoclast activity in organ culture [263]. Once again the ability to increase RANK-L and concomitantly decrease OPG explains the resorptive impact of PGE [264,265]. In contrast to the ability of PGE to stimulate osteoclast formation via upregulation of RANK-L on stromal cells, the eicosinoid inhibits the activity of preformed osteoclasts [81,266]. Isolated osteoclasts, when exposed to PGE, cease resorbing bone [266]. The cells transiently contract in a manner similar to that attending calcitonin exposure [267], a finding consistent with the fact that, like calcitonin, prostaglandins express their biological properties through cAMP generation. In fact, many of the resorptive effects of prostaglandins are mimicked by cell-permeant analogues of the nucleotide [81,268]. Interestingly, while prostaglandins and calcitonin both activate adenylate cyclase, the two agents appear to impact the enzyme differently [269].

H. Calcitonin Calcitonin, when administered to vertebrates, dampens bone resorption by directly targeting osteoclasts [161,270]. Thus, the hormone prompts a rapid decline in circulating Ca2 activity [271] and urinary hydroxyproline excretion [272], phenomena indicative of suppressed bone degradation. Furthermore, addition of calcitonin to any in vitro mammalian osteoclast model blunts the capacity of the cell to degrade bone. In actuality, the failure of calcitonin to suppress resorption in a mammalian system raises concern as to whether the model contains bona fide osteoclasts. However, there is no convincing evidence that endogenously secreted calcitonin directly modulates osteoclast activity (see Chapter 73). Reflecting the rapid biological effects of the hormone, a characteristic feature of mammalian osteoclasts is the expression of calcitonin receptors [273]. In fact, calcitoninbinding sites are the only peptide hormone receptors unequivocally resident in these cells. While avian osteoclasts probably lack calcitonin receptors [268], mammalian polykaryons failing to express them are not osteoclasts. However, calcitonin receptors are not unique to the osteoclast, as they also appear in macrophages derived from nonskeletal sources [274], such as lung [275]. This finding is in keeping with the capacity of various macrophage precursors, including those of alveolar origin, to differentiate

in vitro into polykaryons with the complete osteoclast phenotype [11]. When added to organ culture, calcitonin decreases the number of osteoclasts [276] and nuclei per cell [277]. The ruffled membrane disappears, a process reflecting its internalization and vacuolar conversion [123]. Proteins destined for the ruffled membrane, such as the mannose-6-phosphate receptor, are rerouted to intracellular vesicles [123]. TRAP secretion is altered by calcitonin, transiently increasing but ultimately declining [278]. Similarly, osteopontin mRNA expression by osteoclasts is blunted by the hormone [279]. Within seconds of calcitonin exposure, osteoclasts become immobile and contract [51,280,281], a phenomenon associated with changes in the microtubule [282] and microfilament [267] cytoskeleton (Fig. 12). The arrested motility of the cell has been attributed to two distinct events termed quiescence and retraction, each of which may be mediated by distinct signaling pathways [283]. Prolonged exposure to calcitonin results in a declining sensitivity of osteoclasts to the hormone [284,285]. The escape phenomenon, which reduces the therapeutic efficacy of the hormone, has been attributed to induced PTH secretion [286] and anticalcitonin antibody generation [287]. Takahashi and co-workers [288] have shown, however, that calcitonin downregulates its own receptor in osteoclasts. Calcitonin exposure is followed in the osteoclast by an immediate increase in intracellular Ca2 activity and generation of cyclic AMP [289]. The hormone also activates protein kinase C [287]. These accumulated data suggest that calcitonin mediates a number of intraosteoclastic events via distinct signaling pathways. The effects of PTH and calcitonin are discussed further in Chapter 12.

I. Superoxide and Nitric Oxide Oxygen-derived free radicals and nitric oxide (NO) alter osteoclastic bone resorption dramatically [290]. Superoxide is produced by osteoclasts resorbing bone [71,291,292] and therefore may function in an autocrine manner. These anions are located in the ruffled membrane [290] and are induced when osteoclasts contact skeletal matrix [71]. Osteoclasts generate superoxide via NADPH oxidase [292]. The cells also contain superoxide dismutase [70,293], which, when added to osteoclast cultures, decreases resorptive activity [294]. Superoxide anion generation may also play a role in regulated bone resorption. For example, IL-1 and PTH-induced osteoclast activity is blunted by superoxide dismutase [71], and calcitonin diminishes superoxide generation [71,291]. In contrast to superoxide, hitric oxide is antiresorptive [295,296], and the two molecules may act together to regulate osteoclast function [290]. NO targets directly to osteoclasts [295,296] and, like calcitonin, inhibits spreading

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FIGURE 12 Osteoclasts exposed to cyclic AMP-inducing agents, including calcitonin and prostaglandin E, undergo rapid contraction and motility arrest. Here, an untreated, isolated rat osteoclast (A) was exposed for 2 h to 108M dibutyryl cyclic AMP (B), inducing immediate contraction and immobility (phase contrast).

[295]. The molecular mechanisms responsible for the osteoclast-inhibiting effects of NO and CT differ [295]. NO is produced by osteoclasts via NO synthase activity [290]. The enzyme in this cell is both constitutively present and regulated [290]. Thus, like superoxide, NO may autoregulate osteoclast function. However, this short-lived reactive radical is also synthesized in abundance by endothelial cells that are in close proximity to, and may govern the activity of, osteoclasts [295]. Perhaps reflecting their known capacity to modulate osteoclasts, osteoblasts also secrete NO in response to inflammatory cytokines [297,298]. Interestingly, osteopontin dampens NO synthesis by renal cells, an event perhaps mediated through the integrin v3 [299]. Whether the bone matrix protein also has an impact on osteoclastic production of NO is unknown.

J. Osteoclast Apoptosis Net bone resorption represents the sum of osteoclast activity and number, with the latter parameter itself dependent on the rates of cell formation and death. Treatment of mature osteoclasts with estrogen results in the rapid initiation of apoptosis, an event mediated by transforming growth factor  [300]. Nitrogen-containing bisphosphonates induce osteoclast death, an event resulting from the blunted synthesis

of farnesyl and geranylgeranyl pyrophosphate, two moieties central to the acylation, and thus activation, of members of the Ras superfamily [301 – 303]. Signaling through Ras proteins modulates cell survival. Separately, bisphosphonates lacking the nitrogen atom stimulate osteoclast death by an undetermined mechanism, but one that does not involve small GTPases [303]. Caspases, a family of cysteine proteases [304], are involved in both the initiation and the execution phases of apoptosis, and these molecules modulate the life span of the osteoclast. Thus, treatment of osteoclast cultures with a pan inhibitor of caspase action, Z-VAD-Fmk, decreases the number of apoptotic cells [305]. Preliminary data indicate the likely involvement of caspases 3 and/or 7, both executor, or downstream caspases [306]. The proximal signals leading to the activation of these distally acting proteases, in osteoclasts, are unknown.

VIII. DISEASES OF THE OSTEOCLAST A. Osteopetrosis The osteopetroses are a group of congenital diseases characterized by increased skeletal mass due to dysfunctional osteoclasts. Despite pathogenetic heterogeneity, all

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FIGURE 13

Molecular mechanisms of osteopetrosis. See text for details. (See also color plate.)

forms of osteopetrosis are distinguished by radiopaque bones, with loss of differentiation between the cortex and the marrow. The molecular defect of a number of osteopetrotic mutants, particularly of murine origin, are now defined and serve as a rich source of information regarding the origin and the function of osteoclasts (Fig. 13 see also color plate). 1. OSTEOPETROSIS DUE TO DEFECTIVE OSTEOCLAST PROGENITORS Initial evidence regarding the origin of osteoclasts was provided by Walker [307,308] wherein the circulation of a normal mouse was joined to that of an osteopetrotic, microphthalmic (mi/mi), or gray-lethal (gl/gl) littermate. By 6 weeks of age all of the excess mutant skeleton was resorbed. Walker concluded that mature osteoclasts, or their progenitors, were introduced from the normal mouse into the mutant’s marrow and subsequent experiments support this hypothesis. For example, the skeletal lesion of irradiated gl/gl or mi/mi mice is cured by infused, normal marrow or spleen cells. Conversely, mi/mi or gl/gl spleen cells induce osteopetrosis in irradiated normal mice [309]. Together, these experiments provide direct evidence for the hematopoietic origin of osteoclasts. These studies also establish that ultimate proof of the mutated gene of interest is, in fact, essential for osteoclast differentiation and requires rescue of osteopetrotic mice with normal osteoclast precursors, or administration of the gene product. The technique of homologous recombination has yielded a plethora of osteopetrotic knockout mice and major insights into the genes regulating osteoclastogenesis. For example, deletion of the macrophage and B-cell-specific PU.1 gene generates mice completely devoid of osteoclasts and their precursors [310]. These animals represent the earliest known developmental defect in osteoclastogenesis. c-fos, deletion of which prompts osteopetrosis [143], is a widely expressed nuclear protooncogene belonging to a multigene family including fosB, fra-1, and fra-2 [311]. While the fos mutant lacks osteoclasts, marrow macrophages are actually increased relative to wild type. Thus, c-fos may exert its osteoclast differentiating effect distal to PU.1. Similarly, deletion of the p50 and p52

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subunits of NF-B prompts osteoclast-deficient osteopetrosis in which macrophage differentiation is extant [312]. NF-B, therfore, impacts osteoclastogenesis in a manner reminiscent of c-fos. The osteopetrotic phenotype of the NF-B knockout indicates cell surface receptors activating this transcription complex may also participate in the osteoclastogenic process. Thus, deletion of RANK-L [135] or its functional blockade by the overexpression of osteoprotegerin [91] each results in osteopetrotic mice devoid of osteoclasts. 2. OSTEOPETROSIS DUE TO DEFECTIVE OSTEOCLAST FUNCTION Among the most interesting of the osteopetrotic mutants is the c-src -/- mouse [59]. This animal, produced by homologous recombination, exhibits no detectable abnormalities in brain or platelets where pp60c-src is normally abundant, but is affected by a form of osteopetrosis in which osteoclasts are numerous. This latter finding suggests that the osteoclast lineage is intact but that the cell is functionally impaired [109]. In fact, electron microscopic examination reveals that src -/- osteoclasts contain no ruffled membranes (Fig. 14). Moreover, osteoclasts generated in vitro from src mutant marrow cells fail to pit bone. These observations signify that lack of bone resorption by src -/- osteoclasts is intrinsic to this cell and not reflective of the bone microenvironment [313]. Similar results were obtained with deletion of TRAF6 [151], a protein associating with the intracellular domain of RANK and which signals following association with c-src [152]. The absence of ruffling in src mutant osteoclasts suggests that pp60c-src is fundamental to osteoclast polarization and may participate in transporting vacuolar H -ATPase from internal pools of acidic vesicles to the plasma membrane. Although this hypothesis lacks direct proof, it is supported by several lines of circumstantial evidence. For example, pp60c-src is a specific marker of macrophages committed to the osteoclast as compared to the host defense phenotype [145]. Both pp60c-src and the vacuolar H -ATPase associate with microtubules in the putative pathway toward the formation of the ruffled membrane [20]. The osteoclast src protein is increased and decreased, respectively, in response to parathyroid hormone and calcitonin [314]. Moreover, bovine adrenal gland chromaffin granules are highly enriched in pp60c-src [315 – 317]. Because these structures are secretory organelles, they provide indirect evidence that the tyrosine kinase has a role in vesicle transport and/or secretion, events akin to ruffled membrane formation. Interestingly, the role of pp60c-src in osteoclast function appears to involve its capacity both as a tyrosine kinase and docking protein, as a kinase-inactive construct substantially, but not completely, rescues the c-src-/- osteoclast [318]. With the realization that the v3 integrin and cathepsin K may be critical to optimal osteoclast function, mice

CHAPTER 3 Osteoclast Biology

FIGURE 14 Osteoclasts from normal and c-src-/- mice. Wild-type cells (A) contain a well-developed ruffled membrane (r), which is lacking in the osteopetrotic mutant (B). Reproduced from J. Clin. Invest. 90, 1622 – 1627 (1992). Copyright permission of The Society for Clinical Investigation.

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92 devoid of one or the other molecule have been generated [101,125]. While each lack the characteristic pathological features of osteopetrosis, they both contain dysfunctional osteoclasts eventuating in failure to normally resorb bone and, hence, enhanced bone mass. Two general phenotypes of osteopetrosis have been described in humans. One is relatively asymptomatic, whereas the other, usually present at birth, is fatal in infancy or early childhood. While the molecular mechanisms underlying the malignant forms of osteopetrosis are not yet resolved, there is information regarding the “benign’’ phenotype. One such example is characterized by renal tubular acidosis. Consistent with the capacity of the isoform’s inhibitors to also block parathyroid hormone-induced bone resorption, mutation of the carbonic anhydrase II gene is responsible for this form of benign osteopetrosis [319]. Carbonic anhydrase II, in normal osteoclasts, catalyzes the hydration of CO2 to carbonic acid. Dissociation of carbonic acid into bicarbonate ions and protons permits formation of the resorptive microenvironment, which is probably defective in carbonic anhydrase II-deficient patients. Considering the importance of proton secretion in bone degradation, one might expect inactivating mutations of the osteoclast H -ATPase to impair the resorptive process. Indeed, osteoclasts generated in vitro from marrow cells of a patient with craniometaphyseal dysplasia, a rare genetic sclerosing bone disease, fail to resorb bone [320]. These osteoclasts do not express the H -ATPase as determined by immunohistochemistry. 3. OSTEOPETROSIS DUE TO DEFECTIVE OSTEOCLASTOGENIC MICROENVIRONMENT The murine recessive op mutation prompts an osteopetrotic phenotype characterized by a failure to generate monocytes, macrophages, and osteoclasts [321,322]. These animals are not cured by marrow transplantation. Alternatively, op/op hematopoietic progenitors, administered to wild-type mice, differentiate into normal osteoclasts. These experiments prove that the op mutation does not target macrophage and osteoclast progenitors but rather that the microenvironment in which these cells develop is defective [323,324]. In fact, op/op bone cells and fibroblasts fail to produce competent M-CSF [84,325]. Furthermore, the op mutation maps to a single base pair insertion in the coding region of the M-CSF gene, resulting in a translation frame shift, and insertion of a stop codon 21 bp downstream, producing a truncated, nonfunctional M-CSF protein [326]. The observation that the op/op mouse is rescued by subcutaneous injections of recombinant M-CSF provides additional evidence that lack of this protein is the cause of its osteopetrosis [83,327,328]. The op/op mouse underscores the importance of the hematopoietic microenvironment in osteoclast development and points to the fact that osteoclasts and macrophages share a common lineage.

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Surprisingly, op/op mice progressively recover from lack of both macrophages and osteoclasts with age and, by 22 weeks, the marrow cavity size and cellularity appear unremarkable [329]. The numbers of mononuclear phagocytes, macrophages, and osteoclast precursors progressively increase to normal. Thus, the animal is able to compensate for a lack of M-CSF, presumably by other cytokines with whose function it overlaps. The spontaneous rescue of the op/op mouse has been shown to reflect expression of GM-CSF, a protein with many activities shared, in vitro, with M-CSF [330]. Supporting this contention, GM-CSF administered to op/op mice during their osteopetrotic phase cures the bone phenotype. This observation, taken with the normal skeletons of GM-CSF knockout mice [331], indicates that either M-CSF or GM-CSF, in the absence of the other, will promote osteoclastogenesis. 4. CURE OF HUMAN OSTEOPETROSIS Based on the animal models described earlier, a female patient with autosomal-recessive osteopetrosis was cured after receiving her brother’s marrow. In this instance, the donor origin of osteoclasts was established by following the Y chromosome [332]. The pretransplantation abundance of osteoclasts, albeit dysfunctional, established the patient’s capacity to provide an osteoclastogenic environment for normal marrow. Given an appropriate donor, marrow transplantation is the treatment of choice for malignant osteopetrosis.

B. Paget’s Disease Paget’s disease of bone is a paradigm of remodeling gone awry. As such, the initiator of bone remodeling, namely the osteoclast, is the pathogenetic cell. In fact, despite the array of abnormal histological features attending Paget’s disease, it is the appearance of the osteoclast that is pathognomonic of this condition [333]. The number of osteoclasts, and their nuclei, are increased greatly in Paget’s disease and the cells are enormous (Fig. 15). In contrast to normal osteoclasts in which nuclei polarize toward the nonresorptive surface, those in pagetic cells are distributed diffusely throughout the cytoplasm. Reflecting osteoclast size and number, bone resorption is accelerated greatly. Resorption bay volume and prevalence are increased manyfold [333,334]. Indeed, the initial cellular event in the pagetic skeleton is rapid resorption, often resulting in large, radiographically evident, lytic lesions [335]. Interestingly, one finds histological evidence of enhanced resorption in radiographically uninvolved bone. It is unresolved whether this phenomenon represents subclinical Paget’s disease or, as has been suggested [333,336], mild

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FIGURE 15

Osteoclasts (arrows) in bone biopsy taken from a patient with Paget’s disease. The cells are enormous and contain many nuclei, which, in contrast to normal osteoclasts (see Fig. 1), are nonpolarized (nondecalcified, toluidine blue stain).

hyperparathyroidism. In any event, given the central role that the osteoclast plays in Paget’s disease, it is not surprising that successful treatment entails arresting bone resorption [337,338]. While the precise etiology of Paget’s disease is not yet understood, the measles virus appears to be the most likely candidate. For example, osteoclasts in pagetic bone contain filamentous nuclear [339 – 341] and cytoplasmic inclusions [342] typical of the paramyxovirus family. Most importantly, experiments performed in Roodman’s laboratory demonstrate that targeting this virus to osteoclast precursors in vivo yields a mouse with osteoclasts mimicking those of pagetic patients [343]. This laboratory also found that similar to the in vivo situation, bone marrow derived from patients with Paget’s disease gives rise to numerous, large, hypernucleated osteoclast-like cells [344]. In contrast to polykaryons generated from normal marrow, these multinucleated cells exhibit increased sensitivity to 1,25-dihydroxyvitamin D and produce the osteoclastogenic cytokine IL-6, suggesting that an autocrine event mediates osteoclast proliferation in the disease [345]. Supporting this hypothesis is the fact that IL-6 circulates in increased amounts in pagetic patients [186]. The abundance of osteoclasts in Paget’s disease reflects the proliferative capacity of precursor cells. The number of CFU-GM colonies formed from pagetic marrow is increased, as is replication of CD34 cells, the earliest osteoclast progenitor yet identified in human [41]. It is of interest that pagetic marrow cells not in the osteoclast lineage (i.e., CD34 cells) may serve an accessory function in the

generation of resorptive polykaryons [41]. In this regard, pagetic stromal cells overexpress the RANK ligand, and marrow isolated from affected patients generates increased numbers of osteoclasts in response to the cytokine [346].

C. Cancer Osteolysis is the product of many malignant neoplasms either resident in or distant from bone. In most instances, it appears that tumor-induced osteolysis reflects the recruitment of osteoclasts, by-products of the neoplasm or bone matrix per se to the site of potential bone destruction. Such recruitment may be (i) the result of intimate interactions of a localized solid tumor, in bone, with osteoclast progenitors, (ii) humoral factors secreted by a tumor absent of skeletal metastases, or (iii) bidirectional paracrine stimulation of the tumor and bone marrow cells by cytokines. 1. SOLID TUMORS RESIDENT IN BONE Tumors commonly metastatic to the skeleton include those primary in lung, prostate, thyroid, and kidney [347]. Breast cancer is, however, by far the predominant source of osteolytic lesions. Reflecting excessive bone resorption, carcinoma of the breast is frequently complicated by hypercalcemia and fracture. The evidence that osteoclasts are pivotal to development of metastatic tumor-induced osteolysis includes: (i) in contrast to their ability to degrade soft tissues, cancer cells appear to have a limited, if any, capacity to resorb bone [334];

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FIGURE 16 Osteoclastic bone resorption in skeletal metastasis. Osteoclasts recruited by tumor resorb bone. Breast cancer cells (arrows) are in close proximity to osteoclasts (arrowheads) and follow the polykaryons into resorption bays (hematoxylin and eosin). (ii) breast cancer-induced osteolysis is blunted by osteoclast-inhibiting bisphosphonates [348]; (iii) osteoclasts are abundant in foci of metastatic breast cancer (Fig. 16); and (iv) breast cancer cells secrete potential osteoclastogenic agents [349 – 351]. In fact, evidence shows that parathyroid hormone-related protein is pivotal to the osteolytic properties of metastatic breast cancer. This protein, particularly produced by those breast cancers with a predisposition for skeletal metastasis, prompts marrow stromal cells, or osteoblasts, to synthesize osteoclast-recruiting cytokines [352]. The tumor-recruited osteoclasts resorb bone, releasing stored growth factors, notably transforming growth factor , which induces the neoplasm to proliferate and synthesize additional PTHrP [353]. This being the case, one would expect inhibition of tumor-induced osteoclastic bone resorption to decrease the tumor burden; in fact, bisphosphonates have such an effect [354]. 2. HUMORAL HYPERCALCEMIA OF MALIGNANCY PTHrP is also the active agent secreted by most tumors inducing humoral hypercalcemia of malignancy [355,356]. The amino terminus of the molecule is recognized by the PTH receptor. As this receptor has not been shown

convincingly to function in osteoclasts, the osteoclastogenic, and consequent, hypercalcemic effects of PTHrP are probably mediated by osteoblasts or marrow stromal cells in which the PTH/PTHrP receptor is abundant. 3. MULTIPLE MYELOMA In contrast to other B-cell malignancies, lytic bone lesions are common in patients with multiple myeloma. In fact, myeloma represents one of few malignancies almost always associated with osteolysis without an apparent osteoblastic component [357]. The central role osteoclasts play in the generation of myeloma bone lesions is underscored by the dramatic impact of bisphosphonates [358]. Bone biopsies from myeloma patients contain abundant osteoclasts in close proximity to the tumor [359,360]. Thus, myeloma cells alone, or in combination with surrounding stromal cells, likely produce factors capable of activating or recruiting osteoclasts. Medium conditioned by extirpated myeloma contains candidate molecules, including IL-6, IL-1, TNF, and macrophage inflammatory protein [361,362]. IL-6, which impacts osteoclasts profoundly, is also critical to myeloma proliferation [363,364]. Interestingly, and in keeping with the role of the

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cytokine in osteoclastogenesis attending postmenopausal osteoporosis, IL-6 mRNA is expressed by stromal and not myeloma cells. These observations raise the issue as to how myeloma cells induce cytokine production by accessory cells. Evidence indicates that the osteoclastogenic properties of myeloma cells depend on their contact, via the 41 integrin, with VCAM-1 on stromal cells [365]. 4. INFLAMMATORY OSTEOLYSIS Inflammatory osteolysis is a common form of clinically significant bone loss. This family of conditions includes alveolar bone loss attending periodontal disease, periprosthetic osteolysis, which frequently follows orthopedic implant surgery, and osteopenia accompanying rheumatoid arthritis. Each of these conditions is characterized by abundant osteoclast proliferation, eventuating in dramatic, localized bone destruction. In the case of rheumatoid arthritis, the osteopenia may also be systemic. TNF, interacting with its p55 (type 1) receptor, appears to be central to the osteoclastogenesis of inflammatory osteolysis [145,366] and, hence, newly available drugs inhibiting this cytokine hold therapeutic promise in these disorders. 5. POSTMENOPAUSAL OSTEOPOROSIS Postmenopausal (type 1) osteoporosis is due to an absolute increase bone resorption relative to the premenopausal state [190]. This enhancement of resorptive activity reflects the absence of estrogen’s antiosteoclastogenic effects. Thus, bone biopsies taken following estrogen withdrawal are rich in osteoclasts [190]. The means by which estrogen suppresses osteoclastogenesis was discussed earlier and in Chapter 41.

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ROSS AND TEITELBAUM 318. P. Schwartzberg, L. Xing, C. A. Lowell, E. Lee, L. Garrett, S. Reddy, G. D. Roodman, B. Boyce, and H. E. Varmus, Complementation of osteopetrosis in src-/- mice does not require src kinase activity. J. Bone Miner. Res. 11, S135 (1996). 319. W. S. Sly, D. Hewett-Emmett, M. P. Whyte, Y.-S. Yu, and R. E. Tashian, Carbonic anhydrase II deficiency identified as the primary defect in the autosomal recessive syndrome of osteopetrosis with renal tubular acidosis and cerebral calcification. Proc. Natl. Acad. Sci. USA 80, 2752 – 2756 (1983). 320. T. Yamamoto, N. Kurihara, K. Yamaoka, K. Ozono, M. Okada, K. Yamamoto, S. Matsumoto, T. Michigami, J. Ono, and S. Okada, Bone marrow-derived osteoclast-like cells from a patient with craniometaphyseal dysplasia lack expression of osteoclast-reactive vacuolar proton pump. J. Clin. Invest. 91, 362 – 367 (1993). 321. S. C. Marks, Jr. and P. W. Lane, Osteopetrosis, a new recessive skeletal mutation on chromosome 12 of the mouse. J. Hered. 67, 11 – 18 (1976). 322. W. W. Wiktor-Jedrzejczak, A. Ahmed, C. Szczylik, and R. R. Skelly, Hematological characterization of congenital osteopetrosis in op/op mouse: Possible mechanism for abnormal macrophage differentiation. J. Exp. Med. 156, 1516 – 1527 (1982). 323. N. Takahashi, N. Udagawa, T. Akatsu, H. Tanaka, Y. Isogai, and T. Suda, Deficiency of osteoclasts in osteopetrotic mice is due to a defect in the local microenvironment provided by osteoblastic cells. Endocrinology 128, 1792 – 1796 (1991). 324. S. C. Marks, Jr., M. F. Seifert, and J. L. McGuire, Congenitally osteopetrotic (op/op) mice are not cured by transplants of spleen or bone marrow cells from normal littermates. Metabol. Bone Dis. Rel. Res. 5, 183 – 186 (1984). 325. R. Felix, M. G. Cecchini, W. Hofstetter, P. R. Elford, A. Stutzer, and H. Fleisch, Impairment of macrophage colony-stimulating factor production and lack of resident bone marrow macrophages in the osteopetrotic op/op mouse. J. Bone Miner. Res. 5, 781 – 789 (1990). 326. H. Yoshida, S.-I. Hayashi, T. Kunisada, M. Ogawa, S. Nishikawa, H. Okamura, T. Sudo, L. D. Shultz, and S.-I. Nishikawa, The murine mutation osteopetrosis is in the coding region of the macrophage colony stimulating factor gene. Nature 345, 442 – 443 (1990). 327. R. Felix, M. G. Cecchini, and H. Fleisch, Macrophage colony stimulating factor restores in vivo bone resorption in the op/op mouse. Endocrinology 127, 2592 – 2594 (1990). 328. H. Kodama, A. Yamasaki, M. Nose, S. Niida, Y. Ohgama, M. Abe, M. Kumegawa, and T. Suda, Congenital osteoclast deficiency in osteopetrotic (op/op) mice is cured by injections of macrophage colony-stimulating factor. J. Exp. Med. 173, 269 – 272 (1991). 329. S. J. Begg, J. M. Radley, J. W. Pollard, O. T. Chisholm, E. R. Stanley, and I. Bertoncello, Delayed hematopoietic development in osteopetrotic (op/op) mice. J. Exp. Med. 177, 237 – 242 (1993). 330. Y. Y. Myint, K. Miyakawa, M. Naito, L. D. Shultz, Y. Oike, K. Yamamura, and K. Takahashi, Granulocyte/macrophage colonystimulating factor and interleukin-3 correct osteopetrosis in mice with osteopetrosis mutation. Am. J. Pathol. 154, 553 – 566 (1999). 331. E. Stanley, G. J. Lieschke, D. Grail, D. Metcalf, G. Hodgson, J. A. M. Gall, D. W. Maher, J. Cebon, V. Sinickas, and A. R. Dunn, Granulocyte/macrophage colony-stimulating factor-deficient mice show no major perturbation of hematopoiesis but develop a characteristic pulmonary pathology. Proc. Natl. Acad. Sci. USA 91, 5592 – 5596 (1994). 332. P. F. Coccia, W. Krivit, J. Cervenka, C. Clawson, J. H. Kersey, T. H. Kim, M. E. Nesbit, N. K. Ramsay, P. I. Warkentin, S. L. Teitelbaum, A. J. Kahn, and D. M. Brown, Successful bone-marrow transplantation for infantile malignant osteopetrosis. N. Engl. J. Med. 302, 701 – 708 (1980). 333. P. J. Meunier, J. M. Coindre, C. M. Edouard, and M. E. Arlot, Bone histomorphometry in Paget’s disease. Arthritis Rheum. 23, 1095 – 1103 (1980).

