Vitamin D, Volume 1-2, Second Edition

List of Contributors Numbers in parentheses indicate the page(s) on which the authors’ contributions begin. Steven A...

Author: J. Wesley Pike | Francis H. Glorieux | David Feldman

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List of Contributors

Numbers in parentheses indicate the page(s) on which the authors’ contributions begin.

Steven A. Abrams (811)

USDA/ARS Children’s Nutrition Research Center, Houston, Texas 77030, USA John S. Adams (341, 1379)

Division of Endocrinology, Diabetes, and Metabolism, University of California, 8700 Beverly Blvd, Los Angeles, Cedars-Sinai Medical Center, Los Angeles, California 90048, USA Judith E. Adams (967)

Clinical Radiology, Imaging Science and Biomedical Engineering, Stopford Building, The University, Manchester M13 9PT, United Kingdom Luciano Adorini (631, 1511, 1833)

BioXell SpA, 20132 Milano, Italy Paul H. Anderson (711)

Institute of Medical and Veterinary Science, Adelaide, South Australia, Australia Gerald J. Atkins (711)

Hanson Institute, Adelaide, South Australia, Australia Jane E. Aubin (649)

Department of Molecular and Medical Genetics, Faculty of Medicine, University of Toronto, Toronto, Ontario M5S 1A8, Canada Isabelle Bailleul-Forestier (599)

INSERM E110, Université Paris VII, IFR58, Cordeliers Biomedical Institute, 75270 Paris Cedex 06, France Julia Barsony (363)

Laboratory of Cellular Biochemistry and Biology, NIDDK/NIH, Bethesda, Maryland 20892-0850, USA Thomas K. Barthel (219)

Department of Biochemistry and Molecular Biophysics, University of Arizona, Tucson, Arizona 85721, USA

Norman H. Bell (789)

Department of Medicine, Medical University of South Carolina, Charleston, South Carolina 29425, USA Ariane Berdal (599)

INSERM E110, Université Paris VII, IFR58, Cordeliers Biomedical Institute, 75270 Paris Cedex 06, France Joel J. Bergh (751)

Department of Biological Sciences, University of Delaware, Newark, Delaware 19716, USA Jacqueline L. Berry (1293)

University of Manchester, Vitamin D Research Group, Department of Medicine, Manchester Royal Infirmary, Manchester M13 9WL, UK Daniel D. Bikle (609)

Endocrine Research Unit, Veterans’ Affairs Medical Center, University of California-San Francisco, San Francisco, California 94121-1598, USA John P. Bilezikian (1355)

Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, New York 10032, USA Ernst Binderup (1489)

Biological Research, Leo Pharma, DK-2750 Ballerup, Denmark Lise Binderup (1489)

Biological Research, Leo Pharma, DK-2750 Ballerup, Denmark Nicholas J. Bishop (803)

Academic Department of Child Health, University of Sheffield, Sheffield Children’s Hospital, Sheffield S10 2TH, United Kingdom Ilse Bogaerts (135)

Laboratory Analytische Chemie, Van Evenstraat 4; B-3000 Leuven, Belgium

xiv Ricardo L. Boland (883)

Department de Biología, Bioquímica & Farmacía, Universidad Nacional del Sur, San Juan 670, (8000) Bahía Blanca, Argentina Adele L. Boskey (477)

Mineralized Tissues Laboratory, Hospital for Special Surgery, Affiliated with Weil College of Cornell Medical School, New York, New York 10021, USA Roger Bouillon (135, 429, 1763)

Legendo, Onderwijs en Navorsing 902, U.Z. Gasthuisberg, Herestraat 49, B-3000 Leuven, Belgium Barbara D. Boyan (575)

Department of Biomedical Engineering at Georgia Tech and Emory University, Georgia Institute of Technology, Atlanta, Georgia 30332, USA Philippe Brachet (1779)

INSERM U 643, Centre Hospitalier Universitaire, 30 bd Jean Monnet, 44093 Nantes, France Alex J. Brown (1313, 1449)

Renal Division, Washington University School of Medicine, St. Louis, Missouri 63110, USA Edward M. Brown (551)

Division of Endocrinology, Diabetes and Hypertension, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA Carsten Carlberg (313)

Department of Biochemistry, University of Kuopio, PFIN-70211 Kuopio, Finland Geert Carmeliet (429)

Legendo, Onderwijs en Navorsing 902, U.Z. Gasthuisberg, Herestraat 49, B-3000 Leuven, Belgium Thomas O. Carpenter (1049)

Department of Pediatrics, Yale University, School of Medicine, New Haven, Connecticut 06520-8064, USA Marie-Claire Chapuy (1085)

INSERM Unit 403, Faculty Laennec and Department of Rheumatology and Bone Disease, Edouard Herriot Hosptial, Lyon, France Fredriech K. W. Chan (1355)

Department of Medicine, Queen Elizabeth Hospital, Hong Kong Tai C. Chen (1599)

Vitamin D, Skin, and Bone Research Laboratory, Boston University Medical Center, Boston, Massachusetts 02188, USA Sylvia Christakos (721)

Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, New Jersey 07103, USA

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CONTRIBUTORS

Margaret Clagett-Dame (1543)

Department of Biochemistry, University of Wisconsin–Madison, Madison, Wisconsin 53706, USA Thomas L. Clemens (899)

Department of Cell Biology and Physiology, Department of Internal Medicine, University of Cincinnati School of Medicine, Cincinnati, Ohio 45276, USA Je-Yong Choi (327)

Department of Biochemistry, Kyungpook National University, Daegu, Korea Fredric L. Coe (1339)

Nephrology Section, The University of Chicago Pritzker School of Medicine, Chicago, Illinois 60637, USA Kay Colston (1663)

Department OGEM, St. George’s Hospital Medical School, London, SW17 0RE, United Kingdom Juliet E. Compston (951)

Department of Medicine, Level 5, University of Cambridge, School of Clinical Medicine, Addenbrooke’s Hospital, Cambridge, England CB2 2QQ, United Kingdom Nancy E. Cooke (117)

Department of Genetics and Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6149, USA Clara Crescioli (1833)

Endocrinology Unit, Department of Clinical Physiopathology, University of Florence, Florence 50139, Italy Heide S. Cross (1709)

Department of Pathophysiology, Medical, University of Vienna, A-1090 Vienna, Währingergürtel 18-20, Austria Michael Danilenko (1635)

Department of Clinical Biochemistry, Faculty of Health Science, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel Jean-Luc Davideau (599)

INSERM E110, Université Paris VII, IFR58, Cordeliers Biomedical Institute, 75270 Paris Cedex 06, France Michael Davies (1293)

Vitamin D Research Group, University Department of Medicine, Manchester Royal Infirmary, Manchester, M13 9WL, United Kingdom Hector F. DeLuca (3, 1543)

Department of Biochemistry, University of Wisconsin–Madison, Madison, Wisconsin 53706, USA Marie B. Demay (341)

Endocrine Unit, Department of Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114, USA

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xv

CONTRIBUTORS

Puneet Dhawan (721)

Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, New Jersey 07103, USA Carlos Encinas Dominguez (219)

Department of Biochemistry and Molecular Biophysics, University of Arizona, Tucson, Arizona 85721, USA Diane R. Dowd (291)

Department of Pharmacology–W334, Case Western Reserve University, Cleveland, Ohio 44106, USA Marc K. Drezner (1159)

Department of Medicine, Endocrinology, Diabetes, and Metabolism Section, University of Wisconsin–Madison, Madison, Wisconsin 53792, USA Thomas W. Dunlop (313)

Department of Biochemistry, University of Kuopio, PFIN-70211 Kuopio, Finland Adriana S. Dusso (1313)

Renal Division, Washington University School of Medicine, St. Louis, Missouri 63110, USA Richard Eastell (1101)

University of Sheffield Clinical Sciences Centre, Northern General Hospital, Sheffield South Yorkshire S5 7AU, United Kingdom Michael J. Econs (1189)

Indiana University School of Medicine, Department of Medicine and Medical and Molecular Genetics, Indianapolis, Indiana 46202, USA John Eisman (193)

Bone and Mineral Program, Garvan Institute of Medical Research, St. Vincent’s Hospital, Sydney, New South Wales 2010, Australia Sol Epstein (1253)

Division of Endocrinology, Diabetes, and Bone Diseases, Department of Medicine, Mount Sinai School of Medicine, New York, New York 10029, USA Erik Fink Eriksen (1805)

Osteoporosis Team of Lilly Research Laboratories, Lilly Corp Center, Indianapolis, Indiana 46285, USA Luis M. Esteban (193)

Bone and Mineral Program, Garvan Institute of Medical Research, St. Vincent’s Hospital, Sydney, New South Wales 2010, Australia Dan Faibish (477)

Mineralized Tissues Laboratory, Hospital for Special Surgery, Affiliated with Weil College of Cornell Medical School, New York, New York 10021, USA

Yue Fang (1121)

Department of Internal Medicine, Genetic Laboratory, Erasmus Medical Centre, NL-3015 GE Rotterdam, The Netherlands Mary C. Farach-Carson (751)

Department of Biological Sciences, University of Delaware, Newark, Delaware 19716, USA Murray J. Favus (1339)

Section of Endocrinology, University of Chicago, Chicago, Illinois 60637, USA David Feldman (1207, 1679)

Stanford University School of Medicine, Division of Endocrinology, Stanford, California 94305-5103, USA David Findlay (711)

Department of Orthopedic Surgery and Trauma, University of Adelaide, Adelaide 5000, South Australia, Australia Christian Frank (313)

Department of Biochemistry, University of Kuopio, PFIN-70211 Kuopio, Finland Leonard P. Freedman (263)

Department of Molecular Endocrinology and Bone Biology, Merck Research Laboratories, West Point, Pennsylvania 19486-0004, USA Ryuji Fujiki (305)

University of Tokyo, Institute of Molecular and Cellular Biosciences, 1-1-1 Yayoi-cho, Bunkyo-ku, Tokyo, 113-0032, Japan Masafumi Fukagawa (1821)

Division of Nephrology and Dialysis Center, Kobe University School of Medicine, 7-5-2 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan Robert F. Gagel (687)

Section of Endocrinology, University of Texas M.D. Anderson Cancer Center, Houston, Texas, USA Emmanuel Garcion (1779)

INSERM U 646, 10 rue André Boquel, 49100 Angers, France Edith M. Gardiner (193)

Bone and Mineral Program, Garvan Institute of Medical Research, St. Vincent’s Hospital, Sydney, New South Wales 2010, Australia Marielle Gascon-Barré (47)

Département de Pharmacologie, Faculté de Médecine, Université de Montréal, and Centre de recherche de l’Université de Montréal, Montréal, Québec H2X 1P1, Canada Edward Giovannucci (1617)

Harvard School of Public Health, Department of Nutrition, Boston, Massachusetts 02115, USA Henning Glerup (1805)

Aarhus Kommunehospital, Dept V, Noerrebrogade 44, DK-8000 Aarhus C, Denmark

xvi Francis H. Glorieux (1197)

Genetics Unit, Shriners Hospital for Children, Departments of Surgery, Pediatrics, and Human Genetics, McGill University, Montréal, Québec H3G 1A6, Canada Wagn O. Godtfredsen (1489)

Medicinal Chemistry, Leo Pharma, DK-2750 Ballerup, Denmark David Goltzman (737)

Department of Medicine, McGill University and McGill University Health Center, Montréal, Québec H3A 1A1, Canada Soraya Gutierrez (327)

Departamento de Biología Molecular, Universidad de Concepción, Concepción, Chile Conny Gysemans (1763)

Legendo, Onderwijs en Navorsing 902, U.Z. Gasthuisberg, Herestraat 49, B-3000 Leuven, Belgium Bernard P. Halloran (823)

Division of Endocrinology, Veterans Affairs Medical Center, San Francisco, California 94121, USA Carol A. Haussler (219)

Department of Biochemistry and Molecular Biophysics, University of Arizona, Tucson, Arizona 85721, USA Mark R. Haussler (219)

Department of Biochemistry and Molecular Biophysics, University of Arizona, Tucson, Arizona 85721, USA Robert P. Heaney (773)

Creighton University, Omaha, Nebraska 68131, USA Johan Heersche (649)

Faculty of Dentistry, University of Toronto, Toronto M5S 1A8, Ontario, Canada Helen L. Henry (69)

Department of Biochemistry, University of California–Riverside, Riverside, California 92521, USA Pamela A. Hershberger (1741)

Department of Pharmacology, University of Pittsburgh, Pittsburgh, Pennsylvania 15213, USA Martin Hewison (1379)

Division of Medical Sciences, The University of Birmingham, Queen Elizabeth Medical Centre, Birmingham, B15 2TH, United Kingdom Richard A. Heyman (1557)

X-Ceptor Therapeutics, San Diego, California 92121, USA Kanji Higashio (665)

Research Center for Genomic Medicine, Saitama Medical School, Saitama, 350-1241, Japan

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CONTRIBUTORS

Michael F. Holick (37, 1511, 1791)

Vitamin D, Skin, and Bone Research Laboratory; Department of Medicine; Endocrinology, Nutrition and Diabetes Section; Boston Medical Center and Boston University School of Medicine, Boston, Massachusetts 02118, USA Bruce W. Hollis (931)

Departments of Pediatrics, Biochemistry, and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina 29425, USA Ronald L. Horst (15)

U.S. Department of Agriculture, Agricultural Research Service, National Animal Disease Center, Ames, Iowa 50010-0070, USA Jui-Cheng Hsieh (219)

Department of Biochemistry and Molecular Biophysics, University of Arizona, Tucson, Arizona 85721, USA Karl L. Insogna (1049)

Department of Medicine, Yale University School of Medicine, New Haven, CT 06520-8064, USA Elizabeth T. Jacobs (219)

College of Medicine, University of Arizona, Tucson, Arizona 85721, USA Amjad Javed (327)

Department of Cell Biology, University of Massachusetts Medical School, Worcester, Massachusetts 01655, USA Glenville Jones (1423)

Department of Biochemistry, Queen’s University, Kingston, Ontario K7L 3N6, Canada Candace S. Johnson (1741)

Department of Pharmacology & Therapeutics, Roswell Park Cancer Institute, Buffalo, New York 14263, USA Peter W. Jurutka (219)

Department of Biochemistry and Molecular Biophysics, University of Arizona, Tucson, Arizona 85721, USA Mehmet Kahraman (1405)

Department of Chemistry, Johns Hopkins University, Baltimore, Maryland 21218-2685, USA S. Kaleem Zaidi (327)

Department of Cell Biology, University of Massachusetts Medical School, Worcester, Massachusetts 01655, USA Heidi J. Kalkwarf (839)

Division of General and Community Pediatrics, Children’s Hospital Medical Center, Cincinnati, Ohio 45229, USA

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xvii

CONTRIBUTORS

Shigeaki Kato (305)

University of Tokyo, Institute of Molecular and Cellular Biosciences, 1-1-1 Yayoi-cho, Bunkyo-ku, Tokyo, 113-0032, Japan Anne-Marie Kissmeyer (1489)

Biological Research, Leo Pharma, DK-2750 Ballerup, Denmark Hirochika Kitagawa (305)

University of Tokyo, Institute of Molecular and Cellular Biosciences, 1-1-1 Yayoi-cho, Bunkyo-ku, Tokyo, 113-0032, Japan Lilia M. C. Koberle (1355)

Health Sciences Department, Federal University, Sao Carlos, Brazil H. Phillip Koeffler (1727)

Hematology/Oncology Division, Cedars-Sinai Medical Center, Los Angeles, California 90048, USA Ruth Koren (761)

Felsenstein Medical Research Center, Beilinson Campus, Rabin Medical Center, Petah Tikva 49100, Israel Barbara E. Kream (703)

Department of Medicine, University of Connecticut Health Center, Farmington, Connecticut 06030-1850, USA Richard Kremer (737)

Department of Medicine, McGill University and McGill University Health Center, Montréal, Québec H3A 1A1, Canada Aruna V. Krishnan (1679)

Stanford University School of Medicine, Division of Endocrinology, Stanford, California 94305-5103, USA Noboru Kubodera (1525)

Department of Product Planning, Chugai Pharmaceutical Co. Ltd., 2-1-9 Kyobashi, Chuo-ku, Tokyo, 104-8301, Japan Rajiv Kumar (515)

Departments of Medicine, Biochemistry and Molecular Biology and Mayo Proteomics Research Center, Divisions of Nephrology, and Endocrinology, Diabetes, Metabolism, and Nutrition, Mayo Clinic and Foundation, Rochester, Minnesota 55905-0002, USA Kiyoshi Kurokawa (1821)

Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo, Japan Christopher J. Laing (117)

Department of Genetics and Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6144, USA Jacques Lemire (1753)

Pediatric Nephrology, University of California-San Diego, La Jolla, California 92093-0831, USA

Frédéric Lézot (599)

INSERM E110, Université Paris VII, IFR58, Cordeliers Biomedical Institute, 75270 Paris Cedex 06, France Yan Chun Li (721, 871, 1511)

Department of Medicine/GI Section, University of Chicago, Chicago, Illinois 60637, USA Jane B. Lian (327)

Department of Cell Biology, University of Massachusetts Medical School, Worcester, Massachusetts 01655, USA Alexander C. Lichtler (703)

Department of Genetics and Developmental Biology, The University of Connecticut Health Center, Farmington, Connecticut 06030, USA Paul Lips (1019)

Department of Endocrinology, Vrijie University Medical Center, Amsterdam, 1007 MB, The Netherlands Yan Liu (721)

Department of Biochemistry and Molecular Biology, New Jersey Medical School, Newark, New Jersey 07103, USA Paul MacDonald (291)

Department of Pharmacology–W334, Case Western Reserve University, Cleveland, Ohio 44106, USA Hubert Maehr (1511)

BioXell, Inc., Hoffmann-La Roche, Inc., Nutley, New Jersey 07110-1199, USA Mario Maggi (1833)

Andrology Unit, Department Clinical Physiopathology, University of Florence, 50139 Florence, Italy Peter J. Malloy (1207)

Stanford University School of Medicine, Division of Endocrinology, Stanford, California 94305-5103, USA David J. Mangelsdorf (863)

Department of Pharmacology, Howard Hughes Medical Institute, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390-9050, USA Chantal Mathieu (1763)

Legendo, Onderwijs en Navorsing 902, U.Z. Gasthuisberg, Herestraat 49, B-3000 Leuven, Belgium Brian May (85)

School of Molecular and Biomedical Science, University of Adelaide, South Australia 5005, Australia Andrew P. Mee (1293)

Vitamin D Research Group, University Department of Medicine, Manchester Royal Infirmary, Manchester, M13 9WL, United Kingdom

xviii Pierre J. Meunier (1085)

INSERM Unit 403, Faculty Laennec and Department of Rheumatology and Bone Disease, Edouard Herriot Hospital, Lyon, France Toshimi Michigami (851)

Department of Environmental Medicine, Osaka Medical Center and Institute for Maternal and Child Health, Osaka, Japan Martin Montecino (327)

Departamento de Biología Molecular, Universidad de Concepción, Concepción, Chile Dino Moras (279)

Département de Biologie et de Génomique Structurales, CNRS/INSERM/Université Louis Pasteur 1, BP 10142, 67404 Illkirch Cedex, France Roberta Morosetti (1727)

Pediatric Oncology Division, Catholic University of Rome, Rome, Italy Howard A. Morris (711)

Hanson Institute, Adelaide, South Australia, Australia Daniel L. Motola (863)

Department of Pharmacology, Howard Hughes Medical Institute, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390-9050, USA Josephia Muindi (1741)

Department of Medicine, Roswell Park Cancer Institute, Buffalo, New York 14263, USA Shigeo Nakajima (851)

Department of Developmental Medicine (Pediatrics), D-5, Osaka University Graduate School of Medicine, Osaka 565-0871, Japan Tally Naveh-Many (537)

Minerva Center for Calcium and Bone Metabolism, Hebrew University Hadassah Medical Center, Ein Karem, Jerusalem, 91120, Israel Philippe Naveilhan (1779)

INSERM U 643, Centre Hospitalier Universitaire, 30 bd Jean Monnet, 44093 Nantes, France Isabelle Neveu (1779)

INSERM U 643, Centre Hospitalier Universitaire, 30 bd Jean Monnet, 44093 Nantes, France Anthony W. Norman (381)

Department of Biochemistry, University of California, Riverside, Riverside, California 92521-0129, USA Anders Nykjaer (153)

Institute of Medical Biochemistry, University of Aarhus, Ole Worms Allee, DK-8000 Aarhus C, Denmark James O’Kelly (1727)

Hematology/Oncology Division, Cedars-Sinai Medical Center, Los Angeles, California 90048, USA

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CONTRIBUTORS

John L. Omdahl (85)

Office of Research, University of New Mexico School of Medicine, Albuquerque, New Mexico 87131-5166, USA Peter Ordentlich (1557)

X-Ceptor Therapeutics, San Diego, California 92121, USA Keiichi Ozono (851)

Department of Developmental Medicine (Pediatrics), D-5, Osaka University Graduate School of Medicine, Osaka 565-0871, Japan A. Michael Parfitt (497, 1029)

Division of Endocrinology and Center for Osteoporosis and Metabolic Bone Disease, University of Arkansas for Medical Science, Little Rock, Arkansas 72205, USA Donna M. Peehl (1679)

Stanford University School of Medicine, Division of Endocrinology, Stanford, California 94305-5103, USA Sara Peleg (1471)

Department of Endocrine Neoplasia and Hormonal Diseases, Unit 435, University of Texas, M.D. Anderson Cancer Center, Houston, Texas 77030-4009, USA Xiaorong Peng (721)

Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, New Jersey 07103, USA John M. Pettifor (1065)

Department of Pediatrics, Chris Hani Baragwanath Hospital, Mineral Metabolism Research Unit, P.O. Bertsham, Johannesburg, Gauteng 2013, South Africa J. Wesley Pike (167, 1207, 1403, 1543)

Department of Biochemistry, University of Wisconsin–Madison, Madison, Wisconsin 53706, USA Elizabeth A. Platz (1617)

Department of Epidemiology, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland 21205, USA Lori A. Plum (1543)

Department of Biochemistry, University of Wisconsin–Madison, Madison, Wisconsin 53706, USA Shirwin Pockwinse (327)

Department of Cell Biology, University of Massachusetts Medical School, Worcester, Massachusetts 01655, USA Huibert A.P. Pols (1121, 1571)

Department of Internal Medicine, Erasmus Medical Centre, NL-3015 GD Rotterdam, The Netherlands

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CONTRIBUTORS

Anthony A. Portale (453, 823)

Department of Pediatrics, University of California – San Francisco, San Francisco, California 94121, USA Gary H. Posner (1405)

Department of Chemistry, Johns Hopkins University, Baltimore, Maryland 21218-2685, USA Mehrdad Rahmaniyan (789)

Department of Medicine, Medical University of South Carolina, Charleston, South Carolina 29425, USA Amiram Ravid (761)

Felsenstein Medical Research Center, Beilinson Campus, Rabin Medical Center, Petah Tikva 49100, Israel G. Satyanarayana Reddy (15, 1511)

Brown University, Department of Chemistry, Providence, Rhode Island, USA Jörg Reichrath (1791)

The Saarland University Hospital, Department of Dermatology, Kirrberger Str., 66421 Homburg/Saar, Germany Timothy A. Reinhardt (15)

U.S. Department of Agriculture, Agricultural Research Service, National Animal Disease Center, Ames, Iowa 50010-0070, USA Alfred A. Reszka (263)

Department of Molecular Endocrinology and Bone Biology, Merck Research Laboratories, West Point, Pennsylvania 19486-0004, USA B. Lawrence Riggs (1101)

Division of Endocrinology, Mayo Clinic and Mayo Foundation, Rochester, Minnesota 55905, USA Natacha Rochel (279)

Département de Biologie et de Génomique Structurales, CNRS/INSERM/Université Louis Pasteur 1, BP 10142, 67404 Illkirch Cedex, France Mishaela R. Rubin (1355)

Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, New York 10027, USA Andrew F. Russo (687)

Department of Physiology and Biophysics, University of Iowa, Iowa City, Iowa 52242, USA Philip Sambrook (1239)

Institute of Bone & Joint Research, University of Sydney, Royal North Shore Hospital, Sydney 2065, Australia Adina E. Schneider (1253)

Division of Endocrinology, Diabetes, and Bone Diseases, Department of Medicine, Mount Sinai School of Medicine, New York, New York 10029, USA

Gary G. Schwartz (1599)

Comprehensive Cancer Center, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157, USA Zvi Schwartz (575)

Department of Periodontics, Hebrew University Hadassah Faculty of Dental Medicine, Jerusalem, Israel Jiali Shen (327)

Department of Cell Biology, University of Massachusetts Medical School, Worcester, Massachusetts 01655, USA Nirupama K. Shevde (167, 1543)

Department of Biochemistry, University of Wisconsin–Madison, Madison, Wisconsin 53706, USA Rafal R. Sicinski (1543)

Department of Chemistry, University of Warsaw, ul. L. Pasteura 2, Warsaw 02-093, Poland Justin Silver (537)

Minerva Center for Calcium and Bone Metabolism, Hebrew University Hadassah Medical Center, Ein Karem, Jerusalem, 91120, Israel Eduardo A. Slatopolsky (1313, 1449)

Renal Division, Washington University School of Medicine, St. Louis, Missouri 63110, USA Bonny L. Specker (839)

Martin Program in Human Nutrition, South Dakota State University, Brookings, South Dakota 57007, USA René St-Arnaud (105, 1197)

Genetics Unit, Shriners Hospital for Children, Montréal, Quebec H3G 1A6, Canada Gary S. Stein (327)

Department of Cell Biology, University of Massachusetts Medical School, Worcester, Massachusetts 01655, USA Janet L. Stein (327)

Department of Cell Biology, University of Massachusetts Medical School, Worcester, Massachusetts 01655, USA Paula H. Stern (565)

Department of Molecular Pharmacology, Northwestern University Feinberg School of Medicine, Chicago, Illinois 60611, USA George P. Studzinski (1635)

UMD-New Jersey Medical School, Newark, New Jersey 07103, USA Tatsuo Suda (665)

Research Center for Genomic Medicine, Saitama Medical School, Hidaka-shi, Saitama 350-1241, Japan

xx

LIST

Amelia L.M. Sutton (291)

Department of Pharmacology–W334, Case Western Reserve University, Cleveland, Ohio 44106, USA Peter Tebben (515)

Departments of Medicine, Biochemistry and Molecular Biology and Mayo Proteomics Research Center, Divisions of Nephrology, and Endocrinology, Diabetes, Metabolism, and Nutrition, Mayo Clinic and Foundation, Rochester, Minnesota 55905-0002, USA Harriet S. Tenenhouse (453)

Departments of Pediatrics and Human Genetics, McGill University and Montréal Children’s Hospital Research Institute, Montréal, Québec, H3Z 2Z3, Canada Michelle L. Thatcher (219)

Department of Biochemistry and Molecular Biophysics, University of Arizona, Tucson, Arizona 85721, USA Susan Thys-Jacobs (1355)

Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, New York 10027, USA Dwight A. Towler (899)

Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110, USA Donald L. Trump (1741)

Department of Medicine, Roswell Park Cancer Institute, Buffalo, New York 14263, USA André G. Uitterlinden (1121)

Genetic Laboratory, Department of Internal Medicine, Erasmus Medical Centre, NL-3015 GD Rotterdam, The Netherlands Milan R. Uskokovi´c (1511)

BioXell, Inc., Nutley, New Jersey 07110-1199, USA Sami Väisänen (313)

Department of Biochemistry, University of Kuopio, PFIN-70211 Kuopio, Finland Hugo Van Baelen (135)

Legendo, Onderwijs en Navorsing 902, U.Z. Gasthuisberg, Herestraat 49, B-3000 Leuven, Belgium Sophie Van Cromphaut (429)

Legendo, Onderwijs en Navorsing 902, U.Z. Gasthuisberg, Herestraat 49, B-3000 Leuven, Belgium Johannes P.T.M. van Leeuwen (1571)

Department of Internal Medicine, Erasmus MC, 3000 DR Rotterdam, The Netherlands

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CONTRIBUTORS

Joyce B.J. van Meurs (1121)

Genetic Laboratory, Department of Internal Medicine, Erasmus Medical Centre, NL-3015 GD Rotterdam, The Netherlands Andre J. van Wijnen (327)

Department of Cell Biology, University of Massachusetts Medical School, Worcester, Massachusetts 01655, USA Christel Verboven (135)

Laboratory Analytische Chemie, Van Evenstraat 4; B-3000 Leuven, Belgium Reinhold Vieth (995)

Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Ontario, M5G 1X5, Canada Robert H. Wasserman (411)

Department of Biomedical Sciences, VRT-08-20, Cornell University, Ithaca, New York 14853, USA JoEllen Welsh (1663)

Department Biological Science, University Notre Dame, Notre Dame, Indiana 46556, USA G. Kerr Whitfield (219)

Department of Biochemistry and Molecular Biophysics, University of Arizona, Tucson, Arizona 85721, USA Michael P. Whyte (913)

Center for Metabolic Bone Disease and Molecular Research, Shriners Hospital for Children, St. Louis, Missouri 63131, USA and Division of Bone and Mineral Diseases, Washington University School of Medicine at Barnes-Jewish Hospital, St. Louis, Missouri 63110, USA. Thomas Willnow (153)

Division of Molecular Cardiovascular Research, Max-Delbrueck-Center for Molecular Medicine, Robert-Roessle-Strasse 10, D-13125 Berlin, Germany Didier Wion (1779)

INSERM U318, Centre Hospitalier Michallon, 38043 Grenoble cedex 09, France Hisataka Yasuda (665)

Center for Experimental Medicine, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan Daniel Young (327)

Department of Cell Biology, University of Massachusetts Medical School, Worcester, Massachusetts 01655, USA Chi Zhang (291)

Department of Pharmacology–W334, Case Western Reserve University, Cleveland, Ohio 44106, USA

Preface to the 2nd Edition

Those interested in the vitamin D field will not be surprised that this second edition is considerably larger than the first edition. A great deal of progress has been made since the first edition was published in 1997. However, our goal in planning this updated version remains the same. We have endeavored to provide investigators, clinicians, and students with a comprehensive, definitive, and up-to-date compendium of the diverse scientific and clinical aspects of vitamin D, each area covered by experts in the field. Our hope for the second edition is that this book will continue to serve as both a resource for current researchers, as well as a guide to assist those in related disciplines to enter the realm of vitamin D research. We hope that this book will illuminate the vitamin D field and help investigators identify areas where new research is needed as well as educate them about what is currently known. We believe that the first edition succeeded in stimulating interactions between researchers and clinicians from different disciplines and that it facilitated collaborations. As we move from basic science and physiology to the use of vitamin D and its analogs as pharmacological agents to treat various diseases, the need for cross-collaborations between researchers and clinicians from different disciplines will increase. We hope that this new volume will continue to be a valuable resource that plays a role in this advancement and stimulates and facilitates these interactions. Enormous progress in the study of vitamin D has been made in the approximately eight years since the first edition was written and we hope that this book has contributed in some way to this progress. The first edition proved to be highly valuable to its readers and the chapters have been cited frequently as authoritative reviews of the field. However, it became clear to us that the time was ripe to organize a second edition. Building on the original, we hope this second edition

will incorporate all of the progress made in the field since the first edition was published so that our objective of an up-to-date compendium containing everything you wanted to know about vitamin D will continue. The second edition is essentially a new and reinvigorated book. We have changed the symbol on the cover to reflect its updated content and the field's continued evolution into the molecular world. This new edition now includes 104 chapters. In order to cover the growth of new information on vitamin D, we have had to publish this new edition in two volumes. In addition, the book has undergone some major remodeling. There are 33 completely new chapters and 18 other chapters have had major changes in authorship and are totally rewritten. While approximately half of the chapters have some of the same authors, all have had major updates and many have new co-authors with new perspectives. We have endeavored to attract the leading investigators in each field to author the chapter covering their area. We are especially pleased with the roster of authors who have written for the second edition. They really are the leaders in their respective fields. We wish to express our thanks to Tari Paschall, Judy Meyer, and Sarah Hajduk as well as the rest of the Elsevier/Academic Press staff for their expertise and indispensable contribution to bringing this revised edition to fruition. Most of all, we thank the authors for their contributions. We hope that our readers will find this updated volume useful and informative and that it will contribute to the burgeoning growth of the vitamin D field. DAVID FELDMAN J. WESLEY PIKE FRANCIS H. GLORIEUX

Preface to the 1st Edition

Our reasons for deciding to publish an entire book devoted to vitamin D can be found in the rapid and extensive advances currently being made in this important field of research. Enormous progress in investigating many aspects of vitamin D, from basic science to clinical medicine, has been made in recent years. The ever-widening scope of vitamin D research has created new areas of inquiry so that even workers immersed in the field are not fully aware of the entire spectrum of current investigation. Our goal in planning this book was to bring the diverse scientific and clinical fields together in one definitive and up-to-date volume. It is our hope that this compendium on vitamin D will serve as both a resource for current researchers and a guide to stimulate and assist those in related disciplines to enter this field of research. In addition, we hope to provide clinicians and students with a comprehensive source of information for the varied and extensive material related to vitamin D. The explosion of information in the vitamin D sphere has led to new insights into many different areas, and in our treatment of each subject in this book we have tried to emphasize the recent advances as well as the established concepts. The classic view of vitamin D action, as a hormone limited to calcium metabolism and bone homeostasis, has undergone extensive revision and amplification in the past few years. We now know that the vitamin D receptor (VDR) is present in most tissues of the body and that vitamin D actions, in addition to the classic ones, include important effects on an extensive array of other target organs. To cover this large number of subjects, we have organized the book in the following manner: Section I, the enzymes involved in vitamin D metabolism and the activities of the various metabolites; Section II, the mechanism of action of vitamin D, including rapid, nongenomic actions and the role of the VDR in health and disease; Section III, the

effects of vitamin D and its metabolites on the various elements that constitute bone and the expanded understanding of vitamin D actions in multiple target organs, both classic and nonclassic; Section IV, the role of vitamin D in the physiology and regulation of calcium and phosphate metabolism and the multiplicity of hormonal, environmental, and other factors influencing vitamin D metabolism and action; and Sections V and VI, the role of vitamin D in the etiology and treatment of rickets, osteomalacia, and osteoporosis and the pathophysiological basis, diagnosis, and management of numerous clinical disorders involving vitamin D. The recent recognition of an expanded scope of vitamin D action and the new investigational approaches it has generated were part of the impetus for developing this volume on vitamin D. It has become clear that in addition to the classic vitamin D actions, a new spectrum of vitamin D activities that include important effects on cellular proliferation, differentiation, and the immune system has been identified. This new information has greatly expanded our understanding of the breadth of vitamin D action and has opened for investigation a large number of new avenues of research that are covered in Sections VII and VIII of this volume. Furthermore, these recently recognized nonclassic actions have led to a consideration of the potential application of vitamin D therapy to a range of diseases not previously envisioned. This therapeutic potential has spawned the search for vitamin D analogs that might have a more favorable therapeutic profile, one that is less active in causing hypercalcemia and hypercalciuria while more active in a desired application such as inducing antiproliferation, prodifferentiation, or immunosuppresion. Since 1α,25-dihydroxyvitamin D [1,25(OH)2D] and its analogs are all presumed to act via a single VDR, a few years ago most of us probably would have thought that it was impossible to achieve a

xxiv separation of these activities. Yet today, many analogs that exhibit different profiles of activity relative to 1,25(OH)2D have already been produced and extensively studied. The development of analogs with an improved therapeutic index has opened another large and complex area of vitamin D research. This work currently encompasses three domains: (1) the design and synthesis of vitamin D analogs exhibiting a separation of actions with less hypercalcemic and more antiproliferative or immunosuppressive activity, (2) the interesting biological question of the mechanism(s) by which these analogs achieve their differential activity, and (3) the investigation of the potential therapeutic applications of these analogs to treat various disease states. These new therapeutic applications, from psoriasis to cancer, from immunosuppression to neurodegenerative diseases, have drawn into the field an expanded population of scientists and physicians interested in vitamin D. Our goal in editing this book was to create a comprehensive resource on vitamin D that would be of use to a mix of researchers in different disciplines. To achieve this goal, we sought authors who had contributed greatly to their respective fields of vitamin D research. The book has a large number of chapters to accommodate

PREFACE

TO THE

1ST EDITION

many contributors and provide expertise in multiple areas. Introductory chapters in each section of the book are designed to furnish an overview of that area of vitamin D research, with other chapters devoted to a narrowly focused subject. Adjacent and closely related subjects are often covered in separate chapters by other authors. While this intensive style may occasionally create some redundancy, it has the advantage of allowing the reader to view the diverse perspectives of the different authors working in overlapping fields. In this regard, we have endeavored to provide many crossreferences to guide the reader to related information in different chapters. We express our thanks to Jasna Markovac (Editor-inChief), for encouraging us to develop this book and guiding us through the process; to Tari Paschall (Acquisitions Editor) and the Academic Press staff for their diligence, expertise, and patience in helping us complete this work. Most of all, we thank the authors for their contributions that have made this book possible. DAVID FELDMAN J. WESLEY PIKE FRANCIS H. GLORIEUX

Abbreviations

AA AC ACE ACF ACTH ADH ADHR ADP AHO AI AIDS Aj.AR ALP ANG II ANP APC APD AR ARC ATP ATRA AUC Bmax BARE bFGF BFU BGP BLM BMC BMD BMI BMP BMU bp BPH

arachadonic acid adenyl cyclase angiotensin converting enzyme activation frequency adrenocorticotropin antidiuretic hormone (vasopressin) autosomal dominant hypophosphatemic rickets adenosine diphosphate Albright’s hereditary osteodystrophy adequate intake acquired immunodeficiency syndrome adjusted apposition rate alkaline phosphatase angiotensin II atrial natriuretic peptide antigen presenting cell aminohydroxypropylidene bisphosphonate androgen receptor activator recruited cofactor adenosine triphosphate all-trans-retinoic acid area under the curve maximum number of binding sites bile acid response element basic fibroblast growth factor burst-forming unit bone Gla protein (osteocalcin) basal lateral membrane bone mineral content bone mineral density body mass index bone morphogenetic protein basic multicellular unit base pairs benign prostatic hyperplasia

BSA BUA [Ca2+]i

bovine serum albumin bone ultrasound attentuation internal calcium ion molar concentration CaBP calcium binding protein CAD coronary artery disease CaM calmodulin cAMP cyclic AMP CaSR or CaR calcium sensing receptor CAT chloramphenicol acetyltransferase CBG corticosteroid-binding globulin CBP competitive protein binding assay CC chief complaint CDCA chenodeoxycholic acid CDK or Cdk cyclin-dependent kinase cDNA complementary DNA CDP collagenase-digestible protein Cdx-2 caudal-related homeodomain protein CFU colony-forming unit cGMP cyclic GMP CGRP calcitonin gene-related peptide CHF congestive heart failure CK-II casein kinase-II CLIA competitive chemiluminescence immunoassay cM centimorgans Cm. Ln. cement line CNS central nervous system CPBA competitive protein binding assays cpm counts per minute CRE cAMP response element CREB cAMP response element binding protein CRF chronic renal failure CsA cyclosporin A CSF colony-stimulating factor

xxvi CT CTR CTX CVC CYP CYP24 DAG DBD DBP DBP DC DCA DCT DEXA or DXA 7-DHC DHEA DHT DIC DMSO DR DRIP DSP E1 E2 EAE EBT EBV EC EC50 or ED50 ECaC ECF EDTA EGF ELISA EMSA EP1 ER ERE ERK Et FACS FAD FCS FDA FFA FIT

ABBREVIATIONS

calcitonin or computerized tomography calcitonin receptor cerebrotendinous xanthomatosis calcifying vascular cells cytochrome P450 cytochrome P450, 24-hydroxylase diacylglycerol DNA binding domain diastolic blood pressure vitamin D binding protein dendritic cell deoxycholic acid distal convoluted tubule dual energy X-ray absorptiometry 7-dehydrocholesterol dehydroepiandrosterone dihydrotachysterol or dihydrotestosterone disseminated intravascular coagulation dimethyl sulfoxide direct repeat vitamin D receptor interacting protein dental sialoprotein estrone estradiol experimental autoimmune encephalitis electron-beam computed technology Epstein-Barr virus endothelial cells effective concentration (dose) to cause 50% effect epithelium calcium channel extracellular fluid ethylenediaminetetraacetic acid epidermal growth factor enzyme-linked immunosorbent assay electrophoretic mobility shift assay PG receptor-1 estrogen receptor or endoplasmic reticulum estrogen response element extracellular signal-regulated kinase endothelin fluorescence-activated cell sorting or sorter flavin adenine dinucleotide fetal calf serum U.S. Food and Drug Administration free fatty acid Fracture Intervention Trial

FMTC FP FRAP FS FSK FXR g g G0, G1, G2 GAG GC-MS G-CSF GDNF GFP GFR GH GHRH GIO GM-CSF GnRH GR GRE GRTH HAT HDAC HEK HHRH HIV HNF HPI HPLC HPV hr HRE HSA Hsp HSV HVDRR HVO IBMX IC50 ICA ICMA IDBP IDDM IDM IFN

familial medullary thyroid carcinoma formation period fluorescence recovery after photobleaching Fanconi syndrome forskolin farnesol X receptor gram acceleration due to gravity gap phases of the cell cycle glycosaminoglycan gas chromatography-mass spectrometry granulocyte colony-stimulating factor glial cell-derived neurotrophic factor green fluorescent protein glomerular filtration rate growth hormone growth hormone-releasing hormone glucocorticoid induced osteoporosis granulocyte-macrophage colonystimulating factor gonadotropin-releasing hormone glucocorticoid receptor glucocorticoid response element generalized resistance to thyroid hormone histone acetyltransferase histone deacetylase human embryonic kidney hereditary hypophosphatemic rickets with hypercalciuria human immunodeficiency virus hepatocyte nuclear factor history of present illness high-performance liquid chromatography human papilloma virus hour hormone response element human serum albumin heat-shock protein herpes simplex virus hereditary vitamin D-resistant rickets hypovitaminosis D osteopathy isobutylmethylxanthine concentration to inhibit 50% effect intestinal calcium absorption immunochemiluminometric assay intracellular vitamin D–binding protein insulin-dependent diabetes mellitus infants of diabetic mothers interferon

xxvii

ABBREVIATIONS

Ig IGFBP IGF-I, -II IGF-IR IL i.m. IMCal i.p. IP3 IRMA IU IUPAC i.v. JG JNK Kd Km kb kbp kDa KO LBD LCA LDL Li. Ce. LIF LNH LOD LPS LT LXR M M MAPK Mab MAR MAR MARRS MCR M-CSF MEN2 MGP MHC min MLR Mlt MR MRI mRNA MTC NADH

immunoglobulin IGF binding protein insulin-like growth factor type I, II IGF-I receptor interleukin (e.g., IL-1, IL-1β, etc.) intramuscular intestinal membrane calcium binding complex intraperitoneal inositol trisphosphate immunoradiometric assay international units International Union of Pure and Applied Chemists intravenous juxtaglomerular c-Jun NH2-terminal kinase dissociation constant Michaelis constant kilobases kilobase pairs kilodaltons knock out ligand binding domain lithocholic acid low-density lipoprotein lining cell leukemia inhibitory factor late neonatal hypocalcemia logarithm of the odds lipopolysaccharide leukotriene liver X receptor mitosis phase of cell cycle molar mitogen-activated protein kinase monoclonal antibody matrix attachment region mineral apposition rate membrane-associated rapid response steroid metabolic clearance rate macrophage colony-stimulating factor multiple endocrine neoplasia type 2 matrix Gla protein major histocompatibility complex minute mixed lymphocyte reaction mineralization lag time mineralcorticoid receptor magnetic resonance imaging messenger ribonucleic acid medullary thyroid carcinoma nicotinamide adenine dinucleotide

NADPH

nicotinamide adenine dinucleotide phosphate NAF nuclear accessory factor NBT nitroblue tetrazolium NcAMP nephrogenous cAMP NCP noncollagen protein NGF nerve growth factor NHANES III National Health and Nutrition Examination Survey III NHL Non-Hodgkin’s lymphoma NIDDM non-insulin-dependent diabetes mellitus NIH National Institutes of Health NK cell natural killer cell NLS nuclear localization signal NMR nuclear magnetic resonance NPT sodium/phosphate cotransporter NR nuclear receptor Ob osteoblast Oc osteocalcin or osteoclast OCIF osteoclastogenesis inhibitory factor (same as OPG) OCT 22-oxacalcitriol ODF osteoclast differentiation factor (same as RANKL) 1α-OHD3 1α-hydroxyvitamin D3 25OHD3 25-hydroxyvitamin D3 1,25(OH)2D3 1α,25-dihydroxyvitamin D3 24,25(OH)2D3 24,25-dihydroxyvitamin D3 OHO oncogenic hypophosphatemic osteomalacia Omt osteoid maturation time OPG osteoprotegerin OPN osteopontin OSM oncostatin M OVX ovariectomy Pi inorganic phosphate PA2 phospholipase A2 PAD peripheral arterial vascular disease PAM pulmonary alveolar macrophage PBL peripheral blood lymphocyte PBS phosphate-buffered saline PC phophatidyl choline PCNA proliferating cell nuclear antigen PCR polymerase chain reaction PCT proximal convoluted tubule PDDR pseudovitamin D deficiency rickets PDGF platelet-derived growth factor PEIT percutaneous ethanol injection therapy PHEX phosphate regulating gene with homologies to endopeptidases on the X chromosome PG prostaglandin

xxviii PHA PHP PIC PKA PKC PKI PLA2 PLC PMA PMCA PMH p.o. poly(A) PPAR PR PRA PRL PSA PSI PT PTH PTHrP PTX PUVA QCT QSAR 9-cis-RA RA RA RANK RANKL RAP RAR RARE RAS RBP RCI RDA RFLP RIA RID RNase ROCs ROS RPA RRA RT-PCR RXR

ABBREVIATIONS

phytohemagglutinin pseudohypoparathyroidism preinitiation complex protein kinase A protein kinase C protein kinase inhibitor phospholipase A2 phospholipase C phorbol 12-myristate 13-acetate plasma membrane calcium pump past medical history oral polyadenosine peroxisome proliferator-activated receptor progesterone receptor plasma renin activity prolactin prostate-specific antigen psoriasis severity index parathyroid parathyroid hormone parathyroid hormone-related peptide parathyroidectomy psoralen-ultraviolet A quantitative computerized tomography quantitative structure-activity relationship 9-cis-retinoic acid retinoic acid rheumatoid arthritis receptor activator NF-κB receptor activator NF-κB ligand receptor-associated protein retinoic acid receptor retinoic acid response element renin–angiotensin system retinol-binding protein relative competitive index recommended dietary allowance restriction fragment length polymorphism radioimmunoassay receptor interacting domain ribonuclease receptor operated calcium channels reactive oxygen species ribonuclease protection assay radioreceptor assay reverse transcriptase-polymerase chain reaction retinoid X receptor

RXRE SBP SD SDS SE SEM SH SHBG SLE SOS Sp1 SPF SRC-1 SSCP SV40 SXA t1/2 T3 T4 TBG TBP TC TF TFIIB TG TGF TIO TK TLR TmP or TmPi TNF TPA TPN TPTX TR TRAP TRAP TRP TRE TRE TRH Trk TSH TSS UF US USDA UTR UV VDDR-I

retinoid X receptor response element systolic blood pressure standard deviation sodium dodecyl sulfate standard error standard error of the mean social history sex hormone–binding globulin systematic lupus erythematosus speed of sound selective promoter factor 1 sun protection factor steroid receptor coactivator-1 single strand conformational polymorphism simian virus 40 single energy X-ray absorptiometry half-time triiodothyronine thyroxine thyroid-binding globulin TATA binding protein tumoral calcinosis tubular fluid general transcription factor IIB transgenic transforming growth factor tumor-induced osteomalacia thymidine kinase toll-like receptor tubular absorptive maximum for phosphorus tumor necrosis factor 12-O-tetradecanoylphorbol-13-acetate total parenteral nutrition thyroparathyroidectomized thyroid hormone receptor tartrate-resistant acid phosphatase thyroid hormone receptor associated proteins transient receptor potential thyroid hormone response element TPA response element thyrotropin-releasing hormone tyrosine kinase thyrotropin transcription start site ultrafiltrable fluid ultrasonography U.S. Department of Agriculture untranslated region ultraviolet vitamin D-dependent rickets type I (see PDDR)

xxix

ABBREVIATIONS

VDDR-II VDR VDRE VDRL VEGF VERT VICCs VSMC

vitamin D-dependent rickets type II (see HVDRR) vitamin D receptor vitamin D response element vitamin D receptor ligand vascular endothelial growth factor Vertebral Efficacy with Risedronate Therapy studies voltage-insensitive calcium channels vascular smooth muscle cell

VSSCs WHI WRE WSTF XLH XRD ZEB

voltage-senstive calcium channels Women’s Health Initiative Wilms’ tumor gene, WT1, responsive element Williams syndrome transcription factor X-linked hypophosphatemic rickets X-ray diffraction zinc finger, E box-binding transcription factor

Approximate Normal Ranges for Serum Values in Adultsa Measure

SI Units

Ionized calcium Total calcium Phosphorous, inorganic 25(OH)D 1,25(OH)2D

1.12–1.32 mmol/L 2.17–2.52 mmol/L 0.77–1.49 mol/L 24.9–169.5 nmol/L 60–108 pmol/L

Conventional Units

Conversion Factorb

4.5–5.3 mg/dL 8.7–10.1 mg/dL 2.4–4.6 mg/dL 10–68 ng/mL 25–45 pg/mL

0.2495 0.2495 0.3229 2.496 2.40

Approximate Normal Ranges for Serum Values in Childrena Measure Ionized calcium Total calcium Phosphorous, inorganic 25(OH)D 1,25(OH)2D aNormal

SI Units

Conventional Units

Conversion Factorb

1.19–1.29 mmol/L 2.25–2.63 mmol/L 1.23–1.62 mol/L 34–91 nmol/L 65–134 pmol/L

4.8–5.2 mg/dL 9.0–10.5 mg/dL 3.8–5.0 mg/dL 14–37 ng/mL 27–56 pg/mL

0.2495 0.2495 0.3229 2.496 2.40

ranges differ in various laboratories and these values are provided only as a general guide. factor X conventional units = SI units

bConversion

Useful Equivalencies of Different Units Vitamin D Calcium Phosphorus

1 µg = 40 IU 1 mmol = 40 mg 1 mmol = 30 mg

CHAPTER 1

Historical Perspective HECTOR F. DELUCA Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin

I. Discovery of the Vitamins II. Discovery That Vitamin D Is Not a Vitamin III. Isolation and Identification of Nutritional Forms of Vitamin D

IV. Discovery of the Physiological Functions of Vitamin D V. Discovery of the Hormonal Form of Vitamin D References

I. DISCOVERY OF THE VITAMINS

animals whereas purified materials could not [3]. Funk had found similar results for the prevention of neuritis and reasoned that there were “vital amines” present in foods from natural sources and actually provided the basis for the term “vitamins” used later to describe essential micronutrients [5].

A. Early Nutritional Views The field of nutrition was largely dominated in the nineteenth century by German chemists, led by Justus von Liebig [1]. They taught that adequacy of the diet could be described by an analysis of protein, carbohydrate, fat, and mineral. Thus, a diet containing 12% protein, 5% mineral, 10–30% fat, and the remainder as carbohydrate would be expected to support normal growth and reproduction. This view remained largely unchallenged until the very end of the 19th century and the beginning of the 20th century [2–5]. However, evidence opposing this view began to appear. One of the first was the famous study of Eijkman who studied prisoners in the Dutch East Indies maintained on a diet of polished rice [6]. A high incidence of the neurological disorder beri-beri was recorded in these inmates. Eijkman found that either feeding whole rice or returning the hulls of the polished rice could eliminate beri-beri. Eijkman reasoned that polished rice contained a toxin that was somehow neutralized by the rice hulls. Later, a colleague, Grijns [7], revisited the question and correctly demonstrated that hulls contained an important and required nutrient that prevented beri-beri. Other reports revealed that microorganic nutrients might be present. The development of scurvy in mariners was a common problem. This disease was prevented by the consumption of limes on British ships (hence, the term “Limey” to describe British sailors) and sauerkraut and fruits on other ships. This led Hoist and Frohlich to conclude that scurvy could be prevented by a nutrient present in these foods [8]. Experiments by Lunin, Magendie, Hopkins, and Funk showed that a diet of purified carbohydrate, protein, fat, and salt is unable to support growth and life of experimental animals [2–5]. This suggested that some unknown or vital factor present in natural foods was missing from the purified diets. Hopkins developed a growth test in which natural foods were found to support rapid growth of experimental VITAMIN D, 2ND EDITION FELDMAN, PIKE, AND GLORIEUX,

B. McCollum and Osborne and Mendel’s Discovery of Vitamin A and B Complex A key experiment demonstrating essential micronutrients was one carried out at the Wisconsin Agricultural Experiment Station, engineered by Stephen Moulton Babcock and carried out by E. B. Hart supported by McCollum and Steenbock [9]. Herds of dairy cows were maintained on a diet composed individually only of corn, oats, or wheat or were fed a mixture of all of these grains, all receiving the same amount of carbohydrate, protein, fat, and salts and all providing equal analysis according to the German chemists [1]. The animals on the corn diet did very well, produced milk in large amounts, and reproduced normally. Those on the wheat diet failed to thrive and soon were unable to reproduce or lactate. The oat group was found to be intermediate between the corn and wheat groups, and the mixture approximated the growth and reproduction found with corn. Yet all these diets had the same proximate analysis. The conclusion of the Wisconsin Experiment Station study was that there are unknown nutrients present in corn and not found in wheat that are essential for life and reproduction. This led E. B. Hart, Chairman of Biochemistry at Wisconsin, to conceive that a search for these nutrients must begin. Professor McCollum was asked to search for these nutrients using small experimental animals. McCollum and Davis demonstrated there was present in butter fat a substance that prevented xerophthalmia and was also required for growth. They termed this “a lipin-soluble growth factor” [10]. McCollum later named this factor “vitamin A” [11]. Copyright © 2005, Elsevier, Inc. All rights reserved.

4 This substance was absent from lard and other fats but was found in large amounts in cod liver oil. In constructing the diets, McCollum obtained the carbohydrates and salts from milk whey, which, unknown to him, supplied the vitamin B complex group of micronutrients that permitted him to observe a vitamin A deficiency. McCollum at Wisconsin [11] and Osborne and Mendel [12] at the Connecticut Experiment Station carried out experiments in which cod liver oil was used as a source of fat in the diet, but the minerals were supplied from pure chemicals mixed to approximate the mineral composition of milk. Starch or sugar was used as the carbohydrate. These animals developed a different group of symptoms, namely, neuritis, which could be cured by the provision of the milk components. McCollum and Osborne and Mendel correctly concluded that this activity was due to a different micronutrient called “vitamin B.” This ushered in the concept of the organic micronutrients known as vitamins.

C. History of Rickets The disease rickets was very likely known in antiquity but was described in the 15th century as revealed by later writings. Whistler first provided a clear description of rickets in which the skeleton was poorly mineralized and deformed [13]. Rickets undoubtedly in ancient times appeared only on rare occasions and hence was not considered a problem. However, at the end of the 19th century, the industrial revolution had taken place: A highly agrarian population had become urbanized, and smoke from the industrial plants polluted the atmosphere. Thus, in low-sunlight countries such as England, rickets appeared in epidemic proportions. In fact, it was known as “the English disease” [14]. Some reports of the beneficial action of cod liver oil had appeared. However, they were not given scientific credence. With the discovery of the vitamins, Sir Edward Mellanby in Great Britain began to reason that rickets might also be a disease caused by a dietary deficiency [15]. Mellanby fed dogs a diet composed primarily of oatmeal, which was the diet consumed where the incidence of rickets was the highest (i.e., Scotland). McCollum inadvertently maintained the dogs on oatmeal indoors and away from ultraviolet light. The dogs developed severe rickets. Learning from the experiments of McCollum, Mellanby provided cod liver oil to cure or prevent the disease. Mellanby could not decide whether the healing of rickets was due to vitamin A known to be present in the cod liver oil or whether it was a new and unknown substance. Therefore, the activity of healing rickets was first attributed to vitamin A.

HECTOR F. DELUCA

D. Discovery of Vitamin D McCollum, who had moved to Johns Hopkins from Wisconsin, continued his experiments on the fat-soluble materials. McCollum used aeration and heating of cod liver oil to destroy the vitamin A activity or the ability to support growth and to prevent xerophthalmia [16]. However, cod liver oil treated in this manner still retained the ability to cure rickets. McCollum correctly reasoned that the activity in healing rickets was due to a new and heretofore unknown vitamin that he termed “vitamin D.” On the basis of the experiments of McCollum and of Mellanby, vitamin D became known as an essential nutrient.

II. DISCOVERY THAT VITAMIN D IS NOT A VITAMIN At the same time that Sir Edward Mellanby was carrying out the experiments in dogs, Huldshinsky [17] and Chick et al. [18] independently found that rickets in children could be prevented or cured by exposing them to sunlight or to artificially induced ultraviolet light. Thus, the curious findings were that sunlight and ultraviolet light somehow equaled cod liver oil. These strange and divergent results required resolution. Steenbock and Hart had noted the importance of sunlight in restoring positive calcium balance in goats [19]. At Wisconsin, with McCollum carrying out experiments in small experimental animals (i.e., rats), Steenbock was required to work with larger animals. Steenbock then began to study goats because they would consume less materials and could serve as better experimental animals than cows. Steenbock began to study the calcium balance of lactating goats and found that those goats maintained outdoors in the sunlight were found to be in positive calcium balance, whereas those maintained indoors lost a great deal of their skeletal calcium to lactation [19]. Steenbock and Hart, therefore, noted the importance of sunlight on calcium balance. This work then undoubtedly led Steenbock to realize that the ultraviolet healing properties described by Huldschinsky might be related to the calcium balance experiments in goats. By irradiating the animals and diets, Steenbock and Black found that vitamin D activity could be induced and rickets could be cured [20]. A similar finding was reported soon thereafter by Hess and Weinstock [21]. Steenbock then traced this to the nonsaponifiable fraction of the lipids in foods [22]. He found that ultraviolet light activated an inactive substance to become a vitamin D–active material. Thus, ultraviolet light could be used to irradiate foods, induce vitamin D activity, and fortify foods to eliminate rickets as a major

5

CHAPTER 1 Historical Perspective

medical problem. This discovery also made available a source of vitamin D for isolation and identification.

III. ISOLATION AND IDENTIFICATION OF NUTRITIONAL FORMS OF VITAMIN D From irradiation of mixtures of plant sterols, Windaus and colleagues isolated a material that was active in healing rickets [23]. This substance was called “vitamin D1,” but its structure was not determined. Vitamin D1 proved to be an adduct of tachysterol and vitamin D2, and thus vitamin D1 was actually an error in identification. The British group led by Askew was successful in isolating and determining the structure of the first vitamin D, vitamin D2 or ergocalciferol, from irradiation of plant sterols [24]. A similar identification by the Windaus group confirmed the structure of vitamin D2 [25]. Windaus and Bock also isolated the precursor of vitamin D3 from skin, namely, 7-dehydrocholesterol [26]. Furthermore, 7-dehydrocholesterol was synthesized [27] and converted to vitamin D3 (cholecalciferol) as identified by the Windaus group [28]. Thus, the structures of nutritional forms of vitamin D became known (Fig. 1). Windaus and Bock, having isolated 7-dehydrocholesterol from skin, provided the presumptive evidence that vitamin D3 is the form of vitamin D produced in skin, a discovery that was later confirmed by the chemical identification of vitamin D3 in skin by Esvelt et al. [29] and of a previtamin D3 in skin by Holick et al. [30]. Synthetic vitamin D as produced by the irradiation process replaced the irradiation of foods as a means of fortifying foods with vitamin D and was also rapidly applied to a variety of diseases including rickets and tetany and in the provision to domestic animals such as chickens, cows, and pigs. Windaus’ group provided chemical syntheses of the vitamin D compounds, confirming their structures and

thus ending the era of the isolation and identification of nutritional forms of vitamin D and making them available for the treatment of disease. For his contributions, Windaus received the 1938 Nobel Prize in chemistry.

