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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1

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

This Page Intentionally Left Blank

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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Contents

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.


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

Hematopoietic Stem 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


thrombin receptor

endothelial cell

+ Mg2+

CXCR-4

ICAM-1 LFA-1

SDF-1

hyaluronic acid CD44 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,

Aldosterone

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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

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

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