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105 352. T. A. Guise, and G. R. Mundy, Cancer and bone. Endocr. Rev. 19, 18 – 54 (1998). 353. J. J. Yin, K. Selander, J. M. Chirgwin, M. Dallas, B. G. Grubbs, R. Wieser, J. Massague, G. R. Mundy, and T. A. Guise, TGF-beta signaling blockade inhibits PTHrP secretion by breast cancer cells and bone metastases development. J. Clin. Invest. 103, 197 – 206 (1999). 354. I. J. Diel, E. F. Solomayer, S. D. Costa, C. Gollan, R. Goerner, D. Wallwiener, M. Kaufmann, and G. Bastert, Reduction in new metastases in breast cancer with adjuvant clodronate treatment. N. Engl. J. Med. 339, 357 – 363 (1998). 355. A. A. Budayr, R. A. Nissenson, R. F. Klein, K. K. Pun, O. H. Clark, D. Diep, C. D. Arnaud, and G. J. Strewler, Increased serum levels of a parathyroid hormone-like protein in malignancy-associated hypercalcemia. Ann. Intern. Med 111, 807 – 812 (1989). 356. N. Horiuchi, M. P. Caulfield, J. E. Fisher, M. E. Goldman, R. L. McKee, J. E. Reagan, J. J. Levy, R. F. Nutt, S. B. Rodan, T. L. Schofield, T. L. Clemens, and M. Rosenblatt, Similarity of synthetic peptide from human tumor to parathyroid hormone in vivo and in vitro. Science 238, 1566 – 1568 (1987). 357. J. F. Rossi, R. Bataille, D. Chappard, C. Alexandre, and C. Janbon, B cell malignancies presenting with unusual bone involvement and mimicking multiple myeloma: Study of nine cases. Am. J. Med. 83, 10 – 16 (1987). 358. R. A. Kyle, Maintenance therapy and supportive care for patients with multiple myeloma. Semin. Oncol. 26, 35 – 42 (1999). 359. G. R. Mundy, L. G. Raisz, R. A. Cooper, G. P. Schechter, and S. E. Salmon, Evidence for the secretion of an osteoclast stimulating factor in myeloma. N. Engl. J. Med. 291, 1041 – 1046 (1974). 360. A. Valentin-Opran, S. A. Charhon, P. J. Meunier, C. M. Edouard, and M. E. Arlot, Quantitative histology of myeloma-induced bone changes. Br. J. Haematol. 52, 601 – 610 (1982). 361. X. G. Zhang, R. Bataille, M. Jourdan, S. Saeland, J. Banchereau, P. Mannoni, and B. Klein, Granulocyte-macrophage colony-stimulating factor synergizes with interleukin-6 in supporting the proliferation of human myeloma cells. Blood 76, 2599 – 2605 (1990). 362. M. Kawano, I. Yamamoto, K. Iwato, H. Tanaka, H. Asaoku, O. Tanabe, H. Ishikawa, M. Nobuyoshi, Y. Ohmoto, and Y. Hirai, Interleukin-1 beta rather than lymphotoxin as the major bone resorbing activity in human multiple myeloma. Blood 73, 1646 – 1649 (1989). 363. M. Kawano, T. Hirano, T. Matsuda, T. Taga, Y. Horii, K. Iwato, H. Asaoku, B. Tang, O. Tanabe, and H. Tanaka, Autocrine generation and requirement of BSF-2/IL-6 for human multiple myeloma. Nature 332, 83 – 85 (1988). 364. X. G. Zhang, B. Klein, and R. Bataille, Interleukin-6 is a potent myeloma-cell growth factor in patients with aggressive multiple myeloma. Blood 74, 11 – 13 (1989). 365. Y. Mori, T. Michigami, M. Dallas, M. Niewolna, B. Story, R. Lobb, G. R. Mundy, and T. Yoneda, Anti-4 integrin antibody suppresses the bone disease of myeloma and disrupts myeloma-marrow stromal cell interactions. J. Bone Miner. Res. 14, S173 (1999). 366. K. D. Merkel, J. M. Erdmann, K. P. McHugh, Y. Abu-Amer, F. P. Ross, and S. L. Teitelbaum, Tumor necrosis factor- mediates orthopedic implant osteolysis. Am. J. Pathol. 154, 203 – 210 (1999).

CHAPTER 4

The Biochemistry of Bone JAYASHREE A. GOKHALE AND ADELE L. BOSKEY Hospital for Special Surgery, Weill Medical College of Cornell University, New York, New York 10021

PAMELA GEHRON ROBEY Bone Research Branch, National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland 20892

I. II. III. IV. V.

VI. Other Proteins VII. Requirements for Matrix Mineralization VIII. Pathways of Matrix Mineralization References

Introduction Collagen Glycosaminoglycan-Containing Proteins Glycoproteins Gla-Containing Proteins

in the cells that destroy bone (osteoclasts) and, in addition, the osteoblastic precursors that will replace the calcified cartilage with bona fide bone. The initial bone formed, woven bone, is a rather unorganized conglomeration of collagenous and noncollagenous proteins that induce the precipitation of mineral. Through modeling by osteoclasts, this primordial bone is removed and replaced by the formation of lamellar bone, a more highly organized structure with alternating layers of mineralized extracellular matrix, whose plywood-like structure provides bone with its mechanical strength. This structure is further reinforced by the development of the Haversian canal system centered around a blood vessel and a nerve, providing nutrients and signals to the cells entombed in bone (osteocytes), while maintaining communication through osteocytic cell processes in canaliculae. Initially it was hypothesized that mineralized matrices were composed of a unique set of matrix proteins, the combination of which would initiate the precipitation of hydroxapatite. However, with the development of techniques for isolation of the components without degradation [2,3] and cell culture systems that faithfully recapitulate the osteoblastic phenotype [4 – 9], it became apparent that most,

I. INTRODUCTION A. Bone: The Tissue The skeleton is essentially responsible for providing not only structural support and protection to the body’s organs, but also for serving as a reservoir for calcium, magnesium, and phosphate, ions that are of critical importance in physiology. The fabric of bone is a unique composite of living cells embedded in a remarkable threedimensional structure of extracellular matrix that is stabilized by a mineral, a carbonate-rich analogue of the geologic mineral hydroxyapatite. During development, mesenchymal cells form the skeleton via two basic pathways [reviewed in 1]. Endochondral bone is formed by an initial condensation of mesenchymal cells that induces the expression of the chondrocytic phenotype. The cartilaginous structure that is formed through the sequential expression of a number of genes that regulate morphogenesis serves as a temporary model. As part of that developmental sequence, the cartilage becomes calcified. The provisional calcified cartilagenous precursor is subsequently replaced by bone. Invasion by blood vessels brings

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if not all, of the proteins synthesized by bone-forming cells are also synthesized by nonskeletal cells. There are few, if any, truly bone-specific proteins. However, it is now clear that the composition of bone is quite different: 70 – 90% of bone is composed of mineral with only 10 – 30% represented by protein. The ratio of collagenous to noncollagenous proteins differs greatly from that of other tissues, with collagenous protein composing 90% of the organic matrix (compared to 10 – 20% in other tissues) and noncollagenous proteins accounting for 10% (compared to as much as 80– 90% in soft tissues with the exception of tendon). In addition, all of the known collagenous and noncollagenous proteins in bone studied to date differ from those in other tissues in their chemical nature. These diverse forms result from alternative splicing of mRNA and different posttranslational modifications such as glycosylation, phosphorylation, and sulfation. These chemical differences most likely influence the function of these proteins, and the appropriate mixture provides the extracellular matrix with the ability to calcify. While the mineralized matrix is viewed by many as mere cement, it is actually a fairly dynamic aggregate structure. Although the most abundant proteins in bone matrix play structural roles in providing the scaffolding and binding sites for the regulation of mineral deposition and turnover, these proteins also function in modulating cellular activity. Cell – matrix interactions have become increasingly recognized as important determinants during all stages of development and tissue homeostasis. In addition, extracellular matrix proteins are the secretory products of cells in the osteoblastic lineage and, as such, they represent biochemical markers of the formation process (either as complete forms or precursor molecules) or the resorption process (in their degraded form). Therefore, it is necessary to have an indepth understanding of the biosynthetic process by which these proteins are formed. In addition, in order to decipher intelligently the clues provided by their assay ofin body fluids for determination of the status of the skeleton, it is important to understand their function in bone homeostasis.

B. Bone Matrix Formation: The Role of Maturational Stage It is now well accepted that bone formation is accomplished by cells in the osteoblastic lineage that pass through a series of maturational stages [10 – 13; see also Chapter 2]. The lineage is composed of cells that start off as uncommitted precursors. These precursors may be highly proliferative during development, but most likely at maturity, these stem cells are quiescent, thus preserving a reservoir of cells that have gone through a limited number of mitoses. Upon command, by signals that have yet to be fully identified, they become committed to the osteoblastic lineage and are

recognizable as fibroblast-like, proliferative osteoprogenitors. At some point, their rate of proliferation slows, and they are more aptly termed preosteoblasts, mainly by virtue of their location immediately adjacent to the real workhorse of the lineage, the osteoblast, on the opposite side of where mineralization will occur or is occurring. The term osteoblast is best thought of as a histological definition that describes a particular cell that has a large nucleus with prominent nucleoli indicative of a high rate of genetic expression. Additionally, it has a greatly expanded rough endoplasmic reticulum that is somewhat polarized such that the cell secretes enormous amounts of matrix toward the mineralization front, creating a layer of unmineralized osteoid. For reasons that are not yet known, a limited number of cells disengage themselves from the osteoblastic layer and are left behind as apposition proceeds. As these cells become buried in matrix that becomes mineralized (through a somewhat nebulous stage termed an osteoid osteocyte or an osteoblastic osteocyte), the cell maintains its communications with cells above it in the osteoblastic layer. This occurs via retention of cell contacts as cell processes become surrounded by mineralized matrix. Consequently, the most differentiated member of the lineage, the osteocyte, is still in communication with the osteoblastic layer above it. It is very likely that these embedded osteocytes serve a mechanoreceptor function. Through the cell processes in the canaliculae, they constantly monitor the surrounding environment and signal to the osteoblastic layer or lining cell layer when and where resorption is needed to remove and refurbish fatigued matrix or to respond to alterations in mechanical load. The expression of bone matrix proteins is somewhat stage specific (Fig. 1). However, currently it is still not perfectly clear whether one cell at one particular stage of maturation (the osteoblast) is capable of making all the components of the mineralized matrix or whether the process requires cells at different stages of maturation simultaneously to be present. Considering the amount of coupling that occurs between cells at different stages of maturation through gap and tight junctions, a great deal of cooperativity in the bone-forming process is likely. In vitro model systems have not really sorted out this issue, as none of the cell culture systems currently available go through the different stages of maturation in a synchronous fashion. Uncommitted progenitors, preosteoblasts, osteoblasts, and sometimes even osteocytes can be identified in populations of cells that are in the process of forming bone nodules in vitro (the current state of the art in vitro) [14]. It has been demonstrated that proliferation of a single cell gives rise to the whole spectrum of maturational stages, and nature must have a reason for reestablishing this continuum. Although identification of the various maturational stages and the characterization of the biological properties of the cells within each stage have advanced our under-

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FIGURE 1

Maturational stage and bone matrix gene expression. Osteoblastic cells pass through a series of maturational stages, each of which can be partially characterized by the bone matrix proteins that they produce. In addition, osteoclasts also secrete proteins that become incorporated into mineralized matrix.

standing of bone formation greatly, the mechanism of how and when various factors influence bone matrix protein expression is not clear. The literature is cluttered with numerous reports describing the effects of hormones, growth factors, cytokines, chemokines, and so on osteoblastic metabolism, and the results of these studies are extremely variable. This variability may reflect the fact that the effects that these factors exert most likely depend on animal species, donor age and sex, stage of maturation of the cells, state of transformation, and certain cell culture parameters, such as length of time in culture, density, and nutrient conditions. This chapter focuses on the structural aspects of bone matrix proteins and their genes and only highlights what factors are known to influence gene expression. The topic of what factors influence expression at a particular stage of maturation is addressed in more detail in Chapter 2. Only a few years ago, relatively few human bone matrix genes had been isolated and characterized. The current list is quite lengthy, and most of the information provided in this chapter pertains to human genes and proteins unless information is available only from another animal species.

C. Mineral The mechanical strength of bone is attributable to the presence of mineral, which converts the pliable organic

matrix into a more rigid structure [15,16]. In the composite structure of cells, protein and mineral, the bone mineral (apatite) crystals, approximately 300 Å in their longest dimension, are aligned along the collagen fibril axis [17 – 19]. A variety of structural analyses, including X-ray and electron diffraction [20 – 22], infrared spectroscopy [23], high-voltage electron microscopy [24], nuclear magnetic resonance (NMR), and X-ray absorption fine structure analysis (EXAFS) [25 – 27] have shown that mineral crystals are analogous to the naturally occurring geologic mineral, hydroxyapatite (Ca10(PO4)6(OH)2) (Fig. 2). In bone, mineral includes numerous ions not found in pure hydroxyapatite. For example, HPO42, CO32, Mg2, Na, F, and citrate are adsorbed on the crystal surfaces and/or substituted in the lattice for the constituent Ca2, PO43, and OH ions [28 – 34]. This poorly crystalline apatite, because of its small size and large number of latticesubstituted and surface-adsorbed ion impurities, goes into solution more readily than the larger, more perfect crystals of geologic hydroxyapatite. For example, some ions (HPO42, CO32, Na2, and F) can be in the lattice and on the surface, whereas others (Mg2 and citrate) prefer surface locations. This altered solubility allows bone mineral to play an important role in Ca2, Mg2, and PO43 ion homeostasis [35]. Despite claims of the presence of other mineral phases in bone, e.g., brushite [36], octacalcium phosphate [37],

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FIGURE 2 Crystal lattice structure. A portion of the apatite structure is depicated as it would be viewed along the length (c axis) of the hydroxyapatite crystal, showing the hexagonal arrangement of the Ca2 and PO43ions about the OH position.

amorphous calcium phosphate [38], and whitlockite [39], current evidence supports the viewpoint that bone mineral is predominantly apatitic, with numerous, perhaps unique, impurities [40]. This viewpoint is maintained for the rest of the chapter.

gene activation and regulation, (ii) RNA transcription and processing, (iii) translation into protein, posttranslational modification, and secretion from the cell, and finally (iv) deposition in the extracellular matrix. The following description pertains specifically to the individual  chains of type I collagen. However, virtually all of these processes apply to the other proteins found in bone.

II. COLLAGEN In the vertebrate body, the major structural protein is collagenous in nature. Collagen is defined as a trimeric molecule composed of  chain subunits that are wound together to form a triple helix [41]. A significant feature of component  chains is that their primary sequence is composed almost entirely of a repeating triplet sequence, GlyX-Y, where X is often proline and Y is often hydroxyproline [42]. Collagenous proteins are either homotrimeric, composed of three identical  chains, or heterotrimeric, with two or three different  chains. Currently there are over 23 different  chains that associate to yield 13 different types of collagens, with 6 more potential types that to date have only been identified at the cDNA level [43 – 45]. The various forms of collagen are as diverse as the tissues in which they are found. These variations on a theme convey distinct features such that the collagen type is uniquely suited to carry out particular functions in a given tissue. In each of these tissues, collagen most likely serves a mechanical function. For example, in mineralized tissues, collagen fibrils provide tensile strength [15,46]. Given the complex nature of collagen expression, it serves as a useful example for describing the pathway of (i)

A. Gene Structure The structure of mammalian genes is as varied as the proteins for which they code. An example of a prototype gene structure is shown in Fig. 3. In the 5 region of the gene, there is usually a sequence that is not transcribed from DNA into RNA. As described later, this untranslated region (UTR), the promoter, is the primary site for the regulation of gene activity. Structures can also form due to the presence of palindromic type sequences (mirror image and complimentary) that allow the DNA to take on conformations other than the typical Watson – Crick double helix. These unusual conformations (such as z DNA) may serve as recognition sites for certain factors and enzymes that regulate gene activity [47 – 50]. Following this 5 UTR is the portion of the gene that will be transcribed into an RNA sequence through the action of RNA polymerases. Genes contain sequences that are found in mature RNA and dictate the amino acid sequence of protein (exons), interspersed with sequences that are removed and do not end up in mature mRNA or in the translated protein (introns). The exon – intron structure of a gene

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FIGURE 3 Basic structure of prototype gene. In addition to exons (E), introns (I), and the transcription start site (toward the 3 end), the gene contains a promoter toward the 5 end, upstream of the transcription start site. It includes defined sequences or cis-acting elements such as CCAAT and TATA boxes. Trans-acting factors (proteins) can bind to cis elements to regulate transcription.

can be simple, with only a single exon, or highly complex, with multiple exons as exemplified by the genes for type I collagen. The gene for the constituent  chains of type I collagen, COL1A1, located on chromosome 17q21.3-q22, is 18 kb in length and contains 51 exons [51,52], whereas COL1A2, on chromosome 7q21.3-q22, is 35 kb and has 52 exons [52 – 54]. It is often the case that exons code for functional regions of the resulting protein, and may thereby represent an ancestral gene. In many of the genes for collagen  chains, the exons are often 54 bases, or multiples thereof, that code for six [GLY-X-Y]n triplets. Following the open reading frame that designates the protein sequence is another stretch of DNA sequence that codes for mRNA that again does not end up in protein. This 3-untranslated region (3 UTR) contains sequences that may also influence gene activity. Signals for polyadenylation, another posttranscriptional modification, are also located within this region [55,56].

B. Gene Activation and Transcription The process of gene activation is a complex series of events that are mediated by cis and trans-acting factors (see Fig. 3). Cis-acting elements are defined as sequences present in DNA that are required for the binding of factors resident within the nuclear environment that influence (either positively or negatively) gene transcription. They include the binding sites for enzymes that are required for the synthesis of RNA complementary to the DNA template, such as polymerases. These types of cis-acting elements are exemplified by the sequences TATA and CAAT, which have been postulated to serve as the binding/orientation sites for

polymerases. In addition, GC-rich regions may also regulate polymerase activity, perhaps through the formation of three-dimensional structures. Cis-acting elements are found most commonly upstream (5) from the RNA transcription start site (the promoter region), where polymerase initiates the synthesis of mRNA. However, cis elements can also be located in sites quite distant from the transcription start site and may also reside in intronic sequences, or in the 3 regions of the gene. Cis-acting elements are also the binding sites for transacting factors, which are defined as proteins that either singly or in combination have the ability to up- or downregulate gene activity. Trans-acting elements are extremely diverse, and many are the gene products of protooncogenes or receptors bound to their ligands. These DNA-binding proteins have a number of structural motifs that allow them to bind to the double helix [reviewed in 57], including helix – loop – helix [58,59], and leucine zipper proteins, which can also have important functions in the cytoplasm [60]. Other motifs include HMG-1 box-binding proteins [61] and zinc-binding proteins (fingers, twists, and clusters) [62]. By binding to specific sequences, these complexes can either enhance the activity of the polymerizing enzymes, leading to high levels of a particular mRNA species, or suppress their activity, such that there are very few or virtually no copies produced. The promoters for COL1A1 and COL1A2 have been characterized in detail and contain similar but not identical promoter elements [63 – 67]. At 29 bp from the transcription start site, the COL1A1 promoter contains a TATA box, whereas it is absent in the COL1A2 promoter. Further upstream, both contain a CCAAT sequence (100 bp in COL1A1 and 82 bp in COL1A2), as well as a long stretch of Cs and Ts, which confer S1 nuclease and DNAse

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hypersensitivity, implying a relatively open structure. The COL1A1 sequence then becomes A-G rich, which could indicate another potential change in tertiary structure due to base pairing between these two regions. An SP1 element (the binding site of a constitutive transcription factor) is also present. Studies utilizing rather extended regions of this COL1A1 promoter have demonstrated that it is active. However, maximal activity may require sequences present in the first intron, which also contains SP1 and DNAse hypersensitive regions [68]. This concept remains controversial because both stimulatory [69,70] and inhibitory [70] elements have been described in this region. The 2(I) contains a positive enhancer in the first intron and a CAATbinding protein (CTF/NF1)-binding site [71]. Other elements include a vitamin D response element (VDRE) in COL1A1 [72] and a CAAT-like region that binds to nuclear factor 1 (NF1) in the COL1A2 promoter [73]. It is of interest that the amount of mRNA for COL1A1 is twice the amount of COL1A2, a ratio that is reflected in the final triple helical molecule. Consequently, there must be factors (e.g., ascorbic acid) that regulate the production of these two gene products such that there are never excessive amounts of COL1A2 mRNA [74 – 77].

C. Gene Regulation While there is only one copy of the genes that code for the COL1A1 and COL1A2 in mammalian genomes, the regulation of type I collagen production in bone is somewhat different from that in soft connective tissues. It has been recently found that different parts of the COL1A1 promoter are required to maintain production by bone-forming cells in vitro [78 – 79]. In bone cell and organ cultures, collagen synthesis increased by heparin [80], organic phosphate [81], interleukin (IL)-4 [82], and gallium [83]. In contrast, collagen synthesis is decreased by prostaglandin E2 [84] 1,25-dihydroxyvitamin D3 [85], cortisol [86], parathyroid hormone (PTH) [87], epidermal growth factor (EGF) [88], basic fibroblast growth factor (bFGF) [89], IL10 [90], and lead [91]. Although the COL1A1 promoter contains a VDRE-like element, binding of this element by the VDR that has bound to its ligand inhibits expression. In addition, removing this element from the promoter does not totally abolish the inhibitory effect of 1,25-dihydroxyvitamin D3, indicating that other cis- and/or trans-acting factors are involved [85]. Depending on the concentration and the stage of the cell culture, dexamethasone, can either increase or decrease collagen synthesis [92,93].

D. RNA Processing Once transcription is initiated, a precursor form of RNA (often termed Hn or heteronuclear RNA) is transcribed

from the DNA template. This precursor form contains the intronic sequences that must be removed prior to translation to yield mRNA with only exonic, 5 UTR, and 3 UTR sequences. This process is accomplished through the action of a number of enzymes that associate to form what is termed a spliceosome [94,95]. Several types of splicing reactions can occur based on the sequences that bridge the exon and intron. Splicing occurs by bringing together the junction consensus sequence (GT ........ AT) at both the 5 and the 3 end of the intron and cleavage by enzymes via formation of a lariat-like structure [96]. Differential splicing can also occur via exon skipping, or exon splitting due to the presence of splice junctions that are buried within the exonic sequences. The factors that regulate differential splicing are not well understood but are believed to provide a particular cell type with the ability to generate different mRNA species. The mRNA is further modified by the addition of a methyl cap at the 5 end. The addition of a polyadenyl tail in the noncoding region at the 3 end completes the formation of the mature mRNA. The regulation of polyadenylation (choice between different polyadenylation sites and length of polyadenylation) is not well known but is implicated in regulating the half-life of the mRNA. At this point, the fully processed mRNA species are transported to the cytoplasm (Fig. 4). In the case of COL1A1, the sizes are 7.2 and 5.9 kb, and for COL1A2, the sizes are 6.5 and 5.5 kb.

E. Translation and Secretion Once in the cytoplasm, the mRNA species associate with ribosomes, possibly through the formation of stem loops that have been described in some mRNA species, including the mRNA for COL1A1 [97]. The initiation codon in most proteins is AUG (or CUG) for methionine. The initial translation of secreted proteins produces a signal peptide that allows binding of the ribosome and nascent polypeptide chain to the cytoplasmic surface of the endoplasmic reticulum (ER). As translation proceeds, the protein is extruded into the lumen of the ER and the signal peptide is cleaved. However, it should be noted that, like many other proteins, collagen  chains are synthesized in a precursor form. Noncollagenous sequences are found at both amino (pN) and the carboxyl (pC) termini, resulting in a chain termed a pro chain [reviewed in 98]. During passage of the nascent polypeptides through the ER, posttranslational processing begins by the action of certain enzymes that are resident along the secretory pathway. Prolyl hydroxylases will form hydroxyprolyl residues immediately preceding glycyl residues in the triplet sequence. In most cases, proline is hydroxylated in the 4 position; however, in certain tissues, another enzyme will hydroxylate in the 3 position. Prolyl hydroxylation is required

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FIGURE 4 Gene activation and RNA transcription. Gene activity is regulated by the interaction of cis-acting (DNA sequences) and trans-acting (nuclear factors) elements that either initiate or repress transcription. Once a gene is activated, polymerases translate the antisense genomic sequence, including the exons (which ultimately code for the protein) and the introns (intervening sequences). The initial transcription product, HnRNA, is processed through the action of spliceosomes to remove the intervening sequences. RNA is further modified by methylation of the 5 end and by the addition of poly(A) to the 3 end prior to transport to the cytoplasm.

for the formation of a stable helical structure because the hydroxyl groups participate in intrahelix hydrogen bonding. Certain lysyl residues will also be hydroxylated by lysyl hydroxylase in the ER. Hydroxylysyl residues can be further modified through the action of sugar transferases that add first galactosyl residues, and sometimes an additional glucosyl residue to form galactosyl-hydroxylysine and glucosyl-galactosyl-hydroxylysine, respectively. The function of these sugar modifications is unknown. Once translation of the pro chains has been completed, the noncollagenous precursor sequences at the carboxy termini of two pro1 and one pro2 chains associate and bond through disulfide bridges. This aggregation is then followed by triple helix formation progressing from the C terminus moving toward the N terminus as the protein passes through the ER. Posttranslational modifications continue until the target residues are no longer accessible due to triple helix formation [reviewed in 98]. The formation of this unique conformation is dependent on the GLY-X-Y sequence throughout the helical domain of the molecule. This sequence allows the formation of rodlike triple helices [41], as glycine is the only amino acid small enough to fit within the center of the triple helix. The X and Y amino acids occur on the surface of the triple helix and are arranged in charged clusters, which facilitate the interaction of the individual molecules in the formation of

fibrils [99]. It is currently believed that there are specific domains within the fibrils that interact with cells, fibronectin, decorin, and other matrix molecules, but these have not been well identified. Individual  chains of the collagen molecule coil about one another in an extended rigid helix. The structure is stabilized by hydrogen bonding between OH groups on hydroxyproline and intrachain water [100] and by aldehyde derived cross-links [101]. More details of the collagen structure have bene reviewed by Kuhn [102]. In addition, the crystal structure of a small triple helical peptide [103] has provided confirmation of earlier structure predictions. With the passage of the hydroxylated and glycosylated precursor to the Golgi apparatus, the procollagen molecule is further modified by the addition of N-linked oligosaccharides to certain asparaginyl residues in the carboxy-terminal precursor region. Initially, a high mannose structure is transferred to the acceptor residue, and the oligosaccharide is trimmed and rebuilt by the addition of N-acetylgalactosamine and N-acetylglucosamine to form a complex type oligosaccharide. Again, the function of these sugar modifications is unknown, although they are thought to be important for transport of the molecule from the cell [104]. In the Golgi apparatus, O-linked glycosylation reactions also occur; however, collagen  chains are not modified in this fashion.

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The process by which collagen molecules are moved from one cellular compartment to another is not completely known, but it has been speculated to be mediated by proteins termed chaperonins, some of which are heat shock proteins [105]. Due to the major role that collagen plays in all tissues, and the complicated nature of its biosynthesis, its processing and secretion may be tightly regulated by such proteins. It is likely that a derangement in the secretory apparatus that affects collagen secretion would result in disease. The triple helical and posttranslationally modified procollagen molecules within secretory granules are then transported to the cell surface and extruded from the cell (Fig. 5).

F. Deposition and Fibril Formation Although the exact process of fibril formation is not fully characterized, it has been speculated that it can occur either prior to or during the process of secretion by the formation of deep invaginations of the cell membrane, which provide a secluded environment [106]. It is clear that the nontriple helical precursor extensions must be removed in

FIGURE 5

an orderly fashion in order for normal fibers to form. Specific peptidases remove the N-terminal extension (yielding pC collagen) or the C-terminal extension (yielding pN collagen). When both extensions are removed, the fully mature triple helical collagen molecule still contains short nontriple helical telopeptides at both termini. Interestingly, it has been suggested that both pN and pC propeptides participate in feedback inhibition of collagen synthesis [107 – 111]. In addition, the pN peptide remains, at least in part, within bone matrix and was identified as the 24-kDa phosphoprotein [112,113]. The pC propeptide escapes into the circulation and has been used as a measure of collagen biosynthesis [114]. However, because pC can be contributed from the synthesis by soft tissues as well, it is not totally reflective of bone formation. The mature collagen molecules then form head to tail and lateral associations, and it is thought that the lateral associations are inhibited by the presence of the pN collagen. The addition of pN collagen or pC collagen molecules to the outermost layer of fibers may in fact dictate when fibril growth stops. Small fibers tend to be coated with pN collagen, whereas larger fibers are associated with pC collagen [115,116]. Fiber diameter growth may also be regulated by

Protein translation, modification, and secretion. Once in the cytoplasm, mRNA is translated into protein by ribosomes. In the case of bone matrix proteins (and the majority of secreted proteins), mRNA codes for a signal peptide that allows the ribosome to attach to the endoplasmic reticulum, allowing the nascent peptide to be extruded. In the lumen, hydroxylases modify certain prolyl and lysyl residues. Once the C-terminal portion is completed, the C propeptides of three chains associate and triple helix formation proceeds to the amino terminus. The pathway to secretion proceeds through the Golgi apparatus where the molecule is further modified by the addition of oligosaccharides to the C propeptide, and galactosyl and glucosyl-galactosyl residues to lysine and hydroxylysine residues by sugar transferases. Upon secretion, propeptides at N and C termini are cleaved, and the molecules are deposited in a four-dimensional staggered array and are further stabilized by covalent cross-links.

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the binding of other collagenous or noncollagenous proteins to the outer fibril surface [116 – 119]. This is an important point because it may be that the surface of type I collagen fibrils is not directly accessible for binding, and that collagen may be functioning in a purely structural fashion when intact within bone collagen fibers. Other activities may occur once fibril-associated proteins are removed or when the collagen fibril is degraded. The structure of fibrillar type I collagen has been described in detail [120]. Katz’s structural models, based on low angle X-ray scattering, confirm the structural pattern predicted by Hodge and Petrushka [121]. According to this model, individual collagen fibrils are aligned in a quarterstaggered array, with a 280-nm periodicity. As a result of the quarter stagger, there are gaps (holes) within the fibrillar structures, and it is in these gaps and in the overlapping regions adjacent to them that bone mineral first appears [18,122 – 125]. Specifically, Traub and co-workers [125] have shown that the first mineral crystals appear in a specific region of the collagen fibril known as the e band. However, it should be noted that the majority of these studies have been performed in mineralizing turkey tendon, which may not be identical to other patterns of matrix mineralization. Once fibers have been formed in the extracellular environment, they are further stabilized by the formation of inter- and intramolecular cross-links. This process occurs through the action of lysyl oxidase, which converts lysyl and hydroxylysyl residues in the telopeptide regions to allysine and hydroxyallysine, which are aldehydic in nature. Immature cross-links that are reducible by sodium borohydride are formed by the condensation of the aldehydes with other lysyl and hydroxylysyl residues within the helical region of a neighboring molecule. With time, these crosslinks become more insoluble by condensation with histidinyl residues or its aldehydic derivatives. Through this time-dependent process, as reviewed (126), up to four 

TABLE 1 Collagen

chains can ultimately become cross-linked, leading to great stability and insolubility of the collagenous scaffolding (Fig. 5).

