Vitamins and Hormones, Volume 66 (Vitamins and Hormones)

Vitamins and Hormones VOLUME 66 Editorial Board Tadhg P. Begley Anthony R. Means Bert W. O’Malley Lynn Riddiford Arm...

Author: Gerald Litwack (Editor)

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Vitamins and Hormones VOLUME 66

Editorial Board

Tadhg P. Begley Anthony R. Means Bert W. O’Malley Lynn Riddiford Armen H. Tashjian, Jr.

Vitamins and Hormones ADVANCES IN RESEARCH AND APPLICATIONS

Editor-in-Chief

Gerald Litwack Department of Biochemistry and Molecular Pharmacology Jefferson Medical College Thomas Jefferson University Philadelphia, Pennsylvania

VOLUME 66

Amsterdam Boston Heidelberg London New York Oxford Paris San Diego San Francisco Singapore Sydney Tokyo

This book is printed on acid-free paper. Copyright ß 2003, Elsevier Science (USA). All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the Publisher. The appearance of the code at the bottom of the first page of a chapter in this book indicates the Publisher’s consent that copies of the chapter may be made for personal or internal use of specific clients. This consent is given on the condition, however, that the copier pay the stated per copy fee through the Copyright Clearance Center, Inc. (222 Rosewood Drive, Danvers, Massachusetts 01923), for copying beyond that permitted by Sections 107 or 108 of the U.S. Copyright Law. This consent does not extend to other kinds of copying, such as copying for general dustribution, for advertising or promotional purposes, for creating new collective works, or for resale. Copy fees for pre-2003 chapters are as shown on the title pages. If no fee code appears on the title page, the copy fee is the same as for current chapters. 0083-6729/2003 $35.00 Permissions may be sought directly from Elsevier’s Science & Technology Rights Department in Oxford, UK: phone: (+44) 1865 843830, fax: (+44) 1865 853333, e-mail: [email protected]. You may also complete your request on-line via the Elsevier Science homepage (http://elsevier.com), by selecting ‘‘Customer Support’’ and then ‘‘Obtaining Permissions.’’

Academic Press An Elsevier Science Imprint. 525 B Street, Suite 1900, San Diego, California 92101-4495, USA http://www.academicpress.com

Academic Press 84 Theobald’s Road, London WC1X 8RR, UK http://www.academicpress.com International Standard Book Number: 0-12-709866-6 PRINTED IN THE UNITED STATES OF AMERICA 03 04 05 06 07 08 9 8 7 6 5 4 3

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Former Editors

Robert S. Harris

Kenneth V. Thimann

Newton, Massachusetts

University of California Santa Cruz, California

John A. Lorraine University of Edinburgh Edinburgh, Scotland

Ira G. Wool University of Chicago Chicago, Illinois

Paul L. Munson University of North Carolina Chapel Hill, North Carolina

Egon Diczfalusy Karolinska Sjukhuset Stockholm, Sweden

John Glover

Robert Olsen

University of Liverpool Liverpool England

School of Medicine State University of New York at Stony Brook Stony Brook, New York

Gerald D. Aurbach Metabolic Diseases Branch National Institute of Diabetes and Digestive and Kidney Diseases National Institutes of Health Bethesda, Maryland

Donald B. McCormick Department of Biochemistry Emory University School of Medicine Atlanta, Georgia

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Contents

Contributors Preface xix

xv

1 Molecular Biology of Hematopoietic Stem Cells Ulrich Steidel, Ralf Kronenwett, Simona Martin, and Rainer Haas I. II. III. IV. V. VI. VII.

Introduction 2 Immunological and Functional Characteristics 3 Cell Cycle and Differentiation Control 6 Adhesion Molecules in Hematopoiesis and Stem Cell Trafficking Aging and Telomeres 13 Trandifferentiation and Developmental Plasticity 17 Conclusions 19 References 19

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2 Aldosterone: Its Receptor, Target Genes, and Actions David Pearce, Aditi bhargava, and Timothy J. Cole I. II. III. IV. V. VI. VII. VIII.

Introduction 30 Physiological Actions of Aldosterone 32 Molecular Basis of Mineralocorticoid Action 37 Aldosterone Action in Epithelia: Afforded by 11-Hydroxysteroid Dehydrogenase 2 42 Genetic Mouse Models in the Investigation of Aldosterone Action 46 Aldosterone Target Genes That Mediate Physiological Responses 48 Controversies with Aldosterone 57 Concluding Remarks 61 References 62

3 Corticosteroid Receptors, 11-Hydroxysteroid Dehydrogenase, and the Heart Karen E. Sheppard I. II. III. IV. V. VI. VII.

Introduction 79 Corticosteroid Hormones 79 Corticosteroid Receptors 80 Mechanism of Action of Corticosteroid Receptor Modulators of Corticosteroid Signaling 87 Heart 91 Summary 101 References 102

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4 Forms of Mineralocorticoid Hypertension Paolo Ferrari and Olivier Bonny I. II. III. IV. V. VI. VII.

Introduction 114 Evolution, Salt, and the Renin-Angiotensin-Aldosterone System Key Elements of Mineralocorticoid Activity 117 Mineralocorticoid Hypertension 124 Primary Aldosteronism 125 Genetic Forms of Mineralocorticoid Hypertension 135 Aldosterone–Dependent Essential Hypertension 142 References 143

5 Peroxisome Proliferator-Activated Receptors and the Cardiovascular System Yuqing E. Chen, Mingui Fu, Jifeng Zhang, Xiaojun Zhu, Yiming Lin, Mukaila A. Akinbami, and Qing Song I. II. III. IV. V. VI. VII. VIII.

Introduction 158 Discovery, Structure, and Tissue Distribution of PPARs, PPARs Ligands 160 Mechanisms of Action of PPARs 162 PPAR in the Cardiovascular System 163 PPAR in the Cardiovascular System 170 PPAR in the Cardiovascular System 173 Conclusions 175 References 176

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6 Serotonin and the Neuroendocrine Regulation of the Hypothalamic-PituitaryAdrenal Axis in Health and Disease N. R. Sullivan Hanley and L. D. Van De Kar I. II. III. IV. V. VI.

Overview of Serotonin 190 Neuroanatomy of the Hypothalamic-Pituitary-Adrenal Axis 195 Serotonin and the Hypothalamic-Pituitary-Adrenal Axis 203 Physiological Interactions 212 Pathophysiological Interactions 220 Concluding Remarks 228 References 229

7 The Thymosins Prothymosin , Parathymosin, and -Thymosin: Structure and Function Ewald Hannappel and Thomas Huff I. II. III. IV. V. VI.

Introduction 258 Polypeptide 1 259 -Thymosins and Prothymosin Parathymosin 270 -Thymosins 273 Conclusions 284 References 285

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8 Thymosin 4 Interactions Michael R. Bubb I. II. III. IV.

-Thymosin Structure 298 Thymosin 4 and the Actin Cytoskeleton 299 Assays for Thymosin 4-Actin Interactions 306 Ternary Complexes 309

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V. Thymosin 4 Ligands in Immunity and Inflammation References 313

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9 Polypeptide Hormones: Signaling Molecules in Plants Paul Chilley I. II. III. IV. V. VI. VII. VIII. IX.

Introduction 318 Systemin and Systemin-like Peptides 318 Rapid Alkalinization Factor (RALF) 321 ENOD40 and Root Nodulation 322 CLAVATA3 and Meristem Organization 324 Phytosulfokines 327 Brassica Self-Incompatibility 330 Polaris (PLS) 334 Conclusion 337 References 337

10 Parathyroid Hormone-Related Protein (PTHrP): A Nucleocytoplasmic Shuttling Protein with Distinct Paracrine and Intracrine Roles David A. Jans, Rachel J. Thomas, and Matthew T. Gillespie I. II. III. IV. V. VI. VII.

Introduction 346 Paracrine and Intracrine Actions of PTHrP 347 The Nuclear Import Mechanism of PTHrP 356 Nuclear Transport of Polypeptide Ligands 368 Nuclear Export Pathway of PTHrP 370 Functional Role of PTHrP in the Nucleus/Nucleolus Future Prospects 374 References 375

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11 Nerve Growth Factor-Dependent Regulation of Nade-Induced Apoptosis Jun Mukai, Peter Suvant, and Taka-Aki Sato I. II. III. IV. V. VI. VII. VIII. IX.

Background 386 Structural Features of NADE 389 NADE Isoforms 390 Genomic Structure of NADE Genes 391 Expression of NADE 392 Association of NADE with p75NTR 393 14-3-3 Protein Interacts with NADE 395 NADE Is Involved in NGF-Induces Apoptosis via p75NTR Future Directions 398 References 399

12 Membrane Transport of Folates Larry H. Matherly and David Goldman I. Introduction 405 II. Reduced Folate Carrier (RFC), a Member of the SLC19 Family of Transporters 405 III. Transport of Folates by SLC21 Organic Anion Carriers 427 IV. Folate Transporters That Operate Optimally at Low pH: The Mechanism of Folate Transport in Intestinal Cells 428 V. The Family of Folate Receptors (FRs) 430 VI. Multidrug Resistance-Associated Proteins (MRPs) and Their Impact on the Transport of Folates 434 VII. Transport of Folates by Other ABC Exporters 436 VIII. Factors That Influence Concentrative Folate Transport in Cells 437 IX. The Localization of Folate Transport in Epithelia 439 X. The Role if Folate Transporters in Mouse Development 441 References 441

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13 Vitamin A and Infancy: Biochemical, Functional, and Clinical Aspects Silverio Perrotta, Bruno Nobili, Francesca Rossi, Daniela Di Pinto, Valeria Cucciolla, Adriana Borriello, Adriana Oliva, and Fulvio Della Ragione I. A Premise 458 II. Vitamin A: Intestinal Digestion, Absorption, and Tissue Delivery 459 III. Intracellular Metabolism 464 IV. Retinol and Embryogenesis: Mechanism of Action and Importance 470 V. Retinol and Infancy 499 VI. Altered Vitamin A Levels and Childhood Pathologies 509 VII. Few Final Considerations 535 References 536 Index

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Contributors

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

Mukaila A. Akinbami (157) Cardiovascular Research Institute, Morehouse School of Medicine, Atlanta Georgia. Aditi Bhargava (29) Department of Medicine and Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, California. Olivier Bonny (113) Division of Nephrology and Hypertension, Inselspital, University of Berne, Berne, Switzerland. Adriana Borriello (458) Department of Biochemistry and Biophysics ‘‘F. Cedrangolo,’’ Medical School, Second University of Naples, Naples, Italy. Michael R. Bubb (297) Department of Medicine, University of Florida, Gainesville, Florida. Yuqing E. Chen (157) Cardiovascular Research Institute, Morehouse School of Medicine, Atlanta Georgia. Paul Chilley (317) The Integrative Cell Biology Laboratory, School of Biological and Biomedical Sciences, University of Durham, South Road, Durham. Timothy J. Cole (29) Department if Biochemistry and Molecular Biology, University of Melbourne, Melbourne, Australia. Valeria Cucciolla (458) Department of Biochemistry and Biophysics ‘‘F. Cedrangolo,’’ Medical School, Second University of Naples, Naples, Italy.

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Paolo Ferrari (113) Division of Nephrology and Hypertension, Inselspital, University of Berne, Berne, Switzerland. Mingui Fu (157) Cardiovascular Research Institute, Morehouse School of Medicine, Atlanta Georgia. Matthew T. Gillespie (345) St. Vincent’s Institute of Medical Research, Fitzroy, Australia. David Goldman (403) The Departments of Medicine and Molecular Pharmacology, Albert Einstein Cancer Center, and Albert Einstein College of Medicine, Bronx, New York. Rainer Haas (1) Department of Hematlogy, Oncology, and Clinical Immunology, Heinrich Heine University of Du¨sseldorf, Du¨sseldorf, Germany. Ewald Hannappel (257) Institute for Biochemistry/Faculty of Medicine, University of Erlangen-Nu¨rnburg, Erlangen, Germany. Thomas Huff (257) Institute for Biochemistry/Faculty of Medicine, University of Erlangen-Nu¨rnburg, Erlangen, Germany. David A. Jans (345) Nuclear Signaling Laboratory, Department of Biochemistry and Molecular Biology, Monash University, Australia. L. D. Van de Kar (189) Department of Pharmacology, Center for Serotonin Disorders, Loyola University of Chicago, Stritch School of Medicine, Maywood, Illinois. Ralf Kronenwett (1) Department of Hematology, Oncology, and Clinical Immunology, Heinrich Heine University of Du¨sseldorf, Du¨sseldorf, Germany. Yiming Lin (157) Cardiovascular Research Institute, Morehouse School of Medicine, Atlanta Georgia. Simona Martin (1) Department of Hematology, Oncology, and Clinical Immunology, Heinrich Heine University of Du¨sseldorf, Du¨sseldorf, Germany. Larry H. Matherly (403) The Experimental and Clinical Therapeutics Program, Barbara Ann Karmanos Cancer Institute, and the Department of Pharmacology, Wayne State University School of Medicine, Detroit, Michigan. Jun Mukai (385) Division of Molecular Oncology, Department of Otolaryngology/Head & Neck Surgery and Pathology, College of Physicians & Surgeons, Columbia University, New York, New York. Bruno Nobili (458) Department of Pediatrics, Second University of Naples, Naples, Italy. Adriana Oliva (458) Department of Biochemistry and Biophysics ‘‘F. Cedrangolo,’’ Medical School, Second University of Naples, Naples, Italy.

Contributors

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David Pearce (29) Department of Medicine and Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, California. Silverio Perrotta (458) Department of Pediatrics, Second University of Naples, Naples, Italy. Daniela Di Pinto (458) Department of Pediatrics, Second University of Naples, Naples, Italy. Fulvio Della Ragione (458) Department of Biochemistry and Biophysics ‘‘F. Cedrangolo,’’ Medical School, Second University of Naples, Naples, Italy. Francesca Rossi (458) Department of Pediatrics, Second University of Naples, Naples, Italy. Taka-aki Sato (385) Division of Molecular Oncology, Department of Otolaryngology/Head & Neck Surgery and Pathology, College of Physicians & Surgeons, Columbia University, New York, New York. Karen E. Sheppard (77) Molecular Physiology Laboratory, Baker Heart Institute, Melbourne, Australia. Qing Song (157) Cardiovascular Research Institute, Morehouse School of Medicine, Atlanta Georgia. Ulrich Steidl (1) Department of Hematology, Oncology, and Clinical Immunology, Heinrich Heine University of Du¨sseldorf, Du¨sseldorf, Germany. N. R. Sullivan Hanley (189) Department of Pharmacology, Center for Serotonin Disorders, Loyola University of Chicago, Stritch School of Medicine, Maywood, Illinois. Petro Suvant (385) Division of Molecular Oncology, Department of Otolaryngology/Head & Neck Surgery and Pathology, College of Physicians & Surgeons, Columbia University, New York, New York. Rachel J. Thomas (345) St. Vincent’s Institute of Medical Research, Fitzroy, Australia. Jifeng Zhang (157) Cardiovascular Research Institute, Morehouse School of Medicine, Atlanta Georgia. Xiaojun Zhu (157) Cardiovascular Research Institute, Morehouse School of Medicine, Atlanta Georgia.

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Preface

This volume of Volumes and Hormones begins with a review of a general topic on the Molecular Biology of Hematopoietic Stem Cells by U. Steidl, R. Kronenwett, S. Martin, and R. Haas. A group of reviews follow relating to steroid hormones. The first is entitled Aldosterone: Its Receptor, Target Genes, and Actions by D. Pearce, A. Bhargava, and T. J. Cole. K. E. Sheppard follows with Corticosteroid Receptors, 11-Hydroxysteroid Dehydrogenase, and the Heart. P. Ferrari and O. Bonny write on Forms of Mineralocorticoid Hypertension. A contribution then appears on PPARs and the Cardiovascular System by Y. E. Chen, M. Fu, J. Zhang, X. Zhu, Y. Lin, M. A. Akinbami, and Q. Song. This grouping is completed by a paper entitled Serotonin and the Neuroendocrine Regulation of the Hypothalamic-Pituitary-Adrenal-Axis in Health and Disease by N. R. Sullivan-Hanley and L. D. Van de Kar. Peptide hormones are represented by five reviews, starting with The Thymosins: Prothymosin , Parathymosin, and -Thymosins by E. Hannappel and T. Huff. A complementary review of Thymosin 4 Interactions appears by M. R. Bubb. P. Chilley reviewed plant hormones in Polypeptide Hormones: Signaling Molecules in Plants. Next comes Parathyroid Hormone-Related Protein (PTHrP): A Nucleocytoplasmic Shuttling Protein with Distinct Paracrine and Intracrine Roles by D. A. Jans, R. J. Thomas, and M. T. Gillespie. NGF-Dependent Regulation of NADE-Induced Apoptosis by J. Mukai, P. Suvant, and T. A. Sato ends this group. The volume is completed by two papers on vitamins. The first is Membrane Transport of Folates by L. H. Matherly and I. D. Goldman. The last is

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entitled Vitamin A and Infancy: Biochemical, Functional, and Clinical Aspects by S. Perrotta, B. Nobili, F. Rossi, D. Di Pinto, V. Cucciolla, A. Borriello, A. Oliva, and F. Della Ragione. The Editor-in-Chief would like to draw your attention to the new cover, which appears for the second time after volume 65, and also the format within. The aim is to serve a wide audience with up-to-date reviews of emerging areas of interest. Gerald Litwack Philadelphia November, 2002

1 Molecular Biology of Hematopoietic Stem Cells Ulrich Steidl, Ralf Kronenwett, Simona Martin, and Rainer Haas Department of Hematology, Oncology, and Clinical Immunology, Heinrich Heine University of Du¨sseldorf, D-40225 Du¨sseldorf, Germany

I. II. III. IV.

Introduction Immunological and Functional Characteristics Cell Cycle and Differentiation Control Adhesion Molecules in Hematopoiesis and Stem Cell Trafficking V. Aging and Telomeres VI. Trandifferentiation and Developmental Plasticity VII. Conclusions References

Human CD34+ hematopoietic stem and progenitor cells are capable of maintaining a life-long supply of the entire spectrum of blood cells dependent on systemic needs. Recent studies suggest that hematopoietic stem cells are, beyond their hematopoietic potential, able to differentiate into nonhematopoietic cell types, which could open novel avenues in the field of cellular therapy. Here, we concentrate on the molecular biology underlying basic features of hematopoietic stem cells. Immunofluorescence analyses, culture assays, and transplantation models permit an extensive immunological as well as functional characterization of human hematopoietic stem and progenitor cells. New methods such Vitamins and Hormones Volume 66

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Copyright 2003, Elsevier Science (USA). All rights reserved. 0083-6729/03 $35.00

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as cDNA array technology have demonstrated that distinct gene expression patterns of transcription factors and cell cycle genes molecularly control self-renewal, differentiation, and proliferation. Furthermore, several adhesion molecules have been shown to play an important role in the regulation of hematopoiesis and stem cell trafficking. Progress has also been made in elucidating molecular mechanisms of stem cell aging that limit replicative potential. Finally, more recent data provide the first molecular basis for a better understanding of transdifferentiation and developmental plasticity of hematopoietic stem cells. These findings could be helpful for nonhematopoietic cell therapeutic approaches. ß 2003 Elsevier Science (USA).

I. INTRODUCTION Hematopoietic stem cells are able to maintain a life-long supply of the entire spectrum of blood cells, dependent on the varying systemic demands of the individual. Hematopoiesis physiologically depends on a precisely regulated equipoise of self-renewal, differentiation, and migration of more or less mature hematopoietic stem and progenitor cells. The knowledge of these key features of hematopoietic cells provides the basis for the clinical use of hematopoietic stem cells in the autologous or allogeneic transplant setting for the treatment of patients with malignant or autoimmune diseases. Hematopoietic stem cells for transplantation can be collected either from bone marrow or peripheral blood after mobilization with cytotoxic chemotherapy, cytokines, or both. For example, granulocyte colonystimulating factor (G-CSF) can be given to patients during the period of hematological reconstitution postchemotherapy or to healthy donors during steady state hematopoiesis to increase the number of circulating stem cells. Hematopoietic stem cells from bone marrow for transplantation have been used over the last three decades, whereas transplantation of blood-derived hematopoietic stem cells was performed for the first time in 1986 (Korbling et al., 1986) and has become more and more popular (Haas et al., 1994a,b, 1995a,b, 1997; Hohaus et al., 1997; Voso et al., 1999, 2000; Kobbe et al., 2002a,b). Autologous transplantation of hematopoietic stem cells permits hematological recovery after myeloablative therapy, thus allowing cytotoxic high-dose therapies for patients with multiple myeloma, non-Hodgkin’s lymphoma, breast cancer, lung cancer, or sarcoma. Beyond intensified cytotoxic treatment, allogeneic transplantation of hematopoietic stem cells offers additional therapeutic opportunities. On the one hand, it permits potentially curative treatment of malignant diseases involving hematopoietic stem and progenitor cells; on the other hand, allogeneic transplants bear an immunotherapeutic capacity to control or eradicate residual malignant cells

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of the recipient. Those therapeutic principles are used in the treatment of patients with acute and chronic myelogeneous leukemia, myelodysplastic syndromes, and aplastic anemia. By exploiting the graft-versus-tumor effect, allogeneic blood stem cell transplantation has also been envisaged for patients with solid tumors such as breast or lung cancer. Transplantation of stem cells from peripheral blood comprises several steps, beginning with mobilization and harvest of stem cells, cryopreservation and thawing, transfusion, as well as homing and engraftment of the cells. Ideally, this multistep process leads to hematological reconstitution and maintainance of long-term hematopoiesis. In addition to the profound clinical experience in hematopoietic stem cell transplantation gained during the last 30 years, we have obtained a better understanding of each of these steps at a molecular level. New methods such as cDNA array technology and proteomics have further accelerated our progress in understanding stem cell physiology by allowing diversified molecular insight into functional genomics of hematopoietic stem cells. More recently, several pivotal experiments have demonstrated that hematopoietic stem cells are, beyond their hematopoietic potential, able to differentiate into a variety of nonhematopoietic cell types such as hepatocytes, cardiomyocytes, endothelial cells, and cells of the nervous system (Lagasse et al., 2000; Mezey et al., 2000; Orlic et al., 2001a,b; Hess et al., 2002). These observations could be the basis for the development of new treatments for patients with myocardial or cerebral infarction as well as degenerative disorders (Mezey et al., 2000; Lagasse et al., 2000; Orlic et al., 2001a,b). However, novel data have challenged the transdifferentiation model by suggesting cell fusion rather than plasticity of stem cells (Ying et al., 2002; Terada et al., 2002). Our molecular understanding of the communicational skills and the signaling pathways of hematopoietic stem and progenitor cells initiating and mediating transdifferentiation is still poor. Here, we review immunological and functional characteristics as well as the molecular biology of human hematopoietic stem cells with respect to basic stem cell capabilities such as transcriptional regulation, cell cycling, self-renewal and differentiation, migration and trafficking, as well as cellular aging. In the last section, we describe and discuss findings that contribute to our molecular understanding of stem cell plasticity and transdifferentiation.

II. IMMUNOLOGICAL AND FUNCTIONAL CHARACTERISTICS Several methods have been used to characterize hematopoietic stem and progenitor cells with respect to expression of surface molecules as well as functional properties in cell culture and in vivo models. A widely used phenotypic marker of hematopoietic stem and progenitor cells is the CD34

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antigen (Civin et al., 1984). CD34 is a highly glycosylated transmembrane protein of 115 kDa, which can be phosphorylated by a variety of kinases such as protein kinase C and tyrosine kinases (Lanza et al., 2001). Ligands of CD34 on hematopoietic stem cells have not been identified so far, and its function is still unknown. On average, the CD34 antigen is present on 0.5– 3% of cells in human bone marrow. It is expressed not only on hematopoietic progenitor and stem cells but also on endothelial cells and some stromal cells, which might suggest a common origin from a mesenchymal stem cell (Fina et al., 1990). The population of hematopoietic CD34+ cells is heterogeneous regarding phenotype and function. Further subset analysis, using monoclonal antibodies directed against differentiation- or lineage-specific antigens, can divide the CD34+ cell compartment into primitive hematopoietic stem cells and more mature lineage-committed progenitor cells (Civin and Gore, 1993) (Fig. 1). The subset of early CD34+ hematopoietic stem cells with high selfrenewal and multilineage differentiation capacity is characterized by low, or absent, coexpression of HLA-DR or CD38 without expression of lineagespecific antigens (Sutherland et al., 1989; Terstappen et al., 1991; Weilbaecher et al., 1991; Petzer et al., 1996). Another discrimination between CD34+ cell subsets is based on the expression of Thy-1 (CDw90), as this antigen is present on more primitive progenitor cells, including early stem cells (Craig et al., 1993). The antigenic profile of more differentiated CD34+ hematopoietic progenitors is characterized by the coexpression of CD38 and lineage-specific antigens (Terstappen et al., 1991; Civin and Gore, 1993; Huang and Terstappen, 1994). Myelomonocytic differentiation is associated with CD33 and CD45RA whereas CD34+ cells coexpressing CD71 and Gly-A represent erythroid progenitor cells. CD41 and CD61 are megakaryocyte-associated markers whereas the CD34+/CD19+ and CD34+/ CD7+ immunophenotypes are specific for B lymphoid and T lymphoid progenitors, respectively. Another way to characterize subsets within the CD34+ cell population is by culture assays and mouse transplantation models. Those functional assays permit the investigator to address the two basic features of a hematopoietic stem cell candidate: self-renewal and multilineage differentiation. Functional in vitro assays include (1) colony-forming cell cultures based on semisolid media such as methylcellulose or agar, (2) liquid suspension cultures followed by colony-forming assays, and (3) bone marrow stromal cell-dependent long-term cultures. Using colony-forming cell assays, clonogenic lineage-determined or pluripotent hematopoietic progenitor cells can be assessed. The assays determine the differentiation capacity of individual progenitor cells by their ability to generate mature colonies such as colony-forming units granulocyte/erythrocyte/macrophage/ megakaryocyte (CFU-GEMM), colony-forming units granulocyte/macrophage (CFU-GM), or burst-forming units erythrocyte (BFU-E) (Fauser and

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lineage-determined hematopoietic progenitor cell CD71 Gly-A erythroid multipotent hematopoietic progenitor cell

CD33 CD45RA myeloid

hematopoietic stem cell

CD41 CD61 megakaryocytic

CD19

B lymphoid

CD7

T lymphoid

CD34 Thy-1 HLA-DR CD38

FIGURE1. Immunological characteristics of hematopoietic stem and progenitor cells. Messner, 1979). In contrast, bone marrow stroma cell-based assays were developed to assess early hematopoietic stem cell candidates. The long-term bone marrow culture (LTBMC) was first developed by Dexter and Lajtha (1974) for progenitor cells of mice and was modified for human cells by Gartner and Kaplan (1980). Enumeration of primitive hematopoietic cell subsets was possible by the availability of long-term culture-initiating cell (LTC-IC) assays as well as cobblestone area-forming cell (CAFC) assays (Sutherland et al., 1990; Breems et al., 1994). The culture conditions in these bone marrow stromal-based assays maintain the hematopoietic progenitor

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population within an adherent stromal layer. The latter consists of endothelial cells, adipocytes, macrophages, and fibroblasts as well as an extracellular matrix that contains fibronectin, laminin, collagen, and glycosaminoglycans. A major disadvantage of stromal-based methods is their inability to maintain progenitors of both the lymphoid as well as the myeloid lineage. A myeloid–lymphoid initiating cell (ML-IC) assay has been described that allows assessment of primitive multilineage progenitor cells in vitro (Punzel et al., 1999). In addition, several other culture conditions were used, showing that single CD34+ Lin cells could differentiate into multiple lineages such as myeloid, B lymphoid, T lymphoid, natural killer, or dendritic cells (Hao et al., 1996; Miller et al., 1999). The functional potential of human CD34+ hematopoietic progenitor and stem cells in vivo could be examined by xenogeneic transplantation models including severe combined immunodeficient (SCID) mice or fetal sheep (McCune et al., 1988; Srour et al., 1993; Kollmann et al., 1994; Fraser et al., 1995). Finally, the differentiation and self-renewing capacity of human CD34+ hematopoietic progenitor and stem cells was shown by the use of immunomagnetically enriched autologous or allogeneic CD34+ peripheral blood stem cells to support high-dose therapy in patients with hematological malignancies or solid tumors (Civin et al., 1996; Yabe et al., 1996; Hohaus et al., 1997; Voso et al., 1999; Shpall et al., 1995; Bensinger et al., 1996; Marin et al., 1997; Urbano-Ispizua et al., 1997).

III. CELL CYCLE AND DIFFERENTIATION CONTROL Precise regulation of cell cycling and differentiation of hematopoietic stem cells is a prerequisite for adequate and controlled replenishment of the different subsets of blood cells, dependent on systemic needs. Further, a balance between self-renewal and differentiation of stem and progenitor cells is required to produce sufficient numbers of mature differentiated effector cells and to sustain a pool of pluripotent stem cells as a lifetime reservoir for hematopoiesis. Several models and experimental methods have been used to elucidate the molecular mechanisms underlying these processes. One approach is the comparison of bone marrow-derived CD34+ (BM-CD34+) cells and CD34+ cells from the peripheral blood (PB-CD34+) mobilized by G-CSF because it is known that BM-CD34+ cells are cycling more actively in comparison with their more quiescent counterparts in the peripheral blood (Uchida et al., 1997; Rumi et al., 1997; Steidl et al., 2002a). The comparison of CD34+ cells from bone marrow with the CD34-negative cell fraction is another method by which to search for the expression of genes that might be necessary and characteristic for hematopoietic stem cells (Furukawa, 2002). Comparative analysis of

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developmentally early hematopoietic stem cells and lineage-determined progenitors of the different hematopoietic subsets offers the opportunity to identify genes that play a role in differentiation decisions of hematopoietic cells (Furukawa, 1997). In addition, many researchers have used knock-in or knock-out animal models in order to identify the effects of activation or inactivation of distinct genes on hematopoiesis. Although this reductionistic experimental approach in rodent models seems to provide the most precise and reliable results, it is best to keep in mind that it might be difficult to transfer the knowledge gained to the human system. We examined BM-CD34+ and PB-CD34+ cells by means of cDNA array technology to obtain diversified insight into the genetic program of primary human hematopoietic stem and progenitor cells (Steidl et al., 2002a). In BM-CD34+ cells significantly higher expression of genes for cell cycle progression and DNA synthesis was found, which explains on a molecular basis the greater cycling activity of sedentary BM-CD34+ cells in comparison with circulating PB-CD34+ cells (Fig. 2). For each phase of the cell cycle we identified genes, the activity of which was apparently responsible for the transition from quiescence to active cycling of CD34+ cells. From our data, we concluded that downregulation of genes encoding GATA-2 and N-Myc as well as upregulation of the gene encoding E2F1 transcription factor initiate the entry of human hematopoietic stem cells into the cell cycle,

Cell Cycle

E2F-1

TOP2A LIG1 PCNA MCM7 MCM5 RFC37 MCM2 MCM6 POLD MCM4 LIG3

DNA Synthesis

N-myc GATA-3 GATA-2 TIS11B GABPA BTEB2 Humdp2 CLK1

BM > PB −3

UBCH10 CDC20 B-MYB CDC28 CDC25A PLK Prothymosin a CDC25B CDC25C

−2

−1

Transcription PB > BM 0

1

2

3

Differential Gene Expression log2(PB-CD34+/BM-CD34+)

FIGURE 2. Differentially expressed genes in CD34+ cells from peripheral blood (PB) and bone marrow (BM).

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whereas elevated expression of genes encoding CDC25A and B-MYB facilitates the G1–S transition. Augmented DNA synthesis during the S phase of cycling BM-CD34+ cells is molecularly reflected by the presence of PCNA, LIG1, LIG3, RFC37, TOP2A, POLD1, and MCMs 2, 4, 5, 6, and 7. G2 phase promotion, as well as the G2–M and M–G1 transitions are maintained by CDC25B, CDC25C, PLK, CKS1, and UBCH10. Furukawa et al. examined expression of several cell cycle-related genes in CD34+ cells, in comparison with the CD34-negative cell fraction, from bone marrow, by semiquantitative reverse transcription polymerase chain reaction (RT-PCR) (Furukawa, 1997). They found that expression of several cyclins and cyclin-dependent kinases (CDKs), except CDK4, was suppressed in CD34+ cells in comparison with CD34 cells. The CDK inhibitor p16 was expressed at a higher level in CD34+ cells, whereas p21 and p27 expression was increased within the CD34-negative cellular subset. In a further study, Furukawa and co-workers examined expression of cell cycle control genes in CD34+ cells dependent on differentiation into distinct hematopoietic lineages (Furukawa et al., 2000). They demonstrated a universal upregulation of cdc2, cdk4, cyclin A, cyclin B, and p21, and downregulation of p16, during differentiation irrespective of the commitment of CD34+ cells to the myeloid, erythroid, or megakaryocytic lineage. Upregulation of cyclin D1 and p15 was found solely on myeloid differentiation, indicating the association of those proteins with myeloid determination of hematopoietic progenitors (Della et al., 1997; Schwaller et al., 1997; Teofili et al., 2000). p15 works as a negative regulator of proliferation by inhibiting the activity of cyclin-dependent kinases and consecutively arresting cells in G1 phase of cell cycle. The view that p15 is a crucial element in the regulation of myeloid proliferation is further supported by the observation of reduced p15 expression levels in several leukemias (Drexler, 1998). Dolznig et al. and Dai et al. reported that cdc2 and cyclin A were upregulated during erythroid differentiation of hematopoietic progenitors (Dolznig et al., 1995; Dai et al., 2000). Elevation of p21 expression in erythroid progenitors was independently demonstrated by Hsieh et al. (2000) and Taniguchi et al. (1999). Upregulation of cyclin D3 seems to be characteristic and necessary for megakaryocytic differentiation (Furukawa, 1997; Zimmet et al., 1997; Wang et al., 1999; Furukawa et al., 2000). In summary, the expression of cell cycle control genes is modulated during hematopoietic development and results in lineage-specific expression patterns that are involved in the regulation of differentiation and proliferation of hematopoietic cells. Beside cell cycle control genes, transcription factors are known to play an important role in the differentiation of hematopoietic stem and progenitor cells. In comparing expression patterns of transcription factors of primary human cells, we found that mobilized CD34+ cells from the peripheral blood

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expressed nine transcription factors (GATA-2, N-Myc, TIS11B, CLK1, IRF2, Humdp2, GATA-3, GABPA, and BTEB2) to a significantly higher extent than did bone marrow CD34+ cells (Steidl et al., 2002a) (Fig. 2). GATA-2 and N-Myc have already been demonstrated to keep hematopoietic stem cells in an undifferentiated stage and thereby favoring selfrenewal (Stanton and Parada, 1992; Briegel et al., 1993; Fujimaki et al., 2001). Because PB-CD34+ cells contain a higher number of developmentally early self-renewing cells (Haas et al., 1995a), our data suggest that the other upregulated transcription factors are also involved in the maintainance of a self-renewing population of immature hematopoietic stem cells. The transcription factor Notch-1 plays an important role in early hematopoietic progenitors by inhibiting hematopoietic differentiation (Ohishi et al., 2002). Maintainance of GATA-2 expression has been identified as a molecular mechanism underlying the differentiation arrest caused by Notch-1 (Kumano et al., 2001). HoxB4, a member of the Hox family of transcription factors, has also been recognized as an important regulator of immature hematopoietic cells (Buske et al., 2002). In a mouse transplantation model Antonchuk et al. demonstrated that increased selfrenewal activity mediated by HoxB4 greatly enhanced growth of primitive hematopoietic cells (Antonchuk et al., 2001). The same group showed that HoxB4 induces ex vivo expansion of hematopoietic stem cells (Antonchuk et al., 2002). Furthermore, HoxB4 forced primitive progenitors from yolk sac or embryonic stem cells to switch to definitive hematopoietic stem cells with long-term multilineage potential in primary and secondary recipients, which underlines the central role of HoxB4 in early hematopoiesis (Kyba et al., 2002). Another member of the Hox transcription factor family, HoxA10, is an important regulator of myeloid differentiation as it induces growth of primitive myeloid progenitors in mice (Bjornsson et al., 2001) and contributes to leukemogenesis (Buske et al., 2001; Taghon et al., 2002). Upregulation of PU.1 transcription factor and GATA-1 have been shown to initiate myeloid differentiation (Scott et al., 1994; Nerlov and Graf, 1998; Mueller et al., 2002). In contrast, simultaneous upregulation of PU.1 and interleukin 7 (IL-7) receptor and downregulation of GM-CSF receptor seem to initiate lymphoid development (DeKoter and Singh, 2000; Kondo et al., 2000; DeKoter et al., 2002). Furthermore, Pax5 was shown to be necessary for development of B cell precursors (Nutt and Busslinger, 1999; Nutt et al., 2001). Data of Reddy et al. demonstrated that transcription factor C/EBP physiologically inactivates PU.1 and thereby redirects PU.1-dependent cell development (Reddy et al., 2002). Megakaryocytic differentiation requires expression of GATA-1 and NFE2 as well as BACH2 (Terui et al., 2000). Signal transducer and activator of transcription (STAT3) is upregulated on stimulation with thrombopoietic

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cytokines and contributes predominantly to early events in megakaryopoiesis (Kirito et al., 2002). In conclusion, distinct expression patterns of transcription factors steer the balance between self-renewal and commitment to differentiation of hematopoietic stem cells. Some evidence has been provided for the suggestion that stochastic activation of transcription factor expression is the basis for differentiation steps of hematopoietic cells (Nutt and Busslinger, 1999; Zhu and Emerson, 2002). In this model, the likelihood of defined expression patterns of transcription factors that induce differentiation basically depends on stochastic variations of transcription factor expression levels. However, this ‘‘intrinsic’’ stochastic process can apparently be modulated by ‘‘extrinsic’’ signals, cytokines, for instance, resulting in directed differentiation (Batard et al., 2000; Kirito et al., 2002; Pierelli et al., 2002). The transcriptional determination of the developmental fate of hematopoietic stem cells is probably not an abrupt irreversible event but rather a continuous decision process (Hu et al., 1997; Enver and Greaves, 1998; Rothenberg, 2000; Papayannopoulou et al., 2000). Data indicate that hematopoietic progenitors lose their multilineage differentiation potential gradually during differentiation and maturation (Zhu and Emerson, 2002). The finding that hematopoietic stem cells are able to give rise to nonhematopoietic cells under certain conditions, which is discussed later, also supports the model of a continuous and partially reversible developmental control of hematopoietic stem cells.

IV. ADHESION MOLECULES IN HEMATOPOIESIS AND STEM CELL TRAFFICKING During fetal development, hematopoiesis shifts from liver to the bone marrow, which remains the major site of hematopoietic stem and progenitor cells in adults. Still, small amounts of CD34+ cells are present in the peripheral blood and at other organs such as spleen and liver, suggesting a continuous migration and exchange of hematopoietic stem cells between bone marrow and other organs in adults (Wright et al., 2001). Besides, hematopoiesis is dependent on a close proximity of hematopoietic stem and progenitor cells with a microenvironment in the bone marrow, which provides regulatory growth factors and cellular interactions essential for proliferation, differentiation, and survival of hematopoietic stem cells (Whetton and Graham, 1999) (Fig. 3). The bone marrow stroma consists of stromal cells including fibroblasts, osteoblasts, adipocytes, myocytes, endothelial cells, dendritic cells, and macrophages. These cells produce extracellular matrix proteins such as fibronectin, laminin, vitronectin, or hyaluronic acid as well as hematopoietic cytokines. Normal hematopoiesis is

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Peripheral Blood

CD34+ blood stem cell

CD34+ blood stem cell

thrombin receptor

endothelial cell

+ Mg2+

CXCR-4

ICAM-1 LFA-1

SDF-1

hyaluronic acid CD44 CD34+ blood stem cell

CD34+ blood stem cell

VCAM-1 VLA-4

fibronectin stromal cell Bone Marrow

FIGURE 3. Adhesion molecules involved in hematopoietic stem cell trafficking. regulated by an interaction between hematopoietic cells and stromal cells or extracellular matrix components through specific cell surface receptors and cytokines (Kronenwett et al., 2000). 1-Integrins such as the very late antigens (VLA) VLA-4 (CD29/CD49d) and VLA-5 (CD29/CD49e), which are expressed on CD34+ cells, play a dominant role in adhesive interactions. In particular, VLA-4-mediated interaction between hematopoietic stem cells and bone marrow stroma is of functional relevance for hematopoiesis as well as for stem cell trafficking (Miyake et al., 1991; Prosper et al., 1998; Kronenwett et al., 2000). VLA-4 binds to vascular cell adhesion molecule 1 (VCAM-1) as well as to the extracellular matrix protein fibronectin. Circulating CD34+ cells express VLA-4 at a lower level when compared with CD34+ cells residing in the bone marrow, suggesting that the release of CD34+ cells and the ability to circulate might be regulated by the expression level and affinity state of VLA-4 (Mohle et al., 1995; Prosper et al., 1998; Lichterfeld et al., 2000). Likewise, systemic administration of anti-VLA-4 monoclonal antibodies resulted in an increase of hematopoietic progenitor cells in mice and primates (Papayannopoulou and Nakamoto, 1993; Craddock et al., 1997; Christ et al., 2001b). On the other hand, VLA-4 antibody treatment of mice was associated with inhibition of engraftment of hematopoietic stem cells (Papayannopoulou et al., 1995). All these studies demonstrate that VLA-4-mediated adhesive interactions are necessary for mobilization of hematopoietic stem cells into peripheral blood and their homing and localization in the bone marrow microenvironment. The exact

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molecular mechanisms regulating 1-integrin-mediated cellular interactions are still unclear. One possibility is a switch of the integrins from a functional low-affinity state to a high-affinity state by cytokines, resulting in a more avid binding of the CD34+ cells to the adhesive ligands (Kovach et al., 1995; Levesque et al., 1995). Moreover, Mg2+ ions or contact with endothelial cells results in activation of the VLA-4 receptor, suggesting that modulation of the divalent cation concentration in the vicinity of the 1-integrin alters its activity on CD34+ cells adherent to the endothelial cell lining (Lichterfeld et al., 2000). 1-Integrin-mediated adhesive interactions serve not only to mechanically tie CD34+ hematopoietic stem cells in the bone marrow but also to regulate their growth. The intracellular domain of the 1-subunit can initiate signal transduction cascades such as activation of the Ras/mitogen-activated protein kinase (MAPK) pathway (Schlaepfer et al., 1994), which results in increased expression of c-myc and c-fos (Shaw et al., 1990) and in activation of phosphatidylinositol 3-kinase (PI3-kinase). Thus, engagement of 1-integrins affects proliferation and survival of cells. Whether these signaling pathways are also relevant for hematopoietic cells is still unknown. It could be shown that coculture of CD34+ cells under physiological cytokine conditions with extracellular matrix proteins inhibited proliferation of the progenitors (Hurley et al., 1995). This functional effect was mediated by 1-integrins as assessed by the use of blocking monoclonal antibodies. In another study, the 1-integrin-mediated contact between hematopoietic stem cells and stroma enhanced the proliferative stimulus induced by cytokines (Schofield et al., 1998). This finding might explain why circulating CD34+ cells out of contact with bone marrow stroma are mainly quiescent cells in the G0/G1 phase of the cell cycle (Fruehauf et al., 1998; Steidl et al., 2002a). However, the seemingly converse findings with respect to the functional effects of 1-integrin-mediated cellular interactions can be explained by different environmental conditions in which the hematopoietic progenitor cells reside. Besides 1-integrins, the 2-integrin LFA-1 plays a role in adhesion and migration of CD34+ hematopoietic stem and progenitor cells. Ligands are members of the superimmunoglobulin family, intercellular adhesion molecules 1 and 2 (ICAM-1 and ICAM-2). Adhesion to and migration through an endothelial cell layer could be inhibited by LFA-1-directed blocking monoclonal antibodies, supporting the relevance of the 2-integrin for mobilization, trafficking, and homing of CD34+ hematopoietic stem cells (Mohle et al., 1995, 1997). Hematopoietic stem cells express several other adhesion molecules such as L-selectin (CD62L), CD44, and platelet– endothelial cell adhesion molecule 1 (PECAM-1) (CD31). L-selectin mediates the initial contact of leukocytes with endothelium and thus might play a role in homing of stem cells (Mohle et al., 1997). The highly glycosylated surface molecule CD44, which binds to hyaluronic acid and fibronectin, emerges in different isoforms arising from differences in

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glycosylation and alternative splicing. It is also involved in hematopoiesis and stem cell trafficking as monoclonal antibodies against CD44 inhibited adhesion to bone marrow stroma, mobilized progenitor cells in mice, and abolished hematopoiesis in long-term bone marrow cultures (Miyake et al., 1990; Khaldoyanidi et al., 1996; Christ et al., 2001a). Interestingly, adhesive interactions playing a role in stem cell trafficking are similar to those involved in the recruitment of leukocytes at sites of inflammation (Imhof and Dunon, 1997; Steidl et al., 2000). This suggests common molecular mechanisms of migration and homing for leukocytes of different developmental stages. Besides cytokines and adhesion molecules the interaction between the -chemokine stromal-derived factor 1 (SDF-1) and its receptor CXCR-4 plays a prominent role in stem cell migration and hematopoiesis. SDF1 knock-out mice died perinatally of bone marrow failure, while fetal liver hematopoiesis was not affected (Nagasawa et al., 1996). This suggests that the deficiency in myelopoiesis in the neonatal bone marrow is a result of disturbed migration of stem cells from fetal liver to bone marrow in these knock-out mice. In humans, CXCR-4 is expressed in CD34+ cells, dependent on the differentiation state as the more immature hematopoietic stem cells were brightly positive for CXCR-4 whereas more differentiated progenitors had lower CXCR-4 expression levels (Deichmann et al., 1997; Viardot et al., 1998). SDF-1, produced by bone marrow stromal cells, seems to be a general chemoattractant for hematopoietic stem cells and mediates transendothelial migration (Aiuti et al., 1997; Mohle et al., 1998). The thrombin receptor, which plays an important role in monocyte chemotaxis and migration of malignant cells, was shown to be expressed in human CD34+ hematopoietic progenitor cells (Steidl et al., 2002a). As assessed by cDNA array technology and immunofluorescence analysis, the expression level of thrombin receptor was 3-fold higher in CD34+ cells from peripheral blood than in progenitor cells residing in the bone marrow. This finding suggests a migration-mediating function not only for monocytes but also for hematopoietic progenitor cells. Thrombin might guide circulating CD34+ cells to sites of cellular damage in order to facilitate regeneration. In conclusion, a complex network of adhesion molecules, cytokines, and chemoattractants is involved in stem cell trafficking and hematopoiesis by mediating migration, proliferation, differentiation, and release from the bone marrow with subsequent homing at other body sites.

V. AGING AND TELOMERES The human hematopoietic system responds to considerable replicative demands. On the basis of a daily production of about 1012 blood cells in the adult and about 4 1015 blood cells life long (Lansdorp, 1998), it has been

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estimated that to maintain steady state hematopoiesis throughout human life, stem cells must undergo approximately 52 divisions. Therefore, if stem cells have a replicative limit that exceeds 52 population doublings, normal hematopoiesis would not be affected during the whole life span (Effros and Globerson, 2002). Indeed, it appears that the hematopoietic stem cell population continues to function through old age as there are no signs of anemia or lymphopenia in the elderly under normal conditions (Globerson, 1999; Bagnara et al., 2000; Globerson and Effros, 2000). On the other hand, the reserve in situations of hematological stress declines in old age (Chatta and Dale, 1996). This suggests that either the stem cell population decreases or the capacity of hematopoietic stem cells to respond to replication signals is reduced in older age, suggesting ‘‘replicative senescence.’’ The replicative senescence relates to a definite capacity of cells to divide, as well as to the genetic and functional changes that accompany cell division (Hayflick, 1992). A parameter indicative of cellular aging is the length of telomeres. These are specialized structures consisting of 2- to 15-kb noncoding hexanucleotide (TTAGGG)n repeats that cap the ends of eukaryotic chromosomes (Blackburn and Gall, 1978; Conrad et al., 1990; Zakian, 1995). They prevent degradation, recombination, and fusion of the double-stranded DNA ends (Blackburn, 1991; van Steensel et al., 1998; Smith and Blackburn, 1999), mediate chromosome–nuclear matrix interactions (Mathog et al., 1984; Walker et al., 1991), protect coding DNA from enzymatic breakdown (White and Haber, 1990; Sandell and Zakian, 1993), and may exert effects on regional subtelomeric gene transcription (Levis et al., 1985; Gottschling et al., 1990). In contrast to the coding DNA sequences, telomeres shorten with each round of cell division in normal human somatic cells. Such shortening is due to the ‘‘end-replication problem’’ (Olovnikov, 1973): as DNA polymerase can act only in the 5-to-3 direction, it is unable to completely replicate the 3 end of the DNA lagging strand, resulting in a DNA loss of 40–120 bp per division (McEachern et al., 2000). The progressive shortening of telomere length may result in cell cycle arrest or chromosomal instability, and leads ultimately to loss of the cell’s replicative capacity or ‘‘cellular senescence’’ (Harley, 1991; Sandell and Zakian, 1993). The loss of telomeric DNA provides the basis for the ‘‘telomere hypothesis’’ of cellular aging (Harley, 1991; Harley et al., 1992), because shortening of telomeres was proposed as a biological mitotic clock that determines finite cell replications. Shortening of telomeres in vivo with time was found in several cell types such as leukocytes (Vaziri et al., 1993), dermal and epidermal skin cells (Lindsey et al., 1991), and colon epithelia (Hastie et al., 1990). In hematopoietic stem cells, Vaziri et al. (1994) demonstrated a proliferationassociated loss of telomeric DNA between 35 and 45 bp per population

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0.7 kb

Mean TRF lenght (kb)

10

8

7 CD34+ cells

MNC

FIGURE 4. Telomere shortening during maturation of leukocytes. Telomere length of blood-derived mononuclear cells (MNCs) and CD34+ cells of eight individuals is displayed. The mean difference in telomere length is indicated. TRF, Terminal restriction fragment.

doubling. Telomeres in CD34+ stem cells from adult bone marrow and peripheral blood are shorter than in CD34+ cells from cord blood and fetal liver (Engelhardt et al., 1997), suggesting attrition in the course of normal replication as a function of age. Consistent with that, telomeres in mononuclear cells (MNCs) from peripheral blood are significantly shorter than in CD34+ cells (Fig. 4) (Kronenwett et al., 1996). Looking at the more primitive CD34+CD38lo stem cell subset, Vaziri et al., (1994) also demonstrated age-related changes, as cells with this phenotype purified from adult bone marrow have shorter telomeres than do cells from fetal liver or umbilical cord blood, which indicates again that the proliferation potential of stem cells decreases with age. Considering telomere length as a possible molecular marker of a patient’s hematopoietic reserve, we determined the mean terminal restriction fragment (TRF) length of blood cells from patients with cancer who received cytotoxic chemotherapy with G-CSF support for blood stem cell mobilization (Kronenwett et al., 1996). Assuming that stem cells are forced to undergo an increased number of cell divisions during marrow recovery, we hypothesized that a significant amount of previous cytotoxic chemotherapy would lead to reduced telomere length in patient blood cells. Interestingly, the amount of previous cytotoxic therapy was not related to the mean TRF of mononuclear cells. Therefore, cytotoxic chemotherapy may diminish the number of hematopoietic progenitor cells that contribute to the circulating population during G-CSF-supported marrow recovery, whereas the cumulative myelotoxicity

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is reflected by a decrease in the telomere length of the spared stem cells and their progeny. Further, there was no relationship between the mean TRF length and the level of circulating CD34+ cells. Thus, the telomere length of leukocytes from peripheral blood does not indicate the proliferative capacity of the hematopoietic system. On the other hand, the mean TRF length was related to patient age. The average shortening of telomeric DNA in our patient group was 25 bp/year. This finding is similar to data of Vaziri et al. (1993) and Hastie et al. (1990), who examined peripheral blood cells from normal donors. In the clinical transplantation setting, telomeres help to gain new insights into the processes of engraftment and early hematopoietic reconstitution while they serve as surrogate markers of stem cell behavior in transplanted patients. After autologous transplantation, a telomere loss of 1–2 kb accounts for 20–40 years of premature aging in the recipient (Engelhardt and Finke, 2001). Analysis of peripheral blood granulocytes from bone marrow transplant recipients in comparison with their donors showed a significant shortening of telomere length and that the extent of the reduction inversely correlated with the number of nucleated cells infused (Notaro et al., 1997). The telomere loss in recipients of allogeneic bone marrow transplantation is equivalent to a median of 15 years of aging in healthy controls (Wynn et al., 1998). It thus appears that the transplanted stem cells undergo extensive replication in the process of reconstituting the recipient and this imposed stress of replication may accelerate cell senescence. A mechanism evolved to prevent telomere attrition involves the activation of telomerase, a ribonucleoprotein with reverse transcriptase activity that counteracts the end replication problem by synthesizing new telomeric TTAGGG repeats onto the 3 end of telomeres (Greider and Blackburn, 1989; Lingner et al., 1997; Bodnar et al., 1998). The enzyme was found in significant amounts in a vast majority of tumors as well as in extracts from immortalized human cells (Morin, 1989; Kim et al., 1994; Counter et al., 1995), thereby enabling indefinite cell divisions. Its activity is low or below the limit of detection in normal somatic cells (Counter et al., 1995; Broccoli et al., 1995). Germ line cells are apparently exceptional, because they constitutively express telomerase (Kim et al., 1994). Telomerase activity is regulated throughout human development, undergoing silencing in almost all organ systems from embryogenesis onward (Forsyth et al., 2002). Still, regulated telomerase activity is seen in basal/stem cell populations of highly regenerative tissues, such as those of the immune system, skin, and intestine. Analysis of the baseline levels of telomerase activity in hematopoietic CD34+ cells demonstrated the lowest values in fetal liver followed by cord blood and peripheral blood, with the highest levels in bone marrow (Chiu et al., 1996; Engelhardt et al., 1997). In primitive hematopoietic CD34+CD71loCD45RAlo cells the constitutive level

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is lower than in early CD34+CD71+ progenitor cells (Engelhardt et al., 1997). There is a correlation between telomerase activity and cell cycle status: nonexpanding CD34+ cells have low or undetectable levels of telomerase. Upregulation occurs in response to cell cycle activation and cytokine-induced proliferation (Engelhardt et al., 1997). However, the basal constitutive levels of telomerase are apparently insufficient to prevent the shortening of telomeres during normal replication. In summary, telomeres of hematopoietic stem cells shorten physiologically during ontogenesis as well as on replicative stress. Thus, hematopoietic stem cells age molecularly, resulting in diminished proliferation potential. This observation challenges the idea of self-renewal of hematopoietic stem cells, which implicates identical replications. Further research unraveling the detailed mechanisms involved in stem cell aging will not only help to understand senescence of the hematopoietic system but will also have major implications for gene therapy, stem cell transplantation, and tissue engineering.

VI. TRANSDIFFERENTIATION AND DEVELOPMENTAL PLASTICITY Recent findings suggest that hematopoietic stem cells not only give rise to hematopoietic cell lineages but are also capable of differentiating into non hematopoietic cells. In 1999, Gussoni et al. observed that transplantation of hematopoietic stem cells restored dystrophin expression in mice with Duchenne muscular dystrophy (Gussoni et al., 1999). Human bone marrow cells as well as purified hematopoietic stem cells were shown to be able to differentiate into hepatocytes (Lagasse et al., 2000; Theise et al., 2000; Alison et al., 2000). Orlic and co-workers demonstrated that bone marrow cells as well as cytokine-mobilized cells from the peripheral blood were able to give rise to cardiomyocytes in infarcted hearts and markedly improved organ function and survival of the animals (Orlic et al., 2001a,b). Jackson and colleagues found that adult mesenchymal stem cells from bone marrow regenerated ischemic cardiac muscle and vascular endothelium (Jackson et al., 2001). After bone marrow transplantation cells derived from the marrow were also shown to migrate into the brain and to express neuronal antigens in mice (Mezey et al., 2000). Intracerebral transplantation of adult hematopoietic progenitors into neonatal mouse brain resulted in expression of oligodendroglia-specific markers (Bonilla et al., 2002), while intracranial transplantation of bone marrow resulted in better functional restoration in rats with traumatic brain injury (Mahmood et al., 2001; Zhao et al., 2002). In interpreting the presented data it is necessary to be aware of experimental and methodical limitations. Novel data have challenged the transdifferentiation model by suggesting cell fusion rather than plasticity of

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stem cells (Terada et al., 2002; Ying et al., 2002). Another central question concerns whether transdifferentiation has occurred or whether developmental heterogeneity of the donor cells is the cause of the observed seemingly unrestricted differentiation processes. In most of the studies mentioned previously, unpurified cell populations were used for transplantation experiments so that it is not possible to identify the cell of origin. But even enriched or purified cells as used by Lagasse or Orlic and co-workers still represent heterogeneous cellular populations. Because it is difficult to exclude the coexistence of diverse stem cell types in a cellular subset it is likely that crude bone marrow contains, besides hematopoietic stem cells, other types of multipotent stem cells that not necessarily derive from a common progenitor (Pittenger et al., 1999; Reyes et al., 2001; Jiang et al., 2002). However, both principles of developmental heterogeneity of cellular populations as well as plasticity on a single-cell level may apply. A better molecular understanding of the communicational skills and the signaling pathways of hematopoietic stem and progenitor cells is required to understand the conditions under which non-lineage-restricted differentiation or transdifferentiation of hematopoietic cells may occur. For the first time, Terskikh et al. provided evidence of overlapping genetic programs of hemato- and neuropoiesis in mice (Terskikh et al., 2001). In our own study, assessing gene expression profiles of primary human CD34+ cells from peripheral blood and bone marrow using cDNA arrays (Steidl et al., 2002a), we detected the expression of receptors such as GABA-B receptor, EphA1 receptor, and membrane protein of cholinergic synaptic vesicles (VAT1), which were primarily assigned to the nervous system. Those findings prompted us to apply specialized cDNA arrays, quantitative realtime RT-PCR, and fluorescence-activated cell sorting (FACS) analysis in a search for the expression of genes known to be involved in neurobiological functions. We found expression of neurobiological receptors and assembly molecules, ligand-gated as well as voltage-gated ion channels, and genes involved in synaptic vesicle fusion that has not been described in CD34+ cells so far (Steidl et al., 2002b). Those data suggest a molecular interrelation of neuronal and hematopoietic signaling mechanisms and insinuate a close molecular and ontogenetic propinquity of neuro- and hematopoietic cells. This view is supported by a study reporting the detection of a potential human neurohematopoietic stem cell population (Shih et al., 2001). In summary, several studies imply that hematopoietic stem cells might have differentiation potential beyond their hematopoietic determination, which could open novel therapeutic avenues in the treatment of various degenerative diseases. Still, a better understanding of the molecular framework underlying transdifferentiation and plasticity will be a prerequisite for a purposeful use of the entire therapeutic potential of hematopoietic stem cells.

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VII. CONCLUSIONS Recent experimental results have advanced our understanding of the molecular biology underlying basic features of hematopoietic stem cells such as trafficking, cell cycling, differentiation, and aging. The expanding molecular knowledge notwithstanding, further research will be necessary to establish a coherent molecular model that encompasses functions of hematopoietic stem cells within the hematopoietic system as well as novel observations of developmental plasticity.

REFERENCES Aiuti, A., Webb, I. J., Bleul, C., Springer, T., and Gutierrez-Ramos, J. C. (1997). The chemokine SDF-1 is a chemoattractant for human CD34+ hematopoietic progenitor cells and provides a new mechanism to explain the mobilization of CD34+ progenitors to peripheral blood. J. Exp. Med. 185, 111–120. Alison, M. R., Poulsom, R., Jeffery, R., Dhillon, A. P., Quaglia, A., Jacob, J., Novelli, M., Prentice, G., Williamson, J., and Wright, N. A. (2000). Hepatocytes from non-hepatic adult stem cells. Nature 406, 257. Antonchuk, J., Sauvageau, G., and Humphries, R. K. (2001). HOXB4 overexpression mediates very rapid stem cell regeneration and competitive hematopoietic repopulation. Exp. Hematol. 29, 1125–1134. Antonchuk, J., Sauvageau, G., and Humphries, R. K. (2002). HOXB4-induced expansion of adult hematopoietic stem cells ex vivo. Cell 109, 39–45. Bagnara, G. P., Bonsi, L., Strippoli, P., Bonifazi, F., Tonelli, R., D’Addato, S., Paganelli, R., Scala, E., Fagiolo, U., Monti, D., Cossarizza, A., Bonafe, M., and Franceschi, C. (2000). Hemopoiesis in healthy old people and centenarians: Well-maintained responsiveness of CD34+ cells to hemopoietic growth factors and. J. Gerontol. A Biol. Sci. Med. Sci. 55, B61–B66. Batard, P., Monier, M. N., Fortunel, N., Ducos, K., Sansilvestri-Morel, P., Phan, T., Hatzfeld, A., and Hatzfeld, J. A. (2000). TGF- 1 maintains hematopoietic immaturity by a reversible negative control of cell cycle and induces CD34 antigen up-modulation. J. Cell Sci. 113, 383–390. Bensinger, W. I., Buckner, C. D., Shannon-Dorcy, K., Rowley, S., Appelbaum, F. R., Benyunes, M., Clift, R., Martin, P., Demirer, T., Storb, R., Lee, M., and Schiller, G. (1996). Transplantation of allogeneic CD34+ peripheral blood stem cells in patients with advanced hematologic malignancy. Blood 88, 4132–4138. Bjornsson, J. M., Andersson, E., Lundstrom, P., Larsson, N., Xu, X., Repetowska, E., Humphries, R. K., and Karlsson, S. (2001). Proliferation of primitive myeloid progenitors can be reversibly induced by HOXA10. Blood 98, 3301–3308. Blackburn, E. H. (1991). Structure and function of telomeres. Nature 350, 569–573. Blackburn, E. H., and Gall, J. G. (1978). A tandemly repeated sequence at the termini of the extrachromosomal ribosomal RNA genes in Tetrahymena. J. Mol. Biol. 120, 33–53. Bodnar, A. G., Ouellette, M., Frolkis, M., Holt, S. E., Chiu, C. P., Morin, G. B., Harley, C. B., Shay, J. W., Lichtsteiner, S., and Wright, W. E. (1998). Extension of life-span by introduction of telomerase into normal human cells. Science 279, 349–352. Bonilla, S., Alarcon, P., Villaverde, R., Aparicio, P., Silva, A., and Martinez, S. (2002). Haematopoietic progenitor cells from adult bone marrow differentiate into cells that express oligodendroglial antigens in the neonatal mouse brain. Eur. J. Neurosci. 15, 575–582.

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Steidl, U., Selbod, O., Schroeder, T., Schaub, S., Seres, J., Maercker, C., Rohr, U. P., Fenk, R., Aivado, M., Gattermann, N., Bornstein, S. R., Haas, H. L., Haas, R., and Krouenwett, R. (2002b). Expression and functional activity of neurobiological receptors in primary human CD34+/CD38dim hematopoietic stem cells. Blood 100, 291a, abstract. Sutherland, H. J., Eaves, C. J., Eaves, A. C., Dragowska, W., and Lansdorp, P. M. (1989). Characterization and partial purification of human marrow cells capable of initiating longterm hematopoiesis in vitro. Blood 74, 1563–1570. Sutherland, H. J., Lansdorp, P. M., Henkelman, D. H., Eaves, A. C., and Eaves, C. J. (1990). Functional characterization of individual human hematopoietic stem cells cultured at limiting dilution on supportive marrow stromal layers. Proc. Natl. Acad. Sci. USA 87, 3584–3588. Taghon, T., Stolz, F., De Smedt, M., Cnockaert, M., Verhasselt, B., Plum, J., and Leclercq, G. (2002). HOX-A10 regulates hematopoietic lineage commitment: Evidence for a monocytespecific transcription factor. Blood 99, 1197–1204. Taniguchi, T., Endo, H., Chikatsu, N., Uchimaru, K., Asano, S., Fujita, T., Nakahata, T., and Motokura, T. (1999). Expression of p21Cip1/Waf1/Sdi1 and p27Kip1 cyclin-dependent kinase inhibitors during human hematopoiesis. Blood 93, 4167–4178. Teofili, L., Morosetti, R., Martini, M., Urbano, R., Putzulu, R., Rutella, S., Pierelli, L., Leone, G., and Larocca, L. M. (2000). Expression of cyclin-dependent kinase inhibitor p15INK4B during normal and leukemic myeloid differentiation. Exp. Hematol. 28, 519–526. Terada, N., Hamazaki, T., Oka, M., Hoki, M., Mastalerz, D. M., Nakano, Y., Meyer, E. M., Morel, L., Petersen, B. E., and Scott, E. W. (2002). Bone marrow cells adopt the phenotype of other cells by spontaneous cell fusion. Nature 416, 542–545. Terskikh, A. V., Easterday, M. C., Li, L., Hood, L., Kornblum, H. I., Geschwind, D. H., and Weissman, I. L. (2001). From hematopoiesis to neuropoiesis: Evidence of overlapping genetic programs. Proc. Natl. Acad. Sci. USA 98, 7934–7939. Terstappen, L. W., Huang, S., Safford, M., Lansdorp, P. M., and Loken, M. R. (1991). Sequential generations of hematopoietic colonies derived from single nonlineage-committed CD34+ CD38 progenitor cells. Blood 77, 1218–1227. Terui, K., Takahashi, Y., Kitazawa, J., Toki, T., Yokoyama, M., and Ito, E. (2000). Expression of transcription factors during megakaryocytic differentiation of CD34+ cells from human cord blood induced by thrombopoietin. Tohoku J. Exp. Med. 92, 259–273. Theise, N. D., Nimmakayalu, M., Gardner, R., Illei, P. B., Morgan, G., Teperman, L., Henegariu, O., and Krause, D. S. (2000). Liver from bone marrow in humans. Hepatology 32, 11–16. Uchida, N., He, D., Friera, A. M., Reitsma, M., Sasaki, D., Chen, B., and Tsukamoto, A. (1997). The unexpected G0/G1 cell cycle status of mobilized hematopoietic stem cells from peripheral blood. Blood 89, 465–472. Urbano-Ispizua, A., Rozman, C., Martinez, C., Marin, P., Briones, J., Rovira, M., Feliz, P., Viguria, M. C., Merino, A., Sierra, J., Mazzara, R., Carreras, E., and Montserrat, E. (1997). Rapid engraftment without significant graft-versus-host disease after allogeneic transplantation of CD34+ selected cells from peripheral blood. Blood 89, 3967–3973. van Steensel, B., Smogorzewska, A., and de Lange, T. (1998). TRF2 protects human telomeres from end-to-end fusions. Cell 92, 401–413. Vaziri, H., Schachter, F., Uchida, I., Wei, L., Zhu, X., Effros, R., Cohen, D., and Harley, C. B. (1993). Loss of telomeric DNA during aging of normal and trisomy 21 human lymphocytes. Am. J. Hum. Genet. 52, 661–667. Vaziri, H., Dragowska, W., Allsopp, R. C., Thomas, T. E., Harley, C. B., and Lansdorp, P. M. (1994). Evidence for a mitotic clock in human hematopoietic stem cells: Loss of telomeric DNA with age. Proc. Natl. Acad. Sci. USA 91, 9857–9860. Viardot, A., Kronenwett, R., Deichmann, M., and Haas, R. (1998). The human immunodeficiency virus (HIV)-type 1 coreceptor CXCR-4 (fusin) is preferentially expressed on the more immature CD34+ hematopoietic stem cells. Ann. Hematol. 77, 193–197.

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2 Aldosterone: Its Receptor, Target Genes, and Actions David Pearce,* Aditi Bhargava,* and Timothy J. Cole{ *

Department of Medicine and Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, California 94143 { Department of Biochemistry and Molecular Biology, University of Melbourne, Melbourne, Victoria 3010, Australia, Australia

I. Introduction II. Physiological Actions of Aldosterone A. Mineralocorticoid Target Tissues B. Aldosterone Stimulation of Ion Transport in Tight Epithelia C. Aldosterone Action in Nonepithelial Tissues III. Molecular Basis of Mineralocorticoid Action A. Introduction: Basic Paradigm of Mineralocorticoid Receptor Gene Regulation B. Mechanisms of Mineralocorticoid Receptor Specificity C. Role of Coactivators, Corepressors, and Chromatin in Mineralocorticoid Receptor Gene Regulation IV. Aldosterone Action in Epithelia: Afforded by 11b-Hydroxysteroid Dehydrogenase 2 Physiological Functions of 11b-Hydroxysteroid Dehydrogenase 2

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Copyright 2003, Elsevier Science (USA). All rights reserved. 0083-6729/03 $35.00

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V. Genetic Mouse Models in the Investigation of Aldosterone Action VI. Aldosterone Target Genes That Mediate Physiological Responses A. Aldosterone Action in Brain B. Na+,K+-ATPase C. Epithelial Sodium Channel D. Aldosterone-Regulated Genes Whose Products Alter Epithelial Sodium Channel Localization or Activity E. Aldosterone-Regulated Genes Whose Products Regulate Other Components of Ion Transport Machinery F. Gene Products That Appear to Markedly Alter Components of the Aldosterone-Regulated Network, But That Are Not Direct Targets of Aldosterone Gene Regulation VII. Controversies with Aldosterone A. Aldosterone, Cardiac Fibrosis, and Heart Failure B. Rapid Nongenomic Actions of Aldosterone C. Aldosterone and Insulin Cross-Talk VIII. Concluding Remarks References

I. INTRODUCTION The year 2002 marks the 50th anniversary of the discovery of aldosterone as a mineralocorticoid-acting steroid hormone synthesized and secreted from the bovine adrenal (Grundy et al., 1952). The past 50 years has seen the characterization of the many physiological processes regulated by aldosterone, the elucidation of its mechanism of action via activating mineralocorticoid receptors in target cells, and the demonstration that abnormalities in the aldosterone signaling pathway can cause human disease. Aldosterone is the physiological mineralocorticoid in mammals and, together with another adrenal steroid, the glucocorticoids (cortisol in humans and corticosterone in rodents), helps to maintain homeostasis in a large number of physiological systems. Aldosterone synthesis and secretion by the adernal gland are stimulated by a number of factors including the peptide hormone angiotensin II, high serum potassium, and the pituitary hormone, adrenocorticotropic hormone (ACTH) (Fig. 1) . Physiologically, the major role of aldosterone in epithelial tissues such as the kidney, colon,

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FIGURE 1. Schematic diagram of the renin–angiotensin–aldosterone system (RAAS). Note the synergy between K+ and Ang II in stimulating aldosterone synthesis and release (denoted by converging arrows). ACTH also stimulates aldosterone synthesis but only transiently, and does not synergize with Ang II or K+. Aldosterone in turn acts in multiple tissues, including the classic target epithelia in which it controls ion transport, and nonclassic targets, in which it has a variety of effects related to blood pressure control and tissue remodeling. JGA, Juxtaglomerular apparatus; ASDN, aldosterone-sensitive distal nephron; ACE, angiotensin-converting enzyme. Other abbreviations are as defined in text. and salivary glands is the retention of sodium and water via stimulating unidirectional transepithelial sodium transport (Grunder and Rossier, 1997). However, it also acts in nonepithelial tissues including the brain, vascular smooth muscle, and the heart (Fig. 1). Its actions in these sites produce a variety of effects including elevation of blood pressure, increased salt appetite (Gomez-Sanchez et al., 1990), and in the presence of elevated salt, the production of cardiac fibrosis in the ventricles of the heart (Young and Funder, 1996). Mineralocorticoids exert the majority of their effects via specific intracellular protein receptors in target cells. There are two types of receptors: the mineralocorticoid receptors (MR) and the structurally related glucocorticoid receptors (GR). Together with progesterone receptors (PR) and androgen receptors (AR), they form a receptor subfamily within the superfamily of ligand-dependent nuclear transcription factors, which represents one of the largest family of transcription factors in eukaryotes (Evans, 1988) (Fig. 2) MR has an equal affinity for both physiological glucocorticoids and aldosterone (Sheppard and Funder, 1987), whereas GR has a significantly higher affinity for glucocorticoids. On ligand binding, these receptors undergo a conformational change to form dimers that recognize and bind to regulatory DNA sequences, called hormone response elements (HREs), which are usually located near the promoter of target genes where they activate or repress gene transcription.

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689

604 N-TERMINUS

981

DBD

LIGAND

MR

<15%

94%

60%

GR

<15%

86%

55%

AR

<15%

92%

50%

PR

52%

30%

ER

<15%

FIGURE 2. Strip diagrams comparing the sequences of the steroid receptors. Note that MR and GR are the most closely related, particularly in their DNA-binding domains.

In spite of circulating levels of plasma cortisol at two to three orders of magnitude higher than that of aldosterone, it has been clearly demonstrated that MR in epithelia will bind and respond to aldosterone in mineralcorticoid target tissues in vivo. The mechanism providing protection of MR in epithelia from higher levels of glucocorticoids remained unknown until it was demonstrated that MR-containing epithelia also express an enzyme able to effectively modify and inactivate glucocorticoids. This enzyme was shown to be 11-hydroxysteroid dehydrogenase type II (11HSD2), which is necessary for conferring aldosterone specificity on MR in mineralocorticoid target tissues, by converting biologically active cortisol to inactive cortisone in humans, and corticosterone to 11-dehydrocorticosterone in rodents (Edwards et al., 1988; Funder et al., 1988). This article describes the physiological actions of aldosterone in mammals, presents the current view of its molecular pathway of action in cells, and summarizes the short but expanding list of target genes that mediate physiological change that are directly regulated by aldosterone. We also discuss three current controversies surrounding the action of aldosterone. These are the emerging pathophysiological role of aldosterone in cardiac fibrosis and heart failure; the compelling evidence for rapid nongenomic actions of aldosterone and their physiological relevance; and finally the possible cross-talk of aldosterone with insulin action, particularly in relation to a clinical condition referred to as metabolic syndrome or syndrome X.

II. PHYSIOLOGICAL ACTIONS OF ALDOSTERONE Aldosterone has potent effects on mineral homeostasis through its actions in tight epithelia. It stimulates ion (primarily Na+,K+, and Cl)

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transport in the distal nephron, colon, salivary glands, and sweat glands, resulting in alterations in plasma ion concentrations, as well as in extracellular fluid volume and blood pressure. The control of volume and mineral homeostasis is the essential, and likely phylogenetically the earliest, role of aldosterone. However, aldosterone also acts in a variety of ‘‘nonclassic’’ targets including brain, heart, and vascular smooth muscle. Its actions in some of these targets play obvious roles in homeostasis, whereas in others they provide no discernible benefit, but rather appear only to be harmful. The actions of aldosterone in classic target tissues require changes in gene transcription; there is presently no direct evidence that nongenomic actions of aldosterone are implicated in physiologically relevant actions in epithelia. Some data support the idea that aldosterone acts through nongenomic mechanisms in certain nonclassic targets; these are addressed in Section VII. In this section, the physiologic actions of aldosterone that require changes in gene transcription are addressed, focusing primarily on its effects on ion transport in epithelia.

A. MINERALOCORTICOID TARGET TISSUES

At the outset, it is important to emphasize that the MR is a dual receptor for glucocorticoids and mineralocorticoids. Indeed, workers studying glucocorticoid (GC) action in the brain identified MR as the type I corticosteroid receptor with a Kd for the physiologic GCs, cortisol and corticosterone, of 0.3 nM, 10-fold higher affinity than the type II receptor, which turned out to be GR (Krozowski and Funder, 1983). Independent work in the study of mineralocorticoids (MC) led to the identification of two receptors for aldosterone in kidney extracts. One of these was recognized as GR because of its similarity to GR purified from liver extracts, with a Kd for aldosterone of approximately 30–50 nM, approximately 100-fold higher than that of MR. The other kidney receptor was identified as MR by the correspondence of its ligand-binding profiles to ligand activities in stimulating Na+ transport (Rousseau et al., 1972). The recognition that the type I GC receptor was likely to be the same gene product as the kidney MR came with the demonstration of their identical biochemical and steroidbinding properties (Ermisch and Ruhle, 1978; Krozowski and Funder, 1983). This observation created a new paradox in that endogenous GCs such as corticosterone and cortisol have little intrinsic MC activity in spite of their high affinity for MR. The solution to this puzzle came, of course, with the subsequent recognition that an enzyme, specific to classic MC target tissues, metabolizes GCs but not MCs to inactive metabolites (Edwards et al., 1988; Funder et al., 1988). 11-HSD2, which allowed MR to be adopted as a selective mediator of aldosterone action in a subset of tissues, is discussed in Section III.

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Phylogenetic evidence is uncertain as to whether MR predates aldosterone. There appears to be a single CYP11B enzyme in fish; however, whether it has dual 11- and 18-hydroxylase activity is less certain. CYP11B in amphibia has dual activity and can accomplish the final steps of aldosterone synthesis (Morris, 1981). Paralleling these observations, aldosterone is present and of clear physiologic importance in amphibia and land vertebrates; however, although it has been identified (at variable levels) in fish, its physiologic function and regulators remain uncertain (Reinking, 1983). It has been suggested that cortisol and 1-hydroxycorticosterone are the sole hormones that activate both MR and GR in teleosts (Wendelaar, 1997) and elasmobranchs (Hazon et al., 1999), respectively. Moreover, the precise timing in vertebrate evolution of the appearance of 11-HSD2, which is presumed to be essential for selective occupancy of MR by aldosterone, is also uncertain, as is the timing of distinct MR and GR. There is little question that MR and GR are derived from a common ancestor that was a general corticosteroid receptor (Thornton, 2001), and that both genes are present in teleosts. Moreover, evidence strongly supports the idea that in contrast to earlier impressions, MR and not GR mediates the effects of corticosteroids on gill chloride cells (Sloman et al., 2001). It currently remains uncertain whether MR is present in elasmobranchs or other more ‘‘primitive’’ marine vertebrates (Thornton, 2001). In mammals, MR functions as a receptor for aldosterone in a subset of tissues (those protected by 11-HSD2), whereas in other tissues it likely functions solely as a glucocorticoid receptor or a dual receptor for both mineralocorticoids and glucocorticoids. B. ALDOSTERONE STIMULATION OF ION TRANSPORT IN TIGHT EPITHELIA

As noted in the introduction, 2002 marks the 50th anniversary of the identification of aldosterone as a mineralocorticoid by Grundy, Simpson, and Tait. Its effects on blood pressure, the latent period (recognized later as due to the requirement for new transcription and protein synthesis), and reciprocal effects on Na+ and K+ excretion by the kidney were established by the end of the decade (Ganong and Mulrow, 1958, and references therein). These investigators also identified the latent period, which was later shown to be due to the requirement for new RNA and protein synthesis. Early mechanistic studies of aldosterone effects on ion transport were confined to amphibia, in particular, the bladder of Bufo marinus (Edelman et al., 1963) and the skin of Xenopus laevis (Voute et al., 1969), both of which can be excised and studied by the methods developed by Ussing to determine active ion transport (Ussing, 1965). Studies on colon, using the Ussing method, also provided key insights into the mechanism of aldosterone-stimulated ion transport (Frizzell and Schultz, 1978); however, the development of micropuncture and microperfusion techniques was needed to study

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FIGURE 3. Aldosterone has multiple effects on principal cells of the ASDN. SGK1 is one of the best characterized ARPs (aldosterone-responsive proteins). SGK1 is now known to act on ENaC plasma membrane abundance, at least in part, through Nedd4-2; it also acts on ROMK1 through NHERF2 (see text for details). The dashed arrows represent effects that might be direct or indirect. Increased Na+ entry is likely to be an important mediator of at least some of the downstream effects of aldosterone (Wade et al., 1990). aldosterone action in the distal nephron (Field et al., 1984; Wright and Giebisch, 1978). Salivary and sweat glands are also important sites of aldosterone-regulated ion transport (Butkus et al., 1976; Robertshaw, 1977). Although clear species- and tissue-specific effects can be discerned, all aldosterone-responsive epithelia have high electrical resistance, limited or regulable water permeability, and are capable of generating extremely high electrochemical gradients, the driving force for which is the basolateral K+ATPase. Apical Na+ transport and K+ transport are passive and proceed down their respective electrochemical gradients; however, unlike many electrically ‘‘leaky’’ epithelia, such as proximal renal tubule or small bowel, Na+ and K+ transport across the apical membrane of the aldosteroneregulated tight epithelia proceeds through highly selective channels, and is not directly coupled to that of any other ion or solute (Fig. 3). The major early action of aldosterone appears to be to stimulate the apical entry of Na+, as first suggested by Crabbe (1963). In mammals, the distal nephron is the single most important site of MC action and is the primary focus of the rest of this section. In part for

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historical reasons, and in part for ease of terminology, the aldosteroneresponsive nephron segments are frequently referred to simply as the ‘‘collecting duct’’ (CD) but are more properly called the ‘‘aldosteronesensitive distal nephron’’ (ASDN) (Verrey, 1995). Many of the general features of regulation in this site are applicable to the actions of aldosterone in other epithelia; the reader is referred to Amasheh et al. (2000), Butkus et al. (1976), Crabbe (1991), and Robertshaw (1977) for more detailed discussion of aldosterone action in nonrenal epithelia. Although there is not uniform agreement about nephron sites of MR expression, it is clear that it is expressed at high levels beginning in the late distal convoluted tubule and extending into the connecting tubule and cortical and medullary collecting ducts (Gnionsahe et al., 1989; Marver, 1980; Todd-Turla et al., 1993). It is also clear that MR is not expressed at significant levels in the glomerulus or proximal convoluted tubule; however, expression in the thick ascending limb of Henle’s loop has been found by some (Gnionsahe et al., 1989). The ability of these nephron segments to generate and maintain large electrochemical gradients (between the tubule lumen and the extracellular space) is central to their role in maintaining fluid and electrolyte homeostasis in response to variations in the environment. Importantly, in spite of the lack of direct coupling, ion movements in the ASDN do profoundly influence each other through their effects on electrical gradients. Aldosterone potently stimulates Na+ transport and, although it may have direct effects on K+ and Cl as well, the sole effect on Na+ transport is likely sufficient for its effects on K+ secretion and Cl reabsorption. This conclusion is supported by a variety of evidence, perhaps most notably by the similar phenotypes of primary aldosteronism and Liddle’s syndrome on the one hand, and aldosterone deficiency and type I pseudohypoaldosteroism on the other; Liddle’s syndrome and pseudohypoaldosteronism are caused by mutations (gain and loss of function, respectively) in the genes encoding the epithelial sodium channel (ENaC) (Karet and Lifton, 1997). The increased H+ and K+ secretion, and Cl reabsorption, appear to be due primarily to the increased lumen-negative potential due to the electrogenic movement of Na+ (Wright and Giebisch, 1978). In the ASDN, two major classes of cells, principal cells and intercalated cells (Brenner and Rector, 2000), are recognized that line the luminal surface and comprise a highly regulated pathway for ion movement. Protons constitute the major ion species transported by intercalated cells, whereas sodium and potassium are transported by the principal cells, and chloride is transported by a combined paracellular and transcellular route (Brenner and Rector, 2000; Schuster and Stokes, 1987). Both cell types express MR and respond to aldosterone with changes in ion transport, although the mechanisms appear to be distinct, and only principal cells express SGK1 (Loffing et al., 2001).

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C. ALDOSTERONE ACTION IN NONEPITHELIAL TISSUES

Whereas virtually all tissues in the body express GR, MR is expressed in a much narrower distribution (Funder, 1993). Free aldosterone in the circulation is 20–200 times lower than that of cortisol (or corticosterone), and it seems likely that MR is predominantly occupied by the latter except in cells with a mechanism for reducing glucocorticoid levels. In most parts of the brain, tissue levels of aldosterone are lower than in peripheral tissues because the MC appears to be excluded by relatively poor penetration of the blood–brain barrier (Funder and Myles, 1996; Hendler and Livingston, 1978); this exclusion appears to be mediated in large measure by multidrug resistance gene 1-type P-glycoproteins (Uhr et al., 2002). The bulk of evidence strongly supports the idea that MR in hippocampus and most other brain regions functions as a GR, not as an MR (Joels and de Kloet, 1994). Evolutionary evidence is uncertain as to whether MR predates aldosterone, although current consensus appears to be that MR originally served as a high-affinity glucocorticoid receptor (Funder, 1996). It is likely, however, that MR is regulated by aldosterone in brain regions unprotected by the blood–brain barrier in cells with a mechanism for inactivating corticosterone. There is evidence to support 11-HSD2 expression in brain regions involved in salt appetite and blood pressure (BP) regulation (Gomez-Sanchez, 1991; Roland et al., 1995). Aldosterone actions in the heart and blood vessels also have gained interest because of their importance in the progression of congestive heart failure, and possibly in BP regulation. This interesting area of ongoing basic and clinical research has been reviewed (Young and Funder, 2002).

III. MOLECULAR BASIS OF MINERALOCORTICOID ACTION A. INTRODUCTION: BASIC PARADIGM OF MINERALOCORTICOID RECEPTOR GENE REGULATION

MC effects in epithelia are well characterized, of unequivocal physiologic importance, and require changes in gene transcription. In contrast, MC effects in nonclassic target tissues are less well characterized, of uncertain physiologic relevance, and the role of gene regulation in mediating the actions of aldosterone is less certain (see Section VII). However, nonepithelial effects of aldosterone are almost certainly of pathophysiologic relevance, and there is mounting evidence that some of these effects are nongenomic; it remains to be determined whether they are mediated by the classic MR, a novel receptor, or both (Simoncini et al., 2000; Wehling et al., 1992). The classic steroid receptors clearly can mediate nongenomic actions;

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however, the physiologic relevance remains somewhat controversial (HafeziMoghadam et al., 2002; Kelly and Levin, 2001; Mihailidou et al., 1998; Su et al., 1988). In this section, the molecular mechanisms of MR function as a regulator of gene transcription are addressed. The reader is referred to primary work and reviews on nongenomic steroid actions (Schmidt et al., 2000; Simoncini et al., 2000). The importance of gene regulation to aldosterone action in epithelia was first established by Edelman and colleagues in the early 1960s (Edelman et al., 1963; Edelman and Fimognari, 1968). These seminal studies, along with work by Tomkins and colleagues on the GR, established the basic paradigm of steroid action (reviewed in Tomkins and Martin, 1970). The GR, in particular, became a prototype for the molecular characterization of hormone-regulated gene transcription, and a vast amount is known about its detailed mechanism of action (see, e.g., Yudt and Cidlowski, 2002). Although much of what has been learned about GR is applicable to MR, there are of course significant differences as addressed later. MR binds aldosterone with high affinity (Kd approximately 109 M) and appears to mediate most of the effects of aldosterone within the physiologic range of hormone concentrations (Felig et al., 1995). GR binds aldosterone relatively poorly (Kd approximately 3 108 M) but likely plays a significant role in mediating aldosterone effects at high concentrations, or when glucocorticoids are able to access GR, for example, when 11-HSD2 levels or activity are low (see Section IV). Importantly, when activated by high levels of aldosterone or by glucocorticoids, GR is able to mediate MR-like effects on ion transport in CD cells (S. Y. Chen et al., 1998; Naray-Fejes-Toth and Fejes, 1990; Schmidt et al., 1993; Schulz-Baldes et al., 2001). The significance of subtle differences between GC and MC that have been reported in inner medullary CD cells (Laplace et al., 1992) and colon (Bastl et al., 1989; Turnamian and Binder, 1989) remains unclear. Steroid hormones are lipid soluble and readily enter virtually all cells by simple diffusion. However, steroids can be pumped out of cells (Kralli and Yamamoto, 1996), which is likely, along with 11-HSD2, to contribute to specificity. In any case, MR and GR, like all members of the nuclear receptor superfamily, are intracellular receptors that bind hormone after it has entered the cell; it does not appear that either exists in a membranebound state, although membrane-bound, nonclassic receptors have been proposed. The receptors are complexed with various chaperones, including heat shock proteins hsp90, hsp70, and hsp56, which appear to be essential to prepare the receptor for interaction with hormone (Pratt and Welsh, 1994). The cellular localization of MR and GR has been well characterized: GR is strongly cytoplasmic in the absence of hormone and, on hormone binding, it translocates into the nucleus, where it remains for variable amounts of time depending on cellular conditions (Defranco, 2000). In the presence of agonists but not antagonists, GR is organized into several hundred

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clusters that have been suggested to represent GR bound to specific DNA target sites (Htun et al., 1996; McNally et al., 2000). The pattern of MR has some similarities to GR; however, in the absence of hormone a substantial fraction of the receptor is already nuclear. As for GR, agonists drive the remaining receptor into the nucleus and induce the formation of numerous clusters (Fejes-Toth et al., 1998). Consistent with the idea that the small mobile clusters represent DNA-bound receptor, mutant receptors that are unable to bind DNA enter the nucleus in response to hormone but do not form wild-type clusters. In contrast, MR that is unable to dimerize and hence unable to bind to certain classes of hormone response elements (but can bind to others) still forms clusters that are indistinguishable from wild type (Pearce et al., 2002). The cytoplasmic localization of unliganded MR and GR is distinct from the nuclear receptors (such as thyroid and vitamin D receptors, which are nuclear in the presence or absence of hormone), and its physiologic relevance is unknown. It is appealing to speculate that nongenomic receptor actions are mediated by the cytoplasmic forms. MR and GR share common structural features with other members of the superfamily, particularly in their centrally located DNA-binding domain (DBD) and C-terminally located ligand-binding (LBD) domain (Fig. 2). N-terminal to the DBD is a highly variable region that most clearly distinguishes the members of the superfamily at the level of primary structure. This region varies both with respect to amino acid composition (with only short stretches of significant homology even among closely related family members) and length (varying from as little as 100 to more than 600 amino acids) (Evans, 1988). B. MECHANISMS OF MINERALOCORTICOID RECEPTOR SPECIFICITY

In keeping with its structural variability, the N-terminal domain appears to be a key determinant of receptor specificity, and indeed may be the sole determinant of differences in transcriptional activities between the closely related MR and GR (Pearce and Yamamoto, 1993; Rupprecht et al., 1993). These two receptors appear to have indistinguishable DNA-binding specificities, and hence it is not surprising that their distinct activities are attributable to differences in protein–protein interactions, both in stimulating and repressing gene transcription at certain classes of target genes (Liu et al., 1996; Meijer et al., 2000; Pearce and Yamamoto, 1993; Rupprecht et al., 1993). The early paradigms of steroid receptor specificity, which envisioned the DBD simply as an anchor that held the receptor at appropriate HREs (or glucocorticoid response elements, GREs), attributed transcriptional specificity to the lock-and-key relationship between receptor and DNA. Although some aspects of this picture have been borne out, it is

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incomplete because it does not account for repression, and accounts for specificity only in some cases (e.g., when DNA-binding specificities are nonoverlapping as for MR and ER). MR and GR activate transcription similarly in some cellular and DNA contexts but differently in others (Arriza et al., 1987; Pearce and Yamamoto, 1993); hence, distinct transcriptional specificities must be context dependent and derive by other means. A first step in identifying a molecular basis for receptor specificity came with the recognition that MR and GR differentially repress gene transcription (Meijer et al., 2000; Pearce and Yamamoto, 1993). Another mechanism for specificity is determined by the far lower capacity of MR for synergy in stimulating gene transcription because of an inhibitory domain in its N terminus. Transcriptional synergy, defined as a more than additive increase in gene transcription conferred by multiple transcription factors or multiple DNA response elements acting together, is a fundamental determinant of gene regulatory responses (Courey, 2001; Herschlag and Johnson, 1993). The first evidence that MR and GR have distinct capacities for transcriptional synergy came from observations that MR stimulated transcription of reporters driven by multiple palindromic GREs much less strongly than GR (Rupprecht et al., 1993). Transcriptional synergy increased and the difference between MR and GR was lost when the N terminus was removed. This inhibitory function was localized to a short synergy control (SC) motif with the consensus sequence (I/V)KXE (Iniguez-Lluhi and Pearce, 2000). Disruption of the SC motifs selectively increases GR activity at compound but not single response elements (i.e., the SC motifs act selectively to curtail receptor synergy). Moreover, transfer of SC motifs to an activator devoid of them is sufficient to impose limits on synergy. Interestingly, the SC motif was also identified as a target for modification by a ubiquitin-like protein, SUMO1, that controls the activity and localization of a variety of proteins (Mahajan et al., 1998; Yeh et al., 2000). Although MR and GR share little homology in their N-terminal domains, they both contain SC motifs. One likely determinant of the far lower activity of MR than GR at genes driven by HRE multimers is that its N terminus contains four SC motifs, whereas GR has two; AR also has two and PR one, whereas ER has none (Iniguez-Lluhi and Pearce, 2000). Importantly, the SC motif is found in a variety of other transcriptional regulators, in which it also appears to inhibit transcriptional synergy (Iniguez-Lluhi and Pearce, 2000). The SC motif mutations recapitulate in many respects mutations that disrupt the DBD dimer interface; the latter have the paradoxical effect of markedly increasing MR or GR activity at GRE multimers (Liu et al., 1996). The receptor N terminus, and in particular the SC motifs, are required for the increased activity of the dimer mutants. Although the physiologic implications are uncertain, these observations suggest the interesting possibility that the DBD dimer interface and the SC motifs act in a common pathway that responds to DNA and cellular context to alter receptor activities.

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C. ROLE OF COACTIVATORS, COREPRESSORS, AND CHROMATIN IN MINERALOCORTICOID RECEPTOR GENE REGULATION

Several different classes of coregulators are recognized that play key roles in every stage of transcription initiation. These include (1) the p160 coactivators and CREB-binding protein (CBP) that recruit histonemodifying enzymes that render chromatin transcriptionally competent (Stallcup et al., 2000), (2) the DRIP/TRAP complex and TAFs (TBP-associated proteins) that assist in recruitment of the general transcription machinery (Freedman, 1999), (3) the Swi/Snf family of chromatin-rearranging molecular machines (Peterson and Workman, 2000), and (4) the corepressors, some or all of which recruit histone deacetylases (Collingwood et al., 1999). The p160 coactivators are of particular relevance to MR activity and so are addressed here in greater detail. This group includes GRIP1 (TIF2), SRC1, and pCIP, which were identified by a two-hybrid screen using the receptor LBDs as bait (Stallcup et al., 2000; Torchia et al., 1998). They share the common features of interacting directly with nuclear/steroid receptor LBDs and augmenting receptor activity. They lack DBDs but can activate transcription themselves if brought to the DNA either through interaction with a nuclear receptor or by a heterologous DBD (such as that of Ga14 or LexA) (Hong et al., 1996). The principal nuclear/steroid receptor interaction motif (the ‘‘LXXLL motif ’’) appears to interact selectively with receptor LBDs. Because MR and GR activities are distinguished by their N-terminal regions (Heck et al., 1994; Pearce and Yamamoto, 1993; Rupprecht et al., 1993), the coactivators were thought not to distinguish MR from GR (Hong et al., 1996). More recently, however, several groups have reported interactions of GRIPI and SRCI with the N-terminal AF1 regions of steroid receptors (Bevan et al., 1999; Ma et al., 1999; Webb et al., 1998) and, importantly, GRIP1/TIF2 and p300 potentiate MR AF1 function (Fuse et al., 2000). Of particular interest, observations suggest that GRIP1/TIF2 works in conjunction with RNA helicase A to differentially regulate AF1 function in response to aldosterone versus corticosterone (Kitagawa et al., 2002). Although the p160 coactivators recruit histone acetyltransferases (HATs) and histone acetylation is a central part of their activities (Collingwood et al., 1999), they also appear to function at least partially in the absence of chromatin, implying that they can participate in post chromatin steps of transcriptional regulation (Hong et al., 1997). Although they do appear to differentially interact with MR and GR N termini (O. C. Meijer, personal communication), they do not appear to differentially influence MR and GR synergistic activities, or impact on SC motif function. In keeping with the paradigm suggested by coactivator acetylation of histones, the corepressors appear to function, at least in part, by recruiting histone deacetylases (HDACs). mSin3A, for example, first identified as a

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mediator of repression by the Mad-Max transcription factor (Ayer, 1999), was subsequently found to mediate repression by nuclear hormone receptors and to form a multisubunit complex with the corepressors SMRT and NCoR, and HDAC1, the first histone deacetylase to be cloned (Nagy et al., 1997). Their roles in MR function remain unknown. The general role of chromatin in gene transcription is clearly repressive, and one step in transcriptional activation is derepression of its effects. Modifications of histones such as acetylation and phosphorylation render nearby genes more accessible to transcription initiation, and deacetylation and dephosphorylation are important in gene silencing (Pazin and Kadonaga, 1997). Interestingly, some of the earliest evidence of the role of chromatin in gene regulation comes from investigations into the GC regulation of the murine mammary tumor virus (MMTV) long terminal repeat (LTR) (Zaret and Yamamoto, 1984). More recently, MMTV has been used to show that GR-dependent modification of a phased array of nucleosomes is a prerequisite for transcriptional activation of the MMTV promoter (Fryer and Archer, 1998). The activation of MMTV organized into chromatin is transient with rapid induction and deinduction phases, whereas nonchromatin MMTV templates undergo sustained stimulation (Archer et al., 1994). In this regard, it is interesting to note that SGK1, an important mediator of MC-regulated Na+ transport (see Section VI), is similarly strongly and transiently stimulated by aldosterone acting through MR or GR (Bhargava et al., 2001; Chen et al., 1999; and Bhargava, unpublished data). This type of regulation may be important for a significant subset of steroid-regulated genes (Meisner et al., 1985). The physiologic implications of these observations for aldosterone action remain to be determined; however, one possible role for the deinduction of SGK1 (and possibly other aldosterone-regulated genes) is to avoid excessive Na+ reabsorption during the consolidation (late) phase of aldosterone action, and to allow a more rapid response to subsequent decreases in aldosterone. It is interesting to note that a modest delay in the deinduction phase could contribute to the development of salt-sensitive hypertension. The mechanistic basis and pathophysiologic relevance of these observations require further inquiry.

IV. ALDOSTERONE ACTION IN EPITHELIA: AFFORDED BY 11-HYDROXYSTEROID DEHYDROGENASE 2 A key factor in the physiological action of aldosterone and cortisol is the activity of two enzymes called 11-hydroxysteroid dehydrogenases 1 and 2 (for a review see Krozowski et al., 1999). These enzymes catalyze the interconversion of cortisol and cortisone in humans and of corticosterone

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and 11-dehydrocortisone in rodents. The activity of these enzymes in particular cell types or tissues can critically dictate the presence or level of active GC. This has consequences for both aldosterone and GC signaling. GC circulate in a 20- to 100-fold excess over aldosterone and without the presence of 11-HSD2, which acts predominantly as a reductase, the nonselective MR would never be activated by aldosterone. Hydroxysteroid dehydrogenases were first characterized in 1953, when an enzymatic activity catalyzing the conversion of 11-dehydrocortisone to corticosterone was first identified in rat liver and was named ‘‘11-hydroxy dehydrogenase’’ and eventually renamed 11-hydroxysteroid dehydrogenase (11-HSD). In the 1980s 11-HSD enzymes were studied in more detail because of a link to a newly recognized clinical syndrome called apparent MC excess (AME). This syndrome was characterized by a defect in cortisol metabolism. It was suggested that the normal conversion of cortisol to cortisone did not occur and that cortisol occupied an ‘‘abnormal’’ MR, where cortisol acted as a potent mineralocorticoid (Oberfield et al., 1983). The cDNA for the human MR was subsequently cloned and MR expression studies, together with MR competition binding assays in vitro, indicated that cortisol, corticosterone, and aldosterone all had an equal affinity for MR (Arriza et al., 1987; Krozowski and Funder, 1983). This represented a conundrum and required the explanation for aldosterone specificity to MR in vivo in the face of a 20- to 100-fold higher level of circulating free cortisol. In 1988 two groups independently provided evidence that this specificity in vivo for the MR was conferred by an 11-HSD itself (Edwards et al., 1988; Funder et al., 1988). Thus the inactivation of cortisol and corticosterone within MC target tissues, such as the kidney, distal colon, and salivary glands enabled aldosterone to specifically activate MR. When this protective mechanism is disrupted, as in AME, cortisol is able to activate MR and acts as a potent MC. A cDNA for an 11-HSD was finally cloned and termed 11-HSD1 (Agarwal et al., 1989; Tannin et al., 1991). This was a low-affinity, NADP(H)-dependent, bidirectional dehydrogenase/11-oxoreductase. From studies performed on the kinetics of this enzyme and its expression in different tissues, it became clear that 11-HSD1 was not the enzyme responsible for the protection of MR in the distal nephron (Brown et al., 1993; Mercer and Krozowski, 1992; Rusvai and Naray-Fejes-Toth, 1993). 11-HSD1 is widely distributed, and is expressed in liver, kidney, fat, brain, ovary, and testis (reviewed in Seckl and Walker, 2001). It has been implicated in the etiology of visceral obesity (Masuzaki et al., 2001). In 1994, cDNAs for a novel 11-HSD enzyme called 11-HSD2 were cloned from a human and sheep kidney cDNA library (Agarwal et al., 1994; Albiston et al., 1994). This was followed by cloning of the rabbit (Naray-Fejes-Toth et al., 1994), rat (Zhou et al., 1995), and mouse homologs (Cole, 1995). This enzyme is highly conserved between species (Condon et al., 1996; Krozowski

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et al., 1995) and is a high-affinity, NAD-dependent, unidirectional 11dehydrogenase. It is now established that AME is an autosomal recessive disorder caused by mutations in the 11-HSD2 gene. In patients with AME, cortisol has access to MR, resulting in a variety of symptoms due to the action of inappropriately activated MR. Other features reported in patients with AME include moderate intrauterine growth retardation, postnatal failure to thrive, polyuria and polydipsia, and nephrocalcinosis. It has also been associated with sudden death (Kitanaka et al., 1996). In 1989 a new variant of AME, termed type II AME, was reported in which the same clinical picture of hypertension, hypokalemia, and suppressed renin–angiotensin– aldosterone system was observed, but unlike classic AME patients excreted normal ratios of cortisol to cortisone metabolites in the urine (Ulick et al., 1990). There were, however, abnormal ratios of other steroids in the urine of patients that suggested a possible defect in 5-reduction of the ‘‘A ring’’ of cortisol (Mantero et al., 1994). Type II AME displays an autosomal recessive mode of inheritance, but its molecular basis is yet to be elucidated (White et al., 1997). Substrates for human 11-HSD2 include cortisol, corticosterone, dexamethasone, 9-fluorocortisol, and prednisolone. These steroids are oxidized by 11-HSD2 to form inactive 11-ketosteroids (Escher et al., 1994; Ferrari et al., 1996). Although it is widely accepted that 11-HSD2 is a unidirectional dehydrogenase, there has been some indication that it can also reduce 9-fluorinated steroids, such as 9-fluorocortisone (Diederich et al., 1996). The activity of 11-HSD2 can be inhibited by both exogenous and endogenous substances such as carbenoxolone, glycyrrhetinic acid, 11- or 11-hydroxyprogesterone, and some bile acids, resulting in GC-dependent MC excess, Na+ retention, and hypertension in vivo (Buhler et al., 1994; Souness et al., 1995; Stewart et al., 1987). Glycyrrhetinic acid, along with glycyrrhizic acid, is a major constituent of licorice (Stewart et al., 1987). It is now known that both inhibit 11-HSD2, explaining why symptoms of licorice intoxication are similar to those of apparent MC excess. Apart from these endogenous inhibitors, 11-HSD2 has also been reported to be inhibited to some degree by its own end products and other endogenous steroid hormones (Ferrari et al., 1996). PHYSIOLOGICAL FUNCTIONS OF 11b-HYDROXYSTEROID DEHYDROGENASE 2

The expression of 11-HSD2 was initially thought to be restricted to classic aldosterone target tissues but has now been detected in a large number of other cell types and tissues. Human 11-HSD2 transcripts have been detected in kidney, colon, pancreas, and placenta, and is weakly detected in the ovary, prostate, adrenal gland, and small intestine (Albiston

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et al., 1994). There is sexual dimorphic expression of 11-HSD2 in kidney and colon, and may reflect male–female differences in sodium homeostasis (Condon et al., 1996). In humans there are considerable differences in both the production and metabolism of cortisol in healthy men and women, suggesting a gender difference in the activity of human 11-hydroxysteroid dehydrogenases. Such gender differences in activity suggest that local bioavailability of cortisol is reduced in women relative to men, an effect that is independent of plasma cortisol levels (Raven and Taylor, 1996). The physiological function of 11-HSD2 in MC target tissues, such as kidney, colon, salivary glands, and sweat glands, has been well described (Agarwal and Mirshahi, 1999; Krozowski et al., 1999). 11-HSD2 metabolizes biologically active glucocorticoids to inactive metabolites, thus conferring aldosterone specificity to otherwise nonselective MRs. The action of 11-HSD2 ensures that aldosterone activates MR, thereby regulating sodium homeostasis. Other tissue systems that express 11-HSD2 include the placenta, brain, adrenal gland, and reproductive organs. The syncytial trophoblast cells of the human placenta are rich in 11-HSD2 activity, which is proposed to block transplacental passage of maternal glucocorticoids to the fetus, thereby protecting the fetus from high circulating levels of maternal GCs (Krozowski et al., 1995). Excess GC exposure can disturb normal patterns of growth and differentiation, and variation in 11-HSD2 activity has been linked to abnormal fetal growth (Benediksson et al., 1993) and subsequent development of disease in adult life (Seckl, 2001). The pancreas is not normally considered MC target tissue but the intercalated and interlobular ducts of the pancreas contain MR, and aldosterone appears to be involved in regulating amylase levels in pancreatic exocrine cells (Albiston et al., 1994). The presence of 11-HSD2 activity in these cells may therefore protect MR and allow aldosterone to modulate electrolyte levels in pancreatic cells. Aldosterone and cortisol have a multitude of actions in the brain, mediated via specific corticosteroid receptors. 11-HSD2 was predicted to exist in the brain on the basis of observations of aldosterone-selective actions in the brain in terms of blood pressure and salt appetite. In the rat brain 11-HSD2 mRNA has been detected in cells of the commissural portion of the nucleus tractus solitarius, subcommissural organ, and ventrolateral ventromedial hypothalamus (Roland et al., 1995). Scattered labeled cells were also seen in the medial vestibular nucleus. In the mouse 11-HSD2 has been analyzed during prenatal brain development and this was correlated with expression of GR and MR mRNA (Diaz et al., 1998). High 11-HSD2 activity at midgestation may protect the developing brain from activation of GR by GCs, whereas late in gestation reduction of 11-HSD2 activity may allow increasing glucocorticoid activation of GR and MR, permitting maturation of key GC-dependent neuronal and glial cells.

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V. GENETIC MOUSE MODELS IN THE INVESTIGATION OF ALDOSTERONE ACTION The advent of gene-targeting transgenic technology in mice has led to the development of a number of genetically modified mice specifically designed for the investigation of the underlying mechanisms of action of aldosterone (reviewed by Verrey, 2001). We discuss more recent results from genetargeted deficient mice that investigate aldosterone function at either the level of ligand preference (HSD2), receptor function (MR), or at downstream target genes (ENaC subunits , , or ). A summary of the phenotypes of these mutant mice is shown in Table 1. The 11-HSD2–deficient mouse is an animal model of illicit occupation of MR by glucocorticoids, in epithelial tissues and cells normally considered targets of aldosterone. These mice display the classic symptoms of human AME: sodium retention, hypokalemia, and hypertension (Kotelevtsev et al., 1999). Over 50% of 11-HSD2–/–mice die within a few days of birth, primarily from either a failure to suckle, cardiac arrest from hypokalemia, or abdominal complications. In surviving adult 11-HSD2–/– mice the hypertension is severe (>145 mmHg), causing visible cardiac hypertrophy but no cardiac fibrosis, suggesting that fibrosis is mediated by unprotected MR requiring elevated levels of corticosteroids. There is a striking renal phenotype of hyperplasia and hypertrophy of the distal tubule most likely caused by increased MC activity. The enlarged distal tubular epithelium has

TABLE I. Phenotypes of Mice with a Gene-Targeted Mutation in Members of the Aldosterone Signaling Pathway Gene mutation

Time of lethality

Phenotype

MR

Perinatal/adult (50%/50%) 1–2 weeks postnatal

ENaC

40 h postnatal

ENaC

40 h postnatal

ENaC

48 h postnatal

SGK1

Viable

Hypertension, hypokalemia, renal distal tubule hypertrophy/hyperplasia; model of AME Salt/water wasting, reduced renal/colonic ENaC function; model of PHA-1 Lethal respiratory distress syndrome, renal metabolic acidosis Delayed lung fluid clearance, salt wasting, lethal hyperkalemia Delayed lung fluid clearance, salt wasting, lethal hyperkalemia Salt wasting, elevated aldosterone, weight loss on low-salt diet

11-HSD2

Abbreviations: AME, Apparent mineralocorticoid excess; ENaC, epithelial sodium channel; 11-HSD2, 11-hydroxysteroid dehydrogenase type II; MR, mineralocorticoid receptor; PHA1, pseudohypoaldosteronism type 1; SGK1, serum and glucocorticoid-regulated kinase 1.

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increased total mitochondria and basal membrane surface area, a consequence of increased energy requirement from activated ion transport. 11-HSD2 is expressed in the vessel wall and one study has demonstrated endothelial dysfunction that causes enhanced norepinephrine-mediated contraction (Hadoke et al., 2001). This may independently contribute to the level of hypertension found in 11-HSD2 deficient mice. Mice lacking MR have also been generated by gene targeting. MRdeficient mice display normal embryonic development, but, not surprisingly, 1–2 weeks after birth they develop symptoms of pseudohypoaldosteronism type 1 (Berger et al., 1998). They are unable to maintain normal sodium and water, and finally by day 8–13 they die from the effects of dehydration and hyperkalemia. They have a highly activated renin–angiotensin–aldosterone system and have increased fractional renal excretion of Na+ with reduced transepitheilial function in the colon and kidney. There is hyperplasia of the extraglomerular mesangium, extension of renin-producing granular cells upstream along the afferent arteriole, and hyperplasia of interlobar arteries (Hubert et al., 1999). The rescue of MR-deficient mice by administration of isotonic saline has allowed studies in surviving adult mice (Bleich et al., 1999). A striking feature in both neonatal and adult-rescued MR-deficient mice is the lack of any major change in the expression of a number of wellcharacterized aldosterone target genes. These include the ENaC, and Na+,K+-ATPase, subunits in kidney and colon, and Nedd4 in the kidney. The only significant change was a 30% reduction in the mRNA levels of ENaC in the kidney of adult mice. Any changes in the levels of SGK1, an important direct target of aldosterone, in the kidney or colon of MR-null mice have yet to be reported. Adult MR-deficient mice have also been analyzed for deficits in the central nervous system, where corticosteroids are important in hippocampal functions. MR deficiency caused a 65% reduction in granule cell numbers in the hippocampus, implying a significant role in hippocampal neurogenesis and function (Gass et al., 2000). Of the small number of aldosterone target genes so far described, only the ENaC , , and subunit genes have been fully analyzed in mice by gene targeting. Similar to MR-deficient mice all three types of ENaC subunitdeficient mice die shortly after birth, yet with some differences in individual phenotypes. ENaC-deficient mice develop respiratory distress and die within 40 h of birth because of a failure to clear their lungs of liquid (Hummler et al., 1996). The lung expresses all three subunits of ENaC, and this clearly showed for the first time that the subunit of ENaC plays a critical role in perinatal lung liquid clearance. Interestingly, - or -deficient mice, although having increased lung water content at birth, do not display symptoms of respiratory distress (McDonald et al., 1999; Barker et al., 1998). This is even more remarkable in the -deficient mice as they have only about 15% residual ENaC activity in the lung. In contrast to -deficient mice, both - and -deficient mice instead show a severe defect in renal

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function shortly after birth that leads to death within 2–3 days. There is urinary Na+ loss, hyperkalemia, and increased plasma levels of aldosterone; features not dissimilar to those of patients with pseudohypoaldosteronism type 1 (PHA-1). A second subunit–targeted mouse has been described that expresses reduced levels of a Liddle’s syndrome stop truncation mutation of the ENaC subunit (Pradervand et al., 1999). On a low-salt diet these mice display clinical symptoms of acute PHA-1. Overall these mouse models of ENaC dysfunction are invaluable tools in understanding the role of ENaC in sodium handling and the pathophysiology of PHA-1. The problem of lethality in these mouse models of ENaC deficiency is being overcome via the utilization of the Cre recombinase/loxP gene-targeting system. A conditional allele of the ENaC subunit gene that will allow tissue-selective ablation of -subunit expression has been described and will allow investigation in future of the role of the ENaC subunit in different tissues (i.e., lung vs. kidney) of adult mice (Hummler et al., 2002). This tissueselective gene ablation strategy is also being applied to other members of the aldosterone signaling pathway. SGK1-null mice have been reported (Wulff et al., 2002). These mice are viable but consistent with the role of SGK1 in regulating ENaC (Pearce, 2001), they have a moderately severe form of PHA, with diminished capacity to achieve sodium balance in the face of elevated aldosterone and weight loss when placed on a low-sodium diet. They also have a marked shift in tubular Na+ reabsorption to proximal nephron segments. Importantly, the SGK1 knockout has a milder phenotype than the MR knockout (Berger et al., 1998), suggesting that genomic targets other than SGK1, or potentially nongenomic targets, are implicated in MR function.

VI. ALDOSTERONE TARGET GENES THAT MEDIATE PHYSIOLOGICAL RESPONSES The quest for target genes for aldosterone that could account for changes in ion transport has been a challenge. Changes in either mRNA or protein levels of genes that are directly involved in the Na+ transport pathway (K+ATPase, ENaC, etc.) occur late and are relatively subtle. Other genes whose expression appears to be regulated by aldosterone are mitochondrial citrate synthase and flavokinase. In the intercalated cells of the kidney, aldosterone induces H+, K+-ATPase, possibly contributing to increased elimination of protons. Aldosterone has been documented to regulate expression of its receptor. However, it appears that this autoregulation is controversial; MR levels have been shown to change in response to aldosterone treatment in the brain and kidney (Chao et al., 1998; Claire et al., 1981) and, indeed, in our hands we have observed a robust downregulation of MR after aldosterone

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administration in rat kidney (A. Bhargava and D. Pearce, unpublished observation), whereas others have reported no change in MR levels (Escoubet et al., 1996; Meyer and Schmidt, 1994). Thus, even though the issue remains controversial, in physiological systems, it makes sense for the hormone to downregulate its receptor in order to protect against overstimulation (or repression) and thereby from any toxic effects. Aldosterone also appears to transiently downregulate GR mRNA and protein in rat colon (Meyer and Schmidt, 1994) and hippocampus (O’Donnell and Meaney, 1994). In addition to its effect on epithelial target tissues, aldosterone also regulates gene expression in nonepithelial tissues such as the heart, brain, B lymphocytes, and vascular smooth muscle cells. The array of genes regulated by aldosterone in these target tissues remains largely unidentified. Brown adipose tissue was shown to be a new target for aldosterone action (Zennaro et al., 1998). Aldosterone and MR have been shown to induce adipocyte differentiation with a dose-dependent increase in triglyceride content. In addition, aldosterone has been shown to repress uncoupling protein (UCPI) mRNA and function in brown adipocytes (Le Menuet et al., 2000). In the heart, plasma aldosterone level displays a correlation with left ventricle size and with mortality in patients with heart failure (Rossi et al., 1997; Swedberg et al., 1990). Studies from Delcayre and colleagues find a complex relationship between aldosterone and MR. They demonstrate an increase in MR message and protein levels in left ventricle (but not the right ventricle or the kidney) in aldosterone-salt treated rats. Further, they propose that this induction may also be sensitive to mechanical stimulation. In support of this view, they show an increase in mRNA of atrial natriuretic peptide (ANP, a marker for cardiac hypertrophy) in the left but not the right ventricle. Such an increase in MR levels was also observed in two other models of arterial hypertension, AngII-hypertensive rats and SHR (spontaneously hypertensive rat). In all cases, spironolactone failed to ameliorate these effects (Silvestre et al., 2000). Expression of ENaC in the heart is questionable. The known isoforms of ENaC do not appear to detect any message in heart as assessed by Northern blot studies (Killick and Richardson, 1997). SGK1 is expressed in the heart in both rats (Bhargava et al., 2001; Kobayashi et al., 1999) and mice (Lee et al., 2001) as shown by Northern blot analysis and in situ studies. SGK3, like SGK1, is ubiquitous in its expression pattern, whereas there is no detectable SGK2 in the heart (Kobayashi et al., 1999). SGK1 is unresponsive to aldosterone treatment in the heart (Bhargava et al., 2001), whereas SGK2 and SGK3 appear to be unresponsive to hormone treatment in the systems examined so far (Kobayashi et al., 1999). Aldosterone appears to regulate expression of Na+,K+-ATPase in cultured neonatal and adult rat cardiocytes. mRNA

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for both 1 and the 1 subunits was induced 3-fold within 6 h of aldosterone treatment (Ikeda et al., 1991); this induction was insensitive to cycloheximide treatment, suggesting that this was a primary effect and does not require de novo protein synthesis.

A. ALDOSTERONE ACTION IN BRAIN

MR in brain is found in abundance in the limbic system, which includes the hippocampus and amygdala. In hippocampal neurons, both MR and GR have been shown to be colocalized (Van Eekelen and De Kloet, 1992). A high density of MR is also found in the hypothalamic nuclei. The central MR effects are associated with the regulation of ACTH release, water and electrolyte balance, arousal, salt appetite, and blood pressure (Brody et al., 1991; Janiak and Brody, 1988; Sakai et al., 1996). The presence of aldosterone or low levels of corticosteroids (cortisol or F in humans and corticosterone or B in rodents), as observed during circadian nadir or under conditions of mild stress, results predominantly in activation of MR, whereas high levels of corticosteroids (during circadian peak or under conditions of chronic stress) result in activation of both GR and MR. Occupation of MR versus occupation of both receptors (MR and GR) has profound effects on brain function. These effects have been well studied in the hippocampus, where differential activation of the two receptors has been shown to have opposite effects on neuronal excitability (Joels and de Kloet, 1989; Joels and Vreugdenhil, 1998). These effects can be explained by activation or repression of a large number of overlapping and discrete sets of genes. Both MR and GR are known to regulate calcium homeostasis in neurons. Voltage- and ligand-gated calcium channels are thought to be key players, yet at mRNA and protein levels, their regulation has proved disappointing. Plasma membrane Ca2+-ATPase (or calcium pump) isoform 1, an enzyme involved in extrusion of Ca2+ from neurons, was shown to be regulated by these hormones both at the mRNA and the protein level in rat brain (Bhargava et al., 2000, 2002). Another study has identified more than 200 genes in the brain that are responsive to corticosteroid, using serial analysis of gene expression (SAGE) (Datson et al., 2001). Most of the targets identified are part of cellular metabolism and energy regulation pathways, including enzymes of the glycolytic pathway, ATPases, ATP synthase, and lipid metabolism enzymes. Another subset of genes identified is involved in signal transduction pathways. Although these large numbers of genes have been identified as putative target genes, their exact regulation by these hormones needs to be elucidated. Hormone modulation of target genes in the brain is fairly complex and is beyond the scope of this review.

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B. NA+,K+-ATPASE

The Na+,K+-ATPase and the H+, K+-ATPases are closely related members of the P-type family of ion-transporting ATPases. The Na+,K+ATPase, or sodium pump, is localized to the basoleteral membrane, and provides the driving force to extrude three Na+ out and two K+ into the cells against significantly high concentration gradients. The enzyme has diverse functions; it maintains membrane potentials on the one hand, while on the other hand in the renal and intestinal tissues it regulates solute transport (Horisberger et al., 1991b). The and the subunits of the Na+,K+-ATPase assemble in the endoplasmic reticulum to form a functional moiety. Prevention of this assembly leads to degradation of the unassembled subunit (Beguin et al., 2000). Both the Na+,K+-ATPase 1 and 1 subunits have been shown to harbor GREs in their promoters (Kolla and Litwack, 2000; Kolla et al., 1999) and have been shown to be targets of aldosterone action. The GRE from the 1 subunit appears to be differentially regulated by MR and GR, with MR inducing the promoter with greater potency than GR. The increase in activity of the pump in the early phase does not appear to require de novo RNA or protein synthesis but appears to recruit existing sodium pump subunits in response to enhanced Na+ entry. However, during the later phase, aldosterone does appear to increase both the message and the protein levels (Verrey et al., 1987).

C. EPITHELIAL SODIUM CHANNEL

The amiloride-sensitive ENaC constitutes the rate-limiting step for sodium reabsorption in the target epithelial tissues. The activity of ENaC is modulated by aldosterone, vasopressin, and insulin and contributes toward maintenance of sodium homeostasis, blood volume, and blood pressure (for a review see Snyder, 2002). Vasopressin and oxytocin are thought to increase channel density, either by recruiting new channels or by opening of preexisting quiescent channels via the secondary messenger (cAMP) or phosphorylation pathways (Els and Helman, 1989). The core ENaC channel is composed of three subunits, , , and (Canessa et al., 1993, 1994; Lingueglia et al., 1993), whereas occasional channels comprising , , and subunits have been reported (Waldmann et al., 1995). The ENaC is highly selective for Na+ and Li+. Members of the DEG/ENaC family are expressed in the brain and are frequently referred to as the BNaC or ASIC (GarciaAnoveros et al., 1997; Waldmann et al., 1997). Four subunits of acid-sensing ion channels (ASICs) are found in mammalian brain. ASICs participate in proton-sensitive gating and are widely expressed in the central and peripheral nervous systems (Chen et al., 1998; Price et al., 1996).

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During the early phase of Na+ reabsorption, aldosterone does not appear to alter ENaC expression in the kidney, although ENaC is transcriptionally regulated by aldosterone as an early event in distal colon and by GCs in the lung. The lack of regulation by aldosterone in the kidney suggests control through regulation of other genes whose products control, for example, relocalization of ENaC and/or involvement of other proteins that modulate ENaC structure, trafficking, and function. In this regard, it was shown that when aldosterone levels are low (Na+-replete condition), ENaC is mainly intracellular in its localization, exhibiting diffuse punctate staining throughout the principal cells of the collecting duct (Masilamani et al., 1999). Aldosterone infusion or Na+ restriction caused a marked change in redistribution of ENaC to the apical surface (Loffing et al., 2001). Thus, aldosterone appears to increase Na+ reabsorption by either increasing the number of ENaCs at the cell surface (Garty and Palmer, 1997) or by changing its gating properties (Kemendy et al., 1992). It was demonstrated that the C terminus of the ENaC and the ENaC is phosphorylated at Ser/Thr residues by a putative serine-threonine kinase (Shimkets et al., 1998). Identification of SGK, a serine-threonine kinase involved in rapid increase in epithelial sodium currents (S. Y. Chen et al., 1999; Naray-Fejes-Toth et al., 1999) (see Section D for details) led to the idea that SGK may be the elusive kinase that regulates activity of ENaC subunits. However, this notion was short lived, and although SGK associates with ENaC in vitro (Wang et al., 2001a), it does not appear to phosphorylate ENaC subunits and the mysterious kinase that phosphorylates ENaC still remains to be identified. D. ALDOSTERONE-REGULATED GENES WHOSE PRODUCTS ALTER EPITHELIAL SODIUM CHANNEL LOCALIZATION OR ACTIVITY

1. SGK1 SGK1, a serine/threonine kinase, was first identified as a serum and glucocorticoid-regulated kinase in rat mammary tumor cell line (Webster et al., 1993). Various signal transduction pathways exert distinct effects on transcriptional regulation of SGK1. For example, SGK1 expression is induced by injury in brain (Hollister et al., 1997; Imaizumi et al., 1994) and by hypertonic stress or cell volume changes (but not by GCs) in hepatocytes (Waldegger et al., 1997); hypotonic stress induces SGK1 expression in Xenopus cortical collecting duct cells (A6 CCD) (Rozansky et al., 2002) and follicle-stimulating hormone induces its expression in ovarian cells (Alliston et al., 1997). More recently, SGK1 was shown to be actively involved in memory consolidation of spatial learning in rats (Tsai et al., 2002). In addition to all these signals, MCs and GCs induce its expression in both A6 cells and rat kidney (Chen et al., 1999) and rabbit CCD cells

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(Naray-Fejes-Toth et al., 1999). In vivo, physiological doses of aldosterone have been shown to induce its expression as early as 30 min after aldosterone administration in rat kidney (Bhargava et al., 2001), while both aldosterone and dexamethasone have been shown to regulate its expression in the kidney and the colon (Bhargava et al., 2001; Brennan and Fuller, 2000; Shigaev et al., 2000). SGK1 is a primary target gene for aldosterone; its induction remains unaffected by cycloheximide treatment. Its mRNA is induced within 15 min of hormone treatment and peaks at about 45 min in A6 cells, whereas SGK1 protein levels peak at about 6 h (Chen et al., 1999). In vivo, in rat kidney aldosterone treatment results in focal induction of SGK1 mRNA in the distal nephron. The message appears to peak at about 2 h and is back to near-baseline levels within 24 h (both in vitro and in vivo) (Bhargava et al., 2001; Chen et al., 1999). Immunohistochemical studies have shown that SGK1 protein is expressed in the principal cells but not in the intercalated cells (Loffing et al., 2001). SGK1 mRNA expression pattern and responses to aldosterone are similar in mouse kidney. One interesting observation was that chronic but not acute aldosterone excess appears to result in a modest increase in glomerular SGK1 expression and a significant decrease in the initial portion of inner medulla, probably because of decreased interstitial osmolarity (Hou et al., 2002). Whole embryo mouse in situ analysis shows that SGK1 mRNA exhibits a stage- and tissuespecific expression pattern and first appears on embryonic day E8.5 (Lee et al., 2001). SGK1 mRNA has been shown to be induced by aldosterone in the distal colon (Brennan and Fuller, 2000; Shigaev et al., 2000). It has also been shown that the distal descending colon is sensitive to nanomolar levels of aldosterone, and that expression of other aldosterone-responsive genes (ENaCs) is differentially regulated over the course of the distal colon (Amasheh et al., 2000; Epple et al., 2000). Interestingly, robust induction of SGK1 protein is observed after aldosterone treatment in distal colon (Bhargava et al., 2001). It is thus clear that the epithelial response of SGK1 in vivo to aldosterone can be extended to the colon, as an additional epithelial tissue, suggesting that SGK1 may be an essential mediator of aldosterone-stimulated ion transport in all classic MC target tissues. SGK1 displays 45–55% sequence identity throughout its catalytic domain with protein kinase B (PKB/Akt), protein kinase C, ribosomal protein S6 kinase, and cAMP-dependent protein kinase (Webster et al., 1993). A peptide sequence within SGK1 (Sgktide) was shown to be phosphorylated by 3-phosphoinositide-dependent protein kinase 1 (PDK1) (Park et al., 1999). Further evidence that SGK1 is indeed phosphorylated by PDK1 comes from the observation that specific inhibitors of PI3K (LY and wartmannin) inhibit both SGK1 phosphorylation and MC-induced Na+ transport (Wang et al., 2001a).

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SGK1 was the first aldosterone-regulated gene that was shown to strongly stimulate ENaC-mediated Na+ transport in a Xenopus oocyte coexpression assay (S. Y. Chen et al., 1999; Naray-Fejes-Toth et al., 1999; Shigaev et al., 2000; Wagner et al., 2001). Although SGK1 has been shown to interact with and ENaC in vitro (Wang et al., 2001a), it is becoming increasingly apparent that this association does not result in phosphorylation of ENaC by SGK1 but instead a new target for SGK1 has been identified: Nedd4-2 (Debonneville et al., 2001; Snyder et al., 2002) (see later). In oocyte coexpression studies, SGK1 appears to greatly enhance plasma membrane expression of ENaC (Alvarez de la Rosa et al., 1999; Loffing et al., 2001) whereas a kinase-dead mutant of SGK1 prevents this membrane localization. In agreement with these results, a kinase-dead mutant of SGK1 does not stimulate ENaC current in cultured cells but instead displays dominant negative effects (Alvarez de la Rosa et al., 1999; Loffing et al., 2001). Furthermore, A6 cells stably expressing SGK1 exhibit a 3.5-fold enhancement of basal Na+ transport, whereas cells that stably express the kinase-dead mutant show inhibition of both basal and steroid hormone–stimulated Na+ currents (Faletti et al., 2002). Hence, it appears that SGK1 is both necessary and sufficient to mediate the effects of aldosterone on Na+ transport, at least in cultured cells. Evidence also supports the idea that SGK1, in concert with NHERF2, can stimulate ROMK1-mediated K+ transport, independent of its effects on Na+ transport (Yun et al., 2002). For a more focused review of SGK1 and its effect on ENaCs, readers are referred elsewhere (Fillon et al., 2001; Kamynina and Staub, 2002; Pearce, 2001). As discussed in Section V, the development of SGK1-null mice has confirmed the physiological importance of SGK1 in mediating aldosterone effects on renal sodium handling. Two other isoforms of SGK, namely SGK2 and SGK3, appear to be largely unresponsive to GCs and MCs. SGK3 is fairly ubiquitous in its expression, whereas expression of SGK2 is restricted to certain tissues that include liver, kidney, pancreas, and brain. Both SGK2 and SGK-3 are activated by PDK1 (in vitro), albeit at a slower rate than SGK1, and their activities are only partially suppressed by inhibitors of phosphatidylinositol 3-kinase (PI3K). In addition, both SGK2 and SGK3 preferentially phosphorylate Ser and Thr residues that lie in RXRXXS/T motifs (Kobayashi et al., 1999). 2. K-Ras2 K-Ras2, a small G protein and a proto-oncogene, was identified as an aldosterone-induced gene in A6 cells (Mastroberardino et al., 1998). Subsequently, K-Ras2 was shown to be induced both in A6 cells and in Xenopus kidney (Mastroberardino et al., 1998; Spindler and Verrey, 1999) about 2.5-fold at the message level and about 6-fold at the protein level in A6 cells (Spindler and Verrey, 1999; Stockand et al., 1999) within 2–4 h

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of aldosterone treatment. Overexpression of K-Ras2 with ENaC in Xenopus oocytes or in A6 cells resulted in enhanced Na+ current (Mastroberardino et al., 1998; Stockand et al., 1999). Conversely, antisense oligonucleotide against K-Ras2 resulted in decreased aldosterone-induced Na+ current and K-Ras2 protein (Stockand et al., 1999). K-Ras2 is thought to enhance Na+ current by influencing the activity of SGK1 via the PI3K pathway (Wang et al., 2001b). However, the exact mechanism by which K-Ras2 achieves activation of ENaC and increase in Na+ current remains to be elucidated.