IV. DISCOVERY OF THE PHYSIOLOGICAL FUNCTIONS OF VITAMIN D A. Intestinal Calcium and Phosphorus Absorption Besides bone mineralization, the earliest discovered function of vitamin D is its important role in the absorption and utilization of calcium. The first report of this finding was in the early 1920s by Orr and colleagues [31]. Kletzien et al. [32] demonstrated that vitamin D plays an important role in the utilization of calcium from the diet, and a number of experiments were carried out on the utilization of calcium and phosphorus from cereal diets. Nicolaysen was responsible, however, for demonstrating unequivocally the role of vitamin D in the absorption of calcium and independently of phosphorus from the diet [33]. Nicolaysen also followed the early work of Kletzien et al. [32] in which animals adapted to a low calcium diet were better able to utilize calcium than were animals on an adequate calcium diet. This work was confirmed by Nicolaysen, who postulated the existence of an “endogenous factor” that would inform the intestine of the skeletal needs for calcium [34]. This endogenous factor later proved to be largely the active form of vitamin D, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] [35]. Strong support for this concept was provided by the studies of Ribovich et al. [36] that showed animals maintained on a constant exogenous source of 1,25(OH)2D3 are unable to change intestinal calcium transport in response to changes in dietary calcium levels.

B. Mobilization of Calcium from Bone

FIGURE 1 Nutritional forms of vitamin D.

For many years, investigators have attempted to show that vitamin D plays a role directly on the mineralization process of the skeleton. However, early work by Rowland and Kramer [37], later work by Lamm and Neuman [38], and more recent work by Underwood and DeLuca [39] demonstrated very clearly that vitamin D does not play a significant role in the actual mineralization process of the skeleton but that the failure to mineralize the skeleton in vitamin D deficiency is due to inadequate levels of calcium and phosphorus in the plasma. Thus, the action of vitamin D in mineralizing

6 the skeleton and in preventing hypocalcemic tetany is the elevation of plasma calcium and phosphorus [40]. These discoveries laid to rest the concept of a role of vitamin D in mineralization. However, Carlsson [41] and Bauer et al. [42] were the first to realize that a major function of vitamin D is to induce the mobilization of calcium from bone when required. Thus, in animals on a low-calcium diet, the rise in serum calcium induced by vitamin D is the result of actual mobilization of calcium from bone [43]. This important function is known to be essential for the provision of calcium to meet soft-tissue needs, especially those of nerves and muscle, on a minute-to-minute basis when it is in insufficient supply from the diet. It is likely that the function of vitamin D in mobilizing calcium from bone is an osteoclastic-mediated process [44]. It is clear, however, that both vitamin D and parathyroid hormone are required for this function [45]. Furthermore, it is clear that vitamin D plays an important role in osteoclasticmediated bone resorption [46], which is certainly the first event in bone remodeling and an essential event in bone modeling [47].

C. Renal Reabsorption of Calcium and Phosphorus A final site of vitamin D action to elevate plasma calcium is in the distal renal tubule. Although experiments were suggestive of a role for vitamin D in increasing renal tubule reabsorption of calcium, a clear demonstration of this did not occur until the 1980s at the hands of Yamamoto et al. [48]. The renal tubule reabsorbs 99% of the filtered calcium even in the absence of vitamin D. However, reabsorption of the last 1% of the filtered load requires both vitamin D and parathyroid hormone. Thus, these agents work in concert in the renal reabsorption of calcium as well as in the mobilization of calcium from bone. Both agents are required to carry out this function.

D. Discovery of New Functions of Vitamin D With discovery of the receptor for the vitamin D hormone (described in Section V,G below) came the surprising result that this receptor could be found in a variety of tissues not previously appreciated as targets of vitamin D action. It localizes in the distal renal tubule cells, enterocytes of the small intestine, bone lining cells, and osteoblasts in keeping with its known role in calcium metabolism [49,50]. However, its appearance

HECTOR F. DELUCA

in tissues such as parathyroid gland, islet cells of the pancreas, cells in bone marrow (i.e., promyelocytes), lymphocytes, and certain neural cells raised the question of whether the functions of vitamin D might be broader than previously anticipated [49,50]. As a result of those findings, new functions of vitamin D have been found. For example, vitamin D plays a role in causing differentiation of promyelocytes to monocytes and the subsequent coalescing of the monocytes into multinuclear osteoclast precursors and ultimately into active osteoclasts [51,52]. Suppression of parathyroid cell growth and suppression of parathyroid hormone gene expression represent other new vitamin D actions [53,54]. In keratinocytes of skin, vitamin D appears to play a role in suppression of growth and in cellular differentiation [55]. Likely, discoveries of many new functions of 1,25(OH)2D3 will be made and are well on their way, as described in later chapters of this volume.

V. DISCOVERY OF THE HORMONAL FORM OF VITAMIN D A. Early Work of Kodicek The true pioneer of vitamin D metabolism was Egan MA Kodicek working at the Dunn Nutritional Laboratory in Cambridge U.K. Kodicek used a bioassay at first to study the fate of the vitamin D molecule and found that much vitamin D was converted to biologically inactive products [56]. Clearly, however, this approach of assaying vitamin D activity following administration of known doses of vitamin D was of limited value in determining metabolism.

B. Radiolabeled Vitamin D Experiments Professor Kodicek then began to synthesize radiolabeled vitamin D2. Unfortunately, the degree of labeling was not sufficient to permit the administration of truly physiological doses of vitamin D. Nevertheless, Professor Kodicek continued investigations into this important area. At the conclusion of 10 years of work, he concluded that vitamin D was active without metabolic modification and that the metabolites that were found were biologically inactive [57]. This conclusion was reached even as late as 1967, when it was concluded that vitamin D3 itself was the active form of vitamin D in the intestine [58]. However, chemical synthesis of vitamin D3 of high specific activity in the laboratory of the author proved to be of key importance in the demonstration of biologically active metabolites [59].

7


By providing a truly physiological dose of vitamin D, it could be learned that the vitamin D itself disappeared and instead polar metabolites could be found in the target tissues before those tissues responded [60]. The polar metabolites proved to be more biologically active and acted more rapidly than vitamin D itself [61]. Thus, presumptive evidence of conversion of vitamin D to active forms had been obtained as early as 1967.

C. Isolation and Identification of the Active Form of Vitamin D By 1968, the first active metabolite of vitamin D was isolated in pure form and chemically identified as 25-hydroxyvitamin D3 (25OHD3) [62]. Its structure was confirmed by chemical synthesis [63] that provided it for study to the medical and scientific world. For a couple of years, 25OHD3, was visualized as the active form of vitamin D. However, when it was synthesized in radiolabeled form, it was found to be rapidly metabolized to yet more polar metabolites [64]. By this time, the Kodicek laboratory reawakened their interest in metabolism of vitamin D and began to study the metabolism of lα-tritium-labeled vitamin D [65]. Furthermore, polar metabolites of vitamin D were found by Haussler, Myrtle, and Norman [66]. The Wisconsin group labeled these metabolites as peak 5 [64], the Norman group called it peak 4B [66], and Lawson, Wilson, and Kodicek described it as peak P [65]. Kodicek et al. claimed that the metabolite of vitamin D found in intestine was deficient in tritium at the 1-position [65]. However, Myrtle et al. reported that peak 4B did not lose its tritium [67]. Thus, the suggestion of a modification at the 1-position could not be confirmed. The DeLuca group, however, isolated the active metabolite from intestines of 1600 chickens given radiolabeled vitamin D, and, by means of mass spectrometric techniques and specific chemical reactions, the structure of the active form of vitamin D in the intestine was unequivocally demonstrated as 1,25(OH)2D3 [68]. Of great importance was the finding of Fraser and Kodicek that the peak P metabolite could be produced by homogenates of chicken kidney and that anephric animals are unable to produce the peak P metabolite [69]. They correctly concluded that the site of synthesis of the active form of vitamin D is the kidney. The Wisconsin group then chemically synthesized both 1α25(OH)2D3 [70] and 1β,25(OH)2D3 [71] and provided unequivocal proof that the active form is 1α,25(OH)2D3. Furthermore, this group was able to synthesize lαOHD3, an important

analog that assumed great importance as a therapeutic agent throughout the world [72].

D. Proof That 1,25(OH)2D3 Is the Active Form of Vitamin D Proof that 1,25(OH)2D3 and not 25OHD3 is the active form was provided by experiments in which anephric animals respond to 1,25(OH)2D3 by increasing intestinal absorption of calcium and bone calcium mobilization, whereas animals receiving 25OHD3 at physiological doses did not [73–75]. Furthermore, the experiment of nature, namely, vitamin D–dependency rickets type I, an autosomal recessive disorder, provided final proof [76]. This disease could be corrected by physiological doses of synthetic 1,25(OH)2D3, whereas large amounts of vitamin D3 or 25OHD3 were needed to heal the rickets. The exact defect in this disease is now clearly known and is described elsewhere in this volume. 25OHD3 at pharmacological doses likely acts as an analog of the final vitamin D hormone, 1,25(OH)2D3 (Fig. 2).

E. Discovery of the Vitamin D Endocrine System Immediately after the identification of 1,25(OH)2D3 as the active form of vitamin D came studies in which it could be shown that animals on a low-calcium diet produce large quantities of 1,25(OH)2D3, whereas those on a high-calcium diet produce little or no 1,25(OH)2D3 [77]. A reciprocal arrangement was found for the metabolite 24R,25(OH)2D3. Thus, when calcium is needed, production of 1,25(OH)2D3 is markedly stimulated and the 24-hydroxylation degradation reaction is suppressed. When adequate calcium is present, production of 1,25(OH)2D3 is shut off and the 24-hydroxylation reaction is turned on. This discovery also satisfactorily provided evidence that 1,25(OH)2D3 is the likely endogenous factor originally described by Nicolaysen et al. [34]. The next important step was the demonstration that it is parathyroid hormone that activates 1α-hydroxylation of 25OHD3 in the kidney [78]. Thus, parathyroidectomy eliminates the hypocalcemic stimulation of 1α-hydroxylation and suppression of 24-hydroxylation, whereas administration of parathyroid hormone restores that capability. Fraser and Kodicek also provided evidence that, in intact chickens, injection of parathyroid hormone stimulated the 1α-hydroxylation reaction [79]. Thus, the basic vitamin D endocrine system was largely discovered and reported in the early 1970s, being completed by 1974.

8

HECTOR F. DELUCA

OH

Liver

Kidney

Microsomes (Mitochondria)

Mitochondria

HO

HO Vitamin D3

OH

HO 25-hydroxyvitamin D3

OH 1α,25-dihydroxyvitamin D3

FIGURE 2 Activation of the vitamin D molecule.

F. Other Metabolites of Vitamin D During the course of identification of 1,25(OH)2D3, 21,25(OH)2D3 was reported as a metabolite, as was 25,26(OH)2D3 [80,81]. However, the identification of 21,25(OH)2D3 was in error and was corrected to 24,25(OH)2D3, with the correct stereochemistry as 24R,25(OH)2D3 [82]. Over the late 1970s and early 1980s, as many as 30 metabolites of vitamin D were identified [83]. These are covered in other chapters in this volume. Of great importance was the use of the fluoro derivatives of vitamin D to illustrate that the only activation pathway of vitamin D is 25-hydroxylation followed by 1-hydroxylation [84]. Thus, 24-difluoro25OHD3 supported all known functions of vitamin D for at least two generations of animals [85]. 24-Difluoro25OHD3 cannot be 24-hydroxylated. Furthermore, other fluoro derivatives such as 26,27-hexafluoro-25OHD3 [86] and 23-difluoro-25OHD3 [87] are all fully biologically active, illustrating that 26-hydroxylation, 24-hydroxylation, and 23-hydroxylation are not essential to the function of vitamin D.

G. Discovery of the Vitamin D Receptor Zull and colleagues provided evidence that the function of vitamin D is blocked by transcription and protein inhibitors [88]. Thus, it became clear very early that a nuclear activity is required for vitamin D to carry out its functions. This work confirmed and extended the earlier work of Eisenstein and Passavoy [89]. With the discovery of the active forms of vitamin D came new attention to the idea that vitamin D may work through a nuclear mechanism. Thus, Haussler et al. reported vitamin D compounds to be associated with chromatin [66]. However, these experiments did not

exclude the possibility that the vitamin D compounds might be bound to the nuclear membrane. The first clear demonstration of the existence of a vitamin D receptor was at the hands of Brumbaugh and Haussler [90]. Furthermore, the experiments of Kream et al. [91] provided strong and unequivocal evidence of the existence of a nuclear receptor for 1,25(OH)2D3. Intense efforts toward purification of the receptor and its study appeared with the knowledge that it is a receptor protein with a molecular weight of approximately 55,000. In 1987, a partial cDNA sequence for the chicken vitamin D receptor was determined [92]. This was followed by isolation of the full coding sequence for the human [93] and rat [94,95] receptors. Cloning of the cDNAs encoding the vitamin D receptor in a human and mouse permitted the isolation of the gene encoding the vitamin D receptor [96–98]. The human gene was completely described and the mouse promoter was isolated and shown to be a TATA-less Sp1driven promoter [98]. The human gene appears to have alternate promoters [96,97]. Two groups have prepared receptor Null mutant mice, permitting extensive experiments with vitamin D receptorless animals [99,100]. From a historical point of view, one of the most important discoveries was vitamin D-dependency rickets type II [101], which is now known to be due to a defect in the receptor gene [102,103] (discussed in Chapter 11). This discovery essentially provided receptor knockout experiments in humans, allowing unequivocal proof of the essentiality of the vitamin D receptor for the functions of vitamin D. The nature of the receptor and how it functions are described in subsequent chapters along with current thinking on the molecular mechanism of action of 1,25(OH)2D3. However, the discovery of vitamin D responsive elements in the osteocalcin, osteopontin, preproparathyroid, and 24-OHase genes represent important


historical developments [104–107]. This led to a consensus sequence and, most important, the development of the 3, 4, 5-rule of Umesono et al. [108]. It is now clear that vitamin D–responsive elements represent two imperfect repeat sequences separated by three nonspecified nucleotides. The vitamin D receptor will bind to these response elements, but it requires the presence of another nuclear factor, which proved to be the retinoid-X receptor (RXR) [109,110]. It is quite clear that the vitamin D receptor forms a heterodimer on the vitamin D responsive elements with the RXR protein on the 5′ arm of the responsive element and the vitamin D receptor on the 3′ segment [111]. The work of Rosenfeld, Glass, and colleagues [112] has demonstrated that the RXR protein when complexed with the vitamin D receptor on the responsive elements will not accept an RXR ligand, thus acting as a silent partner. Details of what is known concerning the role of the vitamin D receptor in transcription are described fully in subsequent chapters.

Acknowledgment This work was supported in part by a fund from the Wisconsin Alumni Research Foundation.

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DNA encoding the avian receptor for vitamin D. Science 235:1214–1217. Baker AR, McDonnell DP, Hughes M, Crisp TM, Mangelsdorf DJ, Haussler MR, Pike JW, Shine J, O’Malley BW 1988 Cloning and expression of full-length cDNA encoding human vitamin D receptor. Proc Natl Acad Sci USA 85: 3294–3298. Burmester JK, Maeda N, DeLuca HF 1988 Isolation and expression of rat 1,25-dihydroxyvitamin D3 receptor cDNA. Proc Natl Acad Sci USA 85:1005–1009. Burmester JK, Wiese RJ, Maeda N, DeLuca HF 1988 Structure and regulation of the rat 1,25-dihydroxyvitamin D3 receptor. Proc Natl Acad Sci USA 85:9499–9502. Miyamoto K, Kesterson RA, Yamamoto H, Nishiwaki E, Tatsumi S, Taketani Y, Morita K, Pike JW, Takeda E 1997 Structural organization of the human vitamin D receptor chromosomal gene and its promoter. Mol Endocrinol 11:1165–1179. Jehan F, DeLuca HF 1997 Cloning and characterization of the mouse vitamin D receptor promoter. Proc Natl Acad Sci USA 94:10138–10143. Jehan F, DeLuca HF 2000 The mouse vitamin D receptor is mainly expressed through an Sp1-driven promoter in vivo. Arch Biochem Biophys 377:273–283. Yoshizawa T, Handa Y, Uematsu Y, Takeda S, Sekine K, Yoshihara Y, Kawakami T, Arioka K, Sato H, Uchiyama Y, Masushige S, Fukamizu A, Matsumoto T, Kato S 1997 Mice lacking the vitamin D receptor exhibit impaired bone formation, uterine hypoplasia and growth retardation after weaning. Nat Genet 16:391–396. Li YC, Pirro AE, Amling M, Delling G, Baron R, Bronson R, Demay MB 1997 Targeted ablation of the vitamin D receptor: An animal model of vitamin D-dependent rickets type II with alopecia. Proc Natl Acad Sci USA 94:9831–9835. Brooks MH, Bell NH, Love L, Stern PH, Orfei E, Queener SF, Hamstra AJ, DeLuca HF 1978 Vitamin D-dependent rickets Type II. Resistance of target organs to 1,25-dihydroxyvitamin D. N Engl J Med 298:996–999. Hughes MR, Malloy PJ, Kieback DG, Kesterson RA, Pike JW, Feldman D, O’Malley BW 1988 Point mutations in the human vitamin D receptor gene associated with hypocalcemic rickets. Science 242:1702–1706. Wiese RJ, Goto H, Prahl JM, Marx SJ, Thomas M, Al-Aqeel A, DeLuca HF 1993 Vitamin D–dependency rickets Type II: Truncated vitamin D receptor in three kindreds. Mol Cell Endocrinol 90:197–201. Demay MB, Gerardi JM, DeLuca HF, Kronenberg HM 1990 DNA sequences in the rat osteocalcin gene that bind the 1,25-dihydroxyvitamin D3 receptor and confer responsiveness to 1,25-dihydroxyvitamin D3. Proc Natl Acad Sci USA 87:369–373. Noda M, Vogel RL, Craig AM, Prahl J, DeLuca HF, Denhardt DT 1990 Identification of a DNA sequence responsible for binding of the 1,25-dihydroxyvitamin D3 receptor and 1,25-dihydroxyvitamin D3 enhancement of mouse secreted phosphoprotein 1 (Spp1, osteopontin) gene expression. Proc Natl Acad Sci USA 87:9995–9999. Demay MB, Kiernan MS, DeLuca HF, Kronenberg HM 1992 Sequences in the human parathyroid hormone gene that bind the 1,25-dihydroxyvitamin D3 receptor and mediate transcriptional repression in response to 1,25-dihydroxyvitamin D3. Proc Natl Acad Sci USA 89:8097–8101. Zierold C, Darwish HM, DeLuca HF 1995 Two vitamin D response elements function in the rat 1,25-dihydroxyvitamin D 24-hydroxylase promoter. J Biol Chem 270:1675–1678.

12 108. Umesono K, Murakami KK, Thompson CC, Evans RM 1991 Direct repeats as selective response elements for the thyroid hormone, retinoic acid, and vitamin D3 receptors. Cell 65:1255–1266. 109. Yu VC, Delsert C, Andersen B, Holloway JM, Devary OV, Naar AM, Kim SY, Boutin J-M, Glass CK, Rosenfeld MG 1991 RXRβ: A coregulator that enhances binding of retinoic acid, thyroid hormone, and vitamin D receptors to their cognate response elements. Cell 67:1251–1266. 110. Munder M, Herzberg IM, Zierold C, Moss VE, Hanson K, Clagett-Dame M, DeLuca HF 1995 Identification of the porcine intestinal accessory factor that enables DNA

HECTOR F. DELUCA

sequence recognition by vitamin D receptor. Proc Natl Acad Sci USA 93:2796–2799. 111. Jin CH, Pike JW 1996 Human vitamin D receptor-dependent transactivation in Saccharomyces cerevisiae requires retinoid X receptor. Mol Endocrinol 10:196–205. 112. DiRenzo J, Soderstrom M, Kurokawa R, Ogliastro M-H, Ricote M, Ingrey S, Horlein A, Rosenfeld MG, Glass CK 1997 Peroxisome proliferator-activated receptors and retinoic acid receptors differentially control the interactions of retinoid X receptor heterodimers with ligands, coactivators, and corepressors. Mol Cell Biol 17: 2166–2176.

CHAPTER 2

Vitamin D Metabolism RONALD L. HORST AND TIMOTHY A. REINHARDT U.S. Department of Agriculture, Agricultural Research Service, National Animal Disease Center, Ames, Iowa

G. SATYANARAYANA REDDY Brown University, Department of Chemistry, Providence, Rhode Island

I. Introduction II. Vitamin D Metabolism III. Vitamin D Toxicity

IV. Species Variation in Vitamin D Metabolism and Action V. Conclusions References

I. INTRODUCTION

II. VITAMIN D METABOLISM

In 1919, when the field of experimental nutrition was still in its infancy, Sir Edward Mellanby conducted a classic experiment that for the first time associated the supplementation of various growth-promoting fats with the prevention of rickets [1]. He credited the cure to the presence of a fat-soluble substance called vitamin A. McCollum et al. [2], however, later discovered that the factor responsible for healing rickets was distinct from vitamin A. McCollum named this new substance vitamin D. It was also during this period when scientists realized that there were two antirachitic factors with distinct structures. As discussed by Norman [3], the first factor to be identified was designated vitamin D2 (also known as ergocalciferol), whereas the structure of vitamin D3 (cholecalciferol) became evident some 4 to 5 years later. Vitamins D3 and D2 are used for supplementation of animal and human diets in the United States. Vitamin D3 is the form of vitamin D that is synthesized by vertebrates, whereas vitamin D2 is the major naturally occurring form of the vitamin in plants. Animals that bask in the sun such as amphibia, reptiles, and birds therefore synthesize sufficient endogenous vitamin D3 to meet their daily needs. However, herbivores may have evolved utilizing vitamin D2 as their predominant source. This chapter focuses on the general control and function of key enzymes involved in the regulation of vitamin D2 and vitamin D3 metabolism. Species differences in vitamin D metabolism, as well as vitamin D toxicity, are also discussed. The reader is also directed toward a number of additional reviews regarding vitamin D metabolism and action [3–8]. This chapter gives an overview of vitamin D metabolism; critical steps are discussed in further detail in the subsequent chapters of this section. Metabolism of vitamin D analogs is covered in Chapter 81.

A. Overview

VITAMIN D, 2ND EDITION FELDMAN, PIKE, AND GLORIEUX,

Vitamin D refers to a group of compounds that possess antirachitic activity. Technically vitamin D is classified as a secosteroid. Secosteroids are those in which one of the rings has been broken; in vitamin D, the 9,10 carbon–carbon bond of ring B is broken, and it is indicated by the inclusion of “9,10-seco” in the official nomenclature. There are several known nutritional forms of vitamin D, however, the best known examples are cholecalciferol (vitamin D3), which is produced in the skin, and ergocalciferol (vitamin D2), which is derived from plant tissues (Fig. 1). Therefore, when reference is made to vitamin D, the lack of a subscript usually implies either vitamin D2 or vitamin D3. The vitamin Ds are named according to the rules of the International Union of Pure and Applied Chemists (IUPAC) for steroid nomenclature [9]. Because vitamin D is derived from a steroid, the structure retains its numbering from the parent compound cholesterol. Configurations at asymmetric centers are designated by using the R and S notation applying the sequence-rule procedure [10]. Configuration of the double bonds are notated E for “entgegen” or trans, and Z for “zuzammen” or cis [11]. Thus the official name of vitamin D3, by relation to cholesterol, is 9,10-seco(5Z,7E)5,7,10(19) cholestatriene-3β-ol, and the official name of vitamin D2 is 9,10-seco(5Z,7E)-5,7,10(19), 22-ergostatetraene-3β-ol. Contemporary views categorize vitamin D3 not as a vitamin but, rather, as a prosteroid hormone. This concept is supported by the fact that in mammals vitamin D3 is derived from 7-dehydrocholesterol (the precursor of cholesterol) present in the skin. The direct action of sunlight on 7-dehydrocholesterol results in cleavage Copyright © 2005, Elsevier, Inc. All rights reserved.

16

RONALD L. HORST, TIMOTHY A. REINHARDT, AND G. SATYANARAYANA REDDY

FIGURE 1

Important nutritional forms of vitamin D.

of the B ring of the steroid structure that on thermoisomerization yields vitamin D3 (see Chapter 3). The significance of vitamin D as a prosteroid hormone became clearer in 1967 when Morii et al. [11a] isolated a new metabolite of vitamin D3 from rats that was as effective as vitamin D3 in healing rickets, raising blood calcium, and increasing intestinal calcium transport. This compound acted more rapidly than vitamin D3, requiring only 8 to 10 hr after oral administration to initiate its response. This metabolite was identified as 25-hydroxyvitamin D3 (25OHD3) [11b]. The liver was demonstrated to be important in the production of this most abundant circulating form of vitamin D3 that, under normal conditions, is present at 20 to 50 ng/ml [4]. Shortly following the discovery of 25OHD3, a number of laboratories showed that this metabolite is specifically hydroxylated at the lα-position in the kidney to yield 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] [11c–13]. The latter metabolite is now generally accepted as the hormonally active form of vitamin D3. Its importance is evidenced by the genetic disorder pseudo vitamin D–deficiency rickets (PDDR), which is caused by mutations in the 1α-hydroxylase gene. PDDR results in the inability to produce 1,25(OH)2D leading to severe rickets (see Chapters 71 and 72). In normal human plasma, 1,25(OH)2D3 circulates at approximately 1000-fold lower concentrations than 25OHD3 and is generally present at 20 to 65 pg/ml [14]. This simplistic picture outlined for vitamin D3 activation is complicated by the fact that vitamin D3 can be oxidatively metabolized to a variety of products. Most of these numerous metabolites have no identifiable

biological function, and indeed many have been isolated from animals fed abnormally high amounts of vitamin D3. Nevertheless, the evidence collected to date indicates that 25-hydroxylated vitamin D3 metabolites are preferentially metabolized at the side chain. In particular, carbon centers C-23, C-24, and C-26 are readily susceptible to further oxidation. Figure 2 illustrates products of these oxidative pathways. As indicated, these pathways are shared by both 25OHD3 and 1,25(OH)2D3, and their physiological importance is still a matter of controversy. For example, there is evidence that 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] may function to stimulate bone mineralization [15,16], suppress parathyroid hormone (PTH) secretion [17], and maintain embryonic development [18]. For the most part, however, the C-24 hydroxylation and other side-chain modifications are generally considered to be catabolic in nature and play a key role in maintaining vitamin D homeostasis [19]. Although these side-chain oxidative pathways yield metabolites that are considered “nonfunctional,” the presence of these compounds in circulation could pose serious problems in the analysis for 25OHD3 and 1,25(OH)2D3 [20]. Further complicating the issue of understanding vitamin D activation, catabolism, and metabolite analysis is the presence of vitamin D2. Vitamin D2 has been shown to contribute significantly to the overall vitamin D status in humans and other mammals consuming supplemental vitamin D2 [21–23]. Vitamin D2 can also be metabolized in a similar fashion to produce several metabolites analogous to the vitamin D3 endocrine system, including the hormonally

17

CHAPTER 2 Vitamin D Metabolism

OH

−H2 O

OH (1α)

OH

(3β)HO

OH

OH(1α)

(3α)HO

1,25(OH)2D3

OH(1α)

1,25(OH)2-3-epi-D3 −H2O

OH

OH (1α)

FIGURE 2 Pathways of vitamin D3 metabolism.

active form of vitamin D2, 1,25-dihydroxyvitamin D2 [1,25(OH)2D2] [24]. Simple inspection of the side chain, however, would imply that differences between metabolism of vitamin D2 and vitamin D3 may exist. The presence of unsaturation at carbon centers C-22/C-23, along with the additional methyl group at C-24, would seem to preclude the existence of the same metabolic pathways for the two vitamins. Figure 3 outlines some of the known pathways of vitamin D2 metabolism that have been shown to date. Deviations in the vitamin D2 and vitamin D3 pathways are discussed in detail in the following sections.

B. 25-Hydroxylase The 25-hydroxylation of vitamin D is the initial step in vitamin D activation. The enzyme responsible for production of this metabolite is located in the liver (see Chapter 4). Extrahepatic sources of 25-hydroxylation have been described [25]; however, experiments with hepatectomized rats provided evidence that the liver is the major, if not the sole, physiologically relevant site of 25-hydroxylation of vitamin D [26]. Subsequent studies

also described the existence of the 25-hydroxylase in both liver mitochondria and microsomes [27–31]. In early work, the microsomal enzyme was described as an enzyme of low capacity and high affinity and, therefore, the enzyme of greatest physiological importance [31]. In contrast, the mitochondrial enzyme was described as a high-capacity, low-affinity enzyme thought to be relevant only under conditions of high vitamin D concentration such as vitamin D toxicity [32]. Early evidence that the microsomal enzyme was the physiologically relevant enzyme came from experiments that suggested this enzyme could be regulated by vitamin D status [31]. It is now clear that liver production of 25-hydroxyvitamin D (25OHD) is not significantly regulated. 25OHD production is primarily dependent on substrate concentration. An important consequence of this lack of physiological regulation of 25OHD is that measurement of blood 25OHD is an excellent measure of vitamin D nutritional status. The purification and cloning of putative liver 25-hydroxylases have been reviewed several times [33–37]. Examination of the literature shows that most of the focus is on the mitochondrial 25-hydroxylase

18


FIGURE 3 Pathways of vitamin D2 metabolism.

designated CYP27A1, which is a cytochrome P450 capable of C-26(27) hydroxylation of sterols involved in bile acid synthesis and the 25-hydroxylation of vitamin D3. The rat, rabbit, and human enzymes have been cloned [38–41]. The CYP27A1 clone has been expressed

in COS cells [39,42], and its activity has been isolated from the mitochondria of these cells. The expressed enzyme was found to 27-hydroxylate cholestanetriol and 25-hydroxylate vitamin D3. However, CYP27A1 does not 25-hydroxylate vitamin D2 [39]. Rather, CYP27A1


was found to 24-hydroxylate and 26(27)-hydroxylate vitamin D2. These activities could explain the presence of 24-hydroxyvitamin D2 (24OHD2), 1,24-dihydroxyvitamin D2 [1,24(OH)2D2], and 24,26-dihydroxyvitamin D2 [24,26(OH)2D2] [43–45] in the plasma of rats and cows. Since rats fed vitamin D–deficient diets and supplemented with physiological amounts of vitamin D2 have 25OHD2 as their predominant monohydroxylated vitamin D2 metabolite in the plasma [44] and targeted disruption of CYP27A1 does not decrease serum 25OHD3 [46], CYP27A1 is likely not the physiologic enzyme responsible for the 25-hydroxylation of vitamin D. The rat liver microsomal 25-hydroxylase (CYP2C11) has also been studied, but it has been shown to be male-specific [47]. Data have also been presented indicating that microsomes do [48] or do not [49] possess 25-hydroxylase activity. Therefore, conclusions regarding the importance of the CYP27A1 and other ostensible microsomal 25-hydroxylases require additional research. Data obtained studying pig liver 25-hydroxylation of vitamin D3 [36,37,48,50,51] suggest that a third liver 25-hydroxylase exists that is microsomal in origin. In the pig, this enzyme, CYP2D25, is present equally in males and females and is markedly different from CYP27A1 and CYP2C11 based on a terminal amino acid sequence [51]. Most important is the finding that this pig microsomal enzyme 25-hydroxylates vitamin D2 and vitamin D3 equally. The 25-hydroxylation of vitamin D is not yet completely understood. Several enzymes may play a role in the 25-hydroxylation of vitamin D. Whether one enzyme is more physiologically relevant than others remains to be determined. Studies in primary cultures of pig hepatocytes suggest that both CYP2D25 and CYP27A1 can play a role in 25-hydroxylation of vitamin D3 [51]. Nevertheless, it is clear that mammals can use vitamin D2 as a sole source of vitamin D. Therefore, any 25-hydroxylase proclaimed as the key enzyme(s) in the 25-hydroxylation of vitamin D must be capable of 25-hydroxylating vitamin D2 as well as vitamin D3. If mammals, for example, possessed an enzyme with specificity for the vitamin D2 side chain, this enzyme may be missed due to the almost exclusive use of vitamin D3 or vitamin D3 analogs as substrates for the 25-hydroxylating reaction.

C. 1α-Hydroxylase (CYP27B1) In the late 1960s, 25OHD3 was believed to be the metabolically active form of vitamin D. However, the presence of a more polar metabolite, which accumulated

19 in the intestinal mucosa chromatin of chicks administered 3H-labeled vitamin D3, suggested a new candidate for the active form of vitamin D [52]. Subsequent work by Lawson et al. [53] showed that during the formation of this metabolite the 1α-3H was lost. This led them to suggest that the new metabolite had an oxygen function inserted at C-1 in addition to the hydroxyl group at C-25. The enhanced biological activity of this new metabolite was evident before its structure could be determined [54–56]. Fraser and Kodicek [56] demonstrated that nephrectomy abolished production of the new metabolite, and this active vitamin D compound was synthesized by kidney mitochondria. In 1971, three laboratories identified the active form of vitamin D as 1,25(OH)2D3 [11,12,57]. Subsequently, the vitamin D2 form was also isolated and identified [24]. The CYP27B1 is located in the inner mitochondrial membrane of the proximal convoluted tubule cells of the kidney [58] and is discussed in detail in Chapter 5. Extrarenal sites of 1α-hydroxylation have been reported in bone, liver, placenta, macrophages, and skin [59]. The physiological significance of these sites on systemic calcium metabolism is in doubt, as nephrectomy and/or severe renal failure results in very low to undetectable circulating 1,25(OH)2D3 levels [60]. The regulation of 1,25(OH)2D3 production is reciprocally regulated with respect to 24,25(OH)2D3 [61]. Hypocalcemia caused by calcium-deficient diets, vitamin D deficiency, or pathological factors results in increased production of 1,25(OH)2D3 [61–67]. This hypocalcemic-mediated induction of 1,25-dihydroxyvitamin D [1,25(OH)2D] production is secondary to increased PTH. Administration of PTH to animals results in increased 1,25(OH)2D3 production [64,67,68]. PTH treatment in vitro induces CYP27B1 in renal slices [63] and cultured kidney cells [67,69] and is cAMP dependent [63,67,69,70]. Thyroparathyroidectomy (TPTX) or parathyroidectomy (PTX) results in the loss of the ability to synthesize 1,25(OH)2D3. In humans, acute administration of PTH or primary hyperparathyroidism results in increased production of 1,25(OH)2D [14,71], which is evidenced by elevations in plasma 1,25(OH)2D. However, in animal studies where PTH was administered chronically to goats and calves, a transient rise in plasma 1,25(OH)2D was observed followed by a rapid decline to nearly undetectable levels [72,73]. These results could be attributed to hypercalcemic feedback on the renal CYP27B1. When plasma calcium in these animals reached 13 mg/dl, 1,25(OH)2D production appeared to cease. This same group conducted similar experiments in rats and showed that chronic PTH infusion did not result in a reduction of plasma 1,25(OH)2D3, but rather a

20


modest rise [74]. Clearly, there are varying degrees of direct calcium-mediated feedback on the renal CYP27B1. It is possible that species and age affect the set points at which plasma calcium becomes a direct negative regulator of 1,25(OH)2D3 production. In contrast to the indirect role of plasma calcium in inducing 1,25(OH)2D3 production, the role of plasma phosphate appears more direct. As plasma phosphate declines, animals shift from 24,25(OH)2D production to increased 1,25(OH)2D3 production [75,76]. Since phosphate-deficient animals are hypercalcemic, serum PTH is down and therefore cannot be providing the signal to increase 1,25(OH)2D3 production. Furthermore, TPTX phosphate-deficient animals produce 1,25(OH)2D3 similarly to intact phosphate-deficient animals [66]. Gray [77] demonstrated that hypophysectomy abolished the increase in plasma 1,25(OH)2D concentrations that normally accompanied dietary phosphate deprivation. Gray demonstrated that growth hormone or triiodothyronine replacement to hypophysectomized rats restored elevations in plasma 1,25(OH)2D associated with low dietary phosphorus, therefore suggesting a permissive role of these hormones in regulation of the renal CYP27B1 during phosphorus deficiency. A direct negative effect of 1,25(OH)2D on its own production has been reported. The inhibitory effect of 1,25(OH)2D3 on renal CYP27B1 activity occurs both in vivo and in vitro [64,78]. This repressive activity is probably indirect [79]. In vivo, this effect may be partially mediated through the ability of 1,25(OH)2D3 to inhibit PTH secretion [80]. Similarly, Beckman et al. [81] showed that vitamin D toxicity mildly inhibited renal CYP27B1 activity while low-calcium diets significantly induced the CYP27B1. They further demonstrated that administration of toxic doses of vitamin D to animals fed a calcium-deficient diet reduced CYP27B1 activity by 90%. This result occurred in spite of the fact that these animals were hypocalcemic and had serum PTH levels equal to those of control animals receiving calcium-deficient normal vitamin D diets. These data suggest that high plasma concentration of vitamin D metabolites may act directly to suppress CYP27B1 activity. There are many additional factors such as calcitonin (CT), acidosis, sex steroids, prolactin, growth hormone, glucocorticoids, thyroid hormone, and pregnancy that are potential regulators of 1,25(OH)2D production. One of the most recent and interesting is the requirement for the endocytic receptor megalin in the proximal tubular cells to allow uptake of 25OHD for 1α-hydroxylation [82]. Discussion of these is beyond the scope of this general review of vitamin D metabolites. The characteristics and regulation of the CYP27B1 are described further in several reports [37,83–85].

The cloning of the renal CYP27B1 has been achieved [86–89]. Studies with CYP27B1 knockout mice [90,91] suggest that these animals have abnormalities similar to those observed in PDDR. These knockout models will undoubtedly provide new insight into the functions of 1,25(OH)2D and are the subject of further review in Chapter 7.

D. 24-Hydroxylase (CYP24A1) The 24-hydroxylation of 25OHD3 and 1,25(OH)2D3 to form 24,25(OH)2D3 [92] and 1,24,25(OH)3D3 [93,94] is the primary mechanism and the first step in a metabolic pathway to inactivate and degrade these vitamin D metabolites. It now appears that CYP24A1 is ubiquitous and may be present in every cell and tissue that contains the vitamin D receptor (VDR). In the kidney, CYP24A1 is found on the inner mitochondrial membrane of the renal tubules [95]. The primary regulators of renal CYP24A1 activity are PTH and 1,25(OH)2D3. Normal and TPTX animals receiving injections or infusions of 1,25(OH)2D3 show marked increases in both renal CYP24A1 mRNA levels and activity [63,74,96,97]. Administration of PTH partially or completely blocks expression of CYP24A1 mRNA and activity in these animals [63,68,74,96,97]. PTH acts on the kidney via adenylate cyclase and cAMP, and it has been shown that infusions of cAMP in vivo block l,25(OH)2D3mediated inductions of the renal CYP24A1 [96,98]. Animals on calcium-deficient diets have elevated plasma 1,25(OH)2D concentrations, which are accompanied by suppressed or undetectable renal CYP24A1 activity [78,96], as well as reduced VDR concentrations [99]. The reasons for the inability of 1,25(OH)2D3 to up-regulate renal CYP24A1 during calcium deficiency are not clear. Iida et al. [100] have proposed that the down-regulation of renal VDR during calcium deficiency may be responsible for preventing the l,25(OH)2D3mediated induction of renal CYP24A1. In vivo studies by Reinhardt and Horst [74], however, suggest that under these conditions PTH is probably the more important mediator of renal CYP24A1 regulation rather than downregulation of VDR. In their experiments, Reinhardt and Horst [74] showed that 1,25(OH)2D3 treatment of animals on normal calcium diets resulted in significant up-regulation of renal CYP24A1 as well as VDR. However, when PTH was infused simultaneously with 1,25(OH)2D3, VDR up-regulation was still observed (albeit to a lesser degree), whereas CYP24A1 up-regulation was completely blocked. The importance of PTH in preventing the l,25(OH)2D3-mediated up-regulation of the renal CYP24A1 is also apparent

21


by observation in aged rats. With advancing age, renal PTH receptors are down-regulated [101,102], while VDR remains unchanged [103]. The reduction in renal PTH receptors makes the kidney less responsive to PTH [64], which is associated with significant elevations in CYP24A1 mRNA [103,104]. These data suggest that renal responsiveness to PTH, not a decline in VDR, is the major physiological regulator of the renal CYP24A1. In the intestine, 1,25(OH)2D3 is the primary regulator of CYP24A1. In vivo administration of 1,25(OH)2D3 rapidly induces intestinal CYP24A1 activity [105]. This activity peaks by 6 hr postinjection, and rapidly declines thereafter to control levels 24 hr postinjection. Time-course experiments show that CYP24A1 mRNA peaks 4 to 6 hr post-injection and then rapidly disappears [104]. This is in contrast to the renal CYP24A1 mRNA, which peaks 12 to 24 hr post-l,25(OH)2D3 treatment and declines much more slowly. Shinki et al. [96] proposed that the intestinal CYP24A1 was 100 times more sensitive to 1,25(OH)2D3 stimuli than the renal CYP24A1. However, they examined CYP24A1 mRNA only 3 hr after a 1,25(OH)2D3 dose. Because renal CYP24A1 requires an additional 6 to 12 hr to reach peak expression, they likely underestimated the true sensitivity of the kidney to a 1,25(OH)2D3 dose. In contrast to their effect in the kidney, TPTX, PTH administration, or cAMP infusion does not affect intestinal expression of CYP24A1 induced by 1,25(OH)2D3 [96]. Animals fed low-calcium diets, with the associated secondary hyperparathyroidism and high plasma 1,25(OH)2D3 concentrations, have marked inductions of both intestinal CYP24A1 mRNA and activity [96,105]. Another contrast between intestinal and renal CYP24A1 expression is seen in the aging rat model. Intestinal CYP24A1 mRNA and activity decline or change very little in the aged animal. This contrasts to the largely increased expression of renal CYP24A1 observed in the aged animal [103]. Calcitonin has been shown to be a potent suppressor of intestinal CYP24A1 expression [106]. In these experiments, Beckman et al. [81,106] showed that vitamin D toxicity was a potent inducer of CYP24A1 mRNA and enzyme expression in both intestine and kidney. They also showed that if the hypervitaminosis D3-induced hypercalcemia was prevented by feeding low-calcium diets, the intestinal CYP24A1 expression was enhanced four-fold over hypercalcemic animals receiving the same toxic doses of vitamin D3 but consuming a normal calcium diet. These observations prompted the examination of the possibility that CT released in response to the hypercalcemia may have suppressed the induced expression of the intestinal CYP24A1. In their series of experiments, Beckman et al. [106] clearly demonstrated that CT was a potent suppressor of intestinal CYP24A1 activity. Conceivably, the CT-mediated suppression of

CYP24A1 activity could enhance l,25(OH)2D-mediated activities by prolonging its half-life. This could exacerbate conditions that manifest hypercalcemia, such as hypervitaminosis D, by preventing 24-hydroxylation and catabolism of active vitamin D metabolites. The CYP24A1 has been purified [107,108] and cloned [107,109] and the clone has been expressed [107,109]. Analysis of the amino acid sequence of the rat and human CYP24A1s showed that the sequences were 90% similar. The 21-amino acid heme binding region was found to be 100% identical [109]. Ohyama et al. [110] isolated the gene encoding the rat CYP24A1. This single-copy gene was approximately 15 kb and was composed of 12 exons. Several putative vitamin D response elements have been identified and are currently under study. Details of the purification and cloning of the CYP24A1 have been reviewed previously [35], and additional review of the molecular analysis and regulation of the CYP24A1 can be found in Chapter 6 in this book.

E. Physiological Role of CYP24A1 The major site for 24-hydroxylation appears to be the kidney. This is based on the observation that nephrectomy reduced or eliminated plasma 24,25(OH)2D3 [111]. However, nephrectomy also eliminates the production of 1,25(OH)2D3, the primary stimulator of CYP24A1. Therefore, the possibility remains that 24,25(OH)2D3 may reappear in plasma of nephrectomized subjects treated with therapeutic doses of 1,25(OH)2D3. Some studies suggest that 25OHD3 is the primary substrate for CYP24A1 [112]; however, it is now generally accepted that CYP24A1 is distributed throughout the body and that 1,25(OH)2D3, rather than 25OHD3, is the preferred substrate for CYP24A1 [96,113,114]. Napoli et al. [115] and Napoli and Horst [116] identified the formation of 24-oxo-1,25(OH)2D3 and 24-oxo-l,23,25-trihydroxyvitamin D3 [24-oxo-1,23, 25(OH)3D3] from intestinal homogenates incubated with physiological amounts of 1,25(OH)2D3. The formation of 24-oxo-1,23,25(OH)3D3 from 1,25-(OH)2D3 was enhanced by treatment of experimental animals with exogenous 1,25-(OH)2D3. They suggested that 24-hydroxylation, followed by C-23 oxidation, most likely represents a mechanism for terminating the cellular action of 1,25(OH)2D3. In a review, Haussler [117] proposed a model for the cellular action of 1,25(OH)2D3 in which he suggested that receptor-mediated, selfinduced catabolism of 1,25(OH)2D3 modulates the action of 1,25(OH)2D3. The work of Lohnes and Jones [118], Reddy and Tserng [119], and Makin et al. [120] provided further support for this proposal by showing

22


the ubiquitous presence of catabolic pathways initiated by CYP24A1 in 1,25(OH)2D3 target tissues and the complete destruction of 1,25(OH)2D3 by these pathways. In fact, CYP24A1 has been shown to do more than just initiate this catabolic cascade. Akiyoshi-Shibata et al. [121] expressed the rat CYP24A1 cDNA in Escherichia coli. They found that this enzyme not only 24-hydroxylates but catalyzes the dehydrogenation of the 24-OH group and performs 23-hydroxylation resulting in 24-oxo-l,23,25(OH)3D3 production. Only the cleavage at C-23/C-24 resulting in the 24,25,26,27tetranor-1OH,23COOHD3 was not demonstrated. However, it is now recognized from the work of several groups that CYP24A1 is capable of the complete catabolism of 25OHD3 and 1,25(OH)2D3 via a fivestep reaction process that includes 24-hydroxylation, 24-oxidation, 23-hydroxylation, side-chain cleavage, and subsequent production of the final degradative product, calcitroic acid [121–125]. Direct evidence that self-induced metabolism of 1,25(OH)2D3 suppresses the action of 1,25(OH)2D3 on target cells was reported by Pols et al. [126,127] and Reinhardt and Horst [128,129]. Both laboratories showed that ketoconazole inhibited l,25(OH)2D3induced metabolism. This inhibition resulted in increased specific accumulation of 1,25(OH)2D3 in target cells and a significant increase in the cellular half-life of l,25(OH)2D3-occupied VDR [129]. A result of blocking the self-induced metabolism of 1,25(OH)2D3 was up-regulation of the VDR. Reinhardt and Horst [128] extended these studies by demonstrating that selfinduced metabolism of 1,25(OH)2D3 in target cells limits the response of target cells to a primary 1,25(OH)2D3 stimulus by reducing occupancy of VDR by 1,25(OH)2D3 and by preventing VDR up-regulation. Additionally, their data showed that entry of 1,25(OH)2D3 into the cell is restricted due to extensive metabolism of the 1,25(OH)2D3. In whole-cell VDR assays, hormone was degraded so rapidly that VDR binding was prevented. Reinhardt et al. [130] confirmed the inhibitory effects of self-induced induction of CYP24A1 on the cellular action of 1,25(OH)2D3 in vivo by demonstrating that ketoconazole potentiates the 1,25(OH)2D3 up-regulation of VDR in rat intestine and bone. Clearly, one of the primary roles of CYP24A1 catabolic pathway is terminating the actions of 1,25(OH)2D3. Recent work using CYP24A1 knockout mice has provided additional evidence for the role of CYP24A1 in the regulation of 1,25(OH)2D activity via catabolism [19]. These mice were unable to clear 1,25(OH)2D, as was evidenced by abnormally high circulating concentrations of this metabolite. These data provided support to the contention that the primary role of CYP24A1 is

initiating the catabolic pathway and terminating the actions of 1,25(OH)2D. The role of CYP24A1 as an enzyme responsible for the production of a biologically active compound, i.e., 24,25(OH)2D3, has been controversial. The work of St-Arnaud et al. [19] has also addressed part of this issue. Deficient mineralization of intramembranous bone was found in CYP24A1-ablated mice. The genetic cross of CYP24A1-ablated mice with VDR-ablated mice rescued the defective bone phenotype, strongly suggesting that the high concentrations of 1,25(OH)2D acting through VDR and not 24,25(OH)2D was the cause of the bone defect. Constitutive expression of CYP24A1 in transgenic rats surprisingly resulted in low plasma 24,25(OH)2D and 25OHD with no effect on plasma 1,25(OH)2D [131,132]. These rats developed albumineria and hyperlipidemia and suffered from reduced bone mass. The authors demonstrated that excreted albumin appeared to compete for the binding and reabsorption of the DBP-25-OHD3 complex with megalin, resulting in a loss of 25OHD3 into the urine and subsequent reduction of plasma 24,25(OH)2D3. Supplementation of these rats with 25OHD3 prevented the bone loss without changing plasma 1,25(OH)2D.

F. Other Vitamin D3 Derivatives Functionalized at C-24 In a series of experiments conducted by Wichmann et al. [133,134], a number of 24-hydroxylated derivatives were isolated from plasma of chicks made toxic with vitamin D3. These metabolites included 24OHD3, 23,24,25-trihydroxyvitamin D3 [23,24,25(OH)3D3], and 24,25,26-trihydroxyvitamin D3 [24,25,26(OH)3D3]. These metabolites have not been described in animals receiving physiological amounts of vitamin D3, and their biological significance is unknown; however, it is likely that 23,24,25(OH)3D3 and 24,25,26(OH)3D3 are metabolites of 24,25(OH)2D3. 24,25(OH)2D3 is also the probable precursor to the formation of the side chain cleavage product, 25,26,27-tri-norvitamin D324-carboxylic [135]. This metabolite has been shown to be a product of in vitro kidney perfusion using 25OHD3 as substrate. The analogous pathway, however, could not be demonstrated using 1,25(OH)2D3 as substrate (S. Reddy, personal communication, 1996). Another metabolite isolated in the experiments of Wichmann et al. [133] was 23-dehydro-25OHD3. The immediate precursor, site(s), and biological activity of this compound are unknown. Plausible sources of the 23-dehydro compound are dehydration of 24,25(OH)2D3


or 23,25-dihydroxyvitamin D3 [23,25(OH)2D3]. It is not certain if any of the metabolites are important under physiological conditions.

G. 23-Hydroxylation The discovery of a C-23 oxidative pathway emerged much later than the other pathways and was ushered in by the identification of 23(S),25(R)25OHD3-26,23lactone [136,137], 23(S),25(R)1,25(OH)2D3-26,23lactone [138], and their respective precursors 23(S),25(OH)2D3 and 1,23(S),25(OH)3D3 [139,140]. To date, there has been no specific 23-hydroxylase identified for the vitamin D system. Rather, like other side-chain modifications, 23-hydroxylation is likely carried out by CYP24A1 [122]. The compound, 25OHD3-26,23-lactone, can be detected in plasma from normal rats, pigs, and chicks [140,141]. However, in several species, this metabolite is not expressed unless animals are consuming excessive amounts of vitamin D3 [142]. This metabolite has unique activity in that it is three- to fivefold more competitive than 25OHD3 for binding to the plasma vitamin D-binding protein (DBP) [136]. It was, therefore, the first modification of 25OHD3 that led to enhanced binding to the plasma DBP. The metabolite, 1,25(OH)2D3-26,23-lactone, has also been demonstrated under normal conditions [143], with elevated plasma concentrations occurring during exogenous administration of pharmacological amounts of 1,25(OH)2D3 [144]. The major locus for formation of C-23 hydroxylated derivatives appears to be the kidney. Horst and Littledike [142] and Napoli et al. [140] demonstrated that nephrectomy eliminated or greatly impaired the biosynthesis of 25OHD326,23-lactone when animals were treated with excess vitamin D3 or 25OHD3. They showed that this response was due to the inability of the animals to synthesize 23(S),25(OH)2D3. However, when 23(S),25(OH)2D3 was given to nephrectomized animals, the synthesis of 25OHD3-26,23-lactone was restored. These data suggested that C-23-hydroxylation occurred predominantly, but not exclusively, in the kidney, whereas extrarenal tissues are quantitatively important in the pathway leading to 25OHD3-26,23-lactone synthesis, which includes formation of the lactone intermediates 23,25,26(OH)3D3 [145] and 25OHD3-26,23-lactol [146]. Although ambiguities remain regarding the biological effects of C-24 oxidation, 23-hydroxylation appears to clearly be a deactivation event. 23-Hydroxylation is the first side-chain modification of 25OHD3 noted to substantially reduce its affinity for the plasma DBP [147]. 23-Hydroxylation of 1,25(OH)2D3 also leads to its

23 increased plasma clearance and reduced VDR binding and biological activity [148]. The role of 23-hydroxylation as a primary oxidation event for the further metabolism of 25OHD3 and 1,25(OH)2D3 is relatively minor to its role in the further metabolism of vitamin D3 metabolites that have been previously oxidized at C-24. In other words, very little production of 23,25(OH)2D3 or 1,23,25(OH)3D3 would be expected under normal conditions. Rather, the convergences of the C-24 and C-23 oxidative pathways would lead predominantly to the formation of 24-keto-1,23,25(OH)2D3 and 24-keto-1,23,25(OH)3D3 [116], which subsequently cleave to form C-23 acids [119,149]. Therefore, as indicated in Fig. 2, the C-23 oxidative pathway can lead to two different patterns of side-chain modifications for both 25OHD3 and 1,25(OH)2D3. One pathway, which is relatively minor under physiological conditions and more predominant during hypervitaminosis D3, leads through 23-hydroxylation to formation of the lactones, whereas the other more physiologically significant pathway leads through 24-hydroxylation to 23-hydroxylation of 24-keto metabolites. Other oxidized C-23 metabolites that have been identified include 23-keto derivatives of 25OHD3 and 1,25(OH)2D3. 23-Keto-25-hydroxyvitamin D3 was synthesized in vitro from 23(S)25(OH)2D3 and 23(R),25(OH)2D3 and has unique properties in that it binds with twofold higher affinity than 25OHD3 for binding sites on the plasma DBP [150]. This affinity should be compared to that for 23(S),25(OH)2D3, which binds with 6- to 10-fold less affinity. 23-Keto25OHD3 is also about fourfold more competitive than 25OHD3 for binding to the VDR. 23-Ketonization is, therefore, the first example of a side-chain modification enhancing the affinity of 25OHD3 for the VDR. This high affinity of 23-keto-25OHD3 for VDR prompted biosynthesis of 23-keto-l,25(OH)2D3 to determine if this modification might enhance binding of 1,25(OH)2D3 to VDR. Horst et al. [151] prepared this metabolite by incubating 23-keto-25OHD3 in kidney homogenates prepared from vitamin D–deficient chicks. The major metabolite was isolated and identified as 23-keto-1,25(OH)2D3 and was shown to possess about 40% the activity of 1,25(OH)2D3 for VDR binding. 23-Ketonization of 1,25(OH)2D3, therefore, reduced the affinity of 1,25(OH)2D3 to VDR rather than increased binding. 23-Keto metabolites do not appear to be synthesized under physiologic conditions, as Napoli et al. [115] could not demonstrate the presence of these metabolites from rat intestinal homogenates incubated with 1,25(OH)2D3 or from intestinal extracts from rats dosed with 1,25(OH)2D3.

24


H. C-26 Hydroxylation 26-Hydroxylation of 25OHD3 and 1,25(OH)2D3 produces 25,26(OH)2D3 [152] and 1,25,26(OH)3D3 [153], respectively. The natural product was originally assigned the 25(R) configuration. However, Partridge et al. [154] gave the assignment as 25(S). Ikekawa et al. [155] later discovered that the naturally occurring 25,26(OH)2D3 actually existed as a mixture of 25(S) and 25(R) isomers. Although this assignment seems somewhat trivial, it was important in unraveling a controversy that existed regarding the physiological precursor to the in vivo synthesis of 25OHD3-26,23-lactone. Hollis et al. [156] demonstrated that 25,26(OH)2D3 isolated from in vivo sources could act as a precursor to the formation of the 25OHD3-26,23-lactone. Subsequent research, however, suggested that synthetic 25(S),26(OH)2D3 (which at the time was thought to be the natural configuration) did not act as precursor to the formation of 25OHD3-26,23-lactone [157], but synthetic 25(R),26(OH)2D3 could act as a precursor [140,158]. As naturally occurring 25,26(OH)2D3 is a mixture of the R and S isomers, this research validated the conclusion of Hollis et al. [156] suggesting that formation of 25OHD3-26,23-lactone could indeed proceed through 25,26(OH)2D3. This pathway has been shown to be relatively minor [159,160], with the major pathway to 25OHD3-26,23-lactone synthesis proceeding through 23(S),25(OH)2D3 [140,145,158]. The major locus for the 26-hydroxylase is unknown. Blood concentrations of 25,26(OH)2D3 are not depressed in nephrectomized humans or pigs [142,161,162]. Therefore, production of these metabolites must take place at extrarenal sources. 26-Hydroxylase activity has, however, been demonstrated in microsomes isolated from rat and pig kidneys [163]. The only extrarenal source was reported in liver mitochondria [164]. The physiological role of the C-26 oxidative pathway remains elusive. However, 25,26(OH)2D3 and 1,25,26(OH)3D3 have been shown to possess biological activity with regard to stimulating bone calcium resorption and intestinal calcium absorption, albeit to a lesser degree than either 25OHD3 or 1,25(OH)2D3 [152,165]. Therefore, it seems unlikely that 26-hydroxylation is essential for calcium uptake from the gut or release of calcium from bone.

I. C-3 Epimerization Reddy et al. [159,160] have reported the metabolism of 1,25(OH)2D3 in primary cultures of neonatal human keratinocytes and rat osteosarcoma cells into the novel A-ring modified metabolite, 1,25(OH)2-3-epi-D3.

This epimer is formed as a result of the change in the orientation of the C-3 hydroxy group from β to α. Other investigators also confirmed this finding [166,167]. Epimerization of hydroxy groups is a wellknown phenomenon in bile acid metabolism [168] and the reaction is conducted by bile acid hydroxysteroid dehydrogenase. Figure 4 outlines the proposed pathways of C-3 epimerization of 1,25(OH)2D3 as described by Reddy et al. [159]. Although there are two potential pathways for the production of 1,25(OH)2-3-epi-D3 from 1,25(OH)2D3, the most likely pathway is through keto intermediates. The C-3 epimerization has been shown to play a major role in hormone activation and inactivation in other steroid systems [169]. Indeed, 1,25(OH)2-3-epi-D3 binds to the cellular 1,25(OH)2D3 receptor (VDR) with less affinity [170,171] and has minimal activity at activating intestinal calcium transport and bone calcium resorption than 1,25(OH)2D3 [170]. On the other hand, 1,25(OH)2-epi-D3 is equipotent to 1,25(OH)2D3 at suppressing parathyroid hormone secretion in bovine parathyroid cells [171] and at inhibiting keratinocyte proliferation [170,172]. While 1,25(OH)2-3-epi-D3 does undergo side-chain metabolism [159,171], its conversion to C-23 and C-24 oxidized metabolites occurs at a slower rate than 1,25(OH)2D3 [171]; therefore, enhanced metabolic stability of the 1,25(OH)2-3-epi-D3 has been proposed as a possible explanation for the high in vitro activity in spite of its reduced binding affinity for VDR [171]. Thus, the enzyme(s) responsible for C-3 epimerization appears to play an important role not only in the regulation of intracellular concentration of 1,25(OH)2D3 but also in the formation of metabolites with a different biological activity profile in specific target tissues. These differences in intracellular concentration of 1,25(OH)2D3 and its metabolites from one tissue to another may be one possible explanation for the well-known tissuespecific actions of 1,25(OH)2D3.