G. Collagen Types A broad range in the collagen pattern and molar mixture is displayed from one tissue to another and certain types are concentrated in specific tissues [reviewed in 43,44]. In comparison, bone matrix proper contains a rather limited array of collagen types (Table 1), which will be discussed in detail in this chapter. Based on their structural features, collagens can be roughly divided into two groups: fibrillar and nonfibrillar. Fibrillar collagens (types I, II, III, and V) are by far the most abundant forms and are found in the interstitial spaces of connective tissues throughout the body. Type I collagen, the predominant collagen of skin, tendon, and bone, is composed of two 1(I) and one 2(1) chains and forms the major scaffolding of virtually all the connective tissues (with the exception of cartilage). Cartilage contains predominantly type II collagen ([1(II)]3) with limited amounts of other collagen types as described later. Type III, composed of three identical 1(III) chains, and type V, composed of a combination of 1(V), 2(V), and 3(V) chains, are often codistributed with type I. Fetal tissue contains proportionally more type III collagen, which has been reported to coat the surfaces of type I fibrils or to form thin reticulin-like fibrils. Type V collagen is often pericellular and is also enriched in particular tissues such as smooth muscle and blood vessels. The nonfibrillar collagens (types IV, VI, VII, VIII, IX, X, and XI) are characterized by triple helical domains that are either shorter or longer than those of the fibrillar types and may contain stretches of noncollagenous sequences. Type IV collagen, with the composition [1(IV)22(IV)], is

Collagen Types Found in Bone Matrix Location/function

Molecular structure

Type I:[1(1)2 (1)] and [1(1)3]

Constitutes 90% of matrix in the bone matrix. Acts as scaffolding and binds to other proteins that initiate hydroxyapatite deposition

67-nm banded fibrils

Type III:[1(III)3]

Present only in trace amounts and can regulate collagen fiber thickness

67-nm banded, coats type I fibrils

Type :[1(V)22(V)] and [1(V)2(V)

Their absence can result in collagen fibrils of larger diameter

67-nm banded, coats type I fibrils in some tissues

Type X:[1(X)3]

Present in hypertrophic cartilage and can be involved in matrix organization via formation of the template for type I collagen

Probably fishnet-like lattice

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found in basement membranes, including those that surround vascular endothelial cells that invade bone during osteogenesis. Type VI collagen is significantly shorter than other collagen types and is composed of three distinct  chains. These molecules form rope-like microfibrillar structures. Anchoring fibrils are composed of type VII collagen, which is 1.5 times longer than type I. Another short chain collagen, type VIII, is found in Descemet’s membrane of the eye, is synthesized by endothelial cells in culture, and may be related to type X collagen found in calcified cartilage. The localization of type X to hypertrophic chondrocytes is highly specific, but it does not appear to have a major role in cartilage calcification. Type IX and type XI are homologous to type V and are minor constituents in cartilage. Type IX belongs to the socalled FACIT class (fibril associated collagen with interrupted triplex). Structures of the isolated FACIT collagenous proteins, as distinct from those of the fibrillar collagens, include nontriple helical domains, as predicted from the non-GLY-X-Y repeats. Type IX is composed of three different types of  chains, 1(IX), 2(IX), and 3(IX), that form a short and a long triple helix joined by a flexible hinge region. A glycosaminoglycan chain is also attached to one of the  chains at the amino terminus, making this collagen a proteoglycan as well. Type IX has been found covalently attached to and as a coating to type II collagen fibrils and covalently attached to it. Type XII is similar to type IX but has three projections extending from the triple helix. This type may also be associated with type I fibrils in tendon. Type XIV (as well as XII) is structurally related to the type IX collagen fibrils, which trim type II collagen in cartilage.

H. Bone Collagen(s) Although bone matrix proper has been reported to contain only type I collagen, other types are certainly present but not at the levels found in soft connective tissues. Several FACITs have been detected in bone [127,128], and there are occasional reports of low levels of type III and type V [129,130] molecules as well. The FACIT collagens found associated with type I collagen in other tissues are types XII and XIV. Given the potential role of these low abundance collagens in regulating fibril diameter, it is possible that the collagen fibrils in bone grow to much larger diameters than in soft tissues due to the reduced proportion of these diameter-regulating types. However, it may be that these other collagen forms may originate from the vasculature that infiltrates bone to a great extent and may persist in the bone during chemical extraction prior to demineralization. Based on sequence analysis [131], type XII collagen is predicted to be a Mr 340,000 protein with repeating

domains consisting of regions homologous to the type III motif of fibronectin and a von Willebrand factor domain [132]. In addition, there is a type IX collagen-like domain, and several RGD cell-binding domains [132]. The presence of collagen, cell binding, and matrix-binding domains implies that these FACIT collagens may have novel functions. Type XIV collagen’s sequence indicates that it has a similar series of fibronectin and von Willebrand factor domains [132]. Type XII collagen is a homotrimer with two triple helical domains and a large (Mr 190,000) N-terminal globular domain. The helical domains appear to form arms stretching out from the globular domain [129], resulting in a cruciform appearance [133] with one thin and three thick arms. Like type IX collagen, type XII collagen made by fibroblasts, but not by all cells, contains CS  chains [133]. In bone, type XII collagen is seen in the periosteum [134] and is made by periosteal cells in culture [135]. Type XII-like collagens also trim the surface of type I fibrils [136], as does a type XIV variant [137]. The FACIT collagens seem to have a fundamental role in determining matrix structure, as demonstrated by animals lacking or containing mutated forms of the FACIT collagens [138]. These animals exhibit a spectrum of bone and cartilage disorders, presumably due to abnormal fibril formation. In bone, the proteoglycan decorin, as well as types XII and XIV collagens, may be important for regulating in vitro and perhaps in situ type I collagen fibrillogenesis. The observation that a type XII molecule trims the fibrils at specific sites, while proteoglycans are found at other sites along the collagen fibrils, supports this view. This will be discussed in more detail later. Type I collagen is found not only in bone, tendon, and dentin, but also in the sclera of the eyes, the lungs, the cornea, and the skin. These type I collagens are not identical but differ in the extent of all the posttranslational modifications [102]. The predominant form of glycosylation in bone is galactosyl-hydroxylylysine, whereas in soft tissue it is glucosyl-galactosyl hydroxylysine [139,140]. In bone, the cross-linking pattern is also different and originates from the hydroxyallysine pathway, resulting in the formation of a lysyl-pyridinoline cross-link, as opposed to soft connective tissue where cross-links originate predominantly from the allysine pathway leading to hydroyxlysylpyridinoline [141]. This modification is now the basis of a clinical assay that can measure specifically the degradation of type I collagen from bone [126]. The altered crosslinking pattern in bone has been speculated to be due to the deposition of mineral within collagen fibrils [101]. These altered cross-links may be important for the mechanical properties of the tissue. In all connective tissues, the collagens serve mechanical functions, providing elasticity and structure for the component tissues. Although type I collagen is widely distributed,

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the tissue most affected when type I collagen metabolism is abnormal is bone. In bone, extensive data indicate a link between type 1 collagen and bone strength [142]. Insight into these functions comes from detailed analysis of human and other animal tissues in which collagen synthesis is altered. Classic examples are the various forms of osteogenesis imperfecta (brittle bone disease) in which bone fragility has been associated with spontaneous mutations in the type I collagen genes [99,143 – 147] and the Mov-13 mouse in which a viral insertion within the first intron totally silences the 1(I) gene [148]. The remaining 2(I) chains are unable to form a stable triple helical molecule [although 1(I) can form homotrimers]. These mice die during gestation, indicating a critical role for type I collagen during development [149,150]. Transgenic mice in which other mutations are introduced [151,152] or in which the entire 2(I) chain is ablated [153] provide animal models for various types of osteogenesis imperfecta (OI) [154]. The bone changes in such animal models of OI show features similar to those seen in humans. Specifically, when glycine is substituted by aspartate or cysteine at positions close to the C terminus, the disease mimics the perinatal lethal phenotype [155]. Similar substitutions midmolecule generally result in a less severe phenotype [145,146,154] characterized by the presence of thinner than normal collagen fibrils and active osteoblasts containing numerous collagen fibrils. When the entire 2(I) chain is absent, animals that make an 1(I) trimer show even fewer bone abnormalities [153], similar to reported cases in humans [156]. However, there are many exceptions to this general rule of mutation location within the molecule as being predictive of severity of the phenotype. The mineral crystals in the bones of patients and transgenic animals with OI tend to be smaller than those in agematched control bones [157,158]. In the OI mouse (oim) that lacks the 2(I) chain [153], tendon [159] and bone 160 mineralization is aberrant. In the oim tendon, the crystals occasionally appear outside the collagen matrix, a feature never noted when collagen production is normal [161]. Similarly, in oim bones, the pattern of initial mineral deposition and crystal growth along the collagen differs from normal: the crystals appear outside the collagen matrix and with regions of collagen that are less mineralized than those in normal controls [158]. In addition, the presence of thinner fibrils in OI patients may be insufficient to provide nucleation and scaffolding sites, which can potentially translate into fragile bones [162]. It is not known at this time whether mineral seen away from the collagen fibrils was formed in the absence of a collagen backbone or whether it “broke away’’ and was later seen out in the matrix because the collagen structure was not sufficient to support it. Collagen per se does not cause mineral deposition; i.e., it is not a mineral nucleator, as it lacks the appropriate conformation that matches the ion surface of the deposited mineral surface [163]. Nonetheless, data from OI tissues clearly

demonstrate the importance of collagen for providing a scaffold to organize the mineral. As discussed later, noncollagenous matrix proteins appear to initiate and regulate mineral deposition [reviewed in 164,165]. Thus, an additional function of type I collagen is to provide a site for the retention of noncollagenous proteins, some of which appear to be covalently bound [166] whereas others are more loosely associated through specific collagen-binding domains. In patients with OI, decreases in some matrix proteins [167,168] may be due to the deficit of collagen to which they bind or to absolute decreases in protein production [169,170] associated with “protein suicide” resulting from the destruction of abnormal collagen within the cell [99]. Decreased type I collagen production as seen in these OI models demonstrates the template-like and mechanical functions of collagen. It is likely that animals null for 1(I) are not compatible with life because of the role type I collagen plays in the development of lung, blood vessels, and mesenchyme. However, overexpression of type I collagen also provides insight into collagen function. While finding an increase in type I collagen production in bone is relatively rare, it has been reported that MAV.2-0 (myeloblastosis-associated retrovirus) causes an osteoblastic hyperplasia and a relative increase in cortical bone thickness [171], providing another animal model for the characterization of collagen function.

III. GLYCOSAMINOGLYCANCONTAINING PROTEINS Proteoglycans are a class of macromolecules characterized by the covalent attachment of long  chains of repeating disaccharides that are often sulfated, termed glycosaminoglycans (GAGs). The different types of GAGs, chondroitin sulfate (CS), dermatan sulfate (DS), keratan sulfate (KS), heparan sulfate (HS), and hyaluronan (HA, which is unsulfated), are named based on the sugar composition of the repeating disaccharides (Fig. 6). GAGs, with the exception of HA, are synthesized in the Golgi apparatus by the sequential addition of sugar residues to a sugar transporter molecule, dolichol phosphate. Certain sugar residues become sulfated through the action of sulfotransferases; however, the level of sulfation can be extremely variable even within one GAG chain. Ultimately, the growing GAG is transferred from the dolichol phosphate to an acceptor site on a protein core. These sites are most frequently specific seryl or threonyl residues, but in the case of KS, the acceptor site is an asparaginyl residue. There are many different families of core proteins but the factors that dictate which residues become gagosylated and by what type of GAG are not well understood. In contrast to the other GAG chains, HA has not been found covalently attached to a core

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FIGURE 6 Disaccharide composition of glycosaminoglycans (GAGs). The GAG side chains that are covalently attached to proteoglycan core proteins are composed of repeating disaccharide units. The composition of the disaccharides, along with modifications by acetylation, results in the formation of chondroitin sulfate, which is epimerized to form dermatan sulfate, heparan sulfate, and keratan sulfate. Hyaluronan is the sole GAG that remains unsulfated and is not covalently linked to core proteins.

protein. HA does associate with certain cartilage proteoglycans by a noncovalent linkage. Proteoglycans can be glycosylated by the addition of N- and O-linked oligosaccharides. In addition to sulfation of the GAG  chains, the oligosaccharides can also be phosphorylated and/or sulfated, generating a class of macromolecules with multiple posttranslational modifications.

A. Aggrecan During endochondral bone formation, cartilage first hypertrophies and forms a temporary mineralized tissue, calcified cartilage, which is then invaded by blood vessels. The invading vasculature brings with it (1) cells that destroy the calcified matrix (osteoclasts or a related cell type) and (2) osteoprogenitor cells. Because cartilage macromolecules can be in close proximity to forming bone and may actually be incorporated into the initial bony tissue, it is im-

portant to describe its extracellular matrix constituents. It is also not clear whether all of the large CS-proteoglycans isolated from bones are remnants of those in calcified cartilage or specific bone products. The presence of elevated amounts of CS-proteoglycans in the bones of osteopetrotic animals with defective osteoclasts was linked to the inability of these animals to resorb calcified cartilage [172]. The basic scaffolding upon which cartilage matrix is built is type II collagen. In addition, a number of proteoglycans, including (i) a large CS molecule, termed aggrecan reviewed in 173, have the ability to form aggregates with HA and (ii) two small proteoglycans, decorin and biglycan [reviewed in 174 – 177]. Other proteins, including COMP, CD-RAP, chondroadherin, and matrilin-1, are present, but at lower levels [178,179]. Intact aggrecan has a Mr 2.5 million kDa, with a core protein ranging in apparent Mr between 180,000 and 370,000 with just over 100 GAG chains (mostly CS, but with some KS) of about Mr 25,000 (Fig. 7). Based on enzymatic cleavage and sequence homology, five

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FIGURE 7

A representation of the chemical features of the large hyaluronic acid-binding proteoglycan, aggrecan. GAG, glycosaminoglycan; CS, chondroitin sulfate; KS, keratan sulfate; G1, G2, G3, globular domains (see text for description); EGF, epidermal growth factor; CRP, C-reactive protein.

domains have been defined in the core protein: three globular domains, two of which bind to HA (G1 and G2), an interglobular domain, a domain rich in serine-glycine repeats to which the CS and KS glycosaminoglycan  chains are attached, and the third globular domain, G3, at the C terminus. Each of the globular domains are cysteine rich and stabilized by disulfide bonds [180]. The G1 domain in the N terminus is structurally homologous [181] to “link protein” [reviewed in 182], which stabilizes the interaction between the proteoglycan and HA in cartilage [183]. The adjacent G2 domain provides a flexible hinge, whereas the more linear central domain is the site of KS and CS covalent attachment. The C-terminal G3 domain is also predicted from sequence analysis to be globular and contains a set of epidermal growth factor (EGF)-like and complement regulatory protein (CRP)-like sequences at the carboxy terminus [184,185]. The individual GAG chains form extended flexible structures, whereas the serines in the central domains have -D-xylose attachments with restricted orientation [186]. Whereas NMR analysis of isolated CS chains has provided insight into their conformation in different solutions [187], their actual conformation when attached to the core protein is not known. The human aggrecan gene is located on chromosome 15q26 [188]. However, the complete genomic sequence has been reported only in the rat and is 63 kb in length. There are 18 exons with 30 kb of intronic sequence separating the first and the second exon. The intermediate exons roughly encode for each of the structural domains of the molecule with the exception of G1-B (which contains the hyaluronicbinding domain) and G2-B (which contains link protein-

like sequences), which are split between two exons, and G3, the lectin-binding domain, which is coded for by three exons. The splice junctions are primarily symmetrical phase 1 type (in frame) [189,190]. The rat gene promoter lacks a TATA box, and the major transcription start site is located in close proximity with a number of SP1 sites. In addition, there are four AP2 sites located 120 kb upstream in a GC-rich region, and two of the SP1 sites overlap. A GC-rich region is also found in the first exon, which also has four AP2 sites. A potential NF-b (nuclear factor) site is located between 123 and 103 bp, and another AP2 site is located between 81 and 37 bp [190]. The resulting mRNA species of 8.2 and 8.9 kb predict a 2124 amino acid residue protein, including a 19 residue signal peptide. A stretch of 1164 residues contain Ser-Gly repeats, the CS attachment site [189,190]. Structures of the core proteins, individual domains, and segments of these domains have been determined based on NMR and molecular modeling [186,191] and neutron and X-ray scattering of molecules in solution [192]. These studies reveal that the core protein has a fairly linear structure. The protein core of the aggrecan-like proteoglycans (CS/KS-containing) is fairly homologous in a wide variety of tissues, ranging from tadpole tails to human articular cartilage [118]. The structures of isolated individual large aggregating proteoglycans (aggrecan) from hyaline cartilage and the aggregates which they form have been visualized at the electron microscopic (EM) level by rotary shadowing [193,194]. While EM studies of the cartilage molecule support a bottle brush-like conformation predicted earlier from physical and chemical analysis [183],

120 NMR and light-scattering studies of these molecules in solution indicate that they have a more compact form [191,192]. Whether the aggrecan found in bone is there as a bone product and not as a cartilage remnant has yet to be determined. The function of these large proteoglycans in bone is also unknown. In other tissues, the CS-proteoglycans serve a hydrodynamic function, aiding in the retention of both water and cations, and the exclusion of anions [195]. The large proteoglycans form gels, which can both swell and retain water, contributing to the osmotic pressure of the tissue [196]. These proteoglycans in cartilage are also responsible for matrix organization [185], in part through interactions with the glycosaminoglycan chain of type IX collagen that trims the type II collagen fibrils [197]. Each of these properties, as reviewed elsewhere [198], depend on the arrangement, size, and number of the constituent CS chains. In cartilage, proteoglycans are believed to play a major role in the regulation of calcification [for review see 199]. The large aggregating cartilage proteoglycans, in solution can inhibit hydroxyapatite formation and growth [200 – 203]. They can also chelate calcium [204] and serve as a source of calcium ions for mineralization if they are degraded into non-Ca2-binding fragments. Although there is some debate as to whether this chelation is involved in the inhibition of mineralization, it is clear that proteoglycans and their component GAGs stearically block hydroxyapatite formation and growth [201]. It has also been shown that when proteoglycans are degraded, their inhibitory ability is lost or diminished both in vivo [205] and in vitro [206]. However, because the relative amount of large CS-proteoglycans in bone is low, it seems unlikely that they have a critical role in preventing osteoid mineralization. Unfortunately, this cannot be proven based on existing data because none of the mutant animals in which the large CS-proteoglycan is altered in cartilage were reported to show bone abnormalities. This is the case for the cartilage matrix deficiency (cmd) mouse, which lacks a core protein [207], and the brachymorphic mouse (bm), which has a deficiency of the enzyme required for the sulfation of glycosaminoglycan side chains [208]. Only in the case of the bm was the mineral examined, and no abnormalities in the bone were detected by X-ray diffraction, infrared, or EM, although cartilage calcification was altered [208]. In the nanomelic chick, which contains a mutated core protein [209], studies show that although there is no overt bone defect, certain bones are abnormally shaped, perhaps due to the altered distribution of load applied to cartilage-deficient tissues during development. The likely functions for the large CS proteoglycans in cartilage are maintenance of tissue hydration and the regulation of mechanical [210] and hormonal signal transduction. However, it is not known if aggrecan or a related molecule provides these functions in bone.

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B. Versican Another large CS proteoglycan related to aggrecan is found in cartilage at low levels. It has been termed versican, as it is found in variable forms in a large number of extracellular matrices. The protein core, with a Mr 360,000, has a structure similar to that of aggrecan with the exception that it lacks the G2 domain. In addition, versican contains only 12 – 15 CS side chains (Mr 45,000) in contrast to 100 in aggrecan [184] (Fig. 8). The distribution of versicans is somewhat ubiquitous as it is found in smooth muscle and aorta [211], glomeruli [212], brain [213,214], and epidermis [215]. A versican-like molecule is highly enriched in developing mesenchyme destined to become bone and may serve to capture space [216]. Versicans from soft connective tissues are capable of aggregating with HA [185]. In addition, it is now apparent that PG-M, identified as a constituent of the matrix surrounding condensing limb bud mesenchyme, is a splice variant of versican [217]. However, versican isolated from bone cell cultures apparently does not aggregate with HA (Fedarko and Gehron Robey, unpublished results). The versican gene localizes to human chromosome 5q12 – q14 [218,219]. The human gene has been isolated [220], is over 90 kb in length, and is composed of 15 exons with a splice variant that utilizes an additional exon [221]. The sequence predicts a 20 residue signal peptide and a 2389 residue mature protein [222]. Exon 1 codes for the signal peptide and a few amino acids found in the mature molecule, and the HA-binding region (G1) is in exons 3 – 6. These exons share homology with the other HA-binding protein, the link protein. This region also contains an Ig-like protein conformation whose function is unknown. Subsequently, exons 6 and 7 are differentially utilized and contain GAG attachment sites. The carboxy-terminal domain (G3), which contains homology to selectins, EGF, and CRP, is contained within exons 9 – 14. The 3-untranslated region contains three different potential polyadenylation sites [220]. The promoter region has a TATA box located 16 bp upstream from the transcription start site. Transfection analysis indicates that there is a positive enhancer between 209 and 445 bp and a negative element between 445 and 632. Other elements present include an XRE (xenobiotic responsive element that may downregulate P450 levels), SP1-binding sites, CRE (cyclic AMP responsive element), and a CCAAT transcription factor-binding site. Based on differential splicing and polyadenylation, three mRNA species of 10, 9, and 8 kb are produced [217]. Versican expression in other tissues can be modulated by a number of factors, including transforming growth factor (TGF-) [223,224], PDGF [223], and interleukin-1 [225]. There is little information on the modulation of its expression during bone development, but it appears to be

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FIGURE 8

A representation of the chemical features of the widely distributed proteoglycan that is related to, but not identical with, aggrecan. CS, chondroitin sulfate; G1 and G3, globular domains (see text for description); EGF, epidermal growth factor; CRP, C-reactive protein.

upregulated by TGF- in adult human bone cells and fetal bovine long bone cells (Heegaard and Gehron Robey, unpublished data). A potential function for this molecule is to serve as a bridge between the extracellular environment and the cell by binding to HA via the amino-terminal-binding region and to molecules that have yet to be identified on the cell via the carboxy-terminal domain [184]. Versican may also be involved in cell motility and growth [226]. Proteoglycans are known to interact with growth factors, providing a storage site, or facilitating interactions between the extracellular matrix and the cell. Some structural elements within the molecule may also function directly as signals for cellular growth or differentiation [227]. For example, versican contains EGF-like sequences such that upon its destruction, it may serve to stimulate proliferation of osteoprogenitors. EGF has been reported to stimulate proliferation of osteoblastic cells in vitro [228].

C. Small Leucine-Rich Repeat Progteoglycans Another family of proteoglycans is represented by a group whose protein core is characterized by a smaller size and a leucine-rich repeat sequence that is approximately 24 amino acids in length [reviewed in 176,229]. These small proteoglycans are broadly distributed and are found in the

extracellular matrices of many tissues [230]. In cartilage and bone, there are several members of this family, including decorin and biglycan. While they are highly homologous, they exhibit distinctly different patterns of expression and tissue localization, indicative of divergent functions within these tissues. It has been reported that proteoglycans must be removed prior to matrix mineralization and that they inhibit hydroxyapatite crystal growth in test tube experiments, although these two statements do not really comment on the role of a particular species of proteoglycan during matrix mineralization in vivo. It is apparent that there is a net loss of sulfate during matrix mineralization [231,232]. However, using animals and organ cultures, it has been found that a species of sulfate-labeled material accumulates rapidly at the mineralization front [233,234]. One possibility is that the versicanlike molecule is being removed and replaced by the smaller proteoglycans found in bone [235]. Considering the fact that versican has more GAGs per protein core than the small proteoglycans, this would account for the net decrease in sulfate content; however, these data do not rule out the presence of small proteoglycans during matrix mineralization. The leucine-rich core proteins of decorin and biglycan are quite similar in sequence to that of the ribonuclease inhibitor protein. Their structure (in the absence of Ca2 ions) has been determined using X-ray crystallography to 2.3 Å resolution. The ribonuclease inhibitor protein structure

122 consists of 15 leucine-rich repeats, alternatively 28 and 29 residues long, each forming right-handed -- structural motifs resulting in a horseshoe shape as opposed to a globular structure, with the parallel  sheets in the horseshoe circumference [236]. The -- motif is frequently seen in proteins as a way of connecting two antiparallel  strands; however, it can also be used to connect parallel  strands, resulting in a more open structure [237]. This interesting sequence has also been identified in two morphogenetic proteins in Drosophila, chaoptin and toll, in von Willebrand factor-binding protein (GP 1) and in the leucine-rich protein in plasma [238], and in a number of other proteoglycans, which have formed a family, SLRPs, some of which are also found in bone. 1. DECORIN (PG-II, PG-40) Decorin, so named for its ability to bind to and decorate collagen fibrils [118,119,239,240], has also been termed PG-II and PG-40. In soft connective tissues and bone cell cultures, decorin (and biglycan) has DS side chains, whereas in bone, decorin (and biglycan) bears CS [reviewed in 176,241,242]. This difference is of interest because DS is formed from CS by the action of a specific epimerase, and this epimerization can occur uniformly or focally within the GAG chain. The factors that regulate whether a proteoglycan will bear CS as opposed to DS are not known. As one would expect from its proposed function of binding to collagen fibrils, decorin is fairly widely distributed and is found virtually coincident with type I collagen, although the timing of its appearance may be somewhat different. Histochemical studies show the presence of proteoglycans with low molecular weight in the d and e bands of type I collagen fibrils, which disappeared when mineralization occurred [116,117,239]. These histochemical data first suggested that a small proteoglycan might also play a role in mineralization. In the developing skeleton, it decorin is also distributed more uniformly than in the mature animal. In cartilage, decorin is present in very low levels and is restricted to the interterritorial matrix [243]. As bone is formed, it is produced by preosteoblasts and osteoblasts, but its synthesis is not maintained by osteocytes [243]. Nonglycanated forms of decorin (and biglycan) have been found in the intervertebral disk [244]. Decorin has a core protein of approximately Mr 38,000, which includes 10 of the leucine-rich repeat sequences. Although there are three potential GAG attachment sites, only one is utilized with the attachment of a single GAG chain of Mr 40,000, resulting in a molecule with an apparent Mr 130,000 as determined by sodium dodecyl sulfate –polyacrylamide gel electrophoresis (SDS-PAGE) [245] (Fig. 9). The decorin molecule was predicted to contain loops of  strands [246]. It has been demonstrated by far-UV CD spectroscopy that both decorin and biglycan exist as predominantly

GOKHALE, BOSKEY, AND ROBEY

 sheets, and biglycan has a significantly higher  helical structure and assumes different structures in the solution 247. The human gene for decorin has been localized to 12q23 [248 – 250]. In mouse, it is located on chromosome 10, just proximal to the steel locus [251]. A restriction fragment length polymorphism (RFLP ) is also present in humans [252]. The gene is over 38 kb in length and contains 9 exons. The gene shares 55% homology to and is organized in a similar fashion to the biglycan gene (described later) except that the intronic sequences are much longer (two of which are 5.4 kb and greater than 13.2 kb) [249]. The leucine-rich repeat sequences are not organized specifically within the exons. Although the 5 end of the gene eluded characterization for some time, it is now evident that there are two alternatively spliced leader exons (exons 1a and 1b) [249,250]. Exon 1a contains two GC-rich sequences, whereas 1b contains two TATA boxes and one CAAT box that are in close proximity to the transcriptional start site [253]. In addition, AP1, AP5, and NF-b sites were also identified along with a homopurine/homopyrimidine mirror repeat sequence. This region has been postulated to take on a hairpin triplex structure that is unique to decorin compared to the promoters of the other SLRPs. Gene transcription results in a major mRNA species of 1.6 kb and a minor species of 1.9 kb [254,255]. The sequence predicts a 359 residue protein that includes a 30 residue prepro peptide. The synthesis of decorin can be modulated by TGF- [256 – 258]. Dexamethasone upregulates decorin, but downregulates biglycan [259]. The phytoestrogen ipriflavonemetabolite III has been shown to upregulate decorin [260]. Mechanical loading appears to stimulate the synthesis of decorin, but not of biglycan in cartilage [261]. While it would appear that the propeptide is cleaved from the mature decorin in bone, evidence shows that it is maintained in other tissues such as cartilage [262]. Decorin has been shown to bind to and regulate the fibrillogenesis of type I, type II, and type VI collagens [263,264]. It also has a high affinity for thrombospondin [265], TGF- [266], other growth factors, and fibronectin [267,268]. In bone, the proposed functions of decorin are the regulation of collagen fibril diameter and fibril orientation, and possibly the prevention of premature osteoid calcification. However, it is not clear if decorin within the tissue is actually inhibitory to matrix mineralization. Decorin isolated from skin was initially shown to regulate the rate of collagen fibrillogenesis in vitro [269]. Whether bone decorin has the same effect has not yet been demonstrated. Interestingly, bone decorin must be chemically different from tendon decorin, as a different peptide map can be generated by V8 protease [270]. Bone collagen fibrils are a composite of type I with trace amounts of types III, V, and XI and are also trimmed by decorin. Therefore, it is difficult to determine which of these are essential to the