E. ALDOSTERONE-REGULATED GENES WHOSE PRODUCTS REGULATE OTHER COMPONENTS OF ION TRANSPORT MACHINERY

Channel-inducing factor and the Subunit of Na+,K+-ATPase Channel-inducing factor (CHIF) and the subunit of Na+,K+-ATPase (NKA) are members of a new family of small membrane proteins, the FXYD protein family (Sweadner and Rael, 2000). The proteins of the FXYD family have a signature sequence that encompasses the single transmembrane domain that includes the FXYD motif. CHIF is expressed in colon and kidney papilla but not in heart, brain, or skeletal muscle (Attali et al., 1995). CHIF mRNA is induced by aldosterone, dexamethasone, and under conditions of low Na+ (Attali et al., 1995; Brennan and Fuller, 1999; Capurro et al., 1996; Wald et al., 1997). CHIF message increased 4-fold 2–4 h after hormone treatment in the distal colon but not in the kidney. Interestingly, adrenalectomy also repressed the levels of CHIF in the colon but had no effect on CHIF expression in the kidney (Brennan and Fuller, 1999). Injection of CHIF into Xenopus oocytes led to activation of a slowly activated but strongly voltage-dependent, K+ current (Attali et al., 1995). The subunit of Na+,K+-ATPase is expressed in both the kidney and the colon (Pu et al., 2001) but is not regulated by either aldosterone or dexamethasone (Brennan and Fuller, 1999). Both CHIF and the subunit coimmunoprecipitate with the NKA but not with the H+, K+-ATPases (Beguin et al., 2001). CHIF colocalizes with NKA in the basolateral membrane of the principal cells of the CD and in distal colon (Shi et al., 2001). Association of CHIF with NKA increases its affinity for Na+ while it decreases the apparent K+ affinity because of an increased Na+ competition at the external binding site. In contrast, association of the subunit with NKA appears to decrease the apparent Na+ affinity of the pump. Thus, these regulatory mechanisms contribute toward maintaining the Na+ and K+ homeostasis in aldosterone-responsive tissues by a novel mechanism.

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F. GENE PRODUCTS THAT APPEAR TO MARKEDLY ALTER COMPONENTS OF THE ALDOSTERONEREGULATED NETWORK, BUT THAT ARE NOT DIRECT TARGETS OF ALDOSTERONE GENE REGULATION

1. CAP1 Channel-activating protease 1 (CAP1) was first identified from cultured Xenopus CD cells (Vallet et al., 1997). CAP1 is a member of a serine protease family that is thought to be secreted and/or a glycosylphosphatidylinositol-anchored protein. The mouse homolog, mCAP1, has also been cloned (Vuagniaux et al., 2000). CAP1 was found to be highly expressed in all epithelial tissues that express ENaC such as the kidney, intestine, stomach, skin, and lung in both Xenopus and mouse. MCAP1 was found to be abundantly expressed in the proximal tubule and to a lesser extent in the distal nephron. The coexpression of CAP1 with ENaC in Xenopus oocytes results in a 2- to 3-fold increase in the activity of the channel, while it had no effect on the activity of Na+,K+-ATPase in Na+loaded oocytes (Horisberger et al., 1991a), suggesting that CAP1 specifically activated ENaC. The activity of ENaC was also seen to be enhanced by trypsin and chymotrypsin. Thus, even though CAP1 is not a direct target of aldosterone action, it appears to regulate activity of the amiloride-sensitive ENaC directly or indirectly by acting at the extracellular side of the apical membrane. CAP1 is thought to increase the open probability of ENaC; an effect that does not require activation of a G protein-coupled receptor (Chraibi et al., 1998) but seems to occur via proteolysis of a protein that is either an essential component of the channel or closely associated with it. 2. Nedd4-2 The expression of a set of genes, first identified from neural precursor cells, was downregulated during the development of mouse brain, and hence these genes were termed Nedd: NPC-expressed, developmentally downregulated (Kumar et al., 1992). Subsequently, Nedd4 was shown to be a ubiquitin-ligase that was found to be associated with ENaC, and a regulator of ENaC activity (reviewed by Staub et al., 2000). The Nedd4 family of proteins contains several signature motifs that include a C2 (Ca2+dependent lipid-binding) domain (Plant et al., 1997), two to four WW (tryptophan-rich) domains that interact with the PY motif on ENaC subunits, and a carboxy-terminal HECT (homologous to E6-AP carboxy terminus) domain that mediates ubiquitinylation (Staub et al., 2000). A second isoform of Nedd4, Nedd4-2, was identified that binds to and suppresses ENaC activity in a Xenopus oocyte coexpression assay and is likely the physiologically relevant isoform, not the originally identified Nedd4 (now referred to as Nedd4-1) (Kamynina et al., 2001). Nedd4-2, which lacks the C2 domain characteristic of Nedd4-1, is expressed in a

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variety of ion-transporting epithelia including the kidney, lung, placenta, brain, heart, colon, liver, skeletal muscle, and small intestine. No detectable Nedd4-2 was seen in thymus, spleen, and peripheral blood leukocytes (Kamynina et al., 2001). Interestingly, Nedd4-1 can regulate ENaC activity provided its C2 domain is deleted, although the addition of a C2 domain to Nedd4-2 does not affect its ability to downregulate ENaC activity. Subsequent to Nedd4-2-dependent ubiquitinylation, ENaC proteins are removed from the plasma membrane and either recycled or targeted for proteasome-mediated degradation. Interestingly, Liddle’s syndrome, a form of pseudoaldosteronism, results from mutations in the C terminus of and ENaC subunits that disrupt its ability to bind to Nedd4-2 (Kamynina et al., 2001; Shimkets et al., 1994). The similarities between Liddle’s syndrome and primary aldosteronism coupled with the identification of two consensus SGK1 target sequences in Nedd4-2 (but not Nedd4-1) led to the idea that Nedd4-2 interaction with ENaC might be regulated by SGK1 (Debonneville et al., 2001). Indeed, two articles demonstrate that SGK1 interacts with and phosphorylates Nedd4-2 in a PY motif-dependent manner, leading to reduced interaction between ENaC and Nedd4-2, and hence to elevated ENaC cell surface expression (Debonneville et al., 2001; Snyder et al., 2002). These observations strongly suggest a mechanistic link between Liddle’s syndrome and primary aldosteronism.

VII. CONTROVERSIES WITH ALDOSTERONE A. ALDOSTERONE, CARDIAC FIBROSIS, AND HEART FAILURE

Over the past few decades the treatment of acute coronary events has significantly improved and overall mortality from heart attacks has been reduced. In contrast, however, the prevalence of heart failure has been steadily increasing and now represents a major cause of cardiovascular morbidity and mortality. A potential role for aldosterone in the pathogenesis of heart failure has been suggested from the results of the Randomized Aldactone Evaluation Study (RALES) (Pitt et al., 1999). The RALES trial demonstrated, for patients with moderately severe heart failure given a low dose of spironolactone, an MR antagonist (on top of normal therapy), a 30% reduction in mortality and a 35% reduction in morbidity. The implied pathological pathway of aldosterone, either direct or indirect, in heart failure is poorly understood. MR is found at moderate levels in cells of the myocardium including cardiomyocyes, vascular smooth muscle, and fibroblasts. There is almost no 11-HSD2 activity in cardiomyocytes, leaving MR largely unprotected from GCs that in fact have been shown to act in these cells as MR antagonists (Young and Funder, 1996). In rodent

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models, inappropriately high levels of aldosterone in the presence of high salt promotes severe cardiac fibrosis, with marked vasculitis and tissue remodeling, similar to that found in heart failure (Young and Funder, 2002). Also, a number of studies have demonstrated low-level biosynthesis of aldosterone in the failing human heart, further implicating aldosterone in the progression of heart failure. A study on normal human heart failed to detect in any chamber of the heart expression of aldosterone synthase, the key biosynthetic enzyme required for aldosterone synthesis (KayesWandover and White, 2000). Subsequent studies analyzing tissue from the failing human heart detected low-level expression of aldosterone synthase (Young et al., 2001) and local production of aldosterone from failing heart (Mizuno et al., 2001). Key unanswered questions include the following: (1) What are the early aldosterone-induced events leading to cardiac fibrosis? (2) How does aldosterone directly modulate cardiac function; is it directly via cardiac MR or through other mechanisms (i.e., rapid non-genomic actions)? (3) Does MR antagonism block or reverse the onset of vascular injury and cardiac fibrosis? In the mineralocorticoid–salt rodent model of cardiac fibrosis there is emerging evidence of severe coronary damage and marked perivascular inflammation well before changes in collagen status (Fujisawa et al., 2001). There is evidence of leukocyte and macrophage infiltration, and apoptosis of cells in perivascular areas of the heart. Important markers of inflammation such as cyclooxygenase 2, interleukin 6, and osteopontin have also been demonstrated to be induced in hearts of mineralocorticoid–salt-treated rats (Stier et al., 2002). An important inhibitor of collagen breakdown, PAI1, is also elevated in some models of cardiac fibrosis (Huber et al., 2001). In fact, PAI-1–/–mice show protection against vascular damage associated with models of vascular and cardiac fibrosis, and direct inhibition of PAI-1 may provide therapeutic benefits for prevention of arteriosclerotic and fibrotic cardiovascular disease (Kaikita et al., 2001). MR-null mice normally die 1 to 2 weeks after birth, preventing analysis of cardiac phenotypes in adult MR-null mice (Berger et al., 1998), and saline-rescued adult MR-null mice have not been fully analyzed for cardiac defects (Bleich et al., 1999). The role of cardiac MR in fibrosis and heart failure, however, has been addressed by both overexpression of human MR and conditional antisense RNA knockdown of cardiac MR in transgenic mice (Beggah et al., 2002; Le Menuet et al., 2001). Overexpression of human MR in heart and kidney of transgenic mice produced a mild cardiomyopathy with no evidence of cardiac fibrosis (Le Menuet et al., 2001). Remarkably, a 50% reduction in cardiac MR in antisense RNA transgenic mice produced severe dilated cardiomyopathy and extensive interstitial cardiac fibrosis (Beggah et al., 2002). This phenotype was fully reversible on suppression of MR antisense mRNA expression. These results are not easy

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to reconcile given the benefits of MR antagonism by spironolactone in human heart failure. Perhaps spironolactone acts in an MR-independent manner or in a nonclassic fashion via MR in the heart. Alternatively, the MR antisense murine transgenic phenotype of dilated cardiomyopathy and fibrosis may reflect nonspecific effects unrelated to aldosterone/MR signaling. It has been reported that transgenic overexpression in the heart of green fluorescent protein, a so-called biologically inert molecule, causes a similar dilated cardiomyopathy, and suggests that proper transgenesis controls may be required to differentiate between specific and nonspecific effects of a transgene in the myocardium (Huang et al., 2000). The results of the RALES trial clearly demonstrate the benefit of MR blockade in heart failure. A substudy showed a significant lowering of circulating procollagen type III terminal peptide levels, indicating a direct benefit of spironolactone on reducing collagen turnover (Zannad et al., 2001). It will be of interest to demonstrate direct reduction of vessel damage and cardiac fibrosis by spironolactone and other recent selective aldosterone antagonists, such as eplerenone, in mineralocorticoid–salt animal models of cardiac fibrosis. B. RAPID NONGENOMIC ACTIONS OF ALDOSTERONE

There is strong and compelling evidence that aldosterone can change cellular functions rapidly. These nongenomic actions have a rapid time course measured in seconds/minutes and have been reported in both epithelia and nonclassic target tissues such as leukocytes, vascular smooth muscle (VSM), and cardiomyocytes (for reviews, see Christ and Wehling, 1999; Falkenstein et al., 2000). Rapid cellular effects include changes in intracellular cAMP levels, intracellular calcium, increased intracellular pH, altered sodium currents, and activation of protein kinase C. Aldosterone in the nanomolar range has been shown to rapidly increase intracellular calcium in the mouse M1 cortical CD cell line, a response that could be blocked by the PKC inhibitor chelerthrine chloride (Harvey and Higgins, 2000). In human distal colon, rapid responses to aldosterone include increased activity of K(ATP) channels, perhaps priming cells to normal genomic responses to aldosterone, which therefore may be important in overall blood pressure control (Maguire et al., 1999). There is even evidence in renal principal cells of rapid aldosterone activation of amiloride-sensitive ENaCs by nongenomic pathways leading to higher rates of electrogenic Na+ retention (Zhou and Bubien, 2001). Finally, in porcine VSMCs, aldosterone has been shown to induce rapid calcium-dependent increases in cAMP and phosphorylation of the cAMP response element-binding protein, further evidence that aldosterone is able to rapidly activate important protein kinase–signaling pathways (Christ and Wehling, 1999).

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These actions have until more recently been thought to be mediated by distinct and as yet uncharacterized membrane-bound receptors, but there is now evidence that some of these rapid actions may in fact be mediated nongenomically via MR itself. Strong evidence is provided in vascular smooth muscle cells, where aldosterone rapidly increased intracellular pH within minutes via increasing NHE-1 (sodium–hydrogen exchanger 1) activity (Alzamora et al., 2000). This effect was blocked by RU28318, an MR antagonist, and copied by cortisol after blockade of 11-HSD2 by carbenoxolone, therefore implicating MR in this rapid nongenomic effect of aldosterone. Similar results were obtained for the MR antagonist canrenoate, which blocked the rapid nongenomic effects of aldosterone on the Na+,K+,2Cl cotransporter in preparations of rabbit cardiomyocytes (Mihailidou et al., 2000). Clearly at odds with these conclusion are results from studies with MRdeficient mice, in which rapid effects of aldosterone on intracellular calcium levels and cAMP were observed in skin cells of MR-null mice as well as with samples from wild-type littermate controls (Haseroth et al., 1999). These experiments were performed at low physiological doses of aldosterone, and clearly implicate other undefined receptor/signaling mechanisms that may mediate rapid aldosterone action. It seems likely that both MR and other distinct receptor/signaling systems may mediate rapid aldosterone effects, perhaps in distinct cell types and under different physiological conditions. C. ALDOSTERONE AND INSULIN CROSS-TALK

Insulin has well-described natriferic effects in the renal tubules, which are fully maintained in obese humans with resistance to the metabolic effects of insulin (Rocchini et al., 1989). Although there is disagreement about sites of action, there is little question that insulin acts in the distal nephron (thick ascending limb and beyond) (DeFronzo et al., 1975; Nakamura et al., 1983) but may act in the proximal tubule, as well (Baum, 1987). Abnormalities in the RAAS also have been found in the insulin-resistant state, and aldosterone may be inappropriately high (Egan et al., 1994). With these observations in mind, synergy between aldosterone and insulin in stimulating renal tubular sodium transport has clear pathophysiological implications. The bulk of evidence, although not all, supports the idea that they act synergistically in CD cells (reviewed in Pearce, 2001). SGK1, which constitutes a node in the insulin and aldosterone signaling pathways (Wang et al., 2001b), provides an explanation for their capacity for synergy, as shown schematically in Fig. 4. These observations led to the intriguing possibility that SGK1 (and possibly SGK2 and/or SGK3) is central to the pathophysiology of the metabolic syndrome (Pearce, 2001; Reaven, 1997). According to this view, the insulin-signaling cascade in the distal nephron that proceeds through SGK1 is intact. Insulin resistance in muscle, fat, and

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FIGURE 4. SGK1 integrates effects of aldosterone and insulin on ENaC. A speculative scheme is shown for how SGK1 abundance is controlled by aldosterone, while its activity is controlled by insulin. Phospho-SGK1 (denoted by attached yellow circle) then phosphorylates and inhibits Nedd4-2 activity. Because Nedd4-2 is a ubiquitin (Ub) ligase that stimulates removal of ENaC from the apical membrane and targets it for proteasome-mediated degradation, its inactivation increases ENaC abundance in the apical membrane. Ins, Insulin; PI3K, phosphatidylinositol-3-kinase; PIP2, phosphatidylinositol 4,5-bisphosphate; PIP3, phosphatidylinositol 3,4,5-trisphosphate; N4-2, Nedd4-2. Other abbreviations as in text and other figures.

liver (and possibly islets themselves) results in elevated insulin levels and increased renal sodium retention. The RAAS is downregulated but inadequately because of glucose/insulin effects on renin and aldosterone production. In this case, the kidney would constitute an ‘‘unwilling accomplice’’ in the insulin resistance syndrome (Reaven, 1997).

VIII. CONCLUDING REMARKS Aldosterone research over the past 50 years has uncovered an enormous wealth of information about the physiological role that this steroid hormone plays in solute homeostasis and also about its pathophysiological role in aspects of human disease. It is now clear that dysfunction in aldosterone production or signaling is not only the primary cause of a small number of

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salt-handling conditions but also contributes to more widespread clinical conditions such as heart failure, essential hypertension, and the insulin resistance (‘‘metabolic’’) syndrome. Burning issues include further understanding of the role of aldosterone in nonepithelial tissues (such as the heart where little is known of detailed cellular mechanisms of action), and further definition of the physiological and clinical relevance of the fast nongenomic effects of aldosterone. In research directed at the basic mechanism of aldosterone action, two areas stand out: The first is to further understand the specific nuclear events that distinguish signaling through MR versus GR both at the level of agonist/ antagonist binding and via coactivator/corepressor–receptor interactions. This may lead to specific tissue-selective approaches to aldosterone/MR signaling modulation or blockade. The second is to expand the list of specific aldosterone target genes, and to characterize these targets biochemically and physiologically (particularly in the context of genetargeting approaches). Sometimes considered the poor unexciting cousin of the steroid hormone family, aldosterone is now clearly an important factor in the pathophysiology of some common clinical conditions, and of increasing interest as a focus for basic research. There is a solid foundation of knowledge to build on in designing improved treatments for conditions related to aldosterone excess or abnormal activity of its signaling pathways. The advent of pharmacogenomics and tissue-selective agonists/antagonists provides exciting future prospects for targeted therapies for the treatment of aldosterone dysfunction.

ACKNOWLEDGMENTS The authors thank Drs. Perry White, Scott Nunez, and John Funder for helpful discussions. Work in the authors’ laboratories is supported by NIH Grants DK56695 and DK51151 (D.P.) and by NH&MRC Grant 194253 (T.C.).

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3 Corticosteroid Receptors, 11-Hydroxysteroid Dehydrogenase, and the Heart Karen E. Sheppard Molecular Physiology Laboratory, Baker Heart Research Institute, Melbourne 8008, Victoria, Australia

I. Introduction II. Corticosteroid Hormones III. Corticosteroid Receptors A. Mineralocorticoid Receptor and Glucocorticoid Receptor B. Novel High-Affinity Corticosteroid-Binding Sites C. Low-Affinity Corticosteroid-Binding Sites IV. Mechanism of Action of Corticosteroid Receptor A. Genomic Mechanism B. Rapid Nongenomic Effects of Corticosteroids and Their Putative Receptors V. Modulators of Corticosteroid Signaling A. Plasma Binding Proteins B. Active Export of Corticosteroids C. 11b-Hydroxysteroid Dehydrogenase D. Receptor Modification E. Cellular Milieu and Other Factors

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Copyright 2003, Elsevier Science (USA). All rights reserved. 0083-6729/03 $35.00

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VI. Heart A. Expression of Corticosteroid Receptors and 11b-Hydroxysteroid Dehydrogenase Isoforms in the Heart B. Heart Production of Aldosterone and Corticosterone C. Corticosteroid Effects on the Heart D. Corticosteroid Effects on Cardiac Ion Channels E. Cardiac Hypertrophy F. Myocardial Infarction G. Cardiac Fibrosis H. Transgenic Animals VII. Summary References Mineralocorticoid and glucocorticoid hormones are known as corticosteroid hormones and are synthesized mainly in the adrenal cortex; however, more recently the enzymes involved in their synthesis have been found in a variety of cells and tissues, including the heart. The effects of these hormones are mediated via both cytoplasmic mineralocorticoid receptors (MRs) and glucocorticoid receptors (GRs), which act as ligand-inducible transcription factors. In addition, rapid, nongenomically mediated effects of these steroids can occur that may be via novel corticosteroid receptors. The lipophilic nature of these hormones allows them to pass freely through the cell membrane, although the intracellular concentration of mineralocorticoids and glucocorticoids is dependent on several cellular factors. The main regulators of intracellular glucocorticoid levels are 11-hydroxysteroid dehydrogenase (11HSD) isoforms. 11HSD1 acts predominantly as a reductase in vivo, facilitating glucocorticoid action by converting circulating receptor-inactive 11-ketoglucocorticoids to active glucocorticoids. In contrast, 11HSD2 acts exclusively as an 11-dehydrogenase and decreases intracellular glucocorticoids by converting them to their receptor-inactive 11-ketometabolites. Furthermore, P-glycoproteins, by actively pumping steroids out of cells, can selectively decrease steroids and local steroid synthesis can increase steroid concentrations. Receptor concentration, receptor modification, and receptor–protein interactions can also significantly impact on the corticosteroid response. This review details the receptors and possible mechanisms involved in both mediating and modulating corticosteroid responses. In addition, direct effects of corticosteroids on the heart are described including a discussion of the corticosteroid receptors and the mechanisms involved in mediating their effects. ß 2003 Elsevier Science (USA).

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I. INTRODUCTION Mineralocorticoid and glucocorticoid hormones are predominantly products of the adrenal cortex and collectively are known as corticosteroid hormones. The lipophilic nature of these hormones allows them to pass freely through the cell membrane. To date, two receptors that bind corticosteroids with high affinity have been cloned: the mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR). On binding steroid in the cytoplasm, these receptors undergo a conformational change involving the dissociation of heat shock proteins and nuclear translocation. Once in the nucleus the receptors regulate gene transcription and thus are known as ligand-inducible transcription factors. In addition to the cloned corticosteroid receptors, other high-affinity binding sites have been described in various tissues. Membrane receptors have also been reported and are thought to mediate rapid nongenomic effects of corticosteroid hormones. Enzymes involved in mineralocorticoid and glucocorticoid synthesis have been found in a variety of cells and tissues, including the heart, in which expression is increased during heart failure. In addition, nongenomic rapid actions of corticosteroid hormones have been described. This article details the current status of extraadrenal synthesis of corticosteroid hormones, the novel corticosteroid receptors that have been described, the cellular mechanisms that can modulate the response to these steroids, and both the genomic and nongenomic mechanism of corticosteroid action. In addition, direct effects of corticosteroids on the heart are described, including a discussion of the corticosteroid receptors and the mechanisms involved in both mediating and modulating their effects.

II. CORTICOSTEROID HORMONES Mineralocorticoid and glucocorticoid hormones are predominantly products of the adrenal cortex and collectively are known as corticosteroid hormones. The major glucocorticoid in humans is cortisol and in rodent it is corticosterone, whereas the major mineralocorticoid in both humans and rodents is aldosterone. The distinctions between these steroids were originally based on function, for example, glucocorticoids were first identified as hormones that affect glycogen deposition in the liver, whereas mineralocorticoids were hormones that modulated unidirectional transepithelial sodium transport. Since this early classification of steroid based on action, it is clear that these definitions are too strict. It is now known that glucocorticoids mediate a myriad of responses including roles in development (Bolt et al., 2001), maintenance of blood pressure (Grunfeld, 1990), and modulating both stress (Sapolsky et al., 1986) and immune responses (Morand and Leech, 1999). Mineralocorticoids, in addition to their

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epithelial effect on sodium transport, have nonepithelial actions including a central role in modulating salt appetite and blood pressure (Gomez-Sanchez and Gomez-Sanchez, 2001). Glucocorticoid and mineralocorticoid hormones are synthesized from cholesterol, predominantly in the adrenal cortex. Two of the cytochrome P-450 enzymes, which catalyze the final step of these synthetic pathways, are encoded by two closely related genes: CYP11B1 (11-hydroxylase) and CYP11B2 (aldosterone synthase) (Nomura et al., 1993). P-450 11hydroxylase synthesizes corticosterone from deoxycorticosterone in the adrenal zona fasciculata and reticularis and is mainly regulated by adrenocorticotropic hormone (ACTH). P-450 aldosterone synthase converts deoxycorticosterone into aldosterone in the adrenal zona glomerulosa, and its principal regulators are angiotensin II and plasma potassium levels. In addition to the classic adrenal biosynthetic pathway, extra-adrenal sites of corticosteroid hormone synthesis have been identified. Aldosterone and corticosterone synthesis from deoxycorticosterone has been demonstrated in both rat and human tissue including brain (Gomez-Sanchez et al., 1997; MacKenzie et al., 2000), heart (Silvestre et al., 1998; Kayes-Wandover and White, 2000; Young et al., 2001), and vascular cells (Takeda et al., 1995; Hatakeyama et al., 1996). Extra-adrenal synthesis of corticosteroid hormones does not contribute substantially to the circulating levels of these hormones and most likely mediates autocrine and paracrine effects.

III. CORTICOSTEROID RECEPTORS A. MINERALOCORTICOID RECEPTOR AND GLUCOCORTICOID RECEPTOR

Cloning of the steroid receptors provided identification of a common structure consisting of a highly conserved cysteine-rich DNA-binding domain, a C-terminal region that encompasses the ligand-binding domain, and a variable N-terminal region. Hormone-dependent transcriptional activation domains are located within the ligand-binding domain and the Nterminal domain. The sequence similarity between steroid receptors, and receptors for thyroid hormone and retinoids, led to the concept of a nuclear receptor superfamily (Evans, 1988; Mangelsdorf et al., 1995). In addition to the above-described receptors, this superfamily of structurally related proteins includes receptors for vitamin D3 and fatty acids, as well as a myriad of orphan receptors for which the ligands are unknown. To date, two high-affinity receptors for corticosteroids have been cloned: the MR or type I corticosteroid receptor and the GR or type II corticosteroid receptor. The initial distinction between these two receptors was demonstrated in rat kidney slice studies in which aldosterone was shown

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to bind with high affinity to type I sites and with much lower affinity to type II sites, whereas corticosterone (the endogenous rat glucocorticoid) had a lower affinity to both sites (Funder et al., 1972). Two types of GR were then demonstrated in rat hippocampus; one site had high affinity for the synthetic glucocorticoid dexamethasone, whereas the other had a higher affinity for corticosterone (De Kloet et al., 1975). Subsequent biochemical studies and eventual cloning of these receptors demonstrated that the kidney type II site was the classic GR and had high affinity for both dexamethasone and endogenous glucocorticoids, and the kidney type I receptor (MR) was identical to the hippocampal corticosterone-preferring site and intrinsically has high affinity for both corticosterone and aldosterone. Thus, MR is unique in that it has two distinct physiological ligands, depending on the cell and tissue in which it is expressed. In epithelial cells, or mineralocorticoid target tissue such as kidney and colon, aldosterone specificity is conferred on MR by 11-hydroxysteroid dehydrogenase (11-HSD) (Edwards et al., 1988; Funder et al., 1988). The 11-dehydrogenase activity of this enzyme converts corticosterone and cortisol to their MR- and GR-inactive 11-keto metabolites (11-dehydrocorticosterone and cortisone, respectively) and thus allows aldosterone access to MR. In tissues that express MR but do not possess 11-dehydrogenase activity, the higher circulating levels of endogenous glucocorticoids compared with aldosterone, and the equivalent affinity of MR for these steroids, results in MR binding glucocorticoids and mediating glucocorticoid effects (De Kloet et al., 1993). When human GR was originally cloned, two isoforms were identified: GR , the classic ligand-binding GR, and GR , a smaller, nonbinding form. Initially GR was thought to be either a cloning artifact or a splice variant and it was unknown whether it was synthesized as a protein, thus few studies addressed the physiological role of GR . More recently specific antibodies against the human GR isoform has shown that a GR protein does exist. These studies have now renewed interest in the possible functional role of this GR isoform. There is a consensus that GR does not bind ligand and is transcriptionally inactive; however, there are conflicting data on whether GR is a dominant negative regulator of GR- and MR-induced gene transcription (Carlstedt-Duke, 1999; Vottero and Chrousos, 1999). Transient transfection studies have shown that when GR is overexpressed in cells, it could decrease gene transcription induced by GR and MR (Bamberger et al., 1995; Oakley et al., 1996; Bamberger et al., 1997; Oakley et al., 1999). Other studies, however, have failed to find an effect of GR on GR –induced gene transcription (Hecht et al., 1997). The low level of expression of GR compared with GR in cells also raises the question of whether GR has a physiological role, although studies suggest that GR may be induced in pathological states (Leung et al., 1997; Christodoulopoulos et al., 2000; Honda et al., 2000; Derijk et al., 2001). In addition to GR , other alternative splice variants have been

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reported (Krett et al., 1995; Rivers et al., 1999); the functional role of these splice variants is yet to be determined. Furthermore, protein isoforms of GR and GR exist that are the result of translation from different AUG codons. These two proteins, termed GR-A and GR-B, differ significantly in their biochemical and gene activation potential (Yudt and Cidlowski, 2001). Different MR isoforms have also been described. In humans, alternative transcription of two 50 untranslated exons generates two mRNA isoforms, MR and MR , which are coexpressed in aldosterone target tissues; however, their relative abundance varies in a tissue-specific manner (Zennaro et al., 1995). In addition, several MR splice variants have also been described although their function is unknown (Kwak et al., 1993; Bloem et al., 1995; Zhou et al., 2000). More recently, a novel MR splice variant that lacks the hinge and ligand-binding domain was described. This MR splice variant binds DNA and can dimerize with either MR or GR and in so doing enhances their transactivation potential (Zennaro et al., 2001). B. NOVEL HIGH-AFFINITY CORTICOSTEROID-BINDING SITES

In addition to MR and GR, several other binding sites that have high affinity for corticosteroids have been reported. They are the kidney type III binding site (Feldman et al., 1973), the corticosteroid binder IB, and the dehydrocorticosterone (DHB) receptor (Sheppard and Funder, 1996). All these putative receptors can be distinguished from each other and from both MR and GR by either their biochemical properties or specificities for endogenous and synthetic steroids. On the basis of colocalization of binding and enzyme activity in rabbit renal cortical principal cells, a close correlation was demonstrated between binding specificity of the kidney type III site and inhibition of 11-HSD activity, and also between the activity of 11-HSD and the number of corticosterone-binding sites, suggesting that the type III site was 11-HSD2 (Naray-Fejes-Toth et al., 1994; Naray-Fejes-Toth and Fejes-Toth, 1997). The corticosteroid IB binder and GR have identical steroid specificity; however, the IB binder can be distinguished from GR on the basis of its sedimentation coefficient and noncross-reactivity with GR antibodies. This receptor has been shown to be a proteolytic fragment of the glucocorticoid receptor, and whether this smaller GR form is an in vitro artifact or generated in vivo has not been determined (Eisen et al., 1986). The putative DHB receptor was first described in rat colonic crypt cells. This putative receptor binds 11-dehydrocorticosterone (11-DHB), the 11-keto metabolite of corticosterone produced by 11-HSD (Sheppard and Funder, 1996) with high affinity (10 nM) and has negligible affinity for aldosterone, dexamethasone, estradiol, RU38486, or 5dihydrotestosterone, the classic ligands for the family of steroid receptors. This receptor colocalizes with 11-HSD2 but can be distinguished from this

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enzyme on the basis of steroid specificity and the fact that cells transfected with 11-HSD2 gene express nuclear 11-HSD2 but not a nuclear DHB receptor (Sheppard et al., 1998). The physiological function of a DHB receptor is yet to be defined but given its apparent colocalization with 11HSD2 it may allow mineralocorticoid target tissues to respond to circulating glucocorticoids without compromising the aldosterone selectivity of MR. C. LOW-AFFINITY CORTICOSTEROID-BINDING SITES

Similarities in steroid receptor structure with a conserved DNA-binding domain, flanked by a variable N-terminal domain and C-terminal ligandbinding domain, have allowed the cloning and identification of receptors for many nuclear hormones as well as a myriad of orphan receptors for which physiological ligands are yet to be identified. Of the many orphan receptors that have been cloned, only two appear to be corticosteroid responsive: human steroid/xenobiotic receptor (SXR) (Blumberg et al., 1998) and the rodent ortholog, pregnane X receptor (PXR) (Kliewer et al., 1998). These receptors have a low affinity for corticosterone (5 M) and are activated by a diverse group of compounds, including xenobiotics and synthetic steroids. They induce the expression of both the MDR1 gene (Synold et al., 2001), which encodes a transporter that protects cells from toxicity by rapidly effluxing drugs, and CYP3A genes (Blumberg et al., 1998; Kliewer et al., 1998), which are important in the metabolism and elimination of xenobiotics and steroids. Therefore, PXR and SXR are implicated in both catabolism and clearance of xenobiotics and steroids from cells. In the small intestine, corticosterone binds to a nuclear localized site that has a relatively low affinity for corticosterone (50 nM), a high capacity, and a broad steroid specificity compared with both MR and GR. The steroid specificity profile of this binding site distinguishes it from MR, GR, and other steroid receptors, although the specificity mirrors the potency of various steroids to inhibit 11-HSD2 activity, suggesting that binding might be to the substrate-binding site on an 11-HSD isoform. In discordance with this concept is the difference in both tissue distribution and intracellular localization between the small intestinal binding site and 11HSD2 (Sheppard et al., 2000). The corticosterone-binding site in the small intestine is similar to PXR and SXR in its broad steroid specificity, nuclear localization, and high expression in the small intestine. In contrast with these receptors is the 100-fold higher affinity of corticosterone for the small intestinal binding site (50 nM compared with 5 M), and steroid specificity appears to be restricted to C21 steroids. The small intestinal receptor may be related to PXR and SXR as one of a novel branch of the nuclear receptor family in which the receptors are of low affinity, high capacity, and regulate the metabolism of a broad spectrum of compounds. The physiological role of the small intestinal receptor might be more defined than that of PXR and

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SXR in that it would provide an intracellular environment allowing corticosteroid receptors to respond to endogenous glucocorticoids while at the same time protecting them from ligands in ingested material.

IV. MECHANISM OF ACTION OF CORTICOSTEROID RECEPTOR A. GENOMIC MECHANISM

Unlike peptide hormones and growth factors, which bind to cell surface receptors, the lipophilic nature of steroid hormones allows them to pass through the cell membrane and bind to their cognate receptor. Unliganded GR and MR are found in the cytoplasm as a heteromeric complex containing a dimer of heat shock protein (hsp) 90, an immunophilin protein of the FK506-binding family (FKBP-52 or FKBP-54), a 23-kDa protein (p23), and other heat shock proteins (e.g., hsp 70) and immunophilins (Cyp40). The association of these proteins with the unliganded receptor is thought to be involved in maintaining the receptor in a hormone-binding conformation. Interaction of the corticosteroid receptor ligand-binding domain with the hsp90 moiety of the heteromeric complex is required for optimal hormone binding and also ensures the repression of the receptor in the absence of hormone (Pratt and Toft, 1997). On steroid binding, the receptor undergoes an allosteric change (activation) resulting in heat shock protein dissociation, receptor homo- or heterodimerization, and subsequent binding of the receptor to DNA response elements of target genes. The effects of steroid receptor on gene transcription are mediated through the binding of the receptor complex to the specific DNA sequences and the recruitment of various coactivators and repressors (Beato and Klug, 2000). Heterodimerization of MR and GR results in distinct transcriptional responses compared with MR or GR homodimer formation (Trapp et al., 1994). This extends the potential of corticosteroids in regulating responsive genes, in that the transcriptional response will depend on the relative levels of GR and MR as well as the intracellular concentration of glucocorticoids and aldosterone. GR can also directly interact with other transcription factors and modulate gene transcription in this way (Gottlicher et al., 1998). GR can act as ligand-dependent transrepressors of AP1 (Jun/Fos) activity and reciprocally AP1 can inhibit transactivation of GR (Pfahl, 1993). In addition, GR can also mutually interfere with NF-B activity (Ray and Prefontaine, 1994). Three mechanisms have been proposed to account for the interaction of GR with other transcription factors: (1) binding of GR and the transcription factor to a ‘‘composite DNA element’’ and in doing so either potentiating or antagonizing transcription by GR, (2) direct binding of the

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GR to the transcription factor and thus inhibiting binding to the DNA element, and (3) competition of GR and other transcription factors for common transcriptional modulators. B. RAPID NONGENOMIC EFFECTS OF CORTICOSTEROIDS AND THEIR PUTATIVE RECEPTORS

In contrast to classic steroid effects on gene transcription, nongenomic steroid effects have been reported. These effects can be separated from genomic effects on the basis of their rapid onset of action (within seconds to minutes) and their insensitivity to inhibitors of transcription and protein synthesis. Rapid effects of corticosteroid hormones have been shown in a variety of tissues and cells. Rapid neurophysiological and behavioral effects of systemic glucocorticoid administration have been described in mammalian brain (Feldman and Dafny, 1970) and in the roughskin newt (Rose, 2000). Corticosterone induces a rapid (15 min) but transient increase in extracellular levels of the excitatory amino acids that is not inhibited by GR and MR antagonists (Venero and Borrell, 1999). It also rapidly affects the firing rate of barosensitive cardiovascular neurons (Rong et al., 1999) and changes calcium uptake on depolarization by high potassium in synaptic plasma membranes (Sze and Iqbal, 1994). Many of these rapid neuronal responses appear to involve alterations in ion channel conductance and/or interactions with G-protein signaling (Borski et al., 2002). Other nonneuronal rapid effects of glucocorticoids have also been reported. These include a rapid induction of actin polymerization in human endometrial cells (Koukouritaki et al., 1996), a rapid inhibition of ACTH secretion in corticotroph cells (Widmaier and Dallman, 1984) that is probably via a G protein-mediated mechanism (Iwasaki et al., 1997), and in lymphoid cells, glucocorticoids rapidly stimulate apoptosis (Gametchu et al., 1999). In addition to the genomically mediated effects of mineralocorticoids on ion transport, aldosterone also appears to rapidly modulate ion transport in both epithelial (kidney, colon) and nonepithelial cells including human mononuclear leukocytes (HMLs), vascular smooth muscle cells (VSMCs), and cardiomyocytes. In HMLs, VSMCs, canine kidney cells, and colonic crypts, aldosterone induces a rapid activation of the sodium hydrogen exchanger (Wehling et al., 1991b) that is not inhibited by MR antagonists and is not blocked by inhibitors of transcription and protein synthesis (Gekle et al., 1996; Ebata et al., 1999; Winter et al., 1999). In cardiomyocytes, aldosterone rapidly activates sodium influx through the Na+,K+, 2Cl cotransporter, the response being blocked by the MR antagonist potassium canrenoate (Mihailidou et al., 1998). In addition, aldosterone has been shown to induce rapid effects on intracellular pH, intracellular calcium, generation of inositol 1,4,5-trisphosphate, protein

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kinase C activity, phosphorylation of ERK1/2, and intracellular cAMP levels (Christ and Wehling, 1999; Gekle et al., 2002). For glucocorticoid and mineralocorticoid hormones, three mechanisms for rapid nongenomic actions have been proposed (Buttgereit and Scheffold, 2002): (1) Binding to the classic cytosolic receptors, (2) nonspecific interactions with the cellular membrane, and (3) specific interactions with membrane-bound receptors. High-affinity (0.5 nM) membrane receptors for corticosterone have been identified and characterized in the roughskin newt (Moore and Orchinik, 1994; Moore et al., 1995). These receptors are highly specific for corticosterone and cortisol and display negligible affinity for aldosterone and the synthetic glucocorticoid dexamethasone (Orchinik et al., 1991). The correlation between the pharmacology of the membrane corticosterone-binding site and the potencies of various steroids to inhibit courtship behavior in the roughskin newt suggests that this protein is involved in the rapid behavioral responses to corticosterone (Orchinik et al., 1991; Moore and Evans, 1999). A mammalian equivalent of this membrane corticosterone-binding site has not been identified, although glucocorticoid membrane-binding sites have been described. An antibody against GR detects a GR-like antigen in membranes in murine and human lymphoid cell lines (Gametchu et al., 1999). This site differs from cytosolic GR in its cellular localization, molecular size, and a slightly different steroid specificity. The similarity of the membrane GR to cytosolic GR, in that it is recognized by three different anti-GR antibodies, binds the same class of steroids and heat shock proteins (Gametchu et al., 1999), suggests that the membrane-binding site is a modified form of GR. In addition, a high-affinity membrane glucocorticoid-binding site that appears distinct from classic GR has been described in liver (Trueba et al., 1991; Grote et al., 1993), kidney (Ibarrola et al., 1991), and adrenal cortex (Andres et al., 1997). A specific binding site for corticosteroid-binding globulin (CBG) in the plasma membrane has also been implicated in glucocorticoid-induced intracellular signaling via binding of the CBG–glucocorticoid complex to a specific corticosteroid-binding globulin receptor (Strel’chyonok and Avvakumov, 1991). The rapid actions of aldosterone are thought to occur via a plasma membrane receptor, although this putative receptor has not been identified. In plasma membranes from HMLs, kidney, and liver a high-affinity aldosterone-binding site can be demonstrated (Wehling et al., 1991a; Christ et al., 1994; Meyer et al., 1995). This binding site is distinct from classic MR in its steroid specificity (Wehling et al., 1992; Christ et al., 1994), molecular mass (Eisen et al., 1994), and the fact that the MR antagonists spironolactone and canrenone do not inhibit the rapid actions of aldosterone. Good evidence that rapid aldosterone effects can occur through mechanisms distinct from classic MR is that aldosterone can rapidly increase intracellular calcium and cAMP levels in fibroblasts from MR knockout mice (Haseroth et al., 1999). There is also evidence that the rapid nongenomic actions of aldosterone can

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be mediated via the classic intracellular MR. In uterine artery strips, aldosterone induces a rapid increase in intracellular pH that is not blocked by spironolactone but is blocked by the MR antagonist RU28318. When the enzyme, 11-HSD2, which metabolizes cortisol to an MR-inactive metabolite, is inhibited cortisol mimicks the aldosterone effect, thus behaving like the classic intracellular MR.

V. MODULATORS OF CORTICOSTEROID SIGNALING An important determinant of corticosteroid receptor activation is access of the ligand to the receptor, which depends on total plasma levels of hormone, plasma binding proteins, active export of corticosteroids, and intracellular corticosteroid metabolism. In addition, receptor concentration, receptor modification, and receptor–protein interactions can also impact on the corticosteroid response. A. PLASMA BINDING PROTEINS

Corticosteroids exist in plasma in both unbound (free) and proteinbound forms. The major binding protein in plasma for corticosteroids is CBG; this protein has a high affinity for endogenous glucocorticoids (corticosterone in rodent and cortisol in humans) and a much lower affinity for aldosterone. CBG binds 95% of circulating cortisol and corticosterone in humans (Siiteri et al., 1982) and rats (Henning, 1978), respectively. The general consensus is that only the non-protein-bound hormone is available for movement out of capillaries and into cells, where it either binds to specific receptors and mediates a response or is cleared from the circulation via a variety of metabolic pathways. Thus, CBG effectively lowers the available endogenous glucocorticoid and protects it from metabolism. It has also been suggested that CBG may actively participate in the delivery of steroids to target cells by either interacting directly with a plasma membrane (Strel’chyonok and Avvakumov, 1991) or through a specific interaction with elastase on immune cells, leading to delivery of glucocorticoids to sites of inflammation (Hammond et al., 1991). B. ACTIVE EXPORT OF CORTICOSTEROIDS

P-glycoproteins are members of the ATP-binding cassette (ABC) transporter superfamily. Increased expression of P-glycoproteins results in the development of multidrug resistance in cultured cells and is associated with treatment failure in some human tumors. P-glycoproteins are expressed in normal tissues, particularly on the luminal surface of transporting

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epithelia including kidney, small intestine, colon, and capillary endothelial cells of the brain and testis (Bellamy, 1996). Although there is considerable evidence that P-glycoproteins pump drugs out of tumor cells, the function of these proteins in normal cell physiology is uncertain. Both endogenous glucocorticoids and aldosterone are substrates for P-glycoprotein, although the ability to transport these steroids differs (Ueda et al., 1992). These proteins appear to be involved in specific steroid export and can selectively decrease the intracellular concentration of particular steroids in a cell- and species-specific manner (Ueda et al., 1992; Kralli and Yamamoto, 1996). Pglycoprotein expression in the apical membranes of endothelial cells of the blood–brain barrier is thought to modulate access of both synthetic and endogenous glucocorticoids to both rodent and human brain (Meijer et al., 1998; Karssen et al., 2001). C. 11-HYDROXYSTEROID DEHYDROGENASE

Two isoforms of 11-HSD have been cloned and characterized (Agarwal et al., 1989; Albiston et al., 1994). The first of these, 11-HSD1, is NADP preferring and bidirectional, although it acts predominantly as a reductase in vivo, potentiating glucocorticoid action by forming active glucocorticoids from inactive 11-keto metabolites, and thus increasing the local tissue concentration of endogenous glucocorticoids. The second, 11-HSD2, operates as an exclusive 11-dehydrogenase for endogenous glucocorticoids and, given its colocalization with MR in sodium-transporting epithelia and the increase in sodium retention when enzyme activity is compromised (Ulick et al., 1979; Stewart et al., 1988), it is this enzyme that confers aldosterone specificity on MR. 11-HSD1 is widely expressed, with high levels in liver and moderate levels in other tissues including lung, vasculature, heart, and central nervous system. In liver homogenates 11-HSD1 was originally shown to be bidirectional although the reductase activity of this enzyme was unstable (Lakshmi and Monder, 1988). In contrast, studies in intact cells from various tissues suggest that 11-HSD1 acts as a reductase only (Jamieson et al., 1995; Rajan et al., 1996; Sheppard and Autelitano, 2002). The reason for the difference between cell homogenate and intact cells in terms of 11-HSD1 activity is unknown but may be a function of cofactor availability in vivo and/or enzyme phosphorylation (Seckl and Walker, 2001). The consensus is that 11-HSD1 acts as a reductase in vivo, converting 11-ketoglucocorticoid metabolites to MR- and GR-active glucocorticoids, therefore increasing exposure of 11-HSD1-containing cells to glucocorticoids. 11-Ketoglucocorticoid metabolites in rats and humans are 11-dehydrocorticosterone and cortisone, respectively. They circulate at a concentration that is approximately 4-fold less than the endogenous glucocorticoids corticosterone and cortisol (Nomura et al.,

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1997; Livingstone et al., 2000). Considering that 11-dehydrocorticosterone and cortisone have much lower affinity than corticosterone and cortisol for the two major plasma binding proteins CBG and albumin, and are without pronounced diurnal rhythm, they therefore provide a constant major pool of available glucocorticoid for these tissues. Thus, 11-HSD1 potentiates glucocorticoid action by forming active glucocorticoids from inactive 11-keto metabolites. The reported Km values of 11-HSD1 for endogenous glucocorticoids and their 11-keto metabolites in whole cells are 2 M and 0.2 M, respectively (Moore et al., 1993; Bujalska et al., 1997). The high Km values of 11-HSD1 for 11-ketoglucocorticoids, and the 5- to 10-fold lower circulating concentrations of these steroids, casts into question how this enzyme can effectively function in vivo. A possible explanation has come from data that show that 11-HSD1 can adapt to low nanomolar substrate conditions as well as high substrate concentrations (micromolar) depending on enzyme homodimerization (Maser et al., 2002). This is in keeping with a demonstration that low nanomolar concentrations of 11-dehydrocorticosterone are converted to corticosterone by 11-HSD1 in rat cardiac myocyte cultures, leading to GR-activated gene transcription (Sheppard and Autelitano, 2002). Intrinsically MR has an equally high affinity for both endogenous glucocorticoid and aldosterone; however, in vivo MR is aldosterone selective (Sheppard and Funder, 1987a,b). Given that circulating levels of endogenous glucocorticoids are 100–1000 times higher than those of aldosterone (Holbrook et al., 1980), how aldosterone occupied MR in vivo was an enigma until 11-dehydrogenase activity was shown to exclude endogenous glucocorticoids from mineralocorticoid target tissues (Edwards et al., 1988; Funder et al., 1988). This activity was shown later to be due to 11-HSD2 (Ulick et al., 1979; Stewart et al., 1988). 11-HSD2 acts exclusively as a 11-dehydrogenase, it has a high affinity for endogenous glucocorticoids (low nanomolar), and is NAD dependent. At a cellular level, 11-HSD2 plays a crucial role in modulating corticosteroid action. This is supported by clinical data in which decreased enzyme activity or saturation of enzyme activity leads to symptoms suggesting mineralocorticoid excess (Ulick et al., 1979, 1992; Ferrari et al., 1996). In addition to modulating glucocorticoid access to MR, 11-HSD2 also decreases access of glucocorticoids to GR (Sheppard, 1998). Thus, how glucocorticoids mediate glucocorticoid effects in 11-HSD2-containing cells still remains to be answered. Two potential mechanisms are that glucocorticoid effects are being mediated via the 11-keto metabolite activating the putative DHB receptor, or that GR is intracellularly separated from 11-HSD2. When MR and 11-HSD2 are cotransfected into HEK293 cells, both proteins associate with the endoplasmic reticulum membrane. This differs from the nuclear and cytoplasmic distribution of MR when transfected alone (Odermatt et al., 2001). These data suggest that

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MR is in close intracellular association with 11-HSD2. The intracellular localization of GR in 11-HSD2-containing cells has not been addressed, so whether GR is separated intracellularly from 11-HSD2, thus allowing glucocorticoid access to the receptor, awaits investigation. Data supporting such a situation are the consistently enhanced occupancy of MR by corticosterone compared with GR in colon when 11-HSD2 is inhibited (Sheppard, 1998). D. RECEPTOR MODIFICATION

Both GR and MR are phosphoproteins, and phosphorylation of steroid receptors has been implicated as a potential mechanism for ligandindependent activity via cross-talk with other signal transduction pathways (Weigel and Zhang, 1998). GR is a substrate for several kinases and phosphatases and the impact that changes in receptor phosphorylation has on corticosteroid signaling is still largely unknown. Studies on receptor phosphorylation including mutational analysis of receptor phosphorylation sites suggest that these sites are important in receptor half-life, nuclear– cytoplasmic shuttling, and signaling in a promoter-specific manner (Defranco et al., 1995; Webster et al., 1997; Rogatsky et al., 1998). E. CELLULAR MILIEU AND OTHER FACTORS

The cellular concentration of functional GR or MR can impact on the corticosteroid response (Bloom et al., 1980; Danielseen and Stallcup, 1984; Vanderbilt et al., 1987). Because these receptors can form either homodimers or heterodimers that have distinct transactivation properties (Trapp et al., 1994; Liu et al., 1995), transcription of target genes will be controlled in a specific pattern, which will depend on both the ratio of GR to MR in a cell and the corticosteroid status. The interaction of corticosteroid receptors with other transcription factors leads to changes in the ability of activated receptors to regulate gene transcription, and therefore the corticosteroid response is also influenced by the state of activation of other transcription factors. In addition, the coexpression of coactivators and corepressors that associate with the activated corticosteroid receptor– DNA complex will also predict the corticosteroid response at a cellular level. Changes in the expression of the proteins associated with the unliganded GR and MR can also alter the ability of these receptors to induce a response. Decreased levels of hsp90 dramatically decrease signal transduction by steroid hormones (Picard et al., 1990; Kaufmann et al., 1992), presumably by lowering the level of high-affinity receptor complexes. In contrast, increasing nuclear levels of hsp90 have been shown to attenuate glucocorticoid-induced gene transcription (Kang et al., 1999). Indeed, both p23 and hsp90 were shown to disrupt steroid receptor interactions with

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DNA response elements and with coactivators, resulting in a decrease in gene transcription (Freeman and Yamamoto, 2002). Thus, hsp90 appears to modulate the amplitude of responses to corticosteroids at two levels: first, by modulating the ability of receptors to bind ligand and second, at the transcriptional level. Thus, modulation of the corticosteroid response will be the result of an optimal hsp90:receptor ratio both in the cytoplasm and nucleus. Activation of protein kinases and increasing calcium influx can either potentiate or abrogate corticosteroid action in a cell-specific manner (Oikarinen et al., 1984; Gruol et al., 1989; Nordeen et al., 1994; Sato et al., 1996). One mechanism by which protein kinases influence corticosteroid function is via regulation of receptor concentration (Sheppard et al., 1991). Increasing intracellular calcium can decrease receptor binding and activation. The mechanism is posttranslational and involves the reversible conversion of the receptor to a nonbinding form. This is dependent on the cellular milieu, because on cell disruption GR binding is restored (Sheppard, 1994). Similarly, in colonic crypt cells the cellular milieu imparts an apparent low affinity and reduced binding capacity on colonic GR for both corticosterone and the synthetic glucocorticoid dexamethasone (Sheppard, 1998). Whether these effects on receptor binding and activation are via receptor phosphorylation and/or disruption of the hsp90–heteromeric complex remains to be determined.

VI. HEART A. EXPRESSION OF CORTICOSTEROID RECEPTORS AND 11b-HYDROXYSTEROID DEHYDROGENASE ISOFORMS IN THE HEART

Glucocorticoid receptors are ubiquitously expressed and have been shown to be expressed in rodent and human heart (Katz et al., 1988; KayesWandover and White, 2000; Sheppard and Autelitano, 2002), and in both cardiac myocytes and fibroblasts (Sheppard and Autelitano, 2002). The presence of MR in the heart has also been demonstrated. In rabbit, immunohistochemical studies demonstrated MR to be present in cardiac myocytes, endothelial cells of the four cardiac cavities, and vascular smooth muscle cells of large vessels; however, MR was absent from intramyocardial blood vessels. In addition, other heart interstitial cells were labeled but not identified (Lombes et al., 1992). Human cardiac biopsy also demonstrated MR expression in cardiac myocytes by in situ hybridization and immunohistochemistry. In these studies MR was absent from intramyocardial blood vessels and the limited biopsy sample did not permit the examination of endothelial or fibroblast cells (Bonvalet et al., 1995). In rat,

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MR mRNA and specific MR binding have been demonstrated in cardiac myocytes (Sato and Funder, 1996; Sheppard and Autelitano, 2002) but are absent from cardiac fibroblasts (Sheppard and Autelitano, 2002). Thus across species the MR appears to be expressed in cardiac myocytes and, at least in rat, it is absent from the cardiac fibroblast. The expression of nonclassic corticosteroid receptors in cardiac cells has not been addressed, although receptor-binding studies have not detected either a DHB receptor or a site similar to the small intestinal binding site in cardiac cells (K. E. Sheppard, unpublished data). A membrane receptor mediating rapid nongenomic effects of aldosterone may be present in cardiac myocytes given that aldosterone can induce rapid effects on protein kinase (PKC) activity (Sato et al., 1997) and ion transporters (Mihailidou et al., 1998). The coexpression of MR and 11-HSD2 is essential in conferring aldosterone selectivity on the MR. In rat, 11-HSD2 mRNA has been shown to be absent from rat heart by RNase protection analysis (Sheppard and Autelitano, 2002) and in situ hybridization (Roland and Funder, 1996). In contrast, 11-HSD1 is expressed in heart, and is in both isolated cardiac myocytes and fibroblasts. Studies of 11-HSD activity on intact cardiac cells demonstrated that in both cell types 11-HSD1 acted as a reductase only, converting biologically inactive 11-dehydrocorticosterone to corticosterone (Sheppard and Autelitano, 2002). The presence of NADP- but not NAD-dependent 11-HSD activity in rat heart homogenate confirms the presence of 11-HSD1 and the absence of 11-HSD2 (Walker et al., 1992). These data support in vivo studies in which rat heart MR was shown to bind both corticosterone and aldosterone in vivo (Pearce and Funder, 1988) and thus are not protected by 11-HSD2. Limited studies on human heart samples suggest that both 11-HSD1 and low levels of 11-HSD2 are present in this tissue, although the cellular localization of these enzymes is unknown. Northern blot analysis failed to detect 11-HSD2 mRNA in human heart; in contrast, 11-HSD2 mRNA was detected by reverse transcriptase polymerase chain reaction (RT-PCR) (Slight et al., 1996; Kayes-Wandover and White, 2000). Enzyme activity studies on human heart homogenate (Slight et al., 1996) and pieces (Lombes et al., 1995) show that corticosterone could be converted to 11-dehydrocorticosterone in the presence of either NAD or NADP, suggesting that both 11-HSD1 and 11-HSD2 are present. The low level of 11-HSD2 in human heart suggests that this enzyme is present in a minor heart cell population, potentially endothelial cells lining blood vessels or immune cells. In rat, MR expressed in cardiac myocytes is not protected by 11-HSD2 and therefore these receptors would mediate glucocorticoid effects in vivo. In human, further studies are required to address whether 11-HSD2 is coexpressed with MR in cardiac myocytes and therefore confers aldosterone specificity on these receptors.

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B. HEART PRODUCTION OF ALDOSTERONE AND CORTICOSTERONE

In rat, the main endogenous mineralocorticoid is aldosterone and the glucocorticoid is corticosterone. Synthesis of aldosterone and corticosterone from deoxycorticosterone requires the expression of aldosterone synthase and 11-hydroxylase, respectively. Using RT-PCR, 11-hydroxylase and aldosterone synthase have been detected in rat heart (Silvestre et al., 1998), although differences in expression are seen between rat strains (Rudolph et al., 2000) and between species (Young et al., 2001). Both corticosterone and aldosterone and their precursor deoxycorticosterone are detected in the homogenate and perfusate of isolated rat hearts. Similar to the adrenal gland, angiotensin II and ACTH lead to an increase in the release of these steroids from heart, an increase in their concentration in heart tissue, and an increase in heart mRNA expression for both 11-hydroxylase and aldosterone synthase (Silvestre et al., 1998). Experimental rat myocardial infarction results in a 2-fold increase in aldosterone synthase mRNA and a 4-fold increase in aldosterone content in the noninfarcted myocardium. In contrast, 11-hydroxylase mRNA is decreased by 3-fold and corticosterone content is decreased by 2-fold (Silvestre et al., 1999). These studies suggest that aldosterone synthase may play a role in cardiac remodeling after myocardial infarction. Chronic sodium intake in rats, which leads to cardiac hypertrophy, also increases both cardiac aldosterone production and aldosterone synthase mRNA, in contrast to the decrease in plasma aldosterone levels (Takeda et al., 2000), suggesing that cardiac aldosterone synthesis in response to high salt intake might contribute to cardiac hypertrophy and possibly fibrosis independent of the circulating renin– angiotensin–aldosterone system. Using RT-PCR, mRNA encoding enzymes required to synthesize 11deoxycorticosterone from cholesterol have been demonstrated in all chambers of the normal human heart. 11-Hydroxylase mRNA, which encodes the enzyme required to convert 11-deoxycorticosterone to corticosterone, is expressed in all compartments except the left ventricle, whereas mRNA encoding aldosterone synthase, which converts 11deoxycorticosterone to aldosterone, is not detected. The mRNAs encoding enzymes involved in the three final steps of cortisol synthesis (3hydroxysteroid dehydrogenase, 21-hydroxylase, and 11-hydroxylase) are also present; however, 17-hydroxylase/17,20-lyase, which is required for cortisol synthesis from cholesterol, is absent. Thus the normal adult human heart has mRNAs encoding the enzymes required to synthesize corticosterone from cholesterol and cortisol from either 17-hydroxyprogesterone or 17-hydroxypregnenolone, but does not have the enzyme required for aldosterone synthesis (Kayes-Wandover and White, 2000). In contrast, the fetal human heart expresses mRNA for all the above-described enzymes as

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well as aldosterone synthase mRNA. In a study using RT-PCR and looking at the failing human heart, neither aldosterone synthase mRNA nor 11hydroxylase was detected in normal left ventricular heart samples (Young and Funder, 2002). This is in agreement with others (Kayes-Wandover and White, 2000); however, both enzymes were expressed in all four chambers of the failing heart (Young and Funder, 2002). In agreement with this study, aldosterone synthase mRNA was shown to increase in the left ventricle of heart failure patients (Yoshimura et al., 2002). In support of aldosterone being produced in the failing heart is the demonstration that plasma levels of aldosterone in the anterior interventricular vein, which drains blood from the anterior left ventricle, and in the coronary sinus, which drains blood from the whole heart, were higher than in aortic root from patients with left ventricular systolic and diastolic dysfunction (Mizuno et al., 2001) and in patients with essential hypertension (Yamamoto et al., 2002), but not from control patients. Taken together, both the animal and human studies support the hypothesis that heart synthesis of aldosterone is relevant under pathophysiological conditions and that the favorable response to low-dose spironolactone in the Randomized Aldactone Evaluation study (Pitt et al., 1999) may be through antagonizing both the effects of local aldosterone synthesis, as well as circulating aldosterone. However, there are several questions that still need to be addressed. Although mRNAs for these steroidogenic enzymes have been found in heart we do not know whether they are translated into protein. In animal studies, mRNA levels for the enzyme required for corticosterone synthesis were always higher than those for the aldosterone synthesis enzymes, even when aldosterone synthase was induced. In addition, higher concentrations of corticosterone (143 nM) compared with aldosterone (16 nM) are found in rat heart homogenate (Silvestre et al., 1998). These data, taken together with the absence of 11-HSD2 in rat heart, raise the question of how aldosterone can access the non selective MR in vivo. Aldosterone synthase colocalizing with MR in rat cardiac myocytes might be the answer. Studies in humans have not addressed the regulation of 11-hydroxylase mRNA or glucocorticoid synthesis in the normal or pathological heart, so whether locally produced glucocorticoids are similar to aldosterone and increased in the pathological state is unknown. In humans there is low expression of both MR and 11-HSD2 in heart and the cellular localization of the steroidogenic enzymes is unknown. So whether colocalization of the steroidogenic enzymes with MR in human heart provides a mechanism by which local aldosterone production allows aldosterone to bind MR in the absence of 11-HSD2 is still moot. Local production of either aldosterone or corticosterone may also dramatically affect the transcriptional responses of either GR or MR. In nonepithelial cells there is clear evidence that MR and GR produce different effects and that aldosterone effects via MR can be blocked by

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glucocorticoids and vice versa (Funder, 1995). In the hippocampus, where MR mediates glucocorticoid effects (i.e., MR is not protected by 11HSD2), MR and GR mediate opposing effects on excitability and exitatory outflow (Joels and de Kloet, 1994). Intracerebroventricular infusion of aldosterone elevates blood pressure, whereas corticosterone does not. In addition, corticosterone antagonizes the aldosterone effect (Gomez-Sanchez et al., 1990). Similarly, opposing effects of these receptors in heart may also occur, and thus the local synthesis of aldosterone may antagonize the glucocorticoid response by binding MR or GR. This may be due to differences in the transcriptional responses of either aldosterone- or glucocorticoid-activated GR and MR, which may be mediated either via differences in receptor homo- or heterodimerization (Trapp et al., 1994) or via differences in the ability of these receptors to block transcription of other transcription factors (Pearce and Yamamoto, 1993). C. CORTICOSTEROID EFFECTS ON THE HEART

The importance of aldosterone on cardiovascular disease has been highlighted in the RALES trial (Pitt et al., 1999). In this trial a 30% improvement in morbidity and mortality was seen when severe congestive heart failure patients were given low-dose spironolactone in conjunction with existing therapy including loop diuretics and angiotensin-converting enzyme (ACE) inhibitors that block aldosterone secretion. There were no blood pressure or pulse differences between placebo or spironolactone groups, suggesting the positive result was not secondary to hemodynamic changes. This study suggested that aldosterone plays a major role in the pathophysiology of cardiovascular disease and that this is mediated through a direct effect of aldosterone on cardiovascular tissue. Further evidence that aldosterone plays a role in cardiac pathophysiology is a direct correlation between death and serum aldosterone concentrations in heart failure patients (Swedberg et al., 1990), and a correlation between plasma levels of aldosterone and cardiac left ventricular mass in humans (Schlaich et al., 2000). In the heart, aldosterone promotes cardiac hypertrophy and fibrosis, which are unrelated to its hemodynamic effects (Brilla et al., 1990). In rats with experimentally induced left ventricular hypertrophy, elevated levels of plasma aldosterone led to the development of cardiac fibrosis. It was proposed that this effect was a direct effect on heart, because administration of the aldosterone antagonist spironolactone suppressed it at a dose that did not reduce arterial hypertension (Brilla et al., 1993a). The mineralocorticoid specificity of this effect was confirmed by Young et al. (1994). Experimental evidence suggests that elevated levels of glucocorticoids may also have a direct detrimental effect on the structure and function of the heart (Ito et al., 1979; Czerwinski et al., 1991). Studies in rats have shown that short-term glucocorticoid treatment can induce cardiac hypertrophy (Kurowski et al.,

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1984; Czerwinski et al., 1991) whereas, with prolonged treatment, a catabolic process results in the myocardium, causing a reduction in myosin heavy chain synthesis and total protein synthesis (Czerwinski et al., 1991), which may lead to decreased myocardial contractility (Akatsuka et al., 1974). Glucocorticoids also can protect the heart from ischemic injury (Libby et al., 1973; Spath et al., 1974), presumably via their potent antiinflammatory and immunosuppressive actions. D. CORTICOSTEROID EFFECTS ON CARDIAC ION CHANNELS

Action potential prolongation is a hallmark of hypertrophied and failing myocardium and is a consequence of differential expression and function of membrane currents and transporters. Functional downregulation of potassium currents in the ventricle is a recurring theme in hypertrophy and failure; the reduction in the density of the transient outward current is the most consistent observation, whereas data on the density of the inward and the delayed rectifier currents are more contradictory. The altered intracellular calcium handling of the failing heart prolongs the decay of the L-type calcium current. These changes in ventricular myocyte ion handling create an environment that is highly sensitive to triggers for ventricular arrhythmias (Tomaselli and Marban, 1999). Glucocorticoids induce action potential prolongation. This effect appears to be due to a decrease in the amplitude of the transient outward potassium current and an increase in the density of the L-type calcium current (Takimoto et al., 1997; Wang et al., 1999). Whether these glucocorticoid-induced changes in cardiac ion channels contribute to pathophysiology of the hypertrophied and failing heart is yet to be determined. Aldosterone regulation of ion currents has also been reported. Incubation of cardiac myocytes in culture with very high doses of aldosterone (100 nM– 1 M) for 24 h upregulates calcium transport in cardiac myocytes by increasing L-type calcium channel density. This effect is partially blocked by spironolactone whereas inhibition of transcription prevented the increase, suggesting it was a transcriptional effect (Benitah and Vassort, 1999). In addition, a high dose of aldosterone decreases the transient potassium current after 48 h of treatment with 100 nM aldosterone, an effect that was blocked by the MR antagonist RU28318, but not by the GR antagonist RU38486 (Benitah et al., 2001). In addition to these effects, aldosterone treatment (2 nM) of neonatal and adult rat cardiac myocytes in culture increases both intracellular sodium and Na+,K+-ATPase 1 subunit mRNA levels (Ikeda et al., 1991). Short-term exposure (20–60 min) of rabbit cardiac myocytes to aldosterone (10 nM) activates the Na+,K+,2Cl cotransporter to enhance sodium influx and to stimulate the Na+/K+ pump. This effect is blocked by potassium canrenoate, an MR antagonist, and probably reflects

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a nongenomic effect of aldosterone on cardiac myocytes (Mihailidou et al., 1998). In a study in which cardiac myocytes were cultured long term (6–13 days) in the presence of serum, which would have resulted in a hypertrophied state, 24-h treatment with a high dose of aldosterone (1 M) was shown to increase the activity of the Cl/HCO3 exchanger and the Na+/H+ antiporter, the main ionic transporters that regulate pH in neonatal heart. The aldosterone effects on the pH regulatory system were variable with length of culture. The Cl/HCO3 exchange activity was stimulated at the beginning of culture whereas Na+/H+ antiport was influenced later, suggesting that aldosterone regulation of these channels differs depending on the hypertrophic state of the cardiac myocyte (Korichneva et al., 1995). With many of the aldosterone studies high doses of steroid were used. Given that aldosterone can activate the glucocorticoid receptor and that spironolactone is also a GR antagonist (Couette et al., 1992; Sheppard and Autelitano, 2002), it is difficult to determine whether the aldosterone effects are being mediated via MR or GR, or alternatively via a membrane receptor. E. CARDIAC HYPERTROPHY

Cardiac hypertrophy involves both the hypertrophy of cardiac myocytes and cardiac fibroblast proliferation, and thus factors regulating either of these processes can impact on heart enlargement. Aldosterone treatment of neonatal rat cardiac myocytes in culture induces protein synthesis only if coincubated with either the GR antagonist RU38486 or in the presence of high concentrations of glucose. The potentiation of aldosterone-induced protein synthesis by glucose is mediated via activation of PKC. In contrast, glucocorticoids decrease protein synthesis in cardiac myocytes when occupying GR and induce a small but significant increase in protein synthesis when occupying MR in the presence of high glucose (Sato and Funder, 1996). These data suggest that aldosterone can induce cardiac hypertrophy under conditions of high glucose and possibly potentiate the hypertrophic response induced by factors that stimulate PKC. Aldosterone at moderate to high concentrations (10–100 nM) can also significantly increase proliferation of human cardiac fibroblasts, an effect that is comparable to high d-glucose-induced proliferation. No additive growth stimulation is evident when cells are incubated with both aldosterone and d-glucose (Neumann et al., 2002). Whether this effect of aldosterone was via the GR or MR was not determined in these studies. Further evidence that aldosterone can directly impact on myocyte hypertrophy is the demonstration in neonatal rat cardiac myocytes that aldosterone at a high dose (1 M) potentiated the endothelin 1 (ET-1)induced increase in surface area, brain natriuretic peptide, and skeletal actin

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mRNA. Aldosterone augmented JNK activation by ET-1 but not ERK or p38 MAPK, suggesting that aldosterone modulates cardiac hypertrophy at least in part by synergizing with extracellular signals (Oshima et al., 2002). Caution in the interpretation of this study is needed given that the high dose of aldosterone used is unlikely to be reached in vivo, and that aldosterone would bind both MR and GR; therefore, whether the effect is a result of aldosterone occupying MR or aldosterone occupying GR needs to be determined. Both corticosterone and aldosterone induce serum and glucocorticoidinduced kinase (Sgk) gene transcription in isolated cardiac myocytes and cardiac fibroblasts. This effect is blocked by the GR antagonist RU38486, demonstrating that both glucocorticoids and aldosterone can induce Sgk gene transcription via GR (Sheppard and Autelitano, 2002). The role of Sgk in cardiac physiology/pathophysiology is unknown. Aldosterone-induced Sgk interacts with the ubiquitin protein ligase NEDD4-2, leading to its phosphorylation. This phosphorylation reduces the interaction between NEDD4-2 and the epithelial sodium channel, leading to elevated epithelial sodium channel cell surface expression. This is one mechanism by which aldosterone increases epithelial sodium absorption in epithelial cells (Debonneville et al., 2001; Snyder et al., 2002). Sgk has also been shown to regulate other ion channels (Gamper et al., 2002; Yun et al., 2002) and thus Sgk via NEDD4-2 or related ubiquitin ligases may mediate corticosteroid regulation of ion channels in cardiac cells and possibly cardiac action potential (Abriel et al., 2000). F. MYOCARDIAL INFARCTION

Myocardial infarction occurs in response to an imbalance of oxygen supply and demand. Reperfusion of the previously ischemic myocardium and the associated inflammatory response contribute to the development of cardiac injury. Changes in the left ventricle after myocardial infarction can be arbitrarily divided into acute and chronic phases. The acute phase, termed infarct expansion, typically occurs in the first few days after myocardial infarction and is defined by thinning of the infarcted myocardium and dilation of the ventricular cavity (Eaton et al., 1979; Pfeffer et al., 1991). The second phase, which begins after stabilization of the infarct scar and may continue indefinitely, is characterized by continued ventricular dilation as well as myocyte hypertrophy and reactive fibrosis of the remote noninfarcted myocardium (Weiss et al., 1981). Although glucocorticoids protect the heart against ischemic injury (Libby et al., 1973; Spath et al., 1974), they also exacerbate infarct expansion and reduce both wound healing and scar formation. This leads to the development of aneurisms and potentially fatal cardiac ruptures (Hammerman et al., 1984; Mannisi et al., 1987). The adverse effects of glucocorticoids have been

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attributed to their genomic effects. A high dose of dexamethasone was shown to protect the heart muscle of the mouse against ischemia–reperfusion injury by causing the rapid (within 20–60 min), nontranscriptional activation of endothelial nitric oxide synthase (eNOS) (Hafezi-Moghadam et al., 2002). This effect is via binding to the classic GR because RU38486, the GR antagonist, blocked the effect. Myocardial ischemia and reperfusion also result both in the expression of (in leukocytes) annexin I (La et al., 2001) and in the activation of NF-B (Zingarelli et al., 2002b). There is evidence that inhibition of NF-B exerts cardioprotective effects in rodents (Zingarelli et al., 2002a,b) and that annexin I reduces infarct size and the inflammatory response (La et al., 2001). Thus it is also possible that the cardioprotective effects of glucocorticoids are due to one or more of the following: activation of eNOS, inhibition of the activation of NF-B, and/or enhanced expression of annexin I. G. CARDIAC FIBROSIS

Myocardial infarction, hypertrophy, and hypertension are associated with progressive cardiac fibrosis, which can lead to diastolic dysfunction. In a study on the effects of eplerenone, an MR antagonist, on acute infarct expansion and late-phase remodeling associated with myocardial infarction, it was demonstrated that MR antagonism does not affect myocardial infarct healing but does attenuate the reactive cardiac fibrosis of the noninfarcted myocardium. In addition, eplerenone did not attenuate the hypertrophy associated with myocardial infarction. Interestingly, the model used is not characterized by a chronic activation of the renin–angiotensin–aldosterone system, which suggests that normal plasma aldosterone levels are sufficient to support a role in postmyocardial infarction remodeling (Delyani et al., 2001). Fibrosis occurs in rat heart only if aldosterone and sodium intake are chronically increased, and the role of sodium in the pathogenesis is still unresolved (Funder, 1997). In uninephrectomized rats, drinking 0.9% saline sensitizes rats to exogenous aldosterone. These animals develop cardiac hypertrophy and both perivascular and interstitial fibrosis (Brilla and Weber, 1992; Young et al., 1994), compared with those on a restricted sodium intake. A humoral rather than a hemodynamic effect is suggested as being responsible for fibrosis because the extent of fibrosis was similar in the left and right ventricle and spironolactone, the MR antagonist, at a dose that has no hypertensive effects can prevent cardiac fibrosis (Brilla et al., 1993b). Further proof of humoral effects of aldosterone on cardiac fibrosis was demonstrated in rats infused with aldosterone in which the blood pressure was clamped at normotensive levels by intracerebroventricular infusion of an MR antagonist (RU28318). In these animals, cardiac fibrosis and hypertrophy were indistinguishable from those infused with aldosterone

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alone (Young et al., 1995). Interestingly, infusion of either corticosterone or the glucocorticoid receptor antagonist RU38486 did not induce cardiac hypertrophy but did increase interstitial cardiac fibrosis, albeit to a lesser degree compared with aldosterone. In addition, coadministration of 30-fold excess corticosterone substantially blocked the aldosterone-induced fibrosis (Young and Funder, 1996). Glucocorticoids are well known for their inhibitory effects on fibrosis, so one interpretation of these data is that RU38486 increases cardiac fibrosis by antagonizing the effects of endogenous glucocorticoid, and the increase in fibrosis associated with corticosterone infusion may reflect the additive opposing effects of MR and GR activation. The cellular mechanisms by which aldosterone induces fibrosis are still under investigation. Studies on cultured cardiac fibroblasts are inconsistent, with some investigators showing an increase in collagen I mRNA and collagen synthesis (Brilla et al., 1994; Zhou et al., 1996) and other laboratories unable to confirm these studies (Fullerton and Funder, 1994; Kohler et al., 1996). In addition, the MR has been shown to be absent from rat cardiac fibroblasts (Sheppard and Autelitano, 2002), suggesting a direct effect of aldosterone via MR on cardiac fibroblasts is unlikely. H. TRANSGENIC ANIMALS

Overexpression of human MR in mice results in dilated cardiomyopathy involving increased left ventricular diameter and decreased shortening fraction, which is accompanied by a significant increase in heart rate (Le Menuet et al., 2001). There is an increase in cardiac atrial natriuretic protein expression, which is a recognized factor associated with cardiac hypertrophy. An increase in the frequency of dysrhythmia is also observed, which is in agreement with the observation that spironolactone reduces ventricular tachycardia in congestive heart failure patients (Ramires et al., 2000). Surprisingly, in these animals blood pressure is normal and there is no induction of cardiac fibrosis. In a myocyte-specific MR knockout mouse, animals develop severe heart failure and cardiac fibrosis. These effects are reversible when MR antisense mRNA expression is subsequently suppressed and, thus, endogenous MR expression restored. The myocyte MR knockout mice display dilated cardiomyopathy, hypertrophic cardiac myocytes with marked cardiac interstitial fibrosis, and no obvious perivascular fibrosis. This model indicates that myocyte MR are important in maintaining the myocyte in a nonhypertrophied state and are also important in preventing fibrosis. This appears to be in contrast to both the in vivo studies, in which aldosterone increases myocyte hypertrophy and fibrosis, and also to the studies in the transgenic mice overexpressing MR. As previously discussed, the rat cardiac myocyte MR would bind glucocorticoids in vivo given the absence of 11HSD2 in these cells. Thus the detrimental changes observed in the heart of the

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myocyte MR knockout mouse may be due to the loss of glucocorticoid effects mediated via MR. This would then suggest that the hypertrophy and fibrosis associated with elevated aldosterone in vivo is via aldosterone antagonizing the MR-mediated glucocorticoid effects in cardiac myocytes, and would also explain why increasing MR expression does not induce cardiac fibrosis.

VII. SUMMARY This review has covered insights into extraadrenal corticosteroid synthesis, classic and novel corticosteroid receptors, the genomic and nongenomic mechanism of corticosteroid action, and the direct effects of corticosteroids on heart with an emphasis on the factors involved in both mediating and modulating these cardiac effects. It is predictable that at a cellular level there is a multitude of factors that can modulate the intracellular signals of these hormones, given the contrast between the specific cellular effects of mineralocorticoid and glucocorticoid hormones and both the lack of intrinsic steroid specificity of MR and the ubiquitous expression of GR. The level of intracellular corticosteroid concentrations that impacts on the response to corticosteroids is not only dependent on circulating levels of these hormones but also on local corticosteroid synthesis, P-glycoprotein active export, and 11-HSD activity. At the receptor level, specificity to the corticosteroid response can be via the cellular milieu imparting apparent changes in the steroid-binding capacity and affinity of these hormones for both GR and MR. The mechanisms may involve phosphorylation of the receptor or changes in receptor-bound heat shock proteins. In addition, at the transcriptional level receptor dimerization and the ability of receptors to interact with other transcription factors all contribute to relaying specificity to the corticosteroid response. Furthermore, the discovery that corticosteroids have rapid nongenomic effects, mediated via novel membrane receptors or the classic corticosteroid receptors, expands both the time frame of corticosteroid action and the potential for further cross-talk with other intracellular signaling pathways. In the heart the lack of 11-HSD2 and the presence of MR in cardiac myocytes suggest that these receptors mediate glucocorticoid effects in vivo. 11-HSD1 is present in heart cells, facilitating glucocorticoid action by converting circulating receptor-inactive 11-ketoglucocorticoids to active glucocorticoids. Aldosterone access to myocyte MR may occur if under certain conditions 11-HSD1 acted as an 11-dehydrogenase, or if aldosterone was synthesized in the cardiac myocyte. Cardiac aldosterone synthesis occurs in the failing heart, and thus under this pathological condition, aldosterone may mediate its effects via either MR or GR and therefore change the response to both these receptors. It is unlikely that aldosterone-induced cardiac fibrosis is being directly mediated via MR

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because of the absence of this receptor from the cardiac fibroblast, although a direct effect of aldosterone on cardiac fibroblasts might be mediated by the GR, or via a membrane-linked signaling cascade. Our view of the mechanism of action of corticosteroid hormones has expanded over the past 10 years to include rapid membrane effects of these steroids and a better understanding of the importance of the cellular factors that confer specificity to corticosteroid signaling and thus define the physiological response.

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4 Forms of Mineralocorticoid Hypertension Paolo Ferrari and Olivier Bonny Division of Nephrology and Hypertension, Inselspital, University of Berne, 3010 Berne, Switzerland

I. Introduction II. Evolution, Salt, and the Renin–Angiotensin– Aldosterone System III. Key Elements of Mineralocorticoid Activity A. Aldosterone Synthase B. 11b-Hydroxysteroid Dehydrogenase Type 2 C. Mineralocorticoid Receptor D. Epithelial Sodium Channel IV. Mineralocorticoid Hypertension V. Primary Aldosteronism A. Prevalence B. Clinical and Laboratory Findings C. Screening D. Further Evaluation and Diagnosis E. Subtype Delineation F. Therapy VI. Genetic Forms of Mineralocorticoid Hypertension A. Mutations of the 11b-Hydroxylase or 17a-Hydroxylase Gene: Congenital Adrenal Hyperplasia B. Chimeric 11b-Hydroxlase–Aldosterone Synthase Gene: Glucocorticoid-Remediable Aldosteronism Vitamins and Hormones Volume 66

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C. Mutations of the 11b-Hydroxysteroid Dehydrogenase Type 2 Gene: Apparent Mineralocorticoid Excess D. Mutations of the Mineralocorticoid Receptor Gene E. Mutations of the Epithelial Sodium Channel Genes: Liddle Syndrome VII. Aldosterone-Dependent Essential Hypertension References

Hypertension with hypokalemia, metabolic alkalosis, and suppressed plasma renin activity defines mineralocorticoid hypertension. Mineralocorticoid hypertension is the consequence of an overactivity of the epithelial sodium channel expressed at the apical membrane of renal cells in the distal nephron. This is usually the case when the mineralocorticoid receptor is activated by its physiologic substrate aldosterone. The best known form of mineralocorticoid hypertension is an aldosteroneproducing adrenal tumor leading to primary aldosteronism. Primary aldosteronism can also be caused by unilateral or bilateral adrenal hyperplasia and rarely adrenal carcinoma. Interestingly, most of the inherited monogenic disorders associated with hypertension involve an excessive activation of the mineralocorticoid axis. In some of these disorders, mineralocorticoid hypertension results from activation of the mineralocorticoid receptor by other steroids (cortisol, deoxycorticosterone), by primary activation of the receptor itself, or by constitutive overactivity of the renal epithelial sodium channel. The present review addresses the physiology and significance of the key players of the mineralocorticoid axis, placing emphasis on the conditions leading to mineralocorticoid hypertension. ß 2003 Elsevier Science (USA).

I. INTRODUCTION Mineralocorticoid hypertension refers to a primary, renin-independent activation of the mineralocorticoid axis, leading to hypertension because of excessive sodium and water retention by the distal tubule of the kidney. The resulting blood volume expansion suppresses endogenous plasma renin activity. At the cellular level mineralocorticoid hypertension is the consequence of overactivity of the renal epithelial sodium channel. The channel activity is regulated by aldosterone via the mineralocorticoid receptor; however, other steroids, including cortisol, show mineralocorticoid activity. In mineralocorticoid target tissues the enzyme 11-hydroxysteroid dehydrogenase inactivates cortisol into cortisone, thus preventing

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overactivation of the receptor by glucocorticoids. The paradigm for mineralocorticoid hypertension is aldosteronoma, an aldosterone-producing adrenal tumor. Moreover, the vast majority of inherited monogenic hypertensive disorders involve an excessive activation of the mineralocorticoid axis. In some of these disorders, hypertension is not the sole feature, as is the case for congenital adrenal hyperplasia, in which hypertension is accompanied by abnormal sexual differentiation. In other cases hypertension is the unique or preeminent abnormality of the genetic defect. The molecular basis of several forms of severe hypertension transmitted on an autosomal basis has been elucidated. These disorders are a consequence of either abnormal biosynthesis, metabolism, or action of steroid hormones and are ultimately characterized by an overactivation of the epithelial sodium channel. This review details some aspects of the physiology of the renin–angiotensin–aldosterone system and of the key players of the mineralocorticoid axis. Specific conditions leading to mineralocorticoid hypertension are addressed in the second part of the overview.

II. EVOLUTION, SALT, AND THE RENIN– ANGIOTENSIN–ALDOSTERONE SYSTEM There is little doubt that life on our planet first generated in the sea (Griffith, 1987). This is not surprising, because the sodium concentration in seawater averages 170 mmol/liter, a value remarkably close to the extracellular sodium concentration in mammals. Evolution to terrestrial life meant leaving behind the sea and its continuous source of salt and water. Water on land, when available, was fresh, and thus adaptation to land necessitated the development of mechanisms to preserve salinity (Cirillo et al., 1994). This task of regulating salt and water balance was taken over by the kidney (Frassetto et al., 2001; Smith, 1964). Regulation of salt and water occurs in the renal medulla, which developed differently among species, being more prominent in those with a high urine-concentrating capacity (Kriz, 1981). It is therefore not surprising that the nephron in saltwater fish possesses only primitive segments responsible for concentration of the urine, such as the intermediate segment and the collecting duct, and an overdimensioned proximal tubule, whose function is to warrant isotonic reabsorption. Freshwater fish must deal with an excess supply of free water, and therefore their nephrons have an extremely developed distal tubule, whose function is to dilute the urine. The appearance of well-developed nephron elements, such as the loop of Henle and the collecting duct, in order to provide sufficient concentration of the urine by increasing sodium and water reabsorption can best be observed in humans and other terrestrial mammals. Of interest is the observation that a regulated renal tubular sodium reabsorption can largely be documented only in mammals, because

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TABLE I. Renin–Angiotensin–Aldosterone System across Species Structure

Seawater fish Freshwater fish Amphibians Reptiles Mammals a

Function

Juxtaglomerular cells

Macula densa

Aldosterone

Pressor

Na+ transport

Granules Granules + + +

+ + +

(+)a (+) +

+ + + + +

+

(+), Found in some members, but regulation by renin–angiotensin is not established.

aldosterone can be found almost exclusively in mammals (Table I). This evolutionary adaptation of the nephron indicates the central role of the kidney in the regulation of sodium and water balance and hence of blood pressure. Normal regulation of salt and water homeostasis in mammals is controlled in a negative-feedback loop by the renin–angiotensin–aldosterone system (Corvol et al., 1984). The key players of this system are renin, released by the juxtaglomerular cells of the afferent arterioles and macula densa cells of the kidney, and aldosterone, produced by the adrenal glands (Fig. 1). Renin cleaves the biologically inactive decapeptide angiotensin I from its precursor angiotensinogen, which is released in the circulation by the liver. Angiotensin I is rapidly transformed to the active octapeptide angiotensin II by the angiotensin-converting enzyme present in large amounts in the membrane of endothelial cells of the lungs. Angiotensin II has several important direct and indirect effects for the maintenance of circulatory homeostasis. Direct effects include the vasoconstriction of the renal and systemic circulations and the reabsorption of sodium in proximal segments of the nephron. Indirect effects are mediated by stimulating the adrenal cortex to secrete aldosterone (Fig. 1), which promotes the reabsorption of sodium (in exchange for potassium) in epithelial tissues such as the cortical collecting duct of the kidney, the colon, and the salivary and sweat glands. Plasma concentrations of renin, angiotensin II, and aldosterone rise in response to a contraction of intravascular volume and a reduction in renal perfusion and are suppressed by intravascular volume expansion. Angiotensin II is the principal stimulator of aldosterone production when intravascular volume is reduced (Miller et al., 1968; Tobian, 1967) (Fig. 1). Potassium is also a major physiologic stimulus to aldosterone production; aldosterone secretion is integral to potassium homeostasis because aldosterone has the ability to increase potassium excretion in urine, feces, sweat, and saliva (Silva et al., 1977). Aldosterone thereby serves to prevent hyperkalemia during periods of high potassium

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FIGURE 1. Regulation of aldosterone secretion by renin. A decrease in intravascular volume increases the secretion of renin by the juxtaglomerular apparatus, leading to increased conversion of angiotensinogen to angiotensin I and then to angiotensin II. Angiotensin II acts on the adrenal zona glomerulosa to increase the activity of aldosterone synthase and therefore the secretion of aldosterone. Also, -adrenergic nerves and decreases in NaCl concentration in the macula densa cause stimulation of renin release by the granular cells.

intake. For example, aldosterone secretion rises after the consumption of foods high in potassium content or after vigorous physical activity that causes the release of potassium from skeletal muscle. Although there is some evidence that adrenocorticotropin (ACTH) might regulate the expression of aldosterone synthase in rodents, a physiologic role for ACTH-regulated aldosterone secretion in humans is lacking.

III. KEY ELEMENTS OF MINERALOCORTICOID ACTIVITY A. ALDOSTERONE SYNTHASE

The most potent corticosteroids are 11-hydroxylated compounds. In humans, two cytochrome P450 isoenzymes with 11-hydroxylase activity, catalyzing the biosynthesis of cortisol and aldosterone, are present in the

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17α-OH-progesterone

Dehydroepiandrosterone

Progesterone

17α-OH-progesterone

4-Androstendione

11-Deoxycorticosterone

11-Deoxycortisol

CYP11B1 Corticosterone

Cortisol

CYP11B2 18-OH-corticosterone

Aldosterone

MINERALOCORTICOIDS

Zona glomerulosa

GLUCOCORTICOIDS

Zona fasciculata

ANDROGENS

Zona reticularies

FIGURE 2. The pathways of biosynthesis of aldosterone and cortisol from cholesterol. Adrenal steroid biosynthesis is catalyzed by several forms of cytochrome P450 and two hydroxysteroid dehydrogenases. Inherited forms of mineralocorticoid hypertension can result from genetic variants in the enzymes enclosed in boxes. The last three enzymatic conversions required for aldosterone biosynthesis are mediated by a single enzyme, aldosterone synthase (encoded by CYP11B2). Deficiencies in 17-hydroxylase (CYP17) and 11-hydroxylase (CYP11B2) result in hypertension associated with excess 11-deoxycorticosterone.

adrenal cortex: 11-hydroxylase and aldosterone synthase (Fig. 2). The gene encoding aldosterone synthase (CYP11B2) is expressed in the zona glomerulosa under primary control of the renin–angiotensin system. Aldosterone synthase has 11-hydroxylase activity as well as 18-hydroxylase activity and 18-oxidase activity (Fig. 2). The 11-hydroxylase gene (CYP11B1) is expressed in the zona fasciculata and is under the control of ACTH. The substrate for the CYP11B2-encoded protein is 11-deoxycorticosterone, and that of the CYP11B1-encoded protein is 11-deoxycortisol. The CYP11B2 gene was isolated as a cross-hybridizing gene while cloning and analyzing the CYP11B1 gene (Mornet et al., 1989). The sequence of CYP11B2 is approximately 95% identical to CYP11B1 in coding regions and 90% identical in introns. The 50 -upstream region has diverged considerably from that of CYP11B1, suggesting that this second gene, if expressed, may be regulated differently. Both putative proteins contain

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503 amino acids, including a 24-residue signal peptide. This gene was previously thought to be a pseudogene or a less active gene closely related to CYP11B1. Kawamoto et al. (1992) showed that the nucleotide sequence of the promoter region of the CYP11B2 gene, which encodes steroid 18-hydroxylase, is strikingly different from that of the CYP11B1 gene, although the sequences of their exons are almost identical. Studies in cultured cells demonstrated that CYP11B2 encodes an enzyme with steroid 18-hydroxylase activity to catalyze the synthesis of aldosterone and 18-oxocortisol and exhibits steroid 11-hydroxylase activity as well. On the other hand, CYP11B1 as expressed in the cultured cells exhibited steroid 11-hydroxylase activity exclusively. These findings indicated that the two enzymes are products of two different genes and that the 11hydroxylase enzyme participates in the synthesis of glucocorticoids, whereas the C-18 enzyme participates in the synthesis of mineralocorticoids in humans (Kawamoto et al., 1992). Curnow et al. (1991) likewise identified the CYP11B2 gene as that for the aldosterone-synthesizing enzyme. B. 11-HYDROXYSTEROID DEHYDROGENASE TYPE 2

The 11-hydroxysteroid dehydrogenase (11HSD) enzymes catalyze the interconversion of the endogenous cortisol and cortisone in humans (Fig. 3) or of corticosterone and dehydrocorticosterone in rodents (Funder et al., 1988). Cortisone and dehydrocorticosterone exhibit hardly any biological activity because they have negligible affinity for glucocorticoid and mineralocorticoid receptors. Depending on the equilibrium between the biologically active 11-hydroxysteroids and the inactive 11-ketosteroids in a given cell, it might be predicted which cells do and do not respond to cortisol or corticosterone via either glucocorticoid or mineralocorticoid receptor activation (Edwards et al., 1988; Escher et al., 1997; Funder et al., 1988, 1990). Two kinetically distinct forms of 11HSD (11HSD1 und 11HSD2), differentiated in addition by cofactor specificity and differential tissue expression, have been cloned (Agarwal et al., 1989, 1995; Albiston et al., 1994; Tannin et al., 1991; Walker et al., 1992). 11HSD1 activity and expression is found in most tissues; its Km for cortisol is more than an order of magnitude higher than that of 11HSD2; it is NADP(H) preferring and has been shown to have predominantly reductase activity in vivo (Agarwal et al., 1989; Tannin et al., 1991). The role of 11HSD1 was investigated in mice lacking 11HSD1. These animals are unable to convert 11-dehydrocorticosterone to corticosterone in vivo, confirming 11HSD1 as the sole 11-reductase in the mouse, and show reduced activation of glucocorticoid-induced processes (Holmes et al., 2001; Kotelevtsev et al., 1997). 11HSD1-deficient mice have elevated circulating levels of plasma corticosterone levels and adrenal hyperplasia, but they also have attenuated glucocorticoid-induced activation of gluconeogenic enzymes in response to

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FIGURE 3. Peripheral cortisol metabolism and mineralocorticoid receptor selectivity. Cortisol and aldosterone bind with equal affinity to the mineralocorticoid receptor (MR). Plasma concentrations of cortisol are much higher than those of aldosterone, but in mineralocorticoid-responsive cells 11-hydroxysteroid dehydrogenase type 2 (11HSD2) in the endoplasmic reticulum converts cortisol to cortisone, which is not a ligand for the MR, permitting aldosterone to occupy the receptor. The hemiacetal conformation of the 11-hydroxyl group with the 18-aldehyde group of aldosterone renders this steroid a poor substrate for the enzyme.

fasting, and lower glucose levels in response to obesity or stress. Also, 11HSD1 deficiency produces an improved lipid profile, hepatic insulin sensitization, and a potentially atheroprotective phenotype (Morton et al., 2001). In contrast, 11HSD2 has been identified to date in a limited range of tissues (Agarwal et al., 1995; Albiston et al., 1994; Walker et al., 1992); it has a high affinity for cortisol, is NAD requiring, and appears to show only dehydrogenase activity for endogenous glucocorticosteroids (Albiston et al., 1994; Li et al., 1997; Walker et al., 1992) (Fig. 3), although reduction of dehydrodexamethasone has been demonstrated in vitro (Ferrari et al., 1996c). Importantly, immunohistochemical studies have consistently localized 11HSD2 to the distal tubules (Agarwal et al., 1994; Albiston et al., 1994; Krozowski et al., 1995; Naray-Fejes-Toth et al., 1991; Walker et al., 1992). The microsomal 11HSD2 enzyme has a luminal orientation with the catalytic domain facing the cytoplasm (Odermatt et al., 1999).

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Whereas the relevance of 11HSD enzymes for the regulation of the access of steroid molecules to the glucocorticoid receptor has been established only in cell culture experiments (Escher et al., 1997; Teelucksingh et al., 1990; Whorwood et al., 1993) and in transgenic mice (Holmes et al., 2001; Kotelevtsev et al., 1997), the clinical relevance of 11HSD2 activity for mineralocorticoid receptor activation is well defined for some disease states in humans. The activity of the 11HSD2 enzyme can be reliably assessed in vivo by measuring the ratio of biologically active cortisol (F) to inactive cortisone (E), or their tetrahydrometabolites (THF and THE), in the urine, using gas chromatography with mass spectrometry (Ferrari et al., 2001b; Shackleton, 1993). An increased urinary free F:E ratio or urinary (THF + 5THF):THE ratio indicates decreased 11HSD2 activity. C. MINERALOCORTICOID RECEPTOR

There are two types of classic corticosteroid receptor: the high-affinity type 1 or mineralocorticoid receptor (MR) (Arriza et al., 1987) and the lower affinity type 2 or glucocorticoid receptor (GR) (Hollenberg et al., 1985), which are structurally highly homologous. Glucocorticosteroids, corticosterone in rodents and cortisol in humans, bind to MR with high affinity, similar to that of the mineralocorticoid hormone aldosterone (Krozowski and Funder, 1983), and, conversely, aldosterone binds to the human GR with lower affinity, similar to that of cortisol. Molecular cloning of the GR and MR allowed the determination of their primary amino acid structures and prediction of common functional domains. GR and MR display a high degree of identity in their amino acid sequences, with the exception of the variable N-terminal region. The human MR gene encodes a protein of 984 amino acid residues with a predicted molecular size of 107 kDa (Arriza et al., 1987). Structurally and functionally defined domains are observed within the MR and GR receptors (Mangelsdorf and Evans, 1995). The N-terminal part contains the domain A/B, which is involved in transcriptional activation. The central part includes the DNA-binding domain (DBD or C), which is responsible for DNA binding and recognition of the specific hormone-responsive element (HRE) sequences. Domain E, which represents the ligand-binding domain (LBD or E), also contains sequences that are involved in nuclear translocation, receptor dimerization, hormone-regulated transactivation, and interaction with heat shock proteins. On binding to an agonist, the receptor undergoes a major conformational change (transconformation). A number of contact sites are required for proper transconformation and have been identified by sitedirected mutagenesis (Fagart et al., 1998) and by the discovery of a gainof-function mutation leading to a constitutively active receptor, causing a severe hypertensive phenotype (Geller et al., 2000). Dimerization of steroid receptors is a prerequisite for binding to specific HREs lying in the promoter

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FIGURE 4. Schematic representation of the epithelial sodium channel (ENaC). The model shows the tetrameric assembly of ENaC subunits around the central channel pore and that all ENaC subunits participate in the formation of the channel pore.

region of the target gene. In aldosterone target cells, MR is always coexpressed with GR and it has been proposed that MR can heterodimerize with GR, or other transcription factors such as AP-1, allowing more diversity in the physiological response to mineralo- or glucocorticoid hormones, but the in vivo relevance of the phenomenon remains to be established (Bamberger et al., 1997; Pearce and Yamamoto, 1993).

D. EPITHELIAL SODIUM CHANNEL

The epithelial sodium channel (ENaC) is characterized by a high selectivity for sodium over potassium, a low unitary conductance, long open and closed time, and a high affinity for the potassium-sparing diuretics amiloride and triamterene (Garty and Palmer, 1997). The genes encoding ENaC were identified by functional expression cloning (Canessa et al., 1993, 1994b). ENaC is a heteromultimeric protein made of three subunits, termed , , and ENaC (Canessa et al., 1994b) (Fig. 4). All three subunits share about 35% homology at the amino acid level and adopt the same topology, with two transmembrane domains, short intracellular amino and carboxy ends, and a large extracellular loop corresponding to about 70% of the protein mass. When all three subunits are expressed in the same cell, they assemble according to a preferential heterotetrameric structure (Firsov et al., 1998). All three subunits are expressed in the main aldosterone-sensitive target cells or tissues, namely, the last part of the nephron in the kidney (Duc et al., 1994; Loffing et al., 2000), in the distal colon, in the ducts of salivary and sweat glands (Duc et al., 1994), and in the lung, where they could be expressed differentially along the pulmonary tree (Matsushita et al., 1996; Talbot et al., 1999). A highly conserved region in the cytoplasmic

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N-terminal domain is involved in the gating of the channel (Grunder et al., 1997, 1999). Another important feature of the N-terminal region is the presence of numerous lysine residues. Staub et al. (1997) demonstrated that these residues (especially on the and subunits) can be ubiquitinated, and are key elements determining the half-life of the channel. The extracellular loop is the largest domain of the protein, encoded by 10 different exons, in comparison with the N and C termini, which are each encoded by 1 exon. It contains several glycosylation sites (Canessa et al., 1994a; Snyder et al., 1994). Schild et al. (1997) found point mutations that affect substantially the amiloride sensitivity on the three subunits. The intracellular C terminus contains several functional domains involved in the regulation of the number of channels present at the cell surface. A PPPxY motif is present on all three ENaC subunits. Deletion or missense mutations of this motif on the and subunits are found in patients affected by Liddle syndrome, underscoring its importance in channel regulation (Hansson et al., 1995a,b; Shimkets et al., 1994; Snyder et al., 1995). In Liddle syndrome, the channel is hyperactive because of two factors: an increased number of channels present at the cell surface and an increased intrinsic activity of ENaC. The so-called PY motif is the target of Nedd4, a ubiquitin-protein ligase, which binds to the PY motif through its WW domains (Staub et al., 1996). The binding allows the ubiquitination of ENaC and its degradation. The PY motif could also play a role in the regulation of the number of channels present at the cell surface by acting as an endocytic signal (Shimkets et al., 1997). Another possibility of ENaC regulation is phosphorylation. Aldosterone and insulin were found to increase basal phosphorylation of the and subunits, when channel subunits were stably expressed in MDCK cells (Shimkets et al., 1998). However, the phosphorylated residues are not yet identified and the functional relevance has not been established. ENaC is regulated by intracellular as well as by extracellular signaling pathways. Hormones such as aldosterone, vasopressin, insulin, or glucocorticoids regulate ENaC expression and/or activity by intracellular signaling cascades (Garty and Palmer, 1997; Verrey, 2001). A high intracellular concentration of sodium inhibits ENaC by a feedback mechanism (Abriel and Horisberger, 1999). Several kinases have been implicated in the regulation of ENaC (Garty and Palmer, 1997). In particular, the aldosterone-induced SGK (serum and glucocorticoidregulated kinase) was shown to increase ENaC activity and the number of channels present at the cell surface (Chen et al., 1999; Loffing et al., 2001; Naray-Fejes-Toth et al., 1999) and this effect is mediated by the phosphorylation of Nedd4 (Debonneville et al., 2001). Cytoskeleton elements could also play a role in regulating ENaC function. Actin (Jovov et al., 1999), -spectrin (Rotin et al., 1994), and syntaxins (Saxena et al., 1999) were reported to influence ENaC function.

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Luminal high sodium concentrations have been described to downregulate ENaC by self-inhibition (Palmer et al., 1998). Extracellular serine proteases [trypsin and channel-activating protease (CAP-1)] activate ENaC by an extracellular signaling pathway (Vallet et al., 1997; Vuagniaux et al., 2000), but the molecular mechanisms of their effect has not yet been elucidated.

IV. MINERALOCORTICOID HYPERTENSION Mineralocorticoid hypertension results from excessive sodium and water retention by distal nephron segments because of a renin-independent activation of the mineralocorticoid axis, and it can occur as a sporadic condition (primary aldosteronism) or as a consequence of mutations in key elements of the adrenal–renal axis (Ferrari, 2002; Ferrari et al., 2001a; Stewart, 1999) (Fig. 5 and Table II). The resulting blood volume expansion suppresses endogenous plasma renin activity. Because sodium reabsorption in the distal nephron is coupled with enhanced tubular secretion of potassium and protons, hypokalemia and metabolic alkalosis are also common metabolic features of mineralocorticoid hypertension. Overt hypernatremia possibly related to a reset osmostat is rare (Gregoire, 1994), presumably because intravascular volume expands commensurately with sodium retention. The paradigm for this form of hypertension with sodium retention is aldosteronoma (Ganguly, 1998; Stowasser, 2001), a condition in which overproduction of aldosterone by an adrenal tumor results in excessive stimulation of the MR. At a cellular and molecular level mineralocorticoid hypertension is the consequence of overactivity of the ENaC, the gradient-driven sodium channel located in the apical membrane of the principal cells of the cortical collecting duct of the kidney (Figs. 5 and 6). This is usually the case when the MR is activated by its physiologic substrate aldosterone. The MR is a specific nuclear receptor that on binding with aldosterone enhances the expression of the apical ENaC and basolateral Na+,K+-ATPase (Horisberger and Rossier, 1992) (Fig. 6). Aldosterone is not the sole agonist of the MR, and other steroids, showing mineralocorticoid activity in vivo, are 11-deoxycorticosterone (DOC) and cortisol (Fig. 5). In mineralocorticoid target tissues the microsomal enzyme 11HSD (Funder et al., 1988) converts the biologically active 11hydroxysteroids to their inactive 11-ketosteroid forms (Fig. 3), thus protecting the nonselective MR from excess occupation by glucocorticoids. Abnormalities in steroid synthesis have long been known to cause hypertension in some cases of congenital adrenal hyperplasia. In these patients, hypertension usually accompanies a characteristic phenotype with abnormal sexual differentiation. The molecular basis of four forms of severe hypertension transmitted on an autosomal basis but without additional

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FIGURE 5. Mechanisms of mineralocorticoid hypertension. Mineralocorticoid hypertension results from overactivation of the epithelial sodium channel (ENaC), which occurs mainly when the MR is activated by its physiologic agonist aldosterone. Genetic defects in the synthesis of aldosterone (A) can lead to an overproduction of this mineralocorticoid. Moreover, other steroids can activate the MR. This is the case for the aldosterone precursor deoxycorticosterone (DOC) in some forms of congenital adrenal hyperplasia (B) and for cortisol, when a congenital defect in cortisol oxidation (C) is present. Mutation of the MR causing a constitutive activation of the receptor (D) will also result in increased activity of aldosterone-inducible proteins such as the ENaC even in the absence of aldosterone. Finally, ENaC mutations (E), which result in an increased activity of the channel in the apical membrane of the distal tubular cell, will also cause excessive renal sodium (Na+) reabsorption, volume expansion, and hypertension.

phenotypic features has been elucidated. All these conditions are characterized primarily by low plasma renin, normal or low serum potassium, and salt-sensitive hypertension, indicating an increased mineralocorticoid effect. These disorders are a consequence of either abnormal biosynthesis, metabolism, or action of steroid hormones and are ultimately characterized by an overactivation of the epithelial sodium channel in the distal renal tubule causing sodium retention and salt-sensitive hypertension.

V. PRIMARY ALDOSTERONISM The term primary aldosteronism (PA) is used to describe a heterogeneous group of conditions characterized by an overproduction of aldosterone by the zona glomerulosa of the adrenal gland (Table II). Aldosterone secretion

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TABLE II. Sporadic and Inherited Forms of Mineralocorticoid Hypertension Cause

Primary aldosteronism Aldosterone-producing adenoma Bilateral idiopathic hyperplasia Adrenal carcinoma Deoxycorticosterone-secreting adrenal tumor

Mineralocorticoid Sporadic Aldosterone

Deoxycorticosterone

Genetic abnormalitya

—

—

Inherited Congenital adrenal hyperplasia 11-Hydroxylase deficiency 17-Hydroxylase deficiency Familial hyperaldosteronism type I Glucocorticoid-remediable aldosteronism 11-Hydroxysteroid dehydrogenase type 2 deficiency Apparent mineralocorticoid excess Mineralocorticoid receptor mutations Activating mutation of the mineralocorticoid receptor Liddle syndrome subunit of ENaC subunit of ENaC

Deoxycorticosterone CYP11B1 CYP17 Aldosterone Chimeric (CYP11B1/CYP11B2) Cortisol HSD11B2 Progesterone MR None SCNN1B SCNN1G

a CYP11B1, Cytochrome P450, subfamily XIB, polypeptide 1 (11-hydroxylase); CYP11B2, cytochrome P450, subfamily XIB, polypeptide 2 (aldosterone synthase); CYP17, cytochrome P450, subfamily XVII (17-hydroxylase); HSD11B2, 11-hydroxysteroid dehydrogenase type 2; MR, mineralocorticoid receptor; SCNN1B/G, sodium channel, nonvoltage gated 1, / subunit.

in primary aldosteronism is partially autonomous, and the plasma renin level is low. Known causes of PA are adrenocortical adenoma, bilateral micronodular or macronodular adrenal hyperplasia (idiopathic aldosteronism), unilateral adrenal hyperplasia, adrenal carcinoma, or a genetic form, called glucocorticoid-remediable aldosteronism (Table II). The latter, also known as familial hyperaldosteronism type I, is discussed in detail in Section VI. Moreover, a few cases of extraadrenal aldosterone-producing tumor (Abdelhamid et al., 1996) and some cases of familial hyperaldosteronism (type II) associated with aldosteronoma or hyperplasia have also been described (Stowasser and Gordon, 2001). The most common cause of PA is adrenocortical adenoma (aldosteronoma), a disorder first reported by J. Conn (1955). Aldosteronomas are usually small (<2 cm in diameter), are benign by definition, and represent one of a few potentially curable forms of hypertension. There are at least

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FIGURE 6. Mineralocorticoid action in renal cells of the distal tubule and cortical collecting duct. When aldosterone enters the cell it binds to the mineralocorticoid receptor (MR), and thereafter the ligand–receptor complex is translocated into the nucleus. Binding to its hormone response element (HRE) increases the transcription of genes encoding specific aldosteroneinducible proteins, such as the subunits of the apical epithelial sodium channel (ENaC). In turn, this stimulates sodium (Na+) reabsorption and potassium (K+) excretion. Moreover, the activity of the ENaC is also regulated by Nedd4-mediated channel internalization. The MR is protected by the 11HSD2 from occupation by glucocorticoids.

two functionally and perhaps histologically different types of aldosteronoma: a corticotropin-responsive (and renin-unresponsive) type and a reninresponsive type. In most cases of aldosteronoma, aldosterone secretion cannot be suppressed by volume expansion or increased sodium intake (sodium loading), it appears to be unresponsive to angiotensin II, and is strongly influenced by corticotropin (Espiner and Donald, 1980). This is evident by the abnormal aldosterone response on postural testing, the parallel circadian rhythms of aldosterone and cortisol, and the transient decrease in the plasma aldosterone concentration in response to glucocorticoids such as dexamethasone (Ganguly et al., 1977). However, approximately 20% of patients with aldosteronomas are responsive to small

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increases in the plasma level of angiotensin II and have a normal plasma aldosterone response on postural testing (Espiner and Donald, 1980; Irony et al., 1990; Wisgerhof et al., 1981). Idiopathic aldosteronism, which is characterized by bilateral micronodular or macronodular adrenal hyperplasia, constitutes 20 to 30% of cases of primary aldosteronism (Biglieri et al., 1984; Irony et al., 1990; Jeck et al., 1994; Weinberger et al., 1979), although it is considered by some to be a variant of essential hypertension (Lim et al., 2002). Patients with idiopathic aldosteronism are responsive to small increases in circulating angiotensin II and have a normal plasma aldosterone response on postural testing (Mantero et al., 1976; Wisgerhof et al., 1981). Unilateral adrenal hyperplasia has also been reported (Magill et al., 2001; Morioka et al., 2000). Adrenal carcinomas are usually larger than the more common, benign aldosteronomas; they often, but not invariably, produce other adrenal hormones, and may show evidence of local invasion or distant metastasis (Isles et al., 1987; Sasano et al., 1993). A. PREVALENCE

The prevalence of primary aldosteronism in patients with hypertension has not been systematically assessed. In earlier reports, when aldosteronism was investigated only in the presence of severe hypokalemia and not by means of renin and aldosterone measurements, the prevalence of PA was <1% in unselected hypertensive patients (Bech and Hilden, 1975; Berglund et al., 1976; Danielson and Dammstrom, 1981; Lund et al., 1981; Streeten et al., 1979; Tucker and Labarthe, 1977), although Lund et al. (1981) found that 9.3% of patients with hypokalemia had aldosterone-producing adenomas. The introduction of the plasma aldosterone-to-renin activity ratio (ARR) test by Hiramatsu and co-workers (1981) made it possible to include normokalemic, and not just hypokalemic, hypertensive subjects in a screening for PA. This has led independent groups from several countries to report marked increases in detection rate and to estimate the prevalence of PA to be possibly 10-fold or more higher than was previously assumed (Brown et al., 1996; Fardella et al., 2000; Gallay et al., 2001; Gordon et al., 1993, 1994; Hiramatsu et al., 1981; Lim et al., 1999b, 2000; Lins and Adamson, 1986; Loh et al., 2000; Nishikawa and Omura, 2000; Rayner et al., 2000, 2001) (Table III). Depending on the cutoff for the ARR and on whether unselected hypertensive patients, patients with resistant hypertension or requiring more than two antihypertensives for blood pressure control, or hypertensive patients with or without hypokalemia are considered the prevalence of PA ranges between 5 and 15% (Table III). Nevertheless, it should be noted that the prevalence of aldosteronomas, one of the few forms of surgically curable PA, is only approximately 3% in the reported studies (Table III). Moreover, the prevalence of hypertension in

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TABLE III. Prevalence of Primary Aldosteronism Reported by Screening Studies Performed since Introduction of the Aldosterone-to-Renin Ratio Testa Ref.

Region

Hiramatsu et al. (1981) Lins and Adamson (1986) Gordon et al. (1993) Gordon et al. (1994) Brown et al. (1996) Lim et al. (1999b)

Asia

Loh et al. (2000) Nishikawa and Omura (2000) Fardella et al. (2000) Lim et al. (2000) Rayner et al. (2000) Gallay et al. (2001)

Asia Asia

Rayner et al. (2001)

Europe Australia Australia Australia Europe

South America Europe Africa North America Africa

Number of patients

K 3.5b (%)

ARRc

Prevalence Number of PA (%) of APAs

348

91

>2080

7.4

32

0

>760

50

12

52 199 74 495d 135e 350 1020

100 100 100 NA NA 97 72

>690 >830 >830 >750 >550 >400

12 8.5 6.7 16.6 14.4 4.6 5.4

6 5 2 NA NA 6 45

305

100

>690

5.2

1 5 7 10

465 216 90

95 71 NA

>750 >1000 >2700

9.2 32 17

154

NA

>1000

7.1

9

NA

Abbreviations: APA, Aldosterone-producing adenoma; ARR, aldosterone-to-renin ratio; NA, not available; PA, primary aldosteronism. a Introduced by Hiramatsu et al. (1981) b K3.5, percentage of patients with serum potassium 3.5 mmol/liter. c The ARR is indicated with equivalent figures when converted in (pmol/liter) per (ng/ml per h). d Patient characteristics: Referred. e Patient characteristics: Family practice.

patients with incidentally discovered adrenocortical adenoma (incidentaloma) is higher than in an age-matched control population (Bernini et al., 2002; Mantero et al., 2000; Russell et al., 1972). Using the ARR to identify PA in normokalemic patients with adrenal incidentalomas, Bernini et al. (2002) were able to identify 5.6% of subjects as having aldosteroneproducing adenoma. B. CLINICAL AND LABORATORY FINDINGS

Aldosteronomas are rarely found in children (Rogoff et al., 2001). Clinical features of PA are not specific, some patients are completely asymptomatic or have nonspecific symptoms related to hypertension. Others have symptoms-related hypokalemia, such as muscle cramps or weakness, but only rarely paresthesia, or paralysis (Cain et al., 1972;

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Weinberger et al., 1979; Young et al., 1990). Blood pressure can exhibit moderate or marked elevation, and is often resistant to therapy. A few patients have normal blood pressure (Matsunaga et al., 1983; Stowasser et al., 1999). Retinopathy is almost invariably mild, and exudates or hemorrhages are uncommon. As is the case for other forms of mineralocorticoid hypertension, peripheral edema is uncommon, although hypertension in PA is primarily a consequence of renal sodium and fluid retention. In classic PA, spontaneous hypokalemia with metabolic alkalosis and a serum sodium level at the high end of the normal range are often observed. Hypokalemia can be accentuated or induced in a subject with a normal level of serum potassium by oral sodium loading. An increased urinary excretion of potassium (>30 mmol/day in the presence of hypokalemia) is highly suggestive of classic PA. Thus, routine laboratory data can be suggestive but not diagnostic of classic primary aldosteronism. The use of the ARR as a screening test has made PA due to unilateral aldosteronoma and also that due to bilateral idiopathic aldosteronism increasingly diagnosed (Gordon, 1994). When screening with the ARR is performed, a high incidence of PA with normokalemia is found (Brown et al., 1996; Fardella et al., 2000; Gordon et al., 1994; Hiramatsu et al., 1981; Lim et al., 2000; Loh et al., 2000; Rayner et al., 2000) (Table III). Hypokalemia tends to be more severe in patients with aldosteronoma and less severe, or absent, in patients with idiopathic aldosteronism. Hypomagnesemia or abnormal glucose tolerance can be present. Also, parathyroid hypersecretion is a common feature of PA and seems to be a consequence of increased steroid-mediated distal tubular calcium excretion (Ferrari et al., 2002; Resnick and Laragh, 1985; Rossi et al., 1995).

C. SCREENING

The sensitivity of serum potassium measurements for the screening of PA is poor, although spontaneous hypokalemia in a patient with hypertension is a strong indicator that classic PA is present. Most patients with PA have normal serum potassium levels (Table III), whereas other hypertensive patients may have hypokalemia associated with other forms of mineralocorticoid excess, or as a result of diuretic therapy or secondary aldosteronism. On the other hand, the prevalence of PA in hypertensive patients with severe hypokalemia was reported to be as high as 50% (Lins and Adamson, 1986). Plasma renin activity is suppressed in almost all patients with untreated PA. However, many patients with essential hypertension may present with low-renin, high-aldosterone hypertension (Brunner et al., 1972), although plasma renin levels are sensitive to changes in sodium intake and the intake of various medications in those patients. Thus, neither measurements of serum potassium nor measurements of plasma renin are suitable or reliable methods of screening for PA.

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FIGURE 7. Proposed screening and diagnostic work-up for mineralocorticoid hypertension and primary aldosteronism. In all hypertensive patients with an increased plasma aldosterone-torenin ratio (ARR), radiologic imaging by CT scan to search for an adrenal mass should be performed. If aldosteronoma fails to be demonstrated, a fludrocortisone suppression test (FST) should confirm nonsuppressible aldosteronism. In this case, when absent family history does not suggest glucocorticoid-remediable aldosteronism (GRA), adrenal venous sampling (AVS) is advisable. Aldo, Aldosterone; Lateral, lateralized; Spiro, spironolactone; HTN, hypertension.

Determining the ARR in patients with untreated hypertension seems to be the most appropriate screening method for distinguishing patients with PA from those with essential hypertension (Blumenfeld et al., 1994; Fardella et al., 2000; Gordon et al., 1994; Lim et al., 2000) (Fig. 7 and Table IV). In the presence of severe or symptomatic hypertension, patients should take only antihypertensive medications that are least likely to affect measurements of

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TABLE IV. Recommended Cutoff Values for Aldosterone-to-Renin Ratio in Primary Aldosteronism according to Methods and Units Used Plasma renin Activity

Plasma aldosterone ng/dl pmol/liter

Immunoreactive

ng/ml per h

pmol/liter per min

mU/liter

ng/liter

>27 >750

>2.1 >59

>3.3 >89

>5.2 >145

Patients with ARR 750 (pmol/liter) per (ng/ml per h) have >90% probability of having nonsuppressible plasma aldosterone with FST (Lim et al., 2000).

renin and aldosterone, such as -blockers or calcium channel blockers (Barbieri et al., 1981; Carpene et al., 1989). Accuracy of diagnosis of PA can be increased by the administration of a single dose of the angiotensinconverting enzyme inhibitor captopril, followed by the measurement of the ARR (Castro et al., 2002). Some authors suggest that given the low prevalence of PA, routine measurement of plasma aldosterone and renin to screen for the condition in persons with hypertension would not be costeffective and should be reserved for patients with unexplained hypokalemia, with resistant hypertension or requiring more than two antihypertensives for blood pressure control (Kaplan, 2001). However, there are two reasons for a more liberal approach to screening, with application of the ARR to all patients with hypertension. The first is that even mildly hypertensive individuals deserve at least one chance at a cure. The second is that measurements of the ARR are also valuable if PA fails to be demonstrated, because a raised ARR indicates inappropriate aldosterone activity. Lim et al. (1999a) demonstrated that a vast majority of subjects with increased ARR failed to suppress plasma aldosterone on salt loading and showed a marked response to spironolactone treatment. D. FURTHER EVALUATION AND DIAGNOSIS

All patients with hypertension who have an increased ARR should receive further evaluation (Fig. 7). Clearly, patients with hypertension who have spontaneous or profound diuretic-induced hypokalemia and patients with adrenal incidentalomas (Bernini et al., 2002; Kievit and Haak, 2000) or with resistant hypertension are those who most need further evaluation. Inhibiting and stimulating aldosterone and renin secretion by physiologic or pharmacologic interventions including sodium loading and depletion or by using the MR agonist fludrocortisone can provide the definitive biochemical diagnosis of aldosteronism. Using sodium loading following findings

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establishes the diagnosis of PA: (1) a high plasma aldosterone level after intravenous infusion of normal saline (1.25 liters over a 2-h period in the morning), or (2) a high rate of urinary aldosterone excretion while on a diet high in sodium chloride (6 to 9 g/day for 3 days) (Bravo, 1994; Irony et al., 1990; Weinberger et al., 1979). A plasma aldosterone level of <250 pmol/ liter (<8.5 ng/dl) at the end of saline infusion or a urinary aldosterone excretion of <14 g/24 h after sodium loading rule out PA. Sodium depletion by furosemide challenge has a poor predictive value of 42% in patients prescreened with the ARR (Lim et al., 2000) and is therefore not helpful. Some authors have demonstrated poor sensitivity with saline infusion testing (Gordon et al., 1993; Holland et al., 1984) and therefore suggest that PA should be confirmed by the fludrocortisone suppression test (FST) (Fardella et al., 2000; Gordon, 1995b; Gordon et al., 1993). With the FST the diagnosis of PA is based on failure of aldosterone to be suppressed to <180 pmol/liter (<6 ng/dl) after 4 days of fludrocortisone (0.1 mg every 6 h) (Gordon, 1995b). The major drawback of this test is that it must be carried out with the patient in hospital for 4 days under a strict regimen of sodium intake and potassium substitution. E. SUBTYPE DELINEATION

Once the biochemical diagnosis of aldosteronism has been established, the cause can be determined by a variety of tests and techniques. Subtype delineation is critical to decide on the optimal treatment. Discriminant analyses with using plasma aldosterone and potassium levels have been advocated. A postural test in which the plasma aldosterone level fails to increase in a patient who has maintained an upright posture in the morning after recumbency strongly suggests the presence of an aldosteronoma, whereas subjects with idiopathic aldosteronism almost invariably have a normal increment in plasma aldosterone (Ganguly et al., 1973). However, some patients with unilateral adrenal hyperplasia or primary adrenal hyperplasia may have similar postural responses as patients with aldosteronoma (Espiner and Donald, 1980; Irony et al., 1990; Mantero et al., 1976). Likewise, about 20% of patients with aldosteronoma are responsive to angiotensin II and therefore have a true increase in plasma aldosterone when undergoing this test (Feltynowski et al., 1994; Gordon, 1994; Irony et al., 1990). Therefore, none of these tests is sufficiently specific to warrant appropriate discrimination of the cause of PA. Localization of the abnormal adrenal gland should be undertaken by radiological imaging techniques after the biochemical basis of the aldosteronism has been established. However, because patients with ARR 750 have a >90% probability of having nonsuppressible plasma aldosterone with FST (Lim et al., 2000), radiological imaging to search for adrenal mass in all patients with increased ARR seems to be a reasonable approach, particularly in view of the complexity and costs

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of FST (Gordon et al., 1993, 1994) (Fig. 7). Adrenal computed tomographic (CT) imaging can detect most (Fig. 7), but not all, aldosteronomas, because a unilateral excess of aldosterone secretion in the absence of adenoma or hyperplasia may be caused by an adrenal microadenoma not detectable by radiologic imaging (Omura et al., 2002). Conversely, the appearance of a nonfunctional incidentally discovered adrenocortical adenoma on adrenal CT imaging can occasionally cause confusion regarding the diagnosis of idiopathic hyperaldosteronism or aldosteronoma (Doppman et al., 1992; Fallo et al., 1997). In patients with idiopathic aldosteronism, the adrenal glands appear either bilaterally enlarged or normal in size. In a few of these patients, however, one of the adrenal glands may have a nodule, whereas patients with unilateral aldosteronomas may have bilateral nodules (Doppman et al., 1992; Radin et al., 1992). The finding of a large adrenal tumor (>3 cm in diameter) should raise the possibility of an adrenal carcinoma (Mantero et al., 2000), in which case other adrenal steroids (androgens, cortisol, and estrogen) in the plasma or urine should be measured (Isles et al., 1987; Sasano et al., 1993). Experience with magnetic resonance (MR) imaging is promising, particularly because MR imaging is a reliable method in characterization of benign and malignant adrenal masses (Honigschnabl et al., 2002; Slapa et al., 2000). CT scanning, however, remains preferable because of its reliability and lower cost. Adrenal venous sampling (AVS) for measurement of aldosterone and cortisol is invasive but is the most reliable method (Gordon et al., 1994; Magill et al., 2001; Radin et al., 1992; Young et al., 1996) (Fig. 7). This procedure requires considerable skill and experience on the part of the radiologist and carries some risk of adrenal hemorrhage. Measurement of cortisol as well as aldosterone in samples from both adrenal veins and from the inferior vena cava are crucial for evaluating the accuracy of AVS. It was reported that corticotropin stimulation may increase the specificity of AVS (Weinberger et al., 1979). If the blood sampling is reliable, a unilateral excess of aldosterone secretion usually suggests the presence of an aldosteronoma, although in some cases the diagnosis may be unilateral adrenal hyperplasia. If the radiologic diagnosis is difficult or if AVS cannot be performed, scintigraphic localization of adrenal lesions with radiolabeled iodocholesterol or [131I] iodomethyl-19-norcholesterol can be helpful (Gross et al., 1984; Kazerooni et al., 1990). The uptake of tracer is increased in patients with aldosteronoma and absent in those with idiopathic aldosteronism and usually also in those with adrenal carcinoma. Nevertheless, adrenal scintigraphy without adrenal vein sampling may lead to serious errors in patient management (Mansoor et al., 2002). F. THERAPY

In the presence of an aldosteronoma the best treatment option is removal of the adrenal tumor, which has been found to improve blood pressure

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control or cure hypertension in the majority of patients (Jeck et al., 1994; Weinberger et al., 1979; Young et al., 1990) (Fig. 7). Spironolactone therapy before surgery can be a predictor of surgical outcome in patients with aldosteronoma, may restore potassium levels in the body to normal before surgery, and may minimize postoperative hypoaldosteronism (Spark and Melby, 1968). If blood pressure fails to normalize after adrenalectomy, idiopathic aldosteronism with micronodular hyperplasia should be considered (McLeod et al., 1989). Laparoscopic adrenalectomy is now widely and successfully used to resect aldosteronomas and other adrenal masses, including adrenal carcinomas (Henry et al., 1999; Rossi et al., 2002; Schell et al., 1999). The advantages of the laparoscopic technique are evident both in terms of safety (fewer complications) (Brunt, 2002) and costs (shorter hospitalization and recovery periods) (Schell et al., 1999). In idiopathic aldosteronism, unilateral or bilateral adrenalectomy does not usually achieve satisfactory blood pressure control and, therefore, medical treatment is the option of choice (Fig. 7). In patients with unilateral adrenal hyperplasia surgical treatment may improve hypertension, although a cure is unlikely. The most obvious drug strategy is to antagonize aldosterone at the receptor level, using spironolactone (Kremer et al., 1973; Lim et al., 2001; Young et al., 1990) or eplerenone (Delyani et al., 2001), an investigational selective aldosterone receptor antagonist that has less antiandrogenic and antiprogestational effects than spironolactone. Dosage of spironolactone to provide an effective control blood pressure and hypokalemia varies from 25 to 400 mg/day (Kremer et al., 1973; Lim et al., 2001; Young et al., 1990) and, because of the competitive nature of the compound on the receptor (Fanestil, 1968), high doses (>100 mg/day) are often needed. This is often associated with considerable side effects, especially gastrointestinal symptoms, fatigue, impotence, and gynecomastia. Other potassium-sparing agents, including amiloride, have been tried but are not as effective as spironolactone (Kremer et al., 1973; Lim et al., 2001). Spironolactone can be used in combination with other antihypertensive agents, such as calcium channel blockers or angiotensin-converting enzyme inhibitors (Carpene et al., 1989; Lim et al., 2001; Young et al., 1990).