J. Unique Aspects of Vitamin D2 Metabolism The 24 position of vitamin D2, in contrast to the similar position in vitamin D3, can be considered to be highly reactive. It is a tertiary carbon as well as an allylic position, and the formation of a reactive intermediate (radical, cation) at this position would be highly stabilized. The proximity of this reactive center to the 25 position would afford the possibility of C-24-hydroxylation of vitamin D2, but the presence of the C-24 methyl would preclude further oxidation to C-24-keto compounds as is known to occur in vitamin D3 metabolism. Jones et al. [43] were the first to demonstrate C-24 oxidation when they isolated 24OHD2

25


FIGURE 4

Possible pathways of epimerization at C-3 of 1,25(OH)2D3 (adapted from [159]).

from the plasma of male rats treated with 100 IU of radiolabeled vitamin D2. Engstrom and Koszewski [173] have determined that production of 24OHD2 can occur in liver homogenates from a variety of species, and actually exceeds the formation of 25OHD2. Horst et al. [44] have shown that the concentration of 24OHD2 in plasma was about 20% that of 25OHD2 in rats receiving physiological doses of vitamin D2, and was equivalent to 25OHD2 in rats receiving pharmacological doses of vitamin D2. They also demonstrated that 1α-hydroxylation of 24OHD2 to form 1,24(OH)2D2 represented a minor but significant pathway for vitamin D2 activation. In their experiments, they determined that 1,24(OH)2D2 rivaled both 1,25(OH)2D2 and 1,25(OH)2D3 in biopotency. Both 24OHD2 and 25OHD2 and their 1α-hydroxylated metabolites can undergo subsequent hydroxylation to form 24,25-dihydroxyvitamin D2 [24,25(OH)2D2] and 1,24,25-trihydroxyvitamin D2 [1,24,25(OH)3D2]. The formation of 1,24,25(OH)3D2 represented an unequivocal deactivation of the vitamin D2 molecule [174]. Conversely, the comparable vitamin D3 metabolite, 1,24,25(OH)3D3, maintains significant biological activity and must undergo further side-chain oxidation to be rendered totally inactive [174]. Although vitamin D2 is known to undergo side-chain oxidation, only recently has evidence emerged suggesting that vitamin D2 (like vitamin D3) undergoes side-chain cleavage.

The paucity of information regarding vitamin D2 sidechain metabolism is primarily due to the lack of appropriate radiolabeled and cold substrates. Zimmerman et al. [175] have used [9,11-3H]-1,25-(OH)2D2 and cold 1,25(OH)2D2 as substrates to determine if 1,25-(OH)2D2 undergoes side-chain cleavage. Similar experiments were done using [3α-3H]-25OHD2 as substrate [176]. In these metabolism experiments it became clear that aqueous-soluble metabolites were being produced from 1,25(OH)2D2 [175] and 25OHD2 [176]. Furthermore, Zimmerman et al. [175] demonstrated that calcitroic acid was the major aqueous-soluble metabolite being produced from cell culture and kidney perfusion experiments. In a more recent study Horst et al. [177] studied the metabolism of 1,25(OH)2D3 and 1,25(OH)2D2 using a purified rat CYP24A1 system. As expected, 1,23(OH)2-24,25,26,27-tetranor D and calcitroic acid were the major lipid- and aqueous-soluble metabolites, respectively, when 1,25(OH)2D3 was used as substrate. However, when 1,25-(OH)2D2 was used as substrate, 1,24,25(OH)3D2 was the major lipid-soluble metabolite with no evidence for the production of either 1,23(OH)2-24,25,26,27-tetranor D or calcitroic acid. Apparently, the CYP24A1 was able to 24-hydroxylate 1,25(OH)2D2, but was unable to cause further changes that would result in side-chain cleavage. When the analog 1,25-(OH)2-22-ene-D3 was used as substrate, CYP24A1 was again able to effect 24-hydroxylation

26


but not side-chain cleavage (R. Horst, S. Reddy, and J. Omdahl, 2003, unpublished data). Sunita Rao et al. [178] demonstrated that 1,25(OH)2-22-ene-D3 could be metabolized to calcitroic acid by RWLue-4 cells and rat kidney. They suggested that the 1,25(OH)2-22-ene-D3 was first hydroxylated at C-24, followed by further oxidation to 1,25(OH)2-24-oxo-22-ene-D3 prior to side-chain, double-bond reduction to form 1,25(OH)224-oxo-D3. The 1,25(OH)2-24-oxo-D3 was then further metabolized to calcitroic acid, presumably by CYP24A1. The compound 1,25(OH)2D4 [a.k.a. 22,23 dihydro1,25-(OH)2D2] has also been shown to undergo sidechain oxidation similar to that of 1,25(OH)2D2 in vitro [179] and metabolized to calcitroic acid in vivo [180]. These results suggest that metabolism of 1,25(OH)2D2 to calcitroic acid clearly involves enzymes other than CYP24A1. The compounds 25,26-dihydroxyvitamin D2 [25,26(OH)2D2] and 1,25,26-trihydroxyvitamin D2 [1,25,26(OH)3D2] have been chemically synthesized [181,182]. The metabolite 25,26(OH)2D2 has also been tentatively, but not exhaustively, identified from in vivo sources [141,183], but it could not be demonstrated in kidneys perfused with 25OHD2 [184]. Similarly, 1,25,26(OH)3D2 could not be demonstrated [185]. However, it is clear that 26-hydroxylation does occur when vitamin D2 metabolites have been previously 24-hydroxylated. For example, when 24,25(OH)2D2 and 1,24,25(OH)3D2 were used as precursors, the metabolites 24,25,26(OH)3D2 and 1,24,25,26(OH)4D2, respectively, were produced in significant amounts [184,185]. Similarly, Koszewski et al. [45] and Jones et al. [186] demonstrated that C-26 hydroxylation was the major metabolic pathway for the further metabolism of 24OHD2 and 1,24(OH)2D2. Oxidation at C-24, therefore, appears to be a prerequisite for C-26 oxidation of vitamin D2 compounds. A similar situation also appears to exist for C-28 oxidation as demonstrated by Reddy and Tserng [184,185]. In their experiments, they isolated 24,25,28(OH)3D2 and 1,24,25,28(OH)4D2 from rat kidney perfusions and, through the use of various substrates, were able to show C-28 hydroxylation of vitamin D2 metabolites occurs only after C-24 hydroxylation [187].

III. VITAMIN D TOXICITY A. Overview The first documented reports of vitamin D intoxication were made in the late 1920s by Kreitmeir and Moll [188] and Putscher [189]. These cases resulted from the ingestion of large quantities of vitamin D in

the diet. Vitamin D intoxication, however, has never been reported following prolonged sunlight exposure. Holick et al. [190] suggested that nature has provided various control points that prevent the overproduction of vitamin D3 by the skin. The most important point of control is the diversion of vitamin D3 production from 7-dehydrocholesterol to non–biologically active overirradiation products such as lumisterol and tachysterol. In addition, these authors suggested that skin pigmentation and latitude were also significant determinants (albeit to a lesser degree) that limit the cutaneous production of vitamin D3. As discussed earlier, once vitamin D is in circulation, the conversion to 25OHD is relatively uncontrolled. Normally, 25OHD circulates at 30 to 50 ng/ml in most species [141]. However, when vitamin D is given in excess, plasma 25OHD can be elevated to concentrations of 1000 ng/ml or greater [191,192], while plasma 1,25(OH)2D remains at or below normal concentrations [193]. When circulating at very high concentrations, 25OHD can compete effectively with 1,25(OH)2D for binding to the VDR. Therefore, during vitamin D toxicosis, 25OHD can induce actions usually attributed to 1,25(OH)2D [193]. High circulating 25OHD can, therefore, explain how humans with low circulating concentrations of 1,25(OH)2D can show signs of vitamin D toxicity [193], and why anephric humans [who are incapable of producing 1,25(OH)2D] can become vitamin D toxic [194]. Clinical aspects of vitamin D toxicity are discussed in Chapter 78. Although it is generally accepted that 1,25(OH)2D is reduced during hypervitaminosis, a notable exception to this generalization is the ruminant [195,196]. Horst and co-workers [195,197] have shown that vitamin D3 intoxication initiated by giving 15 million IU of vitamin D3 intramuscularly (i.m.) results in significant elevations in plasma 1,25(OH)2D3. In contrast, pigs given the same i.m. dose showed a reduction in plasma 1,25(OH)2D3, as was observed in other species [197]. Therefore, elevations in plasma 1,25(OH)2D may play a significant role in the pathogenesis of vitamin D toxicity in ruminants.

B. Differences in Toxicity between Vitamins D2 and D3 Most research dealing with utilization of vitamins D2 and D3 assumes that these two forms are equally potent in most mammals. However, when large and potentially toxic doses were administered orally to rhesus monkeys [198] and horses [199], or were used in treating childhood osteodystrophy [200], vitamin D2

27


presented fewer hypercalcemic side effects than vitamin D3. In addition, 1OHD2, which is as effective as the 1OHD3 in standard bioassays, was shown to be five- to 15-fold less toxic than 1OHD3 [201]. Studies by Horst et al. [44] provide some insight into the difference between vitamin D2 and vitamin D3 toxicity. They demonstrated that under physiological conditions the predominant monohydroxylated form of both vitamins D2 and D3 is 25OHD. In the vitamin D2–dosed rats, 24OHD2 accounted for approximately 20% of the monohydroxylated metabolites, whereas 24OHD3 could not be detected in the vitamin D3–dosed rats. When a modest superphysiological dose (800 IU/day) of vitamin D3 was given to rats, 25OHD3 remained the predominant metabolite in vitamin D3–dosed rats and was present at 26.3 ng/ml. Under these conditions, there was still no evidence for the presence of 24OHD3. However, when the same amount of vitamin D2 was given, the concentrations of 24OHD2 (14.1 ng/ml) nearly matched those of 25OHD2 (15.9 ng/ml). Interestingly, the combined concentrations of 24OHD2 and 25OHD2 in the vitamin D2–dosed animals (~30 ng/ml) was similar to the 25OHD3 concentration (26.3 ng/ml) in the vitamin D3–dosed rat. In standard assays, 25OHD2 and 25OHD3 are equipotent at displacing 3H-1,25(OH)2D3 from the calf thymus VDR. However, 24OHD2 has been shown to have at least a twofold lower affinity for binding to the calf thymus VDR (R. L. Horst, unpublished data, 1996). Therefore, the reduced toxicity of vitamin D2 is probably a result of diverting metabolism away from the production of 25OHD2 in favor of 24OHD2, which has a relatively limited affinity for VDR (a step necessary for the initiation of a biological response). Further differences between vitamins D2 and D3 were noted in their ability to up-regulate the VDR. Beckman et al. [202] found that VDR was significantly more enhanced in animals fed excess vitamin D3 relative to those animals receiving an equivalent amount of vitamin D2. Increased VDR would potentially accentuate toxic side effects by enhancing the responsiveness of intestinal tissue to the elevated 25OHD.

C. Factors Affecting Toxicity The severity of the effects and pathogenic lesions in vitamin D intoxication depend on such factors as the type of vitamin D (vitamin D2 versus vitamin D3), the dose, the functional state of the kidneys, and the composition of the diet. Vitamin D toxicity is enhanced by a rich dietary supply of calcium and phosphorus, and it is reduced when the diet is low in calcium and phosphorus [203,204]. Toxicity is also reduced when the

vitamin is accompanied by high intakes of vitamin A or by thyroxine injections [205]. The route of administration also influences toxicity. Parenteral administration of 15 million IU of vitamin D3 in a single dose caused toxicity and death in many pregnant dairy cows [195]. On the other hand, oral administration of 20 to 30 million IU of vitamin D2 daily for 7 days resulted in little or no toxicity in pregnant dairy cows [206]. Napoli et al. [207] have shown that rumen microbes are capable of metabolizing vitamin D to the inactive 10-keto-19-norvitamin D3. Parenteral administration would circumvent the deactivation of vitamin D by rumen microbes and may partially explain the difference in toxicity between oral and parenteral vitamin D. Various measures have been used in human medicine for treatment of vitamin D toxicity. These measures are mainly concerned with management of hypercalcemia. Vitamin D withdrawal is obviously indicated. It is usually not immediately successful, however, owing to the long plasma half-life of vitamin D (5 to 7 days) and 25OHD (20 to 30 days). This is in contrast to the short plasma half-life of 1OHD3 (1 to 2 days) and 1,25(OH)2D3 (4 to 8 hr). Because intestinal absorption of calcium contributes to hypercalcemia, a prompt reduction in dietary calcium is indicated. Sodium phytase, an agent that reduces intestinal calcium absorption, has also been used successfully in vitamin D toxicity management in monogastrics [208]. This treatment would be of little benefit to ruminants because of the presence of rumen microbial phytases. There have also been reports that CT [209], glucagon [210], etidronate [211], and glucocorticoid therapy [212] reduce serum calcium levels or prevent the calcinosis resulting from vitamin D intoxication (see Chapter 78).

IV. SPECIES VARIATION IN VITAMIN D METABOLISM AND ACTION Most concepts of vitamin D metabolism and function have been developed with the rat and/or chick as experimental models. Studying vitamin D metabolism is hampered by the paucity of data on the normal circulating levels of vitamin D metabolites in birds, mammals, and reptiles under normal conditions. Most recent research has focused on the analysis of 25OHD and 1,25(OH)2D as indicators of vitamin D status or aberrant physiological states. Table I summarizes the concentrations of the two metabolites that have been reported for several species by various laboratories. Close inspection of the information suggests that some mammals (mole rat, wild wood vole, horse, and wild wood mouse) and aquatic species (lamprey, carp,

28


TABLE I Plasma 25-Hydroxyvitamin D and 1,25-Dihydroxyvitamin D Concentrations in Several Species of Animals Concentration Species

25OHD (ng/ml) 1,25(OH)2D (pg/ml) Ref.

Human Rhesus monkey Rhesus monkey Marmoset Marmoset Wild woodmouse Wild bank vole Mole rat Lamprey Shark Leopard shark Horned shark Carp Bastard halibut Atlantic cod Bullfrog (mature) Soft-shelled turtle Turkey Chicken Cow Sheep Pig Horse aND,

32 188 50 90 64 30 kb from the BsmI and ApaI sites between exons VIII and IX and nearly 40 kb from the singlet(A) repeat in exon IX) [277]. Nevertheless, results from such studies, even if limited in scope, could provide valuable insights. Several groups have sought to identify polymorphic variants in VDR target genes that are likely associated with human disease, including osteocalcin [278], CYP19 [279], the cyclin-dependent kinase inhibitors p21 and p27 [280], and CYP3A4 [281]. This approach is promising, especially when the target gene is actually involved in a physiologic process related to the disease in question, for example CYP19 (aromatase) in the etiology of osteoporosis in postmenopausal women [279]. In fact, a complete assessment of the genetic component for disease risk would ideally account for variations not only in VDR, but also in RXR, VDR coactivators, and relevant target genes. Finally, disorders with newly reported relationships to VDR polymorphisms include: type I diabetes mellitus (higher risk with genotypes At and Bt) [282], susceptibility to the hemorrhagic form of dengue fever (with the t genotype displaying greater resistance) [283], anemia in hemodialysis patients (the BB genotype has lower hemoglobin levels) [284], and various dental indices (AA patients had the highest rates of alveolar bone loss) [285]. Many of these disorders have an immune component, and renewed interest in the immune effects of the VDR-RXR signaling system has received impetus from the recent observation that VDR knockout mice, contrary to early reports of a normal immune system, display an abnormal T-cell response [86].

B. Emerging Clinically Relevant Actions of VDR in the Colon and Hair Follicle In addition to the noncalcemic functions of VDR that have been discussed earlier in this chapter, such as

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modulation of the immune system and prevention of epithelial cell cancers, two striking new extraosseous actions of VDR have come into focus recently, as illustrated in Fig. 12. One of these is to drive the hair cycle in skin, which is unique in that, as explained later, it requires neither a vitamin D-derived ligand [155] nor the transactivation function of VDR [153] (Fig. 12B). Therefore, VDR impinges on hair cycle signal transduction in keratinocytes either in an unliganded form or perhaps occupied by an undiscovered lipophilic ligand present in skin, and does so apparently by repressing a VDR target gene(s) [157]. This gene repression in the hair follicle may be mediated by the recently demonstrated physical and functional interaction between VDR-RXR and the Hr nuclear receptor corepressor product of the hairless gene [157]. The second novel noncalcemic action of VDR, the detoxification of carcinogenic LCA in the colon (Fig. 12A), entails the binding of the newly discovered LCA ligand to VDR to induce CYPs [98]. Thus VDR has emerged as a secondary bile acid sensor as well as calcemic endocrine nuclear receptor [98]. VDR and vitamin D have been implicated in the prevention of colon cancer through epidemiologic observations that regions of the world with the lowest sunlight exposure coincide with high incidences of both rickets and colon cancer [286]. Furthermore, vitamin D supplementation reduces dietary fat-promoted colon carcinogenesis in rats [287], and VDR-null mice exhibit precocious hyperproliferation in the colon descendens where VDR is normally expressed in levels that rival those in skin and small intestine [288]. The paradigm whereby VDR/vitamin D suppresses colon cancer has not been fully characterized, and could involve the ability of VDR to arrest cells at the G1 stage of the cell cycle via induction of p21 [88] and p27 [289], to repress cell growth transcription factors such as c-myc [90] and c-fos [91], or to elicit apoptosis by diminishing the Bcl-2 anti-apoptotic factor [92]. A newly discovered mechanism through which VDR could prevent colon cancer, particularly that induced by high-fat Western diets, is that of chemoprevention by inducing CYP3A4 to detoxify LCA in the colon cells [56,98]. Figure 12A illustrates the role for VDR-RXR in the regulation of intestinal CYP enzymes, in particular CYP3A4. This concept is based upon the recent appreciation that the secondary bile acid, LCA, and its 3-keto metabolite, are novel VDR ligands [98]. Figure 12A also depicts the hepatic synthesis of primary bile acids, focusing on chenodeoxycholic acid, the precursor of LCA. The rate limiting enzyme in this biosynthetic process, CYP7A1, is under positive as well as negative control by VDR-related receptors LXR and FXR, respectively [290]. High liver cholesterol leads

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CHAPTER 13 Nuclear Vitamin D Receptor

A

Oxysterols

+

LIVER

LXR CYP 7A1 Cholesterol FXR − Chenodeoxycholic acid

Gut bacteria 7-dehydroxylation COO−

LCA HO

H VDR

RXR

LCA

+

Colonocyte

CYP3A4 LCA 6αOH LCA

COLON Colon xenobiotic detoxification

B

ABC transporter 6αOH LCA in lumen

Noggin

Wnt ligand Fz Dsh

BMP4

PTHrP

BMPR

PTHR

Keratinocyte ? β-catenin

SMADs

?

1 BULB

HAIR FOLLICLE

Dermal papilla

2

RXRα

Shh, Hoxc13, msx-1, msx-2

Telogen

FIGURE 12

Hr

Lef1/TCF

novel ligand VDR ?

Anagen

Catagen

Two novel extraosseous actions of the VDR-RXR heterodimer: xenobiotic detoxification in the colon and control of hair cycling. (A) Role of VDR-RXR in detoxification of the secondary bile acid, LCA. The rate-limiting step in the formation of bile acids from cholesterol in liver is catalyzed by CYP7A1 [116]. One of the major primary bile acids, chenodeoxycholic acid, can be converted by gut bacteria to LCA, which may accumulate in the colon since it is not effectively reabsorbed. In the colonocytes, LCA-bound VDR (or 1,25(OH)2D3liganded VDR, not shown), heterodimerized with RXR, activates transcription of the gene encoding CYP3A4. The CYP3A4 enzyme then converts LCA to its 6α-hydroxy form, which is a substrate for the ABC efflux transporter. (B) Proposed action of the VDR-RXR heterodimer in the hair follicle. Keratinocytes or their precursors in the bulge of the follicle are thought to receive signals that stimulate the follicle to exit a resting phase (telogen) and enter a phase of active growth (anagen). Recent research [295,299,300] has identified a number of factors that influence this transition, and a simplified diagram is presented here. Abbreviations not defined in the text are Wnt, ortholog of Drosophila wingless and mouse int-1; Lef1, lymphoid enhancer factor-1; TCF, T cell-specific factor; and msx-1 and msx-2, orthologs of Drosophila muscle-specific homeobox protein. Factors that are membrane receptors are boxed. Solid arrows indicate activation and dotted lines ending in a solid perpendicular line denote inhibition. The black circled 1 and 2 in the BMP/SMAD pathway represent sequential negative and positive signaling as described in the text. The precise point(s) at which VDR-RXR or the RXR-VDR-Hr complex acts has not been characterized, and it is not known whether VDR functions unliganded or occupied by a novel, non-vitamin D ligand.

248 to the production of oxysterols (Fig. 4), which are known to bind to LXR and induce the CYP7A1 gene that catalyzes the first step in bile acid synthesis via 7α-hydroxylation [116]. An opposing regulation of CYP7A1 occurs as a result of high levels of primary bile acids in liver (e.g., chenodeoxycholic acid), which bind to FXR (Fig. 4) and, via an indirect mechanism, inhibit the transcription of CYP7A1 [291]. Formation of the secondary bile acid, LCA, by intestinal bacteria is effected by 7-dehydroxylation of chenodeoxycholic acid [292,293]. LCA then travels to the colon, where its potentially toxic or carcinogenic effects [99,100] are proposed to be ameliorated by the catabolic action of CYP3A4 in the colonocyte (Fig. 12A) [105]. The CYP3A4 gene is transactivated by LCA-bound [98] (or 1,25(OH)2D3bound [55,56]) VDR-RXR, leading to 6α-hydroxylation of LCA [105] and its secretion via an ATP binding cassette (ABC) transporter into the colonic lumen for excretion [290]. Regulation of CYP3A4 was previously known to occur via PXR [59], but experiments with knockout mice have clearly demonstrated that another receptor, presumably VDR [98], is capable of mediating this regulation in PXR−/− mice [55,98]. Therefore, we propose that, in addition to its antiproliferation/ prodifferentiation and proapoptotic actions, VDR specifically operates to prevent high dietary fat-induced colon cancer by detoxifying LCA. This chemoprotective action of VDR via CYP3A4 induction occurs when the receptor is occupied by its alternative ligands, LCA or 1,25(OH)2D3, explaining both how LCA is detoxified, and why this process is amplified in the presence of supplemental vitamin D. A second noncalcemic site of VDR action, in this case clinically relevant to the prevention of hair loss, is the hair follicle (Fig. 12B). As described earlier, alopecia in VDR knockout mice cannot be corrected by excess dietary calcium [79]. Also, vitamin D- or 1,25(OH)2D3deficiency, the latter caused by ablation of the CYP27B1 1α-OHase enyzme [155,156], does not confer alopecia. Thus, the action of VDR to trigger normal hair cycling is independent of vitamin D status, indicating that VDR functions in this capacity either in the unliganded conformation, or occupied by a nonvitamin D-derived lipid constitutively present in skin. Interestingly, conditional knockout of RXRα in skin [128] elicits alopecia indistinguishable from that occurring in VDR-null mice, implying that the functional VDR unit, as with 1,25(OH)2D3-dependent actions, is the VDR-RXR binary complex. Further experiments utilizing VDR knockout animals [294], and with VDR−/− mice in which VDR expression is transgenically targeted to keratinocytes [82], have demonstrated that the absence of VDR function renders hair follicles unable to

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enter the growth (anagen) phase. Therefore, as depicted in Fig. 12B, VDR appears to function in keratinocytes to trigger the telogen (resting phase) to anagen (growth phase) transition in the hair cycle. How does VDR impinge upon hair cycle signaling? According to the hypothetical model presented in Fig. 12B, VDR-RXR could intervene at any of a number of steps in the complex process of anagen initiation. Findings mainly from the laboratory of Fuchs et al. [295] have revealed that the Lef/TCF family of transcription factors, complexed with β-catenin, play a crucial role in the transmodulation of several genes which encode proteins that lead to the initiation of anagen. The list of genes directly or indirectly activated by the Lef/TCF/βcatenin complex includes sonic hedgehog (Shh), Hoxc13, msx-1, and msx-2 [295]. This complex also activates hair specific genes such as keratin 14, and represses E-cadherin, the latter effect diminishing adherens junctions to facilitate hair follicle formation [296]. As illustrated in Fig. 12B, control of Lef1/TCF transcription factor includes at least two upstream pathways, one originating with the binding of an extracellular Wnt ligand to a cell surface receptor of the Frizzled (Fz) family. This signal, acting through the Disheveled factor (Dsh), leads to a stabilization of β-catenin and formation of an active complex with Lef1/TCF. The second pathway involves an inhibitory factor, bone morphogenetic protein-4 (BMP4), that functions in a negative manner to block synthesis of Lef1/TCF, and this inhibition can be relieved by association with noggin, an extracellular ligand secreted by the dermal papilla that acts as an antagonist at the BMP receptor (BMPR) [297,298]. Based upon recent experiments [298] in which the BMPR was knocked out, a second function of BMP/SMAD signaling has been revealed subsequent to its antagonism by noggin to permit Lef1 accumulation in hair follicle matrix cells. Once Lef1 has accumulated in response to the noggin antagonist, this is followed by a positive effect of BMP/SMAD to somehow facilitate the cooperative action of β-catenin and Lef1 to modulate transcription in the cortex cells that generate the hair shaft. Therefore, these pathways all converge in the genesis of the transcriptionally active Lef1/TCF/ β-catenin complex (Fig. 12B). To date, VDR has been studied only in relation to β-catenin in colon cancer cells, and paradoxically, the receptor attenuates β-catenin signaling by competing with Lef1/TCF for β-catenin binding [270]. However, the potential actions of VDRRXR to influence either the Wnt or BMP/noggin pathways in keratinocytes have not as yet been probed, and the receptor heterodimer could affect any number of steps in hair-cycle signal transduction as postulated in Fig. 12B.

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A third pathway initiated by yet another extracellular ligand, PTHrP, may be involved in triggering anagen, although recent reports indicate that PTHrP accelerates entry into catagen (apoptosis of the lower follicle) rather that preventing entry into anagen [299,300]. Regardless of which hypothesis for PTHrP action is correct, effective hair growth during anagen would require either inhibition of PTHrP synthesis and secretion, or repression of its receptor (PTHR), in target cells, and as postulated in Fig. 12B, VDR could act as a repressor at either of these steps. Interestingly, transcription of PTHrP is directly inhibited by the 1,25(OH)2D3-occupied VDR-RXR heterodimer through negative VDREs [301,302] (Table I), rendering PTHrP as one of the most attractive candidate targets for repression by VDR in hair follicle signaling. The mechanism whereby VDR-RXR triggers the hair cycle remains unknown. However, naturally occurring point mutations in hVDR that confer alopecia as well as vitamin D-resistant rickets exist in the hVDR DNA binding [74,303,304] and RXR heterodimerization [147] domains, yet mutations that specifically compromise either 1,25(OH)2D3 ligand [147,305] or coactivator [153] contacts yield rickets without alopecia. Thus, neither 1,25(OH)2D3 ligand contacts nor receptor-mediated transactivation is required for VDR to drive the hair cycle; only the RXR heterodimerization and DNA binding functions of VDR appear to be necessary for this action. A tonic inhibitor of hair growth has been hypothesized to exist in telogen skin [306], and a reasonable molecular scenario is that VDR-RXR functions to repress this inhibitor, which may or may not be BMP4 or PTHrP (Fig. 12B). Mechanisms of gene repression by VDR are not well understood (see Section II), and VDR does not associate strongly with traditional nuclear receptor corepressors such as N-CoR and SMRT [307–309]. Recently, VDR has been observed to interact physically and functionally with a novel corepressor, the Hr product of the hairless (hr) gene [157]. Hr recruits HDACs to repress the actions of both TR [310] and ROR [311], and it dramatically inhibits both basal and ligand-stimulated VDRRXR-mediated transcription [157]. Intriguingly, similar to knockout of the genes encoding either VDR or RXRα, inactivating mutations in the mammalian hr gene result in congenital hair loss in both mice [312] and humans [313,314]. Remarkably, the hair loss phenotype elicited by specific mutations in hVDR resembles the atrichia resulting from mutations in the hr gene [315], suggesting that the VDR, RXRα, and Hr proteins all impact a common signaling pathway, perhaps as a trimeric complex (Fig. 12B). Coexpression of hVDR and Hr mRNAs in the matrix and outer root sheath cells of the

mouse skin hair follicle [157] supports this hypothesis. What remains to be determined is where the putative RXRα-VDR-Hr complex impinges on hair cycle signal transduction to trigger the telogen-to-anagen transition, and several possibilities are illustrated in Fig. 12B. Elucidating the molecular role of VDR in controlling the hair cycle clearly represents an important challenge, one that may lead us to fundamental new insight into novel gene repressive mechanisms of VDR action. Finally, understanding this mechanism may facilitate the development of VDR mimetics to prevent or treat hair loss.

VII. SUMMARY AND PERSPECTIVES The vitamin D receptor is a unique member of the nuclear receptor superfamily in the realm of ligand binding, mediating both the endocrine control of bone mineral metabolism and the detoxification of endobiotics such as LCA. VDR functions as a heterodimer with unliganded RXR, with the binary protein complex required for recognition and high-affinity association with VDREs in the promoters of regulated genes, as well as for the recruitment of transcriptional comodulator proteins that govern the activity of RNA polymerase II. As a nuclear receptor that heterodimerizes with RXR, VDR therefore possesses the molecular character both of its evolutionarily closest endocrine relative, the thyroid hormone receptor, and of its closest xenobiotic detoxifying receptor, PXR. The biology of VDR signaling primarily involves (i) stimulation of intestinal calcium and phosphate absorption to prevent rickets/ osteomalacia, (ii) enhancement of bone remodeling via osteoblast-induced osteoclast maturation, (iii) differentiation of skin cells and maintenance of the hair cycle, (iv) repression/induction of CYP enzymes for 1,25(OH)2D3 hormone synthesis/degradation as well as for the promotion of secondary bile acid detoxification, (v) modulation of the immune system, and (vi) potential anticancer actions via the control of epithelial cell growth, differentiation, and apoptosis. Based upon biochemical mutagenesis experiments, the X-ray crystallographic characterization of the DNAand ligand-binding domains, and naturally occurring mutations that confer on patients the vitamin D-resistant phenotype of rickets and/or alopecia, the structurefunction of VDR is reasonably well understood. Amino acid residues in an α-helical region of the zinc finger domain of VDR that contact specific DNA base pairs in VDRE docking sites have been identified, as have nuclear localization signals in this domain. The more structurally complex LBD of VDR consists of a sandwich

250 of α-helices surrounding a hydrophobic pocket that accommodates lipophilic ligands, as well as surface facets for the attraction of numerous interacting proteins including the RXR dimerization partner. The VDR-RXR heterodimer, which is allosterically influenced by 1,25(OH)2D3 and other VDR ligands, associates with (i) ATP-dependent SWI/SNF chromatin remodeling complexes (e.g., PBAF and WINAC) that apparently slide nucleosomes along DNA and are required for both induction and repression; (ii) p160 platform coactivators (e.g., SRC-1) that recruit CBP/p300 to effect HAT modification of chromatin that disrupts internucleosomal interactions to convert heterochromatin to transcriptionally active euchromatin; and (iii) DRIP205/Mediator complexes that bridge to the C-terminal domain of RNA polymerase II plus TFIIB, which links VDR-RXR to transcriptional initiation. Alternatively, VDR-RXR may associate with corepressors such as Hr that attract HDACs to return chromatin structure to the transcriptionally inactive heterochromatin state in the vicinity of gene promoters negatively regulated by VDR. The functional activity of hVDR appears to be influenced by common gene polymorphisms, with certain 5′ promoter (Cdx-2), translation start site (F/f ), and 3′-UTR (L/S) alleles yielding higher expression of (Cdx-2 A-allele and 3′-UTR L-allele), or intrinsically more transcriptionally active (translation start site F-allele), hVDR. Such polymorphic hVDR variants affect bone mineral density and may relate to the epidemiology of osteoporosis, as well as significantly alter the risk of certain epithelial cancers. In conclusion, in the time elapsed since the last edition of this volume, we have witnessed an explosion of new information enhancing our understanding of VDR, including crystallographic views of both the DBD and ligand binding/heterodimerization domains, availability of the complete human genome sequence, and thorough characterizations of VDR and RXR knockout mice. However, many questions still remain unanswered. A major structure-function breakthrough would be the X-ray crystallographic solution of fulllength VDR, or at least of a closely related nuclear receptor, so that we can begin to understand the interactions between the DBD and the ligand binding/ heterodimerization domains. A second major realm of future investigation is the identification of additional VDR-regulated genes, particularly those supporting intestinal calcium and phosphate transport, as the molecular mechanism whereby VDR exerts this primary action is currently uncharacterized. An equally exciting endeavor will be the discovery of additional natural VDR or RXR ligands, especially in the more recently identified and studied VDR target tissues such as the hair follicle/skin and immune systems. The availability

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of this information would allow for molecular investigations of the VDR transcriptional control cycle (Fig. 10) in the presence of novel ligands and/or in the context of other promoters in addition to those of the rat osteocalcin or mouse osteopontin genes that have heretofore been the focus of VDR transactivation studies. Such experiments will likely confirm and extend our understanding of the variety of conformations and coregulator associations that the VDR-RXR heterodimer is capable of achieving. Moreover, insights from the approaches just outlined may then enable us to comprehend the interactions between two or more VDREs in the same vitamin D regulated promoter, or between the VDR-RXR heterodimer and other transactivators that are bound to the same promoter. Finally, all of these concepts need to be applied to the poorly understood topic of negative regulation by the VDRRXR heterodimer. Transrepression by VDR is likely complex, and no doubt involves a variety of mechanisms, but its characterization will increase our knowledge of such fundamental VDR tenets as suppression of PTH and the 1α-OHase enzyme in relation to calcium/ phosphate homeostasis, control of PTHrP and other hair cycle-related genes with respect to preventing alopecia, and regulation of Bcl-2 and other genes controlling cell growth and apoptosis that may play a role in the action of VDR as a nuclear receptor sentinel in preventing epithelial cell cancers. Clearly, there are many challenges ahead in our molecular probing of VDR and, undoubtedly, a few more surprises as well.

Acknowledgments Supported by NIH grants to M.R.H. The authors thank other members of our laboratory, Hope T. L. Dang, Jamie Dawson, Neal Hall, Magdalena J. Kaczmarska, and Stephanie A. Slater, for their contributions.

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CHAPTER 14

Vitamin D Receptor Cofactors: Function, Regulation, and Selectivity LEONARD P. FREEDMAN AND ALFRED A. RESZKA Department of Molecular Endocrinology and Bone Biology, Merck Research Laboratories, West Point, Pennsylvania

I. Introduction II. The Vitamin D Receptor and the Basal Machinery of Transcription III. Coactivators IV. Integration of Signaling Pathways

V. Molecular Basis for Tissue-Selective Vitamin D Receptor Ligands VI. Conclusions References

I. INTRODUCTION

multistep process initiated at the promoter region of expressed genes. It is catalyzed by RNA polymerase II (RNA Pol II) and requires the assembly of general transcription factors (GTFs) including TFIIA, -B, -D, -E, -F, -H, at the promoter (illustrated in Fig. 1; for review see [1–4]). This process is regulated by a combination of transcription factors recruited to a given promoter by direct DNA binding to specific response elements. DNA-bound factors mediate protein–protein interactions with components of the transcription machinery, ultimately targeting the recruitment and/or control of RNA Pol II. Several direct contacts have been identified between VDR or other nuclear receptors and the basal transcription apparatus. TFIIB was reported to interact directly with VDR-LBD. Interestingly, this interaction does not include the transactivation motif (AF-2) of VDR [5,6]. Moreover, the ligand may have a distinct effect on this interaction, depending on the cell type [5,7,8]. Taken together these data may suggest the requirement of additional targets for VDR transcription activity. For example, another basal factor, TFIIA, can also directly bind to VDR. This effect is strongly stimulated by ligand and occurs in the context of VDR bound to a promoter DNA template [9]. VDR also binds several TBP-associated factors TAFs (TATA box-binding protein [TBP] associated factors) that comprise the basal factor TFIID. TAFII135 and TAFII55 bind to VDR, RAR, and TR and enhance their activity [10,11]. TAFII28 has a potentiating or repressive effect on VDR and ER activity, depending on the cell type (COS versus HeLa cells, respectively) [12]. Finally, VDR binds to a number of newly discovered factors that appear to bridge or recruit other activities important for the activation process, as described at length later.

Vitamin D3 receptor (VDR) regulates transcription in direct response to its hormonal ligand, 1α,25(OH)2D3. Ligand binding leads to the recruitment of coactivators, defined as proteins that potentiate the activity of specific transcription factors. Many of these cofactors, as part of large complexes, act to remodel chromatin through ATP-dependent subunits or intrinsic histone-modifying activities. In addition, other ligand-recruited complexes appear to act more directly on the transcriptional apparatus. This suggests that transcriptional regulation by VDR and other nuclear receptors may involve a process of both chromatin alteration and direct recruitment of key initiation components at regulated promoters. This chapter will review the major cofactor complexes found to have significant effects on VDR function. The vast number of these coactivators and coactivator complexes provide potential targets for the relatively specific effects that have been achieved with some clinically important ligand analogs of nuclear receptors (for example, raloxifene for the estrogen receptor). This has spurred a tremendous amount of interest and effort in the quest to generate highly selective nuclear receptor modulators that would confer as drugs desirable effects on target tissues in the absence of potentially deleterious side effects. We will review efforts to achieve this kind of profile with vitamin D analogs, with an emphasis on how such selective ligands might function at the level of cofactor regulation.

II. THE VITAMIN D RECEPTOR AND THE BASAL MACHINERY OF TRANSCRIPTION Transcriptional activation of genes regulated by nuclear receptors and other transcription factors is a VITAMIN D, 2ND EDITION FELDMAN, PIKE, AND GLORIEUX

Copyright © 2005, Elsevier, Inc. All rights reserved.

264 III. COACTIVATORS A. The SRC/p160 Family of Coactivators Over the past several years, a growing number of proteins have been identified as coactivators for various nuclear receptors (for review see [3,13,14]). Many of these putative coactivators have been cloned by yeast two-hybrid or GST pull-down assays by virtue of their interaction with members of the nuclear receptor family, and their ability to potentiate transcriptional activity. Among the many nuclear receptor coactivators characterized thus far, a homologous family of proteins has emerged. It has been alternatively named SRC, NCoA, or more generically, p160, based on one of its first identified members, the 160-kDa protein steroid receptor coactivator-1 (SRC-1) [15]. The SRC/p160 family comprises three types of factors, on the basis of their homologies, including SRC-1/NCoA-1, GRIP1/ TIF2/NCoA-2, and pCIP/RAC3/ACTR/AIB1/TRAM-1 (for review, see [16,17]). The original enzymatic activity found to be common to these factors is histone acetyltransferase (HAT) activity, which catalyzes the acetylation of lysine residues at the N-terminal tails of histones [18,19]. Acetylation is thought to destabilize the interactions between DNA and the histone cores that form its nucleosomal structure in the nucleus. The SRC/p160 family members would thereby act as coactivators by loosening the repressive effect of chromatin on gene expression. In the original publications describing these proteins, ligand-dependent binding to VDR was only documented with the coactivator ACTR, but subsequent analyses have shown that VDR is also the target of GRIP1/TIF-2 and SRC-1 [20,21] (also our observations). Specific inactivation of SRC-1 by gene targeting reveals that there may be at least partial functional redundancy between the different SRC/p160 family members, since only a partial resistance to hormonal response is observed in SRC-1(−/−) mice. This phenomenon is concomitant with increased mRNA levels of other coactivators such as TIF2, perhaps compensating for the loss of SRC-1 [22]. The coactivator effects of SRC-1 on nuclear receptors have also been demonstrated in vitro in the presence of chromatin assembled templates [23]. Under these conditions, SRC-1 strongly potentiates the ligand-dependent activity of the progesterone receptor (PR-B). Interestingly, this potentiation also occurs to a certain extent on naked DNA, in the absence of chromatin. These results may suggest a dual effect of SRC-1, both on chromatin remodeling and on other activities or interactions yet to be identified. Beyond this homologous family, several proteins that have been found to act as co-regulators of VDR

LEONARD P. FREEDMAN AND ALFRED A. RESZKA

cannot be classified in any of the previous categories. NCoA-62 /SKIP, identified by yeast two-hybrid, coactivates VDR in a ligand-dependent fashion [24]. ChIP analyses reveal that NCoA-62 associates with liganded VDR after the binding of SRC-1 [25]. Interestingly, NCoA-62 was also shown to interact with components of the spliceosome, and a dominant negative NCoA-62 inhibits splicing of 1α,25(OH)2VD3 -induced transcripts, suggesting that it somehow couples VDR-mediated transcription to the process of RNA splicing. TIF1 was found to interact with VDR and other nuclear receptors [26]. TIF1 also interacts with heterochromatin-associated proteins and has a kinase activity that can phosphorylate several general transcription factors (TFIIEa, TAFII28, and TAFII55), suggesting a novel mechanism of transcription regulation for nuclear receptors [27].

B. Large Coactivator Assemblies with Multifunctional HAT Activity The SRC/p160 family of coactivators not only binds to nuclear receptors, such as VDR, but also recruits and forms complexes with CBP/p300 [19]. The cointegrators CBP and p300 also bind to a large panel of transcription factors [28], and although they do bind to nuclear receptors, this interaction is much weaker than is seen with SRC/p160. Indeed, they appear to interact with nuclear receptors cooperatively along with SRC/ p160 and other components such as p/CIP and PCAF, together forming a larger coactivator complex [13,29]. Alternatively, CBP and SRC-1 may be stabilized by a specific RNA coactivator, SRA (steroid receptor RNA activator). Interestingly, SRA was found to be part of a 600–700 kDa ribonucleoprotein structure that includes SRC-1 [30]. CBP/p300 display HAT activity [31,32], which plays an integral role in chromatin remodeling. The fact that different components of the complex possess HAT activity suggests some sort of a cooperative effect or/and an increased array of specificities. Recent reports suggest additional functions for CBP’s HAT activity besides chromatin remodeling. CBP/p300 can acetylate nonhistone proteins, such as the transcription factor p53, thus enhancing its DNA binding activity [33]. Components of the basal machinery (TFIIEa, TFIIF) are also acetylated by p300, PCAF, and TAFII250, but the effects of this modification have not yet been elucidated [34]. Intriguingly, CBP/p300 can regulate the association between ACTR and the estrogen receptor by directly acetylating ACTR at two lysines near to one of its NR boxes, thereby disrupting its association with ER [35]. Currently, our view of how SRC/p160 functions is primarily to act as means of recruiting

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CHAPTER 14 Vitamin D Receptor Cofactors: Function, Regulation, and Selectivity

CBP/p300 to a nuclear receptor. It is CBP/p300 rather than SRC/p160 that appears to be the primary source of HAT activity.

C. The Human Mediator Complex: DRIP 1. IDENTIFICATION OF THE COMPLEX

Besides the SRC/p160 coactivators, several laboratories, including our own, identified a novel type of nuclear receptor coactivator complex, alternatively called DRIP, TRAP, ARC, NAT, or mammalian Mediator (reviewed

in [36]) (Table I), with each name depending on the purification process and the activators tested as targets. These complexes are required for activation of transcription in vitro, in different transcription assays involving purified components of the transcription machinery. At the time of their respective discoveries, each of these complexes was thought to be specific for a distinct transcription factor. DRIP (vitamin D receptor interacting proteins) [37,38] and ARC (activator recruited cofactor) [39,40] complexes were originally purified from nuclear extracts by in vitro pull-down assays using GST fusions with VDR-LBD or using the activation motifs of several

TABLE I Subunit Composition of General Coactivator Complexes. DRIP

DRIP250 DRIP240 DRIP205 DRIP150 DRIP130 DRIP100 DRIP97 DRIP92 DRIP77 DRIP70–2

ARC CBP/p300 ARC250 ARC240 ARC205 ARC150 ARC130 ARC105/TIG-1 ARC100 ARC92 ARC77 ARC70 ARC42

SMCC/TRAP

TRAP240 TRAP230 TRAP220 TRAP170 TRAP150 TRAP100 TRAP97 TRAP95 TRAP93 TRAP80

CRSP

p230 CRSP200 CRSP150 CRSP130

ARC36 ARC34

DRIP33

ARC33 ARC32 p28

p150 p140/hSur2

p95 p90 CRSP77 CRSP70

Mediator (mouse)

Mediator (Yeast S.c.)

p160a p160b Rgr1/p110

Nut1 Gal11 Rgr1

Ring3/p96a p96b

Sin4 Srb4 Med1

p78 p70 p55

hSrb10 DRIP36 DRIP34

NAT

hMed7 hMed6 hTRF hSrb11 hSoh1

hSrb7 hNut2

CRSP34 CRSP33

p56/Cdk8 p45 p37 p36 p33 p31/Cycl.C p30 p23 p22 p21 p17 p14

p34 Med7/p36 Med6/p32 TRF/p28a p28b

Srb7/p21

Subunits with highlight are equivalent in the different complexes, or homologs between mammalian and yeast complexes. Subunits in the same lanes have similar molecular weights regardless of any homology.

Med2 Pgd1/Hrs1 Srb10 Med4 Med7 Srb5 Med6 Med8 Srb11 Rox3/Ssn7 Srb2 Med9/Cse2 Srb7 Med10/Nut2 Med11 Srb6

266 transcription factors (SREBP-1a, NF-kB, and VP16), respectively. The TRAP complex (TR associated proteins [41,42]) was isolated by coimmunoprecipitation of epitope-tagged TR stably expressed in HeLa cells. TRAP was subsequently found to be identical to SMCC (Srb/Med-containing cofactor complex [43], a complex purified by coimmunoprecipitation with antibodies directed against epitope-tagged Srb10. The cloning of these component subunits by independent groups revealed the near-identity of their sequences, suggesting that these different complexes might actually constitute a single, universal one. Importantly, some subunits do differ from complex to complex. For example, TRAP150 has no homology with any of its candidate counterparts. In addition, DRIP/ARC/CRSP130, also identified as hSur2 [44], has not been identified in the TRAP complex. The various subunit compositions are summarized in Table I. Other complexes exist that have close identities with the DRIP, ARC, and TRAP complexes. CRSP (cofactor required for Sp1 activation) [45], purified by multiple chromatographic steps, appears to be a subset of nine subunits of the DRIP/ARC complex and might represent a stable core of subunits or a conserved subcomplex among various functionally related complexes. The CRSP complex differs, however, by its two unrelated 34- and 70-kDa subunits (Table I). The homology of CRSP70’s N terminus with the elongation factor TFIIS is a unique feature among all the complexes described so far. Despite its apparently limited number of subunits, CRSP potentiates the activity of the transcription factor Sp1 in vitro. The NAT complex (negative regulator of activated transcription) was identified by coimmunoprecipitation out of HeLa nuclear extracts with an antibody against hSrb10/CDK8 [46]. NAT shares many common subunits with DRIP, ARC, and TRAP. However, when the NAT complex was tested in vitro in a purified transcription assay in the presence of RNA Pol II, general transcription factors (TFIIA, to -H), and a cofactor activity PC4 (see later discussion), it exhibited a repressing effect on transcription driven by various activators, without any influence on basal transcription. This unexpected result suggests that these complexes may not only have an activation potential, but also repressive activities on transcription in vitro, as discussed later. The mouse Mediator complex was identified [47] through biochemical purification out of nuclear extracts of murine cells. Its name reflects its homologies with a yeast Mediator counterpart (Table I; see later discussion). 2. FUNCTIONALITY OF THE DRIP COMPLEX

The functional role of DRIP and related complexes in human cells can be postulated on the basis of a series


of classic studies in yeast (for review, see [48,49]). Genetic and biochemical analyses in yeast revealed the importance of several types of factors for transcription of target genes by RNA Pol II in response to activators [49]. They include the products of SRB genes, identified in a genetic screen as suppressors of the effect of truncations within the CTD of RNA Pol II [50]. Srb proteins point to the importance of CTD phosphorylation in the transition between transcription initiation at the promoter and elongation of the RNA [51] (transcript, and references therein). Biochemical fractionation resulted in the isolation of Mediator as a multisubunit complex [52] that contained Srb proteins, and another set of components named Med proteins [53,54]. Detailed functional studies in yeast revealed the importance of both Srb and Med components of the Mediator complex in either general transcription (Srb4 [55]), or activation of transcription by specific factors both in vivo and in vitro, such as Gcn4, Gal4, or VP16 (observed for Med2, Med6, Pgd1/Hrs1, and Sin4 [53,54]). This raised the definition of the yeast Mediator as a “global transcription coactivator” [56]. The existence of human homologs of a number of yeast Mediator proteins suggested that a corresponding complex would exist in higher organisms. Mammalian Mediator was first identified in mice by Kornberg’s group [47]. Its homology to the yeast Mediator is also suggested by some of its functional characteristics: the complex can bind the CTD of RNA Pol II, and it stimulates in vitro CTD phosphorylation, catalyzed by TFIIH. Some of its subunits have yeast counterparts (Rgr1, Med6, Med7, Srb7), but several other subunits lack any yeast homologs. Interestingly some, but not all, of the mouse Mediator subunits are also present within DRIP, ARC, TRAP/SMCC, and NAT (see Table I). That DRIP, ARC, and TRAP/SMCC complexes all contain Med components [40,43,57] may suggest that these complexes are likely to be human homologs of the Mammalian Mediator. However, a side-by-side comparison of the respective subunits within human and mouse complexes favors a scenario where “mammalian Mediator” encompasses subcomplexes purified as DRIP, ARC, and TRAP/SMCC (Table I). A larger diversity of complexes in higher eukaryotes relative to yeast was previously suggested for the RNA Pol II holoenzyme on the basis of its different subunit compositions in mammalian preparations (i.e., variations in a subset of components) [48]. The potential differences in composition of the DRIP, ARC, TRAP/SMCC, NAT, and mammalian Mediator complexes may reflect this diversity. Whatever the case, the presence of Srb/Med subunits in DRIP, ARC, and TRAP/SMCC strongly suggests that at least part of how this complex functions is through recruitment of RNA Pol II. Indeed, experiments demonstrate


that DRIP bound to liganded VDR indeed can recruit RNA Pol II and associated subunits [58]. As mentioned previously, activities of all these complexes were tested in highly purified transcription assays in vitro, in response to specific activators. The DRIP complex strongly potentiated ligand-dependent VDR-RXR transcription on DNA templates assembled into chromatin, but interestingly it had little or no effect on the same transcription in the absence of chromatin (naked templates). None of the previously identified SRC/p160 coactivators have been found to be part of the purified DRIP complex [38], and the absence of any HAT activity in the DRIP complex suggests that it may contain distinct chromatin remodeling activities, or may recruit them. The ARC complex tested on chromatin-assembled templates exhibits cooperativity between different activators, such as Sp1 and SREBP-1a, on the same template promoter, in conditions where ARC has no effect on the same activators tested individually [40]. This is an interesting demonstration of the ability of these complexes to integrate multiple transcription pathways into a synergistic effect on gene activation. TRAP/SMCC also enhances activatordependent transcription in a purified in vitro system with RNA Pol II, GTFs, and PC4, but only in the absence of or with limiting concentrations of TFIIH [43]. In a transcription system including both PC4 and TFIIH, the SMCC complex can repress transcription. This effect was specific for the presence of activators such as Gal4-AH, since no strong effect was observed on basal transcription. By analogy with the effect of Mediator in yeast on phosphorylation of RNA Pol II CTD, the kinase activity of SMCC was tested in vitro. Although the CTD was phosphorylated, the major substrate was the cofactor PC4, a single-stranded DNA binding protein that is required for activated transcription [59,60]. PC4 can interact in vitro with TFIIA and VP16 [60]. More importantly, PC4 binding and transcription activities are lost upon phosphorylation [59,60]. The conservation of PC4 in yeast (as Tsp1) highlights its general requirement among eukaryotic systems [61]. Interestingly, the repression effect by SMCC was also observed when a CTD-less form of RNA Pol II was used. Based on this, the authors suggested that regulation of transcription by SMCC could be mediated in concert with PC4, but independently of modifications of the RNA Pol II CTD [43].

D. ATP-Dependent Remodeling Cofactors In addition to HAT and HDAC-containing activities, complexes found biochemically to contain ATPdependent chromatin remodeling activities, which

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could be expected to produce more significant chromatin rearrangements, have also been linked to steroid and nuclear receptors. Yeast genetics originally identified a group of gene products, SNF/Swi, which reduced expression of the SUC2 and HO genes involved in sucrose fermentation and mating-type switching, while among suppressors of these genes, SIN or switchindependent mutations, were identified to be histones and other chromatin-associated proteins. SNF2/Swi2 homologs were subsequently found in Drosophila (Brahma) and mammalian cells (hbrm and BRG-1) [62]. Purified multisubunit SNF/Swi complexes were later found to contain DNA-dependent ATPase activity intrinsic to SNF2/Swi2, which simultaneously disrupted nucleosomes and enhanced transcription factor binding [63,64]. Purification of hbrm and BRG-1 revealed they are present in distinct mammalian complexes associated with selective BRG-1 associated factors (BAFs) [65]. ATP-dependent remodeling by these complexes has been shown to be independent of histone tails and is both persistent upon removal of ATP and reversible upon readdition of SNF/Swi [66,67]. An early connection of SNF/Swi to nuclear receptormediated transcription was revealed by the loss of steroid-induced reporter gene activation by GR and ER in Swi-1, -2, and -3-mutant yeast strains [68]. Additionally, when overexpressed in cells, hbrm was found to cooperate with transfected GR in mammalian cell lines devoid of endogenous hbrm [69] and ER was observed to interact with both hbrm and BRG-1 in yeast in twohybrid experiments [70]. Furthermore, conditional BRG-1 knockout F9 cell lines were found to be inviable, while heterozygote lines could not proliferate yet remained sensitive to RA-induced differentiation [71]. One extensively examined model for ligand-dependent perturbations of chromatin structure in preinitiation complex (PIC) formation, as well as antagonism between the progesterone and glucocorticoid receptors (PR and GR), has been the integrated composite MMTV promoter/enhancer (reviewed in [72]). Transiently transfected MMTV DNA is activated by PR ligands; however, random integration of this same DNA into the mammalian genome with concomitant packaging into a phased array of chromatin eliminated PR-dependent gene activation by preclusion of binding site accessibility, but also induced progestin-specific repression of glucocorticoid-mediated transcription from MMTV, suggesting competition for common molecular targets. One report has described a model for this competition mediated through BRG-1 [73]. Interestingly, both progestins and anti-progestins inhibited GR stimulation of integrated MMTV DNA and decreased its association with limiting amounts of BRG-1, as assessed by coimmunoprecipitation. These same compounds were unable

268 to reduce transactivation of transiently transfected promoters, nor were they able to destabilize GR bound to the p160 coactivators NCoA-1/SRC-1 and NCoA2/GRIP-1, as well as with p300. Thus the observed squelching was a function of recruitment of remodeling complex(es) and not through association of other coactivator complexes, and may reflect an initial binding threshold for target site accessibility. In addition to SNF/Swi, other ATP-dependent remodeling complexes have been isolated, including the distinct yeast RSC complex, and a related family of complexes which share homologs of the SNF2/Swi2related ISWI ATPase, namely the Drosophila complexes NURF, ACF, and CHRAC and the human RSF complex (recently reviewed in [74]). The ISWI-containing complexes were initially purified biochemically based on their abilities to catalyze the assembly of nucleosomal DNA (ACF [75]), physiologically space salt or polyglutamate-deposited nucleosomes (ACF [75], CHRAC [76], RSF [77]), enhance transcription factor binding (NURF [78]), and increase transcription from nucleosomal templates in vitro (NURF [79], RSF [77]). Recently, another distinct ATP-dependent remodeling activity was found associated with an HDAC-containing complex, NURD, which also possesses a subunit with limited homology to N-CoR, MTA-1 [80,81]. Intriguingly, antibodies directed against the CHD4/Mi-2 ATPase subunit of NURD partially relieve ligand-independent TR/RXR repression of the TRβA promoter in Xenopus oocytes by microinjection experiments [80]. Recently, two new chromatin remodeling complexes have been characterized through profound functional effects on VDR. VDR has been found to associate with a novel complex called WINAC, which shares subunits of the SWI/SNF and ISWI complexes [82]. WINAC interacts directly with VDR, albeit in a ligandindependent manner, through one constituent subunit, the Williams syndrome transcription factor (WSTF). Interestingly, the WSTF gene is deleted in patients with Williams syndrome, a neurodevelopmental disorder that, in addition to causing cognitive disorders, also results in congenital heart disease. Kitagawa and coworkers found, using chromatin immunoprecipitation (ChIP) assays, that WINAC is recruited to both negative and positive VDREs by VDR, and that WSTF overexpression potentiated VDR-mediated transactivation or transrepression. Presumably, this occurs through the ability of the WINAC complex to enhance both the assembly and disassembly of nucleosomal arrays. As it contains BRG1, WINAC functions in an ATP-dependent fashion. A second chromatin-remodeling complex recently described also appears to be important for VDR transcriptional function, at least in a purified, reconstituted


transcription system. Using conventional biochemical purification, Lemon and co-workers [83] purified an activity that is required for ligand-dependent transactivation by VDR using a chromatin-dependent reconstituted in vitro transcription assay. This activity was identified as a multisubunit complex called PBAF, which shares many, but not all, of the same subunits as SWI/SNF-A. Using antibody depletion, it was found that only PBAF, but not SWI/SNF, supported VDRdependent transactivation, suggesting that at least in this experimental system, which is dependent on the presence of the DRIP complex, these two chromatin remodeling complexes are not functionally equivalent, nor does one complement the activity of the other. The ever-expanding number of chromatin-remodeling, histone-modifying, and Pol II–recruiting complexes that interact with and are utilized by nuclear receptors in general and VDR in particular makes this a particularly challenging scientific question to study. It also suggests that this wide repertoire could be the basis for tissue selectivity by VDR analogs and other nuclear receptor modulators (see next section). However, it also suggests that the process of transcriptional regulation involves multiple steps mediated by multiple complexes conferring distinct functions, whereby VDR and other nuclear receptors serve as ligand-regulated platforms for these complexes to the DNA in the proximity of regulated gene promoters. Some chromatin immunoprecipitation experiments are consistent with this so-called ordered-recruitment model, but it may also turn out that specific promoters (or more accurately, specific VDREs) impose the recruitment of a selective repertoire of cofactor complexes through allosteric effects perpetuated through DNA-bound VDR. The delineation of tissue selectivity by VDR ligands, then, will be an exceedingly complicated paradigm to define, but it may be that this complexity is actually what nature had in mind to achieve maximal regulatory flexibility.