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FIGURE 9 The two most abundant proteoglycans present in bone matrix are the small chondroitin sulfate/dermatan sulfate proteoglycans, decorin and biglycan. The core protein of each is highly homologous to a number of proteins due to the presence of a leucine-rich repeat sequence. CS, chondroitin sulfate; DS, dermatan sulfate; C – C, disulfide bonding.

regulation of collagen fibrillogenesis and maturation in this tissue. Decorin has been reported to bind with high avidity to type VI collagen [264] and hence may be more concentrated in areas where type VI collagen is more abundant, e.g., in the upper zones of epiphyseal growth plate [271]. Decorin, in solution, has a high affinity for type I collagen (Kd  M10) [272] and, in contrast, has a low affinity for hydroxyapatite (Kd  13 g/ mol). When bound to a solid surface, decorin binds rather nonspecifically to apatite relative to other GAG-containing molecules (N, the number of binding sites  333) [273]. Further, it has no detectable direct effect on solution-mediated hydroxyapatite formation or growth [273]. With a low affinity for Ca2 (0.001 g/mg) [267] and a higher affinity for other divalent cations [275], the disappearance of decorin from the collagen fibrils in bone [117] indicates that it is unlikely that it has a direct role in mineralization. However, once removed, other possible nucleators may be exposed, which in turn would facilitate mineralization. Whether this removal also

affects collagen cross-linking and fibril spacing to facilitate mineralization, as well as unmasking nucleators localized under the decorin, remains to be determined, as does the mechanism responsible for the removal of decorin. It is interesting to note that collagen fibril diameter in bone is somewhat reduced in the decorin knockout mouse (M. Young, personal communication), and decorin expression is reduced in certain skin diseases characterized by excessive keratinization [276], stressing the physiologic importance of decorin in regulating fibril formation and collagen – matrix interactions. It is also possible that a key function of decorin is to serve as a binding site for growth factors in these tissues [277]. This may be relevant in the keratinization diseases, in the kidney [276], and in cartilage repair, as well as in the bone where there are other binding sites for these growth factors (see later). It is also of interest to note that there is a decreased expression of decorin in some patients, with OI [169,170,278]. Decorin has also been proposed to play a role in matrix organization by

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FIGURE 10

Biglycan immunolocalization in newly formed trabecular bone. Biglycan was found predominantly in osteoid (Ost) and, to a lesser extent, in mineralized matrix (MM) in human bone. Osteocytic lacunae (OCy) also contain high levels of biglycan. Courtesy of Dr. Paolo Bianco.

binding to other matrix proteins, such as fibronectin [267,268]. Interestingly, it has been found that decorin (and biglycan) inhibits the attachment of bone cells to fibronectin, perhaps by blocking the fibronectin cell attachment site that mediates binding. Decorin also binds to thrombospondin, another matrix protein that is speculated to serve as a matrix organizer. It has been reported that decorin binds to TGF- and blocks its activity in fibroblasts [266]; however, it may also activate TGF- in bone cells [279]. 2. BIGLYCAN (PG-I, PG-SI) Biglycan, also known as PG-I and PG-S, is highly related to decorin and exhibits 55% homology at the protein level [238,280]. Despite its high homology to decorin, it is evident that biglycan is uniquely distributed and present in a temporal and spatial pattern not coincident with that of decorin, indicative of a distinctly different function. Biglycan has been localized to the pericellular environment of endothelial, epithelial, and muscle cells [243]. However, in skeletal tissues, biglycan does not appear to be associated with the collagen fibrils, although the biglycan mouse has highly abnormal collagen fibrils in boneskin (M. Young, personal communication). In contrast, it is more abundant in the growth plate [243] and is concentrated in the intraterritorial matrix and in preosteogenic cells, implying a role in early bone development. It is highly upregulated in differentiating bone cell cultures [281] and, interestingly, it is maintained (at the mRNA and protein levels) in osteocytic lacunae and in the canaliculae, which contain

the osteocytic cell processes (Fig. 10). This implies that biglycan may be important in osteocytic cell metabolism. In mineralizing osteoblast cultures, biglycan expression begins during the preosteoblastic phase of the culture and is commensurate with calcium uptake in contrast to decorin, which increases gradually and remains high as mineralization progressed [282]. Biglycan has a protein core of Mr 37,000 to which (in most forms) two GAG side chains are attached. The aminoterminal domain contains the GAG attachment sites, followed by 12 of the leucine-rich repeat sequences (Fig. 9). The first and the last repeat contain a characteristic pattern of cysteinyl residues that result in a particular pattern of intramolecular disulfide bonding [238,283]. The carboxy domain has a sequence that is unique to biglycan (and differs from decorin and other leucine-rich repeat sequence containing proteins). Biglycan has the propensity to self-aggregate in solution [284], although the physiological relevance of these aggregated structures is not known. It has also been determined that nonglycanated biglycan can be secreted into the matrix and that the level of the core protein alone increases with age in human articular cartilage [174]. Other than predicted structures from the amino acid sequences of the biglycan core protein [238], little is known about the tertiary and quaternary structure of biglycan. The gene for the biglycan core protein is localized to Xq27-ter in humans, the only matrix protein that is not on an autosomal chromosome [285]. The gene is 7 kb in size and codes for a 368 residue proform that is processed to form a

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mature core with 331 residues [238,285]. There are eight exons with relatively small introns compared to those in decorin. Exon 2 codes for the signal peptide, propeptide, and GAG attachment sites. The bulk of the core, the leucine-rich repeat sequences, are not obviously distributed between the remaining exons. The 3 portion of the gene is rich in CT and CA repeats [286] and may result in the Z DNA conformation. The promoter does not contain a TATA box or a CCAAT box, but has a number of cis-acting elements, including SP1, AP1, AP2, NF1, and NF-b-binding sites [285,287]. A number of in vitro transfection experiments have shown that the SP1 site is active, but that it may be binding to a factor other than those described previously [287]. The final mRNA species are 2.1 and 2.6 kb in size. A number of factors have been reported to regulate biglycan synthesis in bone cell cultures and often the pattern is distinctly different from those affecting decorin. TGF- increases biglycan (Gehron Robey, unpublished results) in normal human bone cells and in MC3T3 [258]. Other cell lines were not stimulated by TGF-, but IGF-I and IGF-II increased biglycan levels [288]. Retinoic acid suppresses biglycan in chondrocytes [289]. Dexamethasone and 1,25-dihydroxyvitamin D3 have been reported to decrease its expression in human bone and marrow cell cultures [290,291]. Fluoride, given to cultures of rat osteoblasts in clinically relevant concentrations, decreased GAG chain length and composition [292]. Biglycan (and decorin) binds to type V collagen [293], a collagen abundant in blood vessels. They both also inhibit the activity of antithrombin by interacting with heparin cofactor II. Thus, it has been suggested that one of the functions of biglycan is to create a thromboresistant vascular surface [293]. Whether this is the same for the chondroitin sulfate containing biglycan of bone remains to be determined. In solution, biglycan binds only low amounts of calcium (0.012 mM/ g) but appreciable amounts of small divalent cations such as zinc [294]. Its in vitro interaction with type I collagen can be blocked by increasing phosphate concentrations, implying that biglycan, as distinct from decorin, which is not affected by solution phosphate, interacts through its anionic residues [295]. Also, in solution, biglycan at low concentrations can promote apatite formation, whereas at higher concentrations it inhibits the growth and proliferation of mineral crystals [273]. These effects appear to be due to the highly specific high-affinity binding of biglycan for apatite (Kd 294 g/ mol). The relative extent of apatite formation induced by biglycan compared to other mineral nucleators and its absence from bone collagen fibrils suggest that its primary function may not be related to mineralization of bone. In calcifying cartilage, however, biglycan may play some role in the mineralization process. Biglycan appears to have a regulatory role in bone development. This suggestion is based on the observation that

patients with Turner’s syndrome (genotype 45, XO; i.e., females that are missing one or part of one X chromosome) have decreased biglycan levels, short stature, and other skeletal deformities, including early onset osteoporosis [296]. The biglycan knockout mouse also has a short stature, altered mechanical properties, and altered mineral distribution 297. In contrast, there is overexpression of biglycan in patients with Klinefelter’s disease (genotype 47, XXX) in which patients are excessively tall [296]. Biglycan, like decorin, also binds to a variety of growth factors in solution [266]; thus, it may have functions similar to those of decorin regarding the storage of these growth factors. Biglycan interacts with a number of extracellular components, including the cell-binding domain of fibronectin (thereby inhibiting attachment mediated by fibronectin) [268,298 – 300], TGF- [266], which may alter its activity, and heparin cofactor II [293]. Nonglycanated biglycan has been found to bind to colony-stimulating factor (CSF), which then stimulates the proliferation of nonadherent thymic and peritoneal exudate cells [301]. 3. FIBROMODULIN Fibromodulin is another SLRP that contains keratan sulfate found predominantly in articular cartilage, but also exists in bone [302,303]. The amount of fibromodulin correlates with the size of collagen fibrils in cartilage [304]. In developing bone induced by demineralized bone matrix, fibromodulin is heavily localized to fibrillar bundles [305]. Observations from the fibromodulin knockout mouse have indicated that in the absence of functional fibromodulin, collagen fibrils in tail tendon are abnormal. In these mice, fibrils are significantly thinner, indicating a role for fibromodulin in collagen fibrillogenesis 306. Similarly, a decrease in fibromodulin content and a alteration in structure have been shown to correlate with the aging-related degeneration of vertebral disks 307. The intact protein is approximately 59 kDa, and the protein core shares a great deal of homology with decorin and biglycan, but bears keratan sulfate GAG chains linked to asparaginyl residues rather than chondroitin or dermatan sulfate linked to seryl/threonyl residues [308]. There is also a considerable amount of tyrosine sulfation at the aminoterminal portion of the molecule. The human gene, which is at least 8.5 kb, has been isolated and partially characterized. It has an intron – exon organization, which differs markedly from that of decorin and biglycan. The first exon contains the untranslated region, and most of the coding sequence is located in the second exon, with the remainder residing in the third exon [308]. Fibromodulin interacts with triple helical types I and II collagen [309] and is capable of binding TGF- [310]. Decorin and fibromodulin are the most active collagenbinding proteins, which bind to distinctly different regions on collagen fibrils [309].

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D. Heparan Sulfate Proteoglycans Although heparan sulfate proteoglycans are not usually found within the extracellular matrix of bone, they are cell surface associated, either by phosphoinositol linkages that are cleavable by phospholipase C or by transmembrane insertions. There are at least two heparan sulfate proteoglycans present on cells in the osteoblastic lineage, and most likely there are more. The most predominant type is similar to the syndecan family, first identified on mammary epithelial cells [311]. The intact syndecan molecule is Mr 400,000 and contains a core protein of Mr 80,000 and several heparan sulfate side chains of Mr 60,000 [312]. Glycan, a helper receptor for the type I and type II TGF- receptors, has also been identified in most connective tissue cells [313,314]. It has also been determined that bFGF binding to its receptor is facilitated by heparan sulfate on the cell surface [315], although the identity of this molecule has not been determined in bone cells.

E. Other SRLPs, Proteoglycans, and LeucineRich Repeat Proteins The structures and functions of the less abundant proteoglycans have been reviewed by Hardingham and Fosang [316]. In addition to decorin and biglycan, which are class 1 SRLPs [229], a third small proteoglycan whose core protein structure has not been described is the hydroxyapatite (HA)-binding proteoglycan, HA-PGIII, isolated from porcine bone by Nagata et al. [317]. An analogous hydroxyapatite binding protein from bovine bone has been isolated by Hashimoto et al. [318] and was shown to be covalently cross-linked to type I collagen. The localization of this protein in bone has not been described nor are there data on its function, its ability to regulate fibrillogenesis, or bind growth factors. A protein that contains the leucine-rich repeat sequence was originally identified as osteoinductive factor (OIF) [319,320]. However, the osteoinduction component of this preparation is in fact TGF-, and the other protein has now been renamed osteoglycin. It is not known whether this protein exists as a proteoglycan. However, it is of interest that all of the three proteins found in bone with the leucinerich repeat sequences have the ability to bind to TGF-. Another proteoglycan, PG-Lb, also known as epiphycan, has been isolated from calcifying cartilage and is very homologous to osteoglycin [321 – 324]. PG-100 was found in fibroblasts and preosteoblasts [325], and HA-PGII has been found to be associated with forming mineral crystals [317]. Given the low abundance and the variability of expression between one animal species and another, it is not clear what role they play in bone formation and/or mineralization. Until details of the structures and distributions of these

proteins in bone are determined, it is difficult to speculate on their functions. However, by analogy with the other small proteoglycans, it is likely that they will play some role in regulating matrix structure, collagen fibril diameter, and interaction with other matrix molecules.

F. Hyaluronan Hyaluronan is a ubiquitous component of connective tissue matrices [326] consisting of repeating sequences of glucuronic acid and N-acetylglucosamine linked by 1-3 and 1-4 glycosidic linkages. Chains may be several thousand residues in length, placing hyaluronan among the highest molecular weight glycosaminoglycans. Comper and Laurent [195] described the structure of hyaluronan based on models of fluid flow in the 1970s, showing that the hyaluronan formed space-filling gels. In mature bone, hyaluronan is only a small percentage ( 5% on a weight basis) of the total glycosaminoglycans present [327]. The properties of hyaluronan have been reviewed by Knudson and Knudson [328]. Unlike other GAGs, the synthesis of hyaluronan occurs outside of the cell by a large complex of sugar transferases that have yet to be fully characterized. Consequently, the synthesis of hyaluronan is most likely regulated by factors that are quite distinct from those that regulate other GAGs. Hyaluronan is produced at least during early phases of osteogenesis in vitro; however, very little is known about its metabolism in bone. Large amounts are produced by bone cells in culture [329]. To date, there have been no reports on the modulation of hyaluronan production by osteoblastic cells in response to growth factors and hormones that are known to affect bone. This paucity of information stems from the fact that there is no available antibody that binds to it specifically, and because it is synthesized by a multienzyme complex, there is no single probe for measuring mRNA. However, there is an indirect histochemical assay that relies on a peroxidase-conjugated link protein that will bind to hyaluronan in a specific fashion [330], and methods for blocking hyaluronan binding to the hyaluronan receptor (CD44) are available [331]. In order to understand the role of hyaluronan in bone formation, it would be of interest to apply these techniques to bone Hyaluronan binds weakly and nonspecifically to apatite, with affinity constants varying as a function of hyaluronan chain length [332,333]. There is some discrepancy in reports of the effects of hyaluronan on apatite formation and growth. However, these discrepancies may be related to the different systems used for the studies. Thus, where only pH was kept constant, hyaluronan of molecular weight 104 was shown to have no effect on apatite growth at low Ca  P products [333]. With higher Ca  P products maintained at constant composition, hyaluronan of molecular weight 108

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inhibited apatite growth slightly [332]. This difference is not attributable to hydrodynamic or size effects, as it has been shown that smaller molecular weight hyaluronans have a similar action [334]. Because the inhibition was small relative to other macromolecules studied, one may conclude that hyaluronan in bone is not apt to have a role in the control of mineralization. The structure and regulation of hyaluronan-binding proteins have been reviewed 335. Hyaluronan forms stable complexes with the large aggregating proteoglycans (aggrecan and versican) of cartilage [336] and with collagen-binding heparan sulfates [337]. The interaction of hyaluronan with the heparan sulfate of cell membranes is believed to be one of the ties that hold cells in place in a variety of tissues and this may also be the case in bone. Hyaluronan has been reported to play a role in cell migration and cell adhesion and invasion, as well as differentiation and proliferation of connective tissue cells [338,339]. An additional function of hyaluronan is the maintenance of tissue hydration, a function that it fulfills in many connective tissues. Removal of this molecule by the enzyme hyaluronidase results in tissue desiccation and facilitates tissue mineralization in cartilage [340]. Because of its abundance in early development, hyaluronan is thought to play a role in embryogenesis. Hyaluronan has been shown to facilitate mesenchymal cell movement [341] and cell adhesion [342] and it appears to bind via CD44 as the receptor [343]. It has been suggested that a major function of hyaluronan in mesenchyme-derived tissues is to act as a space filler, holding water and thereby increasing tissue volume [344]. In chondrocyte cultures, Knudson and Toole [345] have shown that hyaluronan promotes cell proliferation, probably because it provides a space/volume where cells can be anchored and provided with nutrition. Studies also show that hyaluronan can alter the extent of collagen production and proliferation of fibroblasts in culture [346]. Hyaluronan may also function as an organizer of the bone marrow [347] and may regulate hematopoiesis that is supported by steroid [348]. Serum hyaluronan concentrations are elevated in rheumatoid arthritis, osteoarthritis, liver cirrhosis, Werner syndrome, renal failure, psoriasis, and various malignancies; consequently, it may be a useful marker for measuring disease activity [349]. It is also of interest that intercellular adhesion molecule-1 (ICAM-1), a cell surface receptor for hyaluronan found on endothelial cells, is upregulated in inflamed tissue [350]. Consequently, hyaluronan may be involved in this tissue reaction.

IV. GLYCOPROTEINS This class of proteins is characterized by the covalent linkage of sugar moieties attached via asparaginyl or seryl residues as described earlier. Collagen also contains another

form of glycosylation (galactosyl and glucosyl-galactosylhydroxylysine), which is virtually specific to collagen. In bone, the most relevant and abundant glycoproteins are represented by alkaline phosphatase, osteonectin, and the cell attachment proteins, which include, but are not limited to, sialoproteins. These glycoproteins may also be further modified by posttranslational sulfation and phosphorylation.

A. Alkaline Phosphatase Alkaline phosphatase is not typically thought of as a matrix protein. However, studies indicate that under normal conditions, alkaline phosphatase is shed by cells of the osteoblastic lineage in culture as they pass through the G2/M phase of the cell cycle [351], and a Ca2-binding glycoprotein with alkaline phosphatase activity has been isolated from mineralized matrix extracts [352]. It is possible that this protein is indeed alkaline phosphatase that has been shed from the cell surface by cleavage of its phosphoinositol type linkage with phospholipase C or in a membranebound form (matrix vesicles). Although this enzymic activity is shared by many types of tissues, there is no doubt that the induction of alkaline phosphatase activity in uncommitted progenitors marks the entry of a cell into the osteoblastic lineage and is a hallmark in bone formation. However, it should be noted that many studies utilize the enzymatic activity alone to measure the presence or absence of alkaline phosphatase. This may be somewhat misleading, as it is possible that the protein can be present but inactive or, conversely, that the protein can be partially degraded and still maintain enzymatic activity. As in all studies where the most reliable information is obtained by measuring both mRNA levels and protein levels, in studies involving alkaline phosphatase, a clearer picture of the metabolism of this protein may be best obtained by measuring the mRNA and the enzymatic activity, as well as the status of the protein (intact versus degraded). Histological localization of alkaline phosphatase through utilization of a chromophore-generating enzyme substrate marks very specific sites within bone marrow and sites of new bone formation [353 – 356] (Fig. 11). Developmental studies in vivo and in vitro have shown that expression precedes mineralization and is maintained during early stages of hydroxyapatite deposition [10 – 12,357 – 359]. Within marrow, cells that are supportive of hematopoiesis are alkaline phosphatase positive (Westen – Bainton cells) and represent, at least in part, members of the bone marrow stromal stem cell population [360]. It has been reported that these cells are related to preadipocytes. In certain pathological conditions, when hematopoiesis is halted, these cells lose their alkaline phosphatase activity and proceed to form fat cells [361]. During osteogenesis, the alkaline phosphatase reaction product very

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FIGURE 11

Alkaline phosphatase in developing bone. By histochemical staining for alkaline phosphatase activity during development, areas that are destined to become bone, as shown here in developing human subperiosteal bone, can be clearly illustrated. The fibrous layer (F) of the periosteum is negative, whereas preosteoblasts (POb) and osteoblasts (Ob) produce high levels of activity. Although a glycoprotein with alkaline phosphatase activity has been isolated from the bone matrix, it is not easily detected in mineralized matrix (MM) by this histochemical assay. Courtesy of Dr. Paolo Bianco.

clearly demarcates areas that will become bone from those that will not. Initially, the cells are all rather flat and spindle shaped, indicative of preosteoblasts, but as osteogenesis continues, some cells (usually those in close proximity to blood vessels) take on the morphology of osteoblasts (plump, highly biosynthetically active cells). As cells mature further to become osteocytes, alkaline phosphatase activity is lost, presumably due to the fact that its function is no longer required [355]. The enzyme exists as a dimer and the identical monomers have an Mr 50,000 – 85,000 depending on animal species and degree of posttranslational modification. Each monomer binds to two zinc and one magnesium ion in each active site. Although the bone/liver/kidney isozyme is the product of the same gene, there are tissue-specific posttranslational modifications that can be detected by monoclonal antibodies [362 – 364]. The enzyme is glycosylated

GOKHALE, BOSKEY, AND ROBEY

and attached to the cytoplasmic membrane on the external surface through a phosphatidyl-inositol-glycan group, which can be cleaved by phospholipase C, thereby releasing it from the cell surface [365,366]. Based on sequence and structural analyses, the E. Scherichia coli enzyme shows extensive homology with mammalian alkaline phosphatase, the major differences being on the enzyme surface [367]. The structure of the enzyme isolated from E. coli has been determined by X-ray crystallographic analyses of single crystals of the (a) Zn2-containing enzyme [368], (b) enzyme with inorganic phosphate bound to the active site [369], and (c) cadmium salt of the enzyme [369]. From E. coli single crystal data, the enzyme takes the form of a twofold symmetrical dimer (100  50  50 Å in dimensions), with the two active sites separated 30 Å from each other. Each subunit consists of a central 10-stranded  sheet surrounded by 15 -helices of various lengths. The active site is in the carboxyl end of the central  sheet, and all the ligands for the three metal ions come from this structure. One serine has been shown to be involved in phosphorylation and dephosphorylation reactions [369]. The phosphate is closely associated with all three metal ions, and the guanidinium group from an arginyl residue. The coordination of the metal ions in the active site is reported to be very similar to the active site of phospholipase C [370], which shows no other structural homology with alkaline phosphatase. The human gene for alkaline phosphatase is located on chromosome 1 [371]. It contains 12 exons, is over 50 kb in length [372], and has an RFLP [373]. The rat gene is at least 49 kb with 13 exons and has a similar gene organization [374]. The gene predicts a protein with 524 amino acids that includes a 17 amino acid signal peptide. The potential for heterogeneity between the bone/liver/kidney isozymes may reflect the fact that there are five potential glycosylation sites. The C-terminal region is hydrophobic, as would be expected for a protein that is linked to the cell membrane via phosphoinositol. It has been determined that regulation of the bone/liver/kidney isozyme is controlled by two leader exons, 1A and 1B [375], with alternative promoters separated by 25 kb. The upstream promoter is used preferentially by bone cells and facilitates its high level of expression in this cell type [376]. The transcription start site is preceded by a GC-rich region, a TATA box, and three SP1 sites [377,378]. The downstream promoter is constitutively active, produces low levels of activity, and is used in the kidney. Three mRNA species of 2.5, 4.1, and 4.7 kb are produced as the result of differential splicing [379]. The list of factors that regulate alkaline phosphatase is lengthy and the results are extremely variable. Expression can be affected by oxygen tension [380] and, in general, requires the presence of serum [381]. In human bone cells,

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1,25-dihydroxyvitamin D3 stimulates alkaline phosphatase [382]. In immortalized rat osteoblastic cells, retinoic acid also stimulated the enzyme [383], and the region 108 to 45 from the transcription start site was required for this stimulation [384]. Pretreatment of fibroblastic cells with substances that induce cAMP followed by retinoic acid treatment causes an elevation in the mRNA coded for by the exon 1B promoter [385]. IL-4 causes an increase in alkaline phosphatase mRNA in proliferating osteoblastic cells [82]. Dexamethasone increases the utilization of the upstream promoter, resulting in an increase in the tissue-specific expression of alkaline phosphatase [386]. Calcitonin increases alkaline phosphatase expression in osteosarcoma cells [387]. In deer antlers, an interesting model of bone formation, alkaline phosphatase was found to be increased by IGF-I [388]. Ascorbate has also been reported to upregulate alkaline phosphatase expression in chrondrocyte cultures [389]. Alkaline phosphatase activity has been found to be decreased by IL-10 [90] and by lead [91]. Reaction mechanisms for the E. coli enzyme have been predicted from kinetic labeling studies [390], as well as from the single crystal structure [369]. These studies show that the enzyme has phosphate transfer activity and hydrolase activity, transferring a phosphate to or from the serine in the active site from or to the substrate, respectively. The predicted structural identity between mammalian and E. coli enzymes [367] implies a conserved mechanism and suggests that the bone (and cartilage), liver, and kidney isozymes have similar nonspecific phosphomonoesterase activities. The specific function of alkaline phosphatase in bone has been debated for more than 60 years. In the 1920s, Robison, before the existence of the multiple isozymes was known, suggested that the function of this enzyme was to hydrolyze phosphate esters, providing a source of inorganic phosphate [391]. Human studies have shown that different isoform structures are likely to exist due to differences in glycosylation. These studies have also shown that higher alkaline phosphatase activity was present in trabecular bone than in cortical bone [392]. In diseases such as rickets, with impaired mineralization, activity of the bone isozyme is increased [393]. It has also been observed that the commencement of mineralization in culture coincides with increased alkaline phoasphatase activity, indicating that this enzyme was necessary for proper mineralization [see reviews in 394,395]. This hypothesis was confirmed by the discovery of mutations in the alkaline phosphatase gene in hypophosphatasia, a disease characterized by improper mineral deposition [396], and by the observation that cells that do not normally mineralize will form a mineralized matrix when transfected with the alkaline phosphatase gene [397]. Because mineral deposition in vitro will occur (i) when alkaline phosphatase is added to cell-free solutions of calcium ions and phosphate esters [398], (ii) in cell cultures

in which alkaline phosphatase activity is inhibited by levamisole [399,400], and (iii) in the absence of alkaline phosphatase substrates [401,402], the transfection studies do not necessarily identify a definitive function. Mice with null mutations in either the tissue-nonspecific alkaline phosphatase [403] or the bone-specific alkaline phosphatase also provide evidence of the importance of alkaline phosphatase for mineralization [404]. Even the tissue-nonspecific alkaline phosphatase knockout shows increased osteoid and defective growth plate development, leading one to question what the specific mechanism of action of alkaline phosphatase might be. It had been suggested that alkaline phosphatase could also function as a protein phosphate transferase in bone [405] and such transferase activity was noted at physiologic rather than basic pH (required for optimal hydrolytic activity). The transferase activity is in line with the mechanistic studies, and the abundance of phosphorylated proteins implies the need for such an activity. In this light, it should be noted that using non hydrolyzable adenosine triphosphate derivatives retards mineralization in culture [406], perhaps because matrix protein phosphorylation is impaired. A role for alkaline phosphatase in signal transduction may also be speculated based on its analogy to other glycan-linked proteins [365,407]. However, few experimental data support this function. Although a precise function for bone alkaline phosphatase is not known, it seems apparent from hypophosphatasia defects [396] that the enzyme plays a crucial role in mineralization. Because its phosphohydrolase activity is optimal at pH 10 [394], a less direct role than hydrolyzing phosphate esters to produce elevated phosphate concentrations is likely. Part of the function of alkaline phosphatase in mineralization may be associated with its abundance in the membrane-bound bodies, matrix vesicles, believed to be the foci of initial mineralization (vide infra). Another postulated function is the dissolution of calcium pyrophosphate dihydrate crystals in cartilage [408].

B. Osteonectin (SPARC, BM-40) As a result of the development of novel techniques for the extraction of bone matrix proteins in a nondegraded form [2,3], one of the first noncollagenous bone matrix proteins to be isolated and characterized was osteonectin [409]. Osteonectin can constitute up to 15% of the noncollagenous proteins in bone depending on the developmental age and the animal species [410]. Although it was initially thought to be bone specific, with the advent of sensitive antibodies and in situ hybridization, it became apparent that osteonectin is expressed in a number of tissues during development and by many cell types in vitro. In fact, osteonectin was independently identified as SPARC (secreted phosphoprotein acidic

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and rich in cysteine) in parietal endoderm [411], culture shock protein in endothelial cells [412], and BM-40 from the EHS mouse basement membrane tumor [413,414]. By in situ hybridization in mouse and human tissue [415,416], osteonectin was found in skin, tendon, developing whiskers, certain nerve trunks, and parietal endoderm. Steroid-producing tissues such as the adrenals, testis, and ovary also produced osteonectin [417]. Immunohistochemical analysis localized transient osteonectin protein to decidua [418] and developing spermatozoa [419], whereas low, but constant, levels were found in renal distal tubule epithelial cells [420,421] and duct cells of salivary gland [420]. In the skeleton, osteonectin mRNA is found in odontoblasts, periosteal cells, osteoblasts, and, to a lesser extent, in osteocytes and hypertrophic chondrocytes [415,416]. By immunohistochemistry (Fig. 12), osteoblasts and osteoprogenitor cells contain osteonectin [422,423], as do odontoblasts [424], periodontal ligament, and gingival cells [425]. Again, low levels are found in chondrocytes and hypertrophic chondrocytes in the growth plate [422,423,426]. During subperiosteal bone formation, there is no detectable osteonectin antigen in the cells or matrix in the fibrous outer layer. Preosteoblasts have a small amount of cytoplasmic reactivity, but osteonectin cannot be detected by immunostaining. In the osteoblastic layer, there is a dramatic

FIGURE 12

upregulation of expression with intense staining of both the cytoplasm and the osteoid, but expression is much lower in osteocytic cells [12,422]. Woven bone contains less osteonectin than more mature, lamellar bone [410], which may explain the differences in concentration between bones of rats (less lamellar in character) and human (more lamellar and osteonal in nature). By radioimmunoassay (RIA), it is noted that bone contains 1000 – 10,000 times more osteonectin than any other connective tissue, such as skin and tendon [427]. Osteonectin is produced transiently in many tissues undergoing differentiation, and it is produced constitutively only by cell types that appear to have ion transport as a common feature. It is associated with development, remodeling, cell turnover, and tissue repair. In vitro studies have revealed that it is involved in counteradhesion and antiproliferation of cells 428. It accumulates in mineralized matrices (dentin, bone, calcified cartilage) and in the  granules of platelets [429]. It has also been found to be associated with prostate tumors [430]. The identification of a bone matrix protein in platelet  granules was the first in a series of discoveries that many bone matrix proteins are found in platelets and megakaryocytes. The reason for the localization of these proteins in cells of this distinctly different lineage is not known. Osteonectin has an apparent Mr 35,000 without reduction of disulfide bonds and appears to increase in size up to

Osteonectin immunolocalization in trabecular bone. (A) Osteonectin is highly enriched in the osteoid and in osteoblasts (Ob) and, to a lesser extent, in osteocytes (open arrow). In addition, osteonectin is also enriched between lamellae (small arrows), which is better demonstrated by immunofluorescence (B). Courtesy of Dr. Paolo Bianco.