VI. GENETIC FORMS OF MINERALOCORTICOID HYPERTENSION A. MUTATIONS OF THE 11b-HYDROXYLASE OR 17a-HYDROXYLASE GENE: CONGENITAL ADRENAL HYPERPLASIA

Several autosomal recessive disorders can cause congenital adrenal hyperplasia (CAH). The most common type, 21-hydroxylase deficiency, responsible for nearly 90% of all CAH, is not associated with hypertension

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(White and Speiser, 2000), but precursors proximal to the enzyme block accumulate and are shunted into adrenal androgens. The clinical manifestation of CAH, often obvious at birth, varies with the degree of enzymatic deficiency and the mix of steroids secreted by the adrenal glands. When the enzyme block causes androgens to accumulate, the disorder is a virilizing form of CAH, causing varying degrees of virilization of an affected female fetus. If the enzyme block impairs androgen synthesis, it is an undervirilizing form, causing inadequate virilization of an affected male fetus. The two forms of CAH associated with hypertension are 11-hydroxylase deficiency, wherein 11-DOC is present in excess along with adrenal androgens, and 17-hydroxylase deficiency, which also has an excess of DOC but a deficiency of androgen production (Table II). Although these are rare causes of hypertension, partial enzymatic deficiencies have been observed in hirsute women (Lucky et al., 1986), so some hypertensive adolescents may have unrecognized, subtle forms of CAH. An 11-hydroxylase deficiency causes 3 to 5% of all cases of CAH. The condition is usually diagnosed in infancy, because the defect sets off production of excessive androgens, although clinical variability is high (Rosler et al., 1982). The enzyme deficiency prevents the hydroxylation of 11-deoxycortisol to cortisol, resulting in cortisol deficiency (Fig. 2 and Table II). The defect also prevents the conversion of DOC to corticosterone and aldosterone. The characteristic steroid profile is elevation of urinary 17-hydroxycorticosteroids and of DOC (Levine et al., 1980). Because of the mineralocorticoid activity of DOC, patients exhibit salt retention and hypertension with hypokalemic alkalosis. Plasma renin activity is low and virilization also occurs. The enzymatic defect has been attributed to several mutations in the CYP11B1 gene (Geley et al., 1996; White et al., 1991). The syndrome is diagnosed by finding high levels of 11deoxycortisol and DOC in the urine and plasma (Zachmann et al., 1983). The treatment is cortisol replacement; mineralocorticoid replacement may also be necessary. The enzyme that catalyzes the 11-hydroxylation of 11-deoxycortisol to form cortisol is a cytochrome P450 protein encoded by the CYP11B1 gene (Fig. 2). The CYP11B1 gene was cloned by Mornet et al. (1989), who found that the gene is 6.5 kb long from the start of transcription to the polyadenylation site and contains nine exons. Thereafter, several mutations in this gene have been reported in many families throughout the world. Phenotypic expression of these mutations occurs in the homozygous or compound heterozygous state owing to the loss of function of the mutant protein. This distinguishes CAH due to CYP11B1 mutations from glucocorticoid-remediable aldosteronism (GRA), a condition expressed in the heterozygous state, in which a chimeric gene encodes a fused P450 protein consisting of the amino-terminal portion (exons 1–4) of CYP11B1

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and the carboxyl-terminal part (exons 5–9) of CYP11B2 (Lifton et al., 1992a; Miyahara et al., 1992). In GRA the chimeric gene results in a gain of function and is discussed separately (Section VI.B). Joehrer et al. (1997) described a female patient with partial steroid 11hydroxylase deficiency, found to have a compound heterozygosity of the CYP11B1 gene for two missense mutations: Asn-133 to histidine (N133H) and Thr-319 to methionine (T319M). In an analysis of DNA from nine patients with severe manifestations of CAH due to 11-hydroxylase deficiency, Geley et al. (1996) identified seven mutations in the CYP11B1 gene. Curnow et al. (1993) reported eight previously uncharacterized mutations in the CYP11B1 gene causing a hypertensive form of congenital adrenal hyperplasia. They pointed out that 7 of a total of 10 known mutations are clustered in exons 6–8. A 17-hydroxylase deficiency is associated with an absence of sex hormones, leading to incomplete masculinization in males and primary amenorrhea in females (Fig. 2 and Table II). 17-Hydroxylase is necessary for both cortisol and estrogen synthesis. Lack of these hormones results in increases in ACTH and follicle-stimulating hormone (FSH). Production of excessive corticosterone and DOC results in hypertension and hypokalemic alkalosis. Aldosterone synthesis is almost totally absent. Estrogen deficiency results in primary amenorrhea and absent sexual maturation. To date approximately 150 cases of 17-hydroxylase deficiency have been recognized (Hermans et al., 1996; Yanase et al., 1991). However, adolescents with hypertension and hypokalemia or abnormal sexual development should be considered suspect. P45017 is a single enzyme that mediates both 17-hydroxylase and 17, 20-lyase activity; it catalyzes 17-hydroxylation of both pregnenolone and progesterone and 17,20-lysis of 17-hydroxypregnenolone and 17-hydroxyprogesterone. The gene CYP17, which encodes this enzyme, is the sole member of a unique gene family within the P450 supergene family (Chung et al., 1987). Phenotypic expression may be variable depending on the degree of residual activity of mutant enzymes. In some cases hypertension is the main feature, while the signs of abnormal sexual development or differentiation may be discrete (Table II). In some cases patients with a 46,XY karyotype are phenotypic females (Jones et al., 1992). A partial combined 17-hydroxylase/17,20-lyase deficiency identified at the age of 20 years in a female Japanese patient was diagnosed because of hypertension and hypokalemia (Yanase et al., 1989). Menstruation was irregular, the breasts were hypoplastic, and pubic or axillary hair was absent (Yanase et al., 1989). Oshiro et al. (1995) also described a mutation in the CYP17 gene in another adult female referred because of hypertension and amenorrhea.

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B. CHIMERIC 11b-HYDROXLASE–ALDOSTERONE SYNTHASE GENE: GLUCOCORTICOID-REMEDIABLE ALDOSTERONISM

Sutherland et al. (1966) and Salti et al. (1969) described a father and son with hypertension, low plasma renin activity, increased aldosterone secretion responsive to dexamethasone, and normal growth and sexual development. Because this form of aldosteronism was corrected by dexamethasone the condition was termed glucocorticoid-remediable aldosteronism (GRA). The hypertension, variable hyperaldosteronism, and abnormal steroid production are all under the control of ACTH and suppressible by glucocorticoids. In GRA there are high levels of the abnormal adrenal steroids 18-oxocortisol and 18-hydroxycortisol, and plasma aldosterone levels may be variable (Gomez-Sanchez et al., 1984; Jamieson et al., 1996; Lifton et al., 1992b; Mulatero et al., 1998). GRA is the result of aldosterone synthase (CYP11B2) activity under the control of ACTH (which normally regulates CYP11B1) and results from an unequal crossing-over involving the CYP11B1 and CYP11B2 genes (Table II). Aldosterone synthase, like steroid 11-hydroxylase, is expressed in both adrenal fasciculata and glomerulosa; the two genes are 95% identical and lie on chromosome 8q immediately adjacent in a head-to-tail orientation with the CYP11B2 gene 50 to the CYP11B1 gene (Mornet et al., 1989). A chimeric gene duplication between the CYP11B1 and CYP11B2 genes is the cause of GRA (Jonsson et al., 1995; Lifton et al., 1992a,b; Pascoe et al., 1992). This chimeric gene encodes aldosterone synthase (functional elements of CYP11B2) but is under the control of ACTH (regulatory elements of CYP11B1). The number of reported cases with ‘‘classic GRA’’ is small; however, this condition might be underdiagnosed. A report on 21 affected members of approximately 1000 descendants of an English convict in Australia (Gordon, 1995a) revealed an extreme phenotypic heterogeneity in GRA, associated with hybrid genes showing somewhat different cross-over points linking the CYP11B1 and CYP11B2 portions. The affected members were often normokalemic, and some remained normotensive until late in life (Gordon, 1995a). In normotensive subjects, biochemical abnormalities are similar to those of hypertensive siblings (Stowasser et al., 1999). Gates et al. (1996) described two large pedigrees with many subjects who had the abnormal chimeric gene associated with glucocorticoid-remediable aldosteronism. Most of the affected members, who had only mild hypertension and normal biochemistry, were clinically indistinguishable from patients with essential hypertension. Thus, for young hypertensive patients with positive family history, and absent postural increase in plasma aldosterone, treatment with glucocorticoid should be given for 4 to 6 weeks. Abnormal activity of CYP11B2 may be characterized phenotypically by elevated

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urinary excretion of tetrahydroaldosterone and aldosterone (Davies et al., 1999) or by a decline in plasma aldosterone in response to dexamethasone (Litchfield et al., 1997; Mulatero et al., 1998). Preferably, gas chromatography– mass spectrometry analysis of the urine with 18-hydroxycortisol assay should be performed whenever available.

C. MUTATIONS OF THE 11b-HYDROXYSTEROID DEHYDROGENASE TYPE 2 GENE: APPARENT MINERALOCORTICOID EXCESS

In 1974 Werder et al. (1974) described a disorder in the peripheral metabolism of cortisol, manifested by hypertension, hypokalemia, low plasma renin activity, and subnormal aldosterone levels. Although the features suggested primary mineralocorticoid excess, no overproduction of mineralocorticoids could be demonstrated. Biochemical characterization of this condition was provided by New et al. (1977) and Ulick et al. (1979) who found a decreased rate of conversion of cortisol to cortisone in two affected subjects, reflecting a deficiency of an 11HSD enzyme (Fig. 3). This form of mineralocorticoid hypertension was called apparent mineralocorticoid excess (AME). It is an autosomal recessive disorder that results from overactivation of the MR by cortisol (Ulick et al., 1979). Symptoms of the disease respond to spironolactone or amiloride administration or a lowsodium diet. AME is caused by mutations in the 11HSD type 2 enzyme (Table II). The molecular basis of the syndrome of AME has been elucidated (Ferrari et al. 1996a,b; Mune et al., 1995; Obeyesekere et al., 1995; Rogoff et al., 1998; Stewart et al., 1996; Wilson et al., 1995a,b, 1998). Mutations in the 11HSD2 gene result in an enzyme with abolished or markedly decreased activity, which causes renal sodium retention, urinary potassium wasting, and low-renin, low-aldosterone hypertension. So far, 50 patients with ‘‘classic AME’’ in 25 kindreds have had DNA analysis, revealing a total of 20 different mutations in the 11HSD2 gene (Ferrari and Krozowski, 2000; Wilson et al., 1998). We reported on a form of lowrenin hypertension in which a gene mutation produces a mild deficiency in the 11HSD2 enzyme but without other phenotypic features that could lead to the diagnosis of AME (Wilson et al., 1998). Along with other findings (Lovati et al., 1999; Soro et al., 1995; Walker et al., 1993), these data suggest that impaired 11HSD2 activity may play a role in the pathogenesis of essential hypertension in some patients and that this may be genetically determined (Ferrari et al., 2000). The prevalence of mutations in the coding region of the 11HSD2 gene in the general population of patients with essential hypertension is presently unknown, but it has been estimated as <1/250,000 among white individuals (Zaehner et al., 2000).

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D. MUTATIONS OF THE MINERALOCORTICOID RECEPTOR GENE

In a screening for mutation of all coding regions of the MR among 75 independent patients with severe hypertension, suppressed plasma renin activity, low aldosterone, and no other underlying cause of hypertension a 15-year-old boy was found to be heterozygous for a missense mutation, resulting in substitution of a leucine for serine at codon 810 (S810L) (Geller et al., 2000) (Table II). The S810L mutation lies in the MR hormonebinding domain, altering an amino acid that is conserved in all MRs from Xenopus to human but not found in other nuclear receptors. This mutation results in constitutive MR activity and alters receptor specificity, with progesterone and other steroids lacking 21-hydroxyl groups, normally MR antagonists, becoming potent agonists. Spironolactone was also a potent agonist of MR-L810, suggesting that this medication is contraindicated in MR-L810 carriers (Geller et al., 2000). Among the 23 relatives of the index patient analyzed, 11 had been diagnosed with severe hypertension before age 20 years, a rare trait in the general population, whereas the remaining 12 had unremarkable blood pressures. Carriers of the mutant allele revealed a marked increase in blood pressure, suppression of aldosterone secretion, and a nonsignificant trend toward lower serum potassium levels (Geller et al., 2000). Two females later found to be MR-L810 carriers had previously undergone five pregnancies. Because progesterone levels normally increase 100-fold in pregnancy it was not surprising to notice that all pregnancies had been complicated by marked exacerbation of hypertension. To date no further cases of activating mutations of the MR have been reported.

E. MUTATIONS OF THE EPITHELIAL SODIUM CHANNEL GENES: LIDDLE SYNDROME

In the early 1960s Liddle et al. (1963) described a young female with hypertension associated with hypokalemic alkalosis not due to hyperaldosteronism but rather to a renal tubular defect. Renal failure eventually developed in this patient, who received a cadaveric renal transplant in 1989, following which her disorder resolved with normalization of the aldosterone and renin responses to salt restriction (Botero-Velez et al., 1994). This condition, later called Liddle syndrome or pseudoaldosteronism, is characterized by hypoaldosteronism, hypokalemia, and decreased renin and angiotensin (Table II). Further studies demonstrated that amiloride and triamterene, but not spironolactone, were effective treatments for hypertension and hypokalemia in patients with this syndrome as long as dietary sodium intake was restricted (Wang et al., 1981). This form of mineralocorticoid hypertension is inherited as an autosomal dominant trait (Warnock, 1998).

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The cloning of ENaC led to the discovery that this hereditary monogenic form of hypertension was caused by mutations deleting the PY motif present in the C terminus of the or subunits of ENaC (Hansson et al., 1995a,b; Shimkets et al., 1994). Shimkets et al. demonstrated complete linkage of the disorder in the index patient described by Liddle to the gene encoding the subunit of ENaC (Shimkets et al., 1994). Implication of ENaC in tight regulation of salt homeostasis, control of extracellular volume, and blood pressure has opened a new field of investigation and pointed out ENaC and all its regulating factors as candidate proteins potentially involved in salt sensitivity and salt resistance (Rossier et al., 2002). ENaC, expressed on the apical side of the cells from the distal tubule and cortical collecting duct, is the key modulator of sodium transport in the kidney (Canessa et al., 1994a; Shimkets et al., 1994) (Figs. 5 and 6). Expression and function of this transporter are under the control of aldosterone (Horisberger and Rossier, 1992; Palmer and Frindt, 1992). In Liddle syndrome the channel is hyperactive, because of two factors: an increased number of channels present at the cell surface and an increased intrinsic activity of ENaC. Staub et al. (1996) demonstrated that the so-called PY motif is the target of Nedd4, a ubiquitin-protein ligase, which binds to the PY motif through its WW domains (Fig. 6). The binding allows the ubiquitination of ENaC and its degradation. In Liddle syndrome, this interaction between Nedd4 and the PY motif of the and subunits is no longer possible and this leads to a higher number of hyperactive channels at the cell surface (Staub et al., 2000) so that sodium can freely diffuse from the tubular lumen into the cell and is then extruded into the interstitium by the Na+, K+-ATPase. Sodium and water retention leads to an increase in blood volume, hypertension, suppression of plasma renin, and low aldosterone in plasma and urine (Warnock, 1998). The number of patients with ‘‘classic’’ Liddle syndrome is extremely low. However, evidence of a relevant mutation in the subunit of ENaC (T594M) was demonstrated in black ‘‘essential’’ hypertensive subjects (Baker et al., 1998), and a variant in the promoter region of the subunit was described in Japanese hypertensive subjects (Iwai et al., 2002), indicating that mutations of ENaC might be a frequent cause of secondary hypertension. The lack of association between molecular variants of either the or subunit of ENaC and hypertension in unselected hypertensive patients (Chang and Fujita, 1996; Fodinger et al., 1998) suggests that careful patient selection based on phenotypical characteristics, such as plasma renin levels or salt sensitivity, may be crucial in order to increase the probability to detect such mutations. For instance, hypertensive patients with a marked response to a therapy with amiloride but unresponsive to spironolactone represent an ideal target; however, these subjects have not been systematically investigated so far and therefore represent a candidate group for genetic analysis.

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VII. ALDOSTERONE-DEPENDENT ESSENTIAL HYPERTENSION It has been estimated that approximately one-third of the hypertensive population have low renin levels, with a higher proportion of low renin in black than in white subjects (Brunner et al., 1972). Moreover, up to 15% of hypertensive subjects have a raised ARR, and in most of these subjects plasma aldosterone is only partially suppressible on salt loading, the current diagnostic criterion for primary aldosteronism (Coghlan et al., 1972; Gordon, 1995b; Luetscher et al., 1969). As discussed previously, renin– angiotensin system control is intact in patients with idiopathic aldosteronism. The degree of integrity of this feedback regulation, although differing in the degree of sensitivity, is similar to that observed in patients with lowrenin essential hypertension. Data suggest that with angiotensin II stimulation, the predominant AT-1 receptors (Belloni et al., 1998) in the zona glomerulosa of adrenal gland are paradoxically upregulated, thus enhancing angiotensin II adrenal sensitivity (Hauger et al., 1978). If adrenal stimulation by angiotensin II is sufficiently prolonged and sustained it could produce adrenal hyperplasia with increased aldosterone secretion (Lim et al., 2002). It has been suggested that the natural history of hypertension proceeds from essential (high to normal renin) hypertension through to lowrenin hypertension to idiopathic aldosteronism over time, a condition that has been described as tertiary aldosteronism (Lim et al., 2002). However, the rate of this progression may be different depending on genetic susceptibility. Evidence of genetic variants in CYP11B2 has been found in a hypertensive population, suggesting that mutations of this enzyme may be relevant in essential hypertension (Brand et al., 1998; Davies et al., 1999). Davies et al. (1999) described a polymorphism in the promoter of the CYP11B2 gene, with a single nucleotide C-to-T transition at position 344, in association with hypertension. The T allele was significantly more frequent than the C allele in the hypertensive compared with the control group patients and subjects with the genotypes TT or TC had significantly higher aldosterone excretion rates than did those with the CC genotype (Davies et al., 1999). An increased frequency of the T allele and a relative excess of TT homozygosity over CC homozygosity were found in patients with idiopathic low-renin hypertension in comparison with both normal to high-renin hypertensive subjects and normotensive control subjects by two independent European and Japanese groups (Rossi et al., 2001; Tamaki et al., 1999). Interestingly, we found that the CYP11B2 genotype predicted long-term graft function in renal transplant patients, with more patients having the CYP11B2 TT than the CC genotype experiencing worsening renal function (Nicod et al., 2002). This association remained when the effect of the CYP11B2 polymorphism was controlled for potential epistatic interactions with the angiotensinogen M235T mutation, the ACE 287-bp deletion/

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insertion (D/I) polymorphism, and angiotensin receptor A1166C gene polymorphisms (Nicod et al., 2002). Expression of the CYP11B2 gene is regulated by angiotensin II. Angiotensin II acts on the CYP11B2 gene promoter region with its variety of control factors, one of which is steroidogenic factor 1 (SF-1) (Clyne et al., 1997; Honda et al., 1993). The 344 C/T single nucleotide difference at this site alters the sensitivity to angiotensin II (Davies et al., 1999). The reported polymorphism causing increased sensitivity of CYP11B2 to angiotensin II (Davies et al., 1999) can be expected to be more prevalent in hypertensive patients responding to MR antagonists; however, to date, this hypothesis has not been investigated and thus needs to be addressed with an appropriate pharmacogenomic study (Ferrari, 1998).

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associated with defects in the peripheral metabolism of cortisol. J. Clin. Endocrinol. Metab. 49, 757–764. Vallet, V., Chraibi, A., Gaeggeler, H. P., Horisberger, J. D., and Rossier, B. C. (1997). An epithelial serine protease activates the amiloride-sensitive sodium channel. Nature. 389, 607–610. Verrey, F. (2001). Sodium reabsorption in aldosterone-sensitive distal nephron: News and contributions from genetically engineered animals. Curr. Opin. Nephrol. Hypertens. 10, 39–47. Vuagniaux, G., Vallet, V., Jaeger, N. F., Pfister, C., Bens, M., Farman, N., Courtois-Coutry, N., Vandewalle, A., Rossier, B. C., and Hummler, E. (2000). Activation of the amiloridesensitive epithelial sodium channel by the serine protease mCAP1 expressed in a mouse cortical collecting duct cell line. J. Am. Soc. Nephrol. 11, 828–834. Walker, B. R., Campbell, J. C., Williams, B. C., and Edwards, C. R. (1992). Tissue-specific distribution of the NAD+ dependent isoform of 11-hydroxysteroid dehydrogenase. Endocrinology. 131, 970–972. Walker, B. R., Stewart, P. M., Shackleton, C. H., Padfield, P. L., and Edwards, C. R. (1993). Deficient inactivation of cortisol by 11-hydroxysteroid dehydrogenase in essential hypertension. Clin. Endocrinol. 39, 221–227. Wang, C., Chan, T. K., Yeung, R. T., Coghlan, J. P., Scoggins, B. A., and Stockigt, J. R. (1981). The effect of triamterene and sodium intake on renin, aldosterone, and erythrocyte sodium transport in Liddle’s syndrome. J. Clin. Endocrinol. Metab. 52, 1027–1032. Warnock, D. G. (1998). Liddle syndrome: An autosomal dominant form of human hypertension. Kidney Int. 53, 18–24. Weinberger, M. H., Grim, C. E., Hollifield, J. W., Kem, D. C., Ganguly, A., Kramer, N. J., Yune, H. Y., Wellman, H., and Donohue, J. P. (1979). Primary aldosteronism: Diagnosis, localization, and treatment. Ann. Intern. Med. 90, 386–395. Werder, E. A., Zachmann, M., Vollmin, J. A., Veyrat, R., and Prader, A. (1974). Unusual steroid excretion in a child with low renin hypertension. Res. Steroids. 6, 385–389. White, P. C., and Speiser, P. W. (2000). Congenital adrenal hyperplasia due to 21-hydroxylase deficiency. Endocr. Rev. 21, 245–291. White, P. C., Dupont, J., New, M. I., Leiberman, E., Hochberg, Z., and Rosler, A. (1991). A mutation in CYP11B1 (Arg-448!His) associated with steroid 11-hydroxylase deficiency in Jews of Moroccan origin. J. Clin. Invest. 87, 1664–1667. Whorwood, C. B., Sheppard, M. C., and Stewart, P. M. (1993). Licorice inhibits 11hydroxysteroid dehydrogenase messenger ribonucleic acid levels and potentiates glucocorticoid hormone action. Endocrinology. 132, 2287–2292. Wilson, R. C., Harbison, M. D., Krozowski, Z. S., Funder, J. W., Shackleton, C. H., Hanauske-Abel, H. M., Wei, J. Q., Hertecant, J., Moran, A., Neiberger, R. E., Balfe, J. W., Fattah, A., Daneman, D., Licholai, T., and New, M. I. (1995a). Several homozygous mutations in the gene for 11-hydroxysteroid dehydrogenase type 2 in patients with apparent mineralocorticoid excess. J. Clin. Endocrinol. Metab. 80, 3145–3150. Wilson, R. C., Krozowski, Z. S., Li, K., Obeyesekere, V. R., Razzaghy-Azar, M., Harbison, M. D., Wei, J. Q., Shackleton, C. H., Funder, J. W., and New, M. I. (1995b). A mutation in the HSD11B2 gene in a family with apparent mineralocorticoid excess. J. Clin. Endocrinol. Metab. 80, 2263–2266. Wilson, R. C., Dave-Sharma, S., Wei, J. Q., Obeyesekere, V. R., Li, K., Ferrari, P., Krozowski, Z. S., Shackleton, C. H., Bradlow, L., Wiens, T., and New, M. I. (1998). A genetic defect resulting in mild low-renin hypertension. Proc. Natl. Acad. Sci. USA. 95, 10200–10205. Wisgerhof, M., Brown, R. D., Hogan, M. J., Carpenter, P. C., and Edis, A. J. (1981). The plasma aldosterone response to angiotensin II infusion in aldosterone-producing adenoma and idiopathic hyperaldosteronism. J. Clin. Endocrinol. Metab. 52, 195–198.

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Yanase, T., Kagimoto, M., Suzuki, S., Hashiba, K., Simpson, E. R., and Waterman, M. R. (1989). Deletion of a phenylalanine in the N-terminal region of human cytochrome P-450(17 ) results in partial combined 17-hydroxylase/17,20-lyase deficiency. J. Biol. Chem. 264, 18076–18082. Yanase, T., Simpson, E. R., and Waterman, M. R. (1991). 17-Hydroxylase/17,20-lyase deficiency: From clinical investigation to molecular definition. Endocr. Rev. 12, 91–108. Young, W.F., Jr., Hogan, M. J., Klee, G. G., Grant, C. S., and van Heerden, J. A. (1990). Primary aldosteronism: Diagnosis and treatment. Mayo Clin. Proc. 65, 96–110. Young, W.F., Jr., Stanson, A. W., Grant, C. S., Thompson, G. B., and van Heerden, J. A. (1996). Primary aldosteronism: Adrenal venous sampling. Surgery. 120, 913–919 [discussion on pp. 919–920]. Zachmann, M., Tassinari, D., and Prader, A. (1983). Clinical and biochemical variability of congenital adrenal hyperplasia due to 11-hydroxylase deficiency: A study of 25 patients. J. Clin. Endocrinol. Metab. 56, 222–229. Zaehner, T., Plueshke, V., Frey, B. M., Frey, F. J., and Ferrari, P. (2000). Structural analysis of the 11-hydroxysteroid dehydrogenase type 2 gene in end-stage renal disease. Kidney Int. 58, 1413–1419.

5 Peroxisome Proliferator-Activated Receptors and the Cardiovascular System Yuqing E. Chen, Mingui Fu, Jifeng Zhang, Xiaojun Zhu, Yiming Lin, Mukaila A. Akinbami, and Qing Song Cardiovascular Research Institute, Morehouse School of Medicine, Atlanta, Georgia 30310

I. Introduction II. Discovery, Structure, and Tissue Distribution of PPARs III. PPAR Ligands IV. Mechanisms of Action of PPARs V. PPARg in the Cardiovascular System A. PPARg Regulation in the Vasculature B. PPARg and Atherosclerosis C. PPARg and Hypertension D. PPARg Genetic Variants and Cardiovascular Disease E. PPARg and the Heart VI. PPARa in the Cardiovascular System A. PPARa in the Vasculature B. PPARa Genetic Variants and Cardiovascular Disease C. PPARa and the Heart Vitamins and Hormones Volume 66

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VII. PPARd in the Cardiovascular System A. PPARd in the Vasculature B. PPARd Regulation VIII. Conclusions References

Insulin resistance syndrome (also called syndrome X) includes obesity, diabetes, hypertension, and dyslipidemia and is a complex phenotype of metabolic abnormalities. The disorder poses a major public health problem by predisposing individuals to coronary heart disease and stroke, the leading causes of mortality in Western countries. Given that hypertension, diabetes, dyslipidemia, and obesity exhibit a substantial heritable component, it is postulated that certain genes may predispose some individuals to this cluster of cardiovascular risk factors. Emerging data suggest that peroxisome proliferator-activated receptors (PPARs), including , , and , are important determinants that may provide a functional link between obesity, hypertension, and diabetes. It has been well documented that hypolipidemic fibrates and antidiabetic thiazolidinediones are synthetic ligands for PPAR and PPAR, respectively. In addition, PPAR natural ligands, such as leukotriene B4 for PPAR, 15-deoxy-12,14-prostaglandin J2 for PPAR, and prostacyclin for PPAR, are known to be eicosanoids and fatty acids. Studies have documented that PPARs are present in all critical vascular cells: endothelial cells, vascular smooth muscle cells, and monocytemacrophages. These observations suggest that PPARs not only control lipid metabolism but also regulate vascular diseases such as atherosclerosis and hypertension. In this review, we present structure and tissue distribution of PPAR nuclear receptors, discuss the mechanisms of action and regulation, and summarize the rapid progress made in this area of study and its impact on the cardiovascular system. ß 2003, Elsevier Science (USA).

I. INTRODUCTION Nuclear hormone receptors comprise a large superfamily of ligandmodulated transcription factors that, in part, mediate a response to steroids, retinoids, and thyroid hormones and play key roles in nearly every aspect of vertebrate development and adult physiology. During the 1980s, steroid/ thyroid receptors were cloned and found to have extensive structural similarity. To date, 48 members of the nuclear hormone receptor family have been identified in the human genome (Maglich et al., 2001), heralding a new era in physiology.

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II. DISCOVERY, STRUCTURE, AND TISSUE DISTRIBUTION OF PPARS The peroxisome proliferator-activated receptors (PPARs), including , / , and , belong to the steroid/thyroid receptor superfamily. In 1990, Issemann and Green first cloned the PPAR gene from a mouse liver cDNA library. PPAR (peroxisome-proliferator activated receptor ) was so named because this receptor was activated by peroxisome proliferators such as fibrates, which caused the proliferation of peroxisomes and hepatomegaly in rodents. Although only PPAR in rodent species has been documented to promote peroxisome proliferation, the name PPAR has generally been accepted for this subfamily of nuclear receptors. The molecular basis of this species-specific phenomenon is still unclear. To date, PPAR genes have been cloned from many species (Table I). Human PPAR, PPAR, and PPAR have been mapped to chromosomes 22q12-q13, 3p25, and 6p21.2-p21.1, respectively. The PPARs share a domain structure typical of other members of the nuclear receptor superfamily (Fig. 1). The N-terminal (A/B) domain contains a ligand-independent transactivation function (AF-1). This domain can be modulated by mitogen-activated protein kinase (MAPK)

TABLE I. PPAR Genes Cloned from Various Species PPAR gene

Species

Ref.

PPAR

Human Rat Mouse Guinea pig Frog

Sher et al. (1993) Gottlicher et al. (1992) Issemann and Green (1990) Bell et al. (1998) Dreyer et al. (1992)

PPAR

Human Rat Mouse Monkey Pig Chicken Hamster Frog Salmon

Greene et al. (1995) Guardiola-Diaz, et al. (1999) Kliewer et al. (1994) Hotta et al. (1998) Ding et al. (1999) Takada et al. (2000) Aperlo et al. (1995) Dreyer et al. (1992) Ruyter et al. (1997)

PPAR

Human Rat Mouse Rabbit Chicken Frog

Schmidt et al. (1992) Xing et al. (1995) Kliewer et al. (1994) Mano et al. (2000) Takada et al. (2000) Dreyer et al. (1992)

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FIGURE 1. Structure and functional domains of PPARs. The A/B domain includes the activation function 1 (AF-1). The C domain is the DNA-binding domain (DBD). The E/F domain is the ligand-binding domain (LBD) and contains AF-2. Numbers indicate the amino acid identity (%) of the DBDs and LBDs between human PPAR, PPAR, and PPAR.

phosphorylation (Adams et al., 1997; Juge-Aubry et al., 1999). The PPAR DNA-binding domain (C) contains two zinc finger motifs that are highly conserved. The ligand-binding domain (E/F), which contains liganddependent transactivation function 2 (AF-2), shows significant sequence variation across subtypes. The three types of PPARs have different expression patterns: PPAR is most abundantly expressed in liver, kidney, heart, and muscle; PPAR is most abundantly expressed in fat cells, large intestine, and cells of the monocyte lineage; whereas PPAR is expressed in nearly all tissues (Lemberger et al., 1996; Escher et al., 2001). These contrasting expression patterns provided one of the first hints that the three PPARs subserve distinct biologies. All PPARs are expressed in vascular smooth muscle cells (VSMCs), endothelial cells, and monocyte-macrophages (Staels et al., 1998; Marx et al., 1998a,b, 1999a,b; Law et al., 2000; Fu et al., 2001a,b, 2002a,b; Zhang et al., 2002), suggesting that PPARs may play an important role in the cardiovascular system.

III. PPAR LIGANDS Although all PPARs are activated by fatty acids and derivatives, PPAR binds hypolipidemic fibrates and natural ligands including leukotriene B4 (LTB4) and 8-(S)-hydroxyeicosatetraenoic acid (8S-HETE) (Devchand et al., 1996; Kliewer et al., 1997). It is well established that PPAR is a key regulator of lipid metabolism and that fibrates regulate the transcription of a large number of genes that affect lipoprotein and fatty acid (FA) metabolism. Evidence also shows that PPAR null mice exhibit abnormalities in triglyceride and cholesterol metabolism, do not respond to fibrates, and become obese with age (Lee et al., 1995). However, all these compounds are weak ligands for PPAR, which may explain why high doses (300–1200 mg/day) of fibrates are required for the treatment of hyperlipidemia. The development of high-affinity PPAR ligands such as GW9578 (Brown et al.,

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1999) is encouraging and will play a major role in understanding the biology of PPAR. PPAR has been linked to adipocyte differentiation and adult-onset diabetes (type II) since the anti-diabetic thiazolidinediones (TZDs) and the naturally occurring compound 15-deoxy-12,14-prostaglandin J2 (15d-PGJ2) were identified as PPAR ligands (Forman et al., 1995; Kliewer et al., 1995). Moreover, the 15-lipoxygenase metabolites of linoleic acid, 9-hydroxyoctadecadienoic acid (HODE) and 13-HODE, have been shown to function as micromolar PPAR agonists (Nagy et al., 1998). However, caution must be taken in analyzing the biological effects elicited by 15d-PGJ2 because it mediates several PPAR-independent signaling pathways (Straus et al., 2000; Rossi et al., 2000). Most of the TZDs at 1 to 10 M have high affinity for PPAR. However, a series of provocative reports suggests that synthetic PPAR ligands in the TZD class have PPAR-independent mechanisms (Moore et al., 2001; Chawla et al., 2001). It is noteworthy that a rational drug design discovery effort based on the crystal structure of PPAR has led to the development of non-TZD tyrosine analogs, such as GW7845 (EC50 of 0.71 nM), which function as potent PPAR ligands. The finding that non-TZD agonists such as GW7845 have similar antidiabetic actions provides additional support for the role of PPAR as a determinant of insulin resistance (Li et al., 2000). In addition, the identification of resistin (for resistance to insulin) as the TZD target gene revealed the role of PPAR as a determinant of insulin resistance (Steppan et al., 2001). Prostacyclin (PGI2) is a PPAR ‘‘natural ligand,’’ but PGI2 also activates PPAR and PPAR with a level of affinity similar to that shown for PPAR. Carbaprostacyclin (cPGI2), a stable synthetic PGI2 analog, has been shown to activate PPAR and to a much lesser extent activates PPAR and PPAR (Forman et al., 1997). In addition, the saturated fatty acids (C6 to C18) can activate both PPAR and PPAR with similar affinity. The lack of both connections with important clinical manifestations and specific PPAR ligands has hampered scientists in elucidating the function of PPAR for many years. A relatively high-affinity synthetic fibrate molecule, GW2433 (EC50 of 50 nM and 1 M for PPAR and PPAR, respectively), was reported (Brown et al., 1997) and used for studying PPAR function (Gupta et al., 2000; Poirier et al., 2001). It is important to note that an intriguing report by GlaxoSmithKline has identified the first high-affinity PPAR ligand, GW501516 (EC50 of 1.2 0.1 nM and >1000-fold selective for PPAR over other subtypes; Oliver et al., 2001). Taken together, these data will spur new interest in the study of PPAR function. For more detailed information on PPAR ligands, we suggest that readers see comprehensive reviews (Desvergne and Wahli, 1999; Willson et al., 2000; Berger and Moller, 2002).

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IV. MECHANISMS OF ACTION OF PPARS PPARs are ligand-activated transcriptional factors. Together with retinoid X receptors (RXRs), another member of the family, the heterodimers bind to the PPAR-responsive element (PPRE), a DR1 or DR2 element, which is a direct repeat of two similar hexanucleotide (5-AGGTCA-3) half-sites separated by one or two nucleotides on its target genes (Fig. 2). In the presence of both PPAR- and RXR-specific ligands, this type of interaction confers synergistic activation of target genes (Kliewer et al., 1992). In addition to ligand-dependent regulation, several cofactors of PPAR have been described, including proteins such as PPAR-binding protein, steroid receptor coactivator 1, TRAP220, and p300 with intrinsic histone acetyltransferase activity for coactivation (Zhu et al., 1996, 1997; Ge et al., 2002; Gelman et al., 1999). Although there is still some controversy concerning whether PPAR coactivation is ligand dependent or not, it is widely believed that unliganded PPAR recruit corepressor complexes that are displaced on ligand binding are replaced by coactivator complexes (Rosenfeld and Glass, 2001). Interestingly, it has been shown that coactivator p300 could enhance PPAR2-mediated gene expression in HeLa cells in a ligand-independent manner (Gelman et al., 1999). A

FIGURE 2. Schematic view of PPAR action. After a ligand binds to PPAR, PPAR dissociates from corepressors, recruits coactivators, heterodimerizes with 9-cis-retinoic acid receptor, and then binds to a DR1 or DR2 response element, which either activates or represses PPAR target genes. PPAR activity is also controlled by both protein degradation and phosphorylation. PPRE, PPAR-responsive element; RA, retinoic acid; RXR, retinoid X receptor; u, ubiquitin.

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ligand-dependent interaction and activation between the N-terminal part of p300 and the C-terminal AF-2 domain of PPAR2 were required. Surprisingly, the authors found that the N-terminal AF-1 domain of PPAR2 also displayed a docking site for p300 and that this interaction and activation were ligand independent. Therefore, understanding the effects of cofactor interactions with PPARs is a key to understanding the overall transactivation of PPAR-responsive genes. The degradation of PPAR proteins also contributes to PPAR functions. TZDs were reported to induce degradation of the PPAR by enhancing ubiquitination (Hauser et al., 2000). However, fibrates were reported to inhibit PPAR ubiquitination and increased the PPAR protein half-life (Blanquart et al., 2002). More experiments are required to understand the roles of TZDs and fibrates in PPAR function as well as to determine the mechanisms of their actions. The transcriptional activity of PPAR can also be regulated by phosphorylation of PPARs (Adams et al., 1997; Juge-Aubry et al., 1999). PPARs are known to repress gene transcription by negatively interfering with CAAT box/enhancer-binding protein, NF-B, STAT, Smad3, and AP-1 signaling pathways (Ricote et al., 1998; Zhou et al., 1999; Delerive et al., 1999a,b, 2000; Fu et al., 2001a; Gervois et al., 2001). Therefore, investigating the PPAR transrepression mechanism may provide novel insight into our understanding of PPAR action.

V. PPAR IN THE CARDIOVASCULAR SYSTEM PPAR has been linked to adipocyte differentiation and insulin sensitivity. Antidiabetic TZDs, commonly known as ‘‘insulin sensitizers,’’ were originally developed without any knowledge of their mechanism of action. It is now recognized that TZDs are pharmacological PPAR ligands. In addition to activation by pharmacological ligands, the endogenous ligands appear to be polyunsaturated fatty acids and eicosanoids such as 15d-PGJ2. However, it is important to note that these ‘‘natural’’ fatty acid ligands can induce a biological effect via non-PPAR-mediated pathways (Rossi et al., 2000; Vaidya et al., 1999; Straus et al., 2000). Furthermore, it has been observed that two of the major oxidized lipid components of oxidized LDL cholesterol, 9-HODE and 13-HODE, appear to function as endogenous activators and ligands of PPAR (Nagy et al., 1998). Although most of the focus on PPAR was related to lipid metabolism and adipocyte biology, studies suggest that PPAR is expressed in the vasculature and plays an important role in the cardiovascular system (Table II).

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TABLE II. PPAR-Regulated Genes in the Cardiovascular Systema Gene name

Regulation

Cell type

Growth factors, cytokines, and receptors TNF- Cardiomyocyte VEGF + VSMC, macrophage, EC Endothelin 1 EC

Ref.

CNP CTGF AT1R

+

VSMC VSMC VSMC

VEGF receptors 1 and 2 PDGF- receptor CCR2 CD36

EC

Takano et al. (2000) M. Inoue et al. (2001); Yamakawa et al. (2000) Delerive et al. (1999b); Fukunaga et al. (2001) Fukunaga et al. (2001) Fu et al. (2001a) Sugawara et al. (2001); Takeda et al. (2000) Xin et al. (1999)

VSMC

Takata et al. (2001)

+

Monocyte VSMC, aorta

Han et al. (2000) Chen et al. (2001); Matsumoto et al. (2000) Transcriptional factors and cell cycle regulatory proteins c-fos VSMC Benson et al. (2000) Ets-1 VSMC Goetze et al. (2001) p21 VSMC Miwa et al. (2000); Wakino et al. (2001) p27 VSMC Hupfeld and Weiss (2001) Cyclin D1 VSMC Hupfeld and Weiss (2001); Miwa et al. (2000) Cyclin E VSMC Hupfeld and Weiss (2001) Adhesion molecules E-selectin EC Nawa et al. (2000) EC, VSMC Chen and Han (2001); Pasceri et al. ICAM-1 /+ (2000) VCAM-1 EC Pasceri et al. (2000) Matrix proteins Osteoprotegerin VSMC Fu et al. (2002b) Osteopontin VSMC Oyama et al. (2002) MMP-9 VSMC Marx et al. (1998b) Others NOS /+ VSMC Ikeda et al. (2000a); Hattori et al. (1999) IP-10 EC Marx et al. (2000) Mig EC Marx et al. (2000) I-TAC EC Marx et al. (2000) iCOX-2 Macrophage Inoue et al. (2000) PAI-1 /+ EC Kato et al. (1999); Marx et al. (1999a) CuZn-SOD + EC Inoue et al. (2001) MHC-II AtheromaKwak et al. (2002) associated cells Continued

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TABLE II.

(Continued)

Gene name

Regulation

Cell type

Ref.

Thromboxane synthase Thromboxane receptor

Macrophage

Ikeda et al. (2000b)

VSMC

Sugawara et al. (2002)

a PPAR is expressed in critical cardiovascular cells including endothelial cells (ECs), vascular smooth muscle cells (VSMCs), monocyte-macrophages, and cardiomyocytes. A summary of PPAR target genes in the cardiovascular system is as follows: tumor necrosis factor , TNF-; vascular endothelial growth factor, VEGF; C-type natriuretic peptide, CNP; connective tissue growth factor, CTGF; angiotensin II type 1 receptor, AT1R; platelet-derived growth factor, PDGF; CC chemokine receptor, CCR2; intercellular adhesion molecule, ICAM; vascular cell adhesion molecule, VCAM; matrix metalloproteinase 9, MMP-9; nitric oxide synthase, NOS; interferon-inducible protein of 10 kDa, IP-10; monokine induced by interferon , Mig; interferon-inducible T cell -chemoattractant, I-TAC; inducible cyclooxygenase 2, iCOX-2; plasminogen activator inhibitor 1, PAI-1; Cu2+, Zn2+-dependent superoxide dismutase, CuZn-SOD; major histocompatibility complex class II molecule, MHC-II. + and , Upregulation and downregulation, respectively.

A. PPAR REGULATION IN THE VASCULATURE

The human PPAR gene is composed of nine exons spanning more than 100 kb on chromosome 3p25-24 (Fajas et al., 1997). It generates seven different mRNAs, PPAR1 to PPAR7, by alternative promoters and differential splicing (Fajas et al., 1998; Zhou et al., 2002). All PPAR isoforms except PPAR2 have identical amino acid sequences. Human PPAR2 has 28 additional amino acids at the N terminus and is reportedly expressed primarily in adipocytes, whereas PPAR1 is expressed in the vasculature (Law et al., 2000; Fu et al., 2001a,b). The role of PPAR1 in the vasculature has been extensively investigated; however, the transcriptional regulation of PPAR1 gene remains poorly understood. Our group found a putative binding site for early growth response gene 1 (Egr-1) in the human PPAR1 gene promoter and provided the first evidence that Egr-1 is a key mediator activating PPAR1 gene expression in VSMCs (Fu et al., 2002a). In the vasculature, Egr-1 is a welldefined transcription factor, which activates several cytokine and growth factor genes such as platelet-derived growth factor A (PDGF-A) (Khachigian et al., 1997), PDGF-B (Khachigian et al., 1996), basic fibroblast growth factor (bFGF) (Biesiada et al., 1996), tumor necrosis factor (TNF-) (Yao et al., 1997), and interleukin 2 (IL-2) (Skerka et al., 1995). It has also been established that these factors can stimulate Egr-1 expression in the vasculature. This positive feedback loop can then amplify and sustain Egr-1-mediated gene transcription (Silverman and Collins, 1999). Interestingly, PPAR activation in vascular cells inhibits the production of cytokines, including TNF-

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PPAR negatively mediates Egr-1 actions in the vasculature. Several growth factors and cytokines induce Egr-1 expression in vascular cells. In return, Egr-1 activates the transcription of these genes, including PDGF-B and TNF-, which are implicated in the development of atherosclerosis. The positive feedback loop serves to amplify and sustain Egr-1mediated gene transcription. We proposed that PPAR upregulation through Egr-1 may exert negative feedback to decrease growth factor- and cytokine-induced Egr-1 expression, and inhibit Egr-1-induced cell cycle protein expression.

FIGURE 3.

(Takano et al., 2000), IL-2 (Delerive et al., 1999a), and vascular endothelial growth factor (VEGF) (M. Inoue et al., 2001; Yamakawa et al., 2000), and downregulates the expression of several growth factor receptors including angiotensin II type I receptor (Sugawara et al., 2001; Takeda et al., 2000), PDGF receptor (Takata et al., 2001), and VEGF receptor (Xin et al., 1999). In addition, PPAR activation can inhibit the expression of cell cycle proteins such as cyclin D1 (Wakino et al., 2000). Hence, PPAR upregulation induced by Egr-1 could negatively inhibit Egr-1 actions in the vasculature (Fig. 3). B. PPAR AND ATHEROSCLEROSIS

The role of PPAR in the development of atherosclerosis is controversial (for reviews see Kersten et al., 2000; Lazar, 2001; Barbier et al., 2002). Several reports support the postulate that PPAR may function as an antiatherogenic factor (Chinetti et al., 2001; Moore et al., 2001; Chawla et al., 2001). Moreover, there are studies suggesting that synthetic PPAR ligands inhibit neointimal formation (Law et al., 1996) and carotid intima– media thickness in humans (Minamikawa et al., 1998). It was also reported that two structurally different PPAR ligands, rosiglitazone (TZD) and GW7845, improved atherosclerotic lesions in low-density lipoprotein (LDL) receptor-deficient male mice. These effects were striking and revealed a reduction in both the number and size of lesions (Li et al., 2000). Surprisingly, in this study, female mice treated with PPAR ligands did not show any reduction of atherosclerotic lesions, thus suggesting that this sexual difference may be due to the influence of estrogen and/or progestin.

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Similarly, inhibition of early atherosclerotic lesion formation by troglitazone treatment was observed in male LDL-deficient mice (Collins et al., 2001). However, troglitazone inhibits fatty steak formation by enhancing high-density lipoprotein (HDL) cholesterol levels and was reported in both male and female apolipoprotein E (apoE)-deficient mice (Chen et al., 2001). To date, four separate hypotheses have been proposed to explain how PPAR ligands inhibit vascular lesion formation and atherogenesis. They include (1) reducing inflammatory cytokine production by macrophages and T cells (Ricote et al., 1998; Jiang et al., 1998; Marx et al., 2002), (2) decreasing extracellular matrix proteins (Fu et al., 2001a), (3) inhibiting VSMC activation (Marx et al., 1998a,b; Law et al., 2000; Wakino et al., 2000; Bishop-Bailey et al., 2002), and (4) enhancing apoptosis in vascular cells (Patel et al., 2001). It is noteworthy that the description of the role of PPAR in the vasculature is primarily based on studies assessing the biological response to pharmacological doses of synthetic PPAR ligands in the TZD class. The inactivation of PPAR expression by homologous recombination in mice has profound biological significance. PPAR null mice (Rosen et al., 1999; Kubota et al., 1999; Barak et al., 1999) exhibit embryonic lethality in association with perturbations in placental vascularization and myocardial thinning. The elimination of PPAR expression prevents adipocyte differentiation. Thus, it is clear that PPAR plays a fundamental role in development and differentiation. Interestingly, heterozygous PPARdeficient mice were protected from developing insulin resistance (Kubota et al., 1999; Miles et al., 2000). Elevated leptin levels were observed in PPAR-deficient mice. Such increases in leptin levels probably account for the protection against insulin resistance. Although the precise mechanisms involved remain unclear, it is anticipated that additional studies with conditional PPAR knockout mice should provide further insight. Mehrabian et al. (2001) reported a locus for aortic lesion formation on mouse chromosome 6. The locus was confirmed by constructing a congenic strain in which the chromosome 6 segment from CAST/Ei (resistant strain) mice was transferred to a C57BL/6J (susceptible strain) background. The congenic strain was almost completely resistant to diet-induced atherosclerosis. Although this locus contains the candidate gene PPAR, the congenic mice exhibited significantly reduced expression of PPAR. In addition, a new selective PPAR antagonist, SB202, showed antiobesity and antidiabetic effects. These observations suggest that downregulation of PPAR activity has potential therapeutic benefits for obesity, diabetes, and atherosclerosis. Further experiments are required to address this interesting phenomenon. Whether PPAR activation is promoting or inhibiting atherogenesis is still controversial. However, the results of ongoing large placebo-controlled clinical trials should be able to address this issue in the near future.

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Understanding the role of PPAR in the vasculature is important because there are more than 2 million type II diabetic patients currently undergoing TZD treatment in the United States. C. PPAR AND HYPERTENSION

Hypertension is associated with substantial insulin resistance, even in patients without diabetes. In diabetic patients, TZD derivatives act as insulin sensitizers, increasing insulin-mediated glucose disposal without increasing insulin release (Sparano and Seaton, 1998) and reducing circulating free fatty acid (FFA) levels (Komers and Vrana, 1998). There is growing evidence suggesting that insulin resistance is linked to hypertension. It is also interesting that troglitazone and rosiglitazone used for the treatment of insulin resistance and diabetes have been found to decrease blood pressure substantially. This effect has been observed in patients with type II diabetes and hypertension (Ogihara et al., 1995), nonhypertensive persons with type II diabetes (Ghazzi et al., 1997), and obese persons without diabetes (Nolan et al., 1994; Tack et al., 1998). Furthermore, treatment with troglitazone has been found to attenuate stress-induced increases in blood pressure (Sung et al., 1999). In another study, decreases in mean blood pressure correlated significantly with reductions in plasma insulin level (Ghazzi et al., 1997). Thus, it appears that TZDs decreased blood pressure by improving insulin resistance. Other potential mechanisms for the hypotensive effects of TZDs may include improved endothelium-dependent vasodilatation, a decrease in calcium influx and calcium sensitivity of the contractile apparatus (Kawasaki et al., 1998; Song et al., 1997), and inhibition of endothelin 1 expression and secretion in vascular endothelial cells through activation of PPAR (Satoh et al., 1999). It is also essential to note that PPAR activation inhibits angiotensin II–induced hypertension by improving endothelial cell function, correcting vessel structure abnormalities, and reducing inflammatory gene expression (Diep et al., 2002). D. PPAR GENETIC VARIANTS AND CARDIOVASCULAR DISEASE

To date, several PPAR polymorphisms have been identified in the coding region (Fig. 4). Such variants include, first, a silent C161T substitution in exon 6 (Meirhaeghe et al., 1998). Without altering the amino acid sequence, it displayed an association with circulating leptin levels (Meirhaeghe et al., 1998) and relative risk of coronary artery disease (Wang et al., 1999). Second, a mutation (P115Q) immediately adjacent to the phosphorylation site of PPAR2 was reported to be markedly associated with severe obesity (Ristow et al., 1998). Third and fourth variants were associated with changes in the

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FIGURE 4. Schematic illustration of polymorphisms in the coding region of the human PPAR gene. Polymorphism is indicated by start arrows. The variants and their associated phenotypes are shown in boxes. BMI, Body mass index.

amino acids at positions 290 and 467 in the PPAR2 ligand-binding domain and inhibit the action of wild-type PPAR in a dominant-negative manner (Barroso et al., 1999). These two variants were associated with severe insulin resistance and hypertension at an unusually young age before obesity, implying that PPAR2 mutations could affect insulin resistance and blood pressure even before they induce the obesity phenotype. The most common variant is a non-conservative substitution that causes a proline-to-alanine change at codon 12 within the PPAR2-unique ligandindependent activation domain (Yen et al., 1997; Beamer et al., 1998). It was correlated to systolic and diastolic blood pressures and body mass index (BMI) in a Utah population (Hasstedt et al., 2001). This variant was also widely subjected to studies of its association with obesity and insulin resistance; however, the results were not consistent. For example, allele-Ala was associated with higher BMI in some populations (Beamer et al., 1998; Valve et al., 1999) but associated with lower BMI and higher insulin sensitivity in other populations (Deeb et al., 1998). In several other studies, no association was found in study cohorts (Mancini et al., 1999; Hasstedt et al., 2001). These conflicting observations may be associated with small sample sizes or the extent to which linkage disequilibrium may vary in populations with different ethnic backgrounds. As such, our understanding of PPAR2 polymorphism may be sufficient to lead to novel preventive, diagnostic, and therapeutic approaches for improving the management of type II diabetes and cardiovascular diseases.

E. PPAR AND THE HEART

All three PPAR members, including PPAR, PPAR1 (not PPAR2), and PPAR, are expressed in the heart (Wayman et al., 2002). PPAR plays

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a critical role in the inhibition of cardiac hypertrophy (Asakawa et al., 2002). Treatment of mice with a PPAR agonist, pioglitazone, inhibits pressure overload-induced increases in the heart weight-to-body weight ratio, wall thickness, and myocyte diameter in mice. In addition, pressure overload-induced increases in the heart weight-to-body weight ratio and wall thickness were more prominent in heterozygous PPAR-deficient mice than in wild-type mice (Asakawa et al., 2002). Furthermore, it has been shown that activation of either PPAR or PPAR substantially reduces myocardial infarct size, significantly improves aortic flow during reperfusion in both normal and diabetic hearts, and considerably improves post ischemic functional recovery in rats (Yue et al., 2001; Khandoudi et al., 2002; Wayman et al., 2002). These observations suggest an important functional role for both PPAR and PPAR in the heart. PPAR plays a pivotal role in heart disease, but the factors that regulate its expression are poorly defined in cardiomyocytes. In a report from our laboratory, we demonstrated that many growth factors and cytokines upregulate PPAR expression through Egr-1-mediated mechanisms in VSMCs (Fu et al., 2002a). In addition, we documented that PDGF upregulated PPAR by the phosphatidylinositol-3-kinase (P13-kinase)/Akt signaling pathway (Fu et al., 2001b). Intriguingly, the Akt pathway has also been shown to regulate the expression of PPAR coactivator 1 (PGC-1) and PPAR, which may shift cardiomyocytes toward glycolytic metabolism shown to preserve cardiomyocyte function and survival during transient ischemia (Cook et al., 2002). Although these data suggest that Akt may play an important role, further studies are required to explore PPAR regulation in cardiomyocytes.

VI. PPAR IN THE CARDIOVASCULAR SYSTEM The current paradigm of the pathogenesis of atherosclerosis emphasizes the importance of ‘‘activated’’ cells expressing pro-inflammatory mediators in the process of atheroma formation and plaque rupture (for reviews see Libby et al., 1998; Ross, 1999). Although there is a substantial body of literature regarding the role of transcription factors such as nuclear factorB (NF-B) as mediators of the pro-inflammatory state of atherogenesis, little is known about the countervailing transcription factors that mediate the intrinsic anti-atherogenic genetic program within vascular cells. PPAR plays an inhibitory role in modulating the vascular cell response to proatherogenic stimuli (Table III). It has been established that PPAR is also a receptor for fibrates, which are widely prescribed for the reduction of high triglyceride levels, a risk factor for coronary heart disease, and that PPAR is a critical regulator of intra- and extracellular lipid metabolism. It is likely

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TABLE III. PPAR-Regulated Genes in the Cardiovascular Systema Gene name

Regulation

Cytokines and chemokines IL-6 IL-8 IL-1 TNF- MCP-1 Others ET-1 VCAM-1 iCOX-2 TF PAF receptor LOX-1 CuZn-SOD I-B MLC-2 E-selectin p22phox

+ +/ + + + + +

Cell type

EC, VSMC

Ref.

EC EC VSMC EC

Stumvoll and Haring (2002); Delerive et al. (1999a) Lee et al. (2000) I. Inoue et al. (2000) Babaev et al. (2001) Lee et al. (2000); Pasceri et al. (2001)

EC EC VSMC Macrophage Macrophage EC EC VSMC Cardiomyocyte EC EC

Delerive et al. (1999b); Ogata et al. (2002) Marx et al. (1999b); Jackson et al. (1999) Staels et al. (1998) Neve et al. (2001); Marx et al. (2001) Hourton et al. (2001) Hayashida et al. (2001) I. Inoue et al. (2001) Delerive et al. (2000) Hamano et al. (2001) Nawa et al. (2000) I. Inoue et al. (2001)

a PPAR is expressed in critical cardiovascular cells including endothelial cells (ECs), vascular smooth muscle cells (VSMCs), monocyte-macrophages, and cardiomyocytes. A summary of PPAR target genes in the cardiovascular system is as follows: interleukin, IL; tumor necrosis factor , TNF-; monocyte chemoattractant protein 1, MCP-1; endothelin 1, ET-1; vascular cell adhesion molecule 1, VCAM-1; inducible cyclooxygenase 2, iCOX-2; tissue factor, TF; platelet activator factor, PAF; low-density lipoprotein (LDL) receptor 1, LOX-1; Cu2+,Zn2+-dependent superoxide dismutase, CuZn-SOD; myosin light chain 2, MLC-2; phagocyte NAD(P)H oxidase subunit 22, p22phox. + and , Upregulation and downregulation, respectively.

that the therapeutic efficacy of fibrates in inhibiting atherogenesis is related in part to their capacity to function as ligands for PPAR

A. PPAR IN THE VASCULATURE

Studies showed that PPAR is present in endothelial cells (I. Inoue et al., 1998; Lee et al., 2000), smooth muscle cells (Staels et al., 1998), and monocyte-macrophages (Neve et al., 2001). Other evidence showed that PPAR inhibits the IL-1-induced expression of IL-6 and cyclooxygenase 2 (COX-2), as well as thrombin-induced endothelin 1 expression in VSMCs, as a result of a negative transcriptional regulation of the NF-B and activator protein 1 signaling pathways (Staels et al., 1998). In addition, PPAR activation inhibits tissue factor expression in macrophages (Marx et al., 2001; Neve et al., 2001), and reduces vascular cell adhesion molecule 1 (VCAM-1) in endothelial cells (Marx et al., 1999b). The data indicate that

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PPAR plays a critical role in the inhibition of atherogenesis. In fact, hypolipidemic drugs that function as ligands for PPAR have been shown to inhibit atherogenesis in clinical trials (for a review, see Robins, 2001). In addition to other drugs that function as PPAR ligands, it is noteworthy that the potent pro-inflammatory leukotriene B4 (LTB4) was identified as an endogenous natural ligand of PPAR (Devchand et al., 1996). This intriguing observation suggests that LTB4 is bi functional in its capacity to initially promote the inflammatory response and then activate a genetic program that will eventually inhibit inflammation. Indeed, this bi functional property is probably necessary for a tightly regulated inflammatory response that must eventually be abrogated in order to avoid long-term tissue damage once the infectious agent is successfully cleared from the body by the host defense system. In the absence of PPAR, the acute inflammatory response to LTB4 becomes a more chronic state of inflammation (Devchand et al., 1996, 1999; Yokomizo et al., 1997). B. PPAR GENETIC VARIANTS AND CARDIOVASCULAR DISEASE

Three PPAR genetic variants, including L162V (Lacquemant et al., 2000; Vohl et al., 2000; Evans et al., 2001), A268V (Lacquemant et al., 2000), and a G/C polymorphism in intron 7 (Flavell et al., 2002; Jamshidi et al., 2002), have been described in association with lipid metabolism, left ventricle hypertrophy, or coronary artery disease. The V162 allele carriers have a protective effect on atherogenesis, with a 50% increase in HDL level among fibrate-treated patients (Flavell et al., 2002; Bosse et al., 2002). However, intron 7 C allele carriers have a greater progression of atherosclerosis than do G allele homozygotes (Flavell et al., 2002). These contrasting data generated from PPAR genetic studies will spur new interest in the study of PPAR function in the cardiovascular system. C. PPAR AND THE HEART

PPAR is an important regulator of fatty acid oxidation in the heart. It plays an important role in the transcriptional control of cardiac energy metabolism. Left ventricular hypertrophy (LVH) is generally understood to be an adaptive response to increased workload under both physiological and pathological stimuli. During this process, an important molecular adaptation in the hypertrophied heart is the increase in glucose utilization and decrease in fatty acid oxidation (FAO) caused by downregulation of mRNA levels for FAO enzyme (Sack et al., 1996). In human populations, PPAR has been identified as a candidate genetic component in the human cardiac hypertrophic response. A G/C single-nucleotide polymorphism in intron 7 of the PPAR gene is significantly associated with left ventricular (LV)

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growth in response to exercise in a cohort of 144 young white healthy males who were randomly assigned to receive losartan during an intense 10-week physical training program (Jamshidi et al., 2002). The influence of the PPAR genotype on the degree of LVH was independent of losartan treatment and ACE genotype. Meanwhile, this PPAR genetic marker was also significantly associated with LV mass, independent of blood pressure in men, but not women, in the third Monitoring Trends and Determinants in Cardiovascular Disease (MONICA) Augsburg survey (Jamshidi et al., 2002). The causative role of PPAR in the pathogenesis of LVH and maladaptive cardiac substrate utilization has been further established by transgenic and knockout studies (Campbell et al., 2002; Finck et al., 2002). PPAR null mice showed altered cardiac energy metabolism with significantly reduced palmitate oxidation rates, along with remarkably increased cardiac malonyl-CoA levels (Campbell et al., 2002). Cardiacspecific PPAR overexpression directed by the cardiac -myosin heavy chain (MHC) promoter changed the expression profile of PPAR target genes involved in cardiac fatty acid uptake and oxidation pathways. Consequently, PPAR overexpression enhanced myocardial fatty acid oxidation rates and reduced glucose uptake and oxidation in the transgenic mice. Moreover, these MHC-PPAR mice exhibited ventricular hypertrophy and transgene expression-dependent alteration in systolic ventricular dysfunction (Finck et al., 2002). These results demonstrate that PPAR is a critical regulator of cardiac FA uptake and oxidation; it may influence the metabolic switch from FA to glucose during hypertrophy.

VII. PPAR IN THE CARDIOVASCULAR SYSTEM Specific roles for PPAR and PPAR have emerged as important determinants of vascular function and structure. Although PPAR is widely expressed in many tissues, the role of PPAR is unclear. To date, PPAR has been linked to colon cancer (He et al., 1999; Gupta et al., 2000) and to proliferation of colon cancer cells (Park et al., 2001). In addition, PPAR has been shown to promote preadipocyte proliferation (Hansen et al., 2001; Jehl-Pietri et al., 2000) and enhance renal medullary interstitial cell growth (Hao et al., 2002). Furthermore, PPAR is the only subtype expressed in the uterus during the implantation period in mice and probably participates in embryo implantation (Lim et al., 1999). A. PPAR IN THE VASCULATURE

Data from our laboratory showed that PPAR is expressed in VSMCs and upregulated after vascular injury (Zhang et al., 2002). Overexpression of

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PPAR in VSMCs increased postconfluent cell proliferation by increasing cyclin A and CDK2, as well as decreasing p57kip2. These data indicate that PPAR activation in VSMCs is in contrast to the activation of PPAR and PPAR, which inhibits VSMC proliferation. Similarly, PPAR is a potent inhibitor of ligand-induced transcriptional activity of PPAR and PPAR in preadipocytes (Shi et al., 2002). In addition, PPAR activation promotes lipid accumulation in human macrophages by increasing the expression of genes involved in lipid uptake and storage, such as the class A and B scavenger receptors (SRA, CD36) and adipophilin (Vosper et al., 2001). Because promoting VSMC activation and increasing lipid accumulation in macrophage are high risks for atherosclerosis, PPAR activation may promote the development of atherosclerosis and restenosis. A provocative report has shown for the first time that PPAR activation in macrophages by the high-affinity agonist, GW501516, increased expression of the reverse cholesterol transporter ATP-binding cassette A1 and induced apolipoprotein A1-specific cholesterol efflux (Oliver et al., 2001). This observed effect of PPAR is similar to the effects of PPAR and PPAR activation in macrophages (Chinetti et al., 2001; Chawla et al., 2001). Furthermore, GW501516 dramatically increased HDL levels in obese rhesus monkeys, suggesting that the increased cholesterol efflux is taken up by HDL and transported to the liver. In addition, it has been reported that the Merck PPAR ligand, L-165041, inhibits VCAM-1 expression and cytokine-induced MCP-1 secretion in endothelial cells (Rival et al., 2002) and increases HDL levels in db/db mice (Leibowitz et al., 2000). Because increased HDL levels are well associated with decreased risk of atherosclerosis, it appears that PPAR activation may inhibit atherogenesis. Taken together, whether PPAR activation is beneficial or detrimental to the cardiovascular system is still controversial. Further studies of the role of PPAR in the cardiovascular system will be necessary to assess the beneficial effects of PPAR activation. Such studies may include but not be limited to clinical trials with high-affinity PPAR agonists. B. PPAR REGULATION

The PPAR gene is composed of nine exons spanning more than 85 kb on chromosome 6p21.2-p21.1 (Skogsberg et al., 2000), but little is known about PPAR transcriptional regulation. The only report on the transcriptional regulation of the PPAR gene revealed that there are two putative -catenin/ Tcf-4-binding sites located on the promoter (He et al., 1999). Upregulation of PPAR was mediated by -catenin/Tcf-4, and this was identified as one of the mechanisms involved in the initiation of colorectal tumors. Obviously, studying the transcriptional regulation of the PPAR gene will not only explain PPAR gene regulation but will also provide new insights that will define the role of PPAR in obesity, diabetes, atherosclerosis, and cancer.

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The inactivation of PPAR expression by homologous recombination in mice has demonstrated that this receptor is involved in lipid metabolism, like two other PPAR isoforms (Peters et al., 2000). Hence, PPAR null mice were smaller than their control littermates. However, PPAR null mice were not generated with a Mendelian pattern of inheritance among the F2 offspring, suggesting that the absence of PPAR may be lethal. Interestingly, it was reported that PPAR null mice are actually embryonic lethal (>90% frequency) because of defects in placental development (Barak et al., 2002). The controversial results may be resolved by different PPAR genetargeting strategies given that the former was targeted only to the PPAR AF-2 domain, whereas the latter was targeted to most of the PPAR gene. It is well established that targeting of the AF-2 domain abolishes only nuclear receptor ligand binding. Therefore, this truncated PPAR (Peters et al., 2000) may still be able to bind to the PPRE of PPAR target genes and interact with other proteins. In addition, the different mouse strains used to generate PPAR null mice may also contribute to the different phenotypes. Thus, it is clear that PPAR plays a fundamental role in development and differentiation. Generating vascular-specific PPAR knockout mice will provide novel insights concerning PPAR roles in the vasculature.

VIII. CONCLUSIONS The PPARs are now the most extensively studied members of the nuclear hormone receptor family. They have become the therapeutic targets that some believe will have the greatest impact on treating human metabolic diseases since the of discovery PPARs in 1990. To date, fibrate and TZD drugs have been successfully employed in the clinical treatment of hypertriglyceridemia and type II diabetes, respectively. Although certain aspects of the functional roles and mechanisms of PPAR in the cardiovascular system are not well defined in the published literature, it has been generally accepted that activation of both PPAR and PPAR inhibits atherogenesis. Therefore, the development of PPAR and PPAR combination therapy may be effective for treating insulin resistance, hypertension, and dyslipidemia associated with type II diabetes. Ongoing large placebo-controlled clinical trials will determine whether the activation of PPAR and/or PPAR is beneficial or detrimental to the cardiovascular system. The newly identified high-affinity PPAR ligands should be useful in previously hampered PPAR studies. Thus, to provide new insights into the development of novel preventive, diagnostic, and therapeutic approaches to the management of metabolic and cardiovascular diseases, a number of important issues need to be addressed. Such approaches should include (1) investigation of the molecular mechanisms of PPAR action using pharmacological and genetic approaches, (2) global monitoring of PPAR

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target genes in cardiovascular cells, using functional genomics, and (3) identification of the natural hormone ligands and proteins associated with physiological functions of PPARs.

ACKNOWLEDGMENTS The work in our laboratory was partially supported by a starting grant from the Morehouse School of Medicine Cardiovascular Research Institute (Enhancement of Cardiovascular and Related Research Areas, HL03676-02), an institutional grant (NIH/NIHGMS S06GM08248), an NIH grant (R01HL068878; Y.E.C.) and the American Heart Association (Y.E.C.). M.F. (grant 0225214B) and Y.L. (grant 0225323B) are supported by American Heart Association Southeast Affiliate Fellowships.

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lymphocytes: Implications for atherosclerosis and transplantation-associated arteriosclerosis. Circ. Res. 90, 703–710. Matsumoto, K., Hirano, K., Nozaki, S., Takamoto, A., Nishida, M., Nakagawa-Toyama, Y., Janabi, M. Y., Ohya, T., Yamashita, S., and Matsuzawa, Y. (2000). Expression of macrophage (M) scavenger receptor, CD36, in cultured human aortic smooth muscle cells in association with expression of peroxisome proliferator activated receptor-, which regulates gain of M-like phenotype in vitro, and its implication in atherogenesis. Arterioscler. Thromb. Vasc. Biol. 20, 1027–1032. Mehrabian, M., Wong, J., Wang, X., Jiang, Z., Shi, W., Fogelman, A. M., and Lusis, A. J. (2001). Genetic locus in mice that blocks development of atherosclerosis despite extreme hyperlipidemia. Circ. Res. 89, 125–130. Meirhaeghe, A., Fajas, L., Helbecque, N., Cottel, D., Lebel, P., Dallongeville, J., Deeb, S., Auwerx, J., and Amouyel, P. A. (1998). Genetic polymorphism of the peroxisome proliferator-activated receptor gene influences plasma leptin levels in obese humans. Hum. Mol. Genet. 7, 435–440. Miles, P. D., Barak, Y., He, W., Evans, R. M., and Olefsky, J. M. (2000). Improved insulin-sensitivity in mice heterozygous for PPAR- deficiency. J. Clin. Invest. 105, 287–292. Minamikawa, J., Tanaka, S., Yamauchi, M., Inoue, D., and Koshiyama, H. (1998). Potent inhibitory effect of troglitazone on carotid arterial wall thickness in type 2 diabetes. J. Clin. Endocrinol. Metab. 83, 1818–1820. Miwa, Y., Sasaguri, T., Inoue, H., Taba, Y., Ishida, A., and Abumiya, T. (2000). 15-Deoxy12,14-prostaglandin J2 induces G1 arrest and differentiation marker expression in vascular smooth muscle cells. Mol. Pharmacol. 58, 837–844. Moore, K. J., Rosen, E. D., Fitzgerald, M. L., Randow, F., Andersson, L. P., Altshuler, D., Milstone, D. S., Mortensen, R. M., Spiegelman, B. M., and Freeman, M. W. (2001). The role of PPAR- in macrophage differentiation and cholesterol uptake. Nat. Med. 7, 41–47. Nagy, L., Tontonoz, P., Alvarez, J. G., Chen, H., and Evans, R. M. (1998). Oxidized LDL regulates macrophage gene expression through ligand activation of PPAR. Cell 93, 229–240. Nawa, T., Nawa, M. T., Cai, Y., Zhang, C., Uchimura, I., Narumi, S., Numano, F., and Kitajima, S. (2000). Repression of TNF--induced E-selectin expression by PPAR activators: Involvement of transcriptional repressor LRF-1/ATF3. Biochem. Biophys. Res. Commun. 275, 406–411. Neve, B. P., Corseaux, D., Chinetti, G., Zawadzki, C., Fruchart, J. C., Duriez, P., Staels, B., and Jude, B. (2001). PPAR agonists inhibit tissue factor expression in human monocytes and macrophages. Circulation 103, 207–212. Nolan, J. J., Lidvik, B., Beerdsen, P., Joyce, M., and Olefsky, J. M. (1994). Improvement in glucose tolerance and insulin resistance in obese subjects treated with troglitazone. N. Engl. J. Med. 331, 1188–1193. Ogata, T., Miyauchi, T., Sakai, S., Irukayama-Tomobe, Y., Goto, K., and Yamaguchi, I. (2002). Stimulation of peroxisome-proliferator-activated receptor (PPAR) attenuates cardiac fibrosis and endothelin-1 production in pressure-overloaded rat hearts. Clin. Sci. 103, 284S–288S. Ogihara, T., Rakugi, H., Ikegami, H., Mikami, H., and Masuo, K. (1995). Enhancement of insulin sensitivity by troglitazone lowers blood pressure in diabetic hypertensives. Am. J. Hypertens. 8, 316–320. Oliver, W. R.Jr., Shenk, J. L., Snaith, M. R., Russell, C. S., Plunket, K. D., Bodkin, N. L., Lewis, M. C., Winegar, D. A., Sznaidman, M. L., Lambert, M. H., Xu, H. E., Sternbach, D. D., Kliewer, S. A., Hansen, B. C., and Willson, T. M. (2001). A selective peroxisome proliferator-activated receptor agonist promotes reverse cholesterol transport. Proc. Natl. Acad. Sci. USA 98, 5306–5311.

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6 Serotonin and the Neuroendocrine Regulation of the Hypothalamic– Pituitary–Adrenal Axis in Health and Disease N. R. Sullivan Hanley and L. D. Van de Kar Department of Pharmacology, Center for Serotonin Disorders Research, Loyola University of Chicago, Stritch School of Medicine, Maywood, Illinois 60153

I. Overview of Serotonin A. The Discovery of Serotonin B. Anatomy of Serotonergic Pathways C. Serotonin Receptors II. Neuroanatomy of the Hypothalamic– Pituitary–Adrenal Axis A. Hypothalamus B. Pituitary Gland C. Adrenal Gland D. Neural Circuitry That Regulates the Hypothalamic–Pituitary–Adrenal Axis E. Extra Hypothalamic Effects on the Hypothalamic–Pituitary–Adrenal Axis III. Serotonin and the Hypothalamic– Pituitary–Adrenal Axis A. Serotonergic Effects on the Hypothalamic– Pituitary–Adrenal Axis Vitamins and Hormones Volume 66

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B. Hypothalamic–Pituitary–Adrenal Axis Effects on the Serotonergic System IV. Physiological Interactions A. Circadian Rhythm B. Stress V. Pathophysiological Interactions A. Depression B. Anxiety Disorders C. Chronic Fatigue VI. Concluding Remarks References

Serotonin (5-hydroxytryptamine, 5-HT)-containing neurons in the midbrain directly innervate corticotropin-releasing hormone (CRH)containing cells located in paraventricular nucleus of the hypothalamus. Serotonergic inputs into the paraventricular nucleus mediate the release of CRH, leading to the release of adrenocorticotropin, which triggers glucocorticoid secretion from the adrenal cortex. 5-HT1A and 5-HT2A receptors are the main receptors mediating the serotonergic stimulation of the hypothalamic–pituitary–adrenal axis. In turn, both CRH and glucocorticoids have multiple and complex effects on the serotonergic neurons. Therefore, these two systems are interwoven and communicate closely. The intimate relationship between serotonin and the hypothalamic–pituitary–adrenal axis is of great importance in normal physiology such as circadian rhythm and stress, as well as pathophysiological disorders such as depression, anxiety, eating disorders, and chronic fatigue. ß 2003, Elsevier Science (USA).

I. OVERVIEW OF SEROTONIN The serotonergic neurons are located in discrete midline nuclei in the brainstem, termed the raphe´ nuclei. Most ascending serotonergic pathways that innervate the forebrain originate in the dorsal raphe´ nucleus, the median raphe´ nucleus, and a lateral–ventral group of serotonergic neurons known as the B9 cell group (Dahlstrom and Fuxe, 1964).

A. THE DISCOVERY OF SEROTONIN

The first interest in serotonin (5-hydroxytryptamine, 5-HT) began when Stevens and Lee (1884) recognized that there was a substance, in clotting blood, that caused vasoconstriction. Because of its characterization as a ‘‘tonic’’ substance in ‘‘serum,’’ the name serotonin was coined. However,

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more than 60 years would pass before Rapport and colleagues at the Cleveland Clinic (Cleveland, OH) would isolate and purify 5-HT from serum in order to investigate it as a possible cause of high blood pressure (Rapport et al., 1948). At this point Rapport was able to give the first proposed structure of 5-HT (Rapport, 1949). Evidence of the presence of 5-HT in the brain was not available until 1953, during an analysis of various tissues (Twarog and Page, 1953). After this discovery, the role of 5-HT as a chemical messenger in the brain was investigated (Amin et al., 1954; Florey and Florey, 1954; Welsh, 1957), marking the beginning of the neuropharmacological study of 5-HT. B. ANATOMY OF SEROTONERGIC PATHWAYS

With the development of the Falck–Hillarp histochemical technique (Falck et al., 1962), Carlsson and colleagues (1962) demonstrated that after exposing freeze–dried tissue sections to formaldehyde vapors, indoleamine compounds such as serotonin emit a yellow fluorescence. Using this method, Dahlstrom and Fuxe (1964) demonstrated that the highest concentration of 5-HT was found in the brainstem raphe´ nuclei. In their work they described the ascending pathways from the raphe´ as traveling through the medial forebrain bundle to provide the primary serotonergic innervation of the forebrain; they also described descending pathways from the raphe´ to the intermediolateral cell column and the dorsal and lateral horns of the spinal cord. The serotonergic cell bodies located in the midline of the brainstem have been designated B1–B9 (Dahlstrom and Fuxe, 1964). Immunofluorescence techniques have gone further to identify 5-HT cell bodies in the locus coeruleus, subcoeruleus, and the substantia nigra (Liposits et al., 1987b). The primary pathway for serotonergic axons to the forebrain travels through the medial forebrain bundle (Azmitia and Segal, 1978; Steinbusch, 1981; Molliver, 1987; Petrov et al., 1992b). B1–B5 cell groups send projections to the spinal cord and brainstem, while B7–B9 cell groups are the two raphe´ nuclei and ventrolateral mesencephalic cell group that give rise to much of the ascending serotonergic projections to the forebrain (Molliver, 1987). The serotonergic nuclei involved in the regulation of the neuroendocrine system are located in the midbrain and pons. Serotonin fibers extend from the dorsal (B7) and median (B8) raphe´ to the hypothalamus (Azmitia and Segal, 1978; Van de Kar and Lorens, 1979; Van de Kar et al., 1980; Sawchenko et al., 1983). C. SEROTONIN RECEPTORS

In 1979, only one serotonin receptor was believed to exist. Over the next 20 years, however, several serotonin receptors were identified and characterized. The most recent criteria for classification of 5-HT receptors

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were set forth by the International Union of Pharmacology Committee on Drug Classification and Receptor Nomenclature (Hoyer et al., 1994). This rigorous classification led to the modern nomenclature for 5-HT receptors, which now comprises seven families (5-HT1–7) totaling 14 structurally and pharmacologically different receptors (Hoyer et al., 1994; Hoyer and Martin, 1997) (Table I). The criteria are operational, that is, drug-related data such as selective agonists, selective antagonists, and ligand-binding affinities; structural, that is, molecular structure; and transductional, that is, nature of effector system (Humphrey et al., 1993; Hoyer et al., 1994). Much of the interest behind classifying receptors lies in the need to produce more selective drugs. The large family of serotonin receptors can be divided into two distinct families: seven-transmembrane proteins that are G protein-coupled receptors and ligand-gated ion channels. The G protein-coupled receptor family consists of 5-HT1, 5-HT2, 5-HT4, 5-HT5, 5-HT6, and 5-HT7 receptors whereas the 5-HT3 receptors are the sole family consisting of serotoningated ion channels. The G protein-coupled receptor family can be further divided into three diverse families on the basis of their coupling to different G proteins. These families include those that couple to Gi/o/z proteins (5HT1), to Gq/11 proteins (5-HT2), and to Gs protein (5-HT4,6,7). The receptors coupled to Gi/o/z proteins (5-HT1 receptor subtypes) inhibit adenylyl cyclase activity, thus decreasing the formation of cyclic AMP (cAMP). The 5-HT2 receptor subtypes, which couple to Gq/11 proteins, activate phospholipase C, leading to the formation of the two second messengers inositol triphosphate (IP3) and diacylglycerol (DAG). The 5-HT4,6–7 receptor subtypes couple to Gs proteins and activate adenylyl cyclase, leading to an increase in cAMP formation. To date, the coupling of 5-HT5 receptors has not been fully elucidated. The serotonin receptors can also be divided into two groups on the basis of their neuronal location; the receptors can be located on either a target neuron or on the 5-HT neuron itself. Serotonergic receptors expressed by the 5-HT neurons are considered to be 5-HT autoreceptors. Autoreceptors are defined as receptors that respond to the transmitter substance released by their own nerve endings. The serotonergic system has two classes of autoreceptors: somatodendritic (5-HT1A) and presynaptic (5-HT1B/1D). The somatodendritic autoreceptors are defined by their location on the cell bodies and dendrites of serotonergic neurons and are composed of the 5-HT1A receptor subtype. When the somatodendritic 5-HT1A autoreceptors are activated by the release of 5-HT from collaterals of the same neuron or neighboring neurons, there is a decrease in raphe´ cell firing (Barnes and Sharp, 1999). It should be noted that 5-HT1A receptors are also located on target neurons. Presynaptic serotonin autoreceptors are located on serotonergic axon terminals. The 5-HT1B and 5-HT1D receptors are the two 5-HT presynaptic autoreceptors. On activation of these presynaptic

TABLE I. Characterization of Serotonergic Receptors Receptor 5-HT1 5-HT1A

Location

Coupling

Transduction system

Neuronal: primarily in CNS

Gi/Go/Gz

5-HT1B

CNS

Gi/Go

Adenylyl cyclase (–), K+ channel ("), Ca2+ channel (#), MAP kinase (") Adenylyl cyclase (–)

5-HT1D

CNS, vascular smooth muscle

Gi/Go

Adenylyl cyclase (–)

5-HT1E 5-HT1F

CNS CNS

Gi/Go Gi/Go

Adenylyl cyclase (–) Adenylyl cyclase (–)

5-HT2 5-HT2A

5-HT2B 5-HT2C

CNS, vascular smooth Gq/11 muscle, platelets, gastrointestinal tract, lung Stomach, vascular Gq/11 smooth muscle, CNS (?) CNS Gq/11

Agonist

Antagonist

8-OH-DPAT, buspirone, 5-CT, ipsapirone, flesinoxan, gepirone, tandospirone

WAY 100635, WAY 100135, methiothepin, spiperone, pindolol, NAN-190 Sumatriptan, L-694247, RU 24969, SB-224289, SB-236057, 5-CT, CP-93, 129, CP-94, 253 SB-216641, GR 127935, methiothepin Sumatriptan, PNU 109291 BRL 15572, GR 127935, L-694247, RU 24969, 5-CT, methiothepin naratriptan, zolmitryptan 5-HT methiothepin (very weak) LY344 864, LY334 370, methiothepin (very weak) sumatriptan, 5-HT

PI hydrolysis, Ca2+ ("), DOI, DOB, MK-212, quipazine, m-CPP PLA2 ! arachidonic Acid, JAK-STAT (?), MAP kinase (?) PI hydrolysis, Ca2+ (") BW 723C86, -methyl-5-HT, DOI PI hydrolysis, Ca2+ ("), DOI, DOB, PLA2 ! arachidonic Ro 60-0175, m-CPP acid

MDL 100,907, ketanserin, spiperone, mianserin

SB-200646, SB-204741 SB-242084, SB-200646A, SB-206553, mesulergine, RS102221, ritanserin, mianserin Continued

TABLE I.

(Continued )

Receptor

Location

Coupling

5-HT3 5-HT3(A–B) 5-HT3C

CNS, peripheral neurons, gastrointestinal tract

Ligand-gated cation channel

5-HT4 5-HT4(a–h,hb,n) Gastrointestinal tract, CNS, heart, bladder, adrenal gland

Transduction system

Agonist

Antagonist

-Methyl-5-HT, SR 57227, phenylbiguanide

Ondansetron, granisetron, tropisetron, MDL 72222, lerisetron, zacopride

Gs

Adenylyl cyclase (+), Ca2+ channel ("), K+ channel (#)

TS-951, metoclopramide, prucalopride, tegaserod, zacopride, cisapride, BIMU 1, BIMU 8

GR 113808, BJP 118655, SB-207226, SB-207710, SB-204070, SDZ205-557, RS 100235

5-HT5 5-HT6(a–b)

CNS

(?)

Adenylyl cyclase (–) (?)

5-HT, 5-CT, LSD

Methiothepin

5-HT6

CNS

Gs

Adenylyl cyclase (+)

2-methyl-5-HT, 5-HT, LSD

SB-271046, SB-258585, SB-357134, Ro 04-6790, Ro 63-0563, olanzapine, clozapine, methiothepin

5 -HT7

CNS, cardiovascular, gastrointestinal tract

Gs

Adenylyl cyclase (+), Ca2+ (")

5-CT, 5-HT, 8-OH-DPAT

SB-269970, SB-258719, DR 4004, clozapine, methiothepin

5-HT7(a–e)

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autoreceptors, there is a decrease in 5-HT release. All of the 5-HT receptor subtypes are in all likelihood expressed by target neurons.

II. NEUROANATOMY OF THE HYPOTHALAMIC– PITUITARY–ADRENAL AXIS A. HYPOTHALAMUS

The hypothalamus is a division of the diencephalon, the ventral portion of the forebrain. The location of the hypothalamus is above the pituitary gland and ventral to the thalamus, separated from the thalamus by the hypothalamic sulcus in the wall of the third ventricle. The hypothalamus continues from the optic chiasm to the mammillary bodies. The ventral portion of the hypothalamus, which includes the infundibular stalk and mammillary bodies, is visible on the ventral surface of the brain. From rostral to caudal, the hypothalamus can be separated into three regions. The anterior region is the area above the optic chiasm; the mammillary bodies and the area dorsal to the mammillary bodies comprise the posterior region; and the tuberal region is the area between the two. Table II lists the major nuclei found within each hypothalamic region.

TABLE II. Major Hypothalamic Nuclei Found in the Anterior, Tuberal, and Posterior Regions of the Hypothalamusa Region

Medial zone

Lateral zone

Anterior nucleus Medial preoptic nucleus Paraventricular nucleus Suprachiasmatic nucleus Nucleus medianus

Lateral nucleus Lateral preoptic nucleus Magnocellular preoptic nucleus

Tuberal

Arcuate nucleus Dorsomedial nucleus Posterior periventricular nucleus Ventromedial nucleus

Lateral nucleus Lateral tuberal nucleus Supaoptic nucleus

Posterior

Mammillary complex Posterior nucleus Premammillary nucleus Tuberomammillary nucleus

Lateral nucleus

Anterior

Supraoptic nucleus

a The nuclei are further divided into medial or lateral, based on their location relative to the third ventricle

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Hypothalamic inputs originating primarily from the forebrain convey information relevant to the autonomic and somatic facets of affective states. Those that originate from the brainstem and spinal cord convey visceral and somatic sensory information. Hypothalamic efferents include the forebrain, brainstem, spinal cord, pituitary portal vessels in the median eminence, and the posterior (neural) lobe of the pituitary gland. The medial forebrain bundle is a major fiber pathway running longitudinally through the lateral hypothalamus, making several reciprocal connections (Millhouse, 1969; Mizuno et al., 1969; Conrad and Pfaff, 1976). Medial forebrain bundle fibers begin in the olfactory portion of the basal forebrain and the limbic septal nuclei. As the axons pass through the hypothalamus, they project numerous fibers before traveling on to the brainstem; much of the information in this pathway relates to visceral and olfactory information. Conversely, neurons in the brainstem send efferents via the medial forebrain bundle to the hypothalamus. The hypothalamus receives input from the amygdala by way of the amygdalo–hypothalamic pathway (Gray et al., 1989; Gray, 1993; Prewitt and Herman, 1998). The amygdalo–hypothalamic pathway is believed to be involved in the adrenocortical response to a number of somatosensory stimuli (Davis and Whalen, 2001). The key role of this pathway is to process emotional content. The hippocampal–hypothalamic tract, beginning in the hippocampus and traveling to the hypothalamus via the mammillary bodies, shares information among the hippocampus, thalamus, hypothalamus, and cingulate gyrus. The retino–hypothalamic tract sends information originating in the retina to the suprachiasmatic nucleus; this tract conveys information about light and helps regulate the circadian rhythm (Hannibal, 2002). Information traveling from the neocortex passes to the hypothalamus through the cortico–hypothalamic tract (Canteras and Swanson, 1992). The dorsal longitudinal fasciculus sends efferents from the hypothalamus to the midbrain to convey information to the visceral and sympathetic motor neurons (Ban et al., 1975; Bernardis, 1975). The paraventricular nucleus, which is the hypothalamic nucleus most closely associated with the hypothalamic–pituitary–adrenal (HPA) axis, has several afferents. Sensory information arrives from the telencephalic limbic system through the stria terminalis (Moga and Saper, 1994), as well as from the cardiovascular regulatory centers in the hindbrain (Kannan and Yamashita, 1985). Catecholaminergic inputs into the paraventricular nucleus mediate the effects of stress on adrenocorticotropin (ACTH) and glucocorticoid secretion (Van de Kar and Blair, 1999). The hypothalamus receives many of its catecholaminergic inputs as collaterals stemming from catecholaminergic innervations to the central nucleus of the amygdala (Petrov et al., 1993). The arcuate nucleus sends neuropeptide-Y-containing axons (Baker and Herkenham, 1995; Kalra and Kalra, 1996) and -endorphin-containing axons (Russell et al., 1995) directly to the

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paraventricular nucleus. The serotonergic system sends nerve terminals into the paraventricular nucleus as well. The dorsal raphe´, a brainstem nucleus rich in serotonergic cell bodies, sends collateral innervations to both the amygdala and the paraventricular nucleus (Petrov et al., 1992b, 1994). In addition, immunohistochemical studies have shown that serotonergic nerve terminals make direct synaptic contacts with corticotropin-releasing hormone (CRH)-immunoreactive neurons in the paraventricular nucleus (Liposits et al., 1987a). Intrahypothalamic -aminobutyric acid (GABA)ergic neurons innervate CRH neurons to inhibit the activity of the hypothalamic–pituitary–adrenal axis (DiMicco et al., 1996; Herman and Cullinan, 1997). The paraventricular hypothalamic nucleus plays a central role in the regulation of hormone secretion from the pituitary gland. The releasing/ inhibitory factors released from the parvocellular neurons include thyrotropin (TRH), growth hormone-releasing hormone (GHRH), the growth hormone-inhibiting hormone (GHIH, somatostatin), and CRH. CRH, synthesized and released from the paraventricular nucleus (Makara et al., 1981; Olschowka et al., 1982; Bruhn et al., 1984; Reul and Holsboer, 2002), is the primary stimulus activating the HPA axis, increasing the release of adrenocorticotropic hormone (ACTH) from the anterior pituitary gland. Most magnocellular neurons are located in the paraventricular nucleus and supraoptic nucleus. Magnocellular neurons synthesize and directly secrete vasopressin and oxytocin into the systemic circulation via their nerve terminals in the posterior lobe of the pituitary gland. B. PITUITARY GLAND

The pituitary gland is located in the sella turcica, a cavity at the base of the skull, and is connected to the hypothalamus by the infundibular or pituitary stalk. One of the primary responsibilities of the hypothalamus is to control the pituitary gland, which secretes several trophic hormones. The pituitary is composed primarily of anterior and posterior lobes with a small avascular zone located between the two lobes called the pars intermedia (or intermediate lobe). The anterior and posterior lobes of the pituitary play different roles regarding hormone release. Hormones released from the posterior pituitary are not actually synthesized in the pituitary but rather in the hypothalamus. The hypothalamic magnocellular neurons, which are located primarily in the supraoptic and paraventricular nuclei, synthesize and package their hormonal peptides, which travel by axonal flow down the hypothalamic– hypophysial tract through the infundibular stalk into the nerve endings located in the posterior lobe of the pituitary. When an action potential travels from the cells in the hypothalamus to the nerve terminals in the

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TABLE III. Hormones Synthesized and Released from Specific Anterior Pituitary Cells Cell type

Hormone released

Corticotophs Gonadotrophs

Adrenocorticotropinc hormone (ACTH) Follicle-stimulating hormone (FSH) Luteinizing hormone (LH) Prolactin (PRL) Growth hormone (GH) Thyroid-stimulating hormone (TSH)

Lactotrophs Somatotrophs Thyrotrophs

posterior pituitary, oxytocin and vasopressin are released into the systemic circulation. The anterior pituitary however, consists of many different cell types that selectively synthesize and secrete hormones (Table III). The anterior pituitary is the lobe specifically involved in the HPA axis. Corticotrophs located in the anterior lobe of the pituitary synthesize and release ACTH into the circulation. The primary stimulus for ACTH release is CRH, which reaches the pituitary through the pituitary portal vessels. C. ADRENAL GLAND

The adrenal gland comprises the third component of the HPA axis. One adrenal gland is located directly above each kidney. Each adrenal gland is composed of the adrenal medulla surrounded by the adrenal cortex. Located in the core of the adrenal gland, the cells of the adrenal medulla synthesize and release norepinephrine and epinephrine in response to sympathetic nervous system stimulation. The adrenal cortex consists of three layers, listed from most superficial to deepest: zona glomerulosa, zona fasciculata, and zona reticularis. Table IV lists the hormones released by each layer. In regard to the HPA axis, we discuss the actions of ACTH on the zona fasciculata leading to the release of glucocorticoids. D. NEURAL CIRCUITRY THAT REGULATES THE HYPOTHALAMIC–PITUITARY–ADRENAL AXIS

The regulation of the HPA axis is highly integrated. The hypothalamus exerts control of the anterior pituitary, which governs the release of steroid hormones from the adrenal cortex. At each step of the HPA axis there is an amplification of hormone release, and the products of the HPA axis are also able to regulate their own secretion through negative feedback loops (see Fig. 1).

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TABLE IV. Hormones Synthesized and Released from the Different Layers of the Adrenal Cortex Adrenal cortex layer Zona glomerulosa Zona fasciculata Zona reticularis

Hormones released Aldosterone Corticosterone, cortisol androgens Corticosterone, cortisol androgens

FIGURE 1. Summary of the neural circuits involved in regulation of the HPA axis. ACTH, Adrenocorticotropic hormone; BNST, bed nucleus of the stria terminalis; CRH, corticotropinreleasing hormone; 5-HT, serotonin; PVN, paraventricular nucleus.

On stimulation of the parvocellular neurons in the paraventricular nucleus, they release CRH into the portal system. CRH then travels to the anterior pituitary, where it binds to CRH (CRH-R1, type 1) receptors located on corticotrophs. Binding of CRH to the CRH-R1

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receptors activates adenylyl cyclase, to regulate gene expression of proopiomelanocortin (POMC), a prohormone that gives rise to three families of peptide hormones: ACTH, -melanocyte-stimulating hormone (-MSH), and -endorphin. The nature of pro-opiomelanocortin processing varies in different cell locations in the pituitary (Levin and Roberts, 1991). -Melanocyte-stimulating hormone and ACTH are primarily synthesized and secreted from the pars intermedia and anterior lobe, respectively, whereas -endorphin is synthesized and secreted from both lobes. CRH binding specifically results in the cleavage and release of ACTH into the systemic circulation (see Levin and Roberts, 1991). ACTH is the anterior pituitary hormone that controls the size of the adrenal gland and synthesis of glucocorticoids in the adrenal gland (Holsboer and Barden, 1996). ACTH binds with high affinity to specific ACTH receptors located on cells in the adrenal cortex. The ACTH receptor, a member of the melanocortin receptor subfamily, is a seventransmembrane receptor coupled to Gs proteins leading to the activation of adenylyl cyclase (Cone et al., 1993). Exposure to ACTH results in a paradoxical upregulation of ACTH-binding sites in vivo (Durand and Locatelli, 1980) and in vitro (Penhoat et al., 1989; Lebrethon et al., 1994) as well as an increase in ACTH receptor mRNA in vitro (Lebrethon et al., 1994). Two types of receptors have been identified for corticosterone on the basis of their affinity for corticosterone. The type I receptor is similar to the classic mineralocorticoid receptor and it exhibits a higher affinity for corticosterone (Kd ¼ 0.5–1 nM) than the type II receptor. The type II receptor is closer to the classic glucocorticoid receptors and possesses a lower affinity for corticosterone (Kd ¼ 2.5–5 nM) (Veldhuis et al., 1982; Reul and De Kloet, 1985). Both of these receptors also differ in location. The type II receptors are found throughout the brain including the limbic system, frontal cortex, brainstem, pituitary, paraventricular nucleus, and other hypothalamic nuclei (Reul and De Kloet, 1985; Jacobson and Sapolsky, 1991; Deak et al., 1999). The type I receptors, on the other hand, are expressed in fewer brain regions and are limited principally to the limbic system (primarily the hippocampus), pituitary, and a few nuclei in the brainstem (Reul and De Kloet, 1985; Jacobson and Sapolsky, 1991; Deak et al., 1999). Interestingly, type I receptor expression in the paraventricular nuclei is found either in low concentrations or not at all (Reul and De Kloet, 1985; Arriza et al., 1988; Meyer et al., 1998). The HPA axis can also be regulated by its own products in a negative feedback manner. ACTH may act as a negative neuromodulator of the synthesis and secretion of CRH (Suda et al., 1987; Sawchenko et al., 1992). Glucocorticoids not only act to influence the metabolism of proteins, glucose, and fats as well as immune function; they also exert a negative feedback inhibition at almost all levels of the HPA axis

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(Feldman and Weidenfeld, 1995). Glucocorticoids exert their negative feedback inhibition through both the type I and type II receptors. Each receptor may mediate different aspects of the feedback regulation, which is discussed further later with regard to circadian rhythmicity. The actions of glucocorticoids on ACTH secretion can be direct or indirect, by swift (within minutes) or delayed (>2 h) mechanisms (Keller-Wood and Dallman, 1984). Several of the parvocellular neurons in the paraventricular nucleus express not only CRH but also mRNA encoding additional peptide hormones such as vasopressin in rats (Sawchenko et al., 1984a,b; Whitnall, 1988). Vasopressin immunoreactivity was also observed in the parvocellular hypothalamic neurons in humans (Mouri et al., 1993; Raadsheer et al., 1993). Colocalization of CRH and vasopressin within parvocellular neurons may be due to low glucocorticoid levels given that adrenalectomized rats show an increase in coexpression (Sawchenko et al., 1984a). Vasopressin has been identified in the portal blood and is believed to be involved in ACTH secretion (Plotsky, 1991). Vasopressin alone has a weak stimulatory effect on ACTH release, but it may act to potentiate the ability of CRH to stimulate ACTH secretion in vitro (Giguere and Labrie, 1982; Turkelson et al., 1982; Aguilera et al., 1983; Rivier and Vale, 1983) and in vivo (Yates et al., 1971; Graf et al., 1985; von Bardeleben and Holsboer, 1989). In cell culture, the action by which vasopressin can potentiate ACTH release induced by CRH is by way of increasing cAMP levels (Giguere and Labrie, 1982). In the rat, the potentiation of the ACTH-releasing effect of CRH appeared to be greater if it was injected before vasopressin (Graf et al., 1985). In addition, the combination of CRH and vasopressin may be less sensitive to glucocorticoid negative feedback than CRH alone (Holsboer and Barden, 1996). E. EXTRAHYPOTHALAMIC EFFECTS ON THE HYPOTHALAMIC–PITUITARY–ADRENAL AXIS

The amygdala plays a prominent role in the activation and regulation of the HPA axis, especially during stressful situations (see Gray, 1993). Destruction of the amygdala results in a reduction of fear in rats (Campeau and Davis, 1995a,b), and direct stimulation of the amygdala in conscious human subjects elicits strong emotional fear (Chapman et al., 1954). More specifically, the central nucleus of the amygdala appears to be involved in the hypothalamic–pituitary–adrenal stress response. Lesions in the central nucleus of the amygdala inhibit the HPA axis response to conditioned fear stress (Van de Kar et al., 1991a; Campeau and Davis, 1995b; Van de Kar and Blair, 1999). Direct stimulation of the central nucleus of the amygdala in conscious animals results in physiological responses, such as pupillary dilation and increased arterial pressure and heart rate, which are classically

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associated with fear (Kaada, 1951; Iwata et al., 1987). Interestingly, 5-HT in the hypothalamus as well as the amygdala is important for amygdalamediated activation of the HPA axis. The need for 5-HT is demonstrated by a lack of HPA axis response to stressful neural stimulation following 5-HT depletion in either the amygdala or the hypothalamus (Feldman et al., 1998; Feldman and Weidenfeld, 1998). Immunohistochemical studies have located CRH cell bodies within the central amygdala (Swanson et al., 1983; Sakanaka et al., 1987; Gray, 1993). One of the possible targets of the CRH neurons located in the central nucleus of the amygdala includes the paraventricular nucleus (Gray et al., 1989). In turn, a heterogeneous assortment of neurons innervate the CRH cell bodies in the central nucleus of the amygdala, including CRH neurons from the lateral hypothalamus and dorsal raphe´ that project back to the amygdala as well as intrinsic CRH cells in the central nucleus of the amygdala (Gray, 1993). The CRH neurons in the central amygdala also innervate serotonergic neurons in the caudal portion of the dorsal raphe´ nucleus (Price et al., 1998; Kirby et al., 2000). Thus, the amygdala appears to serve as a hub of CRH information in response to stressful situations. The hippocampus is seen mainly as a feedback regulator of the HPA axis. Removing the ability of the hippocampus to interact with the hypothalamus by way of hippocampectomy or lesions in the hippocampus or fornix increases the basal activity of the HPA axis (Herman et al., 1989; Fischette et al., 1980; Sapolsky et al., 1991; see Jacobson and Sapolsky, 1991). Correspondingly, electrical stimulation of the hippocampus results in a decrease in plasma glucocorticoids in cats (Slusher and Hyde, 1961) as well as in humans (Sapolsky et al., 1991; Rubin et al., 1966). Furthermore, microinfusion of glucocorticoid receptor antagonists directly into the hippocampus of rats results in hypersecretion of ACTH (Bradbury and Dallman, 1989). In a stressful situation, it has been postulated that the hippocampus is able to regulate both the peak of stress-induced ACTH release following the activation of the hypothalamic–pituitary–adrenal axis as well as the recovery (Jacobson and Sapolsky, 1991). Rats with a fibersparing kainic acid-induced lesion of cells in the hippocampus responded to restraint stress with an increase in corticosterone concentration twice that of sham animals. However, 1 h poststress, the rats with hippocampal lesions maintained the elevated corticosterone levels, whereas the corticosterone levels of the sham-control animals had returned to basal concentrations (Sapolsky et al., 1984). On the other hand, several investigators report that interrupting hippocampal input by lesions results in an inhibition of glucocorticoid secretion in stressful (Conforti and Feldman, 1976) and unstressful situations (Herman et al., 1989). Similarly, there are reports that electrical stimulation of the dorsal or ventral hippocampus leads to an increase in

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plasma corticosterone concentrations (Smith and Root, 1971; Feldman et al., 1982,1987b). The differences between the view that the hippocampus is inhibitory or stimulatory may result in part from differences in the way the experiments were carried out. In the lesion experiments, there may be variability in recovery time following the production of the lesion prior to the experiment; a longer duration may result in functional recovery of the animal (Fischette et al., 1980; Sapolsky et al., 1991). The extent of the lesion or type of stress may also affect corticosterone levels. For example, Regestein and colleagues (1986) demonstrated that lesions in the posterior hippocampus did not alter the cortisol levels of rhesus monkeys in response to shock avoidance or restraint, as compared with controls; however, near-complete destruction of the hippocampus resulted in an increase in cortisol levels in response to shock avoidance and a decrease in cortisol levels in response to restraint (Regestein et al., 1986). In the experiments involving electrical stimulation, the resulting change in corticosterone levels could result from electrical stimulations in distinctly different areas of the hippocampus. See Jacobson and Sapolsky (1991) for a complete discussion. Neuronal efferents from the hippocampus could influence the HPA axis. However, these connections may not be direct. Although a direct connection from the hippocampus to the paraventricular nucleus has been detected by retrograde transport (Silverman et al., 1981; Sawchenko and Swanson, 1983a,b), this direct connection has not been corroborated by anterograde transport (Sawchenko and Swanson, 1983a,b). If the projections are not direct, it is possible that the hippocampus sends neuronal projections to other brain regions that in turn project to the paraventricular nucleus. Such areas would include the bed nucleus of the stria terminalis, the lateral septum, and the ventromedial hypothalamus (Jacobson and Sapolsky, 1991). The hippocampus expresses both type I and type II glucocorticoid receptors; in fact, the hippocampus has the highest level of glucocorticoid type I receptors within the brain (De Kloet et al., 1975; Reul and De Kloet, 1985; Jacobson and Sapolsky, 1991), making the hippocampus a target for glucocorticoid action. The cells in the hippocampus also express 11hydroxysteroid dehydrogenase, which has been proposed to control the ability of glucocorticoids to regulate their own expression level (Moisan et al., 1990).

III. SEROTONIN AND THE HYPOTHALAMIC– PITUITARY–ADRENAL AXIS The serotonergic system and the HPA axis are able to exert profound effects on one another. In this section, we discuss the changes expressed in one system in response to alterations in the other. The physiological and

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pathophysiological interactions between the two systems are discussed more extensively in Section IV and Section V. A. SEROTONERGIC EFFECTS ON THE HYPOTHALAMIC– PITUITARY–ADRENAL AXIS

Serotonin is a known stimulator of the HPA axis (Dinan, 1996; Fuller, 1996). As mentioned in the previous section, serotonergic nerve terminals make direct synaptic connections with CRH-immunoreactive neurons in the paraventricular nucleus (Liposits et al., 1987a), thus giving anatomical context for the involvement of serotonin in the activation of the HPA axis. The increase in synaptic levels of 5-HT potently stimulates the release of CRH, ACTH, and glucocorticoids (Feldman et al., 1987a; Calogero et al., 1989). Furthermore, electrophysiological recordings coupled with lesion studies have established that the hypothalamic paraventricular nucleus is vital for serotonergic stimulation of ACTH and corticosterone as well as the other pituitary hormones (Kawano et al., 1992; Rittenhouse et al., 1992b,1993,1994; Bagdy and Makara, 1994; Van de Kar et al., 1995). 1. Antidepressant Treatment There are three main classes of antidepressant drugs involving the serotonergic system: monoamine oxidase (MAO) inhibitors that block the metabolism of 5-HT; tricyclic antidepressants that block the reuptake of monoamines; and selective serotonin reuptake inhibitors (SSRIs) that specifically block the reuptake of 5-HT. Each of these drugs acts to increase the amount of 5-HT within the synapse and as a consequence activate presynaptic receptors to control the release of 5-HT and postsynaptic receptors in the hypothalamus to stimulate the release of hormones. Acute treatment with antidepressant drugs may have a different effect on the hypothalamic–pituitary–adrenal axis than chronic antidepressant treatment. For example, acute treatment with clomipramine, which specifically blocks the reuptake of 5-HT, leads to a stimulation of the HPA axis in humans (Laakmann et al., 1984) and rats (Armario and GarciaMarquez, 1987). When clomipramine was given to rats chronically, a tolerance to the drug developed (Armario and Garcia-Marquez, 1987). This dual effect was also observed with SSRI treatment. Several studies in rats have demonstrated that a single injection of the SSRI fluoxetine is able to increase the levels of corticosterone (Fuller et al., 1976; Bianchi et al., 1994). Likewise, acute administration of the SSRI fluoxetine or paroxetine leads to an increase in cortisol levels in humans (von Bardeleben et al., 1989; Reist et al., 1996). Chronic treatment with SSRIs has not led to a consistent change in the basal levels of ACTH or glucocorticoids (Raap and Van de Kar, 1999). These studies indicate that acute effects of these antidepressants may be due to the initial rise in synaptic 5-HT concentrations. After chronic

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treatment with antidepressant drugs, the rise in synaptic 5-HT results in adaptive changes of the serotonergic system as well as other brain systems, leading to a change in receptor signaling. 2. 5-HT Precursors l-Tryptophan and 5-hydroxytryptophan (5-HTP), are the two precursors for 5-HT. The essential amino acid l-tryptophan is converted into 5-HTP, which is then converted to 5-HT by 5-hydroxytryptophan decarboxylase. Infusion of l-tryptophan results in an increase in plasma cortisol levels in humans (Bancroft et al., 1991). Intravenous administration of 5-HTP increases cortisol levels in humans (Power and Cowen, 1992). Likewise, oral administration of 5-HTP to humans also leads to an increase in plasma ACTH levels (Maes et al., 1989) and cortisol levels (Meltzer et al., 1984, 1986, 1997; Jacobsen et al., 1987; Maes et al., 1987, 1989, 1990). Westenberg et al. (1982), however, did not observe a change in cortisol levels following oral administration of 5-HTP or l-tryptophan. 3. 5-HT-Releasing Drugs 5-HT-releasing drugs activate the release of both ACTH and corticosterone. For example, 3,4-methylenedioxymethamphetamine (MDMA, ‘‘ecstasy’’) administration in humans significantly increases plasma ACTH (Grob et al., 1996) and cortisol (Mas et al., 1999) levels. On administration of fenfluramine, there is an associated increase in ACTH (Coccaro et al., 1996) and glucocorticoids (O’Keane et al., 1992; Coccaro et al., 1996; Cleare et al., 1998; Steiner et al., 1999). Other 5-HT-releasing drugs that stimulate ACTH release include p-chloroamphetamine (Fuller, 1992a), 1-(1,3-benzodioxol-5-yl)-2-(methylamino)butane (MBDB), 5-methoxy-6-methyl-2aminoindan (MMAI), and p-methylthioamphetamine (MTA) (Li et al., 1996b). 4. 5-HT1A Receptor Agonists In situ hybridization (Wright et al., 1995; Gundlah et al., 1999; Li et al., 2000) and autoradiograhic (Li et al., 1997a,b; Lu and Bethea, 2002) studies indicate that the 5-HT1A receptors are expressed in the paraventricular nucleus of the hypothalamus. The ability of 5-HT1A receptor agonists to stimulate the release of corticosterone has been well documented. 5-HT1A agonists such as 8hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), buspirone, gepirone, ipsapirone, and LY 165163 increase plasma corticosterone levels in rats (Urban et al., 1986; Koenig et al., 1987,1988; Lorens and Van de Kar, 1987; Raap et al., 2000), and buspirone, gepirone, and ipsapirone also increase plasma cortisol levels in humans (Lesch et al., 1990a,b; Sargent et al., 1997; Schwartz et al., 1999). The increase in corticosterone elicited by 5-HT1A agonists such as 8-OH-DPAT involved CRH neurons within the

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paraventricular nucleus of the hypothalamus (Calogero et al., 1989; Bagdy et al., 1990; Pan and Gilbert, 1992). The effects of 8-OH-DPAT in rats can be blocked by pretreatment with 5-HT1A receptor antagonists such as WAY 100635 and spiperone and the 5-HT1A/-adrenergic receptor antagonists pindolol and propranolol (Koenig et al., 1987; Przegalinski et al., 1989; Vicentic et al., 1998), but not by the 5-HT2 receptor antagonists altanserin, ketanserin, pirenperone, and ritanserin (Koenig et al., 1987; Przegalinski et al., 1989). In humans, the effects of ipsapirone can be blocked by the 5-HT1A/-adrenergic receptor antagonist pindolol (Lesch et al., 1990b). As mentioned in Section I, 5-HT1A receptors are known to couple to the Gi/o protein family. Within the hypothalamus the 5-HT1A receptor specifically couple to Gz, a member of the Gi/o protein family (Serres et al., 2000a). Serres et al. (2000a) demonstrated coupling of hypothalamic 5-HT1A receptors to Gz proteins by injecting Gz antisense oligodeoxynucleotides into rat third ventricles and showing that reduced expression of Gz protein resulted in an inhibition of 8-OH-DPAT-mediated ACTH and oxytocin responses. Gz is the only member of the Gi/o protein family that is pertussis toxin insensitive. When rats were pretreated with pertussis toxin prior to 8-OH-DPAT challenges, ACTH release was not inhibited, oxytocin release was potentiated, and prolactin release was blocked. This suggests that 8-OH-DPAT-induced release of ACTH involves Gz protein and not the other members of the Gi/o protein family (Serres et al., 2000a). Interestingly, 5-HT1A receptor-induced ACTH release is able to undergo heterologous desensitization through 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI)-induced activation of the 5-HT2A receptor; however, the desensitization appears to be distal to Gz protein because there is a lack of GTPSinduced inhibition of 5-HT1A agonist binding (Zhang et al., 2001). 5. 5-HT1B/D Receptor Agonists The 5-HT1B and the 5-HT1D receptors are expressed presynaptically as autoreceptors or postsynaptically to convey serotonergic information to target tissue. Initial studies on the neuroendocrine interaction of the 5-HT1B/ 1D receptor utilized the moderately specific 5-HT1B/1D receptor agonist sumatriptan, used for the treatment of migraine headaches. Sumatriptan administration gave varying results, which may be because it does not readily penetrate the blood–brain barrier (Proietti-Cecchini et al., 1997). For example, Eckland et al. (1992) found that oral administration of sumatriptan led to a reduction of plasma cortisol levels during the first 4 h but to no significant change by 24 h. A transient reduction in ACTH and cortisol levels was confirmed in a following study from the same group (Entwisle et al., 1995). On the other hand, Facchinetti et al. (1994) found an increase in cortisol levels along with no change in prolactin levels following a subcutaneous injection of sumatriptan. Then again, Herdman and

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colleagues (1994) found no change in cortisol levels following a subcutaneous injection of sumatriptan; however, they did observe an increase in growth hormone and a decrease in prolactin levels. Other studies have found that sumatriptan produces an increase in growth hormone levels with no effect on the other anterior pituitary hormones (Franceschini et al., 1994; Coiro et al., 1995; Boeles et al., 1997). With the advent of the newer, more selective 5-HT1B/1D antimigraine drug zolmitriptan, which has greater central nervous system penetration, it became possible to reevaluate the involvement of 5-HT1B/1D receptors within the central nervous system. The first neuroendocrine challenge study involving zolmitriptan found an increase in growth hormone with no change in prolactin levels; there was no report on cortisol concentrations (Whale et al., 1999). Using a lower dose of zolmitriptan, Moeller et al. (2000) also found an increase in growth hormone and no change in prolactin levels; in addition, they report no change in plasma cortisol levels. Given that zolmitriptan has a greater ability to penetrate into the central nervous system (Proietti-Cecchini et al., 1997), it would appear that 5-HT1B/1D receptors do not initiate stimulation of the HPA axis. 6. 5-HT2A/2C Receptor Agonists Autoradiographic (Appel et al., 1990), immunocytochemical (Zhang et al., 2002), and in situ hybridization (Wright et al., 1995; Gundlah et al., 1999) studies indicate that 5-HT2A and 5-HT2C receptors are expressed in the paraventricular nucleus of the hypothalamus. The 5-HT2A and 5-HT2C receptors are well documented as activators of the HPA axis (Koenig et al., 1987; King et al., 1989; Fuller and Snoddy, 1990; Owens et al., 1991; Rittenhouse et al., 1994; Van de Kar et al., 2001). Because there is a lack of sufficiently selective agonists for the 5-HT2 receptors, the determination of specific 5-HT2A or 5-HT2C receptor involvement in hypothalamic–pituitary–adrenal activation has relied on 5HT2 receptor antagonists. For example, the 5-HT2A/2C receptor agonists quipazine and MK-212 are believed to act through the 5-HT2A receptor on the basis of antagonism by the 5-HT2A-selective antagonist MDL 100,907 (Hemrick-Luecke and Fuller, 1996). In rats, the 5-HT2 agonist DOI stimulates the secretion of ACTH as well as corticosterone (Rittenhouse et al., 1994; Van de Kar et al., 2001). The DOI response can be blocked by spiperone (Rittenhouse et al., 1994), suggesting that DOI is acting through 5-HT2A receptors given that spiperone has a higher affinity for 5-HT2A receptors than for the 5-HT2C receptor (Canton et al., 1990). The fact that the selective 5-HT2A receptor antagonist MDL 100,907 completely blocked DOI-induced ACTH and corticosterone secretion gave further evidence that DOI is acting through the 5-HT2A receptor to mediate its neuroendocrine response (Van de Kar et al., 2001). Furthermore, MDL 100,907 was able to block increases in corticosterone secretion brought about by other 5-HT2

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receptor agonists such as quipazine, DOI, and m-chlorophenylpiperazine (m-CPP) (Hemrick-Luecke and Evans, 2002). Selective 5-HT2C antagonists had little effect on the corticosterone response to these 5-HT2 agonists (Hemrick-Luecke and Evans, 2002). As demonstrated by dual immunohistochemical labeling of CRH and c-fos, DOI-induced activation of the HPA axis, through the 5-HT2A receptor, is most likely due to the activation of CRH-containing neurons in the paraventricular nucleus of the hypothalamus (Van de Kar et al., 2001). c-fos is a proto-oncogene (immediate early-action gene) activated on synaptic stimulation (Harbuz et al., 1993). There is also evidence that 5HT2 receptor agonists may act through peripheral receptors to further stimulate corticosterone release (Alper, 1990; Rittenhouse et al., 1994; Welch and Saphier, 1994). Given the 10-fold selectivity of m-CPP for the 5-HT2C receptor (Hoyer, 1988), some researchers assume that the role of the 5-HT2C receptor in the activation of the HPA axis can be determined by administration of m-CPP. Several studies have demonstrated that m-CPP is able to stimulate the release of ACTH and glucocorticoids in humans and rats (Bagdy et al., 1989; Murphy et al., 1989; Kahn et al., 1990b; Seibyl et al., 1991; Meltzer and Maes, 1995b; George et al., 1997; Scheepers et al., 2001). Nevertheless, m-CPP-induced corticosterone secretion could not be blocked by the 5HT2C selective antagonist SB-242084, whereas, as mentioned previously, the 5-HT2A-selective antagonist MDL 100,907 was able to block the m-CPPinduced response (Hemrick-Luecke and Evans, 2002). To date, most evidence supports a role for the 5-HT2A but not 5-HT2C receptors in regulating the HPA axis. 7. 5-HT3 Receptor Agonists A few studies have examined the involvement of 5-HT3 receptors in activation of the HPA axis. Pretreatment of rat primary anterior pituitary cells with the 5-HT3/4 antagonist ICS 205-930 or the more selective 5-HT3 antagonist MDL 72222 blocked 5-HT-induced ACTH release, and the 5HT3 agonist 1-(m-chlorophenyl)-biguanide (m-CPBG) elicited the release of ACTH from the primary cell culture (Calogero et al., 1995). Likewise, intracerebroventricular injection of the 5-HT3 antagonist LY-278584 blocked a 5-HT-induced increase in plasma ACTH levels in rats (Kageyama et al., 1998). In contrast, pretreatment of rats with the 5-HT3 antagonist ondansetron was unable to block 5-HT releaser p-chloroamphetamine (PCA)-induced ACTH and corticosterone release (Levy et al., 1993). Ondansetron was also found to have no effect on ACTH release stimulated by either 5-HT or the combination of 5-hydroxytryptophan and the selective serotonin reuptake inhibitor fluoxetine (Jorgensen et al., 1999). Furthermore, the 5-HT3 receptor agonist 2-methyl-5-HT was found to have either an effect that could not be blocked by ondansetron or no effect on ACTH

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release (Levy et al., 1993; Jorgensen et al., 1999). The lack of effect of central 5-HT3 receptors is not especially unexpected given that there is a relatively low level of expression of 5-HT3 receptors in the hypothalamus (Laporte et al., 1992). 8. 5-HT4 Receptor Agonists The 5-HT4 receptor has been implicated in the release of glucocorticoids; however, 5-HT4 receptor-associated release may not involve the brain or pituitary. Initial studies of frog and human adrenal–cortical slices have demonstrated that the stimulatory effects of 5-HT on adrenal steroidogenesis can be reproduced by the 5-HT4 receptor agonist zacopride, and the effects observed after the combination of zacopride and 5-HT are not additive; together these data suggest that the serotonergic actions are mediated through the 5-HT4 receptor expressed by adrenal cortical cells (Idres et al., 1991; Lefebvre et al., 1992). Yet in vivo, the 5-HT4 receptor agonist zacopride induces the secretion of aldosterone with no effect on cortisol levels (Lefebvre et al., 1993). Furthermore, when the HPA axis is blocked by dexamethasone treatment, aldosterone levels still increase in response to the 5-HT4 agonist zacopride or cisapride, suggesting that the stimulation of aldosterone release is not due to the HPA axis (Lefebvre et al., 1993,1995). One study conducted in conscious male rats has demonstrated that the action of either 5-HT or the combination of 5-hydroxytryptophan with the SSRI fluoxetine produces a dose-dependent increase in ACTH levels (Jorgensen et al., 1999). This effect was attenuated by the 5-HT3/4 antagonists tropisterone but not the selective 5-HT3 antagonist ondansetron, implying that the stimulation of ACTH release could be due to the 5-HT4 receptor (Jorgensen et al., 1999). On the other hand, oral administration of the 5-HT4 agonist zacopride to humans did not elicit an ACTH or cortisol response (Lefebvre et al., 1997). So far, the most convincing evidence suggests that 5-HT4 receptors directly stimulate the release of aldosterone from the adrenal cortex. 9.5-HT7 Receptor Agonists Northern blot analysis, in situ hybridization (Lovenberg et al., 1993; Ruat et al., 1993; Shen et al., 1993), and homogenate binding assays (Sleight et al., 1995; Clemett et al., 1999) indicate that 5-HT7 receptors are expressed in the hypothalamus. Little definitive work has been conducted on the involvement of 5-HT7 receptors in the activation of the HPA axis. This is partly due to the lack of selective 5-HT7 agonists and antagonists. Clemett et al. (1998) probed 5-HT7 receptor involvement by administering 5-HT7 receptor antisense oligodeoxynucleotides directly into rat brain cerebral ventricles. In their study, they demonstrate that there is a reduction in 5-HT7 receptor binding with no associated change in the 5-HT2A receptor. The antisense treatment,

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however, had no effect on basal plasma corticosterone levels or corticosterone levels following a 5-min exposure to the elevated plus maze (stress), suggesting that the 5-HT7 receptor is not involved in the hypothalamic– pituitary–adrenal response to this mild stressor. Koenig et al. (1987) have found similar results showing that the 5-HT7 receptor was not involved in the hypothalamic–pituitary–adrenal response to 8-OH-DPAT by blocking the corticosterone response with the nonselective 5-HT1 antagonist pindolol but not blocking the 8-OH-DPAT response with ritanserin, a 5-HT2 antagonist with moderate affinity for the 5-HT7 receptor (Boess and Martin, 1994). Together, these data suggest that the 5-HT7 receptor is not involved in serotonergic stimulation of the HPA axis. B. HYPOTHALAMIC–PITUITARY–ADRENAL AXIS EFFECTS ON THE SEROTONERGIC SYSTEM

CRH-containing neurons innervate the dorsal and median raphe´ (Austin et al., 1997; Valentino et al., 2001), which in turn serve as the source of serotonergic innervation throughout the forebrain (Dahlstrom and Fuxe, 1964; Azmitia and Segal, 1978). Given this close association, it would seem likely that CRH release will have an effect on the serotonergic system. However, there is much debate on the effects of CRH on the serotonergic system (McAllister-Williams et al., 1998). 1. Effects of CRH on 5-HT CRH administration directly into the amygdala leads to an increase in the accumulation of 5-hydroxytryptophan levels in the amygdala following the inhibition of the aromatic amino acid decarboxylase (Boadle-Biber et al., 1993). An in vivo microdialysis study in the medial hypothalamus and the medial prefrontal cortex demonstrated an increase in extracellular 5-hydroxyindoleacetic acid (5-HIAA) levels following intracerebroventricular CRH administration (Lavicky and Dunn, 1993). In vivo microdialysis provides a direct measure of 5-HT release by measuring the amount of 5-HT released into the extracellular space. There appears to be a different effect on the serotonergic system following chronic versus acute CRH treatment. Within the hippocampus, a dual response to CRH occurs in which acute injection of CRH but not chronic intracerebroventricular injection of CRH increases extracellular 5HT levels in freely moving rats (Linthorst et al., 1997). Price and colleagues (1998) demonstrated by in vivo microdialysis that intracerebroventricular CRH administration has a biphasic effect on extracellular 5-HT within the striatum of freely moving rats; lower doses of CRH (0.1–0.3 g) decrease 5-HT levels and higher doses of CRH (3 g) increase 5-HT levels. However, the increase in 5-HT levels in the striatum following the higher dose of CRH was not confirmed in a later study (Price and Lucki, 2001). Interestingly, in

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roughskin newts, intracerebroventricular injections of corticosterone, but not CRH, led to an increase in the levels of 5-HT and 5-HIAA in the dorsomedial hypothalamus, a hypothalamic center involved in neuroendocrine responses to stress (Bailey and DiMicco, 2001; Lowry et al., 2001; DiMicco et al., 2002). Studies examining directly the activation of dorsal raphe´ firing have been divided. Investigators have shown that intracerebroventricular and intraraphe´ administration of CRH leads to an inhibition of 5-HT release (Price et al., 1998; Kirby et al., 2000; Price and Lucki, 2001). However, Lowry and associates (2000) have demonstrated that CRH is able to induce serotonergic firing in rat dorsal raphe´ slices. The stimulatory effect of CRH on serotonergic neurons within the midline raphe´ has been confirmed in vivo with roughskin newts (Lowry et al., 1996). 2. Effects of Glucocorticoids on 5-HT Glucocorticoids are also able to affect the serotonergic system. Glucocorticoids can affect tryptophan catabolism, increase precursor availability, as well as increase the synthesis of 5-HT (see McAllisterWilliams et al., 1998). Glucocorticoids also have an effect on some serotonin receptors as determined after either adrenalectomy or treatment with glucocorticoid agonists. In the majority of cases, 1 day to 3 weeks following an adrenalectomy there was an increase in postsynaptic 5-HT1A receptor number or mRNA expression in the hippocampus (Mendelson and McEwen, 1992; Chalmers et al., 1993; Kuroda et al., 1994; Le Corre et al., 1997; Neumaier et al., 2000). In one study, however 2 weeks after adrenalectomy, there was a decrease in 5-HT1A receptor mRNA expression in the dentate gyrus that was reversed by dexamethasone treatment (Liao et al., 1993). In the majority of studies, treatment with either aldosterone or a low dose of corticosterone was able to reverse adrenalectomy-induced increase in expression of 5-HT1A receptors or mRNA in the hippocampus (Mendelson and McEwen, 1992; Chalmers et al., 1993; Kuroda et al., 1994; Neumaier et al., 2000). Acute and chronic corticosterone treatment resulted in a decrease in 5-HT1A receptors in the hippocampus (Fernandes et al., 1997; Takao et al., 1997). Nearly all studies reviewed found that somatodendritic 5-HT1A receptor density or mRNA expression does not change following adrenalectomy or corticosterone treatment (Tejani-Butt and Labow, 1994; Holmes et al., 1995a,b; Le Corre et al., 1997; Neumaier et al., 2000). One study, however, found a decrease in dorsal raphe´ 5-HT1A receptor expression within 2 weeks of adrenalectomy (Tejani-Butt and Labow, 1994). In the CA1 and CA3 region of the hippocampus, high doses of corticosterone led to a decrease in 5-HT1A receptor-mediated response (Mueller and Beck, 2000; Okuhara and Beck, 1998). Neumaier et al. (2000) found no change in pre- or postsynaptic 5-HT1B receptor mRNA following adrenalectomy or glucocorticoid

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treatment whereas Mendelson and McEwen (1992) found an increase in 5-HT1B receptor expression following adrenalectomy, which could be reversed by corticosterone treatment. High glucocorticoid levels affect the 5-HT2A receptor. Adrenalectomy does not change the density of 5-HT2A receptors in the cortex or hypothalamus (Kuroda et al., 1992, 1994; Holmes et al., 1995b; Chaouloff et al., 1993). In contrast, chronic corticosterone treatment of rats leads to an upregulation of 5-HT2A receptor expression in the cortex (Kuroda et al., 1992; Fernandes et al., 1997; Takao et al., 1997). Adrenalectomy produces an increase in 5-HT2C receptor mRNA in the hippocampus (Holmes et al., 1995b). Hippocampal 5-HT6 and 5-HT7 receptors are also affected by a lack of glucocorticoids. For example, in rats 5-HT6 receptor mRNA is upregulated in the CA1 region of the hippocampus following chemical adrenalectomy, which can be reversed with corticosterone replacement (Yau et al., 1997). Le Corre and colleagues (1997) found an increase in 5-HT7 mRNA in the CA1 and CA3 regions of the hippocampus following adrenalectomy, whereas Yau et al. (1997) found only an increase in the CA3 region.

IV. PHYSIOLOGICAL INTERACTIONS This section describes the physiological importance of serotonin in the HPA axis.

A. CIRCADIAN RHYTHM

The circadian rhythm is the biological activity pattern of an organism during 24 h. A number of neuronal areas inside the brain behave as circadian clocks entrained by the light/dark cycle. However, the driving force, which organizes the various internal clocks that are engineered to oscillate in a circadian manner, is located in the suprachiasmatic nucleus within the basal hypothalamus. The suprachiasmatic nucleus receives important information about changes in light and dark from the retina via the retino–hypothalamic tract. Furthermore, the autonomic nervous system, via the superior cervical ganglion and other neural structures, regulates the pineal gland and its secretion of melatonin. In turn, melatonin regulates the suprachiasmatic nucleus. The suprachiasmatic nucleus also receives neuroendocrine information from other hypothalamic nuclei by way of intrahypothalamic connections. The suprachiasmatic nucleus sends outputs to hypothalamic nuclei to synchronize the activity of the hypothalamus with the light/dark cycle (Raisman and Brown-Grant, 1977; Moore, 1980; and see Angeli et al., 1992).

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1. Circadian Rhythm of the Hypothalamic–Pituitary–Adrenal Axis The concentration of hormones in the blood changes with the time of the day. Glucocorticoids are released in a rhythmic fashion (Bradbury et al., 1991; Angeli et al., 1992). In humans, plasma cortisol levels rise in sporadic bursts with periods of quiescence (Llorente et al., 1996; Van Cauter and Buxton, 2001; Mormon et al., 2002). In diurnal mammals, such as humans, cortisol levels are at their highest point in the early morning hours and their lowest levels occur in late evening (Krieger et al., 1971; Van Cauter and Buxton, 2001). Some studies have pinpointed the peak of ACTH and glucocorticoids between the first and the second rapid eye movement (REM) stages (Kupfer et al., 1983), whereas others generalize the cortisol peak to occur during non-REM sleep (Born et al., 1986). The suprachiasmatic nucleus sends out multiple efferents to other nuclei in the hypothalamus with clear termination in the paraventricular nucleus (Swanson and Cowan, 1975). In the rat, destruction of the suprachiasmatic nucleus results in a loss of corticosterone level circadian peak in the adrenal gland (Moore and Eichler, 1972) and in plasma (Abe et al., 1979; Buijs et al., 1999). Others found only a change in plasma ACTH levels without a change in corticosterone levels (Szafarczyk et al., 1979). Following the dissection of fiber connections in the anterior and lateral hypothalamus or lesions in the medial basal hypothalamus, the ACTH or corticosterone peak is no longer observed (see Bradbury et al., 1991). The suprachiasmatic nucleus contains vasopressinergic neurons (Swaab and Pool, 1975; Van Leeuwen et al., 1978), and the levels of vasopressin fluctuate along with the circadian rhythm in the rat (George and Jacobowitz, 1975; Noto et al., 1983). Vasopressin was postulated to act as a neurotransmitter in the suprachiasmatic nucleus communicating with the HPA axis (Angeli et al., 1992). As mentioned previously, glucocorticoids exert negative feedback on the HPA axis by binding to either the type I or type II receptors. On the basis of an observed shift to the right for steroid-induced inhibition of ACTH secretion, researchers have proposed that the inhibition of ACTH release during the nadir, or lower trough, of glucocorticoids during the circadian rhythm is due to the occupancy of the high-affinity type I receptor and that the negative feedback during the glucocorticoid peak is due to occupancy of the lower affinity type II receptor (Reul and De Kloet, 1985; Levin et al., 1987; De Kloet et al., 1993). Some researchers have proposed that the type I receptor is involved in the negative feedback at all times of the circadian rhythm (Dallman et al., 1989; Young et al., 1998). The type I receptor participates in the regulation of the HPA axis during the circadian peak and nadir in humans (Young et al., 1998). Blocking the type I receptor with the aldosterone receptor (i.e., type I) antagonist spironolactone prevents cortisol feedback inhibition, resulting in elevated cortisol levels (Young et al., 1998).