IV. INTEGRATION OF SIGNALING PATHWAYS The WINAC, SRC-1/p160, and DRIP complexes represent three unrelated protein complexes, together carrying several distinct activities that have been shown to potentiate transcription activity of VDR (and other nuclear receptors). These activities may function together to provide a synergistic effect of 1,25(OH)2D3mediated activation, or to provide specificity of targeting to its regulation. VDR transcription regulation may require a combination of chromatin remodeling activities, as well as efficient recruitment of the RNA Pol II

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motif in the DRIP complex as a combination of two subunits, and might explain the presence of two unrelated activation functions in the same receptor. In the TRAP/SMCC complex, both p53 and VP16 interact via TRAP80, but binding of VP16 and TR (which binds TRAP220) to the TRAP complex is not mutually exclusive, suggesting that this complex may support activation by a combination of distinct transcription factors. The transactivation domain (conserved region 3, CR3) of E1A interacts directly with hSur-2, which corresponds to DRIP130 [44]. This defines yet another novel and distinct target motif for an activator within the complexes. Several other classes of transcription factors have been found to functionally interact with DRIP subunits, and presumably the entire complex. One noteworthy example is interferon-activated ISGF3 transcription factor. ISGF3 is a heterotrimeric complex comprising STAT1, STAT2, and a DNA-binding subunit, IRF9. Upon induction by interferons, resulting in tyrosine phosphorylation of STATs, the complex enters the nucleus, binds to interferon–stimulated response elements, and activates transcription of regulated promoters. Lau and co-workers [88] have reported that ISGF3stimulated transcription is dependent on direct STAT2 interactions with DRIP150. This interaction leads to the recruitment of other DRIP subunits as well as RNA Pol II to ISGF3 target promoters, as demonstrated by chromatin immunoprecipitation. These results

machinery, via several of its basal factors. We have suggested a stepwise model that combines these distinct activities (Fig. 1), where WINAC and/or CBP/p160 coactivator complexes might be required for chromatin remodeling, followed by the direct recruitment of the transcription machinery by the DRIP complex. Definitive experimental analysis has yet to confirm this view [14,84]. In vitro, however, the SRC/p160 and DRIP coactivators appear to have no real intrinsic difference in their ability to interact with nuclear receptors. They both utilize the AF-2 of nuclear receptors and bind with similar affinities (TRAP220 versus TIF2 for TR [85]; DRIP205 and GRIP1 for VDR [86]. Thus we could envision a cooperative model where both CBP/p160 and DRIP complexes simultaneously occupy a promoter, and through their combined actions facilitate activation of transcription (Fig. 1). The observation that CBP can acetylate ACTR, leading to the latter’s dissociation from liganded ER [35], suggests a mechanism for the sequential model, whereby the first complex functions to acetylate histones and disrupt chromatin structure, whereupon it itself dissociates from the receptor, allowing the DRIP complex to bind and act at the level of direct recruitment. Recent studies have revealed multiple binding motifs for coactivators within the DRIP and TRAP complexes. For example, GR interacts with the DRIP complex via both AF-1 and AF-2 motifs with DRIP150 and DRIP205, respectively [87]. These results define the GR binding

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270 implicate an entirely distinct signaling system as functioning through the DRIP complex, and suggest that diverse pathways, such as the vitamin D and interferon signaling systems, may intersect or compete for limiting cofactors (such as the DRIP complex) by sharing subunits required for their optimal transactivation function.

V. MOLECULAR BASIS FOR TISSUE-SELECTIVE VITAMIN D RECEPTOR LIGANDS A. Established Paradigms The molecular basis for tissue selectivity of various VDR ligands is an area of considerable clinical interest. Most intriguing is our expanding understanding that mechanisms conferring tissue selectivity for one type of nuclear receptor may not fully apply to another. The concept of a tissue-selective ligand for a nuclear receptor was first used to describe ligands for the estrogen receptor (ER) that maintained certain estrogenic effects in the absence of others. To a great extent, such ligands, SERMs such as tamoxifen and raloxifene, act as antagonists in certain tissues and cell types (e.g., breast) while conferring some degree of agonism in others (e.g., bone). For the most part, receptor agonism by these ligands is less than that seen with the natural ligand, estradiol. Ligands for the VDR differ in the respect that superagonists have been described in addition to antagonists. For the superagonists, no equivalent ER or PR paradigm exists to help explain the genesis of such activity. For the antagonists, we find that, as with SERMs, some residual agonism is left intact, suggesting VDR modulator-like activity. Interestingly, the molecular basis for partial VDR agonism seems to differ from that described for ERα. Earlier work with ERα sought to explain tissue selectivity through X-ray crystallography analysis. Crystallography comparing the structure of ERα when liganded with estradiol or raloxifene revealed that the position of helix 12 differed, depending on which ligand (agonist or antagonist) was bound into the LBD [89]. Similar differences are predicted to exist for the progesterone receptor when bound to progesterone and the selective progesterone receptor modulator (SPRM), RU486 [90]. The shifting of H12 after modulator (SERM, SPRM, etc.) binding changes the topology of the AF-2 domain and alters the ability of the receptor to recruit coactivators. It also enhances the ability of the receptor to recruit co-repressors, and thus suppresses gene transcription. Although crystallographic analyses of the VDR have been reported, none have yet examined the


conformation of the receptor in the presence of an antagonist. The X-ray structure in the presence of the natural ligand, 1α,25(OH)2D3, shows that VDR has a somewhat larger ligand-binding pocket (697 Å3) than that for ERα (369 Å3) or PR (422 Å3) [91]. This means that 1α,25(OH)2D3 occupies a smaller proportion of the pocket (56%) than with either ERα (63%) or PR (67%). This may suggest that a number of different ligands could bind to VDR and allow the induction of several conformations upon ligand binding. Although we do not have information regarding the structure of VDR in the presence of an antagonist, data do exist for the binding of superagonists [92]. Interestingly, regardless of whether it is bound by 1α,25(OH)2D3 or by either of two superagonists (MC1288 or KH1060), VDR seems to respond in a similar fashion. The overall protein structure is identical and the A- to D-ring moieties of each ligand form identical contacts, bound in identical orientations, with key residues within the VDR LBD. Conclusions based on this work include that superagonist-bound VDR might be more stable and exhibit a longer half-life than seen with the natural ligand. Other work suggests that superagonists can selectively enhance coactivator recruitment. This does resemble the selective effects of ERα and PR ligands on coactivator and corepressor recruitment, although several fundamental differences separate VDR from these nuclear receptors, as discussed below. Selective coactivator recruitment in and of itself is only part of how SERMs and SPRMs elicit tissuespecific responses. To a certain extent, a ligand can elicit only one conformation of the receptor, and this confers a specific affinity of the receptor for each individual coactivator and co-repressor. After this, the altered responses of nuclear receptors to these partial agonists vary cell-type to cell-type, based on the relative levels of expression of each of the coregulators. Thus in a cell with high levels of the right coactivator, e.g.,SRC-1, an agonistic response might be seen, whereas in other cell types, reduced expression of the required coactivator can result in better co-repressor recruitment and resulting transcriptional repression. This can also be achieved through higher levels of co-repressor expression within a given cell type. As noted above for ERα, the altered positioning of H12 upon binding to raloxifene changes the relative affinity of the receptor for the various co-regulators. The clearest evidence for ligand-induced and tissue-selective recruitment of coactivators was generated using chromatin immunoprecipitation (ChIP) analyses [93]. Using MCF-7 and Ishikawa cells as models for breast and uterine stimulation, estradiol was compared to both tamoxifen and raloxifene. Whereas both SERMs display similar antagonism of breast cancer growth, tamoxifen is


considered to be quite uterotropic. Not surprisingly, both breast cancer antagonists displayed a similar lack of ability to recruit coactivators, such as SRC-1, GRIP1, and AIB1, to estrogen-responsive promoters in MCF-7 breast cancer cells. In addition, both effectively recruited the SMRT and NCoR corepressors, along with two histone deacetylases, all of which act to suppress gene transcription. Interestingly, and consistent with their divergent effects on the uterus, raloxifene maintained this general profile in Ishikawa cells, whereas the response to tamoxifen in these cells resembled that of the natural agonist, estradiol. However, of the two promoters tested (c-Myc and cathepsin D) only the proliferative c-Myc promoter showed the agonist recruitment profile. How then is selectivity generated? In the case of MCF-7 versus Ishikawa cells, the selectivity can be traced back to the relative expression of the coactivator, SRC-1, whose expression is diminished in MCF-7 cells (versus that seen in Ishikawa) [93]. Increased expression (via transfection) of SRC-1, but not GRIP1 or AIB1, enhanced the ability of tamoxifen, but not raloxifene, to induce gene expression off of the c-Myc (and IGF-I) promoter. This type of selective transcriptional response, as influenced by altered ratios of coactivators and co-repressors, was also reported for PR in its response to RU486 [94]. Following the observation that RU486 acts as an agonist in T47D (breast cancer) cells and an antagonist in HeLa (cervical carcinoma) cells, it was observed that the T47D cell line has diminished expression of both NCoR and SMRT corepressors, although expression levels of SRC-1 appeared to be normal. Consistent with its ability to convert tamoxifen into an agonist in MCF-7 cells, overexpression of SRC-1 in HeLa cells can convert RU486 into a partial agonist. RU486 can also be converted into an antagonist in T47D cells, where it otherwise displays partial agonism, by the overexpression of SMRT. This results in the suppression of RU486, but not progesterone, recruitment of SRC-1, to PR and, consequently, suppressed ligand-induced transcription. Distinguished from the SPRM-like activities of RU486, the PR pure antagonist, ZK98, is refractive to SRC-1 overexpression and maintains its pure antagonist profile. Antagonism for this ligand does not require overexpression of any co-repressor in these cells.

B. The VDR Paradigm(s) 1. ANTAGONISTS/PARTIAL AGONISTS

With a paradigm in place for two well-characterized nuclear receptors, it seems logical that VDR should act in an identical fashion in response to partial agonists.

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Agonists and superagonists should favor binding of coactivators over co-repressors, while partial antagonists should favor recruitment of co-repressors. To some extent this paradigm holds true, except where co-repressors are concerned. The binding of VDR to co-repressors in the unliganded state is relatively weak, and the physiological role for this interaction, if any, remains unknown. Indeed, mammalian two-hybrid analyses directly comparing VDR to thyroid hormone receptor (TR) showed that TR recruitment of SMRT is up to 26-fold stronger than that for VDR, while the recruitment of NCoR was an incredible 335-fold stronger [95]. One might expect that the binding of an antagonist ligand to VDR could enhance recruitment of either of these co-repressors, but this has not been seen. Indeed, the antagonists, ZK159222 and TEI-9647, actually displace a weakly bound NCoR in GST pulldown analyses. A similar response has also been reported for the Gemini ligand, which displaces NCoR binding in both GST pull-down and mammalian two-hybrid analyses (Gonzales et al., in preparation, 2003). Interestingly, however, overexpression of NCoR along with VDR and RXR has been found to partially suppress Gemini and EB1436-mediated transcription to a greater extent than that seen with the natural ligand, 1,25(OH)2VD3 [96]. This provides some evidence for ligand-specific effects of a co-repressor on VDRmediated gene transcription. Unresolved is the means by which this transcriptional repression is mediated, given the displacement of the co-repressor in the presence of the Gemini ligand. Meanwhile other recent studies have shown that the hairless co-repressor, which suppresses VDR-mediated transcription in the presence of 1,25(OH)2VD3, also binds to the VDR [97]. Consistent with its ability to suppress agonist-induced transcription, the hairless co-repressor binds VDR with equal affinity in the presence or absence of the natural ligand. Whether or not the hairless co-repressor could play a role in partial antagonism by vitamin D analogs remains to be established. 2. SUPERAGONISTS AND SELECTIVE COACTIVATOR RECRUITMENT

In considering the actions of VDR superagonists, several mechanisms seem to play a role, each changing, depending on the ligand tested (Fig. 2). For the superagonist ligand, numerous effects have been described relating to enhanced VDR-RXR heterodimer formation and enhanced recruitment of several of the coactivators. The formation of a VDR-RXR heterodimer in and of itself is an interesting aspect of VDR biology [98]. For many years, RXR was considered a silent partner for VDR, as transcription off of the heterodimer was dependent on VDR ligands alone. Recently, however,

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several lines of evidence have suggested that RXR plays a prominent role in VDR biology in both determining subcellular localization and, indeed, participating in the control of gene transcription. For instance, the dimerization domain of RXR encodes a nuclear localization sequence that is required for VDR translocation to the nucleus [99]. Mutation of this sequence traps VDR in the cytoplasm when in the unliganded state. Interestingly, however, 1,25(OH)2VD3 triggers the localization of NLS-deficient RXR-VDR into the nucleus. Paradoxically, it also enhances the export rate of the normal heterodimer from the nucleus. Within the nucleus, RXR also contributes to 1,25(OH)2VD3-induced transcription. This has been demonstrated by the ability of RXR-specific LXXLL motif peptides to suppress 1,25(OH)2VD3-induced transcription [100]. Very recently, VDR ligands have been shown to elicit a “phantom ligand effect” on RXR and thus to enable the recruitment of p160 family coactivators to RXR itself [101]. This could play a role in superagonist activity by certain VDR ligands, as suggested by the ability of certain of these ligands to induce heterodimer formation at concentrations well below that required by the natural ligand. This has

been demonstrated for the 20-epi ligands, MC1627, MC1288, and 2-methylene-19-nor-(20S)-1,25-dihydroxyvitamin D3 (2-MD), which bind VDR with equal affinity to 1,25(OH)2VD3, yet induce heterodimer formation at concentrations 10 to 100-fold lower [102–105]. It remains unknown whether or not the RXR moiety of the VDR heterodimer recruits coactivators under these circumstances. Enhanced heterodimer formation may contribute to the enhanced transcriptional activity of MC1288, but this is likely only part of the story (Fig. 2). Perhaps more important is the ability of this ligand to enhance recruitment of the DRIP complex to VDR [102]. Interestingly, while MC1288 induces enhanced DRIP205 recruitment, recruitment of SRC-1 and GRIP-1 coactivators is no greater than that seen with the natural ligand [102,103]. Meanwhile, 2-MD shows enhanced recruitment of DRIP205, SRC-1, and GRIP-1 [105], whereas MC1627 shows enhanced DRIP205 and GRIP-1 recruitment. In the context of myeloid cell differentiation, which is potently induced by both MC1288 and MC1627, it is the recruitment of DRIP205 that best correlates with the phenomenon. With regard to 2-MD, it remains unknown whether or not any specific


coactivator recruitment is responsible for enhanced bone anabolic activity. Selective recruitment of DRIP205 and p160 family members can also be regulated as part of the normal differentiation process, as demonstrated in the keratinocyte [106]. In this study, Vitamin D3 analogs were not examined, but by inference, analogs displaying selective coactivator recruitment are likely to show enhanced tissue selectivity. Using GST-VDR as bait, coactivator complexes were purified from both proliferating and differentiated keratinocytes in a 1α,25(OH)2VD3 dependent fashion. Interestingly, whereas DRIP205 and associated proteins predominated in the proliferating cells, AIB-1 (SRC-3) and GRIP-1 predominated after differentiation. This was associated with reciprocal changes in coactivator expression, DRIP205 declining with differentiation and AIB-1 increasing. Although overexpression of either cofactor could enhance transcription in the proliferation phase, only AIB-1 was effective postdifferentiation. We see this as intriguing, since ligands such as MC1288 can enhance recruitment of DRIP-205 during their induction of differentiation, as described earlier. Putatively, such selective coactivator recruitment, coupled with selective expression in the keratinocyte, could enhance the differentiation response in this system. Indeed, MC1288 suppresses proliferation and stimulates keratinocyte differentiation at concentrations 600- and 700-fold lower than seen with the natural ligand, respectively [107]. The enhanced potency of MC1288 in the keratinocyte versus myeloid cells, whereby MC1288 is approximately 100-fold more potent than 1α,25(OH)2VD3, may suggest a somewhat synergistic effect of the ligand specificity with the heightened and selective expression of DRIP-205 in the proliferating keratinocyte. That VDR should show different capacities to recruit one coactivator versus another in a ligand-dependent state is consistent with somewhat different means of recruitment for each coactivator. Indeed, a direct comparison of GRIP-1 and AIB-1 showed that different sequences within VDR are required to recruit each coactivator [108]. Furthermore, RXR seems to play a more active role in recruitment of AIB-1 than GRIP-1 in VDR-RXR-mediated transcription. Nonetheless, there is greater evidence for enhanced GRIP-1 recruitment by VDR ligands than for any other coactivator. Indeed, whereas several ligands can induce an interaction between VDR and SRC-1, GRIP-1, and AIB-1, OCT (22-oxa 1α,25(OH)2VD3) only poorly recruits either SRC-1 or AIB-1, while its recruitment of GRIP-1 is comparable to that of the natural ligand [109]. Consistent with this, GRIP-1, but not SRC-1, overexpression

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substantially enhances OCT-induced transcription in a fashion reminiscent of SERM effects on ERα, as described earlier. Analyses of a broad panel of vitamin D analogs showed that GRIP-1 was more consistently recruited at levels closer to that of the natural ligand than was AIB-1 [104]. For these ligands, transcriptional activity was enhanced by overexpression of GRIP-1, whereas, AIB-1 overexpression actually suppressed transcription. To date, we know of only one example of cell type–dependent coactivator recruitment by VDR modulators. This type of activity was demonstrated for the noncalcemic analog Ro-26–9228, which recruits GRIP-1 in human osteoblasts, but not in CaCo-2 cells [110]. In this study, the coactivator was tested in vitro as a GST-fusion protein; therefore the differential recruitment of GRIP-1 to VDR in these two different cell types could not be attributed to altered expression of the coactivators. Since similar selective recruitment of DRIP-205 and SRC-1 was also reported, it seems that VDR is somehow modified by its expression in these different cell types. This also distinguishes this analog from numerous others, which show more or less selective recruitment of GRIP-1 over other tested coactivators. In summary, the VDR-RXR heterodimer seems to have its own mechanisms for the genesis of VDRMlike activity. As with ERα and PR, VDR can display somewhat selective coactivator recruitment in the presence of a broad range of tissue-selective ligands. Although the data are limited, the preponderance of evidence suggests that co-repressor recruitment is not at all involved in the genesis of VDRM activity. Binding of NCoR and SMRT is weak in the unliganded state, and this is further reduced by the binding of agonist and antagonist ligands. Coactivator-dependent effects on superagonism are also seen with certain ligands. These ligands display both enhanced VDRRXR heterodimer formation and enhanced recruitment of DRIP205 and, perhaps, GRIP-1 in a ligand-specific manner (Fig. 2). Overall, this suggests that the modulation of VDR transcriptional activity is coactivatorspecific. This sets a new paradigm for VDR that is distinct from that of ERα and PR, which respond to coactivator and co-repressors, depending on relative expression levels.

VI. CONCLUSIONS Transcriptional regulation by 1,25(OH)2D3 can be dissected into several functional activities that are mediated by VDR. Generally, transactivation requires that a repressed state of chromatin has to be disrupted

274 in a given target gene’s regulatory regions, together with the ability to promote productive elongation of RNA products by the RNA Pol II machinery at the site of transcription initiation. Several candidates for such activities that are recruited by nuclear receptors in direct response to ligand binding have been identified, as has been described in this review. The HAT activitycontaining coactivators (SRC/p160 family, CBP/p300, PCAF, etc.) appear at face value to act primarily in the disruption of chromatin through histone modifications, although a series of additional, provocative targets corresponding to ATP-dependent chromatin remodelers have more recently been identified. The regulation of RNA Pol II and its ability to respond to activators appears to be mediated by a distinct type of cofactor complex (DRIP/ARC/TRAP/SMCC/Mediator) whose functions are not yet completely elucidated. Given the fact that many classes of activators interact with DRIP beyond VDR and nuclear receptors, this complex must be considered as a regulatory panel for RNA Pol II rather than an exclusive target for nuclear receptors. DRIP may be therefore viewed as a downstream target of multiple transcription activators, perhaps conferring on RNA Pol II the ability to simultaneously integrate multiple signaling pathways onto a single promoter in vivo. This model may provide a key for an interpretation of some unresolved mechanisms involving the simultaneous cross-talk of several signaling systems.

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CHAPTER 15

Vitamin D Nuclear Receptor Ligand-Binding Domain Crystal Structures NATACHA ROCHEL AND DINO MORAS Département de Biologie et de Génomique Structurales, IGBMC, CNRS/INSERM/Université Louis Pasteur, Illkirch, France

I. II. III. IV. V.

Introduction Biological Properties of hVDR∆ Solution Studies Crystal Structure of hVDR∆ Bound to 1α,25(OH)2D3 Mutant Analysis

I. INTRODUCTION All nuclear receptors (NRs) are modular proteins that harbor one DNA-binding domain and one ligandbinding domain (LBD) (Fig. 1) [1,2]. NRs act as agonistinduced factors that enhance or suppress transcription of their target genes. Certain receptors can act as silencers of transcription in the absence of ligands or the presence of certain antagonists. Agonists induce a change in the structure of the NR that allows interaction with coactivators that can acetylate histones, which prepare target gene promoters through decondensation of the corresponding chromatin [3]. Following decondensation, a second complex, called TRAP (thyroid receptor–associated proteins) or DRIP (Vitamin D receptor–interacting proteins) appears to establish linkage to the basal transcriptional machinery [4,5]. The ligand-binding domain of nuclear receptors harbors ligand-dependent activation function or AF-2, a major interface for dimerization with RXR and interface for coactivators as well as co-repressors. This domain is highly structured and encodes several functions in a ligand-dependent manner. Detailed molecular insights into the structure–function of nuclear receptors have been gained by the elucidation of the crystal structures of the LBD alone or in complexes with agonists, antagonists, and co-regulator peptides (Fig. 2). The first 3D structures reported for NR LBDs were those of the unliganded RXRα [6], the all-trans-retinoic acid–bound RARγ [7], and the agonist-bound thyroid receptor TRβ [8]. Unliganded receptors are referred to as apo receptor forms while liganded referred to as holo forms. To date, the crystal structures of 23 distinct NR LBDs VITAMIN D, 2ND EDITION FELDMAN, PIKE, AND GLORIEUX

VI. VII. VIII. IX.

Crystal Structure of zVDR Bound to 1α,25(OH)2D3 Structure of hVDR Complexed to Superagonist Ligands Structure of zVDR in Complex with Gemini Conclusion References

and four LBD heterodimers have been reported. The later concern the EcR LBD bound to an antagonist and RXRα LBD bound to phospholipid ligand [9] and LXβ and RXRβ bound to agonists ligands [10]. The general fold of nuclear receptors consists of a threelayered α-helical sandwich. The helices have been designated H1 to H12, according to the first crystal structures of RXR and RAR [11]. The different crystal structures of apo and holo forms of RXR LBDs as well as extensive NR mutagenesis has demonstrated that the LBD undergoes a major conformational change upon ligand binding (Fig. 2). Ligand-induced conformational changes for NRs have been likened to the “mousetrap mechanism” described for RAR [7]. Upon ligand binding, helix H11 is repositioned in the continuity of helix H10, and helix H12 swings to seal the binding cavity while the ω-loop flips over underneath helix H6 carrying along the N-terminal part of helix H3. Helix 12, also referred as the activating domain AD of the AF-2 function, stabilizes ligand binding by contributing to the hydrophobic environment, in some cases making additional contacts with the ligand. The structural data reveal that H12, when folded back onto the core of the LBD, forms a hydrophobic cleft together with other surface-exposed residues that accommodates the “NR box” of coactivators such as members of the SRC-1/TIF2 family [12–14]. VDRs have been characterized from mammals [15–17], birds [18], Xenopus laevis [19], Paralichtus olivaceus [20], zebrafish (GenBank accession number AAF21427), and recently from lampreys [21]. Sequence analysis of the VDR subfamily members reveals that VDR presents a large insertion domain at the N-terminal Copyright © 2005, Elsevier, Inc. All rights reserved.

280

NATACHA ROCHEL AND DINO MORAS

A/B

C

D

E

N

F AD

C

DNA binding dimerization

DIMERIZATION NLS LIGAND BINDING

AF-1 Activation function ligand independent

AF-2 Activation function ligand-dependent interaction with cofactor

FIGURE 1 Structural and functional organization of nuclear receptors. NRs consist of six domains (A–F) based on regions of conserved sequence and function.

N

H9

H8

H1

H4 H10

H5

H3 H3 H7

H2

H12 9-cisRA

H11 H11

H6

part of the LBD in the peptide connecting helices H1 to H3. The length of this connecting region varies between 72 and 81 residues compared to 15–25 residues for the other nuclear receptors. This insertion region, for which no biological function has been found, is poorly conserved between the VDR species with only 9% identity. One mutation associated with the genetic disease vitamin D–resistant rickets type II has been found in this region (Cys190Trp of hVDR) with no effect on ligand binding [22]. A phosphorylation site also has been identified at Ser208 [23]. Secondary structure prediction reveals that this region is not structured. Among the NR LBD crystal structures solved, it has been reported frequently that the region connecting helices H1 to H3 is poorly ordered and shows little if any secondary structure. In hVDR, this region is sensitive to proteolysis with a trypsin cleavage site at the C terminus of Arg174 [24]. In order to stabilize the overall structure of the hVDR LBD, a VDR LBD mutant has been engineered by removing 50 residues in the region connecting helices H1 to H3 [25,26]. This VDR mutant, hVDR∆ (118-427∆166-216), stabilized the protein by lowering the number of conformations adopted by the insertion region. This hVDR∆ has been fully characterized in solution and compared to the wild-type hVDR.

H12

II. BIOLOGICAL PROPERTIES OF hVDR∆

FIGURE 2 Superimposition of unliganded (green and yellow) and liganded (blue and red) hRXRα LBD monomers. The main conformational differences affect helices H3, H11 and H12. The arrows show the main structural changes upon ligand binding. Adapted from Fig. 4A of Egea PF, Mitschler A, Rochel N, Ruff M, Chambon P, Moras D 2000 Crystal structure of the human RXRalpha ligand-binding domain bound to its natural ligand: 9-cis retinoic acid. EMBO J 19:2592–2601 [48].

The ability of hVDR∆ to bind ligands was determined by Scatchard analysis. This VDR mutant exhibits the same binding affinity for 1α,25(OH)2D3 and 1α,25(OH)2D3 analogs as the hVDR wild type [25,26]. Functional studies of the hVDR∆ and hVDR wild type were performed in order to compare their transactivation properties. In a first system, the hVDR LBD mutant and wild type were fused to the DNA binding domain of the yeast activator Gal4. The chimeric proteins were

CHAPTER 15 Vitamin D Nuclear Receptor Ligand-Binding Domain Crystal Structures

expressed by transient transfection in COS cells and transactivation was measured with a Gal4-responsive reporter. Both proteins exhibit comparable transactivation [25]. In a second system, the full-length hVDR mutant or wild type was cotransfected with a luciferase reporter plasmid containing the osteopontin gene VDRE under the control of the thymidine kinase promoter [26]. Similar results with regard to activation were obtained for the two VDR constructs, confirming the functional integrity of the hVDR∆ protein.

III. SOLUTION STUDIES Like the wild-type receptor, the hVDR∆ behaves as a monomeric species and is able to heterodimerize with RXR. The hVDR∆/RXRs LBDs complexed to 1α,25(OH)2D3 and 9-cis-retinoic acid form a stable species and the two partners are able to bind their ligands as seen by native electrospray ionization mass spectrometry and analytical ultracentrifugation [26]. Low-resolution structural information on the hVDR structure has been obtained by a small-angle X-ray scattering study [26,27]. The scattering function is time and space averaged. The radii of gyration (Fig. 3) of hVDR and hVDR∆ complexed to 1α,25(OH)2D3 were measured and are 26.2 Å and 23.4 Å, respectively. The difference of 2.8 Å, which would correspond to an increase in volume of 35% for a spherical object, is large and would suggest an appended insertion domain weakly connected to the main core LBD. The longest distance in the protein was also estimated in this study. A difference of 15 Å between the Dmax of hVDR and hVDR∆ suggests again that this insertion region may be positioned along the axis collinear to a N- to C-terminal axis including helix H12. This solution study confirms that the insertion domain is mobile and weakly connected to the rest of the LBD, which is itself structured similarly to that of the hVDR wild type.

IV. CRYSTAL STRUCTURE OF hVDR∆ BOUND TO 1α,25(OH)2D3 Deletion of the hinge region insertion in hVDR∆ stabilizes the protein and allowed the crystallization of the hVDR–1α,25(OH)2D3 complex [25]. The crystal structure was solved at 1.8 Å by a combination of molecular replacement using a homology model based on the retinoic acid receptor RARγ [7] and isomorphous replacement with a mercurial derivative. A higher resolution (1.5 Å) data set was collected later [28]. The overall topology of the hVDR∆ LBD (Fig. 4) is that of the canonical LBD with 13 α helices sandwiched in

281

three layers and a three-stranded β sheet. Helices H1 and H3 are connected by two small helices H2 and H3n, H3n replacing the ω loop of the RARγ structure [7]. The truncation of the hVDR∆ construct is positioned just before H3n as shown in Fig. 4. The VDR LBD structure is closely related to that of the holo hRARγ LBD structure [7] with a root mean square deviation (rmsd) of the superimposed structure of 1.2 Å over 179 residues. The connection between helices H1 and H3 follows a path between H3 and the tip of the β-sheet similar to that of the ERα structure [29]. The tip of the β sheet is consequently shifted outward and enlarges the ligand-binding cavity. The β sheet tip is stabilized by hydrogen bonds with residues of the H2-H3n loop. Helix 12 is in the agonist position and stabilized by two interactions with the ligand. Helix 12 is also stabilized by several hydrophobic contacts with residues of H3, H5, and H11, and two polar interactions with residues of H3 and H4 (Fig. 5). Some of these residues contact the ligand, thus indicating an additional indirect ligand control of the position of helix H12. In the hVDR∆ structure, a strong crystalline contact is observed between helix H3n and helices H3, H4, and H12 of a symmetrically related molecule, with H3n mimicking the coactivator SRC-1 peptide contacts [12–14]. Active vitamin D resides in a chair B conformation with the 19-methylene “up” and the 1α-OH and 3β-OH groups in an equatorial and axial orientation, respectively. A chair A conformation of the A-ring would disrupt the hydrogen bonds formed by the hydroxyl and the protein. The conjugated triene system connecting A-ring to C- and D-rings accommodates an almost trans conformation with the C6–C7 bond exhibiting a torsion angle of –149° that deviates significantly from the planar geometry, which results in the curved shape of the ligand bound to the receptor. The deviation explains the lack of biological activity of the analogs with a trans or a cis conformation of the C6–C7 bond [30]. The α face of the C-ring is lined by Trp286, whereas the methyl C18 on the β face points toward Val234 (H3). The ligand-binding pocket is lined by hydrophobic residues (Fig. 6). The elongated ligand embraces helix H3 with its A-ring oriented toward the C termini of helix H5 and the 25-OH close to helices H7 and H11. The 1-OH group forms two hydrogen bonds with Ser237 (H3) and Arg274 (H5), while the 3-OH group is hydrogen-bonded to Ser278 (H5) and Tyr143 (loop H1-H2). The 25-OH moiety forms two hydrogen bonds with His305 (loop H6-H7) and His397 (H11). The triene is tightly fitted in a hydrophobic channel sandwiched between Ser275 (loop H5-β) and Trp286 (β1) on one side and Leu233 (H3) on the other side. The aliphatic chain at position 17 of the D-ring adopts

282


8.5

In I(Q)

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7.5 6.5 5.5 0

0.002

0.004

Q2 (Å 8

B

9

C

7

8

In I(Q)

In I(Q)

6 5 4 3 2

0.006

−2)

7 6 5 4 3

1 0

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0.1

Q (Å

D

0.15

0.2

0.05

−1)

0.1

Q (Å

E

1 0.9

0.9

0.8

0.8

0.7

0.7

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0.6

0.5

0.2

0.25

0.5

0.4

0.4

0.3

0.3

0.2

0.2

0.1

0.1

0

0.15

−1)

1

P(r)

P(r)

0

0.25

0 0

50

r (Å)

0

50

r (Å)

FIGURE 3 Solution scattering analysis of hVDR∆ (▲) and hVDR wild type (◆) bound to 1α,25(OH)2D3. (A) Guinier analysis. (B) and (C) X-ray scattering curves of hVDR∆ and wild type, respectively. (D) and (E) Distance distribution function P(r). Adapted from Fig. 6 of Rochel N, Tocchini-Valentini G, Egea PF, Juntunen K, Garnier JM, Vihko P, Moras D 2001 Functional and structural characterization of the insertion region in the ligand-binding domain of the vitamin D nuclear receptor. Eur J Biochem 268:971–979 [26].

an extended conformation parallel to the C13–C18 bond with the C13–C17–C20–C22 torsion angle close to 90°; it is surrounded by hydrophobic residues. The ligand binding cavity of hVDR∆ is large (697 Å3) with the ligand occupying only 56% of this volume. A channel of water molecules near position 2 of the A-ring makes an additional space that can accommodate ligands with a methyl group at position 2. The fourfold increase in binding affinity of the 2α−methyl analog is in agreement with this observation [31]. Additional space around the aliphatic chain is also observed.

V. MUTANT ANALYSIS In order to investigate structurally and functionally important amino acid interactions within the ligandbinding pocket of the wild-type hVDR in the presence of several synthetic vitamin D analogs, Vaisanen et al. [32] have combined side-directed alanine mutagenesis with limited proteolytic digestion, electrophoretic mobility shift assay, and reporter gene assay. They have shown that structurally different agonists have distinct ligand–receptor interactions and that residues

283


H4 H1 H9

H8

K264

H4 H10 H3

H5 E420 H3

H12

H11

S235

H12

H2 H7

D232 H6

T415

L414

Loop6–7

H2

H3n M412

R154

Insertion Domain 220ELTVS216................164GGGND160

FIGURE 4 Overall fold of hVDR∆ ligand-binding domain. The helices are represented as cylinders and β sheets as arrows. The whole structure is colored in gray except the helix H12 in purple. The ligand is depicted in yellow. The insertion region location is shown in green. The reconnected residues are indicated, together with their sequence numbering. Adapted from Fig. 1 of Rochel N, Wurtz JM, Mitschler A, Klaholz B, Moras D 2000 The crystal structure of the nuclear receptor for vitamin D bound to its natural ligand. Mol Cell 5:173–179 [25]. (See color plate).

FIGURE 5 Intramolecular interactions of helix H12 in VDR. The backbone of the protein is colored in gray except for helix H12 in purple. The side chains residues involved in polar stabilization of H12 are shown together with the hydrogen bonds (green dots). The ligand is depicted in yellow and red for the oxygen atoms. Adapted from Fig. 3 of Rochel N, Wurtz JM, Mitschler A, Klaholz B, Moras D 2000 The crystal structure of the nuclear receptor for vitamin D bound to its natural ligand. Mol Cell 5:173–179 [25]. (See color plate).

F150

F150

S278

O3

R274

Y147

Y143

Y147

Y143

S278

O3

R274

Y293

Y293 S237

S237 O1 L233

S275

I271

W286

I271 M272

V234

V234

V418

S275

O1 L233

W286

I268

V418

L313

M272 L230

I268

V300

L227

L313 V300

L227 F422

F422 H397 O25

H397 O25 L309

L309 Y401

H305

Y401

H305

FIGURE 6 Stereo view of 1α,25(OH)2D3 in the hVDR binding pocket. The ligand molecule is shown in the experimental electron density omit map contoured at 1.0 standard deviation. The hydroxyl groups are depicted as red spheres. Water molecules are shown as purple spheres. Hydrogen bonds are shown as green dotted lines. Carbon atoms are colored in gray, and oxygen and nitrogen atoms are colored in red and blue, respectively. Adapted from Fig. 3 of Rochel N, Wurtz JM, Mitschler A, Klaholz B, Moras D 2000 The crystal structure of the nuclear receptor for vitamin D bound to its natural ligand. Mol Cell 5:173–179 [25]. (See color plate).

284 H229, D232, E269, F279, and Y295 are critical for the agonist conformation [32]. Using a two-dimensional alanine scanning mutational analysis, Choi et al. [33] have studied the interactions between VDR and various vitamin D ligands. Eighteen alanine mutants of residues forming the ligand-binding pocket were studied. These residues form either hydrogen bonds with the ligands (Y143, S237, R274, S278, H305, H397) or hydrophobic interactions (D144, L233, V234, I238, I268, I271, S275, C288, W286, V300, Q400, Y401). The transactivation potencies of these mutants with the natural hormone and 11 other ligands were evaluated through functional studies with luciferase reporter assay and a mouse osteopontin response element. The comparison of six mutants of hVDR∆ and wild-type hVDR has shown that both LBPs are similar, thus providing additional evidence to support identical structures of VDR mutant and wild type. The importance of these residues in the interactions with specific ligands was evaluated in this study. Eight residues (Y143, D144, L233, I271, R274, W286, H397, Y401) have been shown to be essential for transactivation by vitamin D ligands, and mutations to less bulky hydrophobic residues increase the potency of 20-epi ligands [33].

VI. CRYSTAL STRUCTURE OF zVDR BOUND TO 1α,25(OH)2D3 In order to validate the conclusions made on the hVDR∆ structure and to find another crystal packing, the VDR from zebrafish (Danio rerio) was also studied. The zVDR exhibits 100% identity in the LBP lining residues and its transactivation potency is 50% of that of hVDR [34]. This activity is consistent with that of Xenopus laevis VDR or lamprey VDR, which show 50% [19] and 25% [21] of the activity of the hVDR, respectively. The zVDR LBD exhibits 69% identity and 79% similarity in its sequence with that of the hVDR LBD, while the insertion region (191–252 of zVDR) exhibits only 34% identity and 47% similarity. The zVDR LBD in complex with 1α,25(OH)2D3 was crystallized in presence of a coactivator peptide that contains the second NR box of SRC-1 [34]. The zVDR LBD (Fig. 7) adopts the canonical active conformation observed for agonist-bound receptors. The SRC-1 peptide (687-696 HKILHRLLQE) forms an amphipatic α-helix interacting with a hydrophobic cleft on the LBD surface. These interactions are similar to those described for other NRs [12–14]. In particular, Glu446 from H12 [Glu420] forms hydrogen bonds with the backbone amide nitrogen of Leu690 and Leu691.


H1

H12

FIGURE 7 Overall fold of zVDR ligand-binding domain. The helices are represented as cylinders and β sheets as arrows. The whole structure is colored in gray and the SRC-1 peptide in dark gray. The ligand 1α,25(OH)2D3 is depicted in yellow.

At the other end, Lys274 from H3 [Lys246] forms a hydrogen bond to the main chain oxygen of Leu694. The structures of zVDR LBD and hVDR∆ LBD complexes with 1α,25(OH)2D3 are similar with a root mean square deviation of 0.72 Å over 236 main-chain atoms. The binding pocket is identical and the natural ligand adopts the same conformation and forms the same interactions with the protein in the two structures (Fig. 8).

H11

FIGURE 8 Superimposition of the LBP of hVDR∆-1α,25(OH)2D3

in green and zVDR-1α,25(OH)2D3 in yellow. The side-chain residues forming the binding pocket are shown together with the hydrogen bonds (dots).


Notably the channel of water molecules at position 2 of the ligand, which was observed in the hVDR structure, is also observed in the zVDR. The differences observed when superimposing the two structures are small and primarily involve the loops. The ligandbinding pocket is conserved in both sequence and structure. The VDR-specific insertion region between helices H2 and H3 present in the zVDR construct and deleted in the hVDR mutant construct is not visible in the electron density map. This probably reflects the mobility of this region resulting in a static disorder. In the crystal structure of a VDR subfamily member, PXR [35,36], which also contains an insertion region between helices H1 and H3, the loop is also partially unresolved. The structural similarities between zVDR wild-type and hVDR∆ LBDs bound to 1α,25(OH)2D3 validate the overall biological relevance of the mutant hVDR structure.

VII. STRUCTURE OF hVDR COMPLEXED TO SUPERAGONIST LIGANDS Among the several synthetic analogs of vitamin D, the 20-epi compounds [37], which exhibit an inverted stereochemistry at position 20 in the flexible aliphatic chain, have attracted much attention. They are potent growth inhibitors and inducers of cell differentiation, while showing an affinity similar to that of 1α,25(OH)2D3 for VDR [37,38]. KH1060 (1α,25-dihydroxy20epi-22oxa-24,26,27-trihomovitamin D3 [37]), a member of this 20-epi family, exhibits similar properties with decreased calcemic side effects. The 20-epi compounds induce VDR-dependent transcription at concentrations at least 100-fold lower than the natural ligand and provoke antiproliferative activity several orders of magnitude higher than the natural ligand [37–40]. The differences in biological activity of 1α,25(OH)2D3 and the 20-epi molecules in general, and KH1060 in particular, are known to be VDR-LBD dependent. However, they are not yet understood. The ability of 20-epi analogs complexed to VDR to induce transcription appears to correlate with the ability of these compounds to promote coactivator interaction [40]. Differing proteolytic digestion patterns of VDR in the presence of the natural ligand and the 20-epi compounds have been interpreted to reflect large conformational changes in the receptor upon binding of the later class of molecules. In order to investigate the binding mode of the 20-epi analogs to the VDR-LBD, we have determined the high-resolution crystal structures of the hVDR∆ in complex with MC1288 (20-epi-1α,25(OH)2D3 [37]) and KH1060 and compared these structures to that obtained with the natural ligand [28] (Fig. 9).

285

When compared to hVDR∆-1α,25(OH)2D3 complex, the atomic models show a rms deviation on Cα atoms of 0.08 Å and 0.14 Å for hVDR∆-MC1288 complex and hVDR∆−KH1060 complex, respectively. Variations involve only some side chains located at the surface of the protein. In opposition to the view that the 20-epi analogs induce a different agonist conformation, the overall conformation and especially the position of helix H12 are strictly maintained in all three complexes. Furthermore, the ligand-binding cavity is unique and conserved for all three complexes. The rms deviations of all atoms forming the ligand pocket are 0.09 Å for hVDR∆-MC1288 complex and 0.12 Å for the hVDR∆-KH1060 complex. The sizes of the three ligands are 381 Å3, 375 Å3, and 392 Å3 for 1α,25(OH)2D3, MC1288, and KH1060, whereas the volume of the ligand pocket remains unaltered in the three complexes (660 Å3). The ligands occupy only 57% of the volume of the pocket for the VDR1α,25(OH)2D3 and VDR-MC1288 complexes and 59% for the VDR-KH1060 complex. The A, seco-B, and C/D rings form identical contacts as previously described for the 1α,25(OH)2D3-VDR complex. The hydroxyl groups make the same hydrogen bonds: 1-OH with Ser237 and Arg274, 3-OH with Tyr143 and Ser278, and 25-OH with His305 and His397. The deletion of 1-OH or 25-OH leads to the largest changes with a significant decrease in binding (1/1000), while that of the 3-OH has a smaller effect (1/20) [41]. The specific interactions observed in the three ligand–protein complexes involve the hydrophobic contacts of the 17β-aliphatic chains (Fig. 9B). When comparing the natural ligand and MC1288, the main difference is the positioning of the methyl group C21 which results in different contacts with Val300, Leu309, and His397. In MC1288, the C21 moiety is closer to His397. Other protein–ligand contacts differ, but to a lesser extent they involve the methyl groups at positions 23 (His305), 24 (His397), and 27 (His397 and Val418). In these two complexes, the carbon C22 makes no contact at a distance closer than 4.2 Å. In the case of KH1060, the methyl group C21 is quite close to that of the corresponding atom in 1α,25(OH)2D3. Note that the oxygen atom at position 22 of KH1060 forms a van der Waals contact with Val300. The major differences observed between KH1060 and the two other ligands are the tighter and more numerous ligand–protein contacts. The methyl groups C26α and C27α, specific to KH1060, form additional contacts with H3, loop 6–7, H11, and H12. A weak density is observed for the C26α methyl group, suggesting a structural disorder, whereas the C27α methyl group is clearly defined. In the three complexes, the ligands adopt an elongated conformation (Fig. 9C) similar to that described

286


A

A

3-OH 25-OH

B

1-OH

FIGURE 9 Crystal structures of the VDR LBD complexed to 1α,25(OH)2D3, MC1288, and KH1060. (A) Experimental electron density omit map contoured at 2.0 standard deviation of (A) 1α,25(OH)2D3, (B) MC1288, and (C) KH1060. (B) Closeup view of KH1060 in the ligand-binding pocket. Secondary structure features are represented in blue (α-helices) and green (β-strands). The ligand is colored in yellow with the oxygen atoms in red. The volume of the cavity is represented in gray. (C) Superposition of 1α,25(OH)2D3 (yellow), MC1288 (green), and KH1060 (blue) ligands after superimposed VDR complexes. Oxygen atoms are colored in red. Adapted from Fig. 2 and 3 of Tocchini-Valentini G, Rochel N, Wurtz JM, Mitschler A, Moras D 2001 Crystal structures of the vitamin D receptor complexed to superagonist 20-epi ligands. Proc Natl Acad Sci USA 98:5491–5496 [28]. (See color plate).

C

B

C

1-OH

3-OH A D 25-OH C


in the hVDR∆-1α,25(OH)2D3 structure. In the 20-epi complexes, the aliphatic chain is less constrained, thus allowing alternative conformations of the 17β-chain. The distances between the 25-OH group and the 1-OH and 3-OH are similar for the three complexes. These represent the anchoring points that must be maintained in order to obtain an active conformation. The aliphatic side chain of 1α,25(OH)2D3 adopts an extended conformation parallel to the C13–C18 bond with the C13–C17–C20–C22 torsion angle close to 90°. The two 20-epi analogs use different strategies to fit into the cavity. KH1060 accommodates its longer chain by adopting an eclipsed conformation around the O22–C23 bond, whereas MC1288 adopts a gauche conformation and has to compensate for a chain that is too short to maintain the 25-OH interactions because of the C20 inverted stereochemistry. Our preliminary docking experiments of the 20-epi analogs correctly positioned the methyl C21 in the same cavity as 1α,25(OH)2D3, but failed to give the correct geometry of the aliphatic chains. The crystallographic structures provide the exact information regarding these changes and the details of the interactions with the protein. Compared to the natural ligand, the aliphatic chains of the 20-epi analogs are lining the opposite side of the pocket. The different orientation of the chains is reflected by the different values of the torsion angles C16–C17–C20–C22 and C17–C20–C22(O22 for KH1060)–C23. Farther down the chain, while 1α,25(OH)2D3 adopts an extended conformation, the MC1288 and KH1060 exhibit gauche and eclipsed conformations, respectively, to properly orient the 25 hydroxyl group. The energetically unfavorable eclipsed conformation observed in KH1060 (C20–O22–C23–C24 of KH1060 equal to 16°) is made possible by the strong interactions formed by O22 with C12 (2.9 Å) and C18 (3.24 Å). A methylene group at this position as in MC1301 would be shifted away from the D-ring. A crude modeling analysis suggests that the methylene group would move away from C12, adopting a gauche (+) conformation instead of the eclipsed one observed for KH1060. As a result, additional contacts within the protein pockets are formed (Val300) that may contribute to the higher affinity of MC1301; other contributions such as solvation cannot be excluded. Similarly, the naturally occurring hydroxylation of C24α in KH1060 and C23 in 1α,25(OH)2D3 would form steric clashes with His305, affecting the hydrogen bond network with 25-OH. Based on the crystal structures this activity would depend on the capacity of the pocket to accommodate these additional groups. Both conformations and additional interactions of the 20-epi analogs are predicted to afford higher stability and longer half-life of the active complexes. Indeed,

287

within 3 hr, 60% of 1α,25(OH)2D3 dissociates from the VDR complex, whereas only 5–20% of MC1288 is dissociated [38]. Furthermore, receptors lacking the C-terminal helix 12 exhibit different dissociation behavior for the 20-epi analogs that are still capable of interacting with a mutated receptor [38,39]. Limited proteolytic digestion studies show that the 20-epi analog–receptor complexes are more resistant to digestion, suggesting that they are more stable. Additionally, an important role might be played by the rate of assimilation, i.e., how synthetic agonists are metabolized as compared to the natural ligand in different cells types [42,43].

VIII. STRUCTURE OF zVDR IN COMPLEX WITH GEMINI Among more than 3000 synthetic analogs of 1α,25(OH)2D3 synthesized to increase the potency and specificity of the physiological effects of vitamin D, Gemini (1α,25-dihydroxy-21-(3-hydroxy-3-methylbutyl)vitamin D3) is an interesting molecule with two identical side chains branching at carbon 20. Although Gemini binds less efficiently to VDR (38% compared to 1α,25(OH)2D3 [44]), its transactivation potency is similar to that of 1α,25(OH)2D3 in ROS cells [44] and 10-fold higher in HeLa and COS-7 cells [45]. It has been shown that in presence of an excess of co-repressor, the VDR–Gemini complex shifts from an agonist to an inverse agonist conformation through the recruitment of N-Cor and mediates repression [45,46]. Its stereochemistry and its 25% increase in volume when compared to the natural ligand create a major docking problem using the original LBP as a template. The packing constraints of the crystal form obtained for the hVDR∆ discriminate complexes with even small conformational changes near the ligand-binding pocket. In order to overcome this problem and obtain crystals of complexes that could not be crystallized with hVDR∆, we used the zVDR LBD construct. The zVDR in complex with Gemini and a coactivator peptide was crystallized and the structure solved at resolution 2.6 Å [34]. At the backbone level, the overall structure of zVDR-Gemini is almost identical to that of zVDR1α,25(OH)2D3 with a rms deviation of 0.37 Å over 249 main-chain atoms. However, the binding of the ligand’s second side chain results in a significant adaptation of the binding pocket that would have been difficult to predict [47]. A new pocket is created by the combined effect of a backbone shift and a side-chain conformational reorientation. The positions of helices H11 and H12 are unchanged and the peptide coactivator makes the same interactions with the protein, underlying the similar agonist character of both structures.

288 The most striking effect that emerges from this study is the “formation” of a new channel that extends the original pocket. This ligand-dependent pocket emphasizes the adaptability of the LBD and confirms the induced-fit mechanism of ligand binding. Despite large discrepancies in the shape and size of the ligand pocket, the overall structure of VDR and more importantly the position of H12 and of the coactivator peptide remain unaltered, consistent with the agonist character of Gemini. Thus the molecular mechanism of the agonism of Gemini is probably identical to that of 1α,25(OH)2D3.

IX. CONCLUSION The crystal structures of VDR LBD explain most features of ligand binding. These structures show the adaptability of the ligands and of the pocket in some specific cases. In all experimentally determined structures, the ligands were agonists and the protein conformation, notably H12, is conserved. The available structural information does not permit an understanding of the functional and structural role of the insertion domain. The loop 2-3, truncated in the hVDR and only partially visible in the zVDR structures, apparently does not influence the function of this nuclear receptor. Since this unique feature of VDR has not been lost during evolution, the question of its function remains an interesting one. However, the next step of structural studies should address the more general problem of the full-length receptor structure in different functional states.

Acknowledgment This work was supported by grants from CNRS, INSERM, Ministére de la Recherche et de la Technologie and by the Genopole and SPINE programs.

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CHAPTER 16

Comodulators of Vitamin D Receptor–Mediated Gene Expression DIANE R. DOWD, AMELIA L. M. SUTTON, CHI ZHANG, AND PAUL N. MACDONALD Department of Pharmacology, Case Western Reserve University, Cleveland, Ohio

I. Introduction II. Coactivators III. Co-repressors

IV. Conclusion—Integrated Model of Comodulator Activity References

I. INTRODUCTION

with vitamin D response elements (VDREs) in the promoters of target genes. VDREs are characterized by two direct hexameric repeats with a spacer of three nucleotides (DR-3 elements) (Chapter 14). Although purified recombinant VDR interacts weakly with DR-3 elements, nuclear extracts containing one or more accessory factors were shown to dramatically enhance the binding affinity of the VDR to these VDREs [8,9]. Subsequent studies revealed that the nuclear accessory factor was the retinoid X receptor, or RXR (Fig. 1) [10,11]. RXR is currently well accepted as the biologically relevant heterodimeric partner for the VDR and other class II nuclear receptors (NRs) (reviewed in [12]). The association of 1,25(OH)2D3 with VDR promotes both the heterodimerization with RXR and high-affinity binding to DR-3 VDREs, with the VDR occupying the 3′ half-site and the RXR occupying the 5′ halfsite [13,14]. In this manner, the interaction of the liganded VDR/RXR with a VDRE confers target gene selectivity and ultimately influences the rate of RNA polymerase II (RNA Pol II)–directed transcription (Chapter 14). About 15 years ago, a phenomenon termed “squelching” was observed in which the ligand binding domain (LBD) of one NR interfered with the transcriptional activation mediated by a second NR [15,16]. The theory was that there are limiting quantities of accessory factors or adapter proteins that interact with the receptor’s LBD and are necessary for NR-mediated transcription. Thus, the LBD of one NR competes with the other intact liganded receptor for binding to these proteins. Indeed, it is becoming increasingly clear that additional protein–protein interactions are required to regulate 1,25(OH)2D3-dependent, VDR/RXR-mediated transcription. The regulation of VDR-mediated transcription likely involves a complex series of macromolecular interactions occurring in a temporally coordinated fashion. Association between the liganded

Nearly a century has passed since the discovery of fat-soluble vitamin D as a micronutrient that is essential to maintain appropriate bone mineralization. In 1970, the bioactive form of vitamin D was isolated and identified as 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) [1–3]. Its main physiological role is to promote the intestinal absorption of dietary calcium and phosphate. Thus, 1,25(OH)2D3 ensures that the serum concentration of these ions is optimal for normal mineralization of the collagen matrix. Moreover, 1,25(OH)2D3 may directly affect bone remodeling by causing osteoblasts to terminally differentiate into osteocytes and deposit calcified matrix [4]. 1,25(OH)2D3 also promotes the differentiation of precursor cells into mature osteoclasts, which function to resorb bone and maintain appropriate bone remodeling [5]. In addition to its traditional role in maintaining calcium and phosphate homeostasis, the vitamin D endocrine system is also involved in a number of other physiological processes including blood pressure regulation, immune function, mammary gland development, and hair follicle cycling (reviewed in [6]). 1,25(OH)2D3 is generated by two sequential hydroxylations of vitamin D3 (cholecalciferol), a secosteroid precursor that is obtained in the diet or produced in the skin upon exposure to UV light (Chapter 2). 1,25(OH)2D3 is transported in the serum bound to the serum vitamin D binding protein (Chapters 8 and 9). 1,25(OH)2D3 dissociates from this transport protein and, because of its lipophilic nature, is thought to enter the cell by passive diffusion. Once inside the cell, 1,25(OH)2D3 is bound selectively by the vitamin D receptor or VDR (Chapter 11). VDR was first cloned in 1987 and was found to be a member of the superfamily of nuclear receptors (NRs) that regulates gene expression in a 1,25(OH)2D3-dependent manner [7]. The binding of 1,25(OH)2D3 to VDR enhances the association of VDR VITAMIN D, 2ND EDITION FELDMAN, PIKE, AND GLORIEUX


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RXR

VDR

D

D RXR VDR

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D RXR VDR

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FIGURE 1 1,25(OH)2D3-induced heterodimerization between VDR and RXR. VDR binds to its ligand, 1,25(OH)2D3, heterodimerizes with RXR, and binds to a DR-3 type VDRE in the promoter of 1,25(OH)2D3-responsive genes. VDRs occupy the proximal or 3′ half-site while RXR occupies the distal or 5′ half-site.

VDR/RXR heterodimer and other transcriptional components may be classified into two general categories: general transcription factors and the comodulatory proteins. The interaction of VDR with the first group results in direct contacts with the preinitiation complex (PIC), which may facilitate assembly or recruitment of the PIC and thereby stimulate transcription by RNA Pol II. TFIIB and TATA binding-protein-associated factors (TAFs) are examples of this class of transcription factor, which modulates the activity of liganded VDR. In addition to direct contacts with the general transcription machinery, the liganded VDR is also linked to the transcriptional PIC by the NR comodulatory factors. NR comodulators are proteins that interact directly with NRs and modulate, either positively or negatively, their abilities to regulate their transcriptional activity. NR comodulators are classified either as coactivators or co-repressors, and they aid in the induction or repression, respectively, of ligand/receptormediated transcription. Coactivator proteins function to augment transcription by one of three proposed mechanisms. First, coactivators may function as macromolecular bridges between the liganded receptor and the general transcriptional machinery. In this capacity, they may recruit components of the PIC, aid in the

assembly of the PIC, or promote the stability of the complex. Second, some coactivators possess histone acetyltransferase (HAT) activity or recruit HAT activity to the promoter, which may loosen chromatin packaging, thereby making it more accessible to the basal transcriptional machinery. A third possible mechanism of coactivator function is to increase the rate of coupling between RNA Pol II–directed transcription and more distal events such as transcription elongation and RNA processing. For example, several coactivator proteins express domains that are consistent with RNA processing proteins, thus pointing to a role in both activated transcription and RNA splicing mechanisms (Section II, E). Thus, these multifunctional proteins may be able to interact with the nuclear receptor, general transcription factors, chromatin remodeling activities, and the splicing machinery to couple transcription to RNA processing and dramatically influence the rate at which a gene product is expressed. NR co-repressors are generally defined as proteins that interact with unliganded receptors to repress basal expression of hormone-responsive genes. They are distinguished from other general transcriptional repressors that interfere with NRs through distinct mechanisms such as binding to the receptor or to DNA to disrupt NR response-element binding. In this chapter, NR co-repressors are discussed as nuclear factors that interact with the unliganded NRs and directly modify histones or recruit modifying enzymes, such as histone deacetylases (HDACs), to maintain chromatin in a tightly packaged state and silence transcription from the promoter in an NR-dependent manner. Research spanning the past two decades reveals that 1,25(OH)2D3-mediated transcription is more complex than the simple binding of the receptor to DNA and the recruitment of RNA Pol II to initiate transcription. VDR/RXR-activated transcription involves complex interactions that may occur in a spatially distinct and temporally coordinated fashion to increase the rate at which 1,25(OH)2D3-responsive genes are transcribed and at which the resulting RNA transcript is processed. An integrated model of coactivator/co-repressor function (Fig. 2) proposes that type II NRs maintain a transcriptionally inactive state at a promoter by recruiting co-repressors and their associated HDAC activity. Upon binding ligand, the co-repressors either dissociate from the receptor or are displaced from the complex by the entrance of coactivator molecules. These coactivators then acetylate core histones while recruiting other coactivators and transcription factors, thereby creating a transcriptionally permissive environment at the promoter. At later stages, they may also promote the recruitment or stability of the RNA splicing machinery to enhance the rate at which mature RNAs are made and subsequently translated. The focus of this chapter

CHAPTER 16 Comodulators of Vitamin D Receptor–Mediated Gene Expression

cor

epr

ess

RXR VDR

ors

Ac

VDRE TATA

D

D RXR

coa

ctiv ato

rs

VDR RNA Pol II

VDRE

TBP TATA Ac Ac Ac Ac

FIGURE 2

Model of comodulator activity on VDR-mediated gene expression. The VDR/RXR heterodimer is loosely bound to a VDRE in the absence of ligand. In this state, it may interact with co-repressors, which function to keep gene transcription repressed, in part, by keeping histone proteins deacetylated. Upon binding ligand, the co-repressor is released and replaced by coactivators. The coactivators then function to remodel the chromatin and aid in the recruitment of RNA Pol II and other key components of the preinitiation complex.

is on the roles of the comodulator proteins of vitamin Ddependent transcription in the induction and repression of 1,25(OH)2D3-regulated gene expression.

II. COACTIVATORS The binding of 1,25(OH)2D3 to the VDR initiates an orchestrated cascade of protein assembly ultimately leading to transcriptional activation of select target genes. Ligand-induced coactivator recruitment to the VDR/RXR heterodimer is one of the early events in this transactivation process. These initial interactions between VDR and coactivators are the seed for the assembly of intricate multiprotein complexes that remodel the chromatin structure, recruit the core transcriptional machinery, and induce expression of 1,25(OH)2D3-regulated genes. Thus, understanding the molecular details of 1,25(OH)2D3-induced assembly of VDR and various coactivator complexes is central to the process of 1,25(OH)2D3/VDR-activated transcription.