CHAPTER 4 The Biochemistry of Bone

Mr 40,000 – 46,000 following reduction, indicative of intrachain disulfide bonds (Fig. 13). The estimated size is Mr 32,000 by molecular sieve chromatography and Mr 29,000 by equilibrium sedimentation, suggesting that it takes on a rather compact hydrodynamic conformation [429]. Osteonectin is also phosphorylated and glycosylated, two posttranslational modifications that may be regulated differentially depending on the tissue of origin. Due to the nature of the amino acid composition and the nature of the posttranslational modifications, osteonectin is acidic with a pI of 5 [431]. There is a single gene (20,000 kb) for osteonectin located on human chromosome 5 [432] at 5q31 – q33, and with one RFLP in the 5 region [433]. The gene contains 10 exons, several of which code for potentially functional regions. While the exons are relatively small (130 bases), the first intron is approximately 10 kb in length. The coding sequence predicts a 17 residue signal peptide and a 286 residue mature protein. The signal peptide is contained in exon 2, whereas exon 3 contains the amino terminus. This region varies from one animal species to another; however, it contains a preponderance of acidic residues. If this region takes on an -helical conformation, the orientation of the carboxy side chains away from the helix would result in the generation of 12 low-affinity calcium-binding sites. Subse-

FIGURE 13

The chemical characteristics of osteonectin indicate the presence of two -helical regions at the amino terminus, along with an ovomucoid-like sequence with extensive disulfide bonding and two EF hand structures.

131 quent exons code for a cysteine-rich region with homology to an ovomucoid-like (serine protease inhibitor) sequence. A structure that is highly homologous to the EF hand (highaffinity Ca2-binding structure) found in calmodulin is encoded by exon 10 and another one, although not as highly homologous, is found in exon 9 [434 – 436]. Sequence predictions indicate that the EF hand domain is in the C terminus. Several other domains, in addition to the EF hand region, have been defined, including a disulfide-rich domain and a pentapeptide KKGHK domain [437]. Three additional genes show high homology with osteonectin. One gives rise to a synaptic junction glycoprotein in rat, SCI [438], and hevin in human [439], a neural retina protein, QR1 [440], testican [441], and FRP [442]. These molecules contain the carboxy-terminal portion of osteonectin (three-fourths of the molecule) and substitute different amino-terminal ends. The role of these homologous forms of osteonectin in their respective tissues is not known. The promoter region of the gene has been isolated and shown to be active by transfection experiments [430,435, 443 – 445]. The promoter does not contain TATA or CCAAT sequences but contains a purine-rich region with GA repeats between 55 and 126. The bovine promoter has a complimentary CCCT-rich sequence further upstream that is believed to cooperate with the GGA-rich region to control transcription. This information, along with S1 nuclease sensitivity, suggests that this region may become hinged or form a triplex conformation. A novel nuclear factor from osteoblastic cells that binds to this region has been identified [445]. There are also numerous other sites, including multiple SP1 sites, one AP1, one CRE, a growth hormone element (GHE), a heat shock element (HSE), and a metal responsiveness element (MRE). The first exon also contains four CCTG repeats, sequences that have been implicated in transcriptional control. In connective tissues, the gene is transcribed to form mRNA species of 2.2 and 3.0 kb. The cDNA codes for a 17 residue signal peptide and a 303 residue protein with a 17 residue signal peptide [411,432,434,443], but with no evidence to suggest the existence of a propeptide [245]. Osteonectin may be differentially glycosylated and/or phosphorylated [446], a possible explanation for differences noted in molecular weights and reactivity with different antibody preparations. There are at least two potential Nglycosylation sites that bear diantenary oligosaccharides (an intermediate between high mannose and complex type oligosaccharides that contains variable amounts of sialic acid and fucose) [447]. It has been found that intracellular forms of osteonectin found in megakaryocytes and certain osteosarcomas have a different carbohydrate structure from secreted forms of osteonectin found in bone [448]. These structural differences may also be reflected in functional differences [449] and could explain differences in the reactivity of osteonectin with different monoclonal antibodies [450].

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Factors that regulate the synthesis of osteonectin are not well understood. It appears that the control of osteonectin synthesis is deregulated rapidly, as cells of various tissues begin to produce osteonectin in vitro when they do not normally synthesize it in vivo. Biosynthesis has been studied in a number of cell culture systems, and the regulation and association of osteonectin with mineral formation appear to be dependent on the animal species and culture conditions. In cartilage, IL-1 decreases osteonectin synthesis, whereas TNF- and IL-6 have no effect. TGF-, PDGF, and IGF-I are stimulatory and able to reverse the effect of IL-1 [451]. In bovine bone cell cultures that exhibit extensive mineralization, osteonectin appeared at early stages and remained high thereafter [282]. The effect of TGF- is variable and a stimulation [452] as well as a lack of effect [453] have been reported. The expression of osteonectin by normal human bone cells is not altered dramatically by any treatment (Gehron Robey, unpublished results), although very modest increases with dexamethasone, retinoic acid, IGF-I and dibutyryl cAMP have been reported in other systems [288,411,454]. Interestingly, osteonectin is expressed at constant levels even after heat shock, whereas other proteins decrease after this form of stress [455]. This resistance to heat shock may be related to the presence of a potential heat shock element in the promoter. The only structural studies of osteonectin/SPARC/BM40 other than those predicted from cDNA sequences [e.g., 434,437,456] are NMR evaluations [457] and circular dichroism (CD) studies [71]. The CD measurements were interpreted as showing that the protein conformation had 6%  helix, 26%  sheet, with the remainder nonordered in the absence of Ca2, and less than 3%  helix, 21%  sheet, and 82% nonordered structure in its presence. Analysis of the crystal structure has shown that a follistatin-like

FIGURE 14

domain is related to the serine-protease domain of the Kazal family. There is an insertion into the inhibitory loop in BM-40 and a protruding N-terminal  hairpin with striking similarities to epidermal growth factor 458. NMR evaluations showed the presence of a typical EF hand [457], which in other systems is involved in calcium chelation and calcium transport (Fig. 14). The initial investigations of the function of osteonectin demonstrated that when bound to denatured collagen, osteonectin bound calcium and phosphate ions, suggesting that it was promoting mineral deposition [409]. Osteonectin and its metalloprotease-cleaved fragments also bind to type I collagen [409,459], to types III and V collagens [448,460], and to thrombospondin [461]. Such interactions are likely to be important in determining the organization of the osteoid in bone. Later studies showed that osteonectin was an efficient inhibitor of hydroxyapatite formation in solution [462]. As discussed later, these concepts are not mutually exclusive. Because macromolecules that in low concentration act as nucleators by binding to matrix to provide suitable substrate surface for nucleation can in higher concentrations bind directly to one or more faces on the growing crystal. This can block further growth of the crystal. The tissue distribution of osteonectin within bone suggests, however, that it is not involved in the initiation of mineralization [463]. Expressed by cells in the unmineralized, mineralizing, and mineralized bone, osteonectin accumulates only within the mineralized matrix. Whether it has a specific function in further regulating growth and proliferation of mineral or simply accumulates within the mineralized tissue because of its affinity for hydroxyapatite (Kd  8 108, 11.3 mg osteonectin/gm apatite [460]) is not yet known. A role in the regulation of mineralization does not appear to be a principal function for osteonectin because it is

Structure of an EF hand high-affinity Ca2-binding site. Depiction of the theoretical structure and the amino acid sequence for the EF hand, which has an extremely high affinity for ionized calcium. Courtesy of Dr. Neal S. Fedarko.

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a widely expressed protein and, in other tissues, it appears to be more involved in regulating cell shape and cell matrix interactions [437] via calcium binding. For example, a fragment of the molecule has been shown to regulate the proliferation of endothelial cells [464]; the intact molecule acts to regulate cell shape in an ion-independent fashion [465,466]. Similar to thrombospondin, it modulates cell – matrix interactions [467]. The original description of the young osteonectin knockout mouse focused on the formation development of cataracts [468]. More recent studies of older mice indicate that the mice develop osteopenia with a significant loss of trabecular bone associated withdue to a decreased rate in bone formation [469]. A number of functions have been suggested by are derived from experiments utilizing synthetic peptides from different parts of the molecule and determining their effect on endothelial cell cultures. While this is an extremely useful approach that provides much intriguing information, it is not clear whether the intact molecule has the same functions or if the effects are cell specific. A synthetic peptide influenced endothelial cultures undergoing tube formation by decreasing the synthesis of fibronectin and thrombospondin-1 and by increasing the synthesis of plasminogen activator inhibitor-1 (PAI-1) [470]. The intact protein decreased bFGF-induced endothelial cell migration [471]. The binding of osteonectin to endothelial cells similarly may be through one of the EF hand structures at the carboxy terminus [472]. Osteonectin and a peptide derived from a noncalcium-binding region have the ability to stop endothelial cell cycle progression, although it does not have the same effect on fibroblasts [473]. In endothelial cells, it may play a role in barrier function by regulating endothelial cell shape [474]. In addition a peptide that becomes conformationally constrained by binding to Ca2 inhibits the proliferation of endothelial cells [475]. Overexpression of osteonectin causes attachment and spreading of endothelial cells in calcium-deficient medium [476,477]. The various activities of osteonectin in embryogenesis and during wound repair have been reviewed extensively by Sage and co-workers [428,478]. Osteonectin may also bind to copper and serve as a source of this ion during angiogenesis [479]. Osteonectin is produced during mid- and late stages of wound repair [480] and interacts with PDGF (specifically with the  chain) and may modulate its activity by inhibiting binding to its receptor [481]. Treatment of fibroblasts with osteonectin induces metalloproteinase activity [482] and may anchor plasminogen and increase its activation, pointing to a function in tissue remodeling [483]. The disassembly of focal adhesion may be through a follistatin-like region that contains one of the EF hand structures [484]. Another interesting study utilized antiosteonectin in developing frog embryos and found that neurulation and myotome development were impaired [485]. Overexpression of osteonectin in C. elegans caused devel-

opmental abnormalities as well as paralysis [486]. Each of these studies demonstrate that osteonectin can influence development in a number of connective tissues. However, while it is known to affect bone development in the mouse, a its specific role in bone has yet to be established.

C. Tetranectin Tetranectin is a tetrameric protein with a Mr of 21,000 (subunits with Mr of 5800) that was first isolated from serum and found to bind to the kringle 4 domain of plasminogen [487]. It is immunolocalized in developing woven bone [488] and is expressed by osteoblastic cultures undergoing matrix mineralization [488]. The cDNA has been cloned [489] and the gene has been isolated [490]. The gene is 12 kb in length and contains three exons. The cDNA predicts for a 21 residue signal peptide and a 181 residue mature protein. It has sequence homology with asialoprotein receptor and the G3 domain of aggrecan and versican core proteins [491]. Overexpression of tetranectin by tumor cells caused an increase in matrix mineralization upon implantation into nude mice [488]. These data suggest that tetranectin may play a role in mineral deposition.

D. RGD-Containing Glycoproteins One of the major breakthroughs in the field of cell – matrix interactions was the discovery of a sequence contained within matrix proteins that would bind to cell surface receptors, thereby mediating attachment [492]. These cell surface receptors belong, for the most part, to the family of integrins. These receptors are formed by one  subunit and one  subunit, each of which have a cytoplasmic extension that may associate with intracellular signaling pathways, a transmembrane domain and an extracellular domain. The extracellular domains of the  and  subunits form a binding pocket that recognizes the cell attachment sequence in the extracellular matrix protein [reviewed in 493,494]. While there are a number of amino acid sequences that bind to integrins, the most frequently utilized sequence is ArgGly-Asp (RGD) [492]. In bone there are at least seven matrix proteins that contain RGD: thrombospondin, fibronectin, vitronectin, collagen (described earlier), osteopontin, bone sialoprotein, and fibrillin. The reason for this redundancy is not entirely clear, although a clue may lie in the fact that these proteins exhibit different patterns of expression during bone formation. Consequently, the interaction of cells with an ever-changing matrix may mediate, in part, changes in cellular metabolism. What follows is a description of the RGD-containing proteins identified in bone, presented roughly in the order of their appearance during developmental bone formation.

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1. THROMBOSPONDIN(S) This complex, modular glycoprotein [495 – 498] was first isolated and characterized from the granules of platelets [499]. Since its initial discovery, it has been found that there are at least five members of this family. The various forms are found in a large variety of connective tissues, in particular, in areas of demarcation such as at the dermal – epidermal junction in the skin, surrounding muscle fibers, separating glandular epithelium, and in the lung in peritubular spaces [500,501]. Thrombospondin is relatively less abundant in mineralized matrix relative to other glycoproteins; however, due to its complex chemical nature, it is most likely active in modulating cell metabolism. The expression of thrombospondin during development is sporadic and usually coincides with morphogenetic events such as cell proliferation, migration, and commitment, followed by its disappearance as differentiation continues [502]. In bone, it is found during early stages of osteogenesis. Immunohistochemical localization indicated low levels of expression in the periosteum, with primary localization in developing osteoid [503] (Fig. 15). Osteoblastic cells are stained intensely. There is a moderate accumulation of thrombospondin in mineralized matrix [504] and, by Western blotting, the protein can be detected in bone matrix extracts [503]. Thrombospondin is a highly complex molecule with a Mr 450,000 [reviewed in 505,506] (Fig. 16), composed of three identical subunits ranging in Mr from 150,000 to

180,000 that are held together by disulfide bonds. Each monomer has a number of intramolecular disulfide bonds that give rise to a molecule with a roughly dumbbell shape with distinct functional domains. The small amino-terminal globular domain contains a fibrinogen-like sequence along with a region that may have cell-binding activities [507] and heparin- and platelet-binding sites. In addition to homologies to the propeptide of the (1)I chains of types I and III collagen, von Willebrand factor, and the circumsporozoite protein from Plasmodium falciparum, this small globular domain is attached to an extended stalk region that contains three properdin-like (type I) and three EGF-like (type II) repeat sequences. There is a cluster of cysteine residues in the stalk region that participate in the cross-linking of the monomers and binding sites for types I and V collagens, thrombin, fibrinogen, laminin, plasminogen, and plasminogen activator. A large disulfide-bonded domain makes up the carboxy-terminal region of the molecule and contains sequence homologies to parvalbumin and fibrinogen, with seven calmodulin-like (type III) repeat sequences, although this sequence does not take on the EF hand structure. This region binds to the histidine-rich glycoprotein of serum, activates platelet aggregation, and has multiple Ca2-binding sites. Binding of Ca2 participates in the conformation of the globular domain. The RGD sequence is within the Ca2-binding region; consequently, it is not clear whether the RGD actually is active in mediating cell attachment under normal physiological conditions [508].

FIGURE 15 Thrombospondin immunolocalization in developing bone. Thrombospondin is first expressed in the preosteoblastic layer (POb) in the periosteum of human developing long bone and is highly concentrated in osteoid and osteoblasts (Ob). Somewhat lower levels are maintained in the mineralized matrix (MM). Courtesy of Dr. Wojciech J. Grzesik.

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FIGURE 16 Thrombospondin is a disulfide-linked trimer that has globular domains at the amino and carboxy terminus interconnected by a stalk region. Each of these domains has a number of binding sites for other proteins, suggesting numerous potential functions in cell – matrix interactions. The cell attachment consensus sequence, RGD, is in the carboxy-terminal domain; however, its availability depends on the calcium ion concentration, which is known to affect the conformation of this region.

Initially it was thought that there was only one gene for thrombospondin. However, it is now apparent that in humans there are at least five (TSP-5 being equivalent to the cartilage molecule COMP), located on chromosomes 1 (TSP-3), 5 (TSP-4), 6 (TSP-2), 15 (TSP-1), and 19 (COMP) [509 – 514], all at least 16 kb in length. While the coding sequences are all highly homologous and differ only in the number of times that the type I, II, and III sequences are repeated, they utilize different promoters [515]. The complete pattern of expression of the different thrombospondin genes is not complete to date [516], although it is known that TSP-1, TSP-2, and TSP-3 are expressed in bone [517,518]. A promoter from the thrombospondin 1 gene has been isolated and characterized [519,520]. It contains a TATA box and an Egr1 site that is flanked by overlapping GC boxes, followed by a GC-rich region. Binding sites for NFY, AP2, SP1, and an SRE have also been identified. Based on the inhibition of TSP-1 transcription by c-jun, an AP1 site may also be present [521]. The resulting mRNA is 6.1 kb [495]. The organization of the TSP-2 and TSP-3 promoters is similar [522 – 524]. Thrombospondin synthesis has been demonstrated in several cell culture systems, including those of adult human

trabecular bone cells [503], in cultures of rat marrow stromal fibroblasts undergoing nodule formation [525], MC3T3 [526], and MG-63 cells [527]. Synthesis appears to be inhibited by dexamethasone [527]. While reducing the overall net synthesis of thrombospondin, TGF-, increases the amount of thrombospondin retained by the cell layer/matrix fraction [503]. Mutant (Tsp-2 null) mice have abnormalities in connective tissue structure and function, including fragile skin [528] and bone (Bornstein, personal communication). Mutant mice have increased cortical bone thickness and density, which has been explained in terms of effects on cell adhesion and differentiation, and collagen fibrillogenesis [529]. While the precise functions of the thrombospondins are not known, they have been postulated to play a role in development, angiogenesis, tumorogenesis, and wound healing [516,530 – 536], and bone remodeling 537. Thrombospondins bind to the small proteoglycan, decorin [265], and along with this molecule may bind to growth factors such as TGF-, which later serve as cell signals. Thrombospondins also bind to osteonectin [461], and considering their colocalization within the  granules of platelets, this complex, along with PDGF and TGF-, may have a

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function in repair processes in soft connective tissues as well as in fracture healing. The functions of the various isoforms of thrombospondin have been indicated by several in vitro studies. In an osteoblast-like cell culture (MC3T3 cells), mRNA for one form of thrombospondin increased during differentiation [526], suggesting that thrombospondin has a role in this process. Thrombospondin has been shown to increase osteoclast resorption of dentin slices independent of increases in osteoclast attachment [537]. Thus it might similarly affect differentiation in bone, either by regulating the latter processes or by directly causing the activation of cell signals. Thrombospondin has been shown to bind to TGF- [525]. In vitro, thrombospondin has been found to be active in the attachment of osteoblastic cells. However, cell spreading required the synthesis of other molecules [503]. Thrombospondin has been reported to bind the vitronectin receptor, v3, in an RGD-dependent fashion. However, it was found that attachment and spreading of osteoblastic cells were not inhibited by GRGDS, a competitive inhibitor used routinely to demonstrate the RGD dependency of cell attachment [504,538]. Thrombospondin may also bind to decorin, which is known to interfere with the cell attachment to fibronectin [539]. The precise cell surface receptor responsible for mediating cell adhesion to thrombospondin is not known, although a cell surface receptor that recognizes the sequence VVM in the carboxy terminus of the molecule has been identified [540]. In properdin-like repeats, the VTCG sequence has been found to mediate the

FIGURE 17

attachment of platelets, monocytes, endothelial cells, and certain tumor cells via cell surface CD36 [541 – 543]. Thrombospondin has been shown to interact with fibrinogen/fibrin but not with heparin [544]. Mice that lack thrombospondin have disordered collagen in their fragile soft tissues, increased bone density, and altered fibroblast cell attachment [528]. Bone mineral properties have not yet been determined. However, the properties of these mutant animals confirm the importance of thrombospondin in bone development. Interestingly, unlike other RGD proteins in bone, thrombospondin does not mediate osteoclast cell attachment [545,546]. 2. FIBRONECTIN Fibronectin, one of the most abundant extracellular matrix proteins, is also a major constituent of serum [reviewed in 547,548]. It is produced by virtually all connective tissue cells at some stage of development and accumulates in extracellular matrices throughout the body. Fibronectin appears at an early stage of bone formation during development [504] or during induction by demineralized bone matrix [549,550]. Osteoblasts and osteocytes stain intensely for fibronectin and it is also accumulated in mineralized matrix [504] (Fig. 17). Western blotting analysis of bone extracts indicates that it is relatively abundant. Although fibronectin is a product of osteoblastic cells, it could also be adsorbed from the circulation; consequently, there may be multiple forms from different sources entombed within mineralized matrix.

Fibronectin immunolocalization in developing bone. There is virtually no expression of fibronectin in the periosteum and preosteoblasts (POb) of developing long bone, but there are high levels of expression in the osteoblastic layer (Ob). In addition, high levels of fibronectin are maintained in the mineralized matrix (MM). Courtesy of Dr. Wojciech J. Grzesik.

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Fibronectin is a dimeric protein with a Mr 400,000, composed of two subunits of Mr 250,000 that are highly homologous, but variable depending on the cell source, held together by two disulfide bonds near the carboxy termini (Fig. 18). Each of the subunits has multiple domains that bind to fibrin (domains I and VIII), heparin, and certain bacteria (domain I), gelatin and collagen (domain II), DNA (domain IV), cell surfaces, including the RGD site (domain VI), and another heparin-binding site (domain VII). Each of these functional domains is composed of different combinations of homologous repeats (types I, II, and III). Type I and II repeats are formed of sequences 45 – 50 amino acids in length that contain a disulfide loop. The type III repeat is twice as long but does not contain a loop structure. The RGD sequence is located in a type III sequence in domain VI approximately one-third of the way from the carboxy terminus. Details of the fibronectin structure have been evaluated from combinations of predictions from amino acid sequence, single crystal X-ray [551], and NMR studies of isolated domains [552,553]. Fibronectin is considered to have three types of domains, two unique  sheets containing type I moieties at the N terminus, 12 type II domains each with hydrophobic pockets, and 17 – 19 type III domains. The overall structure consists of 35% anti parallel  sheets and no  helices. There is a uniform distribution of these pleated sheets along a long chain, resulting in independent

FIGURE 18

structural domains [554]. Most of the type III domains, one of which contains the cell-binding RGD sequence, are in a flexible region of the molecule. The NMR structure [552] suggests that the abundant  sheets form stacks separated by linker regions, which stabilize the molecule. Each of these domains has a distinct function [555]. The N-terminal domain seems to be required for extracellular matrix deposition [556], another domain is required for binding chondroitin sulfate [557], and the C terminus appears to be needed to stimulate its own synthesis [558]. Interactions with cells are modulated by a type III domain containing the RGD [559,560]. The linking region between these domains allows the molecule to stretch to up to seven times its native length [554]. The detailed functions of each of these domains, and the way that they interact with the cytoskeletons of connective tissue cells, have been reviewed [548]. What is apparent from each of these structure – function studies is that fibronectin is essential for matrix deposition. The fibronectin gene is located on chromosome 7 and is very complex, with up to 50 exons [561]. The chicken gene is 50 kb [562] and six RFLPs have been identified [563]. The functional domains, composed of type I, II, and III repeat sequences, are each coded for by an exon. Consequently, it would appear that fibronectin arose from the duplication of multiple genes. Nucleic acid sequence analysis indicates that approximately 90% of the entire coding

Fibronectin is composed of nonidentical subunits that are disulfide bonded at their carboxy termini. The molecule is composed of a series of repeating units (types I, II, and III) that give rise to domains with affinities for other proteins. There are several known splice variants (with or without EIIIB, EIIIA, and V; see text for description). The splice variant present in bone is not known. The cell attachment consensus sequence in a type III unit is RGD; however, other sequences that participate in cell attachment have been identified.

138 sequence is composed of these three repeats. The fibrinbinding domains are composed mainly of type I repeats, whereas the collagen domain is mainly type III repeats. The human gene promoter has been identified and it contains TATA and CCAAT boxes, is GC rich, and has an SP1 and a CRE-binding site [564,565]. Promoter analysis indicates that the CCAAT and the CRE located between164 and - 90 are essential for gene activity. However, gelshift analysis indicates that there are different complements of proteins that bind to this region depending on the tissue source [566,567]. Other studies have shown that the factor (a heterodimer of 43 kDa and a 73-kDa protein) termed ATF2, which binds to the CRE, facilitates the recruitment of factors that bind to the CCAAT element [568]. The gene is transcribed to form mRNA of 7.5 kb, but as might be anticipated, there is a great deal of heterogeneity based on differential splicing and up to 20 different mRNA species have been identified [569,570]. Within the mRNA sequences there are three regions, EIIIA, EIIIB, and V, that can be inserted or deleted depending on the tissue. An example is seen in the differences between plasma (void of E, but containing V regions) and tissue fibronectins (which contain various combinations of Es), which are the result of exon skipping. Differences between fibronectin produced by different cell types have also been found to be the result of exon subdivision (splicing within an exon). Factors that regulate differential splicing are not known, nor is the nature of the splice variant produced by bone cells. Fibronectin synthesis has been demonstrated in many osteoblastic cell culture systems. However, there is not much information on the nature of factors that regulate its production by bone cells. In adult human trabecular bone cell cultures, TGF- increases fibronectin synthesis [453]. In rat and human cells, PTH and TGF- increased fibronectin up to 11-fold. Estrogen caused a decrease in PTH-stimulated levels but had no effect on TGF--stimulated levels [571]. Gallium nitrate, currently under investigation as a therapeutic compound for increasing bone mass, also stimulates fibronectin synthesis in rat calvarial cells and ROS 17/2.8 cells [83]. The elimination of the fibronectin gene in transgenic animals (and all its multisphere variants) was lethal in utero; connective tissues did not form, indicating that fibronectin is a component essential for development of these tissues [572]. Fibronectin is capable of mediating cell attachment and spreading of rat osteosarcoma cells and normal human trabecular bone cells in vitro [504,573]. Numerous integrins mediate cell attachment to fibronectin, including the v3 vitronectin receptor. Interestingly, the attachment of normal cells to fibronectin was RGD independent, indicating that another receptor may be responsible. In fact, it was found that the osteoblastic layer in developing bone, which is

GOKHALE, BOSKEY, AND ROBEY

actively producing fibronectin, is also positive for the 4 integrin subunit [504]. This subunit can combine with 1 to form a fibronectin receptor that is RGD independent. The sequence that mediates attachment to 41 is not yet known. These results are somewhat different from what has been reported for rat osteoblastic cells, which were reported to attach to fibronectin in an RGD-dependent fashion. However, examination of data indicates that incubation with extremely high concentrations of GRGDS (200 M) decreased attachment by only 45% [574]. Fibronectin also mediates attachment of osteoclasts; however, it has been shown that osteoclasts utilize the v3 integrin [545,546, 575]. High-resolution electron microscopy studies have demonstrated that fibronectin can play a role in early biological crystal nucleation, which may be of significance in ectopic calcification, primary nucleation in calcified tissue, and bone ingrowth on ceramic implants [576]. Fibronectin has also been shown to cause apatite formation in solution [577]. 3. VITRONECTIN Vitronectin, also termed the S-protein of the complement system, is produced predominantly by the liver. It is found in serum at concentrations of 200 – 400 g/ml and in extracellular matrices [578]. In fact, it was first identified as the “serum-spreading” factor [579]. It is also found in basement membranes, but generally appears in most matrices containing the fibrillar collagens. It is detectable in developing bone by immunohistochemistry and is found in a very limited number of cells lying on the surface of newly formed bone. However, it is not clear that these cells are in fact osteoblasts. In addition, bone matrix is only faintly stained by immunological techniques, indicating accumulation in bone matrix at very low levels [504]. However, prior to mineral deposition, vitronectin is increased in concentration in the unmineralized osteoid 580, implying that it may be involved in preparing the matrix for mineralization. Although vitronectin is synthesized primarily in the liver, it is also produced by mesenchymal cells at low levels [581,582]. It may also be a biosynthetic product of osteoblastic cells [580]. The protein has a Mr 70,000, and the primary structure of human vitronectin was predicted from cDNA analysis by Oldberg et al. [583] and Jenne and Stanley [584]. An RGD sequence close to the amino terminus and a heparin-binding site in the carboxyl terminus were predicted. Ehrlich’s study [585] has defined several additional homologous domains in the mammalian vitronectin sequences obtained from different sources. From the amino to the carboxy terminus there is a “somatomedin B” domain that is rich in cysteines, followed by an RGD cell attachment site, a collagen-binding domain, a cross-linking site for transglutaminase, a plasminogen-binding site, a heparin-binding site, a PAI-binding site, and an endogenous cleavage site. Sites for sulfation and