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Other investigators demonstrated that the type I receptors act to potentiate type II receptor-induced regulation of cortisol during the circadian peak (Bradbury et al., 1994; Spencer et al., 1998). 2. The Effect of Serotonin on the Circadian Rhythm of the Hypothalamic– Pituitary–Adrenal Axis A role for 5-HT in circadian rhythm has been postulated because the suprachiasmatic nucleus receives input from the midbrain raphe´ (Dudley et al., 1999). Serotonin levels in the brain rise and fall with a circadian rhythm (Albrecht et al., 1956; Scheving et al., 1968). However, the role that 5-HT plays in circadian ACTH and glucocorticoid surges has been controversial. The daily rise and fall of glucocorticoids is paralleled by a change in 5-HT levels in the brain (Dixit and Buckley, 1967; Scapagnini et al., 1971). Scapagnini and co-workers (1971) found that the brain regions in which the rise and fall in 5-HT content mirrored the diurnal corticosterone levels in rats were in the hippocampus and amygdala. These observations suggest that the biological clock responsible for 5-HT-induced ACTH secretion may be located outside of the hypothalamus. Indeed, lesions in the hippocampus, by medial fornix ablation, disrupt circadianinduced changes in plasma corticosterone (Fischette et al., 1980). Depletion of 5-HT levels with systemic injections of the 5-HT synthesis inhibitor p-chlorophenyl alanine (PCPA) abolished the daily corticosterone rhythm (Scapagnini et al., 1971). In support of the positive role of 5-HT in the activation of the HPA axis, lesions aimed at the destruction of 5-HT cells located in the dorsal raphe´ abolished the circadian rhythm of corticosterone release (see Scapagnini and Preziosi, 1972). Microinjections of the serotonergic neurotoxin 5,7-dihydroxytryptamine (5,7-DHT) into rat suprachiasmatic nucleus blocked the diurnal rhythm of corticosterone as compared with vehicle-treated rats (Williams et al., 1983) and prevented the beginning of a corticosterone diurnal rhythm in 16-day-old male rats (Banky et al., 1986). Other investigators have demonstrated that 5-HT has no effect on the diurnal rhythm of the hypothalamic–pituitary–adrenal axis (Dixit and Buckley, 1969; Bhattacharya and Marks, 1970; Rotsztejn et al., 1977). For example, Rotsztejn et al. (1977) demonstrated that lesions in the dorsal and median raphe´ nuclei and treatment with PCPA, which both resulted in a significant reduction in whole brain 5-HT levels, did not affect the rhythmic changes in corticosterone in rats. The disparity of these results with those of Scapagnini et al. (1971) may be due to incomplete lesions or to the length of time elapsed between creation of the lesion and obtaining of the corticosterone results. A circadian rhythm of 5-HT receptor density has been described for 5-HT1 and 5-HT2 receptors in the frontal cortex (Akiyoshi et al., 1989; Weiner et al., 1992) and for 5-HT1 and 5-HT2C receptors in the

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hippocampus (Weiner et al., 1992; Holmes et al., 1995a). Others have not found a circadian change in the density of 5-HT receptors in the brainstem, frontal cortex, or hypothalamus as measured by 0.8 nM [3H] spiperone, which binds with high affinity to 5-HT1, 5-HT2, and dopamine D2 receptor sites (Di Lauro et al., 1986). Throughout the ventral hippocampus, the 5-HT2C receptor mRNA level has been shown to be higher when 5-HT and plasma corticosterone levels are low (Holmes et al., 1995a,b, 1997). The rhythmic expression of 5-HT2C receptor mRNA in the hippocampus is not sensitive to adrenalectomy, suggesting that circadian changes in 5-HT2C mRNA expression in the hippocampus are not a result of glucocorticoid circadian changes (Holmes et al., 1995b). However, 5-HT2C receptor mRNA expression is sensitive to elevated glucocorticoids and stress, possibly an adaptive response to desensitize the 5-HT2C receptor in response to chronic stress (Holmes et al., 1995a, 1997). 5-HT2C receptor mRNA expression is highest in the CA1 and subiculum (Holmes et al., 1995a,b, 1997). Both send projections to the paraventricular nucleus, some of which travel thorough the bed nucleus of the stria terminalis (Kiss et al., 1983; Herman et al., 1994). Given that the pathway from the bed nucleus of the stria terminalis is inhibitory (Herman et al., 1994), it is possible that changes in the expression of 5-HT2C receptors may have an effect on the HPA axis via this pathway. As mentioned in Section III.A.6, 5-HT2 receptor agonist-induced regulation of the HPA axis is mediated through 5-HT2A receptors, rather than 5-HT2C receptors. Because the 5-HT2A receptor, unlike the 5-HT2C receptor, does not appear to have a circadian rhythm of expression in the hippocampus or hypothalamus (Di Lauro et al., 1986; Weiner et al., 1992; Holmes et al., 1995a, 1997), perhaps the 5-HT2A receptor-mediated response is more apparent when the experiments are conducted. B. STRESS

Stress can be described as the response to a condition that is capable of disrupting homeostasis. Stressors, conditions that jeopardize or are perceived to jeopardize survival, fall into three broad categories: stressors involving a cardiovascular challenge such as hemorrhage, stressors involving a physical stimulus with a strong psychological element such as pain, and stressors involving a psychological response to an aversive condition such as anxiety. All three stressors lead an organism to respond in a broad manner, which Selye (1936) refers to as the ‘‘general adaptation syndrome.’’ The HPA axis serves as a messenger from the brain to the rest of the body, and plasma glucocorticoid levels are a revealing sign that an organism is undergoing stress. The involvement of 5-HT in the response of an organism may be stressor dependent (Fuller, 1992b).

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1. Neuroanatomy of Stress Cardiovascular stressors primarily rely on information regarding blood volume and pressure originating in baroreceptors, found in the wall of the carotid sinus, and the aortic arch and the atrial stretch receptors in the walls of both atria. A decrease in firing frequency signals a drop in arterial blood pressure or atrial pressure, which signals the release of ACTH and glucocorticoids (Baertschi et al., 1976; Anderson et al., 1994, 1995). The nucleus tractus solitarius sends reciprocal projections to areas such as the caudal raphe´ nuclei (raphe´ obscurus, raphe´ pallidus, raphe´ magnus), periaqueductal gray matter, and the paraventricular and lateral hypothalamus (Loewy and Burton, 1978; Thor and Helke, 1987; Loewy, 1990). Many brain structures are involved in the response to psychologically stressful stimuli. In this review, we focus on the hypothalamic–pituitary– adrenal aspect of the response to stress; however, more thorough reviews of neuroendocrine response to stress have been published elsewhere (Van de Kar et al., 1991b; Van de Kar and Blair, 1999; Sapolsky et al., 2000; Pacak and Palkovits, 2001). Stimuli from conditioned and unconditioned stressors pass through either the reticular activating system or the thalamus before the sensory input is then relayed to the amygdala and sensory cortex (Pezzone et al., 1992; LeDoux, 1995; Bhatnagar and Dallman, 1998; Van de Kar and Blair, 1999). The information from the neocortex is then sent to the basolateral nucleus of the amygdala (Davis et al., 1994; LeDoux, 1995; Van de Kar and Blair, 1999). In the case of learned psychological stressors such as conditioned fear, the information from the basolateral and lateral nuclei of the amygdala is communicated to the central amygdaloid nucleus and transmitted to the CRH neurons in the paraventricular nucleus either directly or via the bed nucleus of the stria terminalis (Weller and Smith, 1982; Moga et al., 1989; Cullinan et al., 1993; Gray et al., 1993; Herman et al., 1994; Van de Kar and Blair, 1999). In addition to the relay from the amygdala, the ACTH response also requires neural inputs from the serotonergic neurons in the dorsal raphe´ nucleus as well as A1, A2, C1, and C2 (nor)adrenergic cell groups located in the brainstem. These brain regions have reciprocal projections with the amygdala (Uryu et al., 1992; Wallace et al., 1992; Petrov et al., 1992a, 1993, 1994). 2. Hemorrhage Stressors that influence cardiovascular function include exercise, heat exposure, and hemorrhage. These cardiovascular stressors can elicit a neuroendocrine response such as an increase in vasopressin release, which results in vasoconstriction and water retention. These stressors also increase plasma renin levels and formation of angiotensin II and III that lead to the constriction of arterioles in order to raise vascular resistance in the face of decreasing blood volume. Cardiovascular stressors can also bring about the

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release of oxytocin, prolactin, and ACTH. For the sake of this discussion, the HPA axis is the focus whereas the other neuroendocrine responses are reviewed extensively elsewhere (Matzen, 1995; Van de Kar and Blair, 1999; Pacak and Palkovits, 2001). Hemorrhage is a potent cardiovascular stressor. As an organism undergoes hemorrhage, the body begins to initiate compensatory actions to maintain blood flow to the brain and to counteract the ensuing blood loss. The overall response to the decrease in blood volume can be characterized in two distinct stages (Schadt and Ludbrook, 1991). In the first stage, the sympathetic nervous system is activated and leads to increased heart rate and contractility to maintain mean arterial pressure. This is termed the normotensive stage. If blood loss continues, the normotensive stage is then followed by a hypotensive stage resulting from the protracted hemorrhage. In the hypotensive stage, there is a decrease in heart rate and mean arterial pressure due to vasodilatation. ACTH is released in response to hypotensive hemorrhage (Matzen, 1995). The hypothalamic–pituitary response is believed to be stimulated by cardiopulmonary and arterial baroreceptors. Lesion studies indicate that the nucleus tractus solitarius, dorsal rostral pons, caudal ventrolateral medulla, and paraventricular nucleus of the hypothalamus play an important role in ACTH release in response to hemorrhage (see Matzen, 1995). In rats, intravenous infusion of 5-HT results in three phases of cardiovascular activity (Fozard, 1982; Kalkman et al., 1984; De Vries et al., 1997). The first phase is a depressor phase and bradycardia, mediated by 5-HT3 receptors. The second phase is a pressor response, initiated by 5HT2 receptors. The last phase is a hypotensive phase related to 5-HT1-like or 5-HT7 receptor activation (Kalkman et al., 1984; De Vries et al., 1997). The involvement of 5-HT in hemorrhage has been studied with the headup tilt model. Head-up tilt is an experimental model used to study hemorrhage in human volunteers in a relatively noninvasive manner (Matzen et al., 1993; Matzen, 1995). In this model, subjects are slowly tilted 50 degrees to mimic blood loss, thus causing the subjects to undergo both cardiovascular stages of hemorrhage. Likewise, the neuroendocrine responses of the head-up tilt mimic hemorrhage (Matzen et al., 1993; Matzen, 1995); therefore, neuroendocrine responses to various drugs can be measured. Administration of methysergide, the 5-HT2 receptor antagonist ketanserin (which is also an 1-adrenergic antagonist), and the 5-HT3 receptor antagonist ondansetron had no effect on head-up tilt-induced plasma ACTH or cortisol levels (Matzen et al., 1993). Although animal studies have demonstrated an interaction between 5-HT and cardiovascular reflexes during hypovolemia (Matzen et al., 1993), it appears that the actions of 5-HT are not mediated by these particular 5-HT receptors. However, the lack of effect of these 5-HT receptor antagonists may be due in part to a heterogeneous expression of 5-HT receptors as well as a lack of

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receptor-specific antagonists available for human investigation. Alternatively, the cardiovascular responses to hemorrhage may be mediated by a different family of 5-HT receptors. 3. Hypoglycemia The brain requires glucose for the production of energy. Hypoglycemia brings about a rapid neurophysiological change, the most severe being loss of consciousness and seizures. The HPA axis and other effector systems respond to glucoprivation by increasing peripheral plasma glucose levels, thereby delivering glucose to the brain. Hypoglycemia can be induced in normal human subjects by a challenge infusion with insulin, which causes cells to take in glucose, leading to a decrease in plasma glucose levels. Insulin-induced hypoglycemia also leads to increased secretion of epinephrine, glucagon, growth hormone, prolactin, vasopressin, CRH, ACTH, and cortisol in humans (Kletzky et al., 1980; Watabe et al., 1987). An increase in 5-HT content in the hypothalamus is also evident following insulin-induced hypoglycemia (Gordon and Meldrum, 1970). However, evidence of 5-HT involvement in ACTH and glucocorticoid release in conjunction with hypoglycemia has been contradictory. For example, when Kletsky et al. (1980) gave the serotonin antagonist cyproheptadine (also a histamine antagonist) to normal human volunteers prior to insulin-induced hypoglycemia, there was no inhibition of the secretion of prolactin, growth hormone, or cortisol. In contrast, when Plonk et al. (1974) administered cyproheptadine, they observed a partial blockade of cortisol release in response to hypoglycemia, and when they used the 5-HT antagonist methysergide, there was no significant blockade of the cortisol response. Much of the controversy stems from inadequate pharmacological tools to examine the involvement of 5-HT in humans. For instance, cyproheptadine possesses anticholinergic, antidopaminergic, and antihistaminic properties (Gilbert and Goldberg, 1975; Stone et al., 1961). Methysergide is a 5-HT2 antagonist but also a dopamine D2 and 5-HT1A receptor agonist (Hoyer et al., 1994). Treatment with the selective serotonin reuptake inhibitor (SSRI) fluoxetine had no effect on the ACTH response to hypoglycemia (Fuller and Snoddy, 1977). In contrast, Prescott et al. (1984) found that the 5-HT2 antagonist (and -antagonist) ketanserin inhibited the ACTH response to hypoglycemia by 30%. More recently, Weidenfeld and colleagues (1994) utilized 2-deoxyglucose to decrease intracellular glucose in order to probe the interaction of 5-HT and the hypothalamic–pituitary–adrenal axis. In this experiment, intracerebroventricular injections of ketanserin completely inhibited the 2-deoxyglucose-induced increase in ACTH and cortisol, suggesting that 5-HT2 receptors are involved in the hypoglycemia-induced hypothalamic–pituitary–adrenal axis response.

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4. Foot Shock and Conditioned Stress Several animal models have been employed to imitate human psychological stress. Conditioned fear and immobilization are two paradigms used to study HPA axis responses to ‘‘psychological’’ stress. Foot shock alone leads to an increase in ACTH and corticosterone (Kant et al., 1983; Paris et al., 1987; Saphier and Welch, 1995). The conditioned fear model trains the animal to associate a noxious stimulus with a neutral stimulus. For example, a foot shock (noxious stimulus) can be paired with a light or simply the placement of a rat in a chamber (neutral stimulus) and eventually the foot shock becomes paired with the light or the chamber (neutral stimulus) in which the shock occurs. Once the two stimuli are associated by the experimental animals, the neutral stimulus will trigger the release of stress hormones. This protocol is known as conditioned fear or conditioned stress response (Fendt and Fanselow, 1999). The conditioned fear response leads to the release of ACTH and corticosterone (Paris et al., 1987) (Rittenhouse et al., 1992a; Gray et al., 1993; Saphier and Welch, 1995; Zhang et al., 2000). Chronic treatment of rats with fluoxetine decreased stress-induced behavior but did not inhibit the neuroendocrine responses to conditioned fear (Zhang et al., 2000). The effects of foot shock on 5-HT levels in the brain have been controversial; the results range from no change in 5-HT content (Paris et al., 1987) to an increase in 5-HT metabolism (Driscoll et al., 1983; Dunn, 1988, 2000; Saphier and Welch, 1995). Photic stimulation is another physiological stressor involving exposure to frequent flashes of light. Feldman et al. (1998) demonstrated that the serotonergic system within the amygdala is necessary for activation of the hypothalamic–pituitary–adrenal axis following photic stimulation. They also demonstrated that 5-HT2 receptors within the amygdala are involved in the stress-induced response when they observed that direct injections of the 5-HT2 receptor antagonist ketanserin into the amygdala inhibited the corticosterone response to photic stimuli. 5. Immobilization Stress Immobilization stress is a ‘‘psychological’’ stressor that, like conditioned fear, leads to the secretion of ACTH and corticosterone (Beaulieu et al., 1986; Rittenhouse et al., 1992a; Gaillet et al., 1993; Gray et al., 1993); however, immobilization stress lacks a learned component. The role of central 5-HT in immobilization stress has been debated. Many investigators found an increase in 5-HT metabolism (Shimizu et al., 1989; Dunn, 1999) as well as 5-HT content in the limbic system (Shimizu et al., 1989; Vahabzadeh and Fillenz, 1994), which accompanies immobilization stress. Others have found no change in 5-HT metabolism in the hypothalamus or amygdala (Morgan et al., 1975; Beaulieu et al., 1986). When rats are given the amino acid valine, which competes with the uptake of the 5-HT precursor

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tryptophan, immobilization-induced increase in plasma corticosterone is prevented; the authors conclude that the response of 5-HT to stress is at least partially dependent on the rise of brain tryptophan (Kennett and Joseph, 1981). Likewise, the ACTH response to immobilization stress can be potentiated by blocking the reuptake of 5-HT with fluoxetine and blocked by the nonselective 5-HT receptor antagonist metergoline as well as the 5-HT2C/2A receptor antagonist cinanserin (Bruni et al., 1982). Yet, a reduction in 5-HT content with the serotonin synthesis inhibitor PCPA did not alter immobilization-induced ACTH secretion, nor did it prevent immobilization-induced expression of c-fos mRNA in the hypothalamic paraventricular parvocellular neurons (Harbuz et al., 1993). c-fos is a protooncogene (immediate early-action gene) activated on synaptic stimulation. To summarize, serotonergic neurons play more of a modulatory than a principal role in stress-induced activation of the HPA axis. This is not entirely surprising considering the importance of glucocorticoids for survival. It is more than likely that multiple neurotransmitter circuits act in a redundant manner to guarantee appropriate secretion of glucocorticoids during life-threatening conditions.

V. PATHOPHYSIOLOGICAL INTERACTIONS Chronic activation or dysregulation of the HPA axis can lead to pathophysiological stress-related conditions such as depression, anxiety, and chronic fatigue syndrome. A. DEPRESSION

Depression should be viewed as a collection of symptoms rather than a disease. Depressive symptoms are precursors of other diseases such as coronary artery disease or sleep apnea (Yantis, 1999; Appels et al., 2000). In addition, stress and depression are closely linked (Kessler, 1997; Gold and Chrousos, 2002). The onset of depressive episodes frequently occurs following psychologically as well as physically stressful events, and depression can bring about or aggravate stressful life events (Post, 1992; Kessler, 1997). 1. The Serotonergic System and Depression The etiology of depression is not fully understood. Research over the past few decades has implicated monoamine dysfunction as a possible cause for depressive symptoms (Delgado, 2000). The fact that not all depressed patients respond to the same antidepressant treatment suggests that dysfunction of many mechanisms or neuronal pathways may be responsible for the precipitation of depressive symptoms.

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Evidence implicating a serotonergic deficiency in depression includes the fact that SSRIs, such as fluoxetine and paroxetine, are as effective as the tricyclic antidepressants for the treatment of depressive symptoms (Hirschfeld, 1999). Furthermore, patients responding to imipramine or the MAO inhibitor tranylcypromine relapsed on treatment following the administration of the serotonergic synthesis inhibitor p-chlorophenyl alanine (PCPA) (Shopsin et al., 1975, 1976), demonstrating that serotonin is necessary to maintain the antidepressant effects in these patients. Likewise, diets deficient in the 5-HT precursor tryptophan can transiently reverse antidepressant therapeutic effects (Delgado et al., 1990; Heninger et al., 1992; Bremner et al., 1997a; Fadda, 2000). In clinical trials in nonmedicated depressed patients, a low-tryptophan diet generally does not exacerbate symptoms (Delgado et al., 1994; Heninger et al., 1996). 2. The Hypothalamic–Pituitary–Adrenal Axis and Depression Hyperactivity of the HPA axis is a consistent observation among many depressive patients. Given this observation, several groups associating the dysregulation of the hypothalamic–pituitary–adrenal axis to the causality of depression believe antidepressants may act to normalize the function of the HPA axis (see Holsboer and Barden, 1996; Holsboer, 2001; Pariante and Miller, 2001). The clinical observations leading to this correlation include an increase in CRH-secreting neurons within the limbic region (Raadsheer et al., 1994), an increase in CRH levels in the cerebrospinal fluid (Traskman et al., 1980; Nemeroff et al., 1984), a decrease in CRH-binding sites within in the frontal cortex in response to increased CRH levels (Nemeroff et al., 1988), and an increase in cortisol levels in plasma (Gibbons, 1964; Fang et al., 1981) and urine (Kathol et al., 1989). An apparent lack of glucocorticoid-induced negative feedback inhibition of the HPA axis in depression has implicated glucocorticoid receptor impairment as a possible cause for HPA axis hyperactivity as well as depression. Much of the research regarding depression and glucocorticoid receptors has focused on the type II receptor, given that type II receptor activation is necessary for HPA axis feedback regulation when glucocorticoid levels are high (De Kloet et al., 1998). This is the case for patients with major depression. Some studies have pointed to genetic alteration of the type II receptor as a possible cause of depression. People who have a high familial risk for depression might inherit a mutated type II receptor (Modell et al., 1998). However, to date five novel polymorphisms of the type II receptor gene have been identified, but no specific variant of type II receptor has been found to be associated with depression (Koper et al., 1997). The type I receptor should not be neglected in depression research because it is now apparent that even at the higher levels of glucocorticoids at the circadian peak, type I receptors may play an important role (Dallman

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et al., 1989; Bradbury et al., 1994; Spencer et al., 1998; Young et al., 1998). With regard to the delayed therapeutic effects of antidepressants, the change in type I receptor function correlates more closely than do changes in type II receptors (Reul et al., 1994). Furthermore, in a double-blind experiment, the behavioral effects of the tricyclic antidepressant amitriptyline in humans were blocked by the type I receptor antagonist spironolactone (see Holsboer, 2001). The best illustration of CRH and glucocorticoid receptor impairment in depression involves the dexamethasone suppression test and the dexamethasone–CRH test. The dexamethasone suppression test entails the administration of a low dose of dexamethasone (1–2 mg) late in the evening and the measurement of cortisol levels at various time points during the subsequent day. Nondepressed subjects respond to dexamethasone treatment by decreasing cortisol levels. Many but not all depressed subjects fail to exhibit dexamethasone suppression of cortisol levels. This inappropriate dexamethasone response in depressed subjects is likely a result of impaired type II glucocorticoid receptors (Holsboer, 2001). The dexamethasone suppression test was supplemented by the administration of CRH to create the dexamethasone–CRH test, which is a more sensitive test for the detection of abnormal functions of the HPA axis (Heuser et al., 1994). The dexamethasone–CRH test takes into account the regulatory role of CRH by demonstrating the impairment of CRH receptors. In this test, CRH is administered intravenously after pretreatment with vehicle or a low dose of dexamethasone. Without dexamethasone, normal subjects respond to CRH administration by increasing plasma ACTH levels, whereas depressed subjects exhibit a blunted ACTH response. When the subjects are pretreated with dexamethasone to activate the negative feedback response of the type II glucocorticoid receptors, the responses of depressed and nondepressed subjects are reversed: in normal subjects, dexamethasone-induced suppression overrides the CRHinduced increase in ACTH levels; depressed subjects, however, undergo a paradoxical increase in ACTH response. 3. Serotonin and Hypothalamic–Pituitary–Adrenal Axis Interactions in Depression Neuroendocrine challenge tests with 5-HT releasers (d-fenfluramine), SSRIs, precursors, or 5-HT receptor agonists illustrate the altered interactions between the serotonergic system and the HPA axis in depression. In depressed patients, a d-fenfluramine-induced increase in cortisol levels is attenuated as compared with healthy control subjects (O’Keane and Dinan, 1991; Lucey et al., 1992; Cleare et al., 1996, 1998). Likewise, depressed patients experience a blunted ACTH and cortisol response to an acute challenge with clomipramine, a tricyclic drug with high affinity for the serotonin transporter (Golden et al., 1992). ACTH and

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cortisol responses to the 5-HT1A agonists buspirone, flesinoxan, and ipsapirone are blunted in depression (Lesch et al., 1990a; Meltzer and Maes, 1994, 1995a; Pitchot et al., 1995). With depression, there is no difference in cortisol response to the 5-HT2 agonists m-CPP or MK-212 (Kahn et al., 1988,1990a; Anand et al., 1994). Antidepressant drugs, which act to increase the amount of 5-HT in the synaptic cleft, have a delayed therapeutic action. Changes in the synapse must occur prior to the therapeutic action of these drugs. One of the proposed changes is that the elevated amount of 5-HT in the synapse, particularly by SSRIs, leads to a desensitization of somatodendritic 5-HT1A autoreceptors (Kreiss and Lucki, 1995; Blier and de Montigny, 1996; Casanovas et al., 1999b; Czachura and Rasmussen, 2000; Le Poul et al., 2000; Hervas et al., 2001; Hensler, 2002) and the desensitization of 5-HTIB/ ID synaptic autoreceptors within 2–3 weeks (de Montigny et al., 1990; Blier and Bouchard, 1994; Blier and de Montigny, 1994; Blier, 2001). Combined, the desensitization of autoreceptors releases the serotonergic system from the negative feedback regulation brought about by these receptors. In addition to the autoreceptor-induced feedback, evidence suggests that forebrain 5-HT1A receptors are involved in the negative feedback regulation of serotonergic neurons in the midbrain raphe´ nuclei. Negative feedback interaction has been demonstrated with 5-HT1A receptors expressed by neurons in the amygdala (Bosker et al., 1997, 2001). Similarly, postsynaptic 5-HT1A receptors in the frontal cortex (Ceci et al., 1994; Peyron et al., 1998; Casanovas et al., 1999a; Hajo´s et al., 1999; Haddjeri et al., 2000; Celada et al., 2001) as well as the striatum (Romero et al., 1994) but not the hippocampus (Hjorth et al., 1996) mediate a negative feedback regulation of serotonergic neurons in the midbrain raphe´ nuclei. An antidepressant-induced increase in synaptic 5-HT results in the desensitization of postsynaptic 5-HT1A receptors (Goodwin et al., 1987; Hensler et al., 1991; Serres et al., 2000b; Bosker et al., 2001). In the amygdala, desensitization of postsynaptic 5-HT1A receptors by the SSRI citalopram is linked to an increase in extracellular levels of 5-HT in the amygdala in rats (Bosker et al., 2001). Therefore, the release from negative feedback induced by the autoreceptors and specific postsynaptic 5-HT1A receptors results in an increase in synaptic 5-HT levels in the forebrain. The elevated levels of 5-HT can then act on other 5-HT receptors, some of which may be involved in the therapeutic effect of some antidepressants. The paraventricular nucleus might also play an integral role in the regulation of 5-HT release from the midbrain raphe´ to the forebrain by way of postsynaptic 5-HT1A receptors. As in the amygdala, postsynaptic 5-HT1A receptors in the paraventricular hypothalamus become desensitized after treatment with antidepressants such as fluoxetine, paroxetine, and venlafaxine (Li et al., 1996a, 1997b; Raap et al., 1999; Newman et al., 2000; Serres et al., 2000b). The paraventricular nucleus innervates the dorsal

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and median raphe´ nuclei (Conrad and Pfaff, 1976; Behzadi et al., 1990; Peyron et al., 1998). In addition, the dorsal raphe´ sends collateral innervation to both the amygdala and the paraventricular nucleus (Petrov et al., 1992b, 1994). Together, the evidence of reciprocal innervation gives anatomical context for the possibility of paraventricular nucleus-induced negative feedback on serotonin release from the raphe´ nuclei. B. ANXIETY DISORDERS

Anxiety disorders, like depression, are symptoms underlying several neuropathological disorders. Just as stress is linked to the development or occurrence of depression (see Section V.A), anxiety disorders can either lead to depressive disorders or be expressed with depression (Kessler et al., 1996). Anxiety disorders currently comprise a number of similar disorders including generalized anxiety disorder, obsessive-compulsive disorder, panic disorder, posttraumatic stress disorder, and social phobia (Nutt, 1996). Although fear is considered a normal response to a threatening situation, anxiety is considered to be an unfounded response or inappropriate fear (Ninan, 1999). The key area in the brain associated with the emotional experience of anxiety is the amygdala (LaBar et al., 1995; LeDoux, 1995; Ninan, 1999). Stimulation of the amygdala in humans leads to the expression of emotions associated with fear and anxiety (Davis and Whalen, 2001). A commonality among anxiety disorders is their distorted output from the central nucleus of the amygdala (Ninan, 1999). 1. The Serotonergic System and Anxiety Benzodiazepines are the most common and popular treatment for several anxiety disorders. Although they provide robust and swift amelioration of anxiety symptoms, their potential for tolerance, physical dependence, and motor and cognitive impairment (Ninan, 1999; Argyropoulos et al., 2000) makes them more of a problem than a solution for anxiety disorders. Benzodiazepines potentiate the effects of the neurotransmitter GABA at GABA-A receptors (Sigel, 2002). The acute actions of benzodiazepines may be due in part to a reduction of 5-HT neuronal firing in the raphe´ induced by local GABA-ergic neurons as demonstrated by electrophysiological studies (Gallager, 1978; Ferraro et al., 1996; Gervasoni et al., 2000; Varga et al., 2001), in vivo microdialysis (Forchetti and Meek, 1981; Tao et al., 1996; Tao and Auerbach, 2000), and behavioral studies (Levine and Jacobs, 1992; Maier et al., 1995a,b; Inoue et al., 1996). Treatment with serotonergic drugs such as 5-HT1A agonists and SSRIs is therapeutically effective in the treatment of anxiety (Zohar and Westenberg, 2000). Chronic treatment with 5-HT1A agonists such as buspirone, gepirone, ipsapirone, and tandospirone results in the desensitization of both somatodendritic 5-HT1A autoreceptors and postsynaptic 5-HT1A receptors

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(Blier and de Montigny, 1987; Blier et al., 1990; Godbout et al., 1991; Bohmaker et al., 1993; Matheson et al., 1996; Berlin et al., 1998; Sim-Selley et al., 2000). Similarly, chronic treatment with SSRIs desensitizes both somatodendritic 5-HT1A autoreceptors and postsynaptic 5-HT1A receptors (Anderson et al., 1996; Li et al., 1997b; Berlin et al., 1998; Lerer et al., 1999; Bosker et al., 2001; Dawson et al., 2002; Hensler, 2002; Pejchal et al., 2002). The actions of these serotonergic drugs suggest that their therapeutic mechanism of action is the desensitization of 5-HT1A receptors (Yocca, 1990). 2. The Hypothalamic–Pituitary–Adrenal Axis and Anxiety Researchers have proposed that stress activates the HPA axis to counteract glucocorticoid inhibition to subsequent stressors (Dallman and Jones, 1973; Akana et al., 1992; Cassano and D’Mello, 2001). The effects of repeated stress on the neuroendocrine system have characteristics similar to those encountered in chronic anxiety (Boyer, 2000). Although most patients with anxiety or depression exhibit an increase in CRH levels within the cerebrospinal fluid, the neuroendocrine response associated with anxiety is as a whole the opposite of that of depression (Fang et al., 1981; Bremner et al., 1997b; Arborelius et al., 1999; Boyer, 2000; Kasckow et al., 2001). For example, patients with depression generally have an elevated level of cortisol, and most patients with stress disorders have a lower level of cortisol (Boyer, 2000). Likewise, in depression dexamethasone treatment fails to suppress cortisol levels (Holsboer, 2001), whereas in anxiety disorders not associated with depression, there is a dexamethasone-induced suppression or exaggerated suppression of cortisol levels, particularly in obsessive-compulsive, panic, and posttraumatic stress disorders (Lieberman et al., 1983, 1985; Sheehan et al., 1983; Coryell et al., 1989; Yehuda et al., 1993, 1995; Orlikov et al., 1994; Stein et al., 1997). Furthermore, most depressed patients have a blunted ACTH release following CRH administration, whereas some patients with anxiety disorders, such as panic and posttraumatic stress, primarily have a normal to exaggerated response to CRH administration (Curtis et al., 1997; Heim et al., 2001). However, a few studies report a blunted ACTH response to CRH administration in patients with panic and posttraumatic stress disorders (Roy-Byrne et al., 1986; Smith et al., 1989). The anxiety-induced hyposecretion of glucocorticoids is thought to be a potentiated feedback due to sensitized glucocorticoid receptors (Boyer, 2000). Animal studies reported an association between neuronal glucocorticoid receptors and lymphocyte glucocorticoid receptors (Lowy, 1989, 1990). Lymphocyte glucocorticoid receptor number is increased with anxiety disorders in humans, suggesting an upregulation of neuronal glucocorticoid receptors (Yehuda et al., 1991, 1995; Boyer, 2000). This upregulation would lead to an increase in negative feedback regulation of ACTH and cortisol

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release, thereby resulting in hypocortisolemia. Boyer (2000) hypothesized a ‘‘neuroendocrine continuum’’ to explain the discrepancy between the neuroendocrine responses seen with depression and anxiety. This hypothesis proposes that anxiety occurs prior to major depression. The hypersecretion of CRH observed in anxiety leads to HPA hyperregulation. In people with genetic vulnerability to depression, the hypersecretion of CRH leads to the desensitization of CRH receptors, thereby resulting in dysregulation of the HPA axis. 3. Serotonin and Hypothalamic–Pituitary–Adrenal Axis Interactions in Anxiety Pharmacological challenge tests are used to study anxiety disorders by precipitating an anxiety attack. Sodium lactate infusion induces panic attacks in patients with panic disorder or generalized anxiety disorder (Cowley and Dunner, 1988; Cowley et al., 1988). Inhalation of 5.5% carbon dioxide for 15 min also brings about panic attacks in many patients with anxiety disorders (Rapee et al., 1992). Acute depletion of the 5-HT precursor l-tryptophan had little effect on the level of anxiety in patients with panic disorder, but it was able to potentiate carbon dioxide-induced anxiety (Anderson and Mortimore, 1999; Miller et al., 2000; Schruers et al., 2000). The tryptophan depletion studies would suggest that 5-HT does not play a major role in anxiety attacks, but rather the lack of 5-HT may potentiate other factors that can trigger anxiety attacks. However, administration of d-fenfluramine resulted in the precipitation of an anxiety attack in patients with panic disorder, although it was able to reduce a carbon dioxide (7%)-induced panic attack (Mortimore and Anderson, 2000). Administration of the nonspecific 5-HT1 and 5-HT2 receptor agonist m-CPP induces a greater amount of anxiety in patients with generalized anxiety, obsessive-compulsive or panic disorders as compared with control subjects (Zohar et al., 1987; Charney et al., 1988; Germine et al., 1992; Broocks et al., 2000). The 5-HT1A receptor agonist ipsapirone will elicit a panic attack in patients with panic disorder as well (Broocks et al., 2000). Together, these studies suggest that there is a dual role for 5-HT in anxiety disorders, in which an acute increase in 5-HT function could lead to an anxiety attack and continued treatment with SSRIs may reduce panic attacks. As mentioned earlier, patients with anxiety disorders are believed to have desensitized 5-HT1A receptors. When ipsapirone was administered to patients with panic disorder, there was a decreased cortisol response (Broocks et al., 2000); however, panic disorder patients given m-CPP had an increased cortisol response (Charney et al., 1988; Kahn et al., 1988; Broocks et al., 2000). This study not only further demonstrates that 5-HT1A receptors are desensitized, it also indicates that 5-HT2 receptors may be supersensitized.

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C. CHRONIC FATIGUE

Chronic fatigue is a disorder defined as a disabling fatigue associated with other symptoms such as low-grade fever, sleep disturbances, and myalgias lasting 6 months or longer with no defined medical cause (Demitrack et al., 1992; Fukuda et al., 1994). Although depression is found in nearly half of the patients with chronic fatigue, patients state that their fatigue is due to physical rather than psychological causes (Kruesi et al., 1989; Broocks et al., 2000). 1. The Hypothalamic–Pituitary–Adrenal Axis and Chronic Fatigue A dysfunctional HPA axis has been cited as a possible contributing factor in chronic fatigue. The main thrust of this theory was based on the fact that many of the symptoms associated with chronic fatigue mirror those of patients with glucocorticoid insufficiency (Demitrack et al., 1991; O’Riordain et al., 1994; Parker et al., 2001). In line with this view, studies involving patients with chronic fatigue report a decrease in cortisol in some subjects (Poteliakhoff, 1981; Demitrack et al., 1991) but not all subjects (Yatham et al., 1995; Scott et al., 1998; Scott and Dinan, 1998; Hudson and Cleare, 1999). The differences observed may relate to the mean length of disease in the patients observed, method of cortisol measurement, as well as the comorbidity of depression (see Parker et al., 2001). Most studies observed a blunted ACTH response to CRH but an exaggerated cortisol response to low doses of ACTH in patients with chronic fatigue (Demitrack et al., 1991; Scott et al., 1998). However, low doses of synthetic ACTH did not result in an exaggerated cortisol response (Hudson and Cleare, 1999). In chronic fatigue, disruption of the HPA axis may be a result of a deficiency in CRH (Demitrack, 1997). The lack of CRH can result in fatigue indirectly through decreased activation of the HPA axis or directly by decreasing behavioral arousal given that CRH administration leads to behavioral arousal (Sutton et al., 1982; Vgontzas et al., 2001). The attenuation of ACTH secretion is not reflected by a change in net cortisol release, which may demonstrate a supersensitization of the ACTH receptors. As mentioned earlier, subjects with depression also have an attenuated response to CRH. However, in depression the attenuation is believed to exist as a result of the high plasma levels of glucocorticoids associated with depression, which is not the case for chronic fatigue syndrome given that there is a hyposecretion of cortisol. 2. Serotonin and Hypothalamic–Pituitary–Adrenal Axis Interactions in Chronic Fatigue Given that 5-HT plays such a close role in the regulation of the HPA axis, it would seem natural that 5-HT is involved in chronic fatigue syndrome. Two studies indicate that the prolactin response to administration of the

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5-HT-releasing drug, d-fenfluramine, is significantly potentiated in patients with chronic fatigue (Cleare et al., 1995; Sharpe et al., 1997). However, another study found no change in prolactin response to d-fenfluramine as compared with control (Bearn et al., 1995). Interestingly, the investigators who did not demonstrate an elevated prolactin response did nevertheless observe an increased ACTH response to d-fenfluramine with no associated change in cortisol levels, suggesting an impairment of the adrenal cortex (Bearn et al., 1995). Another study, which used the racemic mixture of fenfluramine, which is less potent, did not observe a potentiation of the prolactin response as compared with controls (Yatham et al., 1995). Further studies utilized 5-HT1A or 5-HT2C receptor agonists. Neuroendocrine challenge tests with 5-HT1A receptor agonists have reported conflicting results. In patients with chronic fatigue, administration of buspirone resulted in a significant increase in plasma prolactin levels with no report on cortisol levels (Bakheit et al., 1992; Sharpe et al., 1996). However, the prolactin response to buspirone is mediated by antagonism of dopamine D2 receptors in the pituitary gland. Ipsapirone administration was less effective in producing an increase in plasma ACTH in chronically fatigued subjects as compared with controls, which would suggest that 5-HT1A receptors are desensitized (Dinan et al., 1997). Although the majority of studies have found an increase in serotonergic activity associated with chronic fatigue, the results are still equivocal. Given that the majority of studies involving chronic fatigue also find a hypoactive HPA axis, which opposes findings in patients with depression, the most parsimonious interpretation would be that the serotonergic activity would be the opposite of that found in depression. Therefore, chronic fatigue most likely involves a hypoactive HPA axis combined with a hyperactive serotonergic system.

VI. CONCLUDING REMARKS To summarize, the serotonergic system and the HPA axis are closely entwined. Serotonergic innervation regulates the HPA axis under stressful conditions as well as nonstressful conditions such as the circadian rhythm. As illustrated by pathophysiological disorders, a slight change in one system can alter the other. Although it seems unclear which is the underlying cause of disturbances in either system, the fact that serotonergic drugs are of therapeutic use suggests that regulation of the serotonergic system can lead to a normalization of the hypothalamic–pituitary–adrenal axis. Although both systems are known to be dysregulated in pathological conditions, a greater understanding of these interactions would be useful in the understanding and treatment of these disorders.

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7 The Thymosins Prothymosin , Parathymosin, and -Thymosins: Structure and Function

Ewald Hannappel and Thomas Huff Institute for Biochemistry/Faculty of Medicine, University of Erlangen-Nu¨rnberg, 91054 Erlangen, Germany

I. Introduction II. Polypeptide b1 III. a-Thymosins and Prothymosin A. Structure of Prothymosin B. Prothymosin and Cell Proliferation C. Prothymosin and Zn2+ D. Bipartite Nuclear Localization Signal and Caspase 3 Cleavage Site E. Phosphorylation of Prothymosin F. Intracellular Partners of Prothymosin G. Prothymosin and Small RNA H. Extracellular Thymosin a1 and Prothymosin IV. Parathymosin A. Zn2+-Binding Protein B. Bipartite Nuclear Localization Signal C. Phosphorylation of Parathymosin D. Parathymosin and Glucocorticoid Action V. -Thymosins A. Purification of b-Thymosins B. Amino Acid Sequences and Phylogenetic Distribution of b-Thymosins C. b-Thymosins and G-Actin Vitamins and Hormones Volume 66

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D. b-Thymosins E. b-Thymosins F. b-Thymosins G. Thymosin b4 VI. Conclusions References

and F-Actin and Cancer in Angiogenesis and Wound Healing and AcSDKP

The studies on thymosins were initiated in 1965, when the group of A. White searched for thymic factors responsible for the physiological functions of thymus. To restore thymic functions in thymic-deprived or immunodeprived animals, as well as in humans with primary immunodeficiency diseases and in immunosuppressed patients, a standardized extract from bovine thymus gland called thymosin fraction 5 was prepared. Thymosin fraction 5 indeed improved immune response. It turned out that thymosin fraction 5 consists of a mixture of small polypeptides. Later on, several of these peptides (polypeptide 1, thymosin 1, prothymosin , parathymosin, and thymosin 4) were isolated and tested for their biological activity. The research of many groups has indicated that none of the isolated peptides is really a thymic hormone; nevertheless, they are biologically important peptides with diverse intracellular and extracellular functions. Studies on these functions are still in progress. The current status of knowledge of structure and functions of the thymosins is discussed in this review. ß 2003, Elsevier Science (USA).

I. INTRODUCTION The thymus is the site of maturation of T lymphocytes. This maturation requires the thymic microenvironment. Even today, the sequence of this maturation within the thymus is not completely understood. In 1970 it was well accepted that a vital part of the process by which the thymus works occurs via humoral factors (Goldstein and White, 1970). Various cell-free extracts were prepared from thymus and tested for their ability to counteract effects of neonatal thymectomy in rodents. Because little was known at the time about the complex processes involved in the maturation of T lymphocytes, testing the biological activities of extracts was not reliable. Klein and co-workers (1965) studied the enhancement of incorporation of [3H] thymidine into DNA of mouse nodes by thymic extracts. In 1966 the same group presented a standardized fractionation of calf thymus and coined the term ‘‘thymosin’’ for the isolated lymphocytopoietic factor (Goldstein et al., 1966). Thymosin was thought to be a single 12.6-kDa

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polypeptide (Goldstein et al., 1972). The starting material was thymosin fraction 5, which was prepared by a standardized five-step procedure: (1) extraction of thymus in isotonic NaCl solution, (2) heat denaturation from 0 to 80 C within 25 min, (3) acetone precipitation, (4) ammonium sulfate precipitation, and (5) ultrafiltration. However, at least two of these steps are disputable in terms of changing the peptide pattern of the extract by proteolysis: proteolysis will occur during the thawing of frozen calf thymus in the cold as well as during the heat denaturation step. Later it turned out that thymosin fraction 5 or fraction 5A (Goldstein and Low, 1985) prepared by a different ammonium sulfate fractionation contained not only one biologically interesting peptide but consisted of a mixture of small polypeptides ranging from approximately 1 to 15 kDa. On an isoelectric focusing gel, 30–40 polypeptide components or fragments were identified. The large number of peptides present in thymosin fraction 5 may indeed represent fragments of larger polypeptides generated during preparation of thymosin fraction 5A and not genuine thymic peptides. Because of the large number of peptides detected a nomenclature for the family of polypeptides present in thymosin fraction 5 was suggested (Goldstein et al., 1977). The thymosins were divided into three main groups according to their isoelectric points: -thymosins, pI below 5.0; thymosins, pI between 5.0 and 7.0; and -thymosins, pI above 7.0. Numerical subscripts were included simply to denote the chronological order of isolation. The decision concerning which peptides to isolate first was crucial. Depending on the point of view, the wrong and right peptides were chosen. Thymosin fraction 5 contains several peptides that are present in higher amounts and these peptides were selected for further purification (Low et al., 1979). Probably the wrong peptides were chosen with respect to thymic hormones, because the nonlymphoid (stromal) cells situated in the thymus providing nursing to immature T cells (van Ewijk, 1991) are outnumbered by T cells to die (Smith et al., 1989; MacDonald and Lees, 1990). However, it turned out that biologically important peptides were isolated with respect to general cell function. The first two peptides isolated from thymosin fraction 5 were 1 and 1 (Low and Goldstein, 1979). Whereas the former was classified as a thymosin on the basis of its activity in certain assays, the latter was termed polypeptide 1. Polypeptide 1 did not show biological activity in the bioassay systems used, indicating that it might not be an important molecule for T cell maturation.

II. POLYPEPTIDE 1 Polypeptide 1 consists of 74 amino acid residues. At least four independent groups sequenced the polypeptide 1 completely or partially.

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It was isolated from bovine thymus as a polypeptide that has lymphocytedifferentiating properties and was designated as ubiquitous immunopoietic polypeptide, UBIP (Goldstein et al., 1975). It was supposed to induce the differentiation of T and B cells via -adrenergic receptors and adenylate cyclase activation. However, the effect on T and B cells was caused by endotoxin present in the UBIP preparations (Low et al., 1979). Because UBIP was present in various animal cells, yeast, bacteria, and plants (Goldstein et al., 1975; Schlesinger and Goldstein, 1975) as well as insect eggs (Gavilanes et al., 1982) UBIP was renamed ubiquitin(1–74). Ubiquitin(1–74), which is extended at the C terminus by a dipeptide (Gly-Gly), is connected with histone 2A via an isopeptide bond in the chromosomal protein A24, forming uH2A (Goldknopf and Busch, 1977). Today, polypeptide 1 is well known as part of ubiquitin, or ATPdependent proteolysis factor 1 (APF-1) (Ciechanover et al., 1980). Ubiquitin plays a key role in a variety of cellular processes, such as ATP-dependent degradation of cellular proteins (Glickman and Ciechanover, 2002); regulation of transcription (Conaway et al., 2002); spermiogenesis by tagging histones 2A, 2B, and 3 (Jason et al., 2002); apoptosis (Jesenberger and Jentsch, 2002); and ribosome biogenesis (Finley et al., 1989). Ubiquitin is a globular protein but the last four C-terminal residues (–73LRGG76) extend from the globular structure and are accessible (Vijay-Kumar et al., 1987). The bond between arginine and glycine is cleaved during the preparation of thymosin fraction 5, generating polypeptide 1. In the ATP-dependent proteolysis system, the C-terminal glycine residue is used for ubiquitinylation of target proteins via an isopeptide bond.

III. -THYMOSINS AND PROTHYMOSIN Thymosin 1 is a small peptide (28 amino acid residues) isolated from thymosin fraction 5. Its isoelectric point is 4.2 and the molecular mass is 3108 Da (Low and Goldstein, 1985). The N-terminal serine residue is acetylated, and the peptide contains no aromatic amino acid residue (Fig. 1). Later, two peptides related to thymosin 1 were isolated from calf thymosin fraction 5. One, lacking four amino acid residues at the COOH terminus, was designated des-(25–28)-thymosin 1. The other, named thymosin 11, contained seven additional amino acid residues at the COOH terminus. (Caldarella et al., 1983). However, none of these peptides were detectable in larger amounts when strongly denaturing conditions were used during extraction of calf thymus (Hannappel et al., 1982b). When rat thymus was pulverized in a mortar chilled in dry ice and added thereafter to boiling water a peptide of 111 amino acids was isolated that contained the entire sequence of thymosin 1 at its N terminus (Haritos et al., 1984a).

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FIGURE 1. Amino acid sequences of thymosin 1, thymosin 11, and prothymosins from various species: Homo sapiens (human, Q15249), Bos taurus (bovine, P01252), Rattus norvegicus (rat, P06302), Mus musculus (mouse, P26350), Rana esculenta (frog, Q90ZK2), and Brachydanio rerio (zebrafish, Q8QGP0). The numbers in parentheses are the accession codes for the SWISSPROT and TrEMBL databases. The numbering is according to human prothymosin. The bipartite nuclear localization signal is outlined; the caspase 3 cleavage sites (–DxxD–) located inside the bipartite NLS are shown in boldface; additional potential caspase 3 sites of non mammalian prothymosins are shown in italic. Invariant residues are indicated by asterisks and highly conserved residues are indicated by colons. Therefore, the new peptide was designated prothymosin (Fig. 1). If thymosin 1 is produced solely by artificial proteolysis during preparation of thymosin fraction 5 or whether this proteolysis resembles the natural processing of prothymosin to thymosin 1 in certain tissues is still not settled (Freire et al., 1985; Franco et al., 1992; Frillingos et al., 1992; Sarandeses et al., 2003). Prothymosin and its mRNA have been detected almost ubiquitously in a wide variety of tissues (Haritos et al., 1984b; Clinton et al., 1989a). In a cDNA library constructed from human spleen mRNA, a clone was isolated that contained a 503-base pair insert including the entire coding sequence of prothymosin. The presence of an initiator codon immediately preceding the codon for the N-terminal serine residue, and a terminator codon immediately following the codon for the Cterminal Asp-109, suggested that prothymosin is synthesized without formation of a larger precursor. Analysis of the 50 sequence preceding the initiator methionine codon excluded the potential presence of a hydrophobic signal peptide (Goodall et al., 1986). According to a survey of human cDNA libraries the prothymosin gene was among the most abundantly expressed genes together with the 90-kDa heat shock protein, myosin light chain, and ribosomal proteins (Adams et al., 1995). The complete amino acid sequences of human (Eschenfeldt and Berger, 1986; Pan et al., 1986; Gomez-Marquez et al., 1989), calf (Panneerselvam et al., 1988b), rat

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(Haritos et al., 1985a; Frangou-Lazaridis et al., 1988) and mouse (Schmidt and Werner, 1991) prothymosin have been established. These prothymosins vary slightly in length (human and calf, 109; mouse, 110; and rat, 111 amino acid residues) and are different in a few amino acid residues (Fig. 1). Even the presence of a putative prothymosin homolog in yeast (Makarova et al., 1989) and Escherichia coli (Vartapetian et al., 1992) has been reported. This supposed high evolutionary conservation of prothymosin was an argument in favor of a fundamental role for the peptide in eukaryotic and prokaryotic cells. Later, the presence of prothymosin or a prothymosin gene in animals other than mammals was shown to be highly unlikely (Trumbore et al., 1998). More recently, however, cDNA clones encoding prothymosins have been identified in Rana esculenta (Aniello et al., 2002; De Rienzo et al., 2002) and zebrafish (TrEMBL accession code, Q8QGP0). The amino acid sequences are highly conserved (70%). In addition, the bipartite nuclear localization site separated by multiple caspase 3 cleavage sites is conserved in the C-terminal region of all prothymosins (Fig. 1). The cDNA of frog prothymosin is expressed ubiquitously. Because the prothymosin expression varies during the spermatogenetic cycle concomitantly with germ cell maturation, prothymosin might contribute to the efficiency of spermatogenesis in species with seasonal breading (Aniello et al., 2002). A. STRUCTURE OF PROTHYMOSIN

Because structure and function of molecules are related, the peculiar chemical features of prothymosin must be considered. All prothymosins are devoid of cysteine, methionine, and aromatic amino acid residues. Thus, they do not absorb at 280 nm. About half the amino acids residues are acidic amino acids. The ratio between glutamic acid to aspartic acid drops from about 2 in mammalian prothymosin to about 1 in frog and zebrafish. Eight lysine residues are conserved in all prothymosins; four are clustered in the N-terminal region (residues 14, 17, 19, and 201) and the others are found in the C-terminal region (residues 87, 101, 102, and 104). A lysine residue in frog and zebrafish replaces the arginine residue at position 30 in mammalian prothymosins, whereas the second arginine residue at position 88 is conserved. Mammalian prothymosins contain five times more acidic than basic amino acid residues, have isoelectric points below 3.5, and belong to the most acidic proteins known in nature. Because of this amino acid composition, prothymosins are soluble in boiling buffer or diluted perchloric acid and partition to the aqueous phase of a phenol extraction. These properties have been successfully used for purification of prothymosins

1

Numbering according to human prothymosin .

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(Haritos et al., 1985b; Vartapetian et al., 1988; Sburlati et al., 1990; Watts et al., 1990; Evstafieva et al., 1995). Because of the large number of acidic amino acid residues prothymosin is highly negatively charged and adopts a random coil-like conformation with no regular secondary structure (Gast et al., 1995), permitting interactions with cationic structures. However, at low pH (3) and rather low concentrations (5 M) compared with intracellular concentrations (up to 0.3 pg/cell, 100 M 2) (Haritos et al., 1984b; Franco et al., 1992; Sburlati et al., 1993), the natively unfolded prothymosin is capable of forming regular elongated fibrils (Pavlov et al., 2002). The low pH presumably decreases the repulsive forces caused by the high negative charge of the protein at neutral pH and thereby induces its partial folding. Whether this change in structure might occur in vivo under certain physiological or pathophysiological conditions is currently unknown. B. PROTHYMOSIN AND CELL PROLIFERATION

Prothymosin expression is elevated in proliferating tissues including colon cancer (Mori et al., 1993) and hepatocellular carcinoma (Wu et al., 1997) and is correlated with c-Myc mRNA expression (Vareli et al., 1995). Prothymosin was found in proliferating lymphoma and transformed 3T3 cells but not in resting cells (Eschenfeldt and Berger, 1986). Antisense RNA or synthetic antisense DNA oligomers of prothymosin are able to inhibit cell division in myeloma cells (Sburlati et al., 1991). Prothymosin gene transcription is directly regulated by activated c-Myc via an E-box element (CACGTG) localized in the first intron of the gene (Eilers et al., 1991). In estrogen receptor-positive breast cancer cells the prothymosin mRNA was rapidly increased by estrogen accompanied by a 6-fold increase in prothymosin content. Prothymosin was found to selectively enhance estrogen receptor (ER) transcriptional activity by sequestering repressor of ER activity, rendering the estrogen–ER complex accessible to coactivators such as SRC-1 (Martini et al., 2000). It is noteworthy that prothymosin, a protein enhancing estrogen–ER transcriptional effectiveness, is itself upregulated by estrogen. The estrogen effect on prothymosin expression in breast cancer cells is mediated via two upstream half-palindromic TGACC motifs in the prothymosin promotor (Garnier et al., 1997; Martini and Katzenellenbogen, 2001). Despite the fact that overexpression of prothymosin promotes tumor formation, mice were injected with murine bladder cancer cells (MBT-2) in conjugation with replication-defective retroviruses encoding prothymosin. These mice exhibited smaller tumor mass, lower tumor incidence, and higher 2 Assuming an even distribution of the peptide in a volume of 270 fl corresponding to the intracellular volume of human mononuclear leukocytes.

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survival rate, as well as higher antitumor cytotoxic activities compared with those injected with control viruses. This effect was not observed in severe combined immunodeficiency mice, suggesting that prothymosin exerted its effect through its immunomodulatory activities. However, as to be expected, tumor cell growth in monolayer culture and colony formation in soft agar was enhanced in the prothymosin gene-modified MBT-2 clones. The growth-promoting effect was circumvented by removing the nuclear localization signal of prothymosin from the construct (Shiau et al., 2001). C. PROTHYMOSIN AND ZN2+

Mice of the RF/J strain are defective in some aspects of cellular immunity, as evidenced by their susceptibility to infections with Candida albicans. When the mice were fed a high-zinc diet and treated daily with 160 ng of prothymosin, an increase occurred in resistance to infections with Candida (Salvin et al., 1987). In the presence of Zn2+ or small unilamellar vesicles composed of dimyristoylphosphatidylcholine and dimyristoylphosphatidic acid (10:1), thymosin 1 adopts a partially structured conformation. A turn is present between residues 5 and 8, whereas the region between residues 17 and 24 adopts an -helical conformation (Grottesi et al., 1998). Binding of divalent metal cation by proteins is dependent on negatively charged amino acid side chains. Therefore the divalent cation-binding properties of prothymosin were evaluated (Chichkova et al., 2000). Prothymosin could bind up to 3 Zn2+ ions in the presence of 100 mM NaCl (KD of 0.23 mM) specifically and up to 13 Zn2+ ions in the absence of NaCl (KD of 0.04 mM) unspecifically. Zinc ions significantly enhanced the binding of prothymosin to HIV-1 Rev but not to histone H1, two putative binding partners (Papamarcaki and Tsolas, 1994; Kubota et al., 1995). Binding of zinc to prothymosin (reported: KD of 1 mM) induces compaction and considerable rearrangement of protein structure into a compact, partially folded, premolten globulin-like conformation (Uversky et al., 2000). No interactions with Mg2+, Mn2+, Cu2+, Ni2+, or Co2+, and a weak interaction with Ca2+, were observed (Chichkova et al., 2000; Uversky et al., 2000). D. BIPARTITE NUCLEAR LOCALIZATION SIGNAL AND CASPASE 3 CLEAVAGE SITE

In 1988 it was first postulated by sequence comparison with nuclear localization signals (NLSs) of other proteins ‘‘that prothymosin is located inside the cell nucleus and that its activity might be to organize some protein complexes’’ (Gomez-Marquez and Segade, 1988). It was recognized that the

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sequence – 101KKQK104 – might serve as NLS. The nuclear localization of prothymosin was confirmed by several groups (Watts et al., 1989; Clinton et al., 1991; Manrow et al., 1991; Castro and Barcia, 1996) but questioned by others (Tsitsiloni et al., 1989). To localize prothymosin in cells wild-type and mutant human prothymosins were expressed as green fluorescent protein (GFP) fusion proteins in human cells. This study led to the identification of a bipartite NLS (–87KR-X12-KKQK104–). Wild-type prothymosin appeared to be exclusively nuclear and excluded from the nucleoli. Mutations (K87R or K101R) in both parts of the putative bipartite NLS resulted in a marked (but not complete) redistribution to the cytoplasm (Rubtsov and Vartapetian, 1996; Rubtsov et al., 1997). This bipartite NLS is conserved in all described prothymosins, including those from frog and zebrafish (Fig. 1). The bipartite NLS is separated by 12 (mammalian), 13 (frog), or 14 (zebrafish) mainly acidic amino acid residues that display several overlapping amino acids motifs, –DxxD–, conforming to caspase 3 recognition sites. When HeLa cells undergo apoptosis, DNA becomes fragmented, caspases are activated, and prothymosin is cleaved at sites near the C terminus. Consequently, the larger part of the bipartite NLS is removed and the truncated peptide forfeits known functions such as nuclear localization. Caspase 3 attacks prothymosin directly. The truncated peptide is deficient in phosphate. Because phosphorylation of prothymosin is supposed to occur in the nucleus this reflects loss of nuclear compartmentalization. Multiple caspase 3 recognition sites are conserved in all prothymosins between amino acid residues 90 and 100. Prothymosins from human, bovine, mouse, and frog possess the motif three times (DDVD, DEDD, and DDVD), whereas the last motif is lost in rat (DDVE). In zebrafish, five potential caspase 3 recognition sites can be identified in this region (DDDDDEDDVD). In addition, three more putative caspase 3 recognition sites are located between positions 63 and 82 of frog and zebrafish prothymosins (Fig. 1). These sites are lost in the mammalian prothymosins. Prothymosin appears to be a carefully designed substrate for caspase 3 (Enkemann et al., 2000; Evstafieva et al., 2000). Most recently it has been reported that prothymosin inhibits activation of caspase 3 by blocking apoptosome formation (Jiang et al., 2003). Because prothymosin itself is a substrate of caspase 3, the inhibition of apoptosome formation might be abolished by cleavage of prothymosin.

E. PHOSPHORYLATION OF PROTHYMOSIN

Prothymosin is phosphorylated in vivo, but there is still considerable controversy about the amino acid residue(s) phosphorylated. It was noticed that prothymosin contains three motifs that resemble consensus sequences for phosphorylation of serine or threonine by casein kinase-2 (Barcia et al., 1992). In mitogenically stimulated murine splenic lymphocytes, prothymosin

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and thymosin 1 were phosphorylated at Thr-7 and Thr-12/13 and not at serine residues, pointing to a kinase different from casein kinase-2 (Barcia et al., 1993). In 2000, this prothymosin kinase was characterized. Despite the fact that the phosphorylation sites of prothymosin are Thr-7 and Thr-12/13 the identified kinase phosphorylates neither thymosin 1 nor thymosin 11. The kinase is located in the cytosol throughout the cell cycle and its activity increases during the S phase and decreases at entry into the G2 phase. Prothymosin kinase is activated by phosphorylation in a mitogen-initiated pathway that is dependent on protein kinase C; however, protein kinase C does not phosphorylate prothymosin kinase directly (Perez-Estevez et al., 2000). It has also been reported that serine or glutamate residues of prothymosin can be phosphorylated. The phosphorylated serine residue was identified as the N-terminally acetylated amino acid residue. Only about 2% of prothymosin was determined to be phosphorylated throughout the cell cycle under steady state conditions. However, prothymosin is plentiful during rapid growth (0.3 pg/cell, 0.02% of total protein). Thus the number of phosphorylated prothymosin molecules may be about 300,000 per cell (Sburlati et al., 1993). This nevertheless low degree of phosphorylation of prothymosin and, even more, its constancy during the cell cycle were the reasons to scrutinize the source of the phosphate residue found at the acetyl serine residue of prothymosin. On the basis of the careful observation that greater than 90% of the phosphate found initially in prothymosin disappeared rapidly, it was recognized that phosphate groups had been linked to prothymosin in an energy-rich bond. The initial sites of phosphorylation are glutamate residues. The formed glutamyl phosphates are extremely labile and are hydrolyzed (90%) or transferred to serine or threonine to form stable esters in vivo (Trumbore et al., 1997). The free energy of hydrolysis of the mixed acid anhydride glutamyl phosphate is higher than that of ATP, and each prothymosin bears several phosphates simultaneously. Therefore, it might be that the glutamyl phosphate bonds in prothymosin are able to supply energy for processes in the nucleus (Trumbore et al., 1997). Because the phosphate residue could also be transferred back to ADP for energetic reasons, glutamyl phosphorylated prothymosin could serve as an energy buffer in the nucleus. This would be reminiscent of the creatine phosphate–creatine system of fast-fatigable muscle fibers. The high-energy status as well as the unusual phosphorylation of glutamate residues of prothymosin raise questions concerning how this reaction is catalyzed. Until now, no glutamyl kinase of prothymosin has been identified. Cell cycle-specific differences in the half-life of glutamyl phosphorylated prothymosin were observed in NIH 3T3 cells: 35 min in G0, 83–89 min during arrest in or progression through the S phase, and 174 min during

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M-phase arrest (Wang et al., 1997; Tao et al., 1999). As indicated by the long half-life of prothymosin phosphate during the M phase, the glutamyl phosphate of prothymosin is not required for mitosis. The half-life of prothymosin phosphate was about 150 min in quiescent (G0) and rapidly growing cells in the presence of actinomycin D, a potent inhibitor of transcription. Thus, it was proposed that prothymosin glutamyl phosphates fuel an energy-requiring step in the production, processing, or export of RNA (Tao et al., 1999). The short half-life of prothymosin glutamyl phosphates observed in quiescent NIH 3T3 cells can be explained by the lower prothymosin content and the lower number of phosphate groups per prothymosin when compared with rapidly growing cells. Resting NIH 3T3 cells synthesize RNA at about one-ninth the rate of rapidly growing cells. Concomitantly, the amount of prothymosin decreases to one-ninth and the number of phosphates incorporated in prothymosin drops from 4–8 to 2–4 in resting cells (Tao et al., 1999).

F. INTRACELLULAR PARTNERS OF PROTHYMOSIN

Studies have shown that prothymosin is a nuclear protein involved in cell proliferation and associated with the nucleosome linker histone H1 (Papamarcaki and Tsolas, 1994). It was recognized that prothymosin binds to nucleosome core histones, in particular, H3 and H4 (Diaz-Jullien et al., 1996), indicating that prothymosin might be involved in chromatin remodeling (Gomez-Marquez and Rodriguez, 1998; Karetsou et al., 1998). Affinity chromatography on prothymosin–Sepharose columns was used to identify proteins in subcellular extracts of transformed human lymphocytes that interact with prothymosin directly or indirectly. The most abundant prothymosin-binding proteins were histones H2A, H2B, H3, and H4. Of the nuclear transport proteins, karyopherin 1, Rch-1, Ran, and RCC1 were detected at high concentrations; NTF2, nucleoporin p62, and Hsp70 were detected at low concentrations; whereas transportin, CAS, and Ran BP1 were not detected. Of the cell cycle control proteins, PCNA, Cdk2, and cyclin A were detected at high concentrations; cdc2, Cdk4, and cyclin B were detected at low concentrations; and cyclin D1, D3, Cip1, and Kip1 were not detected. In agreement with the involvement of the karyopherin 1–Rch1 complex in the nuclear import of NLS-bearing proteins, prothymosin might be transported by this heterodimer into the nucleus. In the nucleus, prothymosin may interact with proteins involved in DNA metabolism and cell cycle control (Freire et al., 2001). Prothymosin interacts with Rev proteins (HTLV-I Rex and HIV-1 Rev), as has been shown by affinity chromatography experiments (Kubota et al., 1995). Rev proteins may possibly be involved in the export of prothymosin from the nucleus. Prothymosin lacks a leucine-rich nuclear export signal

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(NES) and Rev proteins may function as piggybacks for nuclear export of prothymosin. It has been demonstrated that the Epstein–Barr virus nuclear antigen (EBNA3C) interacts with prothymosin in regulating histone acetylation (Cotter and Robertson, 2000). Amino acid residues between positions 366 and 400 of EBNA3C are responsible for the interaction with prothymosin. Moreover, prothymosin interacts with two domains (CH1 and CH3/HAT) of histone acetyltransferase p300 in EBV-infected cells. These two domains of p300 are also responsible for the interaction with the N and C terminus of EBNA3C (residues 1–207 and 620–992), suggesting that these interactions are important for transcriptional regulation and gene expression of the transformed cells (Subramanian et al., 2002). Prothymosin may remove histone H1 from the nucleosome and be involved in the recruitment of other general transcription factors. Therefore prothymosin seems to be involved in chromatin decondensation and acetylation of core histones as it interacts with the histone acetyl transferase p300 at two domains, interacting additionally with other transcriptional factors including RNA polymerase II. Another histone acetyltransferase has been identified as interacting with prothymosin. Prothymosin interacts physically with the CREB-binding protein (CBP), which is a versatile transcription coactivator. The site of interaction was mapped within the N-terminal domain of CBP (residues 1–771) and a region of prothymosin composed of two polyglutamate stretches (Karetsou et al., 2002).

G. PROTHYMOSIN AND SMALL RNA

Prothymosin has been identified as a 13-kDa protein that is present in RNA–protein complexes in human, bovine, and yeast cells (Makarova et al., 1989). As described earlier, the presence of prothymosin in yeast has not been confirmed (Trumbore et al., 1998). Prothymosin is covalently attached to small, unidentified cytoplasmic RNA in mammalian cells. In E. coli cells overexpressing recombinant rat prothymosin, several bacterial tRNAs were identified to be linked via their 50 terminus (Vartapetian et al., 1997). The points of attachment were mapped by mutational analysis to several sites on prothymosin (positions 14–20, 89–98, and 102–106). The attachment of tRNA to prothymosin occurs via a stable bond resistant to denaturing conditions as well as proteolytic degradation of prothymosin. tRNA attachment seems to occur within a cluster of lysine residues located at both termini of prothymosin. Modification of prothymosin by tRNA between positions 89 and 98 might interfere with the function of the bipartite NLS as well as with the caspase 3 cleavage sites. However, tRNA linking to prothymosin appears to be relatively inefficient, at least in E. coli.

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The biological meaning of attaching tRNAs to prothymosin is unclear at present (Lukashev et al., 1999).

H. EXTRACELLULAR THYMOSIN 1 AND PROTHYMOSIN

Prothymosin was called ‘‘thymic hormone’’ because it was detected in blood serum (60 pM) (Panneerselvam et al., 1987) together with its cleavage product thymosin 1 (80–400 pM) (Naylor et al., 1992; Weller et al., 1992; Molinero et al., 2000). Extracellular thymosin 1 stimulates endothelial cell migration, angiogenesis, and wound healing (Malinda et al., 1998). The stimulation of angiogenesis has also been shown for prothymosin (Koutrafouri et al., 2001). Thymosin 1 is able to increase interleukin 2 receptors on mitogenstimulated human lymphocytes (Sztein et al., 1986; Sztein and Serrate, 1989) and promotes secretion of Th1 cytokines such as interferon (IFN-) (Serrate et al., 1987). Combinations of thymosin 1 and nucleoside analogs (famciclovir) or interferon are still under investigation for treating patients with chronic hepatitis B and C virus infections (Billich, 2002; Lau et al., 2002). In hepatitis B e antigen (HBeAg)-positive Chinese patients, the clearance of HBeAg is significantly greater in those treated with a 6-month course of thymosin 1 (Chien et al., 1998); however, the effect is less pronounced in white patients (Mutchnick et al., 1999). Thymosin 1, together with low doses of interferon or interleukin 2, is highly effective in restoring several immune responses depressed by tumor growth and cytostatic drugs and increases the antitumor effect of chemotherapy while markedly reducing the general toxicity of treatment (reviewed in Bodey et al., 2000; Garaci et al., 2000). It has been reported that thymosin 1 can positively modulate hematopoietic functions of murine bone marrow cells and restore myelopoiesis in tumor-bearing mice (Paul and Sodhi, 2002). Mammary carcinomas were observed 3 months after injection of nitrosomethylurea into rats. Daily administration of thymosin 1 (10 g) reduced carcinoma incidence and prolonged survival time (Moody et al., 2002). All these effects attributed to thymosin 1 require that extracellular thymosin 1 be able to influence the intracellular metabolism of target cells. The mechanism of action might either be by modulating cytokine receptors via interaction of thymosin 1 with negatively charged membranes (Grottesi et al., 1998) or by a specific receptor. Specific receptors with low (15 nM) and high (250 pM) affinity for prothymosin have been identified on the surface of human peripheral blood mononuclear cells (Cordero et al., 1994). Three binding partners (31, 29, and 19 kDa) have been purified by affinity chromatography from membranes of phytohemagglutinin (PHA)-activated lymphoblasts. The putative receptor for prothymosin was localized in a

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caplike structure at one of the poles of blood mononuclear cells (Pineiro et al., 2001).

IV. PARATHYMOSIN Compared with prothymosin much less is known about parathymosin. Research has focused mainly on prothymosin and neglected parathymosin. Because of many similarities, prothymosin and parathymosin are thought to build a protein family with similar functions. However, there are differences in their biological function that strongly recommend treating these two peptides as separate entities. Parathymosin was the first thymosin that was not isolated completely or in a fragmented form from thymosin fraction 5. During the isolation of prothymosin an unknown peptide was purified as a by-product (Haritos et al., 1985b). Because of its structural homology to prothymosin in size and amino acid composition (Fig. 2), it was named parathymosin (Haritos et al., 1985c). The amino acid sequence of rat parathymosin was determined (Komiyama et al., 1986). Parathymosin as prothymosin is present in various nonlymphoid tissues of rat in high concentrations (217 g/g liver; 78 g/g thymus). Tissues high in parathymosin tend to be low in prothymosin (110 g/g liver; 916 g/g thymus) (Clinton et al., 1989a). Parathymosin has been quantified in different rat tissues by an enzyme-linked immunosorbent assay (ELISA). All tissues except erythrocytes contain parathymosin (Brand et al., 1991). Early on in parathymosin research, it was speculated that

FIGURE 2. Amino acid sequences of parathymosins from various species: Homo sapiens (human, P20962), Bos taurus (bovine, P08814), Rattus norvegicus (rat, P04550), and Mus musculus (mouse, Q9D0J8). The numbers in parentheses are the accession codes for the SWISSPROT and TrEMBL databases. The bipartite nuclear localization signal is outlined; potential caspase 3 sites are shown in italic. The question mark points to a potential mistake in the amino acid sequence of bovine parathymosin, because all other parathymosins possess a lysine reside at position 79 (Trompeter et al., 1996). The nucleic acid sequence of bovine parathymosin has not been determined. Invariant residues are indicated by asterisks, and highly conserved residues are indicated by colons.

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parathymosin might modulate the action of prothymosin in protecting sensitive strains of mice against opportunistic infection with Candida albicans. The amino acid sequences of human (Clinton et al., 1989b) and bovine (Panneerselvam et al., 1988a) parathymosin are highly homologous to that of rat prothymosin. Mouse parathymosin has not been characterized at the peptide level; however, its cDNA was identified by annotating 21,076 cDNAs analyzed by the RIKEN Mouse Gene Encyclopaedia Project (Kawai et al., 2001). All parathymosins sequenced are acetylated at the N-terminal serine and consist of 101 amino acid residues (Fig. 2). The isoelectric point of rat parathymosin is 4.15, slightly less acidic compared with a pl of 3.55 in the case of rat prothymosin (Haritos et al., 1985c). The translated part of the rat parathymosin gene is interrupted by one large intron (2589 bp) and three small introns (191, 150, and 167 bp). The presence of an initiator codon immediately preceding the codon for the N-terminal serine residue and a terminator codon immediately following the codon for the C-terminal Ala-101 suggested that prothymosin is synthesized without formation of a larger precursor (Trompeter and So¨ling, 1992). A. ZN2+-BINDING PROTEIN

An acidic zinc-binding protein (ZnBP) of 11.5 kDa, purified from rat liver, inhibits the glycolytic enzyme phosphofructokinase-1. The reversible inhibition results from a dissociation of the tetrameric enzyme into its inactive protomers and is dependent on the presence of 1–20 M Zn2+, whereas at higher concentrations of zinc (100 M) inhibition was completely abolished (Brand and So¨ling, 1986). In 1989 the cDNA sequencing of ZnBP revealed its identity with parathymosin (Trompeter et al., 1989). The protein has four binding sites for zinc (KD 6 M). Two of them were called specific because zinc binding is still observed in the presence of high salt (0.75 mM Mg2+, 100 mM NaCl). Four clusters of acidic amino acid residues responsible for zinc binding and inactivation of phosphofructokinase were identified between positions 35 and 78 of parathymosin. By further proteolytic cleavage the two specific zinc-binding sites were located to amino acid residues between positions 51 and 72, whereas the region between positions 35 and 50 is necessary for binding and inactivation of phosphofructokinase (Brand et al., 1988). The zinc-binding sites of parathymosin are different from zinc finger motifs. It has been shown by parathymosin–Sepharose affinity chromatography that the interaction of parathymosin with many enzymes of carbohydrate metabolism is zinc specific. From liver cytosol the following enzymes were retained: hexokinase/glucokinase, glucose-6-phosphate dehydrogenase, phosphofructokinase-1, aldolase, glycerol-3-phosphate dehydrogenase, glyceral-3-phosphate dehydrogenase, fructose-1,6-bisphosphatase, and the phosphorylated form of pyruvate kinase L. Thus a possible role of

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parathymosin in supramolecular organization of carbohydrate metabolism was proposed (Brand and Heinickel, 1991). B. BIPARTITE NUCLEAR LOCALIZATION SIGNAL

Injection of parathymosin into Xenopus oocytes led to a nuclear uptake of the peptide (Watts et al., 1990), the motif –79KR-X10-KRQK94– comparable to a similar motif of prothymosin (–87KR-X12-KKQT104–), has been identified as a bipartite NLS (Trompeter et al., 1996). The bipartite NLS of prothymosin is separated by 12 amino acid residues, which generate several functional caspase 3 cleavage sites, whereas that of parathymosin is separated by 10 amino acid residues displaying only one potential caspase 3 cleavage site. Yet it has not been studied whether parathymosin can be cleaved by caspases. Immunocytochemical localization of parathymosin revealed a cell-type specific distribution between cytosol and nucleus despite the bipartite NLS. In most cells, the cytoplasm was stained exclusively. In contrast, in duodenal and jejunal crypt cells immunostaining was nuclear, whereas the more mature cells at the top of the villi contained most of the antigen in the cytoplasm. Immunostaining of nuclei was also observed in pancreatic duct cells. Duodenal and jejunal mucosae have a high proliferation rate compared with other somatic tissue; the cells at the top of mucosal villi are terminally differentiated. Thus these data indicated that parathymosin is redistributed between cytoplasm and nucleus depending on the proliferation/differentiation process of the tissue (Brand et al., 1991). Nuclear parathymosin is excluded by nucleoli and correlates with early replication sites, as has been shown by indirect immunofluorescence labeling and confocal scanning laser microscopy (Vareli et al., 2000). Despite the data just presented parathymosin was isolated as a zinc-binding protein inhibiting the cytosolic phosphofructokinase, binding to several enzymes of carbohydrate metabolism, and shown to be located in the cytosol in many rat tissues. The puzzle was solved when it was recognized that the translocation of parathymosin from the cytosol to the nucleus was negatively correlated with cell density. Thinly seeded hepatocytes keep their parathymosin in the nucleus, whereas at high cell density parathymosin is retained in the cytoplasm. Thus at high cell density the bipartite NLS must be inactivated, possibly by covering the NLS with a cytosolic protein. Freshly prepared rat liver cytosol indeed contains a protein with an apparent molecular mass of >250 kDa, which is able to conceal the NLS of parathymosin. No protein inhibiting nuclear import of parathymosin could be isolated from permanent cell lines, in accordance with the fact that in these proliferating cells parathymosin was always observed in the nucleus (Trompeter et al., 1999). The following scenario might be conceivable. Resting cells synthesize the protein inhibiting nuclear import of parathymosin, and thus parathymosin is retained in the

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cytosol and inhibits phosphofructokinase partially. Cells meet their ATP demand mainly by oxidative glucose breakdown. However, when cells in primary culture or tumor cells proliferate, their demand for ATP increases and they metabolize glucose anaerobically to lactate (Brand and Hermfisse, 1997). The concentration of the protein inhibiting nuclear import of parathymosin decreases and parathymosin is translocated into the nucleus (Trompeter et al., 1999). There it binds to histone A1 (Kondili et al., 1996) and supports replication of DNA (Vareli et al., 2000). The inhibition of the glycolytic key enzyme phosphofructokinase is abolished, leading to a stimulation of anaerobic glycolysis. C. PHOSPHORYLATION OF PARATHYMOSIN

No reports exist on potential phosphorylation or any other posttranslational modifications of parathymosin except the acetylation of its N terminus. This is surprising because of the sequence homology to prothymosin. Only a doublet detected by Western blot with specific antibodies against parathymosin might indicate the presence of a rather stable posttranslational protein modification (Brand et al., 1991), which might be a phosphorylated form of parathymosin. D. PARATHYMOSIN AND GLUCOCORTICOID ACTION

Whereas prothymosin was found to enhance estrogen receptor transcriptional activity by acting as an inhibitor of an anticoactivator (Martini et al., 2000), parathymosin inhibits activated glucocorticoid-receptor binding to DNA containing glucocorticoid-response elements. Inhibition of glucocorticoid–receptor binding to nuclei is mediated by the acidic domain(s) of parathymosin located between residues 43 and 79. This inhibitory activity of parathymosin may not require zinc. Glucocorticoids inhibit proliferation and promote differentiation of cells. Thus, in proliferating cells, parathymosin would accelerate proliferation by inhibiting glucocorticoid action (Okamoto and Isohashi, 2000).

V. -THYMOSINS The first -thymosin isolated from thymosin fraction 5 was termed thymosin 4 (Low and Goldstein, 1982). This polypeptide has been reported to have an effect on thymus-dependent maturation of lymphoid stem cells by inducing the terminal deoxynucleotidyltransferase (Low et al., 1981). It also inhibits migration of macrophages (Thurman et al., 1984) and exerts effects on hypothalamus and pituitary (Rebar et al., 1981). Shortly after the characterization of thymosin 4, two highly homologous peptides named 8

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FIGURE 3. Amino acid sequences of -thymosins from various species: 4 (P01253); 4Ala

(P34032); 4Xen (Xenopus laevis, P18758); 9 (P21752); 9Met (P21753); 10 (P13472); 11 (Oncorhynchus mykiss, P26351); 12 (Oncorhynchus mykiss, P26352); 12perch (Lateolabrax japonicus, P33248); 13(Mihelic and Voelter, 1994); 14 (Stoeva et al., 1997); 15 (P97563); scallop (Argopectan irradians; Safer and Chowrashi, 1997); zebrafish (Q9W7M8); and sea urchin (Arbacia punctulata; Safer and Chowrashi, 1997). Other -thymosins have been identified only by nucleic acid sequencing: 4Y (O14604); 4nb (Q99406); 15mouse (Q9D2R9); carp-A (Cyprinus carpio, Q91955); carp-B (Q9I954); 10torp (Torpedo marmorata, Q9I980); quail (Coturnix coturnix japonica, Q9DET5); sycon (Sycon raphanus, Q9GUA6); strongyl (Strongylocentrotus purpuratus, O76538); and gilli (Gillichthys mirabilis, Q9DFJ9). The numbers in parentheses are the accession codes for the SWISS-PROT and TrEMBL databases. Invariant residues are indicated by asterisks, and highly conserved residues are represented by colons.

and 9 were purified by isoelectric focusing (Hannappel et al., 1982a). When thymosin fraction 5 was used as the starting material for purification, thymosin 8 was isolated. However, purification starting with fresh-frozen calf thymus by a procedure that minimizes proteolysis yielded thymosin 9 (Hannappel et al., 1982a). Thymosin 9 is identical to 8 except for the presence of an additional dipeptide (AK) at the C terminus (Fig. 3). This indicates that artificial proteolysis might occur during preparation of thymosin fraction 5.

A. PURIFICATION OF -THYMOSINS

To isolate -thymosins, various purification schemes have been developed in our laboratory with particular emphasis to avoid proteolysis

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during the isolation procedure (Hannappel et al., 1982b; Hannappel, 1986; Huff et al., 1997b). The procedures for purification of -thymosins consist basically of four steps: (1) extraction of cells or tissue and simultaneous denaturation of proteases, (2) concentration and desalting by solid-phase extraction, (3) separation of the peptides according to their isoelectric points (isoelectric focusing or chromatofocusing), and (4) separation according to hydrophobicity [reversed-phase high-performance liquid chromatography (HPLC)]. B. AMINO ACID SEQUENCES AND PHYLOGENETIC DISTRIBUTION OF -THYMOSINS

Currently, 14 other -thymosins (Fig. 3) from various vertebrates and invertebrates have been described (Huff et al., 2001). They form a family of highly conserved polar 5-kDa peptides consisting of 40–44 amino acids. Members of this family have been found in species ranging from mammals to echinoderms but not in yeast or in prokaryotes. The presence of an initiator codon immediately preceding the codon for the N-terminal serine or alanine residue, and a terminator codon immediately following the codon for the C-terminal amino acid residue, suggest that -thymosins are synthesized without formation of a larger precursor. The N-terminal residue is always acetylated. Except for one phenylalanine present at position 12 of all -thymosins and a tyrosine at position 40 of 15, -thymosins do not contain aromatic amino acid residues. Thus, they almost do not absorb at 280 nm and can only be detected by absorption below 220 nm. Because many substances absorb below 220 nm, we recommend that -thymosin be separated by reversed-phase HPLC and that the separated peptides be detected by postcolumn derivatization with fluorescamine (Hannappel, 1986; Huff et al., 1997b). According to searches in expressed sequence tag (EST) and cDNA databases, other species may also contain members of the -thymosin family (Fig. 3). In addition, in Drosophila melanogaster and Caenorhabditis elegans, two larger gene products have been identified as containing three repeats of a -thymosin-like sequence (Fig. 4). The Drosophila protein (ciboulot, Cib) binds to G-actin (see later) but, unlike conventional -thymosins, it participates in actin polymerization and is functionally more similar to profilin (Boquet et al., 2000). -Thymosins are largely unstructured in water, comparable to prothymosin and parathymosin. However, addition of trifluoroethanol promotes the formation of -helical structures involving residues 4–16 and 30–40 (Zarbock et al., 1990; Czisch et al., 1993; Feinberg et al., 1996). In most mammalian tissues, at least two -thymosins are expressed (Fig. 3). Tumor tissue might contain additional -thymosins (Bao et al., 1996, 1998; Gold et al., 1997). Thymosin 4 is usually the main peptide,

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FIGURE 4. Comparison of the three -thymosin-like sequences of Cib protein of Drosophila melanogaster (O97428) and a hypothetical 17.0-kDa protein of Caenorhabditis elegans (O17389). The percentages given are calculated as the sum of invariant (*) and highly conserved (:) residues divided by the number of amino acids of the corresponding segment times 100.

representing about 70–80% of the total -thymosins in normal tissue of adult rats (450 g/g spleen, 11 g/g muscle) (Hannappel, 1986). It is present in high concentrations (up to 400 M) in rodent tissue, tumor cells, and cell lines from various mammalian species (Hannappel et al., 1982c; Xu et al., 1982). EBV-transformed human cell lines contain up to 1 pg of thymosin 4 per cell, and 1% of total protein synthesis is dedicated to the synthesis of thymosin 4 (Hannappel and Leibold, 1985). High concentrations of thymosin 4 have been detected in whole blood (12–19.5 mg/liter), mononuclear leukocytes (183–380 fg/cell), polymorphonuclear leukocytes (269–564 fg/cell), and human platelets (6.9–31.7 fg/cell), whereas serum contained less than 1% of the thymosin 4 present in whole blood. Incidentally, this is still equal to a concentration of about 20 nmol/liter of serum. No -thymosins have been detected in erythrocytes (Hannappel and van Kampen, 1987). The high intracellular concentration and ubiquitous distribution of -thymosins prompted us early to speculate that thymosin 4 is not a thymic hormone but fulfills some general function in cells, for example, as part of the cytoskeletal system. C. -THYMOSINS AND G-ACTIN

In spite of data pointing to a general function of -thymosins inside of cells, the concept of thymosin 4 as a thymic hormone lasted until the early

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FIGURE 5. Schematic representation of G-actin sequestering by thymosin 4. For clarity thymosin 4 has been moved to the right by the length of the arrow. Thymosin 4 forms a 1:1 complex with G-actin. The ATP molecule bound to G-actin is depicted in the center of G-actin formed by its four subdomains (I–IV). The C terminus (C) and the N terminus (N) of G-actin reside in subdomain I. Thymosin 4 interacts with G-actin in an extended form like a clip covering G-actin domains III, I, and II and inhibits G-actin polymerization by steric hindrance (Safer et al., 1997). 1990s, when Safer and co-workers established that the previously isolated 5-kDa actin-sequestering peptide (Fx) is identical to thymosin 4 (Safer et al., 1990, 1991), Thymosin 4 sequesters G-actin in a 1:1 complex and inhibits salt-induced polymerization (Fig. 5). All other -thymosins studied also possess G-actin-sequestering activity in various in vitro systems (Hannappel and Wartenberg, 1993; Heintz et al., 1993; Yu et al., 1993; Jean et al., 1994; Huff et al., 1995). -Thymosins have a 50- to 100-fold higher affinity for MgATP–actin than for MgADP–actin (Carlier et al., 1993; Jean et al., 1994). The dissociation constant of the thymosin 4–ATP– G-actin complex is in the range of 0.5 to 2.5 M. Both potential helices of thymosin 4 seem to be important for complex stability. On the basis of chemical and enzymatic cross-linking a structure was proposed for the thymosin 4–G-actin complex. Thymosin 4 interacts in an extended form with subdomains III, I, and II of G-actin and inhibits polymerization by steric hindrance (Fig. 5) (Safer et al., 1997). The stability of the 4–G-actin complex (Huff et al., 2001) can be altered by changes in the amino acid sequence of thymosin 4. This is most impressive in the case of thymosin 4Ala, in which only the first amino acid residue of thymosin 4 is changed

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from serine to alanine, which results in a 3- to 5-fold higher affinity for G-actin (Huff et al., 1995). On the other hand, oxidation of the methionine residue at position 6 (4-sulfoxide) increases the dissociation constant of the complex by about 20-fold (Jean et al., 1994; Huff et al., 1995; Huff and Hannappel, 1997). Concomitantly, a 20-fold molar excess of thymosin 4-sulfoxide is necessary to inhibit polymerization of G-actin. Whether the oxidation of intracellular thymosin 4 to its sulfoxide is a mechanism to modulate affinity for G-actin is questionable. The herbicide paraquat (1,10 dimethyl-4,40 -dipyridylium dichloride) causes damage to human lung, presumably by oxidative stress. In an in vitro situation incubation with paraquat destabilizes the complex of thymosin 4 with G-actin in a timeand concentration-dependent manner as indicated by an about 50-fold increase in the dissociation constant. However, even in the presence of high paraquat concentrations, HPLC analysis revealed no oxidation of thymosin 4. Thus, paraquat might act directly on G-actin (Huff et al., 1998). Comparable to the oxidation of thymosin 4, truncation of the first 6 or 12 amino acid residues (47–43 or 413–43) increases the dissociation constant by about 20-fold. However, peptides 47–43 and 413–43 are no longer able to inhibit polymerization of G-actin despite a level of binding to G-actin comparable to thymosin 4-sulfoxide. Truncation of the first 23 amino acid residues completely abolishes the interaction with G-actin (Fig. 5) (Huff et al., 1995). G-actin covalently cross-linked to thymosin 47–43 can be polymerized by high salt to fibers even in the absence of F-actin-stabilizing phalloidin (Ballweber et al., 2002). Changes in the C-terminal structure of thymosin 4 also modulate the interaction with G-actin. Truncation of the last 26 amino acids (41–16) wipes out the interaction with G-actin completely. After removing the putative C-terminal helix of thymosin 4, the peptide 41–30 still inhibits saltinduced actin polymerization, although a 25-fold molar excess over G-actin was needed (Fig. 5) (Vancompernolle et al., 1992). The effect of C-terminal truncation is only minute when the last two amino acid residues of thymosin 10 were removed (Huff et al., 1997a). Chimeras of thymosin 4 and 15 were generated because thymosin 15 identified in rat prostatic carcinoma (Bao et al., 1996) binds G-actin with a 2.4-fold higher affinity than does thymosin 4. Replacement of the 10 C-terminal amino acids residues of thymosin 15 with those of thymosin 4 reduced the actin-binding affinity of the chimera relative to thymosin 15. Complementary replacement of the thymosin 4 C-terminal amino acid residues with those of thymosin 15 led to increased G-actin binding (Eadie et al., 2000). The central sequence motif 17LKKTETQEK25 of thymosin 4 seems to be important for the interaction with G-actin. This sequence motif is highly homologous to the well-known actin-binding sequence of actobindin (Safer et al., 1991) and is flanked on both sides by a potential helical region.

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Already the first six amino acid residues of thymosin 4 are indispensable for inhibition of salt-induced actin polymerization. Even minute changes in the amino acid sequences ( 4 vs. 4Ala, 4 vs. 4-sulfoxide, or 10 vs. 101–41) might be reflected in the actin-binding affinity and be important for the biological function(s). D. -THYMOSINS AND F-ACTIN

Thymosin 4 is supposed to be the main G-actin-sequestering peptide in mammals. However, it has been demonstrated that thymosin 4 is able to interact with filamentous actin (Carlier et al., 1996; Sun et al., 1996). The ability of thymosin 4 to depolymerize F-actin decreased with increasing concentrations of thymosin 4. At concentrations >100 M, thymosin 4 stabilized F-actin and was incorporated into filamentous actin at low molar ratios (Carlier et al., 1996). The notion that thymosin 4 is not just a simple G-actin-sequestering peptide was supported by the observation that the number and thickness of actin filaments increased in NIH 3T3 cells overexpressing thymosin 10 (Sun et al., 1996). In the presence of phalloidin, chemically cross-linked thymosin 4 actin can be incorporated into F-actin. As expected, chemically cross-linked thymosin 47–43 actin can polymerize even in the absence of phalloidin. Data suggest that thymosin 4 interacting with actin adopts a structural fold similar to that observed in the presence of trifluoroethanol (Ballweber et al., 2002). In a complementary manner, the structure of G-actin is changed by thymosin 4 binding (De La Cruz et al., 2000). E. -THYMOSINS AND CANCER

G-actin-sequestering -thymosins might be involved in cancerogenesis and metastatic potential of tumors because -thymosins could supply a pool of G-actin when cells need filaments (Fig. 6). It is not clear how -thymosins might influence metastasis but it is likely to relate to the need for cells to migrate (Ridley, 2000). Expression of thymosins 4, 10, and 15 appears to be involved in the manifestation of the malignant phenotype of human tumor cells. In general, increased levels of -thymosins seem to correspond to tumor malignancy. In the human breast cancer cell line MCF-7 the expression of thymosins 4 and 10 is differentially regulated (Otto et al., 2002). Several points should be taken into account when discussing the role of -thymosins in cancerogenesis and the metastatic potential of tumors: first, in most studies, only one -thymosin has been investigated, although most cells express several -thymosins, which may or may not share the same biological function(s). The expression of -thymosins is regulated differentially. However, neither the molecular basis of the gene expression nor the need of cells for different -thymosins is understood (Huff et al.,

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FIGURE 6. Functions attributed to -thymosins. The expression of -thymosins (&, ) seems to be involved in differentiation and dedifferentiation and in the organization of actin cytoskeleton (depicted as lines inside of cells). Thymosin 4 released from cells can exert additional effects. It could be oxidized to its sulfoxide or processed to AcSDKP. AcSDKP is degraded by angiotensin 1-converting enzyme (ACE). There is also evidence that -thymosins can act directly on cells, resulting in a change in gene expression or chemotaxis of certain cells. Neither receptors (?) on cells nor the signal transduction pathway(s) (?) have been characterized. -Thymosins can be cross-linked to proteins (fibrin, etc.) by transglutaminases (blood coagulation factor XIIIa).

2001). Second, often only the change in the mRNA is determined, which does not necessarily give information about the intracellular concentration of the -thymosin (Scho¨bitz et al., 1990, 1991a, b). Third, if the cancerogenic function of -thymosins is related to their G-actin-sequestering ability, it might be helpful to monitor changes in G-and F-actin. Thymosin 10 overexpression, for example, seems to be a general event in a wide variety of human carcinomas (human colon carcinonas, germ cell carcinomas of different histological types, breast carcinomas, ovarian carcinomas, uterine carcinomas, colon carcinomas, and esophageal carcinoma cell lines) (Califano et al., 1998). With the advent of DNA array methodology, there is an increasing number of reports indicating that -thymosin expression is correlated with pathological alterations of blood vessels and cancer (Tung et al., 2001; Sardi et al., 2002). By comparing

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melanoma cells with low and high metastatic potential on DNA arrays, the mRNAs for thymosins 4 and 10 were expressed at higher levels in highly metastatic cells (Clark et al., 2000). Thymosin 15 was described as a novel regulator of tumor cell motility in Dunning rat prostatic carcinoma correlated with metastatic potential (Bao et al., 1996). In a clinical pilot study on human prostate cancer, it has been shown that thymosin 15 might be a useful marker to identify high-risk patients (Chakravatri et al., 2000). This -thymosin is also proposed as a marker for other carcinomas (Gold et al., 1997; Bao et al., 1998). To explore the molecular mechanism of transcriptional regulation of thymosin 15, the rat thymosin 15 gene was isolated and characterized (Bao and Zetter, 2000). Thymosin 15 binds G-actin with a 2.4-fold higher affinity than does thymosin 4 (Eadie et al., 2000), which might be responsible for the metastatic potential of those cells. F. -THYMOSINS IN ANGIOGENESIS AND WOUND HEALING

-Thymosins may be involved in angiogenesis (Grant et al., 1995; Malinda et al., 1997), wound healing (Frohm et al., 1996; Malinda et al., 1999), and apoptosis (Iguchi et al., 1999; Niu and Nachmias, 2000). The mRNA for thymosin 4 increased 5-fold in endothelial cells growing on Matrigel compared with cells growing on plastic (Fig. 6). Endothelial cells transfected with thymosin 4 showed an increased rate of attachment and spreading, as well as an accelerated rate of tube formation on Matrigel. Because an antisense oligonucleotide to thymosin 4 inhibited these effects, thymosin 4 might be involved early in the differentiation of endothelial cells and vessel formation (Grant et al., 1995). Hepatocyte growth factor, exerting motogenic effects on various target cells, induces the expression of thymosin 4 in human umbilical vein endothelial cells (HUVECs) (Oh et al., 2002). Thymosin 4 exhibits chemoattractant activity for HUVECs and keratinocytes (Malinda et al., 1997, 1999). The angiogenic effects of various ‘‘thymosin’’ peptides have been studied in the chick chorioallantoic membrane model. Thymosin 4, prothymosin, and thymosin 1 were found to enhance angiogenesis, whereas parathymosin, thymosin 9, and thymosin 10 were inhibitory (Koutrafouri et al., 2001). Because transglutaminases also participate in all these cellular reactions, we investigated whether thymosin 4 might be participating in transglutaminase-catalyzed reactions. Thymosin 4 serves as a specific glutaminly substrate for guinea pig transglutaminase, using dansylcadaverine as aminyl substrate. Thymosin 4 can be labeled rapidly at two residues (Gln-23 and Gln-36) whereas the third glutamine residue at position 39 reacts slowly (Huff et al., 1999). Gln-23 and Gln-36 are conserved in most or all -thymosins (Fig. 3). Despite the presence of nine lysine residues, thymosin

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4 does not participate as an aminyl substrate in the transglutaminase reaction. Thymosin 4 can be cross-linked by tissue transglutaminase to some proteins (fibrinogen, fibrin, collagen, and actin) but not to others (plasmin, hexokinase, alcohol dehydrogenase etc). After activation of human platelets with thrombin, thymosin 4 is released and cross-linked to fibrin. Because factor XIIIa is coreleased from platelets, this transglutaminase might mediate cross-linking (Fig. 6). This provides a potential mechanism to ‘‘fix’’ thymosin 4 near sites of platelet activation or injury, permitting it to contribute in the extracellular space to biological processes associated with clotting and wound repair (Huff et al., 2002). Glucocorticoids upregulate the expression of antiinflammatory mediators, and thus the characterization of these molecules could give the therapeutic benefits of steroids without toxic side effects. Supernatants from monocytes and macrophages cultured in the presence of glucocorticoids contain thymosin 4-sulfoxide as an active component. Whereas thymosin 4 is not active, thymosin 4-sulfoxide promotes dispersive locomotion of neutrophils, lowers their adhesion to endothelial cells, and inhibits their chemotactic response to fMLP. Thymosin 4-sulfoxide is a potent inhibitor of carrageenin-induced edema in mouse paw. Thymosin 4 seems to act inside of cells in its methionyl form as the main G-actin-sequestering peptide, whereas outside of cells the oxidized, methionyl-sulfoxide form of thymosin 4 attenuates inflammatory response (Fig. 6) (Young et al., 1999). It has been noticed that thymosin 4 promotes wound healing and decreases inflammation in heptanol-damaged corneas (Sosne et al., 2001). Similar effects on wound healing and inflammation were observed following alkali injury of corneas from mice. Mouse corneas topically treated with 5 g of thymosin 4 twice daily demonstrated accelerated reepithelialization and decreased signs of inflammation when compared with controls. mRNA transcript levels were decreased for IL-1, macrophage inflammatory proteins (MIP-1, MIP-1, and MIP-2), and monocyte chemoattractant protein 1. Thus, thymosin 4 may provide a treatment for severe traumatic corneal injuries (Sosne et al., 2002). Thymosin 4 is also active in accelerating wound repair in full-thickness wounds in both db/db diabetic and aged mice (Philp et al., 2003). The FDA has just given the permission to begin phase 1 clinical trials with thymosin 4 in wound healing in the US. When we determine thymosin 4 by reversed-phase HPLC in cells or tissue, we can easily identify -thymosins containing methionine residues, because the oxidized, methionyl-sulfoxide-containing form elutes about 2 to 5 min earlier from the column compared with the nonoxidized form (eluting at about 50 min). Exposing methionine-containing -thymosins to neutral or alkaline pH causes oxidation of the methionine residue by oxygen. To avoid oxidation we keep the peptides at slightly acidic pH or add thiodiglycol. Because it has been demonstrated that thymosin 4 and

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thymosin 4-sulfoxide possess different biological properties in terms of G-actin sequestering inside of cells or modulation of inflammation outside of cells, it becomes more and more important to discriminate between the two forms of thymosin 4. These two forms can be distinguished only by reversed-phase HPLC or MALDI-TOF (matrix-assisted laser desorption ionization time-of-flight mass spectrometry). This shift in retention time or molecular mass is used in our laboratory routinely to identify methioninecontaining -thymosins by oxidation with diluted H2O2.

G. THYMOSIN 4 AND ACSDKP

The tetrapeptide AcSDKP (Seraspenide, Goralatide) is a physiological regulator of hematopoiesis, blocking the transition from G0/G1 to S phase of hematopoietic stem cells (Monpezat and Frindel, 1989). It was originally purified from fetal calf bone marrow (Lenfant et al., 1989). AcSDKP represents the N-terminal sequence of thymosin 4 and can be generated by a single cleavage step employing a mammalian Asp-N-like protease (Fig. 6) (Grillon et al., 1990). The protease responsible for conversion of thymosin 4 to AcSDKP has not been isolated from bone marrow. We noticed that thymosin 4 might be a substrate for caspase 3 because of its N-terminal sequence (AcSDKPD-M . . . ), which is similar to the multiple caspase 3 cleavage sites (–DxxD–) of prothymosin. The generated pentapeptide (AcSDPKD) might then be converted by a carboxypeptidase to AcSDPK. However, we were unable to cleave thymosin 4 with caspase 3 in vitro (our unpublished results). Many indirect data indicate that AcSDKP might originate from thymosin 4; however, the final proof is still missing. Thymosin 4 itself is reported to inhibit normal bone marrow progenitor cell growth. Although the inhibitory effect of thymosin 4 is similar to that of AcSDKP, a truncated form devoid of AcSDKP was also active (Bonnet et al., 1996). AcSDKP concentrations have been determined in blood (2 nM) and testicular interstitial fluid (22 nM) by a highly specific enzyme immunoassay (EIA). The authors speculate that AcSDPK might play a role in the regulation of spermatogenesis (Stephan et al., 2000). The half-life of AcSDKP in human plasma is about 80 min. AcSDKP is hydrolyzed by angiotensin 1-converting enzyme (ACE) to AcSD and KP, which is cleaved rapidly in blood to lysine and proline (Rieger et al., 1993; Rousseau et al., 1995). Consequently, higher concentrations of AcSDKP are found in patients treated with ACE inhibitors (Azizi et al., 1997; Comte et al., 1997). Because ACE inhibitors can cause anemia in some patients, it is currently discussed whether the increased level of AcSDKP caused by ACE inhibitors might be responsible for partial resistance of patients to erythropoietin treatment (Le Meur et al., 2001) or not (Abu-Alfa and Perazella, 2002).

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VI. CONCLUSIONS The work on ‘‘thymosin’’ started in 1965. Since then more than 1800 scientific papers have been published. Thymosin fraction 5 was the starting material for the isolation of many biologically important peptides. However, none of them really kept the promise to be a thymic hormone. Polypeptide 1 turned out to be identical to ubiquitin truncated by a dipeptide at the C terminus. Ubiquitin is indispensable for ATP-dependent proteolysis in cells. Thymosin 1 is generated from a larger polypeptide named prothymosin . Despite its suggestive name it became evident that the main function of prothymosin is not as the precursor of thymosin 1; rather, it has several biological activities of its own. Prothymosin might be involved inside the nucleus in several processes controlling transcription and estrogen receptor activity. More studies will be necessary to understand the molecular mechanism by which prothymosin remodels chromatin structure and modulates transcription. Outside cells, other functions have been attributed to thymosin 1 and prothymosin (stimulation of cell migration, angiogenesis, and wound healing). In addition, thymosin 1 is used in clinical trials for treatment of viral infections (hepatitis). Parathymosin has been isolated as a Zn2+-binding protein that inhibits the glycolytic enzyme phosphofructokinase. It interacts with several other enzymes of carbohydrate metabolism and has therefore been proposed to play a role in the supramolecular organization of this important metabolic pathway. Besides this, it translocates from cytosol to the nucleus, dependent on cell density. At high cell density the bipartite NLS of parathymosin is covered by a cytosolic protein and parathymosin binds to and inhibits phosphofructokinase. At low cell density, parathymosin is translocated to the nucleus, where it binds to histone A1 and supports replication of DNA. In contrast to prothymosin, which enhances estrogen receptor transcriptional activity, parathymosin inhibits glucocorticoid-receptor binding to DNA. -Thymosins and especially thymosin 4 are the main G-actinsequestering peptides in mammals, thus playing an essential role in regulation of the microfilamental system. However, it is not clear why often two -thymosins are expressed in mammalian cells. Their differential expression patterns may indicate that they possess different functions in cells and tissues under normal and pathological conditions. Another interesting part of future research on -thymosins will be to identify other intracellular components interacting with -thymosins. Increased levels of -thymosins seem to correspond to tumor malignancy. However, the underlying molecular mechanism is not completely understood. The activity in angiogenesis and wound healing of -thymosins will become an active field of research. At least for some of the described effects an extracellular function for -thymosins seems to be obvious. It will be a major goal to search for the

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molecular mechanisms [receptor(s), signal transduction, interaction with other biomolecules] mediating the effects attributed to extracellular thymosins. It is important to keep in mind that the thymosins are a group of biologically important peptides with similarities and dissimilarities. They constitute three families at minimum: the prothymosin, parathymosin, and -thymosin families.

ACKNOWLEDGMENTS Our work on the thymosins started during a postdoctoral fellowship (E.H.) from 1979 to 1981 at the former Roche Institute of Molecular Biology in the group of Prof. Dr. B. L. Horecker (Nutley, NJ). The authors’ work has been supported since then by the Deutsche Forschungsgemeinschaft (Grants Ha 1148 and Hu 865) and in part by the Deutsche Krebshilfe.

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8 Thymosin 4 Interactions

Michael R. Bubb Department of Medicine, University of Florida, Gainesville, Florida 32610, and the Research Service, Malcom Randall Department of Veterans Affairs Medical Center, Gainesville, Florida 32608

I. -Thymosin Structure II. Thymosin 4 and the Actin Cytoskeleton A. Overview B. Monomer Sequestration and the Critical Concentration of Actin C. b-Thymosin Actin-Binding Proteins D. Differences between Thymosin b4 and Actobindin Family Proteins E. The Thymosin b4–Actin Interface F. Consequences of Interactions at Subdomain 1 of Actin, a Comparison of Profilin and Thymosin b4 III. Assays for Thymosin 4–Actin Interactions A. Native Gel Electrophoresis B. Pyrene Fluorescence IV. Ternary Complexes A. DNase I, Actin, and Thymosin b4 B. Profilin, Actin, and Thymosin b4 V. Thymosin 4 Ligands in Immunity and Inflammation References

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Thymosin 4 is a small, 5-kDa protein with a diverse range of activities, including its function as an actin monomer sequestering protein, an antiinflammatory agent, and an inhibitor of bone marrow stem cell proliferation. Only the effects of thymosin 4 on the actin cytoskeleton have an explanation based on identified molecular interactions. Thymosin 4 is largely unfolded or perhaps completely unfolded in solution. Based on the paradigm introduced by Wright and Dyson (1999) that unfolded proteins may have multiple functions based on their ability to recognize numerous ligands, the flexible structure of thymosin 4 may facilitate the recognition of a variety of molecular targets, thus explaining the plethora of functions attributed to thymosin 4. Furthermore, if multiple ligands bind to thymosin 4, then it is possible that thymosin 4 has a unique integrative function that links the actin cytoskeleton to important immune and cell growth-signaling cascades. ß 2003, Elsevier Science (USA).

I. -THYMOSIN STRUCTURE The complete thymosin 4 amino acid sequence of 43 residues is highly conserved. The sequence is invariant among known mammalian sources with the exception of rabbit, in which alanine substitutes for the N-terminal serine (Goodall et al., 1983). Several isoforms of thymosin 4 have been identified among vertebrates. The invertebrates have -thymosins that more closely resemble the thymosin 15 isoform (Bao et al., 1996) than thymosin 4 (Fig. 1). Although the primary sequence of thymosin 4 is predictive of extensive helical structure, experimental data fail to detect significant secondary structure in aqueous buffer. However, thymosin 4 does have a tendency to form -helix in fluorinated alcohol at residues 4–16 and 30–40 (Zarbock et al., 1990; Czisch et al., 1993). The mostly unfolded state of thymosin 4 is a feature held in common with several other cytoskeletal proteins, including Tau, MAP, MARCKS, actobindin, and synapsin I (Cleveland et al., 1977; Woody et al., 1983; Matsubara et al., 1998; Vandekerckhove et al., 1990; Ho et al., 1991). A possible explanation for the occurrence of unfolded actinregulatory proteins was provided by Wright and Dyson (1999), who suggested the hypothesis that unfolded, nonglobular proteins control key regulatory checkpoints. Like other proteins lacking intrinsic globular structure in solution, thymosin 4 may become structured upon binding to appropriate ligand. The flexibility of the unfolded state may allow for recognition of multiple ligands, and the structure of the bound state may vary depending on the specific ligand (Dunker et al., 2002).

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FIGURE 1. Primary amino acid sequence of mammalian thymosin 4 and thymosin 15. Previously identified -thymosin sequences among Metazoa include mollusks (Aplysia californica, Argopecten irradians), echinoderms (Arbacia punctulata, Paracentrotus lividus, Strongylocentrotus purpuratus, Echinus esculentus), annilid (Hirudo nipponia), and sponge (Sycon raphanus) (Safer and Chowrashi, 1997; Pancer et al., 1999; Stoeva et al., 1997; Manuel et al., 2000).

In the case of its one identified ligand, actin, there are data to support this hypothesis for thymosin 4. Circular dichroism spectroscopy, nuclear magnetic resonance (NMR), and analysis of substituted peptides imply that an N-terminal helix involving some or all of residues 4–16 becomes structured upon binding to actin (Feinberg, et al., 1996; Safer et al., 1997). Alternatively, an equilibrium between folded and unfolded conformations of thymosin 4 may exist such that only the appropriately folded fraction is competent for binding (De La Cruz, 2000). Truncating thymosin 4 by deletion of residues 1–7 or substituting a helix breaking proline at residue 11 severely affected binding (Simenel et al., 2000). Hydrophobic residues on one surface of this helix (Met-6, Ile-9, and Phe-12) are postulated to interact directly with actin based on analysis of peptides with substitutions at these sites (Van Troys et al., 1996).

II. THYMOSIN 4 AND THE ACTIN CYTOSKELETON A. OVERVIEW

The concept that cells might have proteins that function to sequester a pool of monomeric actin that could be used to support rapid actin filament assembly has evolved dramatically in the past 25 years. For many years, profilin, a monomer-sequestering protein discovered by Lindberg (Carlsson et al., 1977), was the only known intracellular protein with this postulated function. The relevance of monomer sequestration was enhanced by the description of an amplification mechanism by which actin filament capping proteins could regulate the extent of monomer sequestration (Tobacman et al., 1983) and a mechanism by which profilin–actin could participate in

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filament elongation at the end of the filament designated as the ‘‘barbed’’ end (Pollard and Cooper, 1984). A critical hurdle was reached in the late 1980s, when it became established that there was insufficient profilin in some cells to account for the extent of monomer sequestration (Southwick and Young, 1990), but at nearly the same time, Safer et al. (1990) developed a novel assay that allowed for the successful identification of thymosin 4 as a small and elusive actin monomer-sequestering protein. Earlier work had shown that thymosin 4 was extremely prevalent in many cells (Hannappel and Liebold, 1985), and therefore could have a major influence on the amount of nonpolymerized actin inside cells. A theory in which profilin serves as a carrier of actin between its sequestered state, where actin is bound to thymosin 4, and free barbed filament ends, where profilin–actin could participate in filament elongation (Pantaloni and Carlier, 1993) has been widely disseminated. Recent data show that binding of profilin and thymosin 4 to actin is not mutually exclusive (Yarmola et al., 2001). Quantitative evaluation of this ternary complex of profilin, thymosin 4, and actin implies that most monomeric actin in cells should have both profilin and thymosin 4 bound simultaneously, a result implying that the actual functions of thymosin 4 and profilin are more complicated than sequestration and shuttle, respectively. B. MONOMER SEQUESTRATION AND THE CRITICAL CONCENTRATION OF ACTIN

The critical concentration of actin, Acc, is the steady-state amount of unpolymerized, monomeric actin defined by the relationship, Acc ¼ k =kþ

ð1Þ

which follows directly from consideration of the elongation reaction for actin filaments for which k+ and k are the respective elongation and dissociation rate constants, A þ Fðn1Þ Ð F

ð2Þ

When an actin monomer adds (or dissociates) from F-actin, the concentration of filaments remains constant, so that the concentration of filaments that can undergo the forward and reverse reactions in Eq. (2) are equal, that is, ½Fðn1Þ ¼ ½F . For the reaction shown in Eq. (2) at steady state, ½A ¼ Acc . The steady-state relationship equating dissociation and association rates, kþ ½A ½Fðn1Þ ¼ k ½F , then reduces to Eq. (1) in which the critical concentration of actin is a constant. In electron micrographs, actin filaments have the appearance of a double strand of subunits forming a right-handed helix, that is, a two-start helix. The actin filament, however, is geometrically equivalent to a left-handed

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one-start helix in which subunits rotate about the long axis by 166 degrees per subunit, and the visual impression of a two-start helix is created by the fact that this rotation approximates 180 . The double-stranded character of an actin filament complicates the quantitative analysis of polymerization because actin could, and probably does under certain circumstances (Bremer et al., 1991), form linear polymers that are based on the intermolecular connections along a single, long pitch strand of the two-start helix. In this case, other equations in addition to (2) above are required to explain the formation of linear polymers and the manner in which linear polymers might convert to helical, two-start polymers, and the result is discussed extensively by Oosawa and Asakura (1975). The relevance to monomer sequestration is that under conditions in which there is little linear polymer, the transition between actin monomer and polymer is sharp so that there are few small actin oligomers. In contrast, when actin oligomers such as dimers and trimers are prevalent, then an accurate discription of the behavior of a monomer-binding protein also requires information about whether or not it also binds to these small actin oligomers. Some -thymosin-like actinbinding proteins have the ability to recognize specific actin oligomers, giving them potentially unique actin-regulatory functions (Bubb et al., 1994). Actin filament capping proteins have been identified that bind to either end of an actin filament. Because of differences in the kinetics of actin addition at the two ends of an actin filament, capping of the ‘‘barbed end’’ of a filament (as identified by decoration with fragments of myosin that give actin the appearance of having pointed and barbed ends) changes the concentration of monomeric actin relative to filamentous actin at steady state, that is, changes Acc. Capping proteins affect monomer sequestration because of changes in Acc. Because a monomer-sequestering protein, M, forms a complex with actin, MA, as defined by its equilibrium reaction with actin, the amount of actin sequestered is proportional to Acc, according to the relationship ½MA ¼ Acc ½M =Kd . This is the amplification mechanism originally noted by Tobacman et al. (1983) by which changes in the actin critical concentration can affect the amount of sequestered actin and therefore the amount of actin polymer. Because Acc is typically quite low in physiological buffers, and may be very low inside cells (Yarmola et al., 2001), and because the concentration of monomer-sequestering protein is much greater (by a factor of 102 to 104), amplification of changes in Acc can be considerable. C. b-THYMOSIN ACTIN-BINDING PROTEINS

Actobindin from Acanthamoeba castellanii was identified in 1986 by Lambooy and Korn. The occurrence of an LKHAET actin-binding motif in actobindin and other actin-binding proteins was noted later when actobindin was sequenced by Vandekerckhove et al. in 1990. Later data showed that covalently cross-links between actin and actobindin occur at

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the lysine in this hexapeptide sequence (Vancompernolle et al., 1991). The covalent sequence of actobindin revealed that this small 9-kDa protein was composed of nearly identically repeated tandem segments of 33 and 34 amino acid residues. Until recently detected in Dictyostelium, actobindin had not been identified in other organisms. A 5-kDa actin-binding protein in human platelets, discovered in 1990 by Safer et al., was subsequently shown to be thymosin 4. The primary sequence of thymosin 4 also contains the LKHAET motif (with conservative substitutions, becoming LKKTET in thymosin 4) and thymosin 4 was noted to be a single repeat form of actobindin. More recently, proteins containing triplicate -thymosin sequences have been identified in Drosophila melanogaster and Caenorhabditis elegans (Boquet et al., 2000; Van Troys et al., 1999). A recent phylogenetic analysis implies that the -thymosins are limited to Metazoan organisms (Manuel et al., 2000), yet inclusion of the three-repeat -thymosins in this family of proteins would seem unjustified without inclusion of actobindin, the two-repeat -thymosin identified in protozoa. D. DIFFERENCES BETWEEN THYMOSIN b4 AND ACTOBINDIN FAMILY PROTEINS

Biochemical data suggest that proteins made of multiple -thymosin repeats (the actobindin family) have distinct properties from the -thymosins themselves. Monomer-sequestering proteins such as thymosin 4 are inhibitors of actin filament nucleation because the rate of nucleation is proportional to the actin monomer concentration to the nth power, where n is number of actin subunits in the smallest oligomer that is more likely to associate than dissociate. Since filament elongation depends only linearly on the concentration of monomeric actin (more precisely, on the concentration of monomeric actin minus Acc), monomer sequestering proteins inhibit nucleation to a relatively greater extent than elongation. The multiple repeat -thymosins are multivalent for actin and, therefore, depending on the orientation and flexibility of these binding sites, could potentially recognize actin oligomers with higher specificity than they have for monomers. Specific interactions with oligomers could affect actin filament nucleation by mechanisms additional to or independent of monomer sequestration. There is evidence that one such protein, actobindin, does inhibit nucleation to a greater extent than other monomer-sequestering proteins (Lambooy and Korn, 1988). Furthermore, actobindin interacts with high affinity with a transient actin oligomer that forms during actin polymerization (Bubb et al., 1994), suggesting that actobindin possibly inhibits nucleation by targeting actin oligomers in the pathway for assembly of effective filament nuclei (Bubb and Korn, 1995). There are also reported data that suggest that thymosin 4 has actinbinding properties other than monomer sequestration. We and others have

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observed that at high concentrations, thymosin 4 might bind to filament ends or sides, or that more than one thymosin 4 molecule binds to actin at saturation (Carlier et al., 1996; Yarmola et al., 2001). Although it is easy to come up with a model that explains these discrepancies, the problem is that the measurements rely on imprecise assays (discussed later). Some nonlinear effects that have been interpreted as something other than monomer sequestration could be an experimental artifact rather than meaningful observation. E. THE THYMOSIN b4–ACTIN INTERFACE

The LKHAET motif (LKKTET in thymosin 4) covalently cross-links to residue Glu-100 in subdomain 1 of actin (Fig. 2). Glu-100 is at relatively large filament radius in the Holmes and Holmes-derived filament models of F-actin (Holmes et al., 1990), so there is no reason to expect a priori that a surface interaction at or near this residue would block monomer addition by

FIGURE 2. A ribbon and helix drawing of an actin monomer based on the crystal structure of Bubb et al. (2002) depicting the surface interactions of actin and thymosin 4. Subdomains 1 through 4 of actin are numbered. The line to the left represents the long axis of a helical filament if the monomer had the same orientation as subunits in the F-actin model of Holmes et al. (1990). The shaded areas show where subunits make extensive intrafilament contacts, based again on the Holmes model. The locations of actin residues specifically identified in interactions of thymosin 4 and actin are depicted by arrows.

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steric affects. In fact, a previous report makes the very curious claim that a modified hexapeptide, LKEAET or LKETET, binds to actin and stimulates actin polymerization (Vancompernolle et al., 1992), certainly implying that a steric effect at that site would not block polymerization. Why does thymosin 4 sequester monomeric actin? Vancompernolle et al. (1992) also show that the 14 to 16 residues of thymosin 4 N-terminal to the hexapeptide are required for inhibition of polymerization. These residues were shown by Safer et al. (1997) to wrap around the ‘‘barbed end’’ of actin monomer across subdomain I extending to a surface of subdomain 3 of actin that is buried by intrafilament subunit contacts in the Holmes model of F-actin. Other studies have shown significant decreases in the sequestration of actin by thymosin 4 with modification of the N-terminus, particularly at residue Asp-5. The data are therefore consistent with inhibition of polymerization by steric effects of the N-terminus of thymosin 4. Other data confirm that the N-terminus of thymosin 4 extends around subdomain 1. Heintz et al. (1993) showed that modification of actin Cys-374 blocked thiol-specific cross-linking of actin to thymosin 4, indicating that Cys-374 near the C-terminus of actin located within subdomain 1 is close to the thymosin 4-binding site. Reichert et al. (1996a) used cysteinesubstituted derivatives of thymosin 4 to show that Cys-374 at the C-terminus of actin could be cross-linked to an Met-6-Cys derivative of thymosin 4, implying that either this thymosin 4 residue was in a very flexible location in the complex or that it was less than 10 A from Cys-374 of actin. Interestingly, the LKEAET motif reported by Vancompernolle to augment actin polymerization is found in several other actin-binding proteins, including the C-terminus of tropomyosin (Lewis et al., 1983). In calponin, the VKYAEK hexapeptide just C-terminal to the calponin homology domain is critical to binding actin (El-Mezgueldi et al., 1996). The homogoly extends beyond the well-characterized hexapeptides as depicted in Figure 3. There is evidence that substitution of A for S at position 145 in calponin is not a conservative substitution (El-Mezgueldi et al., 1996). Caldesmon has a LKEKQQ motif that also is reportedly part of its actin-binding site. Although the function of the motif has not been

FIGURE 3. The LKHAET actin-binding motif of -thymosins and calponin. The location of the complete homologous region, LKKTETQEK, with the sequence of thymosin 4 is shown in italics. Tyr-144 and Lys-147 in calponin and Val-21 and Val-57 in actobindin are the only nonconservative substitutions within the homology region.

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investigated independently in each case and the original studies of Vancompernolle employed peptide concentrations in excess of 300 M, each of these proteins individually promotes actin polymerization. Of course, to ‘‘promote’’ actin polymerization is a vague concept that could involve effects on the rate constants of actin nucleation, exchange of nucleotide and/or divalent cation, and elongation or dissociation. There are no data that delineate between these effects for the hexapeptide motif so it would be premature to speculate on the structural mechanism. Postulated interactions between thymosin 4 and subdomain 2 of actin remain controversial. Independent binding of thymosin 4 and DNase I as reported by Reichert et al. (1996b) can be most simply explained by unique binding sites for thymosin 4 in subdomain 1 of actin and for DNase I in subdomain 2 of actin. However, as discussed later, the data regarding independent binding have also been disputed. Safer et al. (1997) used transglutaminase to covalently cross-link the C terminus of thymosin 4 to subdomain 2 of actin between residues Gln-41 of actin and Lys-38 of thymosin 4. In addition to this zero-length cross-linking technique, they showed that the fluorescence of actin modified at Gln-41 by dansylcadaverine was partially quenched by thymosin 4. These results and additional cross-linking studies of De La Cruz et al. (2000) support a model in which thymosin 4 interacts directly with subdomain 2 of actin. The kinetics of thymosin 4 binding to actin yields surprising results. Formation of a stable interaction of thymosin 4 and actin requires conformational changes in thymosin 4 and possibly in actin (De La Cruz et al., 2000). A comparison of the binding kinetics of intact thymosin 4 and an N-terminal peptide of thymosin 4 by Yarmola et al. (2001) leads to the conclusion that when the C-terminus of thymosin 4 is bound to actin, the free energy barrier between conformational changes in the N-terminus (presumably between unfolded and helical structures) is lowered, facilitating both more rapid binding and dissociation of intact thymosin 4 compared with the N-terminal peptide. F. CONSEQUENCES OF INTERACTIONS AT SUBDOMAIN 1 OF ACTIN, A COMPARISON OF PROFILIN AND THYMOSIN b4

According to the Holmes model of an actin filament, the long pitch helix of the filament involves interactions of subdomains 1 and 3 with subdomains 2 and 4 of successive subunits. Terminal subunits at the barbed end are predicted to have an exposed surface that includes subdomains 1 and 3; terminal pointed end subunits expose the surface of subdomains 2 and 4. A protein that binds to subdomain 1 or 3 such as profilin might therefore be expected to bind to the barbed end of F-actin, in addition to the ability to bind to monomeric actin, and Pollard and Cooper (1984) provided the first

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experimental confirmation of this hypothesis. The ability of thymosin 4 to bind to both subdomain 1 and 3 and to subdomain 2 implies a potential to bind to either end of an actin filament, and, as mentioned earlier, such interactions have been reported (Carlier et al., 1996). The potential to bind should not be confused with evidence of binding as there are no atomic resolution structural data for F-actin; subunits at the ends may differ considerably in structure from that reported for monomeric actin. By similar reasoning, interactions of profilin and actin monomer do not necessarily result only in sequestration of actin monomer, because the complex of actin and profilin could add to the barbed end of an actin filament. Such an interaction would block further elongation of the filament at the barbed end until profilin dissociated. If profilin dissociation was faster than dissociation of the original complex of profilin and actin, then the successive addition of profilin–actin complex to the barbed end could result in filament elongation. Pollard and Cooper (1984) also provided experimental evidence, subsequently verified by several others, in support of this prediction. In contrast to profilin, the complex of thymosin 4 and actin does not participate in filament elongation at the barbed end. The data of Safer et al. (1997) showing additional thymosin 4–actin interactions at subdomain 2 provide a simple steric explanation for this observation. Ballweber et al. (2002) suggest that cooperative transitions in actin structure due to the interaction at subdomain 1 result in an actin conformation that cannot participate in barbed end elongation, even without direct contact between subdomain 2 and thymosin 4. De La Cruz et al. (2000) provide evidence that there are both conformational and steric factors that preclude barbed end addition.

III. ASSAYS FOR THYMOSIN 4–ACTIN INTERACTIONS A. NATIVE GEL ELECTROPHORESIS

Safer first identified thymosin 4 as an actin-binding protein using native gel electrophoresis (Safer et al., 1991). The technique is still frequently employed but incompletely understood. A sample assay is shown in Fig. 4. The technique requires a buffer system with calcium and ATP that retains the activity of actin. The observed result is that actin and thymosin 4 form a complex that migrates as an apparently discrete band on the gel. The fraction of actin in the complex is at least equal to if not greater than the amount predicted to exist in complex in solution given a known Kd. This is surprising and potentially interesting given that this is not an equilibrium-binding technique. Somehow free actin and free

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FIGURE 4. Native gel assay in which actin is shifted to higher electrophoretic mobility when bound to thymosin 4. In this application, the assay was used to show that thymosin 4 competes with the marine natural product, latrunculin A, to bind to actin. Fluorescently labeled thymosin 4 is near the top of the gel when free and has higher electrophoretic mobility when bound to actin (lane 1). In the presence of latrunculin A, much less thymosin 4 is bound to actin (lane 2). When the gel is stained with Coomassie, the small thymosin 4 peptide is no longer visible, and a single band corresponding to actin is observed without or with latrunculin A in lanes 5 and 6, respectively. Addition of thymosin 4 causes a shift in the actin to higher electrophoretic mobility in lane 3. Addition of latrunculin A and thymosin 4 causes less shift in actin because competition between thymosin 4 and latrunculin A results in less thymosin 4–actin complex (lane 4). The samples in lanes 7–10 are identical to those in lanes 3–6 except that the thymosin 4 is unlabeled. From Yarmola et al. (2000) with permission from J. Biol. Chem.

thymosin 4 are apparently separated during electrophoresis. In spite of low or absent free actin and free thymosin 4, the complex does not dissociate when the complex moves into a zone distant from the free ligands, but rather, continues to migrate as an intact complex. There are two potential explanations for the observation that the complex migrates as a discrete band rather than a smear representing continuous dissociation of the complex. One is that the dissociation of thymosin 4 from actin may be very slow, perhaps slow enough to preclude visible dissociation of complex during the course of the electrophoretic run. Second, the thermodynamic conditions in the gel are much different than those in solution. Perhaps molecular crowding within the gel lowers the free energy change of complex association. One would predict, however, that this thermodynamic effect would be small for a molecule as small as thymosin 4.

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Interestingly, the two explanations may be related. Thymosin 4 has a strong tendency to form an -helix, even though solution studies show that it is unfolded. As mentioned previously, there are data that suggest that the N-terminus of thymosin 4 binds and releases rapidly from actin because the transition to and from the -helix at the N-terminus is catalyzed when actin binds to the C-terminus of thymosin 4. The environment of the gel could shift the equilibrium so as to favor a folded helical state at the N-terminus, augmenting affinity and explaining the slow disassociation rate constant.