A. Mechanism of Interaction VDR and other NRs exhibit a modular structure with three principle domains [17]. The amino terminus

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of most NRs contains a transactivation function, although for VDR, this region is small and its function is poorly understood. Adjacent to this domain is the DNA-binding domain, which contains nine strictly conserved cysteine residues forming two zinc-binding modules that mediate high-affinity and highly selective binding to DNA. The large carboxyl-terminal ligandbinding domain (LBD) of VDR is organized into 12 alpha helices [18]. The LBD mediates association of VDR not only with 1,25(OH)2D3 [17], but also with its heterodimeric partner RXR [10,11,19] and comodulatory proteins [20]. Helix 12 of the VDR LBD contains the ligand-dependent activation function-2 (AF-2), which is essential for transactivation mediated by the VDR [19–21]. The molecular mechanisms involved in AF-2-dependent transactivation mediated by VDR are becoming increasingly clear based on structural analysis of VDR and other NRs. As 1,25(OH)2D3 nestles into the ligand-binding cavity of VDR, the AF-2 helix undergoes a subtle yet important conformational change. Helix 12 folds over the LBD [22] and, together with helices 3, 4, and 5, creates a hydrophobic crevice that selectively interacts with a complementary leucine-rich hydrophobic domain on many nuclear receptor coactivators (Fig. 3; [23]). This domain in the coactivators is termed the NR box and is composed of the consensus core sequence LXXLL [24]. The NR box forms an amphipathic α-helix whose orientation in the hydrophobic crevice of VDR is probably stabilized by a “charge clamp” composed of two conserved charged residues, one in the AF-2 helix 12 (E420) and the other in helix 3 of the VDR (K246) [25]. Besides the three highly conserved leucines, other residues in the NR box are also important for high-affinity binding and selectivity of coactivators for individual nuclear receptors [26–28]. While many coactivators interact with NRs via LXXLL motifs, others do not. Those coactivators that lack LXXLL motifs appear to interact with VDR and other nuclear receptors in an AF-2-independent manner (discussed in Section II,D). Thus, it is apparent that NRs can bind multiple coactivators using distinct motifs.

B. SRC Family of Coactivators The first nuclear receptor coactivator identified was steroid receptor coactivator-1 (SRC-1) [29]. SRC-1 was initially identified as a progesterone receptor–interacting protein that selectively enhanced hormone-dependent transcription. Subsequent studies have shown that SRC-1 is important for the transactivation of nearly all nuclear receptors including VDR [20,30,31]. SRC-1 constitutes the founding member of the p160 or SRC family, which also includes GRIP-1/TIF2/SRC-2 [32,33] and p/CIP/RAC3/ACTR/AIB-1/TRAM-1/SRC-3 [34–38].

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FIGURE 3 The vitamin D receptor undergoes a conformational change upon binding hormone. The VDR binds ligand, and this induces a conformational change in helix 12 of the AF-2 domain. Helix 12 folds to trap the ligand in the binding pocket. This change also creates a hydrophobic cleft or surface on VDR composed of helices H3, H4, H5, and H12 that LXXLL motifs in coactivator proteins use for docking to the VDR.

SRCs are composed of several domains that are highly conserved among the coactivator family (Fig. 4). A basic helix–loop–helix domain and a PAS domain exists in the amino terminus of SRCs. The PAS domain is characteristic of the Per/Arnt/Sim family of transcription

factors. This amino-terminal region exhibits intrinsic transcriptional activity when tethered to a heterologous DNA-binding domain and is thought to mediate protein–protein interactions with other transcription factors, perhaps including other PAS proteins [39]. Three LXXLL-containing NR boxes are located in the mid-region of the SRCs and these mediate the liganddependent interaction with NRs [24]. Although these three NR boxes were first presumed to be functionally redundant, subsequent mutational studies showed that the individual LXXLL motifs and surrounding sequences confer some degree of receptor selectivity [27,40,41]. In the case of SRC-3, NR box III appears to be most important for interaction with VDR [42]. The carboxyl termini of SRCs contain a second autonomous transactivation domain [39]. This domain is characterized by a glutamine-rich region common to many transcriptional activators. The large carboxylterminal region of SRC-1 also mediates interactions with the transcription factors CREB-binding protein (CBP)/p300 [27,43], p300/CBP-associated factor (p/CAF) [44], and coactivator-associated arginine methyltransferase 1 (CARM1) [45]. These interactions involve additional LXXLL motifs distinct from the NR boxes described above. The precise mechanisms of transcriptional activation by SRCs are not entirely clear, but most studies point to the central role of SRCs in modifying the histone acetylation state and chromatin structure of hormoneresponsive promoters [46–48]. Once targeted to the appropriate promoter through interactions with NRs, SRCs are thought to remodel the chromatin, creating a template that is more accessible to the transcriptional machinery. This remodeling occurs through the covalent addition of acetyl groups onto the carboxyl-terminal lysine residues of histones. This acetyl modification weakens the electrostatic interaction between the positively charged histone tails and the negatively charged phosphate backbone of the DNA, thus inducing decondensation of the chromatin (reviewed in [49]). Such HAT activity is contained both in the SRCs themselves [36,47] and in several of the other transcriptional proteins that SRCs recruit, such as p300/CBP [50,51] and p/CAF [52]. In addition, p300/CBP binds directly to nuclear receptors and, together with SRC coactivators, they synergistically stimulate transcription [53,54]. Furthermore, SRCs also promote histone methylation, and presumably loosening of the chromatin structure, through their interactions with CARM1 [45]. Murine knockout models of each of the SRCs have been generated [55–57]. These models show a degree of functional redundancy in the SRC family, especially in the development and maintenance of the female reproductive system. However, the individual SRCs


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FIGURE 4

Schematic of the conserved SRC coactivator domains. bHLH, basic helix–loop–helix domain; PAS, region characteristic of the Per/Arnt/Sim family of transcription factors; NR boxes, nuclear receptor interaction boxes containing the LXXLL motifs; CBP interaction, region that interacts with CREB-binding protein; Q-rich, region rich in glutamine residues.

may also have distinct physiological functions perhaps related to nuclear hormone receptor selectivity. For example, deletion of SRC-2 results in aberrant spermatogenesis and testicular defects, whereas deletion of either SRC-1 or SRC-3 has no effect on the male reproductive tract [56]. Mutation of SRC-3 causes decreased estrogen-mediated protection against vascular damage [57]. Since the impact of these genetic deletions on VDR/ 1,25(OH)2D3-targeted systems has not been investigated, it is unknown which SRCs are important in vivo in VDR-mediated transcription. However, in vitro evidence suggests that SRC-2 may be a favored coactivator for VDR since SRC-2 is expressed in 1,25(OH)2D3 target cells such as osteoblasts and preferentially augments 1,25(OH)2D3-induced expression of the osteocalcin gene [58].

C. Mediator-D Complex In addition to SRCs, a large multiprotein complex called DRIP (vitamin D receptor interacting proteins) is a coactivator for VDR and other nuclear receptors [59,60]. DRIP shares many components with the transcriptional coactivator complexes thyroid hormone receptor (TR) activating proteins, activator recruited cofactor, and the mammalian Mediator complex [61–63]. In fact, these four complexes are most probably one in the same. These complexes interact with a variety of mammalian transcriptional activators, most notably NRs, SREBP-1a, and E1A. Thus, they likely have fundamental roles in activator-induced transcriptional processes well beyond the VDR and other NRs. Because of the considerable similarity between proteins comprising these various complexes, a unified nomenclature was proposed that

uses the Mediator complex as the basis for naming the multiple complexes and subunits [64]. Using this nomenclature, DRIP is referred to as Mediator-D. Mediator-D is proposed to interact with the liganded VDR bound to its enhancer element and then to recruit RNA Pol II, thereby acting as a bridge between VDR and the PIC [64,65]. Although mediator complexes may recruit RNA Pol II to the promoter, the polymerase is not tightly bound, thereby allowing for its release and the efficient initiation of transcription. Moreover, yeast mediator does not interact with the hyperphosphorylated form of RNA Pol II involved in transcriptional elongation [66]. These data suggest that mediator complexes are associated with RNA Pol II only during preinitiation and the transition to elongation. Moreover, in vitro transcription studies indicate that following the release of RNA Pol II, mediator remains bound to the promoter to function in the reinitiation of a second round of transcription [67]. Chromatin immunoprecipitation studies on hormone-responsive promoters allude to an even more dynamic cycling process of unknown function [68]. While the Mediator-D complex efficiently stimulates VDR-mediated transcription in vitro on chromatinized templates, Mediator-D and other mammalian mediator complexes do not contain detectable HAT activity [60]. However, recent evidence from yeast mediator complexes suggests they may assist in maintaining chromatin in a hyperacetylated, open conformation [69]. This raises the question of when and how the recruitment of chromatin remodeling complexes and mediator complexes takes place. A model that addresses this question proposes the sequential recruitment of coactivators and is described in more detail later (Section IV).

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10 36 240 17 cdk8 95 130 230 33 97 150 220 78 34 70 100 D VDR

FIGURE 5 Model of the Mediator-D complex interacting with the liganded VDR. Med-220 uses an LXXLL NR box motif for docking to the VDR.

The Mediator-D complex is composed of at least 10 different proteins anchored by Med220, which interacts directly with ligand-activated VDR/RXR heterodimers through the second of two LXXLL motifs (Fig. 5, [70]). While Med220 promotes interaction with the VDR, another component of Mediator-D, Med130, may stimulate assembly of the PIC on the promoter [71]. The interaction between VDR and Med220 is enhanced by phosphorylation of VDR, and this correlates with an increase in 1,25(OH)2D3-mediated transcription [72]. Further support for the importance of this interaction comes from studies from Yang and Freedman [73] using 20-epi analogs of 1,25(OH)2D3. They showed a link between the increased transcriptional activity of these higher potency 20-epi compounds and increased binding of VDR to Med220 when compared to the effects of the 1,25(OH)2D3 ligand. This enhanced binding and transcriptional activity was also reflected in an increase in cellular differentiation and a decrease in proliferation. No differences were observed between 1,25(OH)2D3 and the 20-epi analogs when comparing the ability of these ligands to promote binding of VDR to SRC-2, thus highlighting the importance of mediator complexes in agonist-induced VDR activity. Genetic ablation of Med220 in mice causes embryonic lethality [74]. Moreover, fibroblasts derived from these animals exhibit attenuated thyroid hormone– stimulated transcription, but these cells retain normal retinoic acid responses, suggesting both receptor selectivity and a functional redundancy of NR coactivators in vivo [74]. Heterozygous mutants display growth retardation, impaired transcription and hypothyroidism, underscoring the primary importance of this mediator complex in TR-regulated processes and possibly other NR-mediated transcriptional events. Since VDRmediated transcriptional processes have not been examined in this model, it is unclear whether Med220 is required for 1,25(OH)2D3 action in vivo.

D. NCoA62/SKIP NCoA62 is a VDR and NR coactivator that was isolated as a VDR-interacting protein by a yeast two-hybrid screen [75]. Expression of NCoA62 in mammalian cells enhances vitamin D-, retinoic acid-, estrogen-, and glucocorticoid-activated transcription. NCoA62 is structurally and mechanistically distinct from SRCs and Mediator-D. However, it is highly related to Bx42, a Drosophila melanogaster nuclear protein putatively involved in ecdysone-stimulated transcription [76]. NCoA62 was independently identified as a factor that interacts with the Ski oncoprotein and was termed Ski-interacting protein, or SKIP [77]. Thus, it is referred to as NCoA62/SKIP throughout this chapter. NCoA62/SKIP has subsequently been shown to interact with a diverse array of transcription regulatory factors including CBF1 and silencing mediator for retinoic acid and thyroid hormone receptors (SMRT) [78], Smad2 and Smad3 [79], poly(A)-binding protein 2 [80], human papillomavirus (HPV-16) E7 oncoprotein [81], MIBP1, a member of the major histocompatibility complex binding protein family [82], and the retinoblastoma tumor suppressor protein [83]. These studies highlight a potential common role for NCoA62/SKIP in a more general aspect of target gene regulation by different classes of transcription factors. Importantly, the interaction between NCoA62/SKIP and VDR is independent of the AF-2 domain [75]. Moreover, ligand is not required for the interaction in vitro, although ligand does enhance the interaction. Unlike the SRC family of coactivators, NCoA62/SKIP does not contain obvious LXXLL motifs. NCoA62 also exhibits a marked preference for binding to the VDR-RXR heterodimer relative to VDR alone [84]. The region of NCoA62/SKIP between residues 274 and 342 mediates the interaction with the VDR-RXR heterodimer, and this region is referred to as the receptor interacting domain or RID (Fig. 6). The highly charged C-terminal domain is responsible for NCoA62/SKIP coactivator function in NR-mediated transcription. This region expresses an autonomous transactivation domain that is referred to as TAD-1 (reviewed in [85]). The nuclear localization sequence (NLS) of NCoA62/SKIP is contained in the C-terminal residues 531–536 and is necessary and sufficient for nuclear targeting of NCoA62/SKIP [86]. A strictly conserved LPXP motif is present in all reported NCoA62/SKIP orthologs from a variety of organisms [87], but the function of this domain or region is unknown at present. The mechanisms through which NCoA62/SKIP may function to augment VDR-mediated transcription are becoming more apparent. For example, selective,


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FIGURE 6 Schematic of NCoA62/SKIP. LPXP, a strictly conserved motif of unknown function; SNW, region containing a central conserved serine, asparagine, and tryptophan, as well as the receptor interaction domain (RID); TAD-1, transcription activation domain; NLS, nuclear localization sequence.

multiprotein complexes form between VDR, RXR, NCoA62/SKIP, and SRC coactivators [84]. NCoA62 and SRCs contact different domains within the VDRRXR heterodimer. Indeed, both NCoA62/SKIP, and SRC simultaneously interact with liganded VDR to form a ternary complex in vitro, which suggests that liganded VDR can act as a bridge to recruit both SRCs and NCoA62/SKIP to the promoter complex (Fig. 7) [84]. Coexpression of NCoA62/SKIP and SRCs leads to a synergistic induction of VDR-mediated transcription, and protein interference studies indicate that both coactivators are required for optimal VDR-mediated transcription [84]. Perhaps the most promising clues to the significance and mechanism of NCoA62/SKIP in VDR-activated transcription are found in recent chromatin immunoprecipitation (ChIP) approaches [86]. These studies show that NCoA62/SKIP is physically recruited in a 1,25(OH)2D3-dependent manner to the 1,25(OH)2D3responsive regions of native VDR target genes in osteoblasts including the osteocalcin and 24-hydroxylase promoters. Interestingly, NCoA62/SKIP is associated with the promoter region after the entry of both VDR and SRC, suggesting it may function at more distal steps of the transactivation process compared to the SRCs. As mentioned previously, the SRCs exhibit histone acetyltransferase (HAT) activity and recruit other proteins such as CBP/P300 that possess HAT

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Hypothetical model for VDR-SRC-NCoA62/SKIP complexes in VDR-activated transcription.

activity [47,88]. No significant HAT activity has been detected for NCoA62/SKIP (Zhang and MacDonald, unpublished observations). Thus, one possible interpretation is that NCoA62/SKIP enters the promoter region after the chromatin remodeling step. Additionally, NCoA62/SKIP is also known to interact with basal transcription factors such as TFIIB [89], suggesting that NCoA62/SKIP may also be involved in connecting VDR to the general transcription machinery. Regardless, NCoA62/SKIP and SRCs apparently function through different mechanisms to enhance VDR-mediated transcription, and the function of both distinct classes of coactivators is needed for appropriate vitamin D– activated target gene expression.

E. Nuclear Receptor Coactivators: Potential Links to RNA Processing In addition to chromatin remodeling and recruiting the transcription machinery, NR coactivators have been implicated in more distal steps of gene expression including transcription elongation and RNA processing. In this regard, NR coactivators have been proposed to be important coupling factors linking transcription and RNA processing [90]. The coupling concept is that RNA processing events (e.g., RNA capping, polyadenylation, and splicing) are physically connected to RNA Pol II and that RNA processing begins on the nascent RNA emerging from the polymerase complex (reviewed in [91–94]). Putative candidates that may aid in this coupling process are predicted to associate with and alter the functional properties of proteins involved in both transcription and RNA processing. In fact, many NR coactivator proteins contain domains or regions that are associated with RNA processing proteins, making some NR coactivators strong candidates as transcription-splicing couplers [90]. Indeed, Auboeuf et al. have demonstrated that progesterone receptor (PR)-activated transcription influences splicing decisions of alternatively spliced transcripts in a PR- and

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progesterone response element–dependent manner [90]. NR coactivators such as CoAA and PGC-1 have been shown to alter RNA processing events in NRmediated transcription [90,95]. Thus, the idea put forth in these papers is that activated steroid receptors bind to target gene promoters and recruit factors that are involved in both transcription regulation and splicing regulation. This is thought to ensure efficient coordination between RNA synthesis and the final mature mRNA. NCoA62/SKIP may also play a central role in coupling transcription to mRNA slicing. Several observations support this hypothesis. First, endogenous NCoA62/SKIP is recruited to native VDR-responsive promoters in a 1,25-(OH)2D3-dependent manner in osteoblast-like target cells proving its physical association with the transcriptional regulatory machinery at the responsive promoter regions [86]. Second, studies characterizing protein components of the spliceosome and interchromatin granules, where many splicing factors are present, identified NCoA62/SKIP as a component that is associated with the splicing machinery in general and at discrete steps in the splicing mechanism [86,96–98]. Finally, a dominant-negative NCoA62/SKIP mutant effectively interferes with appropriate splicing of transcripts derived from a VDR-activated mini-gene cassette [86]. These studies indicate that disrupting the function of endogenous NCoA62/SKIP inhibits 1,25(OH)2D3-activated transcription, in part, by interfering with appropriate splicing of the mRNA transcripts. This supports the hypothesis that NCoA62/SKIP may couple VDR-mediated transcription to RNA splicing and ensure appropriate, efficient processing of vitamin D– regulated mRNA transcripts.

F. Other Coactivators In addition to these three classes of coactivators, several other proteins interact with VDR in a liganddependent manner and stimulate VDR-mediated transactivation. As mentioned earlier, VDR directly associates with several components of the transcriptional machinery. For example, VDR selectively binds to TFIIB [99,100], a component of the basal transcriptional complex whose entry is rate-limiting in the preinitiation complex formation. The LBD and amino-terminal regions of VDR are involved in contacting an aminoterminal region of TFIIB [99–101], and these two proteins function cooperatively to enhance ligand-dependent transcription [99]. VDR also interacts with TFIID and TFIIA, two essential components of the basal transcriptional machinery [102,103]. TFIID consists of the TATA-box-binding protein and associated factors

or TAFs. VDR associates with the TFIID subunit hTAF(II)135, and TAF(II)135 selectively potentiates VDR-mediated transcription. Direct interaction of VDR with these general factors further strengthens the link between VDR and the core transcriptional machinery and thus represents a putative mechanism in VDR-activated transcription. VDR also contacts Smad3, a transcription factor activated by TGF-β signaling [104]. Smad3 interacts with VDR in a ligand-dependent manner and stimulates VDR-mediated transcription. Additionally, SRC-1 enhances the binding of Smad3 to VDR, suggesting cooperation between these two coactivators. Given its function in two diverse signaling networks, Smad3 may serve as a bridge between 1,25(OH)2D3 and TGF-β signal transduction pathways. Interestingly, NCo62/SKIP serves as a coactivator for Smad-dependent transcription, lending additional support for the concept of crosstalk between these two pathways [79]. An additional binding partner for VDR is the helix–loop–helix transcription factor Ets-1 [105]. This interaction occurs between the DNA-binding domains of both proteins. Unlike other VDR coactivators, Ets-1 can stimulate VDR-mediated transcription in the absence of ligand on select promoters, such as the prolactin promoter.

III. CO-REPRESSORS The role of the co-repressor in regulating gene expression is generally the converse of that of the coactivator (reviewed in [106]). Co-repressors interact with DNA-bound transcription factors and play essential roles in silencing or repressing transcription. In the case of NR-regulated gene expression, co-repressors generally function to lower basal promoter activity in the presence of unliganded receptor; however, in some cases, repression can occur in the presence of NRs bound to antagonists.

A. SMRT and NCoR Perhaps the best characterized NR co-repressors are the ubiquitously expressed proteins SMRT [107] and nuclear receptor co-repressor (NCoR) [108]. SMRT and NCoR co-repressors strongly repress basal promoters in systems regulated by TR and the retinoic acid receptor (RAR). While the SRC family of coactivators interact with NRs via NR boxes (described earlier), SMRT and NCoR co-repressor interaction is mediated through co-repressor NR (CoRNR) boxes [109]. CoRNR boxes are generally composed of the


sequence Φ-X-X-Φ-Φ, where Φ is a hydrophobic residue and X is any amino acid. Mutation of these sequences abolishes interaction of the co-repressors with unliganded NR [109]. In contrast to the SRC family of coactivators, SMRT and NCoR interaction with VDR is localized to the ligand binding domain but is independent of the AF-2 domain [110,111]. In fact, deletion of the AF-2 helix enhances co-repressor binding [107]. Although VDR is in the same NR family as TR and RAR, the silencing effects of SMRT and NCoR on VDR-regulated templates are weaker than in the other systems [112]. This is most probably due to a reduced binding of SMRT and NCoR to VDR relative to that of TR and RAR. It is possible that there may be antagonists for the VDR that could enhance the binding of the receptor to the co-repressors and thus increase transcriptional silencing. This means of interaction has been documented for co-repressor recruitment to the ER [113,114] and the PR [115]. The mechanism of repression of the SMRT/NCoR class involves interaction with Sin3, a protein shown to be part of a complex that includes HDAC1 and 2 [116–118]. Thus, the Sin3 proteins Sin3A and Sin3B recruit HDAC activity to target gene promoters and their subsequent activities result in compact or tightly wound nucleosomal packaging that ultimately represses basal expression of the promoter.

B. Hairless As mentioned earlier, the VDR plays a role in mammalian hair follicle cycling and disruption of the VDR gene, in some cases, leads to total alopecia (for review see [119]). Inactivation of the hairless gene (hr) also results in a similar phenotype [120]. Hr can repress transcription mediated by VDR, TR and RARrelated orphan receptor, and this discovery provides a molecular basis for its role in the maintenance of hair growth [121–123]. Although Hr lacks homology with SMRT and NCoR, it functions in a similar manner. Like these other co-repressors, Hr binds to VDR and the other NRs in a region localized to the central portion of the ligand binding domain and independent of AF-2 [123], the docking site for some coactivators. This interaction requires two Φ-X-X-Φ-Φ hydrophobic motifs in Hr [123]. Although Hr does not exhibit histone deacetylase activity, it is present in complexes with multiple HDACs. Hr also is found in matrixassociated deacetylase bodies together with SMRT and multiple HDACs [121]. Whereas SMRT and NCoR are ubiquitously expressed, the expression of Hr is restricted primarily to brain and skin, and thus may serve a more specialized role (reviewed in [124]).

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C. Alien In 1999, Dressel et al. described a novel co-repressor, Alien, which is unrelated to the SMRT/NCoR family of co-repressors [125]. In these early studies, Alien interacted with TR in the absence of ligand and repressed TR-mediated basal transcription. Alien does not interact with the glucocorticoid receptor, RXR, or RAR. In this system, the mechanism of Alien-mediated silencing appears to be through the recruitment of Sin3A and its associated histone deacetylase activity. Alien did not interact with SMRT or NCoR and thus represents an independent pathway to Sin3 recruitment. Polly et al. [126] extended these findings to demonstrate that Alien also interacts with VDR and represses basal transcription from a DR-3 VDRE, but not an atypical IP9-type VDRE, suggesting a level of specificity for repression. The interaction of Alien with unliganded VDR is independent of the AF-2 domain of VDR and appears to use a different interaction surface than does NCoR. In contrast to the studies by Dressel, a deacetylase inhibitor had little effect on the ability of Alien to repress transcription. Thus, in the VDR system, Alien appears to be using alternate molecular pathways to exert its corepressor effect.

IV. CONCLUSION—INTEGRATED MODEL OF COMODULATOR ACTIVITY Gene expression is a complex process involving the coordinated repression and activation of transcription. According to the present model of nuclear hormone signaling, unliganded VDR, like other NRs, are DNAbound and complexed with co-repressor molecules that keep chromatin condensed and the promoter inaccessible to the transcription machinery. Upon binding 1,25(OH)2D3, the co-repressors are displaced by coactivators to begin the transcriptional process. To date, more than 30 nuclear receptor coactivators have been identified and recent effort focuses on distinguishing the redundant and unique functions of these proteins. Immunodepletion and dominant negative approaches indicate that SRCs, Mediator-D, and NCoA62/SKIP have different functions and all are required for robust VDR-mediated transcription. However, the temporal and spatial details of how these diverse coactivators and other accessory transcription factors are assembled onto a 1,25(OH)2D3-responsive promoter remain to be determined. Chromatin immunoprecipitation (ChIP) assays have proven instrumental in providing a limited picture of how endogenous VDR and coactivators convene on a native promoter template by capturing transcriptional complexes assembled on the promoter

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at various times. ChIP assays demonstrate that both nuclear receptors and their coactivators cycle on and off the promoter [68,86,127]. Upon ligand addition, VDR, or other nuclear receptors, enters the transcriptional complex first, followed by SRCs (Fig. 8; [68,86]). SRCs likely loosen the chromatin structure by both bringing intrinsic HAT activity and recruiting extrinsic HAT activity to the complex, through their interaction with p300/CBP. SRCs then dissociate, allowing for binding

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of the Mediator-D multimeric complex [68,128]. Mediator-D is thought to recruit the PIC and RNA Pol II holoenzyme to initiate transcription of target genes [65]. NCoA62/SKIP also enters the VDR complex following SRC-1 [86], but the relative entrance of NCoA62/SKIP and Mediator-D to the transcriptional complex is currently unknown. It is possible that SRCs help to recruit NCoA62/SKIP to the complex as suggested by the data that these two proteins can form a stable ternary complex with VDR [84]. Once NCoA62/SKIP is bound, it may target the spliceosome complex to the actively transcribed gene [86], allowing for efficient splicing of the nascent transcript. Taken together, these ChIP studies suggest a temporal model of coactivator action in which all three major classes of coactivators enter the complex at distinct times and provide three different functions: chromatin remodeling, recruitment of the core transcriptional machinery, and coupled transcriptional activation and RNA splicing. Importantly, one must be cautious not to oversimplify what is obviously a hugely complex process that requires a variety of macromolecular machines and coordination of many complex pathways. The model presented in Fig. 8 is meant to represent one global means to integrate the known actions of comodulator proteins, but the reality will likely turn out to be a much more complicated scenario.

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FIGURE 8 Model of the sequential occupation of the promoter site with coactivators, the PIC and the spliceosome. First, ligandactivated VDR/RXR binds to VDREs in target genes and recruits coactivators such as SRCs and p300/CBP. The HAT activity of these coactivators loosens the chromatin structure, allowing for a more transcriptionally permissive environment. Second, SRCs and p300/CBP dissociate, allowing for binding of the Mediator-D multimeric complex. This complex is thought to recruit RNA Pol II and the core transcriptional machinery to initiate active transcription of the target gene. Finally, NCoA62/SKIP binds to the VDR/RXR complex and tethers the splicing machinery to the activated promoter, allowing for immediate splicing of the nascent transcript.

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CHAPTER 17

Promoter Targeting of Vitamin D Receptor through a Chromatin Remodeling Complex SHIGEAKI KATO,* RYOJI FUJIKI, AND HIROCHIKA KITAGAWA* Institute of Molecular and Cellular Biosciences, University of Tokyo, Japan, and *SOREST, Japan Science and Technology, Saitama, Japan

I. Introduction II. Chromatin Remodeling Is a Prerequisite for Transcriptional Controls by the Vitamin D Receptor III. Purification and Identification of Williams Syndrome Transcription Factor as a Vitamin D Receptor Interactant IV. Purification and Identification of a Novel Williams Syndrome Transcription Factor Complex Associating with Vitamin D Receptor V. WINAC Is a Novel Multifunctional ATP-Dependent Chromatin Remodeling Complex That Rearranges

a Nucleosome Array around a Vitamin D Responsive Element in Vitro VI. Williams Syndrome Transcription Factor Coactivated the Ligand-Induced Transactivation Function of Vitamin D Receptor VII. Molecular Mechanism of VDR Promoter Targeting of Vitamin D Receptor by WINAC and Cooperative WINAC Function with the Co-regulator Complexes References

I. INTRODUCTION

These co-regulatory complexes contain enzymes that modify histones through acetylation, which results in rearrangement of nucleosome arrays [9]. However, prior to such necleosome array reorganization by co-regulator complexes, chromatin remodeling is believed to be coupled with the promoter targeting of VDR, like all of the DNA binding transcription factors (Fig. 1).

The calciotropic hormone 1,25(OH)2D3, the active form of vitamin D3, regulates calcium homeostasis as well as cellular proliferation and differentiation [1]. Most biological actions of 1,25(OH)2D3 are believed to be mediated through transcriptional controls of a particular set of target genes by the vitamin D receptor (VDR) [2,3]. VDR is a member of the nuclear receptor (NR) gene superfamily, which acts as a ligand-inducible transcriptional factor [4]. Like other members of the nuclear receptor superfamily, VDR structure is divided into several functional domains, with the most highly conserved DNA-binding domain (C) located centrally and the less highly conserved ligand-binding domain (E) located at the C-terminal end [5]. Most nuclear receptors harbor both an N-terminal activation function 1 (AF-1) and a C-terminal AF-2 domain [6]. The VDR, however, appears to lack the significant N-terminal AF-1 function because of its relatively short A/B domain. In the promoters of target genes that are controlled by liganded VDR, VDR/RXR heterodimer recognizes and directly binds to cognate vitamin D responsive elements (VDREs) [6]. Liganded VDR bound upon the VDREs induces the recruitment of a number of histone acetyltransferase (HAT) and non-HAT coactivators and coactivator complexes to activate transcription [7,8]. VITAMIN D, 2ND EDITION FELDMAN, PIKE, AND GLORIEUX

II. CHROMATIN REMODELING IS A PREREQUISITE FOR TRANSCRIPTIONAL CONTROLS BY THE VITAMIN D RECEPTOR At transcriptional initiation sites in promoters, rearrangement of the nucleosome array is thought to be indispensable for controlling transcription of sequencespecific regulators such as VDR through chromatin remodeling and histone modifications [9,10]. These complexes (Fig. 2) modify the chromatin configuration in a highly regulated manner. One class contains several discrete subfamilies of transcription coregulatory complexes that harbor either HAT or histone deacetylase (HDAC) activities to covalently modify histones through acetylation [11]. In NR ligand-induced transactivation processes, the complexes containing HDAC first act to corepress transactivation of unliganded NRs. Upon ligand binding, two HAT complexes, p160/CBP and TRRAP/GCN5, are Copyright © 2005, Elsevier, Inc. All rights reserved.

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SHIGEAKI KATO, RYOJI FUJIKI, AND HIROCHIKA KITAGAWA

Co-activator complexes

Co-repressor complexes

HAT complexes

HDAC complexes HDAC1/2

PCAF complex

mSin3

p300

NCoR/ SMRT

non-HAT co-activators

p160 family protein

DRIP/TRAP complexes Chromatin remodeling

Chromatin remodeling

VDR RXR

VDRE

FIGURE 1 VDRs coordinate the activities of chromatin remodeling complexes to facilitate. Because the promoter is assembled into a nucleosomal structure that is transcriptonally inactive the chromatin environment limits the accessibility of DNA-binding transcription factor and their co-regulators to promoter. To conquer this difficulty, an interaction of VDR with unknown chromatin remodeling complexes reorganizes nucleosomal arrays at VDR targeting promoters, which results in stabilized binding of transcription co-regulator.

A SWI/SNF family

ISWI family

ATPase

Bromo

hSWI/SNF complex Brg1/ BAF250 hBrm BAF60a/b/c BAF155 BAF170 BAF47 Ini1 BAF50

B

ATPase

SANT

hSpt3 hPCAF TAF30 hAda3 PAF65β

chromo

ATPase

hNuRD complex

hAcf1

MTA2 HDAC1 MBD3

hISWI

RbA MTA1 p46 Mi-2 RbA HDAC2 p48

HDAC complexes CBP/p160 complex

TAF15 PAF65α hAda2 TRRAP

PHD

hACF complex

HAT complexes hPCAF complex

Mi-2 family

CBP/ p300 p160 family protein

mSin3 complex HDAC1SAP18 RbA p46 mSin3 SAP30 HDAC2 RbA p48

TAF31

FIGURE 2 Chromatin remodeling complexes. (A) ATP-dependent chromatin remodeling complexes. These types of complexes can be divided into three major classes based on catalytic ATPase subunit: Brg1/hBrm, hISWI, and Mi-2. Shown here are some representative members of the SWI/SNF, ISWI, and Mi-2 families of human ATP-dependent chromatin remodeling complexes. (B) Some representative members of the histone modifying complexes. Purple, ATPase; red, histone acetyltransferase; green, histone deacetylase. (See color plate).

CHAPTER 17 Promoter Targeting of Vitamin D Receptor through a Chromatin Remodeling Complex

recruited with dissociation of the HDAC complexes, and coactivate the NR function [12–14]. Another class of complexes contains chromatin remodeling complexes that use ATP hydrolysis to rearrange nucleosomal arrays in a noncovalent manner, thereby making chromosomal DNA accessible for sequence-specific regulators such as the VDR [11]. Besides of transcriptional controls, these ATP-dependent chromatin remodeling complexes are supposed to act on DNA repair and DNA replication. These complexes are further classified into subfamilies based on the major catalytic components, the ATPases (Brg1/hBrm, hISWI, and Mi-2) [15,16]. Recently, the ligand-induced transactivation function of VDR in vitro has been shown to require a SWI2/SNF2-type chromatin remodeling complex containing pBAF180 [17].

III. PURIFICATION AND IDENTIFICATION OF WILLIAMS SYNDROME TRANSCRIPTION FACTOR AS A VITAMIN D RECEPTOR INTERACTANT To search for novel co-regulatory complexes that interact with VDR, HeLa cell nuclear extracts were incubated with a chimeric VDR-hinge-ligand binding domain region (VDR-DEF) fused to glutathione-Stransferase (GST) in the presence or absence of 1,25(OH)2D3 (Fig. 3A). Proteins that interacted with VDR-DEF were separated by SDS-PAGE and silver stained (Fig. 3B). Mass spectrometry coupled with the apparent molecular weights of the different proteins

GST

P11

kDa

HNE

HeLa nuclear extract (HNE)

VDR(D3+)

B

VDR(D3−)

A

TRRAP/PAF400 P11 Phosphocellulose column DRIP240/TRAP230 200 GST

DRIP205/TRAP220 WSTF TRAP130

116 GST–VDR(D3−)

GST–VDR(D3+)

WINAC

WINAC DRIP/TRAPs

307

DRIP100/TRAP100 97 DRIP70 66

WSTF TRAP220 TIF2 Brg1 hSNF2h

FIGURE 3 Purification and identification of human proteins interacting with 1,25(OH)2D3–unbound and bound VDR. (A) Purification scheme for VDR interacting proteins. The eluted fraction from a P11 phosphocellulose column was incubated with immobilized GST-VDR(DEF) in the absence or presence of 1,25(OH)2D3 (D3, 10−6 M). (B) Identification of ligand-independent and -dependent VDR interacting proteins. In the upper panel, fractions were subjected to SDS-PAGE, followed by silver staining. Total HeLa S3 nuclear extract [HNE] (lane 1), a fraction eluted from the P11 column [P11] (lane 2), fractions from GST [GST] (lane 3), and unliganded and liganded GST-VDR(DEF) columns [VDR(−);VDR(+)] (lanes 4 and 5) were examined by mass spectrometry. Identified proteins are indicated at the right side of the panel. The lower panel shows Western blot analysis using specific antibodies shown in the panel.

308


associated with the ligand activated VDR-DEF led to the identification of several known components of the DRIP/TRAP/SMCC complex (Fig. 3B) that were in agreement with previous observations [12,14]. One ligand-independent VDR-specific interactant turned out as the Williams syndrome transcription factor (WSTF)/ WBSCR9/BAZ1B [18,19]. WSTF is a candidate gene whose product is potentially responsible for diverse WS disorders [18,20]. WSTF is highly homologous to hACF1 as one of the Bromodomain-adjacent-to-Zn finger (BAZ) family proteins [19], and hACF1 is a partner of hSNF2h (a Drosophila ISWI homolog), which forms well-characterized ISWI-based chromatin remodeling complexes (see Fig. 2) [21]. These facts raise the possibility that a WSTF-containing complex is an ATPdependent chromatin complex.

than 670 kDa contained WSTF, indicating that WSTF forms a stable nuclear complex. With mass fingerprinting, we identified all the components of the purified complex containing WSTF (Fig. 4C) and designated this complex as WINAC (WSTF including nucleosome assembly complex) [22]. WINAC consists of at least 13 components, but unexpectedly, does not contain hSNF2h as an ATPase subunit (Fig. 4C). Rather, the SWI/SNF type ATPases (Brg1 and hBrm) and several BAF components are shared with the SWI2/SNF2-based complexes [9]. Interestingly, WINAC appears to harbor three additional components (TopoIIb, FACTp140, and CAF-1p150) [23–25], which have not been observed in any of the known ATP-dependent chromatin remodeling complexes, but are supposed to be involved in other nuclear processes such as DNA repair and replication.

IV. PURIFICATION AND IDENTIFICATION OF A NOVEL WILLIAMS SYNDROME TRANSCRIPTION FACTOR COMPLEX ASSOCIATING WITH VITAMIN D RECEPTOR

V. WINAC IS A NOVEL MULTIFUNCTIONAL ATP-DEPENDENT CHROMATIN REMODELING COMPLEX THAT REARRANGES A NUCLEOSOME ARRAY AROUND A VITAMIN D RESPONSIVE ELEMENT IN VITRO

WSTF-containing complexes were purified by sequential columns (Fig. 4A) and fractionated on glycerol density gradients (Fig. 4B, upper panel). The fractionated complex with a molecular weight of greater

A

B Nuclear extract from f–WSTF cells GST

An ATP-dependent chromatin assembly reaction is clearly induced by WINAC as assessed by a standard micrococcal nuclease assay (Fig. 5A), indicating that

C kDa 200 116

BAF250 Brg1/hBrm TopoIIb WSTF

97 GST–VDR(D3−)

Glycerol density gradient

66

BAF170 BAF155 FACT p140 CAF–1 p150

WSTF Brg1

aFLAG M2 resin

WINAC

VDR GST–VDR BAF60a BAF57 BAF53 Ini1 WINAC

FIGURE 4 Purification and identification of a human WSTF-containing multiprotein complex “WINAC”. (A) Purification scheme of WINAC from MCF7 stable transformants. (B) Fractionation of purified complexes on glycerol density gradient. In the lower panel, Western blot analysis of each fraction using specific antibodies is shown. (C) The purified complex was subjected to SDS-PAGE, followed by silver staining and identified by mass spectrometry (indicated in the left of the panel).

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A

ATP WINAC

B

+ − + + − +

Proximal probe

Distal probe

C

Proximal Probe

GAL–VDR −

+

−

+

−

+

−

+

GAL–ERα

+

+

−

−

−

−

+

+

−

−

+

+

GAL–PPAR −

−

+

+

−

+

−

+

WINAC

WINAC

FIGURE 5 WINAC as an ATP-dependent chromatin remodeling complex. (A) Chromatin reconstitution activity of WINAC. The reacted samples were subjected to partial micrococcal nuclease digestion. The molecular mass marker is the 200-bp ladder. (B) Chromatin disruption by WINAC is specifically VDR dependent. Oligonucleotide probe corresponds to either a sequence between the GAL4 sites and the RNA start site (Proximal Probe) or 900 bp upstream of the start site (Distal Probe). (C) WINAC is unable to affect the chromatin architecture through other nuclear receptors (ER and PPAR).

Brg1/hBrm in WINAC serves as an ATPase for this ATP-dependent chromatin remodeling process. WINAC appeared to have a chromatin assembly activity (data not shown) similar to RSF [22,26]. We then examined the ability of WINAC to disrupt nucleosome arrays through VDR bound VDRE since the known ATP-dependent chromatin remodeling complexes are able to recognize a nucleosomal array around binding sites of a sequence specific regulator [27]. Disruption of the nucleosome arrays in the vicinity of the GAL4 binding site was induced only when both VDR and WINAC were present (Fig. 5B), while other regions appeared unaffected in the nucleosome array. Reflecting VDR-specific nucleosome disruption by WINAC (among several tested receptors) (Fig. 5C), WINAC potentiated ligand-induced transactivation in vitro only for VDR, but not for either ERα or PPARγ (data not shown)[22].

VI. WILLIAMS SYNDROME TRANSCRIPTION FACTOR COACTIVATED THE LIGAND-INDUCED TRANSACTIVATION FUNCTION OF VITAMIN D RECEPTOR In a reporter assay, 1,25(OH)2D3 (10−9 M) was effective in inducing VDR AF-2 transactivation function, and WSTF coactivated this ligand-induced AF-2

function of VDR, but not ERα (Fig. 6A). Coactivation by either Brg1 or hBrm was expectedly detected in VDR and ERα (Fig. 6A) [28,29]; however, such coactivator-like activity of WSTF was found only for VDR, but not for ERα, even in the presence of Brg1/hBrm (Fig. 6A). ChIP analysis of the positive VDRE in the human 25-hydroxyvitamin D3 24-hydroxylase gene promoter revealed that associations of VDR and the WINAC components with the promoter did not require ligand binding. In contrast, ligand binding to VDR appeared to be a prerequiste for occupancy of known coactivators TRAP220 and TIF2 on the promoter (Fig. 6B), together with ligand-induced histone H4 acetylation (data not shown) [22] though ligand-induced TRAP220 and TIF2 occupancy was cyclic (data not shown), as already described [28]. Such ligand-dependent and -independent recruitment of factors to the promoter and histone modification were robustly attenuated by WSTF-RNAi expression (Fig. 6B). As the VDR/RXR heterodimer also represses transcription in a ligand-dependent manner through a negative VDRE (nVDRE), the action of WSTF in ligandinduced transrepression was examined in a naturally occurring nVDRE in the human 25-hydroxyvitamin D3 1α-hydroxylase [1 (OH)ase gene promoter [30]. ChIP analysis revealed that VDR and WINAC appear to bind to the nVDRE in a ligand-independent manner, while cyclic ligand-induced recruitments of N-CoR and HDAC2 were

310


B Transcription activity (fold)

A GAL4-VDR-DEF

WSTF

− − − − − +

− − − − +

− − − − +

− − − − − − + − − − − − − + + +

+ − + − − +

− + + − − +

− − − + − +

− + − + − +

− − − − + +

− + − − + +

− − + − − + − − − − −

WSTF-RNAi − − − + − − + − + − + Brg1

− − − − + + + − − − −

TRAP220

− − − − − − − + + − −

TIF2 E2

− − − − − − − − − + + − + + + + + + + + + +

Input

Input

VDR

VDR

fWSTF

fWSTF

Brg1

Brg1

TRAP220

NCoR

TIF2

HDAC2

hSNF2H

hSNF2H

D3

−

+

−

+

+

D3

−

+

−

+

+

WSTF-RNAi

−

−

−

−

+

WSTF-RNAi

−

−

−

−

+

D GAL4-ERα-DEF

1α(OH)ase gene promoter negative distal VDRE

Transcription activity (fold)

− − − − − −

Transcription activity (fold)

WSTF WSTF-RNAi Brg1 TRAP220 TIF2 D3

C 24(OH)ase gene promoter positive distal VDRE

WSTF WSTF-RNAi Brg1 NCoR HDAC2 D3

− − − − − −

− − − − − +

− − − − +

− − − − +

− − − − − − + − − − − − − + + +

+ − + − − +

− + + − − +

− − − + − +

− + − + − +

− − − − + +

− + − − + +

FIGURE 6

Ligand-dependent promoter targeting of co-regulators through WINAC-VDR association. (A) VDR-specific facilitation of coactivator accessibility by WINAC. MCF7 cells were transfected with the expression vectors of a luciferase reporter plasmid containing the GAL4 upstream activation sequence (UAS) [17mer(×2)] driven by the β-globin promoter (0.5 µg). PML-CMV (2 ng), GAL4-DBD-VDR-DEF (0.2 µg), GAL4-DBD-ERa-DEF (0.2 µg), pDNA3-FLAG-WSTF (+; 0.1 µg: ++; 0.3 µg), pSV-Brg1 (0.2 µg), pSV-hBrm (0.2 µg), pcDNA3-TRAP220 (0.3 µg), pcDNA3-TIF2 (0.3 µg), siRNA (+; 0.1 µg: ++; 0.2 µg) of WSTF-RNAi, or control RNAi or their combinations were transfected as indicated in the panels in the absence or presence of ligand (10−9 M). Bars in each graph show the fold change in luciferase activity relative to the activity of the receptor transactivation in the presence of ligand. (B, C) ChIP analysis on the 24(OH)ase promoter and 1α(OH)ase promoter of WSTF stable transformants. Soluble chromatin was prepared from WSTF stable transformants treated with 1,25(OH)2D3 (10−9 M) for 45 min and immunoprecipitated with indicated antibodies. (D) The coregulator-like actions of WSTF on the naturally occurring negative vitamin D response elements. MCF7 cells were transfected with the expression vectors of either the luciferase reporter plasmid containing a human 1α(OH)ase promoter containing a negative VDRE and the factors shown in (A) or together with pcDNA3-N-CoR (0.3 µg), pcDNA3-HDAC2 (0.3 µg).

observed (Fig. 6C). Ligand-dependent repression was dependent on the expression levels of WSTF (Fig. 6D). Thus, it is likely that WINAC association with VDR facilitates targeting of a putative co-repressor complex to the nVDRE. Thus, these findings indicate that WINAC rearranges the nucleosome array around the positive and negative VDREs, thereby facilitating accessibility of the coregulatory complexes accessible to VDR for further transcription controls (Fig. 7).

VII. MOLECULAR MECHANISM OF VDR PROMOTER TARGETING OF VITAMIN D RECEPTOR BY WINAC, AND COOPERATIVE WINAC FUNCTION WITH THE CO-REGULATOR COMPLEXES Both WSTF and the ATPase subunits coactivated the ligand-induced VDR transactivation, a finding similar

311


WINAC WSTF

VDR RXR

VDR RXR

HDAC complexes HAT complexes

HDAC1/2

WINAC

mSin3

PCAF complex

NCoR/ WSTF SMRT VDR RXR

Gene activation

WSTF

VDR RXR

Target gene

gene represseion

WINAC

p300

p160 family protein

1α,25(OH)2D3

Target gene

FIGURE 7

Model of WINAC action in chromatin reorganization. WINAC mediates the recruitment of unliganded VDR to VDR target sites in promoters, whereas subsequent binding of co-regulators requires ligand binding. This recruitment order exemplifies that an interaction of a sequence-specific regulator with a chromatin remodeling complex can organize nucleosomal arrays at specific local sites in order to make promoters accessible for co-regulators.

to the reported coactivator-like actions of the SWI2/SNF2-type complex components, ATPases and BAF57, for the ligand-induced ERα transactivation [28,29]. Notably, VDR coactivation by the liganddependent NR coactivators (TIF2 and TRAP220) was abrogated by WSTF-RNAi expression. However, such WSTF coactivator-like actions were not observed for ERα and the other receptors tested (data not shown), nor were they reduced by WSTF-RNAi expression, supporting the observed direct and selective interaction of WSTF with VDR, though it remains unclear whether promoter targeting of VDR requires only WINAC or the other chromatin remodeling complexes. More remarkably, we found that WSTF could potentiate the ligand-induced transrepression of VDR on the 1α(OH)ase negative VDRE (Fig. 6D), where ablation of endogenous WSTF by RNAi expression led to a significant reduction in ligand-induced co-repressor recruitment (Fig. 6C). Thus, ligand-independent association of WINAC and VDR in VDR target promoters appears to facilitate local nucleosomal array accessibility for ligand-dependent co-regulators, following histone tail modifications by the recruited co-regulator complexes [11] (Fig. 7). From our ChIP analysis, VDR appears to be selectively targeted through WINAC to the promoters in a ligand-independent fashion or following recruitment of co-regulator complexes. This ligand-independent association of VDR with the target promoter is surprisingly distinct from the ligand-induced promoter targeting of many other steroid receptors [28,29]. It is possible that other nonsteroidal receptors such as

RAR, RXR, and TR may associate with their target promoters in the absence of hormone, and as-yetunidentified chromatin remodeling complexes may assist the promoter targeting. As HAT and HDAC complexes appear not to associate with unliganded VDR bound upon the tested promoters, WINAC targeting to the VDR target promoters does not appear to require specific histone tail modifications by other co-regulators. Thus, it is possible that WINAC binding to promoters facilitates VDR recognition and specific binding to VDREs, through nucleosomal mobilization by WINAC, presumably cooperating with the other chromatin complexes [17]. Alternatively, once VDR binds to VDREs during nonspecific chromatin remodeling, WINAC might be recruited to VDR on specific promoters to engage in local nucleosome reorganization. The latter possibility coincides well with a recent report regarding a sequence-specific regulator, SATB1 [16]. As a result of WINAC recruitment, the local chromatin structure near VDREs may transit into an active chromosomal state that appears competent for receipt of both coactivator and co-repressor complexes dependent on the VDRE sequences and the tertiary positions of DNA-bound VDR. This scheme may not be considered, however, with VDREs composed of unrelated DNA sequences with the canonical VDREs. Regardless, our hypothesis is not consistent with current understanding that the chromatin remodeling complexes are recruited only after acetylation/ deacetylation of histone tails by the coregulatory complexes [11]. However, the orders of the complex targetings are supposed to be dependent on regulator type

312


and promoter context [31,32]. This point should be addressed with VDR together with WINAC.

activator interactions with the ATM-related Tra1 subunit. Science 292:2333–2337. Fyodorov DV, Kadonaga JT 2001 The many faces of chromatin remodeling: SWItching beyond transcription. Cell 106:523–525. Yasui D, Miyano M, Cai S, Varga-Weisz P, Kohwi-Shigematsu T 2002 SATB1 targets chromatin remodeling to regulate genes over long distances. Nature 419:641–645. Lemon B, Inouye C, King DS, Tjian R 2001 Selectivity of chromatin-remodeling cofactors for ligand-activated transcription. Nature 414:924–928. Lu X, Meng X, Morris CA, Keating MT 1998 A novel human gene, WSTF, is deleted in Williams syndrome. Genomics 54:241–249. Jones MH, Hamana N, Nezu J, Shimane M 2000 A novel family of bromodomain genes. Genomics 63:40–45. Peoples RJ, Cisco MJ, Kaplan P, Francke U 1998 Identification of the WBSCR9 gene, encoding a novel transcriptional regulator, in the Williams–Beuren syndrome deletion at 7q11.23. Cytogenet Cell Genet 82:238–246. Poot RA, Dellaire G, Hulsmann BB, Grimaldi MA, Corona DF, Becker PB, Bickmore WA, Varga-Weisz PD 2000 HuCHRAC, a human ISWI chromatin remodelling complex contains hACF1 and two novel histone-fold proteins. EMBO J 19: 3377–3387. Kitagawa H, Fujiki R, Yoshimura K, Mezaki Y, Uematus Y, Matsui D, Ogawa S, Unno K, Okubo M, Tokita A, Nakagawa T, Ito T, Ishimi Y, Nagasawa H, Matsumoto T, Yanagisawa J, Kato S 2003 The chromatin remodeling complex WINAC targets a nuclear receptor to promoters and is impaired in Williams Syndrome. Cell 113:1–13. Smith S, Stillman B 1989 Purification and characterization of CAF-I, a human cell factor required for chromatin assembly during DNA replication in vitro. Cell 58:15–25. Varga-Weisz PD, Wilm M, Bonte E, Dumas K, Mann M, Becker PB 1997 Chromatin-remodelling factor CHRAC contains the ATPases ISWI and topoisomerase II. Nature 388:598–602. LeRoy G, Orphanides G, Lane WS, Reinberg D 1998 Requirement of RSF and FACT for transcription of chromatin templates in vitro. Science 282:1900–1904. Loyola A, LeRoy G, Wang YH, Reinberg D 2001 Reconstitution of recombinant chromatin establishes a requirement for histonetail modifications during chromatin assembly and transcription. Genes Dev 15:2837–2851. Ito T, Bulger M, Pazin MJ, Kobayashi R, Kadonaga JT 1997 ACF, an ISWI-containing and ATP-utilizing chromatin assembly and remodeling factor. Cell 90:145–155. Shang Y, Hu X, DiRenzo J, Lazar MA, Brown M 2000 Cofactor dynamics and sufficiency in estrogen receptor-regulated transcription. Cell 103:843–852. Belandia B, Orford RL, Hurst HC, Parker MG 2002 Targeting of SWI/SNF chromatin remodelling complexes to estrogenresponsive genes. EMBO J 21:4094–4103. Murayama A, Takeyama K, Kitanaka S, Kodera Y, Hosoya T, Kato S 1998 The promoter of the human 25-hydroxyvitamin D3 1α-hydroxylase gene confers positive and negative responsiveness to PTH, calcitonin, and 1α,25(OH)2D3. Biochem Biophys Res Commun 249:11–16. Lomvardas S, Thanos D 2002 Modifying gene expression programs by altering core promoter chromatin architecture. Cell 110:261–271. Soutoglou E, Talianidis I 2002 Coordination of PIC assembly and chromatin remodeling during differentiation-induced gene activation. Science 295:1901–1904.

Acknowledgment We sincerely thank all collaborators for the WINAC projects, and the laboratory members for helpful discussions and technical support. We are grateful to Miss Y. Nagasawa for preparation of the manuscript.

References

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1. Deluca HF 1986 The metabolism and functions of vitamin D. Adv Exp Med Biol 196:361–375. 2. Bouillon R, Okamura WH, Norman AW 1995 Structurefunction relationships in the vitamin D endocrine system. Endocr Rev 16:200–257. 3. Walters MR 1992 Newly identified actions of the vitamin D endocrine system. Endocr Rev 13:719–764. 4. Mangelsdorf DJ, Thummel C, Beato M, Herrlich P, Schutz G, Umesono K, Blumberg B, Kastner P, Mark M, Chambon P, et al. 1995 The nuclear receptor superfamily: the second decade. Cell 83:835–839. 5. Huges MR, Malloy PJ, Kieback DG, Kesterson RA, Pike JW, Feldman D, O’Malley BW 1988 Point mutations in the human vitamin D receptor associated with hypocalcemic rickets. Science 242:1702–1705. 6. Haussler MR, Whitfield GK, Haussler CA, Hsieh JC, Thompson PD, Selznick SH, Dominguez CE, Jurutka PW 1988 The nuclear vitamin D receptor: biological and molecular regulatory properties revealed. J Bone Miner Res 13: 325–349. 7. Rachez C, Lemon D, Suldan Z, Bromleigh V, Gamble M, Naar M, Erdjument H, Tempst P, Freedman P 1999 Ligand-dependent transcription activation by nuclear receptors requires the DRIP complex. Nature 398:824–828. 8. Glass CK, Rosenfeld MG 2000 The co-regulator exchange in transcriptional functions of nuclear receptors. Genes Dev 14:121–141. 9. Narlikar GJ, Fan HY, Kingston RE 2002 Cooperation between complexes that regulate chromatin structure and transcription. Cell 108:475–487. 10. Emerson BM 2002 Specificity of gene regulation. Cell 109:267–270. 11. Hassan AH, Prochasson P, Neely KE, Galasinski SC, Chandy M, Carrozza MJ, Workman, JL 2002 Function and selectivity of bromodomains in anchoring chromatinmodifying complexes to promoter nucleosomes. Cell 111: 369–379. 12. Yanagisawa J, Kitagawa H, Yanagida M, Wada O, Ogawa S, Nakagomi M, Oishi H, Yamamoto Y, Nagasawa H, McMahon SB, Cole MD, Tola L, Takahashi N, Kato S 2002 Nuclear receptor function requires a TFTC-type histone acetyltransferase complex. Mol Cell 9:553–562. 13. Lanz RB, McKenna NJ, Onate SA, Albrecht U, Wong J, Tsai SY, Tsai MJ, O’Malley BW 1999 A steroid receptor coactivator, SRA, functions as an RNA and is present in an SRC-1 complex. Cell 97:17–27. 14. Brown CE, Howe L, Sousa K, Alley SC, Carrozza MJ, Tan S, Workman JL 2001 Recruitment of HAT complexes by direct

21.

22.

23. 24.

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27. 28. 29. 30.

31. 32.

CHAPTER 18

Molecular Basis of the Diversity of Vitamin D Target Genes CARSTEN CARLBERG, THOMAS W. DUNLOP, CHRISTIAN FRANK, AND SAMI VÄISÄNEN Department of Biochemistry, University of Kuopio, Kuopio, Finland

I. Molecular Basis of the Genomic Actions of 1,25(OH)2D3 II. Classical Vitamin D–Receptor Binding Sites

III. Complex Vitamin D–Receptor Binding Sites IV. Conclusion References

I. MOLECULAR BASIS OF THE GENOMIC ACTIONS OF 1,25(OH)2D3

the p160-family, such as SRC-1, TIF2, and RAC3 [8]. These CoAs link the ligand-activated VDR to enzymes displaying histone acetyltransferase (HAT) activity that cause chromatin opening. Subsequently, ligand-activated VDR changes rapidly from the CoAs of the p160-family and those of the DRIP/TRAP family. The latter are part of a mediator complex of approximately 15 proteins that build a bridge to the basal transcription machinery [9]. In this way ligand-activated VDR fulfills two tasks, opening chromatin and activating transcription. The LBD of the VDR can be stabilized by 1,25(OH)2D3 or its analogs in an agonistic, antagonistic, or inverse agonistic conformation [10]. The position of helix 12 is the most critically important feature of these conformations, because it determines the distance that separates the charge clamp amino acids K246 in helix 3 and E420 in helix 12 that are both essential for VDR– CoA interaction (see Chapters 13–15). Most VDR ligands have been identified as agonists and only a few as pure or partial antagonists [11]. Two side-chain analogs, such as Gemini and its derivatives, have conditional inverse agonistic properties [12]. Antagonists induce CoR dissociation from the VDR but completely or partially prevent CoA interaction and thus transactivation [13]. At supramolar CoR concentrations inverse agonists actively recruit CoRs to the VDR and thus mediate repression of 1,25(OH)2D3 target genes. These ligand-triggered protein–protein interactions are the central molecular events of nuclear 1,25(OH)2D3 signaling.

A. Ligand-Triggered Protein–Protein Interactions of the VDR The vitamin D3 receptor (VDR) is the only nuclear protein that binds with high affinity to the biologically most active vitamin D metabolite, 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) [1]. The VDR is one of 11 members of the nuclear receptor (NR) superfamily that function as classical endocrine receptors. These include the receptors for the nuclear hormones retinoic acid, thyroid hormone, estradiol, progesterone, testosterone, cortisol, and aldosterol, which bind their specific ligand with a Kd of 1 nM or less [2]. Like most members of the NR superfamily, the VDR contains two zinc finger structures forming a characteristic DNA-binding domain (DBD) of 66 amino acids [3] and a carboxyterminal ligand-binding domain (LBD) of approximately 300 amino acids formed by 12 α-helices [4] (see Chapters 11–15). Ligand binding causes a conformational change within the LBD, whereby helix 12, the most carboxy-terminal α-helix, closes the ligand-binding pocket via a “mousetrap-like” intramolecular folding [5]. The LBD is also involved in a variety of interactions with nuclear proteins, such as other members of the NR superfamily, coactivator (CoA) and co-repressor (CoR) proteins [6]. CoR proteins, such as NCoR, SMRT, and Alien, link nonliganded, DNA-bound VDR to enzymes with histone deacetylase activity that cause chromatin condensation [7]. This provides VDR with intrinsic repressive properties comparable to those of retinoic acid and thyroid hormone receptors (RARs and T3Rs). The conformational change within VDR’s LBD after binding of 1,25(OH)2D3 or one of its agonistic analogs results in replacement of CoR by a CoA protein of VITAMIN D, 2ND EDITION FELDMAN, PIKE, AND GLORIEUX

B. DNA Binding of the VDR An essential prerequisite for a direct modulation of transcription via 1,25(OH)2D3-triggered protein– protein interactions is the location of activated VDR close to the basal transcriptional machinery. This is achieved Copyright © 2005, Elsevier, Inc. All rights reserved.