CHAPTER 4 The Biochemistry of Bone

cAMP-dependent phosphorylation are also present. The human gene is located on chromosome 17q [586]. In vitro, vitronectin is very active in mediating attachment of all cell types. This in vitro finding may be somewhat misleading, as of all the serum proteins, vitronectin is the most active in binding to standard tissue culture plastic. Consequently, when cells are cultured in vitro, there may be a selection process whereby cells that bear the vitronectin receptor v3 have a distinct advantage over cells that do not have this receptor. Integrins other than the v3 may also bind to vitronectin [587]. Bone cells attach very strongly to vitronectin [504,588]. However, it is not clear what role this interaction plays in bone formation. Because osteoclasts have integrins that are vitronectin receptors [589], it would seem likely that the vitronectin in bone is needed for osteoclast adherence. Further, antibodies to the vitronectin receptor inhibit in vitro osteoclast action [589]. However, the vitronectin receptor distribution on the osteoclasts changes depending on whether the cells are attached or motile [590]. In addition, other RGD-containing proteins such as osteopontin or bone sialoprotein may be utilized by the osteoclast’s v3 receptor. Vitronectin has been shown to inhibit secondary nucleation of apatite crystals in vitro [591]; however, a direct effect on mineral deposition has not been established. 4. OSTEOPONTIN (BSP-1, SPP, pp66, Eta-1) This acidic glycoprotein was identified independently in several cell systems. In bone, it was termed BSP-1, now known as osteopontin [592 – 594]; however, it was also described as a secreted phosphoprotein, SPP, and pp66, a protein that is dramatically upregulated by cell transformation [595 – 597] and in association with tumor progression [598 – 599]. In addition to bone where osteopontin is largely accumulated, it is found in the kidney tubule epithelium, especially in the loop of Henle and the distal convoluted tubule [600], and it is secreted into forming urine (perhaps to inhibit crystal formation) [601 – 603]. It is also produced by mammary epithelium, as it is found at high concentrations in milk [604]. Immunolocalization and in situ hybridization have identified osteopontin in the uterus and placental membranes and in metrial gland cells [440,605]. In adult human tissue, its expression is found in epithelial cells of the gastrointestinal tract, gall bladder, pancreas, urinary and reproductive tracts, lung bronchi, mammary and salivary glands, sweat ducts, and smooth muscle [606], as well as in a variety of tumors [607]. Osteopontin is also found within neuronal cells in the brain and in the inner ear [440,608 – 610]. Platelets and megakaryocytes also contain mRNA for osteopontin, although the level of actual protein appears to be very low [611]. In general, data that have emerged in recent years indicate that osteopontin mediates autocrine – paracrine func-

139 tions in the regulation of tissue formation and plays a role in tissue homeostasis [reviewed in 612]. Inspection of osteopontin production at the cellular level during subperiosteal bone formation indicates that it is produced by osteoblasts and, to a lesser extent, by osteocytes, making it a late marker of osteoblastic differentiation and an early marker of matrix mineralization [11,12,613 – 616]. Marrow also contains cells that contain both osteopontin message and protein. In bone marrow ablation studies in which gene expression was measured as a function of new bone formation, osteopontin exhibited a biphasic pattern of expression. Levels were found to be high during the early phase of the process, most likely corresponding to the initial phase of cell proliferation [617,618]. Subsequently, levels fell to baseline, but began to climb to maximal levels just prior to or coincident with matrix mineralization [463,619]. In addition to being produced by osteoblasts, osteopontin is produced by hypertrophic chondrocytes [620 – 622] and osteoclasts [601,609,610,623,624]. Osteopontin has been localized in bone matrix at the EM level and is highly enriched at cement lines [613,625,626]. Osteopontin is also found in dentin [609]. Some studies have reported that osteopontin is not prominent in osteoid and is most prominent in cells close to the metaphyseal/diaphyseal border where there is active resorption, although the cells surrounding the osteoclasts contained the mRNA for osteopontin whereas the osteoclasts did not [627]. The molecular weight of osteopontin is highly variable depending on the method of analysis. By SDS-PAGE, the Mr varies from 44,000 to 75,000 depending on the percentage of acrylamide in the gel [245,582]. By equilibrium sedimentation analysis, it has a Mr of 44,000 (Fig. 19). Due to the nature of posttranslational modifications, it does not stain well with Coomassie brilliant blue, but becomes blue with Stains All [239,628] in agreement with its acidic pI of 5.0. The structure of osteopontin was predicted by Prince from the primary sequence of bovine osteopontin [629,630]. There is an RGD cell-binding domain, and a single poly-aspartyl repeat sequence. This poly-aspartyl sequence is highly conserved in all species, implying a functional importance for this domain. Direct analysis of the protein indicates that the bone form has an N-linked oligosaccharide, 5 – 6 O-linked chains, 12 phosphoserine residues, and 1 phosphothreonine [592]. Chick, rat, mouse, and human proteins show considerable homology, although potential phosphorylation sites vary [631]. In a posttranslational modification, osteopontin becomes cross-linked to fibronectin through the action of transglutaminase [632], which may further stabilize its deposition in bone matrix. The structure of a segment of the osteopontin molecule has been determined by NMR methods [633]. This structural study examined small peptides in the cell-binding (RGD) domain and defined the stereospecificity of the

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FIGURE 19 The osteopontin molecule is composed of numerous stretches of  helix (depicted as cylinders) interconnected in several cases by -pleated sheets, one of which contains the cell attachment consensus sequence (RGD). A stretch of polyaspartic acid (Poly Asp), along with phosphorylated residues (PO4), makes osteopontin a highly acidic molecule. Adapted from Denhardt and Guo, FASEB J. 7, 1475 – 1482 (1993).

serine peptide, GRGDSL. Both the RGD cell-binding domain and a RGD-free cell binding domain in the N terminus have the structures required for integrin interactions needed for cell attachment [634]. Although to date there have been no conformational studies on intact osteopontin, elegant NMR studies of the dentin phosphoprotein’s (phosphophoryn) conformation at different pHs reveal some general features of phosphorylated protein structures that might appear in osteopontin [635,636]. These NMR studies showed that dentin phosphophoryn consisted of structurally organized repeating charged clusters separated by neutral amino acids that were postulated to serve as flexible hinges. The charge clusters are those involved in calcium chelation, and most likely in interactions with the mineral. The availability of expression systems for the native protein [e.g., 637] can facilitate determination of function based on extents of phosphorylation and varying sialic acid contents. Sequence analysis demonstrates that osteopontin and the other phosphorylated sialoproteins have structural features (-pleated sheets containing anionic and phosphorylated residues) that make them well suited for interactions with hydroxyapatite [638]. The osteopontin gene has been isolated in many animal species [639 – 643] and is localized to 4q13 – 21 in humans. The gene contains seven exons and one RFLP [644], and several different alleles [645] have been reported. While the amino acid sequence is highly conserved, there are signifi-

cant differences that appear to be the result of differential splicing of certain exons in different tissues [640,644,646]. In bone, the mRNA predicts a 301 residue protein that includes a 16 residue signal peptide [630,644], whereas osteopontin from osteosarcoma appears to have an insertion due to alternative splicing [646]. The osteopontin promoter is highly complex as would be expected, given the ranges of tissues in which it is synthesized at very precise times and locations. The first kilobase of the mouse osteopontin promoter has been studied intensely. It contains a TATA box, an inverted CCAAT, and a GC box going from 3 to 5 upstream from the transcription start site. There is a positive enhancer between 543 and 253 bp and a negative element between 777 and 543 [642]. There are five PEA-3 (polyoma enhancer activator) sites, multiple TPA sites, SP1, thyroid hormone response (THR), growth hormone factor (GHF), AP4, AP5, AP1, ras activation element (RAE) sites, and a vitamin D response element (VDRE) site [647]. Transcription in bone gives rise to a 1.6-kb mRNA. Due to the correlation of osteopontin production with initial matrix mineralization [648], there have been many studies on the effect of growth factors and hormones on osteopontin synthesis [649]. In ROS 17/2.8 cells, osteopontin is stimulated by 1,25-dihydroxyvitamin D3 [650,651] and TGF- [652]. Long-term treatment with TGF- caused a decrease in expression of osteopontin, indicating a decrease

CHAPTER 4 The Biochemistry of Bone

in osteoblastic phenotype [653]. Osteopontin synthesis is also decreased by dexamethasone [654] and PTH, perhaps due to the induction of cAMP [655]. Endothelin-1, a product of endothelial cells, stimulates osteopontin in osteosarcoma cells [656]. Intermittent compressive forces also stimulate osteopontin [657]. In osteoclast cultures, it has been found that calcitonin inhibits osteopontin mRNA [658]. Phosphoproteins in general have long been linked to the mineralization process based on their accumulation at the mineralization front [106,659] and on the inability of dephosphorylated bone matrices to support mineralization in metastable calcium phosphate solutions [660,661]. The two major non collagenous matrix proteins osteopontin and bone sialoprotein, which accumulate in cement lines and in spaces among mineralized fibrils, show different densities and distribution throughout the tissue. Furthermore, the amount of these proteins generally correlates with the type of bone, speed of tissue formation, and packing density of collagen fibrils [662]. At the molecular level there is indirect evidence from cell cultures deprived of ATP that matrix protein phosphorylation is an essential step in the formation of a mineralizable matrix [406]. Osteopontin is an inhibitor of hydroxyapatite formation and growth in a variety of in vitro systems [663 – 665]. Dephosphorylated osteopontin lacks the ability to inhibit hydroxyapatite formation or growth [663,664], indicating the importance of the phosphate residues and explaining, in part, why osteopontin from different tissues with varying degrees of phosphorylation [e.g., 666] may have diverse effects on mineral formation and growth. Identification of the phosphorylated residues and protein domains required for this inhibition remain to be determined, both in solution and when the protein is bound to a matrix, thereby retaining the conformation it would have in situ. Based on the EM appearance of apatite crystals grown in the presence of 0 – 100 g/ml osteopontin, it appears that this protein blocks crystal elongation [663] rather than secondary nucleation, as is the case for dentin phosphophoryn [667]. This implied that osteopontin binds with high affinity to one or more apatite crystal faces. In fact, it has been shown [613] that osteopontin binds to hydroxyapatite with both high specificity (N  0.026 mol/m2) and high affinity (Kd  1087 g/ mol). It is of interest to note that osteopontin inhibits elastin calcification [668] and in vitro calcification mediated by vascular smooth muscle cells [669]. Thus osteopontin may be important in the prevention of atherosclerotic plaque development. In urine, an osteopontin homologue, uropontin, is similarly believed to prevent the formation and growth of kidney stones based on the in vitro effects of uropontin on oxalate crystal formation and agglomeration [601,602]. Antibodies to this protein localize osteopontin in layers on the kidney stone, suggesting binding to non calcium phosphate minerals. Although it was initially thought that osteopontin might be the phosphorylated

141 protein that was responsible for the nucleation of bone mineral, in solution, osteopontin does not appear to act as a nucleator in any of the in vitro systems studied to date [663,665]. These differences could be due to whether osteopontin is free (in vitro) or bound to the matrix (in vivo). In the latter case, it could be acting as a nucleator. In a number of connective tissue cells in culture, osteopontin appears to be an early marker of cell adherence, being visible immunohistochemically as soon as cells attach to the substratum. Osteopontin promotes osteoblastic cell attachment in vitro [504,573] and therefore may be important in determining the arrangement of cells in the matrix. It has been reported that osteopontin utilizes the vitronectin receptor, v3; however, the evidence is indirect [670]. A fragment of osteopontin also promotes osteoblastic attachment and, interestingly, it does not contain RGD [634]. The evidence for the utilization of the v3 by osteoclasts is much more direct, as attachment could be blocked with a monoclonal specific for the vitronectin receptor [575]. It is possible that this is the initial function of the phosphorylated bone sialoproteins (BSPs) and that later they play different roles by regulating (by osteopontin) and initiating (by BSP) mineralization. Likewise, osteopontin may also play a part later in recruiting osteoclasts to the mineral surface [671] and mediating their activity, as it has been reported that binding of osteopontin by osteoclasts can trigger intracellular signaling [672]. It also appears that osteoclasts dephosphorylate osteopontin, which may in turn alter its activity following bone resorption [673]. It has been shown that osteopontin inhibits the production of nitric oxide synthase (NOS) stimulated by cytokines in kidney cells [674]. This may be of interest in light of the potential role for NOS in osteoblast activity [675]. Another function is in association with osteopontin’s identity to an early T lymphocyte activation-1 (Eta-1) gene. This gene regulates resistance to different strains of viruses, and this resistance has been shown to correlate to different osteopontin alleles [645]. With respect to bone, studies from osteopontin knockout mice indicate that it is not necessary for normal bone development [676]. However, these animals have larger crystals in their bones than age-matched controls [677] and defective osteoclast function [676]. Other functions of osteopontin include enhancement of cell survival by inhibiting apoptosis [676]. This may explain why increased osteopontin expression is associated with metastatic tumor cells 678. In general, whenever ectopic mineral is formed in disease states, many of the bone matrix proteins are found because their expression has been induced by factors that are not yet well understood, or alternatively, due to their affinity for hydroxyapatite. For example, osteopontin has been found in diffuse calcification sites in association with atherosclerosis [679], in mineralized foci within certain mammary tumors [680,681], granulomas

142 of various etiologies [682], and pristane-induced calcified deposits in mice (Gokhale, unpublished data). 5. BONE SIALOPROTEIN (FORMERLY BSP-II) Another glycoprotein, somewhat more bonespecific than osteopontin, is the heavily sialylated glycoprotein, bone sialoprotein (formerly known as BSP-II). A fragment of the protein was first isolated by Andrews and co-workers [683], who initially termed it bone sialoprotein. Subsequently Fisher and co-workers isolated the intact molecule [684] and Oldberg and co-workers later determined the sequence [685]. It can comprise up to 10% of the noncollagenous protein of bone, depending on the animal species. Outside of the skeleton under normal circumstances, examples of BSP expression are very limited. BSP is present in the circulation and may derive in part from platelets [686]. It is possible that BSP is a product of megakaryocytes [687]; however, studies utilizing gray platelets (which lack granules) indicate that BSP may be adsorbed from the serum [686]. BSP is very specific to mineralized tissues and is found in dentin, cementum, and certain regions of hypertrophic chondrocytes [245,628,684, 688 – 691]. In normal healthy tissue, there may be low levels of BSP expression in mammary epithelial cells [692; Van der Pluijm and Gehron Robey, unpublished data] and it is associated with microcalcifications in breast carcinoma [693]. Within the skeleton, BSP expression is also quite limited. During subperiosteal bone formation, the fibrous periosteum and preosteoblastic layers are devoid of expression. Cells in the osteoblastic layer contain BSP, which appears just before or coincident with mineralization. By immunohistochemical staining, BSP localization is not uniform, with only groups of well-defined osteoblasts staining intensely for BSP. The pattern of localization is somewhat atypical in that the cytoplasm is not uniformly stained as seen with other bone matrix proteins. Localization is enriched in the Golgi apparatus. This unique localization was verified at the EM level by the immunogold technique in calcified and undecalcified sections. In tracing the biosynthesis through the secretory pathway, it was noted that BSP, translated and transported through the RER, is concentrated in the Golgi apparatus [353]. Subsequently, it is packaged into secretory vesicles that contain a discrete packet of material that appears to have a honeycomblike substructure. These secretory packets move to the cell surface where their contents are deposited in toto and mineralize immediately in a pericellular area that is relatively devoid of collagen fibrils. These packets (which do not appear to be enclosed within a membrane and are smaller than what has been called an extracellular matrix vesicle) appear to be the first detectable mineralized structures in new bone [353]. Despite this precise localization of BSP with initial matrix mineralization, it is not produced continuously in subse-

GOKHALE, BOSKEY, AND ROBEY

quent stages. After the initial deposition of mineral, the same cells that were previously BSP positive become devoid of BSP, despite the fact that they are identical positionally and morphologically to their previous state of development. These data suggest that the secretion of BSP is not constitutive, but regulated [353]. As shown by in situ hybridization and immunohistochemistry, BSP is also found as chondrocytes begin to hypertrophy during endochondral bone formation, suggesting another example of the osteoblast-like character of hypertrophic chondrocytes [622,689]. The region that separates the developing bone from the cartilage, the lamina limitans, and developing osteoid in this area are also intensely stained [627,689]. In adult trabecular bone, BSP is distributed in a lamellar pattern, and osteoid osteocytes and osteocytes contain the protein (Fig. 20). BSP mRNA and protein were also localized to morphologically well-defined osteoclasts sitting within Howship’s lacunae (Fig. 21). This was the first demonstration of a bone matrix protein being synthesized by both osteoblasts and osteoclasts [689]. BSP has an apparent Mr of approximately 75,000 as judged by SDS-PAGE and is composed of 50% carbohydrate (12% sialic acid, 7% glucosamine, 6% galactosamine) (Fig. 22). It is also rich in aspartic acid, glutamic acid, and glycine, and due to this unique composition, it does not stain well with Coomassie brilliant blue, but is stained by Stains All [684]. BSP, distinct from osteopontin, has two or three sets of polyglutamic acid stretches, each starting with a serine/phosphoserine, and tends to be more highly glycosylated and less phosphorylated [648]. Structure prediction [694] places the polyglutamate stretches in an  helical domain, whereas the proline rich cell-binding RGD containing domain would occur at a V-shaped segment, with the arms of the V highly anionic. The low content of hydrophobic amino acids predicts an open, extended structure [694] analogous to that observed for the bovine BSP in rotary shadowed micrographs [628]. In addition to glycosylation, BSP can also be phosphorylated and sulfated [695]. The sulfate can be localized to either carbohydrate side chains or tyrosine residues [696]. From sequence homologies, a region for such tyrosine sulfation was noted to be between the postulated apatite and the RGD cell-binding sites [694]. Interestingly, in rabbits, BSP is also a proteoglycan. Rabbit bone extracts revealed a protein with keratan sulfate side chains that was identified as BSP after removal of the side chains by keratanase [697]. This keratan sulfate-BSP may also be related to a similar molecule that was identified as a constituent of medullary bone in chickens, which is elevated and decreased during the egg-laying cycle [698]. The functional significance of this posttranslational modification is not known; however, it is an example of how a bone matrix protein can take on different forms, and presumably different functional properties, depending on the animal species. There is also an RGD cell attachment domain in

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FIGURE 20

Bone sialoprotein immunolocalization in trabecular bone. While highly expressed within the osteoblasts in developing bone, BSP in adult bone is found primarily in the matrix between lamellae (small arrows) and is often enriched at cement or reversal lines (not shown). In addition, some, but not all, osteoid osteocytes (Ost Ocy) and osteocytes (Ocy) contain BSP. Courtesy of Dr. Paolo Bianco.

BSP, located near the carboxy terminus, which recognizes the vitronectin receptor [670] and facilitates the in vitro attachment of fibroblasts [699], osteoblastic cells [504,588], and osteoclasts [575]. A S. aureus-binding site is located

near the amino terminus [700], a factor that implicates BSP in the etiology of osteomyelitis [701]. Although NMR data on RGD peptides exist [553], detailed conformational studies of intact BSP are not available.

FIGURE 21 Bone sialoprotein immunolocalization and in situ hybridization in osteoclasts. Osteoclasts contain both BSP (A) and its mRNA (B and C, Nomarski optics), the first demonstration of the dual source of BSP in bone. It is speculated that osteoclasts utilize endogenous BSP to attach to the bone matrix in preparation for forming the sealing zone. It is not yet known if BSP mRNA is transcribed within osteoclastic nuclei or if mRNA is translated. It is possible that osteoclasts gain these moieties by fusing with osteoblastic cells. Courtesy of Dr. Paolo Bianco.

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FIGURE 22 Sequence analysis predicts the presence of multiple stretches of polyglutamic acid (Poly-Glu) in the first half of the molecule and tyrosine-rich regions in the amino- and carboxy-terminal domains. In the carboxy-terminal region, many of these tyrosines are sulfated. The cell attachment consensus sequence (RGD) is flanked by such regions at the carboxy terminus of the molecule. The molecule is composed of 50% carbohydrate, including a high concentration of sialic acid residues. Glycosylation is somewhat restricted to the amino-terminal 50% of the molecule. Adapted from Fisher, McBride, Termine, and Young, J. Biol. Chem. 265, 2347 – 2351 (1990).

However, NMR analysis of a recombinant C-terminal region that contains the RGD indicated the presence of a random coil. It is likely, however, that similar to phosphophoryn, BSP chelation of calcium will involve phosphorylated clusters, and perhaps the sulfated tyrosines are unique to this form of BSP, as contrasted with the other two phosphorylated sialoproteins [317,695,696]. The human gene for bone sialoprotein was initially localized to 4q28-q32 [702 – 704], but now appears to be linked to the osteopontin gene and dentin matrix protein-1 at 4q1321 which have a similar organization and may have arisen by gene duplication (Fisher and Young, personal communication). It is approximately 15 kb in length, containing seven exons, the first six of which are small, with most of the coding sequence located in the last exon [703,704]. The signal peptide and first two amino acids of the mature protein are coded for in exon 2, and exons 3 and 4 contain regions that are rich in tyrosine and phenylalanine. The polyglutamyl acid stretches are contained in exons 5 and 6, and the RGD region is in exon 7. The splice junctions are all type 0, which means that differential splicing would leave the codon intact. Consequently, any splice variant results in an mRNA that will remain within the reading frame and not change the coding sequence. The cDNA codes for a 320 residue protein that includes a 16 residue prepeptide such that the mature protein (unglycosylated) has a predicted molecular weight of 33,600 [702].

The promoter region contains some unusual characteristics [704,705]. There is an inverted TATA and CCAAT box in close proximity to an AP1 site (148 to 142 bp), a CRE (122 to 116 bp), and a homeobox-binding site (200 to 191 bp). A retinoic acid response element (RARE) is present and overlaps with a glucocorticoid response element (GRE, 1038 to 1022 bp). A VDRE overlapping the inverted TATA has also been identified [703]. There is a polypurine (CT rich) stretch that is also found in the osteopontin promoter [643], which can possibly take on DNA triplex conformation [706]. An AC-rich region is also present, which may take on a left-handed helical configuration. This type of structure can either stimulate or inhibit transcription of the gene [reviewed in 707]. A functional YY-1 site has been identified in intron 1 [704,707]. However, the elements that convey tissue specificity to the expression of this gene have not yet been determined. Transcription of the gene results in a mRNA of 2.0 kb, although higher molecular forms have been described [702, Beresford, personal communication]. Studies on the regulation of BSP in vitro have been hampered somewhat by the fact that it is only reproducibly produced in cultures that are actively mineralizing. Because most in vitro model systems are not mineralizing, expression levels are usually low, but detectable. An exception is the cell line UMR-106-BSP, a variant that constitutively produces high levels of BSP [696]. Biosynthesis of BSP is

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tightly coordinated with the maturational stage of osteoblastic cells. Using fetal bovine bone cell cultures, BSP mRNA was increased rapidly at the stage of matrix mineralization. These data suggest that the production of BSP is regulated by autocrine factors that are produced by the cells as they go through different stages of maturation. Studies utilizing 1,25-dihydroxyvitamin D3 have shown that unlike osteopontin, BSP synthesis is decreased. A derivative of ipriflavone (metabolite III) has been reported to increase the synthesis of bone sialoprotein [260]. BSP is relatively unique to mineralizing tissues [702,709], expressed only by those cells forming a mineralized matrix [648]. These observations, combined with solution studies showing that BSP acts as a hydroxyapatite nucleator [665] and immunohistochemical data showing that BSP expression in culture is maximal during early matrix mineralization [282,648], suggest that BSP is involved in bone mineralization [317,710,711]. The solution studies of the effect of BSP on apatite formation suggest which structural features are important in this process. When the effect of BSP on mineralization is monitored in an agar gel or at constant pH in solution, it facilitates hydroxyapatite deposition [665,712], although BSP can also block seed growth [713]. Blocking the carboxylic groups, presumably those in the polyglutamyl domains, destroys the nucleation abilities of BSP, while dephosphorylating the molecule has less of an effect. This suggests that apatite – BSP interactions occur predominantly through the polyglutamyl repeats; however, other portions of the molecule are also involved [713]. Although solution data do not prove that BSP has this same function in situ, it does demonstrate the nature of the interaction between BSP and hydroxyapatite. Effects of the variable phosphorylation of BSP remain to be determined. Numerous studies have shown that BSP is active in mediating cell attachment of a number of different cell types that is dependent on RGD [504,573,575,588,589,714]. In studies where it was noted that endogenously produced enzymes could cleave BSP into more or less defined fragments, it was noted that there is a cleavage site just upstream from the RGD that results in a large fragment that lacks RGD and a small fragment that contains RGD at the amino terminus. Surprisingly, the large fragment, which lacks RGD, was able to support cell attachment, whereas the small fragment, which contains the RGD, was relatively inactive [588]. These data suggest that there is a sequence upstream from the RGD that is capable of supporting the attachment and spreading of bone cells, whereas RGD without this upstream region is not active. Although it was initially thought that these data suggested binding of BSP to an integrin other than the v3, current data suggest that the fragments associate with different regions within the same binding site on the integrin, albeit with different affinity and efficacy 713. Because the RGD region is clustered with tyrosine phosphorylation consensus sequences (tyrosyl

145 residues preceded by an acidic residue and a  turn), it was thought that the degree of sulfation may also influence cell attachment. However, unsulfated BSP (prepared from UMR-106-BSP cells treated with the sulfation inhibitor chlorate) was able to support attachment and spreading [715]. It should be noted, however, that cell attachment is an in vitro assay that may not totally reflect the subtleties of a process that occurs in vivo. Although data are not definitive, it appears that osteoblastic cells attach to BSP via the v3 integrin (vitronectin) receptor. Data that osteoclasts use this receptor are much more definitive and several studies have demonstrated that the attachment of osteoclasts can be blocked with antibodies against it [575,589]. In addition, changes in intracellular Ca2 and pH levels in osteoclasts have been shown to be altered, indicating that the binding of BSP (and osteopontin as well) may initiate a signaling pathway and ultimately cause a change in cellular metabolism. It is not yet known what stage of osteoclast formation and/or activation these two molecules are influencing, as there is some controversy as to whether they mediate formation of the sealing zone, which is required for the formation of the extracellular lysosome-like compartment. Other potential functions include mediating interactions between cells and collagen fibrils [716]. Early studies that utilized the injection of isotopic sulfate into animals along with autoradiography and biochemical isolation techniques described a population of sulfated molecules that moved rapidly from the osteoblastic cells to the mineralization front [233,234]. In addition, microprobe analysis indicated that newly formed hydroxyapatite was in close proximity to sulfate [717]. Because proteoglycans account for virtually all of the free sulfate that is incorporated into macromolecules, it was thought that this population of sulfated macromolecules was proteoglycan(s) [233,234]. However, new data that BSP can also be sulfated may provide strong evidence for the role of BSP in matrix mineralization. There are a few suggestions that abnormal BSP metabolism may result in abnormal bone formation and/or disease. In one case, it was demonstrated that BSP was lacking in a cow with an osteopetrosis-like syndrome (Weintroub, personal communication). Given the potential role of BSP in osteoclastic resorption, this may be an indication that in some cases of osteopetrosis, the matrix required for optimal osteoclastic activity is lacking. It has been demonstrated that the production of BSP by certain primary cancers (breast, prostate, and thryoid, to name a few) coincides with the invasion of the tumor into the surrounding tissue and the appearance of mineralized foci within the primary tumor. Furthermore, the levels of BSP in the primary tumors are prognostic of long-term outcome [718,719]. Studies with carcinoma cell lines have shown that metastatic cells do in fact preferentially attach to BSP [720], and it has been

146 demonstrated that cell-bound BSP binds to factor H, a mediator in the alternate complement pathway, and may be involved in the escape of tumor cells from immunosurveillance [721] BSP knockout mice [722], which have a totally nonfunctional BSP gene, were reported to be indistinguishable from wild-type mice at birth, 8.5 days, and 1 month. However, at 1 year they were 25% smaller than the wild type. Histologically, the predominant observation in long bones and in the calvaria was a decrease in marrow space. Incisors were also elongated. X-ray diffraction of the homogenized bones of the knockout animals revealed no differences in mineral crystal relative to controls (Aubin, personal communication). Detailed analyses of spatial changes in mineral properties have not yet been reported. 6. BONE ACIDIC GLYCOPROTEIN-75 (BAG-75) Another sialoprotein originally isolated from rat bone has an apparent Mr of 75,000 and hence is called bone acidic glycoprotein-75 (BAG-75) [723 – 725]. This protein is heavily glycosylated and contains 7% sialic acid and 8% phosphate. Thirty percent of the residues are acidic in nature. It is found only in bone, dentin, and growth plate cartilage, but in culture, cells from soft connective tissues have also been found to synthesize low levels of this protein [724]. It is not known what percentage of this protein exists in bone. BAG-75 shares sequence homology with the dentin phosphoprotein, phosphophoryn, osteopontin, BSP, and dentin matrix protein and therefore may have similar structural features [723,724,726]. The structures of phosphophoryn fragments, predicted from NMR data, were discussed earlier with osteopontin. The cDNA and the gene have not yet been cloned for this molecule. However, some data are available from direct amino acid sequencing. The amino terminus is about 30% homologous with osteopontin. In addition, it does contain polyacid stretches as do osteopontin and bone sialoprotein [723,727]. However, BAG-75 contains both polyaspartate and polyglutamate domains, as well as several phosphorylation sites and an RGD cell binding site [638]. The protein binds with high affinity to both hydroxyapatite and Ca2 ions [723], as well as to collagen [727]. Immunolocalized next to cells in bone and concentrated in newly formed osteoid, this protein may combine the properties of osteopontin (a mineralization inhibitor) and bone sialoprotein (a nucleator). Preliminary studies with its homologue dentin matrix protein demonstrate that it may have both functions (Boskey, unpublished data). BAG-75 also inhibits the resorptive activity of osteoclasts, presumably by blocking access to bone mineral [728]. Related to BAG-75 is dentin matrix protein-1 [729,730] originally isolated from teeth, but now shown to be expressed by bone marrow stromal cells (Fisher, personal