B. PYRENE FLUORESCENCE

Attempts to label actin at Cys-374 with pyrene iodoacetamide (pyrenyl actin) usually result in 50–80% labeling at this one site. Previous work has shown that pyrenyl actin randomly copolymerizes with unlabeled actin, and there is no reason to think the time course of nucleation or elongation reactions for pyrenyl actin is any different relative to unlabeled actin. Also binding of other actin-binding proteins to pyrenyl actin does not usually affect pyrene fluorescence. However, pyrenyl actin has the very useful property that the fluorescence of filamentous pyrenyl actin is 8 to 30 times higher than that of pyrenyl-labeled monomer. The total fluorescence, F, of a solution of pyrenyl actin solution is therefore ð3Þ F ¼ rfl Acc þ ðrpol ½AF Þ where rpol is the ratio of fluorescence of F- to G-actin and rfl is the instrument- and experiment-specific ratio of measured fluorescence per molar concentration of pyrene-G-actin. Because, at steady state, Acc is often much less than the concentration of F-actin, [AF], F is nearly proportional to [AF], and F serves as a useful quantitative measure of F-actin concentration. There are several problems with this assay relevant to thymosin 4–actin interactions. First, the amount of F-actin is usually an indirect measure of the quantity of interest, the amount of TA complex. For example, determination of Kd requires knowledge of [TA]. Although [TA] can be calculated indirectly from the equation for total actin, ½Atotal ¼ ½TA þ Acc þ ½AF ; ½AF is often much larger than [TA] or Acc, so that a small error in the measurement of [AF] leads to a large percentage error in [TA]. Second, other factors can influence F, for example, bundled filaments will often demonstrate different levels of pyrenyl actin fluorescence depending on the aggregation state and independent of F-actin concentration. Finally, the pyrenyl modification of actin can affect the affinity of actin-binding proteins. In an anisotropy assay for thymosin 4–actin interactions, we found that pyrenyl actin bound to thymosin 4 with lower affinity than unlabeled actin.

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IV. TERNARY COMPLEXES A. DNASE I, ACTIN, AND THYMOSIN b4

Huff et al. (1995) used an equilibrium ultracentrifugation assay to show that DNase I decreases the affinity of actin for thymosin 4 by a factor of about five when actin should have been 99% saturated with DNase I. Adding a much larger excess of free DNase I decreased the apparent affinity of thymosin 4 by another order of magnitude. Because actin was nearly saturated with DNase I prior to the addition of this large excess, the results cannot be explained adequately by any simple model of steady-state binding interactions. Instead, this result could indicate a nonspecific interaction between free DNase I and thymosin 4, a problem with DNase I such that it has low affinity for actin, or some other unanticipated complicating factor in the assay. Contrasting results were reported by Reichert et al. (1996b) using covalent cross-linking and native gel electrophoresis techniques to detect a ternary complex that indicated independent binding of DNase I and thymosin 4 to actin. Ballweber et al. (1998) employed steady-state fluorescence in an assay similar to that shown in Fig. 5 in an attempt to distinguish between competitive and independent binding. The results showed that the amount

FIGURE 5. Steady-state pyrene fluorescence assay showing that this experimental method may not distinguish between competitive and independent binding. F-actin (4% pyrenyl actin) containing gelsolin at a ratio 1:200 was diluted to indicated concentrations in the absence (squares) or presence of 3 M profilin (circles), or 6 M thymosin 4 (up triangles), or both (down triangles). Solid lines represent the best linear fits to data, yielding Acc of 0.72 M. For comparison with data obtained when both sequestering proteins are present, the dashed line represents the calculated theoretical dependence assuming purely competitive binding, and the nearly superimposed dotted line represents the calculated theoretical dependence assuming purely independent binding of the two actin-binding proteins. From Yarmola et al. (2001) with permission from J. Biol. Chem.

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of sequestered actin in the presence of both thymosin 4 and DNase I was nearly equal to the sum of sequestered actin in the presence of the same amounts of thymosin 4 and DNase I when measured separately. This was interpreted as a confirmation of competitive binding. Although these data are consistent with competitive binding, we have previously demonstrated that these results are also consistent with predictions based on independent binding. The investigators may have assumed that competitive binding would result in significantly more sequestered actin than independent binding, and although it may seem to be intuitively correct that independent binding is less efficient at sequestering actin than competitive binding, this is not the case. As we show elsewhere (Yarmola et al., 2001), if [Aseq]0 is the amount of sequestered actin predicted by competitive binding and [Aseq] is the amount of sequestered actin predicted by independent binding, then, ½Aseq ¼ ½Aseq 0 þ ½Atern where ¼ ðKdD KdT A2cc Þ=½ðKdD þ Acc ÞðKdT þ Acc Þ

ð4Þ

[Atern] is the concentration of ternary complex, and KdD and KdT are the respective equilibrium dissociation constants for DNase I and thymosin 4. It follows that the amount of sequestered actin predicted by competitive binding can be greater or less than that predicted for independent binding, as > 0 if ðKdD KdT Þ1=2 > Acc ; < 0 ifðKdD KdT Þ1=2 < Acc ; and ¼ 0if ðKdD KdT Þ1=2 Acc . Using the data supplied by Ballweber et al. (1998) for conditions with Acc equal to 0.23 M, KdD was 0.035 M and KdT was 0.80 M. In this case, is a small negative quantity, and the predicted differences between independent and competitive binding are not experimentally detectable. Much of the discrepancy in the reported results for the effect of DNase I on the affinity of thymosin 4 for actin can probably be explained by the very large differences between the affinities of DNase I and thymosin 4 for actin. Combeau and Carlier (1992) demonstrated that the equilibrium dissociation constant of DNase I to actin is approximately 0.2 nM, considerably lower than prior estimates. Such high affinity leads in many instances to nearly stoichiometric binding, with the result that a 10- to 100fold decrease in affinity in DNase I for actin would be marginally detectable in the assays employed by Reichert et al. (1996b).

B. PROFILIN, ACTIN, AND THYMOSIN b4

The failure to obtain a covalently cross-linked ternary complex of profilin, thymosin 4, and actin was interpreted as evidence that profilin competes with thymosin 4 to bind to actin (Ballweber et al., 1998). However, this result has alternative explanations. The site of covalent crosslinking on actin could have been identical for both profilin and thymosin 4,

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so that both could not cross-link simultaneously even if both were bound. Or, given the substantial evidence that the N-terminus of thymosin 4 binds very near but not overlapping with the crystallographic interface of profilin and actin (Schutt et al., 1993), the interaction of profilin with actin may distort the thymosin 4–actin interaction so that cross-linking reaction is less efficient. Steady-state fluorescence data were also interpreted as showing that binding of profilin and thymosin 4 were mutually exclusive, but as shown in Fig. 5, these data actually fail to distinguish between competitive and independent binding. Recent work from our laboratory provided several lines of evidence that profilin and thymosin 4 can bind simultaneously to actin (Yarmola et al., 2001). This result is consistent with information regarding the binding surfaces of thymosin 4 (Fig. 2) and profilin (Schutt et al., 1993), for which there is minimal if any overlap in subdomain 1, and even if the overlap in subdomain 1 precluded binding by thymosin 4, this would still allow thymosin 4 to bind to subdomain 2. However, there are no published experimental data that can be used to predict the affinity of such an interaction. The marked allosteric effects of profilin on actin, such that binding of profilin can influence nucleotide exchange at a distant binding site, make this theoretical analysis somewhat superficial in the absence of experimental data. Our data show that thymosin 4 binds to profilin–actin approximately 10 times more weakly than it binds to actin alone with a Kd of 2–8 M, that is, profilin is a noncompetitive inhibitor of thymosin 4. Given this Kd, the concentrations of thymosin 4 and profilin in some cells are sufficient to ensure that most sequestered actin will have both proteins bound simultaneously. For example, in human polymorphonuclear cells, the concentrations of profilin and thymosin 4 are approximately 40 and 150 M, respectively (Giehl et al., 1994). Platelets may contain as much as 560 M thymosin 4 and 55 M profilin (Goldschmidt-Clermont et al., 1991; Nachmias et al., 1993). The functional importance of this finding is unclear, as little is known about the effects of the ternary complex of thymosin 4–profilin–actin on nucleation, capping, and filament elongation. However, this finding does present a possible resolution to the interesting paradox of in vivo actin sequestration. Much theoretical evidence and in vitro experimental data suggest that molecular crowding in the cytoplasm may lower Acc in vivo (Fig. 6). If this is true, then because the amount of sequestered actin is proportional to Acc, proteins such as thymosin 4 or profilin acting independently cannot sequester significant amounts of actin. However, if binding is noncompetitive, then because of the disparity between Acc and the equilibrium dissociation constants for profilin and thymosin 4, these proteins will act synergistically to form ternary complex according to Eq. (4). The effect as predicted for polymorphonuclear cells is illustrated in Fig. 6.

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FIGURE 6. Effect of ternary complex formation on the amount of sequestered actin. Given a low value for Acc, the amount of sequestered actin is much greater when binding of thymosin 4 and profilin to actin is independent (3, dotted line) or noncompetitive with Kd for thymosin 4 to profilin–actin of 3.0 M (2, solid line) than when binding is competitive (1, dashed line), in which case, sequestration is virtually absent. The theoretical dependence on total profilin concentration assumes a critical concentration, Acc, of 0.01 M based on data shown in the inset and a total thymosin 4 concentration of 150 M, the amount found in polymorphonuclear cells. The steep dependence of sequestered actin on profilin concentration implies that cells can regulate actin polymerization by altering profilin concentration, for example, by regulation of production or turnover of polyphosphoinositides, another ligand of profilin. Inset: steady-state pyrene fluorescence assay showing decrease of actin critical concentration in the presence of 6% PEG 8000 (triangles), which was used to simulate molecular crowding in the cytoplasm, in comparison with results for no PEG (circles). The critical concentration with PEG is 0.003 0.008 M. From Yarmola et al. (2001) with permission from J. Biol. Chem.

V. THYMOSIN 4 LIGANDS IN IMMUNITY AND INFLAMMATION Thymosin 4 is a component of thymosin 4 fraction V, although it is probably not the active component in chemotherapeutic applications (Spangelo et al., 1998). Thymosin 4 has both immunoregulatory (Baxevanis et al., 1987) and antiinflammatory properties (Young et al., 1999). In contrast to the intracellular actin-binding effects of thymosin 4, these effects are observed investigationally after the extracellular application of thymosin 4. Although the ligands are yet unidentified, the dose–response characteristics are also much different for modulation of these signaling pathways, so that 10–100 M of thymosin 4 in motile cells regulates actin dynamics, but nanomolar and even picomolar concentrations provoke extracellular signaling (Bonnet et al., 1996). Posttranslational modification of Met-6 on thymosin 4 by sulfoxide probably alters its actin-binding properties and its extracellular effects (Young et al., 1999), and therefore

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could have a role in switching thymosin 4 from intracellular to extracellular function. How does an intracellular actin-binding protein without a signal peptide get secreted? Perhaps there is no constitutive pathway for thymosin 4 secretion, but release occurs in response to significant perturbations of the cytoskeleton that are sufficient to alter cytoskeletal– plasma member continuity. One such possibility would be that thymosin 4 release occurs in conjunction with alterations in the cytoskeleton associated with apoptosis that correlate temporally with blebbing and/or cytoplasmic condensation (Mills et al., 1999). The oxidative environment of apoptosis may then potentiate the antiinflammatory effects of thymosin 4 by production of methonine sulfoxide, and the release of such an agent could be causally related to the absence of inflammation seen in response to these dying cells

ACKNOWLEDGMENT Supported by the Medical Research Service of the Department of Veterans Affairs.

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Bubb, M. R., Knutson, J. R., Porter, D. K., and Korn, E. D. (1994). Actobindin induces the accumulation of actin dimers that neither nucleate polymerization nor self-associate. J. Biol. Chem. 269, 25592–25597. Bubb, M. R., Govindasamy, L., Yarmola, E. G., Vorobiev, S. M., Almo, S. C., Somasundaram, T., Chapman, M. S., Agbandje-McKenna, M., and McKenna, R. (2002). Polylysine induces an antiparallel actin dimer that nucleates filament assembly: Crystal structure at 3.5-A resolution. J. Biol. Chem. 277, 20999–21006. Carlier, M. F., Didry, D., Erk, I., Lepault, J., Van Troys, M. L., Vandekerckhove, J., Perelroizen, I., Yin, H., Doi, Y., and Pantaloni, D. (1996). T-beta 4 is not a simple G-actin sequestering protein and interacts with F-actin at high concentration. J. Biol. Chem. 271, 9231–9239. Carlsson, L., Nystrom, L. E., Sundkvist, I., Markey, F., and Lindberg, U. (1977). Actinpolymerizability is influenced by profilin, a low molecular weight protein in nonmuscle cells. J. Mol. Biol. 115, 465–483. Cleveland, D. W., Hwo, S. Y., and Kirschner, M. W. (1977). Physical and chemical properties of purified Tau factor and the role of Tau in microtubule assembly. J. Mol. Biol. 116, 227–247. Combeau, C., and Carlier, M. F. (1992). Covalent modification of G-actin by pyridoxal 5-phosphate: Polymerization properties and interaction with DNase I and myosin subfragment 1. Biochemistry 31, 300–309. Czisch, M., Schleicher, M., Horger, S., Voelter, W., and Holak, T. A. (1993). Conformation of thymosin beta 4 in water determined by NMR spectroscopy. Eur. J. Biochem. 218, 335–344. De La Cruz, E. M., Ostap, E. M., Brundage, R. A., Reddy, K. S., Sweeney, H. L., and Safer, D. (2000). Thymosin-beta(4) changes the conformation and dynamics of actin monomers. Biophys. J. 78, 2516–2527. Dunker, A. K., Brown, C. J., Lawson, J. D., Iakoucheva, L. M., and Obradovic, Z. (2002). Intrinsic disorder and protein function. Biochemistry 41, 6573–6582. El-Mezgueldi, M., Strasser, P., Fattoum, A., and Gimona, M. (1996). Expressing functional domains of mouse calponin: involvement of the region around alanine 145 in the actomyosin ATPase inhibitory activity of calponin. Biochemistry 35, 3654–3661. Feinberg, J., Heitz, F., Benyamin, Y., and Roustan, C. (1996). The N-terminal sequences (5-20) of thymosin beta 4 binds to monomeric actin in an alpha-helical conformation. Biochem. Biophys. Res. Commun. 222, 127–132. Giehl, K., Valenta, R., Rothkegel, M., Ronsiek, M., Mannherz, H. G., and Jockusch, B. M. (1994). Interaction of plant profilin with mammalian actin. Eur. J. Biochem. 226, 681–689. Goldschmidt-Clermont, P. J., Machesky, L. M., Doberstein, S. K., and Pollard, T. D. (1991). Mechanism of the interaction of human platelet profilin with actin. J. Cell Biol. 113, 1081–1089. Goodall, G. J., Morgan, J. I., and Horecker, B. L. (1983). Thymosin beta 4 in cultured mammalian cell lines. Arch. Biochem. Biophys. 221, 598–601. Hannappel, E., and Leibold, W. (1985). Biosynthesis rates and content of thymosin 4 in cell lines. Arch. Biochem. Biophys. 240, 236–241. Heintz, D., Reichert, A., Mihelic, M., Voelter, W., and Faulstich, H. (1993). Use of bimanyl actin derivative (TMB-actin) for studying complexation of beta-thymosins. Inhibition of actin polymerization by thymosin beta 9. FEBS Lett. 329, 9–12. Ho, M. F., Bahler, M., Czernik, A. J., Schielbler, W., Kezdy, F. J., Kaiser, E. T., and Greengard, P. (1991). Synapsin I is a highly surface-active molecule. J. Biol. Chem. 266, 5600–5607. Holmes, K. C., Popp, D., Gebhard, W., and Kabsch, W. (1990). Atomic model of the actin filament. Nature 347, 44–49. Huff, T., Zerzawy, D., and Hannappel, E. (1995). Interactions of beta-thymosins, thymosin beta 4-sulfoxide, and N-terminally truncated thymosin beta 4 with actin studied by

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equilibrium centrifugation, chemical cross-linking and viscometry. Eur. J. Biochem. 230, 650–657. Lambooy, P. K., and Korn, E. D. (1986). Purification and characterization of actobindin, a new actin monomer-binding protein from Acanthamoeba castellanii. J. Biol. Chem. 261, 17150–17155. Lambooy, P. K., and Korn, E. D. (1988). Inhibition of an early stage of actin polymerization by actobindin. J. Biol. Chem. 263, 12836–12843. Lewis, W. G., Cote, G. P., Mak, A. S., and Smillie, L. B. (1983). Amino acid sequence of equine platelet tropomyosin. Correlation with interaction properties. FEBS Lett. 156, 269–273. Manuel, M., Kruse, M., Muller, W. E., and Le Parco, Y. (2000). The comparison of betathymosin homologues among metazoa supports an arthropod-nematode clade. J. Mol. Evol. 51, 378–381. Matsubara, M., Yamauchi, E., Hayashi, N., and Taniguchi, H. (1998). MARCKS, a major protein kinase C substrate, assumes non-helical conformations both in solution and in complex with Ca2+-calmodulin. FEBS Lett. 421, 203–207. Mills, J. C., Stone, N. L., and Pittman, R. N. (1999). Extranuclear apoptosis. The role of the cytoplasm in the execution phase. J. Cell Biol. 146, 703–708. Nachmias, V. T., Cassimeris, L., Golla, R., and Safer, D. (1993). Thymosin beta 4 (T beta 4) in activated platelets. Eur. J. Cell Biol. 61, 314–320. Oosawa, F., and Asakura, S. (1975). ‘‘Thermodynamics of the Polymerization of Protein,’’ pp. 28–55. Academic Press, New York. Pancer, Z., Rast, J. P., and Davidson, E. H. (1999). Origins of immunity: Transcription factors and homologues of effector genes of the vertebrate immune system expressed in sea urchin coelomocytes. Immunogenetics 49, 773–786. Pantaloni, D., and Carlier, M. F. (1993). How profilin promotes actin filament assembly in the presence of thymosin beta 4. Cell 75, 1007–1014. Pollard, T. D., and Cooper, J. A. (1984). Quantitative analysis of the effect of Acanthamoeba profilin on actin filament nucleation and elongation. Biochemistry 23, 6631–6641. Reichert, A., Heintz, D., Echner, H., Voelter, W., and Faulstich, H. (1996a). Identification of contact sites in the actin-thymosin beta 4 complex by distance-dependent thiol cross-linking. J. Biol. Chem. 271, 1301–1308. Reichert, A., Heintz, D., Echner, H., Voelter, W., and Faulstich, H. (1996). The ternary complex of DNase I, actin and thymosin beta4. FEBS Lett. 387, 132–136. Safer, D., and Chowrashi, P. K. (1997). Beta-thymosins from marine invertebrates: Primary structure and interaction with actin. Cell Motil. Cytoskeleton 38, 163–171. Safer, D., Golla, R., and Nachmias, V. T. (1990). Isolation of a 5-kilodalton actin-sequestering peptide from human blood platelets. Proc. Natl. Acad. Sci. USA 87, 2536–2540. Safer, D., Elzinga, M., and Nachmias, V. T. (1991). Thymosin beta 4 and Fx, an actinsequestering peptide, are indistinguishable. J. Biol. Chem. 266, 4029–4032. Safer, D., Sosnick, T. R., and Elzinga, M. (1997). Thymosin beta 4 binds actin in an extended conformation and contacts both the barbed and pointed ends. Biochemistry 36, 5806–5816. Schutt, C. E., Myslik, J. C., Rozycki, M. D., Goonesekere, N. C., and Lindberg, U. (1993). The structure of crystalline profilin-beta-actin. Nature 365, 810–816. Simenel, C., Van Troys, M., Vandekerckhove, J., Ampe, C., and Delepierre, M. (2000). Structural requirements for thymosin beta4 in its contact with actin. An NMR-analysis of thymosin beta4 mutants in solution and correlation with their biological activity. Eur. J. Biochem. 267, 3530–3538. Southwick, F. S., and Young, C. L. (1990). The actin released from profilin–actin complexes is insufficient to account for the increase in F-actin in chemoattractant-stimulated polymorphonuclear leukocytes. J. Cell Biol. 110, 1965–1973. Spangelo, B. L., Farrimond, D. D., Thapa, M., Bulathsinghala, C. M., Bowman, K. L., Sareh, A., Hughes, F. M.Jr., Goldstein, A. L., and Badamchian, M. (1998). Thymosin fraction

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5 inhibits the proliferation of the rat neuroendocrine MMQ pituitary adenoma and C6 glioma cell lines in vitro. Endocrinology 139, 2155–2162. Stoeva, S., Horger, S., and Voelter, W. (1997). A novel beta-thymosin from the sea urchin: Extending the phylogenetic distribution of beta-thymosins from mammals to echinoderms. J. Pept. Sci. 3, 282–290. Tobacman, L. S., Brenner, S. L., and Korn, E. D. (1983). Effect of Acanthamoeba profilin on the pre-steady state kinetics of actin polymerization and on the concentration of F-actin at steady state. J. Biol. Chem. 258, 8806–8812. Van Troys, M., Dewitte, D., Goethals, M., Carlier, M. F., Vandekerckhove, J., and Ampe, C. (1996). The actin binding site of thymosin beta 4 mapped by mutational analysis. EMBO J. 15, 201–210. Van Troys, M., Vandekerckhove, J., and Ampe, C. (1999). Structural modules in actin-binding proteins: Towards a new classification. Biochim. Biophys. Acta 1448, 323–348. Vancompernolle, K., Vandekerckhove, J., Bubb, M. R., and Korn, E. D. (1991). The interfaces of actin and Acanthamoeba actobindin. Identification of a new actin-binding motif. J. Biol. Chem. 266, 15427–15431. Vancompernolle, K., Goethals, M., Huet, C., Louvard, D., and Vandekerckhove, J. (1992). G- to F-actin modulation by a single amino acid substitution in the actin binding site of actobindin and thymosin beta 4. EMBO J. 11, 4739–4746. Vandekerckhove, J., Van Damme, J., Vancompernolle, K., Bubb, M. R., Lambooy, P. K., and Korn, E. D. (1990). The covalent structure of Acanthamoeba actobindin. J. Biol. Chem. 265, 12801–12805. Woody, R. W., Clark, D. C., Roberts, G. C. K., Martin, S. R., and Bayley, P. M. (1983). Molecular lexibility in microtubule proteins: Proton nuclear magnetic resonance characterization. Biochemistry 22, 2186–2192. Wright, P. E., and Dyson, H. J. (1999). Intrinsically unstructured proteins: Re-assessing the protein structure-function paradigm. J. Mol. Biol. 293, 321–331. Yarmola, E. G., Somasundaram, T., Boring, T. A., Spector, I., and Bubb, M. R. (2000). Actinlatrunculin A structure and function. Differential modulation of actin-binding protein function by latrunculin A. J. Biol. Chem. 275, 28120–28127. Yarmola, E. G., Parikh, S., and Bubb, M. R. (2001). Formation and implications of a ternary complex of profilin, thymosin 4, and actin. J. Biol. Chem. 276, 45555–45563. Young, J. D., Lawrence, A. J., MacLean, A. G., Leung, B. P., McInnes, I. B., Canas, B., Pappin, D. J., and Stevenson, R. D. (1999). Thymosin beta 4 sulfoxide is an antiinflammatory agent generated by monocytes in the presence of glucocorticoids. Nat. Med. 5, 1424–1427. Zarbock, J., Oschkinat, H., Hannappel, E., Kalbacher, H., Voelter, W., and Holak, T. A. (1990). Solution conformation of thymosin beta 4: A nuclear magnetic resonance and simulated annealing study. Biochemistry 29, 7814–7821.

9 Polypeptide Hormones: Signaling Molecules in Plants

Paul Chilley The Integrative Cell Biology Laboratory, School of Biological and Biomedical Sciences, University of Durham, South Road, Durham DH1 3LE, UK

I. II. III. IV. V. VI. VII. VIII. IX.

Introduction Systemin and Systemin-like Peptides Rapid Alkalinization Factor (RALF) ENOD40 and Root Nodulation CLAVATA3 and Meristem Organization Phytosulfokines Brassica Self-Incompatibility Polaris (PLS) Conclusions References

Biochemical and genetic studies have identified peptides that play crucial roles in plant defense, growth, and development. The number of known, functionally active, peptides is currently small, but genome sequencing has revealed many potential peptide-encoding genus. A major challenge of the post-genomic era is to determine the function of these molecules. ß 2003, Elsevier Science (USA).

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I. INTRODUCTION For many years, insight into intercellular signaling in plants was confined by the knowledge of a modest number of relatively simple organic molecules called phytohormones. The ‘‘five classic’’ plant hormones are auxin, cytokinin, ethylene, abscisic acid, and gibberellin. Extensive research in animal systems has revealed that intercellular signaling is mainly mediated by chemical signals such as steroids, small bioactive compounds, and peptides. Steroids are now known to be actively used by plants as signaling molecules (the brassinosteroids), but until just over a decade ago plants were not known to utilize polypeptides as regulatory molecules. The discovery of insulin in 1922 by Banting and Best in animal systems established that polypeptides can be used as signaling molecules that regulate a diverse range of physiological processes. It was not until 1991 that the first functional plant peptides were discovered and since then only a small number of plant polypeptides have been characterized. In this article, the evidence for plant polypeptides as signaling molecules with roles in intercellular signaling and development are reviewed.

II. SYSTEMIN AND SYSTEMIN-LIKE PEPTIDES Preexisting physical barriers that limit damage to plants such as the cuticle and woody coverings may provide some defense from herbivores, and thorns and trichomes may restrict pest activity to the more nutritious parts of the plant. Once an injury occurs, however, there is no possibility of mobilizing cells, devoted to wound healing, as occurs in mammals. Consequently plants have adapted to making each cell competent for the activation of defense responses, which largely depends on the activation of specific genes encoding so-called systemic wound response proteins (SWRPs) (Bergey et al., 1996). These wound-activated responses are directed to the healing of damaged tissues and also to the activation of defense mechanisms that aids prevention from further damage. Localized injury to the plant activates defense mechanisms throughout the plant, both in tissues damaged directly (local response) and in the nonwounded areas (systemic response). In solanaceous species such as tomato and potato, serine proteinase inhibitors (I and II) accumulate as part of the local response to damage and have been shown to inhibit insect feeding in transgenic plants (Hilder et al., 1989). Transcriptional activation of the proteinase inhibitors is not only local but also systemic so that undamaged leaves at a distance from the wound site accumulate these inhibitors. This phenomenon suggests the transmission of a signal or signals from the wound site to other parts of the plant. The isolation of this transmissible signal was facilitated by the development of a bioassay using young tomato plants. Tomato leaf juice

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extract applied to excised 14-day-old tomato plants through their cut stems for 30 min caused the leaves to accumulate proteinase inhibitor proteins (Ryan, 1974), indicating that the inducing factor was in the soluble extract of the leaves. A bioassay of protein extracts monitoring the induction of proteinase inhibitors I and II led to the purification of the first plant peptide to show signaling activity (Pearce et al., 1991). The pure substance, called systemin, was found to be an 18-amino acid polypeptide with the sequence AVQSKPPSKRDPPKMQTD. The systemin polypeptide is biologically active at femtomole concentrations, ranking it as among the most powerful plant gene-activating compounds. The movement of systemin from leaf wounds to other locations in the plant was investigated using autoradiographic and biochemical techniques. Narvaez-Vasquez et al. (1995) showed that when [14C]systemin was applied onto fresh wounds after 30 min the systemin was distributed throughout the wounded leaf, then transported to the petiole and finally into the upper leaves within 1 to 2 h of application. Further studies using [3H]systemin demonstrated that systemin moves from the wound site to the vascular system via the apoplasm and xylem, then to the phloem, where it is finally transported to target cells throughout the plant. Tissue-specific expression of prosystemin was investigated using a 2.2-kb promoter region linked to the -glucuronidase (GUS) reporter gene (Jacinto et al., 1997). Tomato plants transformed with this construct exhibited low levels of GUS activity that increased in response to wounding. Histochemical staining showed that GUS activity is associated with cells of the vascular bundles of leaf main veins, petiolules, petioles, and stems (Ryan and Pearce, 1998). Confirmation of this study was achieved by tissue printing, using prosystemin antibodies (Jacinto et al., 1997). The 18-amino acid systemin is not the primary translation product of its gene. By screening a cDNA library using a probe corresponding to the systemin gene, a partial cDNA was isolated (McGurl et al., 1992) and subsequently characterized (McGurl and Ryan, 1992). The gene structure revealed that systemin is processed from the C-terminal region of a 200amino acid precursor called prosystemin. Prosystemin does not possess a signal sequence at the N-terminus and also lacks any posttranslational modification, suggesting that prosystemin is synthesized in the cytosol (Delano-Frier et al., 1999). Transgenic tomato plants expressing an antisense version of prosystemin provided evidence that the polypeptide is an essential component of the defense system against insect pests as these plants exhibited a defective systemic wound response. The resistance of these transgenic plants toward larval attack was compromised in comparison to the wild-type plants demonstrating the crucial role of this inducible defense response. Conversely, transgenic plants constitutively expressing prosystemin at high levels behaved as if they were in a permanent wounded state (McGurl et al.,

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1994). It appears that cleavage of prosystemin is not required for biological activity because the activities of prosystemin and systemin are the same (Dombrowski et al., 1999; Vetsch et al., 2000). However, a prosystemin lacking the systemin sequence, under the same experimental procedure, was totally inactive (Dombrowski et al., 1999). These results suggest that for signaling to occur, the systemin sequence must be present. A systemin-binding protein was identified in the plasma membranes of leaf cells and also on the surface of Lycopersicon peruvianum suspension cultured cells (Meindl et al., 1998; Scheer and Ryan, 1999). The properties of the binding protein are indicative that it is a receptor involved in signal transduction of the systemin response. For example, the dissociation constant of the systemin–receptor interaction is in the range found for polypeptide–receptor interactions in animal systems and the binding to the receptor increases several fold in response to methyl-jasmonate, suggesting that the receptor is a systemic wound-response protein (Scheer and Ryan, 1999). The systemin receptor has now been isolated and characterized (Scheer and Ryan, 2002). The 160-kDa cell-surface protein (SR160) contains a putative amino acid signal sequence, a leucine zipper, 25 leucine-rich repeats (LRRs), a transmembrane domain, and a Ser/Thr protein kinase domain. The identification of the predicted systemin receptor supports the view that this family of receptors may play a major role in the recognition of polypeptide ligands in plants. Another LRR receptor kinase that has been identified in plants is CLV1, which is involved in developmental processes (Trotochaud et al., 2000) and is briefly discussed in this review. A model for the activation of defense genes by systemin can be briefly summarized as follows. On wounding, systemin is transported through the plant as a mobile signal after proteolytic cleavage from its precursor. Systemin interacts with the cell-surface receptor, which initiates a signaling cascade that includes the release of linolenic acid (LA) from plant cell membranes and its subsequent conversion to phytodienoic acid (PA) and jasmonic acid (JA) via the octadeconoid pathway (Vick and Zimmerman, 1984). JAs, together with ethylene (O’Donnell et al., 1996), are considered to be key regulators for stress-induced gene expression in plants and therefore the octadecanoid pathway appears to be a general signaling pathway for plant processes involved in defense and stress (Weiler, 1997). One convincing piece of evidence that the octadecanoid pathway has a role in mediating systemin signaling has come from a tomato mutant line. The defenseless mutant (def1) is deficient in a component of the octadecanoid pathway. On wounding the level of JA is not raised, less proteinase inhibitor is produced, and the plant is almost unresponsive to systemin. On attack by herbivores the def1 mutant is more susceptible to damage than the wild type (Lightner et al., 1993; Howe et al., 1996). However, the mechanism by which defense-related genes are transcriptionally activated by JAs and ethylene remains obscure.

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Tob Sys I

N - RGANLPOOSOASSOOSKE - C

Tob Sys II

N - NRKPLSOOSOKPADGQRP - C

Tom Sys

N - AVQSKPPSKRDPPKMQTD - C

FIGURE 1. Amino acid sequences of tobacco (Tob Sys I, Tob SysII) and tomato (Tom Sys) systemins.

Prosystemin orthologues have also been identified in other solanaceous species, such as potato, bell pepper (Capsicum annuum), and black nightshade (Solanum nigrum) with sequence identities of 73–88% (Constabel et al., 1998). The deduced systemin polypeptides from the three species are relatively similar with differences found primarily in the N-terminal portion of the molecules, whereas the C-terminal amino acids (residues 12–18) were all found to be identical. No orthologous tobacco peptides had been identified until recently. A bioassay, based on the fact that systemin caused alkalinization of cell culture medium, led to the isolation of three 18-amino acid polypeptides (Pearce et al., 2001a). The two most abundant polypeptides designated Tob Sys I and II (Tobacco Systemin I and II), like tomato systemin, can induce the expression of trypsin inhibitors as well as a mitogen-activated protein (MAP) kinase and both precursor proteins are inducible by methyl-jasmonate treatment, indicating the involvement of the octadecanoid pathway. Despite these similarities in function, Tob Sys I and Tob Sys II are not homologous to each other or to tomato systemin (Fig. 1) and in contrast to tomato systemin, Tob Sys I and II are linked to pentose sugars. An interesting feature of Tob Sys I and II is that both polypeptides are generated from a single 165-amino acid preproprotein. Tob Sys I is located at the N-terminal, whereas Tob Sys II is located at the C-terminal and a secretion signal resides upstream of Tob Sys I, demonstrating that multiple polypeptide signals can be generated from a single precursor in plants. Although prohormones (precursor proteins of two or more independent signal molecules) have been identified in animals and yeast (Steiner et al., 1992), the Tob Sys system is the first example in plants.

III. RAPID ALKALINIZATION FACTOR (RALF) During the purification of tobacco systemins a 49-amino acid polypeptide was identified and isolated that caused the medium containing tobacco suspension cells to become rapidly alkalinized. Because of the rapid response in the alkalinization assay, this polypeptide was termed rapid alkalinization factor (RALF) (Pearce et al., 2001a). Similar to the tobacco systemins, RALF induces MAP kinase activity in tobacco cells, but in

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contrast RALF does not appear to be a defensive wound signal as it does not induce the synthesis of trypsin inhibitor in the leaves of tobacco plants (Pearce et al., 2001a). To identify the full-length tobacco RALF, a cDNA library was probed with a tomato expressed sequence tag (EST) probe. The deduced open reading frame (ORF), from the tobacco cDNA, encoded a RALF precursor protein of 115 amino acids (Pearce et al., 2001b). Analysis of this protein revealed a putative 25-amino acid signal peptide at the N-terminus and the 49-amino acid RALF sequence at the C-terminus, suggesting that the precursor proteins are synthesized through the secretory pathway and then further processed. The molecular mass of the RALF polypeptide deduced from the precursor protein matched the molecular mass of the purified RALF polypeptide confirming that RALF is processed from a larger precursor (Pearce et al., 2001b). ESTs, derived from a variety of tissues, coding for RALF have been identified in 16 plant species. The RALF sequence itself and a region just N-terminal of the RALF sequence show a high percentage of identity at the amino acid level. Homology in the primary structure between the divergent plant species and the fact that the ESTs were derived from a variety of tissues suggest that RALF has a fundamental role in plant growth and development (Pearce et al., 2001b). A clue to the possible role of RALF in plants was found by transferring tomato and Arabidopsis seedlings to medium containing micromolar levels of tomato RALF (Pearce et al., 2001b). Root development was immediately arrested, and the cotyledons of the Arabidopsis seedlings exhibited a lighter green color. Visual inspection of the roots revealed that the elongation zones and meristems were arrested and the meristem cells themselves were enlarged compared to untreated plants. When RALF-treated plants were transferred back to RALF-free media the roots resumed growth showing that the response is reversible. The biochemical and physiological mechanisms behind the process of root growth arrest have yet to be determined.

IV. ENOD40 AND ROOT NODULATION Leguminous plants have the ability to enter into symbiosis with nitrogenfixing bacteria (collectively called rhizobia) to form the root nodule. The formation of root nodules involves the division of root cortical cells, typically adjacent to the protoxylem poles. Positional information controlling this formation is probably the signaling molecule ethylene (Heidstra et al., 1997) as ethylene blocks cortical cell divisions and because a key enzyme in its synthesis, ACC oxidase, is transcriptionally upregulated in the pericycle adjacent to the phloem poles. Plant genes that are specifically induced by nodulation factor-secreting rhizobia during early stages of nodule development have been termed early

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nodulin genes (Enod ). Among these genes, ENOD40 is one of the earliest expressed nodulins in the root pericycle adjacent to the phloem poles. The pattern of gene expression complements that of ACC oxidase (Kouchi et al., 1993; Yang et al., 1993) but precedes the mitotic activation of the root cortical cells (Compaan et al., 2001). The implication is that ENOD40 counteracts the effects of ethylene to promote cortical divisions and nodule formation. ENOD40 genes have been identified in many legumes but also in the monocotyledonous plants, rice and maize, suggesting that ENOD40 is widespread throughout the plant kingdom and may have a general biological function (Kouchi et al., 1999). The ENOD40 genes from both legumes and nonlegumes encode a transcript of approximately 0.7 kb that contains no single ORF but does contain two small open reading frames (sORF) in highly conserved regions, called boxes I and II (van de Sande et al., 1996). In alfalfa, conserved box I, found at the 50 end of the gene, encodes a predicted peptide of 13 amino acids, whereas box II, in the central region, encodes a predicted peptide of 27 amino acids (Sousa et al., 2001) (Fig. 2). Recent studies have suggested that the larger sORF might not be sORFII

sORFI alfalfa ENOD40 mRNA

13 a/a polypeptide

27 a/a polypeptide

sORF Soybean ENOD40

mRNA 12 a/a polypeptide 24 a/a polypeptide

FIGURE 2. Nonoverlapping and overlapping small reading frames (sORFs) of alfalfa and soybean ENOD40 genes, respectively.

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translated but may encode a highly stable RNA structure (Compaan et al., 2001). However, using microtargeting techniques it was demonstrated that the translation of the two sORFs, with the presence of the inter-ORF region, is required for the regulation of ENOD40 activity (Sousa et al., 2001). In contrast to systemin, ENOD40 peptides are not produced from cleavage of larger precursors and, as in tomato systemin, there are no signal sequences suggesting that ENOD40 may be synthesized in the cytosol. Functional evidence for a role of ENOD40 in the early stages of nodulation has been studied in Medicago plants in which ENOD40 sequences have been introduced by microbombardment. When bombarded into roots of alfalfa (M. sativa), an M. truncatula ENOD40 gene could induce ectopic cortical cell divisions (Sousa et al., 2001). Furthermore, overexpression of ENOD40 in M. truncatula, inoculated with Sinorhizobium meliloti, increased inner cortical cell divisions, whereas downregulation of ENOD40 reduced the number of nodule primordia and resulted in aberrant nodule development and structure (Charon et al., 1999). The involvement of ENOD40 in nodule development has been implicated in triggering cortical cell divisions during the initiation of nodule morphogenesis. However, it is thought that ENOD40 is not an inducer of cell division per se but rather its activity depends on other factors located within the inner cortex to allow cell cycle activation (Sousa et al., 2001). A further insight into the role of ENOD40 has recently come to light. In vitro translation of soybean ENOD40 mRNA has revealed a second sORF of 24 amino acids that overlaps the box I 12-amino acid (Fig. 2) (Ro¨hrig et al., 2001). The 24-amino acid peptide, but not the 12-amino acid peptide, was detected in nodule extracts using Western blot analysis. Both peptides, however, were found to bind specifically to a protein of 93 kDa, which, on further analysis, was identified as soybean nodulin100, which is a subunit of sucrose synthase (Thummler and Verma, 1987). Symbiotic nitrogen fixation is an energy-demanding process and the rapidly growing nodule tissue requires sucrose for cellulose synthesis. It was proposed that soybean ENOD40 may serve as a regulator in unloading sucrose from the phloem, either by modulating enzyme activity or by directing the enzyme to specific intercellular sites. The sORFs of alfalfa have yet to be investigated for a similar regulatory role. A detailed analysis of promoter sequences of legume and nonlegume ENOD40s together with transgenic studies may provide further insights into ENOD40 functions and regulatory mechanisms.

V. CLAVATA3 AND MERISTEM ORGANIZATION The shoot apical meristem (SAM) is a source of new cells for leaf development and retains a meristematic character analogous to animal stem

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cells. Central to the function of the SAM is that a group of undifferentiated stem cells must be maintained in the center because proliferation of these cells is necessary to provide new cells for organ and new meristem initiation. One striking property of SAMs is their ability to remain relatively constant in size. New organs are generated throughout the life span, thus a pool of undifferentiated stem cells must be maintained at the SAM from which to draw on for new organ initiation. To maintain a functional SAM, the precise coordination between the loss of stem cells from the meristem and their replacement through cell division must be tightly controlled (Brand et al., 2000; Clark, 2001). Genetic studies using Arabidopsis have indicated that the CLAVATA genes (CLV1, CLV2, and CLV3) control the balance between cell proliferation and division (i.e., these genes are required to restrict the amount of stem cell accumulation in shoot apical meristems). Lossof-function mutants, in any of the three different CLAVATA genes, results in a hyperaccumulation of stem cells in the central zone of the shoot apex causing stem overgrowth and production of extra flowers (Clark et al., 1993, 1995; Kayes and Clark, 1998). The phenotypic similarity, together with epistasis experiments, strongly suggests that the CLV genes function in the same pathway. The SAMs of the clv3 and clv1 mutants of Arabidopsis are enlarged compared with the wild type in the mature embryo and continue to enlarge throughout vegetative and inflorescence development, often becoming 1000-fold larger (by volume) than wild type (Clark et al., 1995). Conversely, in transgenic lines expressing high levels of CLV3 mRNA, the shoot meristem ceases to initiate organs after the emergence of the first leaves, indicating that stem cell fate in the meristem center is controlled by the CLV3 signal (Brand et al., 2000). The CLV3 gene encodes a small 96-amino acid peptide with a predicted 18-amino acid secretion signal at the N-terminus (Fletcher et al., 1999). The expression pattern of CLV3, analyzed by RNA in situ hybridization, is first detected in heart stage embryos, in a group of cells between the developing cotyledons, a region predicted to give rise to the SAM (Clark et al., 1995; Barton and Poethig, 1993). Throughout development the expression pattern of CLV3 is always restricted to the most central part of the meristem, the putative stem cells, which are found primarily in the epidermal and subepidermal cell layers of shoot and floral meristems (Fletcher et al., 1999). CLV1 is expressed in the underlying cells in a domain that partially overlaps that of CLV3 (Clark et al., 1997) (Fig. 3), prompting the hypothesis that CLV3 moves from the overlying cell layers to the underlying layer to activate the CLV1 complex. Recent data, using genetic and immunological studies, have shown that the CLV3 protein is transported through the secretory pathway, where it is localized to the apoplast and activates the CLV pathway in the extacellular space (Rojo et al., 2002). The CLV3 polypeptide is therefore a key developmental regulator in plants in which

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CLV3

WUS

CLV1

FIGURE 3. Outline of shoot apical meristem (SAM) showing approximate expression domains of CLV1, CLV3, and WUS. apoplastic localization is required to communicate cell fate information between different regions of shoot and floral meristems. Sequence analysis of the CLV1 gene has indicated that it may function as a receptor kinase with a characteristic extracellular LRR domain and an intracellular serine/threonine kinase domain (Clark et al., 1997). CLV2 also encodes a receptor-like protein without the cytoplasmic domain and forms a membrane-bound complex with CLV1 (Jeong et al., 1999). Biochemical evidence has shown that CLV1 occurs in two distinct complexes of 185 kDa and 450 kDa (Trotochaud et al., 1999). The smaller molecule is a heterodimer of CLV1 and CLV2. The larger multimer contains, as well as the CLV1/CLV2 dimer, a Rho-GTPase-related protein and the kinaseassociated protein phosphatase (KAPP) (Stone et al., 1998). Immunoprecipitation studies have demonstrated that CLV3 coprecipitates with CLV1 in vivo and yeast cells expressing CLV1 and CLV2 bind CLV3 from plant extracts. Crucial for the association of CLV3 with the CLV1/2 complex is the requirement for an active CLV1 kinase as substitution of the Lys-720 residue with an Asp resulted in no CLV3 binding (Trotochaud et al., 2000). Taken together, these results suggest that CLV3 functions as the CLV1 ligand. Recent data, using database searches, have revealed a large family of genes that shares homology with CLV3 (Cock and McCormick, 2001). Most of the predicted polypeptides have N-terminal signal sequences and thus appear to be secreted peptides, although a role for these CLV-like polypeptides in signaling requires further investigation. Also, the fact that the CLV3-like homologs were expressed in a variety of plant organs suggests similar ligand–receptor interactions occur in organs outside the SAM. As outlined earlier, genetic analysis in Arabidopsis has revealed that CLV1/2/3 prevents the overproliferation of undifferentiated stem cells. Several papers have highlighted the close interactions between the CLV genes and other loci, which may suggest mechanisms on how cellular homeostasis is maintained. One of these loci, WUSCHEL (WUS), encodes a homeodomain protein that is expressed in a small group of cells in the central zone (Laux et al., 1996; Mayer et al., 1998). Mutations at the WUS locus exhibit phenotypes

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CLV3

CLV1

CLV1

WUS FIGURE 4. A model for the interaction of CLV1, CLV3, and WUS. The CLV3 protein binds to CLV1, resulting in inhibition of WUS expression. This establishes a feeedback loop that maintains the size of the central zone.

largely opposite those of clv mutants. SAMs can be initiated by wus mutants, but cells within these SAMs are recruited to form lateral organs without replenishment of the stem cell population in the central zone (Laux et al., 1996). Furthermore, wus/clv double mutants have a phenotype indistinguishable from that of the wus single mutants, suggesting that WUS is required for the clv phenotype of meristem growth. Moreover, the expression domain of WUS expands in clv mutants, suggesting that the CLV signaling complex might act to restrict WUS expression (Schoof et al., 2000). These observations suggest the following model (Fig. 4). In wild-type plants the CLV3 polypeptide ligand is secreted from the stem cells at the apex of the meristem through the extracellular space and binds to the CLV1/ 2 receptor complex at the plasma membrane of the underlying cells. The activated CLV signal transduction pathway downregulates WUS activity by restricting its expression to a narrow domain of cells in deeper layers of the meristem. Signaling via CLV3 limits the stem cell-promoting signal, mediated by WUS, back to the stem cell population, maintaining the suitable amount of stem cell activity throughout development.

VI. PHYTOSULFOKINES The relative growth rate of plant cells in culture strictly depends on the initial cell density, which cannot be improved by supplementation of plant hormones such as auxin and cytokinin. However, the relative growth rate can be improved by the addition of conditioned media (CM) prepared from rapidly growing cells in culture, indicating that the growth rate of plant cells is controlled by factors other than auxin and cytokinin (Bellincampi and Morpurgo, 1987; Birnberg et al., 1988). A possible explanation of this phenomenon is the secretion of a mitogenic factor produced by individual

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cells into the CM (Stuart and Street, 1969). For many years the structure of the mitogenic factor remained elusive because of the lack of a sensitive bioassay; however, a sufficiently sensitive bioassay has been used to detect this factor (Matsubayashi and Sakagami, 1996). Using this system, the mitogenic components were identified from conditioned medium derived from mesophyll cell cultures of Asparagus officinalis. These factors, termed phytosulfokines (PSK- and PSK-), were identified and found to be small (five and four amino acids, respectively) disulfated peptides (Fig. 5). PSK- is identical to PSK- except for the loss of the most C-terminal amino acid and is probably a degradation product of PSK-. PSKs have also been identified in CM derived from rice (Matsubayashi et al., 1997), maize (Matsubayashi et al., 1997), zinnia (Matsubayashi et al., 1999), carrot (Hanai et al., 2000a), and Arabidopsis (Yang et al., 2001), suggesting the universal distribution of PSKs throughout plant species. Although the phytosulfokine peptides have not been isolated from intact plants to date, reverse transcription-polymerase chain reaction (RT-PCR) analysis has shown that rice seedlings express the cognate gene most abundantly in the shoot and root apexes, areas with high rates of cell proliferation (G. Yang et al., 1999). This suggests that PSK- might be important for cell proliferation in vivo as well as in cultured cells. PSK- has several other biological activities in addition to plant cell proliferation. For example, in cucumber, PSK- promotes chlorophyll synthesis in etiolated cotyledons (Yamakawa et al., 1998a) and adventitious root formation by hypocotyls (Yamakawa et al., 1998b). PSK- also promotes growth and chlorophyll content of Arabidopsis seedlings under conditions of high nighttime temperature and adventitious bud formation in Antirrhnum majus (G. Yang et al., 1999; H. Yang et al., 1999), as well as reinforcing the frequency of somatic embryogenesis in carrot cultures (Hanai et al., 2000a). PSK- has also been shown to stimulate tracheary element differentiation and regulate the pollen population effect

−C

N− signal peptide

PSK-a Tyr(SO3H)-Ile-Tyr(SO3H)-Thr-Gln

FIGURE 5. PSK- is proteolytically processed from the C-terminal of the preprotein precursor. Removal of the signal sequence and addition of sulfate groups occur through the secretory pathway.

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(Matsubayashi et al., 1999; Chen et al., 2000). The results given earlier suggest that PSKs make cells receptive to signals that ultimately determine the cell density, that is, cell division or cell differentiation. By analogy to animal systems PSK- exerts its effects by activation of genes through a receptor. Evidence for the existence of such a receptor was provided by binding assays with radiolabeled PSKs (Matsubayashi et al., 1997; Matsubayashi and Sakagami, 1999) and further analysis showed that the putative receptors for PSK, in rice plasma membranes, were 120-kDa and 160-kDa glycosylated proteins (Matsubayashi and Sakagami, 2000). Recent work, using carrot microsomal fractions, has shown that the 120kDa protein specifically interacts with PSK (Matsubayashi et al., 2002). Further analysis of this protein showed features found in several hormone receptors from both animals and plants. There is an N-terminal hydrophobic signal sequence, extracellular LRRs, a transmembrane domain, and a cytoplasmic domain. The major extracellular domain of this protein contains 21 tandem copies of a 24-amino acid LRR, which may play a role in protein–protein interactions. The eighteenth LRR also has a 36amino acid island, which has also been identified in the extracellular LRRs of the brassinosteroid receptor BR1 and has been shown to be crucial for its function (Li and Chory, 1997). The cytoplasmic region contains all 12 subdomains found in most eukaryotic serine-threonine kinases, and the kinase region of the protein shares sequence identity with the known plant hormone receptors BR1 and CLV1 (see later). Like systemin, PSK- is derived from the processing of a larger precursor polypeptide (Fig. 5). The preprophytosulfokine precursor (PP-PSK) has recently been identified from rice (H. Yang et al., 1999). The ORF is predicted to encode a PSK precursor of 89 amino acids with a molecular mass of 9.8 kDa. The predicted peptide has an N-terminal signal peptide and a PSK sequence close to the C-terminal. The N-terminal signal peptide resembles a cleavable leader sequence commonly found in animal peptide precursors (Douglass et al., 1984) and is predicted to mediate translocation across membranes of the endoplasmic reticulum during prohormone synthesis and allow secretion into the extracellular space. The PSK- sequences from different species, including Arabidopsis and asparagus, are perfectly conserved; however, the precursors show structural differences, suggesting a complex diversity of peptide-encoding genes in plants that are processed into the same signaling molecule (Yang et al., 2001). Because PSK- strongly stimulates plant cell proliferation in low-density cultures at low concentrations (1 109 M ), deletion and substitution experiments were performed to access PSK- activity. Deletion of the sulfate groups of the tyrosine residues 1 and 3 (see Fig. 5) resulted in very low PSK- activity, indicating that both sulfur groups are important for activity. Further studies showed that by deleting the first and second

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C-terminal amino acids some activity of the parent pentapeptide was retained whereas truncation of the N-terminal showed little or no activity. Substitution experiments also showed that the amino acids Ile-2 and Thr-4, do not merely act as spacers but are necessary for PSK- activity (Matsubayashi et al., 1996). The fact that both tyrosine residues of PSK- are sulfated is interesting as many mammalian secretory proteins have been reported to contain tyrosine sulfate (Huttner, 1984), although so far PSK- is the only peptide in higher plants that possess a sulfated tyrosine. Sulfation of tyrosine residues of secretory proteins in mammalian cells is catalyzed by the enzyme tyrosylprotein sulfotransferase (TPST) (Lee and Huttner, 1983). Using an in vitro TPST assay, based on mammalian studies (Niehrs et al., 1990), it was shown that TPST from several plant lines could indeed lead to the sulfation of PSK peptides (Hanai et al., 2000b). Furthermore, the plant TPST was shown to be localized to the Golgi network, a distribution that is similar to mammalian TPSTs. Because desulfated PSK- shows much reduced activity the presence of TPSTs in plants only emphasizes the importance of sulfation for the physiological function of PSK-. PSK- appears to be a unique and important growth factor in plants. As well as being the first sulfated peptide found in plants, it appears to be widely distributed throughout this kingdom.

VII. BRASSICA SELF-INCOMPATIBILITY Many flowering plants rely on self-incompatibility (SI) to ensure genetic fitness by preventing inbreeding depression. The genetic barrier to SI is based on the ability to discriminate between self-related and genetically unrelated pollen. Developmental arrest occurs when a pollen grain from a self-incompatible plant lands on its own stigma or on a stigma of a genetically related plant where the pollen will either fail to germinate or germinate with failure of the pollen tube to reach the ovules (de Nettancourt, 1977). Thus, SI involves an exchange of information between the haploid pollen and the diploid female reproductive organs to distinguish ‘‘self ’’ and ‘‘nonself ’’ pollen grains. In many cases, this process is controlled by a single multiallelic locus called the S locus. Three different SI systems have been studied in detail at the molecular level, and all three have different mechanisms. The Solanaceae SI trait is genetically controlled by the S locus (de Nettancourt, 1977), where a ribonuclease inhibits incompatible pollen (Anderson et al., 1986). In the Papaveraceae, the stigmatic S gene encodes a protein (Foote et al., 1994), in the presence of incompatible pollen, that triggers a Ca2+ cascade. This signal transduction cascade results in protein phosphorylation, DNA fragmentation, and alterations to the actin cytoskeleton, all of which are thought to contribute to inhibition of pollen tube growth (Jordan et al., 2000;

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Snowman et al., 2000). Both of the previously mentioned systems have been reviewed elsewhere (Silva and Goring, 2001; Golz et al., 1995), but signaling peptides have yet to be found. On the other hand the recent discovery of the pollen component in Brassica (Schopfer et al., 1999) has opened more avenues in peptide signaling in plants and will be focus of this section. Investigations into sporophytic SI has focused on members of the Brassicaceae family—Brassica oleraceae (cabbage, cauliflower, broccoli), B. rapa (chinese cabbage), and B. napus (canola) (Kao and McCubbin, 1996; Cock et al., 2000). The inhibition of self-pollen in Brassica involves a series of complex cell–cell interactions that occur on the stigmatic surface of the pistil. The manifestation of the SI response is rapid, occurring within a few minutes of self-pollination, culminating in inhibition of pollen hydration, pollen germination, or pollen tube invasion of the stigma epidermis (Dickinson, 1995). The female components of the Brassica SI system have been known for some time. As in the previous systems mentioned, SI in Brassica is controlled by a highly polymorphic S-locus. Molecular analysis of SI in Brassica identified two stigmatic components encoded by the S locus; a secreted S-locus glycoprotein (SLG) and an S-locus receptor kinase (SRK), which functions as the female determinant of SI (Nasrallah et al., 1985; Stein et al., 1991). The actual role of the SLG protein in SI is unclear, although, SLG has been proposed to enhance the SI reaction (Takasaki et al., 2000), but in other studies, SLG caused no detectable effects on the SI phenotype (Silva et al., 2001) The SRK protein, which closely resembles animal receptor kinases, is composed of an extracellular domain (S-domain), a single membrane spanning domain, and a cytoplasmic serine/threonine kinase domain (Stein et al., 1991; Delorme et al., 1995). The role of SRK as the sole S specificity determinant in the pistil has been determined (Takasaki et al., 2000; Silva et al., 2001) and consistent with its role in SI, SRK has been shown to be specifically expressed in the stigma (Stein et al., 1991). Since the identification of the female determinants in SI, the male determinant counterpart has been searched for. The search has proved to be difficult due to the complex structure of the S-locus. However, several strategies have been used to identify proteins in the pollen coat. A bioassay for functional pollen coat proteins (PCPs) led to the identification of a basic cysteine-rich peptide of 7 kDa. This, and related peptides, were designated PCPs and are related to a family of cysteine-rich proteins that are characterized by eight conserved cysteine residues (Doughty et al., 1998). Unfortunately, although PCPs were shown to interact with SLG proteins, this interaction was not S-allele specific and was also shown to be unlinked to the S-locus (Schierup et al., 2001; Doughty et al., 1998). PCPs were therefore ruled out as the candidate for the pollen S gene.

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The major problem in identifying the pollen S gene through mapping and sequencing was the sheer size of the S-locus; it is generally believed that the Brassica S-locus spans several hundred kilobases (Boyes et al., 1997). Mapping and sequencing aimed at identifying specific S-linked antherexpressed genes resulted in the identification of several candidates for the pollen S gene. These genes, including SLA (Boyes and Nasrallah, 1995), SLL1 and SLL2 (Yu et al., 1996), which are linked to the S-locus, failed to exhibit features expected of the S pollen determinant. Nevertheless, mapping and sequencing revealed one gene to be implicated in the SI response. SP11 (S-locus protein 11) was first identified as an anther expressed S9-haplotype-specific gene in an SLG/SRK flanking region of the S9-haplotype of Brassica rapa (Suzuki et al., 1999). A different allele of the same gene was identified in Brassica oleracea by sequencing a 13-kb region between the S8 SRK and SLG genes and was designated SCR (S-locus cysteine-rich protein) (Schopfer et al., 1999). The SCR genes from several S-haplotypes have been identified and show much structural polymorphism. All the SCR alleles encode small peptides with a bipartite structure consisting of a relatively conserved hydrophobic N-terminal region with a C-terminal region that is variable and hydrophilic (Schopfer and Nasrallah, 2000). The larger portion of the conserved N-terminus contains a secretion signal suggesting that the SCR polypeptides are processed via a secretory pathway. If no further processing occurs, the products of the SCR genes will be secreted as a mature 8.4- to 8.6-kDa hydrophilic protein consisting of eight cysteine residues. These features are reminiscent of the PCP family, but the positioning of the cysteine residues within the SCR peptides is otherwise distinct. To determine whether the SP11/SCR was the sole pollen S determinant, pollen bioassays and gain-of-function and loss-of-function experiments were performed. A recombinant SP11/SCR protein produced in Escherichia coli was used in the pollination bioassay. The recombinant protein applied onto the papillar cells of S9 stigmas before pollination inhibited the hydration of cross-pollen (S9-haplotype). The same recombinant protein when applied to S8 stigmas did not inhibit hydration of S9-haplotype pollen, demonstrating that SP11/SCR is sufficient to induce the SI response in cells of the same haplotype but not of a different S-haplotype (Takayama et al., 2000). In gain-of-function experiments the SP11/SCR gene was introduced into SI Brassica species. The transgenic plants were shown to have acquired the S-haplotype specificity of the transgene in the pollen but not in the stigma demonstrating that SP11/SCR is the pollen S-determinant (Schopfer et al., 1999; Shiba et al., 2001). In loss-of-function experiments the loss of pollen S-function in a self-compatible Brassica oleracea line was correlated with the deletion of the SP11/SCR gene, again demonstrating SP11/SCR is necessary for pollen SI specificity (Schopfer et al., 1999). Furthermore, because of the sporophytic nature of SI in Brassica, the pollen S-determinant gene is likely

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to be expressed sporophytically in the anther (de Nettancourt, 1977). In situ hybridization of anther sections revealed that SP11 was expressed in the tapetal cells of early stages of flower development and at later stages in the microspores. The tapetal expression pattern alone may explain the sporophytic nature of Brassica SI (Takayama et al., 2000). To confirm the expression pattern of SP11 at the protein level immunohistochemical analyses were carried out using antibodies raised against the SP11 peptide. The results of this experiment showed that SP11 protein expression is confined to the tapetal cells of the anther and in the pollen grains (Shiba et al., 2001). The determinants in the Brassica SI system, SRK, SP11/SRC, and perhaps SLG, initiate the allele-specific rejection of self-incompatible pollen. However, other components are required to carry out cellular responses to reject self-pollen. In a search for downstream signaling molecules using the yeast two-hybrid system, three proteins were identified that interact with the receptor kinase domain of SRK (Bower et al., 1996; Gu et al., 1998). One of these, ARC1, interacts with SRK in a phosphorylation-dependent manner (Gu et al., 1998) and another, THL1, has been reported to regulate autophosphorylation of SRK (Cabrillac et al., 2001). However, other components, still unidentified, must also participate in this complex recognition and rejection system. The identification of genes proposed to be involved in SI has led to a model of self-incompatibility in Brassica (Fig. 6). Prior to pollination, SRK is present in the stigmatic papillae, free to be activated when selfincompatible pollen comes into contact with a stigmata papilla. When this pollen grain lands on the stigma surface, the pollen S-determinant, SP11/ SCR, is thought to diffuse into the papillar cell wall where it interacts with the extracellular domain of SRK presumably in a S-haplotype-specific manner, thus providing the specificity in self-recognition. The activated SRK then initiates a signaling cascade in the stigmatal papillae leading to rejection of incompatible pollen. The involvement of SLG remains elusive but may complex with SRK to aid in stabilization and therefore enhance the SI response (Takasaki et al., 2000; Dixit et al., 2000). The role of ARC1 is again largely unknown but likely acts downstream of SRK, where it may regulate the ubiquination and degradation of another unknown protein in the stigmatic papillae. Although a detailed analysis of the ensuing SRK signaling cascade remains to be resolved, arrest of self pollen may occur at any one of several steps including pollen hydration, germination, or pollen tube growth into the papillar cell wall (Dickinson, 1995). Thus the SRKSP11/SCR triggered pathway may include the activation of a plasma membrane-localized aquaporin-like protein that would result in localized depletion of water and other substances required for pollen germination and tube outgrowth (Ikeda et al., 1997). One ultimate question about SCR is how the peptide might function in SI recognition. The known SCR sequences are

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SCR/SP11

Pollen grain

SRK

Aquaporin ARC

stigmatal papilla

FIGURE 6. The Brassica self-incompatibility system. SCR/SP11 is localized to the pollen coat. On contact with the stigma surface, SSR/SP11 interacts with the SRK recptor kinase complex to activate a signaling cascade that leads to the incompatible SI response. Activation of ARC may lead to the activation of aquaporins to limit the uptake of water necessary for pollen hydration and germination.

devoid of sequence motifs indicative of enzyme activity and therefore are not likely to catalyze a conversion of a precursor molecule into a specific signal molecule. SCR can therefore be presumed to function directly as a ligand molecule (Schopfer and Nasrallah, 2000). As a result of the identification of the SCR peptide, further studies might involve investigations in the origins of the pollen recognition gene and determination of whether polypeptides evolutionary related to SCRs exist that function other than in SI.

VIII. POLARIS (PLS) One experimental system that has attracted much attention as a model to study hormonal interactions in development in plants is the Arabidopsis root; it is well characterized anatomically, physiologically, and developmentally (Dolan et al., 1993), and is readily amenable to genetic screening for

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mutations affecting each of these aspects. Over the years, studies have identified diverse components in the signaling pathways of auxin, ethylene, cytokinins, gibberellins, and abscisic acid, the so-called five ‘‘classic’’ plant hormones. However, there is an incomplete picture of how root growth and development are coordinately regulated by hormones and how hormones interact (‘‘cross-talk’’) to elicit the diverse developmental pathways found in plants. To identify new genes involved in regulation of root development, and to gain new insight into hormonal interactions, a population of Arabidopsis promoter-trap transgenics was screened for root expressed genes and defective root phenotypes. The tagging of genes by promoter-trap insertional mutagenesis potentially facilitates expression analysis of genes (by the characterization of in vivo fusion gene expression), the investigation of tagged gene function by mutant analysis, and the cloning of genes (Topping et al., 1991). This strategy, which has been successful in studying gene expression in Drosophila, mouse, and Caenorhabditis elegans (Bellen et al., 1989; Allen et al., 1988; Hope, 1991), identified a novel GUS-expressing line of which the tagged gene was called POLARIS (Topping et al., 1994). GUS expression, driven by the POLARIS promoter, was first identified in heart-stage embryos, in the basal region of the embryo that corresponds to the embryonic root primordium (Scheres et al., 1994). As the embryo develops, GUS activity is maintained in the embryonic root and in young seedlings and mature plants GUS activity is found most strongly in the tips of both primary and lateral roots with low expression in the aerial parts. GUS expression in the root tips occurs in all the cell types present (columella and lateral root cap, epidermis, meristem, and immature vasculature) and occurs from the earliest stages of the pericycle division in lateral root development (Topping et al., 1994; Topping and Lindsey, 1997). Sequencing of the PLS locus revealed the T-DNA insertion in an ORF encoding a predicted 36-amino acid polypeptide, with a predicted molecular mass of 4.6 kDa and limited homology to known proteins (Casson et al., 2002). The N-terminal 24 amino acids are predicted to form two -sheets whereas the remaining 12 amino acids are likely to form an -helix. Between the two -sheets are three basic arginine residues, which may form a possible cleavage site. The second -sheet contains a repeated SIS separated by four residues that may signify a cAMP and cGMP-dependent protein kinase phosphorylation site (Casson et al., 2002). The C-terminal -helix also contains the repeat KLFKLFK. The lysine residues and a terminal histidine residue represent the only charged residues in this helical region. The three amino acid spacing between each of the lysine residues indicates that they would all lie on the same face of the -helix, creating an amphipathic helix with both hydrophobic and charged faces. The fact that the predicted helical region is leucine rich indicates the potential for a leucine zipper motif, suggesting the possibility for protein–protein interactions. The PLS peptide

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has yet to be detected by Western blotting using polyclonal antibodies to the N-terminal 18 amino acids. However, genetic evidence demonstrates that a partial PLS cDNA is functional and that functionality of the cDNA requires that the PLS ORF has an ATG codon (Casson et al., 2002). These data suggest that the PLS gene encodes a functional polypeptide. A clue to the function of the PLS polypeptide came from the investigation of the pls mutant root phenotype when compared to the wild type. The primary root of the pls mutant was found to be approximately 50% shorter than wild type, and microscopic analysis revealed that the cells of the root meristem and cortex of the primary root were shorter and more radially expanded. Reduced longitudinal cell expansion and increased radial expansion can be caused by increased accumulation or sensitivity to any of several hormones including ethylene (Abeles et al., 1992), auxin (Ljung et al., 2001), or cytokinin (Beemster and Baskin, 2000). To investigate the sensitivity of pls root growth to exogenous hormones, mutant and wild-type seedlings were grown in the presence of different concentrations of exogenous auxin and cytokinin. pls roots were found to be proportionately shorter than wild type in the presence of exogenous cytokinin, over the range of concentrations tested, suggesting that the pls mutant exhibits increased cytokinin responsivness. Consistent with this possibility, the cytokinin upregulated gene IBC6/ARR5 (Brandstatter and Kieber, 1998) was transcriptionally upregulated in the pls background when compared to wild type (Casson et al., 2002). However, there was a significantly reduced growth inhibitory effect of auxin on the pls root compared to wild type and the expression level of the auxin-inducible gene, IAA1, was downregulated, suggesting that the pls mutation causes reduced responses to auxin. Interestingly, although the aerial parts of seedlings and mature plants were not obviously abnormal in their morphology, microscopic analysis revealed that the extent of leaf vascularization in the pls mutant was reduced compared with wild type. Taken together the above results suggest that the PLS peptide is required for correct responses to exogenous cytokinins and auxins, correct root growth, and correct vascular leaf patterning. Because cytokinins are synthesized in root tips and are at relatively high concentrations in roots (Sossountov et al., 1988; Chiapetta et al., 2001), it can be speculated that a mechanism exits to reduce the sensitivity of cells at the root tip to cytokinins. PLS may encode a component of this mechanism. Furthermore, both cytokinins and auxin can induce ethylene biosynthesis (Vogel et al., 1998). It is therefore also possible that the observed increase in cytokininmediated responses in the pls mutant may be mediated by downstream ethylene effects or reduced auxin responses, as auxins and cytokinins interact and often have antagonistic effects, such as in shoot branching, root branching, and vascularization (Ljung et al., 2001). Because it is known that auxin plays an essential role in the patterning of vascular tissues (e.g.,

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Hardtke and Berleth, 1998; Przemeck et al., 1996), possible alterations to auxin–cytokinin interactions in the pls mutant, due to increased cytokinin levels or sensitivity or reduced auxin levels or sensitivity, could account for the observed reduced vascularization in the pls leaf. The results also add support to previous reports that hormonal response pathways are not simple linear pathways but must be robust and diverse enough to integrate a number of separate signals, allowing for the plasticity of plants integrating auxin, cytokinin, and ethylene signals to regulate root growth and leaf growth and development.

IX. CONCLUSIONS It is not surprising that only a small number of functional peptides have been found in plants. Due to their small size, opportunities for peptide gene tagging by insertional mutagenesis, for example, are correspondingly small. Also small cDNAs are often not represented in cDNA libraries, especially if the transcripts are not abundantly expressed. However, there is increasing evidence that such peptides exist and play important roles in a wide spectrum of physiological processes. For example, covered in this article ENOD40, PSK-, RALF, and POLARIS all seem to be associated with some aspect of root growth and development. CLV3 is involved in shoot meristem organization, systemin is involved in wound responses, and the Brassica SCR peptide appears to promote outbreeding. The few known plant peptides found to date have more or less been found by accident, but the field of plant peptide signaling is set to expand. The fully sequenced Arabidopsis genome includes a large number of SORFs encoding possible peptide signal molecules, as well as longer ones for predicted receptor-like proteins with no known ligands. Future work will be directed to the identification of ligands for already cloned receptor-like molecules. The availability of yeast-two hybrid and proteomic technologies can be expected to provide answers to how peptides are used by plants to facilitate intracellular signaling during growth and development.

ACKNOWLEDGMENT I would like to thank Professor K. Lindsey for a critical reading of this manuscript.

REFERENCES Abeles, S., Morgan, and Saltveit, M. (1992). ‘‘Ethylene in Plant Biology.’’ Academic Press, San Diego.

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10 Parathyroid Hormone-Related Protein (PTHrP): A Nucleocytoplasmic Shuttling Protein with Distinct Paracrine and Intracrine Roles David A. Jans,* Rachel J. Thomas,{ and Matthew T. Gillespie{ *

Nuclear Signalling Laboratory, Department of Biochemistry and Molecular Biology, Monash University, Monash University 3800, Australia and, { St. Vincent’s Institute of Medical Research, Fitzroy, Victoria 3065, Australia

I. Introduction II. Paracrine and Intracrine Actions of PTHrP A. Domain Structure of PTHrP B. Paracrine Role of PTHrP C. Intracrine Role of PTHrP III. The Nuclear Import Mechanism of PTHrP A. Nuclear Protein Import Pathways B. The Importin b1-Recognized PTHrP Nuclear Targeting Signal IV. Nuclear Transport of Polypeptide Ligands V. Nuclear Export Pathway of PTHrP A. Nuclear Protein Export Pathways B. The Nuclear Import and Export Pathways of PTHrP Represent an Integrated System VI. Functional Role of PTHrP in the Nucleus/Nucleolus VII. Future Prospects References

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Parathyroid hormone-related protein (PTHrP) was first discovered as a circulating factor secreted by certain cancers responsible for the syndrome of humoral hypercalcemia of malignancy. PTHrP possesses distinct paracrine and intracrine signaling roles. The similarity of its N-terminus to that of parathyroid hormone (PTH) enables it to share PTH’s paracrine signaling properties, whereas the rest of the molecule possesses other functions, largely relating to an intracrine signaling role in the nucleus/nucleolus in regulating apoptosis and cell proliferation. Recent advances have shown that intracellularly expressed PTHrP is able to shuttle in cell-cycle- and signal-dependent fashion between nucleus and cytoplasm through the action of the distinct intracellular transport receptors importin 1 and exportin 1 (Crm1) mediating nuclear import and export of PTHrP, respectively. Together, the import and export pathways constitute an integrated system for PTHrP subcellular localization. Intriguingly, PTHrP nuclear/nucleolar import is dependent on microtubule integrity, transport to the nucleus appearing to occur in vectorial fashion along microtubules, mediated in part by the action of importin 1. PTHrP has recently been shown to be able to bind to RNA, meaning that PTHrP’s nucleocytoplasmic shuttling ability may relate to a specific role within the nucleus/nucleolus to regulate RNA synthesis and/or transport. ß 2003, Elsevier Science (USA).

I. INTRODUCTION Parathyroid hormone-related protein (PTHrP)1 was first discovered as the factor responsible for hypercalcemia produced by solid tumors associated with the head and neck, breast, lung, and kidney (Moseley and Gillespie, 1995), where circulating calcium levels are elevated through bone resorption and renal tubular calcium reabsorption. PTHrP gets its name from the fact that it has homology at its amino-terminus to parathyroid hormone (PTH), and thereby is able to bind to and activate PTH receptor 1 (PTHR1) (Juppner et al., 1988, 1991), and elicit similar biological functions, thereby, to PTH. PTHrP sequences C-terminal to its PTH-like region confer other signaling functions such as renal bicarbonate excretion, transplacental calcium transport, osteoclast inhibition, and regulation of apoptosis (see Lam et al., 2000). 1 Abbreviations: PTHrP, parathyroid hormone-related protein; PTH, parathyroid hormone; PTH1R, PTH/PTHrP receptor type 1; CLSM, confocal laser scanning microscopy; FRAP, fluorescence recovery after photobleaching; GFP, green fluorescent protein; NLS, nuclear localization sequence; NPC, nuclear pore complex; NE, nuclear envelope; TF, transcription factor; NES, nuclear export signal; EGF, epidermal growth factor; PDGF, platelet-derived growth factor; FGF, fibroblast growth factor; IL, interleukin.

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Findings from the last few years have demonstrated that PTHrP is a nuclear/ nucleolar protein in a number of tissues and that this localization is cell cycleregulated, mediated by a nuclear targeting signal within the central portion of the molecule, and involves the specific nuclear import receptor importin 1 (see later). Recent experiments using the technique of fluorescence recovery after photobleaching (FRAP) (Lam et al., 2001a, 2002) indicate that the PTHrP nuclear import pathway is novel in having a requirement for microtubule integrity, and that PTHrP also has a specific pathway for export out of the nucleus. The nuclear import and export pathways for PTHrP appear to represent an integrated system enabling PTHrP to shuttle between nucleus and cytoplasm. The present review examines the paracrine and intracrine signaling properties of PTHrP, and the pathways by which it is imported into and exported out of the nucleus. The new observations indicating that it shuttles between nucleolus and cytoplasm, and is able to bind RNA and modulate RNA synthesis, provide new insights into its role in regulating apoptosis as a malignancy factor.

II. PARACRINE AND INTRACRINE ACTIONS OF PTHrP Molecular cloning of PTHrP and determination of its primary structure were the direct result of the search for the factor mimicking the action of PTH in kidney and bone. PTHrP is encoded, we now know, by a gene distinct from that of PTH, with its gene product detected in 95% of renal carcinomas (Iwamura et al., 1999), 60% of primary breast tumors, and more than 70% of bone metastases, but only 17% of nonbone metastases (see Devys et al., 2001). Under physiological conditions, PTHrP is produced locally in many normal tissues where it has autocrine/paracrine actions during differentiation of many cell types, growth regulation, and embryonic development. The essential role of PTHrP in the latter is indicated by the fact that homozygous deletion of PTHrP leads to death shortly after birth, with major skeletal defects (see later) (Amizuka et al., 1994; Karaplis et al., 1994). A. DOMAIN STRUCTURE OF PTHrP

A schematic of the different domains of the PTHrP polypeptide is included in Fig. 1. PTHrP is encoded by a 15-kb gene, comprising nine exons and three promoter regions. Alternative splicing leads to multiple transcripts, with 139, 141, and 173 amino acid forms, whereas posttranslational processing leads to further diversity. The signal sequence (amino acids 36–1) directs PTHrP to the endoplasmic reticulum where the prohormone

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FIGURE 1. Domain structure of PTHrP and relationship to relevant sequences (single letter code) of PTH (above) and SV40 large T-antigen (the CcN motif—below). The prepro region that includes the signal sequence, the PTH-like region that binds to the PTH1 receptor, the osteostatin (osteoblast-inhibiting) sequence (amino acids 107–111), and region (amino acids 34–86) implicated in placental Ca2+ transport and renal HCO3 handling are all highlighted. Amino acids 108–139 have mitogenic activity in osteoblast and vascular smooth muscle cells (de Miguel et al., 2001), amino acids 122–141 confer binding to -arrestin 1B (Conlan et al., 2002), and the Cterminus has been implicated as possessing RNA-binding capability associated with the GxKKxxK93 core motif (Aarts et al., 1999a, 2001), and a nuclear export signal (NES). Processing leads to multiple forms, including the 139, 141, and 173 amino acid forms. The CK2 and the cdc2/ cdk2 phosphorylation sites are boxed in red and gray, respectively, with the phosphorylation site serines and threonine highlighted in yellow and cyan, respectively, and the NLSs boxed in pink. convertase furin (Hendy et al., 1995) or a similar enzyme removes the preprosequence prior to secretion. Elsewhere in the cell, processing events mediated by other enzymes such as prohormone thiol protease, which appears to be coexpressed with PTHrP in lung cancer cells (Hook et al., 2001), prostate-specific antigen, and kex2 lead to a whole range of PTHrP derivative forms, including 1–36, 38–94, 38–95, and 38–101 forms, as well as the C-terminal fragments 107–138 and 109–138 (Wu et al., 1996). A number of the latter possess distinct signaling functions (see Fig. 1). Specifically, the mid-molecule region of PTHrP (amino acids 67–86) is responsible for placental Ca2+ transport (Kovacs et al., 1996) and inhibition of bicarbonate excretion from the kidney (Ellis et al., 1990), amino acids 107–111 (‘‘osteostatin’’) inhibit osteoclast activity and bone resorption (Fenton et al., 1991; Cornish et al., 1997), and the C-terminus has also been reported to be mitogenic for osteoblasts (Cornish et al., 1999), with amino acids 108– 139 in particular shown to be mitogenic for vascular smooth muscle cells (de Miguel et al., 2001). The GxKKxxK93 sequence appears to represent a core motif responsible for RNA binding similar to that of double-stranded

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RNA-binding proteins such as the human immunodeficiency virus HIV-1 Tat (transactivator) and Rev proteins (Aarts et al., 1999a, 2001), whereas a recent report indicates that residues 122–141 of PTHrP can bind -arrestin 1B, involved in desensitization of response subsequent to the activation of GTP-binding protein-coupled receptors (Conlan et al., 2002). Finally and importantly, PTHrP possesses subcellular targeting sequences within the middle portion and C-terminus, including a functional nuclear localization sequence (NLS) between amino acids 71 and 93 (Fig. 1; see Section III) and a nuclear export sequence (NES) (Fig. 1; see Section V ). The importance of nuclear localization through these targeting sequences to PTHrP function is discussed in Sections II.C.3 and VI.

B. PARACRINE ROLE OF PTHrP

1. Paracrine Actions of PTHrP PTHrP has been found in a whole range of normal tissues, including bone, kidney, liver, skeletal muscle, lung, mammary gland, brain, heart, intestine, ovary, adrenal, parathyroid, placenta, prostate, skin, spleen, stomach, and smooth muscle (see Moseley and Gillespie, 1995). Because it is not detected in the circulation of normal adults, PTHrP’s actions can be concluded to be paracrine, autocrine, or intracrine in most normal tissues. Its paracrine actions on bone and kidney are mediated through its Nterminus, of which 8 of the first 13 residues are identical to those of PTH (see upper part of Fig. 1). PTHrP amino acids 1–36, in fact, are sufficient to mediate PTH1R binding of comparable affinity to PTH amino acids 1–34 (see Clemens et al., 2001). PTH1R is a seven-transmembrane helix GTPbinding protein-coupled receptor closely related to those for calcitonin and glucagon (see Kolakowski, 1994), expressed in a variety of tissues, including kidney and bone, that also express PTHrP, often in the same cells or in those immediately adjacent (Lee et al., 1996). In fact, close juxtaposition of cells expressing PTHrP and PTH1R is a common theme in PTHrP paracrine signaling (see also later). PTH1R activation leads to GTP-binding protein activation, and subsequent stimulation of adenylate cyclase/cAMP/cAMPdependent protein kinase and phospholipase C/ITP/protein kinase C pathways through Gs or Gq, respectively. Downstream effectors include the cAMP-response element binding protein CREB and AP-1 transcription factors (TFs). Renal conservation of Ca2+, a significant factor for the development of hypercalcemia in cancer patents, is likely to be due to the paracrine effects of PTHrP, through the reduction of Ca2+ secretion and promotion of cAMP excretion, as well as vasodilatory regulation (see later). Various cells in the bone microenvironment produce PTHrP, with protein detectable in normal human and rat fetal bone and cartilage (see Moseley et al., 1991; Moseley and Gillespie, 1995), in osteoblasts,

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chondrocytes, and osteoclasts (Moseley et al., 1991; Suda et al., 1996; Kartsogiannis et al., 1997, 1998). The juxtaposition of cells expressing PTHrP and PTH1R is proposed to be required for bone formation (Lee et al., 1996), PTHrP’s primary role in contributing to skeletal integrity being to influence chondrocyte differentiation, proliferation, and apoptosis during skeletal development (Karaplis et al., 1994; Weir et al., 1996; Amling et al., 1997), apparently by acting as a brake to chrondrocyte differentiation and apoptosis. This is in part through increasing expression of the antiapoptotic acting regulator Bcl-2 (Lee et al., 1996; Amling et al., 1997), the knockout phenotype of which is similar to that of PTHrP in many respects. As mentioned earlier, homozygous deletion of PTHrP results in major skeletal defects, with death shortly after birth (Amizuka et al., 1994; Karaplis et al., 1994). Partial rescue of the knockout phenotype is able to be achieved by constitutively expressing active PTH1R from the rat 1 collagen type II promoter (Amizuka et al., 1996; Lee et al., 1996). Although the PTH1Rexpressing PTHrP knockout mice appear normal at birth, a number of aspects of development are abnormal such as tooth eruption and bone development after birth, indicating that these latter functions of PTHrP are almost certainly due to PTH1R-independent actions (Schipani et al., 1997; see below). The ‘‘rescued’’ animals die prematurely at 1–2 months, poignantly indicating that PTHrP’s effects exceed those of PTH1R activation alone. PTHrP is expressed in breast epithelial cells during embryogenesis, where it is found on the periphery of the terminal end buds, the site of ductal growth and morphogenesis, as well as at puberty and during pregnancy (Thiede and Rodan, 1988). PTHrP’s role in breast development is linked to its paracrine functions, where, as for bone (see earlier), short-range signaling between epithelial and mesenchymal cells expressing PTHrP and PTH1R, respectively, is critical to generation of a cAMP response (Dunbar and Young, 1998). Both excess PTHrP or the lack thereof has been reported to disrupt and prevent branching morphogenesis of the mammary gland (Wysolmerski et al., 1995, 1998), whereas the knockout mice for both PTHrP and the PTH1R show failed mammary development (Wysolmerski et al., 1998), clearly supporting the key role of PTHrP paracrine effects in the latter. Reports from several laboratories have demonstrated that exogenous PTHrP inhibits the proliferation of vascular smooth muscle cells, through interacting with the PTH1R on vascular smooth muscle cells (Jiang et al., 1995; Maeda et al., 1996; Massfelder et al., 1997; de Miguel et al., 2001). PTHrP is also a paracrine regulator of smooth muscle tone, helping to maintain the uterus in a relaxed state during pregnancy with levels increasing during pregnancy until a rapid reduction in PTHrP production at the onset of labor (Thiede et al., 1991; Paspaliaris et al., 1992). It can initiate the relax action of smooth muscle, is increased in response to stretch of the bladder and uterus (Moseley and Gillespie, 1995), and is a potent

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vasodilator in uterus, bladder, and stomach, through interaction with the PTH1R, which is also expressed in vascular smooth muscle cells (Maeda et al., 1996). There is thus evidence for PTHrP as a cardiac hormone, a vasodilatory agent, and a local paracrine regulator of smooth muscle tone, but PTHrP is not believed to be essential for cardiovascular development based on PTHrP/ mice, which develop normal cardiovascular systems (Karaplis et al., 1994). Studies in cultured keratinocytes also support a role for PTHrP in inhibiting growth and supporting differentiation (Kaiser et al., 1992, 1994). Whereas epidermal proliferation is inhibited by topical administration of PTHrP (1–34), a PTH/PTHrP-specific antagonist has an opposite effect (Holick et al., 1994), consistent with the idea that these effects of PTHrP, at least in part, are mediated through PTH1R. Targeted expression of PTHrP in the skin of transgenic mice using the keratin 14 promoter results in abnormal differentiation of hair follicles (Wysolmerski et al., 1994), further supporting a role for PTHrP in promoting keratinocyte differentiation. 2. Targeting of PTHrP to the Nucleus Subsequent to Receptor-Mediated Endocytosis Studies of PTHrP–PTH1R trafficking are largely restricted to a study using the PTH1R-expressing rat osteogenic sarcoma line UMR106.01 (Lam et al., 1999a), where cells incubated with fluorescently labeled PTHrP were shown to take up PTHrP. Intriguingly, the internalized PTHrP accumulated rapidly in the nucleus and nucleolus, reaching maximum levels about 14- and 4-fold, respectively, those in the cytoplasm (see Fig. 2A) within less than 40 min. That nuclear/nucleolar uptake was dependent on prior internalization through the PTH1R was shown by the fact that preincubation of the cells with unlabeled PTHrP prevented the uptake of labeled PTHrP due to the downregulation of the receptor by internalization (Lam et al., 1999a). This raises the intriguing possibility that extracellular PTHrP arriving at the nucleus/nucleolus may have a direct action in gene regulation and thus be comparable to other nuclear localizing ligands such as growth hormone (Lobie et al., 1994; Waters et al., 1994), insulin, epidermal growth factor, platelet-derived growth factor (PDGF), members of the fibroblast growth factor (FGF) family, IL (interleukin)-1, IL-2, and IL-5 (Jans et al., 1997a), which has been shown to be capable of targeting its receptor subunits to the nucleus in vitro (Jans et al., 1997b), and a range of others (see Jans and Hassan, 1998). Significantly in this context, there have been reports of PTH1R localization in the nucleus in kidney, liver, gut, and ovary (Watson et al., 2000a), as well as in cultured osteoblast-like cells, such as those of the UMR106 line, in the presence of serum, nuclear/nucleolar localization correlating with the time of DNA synthesis and mitosis (Watson et al., 2000b). Whether this localization is linked to PTHrP action is unclear, as is the nature of the mechanism of the reported PTH1R-binding independent,

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FIGURE 2. Nuclear accumulation of fluorescein-labeled PTHrP in vivo subsequent to receptor-mediated endocytosis (A) and in in vitro in mechanically perforated rat hepatoma cells (B). (A) PTH1R-expressing rat osteosarcoma cells were incubated with FITC-labeled PTHrP and imaged using confocal laser scanning microscopy (see inset; arrows indicate nucleoli) over the time indicated, prior to image analysis of confocal files. Results represent the nucleolar to cytoplasmic (Fnu/c) and nuclear to cytoplasmic (Fn/c) ratios (see Lam et al., 1999a). (B) Mechanically perforated rat hepatoma cell nuclei were incubated with FITC-labeled PTHrP in

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but NLS-dependent (see Section III.B) passage of extracellular PTHrP to the nucleus of PTH1R-receptor-expressing and -nonexpressing chondrocyte lines (see Aarts et al., 1999b). How endocytosed ligands may exit endosomes, either without or with their respective receptor subunits, in order to access the cytoplasm and be subsequently transported to the nucleus is unclear. Endosomal escape mechanisms have been delineated for viruses/virus components such as adenovirus, which exits through endosomal membrane disruption (Curiel et al., 1991), influenza virus, which escapes endosomes through membrane fusion (Plank et al., 1994), and rhinovirus, which induces pore formation in endosomal membranes (Prchla et al., 1995), as well as bacterial toxins. Whether PTHrP possesses an endosomal escape mechanism, perhaps through a modular ‘‘endosomolytic’’ sequence as per the influenza virus hemagglutinin N-terminus (Plank et al., 1994) or the human rhinovirus virus protein VP1 20 amino acid sequence (Prchla et al., 1995), is a matter of speculation at this stage. It is clearly of considerable interest, however, to understand how PTHrP and the other ligands mentioned above are able to access the nucleus so rapidly subsequent to receptor-mediated endocytosis. 3. Other PTHrP Receptors? The existence of cell-surface receptors other than PTH1R able to recognize PTHrP or distinct fragments thereof, such as the mid-molecule region of PTHrP responsible for placental calcium transport (Kovacs et al., 1996), has been suggested. PTHrP or PTHrP mid-molecule fragments, but not PTH, can stimulate placental calcium transport in a sheep model (Rodda et al., 1988), for example, C-terminal PTHrP fragments appear to possess activities that differ from those of the PTH-like domain (see Fig. 1), including inhibition of bone resorption due to the osteostatin region (Fenton et al., 1991), but no candidate receptors for these PTHrP fragments have been identified at the molecular level in mammalian systems as yet. There is another receptor for PTH (PTH2R) that is expressed in a few tissues including brain, vascular tissue, and exocrine pancreas, but it is unable to mediate response to PTHrP (Usdin et al., 1995). C. INTRACRINE ROLE OF PTHrP

1. Receptor-Independent Actions of PTHrP As hinted at earlier, PTHrP also acts as a multifunctional hormone in several systems in a paracrine/autocrine manner through PTH1R-independent the presence of the indicated transport factors together with an ATP regenerating system, and imaged using confocal laser scanning microscopy (see inset) over the time indicated, prior to image analysis to determine the fold nuclear uptake (see Lam et al., 1999a, 2001b).

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mechanisms to influence embryonic development, cell growth, and differentiation (Clemens et al., 2001). Studies in PTHrP knockout mice have shown that homozygous fetuses lacking PTHrP have lower blood Ca2+ and a reduced fetal–maternal Ca2+ gradient (Kovacs et al., 1996). The PTHhomologous N-terminus of PTHrP is not responsible for this transport, as demonstrated by using PTH1R knockout mice in which calcium transport is not decreased. The absence of PTH1R in this system indicates that PTHrP has receptor-independent functions. Further, as indicated earlier, overexpression of constitutively active PTH1R does not complement a number of the characteristics of PTHrP knockout mice (see earlier; Schipani et al., 1997). Clearly, PTHrP has important intracrine roles (see earlier). 2. Intracellular Forms of PTHrP Cells transfected to express GFP–PTHrP demonstrate nuclear/nucleolar PTHrP, as do many tumor and normal tissues with respect to endogenous PTHrP as shown using immunohistochemical approaches (see Lam et al., 2000). Clearly, a pathway for PTHrP intracellular trafficking would appear to exist, additional to vectorial targeting to the trans-Golgi network and subsequent secretion from the cell. One mechanism by which PTHrP may escape secretion and instead be targeted to intracellular compartments is through internal ribosome entry sites (IRESs), which represent regions that may bind a ribosome and a secondary translational initiation site typified by a leucine (CUG codon) (Gan et al., 1998). The nuclear localizing polypeptide ligand FGF2, for example, possesses three potential CUGs upstream of its AUG start site (Vagner et al., 1995), translation from any of which results in the inclusion of an NLS prior to the signal sequence, which leads to nuclear localization instead of secretion. PTHrP also possesses three potential IRESs 50 of its ATG start codon that could function not to effect inclusion of an NLS (because the PTHrP NLS is in the middle of the molecule—see Fig. 1), but to prevent secretion, by producing PTHrP that lacks a secretory signal. PTHrP has indeed been shown to be able to be translated from these alternative sites when the initiation ATG at amino acid 36 is mutated (Amizuka et al., 2000; Nguyen et al., 2001). Significantly, these PTHrP isoforms attained nuclear/nucleolar localization and were not secreted (Amizuka et al., 2000; Nguyen et al., 2001). Another mechanism by which mature PTHrP might escape the secretory pathway is reverse translocation from the endoplasmic reticulum (ER) lumen back into the cytoplasm (Nguyen and Karaplis, 1998); proteins following this pathway are believed to be targeted to the ubiquitinproteosome machinery for degradation (Kopito, 1997). Although proteasome-mediated degradation of PTHrP has been reported (Meerovitch et al., 1998), passage through this pathway would require escape from degradation for intact PTHrP to be targeted intracellularly (Nguyen and Karaplis, 1998).

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Intracellular PTHrP forms lacking the prepro secretory signal have been detected using various approaches and demonstrated to influence proliferation, differentiation, and apoptosis, effects that are dependent on its translocation between the cytoplasm and nucleus (Nguyen and Karaplis, 1998; Henderson et al., 1995). Nuclear/nucleolar localization of endogenous PTHrP has been demonstrated in various cell types such as chondrocytes (Henderson et al., 1995), vascular smooth muscle cells (Massfelder et al., 1997), and keratinocytes (Lam et al., 1997). In the case of the former, nuclear/nucleolar targeting of PTHrP appears to be required for enhancing the survival of chondrocytes in culture under conditions that promote apoptosis (Henderson et al., 1995), and also MCF-7 breast cancer cells (Tovar Sepulveda et al., 2002). PTHrP has similarly been shown to protect prostate cancer cells from apoptotic stimuli (Dougherty et al., 1999), presumably in an intracrine role related to nuclear localization. In other cells, such as the IEC-6 intestinal line, overexpressed PTHrP appears to have an overt proliferative action (Ye et al., 2001a,b), although in fact enhancing apoptosis under conditions of stress. Exogenous PTHrP had no such effect, and mutation of the PTHrP NLS (see later) abrogated the effect (Ye et al., 2001a,b), implying that the intracrine effect was directly linked to PTHrP’s ability to localize in the nucleus. In vascular smooth muscle cells, PTHrP nuclear localization increases cell proliferation, in stark contrast to the effect of adding extracellular PTHrP, which inhibits proliferation (see earlier; Massfelder et al., 1997; de Miguel et al., 2001), emphasizing the difference between PTH1R-dependent actions through extracellular PTHrP in a paracrine role, and intracrine PTHrP signaling independent of PTH1R. Importantly and significantly, the latter seems inextricably linked to PTHrP nuclear location, nuclear PTHrP correlating with an increase in mitogenesis in vascular smooth muscle cells (Massfelder et al., 1997), and enhanced IL-8 expression in prostate cancer cells (Gujral et al., 2001). Similarly, deletion of basic residues of the PTHrP NLS (see Section III.B) results in impaired conferred resistance to apoptosis on the part of transfected CFK2 chondrocytes (Henderson et al., 1995). 3. Cell-Cycle-Dependent PTHrP Localization in the Nucleus and Nucleolus PTHrP protein and mRNA is expressed in cell cycle-dependent fashion, as shown by studies in smooth muscle cells (Okano et al., 1995) and keratinocytes (Lam et al., 1997), with highest expression occuring at the G2 and M phases of the cell as cells approach and undergo mitosis. Further, induction of PTHrP mRNA is responsive to mitogenic factors only when cells are at the G1 phase of the cell cycle (Lam et al., 1997). Interestingly, PTHrP is localized to the nucleus and specifically to the nucleolus at this time (Lam et al., 1997), whereas it is cytoplasmic for much of the rest of the cell cycle. This implies strongly that PTHrP shuttles between nucleus/ nucleolus and cytoplasm (see Section V) and that this is regulated.

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Regulation of PTHrP subcellular localization appears to be mediated through phosphorylation by the cell cycle-regulated, cyclin-dependent kinases (cdks) p33cdk2 and p34cdc2, with the key phosphorylation site (T85PGKK) (see Fig. 1) representing a consensus site for cdk phosphorylation (Lam et al., 1999b). Phosphorylation is a well-established mechanism for regulating nucleocytoplasmic transport (Section III.B; see Jans et al., 1998, 2000), with T85 phosphorylation appearing to effect PTHrP retention in the cytoplasm until the appropriate time in the cell cycle. In terms of subnuclear location, PTHrP appears to concentrate in the dense fibrillar component of the nucleolus (Henderson et al., 1995), where it binds RNA (Aarts et al., 1999a). As already mentioned, PTHrP has also been shown to accumulate in the nucleus subsequent to receptor-mediated endocytosis by rat osteogenic sarcoma cells expressing the shared PTH/ PTHrP receptor (PTH1R) (Lam et al., 1999a), as well as using an in vitro nuclear transport assay (see later).

III. THE NUCLEAR IMPORT MECHANISM OF PTHrP A. NUCLEAR PROTEIN IMPORT PATHWAYS

1. The Nuclear Import Machinery The molecular details of conventional nuclear protein import in mammalian cells have been largely established through the application of reconstituted in vitro semiintact cell systems, meaning that anything other than diffusion-driven processes have been largely ignored until recently (see below; Smith and Raikhel, 1999). Conventionally, the first step of nuclear protein import involves the recognition of targeting signals by members of the importin superfamily and translocation of the proteins carrying them to the cytoplasmic side of the nuclear pore complex (NPC), through which all transport into and out of the nucleus occurs (Jans et al., 2000; Jans and Forwood, 2002). For certain classes of protein such as inducible TFs, the importin /1 heterodimer is involved, whereas most other pathways require only importin 1 or an importin homolog; in all cases, the importin homolog docks the importin/transport substrate complex to the NPC, and mediates interaction with Ran. The latter, dependent on the action of key Ran-interacting and regulating proteins including nuclear transport factor 2 (NTF2), Ran-binding protein 1 (RanBP1), Ran GTPase-activating protein 1 (RanGAP1), and the nucleotide exchange factor RCC1 (Gorlich and Kutay, 1999; Ribbeck et al., 1998; Bischoff and Ponsting, 1991; Melchior and Gerace, 1998), mediates release into the nucleus subsequent to translocation of the import substrate through the NPC. Nucleoporins (nups), the FG (single letter amino acid code)-repeat-containing proteins present in multiple

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copies throughout the NPC, serve as binding sites for different transport factors including importin homologs, Ran, and NTF2, thus representing ‘‘docking bays’’ for transport factors and assemblies as they pass through the NPC via a succession of transient binding interactions (Doye and Hurt, 1997; Rexach and Blobel, 1995; Bayliss et al., 2000; Stewart, 2000). Dissociation of the transport complex at the conclusion of translocation through the NPC is effected by binding to importin of Ran in the GTP-bound form to trigger release of the nuclear import substrate and importin (in the case of importin /1-mediated nuclear import) into the nucleoplasm. The scheme of this basic pathway is illustrated in Fig. 3 (see also, by way of contrast, Fig. 6). Nuclear RanGTP is maintained at sufficiently high concentration by the combined action of NTF2, which transports RanGDP from the cytoplasm to the nucleus through a series of nup/FG-repeat-dependent docking events analogous to those of importin (Bayliss et al., 2000), and RCC1, which converts RanGDP into RanGTP (Ribbeck et al., 1998; Bischoff et al., 1991; Melchior and Gerace, 1998; Izaurralde et al., 1997). Cytoplasmic RanGDP is maintained through RanGAP1, which is predominantly cytoplasmic, as opposed to RCC1, which is nuclear, thus ensuring the asymmetric balance of the guanine nucleotide bound by Ran in the two subcellular compartments (Izaurralde et al., 1997). Although nonhydrolyzable GTP analogs inhibit nuclear transport, no direct role in either import or export has been demonstrated for GTP hydrolysis (Izaurralde et al., 1997; Engelmeier et al., 1999). 2. Nuclear Targeting Signals Conventional nuclear localization sequences (NLSs), the short modular peptide sequences sufficient and necessary for nuclear localization of the proteins carrying them, fall into several broad classes. Two of these are highly basic in nature: those resembling the NLS of the Simian virus 40 (SV40) large tumor antigen (T-ag: PKKKRKV132) (Kalderon et al., 1984), which comprises a short stretch of basic amino acids, and bipartite NLSs, which consists of two stretches of basic amino acids separated by a spacer of 10–12 amino acids (Robbins et al., 1991). Other types include NLSs resembling those of the yeast homeodomain-containing protein Mat2 (Hall et al., 1990) where charged/polar residues are interspersed with nonpolar residues, or the protooncogene c-myc (PAAKRVKLD328) where proline and aspartic acid residues either side of the central basic cluster play a role in nuclear targeting (Makkerh et al., 1996). All of these types of NLS are believed to be recognized specifically by the /1-importin heterodimer, as has been shown for the importins from several species (Efthymiadis et al., 1997; Smith et al., 1997; Briggs et al., 1998; Hu and Jans, 1999; Hu¨bner et al., 1999).

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FIGURE 3. Pathway of conventional importin /-heterodimer-mediated nuclear import.

(1) Signal recognition: the importin / heterodimer recognizes the NLS-containing protein through direct interaction of importin with the NLS. (2) NPC docking: the transport complex docks at the NPC through binding of importin to the FG (single letter amino acid code)repeat-containing nucleoporin components. (3) Translocation: the transport complex moves through the NPC through a series of docking events with different FG-repeat-containing nucleoporins; in parallel, RanGDP is transported through the NPC by NTF2 in analogous fashion. (4) Nucleoplasmic release: Ran in its GTP-bound form, generated by the action of the nuclear-localized nucleotide-exchanger RCC1, binds importin , thereby dissociating the transport complex, and liberating the NLS-containing protein into the nucleoplasm. Importin (importin 1)- or importin homolog-mediated nuclear import pathways (see Section III.A.1) are believed to follow an identical scheme of events, with the difference that importin or a comparable adaptor is not involved (see Fig. 6 top).

Understanding of the molecular details of nuclear targeting signals recognized by importin 1 or other importin homologs is limited to a few examples (see Jans and Forwood, 2002; Jans et al., 2000; Lam et al., 1999a,2001b; Forwood et al., 2001a; Brooks et al., 2002). Importin 1recognized NLSs appear to be largely basic in nature (see Jans and Forwood, 2002; Jans et al., 2000) and thus similar to importin /1-recognized sequences, as do those conferring binding to importin 3 (Pse1p/RanBP5/ Kap121p), 4 (Kap123p/Yrb4p), and the importin 1/7 heterodimer. In contrast, NLSs recognized by importin 2 (transportin) include the hydrophobic 38 amino acid ‘‘M9’’ sequence from hnRNP A1 protein (Pollard et al., 1996), with a consensus core sequence of YNNQSSNFGPMK277 (Bogerd et al., 1999).

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B. THE IMPORTIN 1-RECOGNIZED PTHrP NUCLEAR TARGETING SIGNAL

In considering the cell cycle dependence of nuclear localization of PTHrP (Lam et al., 1997, 1999b), the striking resemblance of PTHrP amino acids 61–95 to the ‘‘CcN motif’’ of SV40 large tumor antigen (T-ag) (see Fig. 1), which confers phosphorylation-mediated regulation of the activity of the T-ag NLS, was noted. In particular, PTHrP contains a T-ag-like NLS (KTPGKKKKAK93) flanked by sites for cdks (T85PGK) and protein kinase CK2 (S61DDE) in identical fashion to T-ag; in the case of the latter, phosphorylation at the cdk site by p34cdc2 kinase inhibits nuclear import through a purported cytoplasmic retention mechanism (Jans et al., 1991a), and the CK2 site enhances interaction of the NLS with importin /1 to increase the rate of T-ag nuclear import (Rihs et al., 1991; Jans and Jans, 1994; Hu¨bner et al., 1997; Xiao et al., 1997). The importance of the CcN motif to PTHrP function is implied by the strong sequence conservation across evolution, comparable to that of the PTH-like PTH1R-binding sequences (amino acids 1–34), even a species as divergent from human as fugu fish retaining 61% identity/76% identity of primary sequence, comparable to the degree of homology in the N-terminus (59% identity/ 79% similarity—see Table 1). In terms of functionality of the sequences in PTHrP, Thr85 is known to be phosphorylated in a cell-cycle-specific manner (see earlier) (Lam et al., 1999b), and almost certainly plays a role in modulating nuclear import, probably through a cytoplasmic retention mechanism, but there is no direct evidence that the CK2 site plays a role in PTHrP subcellular localization. Differences between T-ag and PTHrP CcN motifs became evident when an ELISA-based binding assay was used to assess importin binding to PTHrP directly (Lam et al., 1999a). In stark contrast to the T-ag CcN motif, which is recognized with high affinity by importin /1 [an apparent dissociation constant (Kd) of about 4nM], PTHrP is recognized specifically by importin 1 independently of importin , with a Kd of about 2 nM (Lam et al., 1999a, 2001b). Peptides of varying lengths were used to map the importin 1 binding region of PTHrP to amino acids 67–94 (Lam et al., 1999a) (see Fig. 4), and then alanine (Ala) mutagenesis used to demonstrate that amino acids 84–93 (KTPGKKKKGK) are absolutely essential for importin 1 recognition, with residues 72–83 (TNKVETYKEQPL) additionally required for high-affinity binding (Lam et al., 2001a). The wild-type TNKVETYKEQPLKTPGKKKKGK93 sequence is sufficient to target the heterologous protein streptavidin (about 60 kDa) to the nucleus in a semipermeabilized cell system (Lam et al., 2001a), whereas Ala substitution of residues 84–89 oblates this activity. Clearly, residues 84–93 constitute the minimal importin 1-recognized PTHrP NLS. Consistent with this, competition of nuclear accumulation of labeled PTHrP can be

TABLE I. Conservation of the NLS of PTHrP through Evolution, and Comparison with the PTH-like Region Homology to human sequence Residues 1–34 (PTH-like region)

‘‘CcN motif ’’ Species Mouse/rat Bovine Chicken Fugu a

Primary amino acid sequence of NLS regiona

Identity (%)

Similarity (%)

Identity (%)

Similarity (%)

SDDEGRYLTQETNKVETYKEQPLKTPGKKKKGK93 SDDEGkYLTQETNKVETYKEQPLKTPGKKKKsK93 SeDEGkYLTQETNKsqTYKEQPLKvsGKKKKaK93 SlDrEGtnLpQETNKalaYKdQPLKlatKrKKKar105

100 94 79 61

97 85 76

100 100 79 59

85 79

The single letter amino acid code is used; small letters indicate divergence from the human PTHrP primary sequence.

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FIGURE 4. The PTHrP NLS resides within amino acids 67–94 and is recognized by importin (IMP) as shown using an ELISA-based assay. Microtiter plates were coated with the PTHrP derivatives indicated, and incubated with increasing concentrations of the mouse IMP subunits indicated. Data are fitted for the function B(x) = Bmax (1 ekB), where x is the IMP concentration and B is the amount of IMP bound. The apparent Kds, representing the IMP concentration yielding half maximal binding, are 1.2 nM for PTHrP 35–141 IMP without or with IMP (top left), and 4 and 5 nM, respectively, for PTHrP 67–94 and IMP without or with IMP (bottom left). IMP binding to PTHrP67–94 is inhibited in the presence of RanGTPS (Ran complexed with the nonhydrolyzable GTP analog GTPS) but not RanGDP (bottom right). Results are from a single typical experiment performed in triplicate (SD not greater than 9% of the value of the mean) (see Lam et al., 1999a, 2001b).

effected by pretreatment with saturating synthetic full length PTHrP, or amino acids 74–113, but not amino acids 1–34 or 67–86, in intact cells (Aarts et al., 1999b). This argues strongly that the PTHrP NLS region is critical for PTHrP’s nuclear localization, comparable competition experiments in the semipermeabilized cell system yielding very similar results (Lam et al., 1999a).

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The importance of the NLS to PTHrP function is indicated by the fact that deletion of the basic residues of the NLS results in complete cytoplasmic localization of PTHrP and concomitant impaired PTHrPconferred resistance to apoptosis on the part of transfected CFK2 chondrocytes (Henderson et al., 1995). Mutational studies implicated amino acids 87–107 as responsible for the nuclear localization, whereby mutation of amino acids 87–91 had a considerable inhibitory effect, in contrast to mutations of amino acids 96–100 and 102–106 (Henderson et al., 1995) (see also below). 1. Nuclear Import Pathway of PTHrP PTHrP’s nuclear import properties were demonstrated using fluorescently labeled PTHrP (1–108) in an in vitro nuclear import assay utilizing purified, recombinant nuclear transport components. PTHrP turned out to be one of the first molecules shown to be imported into the nucleus by importin 1 working in concert with RanGTP (see Fig. 2B) and not requiring importin for nuclear accumulation (Lam et al., 1999a, 2001b).The significance is not clear, but as for nuclear import conferred by the high-affinity importin /1-recognized T-ag NLS (Hu and Jans, 1999), PTHrP nuclear import appeared to be inhibited in vitro by NTF2 (Lam et al., 1999a). Binding of importin 1 to PTHrP is reduced in the presence of the GTP-bound but not GDP-bound form of Ran (see Fig. 4, bottom right panel; Lam et al., 2001b), consistent with the idea that RanGTP binding to importin is involved in the release of PTHrP into the nucleus following translocation across the nuclear envelope (NE). Intriguingly, PTHrP appears to be able to bind to components within the nucleus/nucleolus, giving it the ability to accumulate even in the absence of an intact NE in vitro (Lam et al., 1999a). This property is shared with a number of other proteins, in the case of some of which, such as the HIV transactivator Tat (Efthymiadis et al., 1998a), the interferon-induced TF IFi16 (Briggs et al., 2000), and the polypeptide ligand angiogenin (Lixin et al., 2000), the NLS appears to be responsible. Work in the past few years suggests that a range of different proteins can be recognized by and have their nuclear import mediated by importin 1. These include the T cell protein tyrosine phosphatase (Tiganis et al., 1997), a number of constitutively nuclear TFs such as the yeast TF GAL4 (Chan et al., 1998), HMG-box-containing SOX factors, and bZIP TFs such as Jun, Fos, and CREB (Forwood et al., 2001a,b; Preiss et al., 2001), the telomerebinding factor TRF1 (Forwood and Jans, 2002), several related retroviral gene products (Henderson and Percipalle, 1997; Palmeri and Malim, 1999; Truant and Cullen, 1999), and cyclin B1 (Moore et al., 1999). PTHrP is thus not unique in being recognized by importin 1 and transported to the nucleus thereby in the absence of importin ; in terms of polypeptide ligand/ polypeptide ligand receptors (see Section IV), FGF receptor type 1 (FGFR1)

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also appears to be transported to the nucleus by importin 1 (Reilly and Maher, 2001). X-Ray crystallography indicates that importin 1 comprises 19 HEAT repeats (Cingolani et al., 1999), -helix-based structures mediating protein– protein interactions, that resemble the Armadillo (ARM) repeats comprising the majority of the structure of importin (Kobe, 1999). Repeats 7–11 and 12–19 of importin 1 mediate binding to the IBB (importin -binding) N-terminal domain of importin , whereas residues 1–364 and 160–340 (particularly HEAT repeats 5 and 6) mediate RanGTP binding and interaction with the hydrophobic repeats of nucleoporins to effect NPC docking (Bayliss et al., 2000), respectively. In vitro pull-down experiments indicated that residues 380–643 of importin 1 are required for interaction with PTHrP (Lam et al., 1999a, 2001b). PTHrP recognition by importin 1 thus appeared to occur through a region of importin 1 distinct to that recognizing other specific nuclear import substrates such as cyclin B1 (residues 1–462), TRF1 (residues 1–380) (Forwood and Jans, 2002), the BIB (‘‘-importin-binding’’) domain of ribosomal protein rp23 (residues 280– 450) (Jaekel and Go¨rlich, 1998), and CREB and SRY, both of which require the C-terminus of importin 1 for high-affinity binding (Forwood et al., 2001a,b). That the binding site on importin 1 for PTHrP is not competed by CREB has been formally demonstrated using fluorescence polarization measurements (Forwood et al., 2001a). 2. Role of Microtubule Integrity in PTHrP Nuclear Import As indicated in Section III.A.I, the molecular details of the conventional nuclear protein import process in mammalian cells have largely ignored the possibility of vectorial transport, due to the fact that reconstituted in vitro semiintact cell systems have been employed. Several developments have made possible the application of the FRAP technique, used for a number of years to investigate plasma membrane component lateral mobility (Jans et al., 1989, 1990a,b, 1991b; see Jans, 1997), to be applied to examine nucleocytoplasmic flux in living cells. These include improvements in commercial confocal microscopic systems, confocality being necessary to achieve the spatial resolution required for FRAP experiments, and the development of green fluorescent protein (GFP) as a means to label an intracellularly expressed protein fluorescently in vivo. FRAP was performed on rat osteosarcoma cells transfected with a GFP-PTHrP(1–141)-expressing construct where either the nucleus or cytoplasm was bleached, and the return of fluorescence to the bleached compartment, occurring through transport from the other compartment, was monitored over time subsequent to the bleach (see schematic of the FRAP experimental protocol in Fig. 5A– C). It became clear both that PTHrP could shuttle rapidly between nucleus and cytoplasm and that microtubule integrity was required for PTHrP nuclear import (Lam et al., 2001a, 2002). It was found that treatment with

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FIGURE 5. Scheme of fluorescence recovery after photobleaching (FRAP) approach to examine PTHrP nucleocytoplasmic flux. (A) Cells are first transfected with the PTHrP–GFP fusion protein expressing plasmid construct to yield cells with predominantly nuclear/nucleolarlocalized, fluorescent PTHrP containing both NLS and NES targeting signals. (B) Schematic representation of FRAP protocol for nuclear bleaching of transfected cell from (A) to assess nuclear import kinetics (see D, left panel). (C) Schematic representation of FRAP protocol for cytoplasmic bleaching of transfected cell from (A) to assess nuclear export kinetics (see D, right panel). (D) Example of kinetic measurements (half times indicated) of nuclear import (left) and export (right) kinetics from FRAP experiments (see B and C, respectively). The results are for the return of nuclear and nucleolar fluorescence through nuclear import (Fn/c and Fnu/c parameters, respectively) (left panel), and fall of nuclear and nucleolar fluorescence (right panel), through nuclear export (right panel) (see Lam et al., 2001a, 2002).

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FIGURE 5. (Continued)

the microtubule-disrupting agent nocodazole for as little as 30 min reduced PTHrP nuclear/nucleolar import 2-fold, consistent with in vivo colocalization experiments, the filamentous pattern of PTHrP staining in the cytoplasm of endogenously expressing or transfected cells, and visualization of apparent vectorial movement of PTHrP to the nucleus in FRAP experiments (see http://jcsmr.anu.edu.au /dbmb/jans/lam/frap/frap.htm/ Fig2.avi) (Lam et al., 2002). Combining FRAP and microinjection of rhodamine-labeled tubulin enabled visualization of colocalization, as well as of vectorial movement of PTHrP-GFP along rhodamine-labeled microtubules toward the nucleus (Lam et al., 2002) (see http://jcsmr.anu. edu.au/ dbmb/jans/lam/frap/Fig4ii.avi).

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Importin b 1

Import b1mediated nuclear import

b1

cr

Mi

Ran GTP

b1

b1

s

ule

b otu

RCC1 Ran GTP

Ran GDP

PTHrP

PTHrP

RanGTP hydrolysismediated dissociation

CRM1

Ran GDP

RanGTPmediated release

RanGAP1

Ran GDP

PTHrP

NTF2

PTHrP binding to nuclear/nucleolar components (mmobile)

RanBP1 Ran GTP

Ran GTP

CRM1

CRM1

PTHrP

PTHrP CRM1-mediated nuclear export

Ran GTP

CRM1

CRM1

FIGURE 6. Schematic representation of the integrated system for nuclear import and export of PTHrP, including the involvement of microtubules (see Lam et al., 2001a, 2002). PTHrP is imported into the nucleus via an importin 1- and Ran-mediated pathway (Lam et al., 1999a, 2001b, 2002) and exported from the nucleus by CRM1 in LMB-sensitive fashion (Lam et al., 2001a). The roles of RanGAP1 and RCC1 are by analogy with other systems (see Fig. 3), as is the proposed requirement for RanGTP for CRM1-mediated PTHrP nuclear export (see Gorlich and Kutay, 1999). The nuclear import of PTHrP is dependent on microtubule integrity, with transport postulated to be a vectorial process along the microtubule filaments (see Section III.B.2), involving binding mediated by importin (Lam et al., 2002). Nuclear/nucleolar passage of PTHrP involves binding to nuclear/nucleolar components (Lam et al., 1999a, 2001a), dissociation from which is likely to be very slow. Perturbation of PTHrP nuclear export by treatment with the Crm1 inhibitor LMB both blocks nuclear export completely and reduces the rate of nuclear import, demonstrating the integrated nature of the PTHrP nuclear import and export systems (see Section V.B).

In vitro experiments enabled the observations regarding the microtubule dependence of nuclear transport to be linked to the fact that importin 1 was the nuclear import receptor for PTHrP (see Section III.B.2). Microtubules were reconstituted in vitro using taxol, and PTHrP tested for its ability to bind to them in the absence or presence of importins. Significantly, PTHrP not only bound to the microtubules, but binding was enhanced 2-fold by the addition of importin 1 but not importin . The clear implication was that importin 1 facilitates PTHrP microtubule association, presumably as part of the vectorial process of transport along microtubules toward the nucleus (see Fig. 6); clearly, when microtubule integrity is disturbed by nocodazole treatment, nuclear import efficiency is severely impaired.

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3. Role for the Cytoskeleton in Nucleocytoplasmic Trafficking: The ‘‘Fast Track’’ Interestingly, in the context of the link between microtubules and PTHrP nuclear import, the conventional NLS-binding importin subunit has been shown to associate with microtubules/microfilaments in mammalian cells (Barsony et al., 1997; Percipalle et al., 1999), as well as in tobacco protoplasts (Smith and Raikhel, 1998), as well as with microtubules in vitro in an NLS-dependent manner, whereas yeast importin has also been reported to bind directly to the actin-related protein Act2p (Yan et al., 1997). Microtubule/microfilament association of Armadillo repeat-containing proteins (see Section III.B.1) such as catenins and Vac8p (involved in vacuolar protein targeting) has also been reported (Barth et al., 1997). In the case of several viruses, nuclear import appears to be negatively regulated by association with the actin cytoskeleton (Digard et al., 1999), or to involve movement along microtubule filaments (Sodeik et al., 1997) in a fashion analogous to the observations for PTHrP (Lam et al., 2002). The finding that PTHrP binding to microtubules is enhanced by the presence of its NLS receptor importin 1 (Lam et al., 2002) is comparable to the NLS-dependent association of plant importin with microtubules. The close relationship of nuclear import pathways with cytoskeletal components (see Smith and Raikhel, 1999) thus may be a general phenomenon of mechanistic importance. The differences in the requirements of individual substrate/ importin complexes in terms of binding to microtubules indicates the presence of highly selective mechanisms in the transport toward the NPC for different NLS-bearing substrates. A direct link between the cytoskeleton and nuclear import is indicated in the case of the tumor-suppressor protein p53, whose nuclear translocation in response to DNA damage enables transcriptional activation of genes involved in apoptosis and growth arrest. Giannakakou et al. (2000, 2002) showed that in a fashion analogous to PTHrP, p53 associates with tubulin both in vitro (mediated by its N-terminal 25 amino acids) and in vivo, where it localizes to cellular microtubules. Treatment with vincristine or paclitaxel, which depolymerize and stabilize microtubules, respectively, reduces p53 nuclear accumulation in response to DNA damage. Adenovirus type 2 (Ad2) (Giannakakou et al., 2002) and herpes simplex virus (HSV) (Doehner et al., 2002) appear to show the same properties. Overexpression of dynamitin (dissociates the dynactin complex that assists dynein function and association with microtubules) or microinjection of antidynein antibody prevents DNA damage-induced p53 nuclear translocation (Giannakakou et al., 2000); dynamitin overexpression also inhibits passage toward the nucleus of HSV capsid (Doehner et al., 2002). Interestingly, Giannakakou et al. (2002) showed that disruption of microtubule dynamics, but not structure, through treatment with low concentrations of micotubule-targeting agents, actually

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enhanced microtubule-dependent trafficking of p53 and Ad2 toward the nucleus. Functional microtubules and the dynein motor protein thus appear to participate in nuclear transport of p53, HSV capsid, and probably also Ad2. The nature of the importins mediating p53/Ad2 nuclear import, and whether they may play a role in microtubule binding, has not been determined, meaning that the nuclear import pathway of p53/Ad2 cannot, as yet, be assumed to be the same as that of PTHrP. It is intriguing in the case of HSV, however, that importin does appear to play a key role in capsid docking to the NPC, which has a role in triggering release of the viral DNA from the capsid (Ojala et al., 2000), implying that all the proteins mentioned may share a common pathway for microtubule-dependent transport toward the nucleus, with importin playing an integral role.

IV. NUCLEAR TRANSPORT OF POLYPEPTIDE LIGANDS The conventional idea of ligand/receptor signaling is that ligand activates receptor at the plasma membrane, subsequent to which the signal represented by receptor activation is relayed to the nucleus to effect a change in gene expression through kinase phosphorylation cascades/ intracellular second messenger molecules etc. Experimental evidence for transport to and direct signaling in the nucleus of polypeptide ligands, however, is mounting, indicating the likelihood that a number of polypeptide ligands, of which PTHrP can be seen as an example, may have nuclear signaling roles, in addition to that at the membrane in terms of receptor activation (see Jans, 1994; Jans and Hassan, 1998; Pederson, 1998). Although the specific function of ligands in the nucleus is unclear apart from a few exceptions, it seems that one role of nuclear-localizing ligands may be to cotransport their receptors to the nucleus; clearly, in this scenario, the receptor molecule has phosphorylating or other activities that may modulate transcriptional activity either directly or indirectly. A number of polypeptide ligands such as those of the FGF and PDGF classes have well-defined NLSs (see Jans and Hassan, 1998). Where characterized, NLS-dependent polypeptide ligand or ligand/receptor nuclear translocation appears to be through a variety of different pathways, both importin dependent and independent, with a role for importin 1 in several cases. Table II summarizes current knowledge regarding some of these ligands, indicating that there would seem to be a preference for either importin 1 as the nuclear import receptor molecule or for importinindependent pathways. There is obviously insufficient detailed information available to enable general conclusions to be drawn, and the significance is not completely clear, but the implication is that such signaling molecules/ complexes utilize several specific nuclear import pathways.

TABLE II. Signal-Dependent Nuclear Import Pathways of Polypeptide Ligands NLSa

Nuclear import pathway

KVETYKEQPLKTPGKKKKGK93

Importin 1/Ran-dependent; dependent on microtubule integrity (Lam et al., 1999a, 2001b, 2002) Importin 1/Ran-dependent (?) (Reilly and Maher, 2001) Importin 1/Ran-dependent (?) (Schedlich et al., 2000)

Protein Importin-dependent PTHrP FGFR1/FGF (FGF-2-dependent)b IGFBP-3/-5c Importin-independent Angiogenin IL-5 a

GRGRGRPRERVGGRGRGR KKGFYKKKQCRPSKGRKR232 RRRGL35 KKYIDRQKEKCGEERRR109

Single letter amino acid code; residues in italics constitute the minimal NLS FGF-2, N-terminal extension c IGFBP, insulin-like growth factor binding protein. b

Importin/Ran-independent diffusion into and binding in the nucleus/nucleolus (Lixin et al., 2001) Importin/Ran-independent (??); cotransport of receptor (unpublished observations)

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V. NUCLEAR EXPORT PATHWAY OF PTHrP A. NUCLEAR PROTEIN EXPORT PATHWAYS

The nuclear protein export correlates of NLSs, which mediate nuclear protein import, are nuclear export signals (NESs), leucine-rich sequences that are sufficient and necessary for nuclear export of the respective proteins carrying them. NES-mediated nuclear protein export is a rapid, active process (Bogerd et al., 1995; Fischer et al., 1995), mediated by the importin homolog exportin 1 (Crm1p/Xpo1p/Kap124p), and requiring nuclear RanGTP for the initial step of substrate binding (Fornerod et al., 1997; Stade et al., 1997). Passage through the NPC in the export direction is believed to be through a series of transient docking interactions with nucleoporins in a fashion comparable to NPC translocation in the import direction, with cytoplasmic release through dissociation of the exportin– RanGTP–NES-containing substrate being effected by RanGAP1/RanBP1 enhanced Ran-GTPase activity. The nuclear export pathway can thus be considered the exact reverse of the importin -homolog-mediated nuclear import pathway depicted in Fig. 3 (see Fig. 6, bottom). Specific nuclear export receptors analogous to Crm1 include CAS (Cse1p/Kap 109p) (Kutay et al., 1997) and Msn5p (Kap142p), which specifically transport importin (to generate cytoplasmic importin for further cycles of transport), and the yeast TFs Pho4 and Mig1 (Komeili and O’Shea, 1999; De Vit and Johnston, 1999), from the nucleus to the cytoplasm, respectively. That PTHrP is exported from the nucleus through a Crm1-mediated nuclear export pathway was demonstrated in in vivo experiments using the Crm1-specific inhibitor leptomycin B (LMB), which inhibits binding of Crm1 to NES sequences (Kudo et al., 1997). This leads to higher nuclear levels of protein due to the block in export. LMB treatment elevated PTHrP nuclear and nucleolar accumulation about 2-fold (Lam et al., 2001a), FRAP experiments showing that LMB specifically blocks nuclear export, reducing the rate over 10-fold. The Crm1-dependent nuclear export pathway of PTHrP is illustrated in the lower part of Fig. 6. There is no direct evidence as yet, but the PTHrP NES recognized by Crm1 is predicted to be the LSDTSTTSLELDS138 sequence.

B. THE NUCLEAR IMPORT AND EXPORT PATHWAYS OF PTHrP REPRESENT AN INTEGRATED SYSTEM

LMB is a very specific inhibitor of Crm1-NES binding, not affecting any other known importins/exportins or Ran; for example, CAS-mediated recycling of importin to the cytoplasm is not inhibited by LMB. LMB binding to Schizosaccharomyces pombe Crm1 is through very specific binding dependent on Cys529(Kudo et al., 1999), Cys528 being the equivalent

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residue in humans. This is the basis for the fact that Saccharomyces cerevisiae is not a target for LMB-mediated inhibition of nuclear protein export since the budding yeast homolog of Crm1, Xpo1p, lacks Cys at the analogous position, emphasizing LMB’s exquisite specificity. It was thus somewhat of a surprise when it was found that LMB pretreatment reduced the rate of PTHrP nuclear import over 2-fold, in addition to abolishing nuclear export (Lam et al., 2001a). Because LMB is such a specific inhibitor, these results clearly imply the interdependence of the import and export systems. The basis of this does not seem to relate to exhaustion/saturation of transport factors such as RanGTP, which of course is required in the nucleus for both nuclear import (to effect cargo release) and nuclear export (to enable initial complexation of cargo with Crm1) (see Fig. 6), as blocking export would be expected to lead to nuclear accumulation rather than depletion of RanGTP, resulting in more, rather than less, efficient nuclear import through enhancement of the nucleoplasmic release step at the end of the NPC translocation process. The basis of inhibition of PTHrP nuclear import indirectly through blockage of nuclear export may relate, alternatively, to the fact that PTHrP binds to sites within the nucleus and especially nucleolus (see Fig. 6). This is implicated in in vitro experiments where fluorescently labeled PTHrP can accumulate in the nucleus and nucleolus in the absence of an intact NE (Lam et al., 1999a), as well as in FRAP experiments where exchange between bleached and nonbleached areas of the nucleolus is very slow, indicative of ‘‘immobile’’ sites, that is, sites of binding of PTHrP to immobile components in the nucleolus; interestingly, transport of PTHrP into the nucleus appears to be preferentially to these sites (see Lam et al., 2001a). Because these binding sites are likely to be finite in terms of their number, nuclear import may be prevented/reduced if these presumably largely immobile sites are the vectorial end-point of the import process and presumably the starting point of the nuclear export process. Thus, the passage of PTHrP through ‘‘immobile’’/slowly exchanging binding sites within the nucleus/nucleolus could lead to a retardation of the nuclear import process in the absence of nuclear export due to the presence of LMB. Our work on other nuclear import/export substrates indicates that the cytoplasmic concentration of substrate is the critical factor determining the nuclear import rate (Seydel and Jans, 1996; Efthymiadis et al., 1998b; see Lam et al., 2001a), as shown for several different nuclear import substrates including mouse upstream binding factor (mUBF), a nucleolar localizing TF involved in rRNA synthesis, and T-ag. In the scenario where LMB blocks PTHrP nuclear export, it is clear that essentially the only nuclear import substrate available to importin 1 is that produced from de novo synthesis in the cytoplasm. Such a low cytoplasmic concentration is likely to be limiting, possibly close to or even lower than the estimated Kd (about 2 nM) for PTHrP/importin 1 association (see Fig. 4). Thus, even though small

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amounts of PTHrP are present in the cytoplasm, binding with importin 1 is likely to be inefficient, resulting in a reduced nuclear import efficiency. The FRAP studies thus clearly demonstrate for the first time the integrated nature of nucleocytoplasmic transport and subcellular localization through opposing import/export signals/receptors (see also Jans et al., 2000). Many other proteins have been described that contain distinct NLS/ NES sequences recognized by different nuclear transport receptors, including the yeast TF Pho4 (where the import and export receptors are importin 3 and Msn5p, respectively; Komeili and O’Shea, 1999; Kaffman et al., 1998), yeast mitogen-activated protein kinase HOG1 (which requires Nmd5p and Xpo1p for transport into and out of the nucleus, respectively; Ferrigno et al., 1998), and cyclin B1 (Moore et al., 1999; Toyoshima et al., 1998) and HIV Rev (Bogerd et al., 1995; Truant and Cullen, 1999), which, like PTHrP, appear to be transported into and out of the nucleus through independent signals recognized by importin 1 and Crm1, respectively. It can be speculated that all of these proteins, and others showing similar properties (see Jans et al., 2000), are likely to exhibit interdependence of nuclear import and export pathways along lines similar to PTHrP.