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CARSTEN CARLBERG, THOMAS W. DUNLOP, CHRISTIAN FRANK, AND SAMI VÄISÄNEN

through the specific binding of the VDR to a 1,25(OH)2D3 response element (VDRE) in the regulatory region of a primary 1,25(OH)2D3 responding gene [14]. The DBD of the VDR contacts the major grove of a hexameric sequence, referred to as core binding motif, with the consensus sequence RGKTSA (R = A or G, K = G or T, S = C or G). The affinity of monomeric VDR to a single core binding motif is not sufficient for the formation of a stable protein–DNA complex and thus VDR requires formation of homo- and/or heterodimeric complexes with a partner NR in order to allow efficient DNA binding [15]. In most cases the heterodimeric partner of VDR is the retinoid X receptor (RXR), another NR family member. The protein–DNA complex of a VDR-RXR heterodimer sitting on a VDRE therefore can be considered as a molecular switch for primary 1,25(OH)2D3 responding genes. Most NRs are able to dimerize in solution via their LBDs, but the DBDs dimerize only in the presence of DNA [16]. The DBD and the LBD of all NRs are linked by a hinge region of 35 to 50 amino acid residues that form a long α-helical structure according to the crystal structure of the DNA-bound T3R-RXR heterodimer [17]. The loop between this α-helix and the second zinc finger of the DBD contains a short six-amino-acid residue region, referred to as a T-box, which has been suggested to provide a dimerization interface for the interaction with the DBD of RXR [18]. Steric constrains allow dimerization of DBDs only on response elements (REs) with properly spaced core binding motifs. Modeling of the DBDs of VDR and RXR on DNA [17] suggested that an asymmetric arrangement, i.e., head-to-tail, as a direct repeat with three intervening nucleotides (DR3) provides the most efficient interface of the core DBDs. This fits with the 3-4-5 rule of Umesono et al. [19], in which VDR-RXR heterodimers should show optimal binding to DR3-type REs, whereas other NRs prefer DR4-type REs [e.g., T3R, constitutive androstane receptor (CAR) and pregnane X receptor (PXR)] and DR5-type REs (e.g., RARs). On DR3-, DR4-, and DR5-type REs, the different heterodimers bind with the same polarity, in which RXR always binds to the upstream hexamer and the partner receptor, e.g., VDR, to the downstream hexamer [20,21]. This specific and directed dimerization of the DBDs appears to be the major discriminative parameter for selective RE recognition.

II. CLASSICAL VITAMIN D–RECEPTOR BINDING SITES A. Classification of DR3-type REs Numerous studies (e.g., [15,22]) have confirmed Umesono’s suggestion [19] that VDR binds well to

DR3-type REs. Most of these studies also demonstrated that VDR preferentially or even exclusively forms heterodimers with RXR on these REs. DR3-type REs are therefore widely accepted as the classical structure of a VDRE. Every transcriptionally responsive primary 1,25(OH)2D3 target gene has to contain at least one VDRE in its promoter region and these VDREs are generally located relatively close to the transcription start site (TSS) of these genes. It is assumed that matrix attachment regions (MARs) subdivide genomic DNA into units of an average length of 100 kB containing the coding region of at least one gene [23]. DNA looping should be able to bring any DNA site within the same chromatin unit close to the basal transcriptional machinery that is assembled on the TSS (Fig. 1). This model suggests that also very distant sequences can serve as VDREs and that even sequences downstream of the TSS could serve as functional VDR binding sites. The list of the presently known natural VDREs (Table I) indicates that most of them have a DR3-type structure and are located within the first 1000 bp of promoter sequence upstream of the TSS. In addition, this VDRE list suggests that the consensus VDR core binding motif is RGKTSA, although some natural hexameric sequences show a significant deviation from this consensus sequence (Table I). However, one has to take into account that all these VDREs had been identified before the genomes of Homo sapiens and other species had been sequenced, and only limited promoter sequences were available. Moreover, only a very few of these VDREs, such as that of the rat osteocalcin gene, are understood in their promoter context, i.e., in the context of chromatin organization and flanking binding sites for other transcription factors (TFs). Therefore, the present VDRE list (Table I) has to be taken with some precaution. Results from in silico promoter screening and a more detailed understanding of chromatin organization of primary 1,25(OH)2D3 target genes will revise this list soon. On the basis of presently published data, the strongest VDR-RXR heterodimer binding DR3-type VDREs has been identified within the rat atrial natriuretic factor (ANF) promoter [24], the mouse and pig osteopontin promoter [25,26], and the chicken carbonic anhydrase II promoter [27]. These four elements were categorized into class I (see Table I). The DR3-type VDREs of the human and rat 24-hydroxylase (CYP24) promoter [28,29], the human Na+-dependent inorganic phosphate transporter type II promoter [30], the rat osteocalcin promoter [31], the human parathyroid hormone (PTH) promoter [32], and the first VDRE of the rat PTH related peptide (PTHrP) promoter [33] demonstrated only 6–30% of the binding strength of the rat ANF VDRE and were grouped into class II. The binding of VDR-RXR heterodimers to the ten

315

CHAPTER 18 Diversity of Vitamin D Target Genes

VDRE R X R

MAR

V D R

TSS Basal transcriptional machinery

Coding region

? V D R MAR

R X R

VDRE

FIGURE 1

Schematic structure of a chromatin unit. A chromatin unit is a region between two MARs and often contains only one gene. DNA looping should permit that every DNA sequence within the same chromatin unit to be located near the basal transcriptional machinery. The occurrence of VDREs downstream of a coding region has not yet been proven experimentally.

DR3-type VDREs in classes I and II was found to be enhanced by 1,25(OH)2D3 by a factor of 2 to 5 [34]. In contrast, the second VDRE of the rat PTHrP promoter [35], the rat calbindin D9k promoter [36] and the quail slow myosin heavy chain promoter [37], the human growth hormone promoter [38], the chicken integrin β3 promoter [39], the chicken PTH promoter [40], and the human p21WAF1/CIP1 promoter [41] were grouped together into class III, as they showed less than 5% of the affinity for VDR-RXR heterodimers compared to the rat ANF VDRE and no significant ligand inducibility. According to these stringent in vitro criteria, the core sequences of the class III members cannot be considered as functional VDREs. The in vitro binding affinity of VDR-RXR heterodimers to VDREs was shown to be proportional to their in vivo functionality in transiently transfected cells, i.e., to mediate in a heterologous promoter context induction of reporter gene activity [21,42]. In this assay system class III VDREs did not show any in vivo functionality. The hexameric sequences of the class III VDREs show a significant deviation from the RGKTSA consensus and explains their relatively low ability of complex formation with VDR-RXR heterodimers. However, it is possible that class III VDREs may gain responsiveness to 1,25(OH)2D3 in their natural promoter context through the help of flanking partner proteins. Moreover, the functionality of a 1,25(OH)2D3 responding gene will also

depend on a potential cooperative action of two or more VDREs, such as in the case of the CYP24 gene [43].

B. Do DR3-Type REs Differ in Their Function? Since the pleiotropic physiological effects of 1,25(OH)2D3 are based in the final analysis on transcriptional regulation of primary 1,25(OH)2D3 responding genes, the genes should be explained through activation of VDR-RXR heterodimers bound to DR3-type VDREs. Therefore, there have been attempts to explain the various effects of 1,25(OH)2D3 by a multiplicity of 1,25(OH)2D3 signaling pathways that are based on DR3-type VDREs [44]. This leads to the question of whether each DR3-type VDRE has an individual functionality that may explain the specific effects of 1,25(OH)2D3 or whether all DR3-type VDREs function in the same way. The affinity of the known natural DR3-type VDREs for VDR-RXR heterodimers in vitro (at a fixed protein– DNA ratio) seems to be their major discriminating parameter, which allowed the grouping of the VDREs into classes as discussed earlier (Table I). VDR-RXR heterodimers appear to form identical complexes on the 10 VDREs of classes I and II, since indistinguishable VDR-RXR heterodimer conformations were observed on these VDREs [34]. This is in contrast to a report that VDR-RXR heterodimers take different conformations

316


TABLE I Natural VDRCS Gene

Species

Class I DR3-type VDREs ANF Osteopontin Osteopontin Carbonic anhydrase II

Rat Mouse Pig Chicken

AGAGGTCATGAAGGACA AAGGTTCACGAGGTTCA ATGGGTCATATGGTTCA GAAGGGCATGGAGTTCG

−907 −759 −2261 −62

[24] [26] [25] [27]

Human Human Human

CGAGGTCAGCGAGGGCG GGAGTTCACCGGGTGTG CAGGGGCAGCAAGGGCA

−171 −291 −1977

[28] [28] [30]

Human Human Rat Rat Rat Rat

ATGGTTCAAAGCAGACA CCGGGTGAACGGGGGCA CTGGGTGAATGAGGACA CGAGGTGAGTGAGGGCG AGGGTTCAGCGGGTGCG TAAGGTTACTCAGTGAA

−122 −500 −457 −152 −259 −805

[32] [52] [31] [29] [29] [35]

Class III DR3-type VDREs p21 Growth hormone Bone sialo protein PTH related peptide Calbindin D9k Integrin β3 PTH Slow myosin heavy chain

Human Human Rat Rat Rat Chicken Chicken Quail

GTAGGGAGATTGGTTCA TGGGGTCAACAGTGGGA GAAGGGTTTATAGGTCA AGGGTGGAGAGGGGTGA GAGGGTGTCGGAAGCCC GCGAGGCAGAAGGGAGA GAGGGTCAGGAGGGTGT GAAGGACAAAGAGGGGA

−779 −59 −30 −1107 −490 −772 −76 −801

[41] [38] [78] [33] [36] [39] [40] [37]

DR4-type VDREs Pit-1 Calbindin D28k

Rat Mouse

GAAGTTCAGCGAAGTTCA CTGGGGGATGTGAGGAGA

−683 −200

[48] [95]

DR6-type VDREs Osteocalcin Phospholipase C-γ1 CYP24 Fibronectin

Human Human Rat Mouse

TTTGGTGACTCACCGGGTGA GCAGGTCAGACCACTGGACA CGGGTCGAGCCCAGGGTTCA CCGGGTGACGTCACGGGGTA

−514 −805 −231 −152

[51] [56] [55] [54]

ER9-type VDREs Calbindin D9k Osteocalcin c-fos p21

Human Rat Mouse Mouse

TGCCCTTCCTTATGGGGTTCA TGCACTGGGTGAATGAGGACA TGACCCTGGGAACCGGGTCCA TGACCTGAAAGTGGAAGGTGA

−147 −461 −482 −3811

[21] [21] [57] [96]

Complex VDREs Osteocalcin Osteocalcin c-fos

Human Rat Mouse

TTTGGTGACTCACCGGGTGAACGGGGGCA TGCACTGGGTGAATGAGGACA AGGTGAAAGATGTATGCCA AGACGGGGGTTGAAAG

−514 −461 −178

[42] [21] [77]

Class II DR3-type VDREs CYP241 CYP242 Na+-dependent inorganic Phosphate transporter type II PTH Osteocalcin Osteocalcin CYP241 CYP242 PTH related peptide

Sequence

Position

Reference

The core sequence of the different types of VDREs and their position in relation to the TSS are indicated. Hexameric core binding motifs are in bold and deviations from the consensus sequence RGKTSA are underlined.

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on the mouse osteopontin and the rat osteocalcin DR3type VDRE [44]. In that study, nuclear extracts from bone cells provided protein complexes on the two VDREs that were differentially recognized by antibodies. One could assume that the VDR-RXR heterodimers in these complexes interact with different types of cofactors as has been described for estrogen REs [45]. However, VDRRXR heterodimers formed on the different DR3-type VDREs display no significant differences in their interaction with a given CoA or CoR protein. The ligand sensitivity of VDR-RXR heterodimers bound to the different DR3-type VDREs showed no significant deviation from the average value of 0.1 nM. This suggests that the ligand-triggered VDR-RXRVDRE complex formation does not depend on ligand concentration or VDR conformation [34]. In addition, VDR-RXR heterodimers show no significant differences in their 1,25(OH)2D3-triggered interaction with CoA and CoR proteins. These findings suggest that heterodimers are able to differentiate between individual DR3-type VDREs on the basis of the different protein-DNA affinities. However, when the complexes are formed, they appear to function in exactly the same fashion. This means that at least comparative in vitro data cannot support any indications of multiple DR3type VDRE-mediated 1,25(OH)2D3 signaling pathways. Investigations of VDR-RXR heterodimers and their conformations have provided multiple new insights on the functionality of these molecular switches that may well explain their function in living cells [7,46]. This makes it likely that the lack of variation of the in vitro functionality of DR3-type VDREs (from positively as well as negatively regulated genes) can be transferred to the in vivo situation. This facilitates further investigation of the principles of DR3-type VDRE-mediated gene regulation, since an observation that has been made with one specific DR3-type VDRE can be generalized for the whole family of DR3-type VDREs. However, an explanation for the multiplicity of 1,25(OH)2D3 signaling has to be found at a different level.

C. Other DR-Type VDREs Since a variety of DR3-type VDREs seems unable to explain the pleiotropic physiological action of 1,25(OH)2D3 and the dissociated profile that some synthetic 1,25(OH)2D3 analogs display, other VDRE structures such as direct repeats with four and six intervening nucleotides (DR4 and DR6) or everted repeats with nine spacing nucleotides (ER9) may offer an alternative explanation. A comparison of the individual VDRE core sequences (Table I) with their classification according to the affinity for VDR-RXR heterodimers

suggests that the degree of deviation from the core binding motif consensus sequence RGKTSA [47] is proportional to the loss of in vitro functionality [34]. Interestingly, the DR4-type RE of the rat pit-1 gene [48], which contains two perfect core binding motifs, was found to be stronger than any known natural DR3-type VDRE [34]. Does this suggest that DR4-type REs are even better VDREs than DR3-type structures? One has to consider that a DR4-type REs is also recognized by the heterodimeric complexes of T3R, CAR, PXR, and other orphan NRs with RXR [49,50], whereas the same complexes bind to DR3-type REs less tightly than VDR-RXR heterodimers. The competitive situation on DR4-type REs may therefore be the reason why in vivo VDR-RXR heterodimers still prefer DR3-type REs. Moreover, VDR-RXR heterodimers bind to DR4-type REs in the same conformation as to DR3-type REs [50], i.e., there seems to be no differential action of VDR on these elements due to a differential complex formation with RXR. The VDRE of the human osteocalcin promoter was the first identified natural binding site for the VDR [51,52]. It was initially described as a DR6-type structure, but later a third cryptic hexamer was identified at a distance of three nucleotides, so that the whole VDRE is more likely a complex DR6/DR3-type RE (Table I). The DR6 part of the VDRE has been shown to bind VDR homodimers [15] and VDR-RAR heterodimers [53], whereas the DR3 part weakly binds VDR-RXR heterodimers. Other examples of a DR6-type VDREs that bind VDR homodimers and VDR-RAR heterodimers have been identified in the promoters of mouse fibronectin [54], rat CYP24 [55], and human phospholipase C [56]. Their functionality remains to be determined.

D. Everted Repeats Dimerization facilitates cooperative, high-affinity interaction of NRs, such as VDR and RXR, with specific hexameric core binding motifs. As schematically depicted in Fig. 2, VDR-RXR heterodimers bind to DR-type VDREs in a nonsymmetrical head-to-tail tandem arrangement. In contrast, like all palindromic sequences, ER-type VDREs are per se symmetric, since the heterodimeric partner receptors bind in a tail-to-tail arrangement. However, natural ER9-type REs were found to be sufficiently asymmetric in their core binding sequences, in order to allow a polarity-determined binding of heterodimers [21]. So far, ER9-type VDREs have been identified within the promoter regions of the human calbindin D9k gene [21], the mouse c-fos gene [57], and the rat osteocalcin gene [21]. Interestingly, the last VDRE shares one core binding motif with the

318


Direct repeats

Everted repeat 9

3 or 4

DBD

Hinge

LBD Helix 12

Helix 12

RXR

VDR

RXR

VDR

FIGURE 2 DNA complex formation of VDR-RXR heterodimers. The DNA binding of VDR-

RXR heterodimers to DR- or ER-type VDREs is schematically indicated. Some critical α-helices within the LBD of VDR and the DBDs of VDR and RXR are indicated by cylinders.

DR3-type VDRE identified by Demay et al. [31], and there appears to be a complex ER9/DR3-type RE. On DR3-type VDREs, both receptor DBDs are located at roughly the same side of the DNA (tilted by 51.4°), whereas on ER9-type VDREs the DBDs are at nearly opposite sites of the DNA (tilted by 154.3°) [18]. Moreover, the distance between the DBDs along the axis of the DNA is threefold greater on ER9-type VDREs than on DR3-type VDREs (Fig. 2). Computer modeling and experimental studies have both shown that the T-box of VDR contributes to the dimerization interface of the extended VDR DBD with the RXR DBD, i.e., on a DR3-type VDRE the VDR DBD directly contacts the RXR DBD, whereas a direct contact of both DBDs is not possible on an ER9-type VDRE. The lack of DBD dimerization on an ER9-type RE suggests that DNA-driven cooperativity between the partner DBDs is unlikely. In fact, the spacing of the two core binding motifs is less stringent than on DRtype REs, as ER7-, ER8-, and ER10-type structures are also able to bind dimeric VDR complexes [58,59]. However, the Kd value for the binding of VDR-RXR heterodimers to DR3- and ER9-type REs has been determined to be in a similar range of 0.5 to 1 nM [21]. According to our model of multiple 1,25(OH)2D3 signaling pathways [60], the pleiotropic function of 1,25(OH)2D3 is based on a variety of dimeric VDR complexes bound to different types of VDREs. The model assumes that each of these VDR-VDRE

complexes may be representative for a group of primary 1,25(OH)2D3 target genes that are involved in the regulation of a distinct proportion of the pleiotropic actions of this nuclear hormone. In support of the model, some 1,25(OH)2D3 analogs have shown the tendency to preferentially activate VDR-RXR heterodimers that are bound to ER9-type VDREs [61], whereas other analogs prefer DR3-type VDRE-bound VDR complexes [62]. The selective biological profile of the analog EB1089, i.e., having both potent antiproliferative potential and a reduced calcemic effect [63], was associated with a higher selectivity (approximately 15 times) to activate ER9-type VDREs compared to DR3-type VDREs [61]. Because of the relatively low number of known primary 1,25(OH)2D3 target genes with characterized VDREs, this idea has to be proven. However, the genes of mouse c-fos [57] and human and mouse p21WAF1/CIP1 [14] each contain an ER9-type VDRE in their regulatory regions. A VDRE-selective in vitro stabilization of VDR-RXR heterodimers was demonstrated by ligand-dependent gel shift assays and showed that EB1089 mediated the stabilization of VDR-RXR heterodimers on an ER9-type VDREs at approximately eightfold lower concentrations than on a DR3-type VDRE [64]. In contrast, the natural hormone 1,25(OH)2D3 showed no significant selectivity. Taken together, the selective activation of ER9-type VDREs by EB1089, i.e., the observation of promoter selectivity of a 1,25(OH)2D3 analog, seems to be based on the enhanced DNA binding affinity of

319


a subset of all VDR-RXR heterodimers to this type of VDRE.

E. Do Hexameric Core Binding Motifs Provide Sufficient Specificity for DNA Binding of VDR-RXR Heterodimers? It has been known for some time that some NRs, such as T3R, which can also bind as monomers to DNA, have a clear preference for certain 5′-flanking sequences to the hexameric core binding motif [65,66]. In addition, it had been demonstrated that in particular the dinucleotide sequence 5′-flanking of the downstream hexamer of a RE have a significant effect on the complex formation of NR heterodimers, such as RARRXR, T3R-RXR, and VDR-RXR [66–68]. This provides the sequence of the spacer between the hexameric motifs of natural REs with a critical role for determining the specific recognition of the RE and the regulation of the respective gene. The dinucleotide AG, as found, e.g., in front of the downstream motif of the DR4-type RE of the rat pit-1 gene, seems to be optimal for VDRRXR heterodimers [68]. This preference should be independent of the type of RE and apply to all VDRE types. The crystal structure of DNA-bound T3R-RXR heterodimers [17] demonstrated a contact of amino acids of the carboxy-terminal extension of the T3R-DBD with the two 5′-flanking nucleotides of the downstream core binding motif. Similar assumptions could be made for the VDR. Interestingly, variations of the dinucleotide sequence 5′-flanking to the upstream core binding motif also have a lower but still significant influence on complex formation and functional activity of VDR-RXR heterodimers [68]. This suggests that RXR also specifically contacts 5′-flanking nucleotides to its binding motif. In summary, 5′-flanking sequences should be considered as an integral part of a RE, so that more likely octameric motifs instead of hexameric sequences are specific VDR binding sites. A reanalysis of previously characterized VDREs (Table I) for their 5′-flanking sequences will provide a better understanding of their relative strength. Even more important is the possibility of a more accurate prediction of putative VDREs from large amounts of sequence data now available from the human genome.

F. VDR Homodimers and RXR-Independent 1,25(OH)2D3 Signaling VDR homodimers were found to bind and initiate ligand-driven transactivation from DR3- and DR6-type VDREs [15,69]. Moreover, homodimers of VDR DBDs

have even been crystallized on DR3-type REs [3] confirming the view that they may have some physiological relevance. The in vitro binding of VDR homodimers on idealized DR3- and DR6-type REs is not as high as that of VDR-RXR heterodimers on DR3-type REs [42,70]. However, in vitro binding affinity of VDR-RXR heterodimers to natural DR3-type VDRE of class III is also weak [34] and VDR homodimers may be specifically stabilized by CoA proteins [71]. In vitro complex formation experiments as well as yeast or mammalian two-hybrid experiments leave no doubts that within the NR superfamily, RXR is the most efficient protein– protein interaction partner of the VDR. This interaction preference is evolutionarily conserved and also applies to the 15 closest members of VDR within its superfamily [72]. Moreover, at least one of the three RXR subtypes seems to be expressed in every mammalian cell, which makes investigations of putative RXRindependent 1,25(OH)2D3 signaling pathways difficult. In vitro as well as in a Drosophila cell system the formation of VDR homodimers has been shown [15]. Evidence for VDR-RAR and VDR-T3R heterodimers has also been reported [42,53,59]. The concentration of protein was very high, however. VDR-RAR and VDR-T3R heterodimers showed preference for DR5and DR6-type REs [42] and have also been observed on ER7- to ER10-type REs [58,59]. Taken together, on most VDREs and in the vast majority of physiological situations, VDR-RXR heterodimers seem to mediate 1,25(OH)2D3 signaling. There may be exceptions, however. In these cases, their in vitro relevance needs to be studied in the context of the natural promoter in a chromatin environment.

III. COMPLEX VITAMIN D–RECEPTOR BINDING SITES A. Simple versus Complex VDREs With the exception of the VDREs in the human and rat osteocalcin gene, all natural VDREs are formed by only two hexameric core binding motifs in a DR3, DR4, DR6, and ER9 arrangement (Table I). These latter targets may be therefore considered as “simple” VDREs. The question is, whether the occurrence of one simple VDRE within a promoter is sufficient for gene responsiveness to 1,25(OH)2D3. Because of its optimized 5′-flanking dinucleotide and core binding motif sequences the DR4-type RE of the rat pit-1 gene is the most efficient known VDRE in vitro [34,50]. However, the chromatin in the region of the pit-1 gene promoter containing this RE seems to be closed in the adult rat, so that the responsiveness of the gene to 1,25(OH)2D3

320


is lower than expected [73]. This indicates that a high in vitro binding affinity of VDR-RXR heterodimers for a VDRE is not sufficient for responsiveness to 1,25(OH)2D3. When the promoter region that contains the VDRE is covered by condensed chromatin, VDRRXR heterodimers are unable to bind there. This makes sufficiently decondensed chromatin an essential prerequisite for a functional VDRE. Chromatin decondensation is achieved by the activity of HATs, which are recruited to their local chromatin target by CoA proteins. In turn, these CoAs are transiently attracted to a promoter region by ligand-activated NRs and other active TFs. Therefore, the more TF binding sites a given promoter region contains and the more of these TFs are expressed in the respective cell, the higher is the chance that this area of the promoter gets locally decondensed. One example is the VDRE of the rat osteocalcin gene, which is flanked on both sides with a binding site for the TF Runx2/Cbfa1 [74]. By contacting CoA proteins and HATs Runx2/Cbfa1 seems to mediate the opening of chromatin locally, which allows efficient binding of VDR-RXR heterodimers to this decondensed region to occur. This mechanism suggests that VDREs are better targets for VDR-RXR heterodimers, if other TFs are bound to the same chromatin region. In this respect, promoter context and cell-specific expression of other TFs may be of greater importance to VDRE functionality and specificity than its in vitro binding profile. If this idea is true, it will apply to other members of the NR superfamily and will question the validity of isolated simple VDREs. Therefore, in the future VDREs will have to be understood as complex structures with multiple TF binding sites. Some of these TF binding sites will be other NR core binding motifs. The DR6/DR3 and ER9/DR3 structures of the VDREs of the human and rat osteocalcin genes, respectively, are the first examples of complex VDREs. These two complex VDREs show only limited homology to each other, although they are derived from orthologous genes. This suggests that for an important primary 1,25(OH)2D3 responding gene such as osteocalcin, there may be limited evolutionary pressure for a specific VDRE structure. It seems to be more important to guarantee an efficient binding of the VDR to a promoter in competition with the tight packaging of nucleosomes. It is interesting to note that the complex VDRE of the human osteocalcin gene is overlaid by a binding site of the TF AP-1 [75], which provides the RE with an increased activity. These types of REs are also observed for other NRs and often referred to as composite REs [76]. Another interesting example of a complex/composite VDRE has been reported in the mouse c-fos promoter [77]. Within this VDRE three hexameric core binding motifs are forming a DR7/DR7 structure, which contains an internal

binding site for the TF NF-1. Additional examples are the VDRE of the human and mouse fibronectin gene, which contains an internal binding site for the TF CREB [54], or the VDRE of the rat bone sialo protein, which also seems to bind the general TF TBP [78].

B. RE Clusters In mammals, the most responsive known primary 1,25(OH)2D3 target gene is CYP24, which gene product is the key enzyme involved in the catabolism of 1,25(OH)2D3. Activation of this gene provides a negative feedback loop mechanism controlling the level of the hormonal ligand. The reason for the strong responsiveness of the CYP24 gene appears to be that it contains two DR3-type VDREs separated by a distance of less than 100 bp in close proximity to the TSS. These DR3-type VDRE clusters are evolutionarily conserved between humans and rodents [43,79]. The sequences of both VDREs are not optimal and are categorized into class II (Table I). However, these less optimal structures are more than compensated by the fact that they are located in close vicinity to each other and to the TSS of the gene. In addition, binding sites for the TF Ets-1 have been characterized within the CYP24 promoter and seem to interfere synergistically with the two VDREs, i.e., the cluster of VDR and Ets binding sites form another type of complex VDRE. Such types of RE clusters are also known for other genes that are primary targets of additional members of the NR superfamily, such as VDR’s close relatives CAR and PXR. Investigation of natural CAR and PXR target gene, such as CYP2B6 or CYP3A4, respectively [80,81], indicate that a single RE is insufficient for mediating the regulatory role of the receptors and that more likely at least two CAR or PXR REs in close proximity to each other are required. In the case of CAR, these multiple RE clusters are called phenobarbital response enhancer modules (PBREMs), because they mediate the responsiveness to the CAR activator phenobarbital. The CYP2B6 gene contains two DR4-type REs with an additional binding site for the TF NF-1 [80], whereas the PBREM of the UDPglucuronosyltransferase 1A1 gene is formed by three CAR REs [82]. In contrast, the CYP3A4 gene contains an ER6-type RE proximal to the TSS and a more distal DR3/ER6 cluster [81]. Taken together, these assemblies represent two or more simple NR REs together with binding sites for other types of TFs. Their overall function seems to follow the same rules, i.e., the greater number of NRs and other TFs bind to such promoter regions, the greater is the chance that they induce histone acetylation and chromatin decondensation.


Another interesting observation in relation to the RE clusters is that they are rather promiscuously bound by related members of the NR superfamily, such as VDR, CAR, and PXR. VDR was shown to activate the CYP2B6, CYP3A4, and CYP2C9 genes by replacing CAR and PXR within the respective RE clusters [83]. In particular, the CYP3A4 gene, which contains a DR3-type RE within its RE cluster, was shown to be stimulated effectively by 1,25(OH)2D3 and can be considered as a primary 1,25(OH)2D3 responding gene. This suggests that 1,25(OH)2D3 and the VDR may have an impact on the metabolism of prescription drugs, of which 60% are metabolized by the CYP3A4 enzyme [84]. In this context, it is interesting to note that the VDR seems to be able to act as an intestinal bile acid sensor, because certain bile acids have been identified as low-affinity VDR ligands [85]. These actions are also mediated by CYP3A4. It is very likely that future investigations will reveal more of these 1,25(OH)2D3 target genes, thus enlarging the physiological importance of this hormone to an even greater extent.

C. Negative VDREs A relatively undercharacterized aspect of 1,25(OH)2D3 signaling is the mechanism of down-regulation of 1,25(OH)2D3 responsive genes. Microarray experiments have indicated that nearly half of all primary 1,25(OH)2D3 responding genes are down-regulated by the hormone, but few of them have been studied in more detail. It is obvious that only genes which show basal activity can be down-regulated, i.e., these genes exhibit basal activity due to other TFs binding to their promoter. There are several different models that attempt to explain how 1,25(OH)2D3 and the VDR can mediate down-regulation of genes, but the common theme is that VDR counteracts the activity of specific TFs. In the situation where these activating TFs are other NRs or TFs that bind to composite NR REs, VDR could simply compete for DNA binding sites [86,87]. In a similar way, VDR could also compete for binding to partner proteins, such as RXR, or for common CoAs, such as SRC-1 or p300 [88]. In all these situations the down-regulating effects of the VDR should be of general impact, i.e., the mechanism could apply to other genes in the same way. So far, however, no general down-regulating effects of 1,25(OH)2D3 have been reported. The binding of 1,25(OH)2D3 to the VDR results in a ligand-dependent conformational change, which leads to an exchange of proteins that bind to the LBD of the VDR, i.e., altering protein-protein interaction profiles but no changes in VDR-DNA interaction properties.

321 In most cases, however, 1,25(OH)2D3-dependent down-regulation of a gene involves specific DNA binding sites on the specific promoter, which are referred to as negative VDREs. Experiments suggest that DNA binding of the VDR to down-regulated genes such as the PTH gene, does not involve RXR [89,90], whereas other studies, such as on the ANF gene [91], come to opposite conclusions. As discussed above, there are also alternative partner proteins for the VDR, such as RAR or VDR itself, and one might speculate that cell- and gene-specific TFs could help the VDR in binding efficiently to DNA. Investigation of the 1,25(OH)2D3-mediated repression of the PTHrP gene indicates that protein kinases may be involved in the down-regulation of this gene [92]. Some of the natural VDREs in Table I were identified within genes that are down-regulated by 1,25(OH)2D3 and are therefore more likely to be negative VDREs. These sequences are not revealing, however, because in the absence of a natural promoter context and normal chromatin organization, these VDREs behave like positive VDREs. This again highlights the fact that promoter context, the chromatin status, and the cluster of neighboring TFs are important for the function of a simple VDRE. Thus simple VDREs cannot act as negative VDREs. In the case of the calcitonin gene, two separate promoter regions are suggested to be responsible for the down-regulation of gene transcription by 1,25(OH)2D3 [93] (see Chapter 39). Together, this information indicates that mechanisms governing 1,25(OH)2D3-mediated down-regulation is complex. The concept that VDRE clusters together with and other TF binding sites regulate primary 1,25(OH)2D3 responding genes suggests that a promoter may contain both negative and positive VDREs. The activities of the different VDREs are determined by the promoter context and may not be simultaneously active. One might imagine that prior to stimulation with 1,25(OH)2D3 only the negative VDREs bind the VDR and recruit CoRs. This would actively condense the chromatin on a particular promoter region. The addition of ligand induces the release of CoR proteins and reduces chromatin density. The VDR may then be transiently released from the negative VDRE and bind to a positive VDRE, which may be uncovered through 1,25(OH)2D3-dependent local nucleosome acetylation. The VDR then interacts with CoAs of the DRIP/TRAP mediator protein complex on this positive VDRE leading to transient transcriptional activation. After a certain period of time, newly synthesized, unliganded VDR again binds to the negative VDRE, which initiates chromatin closing and inactivation of the positive VDRE. In this or even more complex scenarios, the balance between negative and positive

322


VDREs could explain the time course of the activation of primary 1,25(OH)2D3 responding genes.

have to be included in more effective versions of in silico screening software.

D. Whole-Genome Screening for Putative VDREs

IV. CONCLUSION

Considering both orientations of DNA, the NR core binding motif RGKTSA should be found every 256 bp of genomic DNA. Dimeric assemblies of such hexamers should show up as direct repeats every 65,536 bp of promoter sequence and as everted repeats every 32,768 bp. Since VDR-RXR heterodimers bind comparably well to DR3-, DR4-, and ER9-type REs, an in silico screening is expected to identify a putative VDR binding site every 16,384 bp. This would predict nearly 200,000 putative VDREs within the human genome, and as a consequence on average every gene would contain several VDREs in its promoter and should be responsive to 1,25(OH)2D3. Similar calculations apply to other members of the NR superfamily, and for TFs with a shorter specific binding site even higher numbers would be predicted. A realistic number of 1,25(OH)2D3 responding genes is far less than this, perhaps a few hundred. The number of VDR molecules varies from hundreds to several thousand molecules. These calculations make it obvious that not every putative VDR binding site is used in nature in any cell at any given time. The most obvious reason is that most of these sequences are effectively covered by nucleosomes, so that they are not accessible to the VDR. This applies in particular to those sequences that are isolated from other NR or TF binding sites or lie distant from the promoter. This perspective strongly discourages the idea that isolated, simple VDREs may be functional in vivo. Therefore, the presently identified simple VDREs (Table I) may be parts of more complex VDREs as already demonstrated for the CYP24 and osteocalcin gene. An effective in silico prediction of novel VDREs has to focus on the identification of complex VDREs. Unfortunately, presently available in silico screening software, such as NUBISCAN [94], is unable to identify complex VDREs. Other available programs such as TRANSFAC have the capability to identify larger numbers of REs for different TFs, making the identification of potential complex REs possible. However, even these programs are unable to predict chromatin condensation states and nucleosome positioning, which is essential information in determining the likelihood that a given RE lies in an accessible region of a promoter. General parameters, such as nucleosome positioning and interspecies homology screening of regulatory sequences as well as the binding sites of all other TFs and their cell-specific expression patterns,

The sequencing of the complete human genome and the genomes of other species, i.e., the availability of all regulatory sequences, enables a more mature understanding of the diversity of 1,25(OH)2D3 target genes. Perhaps the idea of simple isolated VDREs, such as those listed in Table I, should shift to the concept of complex VDREs, of which the simple VDRE represents the core. Depending on the temporal presence of cellspecific TFs, these complex REs may act positively or negatively in respect to 1,25(OH)2D3. The coordinated action of these different types of VDREs could explain the individual response of target genes to 1,25(OH)2D3.

Acknowledgment The Academy of Finland (grant 50319) supported the work.

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CHAPTER 19

Intranuclear Organization of the Regulatory Machinery for Vitamin D–Mediated Control of Skeletal Gene Expression GARY S. STEIN, JANE B. LIAN, MARTIN MONTECINO,* JANET L. STEIN, ANDRE J. VAN WIJNEN, AMJAD JAVED, JE-YONG CHOI,** S. KALEEM ZAIDI, SORAYA GUTIERREZ,* JIALI SHEN, SHIRWIN POCKWINSE, AND DANIEL YOUNG Department of Cell Biology and Cancer Center, University of Massachusetts Medical School, Worcester, Massachusetts; ∗Departamento de Biologia Molecular, Universidad de Concepcion, Concepcion, Chile; ∗∗Department of Biochemistry, Kyungpook National University, Daegu, Korea I. Introduction II. Requirements for Physiologically Responsive Control of Skeletal Gene Expression In Vivo III. Gene Expression within the Three-Dimensional Context of Nuclear Architecture IV. Chromatin Remodeling Facilitates Vitamin D–Mediated Promoter Accessibility and Integration of Regulatory Activities V. Nuclear Microenvironments: Accommodating The Rules That Govern In Vivo Transcriptional Control

VI. Scaffolding of Regulatory Elements for Combinatorial Control of Gene Expression VII. Intranuclear Trafficking of Skeletal Regulatory Factors to Subnuclear Sites That Support Transcription: “To Be in the Right Place at the Right Time” VIII. The Regulated and Regulatory Parameters of Subnuclear Organization References

I. INTRODUCTION

factors abrogate competency for vitamin D control of skeletal gene expression during development and fidelity of gene expression in tumor cells.

The architecturally associated subnuclear organization of nucleic acids and cognate regulatory factors illustrates functional interrelationships between nuclear structure and gene expression. Mechanisms that contribute to the spatial distribution of transcription factors within the dynamic three-dimensional context of nuclear architecture control the sorting of regulatory information and the transcriptionally competent or repressed chromatin configuration of gene promoters as well as the assembly and activities of sites within the nucleus that support gene expression. Vitamin D control of gene expression serves as a paradigm for experimentally addressing mechanisms that govern the intranuclear targeting of regulatory factors to nuclear domains where chromatin remodeling and transcription of developmental as well as tissue-specific genes occur. We will provide an overview of molecular, cellular, biochemical, and in vivo genetic approaches that provide insight into the trafficking of regulatory factors that mediate vitamin D–controlled gene expression to transcriptionally active subnuclear sites. Examples will be presented that suggest modifications in the intranuclear targeting of transcription VITAMIN D, 2ND EDITION FELDMAN, PIKE, AND GLORIEUX

II. REQUIREMENTS FOR PHYSIOLOGICALLY RESPONSIVE CONTROL OF SKELETAL GENE EXPRESSION IN VIVO Bone formation during development and skeletal remodeling throughout life requires the complex and interdependent expression of cell growth and phenotypic genes (reviewed in [1,2]). There is a requirement for responsiveness to a broad spectrum of regulatory cues that transduce physiological signals from the extracellular matrix to sites within the nucleus where genes that mediate skeletogenesis reside [3–6]. As our understanding of gene regulatory mechanisms expands, it becomes increasingly evident that there are unique parameters of transcriptional control that support the transient activation and suppression of genes for skeletal development and bone homeostasis. Other mechanisms Copyright © 2005, Elsevier, Inc. All rights reserved.

328 are invoked for long-term obligations to gene expression that sustain the specialized properties of the bone cells. Vitamin D serves as a principal modulator of skeletal gene transcription necessitating an understanding of interfaces between activity of this steroid hormone with regulatory cascades that are functionally linked to the regulation of skeletal genes [7]. There is growing appreciation for the repertoire of factors that influence gene expression for commitment to the osteoblast lineage. It is well documented that sequentially expressed genes support progression of osteoblast differentiation through developmental transition points where responsiveness to phosphorylationmediated regulatory cascades determine competency for establishing and maintaining the structural and functional properties of bone cells [2,4,8,9]. The catalog of promoter elements and cognate regulatory proteins that govern skeletal gene expression offers essential but insufficient insight into mechanisms that are operative in intact cells. Gene promoters serve as regulatory infrastructure by functioning as blueprints for responsiveness to the flow of cellular regulatory signals. But to access the specific genetic information necessitates understanding transcriptional control of skeletal genes within the context of the subnuclear organization of nucleic acids and regulatory proteins. Explanations are required for (1) convergence of multiple regulatory signals at promoter sequences; (2) the integration of regulatory information at independent promoter domains; (3) selective utilization of redundant regulatory pathways; (4) thresholds for initiation or down-regulation of transcription with limited intranuclear representation of promoter elements and regulatory factors; (5) mechanisms that render the promoters of cell growth and phenotypic genes competent for protein–DNA and protein–protein interactions in a physiologically responsive manner; (6) the composition, organization, and assembly of sites within the nucleus that support transcription; and (7) the intranuclear trafficking of regulatory proteins to transcriptionally active foci.

III. GENE EXPRESSION WITHIN THE THREE-DIMENSIONAL CONTEXT OF NUCLEAR ARCHITECTURE Evidence is accumulating that the architectural organization of nucleic acids and regulatory proteins within the nucleus support functional interrelationships between nuclear structure and gene expression (Fig. 1 and 2). There is increasing acceptance that components of nuclear architecture are functionally linked to the organization and sorting of regulatory information in a manner that permits selective utilization [10–20]. The primary

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ET AL .

level of nuclear organization, the representation and ordering of genes and promoter elements, provides alternatives for physiological control. The molecular organization of regulatory elements, the overlap of regulatory sequences within promoter domains, and the multipartite composition of regulatory complexes increase options for responsiveness. Chromatin structure and nucleosome organization reduce distances between regulatory sequences, facilitate crosstalk between promoter elements, and render elements competent for interactions with positive and negative regulatory factors [21]. The components of higher order nuclear architecture that includes nuclear pores [22,23], the nuclear matrix, and subnuclear domains contribute to the subnuclear distribution and activities of genes and regulatory factors (reviewed in [24–27]). Compartmentalization of regulatory complexes is illustrated by focal organization of PML bodies [28–30], Runx bodies [11,12,16,31,32], and the nucleolus, chromosomes [33], as well as by the punctate intranuclear distribution of sites for replication [34–36], DNA repair, transcription [37–42], and the processing of gene transcripts [26,43,44]. There is emerging recognition that nuclear structure and function are causally interrelated. With mounting evidence for organization of nucleic acids and regulatory proteins into subnuclear domains that are associated with components of nuclear architecture, the perception of a dichotomy between nuclear architecture and control of gene expression is difficult to justify. Rather, it is necessary to design experiments to define mechanisms that direct genes and regulatory factors to sites within the nucleus where localization integrates regulatory parameters of gene expression and establishes microenvironments with boundaries between regulatory complexes that are required for fidelity of activity. The bone-specific osteocalcin gene and skeletalrestricted Runx2 (AML3/Cbfa1/PEBP24) transcription factor serve as paradigms for obligatory relationships between nuclear structure with physiological control of skeletal gene expression [32,45–48]. The modularly organized promoter of the bone-specific osteocalcin gene contains proximal and distal regulatory elements that support basal, and tissue-specific as well as growth factor, homeodomain, signaling protein, and steroid hormone responsive transcriptional control (reviewed in [45,46,49–55]) (see Fig. 3). Modulation of osteocalcin gene expression during bone formation and remodeling requires physiologically responsive accessibility of these proximal and upstream promoter sequences to regulatory and coregulatory proteins as well as protein–protein interactions that integrate independent promoter domains. The nuclear matrixassociated Runx transcription factors contribute to the control of skeletal gene expression by sequence-specific

CHAPTER 19 Intranuclear Organization of the Regulatory Machinery for Vitamin D

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FIGURE 1 Multiple levels of chromatin architecture within the nucleus. Higher order chromatin organization in the nucleus results from sequential packaging of DNA from a linear double helix (upper panel). The sequence of the Runx binding element is shown. A loop domain emanating from the highly compact chromatin is schematically illustrated (lower panel). Elements at the base of the loop structure designated MARs [matrix attachment regions, or alternatively locus control regions (LCR) or scaffold attachment regions (SCA)] mediate association of these genomic domains with the nuclear scaffold. Genes within the loop domain undergo local chromatin remodeling to support transcriptional activation or suppression. The nuclear matrix provides anchorage for both nucleic acids and regulatory as well as co-regulatory factors that control transcription.

binding to promoter elements of target genes and serving as scaffolds for the assembly and organization of co-regulatory proteins that mediate biochemical and architectural control of promoter activity.

IV. CHROMATIN REMODELING FACILITATES VITAMIN D–MEDIATED PROMOTER ACCESSIBILITY AND INTEGRATION OF REGULATORY ACTIVITIES It is well recognized that genomic DNA is packaged as chromatin. These “bead on a string” structures designated nucleosomes are structurally remodeled to accommodate requirements for transcription, emphasizing the extent to which architectural organization of genes is causally related to functional activity. The

identification and characterization of proteins that catalyze histone acetylation, deacetylation, methylation, and phosphorylation [56–63], as well as the SWI/SNFrelated proteins [56,64–68] that facilitate chromatin remodeling and potentially the accessibility of promoter sequences to regulatory and coregulatory factors, represent an important dimension in control of the structural and functional activities of genes and promoter regulatory elements [64,69–75]. Relationships of regulatory signaling pathways to enhance activities that modulate gene, chromatin, and chromosome organization can now be directly investigated. Additional levels of specificity are provided by structural modifications of gene promoters that influence competency for factor interactions. Simply stated, changes in the architectural properties of promoter elements determine effectiveness of gene regulatory sequences as substrates for interactions with regulatory factors. The regulatory and regulated parameters of chromatin

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Structural components

Apoptosis

Survivin

Replication sites

RPA

Chromosomal territories

Chromosomes

Nuclear envelope

PML bodies

Nucleoli

Transcription SWI/SNF complex sites

BRCA1

CAF-1

Replication and repair

RUNX domains

VDR SC 35 Domains

Coiled bodies

TLE

Transcription

Splicing FIGURE 2 Components of nuclear architecture are functionally linked to the organization and sorting of regulatory information. Nuclear functions are organized into distinct, nonoverlapping subnuclear domains. Nuclear matrix (shown in the center as revealed by electron microscopy), the underlying network of anastomizing network of filaments and fibers provides structural basis for the functional compartmentalization of nuclear functions. Immunofluorescence microscopy of the nucleus has revealed the distinct subnuclear distribution of vital nuclear processes, including (but not limited to) DNA replication sites and proteins involved in replication such as CAF-1 and RPA; DNA damage/repair as shown by BRCA1; chromatin remodeling, e.g., mediated by the SWI/SNF complex; structural parameters of the nucleus, such as the nuclear envelope, chromosomes, and chromosomal territories; chromatin organization and tissue specific transcriptional control, for example Runx, TLE, and VDR domains; and RNA synthesis and processing involving, for example, transcription sites; SC35 domains, coiled bodies, and nucleoli as well as proteins involved in cell survival e.g., survivin. Subnuclear PML bodies of unknown function have been examined in numerous cell types. All these domains are associated with the nuclear matrix. (See color plate).

remodeling and the rate limiting steps in the relevant signaling cascades are being actively pursued and will unquestionably provide insight into skeletal gene regulatory mechanisms from structural and functional perspectives. The chromatin organization of the osteocalcin gene illustrates dynamic remodeling of a promoter to accommodate requirements for phenotype-related developmental and steroid hormone responsive activity.

Nuclease digestion and ligation-mediated PCR analysis as well as in vitro nucleosome reconstitution studies establish the placement of nucleosomes in the proximal basal/tissue specific domain and at the upstream vitamin D–responsive element, blocking accessibility of these promoter sequences to regulatory proteins in immature bone cells when this skeletal-restricted gene is suppressed [60,76–80]. In response to developmental and skeletal regulatory signals the striking


A

Inactive Osteocalcin Gene

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Basal Transcription OC Gene Distal DHS

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Proximal DHS

Vitamin D Induced Transcription of OC Gene Distal DHS

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Proximal DNasel HS

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FIGURE 3 Remodeling of the Osteocalcin gene promoter during developmental progression of the osteoblast phenotype. The transcriptionally silent rat Osteocalcin gene is schematically illustrated with nucleosomes placed in the proximal tissue-specific and distal enhancer region of the promoter (A). Factors that support basal tissue-specific transcription are recruited to the OC gene promoter and are organized in proximal and distal promoter domains. Modifications in chromatin structure that mediate assembly of the regulatory machinery for the nuclease hypersensitive sites reflect OC gene transcription. A positioned nucleosome resides between the proximal basal and distal enhancer regions of the promoter (B). In response to vitamin D, chromatin remodeling renders the upstream VDRE competent for binding the VDR/RXR heterodimer with its cognate element (C). Higher order chromatin organization permits cross talk between basal transcription machinery and the vitamin D receptor complex that involves direct interactions of the vitamin D receptor, Runx2, and TFIIB (D).

removal of a nucleosome and modifications in chromatin structure render the proximal promoter of the OC gene accessible to regulatory and co-regulatory proteins that support basal level activity [48,48,60,78]. Vitamin D enhancement of osteocalcin gene transcription is associated with removal of the nucleosome at the upstream vitamin D–responsive element that permits binding of the vitamin D receptor–RXR heterodimer [48,60,78,81]. The retention of a nucleosome

between the proximal and upstream enhancer domain reduces distance between the basal and vitamin D–responsive element and supports a promoter configuration that is conducive to protein–protein interactions between the vitamin D receptor and the basal TFIIB transcription factor [82–84]. Interaction of the vitamin D receptor at the distal promoter region of the bone-specific osteocalcin gene requires nucleosomal remodeling [85].

332 Thus, insight into control of skeletal gene expression can be obtained from an understanding of mechanisms that alter osteocalcin gene chromatin organization under biological conditions. Site-directed mutagenesis of osteocalcin genes that are genetically integrated in stable cell lines has established that RUNX elements flanking the proximal and upstream promoter sequences are responsible for developmental and vitamin D– induced chromatin remodeling [48]. The recent demonstration of functional interactions between the VDR and flanking Runx proteins is consistent with linkage of VDRE organization and Runx regulatory elements [86]. Reduced CpG methylation is associated with transcriptional activation of the bone-specific osteocalcin gene in osteoblasts [87]. In vitro and in vivo genetic approaches have demonstrated that RUNX2 controls developmental and steroid hormone–responsive chromatin reconfiguration of the osteocalcin gene promoter [48,80]. Chromatin immunoprecipitation analyses have shown that developmental and vitamin D–linked remodeling of osteocalcin gene promoter organization is accompanied by acetylation of histones in the proximal basal and upstream vitamin D responsive element domains [88,89]. This posttranslational modification of histone proteins reduces the tenacity of histone DNA interactions in a manner that is conducive to an open chromatin organization with increased access to regulatory factors. The most compelling evidence for a functional involvement of chromatin organization in skeletal gene expression is the obligatory relationship of dynamic changes in the biochemical and structural properties of osteocalcin gene promoter organization with competency for bone tissue-restricted and enhanced transcription in response to vitamin D [48]. Yet, despite the cogent support for a central role of chromatin remodeling in transcriptional control of the osteocalcin gene, there are open-ended questions. It is not justifiable to extrapolate from these findings to conclude that all genes that are activated and suppressed during skeletogenesis employ identical mechanisms. From a broader biological perspective there are multiple levels of control that must be mechanistically characterized to explain physiologically responsive regulation of chromatin structure within restricted and global genomic contexts.

V. NUCLEAR MICROENVIRONMENTS: ACCOMMODATING THE RULES THAT GOVERN IN VIVO TRANSCRIPTIONAL CONTROL Key components of the basal transcription machinery and several tissue-specific transcription factor complexes

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are functionally compartmentalized as specialized subnuclear domains [11–13,16,27,39,40,90–102]. Such compartmentalization may, at least in part, accommodate biological constraints on the control of transcription in nuclei of intact bone cells. The low representation of promoter regulatory elements and cognate transcription factors necessitates a subnuclear organization of nucleic acids and regulatory proteins that supports threshold concentrations for the activation and repression of gene expression. From an historical perspective, compartmentalization of the regulatory machinery for ribosomal genes in nucleoli and the organization of chromosomes during mitosis provide paradigms for intranuclear localization of genes and regulatory complexes. During the past several years there has been growing recognition that the organization of nucleic acids and regulatory proteins is functionally linked to the assembly, organization, and activity of gene regulatory machinery. Cellular, molecular, biochemical, and genetic evidence indicates an obligatory relationship between sites within the nucleus where regulatory complexes reside and fidelity of transcriptional control. The biological relevance for the intranuclear distribution of regulatory complexes is directly reflected by aberrant nuclear structure–gene expression interrelationships that are associated with perturbations in skeletal development [18] and leukemia [16].

VI. SCAFFOLDING OF REGULATORY COMPONENTS FOR COMBINATORIAL CONTROL OF GENE EXPRESSION Functional interrelationships between nuclear structure and gene expression are strikingly reflected by dual recognition of regulatory proteins, such as RUNX transcription factors, for interactions with both promoter elements and co-regulatory proteins; such interactions modulate the structural and functional properties of targeted genes at microenvironments within the nucleus. Sequence-specific interactions with promoter elements result in placement of RUNX proteins at strategic sites where they provide scaffolds for protein–protein interactions that mediate the organization of machinery for a broad spectrum of regulatory requirements. These include histone modifications and chromatin remodeling that establish competency for transcription factor binding and genomic conformations that interface activities at proximal and upstream promoter domains, as well as the integration of regulatory cues from signaling pathways that activate or suppress gene expression in a physiologically responsive manner. As a consequence, the RUNX proteins are posttranslationally modified


(e.g., phosphorylated) to further influence the extent to which they engage in regulatory activity. The complexity of RUNX regulatory proteins that assemble as supercomplexes of transcriptional regulatory factors illustrates the potential impact on skeletalrelated gene expression. Recent documentation that RUNX proteins are components of a stable complex that includes basal transcription factors, chromatin remodeling factors, and histone-modifying factors indicates the scope of RUNX-mediated combinatorial control. A key component of the RUNX complex is the p300/CBP coactivator that functions as a transcriptional adaptor. Interactions with several transcription factors results in the formation of multimolecular complexes that regulate expression of a broad spectrum of genes [103]. p300 contains a domain with intrinsic histone acetyltransferase (HAT) activity [104,105], which has been implicated in chromatin structure alterations associated with modulation of gene expression [106]. p300 interacts with additional proteins containing HAT activity that include P/CAF, SRC-1, and ACTR. A basis is thereby provided for formation of large multiprotein complexes that contribute multiple HAT activities with options for specificity [107–111]. It has been established that RUNX2 and p300 are components of the same nuclear complexes in osteoblastic cells [112]. Furthermore, when recruited to the osteocalcin gene promoter by RUNX2, p300 stimulates both basal and vitamin D-enhanced osteocalcin promoter activity. Thus interactions of RUNX2 with p300 supports assembly of multisubunit complexes with several HAT-containing proteins at a series of regulatory regions of the bone-specific osteocalcin gene promoter. In a parallel manner, Kitabayashi et al. [113] have shown that in myeloid cells RUNX1, a homolog of the bonespecific RUNX2, interacts with p300 and together they up-regulate myeloid-specific genes. It was also determined that a C-terminal region of the Runt domain in both RUNX1 and RUNX2, is critical for their interactions with p300 [112,113]. Considering the high degree of homology between these two members of the RUNX transcription factor family it is likely that the structural determinants for RUNX interactions with p300 are conserved. In addition to functioning as transcriptional activators, RUNX proteins suppress gene expression (transcription). Repression requires the recruitment of transcriptional repressors and co-repressors with histone deacetylase activity (HDACs) to promoter regulatory elements of genes that are down-regulated. Combinatorial control that dampens transcription is illustrated by interaction of RUNX2 with the transcriptional co-repressors TLE/ Groucho through a conserved VWRPY domain located at the C terminus of the protein, which represses the

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expression of the bone-sialo protein (BSP) gene in osteoblastic cells [32]. Another example of combinatorial control that results in transcriptional suppression by RUNX2 is down-regulation of the p21CIP/WAF promoter in fibroblastic and osteoblastic cells. Here HDAC6 interacts with a second repression domain that also resides in the C-terminal region of RUNX2 and is recruited to chromatin by RUNX2 [114]. Taken together, these results are consistent with combinatorial control that is mediated by RUNX-dependent recruitment of coactivator and co-repressors proteins that are associated with and organized as multiprotein complexes to activate or repress target genes in a physiologically responsive manner. p300 can also be recruited to gene promoters by the transcription factor C/EBP [115,116]. Interestingly, a C/EBP-responsive regulatory element has been identified in the proximal promoter region of the rat OC gene adjacent to the RUNX2 site C [117]. C/EBPβ physically interacts with RUNX2 and synergistically activates the osteocalcin promoter [117], suggesting that both proteins form a complex with p300 and together up-regulate basal tissue-specific transcription. C/EBPβ has additionally been shown to interact with ATPdependent chromatin remodeling complexes of the SWI/SNF family [118], recruiting these complexes to promoter sequences and activating cell-specific expression. Taken together, these findings indicate that RUNX factors engage in protein–DNA and protein–protein interactions that collectively determine the composition and organization of promoter regulatory complexes. The inclusion of chromatin remodeling activity in these multisubunit complexes provides a biochemical basis for conformational modifications of promoter elements as well as combinatorial specificity for transcription. Transcription factors that function as scaffolds for interaction with co-regulatory proteins provide an architectural basis for accommodating the combinatorial requirements of biological control. Combinatorial control supports replication, transcription, and repair by two mechanisms. Context-dependent combinations and permutations of regulatory proteins are assembled into multipartite complexes that increase specificity. Scaffold-associated protein–DNA and protein–protein interactions permit integration of regulatory activities. Nuclear microenvironments are thereby organized, with gene promoters as focal points, where threshold concentrations of regulatory macromolecules are attained. The complexity that is achieved by these architecturally organized oligomeric factors can maximize options for responsiveness to diverse regulatory requirements for transient and long term biological control.

334 VII. INTRANUCLEAR TRAFFICKING OF SKELETAL REGULATORY FACTORS TO SUBNUCLEAR SITES THAT SUPPORT TRANSCRIPTION: ‘‘TO BE IN THE RIGHT PLACE AT THE RIGHT TIME’’ There is a need to gain insight into mechanisms that direct skeletal factors to subnuclear sites where regulatory events occur. Association of osteoblast, myeloid, and lymphoid RUNX transcription factors that mediate tissue-specific transcription with the nuclear matrix has permitted direct examination of mechanisms for targeting regulatory proteins to transcriptionally active subnuclear domains [12,45–47,95,119–126]. Both biochemical and immunofluorescence analyses have shown that RUNX transcription factors exhibit a punctate nuclear distribution that is associated with the nuclear matrix in situ [11,12,127,128]. Taken together, these observations are consistent with the concept that the nuclear matrix is functionally involved in gene localization and in the concentration and subnuclear localization of regulatory factors [12,93,94,129–132]. The initial indication that nuclear matrix association of RUNX factors is required for maximal activity was provided by the observation that transcriptionally active RUNX proteins associate with the nuclear matrix but inactive C-terminally truncated RUNX proteins do not [6,12,32,128,133] (Fig. 3). This localization of RUNX was established by biochemical fractionation and in situ immunofluorescence as well as by green fluorescent protein tagged RUNX proteins [31] in living cells. Colocalization of RUNX1, 2, and 3 at nuclear matrix–associated sites indicates a common intranuclear targeting mechanism may be operative for the family of RUNX transcription factors [31,32,127]. Variations in the partitioning of transcriptionally active and inactive RUNX between subnuclear fractions permitted development of a strategy to identify a region of the RUNX transcription factors that directs the regulatory proteins to nuclear matrix–associated foci. A series of deletions and internal mutations was constructed and assayed for competency to associate with the nuclear matrix by Western blot analysis of biochemically prepared nuclear fractions and by in situ immunostaining following transfection into intact cells. Association of osteogenic and hematopoietic RUNX proteins with the nuclear matrix is independent of DNA binding and requires a nuclear matrix targeting signal, a 31-amino-acid segment near the C terminus that is distinct from nuclear localization signals [12]. The nuclear matrix targeting signal functions autonomously and is necessary as well as sufficient to direct the transcriptionally

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active RUNX transcription factors to nuclear matrix– associated sites where gene expression occurs [12]. These findings indicate mechanisms involved in the selective trafficking of proteins to specialized domains within the nucleus where they become components of functional regulatory complexes. At least two trafficking signals appear to be required for subnuclear targeting of RUNX transcription factors; the first supports nuclear import (nuclear localization signal) and a second mediates association with the nuclear matrix (nuclear matrix targeting signal). The multiplicity of determinants for nuclear localization and alternative splicing of RUNX messenger RNA may provide the requisite complexity to support targeting to specific sites within the nucleus in response to diverse biological conditions. Furthermore, because gene expression by RUNX involves contributions by factors and co-regulatory proteins that include CBFβ [46,134–139] and C/EBP [117,140], Groucho/TLE [32,141,142], HES, and SMAD [6,143], RUNX may facilitate recruitment of these factors to the nuclear matrix.