GOKHALE, BOSKEY, AND ROBEY

communication). The nonphosphorylated recombinant DMP-1 [731] is a potent apatite nucleator in solution (Boskey, unpublished data). 7. MICROFIBRILLAR PROTEINS As in many connective tissues, bone matrix also contains microfibrillar structures [732]. Although the protein constituents of the microfibrills have not been completely identified, by analogy to other tissues they are likely composed of a family of proteins that include type VI collagen, fibrillins, fibulins, latent TGF-binding proteins (LTBPs), the MAGP proteins, and Big-h3 [733]. Fibrillins and LTGP are cysteine-rich glycoproteins that are related by their composition of repeating EGF motifs. Fibrillin-1 and 2 are similar but not identical components of the microfibrillar aggregates in the skeleton, as well as in eyes and vascular tissues [734,735]. The structure of fibrillin-1 was predicted by Sakai’s group [736,737] to consist of eight repeated EGF-like motifs, an RGD cellbinding region, several cysteine motifs, and a cysteine-poor COOH terminus. The structure as deduced from primary sequence is thought to be stabilized by calcium-binding EGF domains [736,738]. The proteins associate with glycoproteins, lysyloxidase, a 58-kDa microfibril associated protein, and other protein species forming beaded domains [739]. Fibrillin mutations have been found to cause Marfan’s syndrome, a disease characterized by skeletal, ocular and cardiovascular abnormalities [740]. The association of fibrillins with microfilaments and elastin implies an anchoring function. Fibrillins only form microfibrils in the presence of Ca2; the recently reported mutations in Marfan’s patients who lacked this Ca2-binding domain [738] speak to the importance of these microfibrillar structures. How the microfibrils regulate the growth of long bones is not known; however, immunolabeling in the chick embryo demonstrated labeling in the primary axis and the ventral surface of the notochord [741], suggesting a role in early development. Related to the fibrillins are the latent TGF--binding proteins (LTBPs) and, as their name implies, their proposed function includes binding to latent TGF-. However, they also appear to be microfibrillar proteins based on immunohistochemical analysis colocalizing LTPB-1 with fibrillin at light microscopic and EM levels. These studies show that in addition to regulating TGF-1 activity, LTBP1 may function as a structural component of connective tissue microfibrils. LTBP1 may therefore be a candidate gene for Marfan-related connective tissue disorders in which linkage to fibrillins has been excluded [742]. 8. OTHER CELL-BINDING PROTEINS As microanalytical techniques become more refined, both at the protein level by immunohistochemistry, radioimmunoassays, and enzyme-linked immunosorbent

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assays (ELISA) and at the nucleic acid level by reverse transcriptase polymerase chain reaction (RT-PCR), it is certain that the list of glycoproteins, including RGD-containing proteins, will continue to grow. These proteins, while not as abundant as those described earlier, most likely make significant contributions to the development of bone. As transgenic animals are generated that either lack or contain a mutated form of these molecules, and/or as chemical amounts of purified proteins are generated through the use of recombinant techniques for use in cell culture and other model systems, more information will become available on the precise roles of all the glycoproteins in bone development. Tenascin (neuronectin), originally identified in muscle, is also made in bone cells [743,744], bone marrow cells [745], and cartilage cells [746]. Tenascin has a hexameric quaternary structure, with six arms attached to a central globular domain [747]. It has been found to be associated with mesenchyme, which is differentiating into bone or cartilage [748,749], but was absent from mature tissue. This indicates that tenascin may be important for early matrix development, where it acts as a modulator of cell – extracellular matrix interactions [747]. A 36-kDa cartilage matrix protein with an RGD sequence [750], this protein has the highest affinity for fibroblasts of all the bone matrix proteins tested to date [751]. Based on protein and DNA sequencing, the protein consists of a 337 amino acid peptide chain, with 10 leucine-rich repeats surrounded by disulfide-bonded loops at each end. Based on its strong cell-binding ability and its distribution, it is most likely that this protein is involved in regulating cell adherence in a variety of connective tissues. Another proteoglycan, osteoadherin, a member of the leucin-rich repeat family, has been purified from bovine bone [752]. Biochemical analysis has shown that it has a molecular mass of 85 da and contains RGD sequences and keratan sulfate chains. It is synthesized primarily by osteoblasts and can bind to hydroxyapatite with efficiency similar to that of fibronectin. This binding appears to be mediated by integrin V3. It has been demonstrated that depending on the species, osteoadherin and osteomodulin are the same protein/gene [753]. A 90 Da protein, periostin (previously called OSF-2), is expressed preferentially in periosteum and periodontal ligaments 754, suggesting a potential role in bone and tooth formation as well as in maintenance of structure. It is secreted by osteoblasts and osteoblast-like cell lines and exists as several isoforms. Distinct from the other cell-binding proteins discussed here, it does not contain a RGD sequence (L. Bonewald, personal communication). Biochemical analysis has shown that glycosylation is not a major component of this protein. In vitro functional studies have shown that the antiperiostin antibody inhibits attachment and spreading of MC3T3-E1 cells, indicating that periostin

is involved in cell adhesion. In addition, TGF- increased the expression of periostin in primary osteoblastic cells significantly. These results indicated that periostin may play a role in recruitment and attachment of osteoblast precursors in the periosteum.

V. GLA-CONTAINING PROTEINS Bone contains a number of proteins that are modified posttranslationally by vitamin K-dependent enzymes to form the amino acid -carboxy glutamic acid (gla). Due to the sequence requirements of the carboxylating enzymes, the gla proteins of bone share some sequence homology with certain blood coagulation factors that require -carboxylation to maintain their activity.

A. Osteocalcin Osteocalcin was the first bone matrix protein to be isolated by the use of nondegradative techniques from acid demineralized bone [755,756]. It comprises up to 15% of the noncollagenous protein, although the level varies depending on the animal species [410] and accounts for up to 80% of the total gla content of mature bone [757]. It was initially reported to be virtually exclusive to bone and was considered the only bone-specific protein. Consequently, a great deal of effort has gone into trying to determine when and where osteocalcin is synthesized and how it functions in bone metabolism. Extensive screening of protein and RNA extracts [758,759] and tissue sections by immunohistochemistry [613,760 – 762] from virtually all tissues has failed to detect osteocalcin in any tissue other than dentin and bone. The one exception was marrow, which led to the discovery that megakaryocytes and platelets contain mRNA for osteocalcin (along with the mRNAs for many of the other bone matrix proteins). It is still not clear how much osteocalcin protein is actually synthesized by platelets and megakaryocytes or what its function is in these cells [611,763]. During bone development, osteocalcin production is very low and does not reach maximal levels until late stages [764 – 767]. By immunohistochemistry, mineralization fronts stain intensely for osteocalcin, but it has been difficult to demonstrate osteocalcin in osteoid and in cells. However, using an antibody against the precursor form of osteocalcin, the primary cell type that stains in developing human subperiosteal bone is the osteocyte. This antibody intensely stained the cell processes in canaliculae and suggests that perhaps osteocalcin bypasses the osteoid layer by being secreted directly at the mineralization front through the osteocytic cell processes [768] (Fig. 23). However, it should be noted that in other animal species, particularly in

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FIGURE 23

Osteocalcin immunolocalization in developing bone. Localization of osteocalcin using an antibody against the mature secreted form of the protein sharply demarcates the mineralization front (MF) in developing bone (A). Note, however, the lack of localization within cells that should be synthesizing this molecule. However, when utilizing an antibody raised against the precursor peptide (which is not maintained within mineralized matrix), it can be seen that osteoid osteocytes and osteocytes contain high levels of the proform of the molecule (B and C). Courtesy of Dr. Paolo Bianco.

rat, endosteal osteoblasts are positive for osteocalcin [758,760,761]. The protein has a molecular weight of 5300 but migrates with an apparent Mr of 14,000 Da on SDS-PAGE. The entire sequence has been determined by direct protein sequencing [769,770]. Depending on the animal species, there is one intramolecular disulfide bond and three to five residues of -carboxy glutamic acid [756] (Fig. 24). The original structural predictions by Hauschka and Carr [771] based on circular dichroism suggested that osteocalcin had a structure with an extensive (40%)  helix in the presence of calcium ions. As detailed elsewhere [772], the predicted structure of osteocalcin in the presence of Ca2 consists of two antiparallel -helical domains, one containing -carboxyglutamic acid residues and one rich in acidic amino acids. Both these domains were proposed as sites for calcium chelation. The -carboxyglutamic acids were calculated to be 0.5 nm apart, corresponding to the 0.55-nm interatomic spacing of Ca2 ions in the 001 plane of the apatite lattice, suggesting that this domain might be involved in binding to the mineral. A -pleated sheet in the C terminus was suggested as a cell-binding site. More recent insight into the osteocalcin structure comes from comparisons of NMR data for Ca2 and Pb2 salts [773] and Ca2 and Lu3 (lutecium) salts [774]. These NMR studies show that Pb and Lu3 compete for Ca2 binding sites. Because Pb2 blocks the binding of osteocalcin to hydroxyapatite in

FIGURE 24 This small molecule contains two stretches of  helix (depicted as cylinders) and two regions of -pleated sheet (arrows). The carboxylated residues of glutamic acid in the amino-terminal helix orient the carboxy groups to the exterior, thereby conferring calcium ion binding with relatively high affinity. There is one intramolecular disulfide bridge (C – C) in the middle region of the molecule. Adapted from Hauschka and Carr, Biochemistry 21, 258 – 272 (1985).

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solution, such data imply that the osteocalcin – apatite interaction occurs through the same domain as Ca2 chelation in solution. Comparison of Lu3 and Ca2 data for the dog apoprotein demonstrate, the presence of two high-affinitybinding sites for Ca2 and the conformational changes that occur when Ca2 is present. The human gene is localized on chromosome 1 [775] and the structure has been determined [776]. The gene is 1.2 kb with four exons that predict a protein of 125 amino acids. The signal peptide contains 26 amino acids in exon 1, a propeptide of 49 amino acids in exon 2 along with the -carboxylation recognition sequence, two stretches that become -carboxylated in exon 3, and the remainder of the molecule and untranslated region in exon 4 [777,778]. Interestingly, the mouse genome contains three osteocalcin genes, two of which are activated in bone and one activated in the kidney [754]. Although some of the basic elements have been determined in the human promoter, most of the extensive characterization has been done primarily in rodent promoters. It contains a TATA box and a CCAAT box. In addition there is one NF1-binding site and one AP2-binding site, a viral core enhancer, and a CRE. There is a VDRE at 463 to 437 bp [779] that is flanked by other nuclear-binding sites [780 – 783]. A GRE is found in close proximity to the TATA (16 to 1 bp) [784]. A vitamin A response element has also been identified [785]. Apparent AP1 binding sites are also located in close proximity to, or overlapping with, the VDRE and the CAAT box [786]. Because of the highly specific nature of osteocalcin expression, the promoter has been scrutinized intensely to determine what properties convey tissue specificity. This has led to the characterization of the “osteocalcin box” [787,788], located between 99 and 76 bp, which is functionally active [789,790] and contains a binding site for Msx-1 or Msx-2 (homeodomain proteins). Msx-related factors have been found to be synthesized by osteoblastic cells [791]. The first intron has also been characterized and found to have a potential silencer [792,793]. Further characterization of this promoter led to the identification of a binding site, OSE2, located between bp 146 and 132, which binds the transcription factor, cbfa1, the so-called osteogenic “master gene”[794]. The effects of growth factors and hormones on osteocalcin vary somewhat, which most likely reflects differences in the culture systems under investigation (stage of maturity, length of time in culture, etc.). Osteocalcin synthesis is upregulated by 1,25-dihydroxyvitamin D3 [781 – 783,788, 795 – 797] and by 22-oxacalcitriol [798]. In general, most factors decrease osteocalcin expression, such as PTH [799], glucocorticoids [800,801], TGF- [802,803], PGE2 [799], IL-1 [804,805], TNF- [804], IL-10 [90], and lead [91]. Mechanical loading has also been reported to have a negative effect [806].

149 The proposed functions for osteocalcin have been reviewed extensively [807,808]. Because osteocalcin has a high and relatively specific affinity for apatite, probably due to the binding of the gla domain to the 100 (a axis) face of the apatite crystal, the protein has been proposed as a specific regulator of the length of the mineral crystals in bone. Osteocalcin is not expressed in culture until mineralization starts [808,809], which fits the model that it is a regulator of the size and habit of the mineral crystals rather than a promoter of mineral crystal formation. In solution, osteocalcin is an effective crystal growth inhibitor [429,810], whereas -carboxyglutamate-free osteocalcin has no such effect, which is in good agreement with the proposed function. Similarly, during new bone formation, osteocalcin staining and expression occur after mineralization starts [811,812], demonstrating its function in later stages of bone formation and remodeling. Osteocalcin is made by osteoblasts/osteocytes and is considered to be a marker of osteoblastic function [813,814] as well as a coupler of osteoblast – osteoclast action. It also appears to be important for induction of the osteoclast phenotype [815]. Studies probing the function of osteocalcin have shown that osteocalcin-deficient bone particles are not resorbed readily [816], in good agreement with the report that osteocalcin is involved in osteoclast recruitment [817,818]. This concept is also supported by the defective osteocalcin production noted in some humans and animals with osteopetrosis, a severely deforming disease characterized by the failure to remodel bone and calcified cartilage [819 – 821]. Osteocalcin may have other functions. The osteoclastrecruiting function has been questioned based on earlier observations that animals with osteocalcin-deficient bones due to warfarin treatment fail to show significant abnormalities in bone growth and fracture healing [755]. These animals did show excessive growth plate mineralization [822], which could be interpreted as a failure to remodel calcified cartilage. Whether this remodeling effect was due to the failure of osteocalcin to bind to the mineral in calcified cartilage or to a defect in the actions of matrix-gla protein (see earlier discussion), which is produced in the cartilage, is not known. As reviewed here, sufficient data link osteocalcin to a regulatory role in bone mineral maturation. It is likely, however, that several proteins have a similar function, thus it will be difficult to prove this function in vivo. Lambs that lack -carboxylated osteocalcin show decrease in both new bone formation and bone resorption [823], supporting a role for osteocalcin in remodeling. Mice with a null allele (both bone genes deleted) appear normal at birth and at 4 weeks, but by 24 weeks, they have increased bone mass. Preliminary analysis suggested a role for osteocalcin in regulating bone growth in vivo 794. Mineral crystals in the bones of osteocalcin-null animals fail to mature [824], supporting its role in bone remodeling.

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B. Matrix Gla Protein Studies that measured the content of gla residues in bone during development indicated that gla-containing proteins were present at earlier stages of development than could be accounted for by the expression of osteocalcin, the major gla-containing protein in bone. Consequently, the presence of matrix gla protein, MGP, was predicted prior to its actual isolation and purification. It was first isolated from bone due to its copurification with BMP [825,826]. While there is little information on the developmental expression of MGP throughout the body, it is known that it is expressed in lung, kidney, heart, cartilage [827], and smooth muscle cells [828]. MGP is more abundant in cartilage than in bone [829]. In the skeleton, MGP expression appears early and remains at the same level at all subsequent stages of development [765]. However, close examination of early developmental stages indicates that MGP expression is much more limited, found only in lung bud and at the limb bud mesenchyme – epithelial interface [830]. MGP has a Mr 15,000 although it migrates as a substantially larger molecule on SDS-PAGE. The secreted form contains five residues of gla and one disulfide bridge in a 77 to 79 amino acid residue protein. A distinct physical property of matrix gla protein is its insolubility in physiologic solutions (10 g/ml) and its tendency to selfassociate via hydrophobic interactions. It also appears that a propeptide present at the carboxy terminus is removed to form the mature protein [831]. Matrix gla proteins from five different species have been shown to have phosphorylated serine residues [832,833]. Thus the protein is a phosphorylated gla protein. Due to its insolubility, along with difficulties in isolating a purified protein, it has been difficult to predict the potential structure of MGP, and detailed conformational studies are not yet available. However, cDNA sequences for several species allowed the primary structure to be predicted [832,833]. The MGP gene has been localized to 12p [834]. The gene is approximately 3.9 kb long and contains four exons. The signal peptide is coded for by exon 1 and an helical region by exon 2. The recognition sequence for the carboxylating enzymes is found in exon 3, and a sequence that actually becomes -carboxylated is in exon 4. There are a series of AluI repeats in the 3 -untranslated region of the gene. The promoter has been characterized and found to contain a TATA box and a CAAT box, along with a perfect palindromic sequence that is similar to a RARE [835]. Little is known about the factors that regulate the synthesis of MGP. In ROS 17/2.8 cells, 1,25-dihydroxyvitamin D3 was found to stimulate its synthesis, although higher levels were needed than for the induction of osteocalcin. It is also apparent that retinoic acid induces MGP expression greatly, which may be highly significant for cartilage development [835].

The matrix gla protein (along with osteocalcin) was initially suggested to be important for the process of endochondral ossification because warfarin-treated rats showed premature epiphyseal closure [822], indicative of impaired remodeling of calcified cartilage. At the time of the report, the matrix gla protein had not been identified, but because cartilage chondrocytes were not thought to produce osteocalcin, a second gla protein was postulated. When the matrix gla protein was identified as a chondrocyte product [827], warfarin data were reinterpreted to consider a potential role for the matrix gla protein. The findings that retinoic acid, a known teratogen, stimulated matrix gla protein gene expression, coupled with the observation that matrix gla protein copurifies with the bone morphogenetic proteins, led Price to suggest that the matrix gla protein may mediate effects of retinoic acid during cellular differentiation, perhaps by serving as a natural carrier for BMP. A recent discovery indicates that the matrix gla protein can be found in a variety of soft tissues and that its mRNA is abundant in lung tissue, although the gla protein content was low [836]. This suggests that the protein has a more general secretory function. Mice that have their MGP gene deleted die prematurely because of massive tracheal cartilage and blood vessels calcification [828]. The endochondral cartilage in these animals is also excessively mineralized, but trabecular and cortical bones appear comparable in mineral properties to age-matched controls [837]. This is convincing evidence that MGP is an in vivo inhibitor of mineralization. This has further been shown in cell culture studies in which ablation of the MGP to sternal chondrocyte cultures resulted in dystrophic mineralization, whereas the addition of exogenous MGP prevented calcification under conditions in which mineralization is normally observed [838]. Additional insights into MGP function may come from reports that in breast cancer cells there is an increase in the expression of MGP [839]. Interestingly, in prostate cells undergoing apoptosis, there is also an increase in MGP [840]. In light of data in knockout animals and in cell culture, it seems likely that expression of this protein may be a protective action by the cell against unwanted calcification. Expression of MGP in cells from non-onion fractures, but not healing fractures, indicates its importance in bone healing [841].

C. Protein S This gla-containing protein is synthesized primarily in the liver, but has been isolated from bone matrix [842]. Although synthesis was demonstrated in osteosarcoma cells [768,842], it has not been reported to be synthesized endogenously in bone. However, it should be noted that patients with protein S deficiency suffer from osteopenia, and

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consequently this protein, irrespective of its origin, most likely plays a role in bone metabolism.

VI. OTHER PROTEINS A. Proteolipids Proteolipids, as a general class of macromolecules, are membrane components, consisting of a hydrophobic protein component and covalently bound lipid [843,844]. Proteolipids have been isolated from a variety of connective tissues, including bone [845 – 848] and calcified cartilage [849,850], where there are cell and matrix vesicle membrane components [851]. Based on analyses of the apoprotein amino acid compositions, it is clear that there may be more than one type of proteolipid component in bone. Although the primary structures of several of the bone and/or cartilage proteolipid apoproteins have been determined [852 – 854], their detailed structure is not known. Although an oral bacterial proteolipid associated with calcification has been sequenced [855], structures of the bone and cartilage proteolipids have not yet been described. However, the structures of several other proteolipids that appear to have common features have been described in detail. The protein conformations of a variety of proteolipid proteins have been determined using NMR [856], fluorescent labeling [857,858], electron microscopy and quasi-elastic light scattering [859], and Fourier transform infrared spectroscopy [860]. In general, these transmembrane proteins have hydrophobic domains throughout the molecule, including N and C termini. In many cases, the transmembrane domain  helices are highly ordered, although they have abundant hydrophobic residues (e.g., polyvaline). These hydrophobic domains facilitate interactions with the lipids in the membranes in which the proteolipids are contained. In contrast, the N- and C-termini are generally flexibly disordered and contain covalently linked lipids, such as palmitoyl-cysteine or acidic phospholipids. Bone and related cartilage proteolipids have several functions. Based on studies with the bacterial analogue of the chondrocyte proteolipid [848,861], a role in proton transport has been postulated [862,863]. This activity is quite distinct from that of another chondrocyte proteolipid, which is a phosphate ion transporter, taking phosphate ions into and out of the cell [864]. They have also been demonstrated to act as hydroxyapatite nucleators both in vitro [845,847,850], in a gelatin gel [865], and when implanted in a millipore chamber in vivo [866]. The calcifiable proteolipids are associated [867,868] with a complex consisting of decreasing molar amounts of calcium, the acidic phospholipids, and inorganic phosphate [869]. These complexes are capable of acting as hydroxyapatite nucleators in the presence, as well as in the absence, of the proteolipid

apoprotein [867,870,871]. Such complexes were also shown to be components of the membranes of extracellular matrix vesicles [872] and to be able to act as ion transporters [873]. Proteolipids isolated from cartilage matrix vesicles have been shown to include the annexins [874]. Annexins are Ca2 and phospholipid-binding proteins [875] previously known as lipocortin, calpactin, endonexin, chromobindin, anchorin, and so on [876]. Annexins have also been shown to have ion transporter roles [877], but other functions are also known. Lipocortin I is a phospholipase A2 inhibitor [878], and anchorin is a collagen-and cytoskeletal-binding protein [879]. The annexins are synthesized by osteoblasts [880] as well as by chondrocytes [855,881] and are abundant in matrix vesicle membranes where they are involved in the initiation of calcification [882]. Annexins share a 17 amino acid residue homology, which is probably important for the Ca2-dependent phospholipid binding [879]. Wu et al. [851] have reported that the “nucleational core complex,” i.e., the essential structure needed to cause hydroxyapatite formation in matrix vesicles, consists of annexins (i.e., proteolipid), Ca2, Pi2, and phosphatidylserine. This is in good agreement with earlier reports indicating that cartilage proteolipids and their associated complexed acidic phospholipids were hydroxyapatite nucleators. Because proteolipids are concentrated in matrix vesicle membranes [872] and because most are involved in ion transport, these proteolipids seem to be of general importance in accumulating ions within the cell and/or extracellular matrix vesicles. As ions accumulate within vesicles, in the presence of proteolipids and phosphatidylserine, mineral crystals form associated with the membranes. Thus, the two functions of the proteolipids may be closely related.

B. Enzymes and Inhibitors It is apparent from the slow destruction of bone matrix proteins that occurs with increasing age [883,884] that enzymes are present within the mineralized matrix. The origin of these enzymes can be from the circulation due to their affinity for hydroxyapatite or from neighboring cells that are highly associated in bone such as endothelial cells, hematopoietic cells, and cartilage cells. In addition, cells in the osteoblastic lineage also synthesize a number of enzymes and their inhibitors. A source of enzymatic activity that has often been overlooked is the osteoclasts themselves, which secrete lysosomal types of enzymes into the extracellular compartment formed by the sealing zone. 1. METALLOPROTEINASES The matrix metalloproteinases (MMPs) [reviewed in 885] are a family of enzymes that can be roughly divided into collagenases, which produce the initial cleavage of

152 native, triple helical collagen; gelatinases, which further degrade the structurally compromised collagen, and stromelysins, which degrade proteoglycans. However, there is some degree of overlap among these groups. It has long been known that connective tissue cells that are synthesizing extracellular matrix proteins also have the capacity to synthesize proteins that destroy the very same proteins. Osteoblastic cells are no exception, and it has been reported that they synthesize collagenase-1 (MMP-1) [886], collagenase-3 (MMP-13) [887], gelatinase A (MMP-2) [888] and gelatinase B (MMP-9), [889 – 892], and MT1-MMP (vide infra). Some osteoblastic cells have been found to bear a cell surface receptor for collagenase. In addition, matrix vesicles contain MMPs [893] along with a stromelysin-like activity that has the ability to degrade proteoglycans [894,895]. Osteoclasts also have been reported to contain collagenases [896,897]. To date, a number of transgenic animals have been generated that are deficient in an MMP, but generally did not display a skeletal defect. The one exception is the recently reported MT1-MMP knockout mouse, which while normal at birth quickly develops a severe skeletal phenotype characterized by dwarfism, osteopenia, and arthritis [898]. Tissue inhibitors (TIMPs, forms 1 and 2) inhibit the activity of metalloproteinases [899]. However, it is not clear to date at which point this activity is produced. Depending on the cell culture system, it is apparent that there is a great deal of variability in the ability to produce these enzymes and inhibitors. In those cell culture systems producing MMPs, it has been found that MMP activity is stimulated by PTH [891,900], TNF- [901], and retinoic acid [902]. The crystal structures of two of the matrix metalloproteases [903] and one form of collagenase [904] have been resolved. These are both members of the stromelysin III family (to date, 11 such enzymes have been identified). Structural features include a histidine-rich -helical catalytic domain that chelates the metal ion required for activity [905], a C-terminal domain that is involved in linking the enzyme to the membrane, and pre- and pro-N-terminal domains that are thought to be responsible for maintaining the enzyme in inactive form via interaction with TIMP [906]. The catalytic domain is a spherical region with a methionine-based turn containing Zn2 and Ca2-binding sites, with proline-86, phenylalanine-79, and aspartate-232 forming the base of the active site residues [904]. The crystal structure of elastase [906], the enzyme responsible for the degradation of elastin, has similar features. The crystal structure of the active site of collagenase [905,907] shows the presence of a five-stranded -pleated sheet, three  helices, and binding sites for Ca2 and Zn2, but here the zinc is chelated only by histidines. Collagenase cleaves at a unique site in the collagen triple helix [908] and at a minor site in the nonhelical N-terminal

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region. Activated by tyrosine kinase-dependent phosphorylation, collagenase-mediated turnover of the bone matrix seems essential during growth and repair, but not during early development. Thus, homozygous transgenic mice whose type I collagen does not contain the unique cleavage site appear normal at birth, but develop thickened skin, uteri, and bone during growth and have impaired fracture healing [909]. In bone, osteoclasts produce several cysteine proteases as well as metalloproteases. It is believed that during osteoclastic resorption the cysteine proteases that have acidic optimal pHs act first [910,911]. Then, as the mineral is dissolved in the acidic environment and the acidity is neutralized, the matrix metalloproteases function. Specific inhibitors of the cysteine proteases have been used effectively to inhibit osteoclastic resorption [912]. Such inhibitors, while blocking the actions of the cysteine proteases, increase the activities of some lysosomal enzymes [913]. As reviewed in detail elsewhere [911], metalloproteases degrade proteoglycans, collagens, and matrix proteins [914 – 916]. One of the most important of the cysteine protease degradative enzymes in bone may be cathepsin K, as this enzyme is expressed mainly by osteoclasts and appears to initiate the bone degradation process. In this light, cathepsin K knockout animals have osteopetrosis associated with abnormal matrix degradation but normal mineral resorption [917,918]. 2. PLASMINOGEN ACTIVATOR (PA) AND PLASMINOGEN ACTIVATOR INHIBITOR (PAI) Another enzyme system that is represented in bone matrix is plasminogen activator (both urinary and tissue types, uPA and tPA) and PAI-1 [919, reviewed in 920]. PA and PAI have been identified in both transformed and nontransformed cell culture systems [921], (Kopp and Gehron Robey, unpublished results). However, it is also possible that enzymic activity detected in bone matrix can also originate from other neighboring cell types. The proposed functions of these proteins are varied. They range from the controlled activation of other enzymes, such as matrix metalloproteinases, to the activation of growth factors, such as TGF- and the IGFs that are bound and modulated by the IGF-binding proteins. The synthesis of enzymes and inhibitors is regulated by a number of growth factors and hormones, and in general they are modulated in different directions; i.e., if the enzyme goes up the inhibitor goes down, and vice versa. PA is upregulated by PTH [921 – 924], 1,25-dihydroxyvitamin D3 [925], and acidic and basic FGF, EGF, and PDGF [926]. PAI is concurrently decreased by these factors. Activity is either upregulated or downregulated by TGF-, depending on the cell type [927], although there is generally an increase in PAI-1 [926,928]. PA activity is decreased and PAI activity is increased by glucocorticoids [929].

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3. MATRIX PHOSPHOPROTEIN KINASES A third category of enzymes that appear to be critical for the formation of mineralized connective tissues are the extracellular matrix phosphoprotein kinases. Matrix protein kinases isolated from bone and dentin [930 – 934] are casein II kinases, whose activities can be inhibited by heparin and 2,3-diphosphoglycerate. Analogous to some tyrosine kinases found in the extracellular matrix [935], these phosphoprotein kinases are responsible for the extracellular addition of phosphate to noncollagenous matrix proteins [936]. Deficient phosphorylation due to altered casein kinase II activity has been reported in the hypophosphatemic mouse, an animal model of human hypophosphatemic rickets, that is resistant to phosphate and vitamin D treatment [937]. Further, because there are protein kinases in the extracellular matrix, it is likely that phosphoprotein phosphatases are also present.