VI. FUNCTIONAL ROLE OF PTHrP IN THE NUCLEUS/NUCLEOLUS It is clear that PTHrP is able to localize in the nucleus through very specific mechanisms, as part of its intracrine and probably also paracrine signaling pathways. In the former case, the nonsecreted form localizes in the nucleus through the action of importin 1 and requires microtubule integrity, whereas the latter, although almost certainly involving an endosomal escape mechanism subsequent to receptor-mediated endocytosis to access the cytoplasm (see Section II.B.2), most likely follows a nuclear import pathway identical to that of the intracrine form. As for many nuclear localizing polypeptide ligands (see Sections IV), however, the precise signaling role in the nucleus/nucleolus of PTHrP remains largely a matter for speculation. In this context, it is worth considering the fact that PTHrP transport to the nucleus is preferentially to the nucleolus, as indicated in the FRAP experiments (Lam et al., 2001a), and that PTHrP appears to bind to components in the nucleolus, as well as the nucleus (Lam et al., 1999b, 2001a). Apart from being the site of ribosome assembly and rRNA transcription, the nucleolus has more recently been recognized as an important reservoir for regulatory proteins and TFs (Stegh et al., 1998; Aarts et al., 1999a; Visintin et al., 1999; Lohrum et al., 2000). Hence, it is possible that PTHrP may be involved in the transcriptional regulation of ribosomal genes through interactions with nucleolar TFs, or may regulate the activity of other factors by nucleolar sequestration in a manner similar

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to the ARF tumor suppressor and the interacting MDM2 oncoprotein (Lohrum et al., 2000). The fact that PTHrP is a nuclear-cytoplasmic shuttling protein with specific targeting signals recognized by distinct cellular receptors (importin 1 and Crm1) (see earlier), coupled with the fact that it can bind homopolymeric and total cellular RNA (Aarts et al., 1999a), implies a possible role of PTHrP, perhaps in conjunction with other proteins, as a nuclear export factor for RNA. Localization and binding to slowly exchanging sites in the nucleolus could thus be integral to PTHrP’s role in binding to RNAs, prior to transporting them to the cytoplasm. Alternatively, these nucleolar component-binding capabilities and the RNAbinding capability of PTHrP may be important in regulating ribosome assembly (Aarts et al., 2001), whereby PTHrP has been reported to downregulate rRNA synthesis, most likely at an early step in ribosome biogenesis or assembly, resulting in decreased translation. The reduced translation results in a rapid exit from the cell cycle by immature chondrogenic cells, thus preventing apoptosis (Aarts et al., 2001). These observations may relate integrally to those indicating cell-cycle-dependent expression and nuclear localization of PTHrP (see Section II.C.3). Thus, PTHrP may act as a ‘‘competence factor’’ to promote the rapid exit of cells from the cell cycle and prevent their apoptotic cell death in an unfavorable environment, through inhibition of ribosome biogenesis and downregulation of translation (see Aarts et al., 2001). In preliminary experiments, we (Lam et al., unpublished) have shown that treatment of GFP–PTHrP-expressing cells with the RNA-polymerase inhibitor actinomycin D inhibits association of PTHrP with the nucleolus, not inconsistent with the notion of a role of PTHrP either in RNA transport or ribosome assembly. RNA binding on the part of PTHrP may also contribute to its microtubule association, as a large part of cytoplasmic mRNA appears associated with cytoskeletal elements (Taneja et al., 1992; Bassel and Singer, 1997), and nuclear proteins such as mRNA-binding protein mrnp41 (Kraemer and Blobel, 1997) and Cbf5p, a yeast nucleolar protein that regulates rRNA synthesis (Cadwell et al., 1997), have also been demonstrated to associate with the cytoskeleton. Finally, PTHrP appears to have a direct, potentially tumorigenic, intracrine action in inducing IL-8 mRNA and protein production in prostate cancer cells, thereby regulating growth by angiogenic activity and growth-promoting effects (Gujral et al., 2001). IL-8 is not produced in normal prostate cells or in benign prostate hyperplasia, but significant levels are detected in prostate cancer cells (Ferrer et al., 1998). Interestingly, exogenously added PTHrP (paracrine pathway) does not lead to an elevation in IL-8 mRNA expression, in contrast to endogenous production of PTHrP fragments (Gujral et al., 2001). The PTHrP NLS did not appear to be required for this activity, although the fact that small, freely diffusible

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PTHrP fragments were used makes the physiological relevance unclear. Despite this, it seems not inconceivable that PTHrP may be involved in binding DNA or RNA sequences that modulate IL-8 transcription/ translation within the nucleus/nucleolus, with nuclear targeting through the action of the NLS probably integral to this function.

VII. FUTURE PROSPECTS It seems clear that PTHrP is a signaling molecule possessing distinct paracrine and intracrine actions, with the latter integrally linked to its nuclear/nucleolar-localizing ability, related in turn to the action of specific signal-dependent intracellular transport systems and regulation thereof according to the cell cycle. The function of PTHrP in the nucleus remains in question, but the recent observations regarding a role in modulating RNA synthesis linked to ribosome synthesis in the nucleolus are consistent with many other observations, and provide a working hypothesis for further work. Understanding of the specific role of PTHrP in the nucleus/nucleolus may be obtained by employing either the yeast two-hybrid system along the lines of recent work (Conlan et al., 2002), and/or proteomic approaches to search for binding partners of PTHrP within the nucleus/nucleolus. This should definitively establish that PTHrP is involved in signaling complexes in the nucleus/nucleolus and their likely function. Using similar approaches, it should also be possible to identify and isolate the factors regulating PTHrP cytoplasmic retention (Lam et al., 1999b; see Section II.C.2). In terms of PTHrP’s RNA-binding role, the hunt for specific RNA species and sequences using affinity chromatography approaches would appear to be a promising strategy. PTHrP’s role as a growth/malignancy factor (see Moseley and Gillespie, 1995) clearly relates integrally to its ability to localize in the nucleus/ nucleolus and thereby delay apoptosis (Henderson et al., 1995; Tovar Sepulveda et al., 2002) and promote proliferation (Massfelder et al., 1997) in different cell types. Interestingly and importantly, MCF-7 and MDA-MB231 breast cancer cell lines made to overexpress PTHrP possess increased tumorigenic capacity and metastatic potential (Guise et al., 2002; Thomas et al., 1999). That PTHrP nuclear localization is so integral to its function implies that strategies to block PTHrP nuclear localization in cancers overexpressing it could at least potentially lead to increased apoptosis and hence reduced tumorigenic potential. The results, in part summarized here, regarding cytoskeletal and cell cycle control over PTHrP nuclear-cytoplasmic flux thus may have important future applications in anticancer therapies.

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ACKNOWLEDGMENTS Our work has been supported at different times by the National Health and Medical Research Council (NHMRC), the Anti-Cancer Council of Victoria (ACCV) grant, the Australian National University Institute of Advanced Studies bilateral collaborative scheme grant, the Rebecca Cooper Foundation, and the Chugai Pharmaceutical Company, Japan.

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11 Nerve Growth FactorDependent Regulation of NADE-Induced Apoptosis Jun Mukai, Petro Suvant, and Taka-Aki Sato Division of Molecular Oncology, Department of Otolaryngology/Head & Neck Surgery and Pathology, College of Physicians & Surgeons, Columbia University, New York, New York 10032

I. II. III. IV. V. VI. VII. VIII.

Background Structural Features of NADE NADE Isoforms Genomic Structure of NADE Genes Expression of NADE Association of NADE with p75NTR 14-3-3 Protein Interacts with NADE NADE Is Involved in NGF-Induced Apoptosis via p75NTR A. Structure–Function Analysis of NADE B. NADE Is Involved in NGF-Induced Cell Death in Oligodendrocytes C. NADE Is Involved in Neuronal Apoptosis in Ischemia IX. Future Directions References

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The p75 neurotrophin receptor (p75NTR) is a member of the tumor necrosis factor receptor (TNFR) superfamily, and can mediate both cell survival and cell death in response to nerve growth factor (NGF). Based on the structural and functional differences between p75NTR and the related receptors Fas or TNFR, it has been suggested that these receptors have distinct signaling functions. NADE (p75NTR-associated cell death executor) is a p75NTR-associated protein that mediates apoptosis in response to NGF by interacting with the cell death domain of p75NTR. NADE has at least four isoforms, designated as NADE2, NADE3, NADE4/Bex1, and NADE5/Bex2. NADE plays a role in NGF-induced apoptosis in oligodendrocytes and in zinc-induced neuronal death. In this review, we focus on the proapoptotic actions of NADE that regulate p75NTR signaling in response to NGF. ß 2003, Elsevier Science (USA).

I. BACKGROUND Cell growth, cell differentiation, and genetically controlled programmed cell death are required for normal development of the nervous system and for neural plasticity in adult vertebrates. Neurotrophins, including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4/5, play critical roles in cell differentiation, cell survival, and cell death in the mammalian nervous system. Neurotrophins bind to two different types of transmembrane receptors, the Trk (tropomyosin-related kinase) family of receptors and the p75 neurotrophin receptor (p75NTR). Various Trk family members exhibit specific preferences for certain neurotrophins, such that NGF preferentially binds to TrkA, BDNF and NT-4/5 preferentially bind to TrkB, and NT-3 preferentially binds to TrkC (Barker, 1998). Trks contain a tyrosine kinase motif within their intracellular domains. Receptor dimerization and autophosphorylation are required for Trks to generate survival signals. Binding of NGF to TrkA activates the intrinsic tyrosine kinase activity of the receptor and subsequently induces phosphorylation of multiple substrates. This eventually leads to the activation of mitogen-activated protein (MAP) kinase, phosphatidylinositol-3 kinase, and other intracellular signaling cascades (Peng et al., 1995; Yao and Cooper 1995). The TrkA receptor promotes cell survival and initiates differentiation signals in various neuronal cells (Kaplan et al., 1994; Lachyanker et al., 1997). In contrast to the Trks, p75NTR is a member of the tumor necrosis factor (TNF) receptor superfamily, and can promote both cell survival and cell death (see reviews, Roux and Barker, 2002; Hempstead, 2002; Chao and Bothwell, 2002; Bibel et al., 2000). In most cell types, all of the neurotrophins bind to p75NTR with equal affinity (at the nanomolar level)

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(Dechant and Barde, 1997). Recently, it has been shown that the proform of NGF binds to p75NTR with a higher affinity than does mature NGF, and that this results in increased death signaling (Lee et al., 2001). In contrast, pro-NGF binds to TrkA much less strongly than does mature NGF. Several lines of evidence from various systems, including cultured cells as well as knockout and transgenic mice, suggest that p75NTR has a proapoptotic role (Rabizadeh, 1983; Bredesen and Rabizadeh, 1997; Majdan et al., 1997; Miller and Kaplan, 1998; Frade et al., 1996; von Schack et al., 2001; Yoon et al., 1998). Furthermore, NGF induces apoptosis in terminally differentiated primary oligodendrocytes expressing p75NTR, but not TrkA. Other neurotrophins, such as BDNF and NT-3, do not induce apoptosis. NGF increases intracellular ceramide levels and activates c-Jun aminoterminal kinase (JNK), both of which are thought to participate in apoptotic signaling (Casaccia-Bonnefil et al., 1996). Programmed cell death requires activation of various caspases. NGF-induced oligodendrocyte death activates caspase-1, -2, and -3, but not caspase-8 (Gu et al., 1998). The only known consensus motif within the intracellular domain of p75NTR is a death domain (DD), similar to that found in the p55 TNFR (TNFR1) (Tartaglia et al., 1993) and in the related Fas receptor. DDs have been classified as type 1 (e.g., those found in TNFR1, Fas, FADD, RIP, and TRADD) and type 2 (e.g., those found in p75NTR, DAP kinase, p100 and p105 NF-B, and myD88) (Feinstein et al., 1995). Type 1 DDs are involved in protein–protein interactions that occur when receptors homodimerize or heterodimerize. When the Fas receptor binds its ligand, this recognition event is translated into intracellular signals that eventually lead to activation of caspase-8 through the formation of the death-inducing signaling complex (DISC). DISC includes self-aggregations of the Fas DD, oligomerization of DDs between Fas and FADD, and oligomerization of death effector domains (DEDs) between FADD and procaspase-8 (Fig. 1). On the other hand, the type 2 DD of p75NTR does not self-aggregate, as does the DD of Fas (Liepinsh et al., 1997). As p75NTR does not appear to interact with the DDs of FADD or TRADD (Coulson et al., 1999; Wang et al., 2001), DD-dependent signaling mechanisms utilized by p75NTR may differ from those used by Fas and its family receptors. A number of novel p75NTR-binding proteins have recently been identified. NRIF, NRAGE, and NADE induce apoptosis, whereas TRAF6, TRAF4, RhoA, FAP-1, and RIP2 appear to promote cell survival (Casademunt et al., 1999; Salehi et al., 2000; Mukai et al., 2000; Khursigera et al., 1999; Ye et al., 1999; Yamashita et al., 1999; Irie et al., 1999; Khursigera et al., 2001 ) (Fig. 1). Of these, NRIF, NRAGE, and NADE have been reported to function in the p75NTR-mediated apoptotic pathway. NRIF is known to block cell division. The retinas of nrif / mice show reduced cell death, and this reduction is quantitatively similar to that seen in p75/ and ngf / mice (Casademunt et al., 1999). NRAGE

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FIGURE 1. NGF-induced apoptotic signaling pathways through p75NTR. The p75NTR intracellular domain contains a juxtamembrane domain (JM), type II death domain (DD), and a C-terminal PDZ binding domain. SC-1, NRAGE, TRAF4, and TRAF6 interact with the JM. NADE, RIP2, and RhoA bind to the DD. NRIF binds to both the JM and the DD. FAP-1 binds to the C-terminal amino acid SPV in p75NTR. TRAF6-p62-atypical protein kinase C (aPKC) complex links p75NTR to TrkA (Wooten et al., 2001). NRAGE also associates with TrkA. SC-1, NRAGE, and NRIF can disrupt the cell cycle, and NRAGE and NRIF can also promote apoptosis. The NADE–14-3-3 complex promotes apoptosis with caspase activation. TRAF6, RIP2, and FAP-1 promote survival signals through NF-B activation. RhoA promotes neurite outgrowth. Apoptotic signaling at Fas receptor has been well characterized. The death-inducing signaling complex (DISC) contains the adaptor protein Fas-associated death domain protein (FADD) and procaspase-8, which can initiate the process of apoptosis. The binding of the Fas ligand (FasL) to the Fas receptor induces trimerization of Fas. Fas DD recruits a DDcontaining FADD as homophilic association. Procaspase-8 contains two DED at the Nterminus, and recruits to the DED of FADD. Immediately after recruitment, procaspase-8 is proteolytically processed to its active forms that consist of large and small catalytic subunits.

binds to the p75NTR both in vitro and in vivo, and blocks the physical association of p75NTR with TrkA. Overexpression of NRAGE facilitates cell cycle arrest and allows NGF-dependent apoptosis to occur within sympathetic neuron precursor cells (Salehi et al., 2000). NADE was isolated as a p75NTR-binding protein (Mukai et al., 2000). Coexpression of NADE and p75NTR induces cell death in 293T cells. NGF induces a dose-dependent association of NADE with the DD of p75NTR, and p75NTR/NADE-induced cell death required NGF, but not BDNF, NT-3, or NT-4/5 (Mukai et al., 2000). p75NTR and NADE gene expression were concomitantly induced in degenerating rat hippocampal CA1 neurons

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after forebrain ischemia. NADE contributes to p75NTR-induced cortical neuronal death (Park et al., 2000). Furthermore, 14-3-3 proteins associate with NADE and play a role in p75NTR/NADE-mediated apoptosis in HEK293 cells, PC12nnr5 cells, and oligodendrocytes (Kimura et al., 2001). The NGF-dependent actions of p75NTR/NADE-mediated cell death is discussed in this chapter.

II. STRUCTURE FEATURES OF NADE NADE was originally isolated from a mouse embryo cDNA library by a yeast two-hybrid system, in which the DD of p75NTR (amino acids 338– 396) was used as a target (Mukai et al., 2000). NADE is identical to Bex3 (brain expressed X-chromosomal gene 3) (Brown et al., 1999). The NADE protein is comprised of 124 amino acids, with a predicted molecular mass of 14,532 Da; it appears to be a hydrophilic and acidic protein (estimated pI ¼ 5.97). A previously uncharacterized human gene, HGR74 (Rapp et al., 1990), shows significant identity to NADE (92.8% identity, except for the histidine- and asparagine-rich stretch of amino acids at residues 36–48). Thus, HGR74 appears to be the human homolog of NADE. NADE proteins in mouse, rat, and human have three consensus motifs, the leucine-rich nuclear export signal (NES), ubiquitination sequences, and a prenylation site. The leucine-rich NES motif, conserved in NADE and other proteins, is shown in Fig 2A. NES is a recently identified transport signal, which is both necessary and sufficient to mediate nuclear export of large carrier proteins (Gorlich and Mattaj, 1996). Many proteins have been reported to be tightly regulated by their NES, including cZyxin (Sadler et al., 1992), MAPKK (Kosako et al., 1993), PKI- (van Patten et al., 1992), RevHIV-1 (Park et al., 1998), and Gle1 (Murphy and Wente, 1996). The destruction box is an important ubiquitination signal that was discovered in mitotic cyclins and in certain other cell-cycle regulators (Paqano, 1997). Substrates of the ubiquitin/proteasome pathway include a number of cell regulatory molecules, such as cyclins, the Myc oncogene protein, and p53. Regulated degradation of these molecules has been linked to the control of cell proliferation, cell cycle progression, and apoptosis. NADE has two ubiquitination motif boxes, following the general structure R1(A/T)2(A)3 L4(G)5X6(I/V)8(N)9 (Fig. 2B). The CAAX box, localized at the C-terminal end, is a prenylation signal sequence that acts as a target for the posttranslational modification of the protein by the addition of either a geranyl–geranyl or a farnesyl group to the cysteine residue, similar to what occurs in the Ras family of proteins (Brown and Kay, 1999). In vitro prenylation analysis of bacterially expressed GST-NADE (Khosravi-Far and Der, 1995) was not detected using [3H]mevalonolactone, trans, trans-[3H]farnesyl pyrophosphate, and (all-trans)-[3H]geranyl–geranyl

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FIGURE 2. Consensus motif in NADE. (A) Comparison of the NES in various proteins. The consensus sequence for NES is outlined. Genbank Accession numbers are cZyxin, X69190; MAPKK, D13700; PKI-, L02615; TFIIIA, M85211; RevHIV-1, AF075719; RanBP1, L25255; FMRP, L29074; Gle 1, U68475; mouse NADE, AF187066; rat NADE, AF187065; and human NADE/HGR74, AF187064, M38188. (B) Consensus sequence of the ubiquitination signal (Box1 and Box2) in NADE proteins.

pyrophosphate, suggesting that the CLMP sequence in the C-terminal end of NADE may not be a functional prenylation signal sequence (J. Mukai and T-A. Sato, unpublished data).

III. NADE ISOFORMS In the database search for human NADE/HGR74/Bex3 homologs, four previously uncharacterized human genes were identified in the Xq21–22 region (Fig. 3). The full cDNAs for each new gene were compiled from 10 to 30 separate EST sequences. Human Bex1 (Accession number Z70233; 128 amino acids) and human Bex2 (Accession number AF220189; 125 amino acids), tentatively designated as NADE4 and NADE5, respectively, are closely similar to each other at the amino acid level (114/125 identical amino acids and a three-amino acid deletion in NADE5/Bex2). Human NADE4/ Bex1 and human NADE5/Bex2 are also highly similar to mouse Bex1 and mouse Bex2. The high degree of identity between NADE4/Bex1 and NADE5/ Bex2 at the amino acid and nucleotide levels suggests that an ancestral gene in both the mouse and human lineages was independently duplicated to give rise to the NADE4/Bex1 and NADE5/Bex2 genes. The third new human gene, designated NADE2 (112 amino acids), is more closely related to human NADE/Bex3/HGR74 (64/105) than to either NADE4/Bex1 or NADE5/Bex2

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FIGURE 3. Deduced amino acid sequence of NADE family proteins. The amino acid sequences of human NADE/Bex3, NADE2, NADE3, NADE4/Bex1, and NADE5/Bex2 are compared. The consensus line shows the residues conserved between at least two of the sequences. The identical amino acids are highlighted in dark gray and similar amino acids in light gray.

(49/107 and 49/106, respectively). The fourth new gene, NADE3 (121 amino acids), differs from the other NADE peptides in its carboxyl terminal. However, NADE2 still shares a high level of overall pairwise identity with the other four NADE family proteins (40–50%). The most variable region of the NADE peptides corresponded to the amino acids between 70 and 79 in NADE5. All five human NADE peptides shared 21/102 identical amino acids and 23 additional residues are conserved in four of the five sequences. The lysine residue at position 80 is conserved in each of the NADE proteins and has been implicated in NADE ubiquitylation.

IV. GENOMIC STRUCTURE OF THE NADE GENES The cosmids containing genomic sequences for human NADE2, NADE3, NADE4/Bex1, and NADE5/Bex2 were found in BLAST searches of the nonredundant database of GenBank (Accession numbers Z70719.1, AL035494.8, Z70233.1, and AF220189, respectively). All NADE genes

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P 22.3 22.2 22.1 21.3 21.2 21.1 11.4 11.3 11.23 11.22 11.21 11.1 11.1 11.2 12

MRX26 (Robledo et al., 1996)

13

21.1 21.2

XMLR syndrome

21.3

Art's syndrome

22.1 22.2 22.3

(Chudley et al., 1999) (Kremer et al., 1996)

MRX47 (des Portes et al., 1997) MRX35 (Gu et al., 1996) XMLR (Raynaud et al., 1998)

23 24

NADE2 NADE5 NADE4 NADE3 NADE

25 26 27 28

q

FIGURE 4. Genomic localizations of NADE genes. X-chromosomal loci of mental retardation cases and NADE genes. exhibit three exons and two introns, with the last exon covering the entire open reading frame of the peptide. All cosmids containing genes within the NADE family have been mapped to the Xq22.1–23 region by hybrid analysis. The human chromosomal region containing the NADE/Bex genes has revealed at least 16 diseases or syndromes that map to this region, and many of these diseases involve symptoms of hearing loss and and mental retardation (Brown and Kay, 1999). The chromosomal loci of NADE genes and the relevant mental retardation cases are shown in Fig. 4.

V. EXPRESSION OF NADE NADE mRNA is expressed at the highest levels in brain, heart, and lung, with lower levels present in stomach, small intestine, and muscle, and no detectable expression in liver. NADE protein can be detected by Western blotting in PCNA and PC12 cells only after treatment with proteasomal

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inhibitors, such as ALLN (Rock et al., 1994), PSI (Traenckner et al., 1994), or MG132 (Rock et al., 1994). The effect is specific to proteasomal inhibitors, as treatment of cells with the calpain inhibitor ALLM has no effect on accumulation of NADE protein (Rock et al., 1994). These results demonstrate that NADE protein is expressed at a low steady-state level in these cells and is regulated by the proteasome. In 293T, PC12, and PC12nnr5 cells expressing wild-type NADE, two bands (22 and 44 kDa) are detected by Western blotting when sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) is performed under reducing conditions (Mukai et al., 2000). These bands correspond to the monomeric and dimeric forms of NADE, respectively. Dimerization of NADE was confirmed by a two-hybrid system screen and by immunoprecipitation (Mukai et al., 2002). NADE is induced by various types of stimuli in neural cells. NGF induces apoptosis in differentiated rat primary oligodendrocytes expressing high levels of p75NTR (Gu et al., 1999). After NGF treatment, the expression of NADE mRNA and protein is increased in oligodendrocytes (Mukai et al., 2000). When cultured cortical neurons are exposed to 300 M ZnCl2, both NADE mRNA and NADE protein are induced beginning 4 h after a 15-min exposure to zinc (Park et al., 2000). NADE protein expression decreases 20 h after the zinc exposure. NADE is expressed in the CA1 pyramidal cell layer of the hippocampus 48 h after transient ischemia (Park et al., 2000). NADE also appears to play a role in the development of the rodent cochlea. During cochclear development, massive rearrangement of afferent and efferent neural fibers occurs within the first 2 weeks after birth (Knipper et al., 1996). p75NTR-mediated apoptosis of epithelial cells within the inner sulcus of the rodent cochlea has been suggested to be the molecular mechanism by which the inner spiral sulcus is formed (Knipper et al., 1999). NADE protein appears in pillar cells of the basal turn to the mid turn at postnatal Day 2 (PN2) (Sano et al., 2001). Cells in the phalanx of Deiters also stain weakly for NADE at PN2. After PN4, NADE immunoreactivity is detected in all turns of the cochlea and spreads medially in inner pillar cells, which indicates the formation of the tunnel of Corti. Expression of p75NTR is observed in pillar cells at PN0 and PN2. At development stages after PN2, p75NTR immunoreactivity begins to diminish, starting from the basal turn and progressing toward the apical turn. Because of this reciprocal expression pattern of NADE and p75NTR, it remains unclear whether NADE promotes apoptosis during cochlear development.

VI. ASSOCIATION OF NADE WITH P75NTR NADE was isolated as a p75NTR-binding protein with a yeast twohybrid system, using the DD of p75NTR (amino acids 338–396) as a target

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FIGURE 5. Structure–function relationship of various regions of NADE. The structure of full-length NADE is shown, with amino acid numbers indicated below. The nuclear export signal (NES) is localized at amino acids 90–100 and the ubiquitin sequence (US ) is localized at 91–112. The proapoptotic region is located within residues 41–71. The regulatory region resides within residues 72–112 and contains the p75NTR-binding region (81–112) and the self-association region (81–112). The 14-3-3 binding region is located within residues 81–124. (Mukai et al., 2000). NADE protein (translated in vitro from rabbit reticulocytes) can also interact with a p75NTR DD–GST fusion protein, suggesting that NADE directly binds to the p75NTR DD. The p75NTRbinding domain in NADE is located at residues 81–106, an area that includes the NES (Fig. 5). A NES mutant lacking nuclear export cannot bind to p75NTR, suggesting that the NES motif is critical for p75NTR/ NADE binding (Mukai et al., 2002). Although the NES mutant of NADE fails to self-associate, it still remains unclear whether the binding of NADE and p75NTR requires dimerization of NADE. The association of NADE with p75NTR is ligand dependent, as increasing doses of NGF enhance the interaction (Mukai et al., 2000). In 293T cells cotransfected with NADE and p75NTR, NGF exposure rapidly recruits NADE to the DD of p75NTR, peaking after 5–10 min and decreasing to 25% of its peak value after 30 min (J. Mukai and T-A. Sato, unpublished data). This finding is similar to those of other p75NTRbinding proteins. The binding of TRAF6 or RIP2 with p75NTR also peaks within 5–10 min after NGF treatment (Khursigara et al., 1999, 2001). The NGF-induced native protein complex of NADE and p75NTR can be immunoprecipitated from the lysates of PC12 cells treated with a proteasomal inhibitor (Mukai et al., 2000). Although the native protein complex can also be immunoprecipitated from zinc-treated cortical neurons, an antibody that blocks p75NTR function (REX) (Weskamp and Reichardt, 1991) inhibits the coimmunoprecipitation of NADE and p75NTR, suggesting that the formation of the native p75NTR/NADE complex is regulated by NGF during zinc-induced neuronal cell death (Park et al., 2000).

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VII. 14-3-3 PROTEIN INTERACTS WITH NADE To further investigate NADE function, we identified NADE-binding partners with the yeast two-hybrid system. A yeast expression library derived from embryonic cDNA was screened, using a full length of NADE as a bait. Some positive clones contained an overlapping region of 14-3-3e, extending from Thr-91 to Leu-209 (Kimura et al., 2001). This overlapping region contains a motif recognized by other 14-3-3-binding proteins. A previous study showed that 14-3-3e binds to phosphorylated serine residues within the consensus amino acid sequence RSXpSXP (where X is any amino acid and pS is a phosphorylated serine residue). NADE does not contain this motif, although it is present in many proteins that bind to 14-3-3e. ADP-ribosyltransferase and exoenzyme S (ExoS) from Pseudomonas aureginosa also bind to 14-3-3 in the absence of this consensus motif (Masters et al., 1999). However, NADE and ExoS share no similarity in amino acid sequence. The 14-3-3 binding region in NADE lies between amino acids residues 81 and 124, which includes the regulatory region. The native NADE–14-3-3e complex can be immunoprecipitated from lysates of PC12nnr5 cells, which express p75NTR and lack TrkA. This complex interacts with p75NTR in an NGF-dependent manner (Kimura et al., 2001).

VIII. NADE IS INVOLVED IN NGF-INDUCED APOPTOSIS VIA P75NTR When 293T and COS7 cells are cotransfected with NADE and p75NTR and then treated with NGF, TUNEL-positively stained nuclei, morphological changes, and DNA fragmentation typical of apoptosis are observed (Mukai et al., 2000, 2002; J. Mukai and T-A. Sato, unpublished data). In contrast, cells transfected with either NADE or p75NTR alone are similar to cells transfected with the control vector, in that they do not display these morphological characteristics of apoptosis in response to NGF. Cell death is not observed in response to treatment with BDNF, NT-3, or NT-4/5 in cells cotransfected with NADE and p75NTR. PC12 cells (which express both p75NTR and TrkA) and PC12nnr5 cells (which express p75NTR but lack TrkA) also exhibit NGF-induced cell death when transfected with NADE (Mukai et al., 2000). Programmed cell death requires activation of caspases. In cell death signaling mediated via Fas and TNFR1, procaspase-8, which contains a DED, is proteolytically activated by oligomerization of DDs and DEDs following its recruitment to the DISC, whereas procaspase-8 is not activated in p75NTR-mediated apoptosis (Gu et al., 1999; Wang et al., 2001).

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Caspase-1, -2, and -3 are activated during NGF/p75NTR-induced oligodendrocyte death (Gu et al., 1999). NADE also activates caspase-2 and caspase-3 during NGF-induced cell death (Mukai et al., 2000). These procaspases are processed to active forms in response to NGF in 293T cells cotransfected with NADE and p75NTR. Under these conditions, procaspase-8 is not processed to the active form (J. Mukai and T-A. Sato, unpublished data). Activation of caspase-3 is also detected in both parental PC12 cells and in PC12 nnr5 cells transfected with NADE (Mukai et al., 2000).

A. STRUCTURE–FUNCTION ANALYSIS OF NADE

Mutational analysis was used to determine the structural elements of NADE that are required for apoptosis (Mukai et al., 2002). The Nterminal deletion mutant, NADE (81–124), fails to induce apoptosis, whereas a larger deletion of 40 additional N-terminal amino acids [NADE (41–124)] does not affect apoptosis. The C-terminal deletion mutant, NADE (1–71), retains the ability to induce apoptosis. Furthermore, expression of a minimal region of NADE containing only residues 41–71 [NADE (41–71)] is sufficient to induce apoptosis. In cells expressing wildtype NADE, the presence of p75NTR is required to induce apoptosis. In contrast, expression of NADE (1–71) or NADE (41–71), which lacks the p75NTR-binding domain (residues 81–106), retains its apoptotic function even in the absence of p75NTR expression. NADE (41–71) is critical for proapoptotic function, and was therefore designated as the proapoptotic region. NADE (41–124) and NADE (1–112), which can each associate with p75NTR, induce apoptosis in an NGF-dependent manner. Thus, the C-terminal amino acid residues (72–112), designated as the regulatory region, are essential for NGF-dependent regulation of apoptosis (Fig. 5). A series of point mutations were made within the NES motif of NADE, as this subregion (90–100) is located within the regulatory region (Fig. 5). Analogous mutations in other NES-containing proteins have been reported to prevent nuclear export (Bogerd et al., 1996; Kim et al., 1996). A triple NADE NES mutant (L94A, L97A, and L99A) exhibited decreased interaction with p75NTR, decreased self-association, failed to undergo apoptosis, and lacked nuclear export. These findings suggest that the NES motif of NADE plays a critical role in each of these functions. The regulatory region of NADE associates with 14-3-3 proteins, but the triple NADE mutant (L94A, L97A, L99A) fails to associate with 14-3-3e (Kimura et al., 2001). These findings suggest that the NES motif of NADE might be an important platform for switching the conformation of NADE to regulate proapoptotic activity.

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B. NADE IS INVOLVED IN NGF-INDUCED CELL DEATH IN OLIGODENDROCYTES

The expression of NADE mRNA and protein is increased in oligodendrocytes in response to NGF treatment (Mukai et al., 2000). To evaluate NADE-mediated events that induce apoptosis in oligodendrocytes, NADE (81–124) was transfected into mature oligodendrocytes. The protein encoded by this construct associates with p75NTR in response to NGF, but lacks a proapoptotic region. Expression of NADE (81–124) inhibits NGF-induced apoptosis in oligodendrocytes. Thus, NADE (81–124) appears to have a dominant negative effect on NGF-induced apoptosis in oligodendrocytes. 14-3-3e, a NADE-binding protein, is involved in NGF-induced cell death (Kimura et al., 2001). 14-3-3 (1–207), a mutant in which amino acid residues 208–255 have been deleted, maintains association with NADE, and exhibits diminished NGF-induced cell death when expressed both in 293T and in PC12nnr5 cells. In addition, this mutant form inhibits activation of caspase-3 during NGF-induced apoptosis in these cells. In primary oligodendrocytes, expression of exogenous 14-3-3 (1–207), inhibits NGF-induced apoptosis (Kimura et al., 2001). These results suggest that NADE and its binding protein, 14-3-3, are involved in NGF-induced cell death in oligodendrocytes.

C. NADE IS INVOLVED IN NEURONAL APOPTOSIS IN ISCHEMIA

When cultured cortical neurons are exposed to ZnCl2 (300 M for 15 min), neurons degenerate over the following day (Kim et al., 1999). NADE and p75NTR are induced in cortical cultures beginning 4 h after the 15-min exposure to zinc. NGF is also induced after exposure to zinc. A p75NTR function-blocking antibody (REX) (Weskamp and Reichardt, 1991) attenuates zinc neurotoxicity, suggesting that p75NTR may contribute to zinc neurotoxicity. REX inhibits the association of NADE with p75NTR in zinc-exposed neurons (Park et al., 2000). NADE antisense oligonucleotides partially attenuate zinc-induced neuronal death, suggesting that NADE induction contributes at least partially to zinc-induced neuronal death (Park et al., 2000). After transient cerebral ischemia, zinc toxicity contributes to selective neuronal death (Koh et al., 1996). Ischemia in the rat hippocampus induces p75NTR and NADE immunoreactivity in the CA1 pyramidal cell layer beginning 48 h after the ischemia. TUNEL staining of adjacent sections shows that there is a close correlation between p75NTR/NADE coimmunoreactivity and irreversible neuronal damage in CA1 pyramidal neurons. Injection of CaEDTA into the lateral ventricle completely blocks the development of both p75NTR and NADE immunoreactivity throughout

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the hippocampus, and prevents the induction of neuronal death triggered by zinc-induced ischemia (Park et al., 2000).

IX. FUTURE DIRECTIONS We have shown that NADE is a unique member of the family of p75NTR-binding proteins. NADE interacts with the DD of p75NTR and is involved in NGF-induced apoptosis in several cell lines and under various conditions. Recent reports suggest that the signaling pathways responsible for NGF/p75NTR-dependent apoptosis involve the DD, but not the juxtamembrane (JM) region (Wang et al., 2001; Perini et al., 2002; Mukai et al., 2002), whereas other data indicates that the JM, but not the DD, is required for neuronal death (Coulson et al., 2000). Many studies have investigated the role of caspases in NGF-induced cell death. Caspase-1, -2, and -3, but not caspase-8 were found to be activated during NGF-induced apoptosis in oligodendrocytes (Gu et al., 1999). However, activation of caspase-3 and -8 is required in NGF-induced neuroblastoma cell death (Perini et al., 2002). Recent reports show that caspase-9 is activated during NGF/p75NTR-mediated apoptosis (Wang et al., 2001; Troy et al., 2002). p75NTR-mediated neuronal cell death is associated with mitochondrial loss of cytochrome c, and requires Apaf-1 and caspases-9, -6, and -3, but not caspase-8. In particular, caspase-6 plays a central role in mediating apoptosis (Troy et al., 2002). Thus, it still remains unclear whether NGF/ p75NTR-mediated apoptosis requires the DD or JM domains, and how apoptotic signals trigger activation of the caspase cascade after p75NTR activation. Further study of NADE and other adaptor proteins will facilitate our understanding of the signaling cascade that mediates NGF/ p75NTR-dependent apoptosis. Recently, several groups have shown that -amyloid (A) and prion peptides bind to p75NTR and promote neural cell death (Rabizadeh et al., 1994; Yaar et al., 1997, 2002; Perini et al., 2002; Della-Bianca et al., 2001). A binds to trimers and monomers of p75NTR, which leads to activation of JNK and promotes apoptosis (Yaar et al., 2002). Furthermore, A-induced apoptosis requires the DD, but not the JM domain (Perini et al., 2002). These studies suggest that A-induced apoptosis may share a similar signaling pathway with NGF-induced apoptosis via p75NTR. Further study of NADE may provide new insights for delineating the molecular mechanisms underlying these neurodegenerative diseases.

ACKNOWLEDGMENT We thank Ms. Ami Kuno for excellent secretarial assistance and artwork.

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Salehi, A. H., Roux, P. P., Kubu, C. J., Zeindler, C., Bhakar, A., Tannis, L. L., Verdi, J. M., and Barker, P. A. (2000). NRAGE, a novel MAGE protein, interacts with the p75 neurotrophin receptor and facilitates nerve growth factor-dependent apoptosis. Neuron 27, 279–288. Sano, H., Mukai, J., Monoo, K., Close, L. G., and Sato, T. A. (2001). Expression of p75NTR and its associated protein NADE in the rat cochlea. Laryngoscope 111, 535–538. Tartaglia, L. A., Ayres, T. M., Wong, G. H., and Goedel, D. V. (1993). A novel domain within the 55 kd TNF receptor signals cell death. Cell 74, 845–853. Traenckner, E. B., Wilk, S., and Baeuerle, P. A. (1994). A proteasome inhibitor prevents activation of NF-kappa B and stabilizes a newly phosphorylated form of I kappa B-alpha that is still bound to NF-kappa B. EMBO J. 13, 5433–5441. Troy, C. M., Friedman, J. E., and Friedman, W. (2002). Mechanisms of p75-mediated death of hippocampal neurons: Role of caspases. J. Biol. Chem. 277, 34295–34302. van Patten, S. M., Howard, P., Walsh, D. A., and Maurer, R. A. (1992). The alpha- and betaisoforms of the inhibitor protein of the 3, 5-cyclic adenosine monophosphate-dependent protein kinase: Characteristics and tissue- and developmental-specific expression. Mol. Endocrinol. 6, 2114–2122. von Schack, D., Casademunt, E., Schweigreiter, R., Meyer, M., Bibel, M., and Dechant, G. (2001). Complete ablation of the neurotrophin receptor p75NTR causes defects both in the nervous and the vascular system. Nat. Neurosci. 4, 977–978. Wang, X., Bauer, J. H., Li, Y., Shao, Z., Zetoune, F. S., Cattaneo, E., and Vincenz, C. (2001). Characterization of a p75NTR apoptotic signaling pathway using a novel cellular model. J. Biol. Chem. 276, 33812–33820. Wen, W., Meinkoth, J. L., Tsien, R. Y., and Taylor, S. S. (1995). Identification of a signal for rapid export of proteins from the nucleus. Cell 82, 463–473. Weskamp, G., and Reichardt, L. F. (1991). Evidence that biological activity of NGF is mediated through a novel subclass of high affinity receptors. Neuron 6, 649–663. Wooten, M. W., Seibenhener, M. L., Mamidipudi, V., Diaz-Meco, M. T., Barker, P. A., and Moscat, J. (2001). The atypical protein kinase C-interacting protein p62 is a scaffold for NFkB activation by nerve growth factor. J. Biol. Chem. 276, 7709–7712. Yaar, M., Zhai, S., Pilch, P. F., Doyle, S. M., Eisenhauer, P. B., Fine, R. E., and Gilchrest, B. A. (1997). Binding of -amyloid to the p75 neurotrophin receptor induces apoptosis. A possible mechanism for Alzheimer’s disease. J. Clin. Invest. 100, 2333–2340. Yaar, M., Zhai, S., Fine, R. E., Eisenhauer, P. B., Arble, B. L., Stewart, K. B., and Gilchrest, B. A. (2002). Amyloid binds trimers as well as monomers of 75-kDa neurotrophin receptor and activates receptor signaling. J. Biol. Chem. 277, 7720–7725. Yamashita, T., Tucker, K. L., and Barde, Y. A. (1999). Neurotrophin binding to the p75 receptor modulates Rho activity and axonal outgrowth. Neuron 24, 585–593. Yao, R, and Cooper, G. M. (1995). Requirement for phosphatidylinositol-3 kinase in the prevention of apoptosis by nerve growth factor. Science 267, 2003–2006. Ye, X., Mehlen, P., Rabizadeh, S., VanArsdale, T., Zhang, H., Shin, H., Wang, J. J. L., Leo, E., Zapata, J., Hauser, C. A., Reed, J. C., and Bredesen, D. E. (1999). TRAF family proteins interact with the common neurotrophin receptor and modulate apoptosis induction. J. Biol. Chem. 274, 30202–30208. Yoon, S. O., Casaccia-Bonnefil, P., Carter, B., and Chao, M. V. (1998). Competitive signaling between TrkA and p75 nerve growth factor receptors determines cell survival. J. Neurosci. 18, 3273–3281.

12 Membrane Transport of Folates

Larry H. Matherly The Experimental and Clinical Therapeutics Program, Barbara Ann Karmanos Cancer Institute, and the Department of Pharmacology, Wayne State University School of Medicine, Detroit, Michigan 48201

I. David Goldman The Departments of Medicine and Molecular Pharmacology, Albert Einstein Cancer Center, Albert Einstein College of Medicine, Bronx, New York 10461

I. Introduction II. Reduced Folate Carrier (RFC), a Member of the SLC19 Family of Transporters A. Cloning of the cDNAs for RFC B. Patterns of RFC Expression and Occurrence of Heterogeneous RFC Transcripts C. Structure and Function of RFC D. The Role of RFC as a Factor in the Clinical Utility of Methotrexate III. Transport of Folates by SLC21 Organic Anion Carriers IV. Folate Transporters That Operate Optimally at Low pH: The Mechanism of Folate Transport in Intestinal Cells V. The Family of Folate Receptors (FRs) A. Structures and Specificities of FRs , , AND Vitamins and Hormones Volume 66

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VI. VII. VIII. IX. X.

B. Transcriptional and Posttranscriptional Regulation of the FRs C. Physiological and Pharmacological Roles and Mechanisms of FR-Mediated Folate Uptake Multidrug Resistance-Associated Proteins (MRPs) and Their Impact on the Transport of Folates Transport of Folates by Other ABC Exporters Factors That Influence Concentrative Folate Transport in Cells The Localization of Folate Transporters in Cells and Their Roles in Vectorial Transport in Epithelia The Role of Folate Transporters in Mouse Development References

The chapter reviews the current understanding of the transport mechanisms for folates in mammalian cells—their molecular identities and organization, tissue expression, regulation, structures, and their kinetic and thermodynamic properties. This encompasses a variety of diverse processes. Best characterized is the reduced folate carrier, a member of the SLC19 family of facilitative carriers. But other facilitative organic anion carriers (SLC21), largely expressed in epithelial tissues, transport folates as well. In addition to these bi-directional carrier systems are the membrane-localized folate receptors alpha and beta, that mediate folate uptake unidirectionally into cells via an endocytotic process. There are also several transporters, typified by the family of multidrug resistance-associated proteins, that unidirectionally export folates from cells. There are transport activities for folates, that function optimally at low pH, related in part to the reduced folate carrier, with at least one activity that is independent of this carrier. The reduced folate carrier-associated low-pH route mediates intestinal folate transport. This review considers how these different transport processes contribute to the generation of transmembrane folate gradients and to vectorial flows of folates across epithelia. The role of folate transporters in mouse development, as assessed by homologous deletion of folate receptors and the reduced folate carrier, is described. Much of the focus is on antifolate cancer chemotherapeutic agents that are often model surrogates for natural folates in transport studies. In particular, antifolate transport mediated by the reduced folate carrier is a major determinant of the activity of, and resistance to, these agents. Finally, many of the key in vitro findings on the properties of antifolate transporters are now beginning to be extended to patient specimens, thus setting the stage for understanding response to these drugs in the clinical setting at the molecular level. ß 2003, Elsevier Science (USA).

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I. INTRODUCTION The membrane transport of folate compounds in mammalian cells is mediated by a variety of diverse processes. Best studied is the reduced folate carrier (RFC), a member of the major facilitator superfamily (Saier et al., 1999). Recently, other facilitative carriers largely expressed in epithelial tissues have been shown to be folate transporters. Complementing transport fluxes mediated by these bidirectional systems are those involving the membrane-localized folate receptors (FRs), including FR and FR, that mediate folate uptake into cells via an endocytotic process, and assorted transporters typified by the family of multidrug resistance-associated proteins (MRPs) (Borst and Elferink, 2002) that are unidirectional exporters. Further, there are transport activities for folates that function optimally at low pH and play a major role in intestinal transport. Although this activity is related, in part, to RFC, there are folate transport processes optimal at low pH that are RFC independent. This chapter summarizes the current understanding of these various transport routes, their molecular identities, their regulation, and their kinetic and thermodynamic properties.

II. REDUCED FOLATE CARRIER (RFC), A MEMBER OF THE SLC19 FAMILY OF TRANSPORTERS This SLC superfamily of transporters (Saier et al., 1999) is composed of more than 36 families of facilitative carriers in mammalian cells that are distinct from the ATP-binding cassette (ABC) unidirectional exporters (Dean et al., 2001). Folates are transported by a member of the SLC19 family, termed RFC or SLC19A1, first cloned in 1994 (Dixon et al., 1994). More recently, SLC19A2 (Diaz et al., 1999; Dutta et al., 1999; Fleming et al., 1999; Labay et al., 1999) and SLC19A3 (Rajgopal et al., 2001a) were cloned. These transporters have a high degree of homology, and all have predicted secondary structures of 12 transmembrane domains (TMDs), with a large loop between the sixth and seventh TMDs, and the amino- and carboxyl-termini directed to the cytoplasm. Despite their highdegreeofhomology, substratespecificities forSL19A1versus SL19A2 and SL19A3 are very different. SLC19A1 transports folates and not thiamin (Zhao et al.,2000a, 2002). Conversely, SLC19A2andSLCA3transport thiamin but not folates (Diaz et al., 1999; Dutta et al., 1999; Fleming et al., 1999; Labay et al., 1999). However, the phosphorylated derivates of thiamin (e.g., thiamin mono- and pyrophosphate) are substrates for RFC (Zhao et al., 2000a, 2002) (see Section II.C.2). Besides RFC, no other member of the SLC19 family has been identified that transports folates; and other than some members of the organic anion family (e.g., SLC21) (Russel et al., 2002), no other facilitative carriers have been confirmed to transport folates in mammalian cells.

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A. CLONING OF THE cDNAs FOR RFC

The presence of a carrier mechanism for reduced folates has been known for more than three decades, and many aspects of its functional properties have been characterized in detail. However, the cloning of this carrier in a variety of mammalian cells was first reported in the mid-1990s. 1. Restoration of Transport Function to Transport-Impaired Cells and Structural Properties of the cDNA-Encoded Protein In 1989, Flintoff and colleagues reported that tetrahydrofolate (THF) cofactor and methotrexate (MTX) membrane transport activity could be restored in transport-impaired Chinese hamster ovary (CHO) cells by transfections with genomic DNAs from drug-sensitive human or hamster cells (Underhill and Flintoff, 1989). In 1992, this approach was extended to transfections with CHO genomic DNA cosmid clones that included the gene(s) responsible for MTX uptake (Underhill et al., 1992). In 1994, two reports were published almost simultaneously that described the cloning of RFC cDNAs. Cowan and colleagues used expression cloning to isolate a cDNA from L1210 mouse cells that could restore MTX sensitivity and transport function to transport-impaired ZR-75-1 human breast carcinoma cells (Dixon et al., 1994). Flintoff and colleagues used a CHO cosmid clone to isolate the homologous cDNA from a CHO cDNA library that restored MTX transport and binding activity to transport-impaired CHO cells (Williams et al., 1994). In 1995, four reports on the homologous human cDNAs were published (Moscow et al., 1995; Prasad et al., 1995; Williams and Flintoff, 1995; Wong et al., 1995). The sequence for rat RFC was also deposited in Genbank in 1995 with Accession number U38180. This was followed in 1997 by the description of a cDNA clone from human intestine with an open reading frame identical to the human RFC (hRFC) (Nguyen et al., 1997). It soon became apparent that these cDNAs were able to restore MTX sensitivities and a number of properties typical of the endogenously expressed RFCs to transport-impaired mouse (Brigle et al., 1995), hamster (Williams et al., 1994; Williams and Flintoff, 1995; Wong et al., 1995), and human (Dixon et al., 1994; Moscow et al., 1995; Wong et al., 1997) cells. These included characteristic patterns of uptake with radiolabeled MTX and 5-formyltetrahydrofolate (5-CHO-THF), sensitivities to competitive inhibition by established RFC transport substrates (GW1843U89, folic acid, 5-CHO-THF, ZD1694) and to irreversible covalent inhibition by unlabeled N-hydroxysuccinimide (NHS)-MTX, and a capacity for trans-stimulation by intracellular reduced folates (Wong et al., 1997). However, other properties were inconsistent with those expected for RFC function. For instance, in a series of human (K562) transfectants, the restored transport approximated only 3–30% of the high levels expected from affinity labeling or Western blotting assays of the human reduced

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folate carrier (hRFC) protein (Wong et al., 1997, 1998). In other reports, disparities in transport substrate specificities and pH dependencies were described for endogenously expressed RFC-mediated uptake and that in cells transfected with RFC cDNAs (Chiao et al., 1997; Kumar et al., 1998; Rajgopal et al., 2001b; Wong et al., 1995). The amino acid sequences deduced for the isolated cDNAs predicted 58kDa proteins for the RFCs from mouse (mRFC) (Dixon et al., 1994) and rat cells (Genbank Accession number U38180), 59 kDa for the hamster RFC (haRFC) (Williams et al., 1994), and 65 kDa for the full length hRFC (Moscow et al., 1995; Prasad et al., 1995; Williams and Flintoff, 1995; Wong et al., 1995). Upon expression in transport-impaired hamster (Wong et al., 1995) or human (Wong et al., 1997) cells, the full-length human clone encoded an 85-kDa protein that could be identified by photoaffinity labeling with 125 I-APA-ASA-Lys [N-(4-amino-4-deoxy-10-methylpteroyl)-N -(4-azido125 5- l-salicylyl)-l-lysine]. Upon enzymatic deglycosylation, the broadly migrating band shifted to 65 kDa, in close agreement with the size for the deglycosylated hRFC detected by affinity labeling from transport upregulated K562 (Matherly et al., 1991) or CCRF-CEM (Freisheim et al., 1992) cells and with the predicted size for the hRFC from the cDNA sequence. The mRFC was originally reported as 36 kDa by radioaffinity labeling with NHS-3HMTX (Henderson and Zevely, 1984a); however, in later studies, an 46-kDa protein was widely reported (Schuetz et al., 1988; Yang et al., 1988). The disparity between these earlier values and the predicted size from the cDNA sequence (58 kDa) was initially controversial and was suggested to arise from alternative splicing of murine RFC transcripts and the synthesis of smaller RFC proteins; however, these forms were expressed at exceedingly low levels (Tolner et al., 1997). More recent studies with antibodies to the carboxylterminus and TMD6/7 connecting loop of mRFC suggested that the smaller molecular mass forms were likely the result of unchecked proteolysis during the preparation of plasma membranes for Western blots (Zhao et al., 2000b). 2. Analyses of Secondary Structure By hydropathy analysis, RFC proteins from different species each conformed to a model expected for an integral membrane protein, with 11 or 12 stretches of 17–25 mostly hydrophobic, -helix-promoting amino acids, and an internally oriented C-terminus. Figure 1 shows a topology model for hRFC noting the conserved amino acids. There is a striking degree of sequence conservation of the primary amino acids for several of the putative TMDs (i.e., TMDs 1–5, 7, and 8), and there is a nearly complete lack of homology in the large central loop connecting TMDs 6 and 7, and in the amino and carboxylterminal regions. Although the connecting loops are, in general, less conserved, highly conserved patches (e.g., N-terminal segment of the TMD6/7 loop and TMD10/11 loop) are, nonetheless, present in these regions.

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FIGURE 1. Predicted topology structure of the hRFC. A topology model for hRFC [‘‘TMPRED’’ (Hoffman and Stoffel, 1993)] is shown depicting 12 putative TMDs and internally oriented amino-and carboxyl-termini. Conserved residues between the rodent and human RFCs are shown in black. The locations of the functionally important residues noted in the text are indicated including Gly-44, Glu-45, Ser-46, Ile-48, Asn-58, Asp-88 (Asp-86 in mRFC), Trp-108 (Trp-105), Ser-127 (Ser-125), Ala-132 (Ala-130), Arg-133 (Arg-131), Ser-313 (Ser-309), Arg-373 (Arg-366), Lys-411 (Lys-404), and Asp-452 (Asp-446). Also noted is the highly conserved stretch of functionally important amino acids residues 204–214 in hRFC (202–212 in mRFC) between TMDs 6 and 7. The predicted membrane topology of TMDs 1–8 and the carboxyl-terminus of hRFC has been experimentally verified (Ferguson and Flintoff, 1999; Liu and Matherly, 2002).

Finally, hRFC contains 72–79 more amino acids than the rodent RFCs. The overall homologies between the rodent and human carrier are 64–66% Interestingly, the single N-glycosylation consensus site predicted for the hRFC-encoded protein (at Asn-58) is conserved in the haRFC and rat RFC (both at position 56) but not in the mRFC. Glycosylation of hRFC and haRFC has been experimentally confirmed (Ferguson and Flintoff, 1999; Wong et al., 1998). All the rodent sequences contained at least one additional consensus N-glycosylation site not present in the hRFC. A functional 65-kDa protein was detected on Western blots prepared from transport-impaired K562 (K500E) cells transfected with the Gln-58 hRFC cDNA in which Asn-58 was substituted by Gln. Wild-type (Asn-58) and Gln-58 hRFC proteins containing hemagglutinin (HA) epitopes (YPYDVPDYASL) at their carboxyl-termini were transport competent and by immunofluorescence staining of permeabilized cells with rhodamineconjugated anti-HA antibody, were both localized to the cell surface (Wong et al., 1998). Thus, the extent of N-glycosylation plays no significant role in either transport function or surface targeting of hRFC. Studies of hRFC glycosylation also shed light on the membrane topology of hRFC, as they confirm an extracellular orientation for Asn-58, localized in putative TMD2 (Fig. 1). Because immunofluorescence detection of the

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wild-type (Asn-58) and Gln-58 hRFC carboxyl HA-hRFC required cell permeabilization, an internal cytosolic orientation of the hRFC carboxylterminus was also implied (Wong et al., 1998). Most recently, HA-epitope insertion was used to further map cytosolic orientations of the hRFC amino (Pro-20, Gly-17)-terminus, and the connecting loops between TMDs 6 and 7 (Ser-225, Glu-226), and TMDs 8 and 9 (Ala-332) (Ferguson and Flintoff, 1999; Liu and Matherly, 2002). Moreover, extracellular orientations were confirmed by inserting HA epitopes into the connecting loops between TMDs 3 and 4 (Gln-120) and between TMDs 7 and 8 (Ser-225, Glu-226) (Ferguson and Flintoff, 1999; Liu and Matherly, 2002). Mutation of phenylalanine-182 (in putative TMD 5) to asparagine in Gln-58-hRFC generated a new N-glycosylation [Asn-X(Ser/Thr)] consensus sequence that was glycosylated in transfected K500E cells, establishing an extracellular orientation for this region (Liu and Matherly, 2002). Collectively, these results strongly support the computer-generated topology model for TMDs 1–8 in Fig. 1 and the RFC C-terminal domain. However, the topology for TMDs 9–12 awaits further experimental confirmation. Complete resolution of the membrane topology of RFC is essential to understanding the mechanistic roles of individual amino acids and domains in the transport process. 3. Structural Determinants of hRFC Trafficking and Membrane Targeting Recent studies have begun to explore the structural determinants for intracellular trafficking and plasma membrane targeting of RFC using enhanced green fluorescent protein (EGFP) carboxyl-tagged haRFC or hRFC constructs in transfected cells (Marchant et al., 2002; Sadlish et al., 2002a). Thus, removal of 16 amino acids (residues 7–22) from haRFC (Sadlish et al., 2002a) or 27 N-terminal (1–27) or 139 C-terminal (residues 453–591) amino acids from hRFC (Marchant et al., 2002) did not appreciably affect membrane targeting or function. However, there were suggestions of slight effects on intracellular trafficking as deletions of the Cterminus, or the N- and C-termini together, resulted in decreased expression and increased turnover of haRFC, increased intracellular accumulation relative to membrane-localized haRFC, and an increased proportion of unglycosylated haRFC protein (Sadlish et al., 2002a). Larger deletions from the N- or C-termini (e.g., residues 1–301 or 302–591, respectively) of hRFC resulted in a complete ablation of surface targeting (Marchant et al., 2002). Deletion of the C-terminus of mRFC (amino acids 445–512) resulted in an unstable protein (Sharina et al., 2002). The central loop connecting TMD6 and TMD7 has been studied extensively for its role in RFC targeting and function. Thus, deletion of up to 31 of 66 amino acids from the mRFC or up to 45 of 67 residues for the hamster carrier preserved membrane targeting and transport activity; however, larger deletions (57 and 53–55 amino acids, respectively) abolished activity (Sadlish

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et al., 2002a; Sharina et al., 2002). Interestingly, a critical requirement for maintenance of carrier function in these experiments was the preservation of a highly conserved stretch of 11 amino acids (amino acids 204–214 in haRFC and 202–212 in mRFC). Similar results were recently described for the central loop of hRFC (residues 204–214; Fig. 1). When the nonhomologous 73 or 84 amino acid fragments of the structurally analogous human SLCA2 protein (ThTr1; 18% homologous for the TMD6/7 connecting loop) were inserted into hRFCs from which 49 or 60 residues from the TMD6/7 linker were deleted, transport was restored, although maximal activity had an absolute requirement for the conserved 204–214 peptide (Liu et al., 2003). Collectively, these results suggest that the primary role of the RFC central linker peptide betweenTMDs6and7 istoprovide the proper spatial orientation between the two halves of the RFC protein for proper function, and that this role is largely independent of the amino acid sequence. Moreover, the stretch of 11 conserved amino acids starting at position 204 of hRFC appears to be essential for preservation of high levels of transport activity. The lack of a significant effect from deleting the N-terminus, or replacing nearly the entire central loop with a nonhomologous sequence from the ThTr1 protein, strongly suggests that the molecular determinants of plasma membrane targeting and expression are exclusive of these regions and must be located elsewhere in the hydrophobic backbone of RFC. However, because the effects of C-terminal deletions exclusive of the TMDs on membrane localization ranged from slight (Sadlish et al., 2002a) to complete (Sharina et al., 2002), some role for the RFC C-terminus in intracellular targeting is implied. 4. Organization of the RFC Genes from Hamster, Mouse, and Human Cells The isolation of cDNAs for the RFCs from assorted species soon fostered the characterization of RFC genes from hamster (Murray et al., 1996), mouse (Brigle et al., 1997; Tolner et al., 1997), and human (Tolner et al., 1998; Williams and Flintoff, 1998; Zhang et al., 1998a) cells. It quickly became apparent that in spite of the heterogeneous nature of RFC transcripts (see later), there was only a single RFC gene locus. The murine and hamster RFC genes were smaller than that for hRFC (i.e., 23 and 15.3 kb, versus 27 kb, respectively). All RFC genes contained five major coding exons, preceded upstream by multiple (at least two) noncoding exons (see later). The intron– exon junctions for the coding exons are highly conserved, even though there is little similarity between the intron sizes for the human and rodent RFC genes, and all conform to the GT/AG consensus sequence with the exception of the splice donor site for the first coding exon (designated exon 1 in hRFC), which contains a conserved GC. Coding exon 5 is larger (1446 versus 887 bp) in hRFC than for the homologous murine and hamster RFC genes, reflecting the extended carboxyl-terminus of hRFC. As described later, very recent studies (Whetstine et al., 2002a) indicate that the hRFC gene locus is significantly

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larger than originally believed, reflecting the presence of up to seven noncoding exons, spanning more than 35 kb upstream of the translational start site. RFC chromosomal localizations have been established by fluorescence in situ hybridization. The mRFC gene was localized to chromosome 10 (Roy et al., 1998a) and the haRFC was assigned to chromosome 1 at position q2–q3 (Chan et al., 1995). The localization of the hRFC gene to chromosome 21q22.2 (Moscow et al., 1995) is of particular interest, given the clustering of C1 metabolic enzymes on the q arm of chromosome 21. Moreover, enhanced accumulations of MTX and its polyglutamyl derivatives have been reported to accompany increased levels of hRFC transcripts and copies of chromosome 21 in B-precursor ALL specimens (Synold et al., 1994; Whitehead et al., 1992) (see Section II.D.1). A similar explanation may account for the untoward toxicity of MTX toward patients with Down’s syndrome (Peeters and Poon, 1987).

B. PATTERNS OF RFC EXPRESSION AND OCCURRENCE OF HETEROGENEOUS RFC TRANSCRIPTS

1. RFC Is Ubiquitously Expressed in Tissues and Tumors Whereas the family of FRs (Section V.A) exhibits a relatively narrow and selective tissue expression profile, RFC is expressed broadly in both normal and malignant tissues. For instance, hRFC transcripts were widely detected on an array of mRNAs from 68 human tissues and 8 human tumor cell lines probed with the full- length hRFC cDNA (Whetstine et al., 2002a). High hRFC levels were detected in placenta, suggesting a role for hRFC in transplacental transport of reduced folates. Considerable hRFC message was also detected in liver, leukocytes, kidney, lung, bone marrow, and regions of the intestine (i.e., highest in the duodenum), as well as parts of the central nervous system and brain, whereas low but detectable hRFC was seen in the heart and skeletal muscle. Qualitatively similar results were obtained with a human multitissue Northern blot probed with hRFC cDNA (Whetstine et al., 2002a), and by reverse transcription-polymerase chain reaction (RT-PCR) analysis of human tissue RNAs (Gong et al., 1999; Whetstine et al., 2002a). On Northern blots, there were minor differences in the migrations of the major hRFC transcripts (3.1 kb) and additional larger and smaller transcript forms were also detected (Whetstine et al., 2002a). By immunohistochemistry with a polyclonal antibody to the mRFC carboxyl-terminus (Wang et al., 2001), RFC expression could be localized to specific cell membrane segments. For instance, RFC is expressed in the apical brush border membranes of the small intestine (e.g., jejunum, ileum, duodenum) and colon, and in the basolateral membranes of cortical and medullary renal tubular epithelial cells. RFC is expressed in hepatocyte cell

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membranes. Interestingly, RFC was also detected in plasma membranes on the apical surface of the choroid plexus, in axons and dendrites, and on the apical membrane of cells lining the spinal canal (Wang et al., 2001). Thus, the overwhelming evidence is that RFC is ubiquitously expressed in normal and malignant tissues, consistent with its integral role in tissue folate homeostasis, and implying the existence of intricate mechanisms for controlling patterns of RFC expression and function in response to diverse tissue environments. A related feature involves RFC transcript heterogeneity, as suggested by differences in migrations of the major hRFC transcripts and the presence of additional smaller and larger hRFC transcript forms on Northern blots of RNAs from assorted tissues probed with the full-length hRFC cDNA (Whetstine et al., 2002a). The molecular basis for the RFC transcript heterogeneity is considered in the following section.

2. Molecular Basis for RFC Transcript Heterogeneity For the RFCs from mouse and human cells, a number of low-frequency putative splice forms with coding sequence deletions have been reported. For mRFC, putative splice variants were identified in L1210 cDNA libraries (Brigle et al., 1997; Tolner et al., 1997); however, it is unclear whether truncated mRFC proteins are actually synthesized (predicted molecular masses of 53.6 and 43.4 kDa) from these alternatively spliced transcript forms. An hRFC cDNA with a 626-bp (positions 1568–2193) deletion in both the coding sequence and 30 -untranslated region (UTR) (i.e., coding exon 5) was identified in a K562 subline (Wong et al., 1995). Due to this deletion, the normal in-frame translational stop codon (at position 1774) is lost and a new stop codon at position 2205 is used. Upon expression in CHO cells, the glycosylated truncated hRFC protein (85 kDa) was functional (Wong et al., 1995). Another truncated hRFC form was reported in transport upregulated CEM-7A cells and contained a 987-bp deletion resulting from use of a cryptic splice acceptor site within coding exon 4 (Drori et al., 2000a). The resulting loss of the entire carboxyl-terminus and TMD12 rendered this form nonfunctional. At least two upstream noncoding exons were originally reported for the haRFC and mRFC genes that encode transcripts with different 50 -UTRs linked to a common coding sequence (Murray et al., 1996; Tolner et al., 1999). Although hRFC was initially believed to follow a similar pattern (Tolner et al., 1998; Zhang et al., 1998b), very recent studies of the hRFC gene and transcript forms in human tissues and cell lines suggest a remarkable and unexpected level of complexity (Whetstine et al., 2002a). This is described in the following section.

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FIGURE 2. Expanded upstream region of the hRFC gene including noncoding exons and 50 flanking regions. The schematic depicts the upstream region of the hRFC gene including seven noncoding exons (A1, A2, A, B, C, D, and E) and the first coding exon (exon 1) including the ATG translation start site. The approximate distances between each of the noncoding exons are based on those in chromosome 21 contig HS21C102 (Accession number AL163302). As described in the text, promoter activity has been verified for the 50 flanking regions proximal to noncoding exons A, B, and C (Whetstine et al., 2002a; Whetstine and Matherly, 2001; Zhang et al., 1998b). 3. Roles of Alternative Noncoding Exons and Variable Splicing in the Synthesis of hRFC Transcripts with Heterogeneous 50 -UTRs One of the original reports of the cloning of hRFC described the properties of three unique hRFC cDNAs (designated KS6, KS32, and KS43) with 50 noncoding regions of different lengths (93, 373, and 98 bp, respectively) that were completely nonhomologous beginning at position 52 (Wong et al., 1995). The existence of these distinct hRFC transcript forms was subsequently confirmed by 50 -rapid amplification of cDNA ends (50 -RACE) assay and RT-PCR (Gong et al., 1999; Zhang et al., 1998b). These 50 -UTRs for the KS43, KS32, and KS6 hRFC transcripts were localized to two noncoding exons, immediately upstream from an 3.4-kb intron and coding exon 1, including the translational start site. Because both the KS43 and KS6 50 UTRs were separated by only 6 bp, they were assigned to a single exon (designated exon B). The KS32 50 -UTR was localized to exon A. Based on the results of the multitissue mRNA arrays (see earlier), 50 RACE assays were used to extend these early studies of hRFC to an assortment of human tissues including caudate nucleus, lung, fetal lung, fetal liver, bone marrow, small intestine, heart, and placenta, as well as to cultured HT1080 fibrosarcoma and HepG2 hepatoma cells (Whetstine et al., 2002a). When 50 -RACE products were aligned to the chromosome 21 genome sequence (Contig number HS21C102), up to seven distinct 50 -UTRs were identified, including those previously assigned to the original hRFC-A and -B noncoding exons but also five additional 50 -UTRs, designated A1, A2, C, D, and E (Fig. 2). Altogether, these sequences spanned more than 35 kb upstream of the hRFC translation start site. Exon C appears to be identical to a putative hRFC noncodon exon previously reported to map 1.7 kb upstream from exon B (Gong et al., 1999). Apparent alternative splice forms were detected for four of the seven putative noncoding exons, including six for exon A, two for exon B, two for exon C, and five for exon D. Altogether, a total of 18 distinct hRFC transcript forms were identified, with unique 50 -UTRs fused to a common open reading frame that, presumably, encodes the same hRFC protein.

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Although the physiological significance of this large number of different hRFC mRNAs is still evolving, the patterns of 50 -UTR utilization and splice variants by 50 -RACE appeared to be comparatively cell/tissue specific (Whetstine et al., 2002a). For instance, 95% of the 50 -RACE clones from small intestine contained exon D sequence, whereas in placenta, exon C predominated (85% of clones). Interestingly, the major 50 -UTR in the hRFC cDNAs from various laboratories (i.e., exon B) (Prasad et al., 1995; Williams and Flintoff, 1995; Wong et al., 1995) was comparatively rare in most normal human tissues (<15% of 50 -RACE products); however, this form was abudant in heart (60% of 50 -RACE clones). Based on reports with other genes, 50 -UTR variability may be associated with different translation efficiencies (Fiaschi et al., 2000; Kocarek et al., 2000; Roberts et al., 1997), mRNA stabilities (Fiaschi et al., 2000; Fournier et al., 2001), intracellular targeting (Chen et al., 1996), or even synthesis of alternate proteins (Turner et al., 1999). 4. Relationship of RFC Promoters to Tissue-Specific Gene Expression By analogy with other multipromoter genes, the unique noncoding exons for RFC are likely to be transcribed from distinct promoters. For mRFC, the 50 flanking regions immediately upstream of the alternative noncoding exons 1 and 1a exhibited promoter activity by reporter gene fusions and transient transfections of mouse NIH-3T3 cells (Tolner et al., 1999). By site-directed mutagenesis and/or DNase footprint analysis, three Sp1 sites and a poly(GT)21 dinucleotide repeat in the mRFC 1a region were implicated in promoter activity. For the mRFC promoter 1, two adjacent Sp1 elements were, likewise, suggested to be important to basal promoter activity (Tolner et al., 1999). In contrast to the close homologies in the RFC coding sequences (see earlier), other than their high GC contents, there are no apparent sequence similarities in the upstream noncoding exons or regulatory regions for the human and rodent RFCs. Of the seven putative promoters inferred by the identification of up to seven noncoding exons for hRFC, promoter activity has been confirmed only for the 50 flanking regions proximal to exons A, B, and C (Whetstine et al., 2002a; Whetstine and Matherly, 2001; Zhang et al., 1998b). By in vitro binding assays and transient transfections of HepG2, HT1080, and Drosophila SL2 cells, the hRFC-A and -B basal promoters were found to be regulated by distinctly different families of transcription factors including the bZip superfamily (e.g., c-Jun/c-Fos and Creb/ATF1) and the Sp family of transcription factors (e.g., Sp1, Sp3), respectively (Whetstine and Matherly, 2001). For promoter B, an additional regulation by oncogenic factors such as USF and Ikaros was implied (Whetstine et al., 2002b), whereas critical roles for AP2 and Sp1 in the transactivation of promoter A were demonstrated (Whetstine et al., 2002c). Of particular interest was the finding of a novel series of tandem repeats in the hRFC-A

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upstream region, including functionally important Ap2 and Sp1 elements, and the occurrence of a high-frequency 61-bp polymorphism involving deletion of one of the repeat sequences that alters promoter activity (Whetstine et al., 2002c). hRFC promoter multiplicity should serve to provide a potentially powerful means of ensuring adequate levels of hRFC transcripts (and protein) in response to metabolic requirements for folate cofactors and/or in response to cell- or tissue- specific signals. Furthermore, for the transcription factor families involved, differences in the intracellular levels of individual members (e.g., Sp1 and the long and short isoforms for Sp3, bZip proteins, Ikaros, and AP2) may provide an additional level of tissue-specific regulation through direct competitions for binding, and/or through the formation of heterodimeric and homodimeric binding complexes with differing propensities for promoter transactivation or repression. Regulation at this level would be further influenced by the presence or absence of a high-frequency polymorphism in promoter A. It is interesting that the hRFC promoter C activity could not be detected in transient transfections in MCF-7 cells (Gong et al., 1999). In HepG2 cells, promoter activity was detectable following removal of a 342-bp downstream repressor and could be localized to a 453-bp fragment, including 11 bp of exon C (Whetstine et al., 2002a). A number of potential conserved elements were identified in this region including GATA, Ikaros, GC-box, E-box, NF1, and Creelements. Of course, the level of transcription ultimately achieved by a particular cell or tissue depends on multiple determinants in addition to the intracellular concentrations of the individual transcription factors capable of binding the basal promoters. These include the presence of other critical up- and downstream cis elements and cellular levels of transcription factors capable of binding these sites, the presence of gene polymorphisms, as well as the general promoter architecture and chromatin structure. For instance, c-MYC overexpressing Fisher rat 3T3 cells were found to express RFC at levels 10-fold greater than that for wild-type cells (Kuhnel et al., 2000). and hRFC levels and promoter activity were downregulated in K562 cells engineered to overexpress p53 (Ding et al., 2001). A recent report found that hRFC expression in MTX-resistant MDA-MB-231 correlated with methylation and acetylation of the hRFC-B promoter (Worm et al., 2001). Collectively, these results suggest that cell-specific expression of RFC may reflect different intracellular levels of particular family members or alternate signaling pathways that influence the activity of individual transcription factors. The levels of RFC transcripts eventually achieved within tissues likely reflect the effects of multiple transcription factors that respond to specific stimuli to activate or repress transcription, as well as critical epigenetic processes, that all combine to ensure sufficient levels of folate uptake for cell proliferation and tissue regeneration.

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C. STRUCTURE AND FUNCTION OF RFC

1. The Kinetics of RFC-Mediated Transport Much of what is known about the functional properties of RFC came decades before the gene was cloned and was based on studies with the antifolate MTX in murine leukemia cells (Goldman et al., 1968; Sirotnak et al., 1968). This structural analog of folic acid was found to have properties that made it an ideal compound for characterizing this carrier. Unlike the reduced folates that are the physiological substrates for RFC, metabolism of MTX is negligible over the intervals necessary to assess unidirectional fluxes and transmembrane gradients. Transport of the drug into cells is rate limiting to its very rapid and tight binding to dihydrofolate reductase, its target enzyme. This permits very accurate determination of unidirectional fluxes as well as free drug levels that can be quantitated from efflux studies, thus permitting discrimination between total cell antifolate and the tightly bound component (Goldman et al., 1968). Assessment of the unidirectional flux of reduced folates into cells can be obtained by measurements over short intervals. However, transmembrane gradients and efflux kinetics are virtually impossible to obtain for these forms because of their rapid metabolism to a variety of other THF cofactor species and to polyglutamyl derivatives that do not exit cells, and due to their binding to intracellular proteins. On the other hand, some transport parameters for folic acid can be obtained when its reduction to reduced folates is blocked with trimetrexate, an antifolate that can inhibit dihydrofolate reductase without altering transport via RFC (Assaraf and Goldman, 1997). Transport mediated by RFC has a high degree of structural specificity among the folate compounds in murine and human cells. For instance the Kt for MTX and 5-CHO-THF influx is 5 M, and for 5-methyltetrahydrofolate (5-CH3-THF) 2 M; the Kt for folic acid influx is one to two orders of magnitude higher (Goldman et al., 1968; Westerhof et al., 1995). Transport of reduced folates mediated by RFC is not stereospecific for 5-CH3-THF, as the influx Kts for the natural (6S) and unnatural (6R) isomers of 5-CH3-THF are comparable (Sirotnak and Donsbach, 1974; White et al., 1978). However, the unnatural (6R) isomer of 5-CHO-THF is a much weaker inhibitor of MTX influx than the (6S) isomer (Sirotnak et al., 1979). A number of antifolates including aminopterin have substantially higher affinities than MTX for RFC (Matherly et al., 1985; Westerhof et al., 1995; Wright et al., 2000). Interestingly, the benzoquinazoline antifolate, GW1843U89, is a far better substrate for hRFC than mRFC, as reflected in a 12-fold lower Kt and 80-fold greater Vmax/Kt for Molt 4 human leukemia cells as compared to L1210 cells (Duch et al., 1993). As indicated later, there are a variety of organic anions with very different structures that have relatively high affinities for RFC such as thiamin phosphates. Influx of MTX via RFC is highly temperature dependent, inhibited by sulfhydryl reagents, and typically has a pH optimum

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of 7.5 (Goldman et al., 1968; Sierra et al., 1997). RFC-mediated MTX influx is not Na+ dependent but is highly sensitive to the anionic composition of the extracellular compartment (see later). As indicated earlier, RFC has a very low affinity for folic acid, and it is likely that uptake of this folate in tumor cell lines is mediated largely by another process that has not, as yet, been identified. This is supported by a number of observations. First, the folic acid growth requirement is usually only modestly increased under conditions in which RFC-mediated transport is impaired and the 5-CHO-THF growth requirement is markedly increased (Zhao et al., 1999a). Second, the initial rate of highly purified tritiated folic acid uptake in murine leukemia cells is not inhibited by concentrations of unlabeled MTX that abolish RFC-mediated transport and is not altered in cells in which RFC function is impaired (Sirotnak et al., 1987). Third, changes in RFC-mediated influx of MTX, 5-CHO-THF, and 5-CH3-THF that occur upon association of positively and negatively charged liposomes with Ehrlich ascites tumor cells are not observed for folic acid transport (Fry et al., 1979; Fry and Goldman, 1982). Finally, animals in which RFC has been targeted and inactivated by homologous recombination can be brought to live birth with parental folic acid but not 5-CHO-THF, consistent with transport of folic acid by another pathway (Zhao et al., 2001) (see Section X). RFC-mediated transport also exhibits exchange phenomena consistent with carrier kinetics. For instance, influx of tritiated MTX is transstimulated in cells preloaded with 5-CHO-THF or 5-CH3-THF but not into cells loaded with nonlabeled MTX (Dembo and Sirotnak, 1976; Goldman, 1971a). However, trans-stimulation by MTX occurs when cells are incubated in a high K+ buffer (Fry et al., 1980a). Efflux of MTX from cells is not trans-stimulated by the presence of extracellular folate in a physiological buffer but does occur in anion-free buffer, presumably due to the loss of Cl, which also exchanges with this carrier (see later). Addition of 5-CHO-THF to cells at steady steady with MTX produces a rapid net loss of virtually all exchangeable intracellular drug (Goldman et al., 1968). Although this was attributed to an exchange phenomenon, interpretation is complicated by the expression of MRP exporters that pump MTX out of cells under conditions in which influx is blocked. 2. The Energetics of RFC-Mediated Transport RFC facilitates the transmembrane flows of folates, anionic compounds that would otherwise diffuse very slowly through the lipid membrane barrier. RFC lacks an ATP binding domain, and free energy from the hydrolysis of ATP is not utilized directly to translocate the carrier or to alter its conformation and affinity for substrates at the inner or outer cell membrane interfaces. Rather, facilitative carriers such as RFC achieve uphill transport by the interaction with other molecules that are themselves actively transported by an energy-requiring process. For instance, the sodium gradient generated

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by Na+, K+-ATPase is linked to the transport of many organic molecules, such as glucose, via a cotransport mechanism so that the association of Na+ with its binding site on the glucose carrier increases the affinity for glucose at its binding site. The asymmetrical distribution of Na+ across the cell membrane (outside > inside) results in an asymmetrical distribution of carrier in the highaffinity state for glucose (outside > inside). At the steady state, when the unidirectional glucose fluxes are equal, the glucose concentration in the intracellular water must exceed its extracellular level—hence uphill transport into the cell. A proton gradient across cell membranes also drives the uphill transport of a variety of organic molecules (Stein, 1989, 1990). MTX is a bivalent anion at physiological pH, so that the determination of whether its transport is uphill requires knowledge of the membrane potential. From this, the expected transmembrane gradient can be predicted when transport is simply equilibrating (i.e., when energy is not required to sustain the steady-state intracellular drug concentration). The elements required to determine the electrochemical potential difference across the cell membrane include both the transmembrane chemical distribution of MTX and the membrane potential. If there was no energy coupling to RFC at equilibrium, the concentration of MTX in the intracellular water would be far less than that in the extracellular compartment. Accordingly, the degree to which the intracellular concentration exceeds this predicted value is an indication of the extent of uphill MTX transport into cells. This requires very careful measurements of intracellular water, free (unbound) MTX within cells, and the membrane potential. On this basis, electrochemical-potential differences for MTX have been observed; however, only rarely are significant chemical gradients detected (Goldman et al., 1968; Sharif et al., 1998; Zhao et al., 1997). Complicating assessment of the concentrative capacity of RFC is the presence of a variety of ATP-dependent exporters, particularly the MRPs that pump MTX and other folates out of virtually all mammalian cells (Borst and Elferink, 2002). Hence, the transmembrane MTX gradient achieved at steady state is determined by the net effect of all these processes (see later). Concentrative transport is markedly enhanced when energydependent exporters are blocked, revealing the concentrative potential of RFC (Dembo et al., 1984; Goldman, 1969). This is considered in more detail in Sections VI and VIII. RFC-mediated influx of MTX in murine leukemia cells is neither Na+ nor proton dependent. Rather, RFC is highly sensitive to its anionic environment. Substitution of extracellular chloride with inorganic anions or addition of a variety of structurally diverse organic anions, in particular the organic phosphates, inhibits folate influx into cells (Goldman, 1971b; Henderson and Zevely, 1982a, 1983a). Uphill transport of folates has been attributed to the high concentration of organic phosphates that are synthesized within cells and are retained because of their minimal passive diffusion across the cell membrane (Goldman, 1971b). Indeed, these

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nondiffusible anions are a major contributor to the Donnan potential. According to this paradigm, the efflux of organic phosphates downhill out of cells mediated by RFC is linked to the uphill transport of folates into cells by this carrier. Because RFC is a low-capacity transport system, the net loss of organic phosphates via this route would be trivial in relationship to their far greater rate of synthesis. Thus there would be only a minor impact organic phosphate levels within cells (Goldman, 1971b). There is an abundance of evidence to support the notion of an RFCmediated exchange of folates with inorganic and organic anions. RFC-mediated influx is stimulated by the removal of chloride due to a reduction in the MTX influx Kt (Goldman, 1971b; Henderson and Zevely, 1980, 1983b). Uphill transport into cells is enhanced when the extracellular anion concentration is reduced (Henderson and Zevely, 1980). Influx is competitively inhibited by the substitution of chloride with a variety of univalent and multivalent inorganic anions or the addition of organic anions, and the transmembrane MTX gradient is diminished under these conditions (Goldman, 1971b). Among the most potent anionic inhibitors are the organic phosphates such as the adenine nucleotides and, most interestingly, thiamin mono- and pyrophosphate (see later). The Ki for Cl inhibition of MTX influx is 30 mM. In Cl containing buffer, the Ki for inhibition of MTX influx by ATP is 5 mM but in anion-free buffer the Ki values for ATP and AMP are 40–50 M, and for phosphate, 400 M (Henderson and Zevely, 1981, 1982a, 1983a). Further evidence for a linkage between folate and organic anion transport via RFC comes from exchange phenomena. Efflux of MTX from cells suspended into anion-free buffer is slowed because of the lack of exchanging molecules, but is enhanced by the presence of extracellular folates (Henderson and Zevely, 1981, 1983a). Likewise, efflux of MTX is transstimulated by the extracellular presence of phosphate or organic anions such as thiamin pyrophosphate, AMP, ADP, or succinate (Henderson and Zevely, 1981, 1983a). Of course, interpretation of these findings in intact cells is complicated by the presence of high-capacity parallel efflux routes for folates via MRPs or other transporters that are not subject to exchange with extracellular folate species (Borst and Elferink, 2002; Dean et al., 2001). However, using plasma membrane vesicles, trans-stimulation of MTX fluxes was achieved by the presence of a folate, phosphate, or sulfate in the opposite (trans) compartment (Yang et al., 1984). Recent studies on the transport of thiamin and its phosphorylated derivates provide additional compelling evidence for a functional interaction between organic phosphates and folates at the level of RFC. SLC19A2 and SLC19A3, the two other members of the SLC19 family, transport thiamine but not folates (Dutta et al., 1999; Rajgopal et al., 2001a). RFC transports folates but not thiamin. However, the phosphorylated derivatives of thiamin are good substrates for RFC in murine leukemia cells (Zhao et al., 2000a, 2002).

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Thiamine mono- and pyrophosphate influx is negligible in cells that lack RFC activity and is enhanced in cells for which expression of RFC is high. The influx Kt and Ki for thiamin mono- and pyrophosphates are 15 and 26 M, respectively. In the cell, thiamin is rapidly converted to its pyrophosphate anabolite, which accumulates to high levels. The addition of MTX (which enters and leaves cells via RFC but does not inhibit the thiamin carriers) in the presence of tritiated thiamin markedly enhances net accumulation of thiamin pyrophosphate, presumably by blocking its efflux via RFC. Further, net accumulation of thiamin pyrophosphate is enhanced in cells that lack RFC expression and is substantially reduced in cells in which RFC is overexpressed (Zhao et al., 2000a). Also consistent with the utilization of RFC by structurally unrelated anions is the earlier observation that MTX inhibits phosphate transport into murine leukemia cells (Henderson and Zevely, 1982b). Besides the effects that the anions in solution have on the bidirectional fluxes of folates and on concentrative transport mediated by RFC, the function of this carrier is also affected by membrane charge. For instance, the association of positively charged liposomes with Ehrlich ascites tumor cells resulted in a symmetrical increase in the bidirectional RFC-mediated MTX fluxes with an increased influx Vmax but no change in Kt. This is consistent with an increase in the rate of translocation of the carrier within the cell membrane. This did not impact the energetics of transport as there was no change in the steady-state MTX level (Fry et al., 1979). The association of negatively charged liposomes had the opposite effect, namely a symmetrical decrease in the bidirectional fluxes but no change in the steady-state level achieved associated with a decrease in influx Vmax (Fry and Goldman, 1982). 3. Analysis of RFC Structure As described earlier, the predicted membrane topology of RFC has been largely confirmed (Ferguson and Flintoff, 1999; Liu and Matherly, 2002) and a variety of data are emerging that point to domains that are important determinants of substrate specificity and carrier function. These insights have been gleaned from studies with antifolate resistant cells developed under selective pressure and, in some cases, by site-directed mutagenesis. A large panel of MTX-resistant murine leukemia cell lines was generated with chemical mutagenesis, all of which were transport impaired. In all cases, the defect was due to a mutation in amino acids in or adjacent to predicted TMDs (Zhao et al., 1999a). It is becoming increasingly clear that residues in or flanking the first TMD in RFC comprise an important site of interaction between the carrier and its folate substrates (Fig. 1). Mutations in this region have produced a number of very interesting phenotypes in cultured cell models. For instance, a Glu-45-Lys mutation in mRFC resulted in a global decline in carrier mobility, a marked increase in the influx Kt for MTX, and a comparable decrease in the influx Kt for folic acid. The influx Kt for 5-CHO-THF was

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also decreased, although less than for folic acid, yet there was no change in this parameter for 5-CH3-THF (Zhao et al., 1998a). A similar transport phenotype was observed with the same mutation in hRFC except for the lack of change in the MTX influx Kt (Jansen et al., 1998). Interestingly, Glu45-Lys has been detected in a number of MTX-resistant CCRF-CEM tumor lines selected for resistance to GW1843U89, an antifolate inhibitor of thymidylate synthase (Drori et al., 2000b; Rothem et al., 2002). However, this mutation has not been detected in primary acute lymphoblastic leukemia (ALL) specimens (Whetstine et al., 2001). Subsequent studies using site-directed mutagenesis at position 45 in mRFC indicated that the change in function was not related to the reversal of charge, as substitutions at this site with Gln, Arg, or Lys produced similar changes in influx Kt whereas substitution with Asp decreased carrier affinity for all substrates. Increasing bulk at this site with a Trp substitution markedly decreased the affinity for the reduced folates, although the Kt for folic acid influx was still half that of the wild-type carrier (Zhao et al., 2000c). A Ser-46-Asn mutation was also detected in L1210 cells under MTX selective pressure and produced a substrate-specific change in carrier mobility, as indicated by a decreased Vmax, without a change in Kt (Zhao et al., 1998b). The Vmax for MTX fell by a factor of 40, whereas the Vmax for 5-CH3-THF and 5-CHO-THF decreased by a factor of only 7–8. The same mutation has been detected in hRFC in an osteosarcoma following treatment of patients with MTX (Yang et al., 2003), and a Ser-46-Ile mutation was detected in CCRF-CEM cells selected for resistance to GW1843U89 (Drori et al., 2000b). An Ile-48-Phe mutation in mRFC was identified in L1210 cells selected for resistance to 5,10-dideazatetrahydrofolate (DDATHF), an inhibitor of glycinamide ribonucleotide (GAR) transformylase, that produced a marked decrease in the influx Kt for folic acid (Tse et al., 1998). The net effect of this alteration was enhanced uptake of folic acid, expansion of THF cofactor pools, with feedback inhibition of folylpolyglutamate synthetase, resulting in a substantial contraction in the accumulation of active DDATHF polyglutamates (Tse and Moran, 1998). Mutations at Gly44 in hRFC have also been reported that result in marked losses of carrier function (Rothem et al., 2002; Wong et al., 1999; Zhao et al., 1999a). Mutations at other RFC TMDs have also been detected that result in significant effects on carrier function (Fig. 1). For instance, a mutation in the third TMD in mRFC, Trp-105-Gly, similar to Ile-48-Phe described earlier, increased carrier affinity for folic acid (Tse et al., 1998). An Ala-130Pro mutation in the third TMD in mRFC markedly impaired carrier mobility but without a change in the MTX influx Kt (Brigle et al., 1995). Substitution of Ser-127 in hRFC with Asn (TMD 3) resulted in a decreased Vmax and decreased Kt for MTX (Wong et al., 1999). A mutation (ser-297Asn) in the external loop between the seventh and eighth TMD in mRFC decreased the affinity for MTX compared to other antifolates (aminopterin,

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10-deazaaminopterin, or 10-ethyl-10-deazaaminopterin) (Roy et al., 1998b). A Ser-309-Phe mutation in the eighth TMD of mRFC also produced a highly selective transport phenotype with a 5-fold increase in the influx Kt for MTX and 5-CHO-THF but with only a negligible change in affinity for folic acid or 5-CH3-THF (Zhao et al., 1999b). In an analysis of the role of charged residues located in the TMDs of mRFC, several arginines were found to be essential for activity as a total loss of function resulted upon substitution with leucine. These included arginines 131 (fourth TMD), 155 (at the cytosolic boundary of the fifth TMD), and 366 (tenth TMD). On the other hand, substitution of lysine with leucine at residues 26 and 42 (first TMD) and at residues 332 (ninth TMD) and 404 (eleventh TMD) had only a minimal effect on Vmax and MTX and folic acid binding; but there were some differences in binding among the reduced folates (Sharina et al., 2001). Important roles for Arg-133 in hRFC (homologous to Arg-131 in mRFC) (Liu and Matherly, 2001) and Arg-373 in haRFC (homologous to Arg-366 in mRFC) (Sadlish et al., 2002b) were, likewise, reported. In both cases, substitutions with lysines at these positions were well tolerated; however, other substitutions resulted in significant losses of activity. Replacement of Lys-411 in hRFC (analogous to Lys-404 in mRFC) with leucine was accompanied by a significant loss of transport activity for both MTX and 5-CHO-THF (Witt and Matherly, 2002), implying that the structural or functional role of this residue differs between the human and murine carriers. Replacement of lysine 411 in hRFC with arginine resulted in a selective increase in 5-CHO-THF transport over MTX due to an increased affinity for carrier (Witt and Matherly, 2002). Finally, mutations of the highly conserved aspartates in hRFC (positions 88 and 453) resulted in opposite effects, as nonconservative replacements (e.g., valine) totally abolished activity at position 88 but had minimal effect at position 453 (Liu and Matherly, 2001). Mutagenesis studies of Asp-88 and Arg-133 in hRFC (located in TMDs 2 and 4, respectively) also shed light on the tertiary structure of RFC (Liu and Matherly, 2001). Hence, when the charge on Arg-133 (fourth TMD) was neutralized by substitution with leucine or the charge on Asp-88 (second TMD) was neutralized by substitution with valine, the activity of each mutant carrier was markedly impaired. However, when both mutations were present in the same carrier, transport function was restored (Liu and Matherly, 2001). This restoration is consistent with a charge-pairing phenomenon in which the oppositely charged amino acids in the wild-type carrier are required to maintain proper orientation of their two domains and suggests that the fourth and second TMDs in hRFC are in close proximity. When the charge is neutralized in either amino acid, this orientation is lost. However, when the charge is neutralized at both residue sites, the orientation between the two TMDs is restored. Charge-pairing has been applied to explore the tertiary structure of a variety of facilitative carriers

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(Merickel et al., 1997; Sahin-Toth et al., 1992). Most recently, Flintoff and colleagues used oxidative cross-linking of double cysteine mutants (Asp-86Cys–Glu-394-Cys) of EGFP-tagged haRFC to demonstrate juxtaposition of Arg-373 to Glu-394 (Sadlish et al., 2002b). These structure–function studies have also provided some insights into the elements in RFC that are determinants of the sensitivity of this carrier to anions. As indicated earlier, addition of a variety of organic anions or substitutions of chloride with other inorganic anions inhibit transport of folates associated with an increase in influx Kt. The removal of chloride enhances influx of folates due to a decrease in influx Kt. Mutations in RFC can profoundly alter anion sensitivity. For instance, with the Glu-45-Lys mutation, mRFC function became anion dependent rather than anion inhibited. In the absence of chloride, the decrease in 5-CHO-THF influx was due to a marked decrease in Vmax. Function was restored by the addition of chloride, nitrate, or fluoride (Zhao et al., 1998a). The same RFC mutation in human leukemia cells resulted in a similar anion dependence (Jansen et al., 1998). Subsequent studies using sitedirected mutagenesis indicated that it is the introduction of the positive charge, rather than the loss of the negative charge, at this site that results in loss of function. Hence, substitution of Glu with Arg produced the same anion dependence as observed with Lys; but substitutions with Asp, Leu, or Trp sustained the wild-type phenotype (Zhao et al., 2000c). Other mutations also alter anion sensitivity. With the Ser-309-Phe (eighth TMD) and Glu404-Leu (eleventh TMD) mutations, influx was independent of chloride over a broad range of chloride concentrations with a simulatory effect below 50 mM (Sharina et al., 2001; Zhao et al., 1999b). Consistent with these observations, replacement of Lys-411 in hRFC with Leu or Glu only slightly altered the stimulation of 5-CHO-THF uptake in anion-free buffers (Witt and Matherly, 2002). To date, two polymorphisms in the RFC coding sequence have been identified, a G/A transition at position 80 in hRFC that occurs at allelic frequencies of approximately 40%/60% and a C/T at position 691 that occurs at an allelic frequency of approximately 50%/50% (Chango et al., 2000; Whetstine et al., 2001). The significance of these polymorphisms is considered in Section II.D.2.

D. THE ROLE OF RFC AS A FACTOR IN THE CLINICAL UTILITY OF METHOTREXATE

1. Patterns of hRFC Expression and 50 -UTR Utilization in Childhood Acute Lymphoblastic Leukemia MTX remains a cornerstone in the treatment of a number of malignancies including acute lymbhoblastic leukemia (ALL), osteogenic

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sarcomas, breast cancers, and lymphomas (Chu and Allegra, 1996; Jolivet et al., 1983). In many ways, childhood ALL is the prototypical disease for studying MTX resistance in the clinic, because of the central role of MTX in the treatment of this malignancy (Pui and Evans, 1998) and the comparative ease of obtaining leukemic blasts in high purity from bone marrow or peripheral blood for laboratory studies. Progress in understanding the role of hRFC in clinical response to MTX was initially slowed by the lack of uniformly sensitive and quantitative methods of analysis. Further, hRFC levels and transport activities in patient specimens are generally much lower than those in cultured cells and approach the limits of detection for standard radiotracer drug uptake or blotting assays (Zhang et al., 1998c). In recent years, studies with a fluorescent MTX analog [PT430; N-(4-amino-4-deoxy-N10-methylpteroyl)N-(40 -fluoresceinthiocarbamyl)-l-lysine] that binds avidly to cellular dihydrofolate reductase has fostered flow cytometry studies of MTX resistance markers, including dihydrofolate reductase levels (Matherly et al., 1995, 1997) and RFC (Gorlick et al., 1997; Trippett et al., 1992), in primary leukemias from patients. For assays of RFC, displacement of PT430 bound to dihydrofolate reductase during efflux by added MTX compared to trimetrexate (a lipid-soluble inhibitor that does not use RFC for cellular uptake) has been successfully used to indirectly evaluate carrier function including impaired transport in cells obtained from patients with leukemia (Gorlick et al., 1997; Trippett et al., 1992). With the cloning of hRFC, molecular-based approaches were used for studying carrier expression in patient specimens including Northern blotting (Gorlick et al., 1997) and quantitative RT-PCR (Gorlick et al., 1997; Rots et al., 2000; Zhang et al., 1998c). Importantly, leukemia samples that exhibited impaired MTX transport by PT430 and flow cytometry or uptake of 3H-MTX frequently showed low levels of hRFC transcripts by these methods (Gorlick et al., 1997; Zhang et al., 1998c). From the perception that B-precursor ALL in children is responsive to MTX-based chemotherapy, it was initially surprising that this group of patients exhibited a remarkably wide range of hRFC transcripts. For instance, in 28 children with B-precursor ALL, an 88-fold range of hRFC transcript levels was detected (Zhang et al., 1998c). Similar results were reported by others for leukemias (Gorlick et al., 1997; Rots et al., 2000; Zhang et al., 1998c) and for osteosarcomas (Guo et al., 1999). Interestingly, for osteosarcomas, the frequent loss of hRFC transcripts was generally accompanied by a poor prognosis (Guo et al., 1999). With B-precursor ALLs, hRFC expression, measured by quantitative RT-PCR, quickly became a benchmark of carrier function as transcripts were generally predictive of steady state accumulations of 3H-MTX (Zhang et al., 1998c). However, in a few specimens, hRFC transcripts were notably disproportionate to levels of MTX uptake. For 12 T-ALL samples, MTX

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uptake was generally lower than for B-precursor ALL, and there was no obvious relationship between hRFC transcripts and the low levels of uptake. The basis for this lack of correlation between MTX uptake and hRFC transcripts for a subset of B-precursor ALLs and the majority of T-ALLs is unclear. However, possible explanations include inefficient translation of hRFC transcripts, premature termination of nascent carrier protein, the existence of mutated or otherwise nonfunctional hRFC proteins, and/or increased rates of mutant hRFC degradation, analogous to alterations described for cultured cells (Drori et al., 2000a,b; Gong et al., 1997; Jansen et al., 1998; Sadlish et al., 2000; Wong et al., 1999). Alternatively, low-level steady-state MTX accumulation in ALL specimens may reflect increased levels or activities of the family of MRPs (see Section VI) or other systems involved in efflux of MTX. For B-precursor ALL in children, the wide range of hRFC transcripts appears, at least in part, to reflect the occurrence of subgroups of patients with unique patterns of hRFC expression. Indeed, approximately one-half of children with B-precursor ALL exhibit alterations involving the 21q22.2 chromosomal locus including hRFC (Pui and Evans, 1998). These include hyperdiploidy (97% with increased copies of chromosome 21) (Raimondi et al., 1996) and t(12;21)(p13;q22) (Pui and Evans, 1998). Hyperdiploidy is associated with increased accumulations of MTX and MTX polyglutamates relative to diploid B-precursor blasts (Synold et al., 1994; Whitehead et al., 1992) and an excellent prognosis (Pui and Evans, 1998). This may reflect increased (3 to 5) copies of chromosome 21 and hRFC gene copies, transcripts, and transport protein. Consistent with this notion was a 3-fold higher median level of hRFC transcripts for 11 hyperdiploid B-precursor lymphoblast specimens (54–59 chromosomes) compared to 17 diploid blasts (Zhang et al., 1998c). Similar results were subsequently reported by another group (Belkov et al., 1999). 2. Sequence Variants in the hRFC Gene Locus and Evidence for Alternative hRFC Splicing in Pediatric ALL A major goal of human genetics is to establish the roles of genetic variants that result in altered susceptibilities to disease. This requires identifying gene variations in human populations and assembling an extensive catalog of single nucleotide polymorphisms (SNPs), and characterizing their functional significance and associations with particular diseases. The identification of SNPs that result in altered proteins can provide molecular insights into the bases for interpatient variability in therapeutic responses and drug toxicities. There is a burgeoning interest in the identification of functional polymorphisms that could influence the activity or expression of folatedependent gene products. For instance, a high-frequency ‘‘low-function’’ polymorphic variant of 5,10-methylene tetrahydrofolate reductase (C677T)

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has been associated with elevated serum levels of homocysteine and increased risk of cardiovascular disease and neural tube defects and a lower risk of colon cancer (Ueland et al., 2001). For human thymidylate synthase, double or triple tandem repeats of a 28-bp cis-acting enhancer element has been associated with enhanced levels of gene expression and response to and toxicity of fluorouracil (Pullarkat et al., 2001). For RFC, sequence variants that result in amino acid substitutions could potentially alter carrier activity or stability, whereas alterations in the upstream regulatory regions could possibly have a profound effect on promoter transactivation and hRFC transcript levels. In either case, hRFC sequence variants might result in differences in uptake of MTX in ALL blasts or other malignancies treated with MTX chemotherapy, thus contributing to interpatient variations in treatment response. In normal tissues, alterations in membrane transport of reduced folate cofactors could result in downstream effects on folatedependent anabolic pathways, so as to contribute to interindividual differences in susceptibilities to cardiovascular disease, fetal abnormalities, and/or cancer. The occurrence of hRFC sequence variants could, in part, contribute to the disproportionate patterns of hRFC expression to activity in primary ALL blasts noted earlier. In an initial recent study of a 54 ALL (diagnostic and relapse; both B-precursor and T-ALL) and 51 normal patient DNAs, the presence of SNPs was confirmed at positions 80 (G/A), 696 (T/A), and 1242 (C/T) in the hRFC coding sequence (Whetstine et al., 2001). Of these, only G/A80 resulted in an amino acid change (i.e., Arg/ His-27). Although reports have implied effects of G/A80 (in combination with C677T in 5,10-methylene tetrahydrofolate reductase) on plasma folate levels and homocysteine pools (Chango et al., 2000) and on the risk of neural tube defects (De Marco et al., 2001; Shaw et al., 2002), in transfected cells there were only minor differences in the kinetic transport properties for Arg-27 versus His-27 hRFC (Whetstine et al., 2001). Although similar findings of a low frequency of hRFC sequence alterations in ALLs have been reported (Merola et al., 2002), a surpisingly high frequency of hRFC sequence alterations was reported for osteogenic sarcomas (Yang et al., 2003). A possible role for variant hRFC splice forms in MTX resistance was suggested by the identification of a CATG frame-shift insertion (position 191 in the coding sequence) that resulted in a loss of functional hRFC protein and MTX transport (Wong et al., 1999). Because the CATG insertion occurs at the splice junction for coding exons 1–2, this likely involves alternative splicing of hRFC transcripts. The CATG-191 insertion was also detected in 10–60% of hRFC transcripts from 10 of 16 ALL specimens, by RT-PCR-restriction fragment-length polymorphism analysis and sequencing of hRFC cDNAs (Whetstine et al., 2001). For one ALL specimen with disproportionate levels of hRFC transcripts to

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transport activity, 60% of the transcripts included CATG-191. Of interest will be the results of studies that further assess the relationships between the presence of CATG-191 and hRFC levels and function in primary ALLs. As described in Section II.B.4, recent studies show that up to 78% of hRFC alleles in a small cohort of patients have an additional 61-bp insertion in a region of the hRFC-A promoter originally reported to contain three tandem repeats (Whetstine et al., 2002c). The net effect of this additional repeat sequence is an increased promoter activity that could potentially impact net hRFC levels and transport activities in tumor cells and normal tissues. The effect of the repeat sequence would be further exacerbated in homozygous genotypes and/or accompanying increased chromosome 21 ploidy as commonly found in hyperdiploid B-precursor ALL (see earlier).

III. TRANSPORT OF FOLATES BY SLC21 ORGANIC ANION CARRIERS In the early 1990s, members of a new family of facilitative carriers (SLC21) began to be cloned that transport organic anions in epithelial tissues. Many of these carriers transport folates (Russel et al., 2002). The rat OAT- K1, expressed in liver and kidney, transports MTX with a higher affinity than for a variety of other organic anions including other folates, bromosulfopthalein (BSP), taurocholate, and probenecid. The MTX influx Kt for this Na+-independent system is 1 M (Saito et al., 1996). Rat OATK2, expressed in kidney tubules, also transports MTX and folic acid with apparent comparable affinities but with a lower affinity than for taurocholate. Both transporters are localized to the apical brush border membrane of renal tubular epithelial cells and appear to be involved in the reabsorption of folates from the glomerular filtrate (Masuda et al., 1999). hOAT2, within the group of OAT1–5, is expressed in liver and kidney and transports MTX and a spectrum of other organic anions (Sun et al., 2001). hOAT3 protein is expressed primarily in the kidney (basolateral membrane) and transports MTX (Na+ independent) with a Kt of 11 M (Cha et al., 2001). Both hLST2 (expressed in liver, primarily the basolateral membrane, and a variety of hepatic, colon, pancreatic, and gallbladder cancers) and hLST1 (not widely expressed in tumors) transport MTX (Abe et al., 2001). In the latter case, the transport Kt for MTX is 10–20 M. Hence, these organic anion transporters, along with others not as yet identified or fully characterized, likely play an important role in the transport of folates in the kidney, biliary tract, and some tumors. Although their specificity is broad, these carriers have affinities for folates that are comparable to that of RFC.

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IV. FOLATE TRANSPORTERS THAT OPERATE OPTIMALLY AT LOW pH: THE MECHANISM OF FOLATE TRANSPORT IN INTESTINAL CELLS Although RFC-mediated transport in murine leukemia cells has a pH optimum of 7.5 (Sierra et al., 1997; Sirotnak et al., 1968), this is not the case for folate transport in many other tissues that express RFC. The basis for this discrepancy is not clear. This issue is of special relevance to the mechanism of absorption of folates in the small intestine. RFC is highly expressed in intestine (Chiao et al., 1997) and is localized to the apical brush border of large and small intestinal cells of the mouse (Wang et al., 2001), whereas FRs are not expressed at all in intestine (Said et al., 1997, 2000; Weitman et al., 1992a). However, transport of folates into everted intestinal segments and membrane vesicles and into intestinal cells in vitro at physiological pH is minimal but increases as the pH is decreased to below 5.5 (Said et al., 1987a, 1997; Said and Strum, 1983; Selhub and Rosenberg, 1981). Increased transport at low pH is associated with a marked increase in the transmembrane folate gradient (Schron et al., 1985) and has been associated with a decreased transport Kt (Mason et al., 1990). Other properties of intestinal transport have notable differences from RFCmediated transport in murine leukemia cells. Whereas the affinity of RFC in murine leukemia cells for folic acid is one to two orders of magnitude less than that of MTX (Goldman et al., 1968) in intestinal cells and brush border membrane vesicles, the Kt for folic acid transport is equal to or lower than that of MTX or reduced folates (Said et al., 1987a, 1997). Whereas RFCmediated influx in murine leukemia cells is unchanged or slightly increased by metabolic poisons (Goldman, 1969), influx of MTX is substantially decreased in intestinal cells by these inhibitors (Rajgopal et al., 2001b; Said et al., 1997). Further confounding an understanding of these phenomena is the outcome of transfections of RFC into intestinal epithelial cells. Hence, transfection of mRFC into rat IEC-6 cells increased MTX transport activity at both low (5.5) and physiological (7.5) pHs. Further, the basal and transduced transport activities at pH 5.5 were suppressed with folic acid, but the activity at pH 7.5 was not (Rajgopal et al., 2001b). In another study, transfection of RFC into IEC-6 cells produced a marked increase in folic acid and 5-CH3-THF uptake at low pH but no change at physiological pH. The enhanced component of uptake at low pH was abolished when transfected cells were treated with RFC antisense oligonucleotides (Kumar et al., 1998). However, antisense did not reduce transport of folates below the level in the wild-type cells, raising the possibility that basal transport might be mediated, at least in part, by another process. Further evidence for the role of RFC in intestinal transport is the observation that an antibody to the amino- or carboxy-termini of RFC inhibited folate transport at low pH

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(Chiao et al., 1997). However, it is unclear as to how this occurs as these domains appear to be localized to the cytoplasm (Ferguson and Flintoff, 1999; Liu and Matherly, 2002). Finally, dietary deficiency of folate in the rat results in increased mucosal to serosal transport of folic acid in rat jejunal everted sacs. This is associated with increased expression of RFC message and protein; FR was not expressed under these conditions (Said et al., 2000). RFC is also localized to the membrane of hepatic cells (Wang et al., 2001), but transport of reduced folates and MTX appears to be mediated, at least in part, by different processes. Reduced folate transport increased at low pH and appeared to be coupled to proton transport, whereas the pH optimum for MTX transport was higher in basolateral membranes or intact hepatic cells (Horne, 1990, 1993; Horne et al., 1993; Horne and Reed, 1992). Transport of MTX in freshly isolated rat hepatocytes at physiological pH has properties distinct from RFC, in that it is Na+ dependent, is inhibited by ouabain, but is not inhibited by reduced folates (Gewirtz et al., 1980; Horne et al., 1976). On the other hand, there is evidence for an Na+-dependent transport route in freshly isolated hepatocytes for 5-CH3-THF at physiological pH that is inhibited by MTX (Horne et al., 1978). These studies are complicated by the presence of other facilitative organic anion carriers as well as MRP exporters expressed in hepatic cells. RFC is localized to the apical membrane of retinal pigment epithelial cells, and brush border membrane vesicles from these cells showed negligible transport of 5-CH3-THF at physiological pH. However, there was a marked increase in the initial rate of uptake and net transport as the pH was reduced below 6.0, consistent with proton cotransport (Chancy et al., 2000; Huang et al., 1997). A low pH transport route for folates has also been identified in a variety of other cells. Folate transport in a human colonic epithelial cell line is enhanced at low pH, due to a decrease in the influx Kt (Kumar et al., 1997). Net uptake of MTX was much greater at low pH than physiological pH in human prostate cancer cells (Horne and Reed, 2001). Both low and physiological pH folate transport activities were detected in rat 3T3 cells. Transfection with c-Myc and h-Ras increased RFC expression and transport at physiological pH, whereas transport at the low pH was decreased, suggesting that the latter process is distinct from RFC (Kuhnel et al., 2000). Evidence for a low pH transporter in murine leukemia cells has come from studies with MTX-resistant cell lines with impaired RFCmediated transport, also suggesting that this activity is unrelated to RFC. In one such murine leukemia cell line, influx of MTX, folic acid, and 5-CHOTHF increased as the pH was decreased to 6.2, due to a decrease in the influx Kt and an increase in influx Vmax. This was not observed for 5-CH3THF. The Ki for folic acid influx was comparable to that of the other folates and the transport activity at low pH was markedly diminished with energy

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inhibitors (Henderson and Strauss, 1990). A low pH folate influx activity was also identified in another murine leukemia cell line in which RFC activity was abolished due to a point mutation in the third transmembrane domain (Sierra and Goldman, 1998). In this case, transport of MTX, 5CHO-THF, folic acid, and 5-CH3-THF was all enhanced at low pH, although the pH optimum varied among the folates. The Kt for MTX influx at pH 6.0 was an order of magnitude lower than at physiological pH, but the Kt for 5-CH3-THF influx was not changed. This influx activity was suppressed by metabolic poisons and was present for both folic acid and MTX at the same levels at low pH in both wild-type cells and the mutated line. Further, the low pH transport activity was not appreciably increased in a murine leukemia cell line transfected to high levels of RFC (Sierra and Goldman, 1998). Finally, other studies demonstrate conclusively that there is a low pH folate transport route independent of RFC, in that this activity is present in embryonic fibroblasts (R. Zhao and I. D. Goldman, unpublished observation) obtained from embryos of RFC / genetargeted mice (Zhao et al., 2001). Taken together, these observations indicate that RFC expression can result in transport activities at both physiological and low pH. There is, in addition, at least one other folate transport activity at low pH that is independent of RFC. It is not clear at this point as to how all these observations can be reconciled; however, there are several possibilities. One of the low pH activities could represent a tissue-specific posttranslationally modified form of RFC, or it could be the result of an association of RFC with another protein that modifies its function. Alternatively, a low pH transporter distinct from RFC could have a basal level of activity that is enhanced by an interaction with RFC or could function totally independent of RFC. The physiological utility of a folate transport system that has a low pH optimum is best explained for the gut where there is a low pH (6) microclimate at the absorptive surface of the small intestine that is different from the much higher pH present in the bulk fluid within the intestinal lumen (Lucas et al., 1975; Said et al., 1987b).

V. THE FAMILY OF FOLATE RECEPTORS (FRS) A. STRUCTURES AND SPECIFICITIES OF FRS a, b, AND Y

FR designates a family of high-affinity folate-binding proteins encoded by three distinct genes designated , , and (Brigle et al., 1992; Elwood, 1989; Lacey et al., 1989; Ratnam et al., 1989; Sadasivan and Rothenberg, 1989; Shen et al., 1994), localized to chromosome 11q13.3–q13.5 (Ragoussis et al., 1992). The cDNA for FR was originally isolated from human KB cells, placenta, and CaCo-2 cells (Elwood, 1989; Lacey et al., 1989;

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Sadasivan and Rothenberg, 1989), whereas those for FR and were obtained from human placenta (Ratnam et al., 1989) and malignant hematopoietic cells (Shen et al., 1994), respectively. The mature FRs are homologous (68–79% identical in amino acid sequence) proteins that contain from 229 to 236 amino acids and 2 (, ) or 3 () N-glycosylation sites. The molecular masses of the mature glycosylated proteins approximate 40 kDa (Luhrs, 1991). FR and FR are both glycosylphosphatidylinositol (GPI)-anchored proteins whereas FR contains no GPI anchor due to the lack of an efficient signal sequence for GPI modification; this receptor is secreted (Shen et al., 1995). An additional form, designated FR 0 , is identical to FR except for the presence of a 2-bp deletion that results in a truncated polypeptide due to the deletion of 138 carboxyl-terminal amino acids (Shen et al., 1994). The FRs typically show a high affinity for folic acid (Kd 1 nM ) and a comparatively low affinity for MTX (Kd > 100 nM) (Jansen, 1999). FR and from both human and murine sources (Brigle et al., 1994; Wang et al., 1992) exhibit differing specificities for the natural (6S) versus nonphysiological (6R) diastereomers of 5-CH3-THF and 5-CHO-THF. Thus, FR has a binding preference for the (6R) over the (6S) 5-CH3-THF stereoisomer and shows a significantly lower binding affinity for (6S) 5-CH3-THF than the FR. FR also had an increased affinity over FR for a number of antifolate structures including MTX and 5,10-dideazatetrahydrofolate (Brigle et al., 1994). These differences in binding affinities have been localized to Leu-49, Phe-104, and Gly-164 in FR as replacement of these residues with the corresponding amino acids from FR (Ala, Val, and Glu, respectively) effectively reconstitutes the FR binding phenotype (Maziarz et al., 1999). B. TRANSCRIPTIONAL AND POSTTRANSCRIPTIONAL REGULATION OF THE FRs

The FR genes are similar in size (5–6 kb) and in their structures for the coding exons (Fig. 3). However, they diverge in the structures of the upstream noncoding and regulatory regions (Elwood et al., 1997; Sadasivan et al., 1994; Saikawa et al., 1995; Wang et al., 1998). Thus, FR and each consists of five exons and four introns, with the translation initiation site in exon 2 (Sadasivan et al., 1994; Wang et al., 1998); FR contains seven exons and introns, with the translation start site in exon 4 (Elwood et al., 1997; Saikawa et al., 1995). As noted earlier, FR 0 appears to result from a polymorphism in the FR gene (Wang et al., 1998). The basal promoters for the FR, , and genes have been characterized. The 50 transcript heterogeneity reported for the FR gene results from at least two TATA-less promoters, designated promoter P1, upstream of exon 1, and promoter P4, upstream of exon 4 (Elwood et al., 1997; Saikawa et al.,

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FIGURE 3. Organization of the FR genes. A schematic is presented depicting the organization of the genes for the human FR, , and isoforms based on published studies (Elwood et al., 1997; Sadasivan et al., 1994; Saikawa et al., 1995; Wang et al., 1998). The numbers indicate the lengths of the exons and introns in base pairs. P4 and P are TATA-less promoters. Functional cis elements required for basal promoter activity are indicated in parentheses. (Courtesy of Dr. Manohar Ratnam, Medical College of Ohio.)

1995). Only P4 has been significantly characterized. Transcriptional regulation of P4 involves three clustered GC-rich sequences that bind Sp1 (Saikawa et al., 1995). FR promoter usage is apparently tissue selective as, by RNase protection, transcripts transcribed from promoter 4 were highest in KB cells and lung, and transcripts from the P1 promoter were most prevalent in kidney and cerebellum (Elwood et al., 1997). FR has been reported to be regulated by a single noncanonical Sp1 binding sequence linked to a tandemly repeated GGAAG motif that binds an ets GA-binding protein (Sadasivan et al., 1994). Critical regulatory roles for Sp1 and ets elements in the FR basal promoter were also demonstrated (Wang et al., 1998). Differences in promoter structures and cis elements may partly contribute to the specific tissue expression patterns reported for the FRs. Thus, FR is expressed on the luminal surface of normal epithelial cells and is frequently overexpressed in ovarian and uterine carcinomas (Coney et al., 1991; Weitman et al., 1992a). FR exhibits a comparatively narrow tissue

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distribution, being restricted to hematopoietic tissues (Shen et al., 1994, 1995). FR is not expressed in most normal tissues but is expressed in placenta, thymus, and spleen, and in a large percentage of acute myeloid leukemias (AMLs) (Ross et al., 1999; Shen et al., 1994). A nonfunctional form of FR is expressed in CD-34+ human hematopoietic cells (Reddy et al., 1999). In AML, expression of FR can be markedly increased by retinoid receptor agonists including all-trans-retinoic acid (ATRA) (Wang et al., 2000). This response did not involve terminal differentiation nor inhibition of cell proliferation and was not observed in nonmyeloid cells. A characteristic of FR involves its inducibility in cells cultured in low nanomolar concentrations of folates, a response reflecting increased transcription (Sadasivan et al., 2002), decreased promoter hypermethylation (Hsueh and Dolnick, 1994), and posttranscriptional effects on transcript stability (Hsueh and Dolnick, 1993; Sadasivan et al., 2002). In KB cells, increased FR transcript stability was associated with binding of cytosolic proteins to both the 50 and 30 regions (Sadasivan et al., 2002). Moreover, a 46–kDa cytosolic protein in cervical carcinoma cells appeared to regulate FR at the translational level by binding an 18-bp cis element in the 50 -UTR (Sun and Antony, 1996). C. PHYSIOLOGICAL AND PHARMACOLOGICAL ROLES AND MECHANISMS OF FR-MEDIATED FOLATE UPTAKE

In general, the physiological roles of the FRs are unclear except in a few cases. For certain tissues, FRs likely function to internalize folates. For instance, FR that is highly expressed on the apical brush border of renal tubular epithelial cells might play a role in renal absorption of folates (Weitman et al., 1992b). FR may participate in folate uptake across the placenta (Antony, 1996). Although cellular uptake of folates by FRs is inefficient compared to RFC because of its very low rate of cycling (Sierra et al., 1997; Spinella et al., 1995), an unambiguous transport role manifests at high levels of FR expression as is found in certain malignant tissues (Coney et al., 1991; Ross et al., 1994, 1999; Weitman et al., 1992a). Further, FR (but not FR) knockout mice exhibit fatal morphological abnormalities consistent with their critical role as folate transporters in mouse development (Piedrahita et al., 1999). Mechanistically, folate uptake by membrane bound FRs is envisaged to involve an endocytotic process whereby folates bind to FRs on the plasma membrane, which then invaginate to form vesicles that migrate to the cytosol. Following this, the vesicles acidify, resulting in the dissociation of the folate–FR complex and, finally, the entry of the folate ligand into the cytosol (Kamen et al., 1988; Rothberg et al., 1990). The classic receptormediated endocytotic pathway involves clathrin-coated pits on the cell surface and there is some evidence that FR is also localized, in part, to

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these sites (Mukherjee et al., 1997; Rijnboutt et al., 1996). However, based on studies in MA104 monkey kidney epithelial cells, a hypothesis for another uptake process termed ‘‘potocytosis’’ was proposed. This involves the recycling of clusters of FRs in membrane-associated vesicular structures, termed caveolae, between the cell surface and acidic endocytic compartments with release of ligand into the cytosol (Anderson et al., 1992). Although this process was initially proposed to be linked to RFC (Kamen et al., 1991), subsequent studies demonstrated FR transport activity in cells in which RFC is not functional (Spinella et al., 1995). Further, FRs have also been shown to cluster in membrane sites other than caveolae (Rijnboutt et al., 1996; Wu et al., 1997). Indeed, although many of the basic features of FR endocytotic uptake are generally accepted, the concept of potocytosis and the association of FRs with caveolae remains controversial. The current status of the understanding of potocytotosis was recently reviewed (Mineo and Anderson, 2001). The elevation of FR in certain tumor types (e.g., ovarian) has prompted an interest in potentially using the receptor as a means of tumor-specific delivery of cytotoxic antifolates or folate-conjugated radiopharmaceuticals, imaging agents, small molecule cytotoxins, antisense oligonucleotides, or liposomes (reviewed in Leamon and Low, 2001). Because FR is uniquely expressed at high levels on myeloid leukemia cells, this has been a focus for the development of targeted therapies. Of particular interest is the application of folate-coated liposomal doxorubicin directed to FR expressing AML cells and the in vitro and in vivo potentiation of antileukemic activity upon treatment with ATRA (see earlier) (Pan et al., 2002).

VI. MULTIDRUG RESISTANCE-ASSOCIATED PROTEINS (MRPS) AND THEIR IMPACT ON THE TRANSPORT OF FOLATES At the same time that the characteristics of RFC-mediated transport began to emerge, data were also appearing indicating that there were energydependent processes that limit MTX accumulation in cells. This was based upon the observation that energy inhibitors augmented net uptake of MTX and aminopterin, an effect due almost entirely to inhibition of MTX efflux (Goldman, 1969; Hakala, 1965). It soon became apparent that these observations were related to inhibition of exporters distinct from RFC that transport a variety of molecules out of cells. Using a spectrum of structurally diverse inhibitors, it was possible to dissect export into several distinct mechanisms in intact cells; the characteristics of these processes were established by studies of ATP-dependent folate transport in inside-out membrane vesicles (Henderson et al., 1986, 1994; Henderson and Tsuji,

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1987; Henderson and Zevely, 1984b; Saxena and Henderson, 1996; Schlemmer and Sirotnak, 1992). It is now evident that the transporters identified in this way were members of the MRP family of exporters, nine of which have been cloned to date (Borst and Elferink, 2002; Dean et al., 2001; Litman et al., 2001). Evidence for a role of MRPs in the transport of folates comes from studies on cells transfected with cDNAs for these genes and with inside-out membrane vesicles prepared from these cells. It is clear from these studies that MRPs 1–4 transport folates and antifolates (Chen et al., 2002; Hirohashi et al., 1999; Hooijberg et al., 1999; Kusuhara et al., 1998; Zeng et al., 2000, 2001). Vesicles from Sf9 insect cells expressing MRP4 transport folates; however in another study with MRP4-transfected MCF-7 cells, MTX did not seem to be transported by this exporter (Adachi et al., 2002). This suggests that cell-specific mechanisms may influence transport by MRP4. There is no evidence to date indicating that MRPs 5–9 transport folates. In studies with membrane vesicles from cells transfected with MRPs 1–4, low-affinity (i.e., millimolar Kts) ATP-dependent transport was detected for assorted folate substrates, including MTX, folic acid, and 5CHO-THF (Chen et al., 2002; Zeng et al., 2000, 2001). The affinities of the MRPs for folates and antifolates are orders of magnitude lower than those of RFC and some organic anion carriers. Despite this, their high capacities for transport and their elevated levels in many tissues and tumors should confer an ability to produce a substantial depression of concentrative transport of folates. Indeed, inhibitors of the MRPs have been shown to enhance net cellular uptake of these compounds, suggesting that these exporters are widely expressed and function to exclude folates and antifolates, along with a broad spectrum of metabolites and xenobiotics, from cells. Virtually all natural folates within cells represent polyglutamyl derivatives, which are the active THF cofactors. In most cases, these are the preferred substrates in folate-dependent reactions (Schirch and Strong, 1989). The formation of polyglutamate derivatives of folates and antifolates is mediated by folylpolyglutamate synthetase (Andreassi and Moran, 2002). Polyglutamate derivatives of folates and antifolates accumulate to levels in cells that far exceed their extracellular concentrations, because these congeners are retained within cells and are poor substrates for RFC (Fry et al., 1982a; Jolivet and Chabner, 1983). As expected, studies with membrane vesicles derived from cells transfected to achieve high expression of MRPs 1–4 indicate that the diglutamate and higher polyglutamate derivatives of folates are not substrates for these exporters (Chen et al., 2002; Zeng et al., 2001). However, it is likely that MRPs influence the rate and extent of folate and antifolate polyglutamate formation by regulating the intracellular concentration of free monoglutamyl substrate available for folylpolyglutamate synthetase. This is consistent with the observation that

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resistance to antifolates is enhanced in cells transfected with these transporters (Hooijberg et al., 1999). The major impact of MRPs is to enhance substrate export from cells, and this is associated with a decrease in steady-state levels. However, as substrates enter cells, ABC exporters can associate with substrate either within the cell membrane or after the substrate is released into the cytosol. In the case of the P-glycoprotein family of exporters that transports lipidsoluble molecules, the transporter can capture and eject its substrate within the membrane and thereby slow its unidirectional flux into the cell (Stein, 1997). On the other hand, MRPs appear to interact with their hydrophilic substrates within the cytosol after release from the carriers that mediate their influx. Hence, for P-glycoprotein, metabolic poisons can markedly increase influx (Stein, 1997), whereas for MRPs, the stimulatory effect on influx is often small (Fry et al., 1980b; Goldman, 1969). Studies have begun to explore the contributions of MRP expression to clinical resistance to antimetabolites including MTX and thiopurines. RTPCR analyses of 34 B-precursor and 16 T-ALL specimens showed detectable MRPs 1, 4, and 5 in virtually all specimens (R. M. Flatley and L. H. Matherly, unpublished); however, the levels of MRPs 4 and 5 were particularly variable. MRPs 2, 3, and 6 were nearly uniformly undetectable in primary ALL samples. Thus, the extent to which variations in the levels of individual MRPs may contribute to differential patterns of MTX (and thiopurine) accumulations and drug resistance in ALL is uncertain. However, variations in relative drug efflux, in general, could easily contribute to the poor correlations between hRFC transcript levels and steady-state MTX accumulations in a small number of B-precursor ALLs and the majority of T-ALLs (Zhang et al., 1998c). MRPs are expressed widely in epithelial cells and their expression at the apical versus the basolateral membranes is a critical determinant of the vectorial flow of their substrates. This is considered in Section IX.

VII. TRANSPORT OF FOLATES BY OTHER ABC EXPORTERS There is now evidence emerging that there are other exporters of the ABC gene family that transport MTX and, likely, folates, as well. Of potential importance is the ABC half-transporter, breast cancer resistance protein (BCRP) (Rocchi et al., 2000). Over expression of this gene has been identified in tumor cell lines selected for resistance to the antracycline, mitoxantrone, that are also cross-resistant to MTX due to decreased net accumulation of MTX (Volk et al., 2000). Arg-482 was implicated as a key determinant of substrate specificity for BCRP (Allen et al., 2002; Honjo et al., 2001). Mutations at position 482 appear to enhance export of

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anthracyclines and increase resistance; however, they appear to decrease export of MTX (Allen et al., 2002; Volk et al., 2002). P-glycoprotein MDR1 has been evaluated as a potential route of folate and antifolate export. In cells that express RFC, virtually no folate efflux is mediated by MDR1, even when this protein is overexpressed. For instance, when cells overexpress MDR1 after selection for resistance to trimetrexate (a lipid-soluble antifolate that does not utilize RFC) or doxorubicin, resistance to a variety of cytotoxins that are substrates for this exporter is observed; however, there is no cross-resistance to MTX (Arkin et al., 1989; Assaraf et al., 1989). Nonetheless, when high concentrations of MTX are employed so that passive diffusion is a major component of uptake, overexpression of MDR1 has been associated with MTX resistance (Norris et al., 1996). MDR1-mediated resistance to MTX is most likely to occur under conditions in which RFC function is impaired (Gifford et al., 1998). Hence, when MDR1 was transfected into cells with and without functional RFC, resistance was augmented only in the cells with impaired RFCmediated transport (De Graaf et al., 1996).

VIII. FACTORS THAT INFLUENCE CONCENTRATIVE FOLATE TRANSPORT IN CELLS Concentrative transport of folates is determined by the net effect of complementary and opposing transporters that determine the free monoglutamyl folate level in cells (Fig. 4). Several studies have evaluated the impact of alterations in expression of RFC on concentrative transport. When RFC was transfected into murine leukemia cells that express both RFC and energy-dependent exporter activity, influx of MTX was markedly enhanced, export was also increased but only by a factor approximately half that of influx, and the rate of achievement of steady state was accelerated. However, the steady-state level was increased by a factor of only two. Hence, high expression of carrier produced a substantial increase in bidirectional folate fluxes, but this was accompanied by only a small effect on net concentrative transport (Zhao et al., 1997). If this was a closed system in which RFC was the sole route of MTX transport, then an increase in carrier expression could accelerate only the rate at which the steady-state was reached, but not the absolute steady-state level achieved, assuming that the organic phosphate gradient or other driving forces were not altered. This changes if there are additional transport routes, in this case energydependent exporters. Under these conditions, as RFC expression is increased, the ratio of transport via this pathway to MRP-mediated export increases and net concentrative transport increases. The extent to which overexpression of RFC modulates concentrative transport varies from cell

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FIGURE 4. A schema of the currently known transport pathways for folates. There are two classic facilitative carrier systems that are bidirectional folate transporters. These include the reduced folate carrier (RFC) that transports folates uphill into cells by a cotransport with intracellular anions, in particular, organic phosphates (Section II.C.2). Several organic anion transporters (OATs) have affinities for folates comparable to RFC and, to the extent to which they are concentrative, may produce a transmembrane gradient for folates (Section III.) The folate receptors (FR and FR) transport folates unidirectionally into cells by an endocytotic mechanism that has been termed potocytosis (Section V.C). There are at least two processes with low pH optima that transport folates into cells. One is linked to the expression of RFC and at least one other is not. It is unclear as to whether these processes are uni- or bidirectional (Section IV). Four members of the family of multidrug resistance-associated proteins (MRPs) export folates (Section VI) along with a member of another family of half-transporters, the breast cancer resistance protein (BCRP) (Section VII). The relative magnitudes of transport fluxes mediated by processes that are concentrative versus fluxes mediated by transporters that actively export folates will determine the transmembrane gradients for folates achieved across the cell membrane (Section VIII). The localization of these transporters at the apical versus basolateral membranes will determine the net fluxes of folates across epithelia (Section IX).

line to cell line and is due, at least in part, to the relationship between the relative rates of RFC and MRP transport fluxes. This likely explains the results in ZR-75-1 breast cancer cells for which transfection of RFC produced an increase in the steady-state MTX level that was comparable to the increase in influx (Sharif et al., 1998). In both this and in the case of the murine transfectants (see earlier), the impact of overexpression of RFC on concentrative transport increased as the extracellular level of transport substrate was elevated (Sharif et al., 1998; Zhao et al., 1997). However, the ratio of steady-state intracellular to extracellular drug concentration decreased under these conditions, as efflux mediated by RFC and the MRP exporters saturates at much higher levels than RFC-mediated influx. An interesting human leukemia (CEM-7A) cell line was identified in which RFC was overexpressed, but the transport phenotype was quite

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different than observed for murine leukemia cells transfected with RFC. These cells were selected for growth in low levels of 5-CHO-THF and developed a marked and comparable increase in influx and net transport of MTX. There was an increase in influx Vmax but no change in influx Kt or efflux kinetics. Affinity labeling was consistent with increased expression of RFC (Jansen et al., 1990). However, based on the model described earlier, these findings were unlikely to be attributed to increased activity of RFC, alone, which should have produced some increase in the bidirectional fluxes. Rather, the data suggested that during the process of selection there was another alteration—such as a decrease in MRP activity that slowed the efflux process. Indeed, a recent study substantiates this prediction with the demonstration that there is a marked reduction in the expression of MRP1 in these cells (Assaraf et al., 2002). Of further interest was the observation that incubation of CEM-7A cells for brief intervals with physiological levels of reduced folates markedly decreased MTX transport, an effect that required preservation of dihydrofolate reductase activity (Jansen et al., 1990). The basis for this apparent regulatory change remains to be clarified. The important role that MRPs play in suppressing concentrative transport of antifolates has led to approaches to inhibit their activity in order to augment net uptake of drug. Accordingly, probenecid, which has only a low affinity for RFC and FRs but is an MRP substrate, has been used to augment the net transport of MTX and its polyglutamation in tumor cells (Fry et al., 1982b; Henderson and Zevely, 1985; Sirotnak et al., 1981). Other antineoplastics, such as etoposide and vincristine that are transport substrates for the MRPs, have also been shown to block MTX export (Fry et al., 1982b; Yalowich et al., 1982). This consideration becomes more complex when cells express folate transporters in addition to RFC and the MRPs, such as the FRs and/or members of the SLC21 family of organic anion transporters (Section III). In the latter case, this would occur primarily in epithelial cells and not only would these additional elements affect concentrative transport, but they would also alter vectorial flows across epithelia. This is described in the next section.