A. Properties of Transcriptionally Active Subnuclear Compartments Association of genes and cognate factors with the nuclear matrix may support the formation and/or activities of nuclear domains that facilitate transcriptional control [10,45,55,132,144–151]. Results from our laboratory indicate that the association of RUNX transcription factors with the nuclear matrix is obligatory for activity [11,18]. The promoter recognition function of the runt homology domain of RUNX, and thus the consequential interactions with RUNX-responsive genes, is essential for formation of transcriptionally active foci containing RUNX and RNA polymerase II that are nuclear matrix associated [11]. Additionally, the nuclear matrix targeting signal supports transactivation when associated with an appropriate promoter, and transcriptional activity of the nuclear matrix targeting signal depends on association with the nuclear matrix [11]. Taken together, targeting of RUNX transcription factors to the nuclear matrix is important for their function and transcription. However, components of the nuclear matrix that function as acceptor sites remain to be established. Characterization of such nuclear matrix components will provide an additional dimension to characterizing molecular mechanisms associated with gene expression—the targeting of regulatory proteins to specific spatial domains within the nucleus. An initial indication of transcription factor interactions with the nuclear matrix is provided by crystal structure of the


RUNX nuclear matrix targeting signal that was determined by X-ray diffraction analysis at 2.7 Å [127,152].

B. Subnuclear Targeting and Integration of Signaling Pathways Gene expression during skeletal development and bone remodeling is controlled by a broad spectrum of regulatory signals that converge at promoter elements to activate or repress transcription in a physiologically responsive manner. The subnuclear compartmentalization of transcription machinery necessitates a mechanistic explanation for directing signaling factor to sites within the nucleus where gene expression occurs under conditions that support integration of regulatory cues. The interactions of YAP and SMAD co-regulatory proteins with C-terminal segments of the RUNX2 transcription factor permit assessment of requirements for recruitment of cSRC and BMP/TGFb-mediated signals to skeletal target genes. Our findings indicate that nuclear import of YAP and SMAD co-regulatory factors is agonist dependent. However, there is a stringent requirement for fidelity of RUNX subnuclear targeting for recruitment of these signaling proteins to transcriptionally active subnuclear foci. Our results demonstrate that the interactions and spatial–temporal organization of RUNX and SMAD as well as YAP co-regulatory proteins are essential for assembly of transcription machinery that supports expression of skeletal genes [6,128]. Competency for intranuclear trafficking of RUNX proteins has similarly been functionally linked with the subnuclear localization and activity of TLE/Groucho co-regulatory proteins [32]. These findings are consistent with proteins serving as a scaffold for interactions with co-regulatory proteins that contribute to biological control.

C. In Vivo Consequences of Aberrant Intranuclear Trafficking of RUNX Transcription Factors Using RUNX2 and its essential role in osteogenesis as a model, we investigated the fundamental importance of fidelity of subnuclear localization for tissue differentiating activity by deleting the intranuclear targeting signal via homologous recombination. Mice homozygous for the deletion (RUNX2∆C) do not form bone because of perturbed maturation or arrest of osteoblasts. Heterozygotes do not develop clavicles, but are otherwise normal. These phenotypes are

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indistinguishable from those of the RUNX2 homozygous and heterozygous null mutants, indicating that the intranuclear targeting signal is a critical determinant for function. The expressed truncated RUNX2∆C protein enters the nucleus and retains normal DNA-binding activity, but shows complete loss of intranuclear targeting. These results establish that the multifunctional N-terminal region of the RUNX2 protein is not sufficient for biological activity. Our results demonstrate that subnuclear localization of RUNX factors in specific foci together with associated regulatory functions is essential for control of RUNXdependent genes involved in tissue differentiation during embryonic development [18]. The importance of subnuclear localization of RUNX transcription factors for biological control is further indicated by compromised subnuclear organization and activity of RUNX1 hematopoietic regulatory proteins in acute myelogenous leukemia [16].

VIII. THE REGULATED AND REGULATORY PARAMETERS OF SUBNUCLEAR ORGANIZATION Multiple lines of evidence suggest that components of nuclear architecture contribute both structurally and enzymatically to control gene expression during osteoblast differentiation. Sequences have been identified that direct RUNX transcription factors to nuclear matrix–associated sites that support transcription in a cell cycle–dependent manner [153]. Insight is thereby provided into mechanisms linked to the assembly and activities of subnuclear domains where transcription occurs. In a restricted sense, the foundation has been provided for experimentally addressing intranuclear trafficking of gene regulatory factors and control of association with the nuclear matrix to establish and sustain domains that are competent for transcription. The unique sequences [11,12] and crystal structure for the 31-amino-acid nuclear matrix targeting signal of RUNX transcription factors [127,152] support specificity for localization at intranuclear sites where the regulatory machinery for gene expression is assembled, rendered operative, and/or suppressed. In a broader context, there is a growing appreciation for involvement of nuclear architecture in a dynamic and bidirectional exchange of gene transcripts and regulatory factors between the nucleus and cytoplasm, as well as between regions and structures within the nucleus [13,27,35,154–156]. It would be presumptuous to propose a single model to account for the specific pathways that direct transcription

336 factors to sites within the nucleus that support transcription. However, findings suggest that parameters of nuclear architecture functionally interface with components of transcriptional control. The involvement of nuclear matrix–associated transcription factors with recruitment of regulatory components to modulate transcription remains to be defined. Working models that serve as frameworks for experimentally addressing components of transcriptional control within the context of nuclear architecture can be compatible with mechanisms that involve architecturally or activitydriven assembly of transcriptionally active intranuclear foci. The diversity of targeting signals must be established to evaluate the extent to which regulatory discrimination is mediated by encoded intranuclear trafficking signals. It will additionally be important to biochemically and mechanistically define the checkpoints, which are operative during subnuclear distribution of regulatory factors, and the editing steps, which are invoked to ensure that structural and functional fidelity of nuclear domains, where replication and expression of genes occur. There is emerging recognition that placement of regulatory components of gene expression must be temporally and spatially coordinated to optimally mediate biological control. It is realistic to anticipate that further understanding of mechanisms that position genes and regulatory factors for establishment and maintenance of the bone cell phenotype will clarify nuclear structure–function interrelationships that are operative during osteoblast differentiation and vitamin D modulation of regulatory activity.

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4. 5.

6. 7.

8. 9.

10. 11.

12.

13. 14.

Acknowledgments 15.

Results presented in this chapter were in part supported by grants for the National Institutes of Health (AR45688, PO1CA82834, DE12528, AR39588, AR45689, PO1AR48818, FIRCA R03 TW00990, FONDECYT 1030479/DK32520). The authors appreciate the editorial assistance of Elizabeth Bronstein and Karen Concaugh with the preparation of this manuscript.

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CHAPTER 20

Mouse Models of Vitamin D Receptor Ablation MARIE B. DEMAY

I. II. III. IV.

Endocrine Unit, Department of Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts

Introduction Effect on Growth and Mineral Ion Homeostasis Effect on Vascular System Effect on Reproduction

I. INTRODUCTION The absence of functional vitamin D receptors (VDRs) has been shown to be the molecular basis for the human disease HVDRR (see Chapter 72). Data from affected patients and their families have provided significant insight into the role of the VDR in vivo. However, the limitations of human studies have precluded detailed investigations of the abnormalities in mineral ion homeostasis and determinants of alopecia in these kindreds. Four laboratories have independently generated mice with targeted ablation of the VDR [1–4]. The resultant phenotype mirrors that seen in humans affected with HVDRR. The generation of these mice lacking functional VDRs has permitted detailed investigations into abnormalities in calcium absorption, bone formation, and parathyroid function that cannot be performed in humans. Furthermore, they have permitted novel investigations into the role of the VDR in nontraditional target organs such as the skin and vascular system.

II. EFFECT ON GROWTH AND MINERAL ION HOMEOSTASIS Mice homozygous for targeted ablation of the VDR are born with the expected Mendelian frequency, demonstrating that the VDR is not essential for embryonic development. The pups are phenotypically normal and indistinguishable from their wild-type and heterozygous littermates until the third week of life [1–4]. From 21 days of age, however, the knockout mice maintain a body weight approximately 10–15% lower than that of their sex-matched control littermates until approximately 6 to 8 months of age. From that point on, they VITAMIN D, 2ND EDITION FELDMAN, PIKE, AND GLORIEUX

V. Effect on the Immune System VI. Integument VII. Conclusions References

gradually begin to lose weight, and by 1 year of age are approximately half the size of their sex-matched littermates (Fig. 1). Correlating with normal immunoreactive PTH (iPTH) levels, the knockout mice are normocalcemic the first 2 weeks of life, although, by 21 days, secondary hyperparathyroidism develops, presumably as a consequence of impaired intestinal calcium absorption [1]. This time of onset of impaired intestinal calcium absorption correlates well with data obtained from studies in vitamin D–deficient rats [5]. These investigations demonstrated that intestinal calcium absorption was vitamin D–independent the first 2 weeks of life, then was gradually replaced by a vitamin D–dependent transport system, which predominated by 35 days. Studies in 10-week-old VDR-null mice confirmed a marked impairment in duodenal calcium absorption [3]. This was accompanied by a significant reduction in mRNAs encoding the calcium channels that are thought to regulate calcium entry into the enterocyte, CaT1 and ECaC (see Chapters 24 and 25). There was also a dramatic decrease in the expression of calbindin 9K, a 1,25-dihydroxyvitamin D– inducible gene thought to play a role in intracellular calcium transfer [2–4, 6]. This suggests that the 1,25-dihydroxyvitamin D–induced expression of these calcium channels and of calbindin 9K is a key determinant of intestinal calcium absorption and that this hormonal regulation requires the presence of the nuclear VDR. Interestingly, although ECaC and CaT1 mRNA levels in the kidney are unaffected by VDR status [3], renal calbindin 9K levels are dramatically decreased in the VDR-null mice [2–4, 6], correlating with an increase in renal calcium clearance. These data suggest that calbindin 9K deficiency likely plays an important role in the pathophysiology of renal calcium loss in the VDR-null mice. Copyright © 2005, Elsevier, Inc. All rights reserved.

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FIGURE 1 Phenotype of VDR-null mice. At 1 year of age, the VDR-null mice (foreground) are significantly smaller than their wild-type littermates and demonstrate severe alopecia.

As a result of the PTH–mediated increase in urinary phosphorus clearance, the VDR knockout mice develop hypophosphatemia and by 21 days of age have serum phosphorus levels in the range of 6.5 mg/dl versus 10.5 mg/dl in control littermates. Initially, the secondary hyperparathyroidism is able to compensate for the decrease in intestinal calcium absorption and the knockout mice maintain normal serum ionized calcium levels until day 28. However, from that point onward, ionized calcium levels are decreased by 30%: those of the VDR knockout mice stabilizing at approximately 1.0 mmol/liter versus 1.3 mmol/liter in control littermates [1–4]. At 15 days of age, there is no detectable abnormality in parathyroid function in the VDR knockout mice. They have normal serum iPTH levels and the size of their parathyroid glands is not increased [1], suggesting that the transcription-repressing effects of 1,25-dihydroxyvitamin D on the PTH gene and its antiproliferative effects on parathyroid cells observed in vitro (see Chapter 30) are not required for normal parathyroid development or do not require the actions of the nuclear VDR. However, in association with the development of impaired intestinal calcium absorption, secondary hyperparathyroidism is observed in the VDR-null mice. There is a gradual onset of parathyroid hyperplasia due to increased parathyroid cellular proliferation, evidenced by a twofold increase in the

number of parathyroid cells expressing proliferating cell nuclear antigen at 35 days of age [7]. By 70 days of age, when the iPTH levels are approximately 16-fold elevated, the VDR knockout mice have a 10-fold increase in parathyroid glandular volume, accompanied by an increase in PTH mRNA levels, as assessed by in situ hybridization analyses [1]. When the VDR-null mice are treated with a special diet that enables maintenance of normal mineral ion homeostasis, hyperparathyroidism is not observed, nor is parathyroid glandular hyperplasia [7] (Fig. 2). This suggests that the nuclear VDR is not required for normal parathyroid function when serum calcium and phosphorus levels are normal. Perhaps the role of the VDR in the parathyroid cell is important as a response to hypercalcemic or hypocalcemic stress, or alternatively its role may be redundant. The lack of parathyroid abnormalities in normocalcemic VDR-null mice does, however, suggest that hypocalcemia is the major stimulus to PTH gene transcription and parathyroid glandular proliferation when the actions of 1,25-dihydroxyvitamin D are impaired. Analogous to findings in humans with VDR mutations and in states of vitamin D deficiency during growth (see Chapter 63), VDR-null mice develop rickets and osteomalacia [1–4]. The skeletons of 2-week-old VDR knockout mice are indistinguishable from those of their control littermates, both radiologically and histologically, demonstrating that the VDR is not required

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CHAPTER 20 Mouse Models of Vitamin D Receptor Ablation

A B C FIGURE 2 Parathyroid hyperplasia in the VDR-null mice is prevented by normalizing mineral ions. (A) Parathyroid gland from a 70-day-old wild-type mouse; (B) from a 70-day-old VDR-null littermate fed regular chow; (C) from a 70-day-old VDR-null mouse with normal mineral ion homeostasis. The asterisk marks the parathyroid gland. (See color plate).

for skeletal development. However, expansion of the hypertrophic chondrocyte layer of the growth plate is evident histologically at 21 days [8], and by 35 days of age, there is a dramatic expansion in this layer with disruption of the columnar alignment of the hypertrophic chondrocytes (Fig. 3). Histological analyses at this time also reveal osteomalacia, characterized by a decrease in bone mineralization and an increase in osteoid volume. Histomorphometric analyses of the skeletons of 70-day-old mice reveal a 30-fold increase in osteoid volume in the VDR knockout mice, compared to that observed in their wild-type and heterozygous littermates. This is associated with an increase

A

B

in bone volume/tissue volume, a consequence of an increase in both trabecular thickness and trabecular number (Table I). As a result of the dramatic impairment in bone mineralization, the bones of the VDR knockout mice demonstrate reduced stiffness and decreased strength during biomechanical testing [9]. Analyses of cellular content reveal an increase in osteoblast number, presumably due to increased bone turnover associated with secondary hyperparathyroidism. Despite the marked secondary hyperparathyroidism, osteoclast number is not significantly increased in the VDR-null mice [9]. Since osteoclast differentiation in response to PTH has been shown to be normal in vitro

C

FIGURE 3 Prevention of rickets by normalizing mineral ion homeostasis. (A) Tibial growth plate from a 35-day-old wild-type mouse; (B) from a 35-day-old VDR-null littermate fed regular chow, demonstrating marked expansion of the hypertrophic chondrocyte layer with distortion of the normal columnar structure; (C) from a 35-day-old VDR-null mouse with normal mineral ion homeostasis. (See color plate).

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TABLE I Histomorphometric Parameters in VDR Knockout Mice On regular chow Wild-type OS/BS OV/BV BV/TV

20.9 + 2.9% 1.7 + 0.16% 10.3 + 2.4%

Knockout 86.4 + 2.8% 51.3 + 3.4% 54.5 + 2.9%

On diet that normalizes mineral ions Wild-type

Knockout

22.2 + 3.6% 1.4 + 0.3% 13.1 + 1.4%

23.1 + 1.7% 1.2 + 0.2% 14.3 + 1.2%

OS, osteoid surface; BS, bone surface; OV, osteoid volume; BV, bone volume; TV, tissue volume. Adapted from [9].

in the VDR-null mice [10], an increase in osteoclast number would have been anticipated. The inappropriately normal osteoclast number may be due to an impairment in osteoclast attachment to bone in the VDR-null mice, since the osteoclast αvβ3 integrin is highly induced by 1,25-dihydroxyvitamin D [11,12]. Alternatively, since osteoclasts are unable to resorb osteoid, their attachment to this unmineralized matrix may be impaired. As a consequence of the dramatic decrease in bone mineralization in the VDR knockout mice, their bones demonstrate reduced stiffness and strength on biomechanical testing compared to those of control littermates [9]. When normal mineral ion homeostasis is maintained in the VDR-null mice, all of these skeletal abnormalities are prevented [9]. Notably, there is normalization of the mineral apposition rate and osteomalacia is not observed. There is normalization of osteoid volume and of bone volume/tissue volume (Table I), suggesting that this abnormality in the hypocalcemic VDR-null mice is a consequence of an increase in matrix synthesis due to the anabolic effects of PTH, coupled with impaired bone resorption, since osteoclasts cannot resorb unmineralized matrix. It is notable that in the setting of normal serum calcium and phosphorus levels, histomorphometric analyses and biomechanical testing demonstrate that there is no detectable consequence of VDR ablation. These studies suggest that the VDR is not required for skeletal homeostasis. However, they do not exclude the possibility that the role of the VDR in the skeleton is redundant and that, in its absence, a second receptor or alternative homeostatic pathway performs its normal functions. Notable in this respect is the effect of 1,25-dihydroxyvitamin D on the regulation of RANK ligand production by the osteoblast. Although 1,25-dihydroxyvitamin D has been shown to play an important role in inducing the synthesis of this key regulator of osteoclast differentiation [13], histomorphometric analyses in the normocalcemic VDR-null mice reveal normal osteoclast numbers and

resorption surfaces. However, in vitro studies demonstrate that osteoblasts from VDR-null mice cannot support osteoclastogenesis when cocultured with normal spleen cells and 1,25-dihydroxyvitamin D, although when these cocultures are performed with PTH and interleukin 1α, osteoclasts with resorbing activity were formed [10]. These data clearly demonstrate that the VDR plays a key role in the skeleton; however, in its absence, other regulatory molecules, including cytokines, hormones, or alternative receptors, may be called upon to maintain skeletal homeostasis. Notable in this respect is the notion that the rapid actions of 1,25-dihydroxyvitamin D, such as the rapid increase in intracellular calcium and activation of second messengers, are mediated by an alternative receptor (see Chapters 23 and 33). However, studies in osteoblasts isolated from mice lacking the DNA binding domain of the VDR demonstrate that the rapid increase in intracellular calcium in response to hormone is abolished [4]. These data suggest that at least some of the rapid actions of this steroid hormone are dependent upon the presence of a functional nuclear receptor and challenge the hypothesis that the actions of a membrane receptor are responsible for the maintenance of skeletal homeostasis in the normocalcemic mice lacking functional nuclear receptors. The presence of a normal growth plate prior to the development of impaired mineral ion homeostasis suggests that the expansion of the late hypertrophic chondrocyte layer, characteristic of rickets, is a consequence of impaired intestinal calcium absorption rather than the absence of a functional receptor in the chondrocytes of the VDR-null mice. Furthermore, maintenance of normal mineral ion homeostasis prevents the development of rachitic changes [7] (Fig. 3), demonstrating that, in an ideal metabolic environment, the VDR is not essential for the development or maintenance of a normal growth plate. Since extracellular calcium promotes the expression of markers of terminal chondrocyte differentiation and increases the production of mineralized matrix in chondrocytic cell lines [14], studies

345


were performed to address whether impaired mineral ion homeostasis led to abnormalities in chondrocyte differentiation. These studies demonstrated that the growth plate changes were not associated with a disruption in the acquisition of markers of chondrocyte differentiation, as assessed by in situ hybridization of long bones [8]. Interestingly, expansion of the late hypertrophic chondrocyte layer was observed at a time when the VDR null mice were normocalcemic, but had hypophosphatemia due to secondary hyperparathyroidism. Although no detectable change in chondrocyte proliferation was observed, there was marked impairment in apoptosis of the late hypertrophic chondrocytes in the hypophosphatemic VDR null mice [8]. In vitro studies have demonstrated that inorganic phosphate induces chondrocyte apoptosis in a time and dosedependent manner [15–17], suggesting that the development of hypophosphatemia is a critical event in the etiology of rickets in the VDR-null mice.

III. EFFECT ON VASCULAR SYSTEM Based on epidemiological studies demonstrating an inverse relationship between plasma 25-hydroxyvitamin D levels and blood pressure, investigations were performed in the VDR knockout mice to determine whether, in fact, these mice were predisposed to developing hypertension. The mean blood pressure in the VDR knockout mice was found to be 20 mmHg higher than that of wild-type mice [18]. This was associated with an increase in renin mRNA levels and in plasma angiotensin II levels. Despite an increase in these parameters under basal conditions, salt loading, a suppressor of renin expression, was capable of decreasing renin mRNA levels in the VDR knockout mice. However, the VDR knockout mice still maintained higher renin mRNA levels than controls treated in the same fashion. These data suggest that the regulatory mechanisms required for normal salt and water balance are operative in the VDR knockout mice, but that VDR ablation leads to an increase in basal renin activity. To determine whether this increase in renin was due to hormone deficiency, wild-type mice were rendered vitamin D deficient. Interestingly, a 50% elevation in renin mRNA levels was observed in the vitamin D–deficient mice with functional VDRs, demonstrating that this was a ligand-dependent effect [18]. Studies in cell models demonstrated that overexpression of the VDR in renal cells resulted in a decrease in renin mRNA levels and that regulatory regions in the renin gene could mediate transcriptional repression in response to 1,25-dihydroxyvitamin D (see Chapter 54).

IV. EFFECT ON REPRODUCTION Initial evaluation of the reproductive potential of VDR-null mice revealed variability among the knockout lines generated. Female mice with deletion of the second zinc finger were reported to be fertile [1], whereas those with deletion of the first zinc finger had infertility associated with uterine hypoplasia and impaired folliculogenesis [2]. Subsequent studies revealed that male VDR-null mice from this same line also had reproductive dysfunction, characterized by a decreased sperm count and a reduction in sperm motility. Aromatase activity in the ovary and testis were found to be reduced and evaluation of gonadotropins revealed hypergonadotropic hypogonadism, pointing to a defect at the level of the gonad, rather than reproductive dysfunction secondary to decreased body mass or intercurrent illness [19]. However, studies in mice lacking the DBD of the VDR showed normal uterine, testicular, and seminal vesicle weight as well as normal circulating levels of testosterone or estradiol [4]. Folliculogenesis and spermatogenesis were not impaired in this line of mice, nor was fertility. Interestingly, subsequent analyses revealed that calcium supplementation partially reversed the decrease in aromatase activity and normalized estrogen levels in the mice with ablation of the first zinc finger [19]. When VDR-null females from this same line of mice were fed a high-calcium diet, they had normal-sized litters and normal pup survival [20]; their fertility was shown to be directly related to the calcium content of the diet [21]. These studies once again point to the important contribution of abnormal mineral ion homeostasis to the phenotype of the VDR-null mice and raise the question as to what specific abnormality is responsible for the development of hypergonadotropic hypogonadism in the hypocalcemic mice with targeted ablation of the first zinc finger. Studies of mammary gland development have demonstrated an enhancement of growth and budding in the absence of a functional VDR. The glands of the VDR-null females were shown to have an increase in the number of secondary branch points and as well as in the number of terminal end buds [22]. The lack of VDR also enhanced the proliferative response to estrogen and progesterone. The effect of 1,25-dihydroxyvitamin D on growth of mammary glands in vitro demonstrated a receptor-dependent suppression of the sex steroid– induced branching enhancement in the wild-type mice, but not in those lacking a functional VDR [22]. These observations suggest that hormone-dependent effects of the VDR serve to attenuate mammary development and raise the question of the long-term consequences

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of VDR ablation or vitamin D deficiency on mammary gland development and involution (see Chapter 93).

V. EFFECT ON THE IMMUNE SYSTEM A number of investigations have demonstrated that 1,25-dihydroxyvitamin D induces the expression of several genes with immunomodulatory properties in vitro as well as having immunomodulatory effects in vivo (see Chapters 98 and 99). These studies have demonstrated that 1,25-dihydroxyvitamin D and its analogs are able to prevent the development of autoimmune disorders and synergize with agents that prevent graft rejection in experimental models. Vitamin D–deficient animals have impaired chemotaxis and cell-mediated immunity, but the postulated immunomodulatory properties of the ligand-bound serum vitamin D binding protein raise the question as to what role the nuclear VDR plays in the immune dysfunction observed in vitamin D deficiency (see Chapters 8 and 9). Studies in the VDR-null mice have shown modest consequences of VDR ablation, most of which seem to be reversed by normalization of mineral ion homeostasis [23]. Splenocyte proliferation in response to antiCD3 stimulation is mildly impaired, as is macrophage chemotaxis [23]; however, macrophage phagocytosis and killing are not affected. Leucocyte and lymphocyte subset composition is also normal. In vivo rejection of transplanted islet cells was comparable in the knockout and wild-type mice, demonstrating that absence of the VDR did not enhance graft rejection [23]. Since vitamin D analogs have been shown to delay the onset of diabetes in NOD mice, the effect of VDR status on susceptibility to diabetes was examined. Interestingly, the

A

B

VDR-null mice were protected from low-dose streptozotocin-induced diabetes. All these immune defects, as well as protection from streoptozotocininduced diabetes, were reversed in the setting of normal mineral ion homeostasis [23]. VDR-null mice were shown to have an increase in mature splenic dendritic cells, consistent with the known effects of 1,25-dihydroxyvitamin D on inhibiting dendritic cell maturation [24]. Although studies in cells from the VDR-null mice have demonstrated that the VDR is required for the effects of 1,25-dihydroxyvitamin D on the differentiation of bone marrow progenitors into macrophages and monocytes [25], the lack of significant in vivo consequences of VDR ablation suggest that the role of the nuclear VDR in the immune system is redundant.

VI. INTEGUMENT Analogous to what is seen in numerous HVDRR kindreds, the VDR knockout mice develop progressive alopecia. Clinical evidence of alopecia is seen as early as the 4th week of life, beginning in the periorbital region and progressing dorsally, eventually resulting in total loss of fur by 3 to 4 months of age [1–4]. Histological examination of the skin reveals dilatation of hair follicles with formation of dermal cysts (Fig. 4). This histological phenotype is analogous to that seen in mice with mutations in the hairless gene, in which progressive loss of fur is seen from 2 weeks of age [26]. However, expression of hairless mRNA in the skin and keratinocytes of neonatal VDR null mice is not decreased [27], suggesting that, if these two genes are in a common pathway, the VDR is likely to be downstream of hairless.

C

FIGURE 4 Normalization of mineral ion homeostasis does not prevent skin changes in the VDR-null mice. (A) Skin section from a 70-day-old wild-type mouse; (B) from a 70-day-old VDR-null littermate fed regular chow, demonstrating dermal cysts and dilatation of hair follicles; (C) from a 70-day-old VDR-null mouse with normal mineral ion homeostasis. (See color plate).


Because normalization of mineral ion homeostasis failed to prevent the development of the skin phenotype in the VDR-null mice [7] (Fig. 4), studies were undertaken to address whether the loss of fur was a consequence of an abnormality in keratinocytes, which give rise to the epidermal component of the hair follicle. Numerous studies have demonstrated that 1,25-dihydroxyvitamin D plays a significant role in decreasing keratinocyte proliferation and promoting keratinocyte differentiation (see Chapter 35). Studies in primary keratinocytes isolated from neonatal VDR knockout mice failed to demonstrate abnormal keratinocyte proliferation or impaired differentiation [27]. However, investigations in growing mice demonstrated a decrease in expression levels of epidermal differentiation markers, including involucrin, profilaggrin, and loricrin, from birth to 3 weeks of age [28]. Taken together, these data suggest that the effects of the VDR on keratinocytes are not essential during development, but that they are required for keratinocyte differentiation and skin homeostasis postnatally. Hair follicle development begins at embryonic day 14.5 and is dependent on reciprocal interactions between the epidermal and mesodermal components of the hair follicle. This morphogenic period lasts until the 3rd week of life, which marks the end of the first hair cycle. After this period, the hair follicle continues to cycle, characterized by a phase of rapid growth (anagen), followed by a regression phase (catagen), a quiescent phase (telogen), and reentry into anagen to generate a new hair shaft. Regulation of this postmorphogenic hair cycling is also thought to require continued epidermal–mesodermal communication. Abnormalities in the dermal papilla, the mesodermal component of the hair follicle, or in epidermal–mesodermal communication, required for the maintenance of the normal hair cycle, could also lead to the development of alopecia. Depilation of fur in control mice results in initiation of a new hair cycle with regrowth of fur. Such studies, performed at 18 days of age, a time when there is no detectable histological abnormality in the skin of the VDR-null mice, demonstrated that the VDR-null mice had a defect in anagen initiation [27]. The question remained as to whether this was due to a cellular or a metabolic defect. Because 1,25-dihydroxyvitamin D down-regulates its own biosynthesis by repressing the 25-hydroxyvitamin D-1α-hydroxylase gene [29] and increases its metabolism by up-regulating the 24-hydroxylase gene through VDR-dependent actions [30, 31], VDR-ablated mice have very high levels of 1,25-dihydroxyvitamin D even when mineral ion levels are normalized. Since 1,25-dihydroxyvitamin D has been shown to inhibit keratinocyte proliferation and to promote keratinocyte

347 differentiation [32], very high levels of this hormone or its metabolites may lead to alopecia by toxic interactions with an alternative receptor. However, VDR null mice with undetectable circulating levels of both 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D still developed alopecia, demonstrating that this phenotype was not a consequence of toxic levels of circulating hormone [33]. Interestingly, wild-type littermates with undetectable circulating levels of 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D did not develop alopecia [33]. This observation suggested that absence of receptor per se rather than absence of ligand-dependent receptor effects was responsible for the alopecia in the VDR-null mice and that different skin phenotypes are seen in absence of ligand versus absence of receptor. Since the VDR is widely expressed under normal circumstances and is missing from all cells of the VDR-null mice, it was not evident whether lack of VDR expression in keratinocytes, dermal papilla cells, or other organs of the VDR null-mice was responsible for the abnormality in the hair cycle. Hair reconstitution assays, in which implantation of a mixture of keratinocytes and activated dermal papilla cells into a nude mouse host recapitulates the process of hair follicle morphogenesis [34] leading to a functional hair follicle, provided a method to address whether the VDR was required in either the epidermal or mesodermal component of the hair follicle, or both. By implanting wild-type or VDR-null dermal papilla cells with wildtype or VDR-null keratinocytes, both isolated from neonatal mice in which the hair cycle is still in the morphogenic period, hair follicles were generated, with epidermal and mesodermal components differing in VDR status (wild-type versus null) [33]. These studies confirmed that the VDR was not required for hair follicle morphogenesis. However, hair follicles reconstituted with VDR-null keratinocytes were unable to sustain postmorphogenic hair cycles. In contrast, when dermal papilla cells of either genotype were mixed with wildtype keratinocytes, a normal response to anagen induction, including generation of new hair shafts, was observed. These studies demonstrated that expression of the VDR in the keratinocytes was essential for normal postmorphogenic hair cycling. To address whether keratinocyte-specific expression of the VDR was sufficient to maintain hair follicle homeostasis, transgenic mice expressing the VDR under the regulation of a keratinocyte-specific promoter were generated [35, 36]. These mice were crossbred to the VDR-null mice to generate mice that expressed the VDR only in keratinocytes. These mice did not develop alopecia and had a normal response to anagen-initiating stimuli, providing definitive proof that expression of

348 the VDR in keratinocytes was both necessary and sufficient for the maintenance of postmorphogenic hair cycling. Future investigations will be required to determine which regions of the VDR are required for skin homeostasis, whether the actions are dependent on an unique subset of nuclear receptor coactivators or an intact ligand binding domain, and, ultimately, what molecular pathway is disrupted by VDR ablation.

VII. CONCLUSIONS Studies in mice lacking functional VDRs have clearly demonstrated that the major roles of this nuclear receptor in vivo are the promotion of intestinal calcium absorption and the maintenance of skin homeostasis. Interestingly, effects of the VDR on two traditional target organs, the parathyroid and the skeleton, seem to be redundant, in that no abnormalities are observed in these organs in VDR-null mice with normal mineral ions. The effects of the VDR on intestinal calcium absorption seem to be classical ligand-dependent effects, and significant progress has been made in the identification of genes involved in VDR-mediated intestinal calcium absorption. In contrast, data point to potentially hormone-independent effects of the VDR on the hair cycle, and the molecular pathways that mediate the actions of this nuclear receptor in the skin are yet to be identified.

References 1. Li YC, Pirro AE, Amling M, Delling G, Baron R, Bronson R, Demay MB 1997 Targeted ablation of the vitamin D receptor: an animal model of vitamin D-dependent rickets type II with alopecia. Proc Natl Acad Sci USA 94:9831–9835. 2. Yoshizawa T, Handa Y, Uematsu Y, Takeda S, Sekine K, Yoshihara Y, Kawakami T, Alioka K, Sato H, Uchiyama Y, Masushige S, Fukamizu A, Matsumoto T, Kato S 1997 Mice lacking the vitamin D receptor exhibit impaired bone formation, uterine hypoplasia and growth retardation after weaning. Nat Genet 16:391–396. 3. VanCromphaut S, Dewerchin M, Hoenderop J, Stockmans I, VanHerck E, Kato S, Bindels R, Collen D, Carmeliet P, Bouillon R, Carmeliet G 2001 Duodenal calcium absorption in vitamin D receptor-knockout mice: Functional and molecular aspects. Proc Natl Acad Sci USA 98:13324–13329. 4. Erben R, Soegiarto D, Weber K, Zeitz U, Lieberherr M, Gniadecki R, Moller G, Adamski J, Balling R 2002 Deletion of deoxyribonucleic acid binding domain of the vitamin D receptor abrogates genomic and nongenomic functions of vitamin D. Mol Endocrinol 16:1524–1537. 5. Dostal LA, Toverud SU 1984 Effect of vitamin D3 on duodenal calcium absorption in vivo during early development. Am J Physiol 246:G528–G534.

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6. Li YC, Pirro AE, Demay M 1998 Analysis of vitamin Ddependent calcium-binding protein messenger ribonucleic acid expression in mice lacking the vitamin D receptor. Endocrinology 139:847–851. 7. Li YC, Amling M, Pirro AE, Priemel M, Meuse J, Baron R, Delling G, Demay MB 1998 Normalization of mineral ion homeostasis by dietary means prevents hyperparathyroidism, rickets, and osteomalacia, but not alopecia in vitamin D receptor–ablated mice. Endocrinology 139:4391–4396. 8. Donohue MM, Demay MB 2002 Rickets in VDR null mice is secondary to decreased apoptosis of hypertrophic chondrocytes. Endocrinology 143:3691–3694. 9. Amling M, Priemel M HT, Chapin K, Rueger JM, Baron R, Demay MB 1999 Rescue of the skeletal phenotype of vitamin D receptor ablated mice in the setting of normal mineral ion homeostasis: Formal histomorphometric and biomechanical analyses. Endocrinology 140:4982–4987. 10. Takeda S, Yoshizawa T, Nagai Y, Yamato H, Fukumoto S, Sekine K, Kato S, Matsumoto T, Fujita T 1999 Stimulation of osteoclast formation by 1,25-dihydroxyvitamin D requires its binding to vitamin D receptor (VDR) in osteoblastic cells: Studies using VDR knockout mice. Endocrinology 140:1005–1008. 11. Cao X, Ross FP, Zhang L, MacDonald PN, Chappel J, Teitelbaum SL 1993 Cloning of the promoter for the avian integrin β3 subunit gene and its regulation by 1,25-dihydroxyvitamin D3. J Biol Chem 268:27371–27380. 12. Medhora MM, Teitelbaum S, Chappel J, Alvarez J, Mimura H, Ross FP, Hruska K 1993 1α,25-Dihydroxyvitamin D3 up-regulates expression of the osteoclast integrin αvβ3. J Biol Chem 268:1456–1461. 13. Yasuda H, Shima N, Nakagawa N, Yamaguchi K, Kinosaki M, Mochizuki S, Tomoyasu A, Yano K, Goto M, Murakami A, Tsuda E, Morinaga T, Higashio K, Udagawa N, Takahashi N, Suda T 1998 Osteoclast differentiation factor is a ligand for osteoprotegerin/osteoclastogenesis-inhibitory factor and is identical to TRANCE/RANKL. Proc Natl Acad Sci USA 95:3597–3602. 14. Chang W, Tu C, Pratt S, Chen T, Shoback D 2002 Extracellular Ca2+-Sensing receptors modulate matrix production and mineralization in chondrogenic RCJ3.15.18 cells. Endocrinology 143:1467–1474. 15. Adams C, Mansfield K, Perlot R, Shapiro I 2001 Matrix regulation of skeletal cell apoptosis: Role of calcium and phosphate ions. J Biol Chem 276:20316–20322. 16. Mansfield K, Rajpurohit R, Shapiro I 1999 Extracellular phosphate ions cause apoptosis of terminally differentiated epiphyseal chondrocytes. J Cell Physiol 179:276–286. 17. Mansfield K, Teixeira C, Adams C, Shapiro I 2001 Phosphate ions mediate chondrocyte apoptosis through a plasma membrane transporter mechanism. Bone 28:1–8. 18. Li YC, Kong J, Wei M, Chen ZF, Liu SQ, Cao LP 2002 1,25Dihydroxyvitamin D3 is a negative endocrine regulator of the renin-angiotensin system. J Clin Invest 110:229–238. 19. Kinuta K, Tanaka H, Moriwake T, Aya K, Kato S, Seino Y 2000 Vitamin D is an important factor in estrogen biosynthesis of both female and male gonads. Endocrinology 141:1317–1324. 20. Johnson LE, DeLuca HF 2001 Vitamin D receptor null mutant mice fed high levels of calcium are fertile. J Nutr 131: 1787–1791. 21. Johnson LE, DeLuca HF 2002 Reproductive defects are corrected in vitamin D-deficient female rats fed a high calcium, phosphorus and lactose diet. J Nutr 132:2270–2273.


22. Zinser G, Packman K, Welsh J 2002 Vitamin D3 receptor ablation alters mammary gland morphogenesis. Development 129:3067–3076. 23. Mathieu C, VanEtten E, Gysemans C, Decallonne G, Kato S, Laureys J, Depovere J, Valckx D, Verstuyf A, Bouillon R 2001 In vitro and in vivo analysis of the immune system of vitamin D receptor knockout mice. J Bone Miner Res 16:2057–2065. 24. Xing N, L-Maldonado ML, Bachman LA, McKean DJ, Kumar R, Griffin MD 2002 Distinctive dendritic cell modulation by vitamin D3 and glucocorticoid pathways. Biochem Biophys Res Commun 297:645–652. 25. O’Kelly J, Hisatake J, Hisatake Y, Bishop J, Norman A, Koeffler HP 2002 Normal myelopoiesis but abnormal T lymphocyte responses in vitamin D receptor knockout mice. J Clin Invest 109:1091–1099. 26. Mann SJ 1971 Hair loss and cyst formation in hairless and rhino mutant mice. Anat Rec 170:485–499. 27. Sakai Y, Demay MB 2000 Evaluation of keratinocyte proliferation and differentiation in vitamin D receptor knockout mice. Endocrinology 141:2043–2049. 28. Xie Z, Komuves L, Yu QC, Elalieh H, Ng DC, Leary C, Chang S, Crumrine D, Yoshizawa T, Kato S, Bikle DD 2002 Lack of the vitamin D receptor is associated with reduced epidermal differentiation and hair follicle growth. J Invest Dermatol 118:11–16. 29. Takeyama K, Kitanaka S, Sato T, Kobori M, Yanagisawa J, Kato S 1997 25-Hydroxyvitamin D3 1α-hydroxylase and vitamin D synthesis. Science 277:1827–1830.

349 30. Kerry DM DP, Hahn CN, Morris HA, Omdahl JL, May BK 1996 Transcriptional synergism between vitamin D-responsive elements in the rat 25-hydroxyvitamin D 24-hydroxylase (CYP24) promoter. J Biol Chem 22:29715–29721. 31. Ohyama Y, Ozono K, Uchida M, Shinki T, Kato S, Suda T, Yamamoto O, Noshiro M, Kato Y 1994 Identification of a vitamin D–responsive element in the 5′-flanking region of the rat 25-hydroxyvitamin D3 24-hydroxylase gene. J Biol Chem 269:10545–10550. 32. Bikle DD, Pillai S 1993 Vitamin D, calcium, and epidermal differentiation. Endocr Rev 14:3–19. 33. Sakai Y, Kishimoto J, Demay MB 2001 Metabolic and cellular analysis of alopecia in vitamin D receptor knockout mice. J Clin Invest 107:961–966. 34. Kishimoto J, Ehama R, Wu L, Jiang S, Jiang N, Burgeson RE 1999 Selective activation of the versican promoter by epithelial-mesenchymal interactions during hair follicle development. Proc Natl Acad Sci USA 96:7336–7341. 35. Chen C, Sakai Y, Demay MB 2001 Targeting expression of the human vitamin D receptor to the keratinocytes of vitamin D receptor null mice prevents alopecia. Endocrinology 142: 5386–5389. 36. Kong J, Li XJ, Gavin D, Jiang Y, Li YC 2002 Targeted expression of human vitamin D receptor in the skin promotes the initiation of the postnatal hair follicle cycle and rescues the alopecia in vitamin D receptor null mice. J Invest Dermatol 118:631–638.

CHAPTER 21

Intracellular Vitamin D Response Element Binding Proteins JOHN S. ADAMS

Burns and Allen Research Institute and the Division of Endocrinology, Diabetes and Metabolism, Cedars-Sinai Medical Center, The David Geffen School of Medicine at UCLA, Los Angeles, California

I. Introduction II. New World Primates III. The Biochemical Nature of Vitamin D Resistance in New World Primates IV. New World Primate–like Vitamin D Resistance in Man V. Characterization of the Human Response Element Binding Protein (REBiP)

I. INTRODUCTION Since the publication of the first edition of Vitamin D, much has been learned regarding the cellular machinery for intracellular vitamin D trafficking (see Chapters 10 and 22), genomic action (see Chapters 11–20), and metabolism (see Chapters 4–7). This chapter will chronicle the discovery of a family of heterogeneous nuclear ribonucleoproteins (hnRNPs) that constitute one such previously unrecognized mode of control over the transactivation of vitamin D–regulated genes. As so often happens in science and medicine, this discovery evolved from the molecular analysis of a successful “experiment of nature.” The story begins with this author’s investigation of a man-made disturbance in vitamin D homeostasis in several species of New World primates resident at the Los Angeles Zoo in the mid1980s and concludes with the discovery of a previously unrecognized form of human vitamin D–resistant rickets.

II. NEW WORLD PRIMATES A. Early Primate Evolution In the Eocene period the great southern hemispheric landmass, Pangea, ruptured. This tectonic event resulted in the American landmass and Madagascar moving away from Africa. This continental separation occurred early in the process of primate evolution, trapping primordial primates in South America, Africa, and Madagascar, respectively. As a consequence, the three major primate infraorders, platyrrhines or New World primates, catarrhines or Old World primates, and lemurs, evolved independently of one another [1] (Fig. 1). VITAMIN D, 2ND EDITION FELDMAN, PIKE, AND GLORIEUX

VI. Heterogeneous Nuclear Ribonucleoproteins (hnRNPs) VII. Compensation for the Dominant-Negative Acting, Response Element Binding Proteins VIII. Conclusion Reference

Unlike Old World primates, including our own species, which have populated virtually every landmass on our planet, New World primates have remained confined to Central and South America for the past 50 million years. Compared to Old World primates, especially some of the terrestrial species such as gorilla, New World primates are smaller in stature, a trait well-suited to their lifestyle as plant-eating arboreal sunbathers residing in the canopy of the periequatorial rain forests of the Americas.

B. Simian Bone Disease The appearance of generalized metabolic bone disease in captive primates has been recognized for the past 150 years [2]. The disease, which has not been well studied from a histopathological standpoint, carries the clinical and radiological stigmata of rickets and osteomalacia [3]. Compared to Old World primates reared in captivity, New World primates or platyrrhines are particularly susceptible to the disease. The disorder affects primarily young, growing animals and results in muscle weakness, skeletal fragility, and in many instances death of the affected individual. Rachitic bone disease of this sort has long presented a problem to veterinarians caring for captive platyrrhines, particularly in North American and European zoos [4], because death of preadolescent and adolescent primates prior to sexual maturity severely limits on-site breeding programs. Because the disease was reported to be ameliorated either by the oral administration of vitamin D3 in large doses or by ultraviolet B irradiation of affected Copyright © 2005, Elsevier, Inc. All rights reserved.

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Old World

New World

Madagascar

Hominoidea Platyrrhini

Superorder

Catarrhini

ANTHRIPOIDEA PRIMATES

Lemuridae

Infraorder

PROSIMII

Suborder Order

FIGURE 1 New World primate evolution. Shown in geographic terms is the independent evolution of the three primate infraorders, Platyrrhini, Catarrhini, and Lemuridae, in South America (the New World), Africa (the Old World), and Madagascar, respectively. The superorder Hominoidea represents the human precursors.

primates, it was presumed to be caused by vitamin D deficiency [4]. The frequent occurrence of rickets and osteomalacia in New World primates was also ascribed to the relative inability of platyrrhines to effectively employ vitamin D2 in their diet [5]; a similar observation had been made for chickens [6]. Using assay technology that does not discriminate between 25-hydroxylated vitamin D2 and vitamin D3 metabolites, investigators [7] determined that 25-hydroxyvitamin D (25-OHD) levels were two- to threefold higher when platyrrhines were dosed with supplemental vitamin D3 than with vitamin D2. These data suggested that 25-hydroxylation of vitamin D substrate in New World primates was much more effective when vitamin D3 was employed as substrate. However, in the same study two species of Old World primates demonstrated similar discrimination against vitamin D2, in favor of vitamin D3. In summary, these results seemed to indicate that all subhuman primates, whether Old or New World, were relatively resistant to vitamin D2 in terms of its ability to engender an increase in serum levels of 25-OHD. Finally, Hay [8] suggested that New World primates may transport 25-OHD in the serum by means and via proteins that are dissimilar from those encountered in Old World primate species. This hypothesis was disproven by Bouillon et al. [9], who showed that the

vitamin D binding protein was the major carrier of 25-OHD in their serum of both New and Old World primates. The question of why platyrrhines were more susceptible to vitamin D deficiency than were catarrhines began to be answered with the detection of extraordinarily high circulating levels of the active vitamin D metabolite, 1,25-dihydroxyvitamin D, in New World primates [10–12]. These data confirmed that New World primates were resistant to the vitamin D hormone.

C. Outbreak of Rickets in the New World Primate Colonies of the Los Angeles Zoo The index case in the original studies was a preadolescent New World primate of the Emperor tamarin species (Fig. 2A). When investigated radiographically (Fig. 2B), this tamarin and those like him displayed classical rickets complete with growth retardation and metaphyseal cupping and fraying characteristic of rickets. In order to investigate this rachitic syndrome, blood and urine was collected from involved monkeys as well as from control, nonrachitic New and Old World primates. As shown in Fig. 3, that comparison yielded a biochemical phenotype that was most remarkable for

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CHAPTER 21 Response Element Binding Proteins

A

B

FIGURE 2 A rachitic New World primate resident of the Los Angeles Zoo. (A) A preadolescent emperor tamarin with rickets. (B) The characteristic rachitic “cupping” and “fraying” of the tibial metaphysis (arrows) of this monkey.

an elevated serum 1,25-(OH)2D in rachitic New World primates [11]. In fact, with the exception of nocturnal primates in the genus Aotus, New World primates in all genera had vitamin D hormone levels ranging to 100fold higher than that observed in Old World primates including man [13–16]. In the initial analysis New World primates affected with rickets were those with the lowest 1,25-(OH)2D levels, while their healthy counterparts were those with the highest serum 1,25-(OH)2D. These data were interpreted to mean that most New World primate genera were naturally resistant to the vitamin D hormone, and that the resistant state could be compensated by maintenance of high 1,25-(OH)2D levels. If this was true, then an increase in the serum 1,25-(OH)2D concentration in affected primates should result in biochemical

compensation for the resistant state and resolution of their rachitic bone disease. When rachitic New World primates were exposed to 6 months of artificial sunlight, both serum 25-hydroxyvitamin D (25-OHD) and product 1,25-(OH)2D levels rose significantly (P ≤ 0.02), resulting in cure of rickets [15]. In summary, New World primates are periequitorial sunbathers for a reason. As depicted by the oversized arrows in a simplified scheme of vitamin D synthesis and metabolism in Fig. 4, New World primates require much cutaneous vitamin D synthesis in order to push their 25-OHD and 1,25-(OH)2D levels high enough to effectively interact with the VDR. The question remained: why were these primates resistant to all but the highest levels of the vitamin D hormone?

7-DHC

Rachitic Phenotype

Cholesterol

UVB Blood calcium.............

Slightly decreased

Urine calcium..............

Slightly decreased

Blood phosphate................

Normal

Urine phosphate.................

Normal

Serum creatinine................

Normal

Liver function......................

Normal

25-OHD..............................

Normal

1,25-(OH)2D.......................

Very high

FIGURE 3 Biochemical phenotype of rachitic New World primates. Demonstrated are the biochemical indices of bone health in New World primates suffering from rickets compared to developmental age- and sex-matched, nonrachitic Old World primates. The outstanding characteristic is a 1,25-dihydroxyvitamin D (1,25(OH)2D) level two to three orders of magnitude greater than that observed in Old World primates, including man.

Vitamin D3

25-OHase 25-OHD3

24,25-(OH)2D3

1-OHase 1,25-(OH)2D3

1,24,25-(OH)3D3

Calcitroic acid

VDR

FIGURE 4 Simplified scheme of vitamin D synthesis and metabolism in New World primates. The bold arrows describe the means by which high 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) levels are achieved and maintained in New World primates compared to their Old World and human primate counterparts.

354

JOHN S. ADAMS

III. THE BIOCHEMICAL NATURE OF VITAMIN D RESISTANCE IN NEW WORLD PRIMATES In order to answer the question of resistance in New World primates, cultured fibroblasts and immortalized cell lines from both resistant and hormone-responsive New and Old World primates were used to track, step by step, the path taken by the vitamin D hormone from the serum vitamin D binding protein (DBP) in the blood in route to the nucleus and transactivation of hormone-responsive genes [11,13–22]. It was shown that the movement of hormone from DBP, across the cell membrane and through the cytoplasm and nuclear membrane was indistinguishable from that observed in Old World primate cells. It was also determined that the ability of the New World primate VDR to bind 1,25-(OH)2D3 or 1,25-(OH)2D2 and induce receptor dimerization with the retinoid X receptor (RXR) was normal. In fact, when removed from the intranuclear environment and in distinction to previous reports [12], the VDR in New World primates was similar to the Old World primate VDR in all biochemical and functional respects [22]. That which was not the same was the reduced ability of VDR-RXR complex to bind to its cognate cis element and transactivate the expression of genes. In order to elucidate nuclear receptor events in New World primate cells, the nuclei of New World primate cells were isolated and extracted. In addition to the VDR-RXR, it was determined that these extracts contained a second protein bound to VDREs. This protein

A

was coined the vitamin D response element binding protein or VDRE-BP [21]. In electrophoretic mobility shift assay (EMSA) using the VDRE as probe, Old World primate cell extract contained only the VDR-RXR bound to the VDRE probe, while the New World primate extract contained two probe-reactive bands, one compatible with the VDR-RXR and a second, more pronounced VDRE-BP-VDRE band. This VDRE-BP-VDRE binding reaction was specific, as the VDRE-BP was competed from VDRE probe by the addition of excess unlabeled VDRE. These data suggested that VDRE-BP might function as a dominant-negative inhibitor of receptorresponse element binding by competing in trans with receptor, “knocking it off” the VDRE (Fig. 5), thus preventing VDR-RXR binding to the VDREs. When recombinant human VDR and RXR were permitted to interact in EMSA with increasing amounts of nuclear extract from vitamin D-resistant cells containing a VDRE-BP or from normal vitamin D–responsive cells, the addition of more control extract only amplified the VDR-RXR-retarded probe on the gel. By contrast, increasing amounts of the hormone-resistant extract competed away receptor–probe binding in favor VDRE-BP–probe binding. To date, two distinct VDRE-BPs have been identified, purified, cloned, and characterized in New World primates [21]. Both are members of the heterogeneous nuclear ribonucleoprotein A (hnRNPA) family of single strand mRNA binding proteins [23]. However, as just pointed out, VDRE-BPs can also bind specifically to double-strand DNA. In fact, it is by virtue of their ability to bind DNA that they can be distinguished from

B 1,25-D

RXR

VDR

VDRE

Wildtype

VDRE-BP

VDRE

Dominant-negative

FIGURE 5 The dominant-negative action of the New World primate vitamin D response element binding protein (VDRE-BP). (A) The “wild-type” events by which the retinoid X receptor (RXR) and hormone-liganded vitamin D receptor (VDR) dimer pair interact in trans with the vitamin D response element (VDRE) to regulate transcription of 1,25-dihydroxyvitamin D (1,25-D)-responsive genes. (B) The proposed “dominant-negative” event leading to competition for binding to the VDRE between the VDRE-BP and RXR-VDR; the net result is a blockade of transcriptional regulation.

355

CHAPTER 21 Response Element Binding Proteins

A. Index Case

Luciferase activity (arbitrary units)

500

*p < 0.001 250

0 OWP

+VDRE-BP

NWP

FIGURE 6 Squelching of RXR-VDR-directed, VDRE-reporterdriven transactivation. Shown is the significant decrease in VDREdirected luciferase reporter activity, assayed in relpicate (n = 4), in a subclone of Old World primate (OWP) cells after stable transfection with the New World primate VDRE-BP-2 (solid bar) compared to reporter activity in vector-alone transfected OWP cells (open bar) and New World primate (NWP) cells (shaded bar). Data are shown as luciferase units relative to an internal β-galactosidase standard.

traditional co-repressor proteins [24]. When overexpressed, they can effectively squelch VDR-directed transactivation. Depicted in Fig. 6 is VDRE-directed reporter activity in a subclone of wild-type Old World primate cells stably overexpressing the New World primate VDRE-BP-2 as well as in naturally hormone-resistant New World primate cells. Stable overexpression of VDRE-BP-2 squelched luciferase activity substantially compared to untransfected, wild-type cells to levels observed in hormone-resistant New World primate cells that naturally overexpress the protein; VDRE-BP-1 overexpression reduced, but not significantly, transactivation. This is strong confirmatory evidence that when overexpressed in vivo, VDRE-BP-2 is the cause of vitamin D resistance in these monkeys.

IV. NEW WORLD PRIMATE–LIKE VITAMIN D RESISTANCE IN MAN The Adams laboratory has been studying a vitamin D– resistant state in New World primates for more than 15 years with the expectation that it would provide valuable insight into how steroid hormones regulate gene expression in human primates. Prior to this time it was unknown whether there existed a human homolog to the vitamin D–resistant state in New World primates.

In 1993 a patient with the classical signs of type II hereditary vitamin D–resistant rickets (HVDRRII), including alopecia, was reported [25]. The biochemical phenotype of this patient included hypocalcemia (2.03 mmol/L corrected for albumin [normal range, 2.25–2.55 mmol/L]); raised serum alkaline phosphatase (1101 U/L [normal range, 1200 pg/ml and serum phosphorus < 6.5 mg/dl). Ten patients with a mean PTH of 1826 ± 146 pg/ml, were treated for a minimum of 48 weeks with an intravenous dose of calcitriol commensurate with the levels of PTH. The initial calcitriol dose had to be increased in seven patients. The mean maximum dose of calcitriol was 3.8 µg three times a week. The authors concluded that patients with severe hyperparathyroidism respond well to intravenous calcitriol; however, the dose of this vitamin D metabolite should be adjusted according to PTH levels, and hyperphosphatemia should be kept under control. Thus, it seems that although there is no agreement with regard to the route and frequency of administration, the oral pulse and intravenous administration are the most accepted therapies. However, the meta-analysis of four trials that compared intermittent intravenous calcitriol with oral calcitriol, in randomized controlled studies [165,166] or cross-over trials [167,168], indicated that intravenous therapy was more effective than oral treatment (either daily or “pulse” treatment) for the suppression of intact PTH levels. Beneficial results also have been observed in patients maintained on continuous ambulatory peritoneal dialysis. Salusky et al. [169] studied the pharmokinetics of calcitriol in continuous ambulatory and cycling peritoneal dialysis patients. The kinetics of calcitriol was evaluated after a single dose of 60 ng/kg body weight (equivalent to 4.2 ug for a 70-kg man) given orally, intravenously, or intraperitoneally in six patients. The area under the curve for the increment of serum calcitriol concentration above baseline levels for the 24 hr after a single dose of calcitriol was 62% greater following intravenous injection (2340 ± 115 pg/ml) than after either oral (1442 ± 191 pg/ml) or intraperitoneal (1562 ± 195 pg/ml) administration. These investigators, using a radioisotope tracer of calcitriol, found that 30 to 40% of the hormone adheres to plastic components of the peritoneal dialysate delivery system. By modifying the technique of intraperitoneal calcitriol administration, the authors found that they could obtain a dosage effect comparable to intravenous administration. Thus, it would seem that intraperitoneal administration of calcitriol also is very effective in the control of secondary hyperparathyroidism if precautions to prevent adherence to plastic are taken. It is recommended now that in patients with CKD Stage 5 (GFR < 15 ml/min/1.73 m2) or dialysis, serum PTH be determined at least every three months and serum calcium and phosphorus once a month. The ideal serum intact PTH levels should be 150 to 300 pg/ml. Patients with serum intact PTH greater than 300 pg/ml should receive calcitriol or vitamin D analogs, providing

that the serum phosphorus is less than 5.5 mg/dl and the serum calcium less than 9.8 mg/dl. Ideally, the serum phosphorus should be 3.5 to 5.5 mg/dl, and the Ca × P product less than 55 mg2 × dl2. The administration of calcitriol or its analogs to patients with CKD Stage 5 depends on the levels of circulating intact PTH. For patients with circulating levels of intact PTH of 300–600 pg/ml, the dose of intravenous calcitriol should be 0.5 to 1.5 µg I.V. × 3 per week. For those patients with more severe secondary hyperparathyroidism (PTH 600–1200), the dose of calcitriol should be 2 to 4 µg I.V. three times per week.