C. Bone Morphogenetic Proteins (BMPs) The existence of BMPs (as a concept rather than as isolated proteins) was first suggested by Huggins [938] and later by Urist [939] who observed that the demineralized bone matrix, when implanted in an ectopic site, would cause cartilage and bone formation. Their observations led to an intense search to purify and characterize the factor present in bone. What followed was the identification of a class of proteins that is now recognized as the TGF- superfamily, which include the BMPs, and are capable of inducing bone when implanted into various sites within test animals. The BMPs, with or without carriers, alone or in combination, all seem to have the ability to cause mesenchymal cells to differentiate, and hence to induce bone formation (see Chapter 5 by Olsen). Effects of BMPs on cell morphology and function, proliferation, and differentiation have been reviewed in detail elsewhere [940 – 942]. However, it is now clear that there is a great deal of redundancy and that these proteins are not specific to bone. It is also not yet known how the temporal and spatial pattern of expression of these proteins controls developmental processes. The recognition that the response to these proteins is mediated by cell surface receptors [943] has opened the door to a rapidly expanding field, which will provide a great deal of insight into how this class of proteins functions. Six of the seven well-characterized BMPs are members of the TGF- superfamily whose primary structures have been elucidated by molecular cloning [941]. Of these, only BMP-1 does not have the TGF- structure. The carboxy-terminal regions of BMP-2 – BMP-7 have high structural homology with seven cysteine residues at conserved locations. The mature protein (lacking the propeptides) forms dimers. As reviewed by Wozney and co-workers [941,944,945],

these may exist as hetero-or homodimers, each with different activities. All of the mature BMPs have basic amino termini, which persist following the removal of long propeptides. The mature chains are also glycosylated. BMP-1, in contrast, has domains resembling those in metallo endoproteases and the C1 and C1q of components of complement. The crystal structure of one of the TGF-s (TGF-2) has been determined [946]. It has been shown by NMR [947] that it has an extended rather than a globular structure without a hydrophobic core due to the arrangement of the four disulfide bonds, which form the so-called “TGF- knot’’ in one section of the molecule. Such extended structures common to the TGF- superfamily may be important for interaction with the receptors of the TGF s, which are transmembrane serine-threonine kinases [948]. Based on sequence analyses, it has been found that all of the BMPs have hydrophobic leader sequences, suggesting that they are secreted proteins. This is in line with their proposed functions as growth factor bone-inducing agents. The identification of the defect in the short-ear mutant mouse [949] as an abnormality in the BMP5 gene led to the suggestion that BMPs are involved in the formation, patterning, and repair of specific morphological features. BMP has also been linked to the patterning in the developing chick limb [950]. Another illustration of effects of BMPs is seen in mouse model of fibrodysplasia ossificans progressive, which lacks this protein [951]. Studies have identified BMP receptors on mesenchymal cells, providing a clue as to how these soluble bone derived proteins may function as growth factors in situ. These types of studies have shown that the BMP family is functionally redundant in that phenotypic changes often are not apparent unless more than one of these factors is knocked out [952] (see Chapter 5).

D. Growth Factors The skeletal matrix and mineral are repositories for growth factors [953 – 956], such as the transforming growth factor- family [941], insulin-like growth factors [957], platelet-derived growth factor [958], basic and acidic fibroblast growth factors [953], interleukins [959,960], granulocyte – macrophage colony-stimulating factor [961], tumor necrosis factor [962], and the bone morphogenetic proteins (see earlier discussion). Many of these growth factors are products of bone cells as well as other cells and have specific receptors on osteoblasts, osteoclasts, and osteocytes [963]. These receptors include cell surface receptors that are linked to G-proteins, have directly linked kinase activities [964], and/or that are channel linked [965]. Thus they serve autocrine (self-regulating) and paracrine (regulating other cells) functions [966]. Their actions are regulated by factors such as systemic hormones and by mechanical stress.

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The actions of these growth factors in bone are being reported at an exponential rate and have been reviewed by Gowen et al. [962], Rosen et al. [957], and Yoneda [967] (see also Chapter 14). In general these growth factors have mitogenic and proliferative activities in bone, and many influence the expression of specific phenotypic proteins, thereby affecting both bone formation and resorption. The ability to affect these processes depends on the presence of receptors for the particular growth factor or cytokine. The mechanism of action of cytokine binding to such receptors generally involves action of a transmembrane protein kinase, which in turn results in phosphorylation or dephosphorylation of multiple proteins in the signal transduction cascade [948].

E. Serum Proteins (2-HS-Glycoprotein, Fetuin, Albumin, etc.) The list of nonstructural proteins that have been identified in bone that originate from serum and become entrapped in bone is quite lengthy [968]. This ability of bone to adsorb such a broad spectrum of proteins makes it uniquely suited to be the reservoir of multiple growth factors or proteins that may be required and liberated at least in part by bone resorption. Albumin, 2-HS-glycoprotein, fetuin, transferrin, 1-antitrypsin, 1-antichymotrypsin, IgG, haptoglobulin, hemopexin, serum cholinesterase, and soluble fibronectin are among the plasma proteins that accumulate in bone in detectable amounts [969 – 971]. Their accumulation is most likely due to their binding to hydroxyapatite. This affinity for hydroxyapatite is frequently the basis of the separation of different classes of the immunoglobulins [972], removal of interfering plasma constituents [973] and other purification schemes. Human 2-HS-glycoprotein is produced in the liver and circulates in the bloodstream [974]. There is some debate as to whether connective tissue cells can produce the protein, but it is apparent that it specifically accumulates in mineralized tissues. The high concentration of 2-HS-glycoprotein in bone cannot be due entirely to the presence of blood in the tissue, as the relative amount is too high and the ratio of the protein to albumin is enhanced 100-fold in bone relative to blood [970]. Chondrocytes have been shown to express 2-HSglycoprotein where the protein appears to enhance endochondral ossification. The message is absent from normal human bone cell lines [975], indicating that it probably accumulates in human bone by adsorption from the circulation. In rodents a bone sialoprotein has been shown to have high homology with the 2-HS-glycoprotein [976,977]. However, while Ohnishi et al. [977] found that the protein was expressed by osteoblasts, immunolocalization of the analogous protein by Mizuno et al. [978] failed to show its

presence within the cell per se, implying that like the human homologue it is deposited in bone from other sources. 2-HS-Glycoprotein consists of two nonidentical glycosylated peptide chains (chains A and B) that are held together by disulfide bonds [979,980]. These subunits are characterized by repeating Ala-Ala and Pro-Pro sequences. The initial biosynthetic product is a single polypeptide that is cleaved to form the A and B chains of the mature molecule [981]. The major glycoside is sialic acid [982]. Prediction of the primary structure and chemical digestion studies have led to descriptions of the domain structure of the protein [983,984]. In addition to the single disulfide bond linking the two individual chains by their extreme NH2 and COOH ends, there are five intradisulfide bonds in the A (heavy) chain. The light B chain has no intrachain S – S bonds. The A chain is composed of three domains, consisting of S – S linked loops. Of the five loops that span 4- to 19-amino-acid residues, two highly homologous loops form one domain, flanked on either side by the other tandem repeats. This structure is typical of that of bovine fetuin [985], a member of the cystatin superfamily, and of the histidine-rich glycoprotein family [984]. The cystatin family proteins all have “cystatin-like” domains of linearly arranged disulfide linked loops [984,986] and a variety of N-glycosides [987] that vary within the family, and for the same protein they vary with species. Members of the cystatin family are inhibitors of cysteine proteases. The 2HS-glycoprotein is also homologous to a nonphosphorylated sialoprotein found in rodent bone [976,977]; however, the rodent counterpart of 2-HS-glycoprotein consists of one rather than two chains. 2-HS-Glycoprotein can bind TGF-/BMP cytokines and block their osteogenic activity in culture. Studies with dexamethasone-treated rat bone marrow cell cultures have shown that recombinant fetuin as well as native serum protein can inhibit osteogenesis by similar efficiency. Northern blots showed that both 2-HS-glycoprotein and high doses of TGF- suppressed transcripts of alkaline phosphatase, osteopontin, collagen type I, and bone sialoprotein. These data suggest that 2-HS-glycoprotein (together with TGF) is involved in regulation of osteogenesis in remodeling bone [988]. The human gene sequence for 2-HS-glycoprotein is known [989]. The gene is on chromosome 3, and two RFLPs have been identified. The single mRNA species predicts an 18 residue signal peptide, followed by a sequence that codes for the A and B peptides with an intervening sequence between them. This sequence is presumably lost during cleavage of the precursor to form the mature molecule. In tissues other than bone, 2-HS-glycoprotein has several functions. It has been shown to have opsonic properties [971], to promote endocytosis, and to bind DNA [990,991].

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In liver, 2-HS-glycoprotein (pp63) plays an essential role in insulin signaling, inhibiting the insulin-dependent tyrosine kinase activity of the insulin receptor [992] without competing with insulin binding. It is also believed to be involved in the immune response [993,994]. 2-HS-Glycoprotein has been suggested to play a variety of roles in bone, but none of these has been proven conclusively. It appears at selected times during development and may be involved in differentiation [995]. This idea is supported by earlier studies demonstrating that 2-HS-glycoprotein is found in higher concentrations in young bone as opposed to mature bone [996,997]. The protein appears to have a higher affinity for calcium phosphates than other serum proteins, as the addition of calcium and phosphate to serum led to the removal of all the 2-HS-glycoprotein but removed less than 1% of the albumin [970]. 2-HS-Glycoprotein is also believed to be involved in bone mineralization [998], as decreased levels of this protein are seen in bones with abnormal mineralization pattern due to malnourishment, certain malignancies [993], and Paget’s disease [970]. In contrast, 2-HS-glycoprotein levels are high in some patients with OI [996]. The suggestion that this glycoprotein might affect mineralization is also supported by solution studies. In fact, the ability of serum to inhibit the solution-mediated conversion of amorphous calcium phosphate to hydroxyapatite [999] was attributed to the presence of this high-affinity glycoprotein. This was later confirmed in unpublished in vitro studies where purified 2-HS-glycoprotein was seen to be a much better inhibitor than other serum proteins. The 2-HS-glycoprotein-deficient mouse did not show skeletal abnormalities [1000]; however, the serum from these animals did not inhibit apatite formation as efficiently as that from wildtype animals. The mutant animals also developed ectopic calcifications, confirming the role of this protein as a serum inhibitor of calcification. Other studies suggest that the protein is involved in the recruitment of osteoclast precursors to resorptive sites [817,1001]. Because of its homology to cysteine protease inhibitors, a role in regulating bone resorption seems likely, and it may be that the high concentrations in very young bone may be related to the need to limit remodeling during development. Whole serum [1002] and albumin have been shown to inhibit hydroxyapatite growth in solution [1002 – 1004]. The ability of albumin to inhibit apatite growth is attributed to the affinity of albumin for apatite [973,1005 – 1007]. Specifically, albumin at 50 – 250 g/ml alters the linear rate of growth of apatite seed crystals by binding to the mineral on several faces [1003] and blocking the growth of crystal agglomerates [1004]. Although the primary function of albumin in bone is not apt to be one of regulation of mineralization, the extent of inhibition of hydroxyapatite growth in solution indicated that phosphoproteins were more effective

inhibitors than albumin. Ions such as citrate and magnesium were also less effective in retarding apatite growth in solution [1003]. Transferrin [1008], IgG, IgE, and the other serum proteins also bind to apatite. Studies from the Laboratories of Borkey and Heywood indicated that IgG had no effect on hydroxyapatite formation, morphology, or growth. None of the other serum proteins has been reported to have any effect on either inhibition or promotion of mineralization. Effects on other events in bone matrix deposition are not known; however, because these serum proteins increase in concentration as mineralization progresses [970], it is unlikely that they play significant roles in the bone deposition process.

VII. REQUIREMENTS FOR MATRIX MINERALIZATION Analyses of diseased tissues and tissues from transgenic animals indicate that there are a number of cellular and extracellular factors essential for physiologic mineral deposition. Physiologic mineral deposition is distinguished here from dystrophic or pathologic mineral deposition by its ordered, characteristic appearance on an oriented collagenous matrix. In contrast, in dystrophic calcium phosphate deposition in general, and dystrophic apatite deposition in particular, the mineral crystals are often not collagen associated. They surround and engulf cells and may be longer in size and different in included ion composition from physiologic mineral [1009,1010]. From the just described definition, it is apparent that for physiologic mineral deposition, there must be an appropriate collagen-based matrix. This is emphasized by (a) the smaller size of hydroxyapatite crystals in OI bone [157] and (b) the relative abundance of mineral that is not associated with collagen in these bones with deficient and/or impaired collagen production [158]. Although in some cases the defective mineralization in the OI bones may also be attributed to altered matrix protein production [167] or retention, collagen is clearly an absolute requirement for physiologic bone mineralization. Similarly, because fibronectin forms the basis on which collagen is deposited, it must be also a requirement. Equally apparent from this definition is the essential presence of Ca2+ and Pi ions for mineralization. Calcium ions may be supplied from the cells or from circulating or localized calcium-binding proteins. Phosphate ions may be derived from the breakdown of pyrophosphate, an abundant metabolic product, from hydrolysis of phosphoesters or phosphoproteins, or from circulating Pi ions. The exact Ca2 and Pi content of the extracellular fluid of bone is not known, but in cartilage, micropuncture studies showed the pH to be 7.58, and total Ca2 and total Pi to be 1 – 12 and 3 – 12 mg/dl, respectively [231]. For the formation of

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apatite, a basic environment is also required, and many of the highly anionic matrix proteins probably contribute to creating this environment. Which of these matrix proteins is truly essential for mineralization of bone can only be guessed until the sequence of protein expression is determined precisely and appropriate knockout and transgenic models are developed. Even in these cases it may be difficult to prove an essential role for mineralization, as it is already apparent that there are redundant controls of this critical process. It is certain that the cells are required for the production of a physiologic matrix, synthesizing and exporting necessary enzymes, growth factors, and matrix molecules. As discussed later, the formation of extracellular matrix vesicles is also apt to prove critical for the initiation of mineralization in some cases.

VIII. PATHWAYS OF MATRIX MINERALIZATION Apatite crystals found in bone are distinct from most naturally occurring geological crystals, being smaller and containing more impurities. In addition, bone apatite crystals have a plate-like habit [1011], are arranged in an oriented fashion on a collagen-based matrix, and have a very limited size distribution [19]. In general, the mineral crystals in bone (and dentin) are smaller than those in enamel [1012] and in dystrophic deposits in severely atherosclerotic plaques [1013] or other soft tissue calcifications [202]. The bone mineral crystals do vary in size with tissue site, age, and disease [157,1014], but the range in the lengths of the smallest bone mineral crystals and their orientation implies that their growth must be regulated. Bone mineralization is thus distinct from solution-mediated Ca2 phosphate precipitation where similarly sized, nonoriented small crystals that are formed can ripen to appreciably larger sizes [1015]. This is also distinct from geologic apatite formation where high temperatures and pressures yield extremely large single crystals.

A. Physical Chemistry of Mineralization Calcium phosphate precipitation from solution can yield a variety of phases, depending on the pH, Ca to Pi ratio, and solution supersaturation [21,1015,1016]. When the pH is in the physiologic range (7.4 – 7.8), apatite formation occurs with solution Ca:P molar ratios as high as 2 to 1 and as low as 1 to 1 as long as the solution is supersaturated with respect to apatite (has a Ca P product that exceeds the solubility product for apatite). Depending on the supersaturation, intermediate phases such as amorphous calcium phosphate [1017], octacalcium phosphate [1018 – 1020], or

other intermediates may form [21,1021], but in all these cases, the final product is apatitic. Apatite crystals develop in solution when individual ions or ion clusters associate in the same orientation as in the crystal lattice that they are trying to form. When sufficient ion clusters are oriented correctly, they can persist in solution and can serve as a “critical nucleus” for further crystal growth. Homogeneous nucleation, in which crystals form de novo, is a rare process [1015]. Thus it is likely that in most instances of solution-mediated apatite deposition, nucleation occurs on foreign materials such as dust, scratches on the container, and buret tips. Such heterogeneous nucleation yields the initial crystals, which then facilitate additional growth by the process of secondary nucleation. In secondary nucleation, growth sites on the preformed apatite crystals serve as branch points for the formation of new crystals, analogous in many ways to the branching of the growing glycogen molecule during glycogenesis. Proliferation by secondary nucleation results in numerous small crystals. Crystal growth, in the absence of secondary nucleation, would result in fewer, but larger crystals. This suggests that most of the crystals in bone form by a secondary nucleation-like process or by growth from individual nuclei. Unfortunately, what regulates crystal size in bone cannot be determined from studies of proteinfree solutions. The mineral in bone, as in other physiologically calcified tissues, is associated with an organic matrix (Fig. 25). The protein(s) within such matrices can alter the nucleation and growth of mineral crystals in several ways. When isolated in an environment relatively free of body fluids, the protein(s) can chelate Ca2 or Pi ions, reducing the fluid supersaturations, which in turn would prevent crystal nucleation and/or growth. The protein(s) can form a protected environment around the crystal nucleus, sequestering it and thus preventing crystal growth, or stabilizing the nucleus, protecting it from the external environment. The protein(s) may bind Ca2 and/or Pi ions, forming a surface that resembles the apatite surface. In this manner, the protein serves as an epitaxial (similar surface) nucleator, thereby providing a surface for the start of nucleation. The protein(s) may also bind to one or more faces of the growing crystal because its side chains match positions in the lattice, thereby blocking growth in one or more directions or even blocking growth beyond a specific size. The protein(s) might also bind to other proteins, changing their conformation and their ability to affect crystal nucleation and growth according to the pathways described earlier, or might associate with cells resulting in a change in the extracellular Ca P concentration, or pH. The elegant ultrastructural studies of Addadi and colleagues that combined X-ray crystallographic and electron microscopic techniques provide illustrations for each of these mechanisms for the formation of larger crystals of

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FIGURE 25

Cell-mediated matrix mineralization in developing bone. Early mineralization in chick bone. Electron micrograph showing a 17-day-old embryonic tibia, stained with uranyl acetate and lead citrate. Mineral clusters (C) outside the osteoblast (OB) are associated with collagen (thin arrows) and extracellular matrix vesicles (inset). Empty vesicles (thick arrows) as well as vesicles with mineral are seen. Courtesy of Dr. Steven B. Doty.

calcium carbonates, calcium sulfates, brushite, and octacalcium phosphate [163,1022,1023]. For example, fibronectin has been shown to bind to the ionic surfaces of calcite that did not include water molecules, but does not bind at all to brushite whose surfaces all have bound water [1024]. The acidic macromolecules from sea animals have been shown to determine the shape of calcite crystals [1025]. Cells have been shown to interact with specific faces on such crystals in the presence and absence of RGD-containing macromolecules [1026]. Scanning electron micrographs have similarly been used to identify the binding sites for polyaspartic acid, mollusk shell proteins, and rat dentin phosphoprotein on the surface of octacalcium phosphate [1024]. There are also examples of each of these mechanisms from solution studies of apatite formation. For these, to date, there is no direct evidence of the exact nature of the protein – mineral interaction. Studies of the effects of bone matrix proteins on apatite formation include studies in which preformed seed crystals are added to Ca P solutions, and the rate of crystal growth is determined at fixed Ca P and fixed pH [21] or variable Ca P OH [1021].

Other studies have looked at the formation (nucleation and growth) of apatite from solutions in the presence of insoluble proteins, proteins immobilized on polyanionic beads [1027], or proteins in solution [462]. Diffusion studies in which the protein is held within an agarose [664], silicate [712], or denatured collagen [202] gel have also provided insight into apatite nucleation and growth. From such studies, one can find examples of the mechanisms listed earlier. It should be emphasized, however, that a protein, because of its affinity for apatite, may, in low concentrations, act as a nucleator and in higher concentrations serve to regulate crystal growth. The multifunctional roles of each of the bone matrix proteins should be apparent from the previous sections of this review. The extracellular matrix vesicles (Fig. 25, inset, discussed later) and their component lipids may facilitate Ca and P accumulation, while shielding the apatite nucleus. As reviewed in detail elsewhere [1028], illustration of this behavior in vesicles is seen in the iron oxide forming bacteria and in model liposomes. In model liposomes, where Ca2 accumulation is facilitated by an ionophore, initial mineral

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crystals form inside the liposomes in association with the liposome membrane where they eventually grow and puncture the liposome membrane and become exposed to the external solution [1029 – 1032]. Proteins that can prevent apatite nucleation by chelating Ca2 include aggrecan and its associated proteoglycan aggregates [200]. This may be more complicated in vivo where free Ca2 levels may be affected by Donnan equilibrium effects. However, it has been shown that phosphate can then cause the release of this Ca2, facilitating the mineralization process [664,1028]. An example of a protein that appears to block crystal growth by binding to a specific surface or surfaces is osteopontin, which binds with high specificity and decreases the length of the crystals formed in the collagen-gel system [663]. This is in contrast to the dentin phosphoprotein phosphophoryn, which blocks secondary nucleation by binding to growth sites [667]. Immobilized albumin, osteocalcin, and osteonectin appear to act as nucleators [1027], although evidence shows that the beads on which these proteins are immobilized are themselves nucleators. Fibronectin has also been shown to be capable of nucleating apatite in solution [577]. The only bone/dentin specific matrix proteins shown to act as nucleators are bone sialoprotein [665,712,1033], biglycan [1034], and dentin matrix protein 1 (Boskey, unpublished data). When stripped of its associated matrix proteins, collagen itself, does not nucleate apatite, but when its associated phosphoproteins are present, apatite nucleation on collagen occurs in a dose-dependent manner directly related to the extent of protein phosphorylation [124,660]. Collagen peptides containing the carboxylate region, but not the Nterminal domain can also nucleate apatite in solution [1035].The complexed acidic phospholipids and their associated proteolipids [848] and lipoproteins [851] also nucleate apatite formation in solution [851,1036] and when implanted in a cell-free environment in vivo [866].

B. Cell-Regulated Mineralization In each of the just mentioned solution-mediated studies, hydroxyapatite formation was performed in the absence of cells. However, it is the cells that regulate bone mineralization by producing and secreting the appropriate extracellular matrix components, regulating their interactions, and controlling the flux of Ca, P, and OH ions in their extracellular environments. One must look to cell-mediated studies to understand the pathways of matrix mineralization in vitro [reviewed in 1037]. Unfortunately, most of these cellmediated studies have been concerned with understanding the origin and differentiation of the bone cells rather than with the mineralization process per se. However, from analyses of the cell culture systems, one can learn much about mineralization. Another complicating issue with the

cell culture studies is that most rely on 10 mM -glycerolphosphate, an excellent substrate for alkaline phosphatase, as a source of inorganic phosphate. The hydrolysis of 10 mM -glycerophosphate results in inorganic phosphate concentrations two to five times higher than physiologic levels, in turn producing a medium Ca P product much higher (10 mM2) than physiologic (2 – 5 mM2). In such high Ca P medium, larger, nonoriented mineral crystals distinct from those that exist in situ may be formed [1010,1038]. Organ culture for the study of bone formation was first described by Dame Honor Fell in 1925 [1039]. Similar studies of cultured limb rudiments have been used more recently to show stages of differentiation and the effects of a variety of hormones and growth factors on bone formation [1010,1040]. Tenenbaum et al. used an inverted folded chick periosteum model as their organ culture system [1041,1042] with similar results. The limitation of the organ culture systems for the study of the mineralization process is the variety of cell types present. For that reason, isolated osteoblast cultures or cultures of cells that differentiate into osteoblasts and form a mineralized matrix may be preferable. Based on the system described by Friedenstein et al. [1043], Owen [1044], Ashton et al. [1045], and Benayahu et al. [1046], many investigators have stimulated marrow stem cell cultures to differentiate into osteoblasts [reviewed in 1047]. In these, and in isolated osteoblast systems, steroids such as dexamethasone [1048 – 1050], retinoic acid [383], BMP [1051], and -glycerophosphate [1052] are thought to enhance this differentiation. Osteoblasts isolated from fetal tissues, e.g., fetal rat calvariae, are commonly used to study osteoblasts, as are tumor-derived osteoblast-like cells. Examples of these include ROS17/2.8 [6], MG-63 [1053], SaOS [1054], and MC3T3-E1 [1055] cell lines. Methods have also been developed for isolating and culturing cells from more mature human (and animal) bones [9] and immortalized cell lines [1056 – 1058]. As techniques emerge, there will undoubtedly be an increase in the number of cell culture model systems that will exhibit several phenotypic traits. However, it will have to be determined by stringent criteria (physiological matrix mineralization verified at the EM level) whether these model systems will be useful in studying late stages of bone formation (i.e., matrix mineralization). Rat calvarial cells plated at low density yield isolated colonies, some of which are “osteoblast like” based on morphology, matrix composition, alkaline phosphatase expression, and mineralization [525,1059,1060]. In these osteoblast nodules/colonies, active type I collagen expression follows cell proliferation and then is reduced to baseline levels, whereas alkaline phosphatase activity and expression, osteopontin, collagenase, and osteocalcin expression commence after this. In fact, in such cultures, osteocalcin expression is highly correlated with mineral accretion

CHAPTER 4 The Biochemistry of Bone

[814]. BSP, osteopontin, and osteocalcin expression continue to increase as mineralization proceeds [10]. Mineral deposition in such cultures has been characterized by electron microscopy and wide angle X-ray diffraction [1061 – 1063] and shown to contain hydroxyapatite crystals, although not always oriented in the same direction as the collagen fibrils. These types of cell and organ culture systems have allowed the definition of the proposed sequential events involved in the recruitment and proliferation of osteoprogenitor cells and their differentiation into osteoblasts, and the temporal changes in the expression of bone matrix proteins [10]. Consistently, in each of the different model systems [9,11,608,814,1064,1065], osteopontin, collagen, osteonectin, and BSP expression have been shown to precede mineral deposition, whereas osteocalcin expression occurs after mineral deposition has commenced [10]. This pattern is consistent with the functions suggested for these proteins in this chapter, and although such cultures have not yet defined which of these proteins are necessary for mineralization, they did shed some light on the debate concerning the site of initial calcification in bone. In the 1960s the identification of extracellular matrix vesicles (MV) [1066,1067] as the location of the first mineral deposits in a variety of mineralizing tissues, including membranous bone [1068] and calcifying cartilage [1066,1067,1069], led to the suggestion that these membrane-bound bodies were the site of initial calcification in these tissues. Later studies, reviewed by Anderson [1070,1071] and Wuthier et al. [1072] revealed that these bodies were enriched in enzymes associated with mineralization, among them alkaline phosphatase, ATPase, and proteoglycan-degrading enzymes. The MV membranes contained proteolipids capable of facilitating Ca ion transport into the vesicles, and also had matrix collagens (type II, IX, and X) associated with them [1073]. Tissue slices, suspensions of isolated vesicles, and liposome models of matrix vesicles, in solutions of calcium and phosphate, each led to the deposition of apatite. The mechanism seemed to involve the transport of Ca ions into a vesicle that already contained a high concentration of phosphate ions [1073], formation and growth of vesicle associated mineral crystals, and rupture of the vesicle and the spread of larger mineral crystals into the extravesicular environment. As discussed earlier, the vesicle in solution served the purpose of providing a protected/stabilizing environment for the formation of the first crystals. When inhibitors of mineralization were included with the vesicles, mineralization was facilitated over vesicle-free controls [1074]. Vesicles also serve to provide enzymes that can modify the extracellular matrix, facilitating the proliferation of crystals. Extracellular matrix vesicles have been reported to be the site of initial mineralization in bone and cartilage and organ culture systems as described earlier. In such systems,

159 the mineral-associated vesicles are seen prior to the appearance of bulk mineral. One of the major issues in the debate between those that believe that collagen-based matrix is the initial site of mineralization and those who believe mineralization starts in vesicles concerns how the mineral would travel from the vesicles to its highly aligned/oriented sites on the collagen. Culture data support the matrix vesicle theory but do not prove a link between vesicle and collagenmediated mineralization. An association between vesicles and collagen suggests such a link may exist [1075]. The application of computerized axial tomographs to high-resolution electron micrographs by Landis has provided some explanations. Looking at the highly oriented calcified turkey tendon [159], mineral was seen in vesicles at sites different from mineral associated with the collagen. However, at certain sites, the mineral could be seen branching (showing the link between vesicle and collagen associated mineral). Thus when viewed in three dimensions, it appeared that the mineral does not “swim” but rather grows out radially, extending to the collagen fibril. The question remains whether this mineral associates with mineral already in the collagen or whether mineral from the vesicle manages to grow out to specific sites along the collagen fibril. From the simplistic point of view, the former seems more likely, as annealing of crystals does take place. To date, there is no evidence that collagen-based mineralization cannot occur in the absence of vesicles. Further, because mineralization starts at numerous discrete sites within the collagen fibrils, it has been argued that there would not be sufficient space for matrix vesicles to serve as so many distinct nuclei [19]. In contrast, from solution-based data, it is clear that there are matrix proteins associated with the collagen that can act as nucleators, implying that the latter mechanism may predominate. Collagen fibrils depleted of matrix proteins are not mineralized readily in solution. Collagen mineralization as seen at the EM level starts at specific sites, the so-called e bands, where several matrix proteins are known to localize [125]. Some of the matrix proteins in these sites may shield others, preventing nucleation. Others associated with the collagen may serve as nucleators, as described earlier, allowing the oriented deposition of mineral crystals. The growth of these crystals may also become limited by the space available in the collagen fibrils and by the adsorption of other matrix proteins. From the material presented in this review, we can suggest that (a) decorin, which trims the collagen fibrils and disappears from the e bands in mineralized fibrils [117], matrix gla-protein, and 2-HS glycoprotein are among the shielding agents, (b) bone sialoprotein and other phosphorylated proteins, which are closely associated with the collagen hole zones, serve as nucleators, and (c) several of the other matrix proteins produced by osteoblasts may play a role in regulating crystal size.

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Acknowledgments The authors thank Drs. Paolo Bianco, Wojciech J. Grzesik, Neal S. Fedarko, and Steven B. Doty for providing photographic materials and Luz Ingles for secretarial assistance. Dr. Boskey’s research as discussed in this review was supported by NIH Grants DE04141, AR037661, and AR41325.

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CHAPTER 4 The Biochemistry of Bone

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