IX. THE LOCALIZATION OF FOLATE TRANSPORTERS IN CELLS AND THEIR ROLES IN VECTORIAL TRANSPORT IN EPITHELIA The relative localization of transporters in apical versus basolateral membranes is an important determinant of the vectorial flows of substrates across epithelia (Cui et al., 2001). At least one of the transporters involved in

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this process must provide the driving force for this flow. Some information is emerging on the localization of folate transporters in epithelial cells, and this can now be integrated with information on the localization of other transporters in these tissues. FR is localized to the basolateral membrane of retinal pigment epithelial cells, whereas RFC is localized to the apical brush border membrane, an orientation that would result in the vectorial flow of folates from the choroidal blood to the neural retina (Chancy et al., 2000). However, in other cases, these relationships can be very complex in that they offer possibilities for bidirectional fluxes of folates. For instance, FR is expressed in the brush border of renal tubular epithelial cells (Weitman et al., 1992b) as is Oat-K1 (Masuda et al., 1997), whereas the rOAT1 and hOAT3 organic anion transporters are localized to the basolateral membrane (Cha et al., 2001; Tojo et al., 1999). MRP1 and MRP3 are also localized to the basolateral membrane of renal tubular epithelial cells (Evers et al., 1996; Kool et al., 1999; Raggers et al., 1999; Scheffer et al., 2002). The location of RFC at the basolateral membrane represents a parallel pathway to the perinephron compartment (Wang et al., 2001). MRP2 is localized to the brush border membrane of proximal renal tubular cells (Schaub et al., 1997). Hence, MRP2 would provide a driving force for the secretion of folates into the nephron lumen whereas MRP1 and MRP3 would provide a driving force for reabsorption. Because folates are reabsorbed in the kidney, the net effect of these opposing forces must favor reabsorption. RFC is expressed in the apical brush border membrane of intestinal cells (Wang et al., 2001), whereas FRs are not (Said et al., 2000). However, MRP2 is expressed in the intestinal apical brush border membrane, thus opposing the actions of RFC (Fromm et al., 2000; Mottino et al., 2000). Clearly, there are many potential transporters that can participate in the flux of folates across the intestinal epithelium that in sum represent a strong absorptive driving force. RFC is also highly expressed at the apical brush border of the choroid plexus (Wang et al., 2001) whereas FR is localized to the basolateral membrane (Weitman et al., 1992b), both of which favor the intracellular accumulation of folates. However, a variety of other transporters present in these cells are likely to provide a net driving force for vectorial flows of folates. For instance, MRP1 is localized to the basolateral membrane and exports drugs out of the cerebrospinal fluid (Rao et al., 1999; Wijnholds et al., 2000). MRPs are localized to hepatic canalicular membrane and play a key role in the biliary excretion of folates (Keppler and Konig, 1997; Konig et al., 1999). Indeed, in the absence of MRP2, there is markedly impaired biliary excretion of a variety of organic anions including MTX (Paulusma et al., 1996). Although RFC is expressed on hepatic cells, its localization in apical versus basolateral membranes, based upon histochemical analysis, is not clear (Wang et al., 2001).

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X. THE ROLE OF FOLATE TRANSPORTERS IN MOUSE DEVELOPMENT Both FR and RFC are required for mouse development. For the former, homozygous deletion of the gene was embryonic lethal at the stage of neural tube formation (Piedrahita et al., 1999). Deletion of FR or the deletion of one FR allele resulted in normal development. Likewise, although deletion of one RFC allele produced no phenotype, targeting of both alleles was embryonic lethal at a very early stage of development (Zhao et al., 2001). Some of the latter animals could, nonetheless, be brought to live birth by supplementing the dams with high levels of folic acid. Those animals went on to die within 1–2 weeks due to failure of the hematopoietic organs (bone marrow, spleen, thymus) without any pathological changes in other tissues including the intestine. These data are consistent with the presence of other pathways for folate uptake in the tissues that were spared. This is also suggested by studies in which the folic acid growth requirement is minimally changed in cell lines for which RFC has been mutated and transport function has been markedly impaired (Zhao et al., 1999a).

ACKNOWLEDGMENT This work was supported by Grants CA53535, CA76641, and CA82621 from the National Cancer Institute, National Institutes of Health.

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Williams, F. M. R., Murray, R. C., Underhill, T. M., and Flintoff, W. F. (1994). Isolation of a hamster cDNA clone coding for a function involved in methotrexate uptake. J. Biol. Chem. 269, 5810–5816. Witt, T. L., and Matherly, L. H. (2002). Identification of lysine-411 in the human reduced folate carrier as an important determinant of substrate selectivity and carrier function by systematic site directed mutagenesis. Biochem. Biophys. Acta 1567, 56–62. Wong, S. C., Proefke, S. A., Bhushan, A., and Matherly, L. H. (1995). Isolation of human cDNAs that restore methotrexate sensitivity and reduced folate carrier activity in methotrexate transport-defective Chinese hamster ovary cells. J. Biol. Chem. 270, 17468–17475. Wong, S. C., McQuade, R., Proefke, S. A., Bhushan, A., and Matherly, L. H. (1997). Human K562 transfectants expressing high levels of reduced folate carrier but exhibiting low transport activity. Biochem. Pharmacol. 53, 199–206. Wong, S. C., Zhang, L., Proefke, S. A., and Matherly, L. H. (1998). Effects of the loss of capacity for N-glycosylation on the transport activity and cellular localization of the human reduced folate carrier. Biochem. Biophys. Acta 1375, 6–12. Wong, S. C., Zhang, L., Witt, T. L., Proefke, S. A., Bhushan, A., and Matherly, L. H. (1999). Impaired membrane transport in methotrexate-resistant CCRF-CEM cells involves early translation termination and increased turnover of a mutant reduced folate carrier. J. Biol. Chem. 274, 10388–10394. Worm, J., Kirkin, A. F., Dzhandzhugazyan, K. N., and Guldberg, P. (2001). Methylationdependent silencing of the reduced folate carrier gene in inherently methotrexate-resistant human breast cancer cells. J. Biol. Chem. 276, 39990–40000. Wright, J. E., Vaidya, C. M., Chen, Y., and Rosowsky, A. (2000). Efficient utilization of the reduced folate carrier in CCRF-CEM human leukemic lymphoblasts by the potent antifolate N(alpha)-(4-amino-4-deoxypteroyl)-N(delta)-hemiphthaloyl-L-ornithine (PT523) and its B-ring analogues. Biochem. Pharmacol. 60, 41–46. Wu, M., Fan, J., Gunning, W., and Ratnam, M. (1997). Clustering of GPI-anchored folate receptor independent of both cross-linking and association with caveolin. J. Membr. Biol. 159, 137–147. Yalowich, J. C., Fry, D. W., and Goldman, I. D. (1982). Teniposide (VM-26)- and etoposide (VP-16-23)-induced augmentation of methotrexate transport and polyglutamylation in Ehrlich ascites tumor cells in vitro. Cancer Res. 42, 3648–3653. Yang, C.-H., Sirotnak, F. M., and Dembo, M. (1984). Interaction between anions and the reduced folate/methotrexate transport system in L1210 cell plasma membrane vesicles: Directional symmetry and anion specificity for differential mobility of loaded and unloaded carrier. J. Membr. Biol. 79, 285–292. Yang, C. H., Sirotnak, F. M., and Mines, L. S. (1988). Further studies on a novel class of genetic variants of the L1210 cell with increased folate analogue transport inward. Transport properties of a new variant, evidence for increased levels of a specific transport protein, and its partial characterization following affinity labeling. J. Biol. Chem. 263, 9703–9709. Yang, R., Sowers, R., Mazza, B., Healey, J. H., Huvos, A., Grier, H., Bernstein, M., Beardsley, G. P., Krailo, M. D., Devidas, M., Bertino, J. R., Meyers, P., and Gorlick, R. (2003). Sequence alterations in the reduced folate carrier are observed in osteosarcoma tumor samples. Clin. Cancer Res. 9, 837–844. Zeng, H., Liu, G., Rea, P. A., and Kruh, G. D. (2000). Transport of amphipathic anions by human multidrug resistance protein 3. Cancer Res. 60, 4779–4784. Zeng, H., Chen, Z. S., Belinsky, M. G., Rea, P. A., and Kruh, G. D. (2001). Transport of methotrexate (MTX) and folates by multidrug resistance protein (MRP) 3 and MRP1: Effect of polyglutamylation on MTX transport. Cancer Res. 61, 7225–7232. Zhang, L., Wong, S. C., and Matherly, L. H. (1998a). Structure and organization of the human reduced folate carrier gene. Biochim. Biophys. Acta 1442, 389–393.

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Zhang, L., Wong, S. C., and Matherly, L. H. (1998b). Transcript heterogeneity of the human reduced folate carrier results from the use of multiple promoters and variable splicing of alternative upstream exons. Biochem. J. 332, 773–780 (pt.3). Zhang, L., Taub, J. W., Williamson, M., Wong, S. C., Hukku, B., Pullen, J., Ravindranath, Y., and Matherly, L. H. (1998c). Reduced folate carrier gene expression in childhood acute lymphoblastic leukemia: Relationship to immunophenotype and ploidy. Clin. Cancer Res. 4, 2169–2177. Zhao, R., Seither, R., Brigle, K. E., Sharina, I. G., Wang, P. J., and Goldman, I. D. (1997). Impact of overexpression of the reduced folate carrier (RFC1), an anion exchanger, on concentrative transport in murine L1210 leukemia cells. J. Biol. Chem. 272, 21207–21212. Zhao, R., Assaraf, Y. G., and Goldman, I. D. (1998a). A mutated murine reduced folate carrier (RFC1) with increased affinity for folic acid, decreased affinity for methotrexate, and an obligatory anion requirement for transport function. J. Biol. Chem. 273, 19065–19071. Zhao, R., Assaraf, Y. G., and Goldman, I. D. (1998b). A reduced carrier mutation produces substrate-dependent alterations in carrier mobility in murine leukemia cells and methotrexate resistance with conservation of growth in 5-formyltetrahydrofolate. J. Biol. Chem. 373, 7873–7879. Zhao, R., Sharina, I. G., and Goldman, I. D. (1999a). Pattern of mutations that results in loss of reduced folate carrier function under antifolate selective pressure augmented by chemical mutagenesis. Mol. Pharmacol. 56, 68–76. Zhao, R., Gao, F., and Goldman, I. D. (1999b). Discrimination among reduced folates and methotrexate as transport substrates by a phenylalanine substitution for serine within the predicted eighth transmembrane domain of the reduced folate carrier. Biochem. Pharmacol. 58, 1615–1624. Zhao, R., Gao, F., Wang, Y., Diaz, G. A., Gelb, B. D., and Goldman, I. D. (2000a). Impact of the reduced folate carrier on the accumulation of active thiamin metabolites in murine leukemia cells. J. Biol. Chem. 276, 1114–1118. Zhao, R., Gao, F., Liu, L., and Goldman, I. D. (2000b). The reduced folate carrier in L1210 murine leukemia cells is a 58 kDa protein. Biochim. Biophys. Acta 1466, 7–10. Zhao, R., Gao, F., Wang, P. J., and Goldman, I. D. (2000c). Role of the amino acid 45 residue in reduced folate carrier function and ion-dependent transport as characterized by sitedirected mutagenesis. Mol. Pharmacol. 57, 317–323. Zhao, R., Russell, R. G., Wang, Y., Liu, L., Gao, F., Kneitz, B., Edelman, W., and Goldman, I. D. (2001). Rescue of embryonic lethality in reduced folate carrier-deficient mice by maternal folic acid supplementation reveals early neonatal failure of hematopoietic organs. J. Biol. Chem. 276, 10224–10228. Zhao, R., Gao, F., and Goldman, I. D. (2002). Reduced folate carrier transports thiamine monophosphate: An alternative route for thiamin delivery into mammalian cells. Am. J. Physiol. Cell Physiol. 282, C1512–C1517.

13 Vitamin A and Infancy Biochemical, Functional, and Clinical Aspects

Silverio Perrotta,* Bruno Nobili,* Francesca Rossi,* Daniela Di Pinto,* Valeria Cucciolla,{ Adriana Borriello,{ Adriana Oliva,{ and Fulvio Della Ragione{ *

{

Department of Pediatrics and Department of Biochemistry and Biophysics ‘‘F. Cedrangolo,’’ Medical School, Second University of Naples, Naples, Italy

I. A Premise II. Vitamin A: Intestinal Digestion, Absorption, and Tissue Delivery A. The Digestion of Retinyl Esters B. The Uptake and Reesterification of Retinol in Enterocytes C. Incorporation of the Resulting Retinyl Esters into Chylomicrons and Their Further Metabolism III. Intracellular Metabolism A. Synthesis of Retinoic Acid and Additional Derivatives of Oxidative Pathway B. Mechanisms of Biosynthesis of Retroretinoids IV. Retinol and Embryogenesis: Mechanism of Action and Importance A. Molecular Bases of Retinoids Activity B. Vitamin A as a Critical Molecule in Human Development

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V. Retinol and Infancy A. Recommended Dietary Allowances B. Vitamin A Status in Pregnant and Lactating Women C. Very Low Birth Weight VI. Altered Vitamin A Levels and Childhood Pathologies A. Vitamin A Deficiency B. Vitamin A Deficiency in Pregnant and Lactating Women C. Hypervitaminosis A in Childhood D. Teratogenic Effects of Vitamin A E. A Case of Hypervitaminosis A in an Infant VII. Few Final Considerations References

Vitamin A is a very intriguing natural compound. The molecule not only has a complex array of physiological functions, but also represents the precursor of promising and powerful new pharmacological agents. Although several aspects of human retinol metabolism, including absorption and tissue delivery, have been clarified, the type and amounts of vitamin A derivatives that are intracellularly produced remain quite elusive. In addition, their precise function and targets still need to be identified. Retinoic acids, undoubtedly, play a major role in explaining activities of retinol, but, recently, a large number of physiological functions have been attributed to different retinoids and to vitamin A itself. One of the primary roles this vitamin plays is in embryogenesis. Almost all steps in organogenesis are controlled by retinoic acids, thus suggesting that retinol is necessary for proper development of embryonic tissues. These considerations point to the dramatic importance of a sufficient intake of vitamin A and explain the consequences if intake of retinol is deficient. However, hypervitaminosis A also has a number of remarkable negative consequences, which, in same cases, could be fatal. Thus, the use of large doses of retinol in the treatment of some human diseases and the use of megavitamin therapy for certain chronic disorders as well as the growing tendency toward vitamin faddism should alert physicians to the possibility of vitamin overdose. ß 2003, Elsevier Science (USA).

I. A PREMISE This chapter discusses hypervitaminosis A in infancy. It is well known that a severe insufficient intake of retinol, which frequently occurs in

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children of the so-called ‘‘third world,’’ causes a clearly defined syndrome termed hypovitaminosis A. A strict connection between basic science and clinical investigation has allowed us to unravel the complex molecular mechanisms of the symptoms observed. This interplay not only makes it possible to understand the causes of this disease, but also helps us to acquire data on the functions and molecular effects of vitamin A and its numerous derivatives (retinoids). In contrast to hypovitaminosis A, an excessive intake of retinol has infrequently been observed in adults and children. The possibility of analyzing the clinical picture of hypervitaminosis A in infancy might be very intriguing, especially given the important role of the molecule and its derivatives in embryogenesis and development. Recently, we observed a newborn who, starting from delivery, had erroneously been treated with large amounts of retinol and had developed severe clinical consequences. The clinical symptoms were rapidly reverted by the removal of the molecule, thus resulting in an extremely fast recovery. A series of in vitro studies and biochemical investigations clarified, at least in part, the molecular mechanisms of retinol toxicity, and allowed new hypotheses on the function of vitamin A to be proposed. This chapter will review briefly the major aspects of vitamin A metabolism in humans, its mechanism of action and its physiological roles. We will then discuss the clinical consequences of an altered intake of retinol with a major emphasis on childhood hypervitaminosis A. Finally, we will conclude with some suggestions for a discussion on possible emerging diseases linked to an excessive and pathological use of dietary vitamin integrators.

II. VITAMIN A: INTESTINAL DIGESTION, ABSORPTION, AND TISSUE DELIVERY All vitamin A in the human body is acquired from the diet, either as the provitamin A carotenoids, occurring in vegetables and fruits, or as retinyl esters present in foods of animal origin (Howles et al., 1996; Weng et al., 1999; van Bennekum et al., 1999, 2000). Moreover, a much smaller amount occurs as retinoic acid (RA) (Fig. 1). Generally, carotenoids are cleaved to generate retinol (Fig. 1), or, alternatively, are absorbed intact. On the other hand, retinyl esters are hydrolyzed in the intestinal lumen and retinol is then taken up by enterocytes (Blomhoff et al., 1991; Weng et al., 1999). In this section we will discuss the mechanisms involved in the digestion of retinyl esters, the uptake and reesterification of retinol in intestinal cells, and the incorporation of the resulting retinyl esters into chylomicrons and the secretion of these lipoproteins from the enterocytes.

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OH

O

all-trans-retinol

all-trans-retinal

O

all-trans-retinoic acid OH

11-cis-retinal O

9-cis-retinoic acid O

OH

FIGURE 1. Structure of retinol (all-trans-retinol) and some of its major derivatives.

A. THE DIGESTION OF RETINYL ESTERS

In vitro experiments indicate that retinyl palmitate (the most abundant retinyl ester) might be hydrolyzed by two enzymatic families, i.e., carboxyl ester lipase (CEL) (van Bennekum et al., 1999) and pancreatic triglyceride lipase (PTL). However, it has been demonstrated that mice ablated of CEL take up the same amount of retinol (as retinyl ester) as do wild-type mice (Weng et al., 1999; van Bennekum et al., 1999). Thus, because the hydrolysis of retinyl esters is a necessary step for vitamin absorption, CEL is not the enzyme involved (or is not the only enzyme necessary), and at least one (or more) additional retinyl ester hydrolase (REH) enzymes must be operative in the gut lumen. Extensive biochemical and genetic evidence argues in favor of the idea that this REH activity is due mainly to PTL (Harrison, 1988, 2000), although conclusive proof is still lacking. At present, three pancreatic enzymes might be involved in the hydrolysis of the retinyl esters: PTL and pancreatic lipase-related proteins (PLRP) 1 and 2. PLRP1 has been cloned; the enzyme is 68% homologous to PTL, but its substrate remains unknown

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FIGURE 2. Schematic representation of vitamin A absorption, metabolism, and tissue delivery. The principal dietary sources of retinol are provitamin A carotenoids (such as -carotene) and vitamin A, primarily retinyl esters and retinol. Within the intestinal cells, retinyl esters are hydrolyzed to retinol through retinyl ester hydrolases (REHs). Provitamin A carotenoids are cleaved within the enterocytes to retinal, which can then be metabolized to retinol. Then, retinol is bound to cellular retinol-binding protein, type II (CRBP-II) and, through the action of lecithin:retinol acyltransferase (LRAT), esterified to retinyl esters. Subsequently, retinyl ester is organized (with other dietary lipids) into chylomicrons and secreted into the lymphatic system. The chylomicron retinyl esters are then taken up by hepatocytes, where they are hydrolyzed by retinyl ester hydrolases (REH) to retinol. Within hepatocyte and hepatic stellate cells, retinol interacts with cellular retinol-binding protein, type I (CRBP-I). CRBP-I has been proposed to carry retinol to newly synthetized serum retinolbinding protein (RBP). The RBP–retinol (Holo RBP) complex is then secreted into the circulation to meet tissue needs of vitamin A. After transfer from hepatocytes, retinol is also stored as retinyl esters in stellate cells.

(Giller et al., 1992). PLRP2 is 65% identical to PTL (Jennens and Lowe, 1995). At present, the contribution of PLRP2 to REH activity is not known. Thereby, the emerging picture suggests that several enzymes might be responsible for the complete hydrolysis of retinyl esters in the intestinal lumen (Fig. 2). In addition to the above cited enzymes, the occurrence of REH activity intrinsically located in the brush border membrane of human and rat enterocytes has been reported (Rigtrup and Ong, 1992; Rigtrup et al., 1994). So far, it is possible that this activity corresponds to an intestinal phospholipase B.

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To now, the relative contribution of the reported enzymes to the digestion and absorption of vitamin A retinyl esters has not yet been established. Retinyl ester absorption experiments in appropriate knockout mouse strains and in mice deficient in more than one enzyme might help answer this question. Dietary provitamin A carotenoids are absorbed by the mucosal cells and are converted to retinaldehyde through the action of carotene-15,150 dioxygenase. Upon reduction of this retinaldehyde to retinol by retinaldehyde reductase, retinol derived from dietary carotenoids is metabolically indistinguishable from retinol assumed with the diet. B. THE UPTAKE AND REESTERIFICATION OF RETINOL IN ENTEROCYTES

Studies on retinol uptake indicate that the molecule either at physiological or at pharmacological concentrations is taken up by two different mechanisms, a saturable carrier-mediated process and a nonsaturable diffusion-dependent process (Hollander and Muralidhara, 1977; Hollander, 1981; Said et al., 1989; Quick and Ong, 1990; Dew and Ong, 1994; Nayak et al., 2001). So far, no protein has been characterized that appears uniquely involved in retinol uptake. It is conceivable, however, that three different proteins, isolated as involved in fatty acid uptake, may play a role in retinol transport: (1) the membrane-bound fatty acid-binding protein, (2) a fatty acid transport protein, and (3) CD36 (for review see Abumrad et al. [1998]). After cell entry, free retinol probably interacts with (and is sequestered by) cellular retinol-binding proteins (CRBPs). Two CRBPs, CRBP-I and CRBP-II, have been identified, purified, and characterized. They belong to a family that includes proteins capable of binding fatty acid. These proteins have a remarkable sequence homology and share biochemical and genetic properties. However, their cellular expression pattern is very different. CRBP-I is almost ubiquitous, whereas CRBP-II is expressed mostly in the absorptive enterocytes. Interestingly, CRBP-II is an abundant protein and accounts for 1% of the total soluble proteins of the jejunal mucosa. This tissue distribution indicates that CRBP-II is uniquely suited for intestinal retinol absorption (for reviews, see Ong, 1994; Li and Norris, 1996; Newcomer et al., 1998). CRBP-II seems to play a number of roles in the cellular trafficking of retinol. It can bind to transporters on the brush border membrane and allow mediated diffusion. Moreover, the protein can act as a reservoir to maintain a very low level of free retinoids. An additional function of CRBP-II is to direct the retinol toward specific metabolism, namely esterification. In fact, once inside the enterocyte, retinol is reesterified mainly with palmitic and oleic acids (Quick and Ong, 1990; Levin, 1993).

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In intestinal cells, two enzymes, lecithin:retinol acyltransferase (LRAT) and acyl-CoA:retinol acyltransferase (ARAT), have been identified that are involved in the esterification of free retinol (Fig. 2). It was suggested (but not shown) that retinyl esters formed by LRAT and ARAT could be targeted for secretion with chylomicrons and storage, respectively (Blomhoff et al., 1990). Successive studies have shown that retinol bound to CRBP-II reacts primarily with LRAT, but not with ARAT, and then is directed for secretion (Ong, 1994) (Fig. 2). Further studies demonstrated that the modulation of CRBP-II levels represents a mechanism of control of vitamin A metabolism (Rajan et al., 1991; Suruga et al., 1995; Lissoos et al., 1995; Levin and Davis, 1997; Takase et al., 1998; Suruga et al., 1999). From a quantitative point of view, the general idea that vitamin A is efficaciously adsorbed at the intestinal level and then included in chylomicrons requires reevaluation. First, the recovery of ingested retinol in lymph varies between 20% and 60% (Huang and Goodman, 1965; Goodman et al., 1966; Blomhoff et al., 1991). Second, Hollander (1980) showed that 60% and 30% of the absorbed retinol is secreted into the lymph and portal circulation, respectively. Furthermore, he showed that the secretion of retinol into lymph was modulated by the presence of different concentrations of taurocholate and fatty acids. Third, oral supplementation of retinol to abetalipoproteinemia patients, who are not able to assemble and secrete chylomicrons, resulted in partial recovery from symptoms of retinol deficiency (Kane and Havel, 1995). Finally, cell culture studies showed that free retinol or its metabolites are transported across the cells independent of the assembly and secretion of lipoproteins (Nayak et al., 2001). Thus, the majority of the absorbed retinol is secreted into the lymph in esterified form. However, a small but significant amount is also secreted into the portal circulation, probably as free retinol. The transport of free retinol to the portal circulation is expected to be important in pathological conditions that affect the secretion of chylomicrons. Thus, the limited transport of free retinol may be an essential back-up mechanism for the homeostasis of vitamin A under some conditions. C. INCORPORATION OF THE RESULTING RETINYL ESTERS INTO CHYLOMICRONS AND THEIR FURTHER METABOLISM

The esters of retinol, after their intestinal synthesis, are included along with other dietary lipids into nascent chylomicrons. It is generally believed that retinol is secreted into the lymph mainly as retinyl palmitate. During metabolic studies, analysis of the plasma revealed that most of the retinyl esters are present in small chylomicrons (Lemieux et al., 1998). Substantial amounts of retinyl esters are also found in large chylomicrons followed by smaller amounts in very low-density lipoproteins (VLDL) (Lemieux et al.,

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1998). After the release of chylomicrons into the lymphatic system and, then, in the general circulation, about 75% of vitamin A is taken up by hepatocytes. A minor percentage of the postprandial vitamin A is incorporated by other organs of the human body. After reaching liver cells, the chylomicrons retinyl esters are again hydrolyzed. Subsequently, vitamin A follows two different pathways. A part is released into the circulation bound to its specific transport protein, the so-called retinolbinding protein (RBP). Alternatively, vitamin A is transferred and stored into hepatic stellate cells (also known as fat-storing cells, lipocytes, or Ito cells) (Fig. 2). In the liver, retinol is mostly stored as retinyl esters. Few data are available that explain the mechanisms by which hepatocytes transfer their retinol to the stellate cells. When dietary vitamin A is plentiful, the stellate cells account for more than 80% of retinol contained in the liver. However, when vitamin A is limiting, Ito cells contain a relatively scarce amount of hepatic retinol. Both the cell phenotypes contain high amounts of REH, LRAT, and CRBP-I. The last protein is required to maintain free retinol in an aqueous cellular environment. In the blood of a fasting subject, retinol bound to RBP is more than 95% of the vitamin A occurring in circulation (Fig. 2). Such a complex is bound to an additional protein named transthyretin (TTR). The blood concentration of RBP/vitamin A is maintained within a narrow limit, and changes only when a remarkable hypovitaminosis A or a disease condition occurs (van Bennekum et al., 2000). This complex represents the source of vitamin A for a number of tissues. However, there are low amounts of retinyl esters interacting with VLDL and LDL and a small amount of RA bound to serum albumin. The mechanism through which cells take up retinol from the circulating complexes has not yet been clarified. A possible hypothesis is the occurrence of cell membrane receptors that recognize the retinol/RBP/TTR complex; retinol is then transferred into the cell membrane. Subsequently, vitamin A is oxidized first to retinaldehyde and then to RAs (Fig. 2). Several enzymes are able to catalyze the oxidation of retinol to retinaldehyde and a number of enzymes might catalyze the oxidation of retinaldehyde to RA. Because all the three molecules (vitamin A and its derivatives) are lipids, they are generally bound to proteins within cells. These aspects will be described in details in the next section.

III. INTRACELLULAR METABOLISM RAs are thought to be the major retinol derivatives, nevertheless not all vitamin A actions (outside vision) might be explained by their effects. In vitamin A-deficient animals, for example, RA is less effective to retinol in

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reversing defects in spermatogenesis (Howell et al., 1967) or in the immune response (Davis and Sell, 1983). The fact that retinol can act in a manner independent of RA has been known for many years, yet there has been little progress in understanding the mechanisms underlying such RA-independent activities. A significant advance in this area was the characterization over the past few years of the new bioactive retinol metabolites, namely 14-hydroxyretroretinol (14-HRR) (Tzimas et al., 1996b) and anhydroretroretinol (anhydroretinol, AR) (Bhat et al., 1979) (for a detailed description of these compounds, see Section III.B). These two retroretinoids were initially identified for their putative role in lymphocyte physiology, and subsequent studies revealed that they may have a more general role in cell growth and proliferation. In addition to RAs and retroretinoids, two nonacid derivatives of retinol have been identified, i.e., 4-oxo-retinol (Achkar et al., 1996) and 4-oxoretinal (Blumberg et al., 1996). These compounds have been shown to transactivate RA receptors. These receptors are members of the steroid/ thyroid receptors superfamily and regulate transcription of the target genes upon binding of RAs. Although the mechanism of action of the new retinol derivatives remains to be elucidated, present evidence suggests that they can act through novel pathway(s) (Achkar et al., 1996). The biosynthesis of such a large number of retinoid metabolites requires the presence of many enzymes catalyzing a variety of chemical reactions. Despite the fact that such enzyme activities have long been known, progress in the purification of proteins and in the cloning of their genes has been remarkably slow (reviewed in Napoli, 1996). LRAT, for example, the microsomal enzyme responsible for the esterification of retinol, has been studied extensively for its biochemistry, but the cloning of cDNA was only recently reported (Ruiz et al., 1999). Genes have been isolated for retinol dehydrogenases, which are responsible for the oxidation of retinol to retinaldehyde (Chai et al., 1995, 1996; Kedishvili et al., 1995; Simon et al., 1996), as well as for retinaldehyde dehydrogenases, which can oxidize retinal to RA (Chen et al., 1994; Wang et al., 1996; Penzes et al., 1997). Studies of some of these enzymes have led investigators to favor a model of retinol metabolism in which the preferred substrate is retinol bound to cellular retinol-binding protein (Posch et al., 1991; Herr and Ong, 1992). Characterization of 14-HRR and AR, the novel retinol metabolites, provides yet another example of vitamin A as a prohormone. The fact that various retinol metabolites can act through distinct pathways to produce their specific cellular effects implies that vitamin A metabolism must be a critical point in the regulation of its activities. Therefore, to understand the pleiotropic effects of retinol it is important to characterize the retinolmetabolyzing enzymes and to study their mechanisms of action, their modes of regulation, and the patterns of their spatial and temporal expression.

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A. SYNTHESIS OF RETINOIC ACID AND ADDITIONAL DERIVATIVES OF OXIDATIVE PATHWAY

1. Enzymatic Synthesis of Retinoic Acid The oxidation of retinol is catalyzed by members of the family of cytosolic medium-chain alcohol dehydrogenases (ADHs) (Goodman and Blaner, 1984; Blaner and Olson, 1994; Napoli, 1996; Boleda et al., 1998; Vogel et al., 1999; Duester, 2000; AAVV., 2000). These enzymes are able to oxidize only free retinol and not vitamin A bound to CRBP (Napoli, 1996; Duester, 2000). A second group of enzymes (Ro1DH I, II, and III), belonging to the short-chain alcohol dehydrogenase (SCAD) or short-chain dehydrogenase/reductase (SCDR) family, is supposed to play a role in retinol oxidation (Napoli, 1996; Duester, 2000; AAVV., 2000). SCDRs are present in cells at a scarce concentration and are associated with membrane fractions. Several SCDRs prefer retinol bound to CRBP, or in the case of the 11-cis-retinol dehydrogenase present in the eye, cellular retinaldehydebinding protein (CRalBP) to free molecule as substrate (Napoli, 1996; Duester, 2000; AAVV., 2000). Cytosolic retinol dehydrogenase activity has been linked to medium-chain ADHs, which are formed of 40-kDa subunits (Boleda et al., 1998). The members of the ADH enzyme family are dimeric zinc-containing enzymes requiring NAD+ to catalyze oxidation of a variety of primary, secondary, and various cyclic alcohols (Joernvall and Hoeoeg, 1995). There are several ADH isozymes that oxidize retinol to retinal in vitro (Napoli, 1996; Blaner et al., 1999; Duester, 2000; AAVV., 2000). ADH4 appears to be the most efficient, thus allowing the hypothesis that it can act in vivo. This enzyme, unlike the microsomal retinol dehydrogenases (RolDH I, II, and III), does not catalyze oxidation of retinol bound to purified CRBP-I. Nevertheless, ADH1 and/or ADH4 are expressed in embryonic tissues where RA is essential for development. The pattern of expression of ADH4 overlaps both temporally and spatially the pattern of RA distribution in the developing mouse embryo (Napoli, 1996; Blaner et al., 1999; Duester, 2000; AAVV., 2000). Two reports have recently appeared describing the targeted disruption of the ADH1 and ADH4 genes (Deltour et al., 1999). ADH1/ and ADH4/ mice show normal prenatal and postnatal survival. On the other hand, kidney levels of active retinoids after retinol oral administration were remarkably lower than wild type in the deficient mice. ADH4/ mice maintained on a diet lacking vitamin A during pregnancy had a high rate of stillbirths. These studies indicate that ADH1 and ADH4 play a role in catalyzing retinol oxidation in vivo, but that neither enzyme is indispensable. Thus, ADH1 and ADH4 might be redundant both with each other and with microsomal retinol dehydrogenases. Oxidation of all-trans-retinol to all-trans-retinal may also be due in vivo to members of the SCDR family of microsomal enzymes that are expressed

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principally in liver and are able to employ all-trans-retinol bound to CRBP-I as a substrate (Napoli, 1996; Blaner et al., 1999; Duester, 2000; AAVV., 2000). These enzymes, which possess a molecular mass of 28–‘‘32 kDa, might also catalyze the oxidation/reduction of hydroxy-steroids and prostaglandins (Baker, 1994; Joernvall et al., 1995). Indirect observations, like colocalization with CRBP-I, argued for their importance in vivo. However, the fact that CRBP-I-deficient mice are phenotypically normal critically weakens this possibility. Various aldehyde or retinaldehyde dehydrogenases (RALDH) have been proposed to promote the synthesis of RA in vivo (Blaner and Olson, 1994; Napoli, 1996; AAVV., 1998; Blaner et al., 1999; Vogel et al., 1999; Duester, 2000). One of these, RALDH-2, an NAD+-dependent aldehyde dehydrogenase, appears essential in catalyzing RA formation from retinaldehyde in the embryo (Niederreither et al., 1999). In situ hybridization histochemistry on the embryonic trunk reveals RALDH-2 mRNA both in mesoderm and neuroectoderm, with highest neuroectodermal expression in the ventral horn of the spinal cord at two restricted locations along the anteroposterior axis, presumably the subpopulation of motoneurons that innervates the limbs (Zhao et al., 1996). In the adult, RALDH-2 is expressed in testis and other tissues. RALDH-2 expression may be downregulated in mouse embryos by teratogenic doses of all-trans-RA (Niederreither et al., 1997) Thus, the synthesis of RA may be regulated by negative feedback via RALDH-2 expression. Targeted disruption of the RALDH-2 gene (Niederreither et al., 1999) yields embryos that die at midgestation without undergoing axial rotation. The mutant embryos are shortened along the anteroposterior axis and do not form limb buds. Heart development is impaired, the frontonasal region is truncated, and otocyst size is severely reduced. These malformations are caused by the lack of RA because (1) expression of RA-dependent homeobox genes was reduced in the mutants and (2) the mutant phenotype was suppressed by maternal RA administration. The phenotype of mutant mice provides strong evidence that RALDH-2 plays an indispensable role in catalyzing RA formation in the embryo. The effect of RALDH-2 deficiency on the development of the hindbrain and associated neural crest has recently been reported by Niederreither et al. (2000). They conclude that endogenous synthesis of RA within the embryo is required to direct posterior cell fates in the developing mouse hindbrain and confirm the pivotal role of RALDH-2 in RA production. 2. Enzymes Involved in cis-Retinoids Synthesis cis-Retinoids are needed both to maintain vision and to regulate transcription. Indeed, 11-cis-retinal is the chromophore in the visual pigment rhodopsin. Moreover, 9-cis-RA through different RA receptors

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is necessary for retinoid responsive gene activity. The formation of 9-cisretinoid isomers has been scarcely understood, whereas the 11-cis-retinoid isomers are known to be formed by isomerization of all-trans-retinoids. This reaction is catalyzed by a specific isomer hydrolase that catalyzes the isomerization and hydrolysis of all-trans-retinyl esters to 11-cis-retinol. Since the first reports in 1992 (Mangelsdorf et al., 1992) that 9-cis-RA is a ligand for RXRs (a class of RA receptors, for a detailed description see Section IV.A), several studies have explored possible pathways for 9-cis-RA formation. Three such pathways have been proposed: (1) isomerization of all-trans-RA (ATRA) to 9-cis-RA, probably through nonenzymatic processes, (2) enzymatic oxidation of 9-cis-retinol to 9-cis-RA through a pathway similar to the oxidation of all-trans-retinol to ATRA, and (3) cleavage of 9-cis--carotene either directly to 9-cis-RA or to 9-cis-retinol or 9-cis-retinal that are then directly oxidized to 9-cis-RA. Again, in vitro data supporting these reactions are individually convincing, but it remains unclear whether any of these pathways is important for 9-cis-RA formation in vivo. So far, a human NAD+dependent retinol dehydrogenase that specifically oxidizes 9-cis-retinol but not all-trans-retinol has been cloned and characterized (Mertz et al., 1997). B. MECHANISMS OF BIOSYNTHESIS OF RETRORETINOIDS

The term ‘‘retro’’ is used to describe the retinoid skeleton of these compounds, which is characterized by a shift in the conjugated double bond system resulting in the presence of a double bond between carbon 6 and 7 (Fig. 3). In the series retinoids (e.g., retinol, retinal, RA) there is a rotation about the 6–7 carbon bond that allows the side chain to be out of the plane of the ring. In contrast, the side chain of retroretinoids is forced in the same plane as the cyclohexene ring, and the double bond on the ring becomes part of the conjugated double bond system. Hence retroretinoids have a different three-dimensional structure from the -series retinoids, and their ultraviolet spectrum exhibits a distinct and characteristic vibrionic fine structure (Fig. 3). It is interesting to note that a coplanar conformation between the ring and the side chain has also been shown for retinal when it is bound to protein in bacterorhodopsin, and for retinol when it is bound to the cellular binding protein I. 14-HRR and AR can bind to retinol-binding protein in the plasma and to CRBP-I but do not bind to CRABP-I. The proposed functions for RBP and CRBP include transporting and sequestering retinol and preventing nonspecific hydrophobic interactions. It will be interesting to investigate the physiological roles of these proteins in retroretinoid transport and metabolism and to see whether they serve similar functions for 14-HRR and AR as they do for retinol.

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11-cis-retinal

all-trans-retinoic acid

O

OH

all-trans-retinol

OH OH

14-Hydroxy-Retro -Retinol (14-HRR)

Anhydroretinol (AR)

FIGURE 3. Structures of retroretinoids and relationship with other retinol derivatives.

1. 14-Hydroxyretroretinol 14-HRR was originally purified from HeLa cells and has been subsequently found in a large number of cell types from insects to mammals, including human B lymphocytes, fibroblasts, and leukemia cells. 14-Hydroxyretroretinol is also present as a physiological retinol derivative in human plasma (Arnold et al., 1996) and in several tissues of mice, rats, and rabbits (Tzimas et al., 1996a,b). When pregnant mice were fed with teratogenic amounts of retinol, their embryos were shown to contain relatively high levels of 14-HRR (Tzimas et al., 1996a,b). This is intriguing because so far all of the teratogenic effects of retinol have been attributed to RA. The elevated concentration of 14-HRR suggests it may account for some of the teratogenic potential of vitamin A. Very little is known about the biochemical pathway(s) of 14-HRR production. All tested cell lines convert radiolabeled retinol to 14-HRR in a reaction that appears to be enzymatic. Indeed, phorbol esters activate 14-HRR production 2- to 4-fold (Derguini et al., 1995), and in B and T lymphoblastoid cells, disulfiram, which modifies function by binding to thiol groups, inhibits the synthesis of 14-HRR in a dose-dependent manner.

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2. Anhydroretinol AR was initially purified from Drosophila S2M3 cells and has been identified in other insect and mammalian cells. AR was found in significant amounts in the stellate cells of mammalian liver, and it was detected in mouse lung tissue. With the exception of liver, the amounts of AR present in mammalian cells were lower than the micromolar concentrations of AR needed to induce cell death in bioactivity assays. However, in vitro experiments may require amounts of AR higher than physiological given the fact that AR is a very lipophilic compound. In addition, the production of AR can be tightly regulated within cells, allowing only transient increases in intracellular AR that are difficult to detect. Enzymatic production of cellular AR was suggested for the first time in 1979 when it was shown that spontaneously transformed mouse fibroblasts can metabolize radiolabeled retinol to AR and that this dehydratase activity was destroyed by heat treatment (Bhat et al., 1979). Both the native and the recombinant enzyme have very high affinity for retinol with the Km and Kd values for retinol being in the low nanomolar range. This implies that retinol dehydratase can utilize retinol at its physiological intracellular concentrations. Moreover, retinol dehydratase seems to prefer free retinol over protein-bound retinol as its substrate. This is an important finding because it is contrary to what has been reported previously for other retinol-metabolizing enzymes. Retinol dehydratase is homologous to the family of cytosolic sulfotransferase. They catalyze a bisubstrate reaction in which a sulfonate group is transferred from 30 -phosphoadenosine-50 -phosphosulfate (PAPS) to the target substrate. Accordingly, retinol dehydratase uses PAPS as a cosubstrate (with a Km value in the low micromolar range) (Grun et al., 1996). Because retinol dehydratases appears to be a sulfotransferase, it is reasonable to assume that AR production proceeds via retinyl sulfate, although the existence of such an intermediate remains to be proven. Moreover, it is also likely that mammalian retinol dehydratase is a sulfotransferase.

IV. RETINOL AND EMBRYOGENESIS: MECHANISM OF ACTION AND IMPORTANCE It is beyond the scope of this chapter to review or summarize the complexity of the biological effects of retinol and its derivatives. However, to discuss the role of retinoids in infancy and to explain mechanistically the symptoms of an altered intake of vitamin A, it is useful to unravel, in detail, the molecular bases of the action of retinol, as well as to examine its role in embryogenesis. This section is divided into two parts. First, we will describe,

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from a biochemical point of view, how vitamin A and retinol derivatives function. In the second part, we will analyze the role of these molecules in the development of various tissues. As discussed before, it is clear that vitamin A plays a key role in the regulation and timing of many important biological processes, which range from proliferation to differentiation, from apoptosis to development, morphogenesis, and homeostasis. Because of these roles, it appears conceivable that natural (or synthetic) retinoids might be employed as therapeutic agents. This further increases the importance of clarifying the mechanism of action of vitamin A and retinoids.

A. MOLECULAR BASES OF RETINOIDS ACTIVITY

Consistent with the pleiotropic biological effects, understanding the retinoid signaling pathways has been a particularly complex process. Part of these mechanisms relies on the ability of some RAs to interact with and activate members of a class of DNA-binding proteins, now well known as the nuclear receptor superfamily. However, a wealth of data indicates that other retinoids (and perhaps vitamin A itself) are able to modulate cellular events by retinoic receptor-independent mechanisms that are not well understood. The next sections will describe some aspects of the cellular interactions of vitamin A derivatives. 1. The Retinoic Acid Receptor-Dependent Mechanisms a. The Biochemistry Six RA receptors were discovered, which belong to two classes, the RA receptors (RARs) , , and (Petkovich et al., 1987; Giguere et al., 1987; Benbrook et al., 1988; Brand et al., 1988; Krust et al., 1989) and the retinoid X receptors (RXRs) , , and (Hamada et al., 1989; Mangelsdorf et al., 1990, 1992; Yu et al., 1991; Leid et al., 1992). Each receptor is codified by a specific gene from which frequently multiple isoforms can be obtained by differential splicing and multiple promoters (Lehmann et al., 1991; Leroy et al., 1992). The different receptors are expressed in tissue- and developmental-specific fashion, thus suggesting that they possess a peculiar importance in the regulation of biological processes. The two classes of receptors (i.e., RARs and RXRs) are not remarkably homologous. Although, like all nuclear receptors, they present typical domains including a DNA-(DBD) and a ligand-binding domain (LBD), the two classes seem to have evolved independently, showing a low amino acid homology in their LBD (Pfahl et al., 1994). The structural organization of nuclear receptors is similar, despite wide variation in ligand sensitivity (Fig. 4). With few exceptions, these proteins contain (1) an NH2-terminal region that harbors a ligand-independent

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FIGURE 4. Schematic organization of retinoic acid receptors. transcriptional activation function (AF-1); (2) a core DNA-binding domain, containing two highly conserved zinc finger motifs that target the receptor to specific DNA sequences, known as hormone response element; (3) a hinge region that permits protein flexibility to allow simultaneous receptor dimerization and DNA binding; and (4) a large COOH-terminal region that encompasses the ligand-binding domain, dimerization interface, and a ligand-dependent activation function (AF-2). Upon ligand binding, nuclear receptors undergo a conformational change that coordinately dissociates corepressors and facilitates recruitment of coactivator proteins to enable transcriptional activation (McKenna et al., 1999). Not surprisingly, the specific ligands of the receptors are different. RARs interact with ATRA and 9-cis-RA, whereas RXRs bind solely 9-cis-RA. Importantly, natural high-affinity ligands selective for RXR have not been identified up to now. These findings strongly suggest that RARs represent the major vitamin A signal transducers. To allow RA to modulate gene transcription, RARs must bind to RXRs forming heterodimers. Indeed, only the RXR–RAR heterodimers were found to bind effectively their DNA recognition sequences, the so-called RA response elements (RAREs) (Zhang et al., 1992; Kleiwer et al., 1992; Marks et al., 1992; Bugge et al., 1992). Moreover, in vivo gene studies in knockout mice (Kastner et al., 1997a) and in vitro experiments with cell lines (Chiba et al., 1997) have definitely demonstrated the essential role of the RXR– RAR heterodimer for retinoid signal transduction. Activation of the heterodimer seems to be under the control of RARs, as the RXR– RAR" heterodimer responds only to RAR ligands and not to RXR selective ligands (Kurokawa et al., 1994). Once the RAR has been activated by the binding with a ligand, RXR ligands can allow a further activation of the hetorodimer (Minucci et al., 1997). Thereby, at least in this case, it seems that RXRs act just as enhancers of retinoid signals. However, subsequent investigations on RAR and RAR (La Vista-Picard et al., 1996) did not confirm the strong dominance observed for RAR over RXR (Kurokawa et al., 1994). Indeed, heterodimers containing either RAR or RAR are partially activated by RXR selective ligands (La Vista-Picard et al., 1996).

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A major problem in evaluating these data is that the occurrence of RXR selective ligands has not yet been conclusively demonstrated. Thereby, the true meaning of RAR dominance is of limited significance, because although ATRA activates only RARs, 9-cis RA might interact with RAR and RXR, the two members of the heterodimer. On the other hand, the RAR dominance over RXR may be crucial in some retinoid therapies, approaches where RXR selective retinoids can be used without activating the general retinoid response. In addition to being necessary for efficient RARs-dependent DNA binding and transactivation, it has been demonstrated that RXR is an essential cofactor for several nonsteroidal nuclear receptors, including the thyroid hormone receptors and vitamin D receptor (VDR). In these cases, RXRs appear to act mostly as silent (namely not ligand activated) partners. RXRs also function as coreceptors for an increasing group of nuclear receptors that includes novel ligand-binding receptors as well as orphan receptors, many of which may not interact by themselves with high-affinity ligands. In most of these cases, the RXRs appear to function as ligandresponsive receptors. When RXR heterodimerizes with peroxisome proliferator-activated receptor (PPAR), liver X receptor (LXR), or farnesoid X receptor (FXR), these heterodimers can be activated by either RXR selective ligands or by partner ligands: fatty acids and thiazolidinediones for PPAR and oxysterol and farnesoids for FXR. Furthermore, RXRs can bind and activate DNA as homodimers, which are induced in the presence of their natural ligand, 9-cis-RA. It should be pointed out here that because RXR homodimers and heterodimers bind some of the same response elements, it is often difficult to determine whether the increased activation observed at high RXR ligand concentration results from the RXR molecule in the heterodimer or from RXR homodimers. Nevertheless, it is clear that the RXRs have a wide role as coreceptors, thereby allowing cross-talk between certain retinoid signals and other hormonal and vitamin signals. This is likely to occur not only through the RXR-containing heterodimers, but also through competition between various RXR partners, when the RXR molecules are in limited supply. In the presence of high concentrations of RXR ligands, competition between RXR homodimers and heterodimers may also occur. Thus, retinoid receptors participate in a complex network that allows interactions and cross-talk between a large number of nuclear receptors and, obviously, their ligands. b. The RAREs: The DNA Recognized Sequences The nuclear receptors modulate transcription by binding specific DNA sequences, defined as hormone response element (HREs) or, in the case of the RA receptors, RAREs. RAREs are generally located in the promoter region of the genes responsive to retinoids. They consist of two hexameric half sites (AGGTCA), which can be organized as direct repeat (DR),

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palindromes (Pal), or inverted repeats (IP), separated by a spacer of a variable number of nucleotides. The different arrangement of the half sites and the distance between the sequences are the pivotal features that identify the specificity and mode of binding of the receptors. RARs–RXRs recognize direct repeats separated by one, two, or five nucleotides. Moreover, the heterodimers can bind palindromes with no spacer or with a nine-nucleotide spacer, as well as inverted palindromes with six or eight bases in the spacer. Homodimers consisting of RXRs bind and activate palindromic elements and DR separated by 1 nucleotide (DR-1 elements). It is important to stress that except for some steroid receptors, the interaction of nuclear receptors with DNA is ligand-independent. This means that RARs show a dual functions: they act as transcriptional repressors in the absence of ligand and as activators in the presence of ligands. Such an effect has been clarified by resolution of the three-dimensional structure of both unligated RXRLBD (Bourguet et al., 1995) and the RARLBD bound to ATRA (Renaud et al., 1995), which confirmed that significant conformational changes occur after ligand binding. A further aspect to be considered is that some of the RAREs are also recognized by other receptors, including COUP-TF (and its subtypes), TOR, and Tak1. This means that the temporal and tissutal coexpression pattern of different receptors can lead to a competition for the same DNA regulatory sequence. COUP-TF, for example, occurs at high levels during the development of brain and is, thereby, probably able to efficaciously repress the transcription of genes regulated by RA. Again, the complexity of responsivness might explain the enormous diversity of RAREs. c. Additional Factors Controlling the Receptor-Linked Mechanisms The intricacy of retinoid-dependent transcriptional modulation, which requires either conformational change of nuclear receptors or a regulated interaction between heterodimeric receptors and specific DNA sequences, suggests the probable presence of other regulatory proteins. These factors have been, at least in part, identified both as components of general transcription machinery and as putative positive (coactivator) and negative (corepressor) proteins. Ligand-dependent interaction of nuclear retinoid receptors with a number of basal transcription factors has been described, allowing the proposal of models in which ligand–receptors recruit the TATA boxbinding protein (TBP) and other TBP-associated factors (TAFs). These events promote the assembly of the preinitiation complex and allow gene transcription by RNA polymerase II. The existence of putative corepressors or silencing factors necessary for the ligand-independent repression by nuclear receptors was suggested in several studies (Casanova et al., 1994; Baniahmd et al., 1995; Tong et al., 1996). Two pivotal proteins, nuclear receptor corepressor (N-Cor) (Horlein et al., 1995) and silencing mediator

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for retinoid and thyroid hormone receptors (SMRT) (Chen and Evans, 1995), have been identified and demonstrated to interact with free RAR and TR, but not with ligand-bound receptors. Several coactivators have been identified: they interact with nuclear receptors in a ligand-dependent manner and mediate their transcriptional activity (Fig. 4). Trip1 was isolated as thyroid hormone receptor interacting protein by yeast two hybrids system, and shown to be very similar to the yeast transcriptional mediator Sug1 (Swaffield et al., 1992, 1995; J. W. Lee et al., 1995). The murine homologue, mFUG1, has been recently cloned and shown to interact both in yeast and in vitro with RAR, TR, VDR, and ER, but not with RXR. Several lines of evidence point to a role of CBP/p300 as an important coactivator of retinoid receptors (and more generally, nuclear receptors). CBP was identified as a phosphoCREB-binding protein (CREB is the cAMP-responsive element binding protein), and is required for transcriptional activation by CREB as well as by AP-1 (Chrivia et al., 1993; Arias et al., 1994; Bannister et al., 1995; Bannister and Kouzarides, 1995). Another protein, p300, was independently isolated as an E1A-associated protein and found to be highly homologous with CBP (Arany et al., 1995). The two proteins are functionally interchangeable, thus they are usually named CBP/p300 (Lundblad et al., 1995). Both CBP and p300 are large proteins with over 2400 amino acids and a complex and well-defined structure. CBP/p300 are characterized by a Q-rich domain at the carboxyl-terminus of the protein, the presence of three zinc fingers (C/H) localized at different sites along the molecule, and a bromodomain in the center region. Several regions along the protein are required for the interaction of CBP/p300 with nuclear receptors and other transcription factors such as CREB and AP-1, STAT, myb, myoD, p45/NFE2, p53, and basal transcription factors such as TBP and TFIIB (Chrivia et al., 1993; Arias et al., 1994; J.-S. Lee et al., 1995; Bannister et al., 1995; Bannister and Kouzarides, 1995; Dai et al., 1996; Eckner et al., 1996; Bhattacharya et al., 1996; Avantaggiati et al., 1997; Horvay et al., 1997; Cheng et al., 1997). Interaction of CBP/p300 with the LBD of RAR, RXR, TR, and ER has been demonstrated in in vitro and in vivo experiments. This interaction has a functional meaning, as cotransfection of CBP/p300 improves hormonedependent transcriptional activation by nuclear receptors, and also removes inhibition of AP-1 by RAR or GR (Kamei et al., 1996; Chakravarti et al., 1996). Because CBP/p300 can interact with several transcription factors and is required for their activity, it has been proposed that CBP/p300 is an integrator of multiple signaling pathways. CBP/p300 is expressed in very limited amounts, and is of pivotal importance for normal cellular functions. Indeed, it has recently been demonstrated that the loss of one functional copy of CBP causes the Rubinstein–Taybi syndrome (Petrij et al., 1995).

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Competition between transcription factors for CBP/p300 binding may explain the observed negative interference between them, for instance, the well-known inhibition by retinoid receptors of several transcription factors, such as AP-1 and CREB. Finally, it is important to stress that the level of each single protein might modulate the effect of retinoids. Moreover, these proteins could regulate the specificity of the ligand-activated receptor in choosing the RARE sequence to be modulated. d. The Importance of Histone Acetylation in Retinoid Receptor-Mediated Transactivation It has definitely been demonstrated that chromatin structure plays a key role in gene expression, and that the degree of histone acetylation is involved in chromatin remodeling (see reviews by Grunstein, 1997; Wade et al., 1997). Histone acetylation indeed affects genome transcription in vivo (Durrin et al., 1991) and thereby histone acetyltransferase (HATs) might be involved in gene regulation. Recent cloning of HATs allowed the identification of several transcription factors or coactivators with histone acetyltransferase activity: GCN5, CBP/p300, and hTAFII250 (Brownell et al., 1996; Ogryzko et al., 1996; Mizzen et al., 1996). P/CAF, which shows high homology with GCN5, has also HAT activity (Yang-Yen et al., 1991). Interestingly, it was recently shown that several of the reported proteins interact each other. Thus, a multimeric complex with HAT activity (containing P/CAF, CBP/p300, and other coactivators) is required for transcriptional activation by nuclear hormone receptors, providing strong evidence for a role of histone acetylation in gene expression. Histone deacetylation is obviously associated with transcriptional repression, probably by returning nucleosomes to a tightly inhibited conformation (Pazin and Kadonaga, 1997). Several histone deacetylases have been recently isolated (Vidal and Gaber, 1991; Ayer et al., 1995; Schreiber-Agus et al., 1995; Taunton et al., 1996; Rundlett et al., 1996), which can form a trimeric complex with N-CoR/SMRT and mSin3 (a corepressor for Mad) (Heinzel et al., 1997; Alland et al., 1997; Nagy et al., 1997). Microinjection of antibodies against each of the components of this complex prevents transcriptional repression, indicating that all of them are required for the repressor activity of nuclear receptors (Heinzel et al., 1997). The importance of histone deacetylation in repression by unliganded nuclear receptors was further supported by the use of histone deacetylase inhibitors (Heinzel et al., 1997; Nagy et al., 1997). Thus, a role for histone deacetylation/acetylation can be proposed for the regulation of transcription for nuclear receptors. In this model, binding of an unliganded RXR–RAR heterodimer to a DR-5 HRE recruits a corepressor complex with histone deacetylase activity, promoting nucleosome assembly and therefore potentiating transcriptional repression.

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N-CoR/SMRT serves as a bridge to link the histone deacetylase activity to the DNA-bound receptors. In the presence of ligands, N-CoR/SMRT dissociates from RXR–RAR heterodimers, resulting in displacement of the mSin3/HDAC complex from the DNA. The liganded receptor heterodimer now recruits a coactivator complex with HAT activity, formed by N-CoA, CBP, and probably P/CAF. This complex, which contains histone acetyltransferase activity, acetylates the core histones, which alters the nucleosome structure facilitating the entrance and assembly of the transcriptional machinery and allowing transcription by RNA polymerase II. 2. Retroretinoids: Vitamin A Derivatives Affecting Cell Phenotypes Independently on RA Receptors Vitamin A is a prohormone from which three subclasses of active metabolites are derived: the aldehydes, the carboxylic acids, and the retroretinoids. Although these three families are united under the rubric of signal transduction, they act by different molecular mechanisms. The 11-cisretinaldehyde combines with opsin to form the universal visual pigment rhodopsin, and the RAs form ligands for transcription factors, as discussed in detail before. The retroretinoids intersect with signal transduction at a cytoplasmic or membrane site. As already mentioned, several different retroretinoids have been identified, including AR, 14-HRR, and 14hydroxy-4,14-retroretinol. AR is able to cause cell death that was morphologically indistinguishable from the cell death caused by retinol deficiency (Buck et al., 1991). The acceleration of cell death by AR was dose-dependent, inducing widespread surface blebbing, ballooning, and osmotic bursting of cells within 4–8 h. The cell cytoplasm disintegrated and vacuoles fused, while the nuclei, with their nucleoli, remained intact for prolonged periods of time (O’Connel et al., 1996). These observations were true for all the cell types tested, including B lymphoblastoid cells (Buck et al., 1991), T cells (O’Connell et al., 1996), and NIH-3T3 cells. The finding that retinol and 14-HRR can rescue cells from death following AR treatment argues against generalized AR toxicity. Cell death due to AR appears to be a regulated process, but its features are distinct from those of classic apoptosis. In a mouse thymoma cell line (ERLD cells) that is remarkably responsive to AR, the treatment with the molecule does not cause DNA fragmentation or chromatin condensation, typical hallmarks of apoptosis. There was, however, DNA damage as detected by TUNEL assay. Detailed microscopic analyses demonstrated that cell selling and rupture occurred contemporaneously or prior to DNA damage (O’Connell et al., 1996). Subsequent biochemical investigations (using, for example, Bcl-2 transfection and caspase inhibitors) demonstrated that the mechanism of AR-dependent cell death does not follow traditional apoptotic pathways (reviewed in White, 1996).

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These findings stimulate the investigations on the molecular mechanism of cell death induced by AR. Some important data were obtained that allowed different hypotheses to be proposed: 1. AR kills cells by generating reactive oxygen species. Direct measurements showed that the addition of AR to lymphoblastoid cells increases the intracellular oxidative stress in a time- and dose-dependent manner. Furthermore, the amount of induced oxidative stress directly correlates with the number of dying cells. The addition of retinol, 14-hydroxy-4,14retroretinol, or the antioxidant -tocopherol (vitamin E) decreases ARinduced oxidative stress and proportionally reduces AR-induced cell death. 2. Several pharmacological blockers of signal transduction, including staurosporin (an inhibitor of protein C), methyl-2,5-dihydroxycinnimate, lavendustin A, and genistein (inhibitors of tyrosine kinases), were also found to have no effect on AR-induced cell death. However, herbimycin A, an inhibitor of src-like tyrosine kinase, prevented cell death (O’Connell et al., 1996). This finding supports the hypothesis that AR does not cause generalized cytotoxicity, but determines cell death by acting on specific molecular targets. It also suggests that its signaling pathway includes at least one tyrosine kinase, although it is possible that herbimycin A exerts its inhibitory effect via a yet unidentified mechanism. 3. A number of retinoids, including retinol, 14-hydroxyretroretinol, anhydroretinol, and RA, bind the c-Raf cysteine-rich domain (CRD) with equal affinity in vitro as well as in vivo. However, they displayed opposing effects on UV-mediated kinase activation: retinol and 14-hydroxyretroretinol augmented responses, whereas RA and AR were inhibitory. Oxidation of thiol groups of cysteines by reactive oxygen, generated during UV irradiation, was the primary event in c-Raf activation, causing the release of zinc ions and, by inference, a change in CRD structure. Retinoids modulated these oxidation events directly: retinol enhances, whereas AR suppresses, zinc release, precisely mirroring the retinoid effects on c-Raf kinase activation. Oxidation of c-Raf was not sufficient for kinase activation, productive interaction with Ras being mandatory. Further, canonical tyrosine phosphorylation and the action of phosphatase were essential for optimal c-Raf kinase competence. Thus, retinoids bind c-Raf with high affinity, priming the molecule for UV/reactive oxygen speciesmediated changes of the CRD that set off GTP–Ras interaction and, in context with an appropriate phosphorylation pattern, lead to full phosphotransferase capacity. 14-HRR and anhydroretinol were initially thought to act similarly to ATRA, i.e., binding nuclear receptors to affect transcription of target genes. However, physiological concentrations of retroretinoids were unable to transactivate not only the RARs and RXRs but also a number of other orphan nuclear receptors. In addition, AR-induced death was not inhibited

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by either actinomycin D, which inhibits transcription, or cycloheximide, which inhibits mRNA translation, indicating that macromolecular synthesis is not a requirement for cell death (O’Connell et al., 1996). Retroretinoids are relatively lipophilic compounds that can readily cross membrane barriers. The studies performed to date suggest that they may act on a membrane or on a cytoplasmic receptor to activate (14-HRR) or inhibit (AR) a growth-stimulating signaling pathway. The components of this pathway are the subject of present research and their identification is likely to reveal new mechanisms of vitamin A action.

B. VITAMIN A AS A CRITICAL MOLECULE IN HUMAN DEVELOPMENT

1. A Prologue A critical aspect of the interplay between retinol and infancy is the central role that this vitamin plays in the coordinate and harmonious development of embryo and fetus. In this part of the chapter, we will discuss the biological role of retinol in embryogenesis with an obvious major emphasis on its importance in humans. The importance of vitamin A throughout the life cycle has been well established (Moore, 1957; Wolf, 1984). Recently, it has become more clear that the requirement for this vitamin begins with embryonic life, as also noted by early nutritionists (Wolf, 1984; Blomhoff, 1994a; Zile, 1998, 2001; Ross et al., 2000; Maden, 2000), and that retinoids are critical signaling molecules during development. As described above, the biological effects of vitamin A are mainly (although not exclusively) mediated by its active form, all-trans-RA, and considerable work has addressed the mechanisms whereby ATRA affects embryogenesis and organogenesis (for detailed reviews see Hofmann and Eichele, 1994; Dolk et al., 1999; Maden, 1999; Lu et al., 1999; Ross et al., 2000; Zile, 1999, 2001). On the other hand, the control of vitamin A activity is inseparable from its metabolism because RA biosynthesis and removal are critical steps in RA-regulated signaling pathways. All of the physiologically important vitamin A metabolites and enzyme systems that regulate vitamin A metabolism have been identified in embryos (Hofmann and Eichele, 1994; Maden, 1999; Swindell et al., 1999; Lu et al., 1999; Niederreither et al., 1999; Ulven et al., 2000; Ross et al., 2000; Gavalas and Krumlauf, 2000; XavierNeto et al., 2000; Zile, 1999, 2001). This metabolic regulation is critical in embryonic development, where exogenous RA might have teratogenic effects on almost every developing tissue or organ system (Shenfelt, 1972; Osmond et al., 1991; Hofmann and Eichele, 1994; Wood et al., 1994; Avantaggiato et al., 1996; Zhang et al., 1996). During normal embryonic development only small but physiologically effective amounts of RA are

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present in the embryo, as demonstrated by direct analysis of the early quail embryos (Dong and Zile, 1995). Indeed, analysis of retinoid metabolites by high-performance liquid chromatography (HPLC) in the neurulation stage of quail embryos revealed the presence of the bioactive all-trans-RA and AR, as well as the upstream metabolites retinol, retinal, and retinyl esters, attesting to the enzymatic capability of the early embryo to fully process vitamin A (Dong and Zile, 1995). Obviously, all the studies on the role of vitamin A in embryo development have been performed in animal models, making it difficult to extrapolate the conclusions to humans. However, the absolute essentialness of retinol is definitely illustrated in the avian embryo model in which vitamin A deficiency (VAD) is embryolethal (Thompson, 1969; Dersch and Zile, 1993). From the first cell division, a complicated set of interrelated processes allows tissue organization to take place through specific pathways, leading initially to a developing embryo and then to a mature fetus. These events include the establishment of axial polarity and cell differentiation in response to signaling molecules whose concentrations vary according to the region of the embryo (Melton, 1991). Several signaling molecules, including RA, have been identified. In a number of experiments, the compound was applied directly to chick embryo limb buds and modified their development in a dose- and zone-dependent manner (Brickell and Tickle, 1989). Comparable findings suggested that RA is involved directly in establishing the specific pattern of gene expression along the anteroposterior body axis and also in forming the limbs (Morriss-Kay and Ward, 1999). A vast amount of information has been gathered on vitamin A function at the molecular level from cell culture (Gudas et al., 1994; Clagett-Dame and Plum, 1997; Ross et al., 2000) and from in vivo studies by perturbing normal embryonic development with exogenous retinoid levels (Hofmann and Eichele, 1994; Clagett-Dame and Plum, 1997; Zile, 1999; Maden, 1999; Lu et al., 1999; Ross et al., 2000). A recent approach to answering questions about the function of vitamin A in development has been the use of transgenic mice with changes in retinoid receptor gene structure (Chambon, 1993; Kastner et al., 1994; Sucov et al., 1994; Boylan et al., 1995; Giguere et al., 1996). When the embryonic development of RAR or RXR null mutant mice is monitored (Kastner et al., 1995; Ghyselinck et al., 1997), the abnormalities vary according to the receptor (or receptors) suppressed. A significant number of the observed alterations are consistent with investigations made in the offspring of vitamin A-deficient mice reported earlier (Wilson et al., 1953). When mutation affects only one receptor, the mice survive and the abnormalities are limited, suggesting a functional redundancy between the various subtypes and isoforms of the receptors (Li et al., 1993; Lohnes et al., 1993; Lufkin et al., 1993). In double-mutant mice, lacking either two RAR

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subtypes or both an RAR and RXR, the animals do not survive and the abnormalities are more pronounced and concern some specific ontogenic events (Lohnes et al., 1994; Mendelsohn et al., 1994). Moreover, the spatial and temporal expression of the various subtypes and isoforms of RARs and RXRs in wild-type organisms is important for directing the correct phenotype of the future differentiated cell. Indeed, much research has shown that the expression of RAR and RXR is widespread, whereas the expression of the other subtypes and isoforms is restricted to specific tissues and specific periods of embryonic development (Dolle et al., 1990; Mangelsdorf et al., 1992). Such patterns of expression are also observed in genes coding for the proteins involved in retinol metabolism. To achieve harmonious tissue organization, a given quantity of RA, in its all-trans or 9cis form, should bind specific receptors in due course. This implies that RA is available in the considered cell in an adequate amount and at the specific time; an imbalance in vitamin A status disrupts this equilibrium. In addition to the receptors there are cytoplasmic proteins that bind retinoids. One of them, CRBP-I, is found in the yolk sac, where it binds retinol from the maternal circulation (Ruberte et al., 1991). Subsequently, it delivers retinol to tissues that metabolize the molecule to RA. Once RA is in the cytoplasm, it can enter the nucleus and can bind the specific receptor or, alternatively, CRABPs. One of these, CRABP-I, interacts with RA and prevents it from entering the nucleus. In cells in which CRABP-I is not present, RA might easily go into the nucleus (Denker et al., 1990; Vaessen et al., 1990). It is probable that exogenous RA works as a teratogen in those tissues that have both RAR and CRABP-I protein: cranial neural crest cells and hindbrain. Another important approach for the examination of the molecular mechanisms of retinoid action in developmental regulation is the use of vitamin A-deficient animals, in which gene expression can be studied by regulating the presence of the receptor ligands, the retinoids. With rodent models it is possible to target VAD to distinct gestational windows and address its role in fetal development (Thompson, 1969; Wellik and De Luca, 1995, 1996; Wellik et al., 1997; White and Clagett-Dame, 1996; Smith et al., 1998; White et al., 2000). These rat embryos exhibit specific cardiac, limb, ocular, and central nervous system (CNS) abnormalities, some of which have certain features similar to those reported in retinoid receptor knockout mice (Smith et al., 1998). Several studies have also revealed the importance of vitamin A in fetal lung and kidney development (Wellik et al., 1997; Zachman and Grummer, 2000; McGowan et al., 2000). However, to study the function of vitamin A in development, the rodent models are inadequate in addressing later gestational stages. Indeed, so far, it is also not clear if the molecular functions of vitamin A (during the later stages of gestation) are via RA, or if retinol itself or another active retinal metabolite acts by an unidentified mechanism.

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An ideal system for studying early embryonic development is the vitamin A-deficient avian embryo, i.e., the quail embryo retinoid ligand knockout, which is completely dependent on vitamin A for its early development and which, without vitamin A supplementation, dies at Day 3 of embryonic life (Thompson, 1969; Dersch and Zile, 1993; Kostetskii et al., 1998, 1999; Zile et al., 2000). The completely VAD embryos develop gross abnormalities in the cardiovascular and central nervous systems and trunk. Importantly, these embryos can be ‘‘rescued,’’ and the normal development restored, by administration of ATRA, or its precursor, retinol. Recent studies provide convincing evidence that this model offers a unique opportunity to elucidate the physiological functions of vitamin A in early phases of development (Maden et al., 1998; Zile, 1998; Kostetskii et al., 1998, 1999; Zile et al., 2000). Another in vivo approach to eliminate vitamin A-active forms is to block RA with an anti-RA antibody (Twal et al., 1995) or to inactivate the RA biosynthetic pathways (Maden, 1999; Ross et al., 2000). Knockout mice embryos of the RA-synthesizing enzyme RALDH2 (Niederreither et al., 1999) have many abnormalities similar to those of the VAD quail embryos, but complete VAD was not obtained, probably due to the presence of other RA-generating systems. Partial depletion of RA may also be achieved by the overexpression of CYP26, an enzyme that degrades RA (Swindell et al., 1999; Ross et al., 2000). The role of vitamin A in the development of various tissues will now be described. 2. Heart and Blood Vessels Maternal insufficiency of vitamin A during pregnancy results in fetal death or severe abnormalities in the offspring, including abnormal heart development (Mason, 1935; Wilson and Warkany, 1949; Wilson et al., 1953). Many of the heart defects have been recapitulated in fetuses generated from various combinations of retinoid receptor knockouts. The malformations include a thin-walled dilated heart cavity, abnormalities. in ventricular chambers, and defects in the outflow tract (Kastner et al., 1994; Luo et al., 1996; Kostetskii et al., 1998, 1999; Zile, 1998, 1999; Zile et al., 2000; Smith et al., 1998; Kubalak and Sucov, 1999; Ross et al., 2000; Botto et al., 2001). Studies suggest that vitamin A is critically involved in the formation of the primitive heart (Mason, 1935; Wilson and Warkany, 1949; Wilson et al., 1953; Thompson, 1969; Heine et al., 1985; Dersch and Zile, 1993; Kostetskii et al., 1998, 1999; Zile, 1998, 1999, 2001; Xavier-Neto et al., 1999; Colbert, 2002), likely via the retinoid receptors. RAR2 is expressed in the heart-forming region and is downregulated in the absence of vitamin A (Kostetskii et al., 1996, 1998). Recently, it has been demonstrated that other retinoid receptors are also expressed in the heart-forming regions (Kostetskii et al., 1998). The developing heart, in the absence of vitamin A, is grossly abnormal, thin-walled, dilated, and distended, without chambers, but it contracts until the embryo dies. This is not surprising because the

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expression of early cardiogenic genes that regulate heart precursor cell differentiation into cardiomyocytes is not affected by the lack of vitamin A (Kostetskii et al., 1999). In contrast, exogenous treatments with RA induce differentiation of precursor cells into cardiomyocytes (Kubalak and Sucov, 1999). Among the few transcription factors that are expressed predominantly in the heart, there are GATA-4, -5, and -6, a subfamily of proteins identified in the developing heart and gut of the chicken (Laverriere et al., 1994; Doevedans and VanBilsen, 1996; Evans, 1997; Kostetskii et al., 1999). GATA-4 expression has been reported to be RA responsive (Arceci et al., 1993; Boylan et al., 1995; Zile, 1998; Kostetskii et al., 1999). RA also disrupts the differentiation of cardiac neural crest cells into ganglionic cells destined to contribute to the parasympathetic innervation of the heart, by regulating the expression of Phox2 (Shoba et al., 2002a) and Mash-1 and c-Ret (Shoba et al., 2002b). Recently, molecular cloning and subsequent studies on RALDH2 (for details on its function, see Section III.A.), provided the missing link between teratogenic studies on RA deficiency and morphogenesis (Xavier-Neto et al., 2001). It is of great interest that in the majority (75%) of the retinoid ligand knockout quail embryos the heart is on the wrong side, i.e., in situs inversus position (Dersch and Zile, 1993; Twal et al., 1995; Zile, 1998). The anticipated situs inversus can be ‘‘rescued’’ by administration of vitamin Aactive compounds (Dersch and Zile, 1993; Zile, 1998). However, recent evidence points to vitamin A having a general rather than a specific role, i.e., it appears that vitamin A is required to provide a proper environment for the expression of adequate levels of heart asymmetry genes (Wasiak and Lohnes, 1999; Zile et al., 2000). This concept is supported by evidence from the literature that the generation and distribution of RA in the embryo as well as the expression patterns of vitamin A metabolism enzymes and retinoid receptors are symmetric (Kostetskii et al., 1998; Maden, 1999; Niederreither et al., 1999; Swindell et al., 1999; Ulven et al., 2000; Gavalas and Krumlauf, 2000). The retinoids also act on the already developed heart. Recently, links have been established between (1) the reduced expression of RXR and the switch toward a severe heart failure. (Osorio et al., 2002) and (2) external stress and cardiovascular remodeling (arterial-wall thickening, angiogenesis, cardiac hypertrophy, and interstitial fibrosis) (Shindo et al., 2002). A major developmental defect that may be linked directly to the early embryolethality of the VAD quail embryo is the absence of a cardiac inflow tract (Zile, 1998, 1999; Kostetskii et al., 1999), i.e., the VAD heart has no opening at its caudal end where the extraembryonal blood vessels converge into vitelline veins to deliver blood to the embryonic heart. This defect has not been reported in any of the mammalian models in vivo addressing vitamin A function during embryogenesis. The formation of the cardiac inflow tract may be linked to the expression of the retinoid-regulated cardiac

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transcription factor GATA-4 in the posterior heart-forming area where this gene is involved in a BMP2 pathway (that specifies the endodermal structures of the heart and the underlying foregut primordial), and where apoptosis is observed in the VAD embryo (Ghatpande et al., 2000). In these embryos, the extraembryonal vascular networks are sparse and fail to converge at the level of the cardiac inflow tract (Carlson and Zile, 2001). Regulation of vascular smooth muscle cell (SMC) growth and differentiation is critical for vasculogenesis and for the maintenance of homeostasis in the mature vessel wall (Owens, 1995; Hungerford and Little, 1999). Retinoids and the receptors they bind are gaining an increasingly important role in both cardiovascular development and response of blood vessels to injury (Miano, 2000). Studies on cultured SMCs and adult aorta have documented the mRNA expression of all retinoid receptors except for RXR (Miano et al., 1996). Moreover, retinoid receptor activity was demonstrated in SMCs using a transiently transfected reporter gene assay (Miano et al., 1998). Although the expression of retinoid receptors in vascular SMCs has not been reported during development (Dolle et al., 1990, 1994; Ruberte et al., 1991; Mangelsdorf et al., 1992), receptor expression in developing vessels is inferred on the basis of phenotypes observed in states of retinoid deficiency and in retinoid receptor knockout mice. The effects of retinoids on vasculogenesis have been described in several species. Early studies using retinoid-deficient avian embryos revealed an important role for ATRA in the establishment of an intact intraembryonal– extraembryonal circulatory network; in the absence of ATRA, there was no vitelline artery or omphalomesenteric vein and the embryos died (Thompson et al., 1969; Heine et al., 1985). Null mice for the RALDH2 gene display severe extraembryonic vascular defects and die at midgestation (Niederreither et al., 1999). On the other hand, ATRA excess induces an avascular yolk sac in mouse embryos through a protease-mediated reduction of basic fibroblast growth factor expression (Yasuda et al., 1992). Moreover, administration of 13-cis-RA to pregnant women resulted in an RA embryopathy characterized by malformations of the great vessels (Lammer et al., 1985). Thus, levels of retinoids in developing embryos appear to be of critical importance for proper vasculogenesis to proceed. Additional evidence implicating ATRA in vasculogenesis is reported in studies on retinoid receptor knockout mice. With the exception of RXR null mice, which show hypoplastic thinning of the developing myocardium leading to midgestation lethality (Sucov et al., 1994; Kastner et al., 1994), none of the single-retinoid receptor knockout mice shows discernible defects in the cardiovascular system. On the other hand, multiple retinoid receptor knockout mice (e.g., RAR//RXR/) exhibit multisystem defects and vascular malformations, including a persistent truncus arteriosus, absence of the stapedial artery (second aortic arch derivative), and alterations in the third, fourth, and sixth aortic arches (Kastner et al., 1995; Mark et al., 1998).

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The only in vivo evidence favoring a role for activated retinoid receptors in SMC differentiation is a study from Colbert et al. (1996), who showed that a RARE-lacZ transgene colocalized with the expression of the adult isoform (SM2) of SM myosin heavy chain in the ductus arteriosus. A number of in vitro studies have shown that ATRA can positively influence the SMC differentiation program (Hayashi et al., 1995; Hautmann et al., 1997). Endogenous RA may play a role in inducing smooth muscle differentiation in the fetal ductus arteriosus and maternal administration of RA may accelerate the development of its O2-sensing mechanism (Wu et al., 2001; Shaul, 2001). Retinoids have variable effects on SMC growth modulation in vitro, depending on study design (Chen and Gardner, 1998; Wang et al., 1997). In contrast to these variable findings, several studies have shown retinoids to attenuate growth factor-stimulated SMC proliferation (Kato et al., 1993; Miano et al., 1996; Chen and Gardner, 1998). More recently, several retinoids were shown to inhibit serum- and serotonin-induced canine SMC growth (Pakala and Benedict, 2000). RAR-selective retinoids were more effective in inhibiting serotonin-induced SMC proliferation than other retinoid receptor agonists. Although RAR expression has been reported to be restricted to lung and skin, SMCs express high levels of RAR mRNA (Miano et al., 1996). Several papers have recently showed that retinoids are effective in promoting a larger luminal area after mechanical injury of the vessel wall (Miano et al., 1998; Chen et al., 1999). Lee et al. (2000) found that ATRA attenuated neointimal formation and accelerated reendothelialization in the balloon-injured rat aorta. Importantly, all of the in vivo studies involved near-uniform removal of the endothelium. Retinoids impede neointimal formation, most probably by means of a retinoid receptor-mediated modulation of gene expression. Several genes involved in growth regulation could be targets of activated retinoid receptors (Kato et al., 1993; James et al., 1993). Induction of the PAI-1 gene expression by ATRA may at least partly account for the role of ATRA as an important inhibitor of neointima formation (Watanabe et al., 2002). Retinoids have also been shown to inhibit cell growth, which can result in an antiatherosclerotic action at the vasculature level. Endothelin-1 (ET-1), a potent vasoconstrictor peptide produced in endothelial cells, plays an important role in inducing proliferation of vascular smooth muscle cells. ATRA suppressed ET-1 mRNA expression in cultured endothelial cells (Yokota et al., 2001). Finally, the growing number of synthetic retinoids should be exploited to carefully dissect out which retinoid receptors mediate changes in SMC biology. Several studies have already begun such an analysis, which could be extremely useful in unraveling this important chapter of embryology (Chen and Gardner, 1998; Neuville et al., 1999; Pakala and Benedict, 2000; Takeda et al., 2000; Sirsjo¨ et al., 2000).

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3. Nervous System Vitamin A (and RA) plays an important role in the development of the central nervous system (Hofmann and Eichele, 1994; Maden, 1994, 1999; Maden and Holder, 1992; Maden et al., 1996, 1997, 1998; Eichele, 1997; Clagett-Dame and Plum, 1997; Gale et al., 1999; Gavalas and Krumlauf, 2000; White et al., 2000; Ross et al., 2000; Wendling et al., 2000; Zile, 2001; Cheung et al., 2001; Villanueva et al., 2002). RA value is demonstrated by (1) the distribution of retinoid-binding proteins and retinoid receptors, (2) the effects of interference with RA signaling pathways, (3) the consequences of retinoid excess or deficiency, (4) the detection and evaluation of endogenous retinoids, and (5) the distribution of retinoid-metabolizing enzymes. RA in embryonal carcinoma cells induces, at low doses, differentiation toward cardiac muscle cells, at intermediate doses to the skeletal muscle, and at high doses toward neurons and astroglia (Edwards and McBurney, 1983). Therefore, different concentrations of endogenous RA might be responsible for the induction of the different parts of the embryo such as the central nervous system, the mesoderm, or the heart. The development of the CNS and the peripheral nervous system depends upon the presence and the controlled levels of RA. It is not yet known where and how the RA is generated, but its presence is an absolute requirement. The retinol-binding protein CRBP-I (Maden et al., 1989; Hunter et al., 1991; Maden and Holder, 1991) and the RA-binding proteins CRABP-I (Perez-Castro et al., 1989; Momoi et al., 1989; Maden et al., 1989, 1996; Vaessen et al., 1990; Dencker et al., 1990; Maden and Holder, 1991; Ruberte et al., 1992, 1993; Shiga et al., 1995) and CRABP-II (Ruberte et al., 1992; Lyn and Giguere, 1994; Leonard et al., 1995) are present in the CNS with a distribution suggesting specific functions for retinol and RA in the development of peculiar neuronal populations. In early CNS development of the mouse embryo, RAR, RAR, and RAR are expressed (Ruberte et al., 1991, 1993). Concerning the neural crest and its derivatives, RAR is expressed in the migrating neural crest at a higher level than the other RARs (Ruberte et al., 1991; Osumi-Yamashita et al., 1990). However, it seems that there is a weak and ubiquitous expression of RXR and RXR in mouse embryos (Dolle et al., 1994). RXR is expressed in the organ of Corti and the spiral ganglion in the developing ear and in the neural retina of the eye (Dolle et al., 1994). In light of the binding protein and receptor distribution, it is possible to suggest some clear functions for these molecules: CRABP-I, for example, should control the commissural neurite outgrowth, RAR motoneuron development, RAR neural tube closure, RXR development of eye, ear, and individual forebrain regions, etc. Amazingly, when they were knocked out by homologous recombination in mouse embryo, the resulting embryos were, with few exceptions, perfectly normal with regard to the nervous

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system (Kastner et al., 1995). Thus, CRABP-I null mutants, CRABP-II null mutants, and CRABP-I/II double null mutants are not only normal in their CNS, but in the rest of the body, except for an extra postaxial digit (Gorry et al., 1994; Fawcett et al., 1995; Lampron et al., 1995). Similarly, it has been observed in RAR, RAR, and RXR null mutants (Kastner et al., 1995). Only the RAR and RXR knockouts have many defects that are all related to the CNS and primarily ocular alterations. Widespread eye defects including those of the anterior eye are seen when double RAR null mutants are created such as RAR/ or RAR/ (Lohnes et al., 1994; Grondona et al., 1996; Ghyselinck et al., 1997). Neural crest abnormalities are also seen in double null mutants. In particular, the following alterations have been reported: (1) periocular mesenchymal abnormalities in RAR/ mutants (Grondona et al., 1996; Ghyselinck et al., 1997), (2) alterations of the craniofacial and branchial arch skeleton in all combination mutants (Lohnes et al., 1994; Ghyselinck et al., 1997), (3) abnormalities of the thyroid, parathyroid, and thymus glands (derived from the hindbrain neural crest) in RAR/ and RAR/ mutants (Mendelsohn et al., 1994), and (4) some heart defects (aorticpulmonary septation and aortic arch abnormalities) due to the defective cardiac neural crest in RAR/ and RAR/ mutants (Mendelsohn et al., 1994). These defects thus confirm the role of the RARs in neural crest migration and/or survival. The likely targets of RA action in the CNS include neurite outgrowth, neural crest survival, and pattern specification of the posterior hindbrain (Maden et al., 1996, 1997, 1998; Maden, 1999; Gale et al., 1999). RA in neuronal cells in culture can increase neurite number and length but can also change the differentiated phenotype with regard to the neurotransmitter expressed (Haskell et al., 1987; Qiunn and De Boni, 1991; Wuarin and Sidell, 1991; Berrard et al., 1993). The neural crest is a group of migratory cells that arises from the dorsal surface of the neural tube and migrates out into the body of the embryo along well-defined pathways. The crest cells give rise to an amazing array of differentiated structures in the body including the skull and connective tissue of the head, the autonomic and sensory neurons of the peripheral nervous system, melanophores, Schwann cells, and glandular tissue. RA stimulates the differentiation of neurons by promoting neuronal precursor proliferation and survival (Henion and Weston, 1994). Furthermore, RA stimulates one particular type of neuron, the adrenergic neuron, to differentiate (Dupin and Le Douarin, 1995; Rockwood and Maxwell, 1996), and this effect seems specific to the RAR pathway (Rockwood and Maxwell, 1996). RA signaling during presegmentation stages is necessary for anteroposterior patterning of the CNS (Grandel et al., 2002). The production and function of RA in adult brain are unclear. Because RA is not transported

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preferentially to brain, this tissue likely synthesizes RA more efficiently than other target tissues (Werner and De Luca, 2002). 4. Limb Several lines of evidence suggest that retinoids are involved into the development of limb (Tickle et al., 1982; Thaller and Eichele, 1987, 1990; Smith and Eichele, 1991; Rutledge et al., 1994; Lohnes et al., 1994; Hayamizu and Bryant, 1994; Seleiro et al., 1995; Niederreither et al., 1996, 1999; Helms et al., 1996; Kastner et al., 1997b; Lu et al., 1997; Power et al., 1999). Limbs originate from small buds protruding from the body wall that consist of mesenchymal cells encased in an ectodermal hull (Tickle and Eichele, 1994; Tabin, 1995; Johnson and Tabin, 1997). Experimental manipulations of chick limbs have led to the identification of two major signaling regions that mediate patterning and growth of developing limb buds. The apical ectodermal ridge (AER) is a specialized epithelial structure that is located at the margin of the limb bud. The primary function of the AER is to provide signals that through interaction with the underlying mesenchymal tissue promote limb outgrowth (Saunders, 1948; Summerbell, 1974; Rowe and Fallon, 1982). When the AER is removed, the limb stops growing. The second signaling region consists of a small group of posterior mesenchymal cells, known as the zone of polarizing activity (ZPA) (Saunders and Gasseling, 1968). When the ZPA is removed, the limb fails to develop a distinct pattern along the anteroposterior axis (Pagan et al., 1996; Eichele, 1989). The ZPA, therefore, acts as an organizer for the anteroposterior axial pattern of the developing limb (reviewed in Tickle and Eichele, 1994; Tabin, 1995; Johnson and Tabin, 1997). A third region of interest in the limb bud is called the progress zone that resides at the distal tip of the bud (Summerbell et al., 1973). Cells in the progress zone receive growth signals from the AER and the ZPA. The ZPA, the AER and the progress zone are all required for limb development; removing of any of them results in a truncated limb. We discuss the ZPA in more detail as there is evidence that its formation is dependent on retinoids. Although definite genetic proof is still lacking, various experiments support the hypothesis that retinoids are required for the establishment of a ZPA. Briefly, around Day 2, RA is synthesized in the lateral plate mesoderm of the presumptive wing region. At this stage there is no limb bud. The local RA increase in this region triggers an RAR–RXR-mediated signal transduction cascade that results in transactivation of RA target genes such Hoxb-6 and Hoxb-8. Expression of Hox genes initiates directly or indirectly the expression of Sonic hedgehog (Shh) in the posterior mesenchyme of the limb bud. The expression of bmp-2, a gene downstream of Shh, is also induced. This establishes the ZPA, and a graded signal emanating from the ZPA, possibly along BMP pathways (Yang et al., 1997), assigns distinct identities to the cells of the limb bud mesenchyme. This

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process of cell fate specification provides the basis for the subsequent formation of distinct skeletal elements that characterize the vertebrate limb. A pattern can be established only if the limb bud grows out. Thus, simultaneous with the above specification processes, a signaling cascade regulating limb bud outgrowth is initiated. Fibroblast growth factors (FGFs) are candidates for mediating cell proliferation in this system, because ectopic application of FGF1, 2, 4, 8, and 10 proteins to the interlimb flank induces the formation of an ectopic limb (Cohn et al., 1995; Vogel et al., 1996; Crossley et al., 1996; Ohuchi et al., 1997). Around Day 2, when RA induces Hoxb-8, fgf-10 begins to be expressed in the mesenchyme of the nascent limb bud and maintains this tissue in a state of high proliferation (Ohuchi et al., 1997). fgf-10 then turns on the expression of fgf8 in the nascent AER (Crossley and Martin, 1995; Crossley et al., 1996). After the AER is formed, fgf-4 begins to be expressed in the posterior portion of the AER. FGF2, 4, and 8 in the AER and FGF-2 in the progress zone are thought to maintain cell proliferation in the progress zone and, at the same time, to preserve the expression of Shh in the ZPA. Conversely, Shh maintains the expression of FGFs in the AER. Thus, there is a cross-signaling between the AER and the ZPA mediated by Shh protein from the ZPA signaling to the AER and FGF-4 from the AER signaling to the ZPA (Laufer et al., 1994; Niswander et al., 1994). Stratford et al. (1999) has recently identified target genes in the development of the early limb bud of the quail, employing a retinoid ligand knockout model. Retinoids also influence the later stages of limb development. Embryonic VAD in rats results in a reduction or deletion of long bones and in fused digits (Warkany and Nelson, 1940). Truncations and deletions of the long bones (the humerus, radius, and ulna in the forelimb) and digit deletions and fusions occur after RA treatment of mammalian embryos (Kochhar, 1973, 1985). Mouse embryos lacking RXR have normal limbs and display resistance to limb malformations normally induced by teratogenic RA exposure (Sucov et al., 1995). RA treatments that cause limb defects in 100"% of wild-type embryos failed to elicit malformations in RXR null homozygotes, implicating RXR as a component in the teratogenic process in the limbs. Furthermore, heterozygous embryos are intermediate in their sensitivity to RA, suggesting the importance of RXR gene dosage in limb teratogenesis. These investigations, however, suggest that expression of the RA-inducible gene RAR2 is equivalent between wild-type and homozygous RXR embryos after RA treatment. The spatial expression of Shh and Hoxd-12 is also similar for both wild-type and RXR embryos following RA treatment. Hoxd-12 expression, however, is elevated in RXR embryos. These observations indicate that transcriptional processes, which are inappropriately influenced in the mouse limb by exogenous RA, require RXR for their execution.

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Results from double mutant mice provide additional information on the role of RA in limb patterning (Kastner et al., 1997). Because both RAR and RAR transcripts are uniformly expressed in the early stage of mouse limb bud (Dolle et al., 1989), it was surprising that neither RAR nor RAR single knockout mice produced limb malformations (Lohnes et al., 1993; Lufkin et al., 1993). These findings, however, suggest a functional redundancy between these two receptors. This may well be the case as limbs from RAR/RAR double mutants consistently exhibited malformations, including size reduction of the scapula, perforated scapula, radius agenesis, and abnormal digit number (Lohnes et al., 1994). Many of these limb defects appear to be fairly restricted to the forelimb skeleton and may reflect a requirement of RA to generate the proper amount of limb mesenchyme, as a deficit of mesenchyme leads to a preferential loss of anterior skeletal elements in frog hindlimbs and of posterior skeletal elements in salamander hindlimbs (Alberch and Gale, 1983). The defects do not appear to result from an early zone of polarizing activity defect because limbs displayed a clear A/P asymmetry. The investigators suggest this does not exclude a role for RA in normal A/P limb patterning, as RAR transcripts are expressed in a region that overlaps with the ZPA and appear to be unaffected in the limbs of RAR/RAR double mutants (Lohnes et al., 1994). The conclusions from the limb defects in RAR/RAR double mutants suggest that either RA plays different roles in forelimb and hindlimb development, or they are involved in events occurring at different developmental time periods for the two limbs. It has also been suggested that inactivation of all three RARs might result in more dramatic effects on limb patterning (Kastner et al., 1995). Interestingly, mice knockouts of the RALDH2 gene do not appear to have limb buds (Blomhoff, 1994), suggesting species differences. It has long been known that limbs and tails of amphibians can regenerate following amputation. In mammals this ability is largely lost, although the distal tip of fingers can regenerate (Reginelli et al., 1995). The molecular mechanisms underlying regeneration are still elusive, but progress has been made in understanding this process by means of the analysis of limb regeneration in newts (Brockes, 1994, 1997). Newts are capable of regenerating amputated limbs, even as adults. As a first reaction to amputation, epithelial cells from the wound circumference migrate to seal the wound and form a transient wound epidermis, essential for regeneration. The cells of the mesenchyme underlying the wound epidermis dedifferentiate and form a blastema in which cells proliferate. The first critical feature of regeneration is the ability of differentiated cells to reenter the cell cycle from a postmitotic state. This reentry is due to hyperphosphorylation of retinoblastoma (Rb) protein, which controls the G1–S checkpoint. The factors that alter the phosphorylation state of Rb in newt blastema are presently unknown. A second critical aspect of regeneration is that the blastema gives rise only to missing structures. Thus, amputation at wrist

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level leads to the regeneration of a hand whereas amputation at shoulder level results in the generation of a whole arm. Thus, blastema cells know their position along the proximodistal limb axis and regenerate only structures that are distal to the site of amputation. There is as yet no conclusive evidence that retinoids are necessary for normal regeneration but there are compelling data. First, endogenous retinoids are present in limb blastema (Scadding and Maden, 1994). Second, several retinoid receptors are expressed in the blastema (Ragsdale et al., 1989, 1992; Hill et al., 1993). Third, an RA reporter gene introduced into the blastema is differentially activated along the proximodistal axis (Brockes, 1992, 1994). In summary, exogenous retinoids have profound effects on limb regeneration and evidence is accruing that suggests a role of endogenous retinoids in this process. As to the nature of target genes, little is known, but it is encouraging that proximalization is mediated by only one receptor, RAR2, which should facilitate the search for target genes. Finally, Weston et al. (2002) identified a loss in retinoid receptormediated signaling as being necessary and sufficient for expression of the chondroblast phenotype and demonstrated a close association between RA receptor activity and the transcriptional activity of Sox9, a transcription factor required for cartilage formation (Weston et al., 2002). 5. Hematopoiesis The hematopoietic process begins in the extraembryonic yolk sac strictly associated with initial endothelial cells. The cluster of cells, all of mesodermic origin, forms the so-called ‘‘blood islands,’’ which include erythrocytes and the surroundings endothelial structure. The occurrence in these islands of cells of different embryonic origins led to the hypothesis of the presence of a common progenitor, defined as a hemangioblast (Murray, 1932). This is experimentally confirmed by similar expression programs and by in vitro lineage investigations employing embryonic stem cells (Choi, 1998). However, the precise relationship among hemangioblasts, hematopoietic precursors, and the mature hematopoietic stem cells (occurring in adult or ‘‘definitive’’ bone marrow) has not been elucidated. Intriguingly, the cytokines (or more general signaling molecules) that control the development of blood islands and primitive hematopoiesis are not known, albeit they are distinct from definitive hematopoietic cytokines. Primitive erythrocytes are thought of as a transient population appearing as hyperchromatic large cells, which express embryonic globins. Intriguingly, these cells do not express the regulatory genes that are required for definitive hematopoiesis (for example erythropoietin) (Wu et al., 1995). As emphasized before, vitamin A and its derivatives (particularly ATRA and 9-cis-RA) regulate normal embryogenesis and tissue maintenance (Kastner et al., 1994). In the mouse, the ablation or mutations of RAR or RXR genes have definitely confirmed many of the developmental functions

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ascribed originally to retinoids on the basis of phenotypes due to a deficiency of vitamin A (VAD) (Kastner et al., 1994, 1997). However, a role for retinoids in primitive hematopoiesis has not been suggested by mouse receptor knockouts, indicating that RA signaling is apparently not essential in the process of blood cell formation. Moreover, a transient defect in definitive hematopoiesis in the mutant mouse is caused by a delay in fetal liver hematopoietic development (Makita et al., 2001). On the other hand, there is evidence from nutritional studies suggesting a function for vitamin A during hematopoiesis (Hodges et al., 1978). In fact, either clinical studies employing human volunteers or nutritional surveys of populations from underdeveloped countries find a significant correlation between VAD and anemia. Studies using a rat model of VAD led to contrasting results that support either increased (McLaren et al., 1965) or decreased (Hodges et al., 1978) hemoglobin and hematocrit (it has been suggested that such studies can be complicated by dehydration) (Mejia et al., 1979). Bone marrow hematopoiesis in mice is affected by the absence of retinoids, causing myeloid cell expansion (Kuwata et al., 2000). Several in vitro investigations have studied the effect of RAs on hematopoietic progenitors and differentiation and, in some cases, indicate a positive role for clonal proliferation of progenitors (Douer and Koeffler, 1982). However, it is unclear whether the in vitro approach corresponds to the normal physiological function of retinoids. Recently, employing VAD quail embryo as a model system, it has been demonstrated that RA is required for the development of a primitive hematopoietic process. Indeed, this system overcomes inherent problems in investigating vitamin A function because the embryo represents a complete knockout of the nonredundant ligand as the only source of retinoids provided to the adult is RA that is not transferred to the egg (Thompson et al., 1969; Dersch and Zile, 1993). Using this experimental approach, it has been demonstrated that that retinoids are required for normal primitive erythropoiesis, and that the mechanism of RA action is through a bone morphogenetic proteindependent pathway controlling early expression of the hematopoietic regulatory gene GATA-2. Blood island development initiates normally, but in the absence of RA many of the primitive clusters start a program of apoptosis before terminal differentiation, resulting in significantly reduced numbers of primitive erythrocytes. This, finally, results in a remarkably impaired hematopoiesis. 6. Skin Vitamin A, or retinol, has long been known to play a critical role in epithelial homeostasis. Early observations (Mori, 1992; Wolbach and Howe, 1925) and in vitro investigations (Fell and Mellanby, 1953; Barnett and

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Szabo, 1973; Yuspa and Harris, 1974) clearly established the need for vitamin A in sustaining normal development and differentiation of the epidermis. RARs and RXRs are both expressed in premigratory and migratory neural crest cells that give rise to components of the epidermis. The involvement of RA and RARs in the formation of epidermis as well as hair follicles, sebaceous glands, and other associated appendages has been previously reviewed (Hardy, 1992; Viallet et al., 1992), and it is clear that both vitamin A and RA have effects upon skin differentiation during development by switching between differential development pathways. When the receptor types are specifically studied, it appears that both RAR and RAR are expressed within the developing epidermis and dermis. In contrast, in mature epidermis, the major receptor forms are RAR together with RXR. Although RAR is expressed in many other adult epithelial tissues, it is not expressed in the epidermis at detectable levels within the developing or the adult epidermis. Recently, RXR and RXR were found in dermal fibroblasts (Tsou et al., 1997). All RAR-mediated signaling pathways are dispensable in epidermis for homeostatic keratinocyte renewal. However, topical treatment of mouse skin with selective retinoids indicated that RXR–RAR heterodimers were required for retinoidinduced epidermal hyperplasia, whereas RXR homodimers and RXR– RAR heterodimers were not involved. Chapellier et al. (2002) demonstrate that the topical retinoid signal is transduced by RXR–RAR heterodimers in suprabasal keratinocytes, which, in turn, stimulate proliferation of basal keratinocytes via a paracrine signal that may be heparin-binding EGF-like growth factor. Li et al. (2000) showed that RXR has key roles in hair cycling and in epidermal keratinocyte proliferation and differentiation. Qualitatively, RA is the form of vitamin A that is important for normal epidermal homeostasis (Kang et al., 1995). Despite the fact that the epidermis is an avascular tissue, it contains significant quantities of vitamin A. Vitamin A and its metabolites that are present in the epidermis are derived either directly from retinoids in the blood supply of the dermis, or indirectly via metabolism of the blood retinoids. The retinyl ester present in epidermis is probably the result of synthesis by LRAT. The retinol esterification reaction in keratinocytes has potential to regulate the synthesis of RA because both reactions utilize retinol as a substrate (Randolph and Simon 1995, 1996; Kurlandsky et al., 1996). It is for this reason that the distribution of retinol-esterifying activity across the strata of differentiating cells of the epidermis is important. The majority (about 75%) of total epidermal LRAT activity resides in the basal cells of the epidermis (Kurlandsky et al., 1996). The remaining LRAT activity is located in differentiating suprabasal cells. This distribution of retinol-esterifying activity is also consistent with the retinyl ester content of different epidermal

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strata (To¨rma and Vahlquist, 1990). As plasma retinol is transported from the blood supply in the dermis to the epidermis, it is taken up by and esterified in basal keratinocytes. Some retinol is not taken up in the basal layer and permeates the suprabasal layers of the epidermis, where it is taken up and esterified in differentiating keratinocytes. Thus, retinyl ester continues to accumulate in keratinocytes with increasing suprabasal location (To¨rma and Vahlquist, 1990). This trend toward increasing retinyl ester content and fairly constant retinol content with an increasingly suprabasal position suggests that the rate of retinol transport, uptake, and esterification exceeds the rate of retinol utilization, i.e., RA synthesis, throughout the differentiation process. As with retinal, RA is accessible (potentially) to the epidermis from the circulatory supply of underlying dermis. However, there are at least two sources for the low quantities of RA in the epidermis: (1) plasma RA may be transported to the epidermis bound to albumin from the blood supply of the dermis, and (2) via metabolism of retinol in situ. The characterization of the biochemical pathway(s) of RA synthesis in the epidermis will most certainly be the subject of future investigation. In many tissues, intracellular retinol is bound to CRBP type I (Ong et al., 1994). The significance of CRBP in a tissue is that it coordinates and directs the metabolism of retinol toward pathways of esterification or RA synthesis. Cellular retinal-binding protein has been quantitated in epidermal tissue. The CRBP concentration in neonatal epidermis is very high (Gates et al., 1987) compared to adult epidermis. The reason for this higher level of CRBP in neonatal skin is not known. Cellular retinol-binding protein is expressed in the cytoplasm of keratinocytes in all strata of the epidermis. More CRBP is present in differentiating suprabasal keratinocytes than in the basal layer (Siegenthaler and Saurat, 1991; Busch et al., 1992). The presence of CRBP in all of the living layers of the epidermis is consistent with both retinol esterification and RA synthesis. Consistent with a role for CRBP in modulating the production of RA from retinol, the expression of CRBP in the epidermis is regulated by retinoids. Increasing the content of retinol in the epidermis by topical treatment of skin with retinol increases CRBP mRNA levels throughout the epidermis, presumably via the synthesis of RA (Kang et al., 1995). Concomitant with this increased CRBP expression, topical retinol also increases the epidermal content of retinyl ester (Duell et al., 1996). This response suggests that the epidermis responds to increased retinol levels by increasing the quantity of CRBP available to bind retinol. The resulting increased concentration of CRBP – retinol stimulates the esterification of retinol by LRAT, decreasing retinol levels and the synthesis of RA. Topical treatment of skin with RA increases the expression of CRBP mRNA in a manner similar to that observed for retinol (Fisher et al., 1995). Consequently, keratinocytes throughout the epidermis possess the potential

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to respond to and control their supply of retinol, and the synthesis of RA, by regulating CRBP expression. Keratinocytes also contain cytosolic-binding proteins for RA. Two cytosolic-binding proteins for RA, cellular RA-binding proteins (CRABP) type I and type II, are known (Ong et al., 1994). The incremental abundance of CRABP-I in keratinocytes in progressively later stages of differentiation suggests that it plays an important role in the differentition process with regard to RA. It is not clear, however, what this role might be. The expression of CRABP-II increases dramatically at both the mRNA and protein levels in epidermis exposed to topical RA (Hirschel-Sholz et al., 1989a,b; Astrom et al., 1991; Elder et al., 1992a,b, 1993; Tavakkol et al., 1992; Saurat et al., 1994; Karlsson, 2002), retinal (Saurat et al., 1994), and in epidermis of individuals taking oral retinol (Saurat et al., 1994; Kang et al., 1995) or retinoids (Hirschel-Sholz et al., 1989b). This regulation in response to exogenous retinoids suggests that CRABP-II may function under these circumstances to buffer keratinocyte nuclear RA receptors against sudden or extreme changes in the availability of exogenous RA. It is not clear, however, whether this is the function of CRABP-II in the normal differentiation process. CRABP-II seems to play a facilitating role, enhancing the access of newly synthesized RA to retinoid receptors in the nucleus during differentiation (Jing et al., 1997). As cited above, the RA concentration of epidermis depends on the balance between input via the blood supply of the dermis, synthesis in situ, and output via metabolism. The qualitative importance of cytocrome P-450linked RA metabolism to maintaining low epidermal RA concentrations is evident from in vivo studies in which an inhibitor of cytocrome P-450 function is applied topically to skin (Kang et al., 1996) and in cultured keratinocytes (Randolph and Simon, 1997). The broad mechanism of action of vitamin A leads to drawbacks if the goal of retinoid use is therapeutic. Natural retinoid acids, including ATRA, 9-cisRA, 13-cis-RA, and other retinoids such as 4-oxo-RA, 3,4-didehydroretinol, and 14-hydroxy-4,14-retroretinol have been shown to be biologically active. In particular, ATRA, 13-cis-RA, and the first generation analogues (Etretinate and Acitretin) have been used succesfully in controlling the symptoms of many skin diseases, including acne, rosacea, photodamage, skin-related malignances, and other miscellaneous skin disorders (Orfanos et al., 1997). In addition to affecting proliferation and differentation, topical application of some retinoids leads to erythema, peeling, and dryness (Orfanos et al., 1997) that often results in a cessation of treatment. Many side effects that have been observed can be linked to the nonselective activation of retinoid signaling pathways in vivo. The design of receptorselective ligands, in concert with a better understanding of the mechanism of retinoid action, provides novel approaches for new retinoid or retinoidassociated therapies (Bernard et al., 2002). Specific RXR and RXR ligands

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in skin therapy can now be investigated (Li et al., 1997). Two substances, Adapalene and Tazarotene, have been developed and are presently used for acne and psoriasis, respectively (Shroot and Michel, 1997; Cunliffe et al., 1997; Esgleyes-Ribot et al., 1994; Weinstein, 1996; Thacher et al., 2000; Egan et al., 2001; Ioannides and Katsambas, 2002; Guenther, 2002). Current basic research is also focused on developing receptor-selective retinoids that would have a higher therapeutic index for the treatment and chemoprevention of skin cancer (Levine, 1998; Stratton et al., 2000; Niles, 2002). 7. Lung Several studies have shown that retinoids are important for proper lung morphogenesis and for differentiation of the respiratory epithelium (Malpel et al., 2000). Using partially vitamin A-depleted rodent models, in which the fetuses survive longer and later gestational windows can be examined, it was demonstrated that vitamin A is specifically required during midgestation for fetal lung development and neonatal survival (Wellik et al., 1997; Antipatis et al., 1998; Zachman and Grummer, 2000). Retinoids have received considerable attention as alveolar morphogens and potential therapeutic agents (Mao et al., 2002), after RA was shown to ameliorate the emphysema observed in rats after an intratracheal instillation of elastase (Massaro and Massaro, 1997; McGowan, 2002; Hind et al., 2002a,b). Abnormal cell differentiation and diminished expression of elastin gene and a growtharrest gene were associated with abnormal lung morphology (Antipatis et al., 1998), also seen in mice with deletions in RAR genes (McGowan et al., 2000). Mutants of RAR also exhibit congenital respiratory tract abnormalities (Luo et al., 1996). It is likely that other genes are also influenced by retinoids, and alterations of elastin expression may be secondary to a more generalized effect on cellular proliferation or differentiation. Among the other genes that may be targets of retinoid action that may secondarily alter elastin gene expression are the growth factors, transforming growth factor- and platelet-derived growth factor and its receptor. Other potential targets are the nuclear receptors for homeodomain proteins, such as Hox B5 (Volpe et al., 2000) and GATA family members such as GATA-4, GATA-5, and GATA-6. 8. Kidney Like other tissues and cell system, retinoids play an essential role in kidney organogenesis, and it has only recently been recognized that even mild fetal VAD syndromes can result in a reduction in nephron number (Gilbert and Merlet-Benichou, 2000; Burrow, 2000). Recent studies have also begun to define the cellular and molecular events associated with retinoid actions in the fetal kidney and have demonstrated the essential function of retinoids in branching growth of the ureteric bud. During

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embryogenesis, RA influences tubulogenesis and codetermines the number of glomeruli per kidney (Vilar et al., 1996). In the kidney both RARs and RXRs are cell-specifically expressed. This organ is, therefore, potentially responsive to activation of both receptors by their respective agonists (Sugawara et al., 1997; Wagner, 2001; Yang et al., 1999). Double knockout models have several malformations, including kidney agenesis, hypoplasia, or aplasia of the ureteral bud (Mendelsohn et al., 1994). Importantly, characterization of the renal developmental effects of knockout models combined with metanephric organ culture studies have together shown that one essential function of retinoid action in the developing kidney is the maintenance of c-ret expression in the tips of the ureteric bud. However, many other potential retinoid target genes including midkine, Shh, Hoxd-11, matrix metalloproteinases, and tissue inhibitors of metalloproteinases appear to play important roles in renal development and might be pivotal downstream mediators of retinoid effects in the developing kidney (Burrow, 2000; Wagner, 2001; Vilar et al., 2002). Genetic experiments indicated a role of retinoids in renal development. Surprisingly, however, the action of retinoids in the adult kidney has not been studied, and the kidney is usually not considered a relevant primary target for retinoids. However, retinoids interfere with cell proliferation, inflammation, and the extracellular matrix, and all these processes are relevant in the genesis of inflammatory renal disease. The antiinflammatory and antiproliferative actions of retinoids have long been known. Retinoids interfere with factors such as endothelin, angiotensin II, platelet-derived growth factor, nitric oxide, and transforming growth factor- (Wagner, 2001; Haxsen et al., 2001). Recently, information on the action of retinoids in renal damage models, i.e., anti-Thy1.1 nephritis, ureteral ligation, and subtotal nephrectomy, has become available. In anti-Thy1.1 nephritis, treatment of nephritic rats with ATRA or isotretinoin (13-cis-retinoic acid) effectively limited renal damage and mesangial cell proliferation (Wagner et al., 2000; Schaier et al., 2001), reducing renin–angiotensin system activity (Dechow et al., 2001; Shi et al., 2001), lowering endothelin-1 expression, and downregulating the endothelin A and B receptors in the kidney (Lehrke et al., 2002). Recently, less glomerular expression of TGF-1 and TGF receptor II expression was found in glomeruli of retinoid-treated rats compared to vehicle-treated ones (Morath et al., 2001). 9. Immunity Many reviews summarized the relationship between VAD and increased morbidity and mortality due to infection in animals and humans (Hayes et al., 1994; Ross and Ha¨mmerling, 1994; Kolb, 1995; Ross and Stephensen, 1996; Villamor and Fawzi, 2000). Vitamin A is required by the natural killer (NK) cells and by phagocytic cells, which both mediate innate immunity. The amount and activity of NK

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cell were reduced in VAD animals and dietary retinoid repletion restored this activity (Goldfarb and Herberman 1981; Santoni et al., 1986; Goettsch et al., 1992; Dawson, 1999). Macrophage and neutrophil function are retinoid dependent. Phagocitic cell numbers were unchanged, but phagocytosis and intracellular killing mechanisms were lower in VAD rats than in controls (Ongsakul et al., 1985; Wiedermann et al., 1996; Twining et al., 1997). The neutrophils from VAD rats had abnormal, hypersegmented nuclei (Twining et al., 1996), poor in vitro chemotactic ability, and reduced production of oxidative molecules (Twining et al., 1997). VAD diminishes nearly all immunoglobulin (Ig) isotype responses to T-dependent antigens (Smith and Hayes, 1987) and is associated with defective mucosal IgA responses (Palmer, 1978; Karalliedde, et al., 1979). The repletion restored the Ig responses (Stephensen et al., 1996). Most studies reported normal or nearly normal B cell numbers in VAD animals, indicating adequate B lymphopoiesis (Ross and Ha¨mmerling, 1994). Memory B cell formation was undisturbed in VAD rats (Ross, 1996). Normal IgM responses were obtained in VAD rats immunized with the T-dependent antigens (Wiedermann et al., 1993; Ross, 1996; Arora and Ross, 1994). Together, this evidence strongly suggests that B lymphopoiesis, activation, Ig synthesis and secretion, Ig isotype diversification, and memory formation are not explicitly retinoid dependent. Conversely, Carman et al. (1989) provided definitive evidence for a Th2 cell dysfunction in VAD mice. The macrophage capacity to stimulate antigen-specific T cell proliferation was unaltered in VAD rodents (Carman et al., 1989; Wiedermann et al., 1993). Lymphocyte turnover is tightly controlled, and vitamin A may be one of the regulators. Bone marrowderived lymphopoietic cells migrate into the thymus where they generate mature T lymphocytes. Thymic atrophy occurred during VAD in some species, and retinoid administration restored the intrathymic small lymphocyte numbers (Ross and Ha¨mmerling, 1994). The decrease in CD4+CD8+T cell development may reflect retinoid downregulation of Bcl-2 gene expression (Agarwal and Mehta, 1997; Bruel et al., 1997). The Bcl-2 gene is required for thymocyte maturation. Retinoid also controls some Th1 cell cytokine responses. There is a negative effect of ATRA on interferon (IFN)- synthesis in all cells that produce it (Cantorna et al., 1995). Retinoid dependence of Th2 cytokine synthesis has been also reported (Carman et al., 1992; Cantorna et al., 1994; Racke et al., 1995). Despite the considerable progress partly reported above in understanding the immunobiological role of vitamin A in supporting the immune system, detailed molecular mechanisms and identified effects are largely lacking. A mechanistic explanation is needed for retinoid support of basal NK cell number, and for the decreased chemotaxis, phagocytosis, and oxidative burst in retinoid-depleted macrophages and neutrophils. Detailed studies on

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antigen-presenting cells are needed to understand how retinoids inhibit proinflammatory tumor necrosis factor (TNF)- and interleukin (IL)-6 synthesis and excessive Th1 stimulating capacity. For unknown reasons, Th2 cell and perhaps CTL development and/or cytokine synthesis are retinoid dependent, suggesting additional areas to be explored. The relationship between VAD and infections (measles, HIV, parasites, etc.) is described in another section of this chapter. Few studies has been performed on VAD and autoimmunity. Susceptibility to rheumatoid arthritis depends on genetic and environmental factors (Feldman et al., 1996). VAD exacerbated arthritis in rodents (Wiedermann et al., 1995; Cantorna and Hayes, 1996). Vitamin A has shown some value in treating arthritis. Retinoid inhibition of collagenase gene expression may partially explain suppression of joint destruction (Vincenti et al., 1996). Retinoids may also decrease arthritic inflammation through combined effects on innate immunity and on epithelial, endothelial, and connective tissue cells in the inflammation site. Two clinical trials have tested retinoids in patients with arthritis. Etretinate treatment reduced joint swelling and pain in patients with psoriatic arthritis (Ciompi et al., 1988). However, N-(4-hydroxyphenyl)-retinamide provided no benefit to patients with severe, long-standing rheumatoid arthritis (Gravallese et al., 1996).

V. RETINOL AND INFANCY A. RECOMMENDED DIETARY ALLOWANCES

Vitamin A adequacy is usually discussed in terms of recommended dietary allowances (RDAs). The RDA recommends the daily dietary intake that is sufficient to meet the nutrient requirements of nearly all (97–98%) healthy individuals in each age and gender group. Vitamin A is expressed either as international units (IU) or retinol equivalents (RE). Conversion of vitamin A IU to RE, the micrograms of retinol with equal biological activity, depends on the chemical form of vitamin A, as reported in Table I (Bendich and Langseth, 1989). Thus, the calculation of RE supplied by a diet requires knowledge of the proportions of preformed vitamin A, -carotene, and other vitamin A precursors. The RE cannot be calculated only from the total dietary vitamin A activity in IU. The differences between RE and IU values are based on the assumption that RE for infants younger than 6 month (who are breast milk fed) is all as retinol. All subsequent intakes are assumed to be half as retinol and half as -carotene when calculated as IUs; as REs, three-fourths are retinol and one-fourth is -carotene. For vitamin A the 2001 RDAs in migrograms RE and IU are listed in Table II (National Institutes of Health Clinical Center Web site, available at http://www.cc.nih.gov/ccc/supplements).

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TABLE I. International Unit (IU) and Retinol Equivalent (RE) 1 IU

1 RE

0.30 mg preformed retinol 0.60 mg -carotene 1.20 mg other mixed carotenoids 1.00 6.00 12.00 3.33 10.00

mg retinol mg -carotene mg other provitamin A carotenoids IU vitamin A activity as retinol IU vitamin A activity as -carotene

These recommendations were set in relation to nutrition adequacy plus a margin of safety. Adequacy is based on maintenance of the normal range of vitamin A in serum or plasma (0.70–2.79 mmol/liter). On the basis of research on requirements and concern about possible human toxicity, recommended dietary intakes (RDI) for vitamin A were suggested by Olson (1987). These and other different recommendations (Anonymous, 1976; Underwood, 1984), all intended to support good nutritional status with an appropriate margin of safety, do not define the thresholds above these intakes at which an unacceptable risk of adverse effects can occur. The habitual vitamin A intake of most people in North America and western Europe is sufficient to result in a rise in liver vitamin A concentrations within each decade of life (McLaren, 1984). Nevertheless, some individuals are deficient in vitamin A and the problem may be unrecognized. In two major nutrition surveys—the First Health and Nutrition Examination Survey (NHANES I, 1971–74) (AAVV., 1979) and the U.S. Department of Agriculture (USDA) Nationwide Food Consumption Survey (1977–78) (AAVV., 1980a)—vitamin A was found to be a nutrient problem. Indeed, 20% or more of the population surveyed was obtaining less than 70% of the RDA (AAVV., 1980b). These data were confirmed in NHANES II data, which were extended to include black and Hispanic populations (Carrol et al., 1983). Results of two recent national surveys, the third National Health and Nutrition Examination Survey (NHANES III, 1988–91) and the Continuing Survey of Food Intakes by Individuals (CSFII 1994) (Balluz et al., 2000), suggested that the dietary intake of some Americans does not meet recommended levels for vitamin A. These surveys highlight the importance of encouraging all Americans to include dietary sources of vitamin A in their daily diets. Although classic VAD syndrome is now uncommon in western societies, specific clinical conditions can represent major risk factors. These include, for example, poor intake (food faddism, elderly populations, malabsorption, parenteral nutrition), abnormal losses (hemodialysis), or abnormal metabolism

Age

Children

<6 months 6 months–1 year 1–3 years 4–8 years 9–13 years 14–18 years >19 years

Ages <6 months 500 mg or 1665 IU 300 mg or 1000 IU 400 mg or 1333 IU 600 mg or 2000 IU

Men

Women

Pregnancy

Lactation

900 mg or 3000 IU 900 mg or 3000 IU

700 mg or 2330 IU 700 mg or 2330 IU

750 mg or 2500 IU 770 mg or 2565 IU

1200 mg or 4000 IU 1300 mg or 4335 IU

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TABLE II. Recommended Daily Dietary Allowances of Vitamin A for Children and Adults in Micrograms (mg) Retinol Equivalents (REs) and International Units (IUs)

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(alcoholism). In addition, pregnancy or alcohol abuse may increase vitamin A requirements. Despite the difficulty in comparing results of surveys on supplement usage, there is no doubt that prevalence of supplement use is increasing among males and females, among whites and blacks, and in income groups below and above the poverty level. Depending on the source of data, at least 30% of U.S. residents use vitamin supplements regularly, suggesting that physicians need to be informed about available preparations and prepared to counsel patients in this regard (McDonald, 1986; Moss et al., 1989; Balluz et al., 2000); at the least, patients should be queried about their usual diet and use of vitamin supplements. However, data for certain subgroups of the population, e.g., vegetarians (Read and Thomas, 1983), pregnant and lactating women (Costas et al., 1987), and professional athletes (Grandjean, 1983), indicate a much higher prevalence of supplement use. It is more common among females than males in almost every age group, is higher in whites than in blacks, and increase with higher education and income (Schutz et al., 1982; Stanton, 1983; Stewart et al., 1985; Hartz et al., 1988; Moss et al., 1989). Approximately one-fourth of the U.S. population and two-thirds of users of vitamin and mineral supplements ingest products containing vitamin A (Yearick et al., 1980; Garry et al., 1982; Stanton, 1983; Stewart et al., 1985; Costas et al., 1987; Moss et al., 1989). Survey data indicate that people use supplements for reasons that vary widely. The most commonly stated reasons are (1) to supplement the diet (i.e., nutrition insurance), (2) to improve general health, and (3) to increase energy and vitality (Hartz et al., 1980; Read and Thomas, 1983; Stanton, 1983; Raab, 1987). The widespread consumption of vitamin and mineral supplements is likely to continue and probably even increase with the popular interest in self-care and concern about dietary adequacy in combination with advertising pressure and an increase in lay publications on nutrition, health, and disease prevention. The likelihood of an increase in supplemental consumption is even greater for certain nutrients, including vitamin A, which some studies have indicated may help to reduce the rate of certain cancers. However, both intake and blood levels of vitamin A have generally been shown to be unrelated to the risk of cancer (AAVV., 1997c). Supplemental -carotene has consistently failed to reduce the risk of cancer in randomized trials. Guidelines from some professional societies or governmental panels recommend attempting to obtain vitamins and minerals from food sources rather than from supplements. Sources of vitamin A can be divided in two groups, vegetable and animal foods. In animal foods the vitamin A is present as retinol, and in vegetables it is present as provitamin A. It is necessary to eat six times as much provitamin A to get the same amount of vitamin A as in retinol. It is easier for the body to take up vitamin A if the food is cooked and eaten together with some fat or oil. The animal food

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sources are liver, fish liver oil, egg yolk, milk and milk products, food with milk fat, e.g., margarine and butter, and small fish with liver, e.g., sardines. Vegetable food sources are dark green leaves, for example, spinach, cassava, and mustard; yellow and orange vegetables, for example, carrot, colored yams, yellow squash, and sweet potatoes; yellow and orange fruits, for example, papaya, apricots, and mangoes (but not citrus fruits); and red palm oil. However, the American Dietetic Association and the U.S. Dietary Guidelines also note that some people may need vitamin supplements in addition to a good diet to ensure that their nutritional needs are met (AAVV., 2000, 2001). As a result of the adverse health effects of VAD in children, the World Health Organization (WHO) and the United Nations International Children’s Emergency Fund (UNICEF) issued joint statements about vitamin A and children’s health. Both agencies recommend vitamin A administration for all children diagnosed with measles in communities where VAD is a serious problem and where death from measles is greater than 1%. In 1994, the American Academy of Pediatrics recommended vitamin A supplementation for two subgroups of children likely to be at high risk for subclinical VAD. These subgroups were children 6–24 months of age who had been hospitalized with measles and hospitalized children older than 6 months. Fat malabsorption can promote diarrhea and prevent normal absorption of vitamin A. This is most often seen with cystic fibrosis, sprue, pancreatic disorders, and after intestine surgery. Healthy adults usually have a 1-year reserve of vitamin A stored in their livers and should not be at risk of deficiency during periods of temporary or short-term fat malabsorption. Long-term problems of fat absorbing, however, can result in deficiency, and in these instances physicians may advise vitamin A supplementation. Children may have enough stores of vitamin A to last several weeks. Physicians treating children with fat malabsorption may recommend vitamin A supplementation. Vegetarians who do not consume eggs and dairy foods need greater amounts of provitamin A carotenoids to meet their need for vitamin A. It is important for vegetarians to include a minimum of five servings of fruits and vegetables daily and to regularly choose dark green leafy vegetables and orange and yellow fruits to consume recommended amounts of vitamin A. Intake of up to twice the RDA of vitamin A of 5000 IU is thought to be safe. However, an intake of preformed vitamin A (retinol) in the range of 10,000 IU per day or higher—which can be attainable from foods rich in vitamin A in combination with a multivitamin containing the RDA of retinol—might be undesirable. Intakes of preformed vitamin A in this range have been associated with an increased risk of hip fracture (Melhus et al., 1998), and daily intakes of approximately 10,000 IU during pregnancy have been associated with specific birth defects (Rothman et al., 1995), but confirmation of these associations is needed. The Institute of Medicine has established tolerable upper levels (UL) of intake for vitamin A from

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supplements to help prevent the risk of toxicity. The risk of adverse health effects increases at intakes greater than the UL (Table III). In conclusion, there is no separate RDA for -carotene or other provitamin A carotenoids. The Institute of Medicine report suggests that consuming 3–6 mg of -carotene daily will maintain plasma -carotene blood levels in the range associated with a lower risk of chronic diseases. A diet that provides five or more servings of fruits and vegetables per day and includes some dark green and leafy vegetables and deep yellow or orange fruits will provide recommended amounts of -carotene. B. VITAMIN A STATUS IN PREGNANT AND LACTATING WOMEN

Vitamin A metabolism does not seem to be considerably affected during pregnancy. However, retinol-binding protein (RBP) is found in urine during normal pregnancies, whereas its excretion outside of pregnancy is considered to be a clinical symptom of renal failure (Gero¨ et al., 1986). Vitamin A is transferred from the mother to the embryo across the placenta; vitamin A concentrations in fetal blood are approximately half those in the mother. RBP is involved in this transfer from mother to embryo; nevertheless, its specific metabolism and the existence of other yet unknown binding proteins in the placenta and in maternal and fetal blood require further study (Sklan et al., 1985; To¨rma and Vahlquist, 1986; Dancis et al., 1992). Little information is available about the biogenesis of RA in pregnancy or in the embryo. Studies in humans are scarce; nevertheless, some authors have reported an increase in circulating concentrations of RA and of 4-oxoRA (in their all-trans and 13-cis form) after the intake of vitamin A (Eckhoff et al., 1991; Buss et al., 1994; H. Chen et al., 1996; Miller et al., 1998). Moreover, the teratogenic threshold of circulating RA is not known, nor is the nature of the specific teratogenic metabolite in humans. Uncertainty also remains concerning the extent of transplacental transfer of vitamin A, RA, and their derivatives, as well as about the embryonic or fetal metabolism of these compounds in humans (Creech-Kraft et al., 1989). A better understanding of RA metabolism in human tissue is a prerequisite for estimating potentially teratogenic doses of vitamin A during human pregnancy (Sapin et al., 2000). Although the need in pregnancy is increased above the nonpregnant state, this additional amount is relatively small and confined mostly to the last trimester. The increment can be adequately provided from maternal reserves of women introducing concentrations above the United States RDA of 800 mg retinol equivalents (about 2700 IU) (Food and Nutrition Board, 1989; Tyler et al., 1991). The dietary intake data (Olson, 1987; Reifen and Ghebremeskel, 2001) indicate that vitamin A needs are adequately met in western industrialized countries. Even in these countries, however, a small

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TABLE III. Upper Limits (UL) in Micrograms (mg) and International Units (IU) for Retinal Equivalents Age 0–12 months 1–3 years 4–8 years 9–13 years 14–18 years >19 years

Children 600 600 900 1700

mg mg mg mg

or or or or

2000 2000 3000 5665

Men

Women

Pregnancy

Lactation

2800 mg or 9335 IU 3000 mg or 10,000 IU

2800 mg or 9335 IU 3000 mg or 10,000 IU

2800 mg or 9335 IU 3000 mg or 10,000 IU

2800 mg or 9335 IU 3000 mg or 10,000 IU

IU IU IU IU

505

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percentage of women consumes less than the minimum requirement of vitamin A from food because of religious or cultural reasons, ignorance, or other peculiar circumstances. The study by Duitsman et al. (1995) identifies a population of pregnant women in the United States at high risk of vitamin A inadequacy (Underwood, 1994). In contrast, the median food intake of most women in many nonindustrialized countries is chronically lower than the safe dietary intake and their usual nutrition does not change during pregnancy. They are also likely to have limited body reserves. Mean serum retinol concentrations of about 1.05 mmol/liter (300 mg/liter) have been reported during pregnancy among diverse groups of southasian women (Basu and Arulanantham, 1973; Panth et al., 1990; Sivakumar et al., 1997; Christian et al., 1998a) in comparison with values of 1.57–1.75 mmol/liter (450–500 mg/liter) in better nourished populations (Morse et al., 1975; Yamini et al., 2001). Vitamin A needs during lactation exceed those of pregnancy not because of increased demand by maternal tissues but to replace that lost daily in breast milk. Newman (1993) summarized average daily intake data of unsupplemented lactating women from 19 studies in developed countries and 32 studies from developing countries and reported a difference of more than 2-fold. The average intake in developing countries, therefore, is about two-thirds of the recommended safe daily intake and less than half the average intake of lactating women in developed countries. Evidence from Indonesia indicates subclinical VAD among both pregnant (Suharno et al., 1992) and lactating women (Stoltzfus et al., 1993). The regulated delivery of vitamin A to the fetus during pregnancy limits infant body stores of the vitamin at birth. Infants born prematurely are especially vulnerable to limited body stores. Preterm infants are at a high risk for bronchopulmonary dysplasia, and vitamin A supplementation protects against this condition (see Section V.C). Without continued supplementation, suboptimal vitamin A status may persist for many preterm infants, possibly with health consequences (Peeples et al., 1991). The limited body stores at birth may increase rapidly postpartum, depending on the diet. Breast milk is the only source of vitamin A during the neonatal period for the exclusively breast-fed infant, and it is the principal source for many infants from developing countries as long as breast-feeding continues. The ability to meet infant requirements, therefore, depends on the concentrations and volume consumed, both of which are influenced by maternal vitamin A status and dietary intake. (Ortega et al., 1997). Colostrum and early milk are extremely rich in vitamin A (Wallingford and Underwood, 1986; Newman, 1993). Early feeding practices can therefore significantly augment the neonatal body stores. The preformed vitamin A in breast milk, even from a poorly nourished mother, is adequate to meet basic physiological needs and to avoid clinical deficiency during the first half of infancy. Breast milk from a malnourished mother, who is deficient in vitamin A, however, may not be adequate to maintain and increase body

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reserves in the rapidly growing infant. Infants from age 6 months to 6 years who are depleted of vitamin A body stores are at increased risk of death if they become infected. Diet and/or supplementation of vitamin A of women during pregnancy and lactation could improve their status and that of the infants. The preferred intervention is through a diet that provides a safe concentration of intake throughout pregnancy and lactation. Supplement use during pregnancy presents a logistical problem in most developing countries because it is only safe to give a near physiological dose daily, but few health systems have such frequent contacts with mothers. High-dose supplementation of the lactating mother immediately postpartum may be the safest and most effective approach to improving her status and that of the nursing infant (Wallingford and Underwood, 1986). C. VERY LOW BIRTH WEIGHT

Vitamin A promotes normal growth and differentiation of epithelial cells. VAD results in a progressive sequence of histopathological changes in the epithelial lining of pulmonary conducting airways (Wolbach and Howe, 1925; Wong and Buck, 1971; McDowell et al., 1984a,b). Necrotizing tracheobronchitis is characteristic of early stages of VAD. In more advanced stages of VAD a squamous metaplasia develops. The histopathological changes of VAD and the associated pathophysiological consequences are reversible with restoration of normal vitamin A status (Wolbach and Howe, 1933; McDowell et al., 1984a,b). Recently, Zolfaghari and Ross (2002) showed that LRAT expression and vitamin A storage are regulated by vitamin A status and by treatment with ATRA in the lung of adult rats. These results suggest that the regulated storage of vitamin A may be important for maintaining the integrity and physiological functions of the lung. Very low birth weight (VLBW) neonates are susceptible to acute, subacute, and chronic lung injury (Northway et al., 1967). When the pulmonary conducting airways are injured, the stimulus to epithelial regeneration is triggered (Stahlman et al., 1988a,b). Simultaneous with this injury, if VAD is present, normal development does not occur and chronic bronchopulmonary dysplasia (BPD), the most prevalent form of chronic lung disease in infancy, is the result. VLBW neonates have an increased risk to develop BPD. Among the 3.9 million births in the United States in 1997, approximately 55,000 newborn infants weighed less than 1500 g at birth (Guyer et al., 1998). The pathogenesis of BPD involves factors causing injury to an immature lung and factors inhibiting its healing (Northway et al., 1967; Stahlman et al., 1988a,b). The lung injury can result from such insults as hyaline membrane disease, barotrauma or volutrauma from mechanical ventilation, oxygen toxicity, and airway infection associated with prolonged tracheal intubation (Stahlman et al., 1979). The lung healing can be influenced by nutrients, antioxidants, inflammatory cells, eicosanoids, growth factors,

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peptide hormones, and components of extracellular matrix (Stahlman and Gray, 1990). The role of the essential micronutrient vitamin A in the promotion of orderly growth and differentiation of regenerating epithelial tissues makes vitamin A an important nutrient during recovery from lung injury (Wolbach and Howe, 1925, 1933). This rationale provides the basis for studying vitamin A status in relation to BPD in VLBW neonates. Most VLBW neonates are born with a deficiency of plasma vitamin A and plasma RBP concentrations (Brandt et al., 1978; Shenai et al., 1981; Bhatia and Ziegler, 1983) and with low liver vitamin A stores (Iyengar and Apte, 1972; Olson, 1979; Montreewasuwat and Olson, 1979; Olson et al., 1984; Shenai et al., 1985a, 1990). It is likely that the VLBW neonates are vitamin A deficient at birth because of deprivation of transplacental vitamin A supply resulting from their delivery at an early gestational age. In the absence of an adequate intake of vitamin A in the postnatal period, therefore, these infants are at added risk for becoming vitamin A deficient. VLBW neonates have evidence of VAD (Hustead et al., 1984; Shenai et al., 1985a,b; Shenai et al., 2000). BPD infants typically show a biphasic pattern of plasma vitamin A concentrations (Shenai et al., 1985a). The initial phase is characterized by declining plasma vitamin A concentrations and the subsequent phase is characterized by improving plasma vitamin A concentrations, which nonetheless remain suboptimal for extended periods. A vitamin A intake of at least 1500 IU/kg/day is necessary for normalization of plasma concentrations of vitamin A in VLBW neonates (Shenai et al., 1985a,b). However, the plasma RBP response to vitamin A administration may continue to reflect persistence of VAD, particularly among the more immature infants with significant lung disease (Shenai et al., 1990, 1995). It is possible that the requirement of vitamin A in infants with BPD relative to those with no lung disease is higher because of the ongoing need for regenerative healing from lung injury. It is also possible that the more immature the infant and the more injured the lung, the less efficient is the utilization of available vitamin A at the cellular level. Several clinical trials have tested the efficacy of vitamin A supplementation in preventing the development of BPD in VLBW neonates (Shenai, 1999; Atkinson, 2001). The first randomized, blinded, placebo-controlled clinical trial (Shenai et al., 1987) showed that vitamin A supplementation from early postnatal life in VLBW neonates can improve their vitamin A status and also ameliorate the lung disease, as evidenced by a decreased incidence of BPD and of the associated morbidity. Similar results are showed by Papagaroufalis et al. (1988). Both these trials showed that vitamin A supplementation was associated with a 31% reduction in relative risk and a 25% reduction in incidence of BPD (Ehrenkranz and Mercurio, 1992). In contrast to these trials, two other trials showed no beneficial effect of vitamin A supplementation in prevention of BPD (Bental et al., 1990;

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Pearson et al., 1992). The differences in the results between these clinical studies can be explained on the basis of differences in patient populations, postnatal therapies including surfactant and dexamethasone, and vitamin A intake (Avery et al., 1987; Georgieff et al., 1989; Shapiro, 1990). A recent multicenter trial (Tyson et al., 1999) of vitamin A supplementation has shed further light on this important nutrient in relation to lung disease in VLBW neonates. This trial involved 14 centers in the United States, a large study sample size of 807 infants, and a randomized, blinded, nonplacebo-controlled study design. Infants who weighed 401–1000 g at birth and who received mechanical ventilation or supplemental oxygen at 24 h after birth were studied. Vitamin A supplementation in this trial was effective in lowering the incidence of death or chronic lung disease. Dexamethasone, a glucocorticosteroid hormone, is being used increasingly in the postnatal treatment of VLBW neonates with BPD (Ng, 1993). Postnatal dexamethasone treatment causes a significant, yet short-term, increase in plasma concentrations of vitamin A and RBP in newborn infants (Georgieff et al., 1989). Shenai et al. (2000) in a prospective cohort study suggests that the beneficial pulmonary response to dexamethasone is influenced, at least in part, by the vitamin A status, and that gender plays a role in this response. Oral supplementation with 5000 IU vitamin A in extremely low birth weight infants does not significantly alter the incidence of chronic lung disease (Wardle et al., 2001). However, this dose may be inadequate to achieve optimal serum retinol concentrations (Rush et al., 1994).

VI. ALTERED VITAMIN A LEVELS AND CHILDHOOD PATHOLOGIES A. VITAMIN A DEFICIENCY

Night blindness apparently was first described in Egypt around 1500 b.c. and the Egyptians discovered that eating animal liver was good for its prevention. Also fishermen from New Foundland have known for a long time that they navigate better at night if they eat cod liver. A host of animal studies and anecdotal clinical reports during the first third of the century suggested a close, potentially causal relation between vitamin A status and morbidity and mortality from infection. These are detailed elsewhere (Sommer and West, 1996). Clinical and experimental VADs were recognized to be related during World War I, when it became apparent that xerophthalmia in human beings was a result of a decrease in the content of butterfat in the diet. The evidence for an association between deficient vitamin A status and the occurrence of adverse health outcomes, potentially related to the immune response, was compiled by Scrimshaw (1968) about 30 years ago. Since then, observational studies in humans and intervention trials have been published (Sommer and West, 1996).

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VAD is a major public health problem, particularly in underdeveloped countries. Worldwide it is the second most prevalent nutritional disease after protein–calorie malnutrition. It is estimated that about 750 million people are affected by VAD globally. Vulnerable groups include children from 6 months to 6 years of age and pregnant and lactating women. In particular, there are 125–190 million children with VAD, 5–10 million of whom will develop xerophthalmia, resulting in 1–2.5 million child deaths annually (AAVV., 1997a,b). VAD is especially common in tropical and subtropical regions of Africa, Asia, and Western Pacific. VAD is termed ‘‘primary’’ when it results from an inadequacy of vitamin A or its precursor, carotene, in the diet. It is called ‘‘secondary’’ when it occurs as a result of disorders that interfere with the absorption or storage of the vitamin or provitamin, as in celiac disease, cystic fibrosis of the pancreas, sprue, giardiasis, congenital absence or obstruction of the bile duct, cirrhosis of the liver, ulcerative colitis, prolonged and severe diarrhea, disorders interfering with the conversion of carotene to vitamin A (as may occur in diabetes mellitus and hypothyroidism), and factors causing unusually rapid utilization or loss of vitamin A in the body (such as acute or chronic infections with associated high and sustained fever). The vitamin A storage in well-nourished individuals is of such magnitude that at least 2 to 3 years of severe deprivation would be required to bring about clear-cut deficiency symptoms and pathological lesions. For this reason, VAD occurs rarely in adult man and usually in association with a generally inadequate and unbalanced diets deficient, to a variable degree, in other dietary essentials; in other words, VAD is usually a more or less a predominant phase of a ‘‘multiple deficiency’’ state. In the case of infants and young children, where prior storage of the vitamin is more limited, outright VAD is more common but it is not often a simple deficiency state. Much of what we know regarding symptoms and pathological changes in tissues resulting from VAD in human beings is based upon clinical observations on infants and young children. In human the most obvious and clinically important manifestation of VAD is the eye disease xerophthalmia (Wittpenn and Sommer, 1986). Xerophthalmia is a range of disorders that affects the eye and that can lead to blindness. The symptoms of xerophthalmia occur in approximately the following order: . Nightblindness: the child cannot see in dim light, making the child fall over things and unable to see for eating. . Bitot’s spots: superficial foamy patches composed of epithelial debris and secretions on the exposed bulbar conjunctiva. They do not affect sight in daylight. . Conjunctiva xerosis: dryness, slight roughness, or wrinkles in the white part of the eye, which are usually wet, smooth, and shiny. Changes of the tear ducts also lead to reduced wettability of the eye.

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. Corneal lesions: if the clear part of the eye is damaged the person cannot see properly and may go blind. Blindness is a seriously irreversible change.

Today, there is increased interest in subclinical forms of VAD, described as low storage levels of vitamin A that do not cause overt deficiency symptoms. This mild degree of VAD may increase children’s risk of developing respiratory and diarrheal infections, decrease growth rate, slow bone development, and decrease likelihood of survival from serious illness. Children living in the United States who are considered to be at increased risk for subclinical VAD include . . . . .

toddlers and preschool age children, children living at or below the poverty level, children with inadequate health care or immunizations, children living in areas with known nutritional deficiencies, recent immigrants or refugees from developing countries with high incidence of VAD or measles, and . children with diseases of the pancreas, liver, intestines, or with inadequate fat . digestion/absorption. Vitamin A supplementation was found to decrease mortality among measles patients as early as the 1930s (Ellison, 1932). During the 1960s, it was observed that the increased mortality rate among severely malnourished children was particularly high for those with eye signs of VAD (McLaren et al., 1965; Pereira et al., 1966; Kuming and Politzer, 1967). This was confirmed by longitudinal studies in Indonesia, where preschool children with mild xerophthalmia followed for 18 months had 3 to 12 times the risk of death than those without clinical VAD (Sommer et al., 1983). Positive associations between mild VAD and the risk of respiratory disease and acute diarrhea were also reported (Sommer et al., 1984). Some of these observational studies, however, have limitations, including small sample sizes and lack of adjustment for confounding variables such as socioeconomic and nutritional status. The efficacy of vitamin A supplements on mortality and morbidity end points has been reported in controlled trials (Villamor and Fawzi, 2000). The supplementation of vitamin A can be done (1) as medical therapeutic dosing, given to a person with acute medical problem related to VAD, (2) as universal prophylactic dosing, when VAD is widespread in population, and (3) as target prophylactic dosing, for specific age socioeconomic or ethnic vulnerable group, e.g., people in refugee camps. Other prophylactic intervention measures are vitamin A fortification of food (sugar, bread, monosodium glutamate, dried skimmed milk and margarine), increased consumption of fruits and yellow and dark green leafy vegetables (mango

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and papaw), improved preservation and cooking methods to decrease losses and to improve bioavailability encouragement of breast feeding as the first milk (colostrum) contains a lot of vitamin A, and use mass media to educate people on the importance of vitamin A. Some people advocate direct infant supplementation in the first half of infancy as a more direct means of ensuring adequate vitamin A status by 6 months of age. A cost-effective delivery system exists through immunization systems, such as the WHO Expanded Program of Immunization (EPI). The EPI reaches more infants in repeated contacts (up to five in the first year) than any other public health program in existence (Sutanto and Hutter, 1993). A double blind placebo-controlled trial showed that this supplementation within the EPI is safe with no side effects (de Francisco et al., 1993). Semba et al. (1995) reported that 30 mg (100,000 IU) of vitamin A coadministered with a measles immunization at 6 months of age reduced seroconversion compared to a placebo, causing serious concern. However, other authors reported that vitamin A supplementation with measles vaccine at age 9 months increases measles-specific antibody concentrations (Benn et al., 1997; Bahl et al., 1999) and had a long-term effect on measlesspecific antibody levels contributing to improved measles control in lessdeveloped countries (Benn et al., 2002). 1. Mortality Vitamin A supplementation to children decreases the overall risk of mortality by about 30% (Fawzi et al., 1993). In hospitalized children with measles, the mortality reduction attributable to vitamin A supplementation is 60%, on average. However, in community-based studies conducted among children older than 6 months, the protective effect of vitamin A varied. The first of several controlled double-blind supplementation trials with mortality as their end point was carried out by the Aceh Study Group in Indonesia in the early 1980s. The outcome was a significant 27% reduction in mortality from all causes attributable to vitamin A in the supplemented group of children (Sommer et al., 1986). However, in studies in India (Vijayaraghavan et al., 1990) and the Sudan (Herrera et al., 1992) only modest or no effect was observed. The Hyderabad trial (Vijayaraghavan et al., 1990) had a series of problems that became apparent only after publication from an exchange of letters to the editor: children were routinely examined and treated for disease each week by specially trained health workers. This may explain why both the treatment and control arms experienced mortality much lower than anticipated. This general reduction in mortality drastically reduced the power of the study to detect an effect attributable to the vitamin A supplement (mean reduction 6%) (Beaton et al., 1992). In addition, the study had a large and differential loss of follow-up and low levels of compliance. The Sudan trial (Herrera et al., 1992) probably did not establish a meaningful difference in vitamin A status between the two study

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groups. It is likely that the children analyzed were not particularly vitamin A deficient, and almost half of them lived in homes with sanitary facilities and piped-in water. Thus, they were hardly representative of the economic status and living conditions of most children in the developing world. Importantly, the senior authors of both these studies have subsequently supported the value of improving vitamin A status as a means of reducing childhood mortality (Fawzi et al., 1993; Sommer and West, 1996). In other trials that used vitamin A administered every 4 months, the supplements resulted in a significant reduction in mortality: 19% in Ghana (Ghana VAST Study Team, 1993) and 30% in Nepal (West et al., 1991). Trials of weekly supplementation with a moderate dose in southern India (Rahmathullah et al., 1990) and food fortification in Indonesia (Muhilal et al., 1988) showed risk reductions of 54% and 45%, respectively. When results of eight studies (Sommer et al., 1986; Muhilal et al., 1988; Rahmathullah et al., 1990; Vijayaraghavan et al., 1990; West et al., 1991; Kothari, 1991; Daulaire et al., 1992; Herrera et al., 1992) were pooled, vitamin A supplements significantly reduced total mortality by 30% (Fawzi et al., 1993): the effect varied with the dosage and frequency of administration. In a trial completed in Tanzania after the publication of this meta-analysis, a 50% reduction in mortality was noted both among children infected with the human immunodeficiency virus (HIV) and among those not HIV infected who received vitamin A (Fawzi et al., 1999). The effect could be mediated through a vitamin A-related enhancement of the immune function. A few studies have examined the efficacy of vitamin A supplements among children younger than 6 months old. The supplementation to Indonesian neonates resulted in a significant reduction in the risk of death during the first year of life (Humphrey et al., 1996) with no evidence of biologically significant adverse growth or developmental sequelae (Humphrey et al., 1998); however, no effect was observed on short-term survival in a similar trial in Nepal (West et al., 1995). Morbidity and mortality associated with pneumonia during infancy were not affected by vitamin A supplementation in a meta-analysis that included nine trials (AAVV., 1995), in a large placebo-controlled trial (WHO, 1998), and in a recently randomized placebo-controlled clinical trial (Semba et al., 2001). A meta-analysis (Glaszious and Mackerras, 1993) aimed at identifying and combining mortality and morbidity data for infectious diseases from 20 randomized controlled trials of vitamin A supplementation showed that the supplement has a major role in preventing morbidity and mortality in children in developing countries. In developed countries vitamin A may also have a role in those with life-threatening infection such as measles and those who may have a relative deficiency, such as premature infants. The variability in the results across trials has a number of potential explanations (Fawzi, 1997). The existence of concomitant nutrient

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deficiencies (including fat, protein, and zinc) may impair the bioavailability of the supplement. The effect also varies with the regimen characteristics, such as the dosage and interval of administration: smaller and more frequent doses of vitamin A seem to be more protective than large periodic doses (Muhilal et al., 1988; Rahmathullah et al., 1990). The particular epidemiological profile of the population under study is a relevant factor as well (e.g., the prevalence of infectious diseases at the time of supplementation and their incidence during the follow-up period). 2. Measles, Diarrhea, Respiratory Infections, and Malaria Several hospital-based trials have assessed the efficacy of vitamin A supplements on measles-associated morbidity and mortality (West, 2000). In 1932, Ellison reported a 60% reduction in the risk of death among children receiving vitamin A. In a meta-analysis that included Ellison’s and three more recent trials (Barclay et al., 1987; Hussey and Klein, 1990; Coutsoudis et al., 1991), large doses of vitamin A given on admission resulted in a significant reduction (about 60%) in the risk of death overall (Fawzi et al., 1993). Mortality in children with measles–pneumonia was reduced by about 70% when compared with the control children. In a trial in Kenya, vitamin A supplements had no effect on mortality; however, the study had limited power to examine this question (Ogaro et al., 1993). Another placebo-controlled trial was conducted in Zambia among children who had no severe measles (Rosales et al., 1996): children without pneumonia at baseline who were given vitamin A supplements were at lower risk of developing the disease. A later meta-analysis of six randomized controlled trials, five of which were conducted in hospitals and one in a community setting, concluded that vitamin A has a beneficial effect on morbidity associated with measles and should be used as a treatment for hospitalized measles cases (D’Souza and D’Souza, 2002). Several mechanisms are likely for these positive effects on measles, including a protective action of the vitamin on the epithelial lining of the gastrointestinal tract, increased mucus secretion, and enhanced local barriers to infection (Wolbach and Howe, 1925). The correction of VAD through supplementation may also improve humoral and cellular immune functions, including increases in B lymphocyte activation, proliferation and production of IgM and specific IgG, improvements in the T cell-helping response and cytokine synthesis, and enhancements in the function of natural killer cells and the monocyte/macrophage lineage (Semba, 1998; Jason et al., 2002). The vitamin A supplementation may play a role in lowering morbidity rates associated with pneumococcal disease by delaying the age at which colonization occurs (Christian et al., 2001). However, the generalization of the findings to nonmeasles episodes of these infections was not possible. A number of trials have examined the

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effect of vitamin A supplementation on diarrhea and respiratory infections in children not infected with measles. Several vitamin A efficacy trials have been done among children hospitalized with diarrhea. Two placebo-controlled studies among children in Bangladesh (Henning et al., 1992; Hossain et al., 1998) showed contrasting efficacy of vitamin A supplements: it might reflect differences in the specific etiological factors of diarrhea (rotavirus and Escherichia coli vs. Shigella). Vitamin A supplements had no overall effect on the duration of diarrhea among children in India (Dewan et al., 1995) or malnourished children from the Congo (Donnen et al., 1998). The etiology of diarrhea was not examined in either study. Bhandari et al. (1994) showed that administration of vitamin A during acute diarrhea may reduce the severity of the episode and the risk of persistent diarrhea in non-breast-fed children. Similar benefit was not seen in breast-fed children (Nita et al., 1997). VAD and acute lower respiratory tract infections coexist as important public health problems in many developing countries. About 190 million preschool children live in areas where they are at risk of vitamin deficiency, whereas 4 million children die each year because of acute lower respiratory tract infections and many more suffer from nonfatal respiratory infections (Stansfield and Shephard, 1991). VAD is associated with impaired humoral and cellular immune function, keratinization of the respiratory epithelium, and decreased mucus secretion, which weaken barriers to infection (Ross, 1996). The relation between vitamin A status and nonmeasles acute lower respiratory tract infections is controversial. In Guatemala (Kjolhede et al., 1995) and in Tanzania (Fawzi et al., 1998), the vitamin A supplements had no effect on the duration of hospital stay or on the number of days of several signs of respiratory disease (e.g., hypoxia, fever, or rapid respiratory rate). Two other trials from Brazil (Nacul et al., 1997) and Vietnam (Si et al., 1997) found no effect of vitamin A on the course of pneumonia. Given the protective effects of vitamin A supplements on mortality, it was presumed that vitamin A would have beneficial effects on morbidity. That was not the case, however, in a number of community trials (Beaton et al., 1993). In two trials in Indonesia (Sommer et al., 1986; Abdeljaber et al., 1991) and India (Rahmathullah et al., 1990, 1991) that found an effect on mortality, there was no effect on the incidence of diarrheal or respiratory infections after the intervention. Several studies specifically examined the effect of the supplements on the incidence and/or severity of infections. In several trials, beneficial effects of vitamin A were noted with regard to diarrhea, but no effect was observed on the risk of pneumonia (Ghana VAST Study Team, 1993; Barreto et al., 1994; Bhandari et al., 1994; Kartasasmita et al., 1995; Nacul et al., 1998; Fawzi et al., 2000). However, in a trial in South Africa among children born to HIV-infected women, vitamin A supplements resulted in an apparent but nonstatistically

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significant reduction of both the incidence of total and severe diarrhea and in lower respiratory infection (Coutsoudis et al., 1995). Results from two trials from Australia and one in Haiti support the hypothesis that vitamin A supplements may increase signs of infection, particularly respiratory signs, such as cough (Pinnock et al., 1986, 1988; Stansfield et al., 1993). Findings of placebo-controlled trials from Indonesia (Dibley et al., 1996), India (Ramakrishnan et al., 1995), and Tanzania (Fawzi et al., 2000) suggest that large doses of vitamin A may be harmful when given to children who are well-nourished or specifically vitamin A sufficient. In these children the supplementation may be associated with adverse effects, e.g., lower blood oxygen saturation (Nacul et al., 1997; Si et al., 1997; Fawzi et al., 1998; Stephensen et al., 1998; Sempertegui et al., 1999). The increased occurrence of signs of respiratory infection associated with vitamin A supplements may indicate an improvement in the inflammatory response, attributable to a pharmacological effect of the supplement, but the long-term implications of these increased respiratory signs are not clear. Vitamin A supplements should not be given during episodes of pneumonia without measles, unless there is evidence of VAD. The conditions under which vitamin A supplements can be harmful need to be examined further. Given that vitamin A supplements may be beneficial in reducing diarrheal disease in the period after discharge from hospital, the supplements could be given after recovery from pneumonia and at the time of hospital discharge. However, Rahman et al. (1996) showed that 61% of the supplemented infants remained vitamin A deficient despite supplementation and the acute respiratory infections were more frequent in this group. These results should suggest that these infants remain deficient because of frequent respiratory infections, particularly those accompanied by fever. In contrast to all of the above cited studies, two reports from China (Lie et al., 1993) and Thailand (Bloem et al., 1990) demonstrated significant reductions in the incidence of both respiratory infection and diarrhea in vitamin A-supplemented children compared with control children. However, a main limitation of both these studies was that the control group did not receive a placebo. Three trials specifically examined the efficacy of vitamin A supplements among children hospitalized with pneumonia due to respiratory syncytial virus (RSV). RSV is a paramyxovirus similar to measles and is an important cause of bronchiolitis and pneumonia among infants and children. Two U.S. trials reported no statistical differences regarding the days of intensive care and received supplemental oxygen (Quinlan and Hayani, 1996; Bresee et al., 1996). In contrast, in a trial in Santiago, vitamin A supplementation resulted in an apparently more rapid recovery from tachypnea among children with the most severe hypoxemia at baseline (Dowell et al., 1996).

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Each year there are 400 million individuals affected by Plasmodium falciparum malaria and 2.5 million related deaths (Krogstad, 1996). Many of the 2 billion people living in endemic areas are at risk of having nutrient deficiencies that could impair the development of immunity against P. falciparum and exacerbate the disease. Several studies suggest that vitamin A could play a part in potentiating resistance to malaria (Shankar, 1995, 2000; Varandas et al., 2001). Recently, Shankar (2000) showed that the number of episodes of falciparum malaria among young children in Papua New Guinea was 30% lower in those who received vitamin A than in placebo recipients. Serghides and Kain (2001, 2002) found reduced secretion of TNF-, upregulated CD36 expression, and increased phagocytosis of P. falciparum-parasitized erythrocytes with 9-cis-retinoic acid. It might partly explain the beneficial effects of vitamin A supplementation in malaria. 3. Zinc and Vitamin A Several studies have failed to show that vitamin A supplementation reduces morbidity. One possible explanation for the inconsistent findings is that multiple nutrient deficiencies affect the bioavailability of vitamin A and thereby prevent its beneficial effect. Among these micronutrient deficiencies, zinc is a likely suspect because of its interaction with vitamin A (Christian and West, 1998). Experimental studies have shown that serum retinol concentration is reduced in zinc-deficient animals, and vitamin A supplementation failed to increase the low serum retinol to a normal concentration (Smith et al., 1973). However, when the animals were supplemented with zinc, either alone or in combination with vitamin A, the serum retinol concentration increased. In children with severe malnutrition, zinc supplementation improved serum retinol-binding protein and retinol concentration (Shingwekar et al., 1979). Deficiencies of zinc and vitamin A often coexist in malnourished children, so supplementation with zinc might overcome the failures with vitamin A supplementation observed in several studies. Rahman (2002) showed that vitamin A alone failed to reverse VAD, as determined by measurement of vitamin A concentrations, but that combined zinc and vitamin A supplementation successfully reversed this deficiency. This improved vitamin A status in the children supplemented with both zinc and vitamin A indicates the existence of a biological interaction between zinc and vitamin A (Christian and West, 1998). Faruque et al. (1999) and Khatun et al. (2001) reported that zinc reduced the proportion of children with acute diarrhea who went on to have prolonged episodes, but no additional benefit was observed in children given combined zinc and vitamin A. However, recent studies showed a significant reduction in the incidence and prevalence of persistent diarrhea and dysentery, and acute lower respiratory tract infections in children supplemented with both zinc and vitamin A (Rahman et al., 2001; Bhandari et al., 2002; Datta, 2002). On the contrary, children with severe measles accompanied by

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pneumonia treated with antibiotics and vitamin A did not show any additional benefit from also receiving a zinc supplement (Mahalanabis et al., 2002). Finally, supplementation with iron and zinc (to improve vitamin A status) in preschool children (Mun˜ oz et al., 2000) and combined supplements of vitamin A and iron increase hemoglobin levels above the increments attributable to only one of the nutrients in pregnant women (Suharno et al., 1993) and in anemic preschool children (Mejı´a and Chew, 1988). 4. Conclusion Large doses of vitamin A provide an effective solution to the problem of VAD in areas of the world in which this is a public health problem. Even though periodic large doses of vitamin A are beneficial in the short term, their use as the only approach to the problem of VAD has limitations. VAD coexists with other nutrient deficits that are not addressed by the supplementation program. In addition, the effectiveness of this approach is limited to the duration of the program, and children who live in distant places and who probably need the supplement most may be difficult to reach consistently at 1- to 6-month intervals. Furthermore, large programs can put financial and logistical strains on the health care systems in many developing countries. Toxicity due to ingestion of multiple large doses over a short period is also a real possibility that needs to be guarded against. A more sustainable solution to the problem of VAD is to guarantee that the population has an adequate consumption of vitamin A in the diet. Small frequent doses (in amounts corresponding to those in diet) may be more protective against mortality and morbidity than large periodic doses. Most communities in which VAD is a serious problem have abundant supplies of vegetables and fruits rich in carotenoid with provitamin A activity. Dietary vitamin A intake is associated with a significant reduction in mortality (Fawzi et al., 1994), diarrheal and respiratory infections (Fawzi et al., 1995), and risks of stunting or wasting (Fawzi et al., 1997). Programs aimed at increasing consumption of dietary vitamin A in these communities should be undertaken in addition to the administration of supplements if the latter strategy is implemented. In areas in which vitamin A-containing foods are not so abundant, horticultural approaches should be considered. Food fortification programs can also be useful in improving the vitamin A status of populations, provided that the groups at highest risk of deficiency are reached by these programs. Over 60 countries are now planning, or have instituted, programs to control VAD. Periodic supplementation as a special endeavor generally achieves sustainable coverage rates of 40–60%, though some countries (and most demonstration projects) attain far higher levels. In Indonesia, where distribution has been integrated into a burgeoning health service system and mass media has been used to educate the public and

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change dietary patterns, a 90% reduction in the prevalence of overt deficiency has been achieved. Unquestionably, the major challenge remains the design and implementation of effective population-based intervention programmes. There is substantial evidence that low serum retinol levels (<1.05 mmol/ liter) are common in children infected with parasites, but no casual relationship between the parasitic infection and the deficiency has been demonstrated. Vitamin A deficiency was seen during infection with Plasmodium species (malaria) (Wolde-Gebriel et al., 1993; Davis et al., 1994; Binka et al., 1995; Das et al., 1996; Friis et al., 1997), Schistosoma mansoni (schistosomiasis) (Friis et al., 1996, 1997), Ascaris lumbricoides (Curtale et al., 1994), and Onchocerca vovulus (river blindness) (Storey, 1993). Ahmed et al. (1993) reported that less than 1% of a vitamin A supplement given to ascaris-infected children was recovered in their stools, suggesting good absorption. Although vitamin A supplementation improved the serum retinol levels, it did not reduce mortality due to malaria (Binka et al., 1995) or ascariasis (Tanumihardjo et al., 1996). Taken together, these results seem to indicate no beneficial effects of vitamin A on immunity to parasites. B. VITAMIN A DEFICIENCY IN PREGNANT AND LACTATING WOMEN

An early trial in England reported that maternal vitamin A supplementation in late pregnancy through the first week postpartum could reduce the incidence of puerperal sepsis (Green et al., 1931), but this lead was ignored. Another study reported lower vitamin A concentrations in placental abruption pregnancies than in normal pregnancies, but no cause-and-effect relation was established; moreover no link has been established between vitamin A-deficient status and partial molar pregnancy (Berkowitz et al., 1995), premature rupture of membranes, or eclampsia (Westney et al., 1994). Maternal night blindness, an indicator of VAD (West and Christian, 1997), has been associated with increased risks of urinary or reproductive tract infections, diarrhea or dysentery (Christian et al., 1998a), and increased acute phase protein concentrations during infection in pregnant women (Christian et al., 1998b). A community-based, randomized trial of vitamin A and -carotene supplementation to women of reproductive age in Nepal yielded the following four key findings: (1) supplementation resulted in a 40–49% reduction in pregnancy-related mortality (West et al., 1999); (2) night blindness during pregnancy was associated with a significantly higher risk of subsequent mortality in women, a risk that was largely ameliorated with supplementation (Christian et al., 2000a), (3) there was no effect of both supplements on fetal loss and mortality up to 6 months of life (Katz et al.,

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2000), and (4) the mortality in the first 6 months of life was higher among infants of women who suffered from night blindness during pregnancy and vitamin A treatment of mothers reduced the risk (Christian et al., 2001a). It has been reported that a vitamin A intake approaching a recommended amount for pregnancy markedly reduced but did not eliminate maternal night blindness in women in Nepal (Christian et al., 1998c, 2000b). Vitamin A supplementation also improved dark adaptation assessed by pupillary threshold detection, a noninvasive testing technique, in pregnant and lactating women (Congdon et al., 2000). Recently, Christian (2001b) showed that zinc increases the effect of vitamin A in restoring night vision among night-blind pregnant women but only in subjects with low initial serum zinc concentrations. Finally, vitamin A supplementation does not appear to increase hemoglobin and plasma erythropoietin concentrations among pregnant women (Semba et al., 2001; Muslimatun et al., 2001). Low vitamin A status does not seem to be related to a higher incidence of intrauterine growth retardation (Rondo et al., 1995; Hasin et al., 1996), although one British study reported a significant correlation between birth weight and anthropometric indexes in a low-income area of London (Ghebremeskel et al., 1994). Supplementation with vitamin A together with iron of pregnant women benefits vitamin A and iron status of their infants (Schmidt et al., 2001; Tanumihardjo, 2002) but did not improve growth or reduce morbidity of their infants during the first year of life (Schmidt et al., 2002). More worrying is the link consistently established between low vitamin A status and high mother-to-child transmission of HIV (Semba, 1997; Kennedy et al., 2000; Dreyfuss and Fawzi, 2002). Mother-to-child transmission of HIV is the dominant mode of acquisition of HIV infection for children, currently resulting in about 1800 new pediatric HIV infections each day worldwide. Semba’s group, working in Melawi (Southern Africa), reported an increased risk of mother-to-child transmission of HIV in maternal VAD (Semba et al., 1994) and a reduction in incidence of low birth weight deliveries among HIV-infected women supplemented with vitamin A (Semba et al., 1997). Fawzi’s group in Tanzania found large positive effects of multivitamins on perinatal outcomes (fetal mortality, low birth weight, preterm births) in HIV-infected women, although vitamin A supplementation alone had no effect (Fawzi et al., 1998). A study from Nepal in which all women (not only those infected by HIV) were supplemented reported no effects of vitamin A on newborn characteristics but a major impact on maternal mortality (reduced by 44%) (Keith et al., 1999). Coutsoudis et al. (1999) showed that vitamin A supplementation does not appear to be effective in reducing overall mother-to-child transmission of HIV, also if a decreased number of pretern births and a reduced mother-to-child transmission of HIV in preterm babies was proved. However, no conclusive evidence exists until today that vitamin A supplementation reduces mother-to-child

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transmission of HIV infection (Fawzi et al., 2000; Shey et al., 2002; French et al., 2002). Finally, Filteau (2001) showed that vitamin A supplementation of HIV-infected pregnant women may prevent the deterioration of gut integrity in their infants who themselves become infected; the improved vitamin A status of HIV-infected infants may decrease their gastrointestinal morbidity. An adequate vitamin A status, one that is neither too low nor too high, is needed for harmonious fetal and child development. Useful specific guidelines were published by the World Health Organization (WHO, 1998). In industrialized countries, there is no endemicity of low vitamin A status, and consequently no need for vitamin A supplementation of pregnant women or women of childbearing age. Such a measure could even be harmful because of the potential risk of teratogenesis with high doses of vitamin A (see Section VI.D). The World Health Organization recommends that a daily vitamin A supplement taking during any part of the fertile period be limited to 10,000 IU (3000 RE) (WHO, 1998). The Teratology Society of the United States recommends that vitamin A supplements or total intake not exceed 8000 IU/day (2400 RE/day) (Anonymous, 1987). In France, the vitamin A content of a supplement for the general population cannot exceed 3000 IU (900 RE). Physicians and gynecologists should be aware of all aspects of the vitamin A–pregnancy issue. They can then prescribe either a well-balanced diet rich in -carotenecontaining vegetables or vitamin A supplementation to women with suspected low vitamin A status. Recently, Huerta et al. (2002) described a mother and her newborn who developed VAD as a result of iatrogenic maternal malabsorption after biliopancreatic diversion for obesity. In areas of endemic VAD, the problem and its solutions are quite different. The recommendations of the World Health Organization can be summarized as follows: 1. During pregnancy, a daily supplement should not exceed 10,000 IU (3000 RE) and a weekly supplement should not exceed 25,000 IU (7500 RE). 2. During the first 6 month postpartum, supplementation is safer if the mother is breast-feeding, which reduces fertility. Otherwise, the supplement given after 6 week postpartum should not exceed 10,000 IU (3000 RE). 3. During the first 6 months of age, the infant can receive a direct supplement of 50,000 IU (15,000 RE) or, preferably, two doses of 25,000 IU (7500 RE) or more if not breast-fed. A distinction between areas in which VAD is endemic (i.e., some lowincome countries) and those in which it is not (i.e., industrialized countries) is conveninent, but may not reflect the real situation. It is quite possible that a significant portion of the low-income population suffers from

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undiagnosed low vitamin A status (Radhika et al., 2002). These women, who often do not undergo prenatal examinations, would benefit from a safely designed vitamin A supplementation protocol. However, to our knowledge, there has been no attempt to identify these women or to correct their nutritional deficiencies. Today, vitamin A supplementation is the most efficient way of correcting VAD. Its only drawback is the potential risk of teratogenesis. Identification of predictors of vitamin A status in pregnant women is of importance in terms of rationally designing and targeting appropriate interventions. Recently, Friis et al. (2001) reported that HIV infection, malaria, gravidity, and gestational age were predictors of serum -carotene and retinol. Relatively new technologies are also being used to determine how best to combat VAD in a field setting: the use of stable isotopes and the analysis of samples with gas chromatography–mass spectrometry. Interesting attempts have been made to replace vitamin A with the provitamin -carotene, which has never been associated with any teratogenic risk (de Pee et al., 1996). When -carotene was provided as a synthetic supplement, it was as efficient as vitamin A in reversing abnormal eye cytology, a clinical marker of VAD (Carlier et al., 1993). Other authors found that the -carotene of orange fruit (de Pee et al., 1998) was a more efficient source of vitamin A than darkgreen leafy vegetables (Bulux et al., 1994; de Pee et al., 1995), probably because of the lower bioavailability of -carotene in the latter. Fostering the local production and utilization of sources of vitamin A is promising, although the problem lies not only in the availability of vitamin A sources, but also in the economic status of the population. C. HYPERVITAMINOSIS A IN CHILDHOOD

Worldwide the incidence of the vitamin A excess, or hypervitaminosis A, is a very minor problem compared with the incidence of VAD. An estimated 200 cases of hypervitaminosis A occur annually whereas an estimated 1 million people develop VAD each year (Bendich and Langseth, 1989; Hathcock et al., 1990). Toxicity has been usually associated with abuse of vitamin A supplements rather than from ingestion of food sources. The lowest reported intakes causing toxicity have occurred in persons with liver function compromised by viral hepatitis, protein-energy malnutrition, or cirrhosis and in patients undergoing hemodialysis for renal failure (Bendich and Langseth, 1989; Hathcock et al., 1990; Fishbane et al., 1995). Especially vulnerable groups include children, with adverse effects occurring with intakes as low as 1500 IU/kg/day. Children can be intoxicated by total daily intakes of vitamin A lower than those necessary to cause adverse effects in adults. Part, if not all, of this difference seems to be due to the smaller body size of children. However, in children hypervitaminosis A develops quickly and usually resolves quickly (Bendich and Langseth, 1989; Hathcock et al., 1990).

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TABLE IV. Signs and Symptoms of Vitamin A Toxicity Acute toxicity

Signs and symptoms

Children

Anorexia, bulging fontanelles, drowsiness, increased intracranial pressure, irritability, vomiting

Adults

Abdominal pain, anorexia, blurred vision, headache, hypercalcemia, irritability, muscle weakness, nausea and vomiting, peripheral neuritis, skin desquamation

Chronic toxicity Children

Adults

Alopecia, anorexia, bone pain, tenderness, bulging fontanelles, craniotabes, fissuring at lip corners, hepatomegaly, splenomegaly, hyperostosis, premature epiphyseal closure, photophobia, pruritis, pseudotumor cerebri, skin desquamation and/or erytema, anemia, thrombocytopenia Alopecia, anemia, anorexia, ataxia, joint pain, bone abnormalities, osteoporosis, brittle nails, cheilitis, conjunctivitis, diarrhea, diplopia, dryness of mucous membranes, edema, elevated CSF pressure, epistaxis, exanthema, facial dermatitis, fever, headache, hepatomegaly, splenomegaly, hyperostosis, insomnia, irritability, menstrual abnormalities, muscular stiffness and pain, nausea, negative nitrogen balance, nervous abnormalities, papilledema, petechiae, polydypsia, pruritis, pseudotumor cerebri, skin desquamation and/or erythema and/or rash, vomiting, weight loss

Hypervitaminosis A can be divided into two categories: acute, resulting from ingestion of a very high dose over a short period of time, and chronic, resulting from continued ingestion of high doses for months or even years (Bendich and Langseth, 1989; Hathcock et al., 1990). Acute and chronic hypervitaminosis A has been observed in both children and adults and Table IV lists the signs and symptoms of vitamin A toxicity (Olson, 1983; Bendich and Langseth, 1989; Hathcock et al., 1990). Most reports of vitamin A toxicity in children involve intakes in the multiple hundreds of thousands of IU per day (Gribetz et al., 1951; Arena et al., 1951; Marie and See, 1954; Persson et al., 1965; Oliver 1985), but a substantial number of intoxications have occurred with much lower intakes (Hathcock et al., 1990). Accuracy of patient and parental reporting of vitamin A dosage after hypervitaminosis A has been diagnosed must be cautiously evaluated (Rosenberg et al., 1982). A variable tolerance of infants to excess vitamin A has been hypothesized to have a genetic predisposition (Carpenter et al., 1987). Over the past 50 years the number of reported cases of hypervitaminosis A has remained relatively constant despite the significant growth in production and use of vitamin A supplements. Higher incidences occurred in 1952–55 and again in 1970–72 (Bendich and Langseth, 1989; Hathcock et al., 1990). The earlier rise coincided with the use in Europe of two very potent vitamin A and vitamin D supplement preparations administered by

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prescription to infants primarily in France and Spain. The second involved many cases in which high doses of vitamin A were taken for dermatological disorders either by prescription or by self-medication. Several scientists (Bendich and Langseth, 1989; Hathcock et al., 1990; Bates, 1995) expressed concern over the possibility that publicity about the use of vitamin A and its synthetic analogues in the treatment of acne and other dermatological problems (Orfanos et al., 1997) and certain cancers as well as its possible role in cancer prevention might lead to inappropriate self-medication with vitamin A supplements. Public interest in vitamin supplement is still enormous, with 30% of the population of the United States currently using such supplements (Balluz et al., 2000). An intake of one or two capsules daily (7500–15,000 RE) is not rare, but it vastly exceeds the recommended dietary allowance for vitamin A. Political pressures have led to a highly unregulated industry with limited control by the Food and Drug Administration over marketing and quality. 1. Acute Hypervitaminosis A It is not assumed that results from experimental animals treated with excess vitamin A can be definitively extrapolated to humans, but the assumption is made that its toxic effects in animals should be taken as indicators of the possible occurrence of similar effects in humans. This index is often expressed as an LD50, the amount of substance in a single dose required to kill 50% of a populations of animals. The LD50 in young monkeys was estimated to be 560,000 IU retinol/kg (Macapinlac and Olson, 1981). High intakes (>300,000 IU) of vitamin A in humans over short periods of times (2–3 weeks) raise steady-state serum vitamin A values from a normal range between 0.70 and 2.79 mmol/liter to levels >2.79 mmol/liter. Levels >62.8 mmol/liter have been reported. When vitamin A intake is discontinued, levels rapidly return to normal. Toxicity appears to occur only when the amount of vitamin A present exceeds the capacity of RBP to bind to it. Vitamin A that is not bound to RBP binds to lipoproteins, and in this form it has toxic effects. In other words, in vitamin A toxicity, plasma RBP levels are normal but concentrations of vitamin A not bound to the specific RBP are increased (Smith and Goodman, 1976). In particular, the prescription of supplemental vitamin A has been reported to induce intracranial hypertension (Gangemi et al., 1985; Sharieff and Hanten, 1996). A bulging fontanelle is often reported in infants and young children (Baqui et al., 1995). Other symptoms of acute hypervitaminosis A include headache (presumably resulting from increased intracranial pressure), nausea, vomiting, and occasionally fever, vertigo, and visual disorientation. Peeling of the skin may also occur (Bendich and Langseth, 1989; Hathcock et al., 1990). Within a few hours of ingesting several million units of vitamin A in polar bear or seal liver, arctic explorers developed

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drowsiness, irritability, headache, and vomiting with subsequent peeling of the skin. The symptoms are generally transient and do not lead to permanent adverse effects (Bendich and Langseth, 1989; Hathcock et al., 1990). Cases of hypervitaminosis A caused by natural food sources are rare but they have a very long history. It has been hypothesized that pathological changes found in a Homo erectus skeleton from 1.5 million years ago were caused by hypervitaminosis A attributable to the consumption of carnivore livers. Consumption of the liver carnivorous animals or large fish has caused severe acute illness. The livers of herbivores (cows and lambs) are generally safe when consumed in moderation as part of a mixed diet; however, regular consumption of large quantities of liver might contribute to excess vitamin A intakes (Walker et al., 1982; Bernstein, 1984; Nagai et al., 1999). The majority of cases of hypervitaminosis A resulted either from misuse of vitamin A supplements by the consumer or from overprescribing of supplements by a physician. Overdosing of children by parents or grandparents was the primary example of hypervitaminosis A caused by the consumer, accounting for 25% of cases. Prescription-related hypervitaminosis A in children occurred when physicians failed to stress to patients or parents the dangers of excessive vitamin A levels (Bendich and Langseth, 1989; Hathcock et al., 1990). Bush and Dahms (1984) described a fatal case of hypercalcemia, hyperphosphatemia, bleeding disorders, and pulmonary insufficiency in a newborn who had received more than 60 times the recommended amount of vitamin A per day for 11 days. The baby’s parents had administered an incorrect amount of a prescribed supplement of vitamin A in an aqueous solution. 2. Chronic Hypervitaminosis A The chronic toxicity in animals of vitamin A preparations and retinol has been extensively reported. Hair loss, localized erythema, thickened epithelium, fatty infiltration of the liver and heart, kidney and testicular defects, anemia, hypercholesterolemia, and sometimes hypertriglyceridemia have been found in animals (Singh and Singh, 1978; Kamm et al., 1984). Skeletal alterations with fractures and reduced formation of dentine and atrophy of lingal odontoblasts have been also reported (Nieman and Obbink, 1954; Hough et al., 1988). Chronic hypervitaminosis A in humans is more common than acute hypervitaminosis A. Serum levels of vitamin A are generally >3.49 mmol/ liter and there are increased levels of the unbound retinol resulting in a change in the ratio of free retinol to retinol bound to RBP as well as increase in retinyl esters (Bendich and Langseth, 1989). Chronic hypervitaminosis A usually develops after doses of >100,000 IU/day have been taken for months. In infants who are given 20,000–60,000 IU/day of water-miscible

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vitamin A, evidence of toxicity may develop within a few weeks. Massive doses (150,000–350,000 IU) of vitamin A or its metabolities are given daily to persons with globular acne. Although the treatment is effective, it puts the patient at risk for vitamin A toxicity. Its symptoms are highly variable but anorexia, dry itchy skin, loss of hair, increased intracranial pressure, and hepatomegaly (more prominent in children) are among the most common manifestations. After high doses of vitamin A children often show bone changes (subperiosteal new bone growth and cortical thickenings, especially of the small bones of the hands and feet and the long bones), rheumatological manifestations (arthritis, myopathy, and vasculitis) (Nesher and Zuckner, 1995), elevated blood lipid levels (Murray et al., 1983), and liver damage (Russel et al., 1974; Geubel et al., 1991; Lettinga et al., 1996; Erickson et al., 2000; Vollmar et al., 2002). Other symptoms related to chronic hypervitaminosis A are observed after supplementation with low to moderate doses of vitamin A on a regular basis over a long period of time. This may induce severe, yet usually reversible, liver damage (Sarles et al., 1990; Geubel et al., 1991). There are great variations in response between individuals and, in addition, a wide range of health and dietary factors can influence susceptibility to chronic hypervitaminosis A. The data on interactions are not sufficient to allow distinction between additive, synergistic, and potentiative effects. An important consideration, however, is that any of these types of interactions may enhance toxic potency, and consequently adverse effects may occur at doses that would be safe otherwise. Among these factors are dosing regimen (high doses develop symptoms in a short period) and form (aqueous dispersions cause higher plasma vitamin A levels than oily solutions) in which the vitamin is given, age and body weight of the consumer, general health status and concurrent health problems, such as anemia, cystic fibrosis, protein-energy malnutrition, and liver and kidney disease, dietary factors, such as ethanol and vitamin (D, E, C, and K) intake, and drugs, e.g., tetracycline (Bendich and Langseth, 1989; Hathcock et al., 1990; Eid et al., 1990). In most cases, when vitamin A intake is discontinued, many symptoms of hypervitaminosis A are relieved within a few days or a week. Full recovery usually follows within weeks or months (Bendich and Langseth, 1989; Hathcock et al., 1990), although there appear to be individual differences in the recovery time from hypervitaminosis A even when patients have all consumed similar doses. Long-term or irreversible effects of hypervitaminosis A include bone changes and cirrhosis. Of all the symptoms of chronic hypervitaminosis A, bone changes are among the most lasting, although permanent bone malformation appears to be relatively rare. There is also some evidence that hypervitaminosis A can cause irreversible damage to the liver (Hruban et al., 1974).

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D. TERATOGENIC EFFECTS OF VITAMIN A

Embryonic exposure to either an excess of retinoid or a deficiency of vitamin A leads to abnormal development (Maden, 1994; Nau et al., 1998). From these results, it was understood very early that the embryo requires a precisely regulated supply of retinoids. Although the spectrum of malformations that have been observed due to either VAD or excess appears to overlap, there are examples of selective responses to only a single condition. No unique mechanism is likely to explain the action of retinoids in normal or teratogenic development. It is more likely that each tissue utilizes RA in a specific process during development. Much work has been performed to relate the process of malformations due to an excess of exogenous retinoids to the role that retinoids play in normal developmental processes. It was recognized as early as the 1930s that maternal insufficiency of vitamin A during pregnancy results in incomplete pregnancies, fetal death, and severe congenital malformation (Mason, 1935; Hale, 1937). In subsequent studies, Wilson et al. (1953) identified a spectrum of congenital abnormalities that results from lack of vitamin A during gestation (Thompson, 1969; Bates, 1983); these abnormalities have been observed in different animal species (Morriss, 1972; Maden, 1994). The major target tissues of VAD include the heart, the ocular tissues, and the circulatory, urogenital, and respiratory systems. Including vitamin A in the diet of the VAD female during specific times of pregnancy prevented the occurrence of these abnormalities, suggesting that vitamin A is required at various distinct stages of development, and clearly establishing the important role of vitamin A in normal embryonic development. So far, critical information is lacking as to how maternally derived vitamin A is processed by the embryo to generate physiological amounts of the bioactive retinoids in a developmental stage- and tissue-specific fashion. In humans, the teratogenic effects of VAD during gestation are often masked by general malnutrition and it is not possible to link an abnormality to a single nutrient or micronutrient (Gerster, 1997). Nevertheless, a pattern has emerged that suggests that children of mothers with xerophthalmia, a symptom of severe VAD, frequently have ocular abnormalities, and that premature neonates with low vitamin A stores are at high risk of respiratory abnormalities (Shenai et al., 1995). It has also been suggested that vitamin A supplementation contributes to the reduction in neural tube defects (Wallingford and Underwood, 1986). In light of these findings, a higher incidence of malformed babies would be expected in areas of endemic VAD, but this is not the case. Although this apparent discrepancy may arise from the failure to report birth defects in these countries, the number of reported cases remains surprisingly low. The few cases reported occurred in India. One study reported 15 cases of microphthalmia or anophthalmia over a 10-year period, which is not considerably excessive (Kapoor and Kapoor, 1977).

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A relationship between high levels of retinoids and human birth defects has been known for some time (Hathcock et al., 1990; Peck and Di Giovanna, 1994; Nau et al., 1998). The teratogenic potential of an excessive intake of retinoid was shown clearly in experimental animals, as reviewed by Soprano and Soprano (1995). The negative effects of retinoids become manifest during the first trimester of embryonic development, and many defects likely arise from the abnormal migration of cranial-neural crest cells or a defect in early axial patterning. The threshold dosage of retinoids required to cause these malformations is unknown (Nau et al., 1998). Evidence that excessive vitamin A is a human teratogen falls into the following three categories: . Extrapolation of the unequivocal data from several animal species strongly suggests that vitamin A at sufficiently high amounts would have teratogenic effects in humans. . There is a temporal association between high intakes of vitamin A by pregnant women and birth of babies showing defects characteristic of vitamin A-dependent animals. This temporal association together with the unequivocal evidence from animals leaves little doubt that a sufficiently high intake of vitamin A by a women at a critical stage of pregnancy could have teratogenic effects. There is much uncertainty about the dose of vitamin A needed to produce such effects. . The teratogenicity of 13-cis-RA has been established in epidemiological studies as well as in animal experiments.

RA has been useful in treating severe cystic acne and has been available (under the name Accutane) since 1982. Because the deleterious effects of administering large amounts of vitamin A or its analogues to pregnant animals have been known since the 1950s (Cohlan, 1954a), the drug carries a label warning that it should not be used by pregnant women. However, most women of childbearing age (15–45 years) have taken this drug since it was introduced, and some of them have used it during pregnancy. This results in malformations that include, as major target tissues, heart, skull, skeleton, limbs, central nervous system, brain, eyes, and craniofacial structures (Cohlan 1954b; Shenefelt 1972; Brockes, 1989; Kochhar et al., 1993; Helms et al., 1997; Kochhar and Christian, 1997). The typical pattern of malformation caused by treatment with excess vitamin A in its general features resembles that caused by VAD (Maden 1994; Gerster 1997). Lammer et al. (1985) studied a group of women who inadvertently exposed themselves to RA and who elected to remain pregnant. Of their 59 fetuses, 26 were born without any noticeable anomalies, 12 aborted spontaneously, and 21 were born with obvious anomalies. The malformed infants had a characteristic pattern of anomalies, including microtia/anotia, micrognathia, cleft palate, conotruncal heart defects and aortic-arch abnormalities, thymic defects, retinal or optic-nerve

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abnormalities, and CNS malformations (see below). More than 1000 cases of congenital deformities may be linked to an exposure of mothers to retinoids (Ellis and Voorhees, 1987). Synthetic retinoids have also been implicated in teratogenesis. For example, human exposure to etretinate, a synthetic, aromatic retinoid prescribed for the treatment of psoriasis, has resulted in spontaneous aborts or in the manifestation of severe malformations (e.g., craniofacial and skeletal) in offspring (Rosa et al., 1986). One case of etretinate embryopathy occurred in an infant conceived 1 year after termination of treatment, probably due to the amassing of the molecule in maternal adipose tissue (Kochhar and Christian, 1997). Although there is a characteristic set of birth defects with ingestion of these retinoids, the pattern of malformations is too complex to reconcile, at present, with the known effects of retinoids on morphogenesis or with the molecular mechanisms elucidated to date (Kochhar and Christian, 1997). The alterations at the CNS level merits a more detailed description. Indeed, many reports have confirmed the teratogenicity of retinoids on the CNS, which mainly results in microphthalmia, encephalocoel, exencephaly, spina bifida, and microcephaly. In addition, there are also characteristic defects of the head and neck that can be attributed to effects on the neural crest, hypoplastic maxilla or mandible, microtia, and tyroid and thymus malformations (Langman and Welch, 1967; Shenfelt, 1972; Yasuda et al., 1986; Alles and Silik, 1990; Sulik et al., 1995; Mulder et al., 2000). The same spectrum of CNS abnormalities is seen in humans after inadvertent administration of 13-cis-RA to the embryo (Lammer et al., 1985; Rosa et al., 1986; Lammer and Armstrong, 1992; Die-Smulders et al., 1995). Classic teratological studies have moved on from descriptions of defects to experimental studies examining the patterns of gene activity and their alteration soon after the teratogenic insult. These have provided surprising revelations and it seems that there are three effects on patterning in the CNS. The first effect is generally referred to as posteriorization of the embryo and it consists in the fact that the front end of the embryo (forebrain) is missing, the domains of expression of anterior genes are extinguished, and the domains of expression of posterior genes are expanded (Durston et al., 1989; Cho and De Robertis, 1990; Sive et al., 1990; Dekker et al., 1992; Leroy and De Robertis, 1992; Lopez and Carrasco, 1992; Lopez et al., 1995). RA acts both directly on the CNS and indirectly via the underlying mesoderm (Sive et al., 1990; Ruizi Altaba and Jessel, 1991; Sive and Cheng, 1991). The posteriorizing effect has been seen in all vertebrate embryos studied (Cunningham et al., 1994; Simeone et al., 1995; Avantaggiato et al., 1996). The loss of Emx1, Emx2, and Dlx1 gene expression domains (normally expressed in the forebrain) and the anteriorization of the Wnt-1, En-1, En-2, Otx2, and Pax-2 (normally expressed more posteriorly in the midbrain and hindbrain) confirmed the morphological observations (Avantaggiato et al., 1996; Ang et al., 1996).

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The external phenotype of embryos showing the second effect has long been recognized: an abnormally rostral position of the otocyst and a shortened preotic hindbrain (Morriss, 1972). It results from the loss of a section of CNS tissue in the posterior midbrain/anterior hindbrain region and is a universal teratological finding (Morriss, 1972; Morriss-Kay et al., 1991; Papalopulu et al., 1991; Holder and Hill, 1991; Sundin and Eichele, 1992; Cunningham et al., 1994; Y. M. Lee et al., 1995; Leonard et al., 1995; Simeone et al., 1995; Lopez et al., 1995; Zhang et al., 1996). The two other characteristic features of this effect are the fusion of the trigeminal ganglion and facial ganglion and the fusion of the first and second branchial arches and mismigration of the neural crest (Thorogood et al., 1982; Pratt et al., 1987; Smith-Thomas et al., 1987; Seegmiller et al., 1991; Kessel, 1993; Cunningham et al., 1994; Y. M. Lee et al., 1995; Leonard et al., 1995; Gale et al., 1996). The third phenotype is a fascinating combination of both effects 1 and 2 in that the anterior hindbrain is affected (effect 2) and it is posteriorized (effect 1) (Marshall et al., 1992; Conlon and Rossant, 1992; Wood et al., 1994; Marshall et al., 1994; Studer et al., 1994; Zhang et al., 1994; Y. M. Lee et al., 1995; Simeone et al., 1995; Hill et al., 1995; Alexandre et al., 1996). There are three other systems or cell types associated with the CNS that are affected by RA during their development. The first is the differentiation of the retina. The effects of excess RA (and, conversely, a deficiency of RA) on the developing eye as a whole have been referred to several times, generating such abnormalities as microphthalmia or anophthalmia (Stenkanp et al., 1993; Kelley et al., 1994). The second system is the stimulation of regeneration of mammalian auditory hair cells by RA. In a damaged organ of Corti, by the administration of ototoxic drugs, 78% of the hair cells had been regenerated by RA treatment (Lefebvre et al., 1993; Kelley et al., 1993). This raises the exciting possibility of the regeneration of hair cells in the human. An additional system concerns the differentiation of oligodendrocytes, which are cells that myelinate CNS axons (Noll and Miller, 1994; Barres et al., 1994). Because retinoids are important factors of the developing CNS rather than being simple teratogenic agents, depleting the embryo of vitamin A should result in abnormal CNS development. The abnormalities in the mammalian CNS due to VAD include hydrocephalus, spina bifida, anophthalmia, microphthalmia, and retina defects and in the head and neck region, ear and teeth abnormalities, cleft palate, and hare lip (Hale, 1933; Warkany, 1945; Wilson et al., 1953; Kalter and Warkany, 1959). Retinoid-deficient embryos by a dietary method resulted in an underdeveloped hindbrain, absent cranial flexure, microphthalmia, and foreshortening of the snout and branchial arches. In the eye, the lens showed apoptosis and retina failed to invaginate to form the optic nerve. The cranial nerves were defective, the neural tube was reduced in size and cellularity, and the brain

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had reduction in differentiating neuronal populations due to reduced proliferation (Dickman et al., 1997; Ross et al., 2000). In deficiency studies using the quail embryo three defects have been observed (Maden et al., 1996). The first defect is that the posterior part of the hindbrain is completely missing (Maden et al., 1997). The second defect is that the axon of neurons does not appear within the surrounding mesoderm and the axon trajectories within the neural tube itself are unorganized and chaotic (Maden et al., 1998). The third defect is widespread apoptosis in the neural crest, as seen in rat embryos (Maden et al., 1996; Maden, 1999). Pasqualetti et al. (2001) identified a temporal window of susceptibility to retinoids that is critical for mammalian inner ear specification, and provided the first evidence that a subteratogenic dose of vitamin A derivative can be effective in rescuing a congenital defect in the mammalian embryo. E. A CASE OF HYPERVITAMINOSIS A IN AN INFANT

This section reports a rare case of chronic hypervitaminosis A in childhood that we recently described (Perrotta et al., 2002). Starting from clinical observations, several in vitro studies were performed that allow a clear demonstration that vitamin A also acts independently of its role as a retinoic acid precursor. A 3-month-old white infant with unrelated parents was admitted to the Department of Pediatrics, Second University of Naples, for investigation on anemia and thrombocytopenia. He had severe normochromic-normocytic anemia, with hemoglobin level of 59 g/liter, mean cell volume of 98 fl, and mean cell hemoglobin concentration of 310 g/liter; the reticulocyte count was 8 109/liter; the leukocytes were 6.2 109/liter with lymphocytes 74%, neutrophils 17%, eosinophilis 3%, and monocytes 6%; the platelets were 30 109/liter. Anisopoikilocytosis was observed in blood smear. The child was born from normal delivery. Birth weight was 2700 g, length 49 cm, and head circumference 32 cm. Dietary history included artificial nursing with prepared formulas. The patient had been well until 10 days earlier, when he began to be anorexic and feverish. He had no history of preceding viral illness or exposure to toxins associated with bone marrow suppression. Physical examination revealed an extremely pale child who was very irritable with signs of increased cardiac output (tachycardia and systolic murmur), bulging anterior fontanel, dry itchy skin with petechiae, angular fissures of the lips, and hepatosplenomegaly. The liver edge was 6 cm below the right costal margin and the spleen edge was 9 cm below the left costal margin. Standard measurements were length 62 cm (50th–75th percentile), weight 5200 g (10th–25th percentile), and head circumference 45 cm (> 97th percentile). Neurological examination was normal with physiological optic discs. The marrow feature showed reduced nucleated red cells with evidence

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of diserythropoiesis in the form of vacuolated or binucleate erythroblasts, almost complete absence of megakariocytopoiesis with micromegakariocytes, and slightly reduced granulopoiesis (myeloid/erythroid ratio 1.4:1). No atypical myeloid or lymphoid cells were seen. Serum bilirubin and haptoglobin levels, hemoglobin electrophoresis, erythrocyte enzyme levels, and osmotic fragility with incubated and nonincubated erythrocytes were normal. Serum iron levels were high (36.6 mM), probably because of decreased utilization. Aspartate transaminase, alanine transaminase, and -glutamyl transpeptidase were slightly increased. Direct and indirect Coombs tests, i-antigen expression on the erythrocyte surface, and the Ham test were negative. Karyotype analysis of bone marrow and peripheral blood cells revealed a normal male pattern without significant spontaneous or diepoxybutane-induced chromosomal breakages. There was no serological evidence of Epstein–Barr virus, human cytomegalovirus, HIV virus, or hepatitis virus infection. No IgG or IgM antibodies to parvovirus B19 were found in serial measurement. Chest x-ray findings were normal. Abdominal ecography was normal, except for hepatosplenomegaly. Following detailed questioning about drug ingestion, it was discovered that the child, owing to misinterpretation of the pediatrician’s prescription, had been given 12 gtt/day of Arovit (an aqueous solution of vitamin A palmitate, Roche) since the tenth day of his life. Thus, the total daily retinol ingested was about 62,000 units, formed by 60,000 units from Arovit plus the vitamin A contained in the prepared formulas. This treatment lasted about 80 days. Increased levels of the unbound retinol resulting in a depressed retinolbinding protein/retinol ratio, as well as high levels of retinyl esters were found. On the basis of these findings a diagnosis of chronic hypervitaminosis A was made and the vitamin ingestion was immediately discontinued. Fifteen days after drug interruption, the hemoglobin level and platelet count rose to 76 g/liter and to 105 109/liters, respectively. The reticulocyte count increased from 8 up to 300 109/liter. The bone marrow examination performed 1 month after stoppage of vitamin ingestion showed a normal population of erythroid and myeloid cells, as well as of megakaryocytes. The retinyl esters were reduced about 60% after 3 months. The liver and spleen returned to normal size after 6 months. Now, after 3 years, the child is in good general condition with normal complete blood cell count. The observed hematological damages characterized by a specific alteration of the erythropoiesis and megakaryocytopoiesis suggested evaluating the effects of Arovit and retinol on the growth and differentiation of two different cell populations. Two cell phenotypes were selected: the K562 cell line and a primary culture of bone marrow mesenchymal stem cells (or marrow stromal cells, MSCs). The cell line, established from an erythroleukemia, is able to differentiate either in megakaryocytes and in erythroblasts. Intriguingly, K562 cells do not respond to RA treatment (Douer and Koeffler, 1982).

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MSCs were chosen as these cells are multipotential in that they can originate precursors for bone marrow stroma, bone, cartilage, and adipose tissue. In addition to progenitors for connective tissues, MSCs also have the potential, under an appropriate microenvironment and stimuli, to differentiate into myogenic precursors. Furthermore, more recently, marrow stromal cells, overcoming their mesenchymal commitment, have been shown to have the capacity to differentiate into neural cells and to express neuron-specific markers. The hematopoietic and nonhematopoietic stem cells not only coexist in the bone marrow, but they also functionally cooperate and are strictly interdependent of each other. Particularly, a series of studies has suggested the importance of cytokine production and cell-to-cell contact by bone marrow stromal cells in the growth and survival of hematopoietic cells. The stromal cell compartment produces not only matrix proteins, but also essential growth factors, which initiate and support the differentiation of primary quiescent, but eventually activated CD34() stem cells into CD34(+) hematopoietic progenitors. Figures 5 and 6 show the effect of increasing amounts of vitamin A and Arovit on the growth of K562 cells (Fig. 5) and bone marrow MSCs (Fig. 6). The range of concentrations used (from 20 to 80 mM) corresponds to that probably reached in vivo, taking into account the daily uptake of the drug, the weight of the infant (about 4 kg), and the reported accumulation of the compound at bone marrow level. In particular, the concentration of vitamin A occuring in Arovit was determined by assuming that 1 mM corresponds to 1.4 IU/ml.

FIGURE 5. Effect of retinol and Arovit on the growth of K562 cells. K562 cells were plated at 300,000 cells/ml and incubated with or without different Arovit or vitamin A amounts. The values are expressed as the percentage of control cell growth after 48 h treatment. The reported results are the media of three different experiments with less than 5% variation.

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FIGURE 6. Effect of retinol and Arovit on the growth of mesenchymal stem cells. MSCs were plated at a density of 5 103 cells/cm2 and incubated with or without different Arovit or vitamin A amounts. The values are expressed as the percentage of growth of control cell after 48 h treatment. The reported results are the media of three different experiments with less than 5% variation.

Arovit potently inhibits K562 proliferation when added to the culture medium. A completely superimposable effect was obtained by using pure vitamin A (Fig. 5). These data clearly suggest that the biological activity of the drug on cell proliferation is almost uniquely due to its vitamin A content. Moreover, the addition of retinol did not significantly modify the morphology of the cells. Finally, incubation of cells with retinol did not cause a definite growth arrest. Indeed, when cells were incubated for 48 h with 40 mM vitamin A and then the drug was removed, a normal rate of proliferation was recovered. The results obtained on the growth of MSCs (Fig. 6) demonstrate that the proliferation rate of this cellular population is noticeably, and almost equally, affected by the two preparations, Arovit and pure vitamin A. Interestingly, a high dosage of Arovit (120 mM) completely inhibits the cell growth. Because it is possible that similar concentrations are reached in vivo, the effect of the molecule is remarkably important. The cell growth inhibition seems unrelated to the activation of the apoptotic process. Indeed, we did not substantiate any morphological alteration at microscope analysis. Moreover, biochemical analysis of polyADP-ribose polymerase hydrolysis, an early marker of apoptosis, confirmed the absence of programmed cell death activation. The effect of vitamin A on cell division cycle engine was also investigated. In particular, we analyzed the content of various protein components of the cell division cycle (cyclins, cyclin-dependent kinases, and cyclin-dependent kinase inhibitors). A significant increase of specific cyclin-dependent kinase inhibitors (p27Kip1 and p21Cip1) was observed, suggesting that the activity of

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the compounds is due to the accumulation of inhibitors of cyclin-dependent kinases. It is well known that the accumulation of p27Kip1 and p21Cip1 might cause a block at the level of either G1–S or S–G2 phase transition. The analysis of cyclin levels revealed an increase of cyclin E and A. This suggests that the vitamin A-dependent proliferation arrest mainly occurs via an elongation of the S phase and/or an interference at level of the G2 checkpoint. In conclusion, the reported case, an infant erroneously treated with a high dose (62,000 IU/day) of vitamin A for about 3 months, represents one of the first examples of anemia and thrombocytopenia due to chronic intoxication of vitamin A. The close temporal relationship between suspension of vitamin A intake and bone marrow recovery clearly suggests that the renewed functionality of hematopoiesis was strictly related to the interruption of vitamin A treatment. Moreover, in vitro studies demonstrated that the antiproliferative activity of vitamin A on different bone marrow cell populations is probably due directly to vitamin A and not to its most important derivatives, namely RAs.

VII. FEW FINAL CONSIDERATIONS This chapter has updated information on vitamin A in infancy, giving particular emphasis to the importance of altered retinol levels as the cause of childhood pathologies. A major premise in evaluating the function of vitamin A is the enormous and wide importance of some retinoids (i.e., ATRA and 9-cis-RA) in the control of gene transcription. Indeed, the RA receptors (RARs and RXRs) not only bind RARE sequences localized in the promoter region of several target genes but also modulate the function of several additional receptors. However, retinol does not act exclusively by RAs in that a number of derivatives, particularly retroretinoids and vitamin A itself, have been demonstrated to work independently on RA receptors by means of not well established pathways. The involvement of retinol and its derivatives in such a large number of processes has been briefly reviewed, with special attention to embryogenesis, a unique, spectacular, and astonishing process. The large role of vitamin A represents a critical problem in that it is necessary to strictly manage the level of the vitamin to avoid negative effects due to hypo- and hypervitaminosis. Importantly, these conditions are more easily reached during infancy. It appears clear that hypovitaminosis A represents the most frequent condition, particularly in developing countries. Hypervitaminosis is more rare, although it also causes important pathological consequences. Understanding the possible toxicity associated with hypervitaminosis A becomes increasingly important in view of the popularity of vitamin A supplementation. The use of large doses of retinol in the care of skin conditions and in the possible prevention of cancer, the large use of

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megavitamin therapy in certain chronic disorders, and the growing tendency toward vitamin faddism should alert physicians to the possibility of vitamin overdose. The National Center of Health Statistics and the Food and Drug Administration found that approximately one-fourth of the U.S. population and two-thirds of users of vitamin supplements ingest products containing vitamin A. Furthermore, retinol is added to several foods, particularly those administered to infants and young children. With food faddism rampant in our society, it is possible that an increasing number of people (particularly children) have an excess of hepatic reserves of vitamin A and risk clinical vitamin A toxicity. Thus, clinicians should request dietary information and consider the diagnosis of hypervitaminosis A in any patient with unexplained signs and symptoms that may be consistent with this condition.

ACKNOWLEDGMENTS This work was supported in part by grants from Associazione Italiana per la Ricerca sul Cancro (AIRC), MURST (Progetti di Rilevante Interesse Nazionale), and Progetti di Ricerca di Ateneo (Seconda Universita` di Napoli) e Progetto Sanita` della regione Campania.

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Index

Page numbers followed by f and t indicate figures and tables, respectively.

A Accutane, teratogenicity of, 528 Acid-base equilibrium, in primary aldosteronism, 130 Acid-sensing ion channels (ASIC), in brain, 51–52 AcSDKP, thymosin b4 and, 283 Actin(s) critical concentration of (Acc), 300 filaments capping proteins, 301 structure of, 300–301 monomers, sequestration of, 300 subdomain 1 of, 303–305, 303f interactions at profilin and, 305–307 thymosin b4 and, 305–307 subdomains 2–4 of, 303f, 304 thymosin b4 and, 275–279, 277f, 284, 299–307 Actobindin, 298, 301 and thymosin b4, comparison of, 302–303 Acute lymphocytic leukemia (ALL) childhood, reduced folate carrier (RFC) expression in, 423–425 drug resistance in, multidrug resistance-associated proteins (MRPs) and, 436 Acute myeloid leukemia (AML), folate receptor expression in, 433

Acyl-CoA:retinol acyltransferase (ARAT), 461f, 463 Adenylyl cyclase, activation of, 200 Adhesion molecules, in hematopoiesis and stem cell trafficking, 10–13, 11f Adrenal carcinoma aldosteronism caused by, 126, 126t, 128 diagnosis of, 134 Adrenalectomy, laparoscopic, 135 Adrenal glands. See also Hypothalamicpituitary-adrenal axis deoxycorticosterone-secreting tumor of, 126t hormone synthesis and release by, 198, 199t imaging of, 134 neuroanatomy of, 198 scintigraphy of, 134 Adrenal hyperplasia. See also Congenital adrenal hyperplasia aldosteronism caused by, 126, 126t, 128 diagnosis of, 133 treatment of, 135 Adrenal venous sampling (AVS), 134 Adrenocortical adenoma aldosteronism caused by, 126, 126t and hypertension, 128–129 Adrenocorticotropic hormone (corticotropin, ACTH) actions of, 200

593

594 Adrenocorticotropic hormone (corticotropin, ACTH) (continued) release of in chronic fatigue, 227–228 in conditioned stress, 219 in hemorrhage, 217 in immobilization stress, 219–220 in response to CRH, 225–226, 227 serotonin-releasing drugs and, 205 Aging, cellular, telomere hypothesis of, 14 Alcohol dehydrogenases, in retinoic acid synthesis, 466 Aldosterone, 79 actions of in brown adipose tissue, 49 cellular, rapid, 59–60, 85-87 in collecting duct, 35–36, 35f, 124, 127f in distal nephron, 35–36, 35f in epithelial tissue, 30–31, 35–36, 42–46, 85 gene regulation and, 37–39 in ion transport, 34–36 in mineral homeostasis, 32–33 molecular basis of, 37–39 mouse models of, 46–48, 46t in nonepithelial tissue, 31, 37, 49, 62, 85 nongenomic, 33, 37–39, 96–97 rapid, 59–60, 62, 85–87, 92 physiological, 32–37 on sodium transport, 35f, 36, 85 SGK1 and, 54, 123 studies of, mouse models in, 46–48, 46t, 86–87 in tight epithelia, 32, 34–36 in volume control, 32–33 in amphibia, 34 biosynthesis of, 117–118, 118f and blood pressure, 34, 37 in brain, 37, 45, 50–51 and cardiac fibrosis, 32, 57–59, 95, 99–100 and cardiac hypertrophy, 95, 97–98 deficiency of, 36 in fish, 34 in heart, 32, 49, 57–59, 93–95, 96–97 and heart failure, 32, 57–59, 94, 95 and insulin, cross-talk of, 32, 60–61 and ion currents, in heart, 96–97 and latent period, 34 overproduction of. See Aldosteronism, primary pathophysiologic role of, 32, 61–62 in cardiovascular disease, 95 physiologic role of, 48–57

Index production of by extra-adrenal tumor, 126 in heart, 93–95 receptor for. See also Mineralocorticoid receptors (MR) expression of, autoregulation, 49 plasma membrane, 86 secretion of, 30–31 regulation of, by renin, 116, 117f synthesis of, 30–31 target genes for, 48–57 products of and epithelial sodium channel, 52–55 and ion transport other than epithelial sodium, 55 research on, directions for, 62 Aldosterone-sensitive distal nephron (ASDN) intercalated cells of, 36 ion movements in, 35–36, 35f principal cells of, 35f, 36 ENaCs of, rapid activation by aldosterone, 59 SGK1 in, 53 Aldosterone synthase, 80, 138 activity of, 117–119 colocalization with mineralocorticoid receptors, 94 expression of, 117 in heart, 93 in heart failure, 58, 94 Aldosteronism glucocorticoid-remediable, 126, 126t, 136–137, 138–139 idiopathic, 126, 126t, 128 diagnosis of, 133–134 management of, 135 primary, 36, 125–135, 126t clinical findings in, 129–130 diagnosis of, 132–133 laboratory findings in, 129–130 Liddle’s syndrome and, 57 screening for, 130–131, 131f subtype delineation, 133–134 treatment of, 134–135 Aldosteronoma aldosteronism caused by, 126–128 clinical findings with, 129–130 corticotropin-responsive (renin-unresponsive), 127 detection of, 133 laboratory findings with, 129–130 mineralocorticoid hypertension with, 115, 124

595

Index prevalence of, 128–129 renin-responsive, 127 treatment of, 134–135 Amiloride, for Liddle syndrome, 140 Amphibians, renin-angiotensin-aldosterone system in, 115, 116t Amygdala in anxiety, 224 and hypothalamic-pituitary-adrenal axis, 201–203 b-Amyloid, and p75NTR, in apoptosis, 398 Androgen receptors (AR), 31 Anemia, in hypervitaminosis A in infancy, 531–535 Angiogenesis, b-thymosins in, 281–283, 284 Anhydroretinol (AR) biosynthesis of, 470 cell death caused by, 477–479 distribution of, 470 structure of, 469f Annexin I, cardioprotective effects of, 99 Antidepressant therapy delayed onset of, 223 effects on hypothalamic-pituitary-adrenal axis, 204–205 mechanism of action of, 221 Antihypertensives and diagnosis of primary aldosteronism, 131–132 for mineralocorticoid hypertension, 135 Anxiety disorders and depression, 224 hypothalamic-pituitary-adrenal axis and, 225–226 serotonergic system and, 224–225 serotonin–HPA interactions in, 226–227 AP1, and transactivation of glucocorticoid receptors, 84, 122 Apical ectodermal ridge (AER), in limb development, vitamin A and, 488–489 Apoptosis b-amyloid-induced, p75NTR in, 398 nerve growth factor (NGF)-induced caspases in, 398 NADE and p75NTR in, 387–389, 388f, 395–398 research on, advances in (future directions for), 398 neuronal, in ischemia, NADE in, 397–398 in oligodendrocytes, NADE in, 397 Apparent mineralocorticoid excess (AME), 43, 44, 126t, 139 mouse models of, 46–47, 46t

variant (type II), 44 Ascariasis, resistance to, vitamin A supplementation and, 519 Atherosclerosis, PPARg and, 166–168 ATP-dependent proteolysis factor 1 (APF-1), 260 Autoimmunity, vitamin A deficiency and, 499

B B1-B9 cell bodies, 191 Benzodiazepines, mechanism of action of, 224–225 Bitot’s spots, 510 Blood, vitamin A in, 464 Blood pressure. See also Hypertension aldosterone and, 37, 45 hormonal regulation of, 80 Blood vessels embryonic development of, vitamin A and, 484–485 PPARa in, 171–172 PPARd in, 173–174 PPARg in, 165–166 Bone marrow long-term culture of, 4–5 stroma of, 10–11 interactions with hematopoietic stem cells, 10–11 Brain. See also Hypothalamic-pituitaryadrenal axis acid-sensing ion channels (ASIC) in, 51–52 aldosterone action in, 37, 45, 50–51 11b-HSD2 in, 37, 45–46 glucocorticoid receptors in, 50, 95, 203 glucocorticoids in, 88 mineralocorticoid receptors in, 37, 50–51, 95 serotonin in, 190, 191 sodium channel (BNaC) in, 51–52 Brassica SCR peptide, 330–334, 334f self-incompatibility in, 330–334, 334f Breast cancer resistance protein (BCRP), and folate transport, 436–437, 438f Bronchopulmonary dysplasia, vitamin A and, 507–509

C Ca2+-ATPase. See Calcium pump Caenorhabditis elegans, b-thymosin-like protein of, 275, 276f, 302

596 Calcium homeostasis, in neurons, 50 intracellular aldosterone and, 59–60 and corticosteroid actions, 91 in heart failure, corticosteroids and, 96 Calcium currents, cardiac, corticosteroids and, 96 Calcium pump, isoform 1, in neurons, 50 Caldesmon, LKEKQQ motif, 304–305 Calponin, VKYAEK motif, 304, 304f Cancer adrenal aldosteronism caused by, 126, 126t, 128 diagnosis of, 134 b-thymosins and, 279–281 colon, prothymosins and, 263 hepatocellular, prothymosins and, 263 targeted therapy for, folate receptors and, 434 Captopril, in diagnosis of primary aldosteronism, 132 Carboxyl ester lipase (CEL), 460 Carcinoma. See Cancer Cardiac fibrosis, aldosterone and, 32, 57–59, 95, 99–100 Cardiac hypertrophy, aldosterone and, 95, 97–98 Caspases, in NGF-induced apoptosis, 398 CBP/p300, as coactivator of nuclear receptors, 475 CD34, 3–4. See also Hematopoietic stem/ progenitor cells, CD34+ CD44, in hematopoiesis and stem cell trafficking, 12–13 Cell cycle control proteins, prothymosin binding to, 267 Cellular milieu, and corticosteroid actions, 90–91 Cellular retinaldehyde-binding protein (CRalBP), 466 Cellular retinol-binding proteins (CRBPs), 462, 464, 466–467 Central nervous system (CNS). See also Brain abnormalities with retinoid deficiency, 530 retinoids causing, 528–531 embryonic development of, vitamin A and, 486–488 c-fos, immobilization-induced expression of, 220 Channel-activating protease 1 (CAP1), and epithelial sodium channel, 56–57, 124

Index Channel-inducing factor (CHIF), and g subunit of sodium pump, 55 Chemoattractants, in stem cell migration and hematopoiesis, 10–13 Children. See also Acute lymphocytic leukemia (ALL) ascariasis in, vitamin A supplementation and, 519 diarrhea in, morbidity and mortality in, vitamin A supplementation and, 514–516, 517 HIV-infected, morbidity and mortality in, vitamin A supplementation and, 513, 515–517 hypervitaminosis A in, 458–459, 522–526 infections in, vitamin A supplementation and, 512–517 malaria resistance in, vitamin A supplementation and, 517, 519 mortality in, vitamin A supplementation and, 512–514 parasitic infestations in, and vitamin A status, 519 respiratory infections in, morbidity and mortality from, vitamin A supplementation and, 514–517 Chimeric 11b-hydroxylase–aldosterone synthase gene, 138–139 Chromatin decondensation of, prothymosin and, 268 in mineralocorticoid receptor gene regulation, 41–42 remodeling, prothymosin and, 267 Chronic fatigue clinical features of, 227 and depression, 227 hypothalamic-pituitary-adrenal axis and, 227–228 serotonin–HPA interactions in, 228 Chylomicrons retinyl esters in, 463–464 vitamin A in, 462, 463 Ciboulot (Cib), of Drosophila melanogaster, 275, 276f, 302 Circadian rhythm, serotonin and HPA interactions in, 212–215 CLAVATA3, in plants, and meristem organization, 324–327 Clomipramine, effects on hypothalamicpituitary-adrenal axis, 204 CLV1/2/3, in plants, and meristem organization, 324–327, 326f, 327f

Index Cobblestone area-forming cell (CAFC) assays, 5 Colon cancer of, prothymosins and, 263 SGK1 in, 53 Computed tomography (CT), of adrenal glands, 134 Conditioned stress, serotonin and hypothalamic-pituitary-adrenal axis in, 219 Congenital adrenal hyperplasia, 135–137 mineralocorticoid hypertension with, 115, 124, 126t undervirilizing, 136 virilizing, 136 Conjunctiva xerosis, 510 Cornea, lesions of, in vitamin A deficiency, 511 Corticosteroid(s), 79–80. See also Aldosterone; Glucocorticoid(s); Mineralocorticoid(s) actions of, rapid nongenomic, 59–60, 62, 79, 85–87 active export of, 87–88 and cardiac ion channels, 96–97 effects on heart, 79, 95–96 high-affinity binding sites for, 82–83 low-affinity binding sites for, 83–84 plasma binding proteins and, 87 receptors for, 80–84, 121. See also Steroid receptors coactivators of, 90 corepressors of, 90 mechanism of action of, 84–87 genomic, 84–85 rapid nongenomic, 85–87 and transcription factors, interactions of, 84–85, 90 type I (high-affinity). See Mineralocorticoid receptors (MR) type II (lower affinity). See Glucocorticoid receptors (GR) signaling by, modulators of, 87–91 Corticosteroid binder IB, 82 Corticosteroid-binding globulin (CBG), 87 binding site for, 86 Corticosterone, 33, 79 production of, in heart, 93–95 receptors for, 200 release of in conditioned stress, 219 in immobilization stress, 219 synthesis of, 80

597 Corticosterone-binding site, in roughskin newt, 86 Corticotropin-releasing hormone (CRH), 199, 201, 202 deficiency of, in chronic fatigue, 227 in depression, 222 effects on serotonergic system, 210–211 Cortisol, 31–32, 33, 79, 114–115 actions of, 124, 125f in brain, 45 in anxiety disorders, 225 bioavailability of, sex differences in, 45 biosynthesis of, 117–118, 118f peripheral metabolism of, 120, 120f Cortisone, 88–89 CREB-binding protein (cAMP response element binding protein (CBP)), 41 prothymosin and, 268 CXCR-4, in stem cell migration and hematopoiesis, 13 CYP11B1 gene, 138 mutations of, 136–137 CYP11B2 gene, 138 expression of, regulation by angiotensin II, 143 variants in, 142–143 CYP17 gene, mutations of, 137 Cyproheptadine, properties of, 218 Cytochrome P-450 CYP11B, 34 CYP11B1, 80 CYP11B2. See Aldosterone synthase P45017, 137 Cytokines, in hematopoiesis and stem cell trafficking, 12

D Death-inducing signaling complex (DISC), 387 11-Dehydrocorticosterone, 88–89 Dehydrocorticosterone (DHB) receptor, 82–83 colocalization with 11b-hydroxysteroid dehydrogenase 2, 82–83 11-Deoxycorticosterone, 124, 125f excess of, 136 Depression and anxiety disorders, 224 and chronic fatigue, 227 hypothalamic-pituitary-adrenal axis and, 221–222 serotonergic system and, 220–221 serotonin–HPA interactions in, 222–224

598 Dexamethasone–CRH test, 222 Dexamethasone suppression test, 222 Diarrhea, in children, morbidity and mortality in, vitamin A supplementation and, 514–517 DNase I, and actin and thymosin b4, ternary complex formation, 309–310 Drosophila melanogaster, b-thymosin-like protein of (ciboulot, Cib), 275, 276f, 302

E Early growth response gene 1 (Egr-1), PPARg and, 165–166, 166f Electrophoresis, native gel, of actin–thymosin b4 interactions, 306f, 307–308 End-replication problem, telomeres and, 14, 16 ENOD40 gene, in plants, and root nodulation, 322–324, 323f Epidermis, vitamin A and, 492–496 Eplerenone, in primary aldosteronism, 135 Epstein-Barr virus nuclear antigen (EBNA3C), prothymosin and, 268 Estrogen receptors (ER), transcriptional activity of, prothymosins and, 263, 284

F F-actin, thymosin b4 and, 279 Fatigue. See Chronic fatigue Fear conditioned, serotonin and hypothalamicpituitary-adrenal axis in, 219 hypothalamic-pituitary-adrenal axis in, 201–203 Fish, renin–angiotensin–aldosterone system in, 115, 116t Fludrocortisone suppression test, 133 Fluorescence recovery after photobleaching (FRAP) technique, and PTHrP nuclear import pathway, 347, 363–366, 364f–365f Fluoxetine effects on hypothalamic-pituitary-adrenal axis, 204 mechanism of action of, 221 and response to hypoglycemia, 218 Folate, transport of. See also Folate receptors (FR); Reduced folate carrier (RFC) by ABC exporters, 436–437, 438f factors affecting, 437–439 multidrug resistance-associated proteins (MRPs) and, 434–436

Index pathways for, 437, 438f Folate receptors (FR), 430–434 folate uptake mediated by mechanisms of, 433–434 pharmacological role of, 434 physiological role of, 433–434 FRa localization of, in cells, 440 in mouse development, 441 promoters, 431–432 specificity of, 430–431 structure of, 430–431, 432f transcriptional and posttranscriptional regulation of, 431–433 FRb specificity of, 430–431 structure of, 430–431, 432f transcriptional and posttranscriptional regulation of, 431–433 FRg specificity of, 430–431 structure of, 430–431, 432f transcriptional and posttranscriptional regulation of, 431–433 functions of, 404 genes for, organization of, 430–431, 432f and targeted cancer therapy, 434 Folate transporters. See also Folate receptors (FR); Reduced folate carrier (RFC) in intestinal cells, 428–430 localization of, in cells, 439–440 with low pH optima, 428–430 in mouse development, 441 in vectorial transport in epithelia, 439–440 Foot shock, and conditioned stress, serotonin and hypothalamic-pituitary-adrenal axis in, 219 Functional residual capacity (FRC), localization of, in cells, 439–440 FXYD protein family, 55

G G-actin, thymosin b4 and, 275–279, 284 GATA-1, and hematopoietic stem/progenitor cell regulation, 9 General adaptation syndrome, 215–216 Gene transcription, modulation by retinoic acid, 472–473, 535 regulatory proteins and, 474–476 Glucocorticoid(s), 79. See also Corticosterone; Cortisol actions of, 79–80, 200–201

Index in collecting ducts, 38, 124, 127f neuronal, 85 parathymosins and, 273, 284 rapid nonneuronal, 85 in brain, 88 cardioprotective effects of, 99 effects on serotonergic system, 211–212 membrane-binding sites, 86 and myocardial infarction, 98–99 regulation of mouse mammary tumor virus (MMTV) long terminal repeat (LTR), 42 secretion of, in anxiety, 226 synthesis of, 80, 200 Glucocorticoid receptors (GR), 31, 38, 79, 80–82 a, 81 aldosterone binding to, 38 alternative splice variants of, 82 amino acid sequence of, 32f in anxiety, 226 b, 81–82 in brain, 50, 95, 203 cellular concentration of, 90 cellular localization of, 38–39, 84 in depression, 221–222 DNA-binding domain of, 32f, 39, 80 evolution of, 34 expression of, regulation by aldosterone, 49 in heart, 91–92 isoforms of, 81 ligand-binding domain of, 32f, 39, 80 and NF-B activity, 84 N-terminal variable region of, 32f, 39, 80 parathymosins and, 273 phosphorylation of, 90 signaling through, research on, directions for, 62 specificity of, 40 structure of, 39, 80 synergy control (SC) motif of, 40 transcriptional responses of, 94–95 and transcriptional synergy, 40 and transcription factors, interactions of, 84–85 as transrepressors of AP1, 84 Glucocorticoid regulatory element (GRE), 51 Glucocorticoid response elements (GREs), 39 Glucose tolerance, abnormalities, in primary aldosteronism, 130 Glycyrrhetinic acid, inhibition of 11b-HSD2, 44 Glycyrrhizic acid, inhibition of 11b-HSD2, 44

599 H Hair cells, auditory, regeneration of, retinoic acid and, 530 Heart. See also Cardiac fibrosis; Cardiac hypertrophy aldosterone actions in, 32, 49, 57–59, 97–98 aldosterone production in, 93–95 11b-hydroxysteroid dehydrogenase isoforms in, 88, 91–92 corticosteroid receptors in, 91–92 corticosteroids and, 79, 95–96 corticosterone production in, 93–95 embryonic development of, vitamin A and, 482–485 epithelial sodium channel in, 49 PPARa and, 172–173 PPARg and, 169–170 SGK1 in, 49–50 Heart failure aldosterone and, 32, 57–59, 94, 95 corticosteroids in, 79 Heat shock proteins, and nuclear receptors, 38, 84, 90–91 Hematopoiesis adhesion molecules in, 10–13, 11f embryonic, retinoids and, 491–492 fetal, 10 physiology of, 2 Hematopoietic stem/progenitor cells aging of, 13–17 blood-derived cell cycling and differentiation of, 6–10, 7f transplantation of, 2–3 from bone marrow, 2 cell cycling and differentiation of, 6–10, 7f transdifferentiation of, 17 CD34+, 3–4 blood-derived, cell cycling and differentiation of, 6–10, 7f bone marrow-derived, cell cycling and differentiation of, 6–10, 7f subsets of, 4–5 cell cycling and differentiation of, 6–10 gene expression during, 7–8, 7f transcription factors and, 8–10 collection of, 2 developmental control of, 6–10 functional characteristics of, 3–6 functional genomics of, 3 functional in vitro assays of, 4 immunological characteristics of, 3–6, 5f

600 Hematopoietic stem/progenitor cells (continued) multilineage differentiation of, 4–6, 17–18 and neuronal development, 17–18 plasticity of, 3, 17–18 replicative senescence of, 14 self-renewal of, 4, 6, 9–10, 17 telomeres of, shortening of, 14–17 trafficking of, adhesion molecules in, 10–13, 11f transdifferentiation by, 3, 17–18 transplantation of, 2–3 allogeneic, 2 telomere loss in, 16 autologous, 2 telomere elomere loss in, 16 and graft-versus-tumor effect, 3 Hemorrhage response to hypotensive stage of, 217 normotensive stage of, 217 serotonin and hypothalamic-pituitaryadrenal axis in, 215–218 Hepatocellular carcinoma (HCC), prothymosins and, 263 Hippocampus, and hypothalamic-pituitaryadrenal axis, 202–203 Histone acetyltransferase (HAT), 41, 476–477 p300, in EBV-infected cells, prothymosin and, 268 prothymosin and, 268 Histone deacetylases, 41–42, 476–477 Histones acetylation of, 41–42 prothymosin and, 268 deacetylation/acetylation, and transcriptional regulation, 476–477 phosphorylation of, 42 prothymosin binding to, 267 Hormone response elements (HREs), 31, 39, 121 Hormones, polypeptide, in plants, 317–337 HoxA10, and hematopoietic stem/progenitor cell regulation, 9 HoxB4, and hematopoietic stem/progenitor cell regulation, 9 5-HT. See Serotonin (5-HT) Human immunodeficiency virus (HIV), mother-to-child transmission of, vitamin A status and, 520–521 Human steroid/xenobiotic receptor (SXR), 83 11b-Hydroxylase, 80 deficiency of, 136

Index in heart, 93 in heart failure, 94 mutations of, 126t, 135–137 17a-Hydroxylase deficiency of, 136, 137 and 17,20-lyase deficiency, combined, 137 mutations of, 126t, 135–137 21-Hydroxylase, deficiency of, 135–136 14-Hydroxyretroretinol (14-HRR) biosynthesis of, 469 and cell phenotype, 477–479 distribution of, 468–469 structure of, 469f 11b-Hydroxysteroid dehydrogenase (11b-HSD) activity of, 81, 203 type I, 88, 119 activity of, 42–43, 88–89, 119 deficiency of, 119–120 in heart, 88, 92 tissue distribution of, 43, 88, 92 type II, 88 activity of, 32, 33, 38, 42–43, 89, 119–121 in brain, 37, 45–46 colocalization with DHB receptor, 82–83 colocalization with mineralocorticoid receptors, 88–90 deficiency of, 126t evolution of, 34 gene for, mutations of, 139 gene-targeted mutation in, mouse models of, 46–48, 46t in heart, 92 inhibition of, 44 mutations of, 44 physiologic functions of, 44–46 in protection of mineralocorticoid receptors in distal nephron, 43–44 sexually dimorphic expression of, 45 species distribution of, 44 substrates for, 44 tissue distribution of, 44–45, 92 5-Hydroxytryptamine (5-HT). See Serotonin (5-HT) Hyperaldosteronism, familial type I, 126, 126t type II, 126 Hypertension essential aldosterone-dependent, 139, 142–143 HSD11B2 gene mutations and, 139 mineralocorticoid. See Mineralocorticoid hypertension

Index PPARg and, 168 salt-sensitive, pathogenesis of, 42 Hypervitaminosis A, 535–536 in children, 458–459, 522–526 acute, 523–525 chronic, 523, 525–526 long-term effects of, 526 epidemiology of, 523–524 in infancy, 531–535 Hypoglycemia, serotonin and hypothalamicpituitary-adrenal axis in, 218–219 Hypothalamic-pituitary-adrenal axis circadian rhythm of, 213–214 serotonin and, 214–215 effects on serotonergic system, 210–212 extrahypothalamic effects on, 201–203 neuroanatomy of, 195–203 regulation of negative feedback in, 200–201 neural circuitry for, 198–201, 199f serotonin and, 191, 203–212 Hypothalamus. See also Hypothalamicpituitary-adrenal axis neuroanatomy of, 195–197, 195t serotonergic inputs to, 191 Hypovitaminosis. See also Vitamin A, deficiency of in children, 458–459

I Imipramine, mechanism of action of, 221 Immobilization stress, serotonin and hypothalamic-pituitary-adrenal axis in, 219–220 Immune system thymosin b4 ligands and, 312–313 vitamin A and, 497–499 Immunophilins, and nuclear receptors, 84 Infection(s) in children, mortality from, vitamin A supplementation and, 512–517 vitamin A and, 497–499, 509 Inflammation, thymosin b4 ligands and, 312–313 Insulin, and aldosterone, cross-talk of, 32, 60–61 Insulin resistance syndrome, 60–61, 61f Integrins b1–, in hematopoiesis and stem cell trafficking, 11–12 b2–, in hematopoiesis and stem cell trafficking, 12

601 Iron supplements for pregnant women, 520 vitamin A supplementation and, 518 Ischemia-reperfusion, myocardial, cardioprotection in, glucocorticoids and, 99 Ito cells, retinol in, 463–464

K Karyopherin b1–Rch1 complex, 267 Ketanserin, and response to hypoglycemia, 218–219 Kidney embryonic development of, vitamin A and, 496–497 in regulation of salt and water balance, 115 Kidney type III binding site, 82 K-Ras2, and epithelial sodium channel, 54–55

L Lactation, vitamin A in, 504–507, 519–522 Lecithin:retinol acyltransferase (LRAT), 461f, 462, 464 Leukocytes migration and homing of, molecular mechanisms of, 13 telomeres of, shortening of, during maturation, 14–15, 15f LFA-1, in hematopoiesis and stem cell trafficking, 12 Licorice intoxication, 44 Liddle’s syndrome, 36, 48, 57, 126t, 140–141 Ligand-inducible transcription factors, 79 Limb(s) embryonic development of, vitamin A and, 488–491 regeneration of, in amphibians, retinoids and, 490–491 Liver, vitamin A uptake by, 463–464 Long-term bone marrow culture (LTBMC), 4–5 Long-term culture-initiating cell (LTC-IC) assays, 5 L-selectin, in hematopoiesis and stem cell trafficking, 12 Lung, embryonic development of, vitamin A and, 496 Lung disease, in very low birth weight infants, vitamin A and, 507–509 17,20-Lyase, 137

602 Lymphoid cells, development of, 9 Lymphoma, prothymosins and, 263

M Magnesium imbalance, in primary aldosteronism, 130 Magnetic resonance imaging (MRI), of adrenal glands, 134 Malaria, resistance to, vitamin A supplementation and, 517, 518 Mammals, renin-angiotensin-aldosterone system in, 116, 116t Measles morbidity and mortality in, vitamin A supplementation and, 512–514, 517 vaccine, vitamin A supplementation and, 512 Megakaryocytes, differentiation of, 9–10 Melanocyte-stimulating hormone (MSH), 200 Metabolic syndrome, 60–61, 61f aldosterone in, 32 Methotrexate (MTX) clinical utility of, reduced folate carrier (RFC) and, 423–427 MDR1-mediated resistance to, 436–437 membrane transport of, reduced folate carrier (RFC) and, 405–407, 415–422 resistance to, MRPs and, 434–436 Methysergide, properties of, 218 Mineralocorticoid(s), 79 actions of, 79–80, 117–124 in collecting ducts, 38, 124, 127f in distal nephron, 35–36, 35f molecular basis of, 37–42 synthesis of, 80 target tissues for, 33–34 Mineralocorticoid hypertension, 124–125 causes of, 114–115, 124 clinical findings in, 129–130 definition of, 114 inherited forms of, 126t, 135–141 laboratory findings in, 129–130 mechanisms of, 124, 125f prevalence of, 128–129, 129t screening for, 130–131, 131f sporadic forms of, 126t Mineralocorticoid receptors (MR), 31, 79, 80–82, 121–122 a, 82 aldosterone binding to, 38, 81

Index aldosterone specificity of, 43, 88 amino acid sequence of, 32f b, 82 in brain, 37, 50–51, 95 cardiac, 57, 91 in fibrosis and heart failure, 58–59 cellular concentration of, 90 cellular localization of, 38–39, 84 DNA-binding domain of, 32f, 39, 80, 121 as dual receptor for glucocorticoids and mineralocorticoids, 33 in epithelia, 32 evolution of, 34 expression of, autoregulation, 49 gene for, mutations of, 140 gene regulation by, 37–39 activators in, 41–42 chromatin in, 41–42 corepressors in, 41–42 gene-targeted mutation in, mouse models of, 46–48, 46t, 86–87, 100–101 isoforms of, 82 knockout, cardiac effects of, 100–101 ligand-binding domain of, 32f, 39, 80, 121 ligands for, 81 in mammals, 34 mutations of, mineralocorticoid hypertension caused by, 126f N-terminal variable region of, 32f, 39–40, 80, 121 overexpression of, cardiac effects of, 100 phosphorylation of, 90 in rapid nongenomic effects of aldosterone, 60, 92 selectivity of, 120, 120f signaling through, research on, directions for, 62 specificity of, 89 mechanisms of, 39–40 splice variants of, 82 structure of, 39, 80 synergy control (SC) motif of, 40 tissue distribution of, 37 transcriptional responses of, 94–95 and transcriptional synergy, 40 Monoamine oxidase inhibitors (MAOIs), and HPA, 204 Multidrug resistance-associated proteins (MRPs), 434–436 inhibition of, 439 localization of, in cells, 440 Myeloid–lymphoid initiating cell (ML-IC) assay, 6

Index Myocardial infarction acute phase, 98 chronic phase, 98 glucocorticoids and, 98–99

N NADE amino acid sequence of, 390–391, 391f and 14-3-3e protein, interactions of, 395, 397 expression of, 392–393 genes for, genomic structure of, 391–392, 392f isoforms of, 390–391 NES in, 389, 390f, 394, 394f in neuronal apoptosis in ischemia, 397–398 and p75NTR, 387–389, 388f, 393–394 in NGF-induced apoptosis, 387–389, 388f, 395–398 prenylation site in, 389 research on, advances in (future directions for), 398 structure–function relationships of, 394, 394f, 396 structure of, 389–390 ubiquitination signal in, 389, 390f Na+,K+-ATPase. See Sodium pump Nedd4-2, and epithelial sodium channel, 56–57, 98, 123, 127f, 141 Nerve growth factor (NGF), apoptosis induced by caspases in, 398 NADE and p75NTR in, 387–389, 388f, 395–398 in oligodendrocytes, NADE in, 397 research on, advances in (future directions for), 398 Nervous system, embryonic development of, vitamin A and, 486–488 Neuronal apoptosis, in ischemia, NADE in, 397–398 Neurotrophins, 386 receptor for, p75. See p75NTR Night blindness, 509, 510 in pregnant women, 519, 520 Notch-1, and hematopoietic stem/progenitor cell regulation, 9 NRAGE, 387–388, 388f NRIF, 387, 388f Nuclear export signals (NESs), 370 in NADE, 389, 390f, 394, 394f

603 Nuclear factor NF-kB, inhibition of, cardioprotective effects of, 99 Nuclear localization sequences (NLSs), 357–359, 358f of parathyroid hormone-related protein (PTHrP), 359–362, 360t, 361f polypeptide ligands, 368, 369t Nuclear protein import cellular machinery for, 356–357 and nuclear targeting signals, 357–359, 358f of parathyroid hormone-related protein (PTHrP), 356–368 cytoskeleton and, 367–368 FRAP examination of, 347, 363–366, 364f–365f microtubule integrity and, 363–366, 364f–366f pathway for, 362–363 pathways for, 356–359 of polypeptide ligands, 368, 369t Nuclear receptor corepressor (N-Cor), 474–476 Nuclear receptors, 80, 158 coactivators, 474–475 corepressors, 474–475 structure of, 472f, 473 transcriptional modulation by, 471–473 histone deacetylation/acetylation and, 476–477 regulatory proteins and, 474–476 Nuclear transcription factors, 31 Nuclear transport proteins, prothymosin binding to, 267

O Oligodendrocytes, NGF-induced apoptosis in, NADE in, 397 4-Oxo-retinal, 465 4-Oxo-retinol, 465

P p23, and steroid receptors, 90 PAI-1, in cardiac fibrosis, 58 Pancreatic lipase-related proteins (PLRP), 460–461 Pancreatic triglyceride lipase (PTL), 460–461 Parasitic infestations, vitamin A deficiency and, 519 Parathymosin(s), 270–273, 284 amino acid sequences of, 270–271, 270f and angiogenesis, 281

604 Parathymosin(s) (continued) bipartite nuclear localization signal, 270f, 272–273 in carbohydrate metabolism, 271–272, 284 and glucocorticoid action, 273, 284 posttranslational modification of, 273 species distribution of, 270–271, 270f and zinc, 271–272, 284 Parathyroid hormone (PTH), in primary aldosteronism, 130 Parathyroid hormone-related protein (PTHrP) domain structure of, 347–349, 348f functional role of, in nucleus/nucleolus, 372–374 as growth/malignancy factor, 374 intracellular forms of, 354–355 intracrine role of, 353–356 localization in nucleus and nucleolus, cell-cycle-dependent, 355–356 nuclear export of, integrated system for, 366f, 370–372 nuclear import and export of, integrated system for, 366f, 370–372 nuclear import of, 356–368 cytoskeleton and, 367–368 FRAP examination of, 347, 363–366, 364f–365f microtubule integrity and, 363–366, 364f–366f pathway for, 362–363 nuclear localization sequences (NLSs) of, 359–362, 360t, 361f nuclear targeting signal for, importin b1-recognized, 359–368 paracrine role of, 349–353 properties of, 346–347 receptor-independent actions of, 353–354 receptor-mediated endocytosis of, and nuclear targeting, 351–353, 352f receptors for other than PTH1R, 353 PTH1R, 351–353 research on, advances in (future directions for), 374 Paroxetine effects on hypothalamic-pituitary-adrenal axis, 204 mechanism of action of, 221 p160 coactivators, in mineralocorticoid receptor gene regulation, 41 Peroxisome proliferator-activated receptor(s) (PPARs)

Index a in cardiovascular system, 170–173, 171t chromosomal location of, 159 genetic variants, and cardiovascular disease, 172 and heart, 172–173 ligands for, 160–161 species distribution of, 159t in vasculature, 171–172 actions of, 162, 162f activation function 1 (AF-1), 159, 160f activation function 2 (AF-2), 160, 160f cofactors for, 162–163 d in cardiovascular system, 173–175 chromosomal location of, 159 ligands for, 161 regulation of, 174–175 species distribution of, 159t in vasculature, 173–174 degradation of, 163 discovery of, 159 DNA-binding (C) domain of, 160, 160f domain structure of, 159–160, 160f g and atherosclerosis, 166–168 in cardiovascular system, 163–170, 164t–165t chromosomal location of, 159 and early growth response gene 1 (Egr-1), 165–166, 166f genetic variants, and cardiovascular disease, 168–169 and heart, 169–170 and hypertension, 168 ligands for, 161 species distribution of, 159t in vasculature, 165–166 ligand-binding (E/F) domain of, 160, 160f ligands for, 160–161 mechanisms of action of, 162–163 N-terminal (A/B) domain of, 159, 160f phosphorylation of, 163 species distribution of, 159t tissue distribution of, 160 transcriptional activation of, 163 P-glycoproteins MDR1, in folate transport, 437 in steroid export, 87–88 Photic stimulation, serotonin–HPA interactions in, 219 Phytosulfokines, 327–330, 328f

Index Pituitary gland. See also Hypothalamicpituitary-adrenal axis hormone synthesis and release by, 197–198, 198t neuroanatomy of, 197–198 Plants CLAVATA3, and meristem organization, 324–327 CLV1/2/3, and meristem organization, 324–327, 326f, 327f ENOD40 gene, and root nodulation, 322–324, 323f POLARIS (pls) in, 334–337 polypeptide signaling molecules (hormones) in, 317–337 rapid alkalinization factor (RALF) in, 321–322 self-incompatibility in, 330–334, 334f systemin in, 318–321, 321f systemin-like peptides in, 318–321, 321f WUSCHEL (WUS), and meristem organization, 326–327, 326f, 327f Plasma aldosterone-to-renin activity ratio (ARR) test, 128–131 in aldosterone-dependent essential hypertension, 142 cut-off values for, in primary aldosteronism, 132t Platelet-endothelial cell adhesion molecule 1 (PECAM-1), in hematopoiesis and stem cell trafficking, 12–13 Pneumonia pneumococcal, in children, morbidity and mortality in, vitamin A supplementation and, 514 respiratory syncytial virus (RSV), in children, vitamin A supplementation and, 516 p75NTR binding proteins for, 387–388 death domain (DD) of, 387, 398 NADE and, 387–389, 388f, 393–394 in NGF-induced apoptosis, 387–389, 388f, 395–398 proapoptotic role of, 387 properties of, 386–387 research on, advances in (future directions for), 398 p75NTR-associated cell death executor. See NADE POLARIS (pls), in plants, 334–337 Postural test, in primary aldosteronism, 133

605 Potassium and aldosterone production, 116 homeostasis, 116–117 in aldosterone-sensitive tissues, 55 imbalance, in primary aldosteronism, 130 transport, SGK1 and, 54 Potassium currents, cardiac, corticosteroids and, 96 PPAR-responsive element (PPRE), 162 Pregnancy, vitamin A in, 504–507, 519–522 Pregnane X receptor (PXR), 83 Profilin and actin and thymosin b4, ternary complex formation, 310–311, 312f and subdomain 1 of actin, 305–307 Progesterone receptors (PR), 31 Proopiomelanocortin (POMC), 200 Protein kinase C, aldosterone and, 92 Protein kinases, and corticosteroid actions, 91 Prothymosin(s) amino acid sequence of, 261–262, 261f, 263 in angiogenesis, 281 binding to nucleosome core histones, 267 bipartite nuclear localization site of, 262, 264–265 caspase 3 cleavage site, 262, 265 in cell proliferation, 263–264 chemical features of, 262–263 divalent cation binding by, 264 evolutionary conservation of, 262 expression of, estrogens and, 263 extracellular, 269–270, 284 immunomodulatory effects of, 264 intracellular, biological activities of, 284 intracellular partners of, 267–268 phosphorylation of, 265–267 phylogenetic distribution of, 261–262, 261f and small RNA, 268–269 and spermatogenesis, 262 structure of, 262–263 synthesis of, 261 and zinc, 264 Pseudoaldosteronism, 140–141 Pseudohypoaldosteronism, type I, 36 mouse models of, 46t, 47, 48 PU.1, and hematopoietic stem/progenitor cell regulation, 9 Pyrene fluorescence assay, of actin-thymosin b4 interactions, 307–308, 309f

606 R Randomized Aldactone Evaluation Study (RALES), 57, 59, 94, 95 Raphe´ nuclei, serotonin in, 190, 191 Rapid alkalinization factor (RALF), in plants, 321–322 Reduced folate carrier (RFC), 405–427 cDNAs for, cloning of, 405–411 in clinical utility of methotrexate, 423–427 expression of, patterns of, 411–412 functions of, 404, 405 factors affecting, 437–439 hamster, gene for, 410–411 human alternative splicing, in childhood ALL, 425–427 gene for, 410–411 sequence variants, in childhood ALL, 425–427 membrane targeting, structural determinants of, 409–410 trafficking, structural determinants of, 409–410 transcript heterogeneity, alternative noncoding exons and variable splicing in, 412–414, 413f 50 -UTRs in childhood ALL, 423–425 heterogeneity of, 412–414, 413f in intestinal cells, 428–430 mouse, gene for, 410–411 in mouse development, 441 promoters, and tissue-specific gene expression, 414–415 properties of, 405 secondary structure of, analysis of, 407–409, 408f structure of, 405–407 analysis of, 420–423 tissue distribution of, 411–412, 414–415 topology model for, 408–409, 408f transcript heterogeneity, molecular basis of, 412 transport mediated by energetics of, 417–420 kinetics of, 415–417 in tumors, 411–412 Renin–angiotensin–aldosterone system, 31f, 115–117 abnormalities of, in insulin resistance, 60 across species, 115–116, 116t Reptiles, renin–angiotensin–aldosterone system in, 115, 116t

Index Respiratory infections, in children, morbidity and mortality from, vitamin A supplementation and, 514–517 Respiratory syncytial virus (RSV), pneumonia, in children, vitamin A supplementation and, 516 Retinal, structure of, 468 Retinaldehyde dehydrogenases (RALDH), in retinoic acid synthesis, 466–467 Retinoic acid, 459, 460f bioactivity of, 464 in embryo, 479–480 enzymatic synthesis of, 466–467 teratogenicity of, 528–529 transcriptional modulation by, 472–473, 533 regulatory proteins and, 474–476 Retinoic acid receptors (RAR), 466 biochemistry of, 471–473 in embryo, 481–482 gene for, 471 ligands for, 471–473 and retinoid X receptors, heterodimer formation, 471–473 structure of, 471, 472f Retinoic acid response elements (RAREs), 472, 473–474 Retinoids activity of molecular basis of, 471–479 retinoic acid receptor–dependent, 471–473 biological effects of, 471 teratogenicity of, 529 cis-Retinoids, synthesis of, enzymes in, 467–468 Retinoid X receptors (RXR), 162 biochemistry of, 471–473 as cofactor for nonsteroidal nuclear receptors, 473 in embryo, 481–482 heterodimer formation with farnesoid X receptor, 473 with liver X receptor, 473 with PPAR-g, 473 with retinoic acid receptors, 472–473 ligands for, 472–473 Retinol, 459, 460f bioactive metabolites of, 464–465 bioactivity of, 464 circulating, cellular uptake of, 464 from dietary carotenoids, 462 and embryogenesis, 470–499

Index in embryogenesis, 479–499 nonacid derivatives of, 465 oxidation of, 466–467 reesterification in intestinal cells, 462–463 secretion of into lymph, 463 into portal circulation, 463 serum levels of, and parasitic infestations, 519 structure of, 469 uptake in intestinal cells, 462–463 Retinol-binding protein (RBP), 461–462 Retinol dehydratase, 470 Retinol dehydrogenases, 465, 468 cytosolic, 466 Retinol equivalents (RE), 499–500 Retinyl ester hydrolase (REH), 460–461, 468 Retinyl esters, 459, 460f in chylomicrons, 463–464 digestion of, 459–461 enzymes in, 460–461, 461f in liver, 464–465 Retroretinoids, 465–466 biosynthesis of, mechanisms for, 468–470 and cell phenotype, 477–479 structure of, 469 Rev proteins, prothymosin and, 267–268 River blindness, vitamin A deficiency and, 519

S Salt appetite, 37 aldosterone and, 45 hormonal regulation of, 80 Schistosomiasis, vitamin A deficiency and, 519 Selective serotonin reuptake inhibitors (SSRIs) effects on hypothalamic-pituitary-adrenal axis, 204–205 mechanism of action of, 221, 225 Senescence, cellular, 14 Serotonergic pathways anatomy of, 191 regulation of, negative feedback in, 223–224 Serotonin (5-HT) and amygdala-mediated activation of HPA, 202 in brain, 190, 191 discovery of, 190–191

607 drugs releasing, effects on hypothalamicpituitary-adrenal axis, 205 and hypothalamic-pituitary-adrenal axis, 203–212 in anxiety disorders, 224–227 in chronic fatigue, 227–228 in circadian rhythm, 212–215 in depression, 220–224 pathophysiological interactions of, 220–228 physiological interactions of, 212–220 in stress, 201–203, 215–220 precursors, effects on hypothalamicpituitary-adrenal axis, 205 Serotonin (5-HT) autoreceptors, 192 desensitization of, 223 presynaptic, 192–195 somatodendritic, 192 Serotonin (5-HT) receptors, 191–195 agonists, 193t–194t antagonists, 193t–194t characteristics of, 192–195, 193t–194t classification of, 192 density, circadian rhythm of, 215 G protein-coupled, 192, 193t–194t 5-HT1 characteristics of, 192, 193t density, circadian rhythm of, 215 5-HT1A agonists, effects on hypothalamicpituitary-adrenal axis, 205–206, 225, 228 characteristics of, 192, 193t desensitization of, 223–224, 225, 227 glucocorticoids and, 211–212 5-HT1B, glucocorticoids and, 212 5-HT1B/1D agonists, effects on hypothalamicpituitary-adrenal axis, 206–207 characteristics of, 192–195, 193t 5-HT1E, characteristics of, 193t 5-HT1F, characteristics of, 193t 5-HT2 characteristics of, 192, 193t density, circadian rhythm of, 215 5-HT2A characteristics of, 193t glucocorticoids and, 212 and 5-HT2 receptor agonist-induced regulation of HPA, 215 5-HT2A/2C, agonists, effects on hypothalamic-pituitary-adrenal axis, 207–208

608 Serotonin (5-HT) receptors (continued) 5-HT2B, characteristics of, 193t 5-HT2C agonists, effects on hypothalamicpituitary-adrenal axis, 228 characteristics of, 193t density, circadian rhythm of, 215 glucocorticoids and, 212 5-HT3 agonists, effects on hypothalamicpituitary-adrenal axis, 208–209 characteristics of, 192, 194t 5-HT3(A-B), characteristics of, 194t 5-HT3C, characteristics of, 194t 5-HT4 agonists, effects on hypothalamicpituitary-adrenal axis, 209 characteristics of, 192, 194t 5-HT4(a-h,hb,n), characteristics of, 194t 5-HT5, characteristics of, 192, 194t 5-HT6 characteristics of, 192, 194t glucocorticoids and, 212 5-HT7 agonists, effects on hypothalamicpituitary-adrenal axis, 209–210 characteristics of, 192, 194t glucocorticoids and, 212 as ligand-gated ion channels, 192, 194t location of, 193t–194t neuronal location of, 192 nomenclature for, 192 transduction systems, 193t–194t Serum and glucocorticoid-regulated kinase. See SGK SGK, 52, 123 gene for, transcription in cardiac myocytes, corticosteroids and, 98 isoform 1 (SGK1) and aldosterone and insulin effects on ENaC, 60–61, 61f, 123 and aldosterone effects on sodium transport, 54, 123 catalytic domain of, sequence identity with protein kinase B, 53–54 and epithelial sodium channel, 52–54 gene-targeted mutation in, mouse models of, 46t, 48, 54 in heart, 49–50 in mineralocorticoid-regulated sodium transport, 35f, 36, 42 and Nedd4-2 interaction with ENaC, 57

Index phosphorylation of, by PDK1, 53–54 transcriptional regulation of, 52–53 isoform 2 (SGK2), 54 in heart, 50 isoform 3 (SGK3), 54 in heart, 49–50 Short-chain dehydrogenase/reductase (SCDR), 466–467 Silencing mediator for retinoid and thyroid hormone receptors (SMRT), 475, 477 Situs inversus, vitamin A and, 483 Skin, embryonic development of, vitamin A and, 493–496 SLC19 family of transporters, 405 SLC21 organic ion carriers, 405 folate transport by, 427 Small intestine, corticosterone binding site in, 83–84 Smooth muscle, vascular, retinoids and, 484–485 Sodium balance, mineralocorticoids in, 45 renal handling of, 115–116, 127f aldosterone-mediated, SGK1 and, 54 transport, aldosterone-mediated, SGK1 and, 54 Sodium channel brain (BNaC), 51–52 epithelial (ENaC), 122–124, 122f aldosterone action and, 51–52, 98 aldosterone-regulated gene products and, 52–55 CAP1 (channel-activating protease 1) and, 56–57, 124 gene for, mutations of, 140–141 gene-targeted mutation in, mouse models of, 46–48, 46t in heart, 49 Nedd4-2 and, 56–57, 98, 123, 127f, 141 in principal cells, rapid activation by aldosterone, 59 regulation of, 123–124 renal, 52, 124, 127f in mineralocorticoid hypertension, 114, 124, 127f subunits of, 46, 46t, 47–48, 51–52, 122, 122f mutations of, 141 Sodium loading, in diagnosis of primary aldosteronism, 132–133 Sodium pump aldosterone action and, 51 in cardiocytes, 50

Index g subunit of, channel-inducing factor (CHIF) and, 55 Spironolactone adverse effects and side effects of, 135 contraindications to, 140 in heart failure, mechanism of action of, 57–59 in primary aldosteronism, 135 Stellate cells, hepatic, retinol in, 461–464 Steroid receptors. See also Corticosteroid(s), receptors for specificity of, 39 structure of, 80 and transcription, 84 and transcriptional synergy, 40 Stressors cardiovascular, 215–218 physical, 215, 218–219 psychological, 215, 219–220 types of, 215 Stress response definition of, 215 and depression, 220 hypothalamic-pituitary-adrenal axis in, 201–203, 215–220 neuroanatomy of, 216 Stromal-derived factor 1 (SDF-1), in stem cell migration and hematopoiesis, 13 Sulfotransferase, 470 Syndrome X, aldosterone in, 32 Synergy control (SC) motif, 40 Systemin, in plants, 318–321, 321f Systemin-like peptides, in plants, 318–321, 321f

T Telomerase activity of, 16 and cell cycle status, 17 regulation of, during development, 16 Telomeres and end-replication problem, 14, 16 functions of, 14 length of cellular aging and, 14 cytotoxic chemotherapy and, 15–16 as marker of hematopoietic reserve, 15 progressive shortening of, 14 as marker for stem cell behavior in transplanted patients, 16 Thiazolidinediones (TZD) hypotensive effects of, mechanism for, 168

609 and PPARs, 161, 163 Thrombin receptor, in stem cell migration, 13 Thrombocytopenia, in hypervitaminosis A in infancy, 531–535 Thymosin(s) a, isoelectric point of, 259 a1, 259, 284 amino acid sequence of, 260, 261f in angiogenesis, 281 clinical trials of, in viral infections, 284 extracellular, 269–270 isoelectric point of, 260 properties of, 260 a11, amino acid sequence of, 260, 261f b, 273–283 amino acid sequences of, 274f, 275–276, 276f in angiogenesis, 281–283, 284 and cancer, 279–281 functions of, 279, 280f historical perspective on, 273–274 isoelectric point of, 259 phylogenetic distribution of, 274f, 275–276, 302 purification of, 274–275 in wound healing, 281–283, 284 b4, 273 and AcSDKP, 283 and actin, interactions of, 275–279, 277f, 284, 299–307 assays for, 306f, 307–308, 309f DNase I and, 309–310 interface in, 303–305, 303f, 304f profilin and, 310–311, 312f in ternary complex formation, 309–311, 312f and actobindin, comparison of, 302–303 amino acid sequence of, 274f, 275–276, 276f, 298, 299f, 302 in angiogenesis, 281 antiinflammatory properties of, 312–313 and cancer, 279–281 immunoregulatory properties of, 312–313 LKEAET motif, 304 LKETET motif, 304 LKHAET motif, 302, 303, 304f LKKTET motif, 302, 303, 304f phylogenetic distribution of, 275–276, 298, 299f structure of, 298–299 and subdomain 1 of actin, 305–307 in wound healing, 282–283, 284

610 Thymosin(s) (continued) b8, 273–274 b9, 274 amino acid sequence of, 274f and angiogenesis, 281 b10 and actin binding, 279 amino acid sequence of, 274f and angiogenesis, 281 and cancer, 279–281 b12, amino acid sequence of, 274f b13, amino acid sequence of, 274f b14, amino acid sequence of, 274f b15 amino acid sequence of, 274f, 275, 298, 299f and cancer, 279, 281 and G-actin, 278 g, isoelectric point of, 259 historical perspective on, 258–259 nomenclature for, 259 polypeptide b1, 259–260, 284 Thymosin b4-sulfoxide biological properties of, 278–279, 283 identification of, 283 Thymosin fraction 5, 259, 273–274 Thyroid hormone receptor interacting protein, 475 Transcriptional synergy definition of, 40 and steroid receptors, 40 Transcription factors, and hematopoietic stem/progenitor cell regulation, 9–10 Transfer RNA (tRNA), prothymosin and, 268–269 Transthyretin (TTR), 464 Tranylcypromine, mechanism of action of, 221 Triamterene, for Liddle syndrome, 140 Tricyclic antidepressants, effects on hypothalamic-pituitary-adrenal axis, 204 Trip1, 475 Tropomyosin-related kinase (Trk) receptors, 386 Tryptophan, deficiency of, 221

U Ubiquitin, 260, 284 Ubiquitous immunopoietic polypeptide (UBIP), 260

Index V Vasculature. See Blood vessels Vasculogenesis, embryonic, vitamin A and, 484–485 Vasopressin, actions of, 201 Very late antigen (VLA), in hematopoiesis and stem cell trafficking, 11–12 Vitamin A biological effects of, 470, 471, 479 in blood, 531 deficiency of and autoimmunity, 499 and childhood pathology, 458 embryolethality of, 483 epidemiology of, 509–510 immune dysfunction in, 498 in infants, pulmonary effects of, 505 and infections, 499, 510–511, 514–520 parasitic infestations and, 519 in pregnant and lactating women, 458, 502, 504, 510, 519–520 prevention of, 508, 535–536 primary, 510 risk factors for, 500 secondary, 510 subclinical, 511 derivatives of, cellular interactions of, 471 dietary intake of, 499–500, 504, 506 excessive, 502 and embryogenesis, 470–499, 524–525 studies of, models for, 480–482 excess. See Hypervitaminosis A in human development, 457, 479 international units (IU) of, 499 metabolism of, 493–494, 504 in embryos, 479 intracellular, 464–470 overdose of. See Hypervitaminosis A in pregnant and lactating women, 502–504 recommended dietary allowances for, 499–502 recommended dietary intakes for, 500 retinol equivalents (RE) of, 499, 504 sources of, 459, 501, 516 for vegetarians, 502 supplements, 516–517 for children adverse effects of, 513 effects on morbidity and mortality, 509, 510–512 recommendations for, 501–502 zinc supplementation and, 515–516

611

Index for infants, 509–510 misuse of, 521, 522–523 for neonates, 519 for pregnant and lactating women, 458, 502, 504, 510, 519–520 prophylactic use of, 509–510 types of, 526 use of, 502 teratogenicity of, 528–529 tissue delivery of, 463–464 toxicity of, 502, 520, 533 in very low birth weight infants, 505–507

W Wound healing, b-thymosins in, 281–283, 284 WUSCHEL (WUS), in plants, and meristem organization, 326–327, 326f, 327f

X Xerophthalmia manifestations of, 526, 536 vitamin A and, 507, 508–509

Z Zinc neuronal death induced by, NADE in, 397–398 parathymosins and, 271–272, 284 prothymosins and, 264 supplementation, and vitamin A supplements for children, 515–516 Zone of polarizing activity (ZPA), in limb development, vitamin A and, 488–489