C. Use of New Less Calcemic Analogs of Calcitriol In an effort to utilize the actions of vitamin D on the parathyroid gland and minimize the toxicities of such therapy, structural alterations of the vitamin D molecule were undertaken to try to develop vitamin D analogs that may retain the effects on the parathyroid glands but have a lesser effect on the calcium and phosphate metabolism [170]. These analogs would be relatively selective in suppressing parathyroid hyperfunction and therefore, more useful therapeutic agents. This subject is extensively discussed in Chapters 80–88. Currently, there is experimental and clinical evidence for the efficacy of four of such vitamin D analogs, which have been approved for the treatment of secondary hyperparathyroidism. Two of these analogs have been developed in Japan, 22-oxacalcitriol and falecalcitriol and the other two in the United States, 19-nor-1,25(OH)2D2 and 1-alpha hydroxy D2. 22-Oxacalcitriol (OCT) differs from calcitriol by an oxygen substitution at position 22 (see Chapter 86). This structure modification appears to reduce the affinity of OCT for the vitamin D receptor, as well as for DBP. The decreased affinity for DBP results in rapid clearance from the circulation, and this may be a mechanism that accounts for low calcemic and phosphatemic effect of OCT [171]. Falecalcitriol is an analog in which the hydrogens of carbons 26 and 27 have been substituted by fluorine atoms. This vitamin D analog has greater activity than calcitriol and is considerably more calcemic and more potent in calcifying epiphyseal cartilage in rats [172]. The increased potency is likely due to a decreased catabolism of this sterol. In patients with chronic renal failure, falecalcitriol was effective in decreasing PTH and appeared to be somewhat more effective than alfacalcidol in suppressing secondary hyperparathyroidism [173]. In the United States, 19-nor-1,25(OH)2D2 (paricalcitol) has been released into the market with the name

ADRIANA S. DUSSO, ALEX J. BROWN, AND EDUARDO A. SLATOPOLSKY

of Zemplar®. This vitamin D analog lacks the carbon at position 19. Zemplar® has been studied extensively and demonstrated to suppress PTH secretion in vitro as potently as calcitriol. Studies in experimental animals have shown that 19-nor-1,25(OH)2D2 is effective in suppressing PTH levels with less hypercalcemia and hyperphosphatemia than that observed with calcitriol treatment. Indeed 19-nor-1,25(OH)2D2 is approximately 10 times less active than calcitriol in mobilizing calcium and phosphate from bone [174]. This vitamin D analog is in widespread clinical use in patients on hemodialysis in the United States, and has been demonstrated to be effective in suppressing PTH levels. Thus, while 3 times more 19-nor-1,25(OH)2D2 than calcitriol is required to achieve equivalent suppression of PTH in animals, studies in patients indicate that a ratio of 3 to 4 is required [175–177]. Similarly, while paricalcitol is 10 times less calcemic and phosphatemic than calcitriol in animals studies, in patients with end-stage renal disease on a low calcium diet, at least 8 times more paricalcitol is required to achieve a similar increment in serum calcium, presumably representing calcium mobilized from bone [178]. Sprague and collaborators demonstrated less severe hyperphosphatemia in patients treated with paricalcitol compared to calcitriol [176]. Recent studies indicate a 16% decrease in mortality in a retrospective study over a period of three years, in a large group of patients treated with paricalcitol (29,025) when compared to those receiving calcitriol (38,378) [179]. Moreover, the two-year survival rate among patients who switched from calcitriol to paricalcitriol was 73% as compared to 64% among those who switched from paricalcitol to calcitriol. The exact mechanism for this effect is not clear at the present time. Another analog of vitamin D is 1-alpha hydroxy D2, commercially known as Hectorol®. This analog is used in the United States for the treatment of secondary hyperparathyroidism. This compound is considered a prohormone since it lacks the 25-hydroxyl group, and it is 25-hydroxylated in the liver to 1,25(OH)2D2. Comparative studies in normal and uremic animals (see Fig. 12) have shown [180] that 1-alpha hydroxy D2 is more hypercalcemic and hyperphosphatemic than 19-nor-1,25(OH)2D2. Further studies in patients are necessary to corroborate this initial experimental observation.

VI. SUMMARY PTH and calcitriol are the major factors responsible for maintaining extracellular calcium homeostasis

125 Ca × P product (mg2/dl2)

1332

100 75 50 25 0

Vehicle

50 ng

100 ng 250 ng

19-norD2

50 ng

100 ng

250 ng

1α-OHD2

FIGURE 12 Effects of vehicle, 19-nor-1,25(OH)2D2 [19-nor; 50, 100, or 250 ng; n = 10] and 1α(OH)D2 [1α(OH)D2; 50, 100, or 250 ng; n = 9] on the Ca × P product in uremic rats. Rats were treated three times a week for two weeks. * and ** indicate p < 0.01 or p < 0.001 versus control rats. Adapted from Slatopolsky et al. [180].

within narrow limits despite the large bidirectional fluxes of calcium across the intestine, bone, and especially the kidney. In chronic kidney disease, the progressive reduction in kidney function not only causes defective renal handling of calcium and phosphorus, but a decrease in renal calcitriol synthesis, which is proportional to the reduction in functional renal mass. In addition to reduced renal 1α-hydroxylase, the enzyme synthesizing calcitriol, several mechanisms contribute to worsen renal calcitriol synthesis. These mechanisms include: impaired substrate availability to renal 1α-hydroxylase; inhibition of renal 1α-hydroxylase activity by hyperphosphatemia, metabolic acidosis and accumulation of uremic toxins; and a blunted response to PTH induction of 1α-hydroxylase activity. The low serum calcitriol levels result in a marked reduction in intestinal calcium absorption with concomitant hypocalcemia, as well as a proportional reduction in VDR levels in critical targets, such as the parathyroid glands. Chronic kidney disease also impairs the activity of the calcitriol/VDR-complex as a transcriptional regulator of the expression of calcitriol responsive genes. Two mechanisms were identified: A reduction in the cellular levels of the VDR-transcriptional partner, the retinoidX-receptor, RXR, and impaired VDR/RXR-DNA– binding interactions. Accumulation of uremic toxins; hypocalcemia- and/or hyperphosphatemia-induced increases in nuclear calreticulin; and uremia-induced activation of transcription factors from VDR-unrelated pathways reduce VDR binding to vitamin D responsive elements on the DNA.

1333

CHAPTER 76 Vitamin D and Renal Failure

In the parathyroid glands, the reduction in calcitriol/ VDR expression and transcriptional activity results in a defective inhibition of PTH synthesis as well as the potent mitogenic signals emerging from overexpression of TGFα and EGFR; impaired induction of the antiproliferative molecules p21 and p27; and defective induction of the CaSR. These defects cause parathyroid hyperplasia, high serum PTH, and reduced sensitivity of the parathyroid gland to suppress growth and PTH secretion in response to calcitriol or calcium. High serum PTH levels lead to osteitis fibrosa and bone loss and systemic toxicities, all of which increase the morbidity and mortality in patients with CKD. Early therapeutic interventions with calcitriol are recommended to delay the onset of calcitriol resistance by preventing the decreases in parathyroid-VDR and CaSR content. In established secondary hyperparathyroidism, the new less-calcemic vitamin D analogs are the treatment of choice. Four of these analogs are available in the USA and Japan. Although not all analog formulations are equally effective in controlling hypercalcemia and hyperphosphatemia in patients with advanced kidney disease, they offer a wider therapeutic window to counteract vitamin D resistance without causing adynamic bone disease. In the case of paricalcitol, there is a survival advantage over exclusive calcitriol therapy. The 2003 recommendations by the National Kidney Foundation of the USA provide optimal dosage of calcitriol and its less calcemic analogs for the different stages of CKD, as well as the corrections in the therapeutic approach based on a close control of serum PTH, P and Ca levels to maximize the efficacy of treatment avoiding adynamic bone disease and the risk of vascular calcifications.

7.

8. 9.

10.

11.

12. 13.

14. 15.

16.

17.

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154. Nordal KP, Dahl E, Halse J, Attramadal A, Flatmark A 1995 Long-term, low-dose calcitriol treatment in predialysis chronic renal failure: can it prevent hyperparathyroid bone disease? Nephrol Dial Transplant 10(2):203–6. 155. Hamdy NA, Kanis JA, Beneton MN, Brown CB, Juttmann JR, Jordans JG, Josse S, Meyrier A, Lins RL, Fairey IT 1995 Effect of alfacalcidol on natural course of renal bone disease in mild to moderate renal failure. Bmj 310(6976):358–63. 156. Maung HM, Elangovan L, Frazao JM, Bower JD, Kelley BJ, Acchiardo SR, Rodriguez HJ, Norris KC, Sigala JF, Rutkowski M, Robertson JA, Goodman WG, Levine BS, Chesney RW, Mazess RB, Kyllo DM, Douglass LL, Bishop CW, Coburn JW 2001 Efficacy and side effects of intermittent intravenous and oral doxercalciferol (1alphahydroxyvitamin D(2)) in dialysis patients with secondary hyperparathyroidism: a sequential comparison. Am J Kidney Dis 37(3):532–43. 157. Slatopolsky E, Weerts C, Thielan J, Horst R, Harter H, Martin KJ 1984 Marked suppression of secondary hyperparathyroidism by intravenous administration of 1,25-dihydroxycholecalciferol in uremic patients. J Clin Invest 74(6):2136–43. 158. Andress DL, Norris KC, Coburn JW, Slatopolsky EA, Sherrard DJ 1989 Intravenous calcitriol in the treatment of refractory osteitis fibrosa of chronic renal failure. N Engl J Med 321(5):274–9. 159. Dunlay R, Rodriguez M, Felsenfeld AJ, Llach F 1989 Direct inhibitory effect of calcitriol on parathyroid function (sigmoidal curve) in dialysis. Kidney Int 36(6):1093–8. 160. Tsukamoto Y, Nomura M, Takahashi Y, Takagi Y, Yoshida A, Nagaoka T, Togashi K, Kikawada R, Marumo F 1991 The “oral 1,25-dihydroxyvitamin D3 pulse therapy” in hemodialysis patients with severe secondary hyperparathyroidism. Nephron 57(1):23–8. 161. Tsukamoto Y, Mariyo R, Nomura Y, Sato N, Faugere MC, Malluche HH 1993 Long-term effect of oral calcitriol pulse therapy on bone in hemodialysis patients. Bone 14(3):421–5. 162. Quarles LD, Yohay DA, Carroll BA, Spritzer CE, Minda SA, Bartholomay D, Lobaugh BA 1994 Prospective trial of pulse oral versus intravenous calcitriol treatment of hyperparathyroidism in ESRD. Kidney Int 45(6):1710–21. 163. Cannella G, Bonucci E, Rolla D, Ballanti P, Moriero E, De Grandi R, Augeri C, Claudiani F, Di Maio G 1994 Evidence of healing of secondary hyperparathyroidism in chronically hemodialyzed uremic patients treated with longterm intravenous calcitriol. Kidney Int 46(4):1124–32. 164. Llach F, Hervas J, Cerezo S 1995 The importance of dosing intravenous calcitriol in dialysis patients with severe hyperparathyroidism. Am J Kidney Dis 26(5):845–51. 165. Bacchini G, Fabrizi F, Pontoriero G, Marcelli D, Di Filippo S, Locatelli F 1997 “Pulse oral” versus intravenous calcitriol therapy in chronic hemodialysis patients. A prospective and randomized study. Nephron 77(3):267–72. 166. Indridason OS, Quarles LD 2000 Comparison of treatments for mild secondary hyperparathyroidism in hemodialysis patients. Durham Renal Osteodystrophy Study Group. Kidney Int 57(1):282–92.

167. Fischer ER, Harris DC 1993 Comparison of intermittent oral and intravenous calcitriol in hemodialysis patients with secondary hyperparathyroidism. Clin Nephrol 40(4): 216–20. 168. Liou HH, Chiang SS, Huang TP, Shieh SD, Akmal M 1994 Comparative effect of oral or intravenous calcitriol on secondary hyperparathyroidism in chronic hemodialysis patients. Miner Electrolyte Metab 20(3):97–102. 169. Salusky IB, Goodman WG, Horst R, Segre GV, Kim L, Norris KC, Adams JS, Holloway M, Fine RN, Coburn JW 1990 Pharmacokinetics of calcitriol in continuous ambulatory and cycling peritoneal dialysis patients. Am J Kidney Dis 16(2):126–32. 170. Slatopolsky E, Brown AJ 2002 Vitamin D analogs for the treatment of secondary hyperparathyroidism. Blood Purif 20(1):109–12. 171. Dusso AS, Negrea L, Gunawardhana S, Lopez-Hilker S, Finch J, Mori T, Nishii Y, Slatopolsky E, Brown AJ 1991 On the mechanisms for the selective action of vitamin D analogs. Endocrinology 128(4):1687–92. 172. Tanaka Y, DeLuca HF, Kobayashi Y, Ikekawa N 1984 26,26,26,27,27,27-hexafluoro-1,25-dihydroxyvitamin D3: a highly potent, long-lasting analog of 1,25-dihydroxyvitamin D3. Arch Biochem Biophys 229(1):348–54. 173. Akiba T, Marumo F, Owada A, Kurihara S, Inoue A, Chida Y, Ando R, Shinoda T, Ishida Y, Ohashi Y 1998 Controlled trial of falecalcitriol versus alfacalcidol in suppression of parathyroid hormone in hemodialysis patients with secondary hyperparathyroidism. Am J Kidney Dis 32(2):238–46. 174. Finch JL, Brown AJ, Slatopolsky E 1999 Differential effects of 1,25-dihydroxyvitamin D3 and 19-nor-1,25-dihydroxyvitamin D2 on calcium and phosphorus resorption in bone. J Am Soc Nephrol 10(5):980–5. 175. Llach F, Yudd M 2001 Paricalcitol in dialysis patients with calcitriol-resistant secondary hyperparathyroidism. Am J Kidney Dis 38(5 Suppl 5):S45–50. 176. Sprague SM, Lerma E, McCormmick D, Abraham M, Batlle D 2001 Suppression of parathyroid hormone secretion in hemodialysis patients: comparison of paricalcitol with calcitriol. Am J Kidney Dis 38(5 Suppl 5):S51–56. 177. Martin KJ, Gonzalez EA, Gellens ME, Hamm LL, Abboud H, Lindberg J 1998 Therapy of secondary hyperparathyroidism with 19-nor-1alpha,25-dihydroxyvitamin D2. Am J Kidney Dis 32(2 Suppl 2):S61–6. 178. Coyne DW, Grieff M, Ahya SN, Giles K, Norwood K, Slatopolsky E 2002 Differential effects of acute administration of 19-nor-1,25-dihydroxyvitamin D2 and 1,25-dihydroxyvitamin D3 on serum calcium and phosphorus in hemodialysis patients. Am J Kidney Dis 40(6):1283–8. 179. Teng M, Wolf M, Lowrie E, Ofsthun N, Lazarus JM, Thadhani R 2003 Survival of patients undergoing hemodialysis with paricalcitol or calcitriol therapy. N Engl J Med 349(5):446–56. 180. Slatopolsky E, Cozzolino M, Finch JL 2002 Differential effects of 19-nor-1,25-(OH)(2)D(2) and 1alpha-hydroxyvitamin D(2) on calcium and phosphorus in normal and uremic rats. Kidney Int 62(4):1277–84.

CHAPTER 77

Idiopathic Hypercalciuria and Nephrolithiasis MURRAY J. FAVUS FREDRIC L. COE

Section of Endocrinology, The University of Chicago Pritzker School of Medicine, Chicago, Illinois Nephrology Section, The University of Chicago Pritzker School of Medicine, Chicago, Illinois

I. Introduction II. Idiopathic Hypercalciuria III. Genetic Hypercalciuric Rats IV. Current View of Human Genetic Hypercalciuria

V. Therapeutics of Idiopathic Hypercalciuria and Effects on Calcium Metabolism VI. Risk of Stone Formation Using Vitamin D Analogs References

I. INTRODUCTION

administration of small doses of 1,25(OH)2D3 to healthy volunteers. An animal model of genetic hypercalciuria has been developed in Sprague-Dawley rats by breeding hypercalciuric male and female animals. The hypercalciuria in genetic hypercalciuric stone forming (GHS) rats is due to an increase in intestinal Ca absorption and bone resorption and decreased renal Ca reabsorption. Elevated vitamin D receptor (VDR) content in intestinal mucosa, renal tubules, and bone cells strongly supports the concept that hypercalciuria is a state of vitamin D receptor-mediated excess. A post-transcriptional dysregulation of VDR is suggested by decreased VDR mRNA and increased accumulation of normal VDR protein that has normal binding affinity for 1,25(OH)2D3. The nature of the genetic defect in GHS rats and in human IH that permit hypercalciuria remains unknown.

This chapter focuses on idiopathic hypercalciuria (IH) as a major cause of hypercalciuria and nephrolithiasis and the potential role of vitamin D. Less frequent causes of hypercalcemia and hypercalciuria may also promote renal stone formation and are discussed in Chapters 78 and 79. IH is found in 5 to 7% of the adult population, is responsible for 50% of calcium oxalate nephrolithiasis, and is the most common 1,25-dihydroxyvitamin D [1,25(OH)2D] excess state. Idiopathic hypercalciuria is characterized by normocalcemia in the absence of known systemic causes of hypercalciuria. Increased intestinal calcium (Ca) absorption is almost always increased, and serum 1,25(OH)2D levels are elevated in one-third to one-half of patients. Serum parathyroid hormone (PTH) levels are elevated in less than 5%. The pathogenesis of IH is unknown but several models have been offered from observations in patients including: a primary increase in intestinal Ca absorption; a primary overproduction of 1,25(OH)2D; and a primary renal tubular Ca transport defect or “renal leak” of Ca. Evidence for each model can be found in some patients with IH, suggesting the disorder may be heterogeneous. There is also evidence that IH is a state of 1,25(OH)2D excess. As already mentioned one-third to one-half of IH patients have elevated serum 1,25(OH)2D levels. The remaining 50% with normal serum 1,25(OH)2D levels cannot be distinguished from those with elevated levels because intestinal Ca absorption is just as high, and negative Ca balance may develop during low Ca intake. Of interest is the observation that all of the changes in Ca metabolism characteristic of IH can be induced by the VITAMIN D, 2ND EDITION FELDMAN, PIKE, AND GLORIEUX

II. IDIOPATHIC HYPERCALCIURIA A. Overview Hypercalciuria is common among patients with Ca oxalate nephrolithiasis and is thought to contribute to stone formation by increasing the state of urine supersaturation with respect to Ca and oxalate. Flocks [1] first commented on the frequency of hypercalciuria among patients with Ca stones; however, it was not until the mid-1950s that Albright and Henneman [2,3] defined the condition of IH as hypercalciuria with normal serum Ca, no systemic illness, and no clinical skeletal disease. The definition of hypercalciuria is arbitrary and based on the distribution of urine Ca excretion values among unselected populations of healthy men Copyright © 2005, Elsevier, Inc. All rights reserved.

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MURRAY J. FAVUS AND FREDRIC L. COE

TABLE I Causes of Normocalcemic Hypercalciuria

for either sex [6]. Using these definitions, hypercalciuria is found in about 50% of calcium oxalate stone formers [6,7] and is the most common cause of normocalcemic hypercalciuric stone formation [7]. The diagnosis of IH requires the exclusion of the known causes of normocalcemic hypercalciuria (see Table I). Surveys of stone formers attending kidney stone clinics report a high proportion with kidney stone formation among first-degree relatives [8,9]. A genetic basis of IH was further suggested by subsequent surveys [10–12] that revealed a strong familial occurrence of IH with high rates of vertical and horizontal penetrance (see Fig. 1) consistent with an autosomal dominant mode of inheritance. IH also occurs in children with the same frequency of occurrence as in adults [13]. That hypercalciuria can have a genetic origin has been clearly demonstrated by breeding experiments in which the offspring of spontaneously hypercalciuric male and female Sprague-Dawley rats are intensely hypercalciuric [14–16]. Other human hypercalciuric genetic disorders have been described, but they differ from IH in

Paget’s disease Sarcoidosis Hyperthyroidism Renal tubular acidosis Cushing’s syndrome Immobilization Malignant tumor Furosemide administration

and women in Western countries [4,5]. The distribution of urine Ca forms a continuum that is clustered about a mean with a long tail of higher values. IH patients are those whose urine Ca exceeds the arbitrary upper limit of normal, which is most commonly defined as greater than 300 mg/24 hr for men, greater than 250 mg/24 hr for women, or greater than 4 mg/kg body weight or 140 mg Ca per gram urine creatinine

Family 3

Family 1 S S

S

S

S

S

S

S

S

S

S

∗ ∗

∗

S

∗

∗ Family 4

Family 2

Family 5 S

S

S

S

S

S S

∗ Family 6

∗

∗

S

∗

Family 7

∗

Family 8 S

S

∗

Family 9 S

S ∗

∗

∗

∗

∗ ∗

FIGURE 1

S

S ∗

S

S

∗

∗

Family pedigrees of nine probands with idiopathic hypercalciuria. Solid circles and solid squares are females and males with hypercalciuria; S is stone formation; * indicates children (younger than age 20). Arrows indicate probands from each family. Dashed symbols are relatives who were not studied. Hypercalciuria occurred in 11 of 24 siblings, 7 of 16 offspring, and 1 of 3 parents of probands. Reprinted by permission of The New England Journal of Medicine (Coe FL, Parks JH, Moore ES. Familial idiopathic hypercalciuria N Engl J Med 300:337–340). Copyright 1979, Massachusetts Medical Society.

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CHAPTER 77 Idiopathic Hypercalciuria and Nephrolithiasis

B. Pathogenesis of Human Idiopathic Hypercalciuria 1. RENAL HISTOPATHOLOGY IN CALCIUM OXALATE NEPHROLITHIASIS

Interstitial crystal deposition at or near the tips of papillae are found in 100% of kidneys of Ca oxalate stone formers who have IH and no systemic cause of hypercalciuria or other cause of stone formation (Table I). Less frequently (43%), nonstone formers may have such papillary depositions [20]. These lesions first described by Randall [21] have recently been found to be composed of calcium phosphate (apatite) and contain no Ca oxalate [22]. The plaques originate in the basement membrane of the thin loops of Henle and spread from there through the interstitium to just beneath the urothelium. There is no Ca phosphate or Ca oxalate crystal depositions within the renal tubule lumen. Rather, the Ca phosphate plaque may serve as a site of heterogeneous nucleation of Ca oxalate crystals that subsequently grow and form Ca oxalate kidney stones [22,23]. The role of hypercalciuria in the development of the Ca phosphate interstitial lesions of Randall’s plaques remains unknown; however, the Ca phosphate plaques are rather specific for Ca oxalate stone formers, as the interstitial lesions are absent in intestinal bypass patients who form Ca oxalate stones [22]. 2. INCREASED INTESTINAL CALCIUM ABSORPTION

Normally, the quantity of Ca absorbed is determined by dietary Ca intake and the efficiency of intestinal Ca absorption [24]. Absorption of Ca across the intestine is the sum of two transepithelial transport processes: a nonsaturable paracellular pathway and a saturable, cellular active transport system [25,26] (also see Chapters 24, 25). Absorption via the paracellular path is diffusional and driven by the lumen-to-blood Ca gradient [24]. The cellular pathway is vitamin D–dependent and is

regulated by the ambient concentration of 1,25(OH)2D. Thus, intraluminal Ca concentration and tissue 1,25(OH)2D levels are the driving forces for Ca translocation via the paracellular and cellular pathways, respectively. Increased intestinal Ca absorption has been found in most patients with IH [27–35]. Using either a single oral dose of Ca isotope to measure fecal isotope excretion or double Ca isotope administration in which the intravenous dose adjusts for isotope distribution, IH patients were shown to have an increase in the Ca absorptive flux (Fig. 2). External Ca balance studies conducted while IH patients and normal nonstone formers ingested diets containing comparable amounts of Ca show net intestinal Ca absorption rates to be greater in IH patients [36]. Biopsies of proximal intestine obtained following oral Ca isotopic administration reveal increased mucosal accumulation of isotope compared to normocalciuric nonstoneformers [37]. Thus, by all techniques used, IH is characterized by increased intestinal Ca absorption. 3. ELEVATED 1,25(OH)2D

Kaplan and colleagues [33] first reported elevated serum levels of 1,25(OH)2D in a group of patients with IH. Subsequently, others have confirmed that, on average, serum 1,25(OH)2D levels are higher in IH (Fig. 3). Increased in vivo conversion of tritiated 25-hydroxyvitamin D3 (3H-25OHD3) to 3H-1,25(OH)2D3 with normal metabolic clearance [38] in a group of IH patients with elevated serum 1,25(OH)2D levels indicate that the increase in serum 1,25(OH)2D levels in some IH patients is the result of increased production. Of note is the considerable overlap of serum 1,25(OH)2D levels between IH patients and nonstone formers (Fig. 3). Normal IH

100 % Calcium absorbed

having either a renal phosphate leak [17] that may lead to rickets (Chapter 69), renal tubular acidosis [18], or X-linked recessive stone formation with early renal failure [19]. Idiopathic hypercalciuria is a common disorder that affects 5 to 7% of otherwise healthy men and women [4]. If 50% of stone formers have IH [6,7], and the frequency of stone disease among adults is 0.5%, then 80 to 90% of IH is asymptomatic and never associated with stone formation. The increased frequency of osteopenia (see Section B.4) suggests that hypercalciuria may be an important pathogenetic factor for development of low bone mass even among those who do not form stones.

80 60 40 20 0 Birge

Wills

Pak

Kaplan

Shen

FIGURE 2 Intestinal Ca absorption in healthy volunteers and patients with IH. Absorption rates are expressed as percentages of dietary Ca absorbed as calculated from the appearance of Ca isotopes in blood or fecal collections. Values are means (horizontal bar) ± 2 standard deviations. Names indicate references: Birge [28]; Wills [29]; Pak [32]; Kaplan [33]; and Shen [34].

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Normal

Serum 1,25(OH)2D3 pg/ml

100

IH

80

60

40

20

Shen Kaplan

Gray Coe Insogna VanDenBerg Breslau

FIGURE 3 Plot of means ± 2 SD of serum 1,25(OH)2D in IH patients and nonstone formers. Horizontal bar is mean of group. Names indicate references: Kaplan [33]; Shen [34]; Insogna [38]; Coe [40]; Gray [42]; Van Den Berg [63]; and Breslau [64].

Thus, for about 50% of IH patients, increased intestinal Ca transport may be caused by increased circulating 1,25(OH)2D. For the remainder, other mechanisms of increased Ca absorption must be considered. The mechanism whereby 1,25(OH)2D production is increased in IH is unknown. The major regulators of renal proximal tubule mitochondrial 25-hydroxyvitamin D 1-hydroxylase (1-hydroxylase) activity include PTH, phosphate depletion, and insulin-like growth factor-I (IGF-I) (see Chapter 5). However, only 5% of IH patients have elevated circulating PTH levels [32,39], and urinary cAMP levels, a surrogate measure of PTH, are also normal in most patients [32,40,41]. Mild hypophosphatemia with reduced renal tubular phosphate reabsorption has been described in as many as one-third of IH patients [33,34,39,42]. A strong inverse association between serum 1,25(OH)2D levels and renal tubular phosphate reabsorption has been reported [34,41]. As elevated PTH or hypophosphatemia accompany elevated serum 1,25(OH)2D in only a minority of patients; the cause of increased serum 1,25(OH)2D in most patients with IH remains unknown. Detailed studies of IGF-I have not been performed. The recent description of sequences of mutations in the q23.3-q24 region of the first chromosome in three kindred with absorptive IH [43] involves a region containing a gene that is analogous with the rat soluble adenylate cyclase gene. This first description of specific

base pair substitutions suggests the possibility of a gene defect associated with IH that may involve altered receptor signaling. Whether this mutation alters functions related to the regulation of the 1-hydroxylase remains to be determined. However, caution has been expressed in accepting this report as conclusively demonstrating that the substitutions or a mutation of this gene causes IH [44]. Serum 1,25(OH)2D values in normals and IH patients overlap extensively in each series reported (Fig. 3). Kaplan et al. [33] found that in patients with absorptive IH [defined as normal fasting urine Ca and normal or elevated serum 1,25(OH)2D, intestinal Ca absorption measured by fecal excretion of orally administered 47Ca was increased out of proportion to the simultaneously measured serum 1,25(OH)2D concentration (Fig. 4B). In contrast, a strong positive correlation between intestinal Ca absorption and serum 1,25(OH)2D is found in normal volunteers, normocalciuric stone formers, patients with primary hyperparathyroidism, and IH patients with fasting hypercalciuria (Fig. 4A). The high intestinal Ca absorption rates with normal or elevated serum 1,25(OH)2D levels suggest that the pathogenesis of IH is heterogeneous, with at least one phenotype resulting from 1,25(OH)2D overproduction. 4. DECREASED RENAL CALCIUM REABSORPTION

A defect in the tubular reabsorption of Ca, a so-called renal leak of Ca, has been postulated as a cause of hypercalciuria in IH. Two reports [45,46] found a greater fraction of filtered Ca excreted in the urine of IH patients compared to nonstone formers. The values were calculated from inulin clearance or creatinine clearance and used blood ionized Ca as an estimate of ultrafilterable Ca. Although urinary sodium (Na) excretion is a major determinant of Ca excretion in normal and IH patients, there is no evidence that patients overingest or overexcrete Na. Hydrochlorothiazide and acetazolamide increase urine Ca, Na, and magnesium (Mg) excretion in IH compared to normals [47], suggesting a generalized defect in proximal tubule electrolyte and water transport in IH patients. The basis for the abnormal renal transport is not known, but increased activity of the erythrocyte plasma membrane Ca2+, Mg2+-ATPase in IH patients and correlation of enzyme activity with urine Ca excretion in families with IH [48] suggests a more widespread genetic defect in monovalent and divalent ion transport. 5. LOW BONE MASS

Abnormal skeletal metabolism in IH has been demonstrated by low bone mineral density of the distal radius [49,50] and lumbar spine [51–53] and by lower skeletal Ca content by neutron activation analysis [54].

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1.0

A Fractional Ca absorption (α)

Fractional Ca absorption (α)

1.0 0.8

0.6

0.4 PHPT RH NN Control

0.2

0

20

40 60 80 100 Plasma 1α,25(OH)2D pg/ml

120

B

0.8

0.6

0.4

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0 20

100 40 60 80 Plasma 1α,25(OH)2D pg/ml

120

FIGURE 4 Relationship of calcium absorption to 1,25(OH)2D levels. (A) Fractional intestinal absorption of oral 47Ca versus serum 1,25(OH)2D level in normal controls (open circles), normocalciuric stone formers (NN, filled circles), IH stone formers who have fasting hypercalciuria and elevated PTH (RH, filled squares), and patients with primary hyperparathyroidism (PHPT, open squares). (B) Fractional calcium absorption of IH patients with absorptive hypercalciuria (normal fasting urine Ca) superimposed on the 95% confidence limits for the relationship in controls. Reproduced from The Journal of Clinical Investigation, 1977, Vol. 59, pp. 756–760 [33], by copyright permission of The American Society for Clinical Investigation.

Reports differ as to possible pathogenesis, with low bone density found only in those with renal leak hypercalciuria in one study [50], and low bone density in those with absorptive hypercalciuria in another study [52]. Information on bone dynamics is limited to one early study in which 47Ca labeling was interpreted as increased bone turnover, with bone resorption and formation both increased [55]. Two studies of bone histology showed reduced bone apposition rate, delayed mineralization of osteoid seams, and prolonged mineralization lag time and formation period [56,57]. These observations suggest defective mineralization, which may be caused by hypophosphatemia in some patients. The observations are also consistent with a defect in osteoblastic function. Measurements of biochemical markers of bone turnover reveal increased urine hydroxyproline excretion in unselected IH patients [58] and increased serum osteocalcin in IH patients with renal but not absorptive hypercalciuria [59]. Whether the low bone density is a result of the lifelong hypercalciuria, habitual low Ca intake, or a genetic defect in osteoblast function independent of urine Ca excretion remains to be determined. In a study of 59 subjects from 11 families with at least one member a hypercalciuric Ca oxalate stoneformer [60], lumbar spine and femoral neck bone density Z scores varied inversely with urine Ca and urine ammonium in the stoneformers but not in the nonstone formers. There were no correlations of Z score for bone turnover markers or serum 1,25(OH)2D levels. Ca consumption was lower in stoneformers, suggesting that the admonition to ingest a low Ca diet to avoid more stones, in fact predisposes to bone loss.

The well-documented low bone mass in IH patients is associated with increased fracture risk [61]. Reduction of urine Ca during thiazide therapy has been studied in a small number of IH patients and found to be effective in improving bone mass (see Section V.B below). 6. PROPOSED PATHOGENETIC MODELS HYPERCALCIURIA

OF IDIOPATHIC

On the basis of the consistent increase in intestinal Ca absorption, normal or elevated serum 1,25(OH)2D levels, and normal or elevated fasting urinary Ca, Pak and colleagues [62] separated IH into three groups: absorptive, renal, and resorptive. In the first, primary intestinal Ca hyperabsorption (Fig. 5A) would transiently raise postprandial serum Ca above normal and increase ultrafilterable Ca. Postprandial hypercalcemia would transiently suppress PTH secretion, resulting in reduced tubular Ca reabsorption and hypercalciuria. In the second, a primary renal tubular leak of Ca (Fig. 5B) would cause hypercalciuria and a transient reduction in serum Ca. Secondary hyperparathyroidism would normalize serum Ca and increase proximal tubule 1,25(OH)2D synthesis, which would stimulate intestinal Ca absorption. PTH secretion would then decline to the extent that serum Ca is normalized. This scenario predicts that serum 1,25(OH)2D would be elevated in renal IH and normal or elevated in absorptive IH [63,64]. A third possibility is based on a primary overproduction of 1,25(OH)2D3 that increases intestinal Ca absorption and bone resorption (Fig. 5C) while PTH remains normal and fasting urine Ca excretion may be normal or elevated.

1344


A

B

Intestinal Ca absorption

Renal tubular Ca transport Hypercalciuria

Transient

Serum Ca Serum Ca

Parathyroid hormone Serum PTH Normal Serum calcitriol

Tubular Ca reabsorption

Serum calcitriol


Hypercalciuria

C

Primary renal calcitriol overproduction

Serum calcitriol

Target tissue calcitriol/vitamin D receptor complex


Bone resorption

?

Renal tubular Ca transport

Hypercalciuria

FIGURE 5 Three proposed models of IH. (A) Absorptive IH with primary intestinal overabsorption, postprandial hypercalcemia, suppressed PTH, and normal fasting urine Ca. Serum 1,25(OH)2D is normal. (B) Primary renal tubular leak of Ca leads to a transient decrease in serum Ca and elevated PTH with secondary increases in serum 1,25(OH)2D and intestinal Ca absorption. Fasting urine Ca is elevated. (C) Primary overproduction of 1,25(OH)2D increases serum 1,25(OH)2D and stimulates intestinal Ca absorption and bone resorption. Serum PTH is normal or decreased, and fasting urine Ca is normal or elevated. 7. TESTS OF THE MODELS

Knowledge of the pathophysiology of IH is fundamental to developing rational therapy for the prevention of recurrent kidney stones. If the model of primary intestinal overabsorption were correct, then dietary Ca restriction would reduce the amount of Ca absorbed and Ca excreted in the urine without altering bone mass. If a renal leak of Ca were the primary event, or if urinary Ca originates from bone rather than diet, then restricting dietary Ca will have little effect on urinary Ca excretion, while worsening Ca balance and promoting bone loss. Testing certain predictions has assessed the accuracy of the absorptive and renal models. a. Fasting Serum PTH, Urine Ca If repeated episodes of postprandial hypercalcemia suppress PTH secretion sufficiently to cause chronic hypoparathyroidism, fasting serum PTH and urine cAMP would be low and fasting urine Ca elevated. Transient suppression of PTH

would permit normal serum PTH, urine cAMP, and fasting urine Ca. In contrast, renal IH requires increased PTH and urine cAMP and increased fasting urine Ca [65]. As existing PTH radioimmunoassays do not differentiate normal from low values, most IH patients have been found to have PTH levels in the normal range. Although normal fasting urine Ca is not unusual among IH patients, only 5% have elevated PTH and, therefore, fail to meet the criteria for renal IH [32,62]. Thus, a primary renal leak of Ca with a secondary increase in PTH cannot account for fasting hypercalciuria in a majority of patients. About 24% of patients meet the criteria of absorptive hypercalciuria by reducing urine Ca during fasting to maintain neutral Ca balance [63]. b. External Ca Balance The relationship between net intestinal Ca absorption and 24-hr urine Ca excretion calculated from 6-day balance studies is different


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FIGURE 6 Urinary Ca excretion as a function of net intestinal Ca absorption. Data are derived from 6-day external mineral balance studies. Solid lines indicate the 95% confidence limits about the mean regression line derived from the data on 195 adult nonstone formers. Individual balance studies performed on 51 patients with IH are shown as open circles. The dashed line represents equivalent rates of urinary Ca excretion and net intestinal Ca absorption (the line of identity). Normal values are from [47,58,68,69]. Values from patients are from References [47,57,71–74], and J. Lemann (personal communication, 1992). Adapted from Asplin et al. [95].

in IH patients compared to normal subjects (Fig. 6) [66–72]. In nonstone formers, urinary Ca excretion is positively correlated with net absorption, and overall Ca balance is positive when net absorption is greater than 200 mg per 24 hr (see 95% confidence limits calculated from balance studies on normal subjects in Fig. 6). Net Ca absorption tends to be greater in IH patients, and for every level of net absorption, 24-hr urine Ca excretion is higher in the patients compared to healthy subjects. In IH patients, a greater portion of absorbed Ca is excreted in the urine. In normal subjects, net Ca absorption exceeds urine Ca excretion, and balance is positive when net absorption exceeds 200 mg/24 hr. In contrast, almost 50% of the IH patients have urine Ca excretion in excess of net absorption and are in negative Ca balance, even when allowance is made for some variability in the balance data (± 50 mg). Thus, at all levels of net Ca absorption, negative Ca balance (above the zero balance or above the line of identity) is common in IH patients but not in healthy subjects. Negative Ca balance in the presence of adequate Ca intake is incompatible with a primary hyperabsorption of dietary Ca or absorptive hypercalciuria and cannot, by itself, account for the hypercalciuria.

1345 c. Urine Ca and Ca Balance during Low Ca Diet The hypothesis of primary intestinal Ca overabsorption predicts that dietary Ca restriction would reduce the amount of Ca absorbed and would therefore reduce urinary Ca excretion. Like normal subjects, IH patients would be in positive or neutral Ca balance when net absorption is above 200 mg/24 hr (Fig. 6). A low Ca diet would reduce urine Ca excretion through an increase in PTH secretion, which would promote distal tubular Ca reabsorption. In contrast, patients with a primary renal Ca leak would be unable to conserve urine Ca at any level of Ca intake and would maintain an excessive or inappropriately high urine Ca excretion even during low Ca diet. As a result, Ca balance during low Ca diet would shift from positive or neutral to negative or become more negative. Serum PTH would be expected to increase to high levels during a low Ca diet. To test whether the responses of IH patients fit these predictions, Coe et al. [40] fed a low Ca diet (2 mg/kg/ day) to nine normal volunteers and 26 unselected IH stone formers. After 10 days on the diet, urine Ca excretion decreased to 2.0 mg/kg body weight or less in both patients and controls, but 17 of the 26 IH patients (Fig. 7) showed values greater than the highest value in normal controls. In patients, urine Ca excretion (CaE) ranged from normal to persistently high levels. It exceeded Ca intake (CaI) (Fig. 7, CaI − CaE) in 11 of the 26 patients and none of the nonstone-forming controls. Thus, almost 50% of the patients had more Ca in the urine than what was provided by the diet and were clearly in negative Ca balance. The results indicate that a chronic low Ca diet may be detrimental for some patients, as the inability to conserve urine Ca would eventually lead to clinically detectable bone loss. The data also suggest that some patients with IH may have diet-dependent hypercalciuria, whereas others have diet-independent hypercalciuria. The two proposed mechanisms cannot be readily distinguished by any clear discontinuity in the distribution of urine Ca values, and serum PTH and 1,25(OH)2D levels do not predict the urine Ca responses during a low Ca diet. Patients with the highest urine Ca and most extreme negative Ca balance had serum PTH and 1,25 (OH)2D levels that were not different from patients who conserved Ca to the levels found in normal subjects. d. Role of 1,25(OH)2D Excess The majority of patients are classified as having absorptive hypercalciuria [62,65], yet negative Ca balance during low Ca diet [40] without elevated PTH, or 1,25(OH)2D is not predicted by the absorptive model (Fig. 5A). Patients who meet the criteria of renal hypercalciuria tend to

1346

MURRAY J. FAVUS AND FREDRIC L. COE 3

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FIGURE 7 Calcium intake and urinary excretion in patients with IH and normal subjects. Urine Ca excretion (UCa) and Ca intake (Cal) are during a low Ca diet. Mean Ca intakes for patients and controls (2.29 ± 0.15 versus 2.31 ± 0.05 mg/kg/day) were not different. Mean urine excretion rates during low Ca intake and values of Cal−CaE (an index of Ca balance) differed significantly between normals and IH patients. Subjects and patients are arranged in ascending order of urinary Ca excretion. Reprinted by permission of the publisher from Coe et al. [40]. “Effects of low-calcium diet on urine calcium excretion, parathyroid function and serum 1,25(OH)2D3 levels in patients with idiopathic hypercalciuria and in normal subjects,” American Journal of Medicine, Vol. 72, pp. 25–32. Copyright 1982 by Excerpta Medica Inc.

have higher serum 1,25(OH)2D levels, but only a small portion have elevated PTH levels. Further, serum 1,25(OH)2D levels do not predict whether patients will be classified as absorptive or renal, and at least onethird of patients have normal serum 1,25(OH)2D levels despite intestinal Ca hyperabsorption. For them, the mechanism of intestinal Ca hyperabsorption remains unexplained.

The model of primary vitamin D excess (Fig. 5C) is supported by elevated 1,25(OH)2D production rates and enhanced biological actions of 1,25(OH)2D, including increased intestinal Ca absorption and bone resorption. Creation of a mild form of 1,25(OH)2D excess was achieved by the administration of pharmacological doses of 1,25(OH)2D3 (3.0 ug/day) to healthy men for 10 days while Ca intake varied from low (160 mg) to normal (372 mg) or high (880 mg) [73–75]. Increased urine Ca excretion and net intestinal Ca absorption led to negative Ca balance as calculated from 6-day metabolic balance studies (Fig. 8). Dietary Ca strongly influenced the response to 1,25(OH)2D3, as Ca balance was more negative during low Ca intake, and the increase in urine Ca resulted primarily from accelerated bone resorption. At low-normal or normal Ca intake, 1,25(OH)2D3 administration increased urine Ca and net intestinal Ca absorption, and Ca balance remained neutral. During normal Ca diet, 1,25(OH)2D3 administration maintained neutral or positive Ca balance. Thus, 3 ug/day of 1,25(OH)2D3, which was insufficient to cause hypercalcemia, during the 10-day study has profound effects on intestinal Ca absorption, urine Ca excretion, and Ca balance. Further, 1,25(OH)2D3 administration caused negative Ca balance only during low Ca intake. Thus, l,25(OH)2D3 induced changes in Ca balance in normal subjects similar to that observed in IH patients on comparable levels of Ca intake. In other experiments, ketoconazole administration to IH patients inhibited renal 1,25(OH)2D biosynthesis [64] and decreased serum 1,25(OH)2D levels, intestinal Ca absorption, and urine Ca excretion. The results of the effects of 1,25(OH)2D3 treatment and the response to ketoconazole provide further support for a primary 1,25(OH)2D excess in at least some patients with IH. The nature of the disordered regulation of renal 1,25(OH)2D production or action remains to be determined, as neither elevated PTH nor hypophosphatemia were present in responders or were absent in nonresponders to ketoconazole.

III. GENETIC HYPERCALCIURIC RATS Tests of the absorptive, renal, and vitamin D excess models of IH have been complicated by difficulty in controlling for potential variables such as inheritance and environmental factors that may influence dietary patterns. The availability of an animal model of IH would permit the testing of the three hypotheses under conditions that exclude genetic and dietary influences. The strong familial occurrence of IH in humans and the high frequency of elevated urine Ca in adult men

1347


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FIGURE 8 Intestinal Ca absorption, urine Ca excretion, and Ca balance in normal men receiving 1,25(OH)2D3 (hatched bars) or controls (open bars) at varying levels of dietary Ca. Values (mg/24 hr) are means ± SEM for 6 men per group. For Ca balance, values above the horizontal line indicate positive balance and those below the line, negative balance. Data from Maierhofer et al. [76] and Adams et al. [77]. Reprinted with permission from Coe FL, Parks JH 1988 Nephrolithiasis: Pathogenesis and Treatment, Second Edition, Year Book Publishers: Chicago 1988 [75].

and women suggested that spontaneous hypercalciuria might also be found in animals.

A. Establishment of a Colony of Genetically Hypercalciuric Rats The distribution of urine Ca excretion in a population of male Sprague-Dawley rats fed a normal Ca diet (0.8% Ca) was similar to that found in a population of healthy humans in that it followed a nonGaussian distribution, with values clustering about the mean and a long tail of higher values [14]. Using an arbitrary definition of hypercalciuria as urine Ca greater than two standard deviations above the mean value, about 5 to 10% of male and female rats were hypercalciuric. Mating males and females with the most severe hypercalciuria resulted in offspring with hypercalciuria. The most hypercalciuric offspring were used for repeated matings, leading to a colony with hypercalciuria that has increased in intensity and frequency with each successive generation [15]. By the twentieth generation,

over 95% of males and females were hypercalciuric. By the fortieth generation, mean urine Ca excretion was 7.0 ± 0.3 mg/24 hr compared to the stable mean excretion of less than 0.75 mg/24 hr by wild-type rats [77]. Hypercalciuria is lifelong and may be detected as soon as the animals are weaned (about 50 g body weight). Weight and growth of the hypercalciuric rats have been comparable to wild-type Sprague-Dawley rats obtained from the same supplier that provided the original spontaneously hypercalciuric animals. No anatomical or structural abnormalities have been identified; however, by 18 weeks of age 100% of the animals have grossly evident Ca-containing kidney stones in the upper and lower urinary tracts [78]. No stones are found in the kidney or urinary tract of wild-type rats.

B. Serum and Urine Chemistries Serum Ca and Mg are within the normal range in the genetic hypercalciuric stone-forming (GHS) male and female rats [15]. Serum phosphate is lower in

1348


C. Mineral Balance Six-day external balance studies performed while the animals were fed a normal Ca diet showed the animals to be in positive balance for Ca, Mg, and phosphorus [15] with greater net Ca absorption in GHS rats. The GHS rats maintained positive Ca balance because the increased urine Ca excretion was matched by a greater net intestinal Ca absorption.

D. Intestinal Calcium Transport To investigate the mechanism of the increased Ca absorption, segments of duodenum were mounted in vitro in modified Ussing chambers, and transepithelial bidirectional fluxes of Ca were measured in the absence of electrochemical gradients [22]. Under these conditions, [15], duodenal segments from GHS rats had a fivefold increase in the mucosal-to-serosal (absorptive) transepithelial flux of Ca (Jms), whereas the secretory flux of Ca from serosa to mucosa (Jsm) was only mildly increased compared to wild type (Table II). As Ca Jms was 10 to 12 times higher than Ca Jsm, changes in Jsm had a nonsignificant effect on net Ca absorption.

E. Serum 1,25(OH)2D Circulating 1,25(OH)2D levels were lower in the fourth generation GHS rats; however, the differences TABLE II

In Vitro Bidirectional Duodenal Calcium Active Transport*

Flux

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*Values are means ± SE for 5 to 11 rats per group. NM and NF are normocalciuric (wild-type) male and female rats, respectively. GHM and GHF are genetic hypercalciuric male and female rats, respectively. Jms and Jsm are mucosal-to-serosal and serosal-to-mucosal fluxes of Ca, respectively. Jnet is net Ca absorption, where Jnet = Jms − Jsm. Adapted from Li et al. [16] and reproduced with permission from The Journal of Clinical Investigation, 1993, Vol. 91, pp. 661–667, by copyright permission of The American Society for Clinical Investigation.

240 200 Duodenal calcium Jnet (nmol/cm2/h)

female rats, and there is no difference between GHS and wild-type males and females. Serum PTH levels in GHS rats are not different from controls. Urine volumes are greater in the GHS rats.

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FIGURE 9 Duodenal Ca net flux (Jnet) as a function of serum 1,25(OH)2D for hypercalciuric and normocalciuric male (open and filled squares, respectively) and female (open and filled circles, respectively) rats. Jnet and serum 1,25(OH)2D were correlated for male and female normocalciuric rats (r = 0.789, n = 12, p < 0.001, solid line) and for male and female GHS rats (r = 0.500, n = 17, p < 0.03, dotted line). The regressions were different (F ratio = 5.469, p < 0.015). Reproduced from The Journal of Clinical Investigation, 1988, Vol. 82, pp. 1585–1591 [15], by copyright permission of The American Society for Clinical Investigation.

disappeared by the tenth generation [at 190 g, mean ± SD serum 1,25(OH)2D was 135 ± 12 versus 174 ± 19 pg/ml, nonsignificant], and no subsequent differences in serum 1,25(OH)2D levels have been observed [16]. In vitro duodenal net flux (Jnet, equal to Jms − Jsm) for Ca was positively correlated with serum 1,25(OH)2D in normocalciuric and GHS male and female rats (Fig. 9). However, the regression coefficients were different for the wild-type and GHS rats, with the latter having a steeper slope. The greater Ca Jnet in GHS rats with serum 1,25(OH)2D levels comparable to the wild-type rats strongly suggests that duodenal Ca-transporting cells in GH rats are more sensitive to 1,25(OH)2D.

F. Role of the Vitamin D Receptor The increased intestinal Ca transport and normal serum 1,25(OH)2D levels in GHS rats suggested either that Ca transport was being stimulated by an unidentified, vitamin D–independent process or that 1,25(OH)2D action was being amplified at the level of vitamin D target tissues. As 1,25(OH)2D stimulates Ca transport by binding to the vitamin D receptor (VDR) to up-regulate vitamin D–dependent genes that encode for proteins involved in transepithelial Ca transport, and because the biological actions of 1,25(OH)2D are directly related to the tissue VDR content [79–81], VDR binding in intestinal epithelial cells was measured. Duodenal cytosolic

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Specific binding of 3H-1,25(OH)2D3 to duodenal cytosolic fractions (VDR) prepared from GHS rats (filled circles) and wild-type controls (open circles) while fed a normal Ca diet. Values are means ± SEM for four observations per concentration point. *, p < 0.05; **, p < 0.01; ***, p < 0.005 vs controls. Reproduced from The Journal of Clinical Investigation, 1993, Vol. 91, pp. 661–667 [16], by copyright permission of The American Society for Clinical Investigation.

fractions prepared in high potassium buffer from male GHS rats bound more 3H-1,25(OH)2D3 than comparable fractions from wild-type control rats [16] (Fig. 10). Cytosolic fractions from kidney cortex and from splenic monocytes also exhibited greater specific binding of 1,25(OH)2D3. Scatchard analysis of the specific binding curves revealed a single class of VDR binding sites in tissues from both wild-type and GHS rats. The number of VDR binding sites in GHS rat duodenal cells was double that found in cells from wild-type rats (536 ± 73 versus 243 ± 42 fmol/mg protein; n = 8 and n = 14; p < 0.001), with comparable affinity of the receptor for its ligand (0.33 ± 0.01 versus 0.49 ± 0.01 nM; nonsignificant). A twofold increase in VDR binding sites was also found in GHS rat renal cortical homogenates [16]. Using Western blotting, homogenates of duodenal mucosa from GHS rats contained a band at 50 kDa that comigrated with duodenal extracts from wild-type rats and with recombinant human VDR. The bands from the GHS rat tissues were more intense compared to controls, confirming that the increase in specific 3H1,25(OH)2D3 binding was due to an increase in VDR protein. Northern analysis of RNA extracts from GHS and wild-type rat tissues revealed a single species of VDR mRNA at 4.4 kb with no difference in migration between the two groups [16]. Duodenal extracts from GHS rats contained less VDR mRNA than controls. Estimates of duodenal cell transcription rates using standard nuclear run-on assays found no clear difference

between GHS rats and controls [16]. The in vivo halflife of the VDR mRNA in GHS rat duodenum was comparable to that of controls (6 hr). Administration of a small dose of 1,25(OH)2D3 (30 ng as a single dose) resulted in a significant elevation of VDR message and prolongation of message half-life in GHS rats but not controls [82]. Thus, in GHS rat intestine, the increased VDR level is not due to an increase in VDR gene transcription. The data are consistent with either an increase in VDR mRNA translation efficiency or changes that result in a prolongation of the VDR half-life. The increased accumulation of the vitamin D–dependent calbindin-D9K found in GHS rat duodenum [16] is evidence that the increased level of VDR is functional and that the increased Ca transport is likely a vitamin D– mediated process. Major questions remain as to the genetic basis of the increased VDR activity. However, sequence of a cDNA prepared from GHS rat duodenal VDR mRNA failed to reveal any difference compared to the sequence of VDR cDNA prepared from wild-type duodenum. The cumulative evidence suggests that the primary genetic defect does not directly involve the VDR gene.

G. Increased Bone Resorption In vitro studies of bone resorption using neonatal calvariae from normal and GHS rats show that Ca efflux (a measure of bone resorption) increases in a dosedependent manner in the presence of 1,25(OH)2D3 or PTH [83]. The dose-response curve is much steeper for 1,25(OH)2D3 in calvariae from GHS rats, whereas the dose-response curves for PTH-stimulated Ca efflux are not different between control and GHS calvariae. Western blotting showed a fourfold increase in VDR protein from GHS neonatal rat calvariae [83]. Thus, the increase in target tissue VDR exerts biological actions that increase l,25(OH)2D3–dependent bone resorption, which likely contributes to the hypercalciuria.

H. Response to Low Calcium Diet To test whether the hypercalciuria in GHS rats is the result of a primary overabsorption of dietary Ca, GHS and wild-type control rats were fed diets either normal (0.6% Ca) or low (0.02% Ca) with respect to Ca. During the low Ca diet, urine Ca excretion decreased in both groups (Fig. 11); however, urine Ca remained higher in GHS rats and resulted in negative Ca balance [84]. The inability of GHS rats to conserve Ca during low Ca intake excludes overabsorption of dietary Ca as the sole cause of hypercalciuria in GHS rats.

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FIGURE 11 Daily urine Ca excretion in nineteenth-generation GHS rats (open symbols) or wild-type control rats (filled symbols) fed a normal Ca diet (NCD, 0.6% Ca, triangles) during days 1–10 followed by either continuation of the NCD (triangles) or feeding of a low Ca diet (LCD, 0.02% Ca, circles). Rats were pair-fed to 13 g of diet per day. Reprinted with permission from Kim et al. [86].

I. Summary of Pathogenesis in the Genetic Hypercalciuric Rat Figure 12 summarizes current knowledge of the pathogenesis of hypercalciuria in the GHS rats. Breeding by selection for hypercalciuria has emphasized a trait in the offspring that likely involves the expression of several genes for full phenotypic expression. To date, none of the genes has been identified. Studies implicate the increased VDR concentration as part of the primary event(s) and a cause of the hypercalciuria; however, a secondary adaptive increase in

Striking similarities in Ca metabolism between GHS rats, IH patients, and human volunteers treated with 1,25(OH)2D3 (Table III) strongly support a primary role of excess 1,25(OH)2D biological action in the pathogenesis of human IH. When deprived of dietary Ca, few patients conserve Ca to the extent that normals do (Fig. 7). The renal IH model predicts ongoing urinary losses of Ca independent of Ca intake, and negative Ca balance during a low calcium diet. However, most patients have normal, not elevated PTH, as renal IH would require. Therefore, the absorptive and renal models of hypercalciuria cannot explain the response of most patients to a low Ca diet. In nonstone formers, 1,25(OH)2D3 administration changes urine Ca and Ca balance to those observed in a majority of IH patients who have either normal or elevated serum 1,25(OH)2D levels. For some patients, elevated serum 1,25(OH)2D3 increases in intestinal Ca hyperabsorption and urine Ca excretion, and causes negative Ca balance during low Ca intake. The source of 1,25(OH)2D excess is more elusive in patients with normal serum 1,25(OH)2D levels. They may be more similar to the GHS rats in that both have normal serum 1,25(OH)2D, increased

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FIGURE 12 Proposed series of events that result from breeding selection for hypercalciuric rats. The renal handling of Ca by GHS rats and the role of increased VDR content, if any, in the transport process remain unknown.

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TABLE III

Pathophysiology of Genetic Hypercalciuria

Parameter Serum Ca Serum phosphate Serum 1,25(OH)2D Urinary Ca on NCD Urinary Ca on LCD Intestinal Ca absorption Ca balance on NCD Ca balance on LCD

Human

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N N I I I I Pos-N N-Neg

N N-D N-I I N-I I N-Neg N-Neg

GHS rats N N N I I I Pos Neg

Values for human controls are responses to treatment with 3 ug 1,25(OH)2D3 daily for 7 days compared to pretreatment. GHS, genetic hypercalciuric stone-forming; NCD, normal Ca diet; LCD, low Ca diet; N, normal; I, increased; D, decreased; Pos, positive; Neg, negative.

intestinal absorption and bone resorption during a low Ca diet, and low bone density. Whether these changes in human IH are due to increased intestinal, renal, and bone cell VDR content that can amplify the biological actions of normal circulating 1,25(OH)2D levels remains to be determined.

V. THERAPEUTICS OF IDIOPATHIC HYPERCALCIURIA AND EFFECTS ON CALCIUM METABOLISM A. Dietary Calcium Restriction Hypercalciuria promotes urine calcium oxalate supersaturation and increases spontaneous crystal formation [85]. The goals of preventive therapy are to reduce Ca oxalate supersaturation by increasing urine volume and decreasing urine Ca excretion. If the pathophysiological role of 1,25(OH)2D excess or VDR excess is borne out, then ideal therapy may eventually include either a specific 1,25(OH)2D antagonist or an inhibitor of VDR function. In the absence of such agents, therapies will continue to concentrate on lowering urine Ca through indirect means. Since the description of IH, dietary Ca restriction has been recommended to lower urine Ca. Dietary Ca restriction or the use of Ca-binding resin to prevent absorption [86] could be efficacious for patients with primary intestinal Ca hyperabsorption (absorptive hypercalciuria). However, it appears that many patients develop negative Ca balance during low Ca intake. For them, chronic dietary Ca restriction and negative Ca balance would eventually cause bone loss, osteoporosis, and increased fracture risk. Reports of lower bone

density in IH patients suggest that Ca restriction may only worsen the existing reduction in bone mass. Therefore, treatment with Ca restriction requires knowledge that the patient will normally conserve urine Ca and not develop negative Ca balance.

B. Thiazides Thiazide and the related chlorthalidone diuretics reduce urine Ca excretion by inducing a NaCl diuresis, which causes volume contraction and decreased Ca delivery to the distal tubule segments [87]. These agents also stimulate distal tubule Ca reabsorption through a direct interaction with the tubule cells [86–88]. Thiazides may decrease or have no effect [31,35,89] on intestinal Ca transport in IH patients, and serum 1,25(OH)2D and PTH levels are not changed by thiazide. In one study, IH patients treated with chlorthalidone for six months improved Ca balance to or toward positive by decreasing both urine Ca and intestinal Ca absorption [90], with urine Ca declining to a greater extent than intestinal absorption. The epidemiological studies suggesting that chronic thiazide therapy reduces fracture risk [91,92] may result from druginduced improvement in Ca balance [89] and reduced bone turnover and improved mineralization [59]. The effects of thiazide on urine Ca and bone metabolism are accompanied by a decrease in new Ca stone formation compared to placebo controls [93]. The beneficial effect of thiazide is evident during the second and third year of therapy, when stone recurrence is reduced by about 50 to 80%. The reduction in new stone formation is due to a decrease in urine Ca oxalate supersaturation, as urine Ca declines while oxalate is unchanged. As thiazides can reduce urine Ca excretion and stone formation rates in all forms of IH [94], knowledge of the pathogenesis of IH in each patient may not be required prior to selecting thiazide therapy.

VI. RISK OF STONE FORMATION USING VITAMIN D ANALOGS A growing research interest in the cell differentiation and immune modulator effects of vitamin D and analogs may result in their use in a variety of disorders [95–97] (also see chapters in Sections VIII and IX). However, the development of hypercalciuria and hypercalcemia may limit the use of the naturally occurring vitamin D metabolites, as well as synthetic analogs [98,99]. While some vitamin D analogs are reported to have little or no hypercalcemic action, hypercalcemia and hypercalciuria may appear at higher doses through

1352 the classic vitamin D actions on intestine, kidney, and bone [97,98]. Low Ca diets have had only modest beneficial effects to limit hypercalciuria and hypercalcemia and could promote bone loss. It remains to be determined whether the newer vitamin D analogs with less calcemic activity will, in practice, cause less calciuria and a lower risk of kidney stone formation. Until such actions of the vitamin D analogs are known, standard approaches to minimize stone formation should be followed. These include (1) assuring sufficient fluid intake to maintain at least 1.5 liters urine output per day; (2) if necessary, increasing urine citrate excretion to normal in those with low citrate [85]; and (3) discontinuing or reducing treatment if significant hypercalciuria develops. The addition of a thiazide may avoid or minimize hypercalciuria, but hypercalcemia may occur because of thiazide-induced Ca retention.

VII. SUMMARY Idiopathic hypercalciuria is the most common cause of Ca oxalate kidney stone formation, and is the most common cause of 1,25(OH)2D3 excess. Several models of hypercalciuria incorporate intestinal Ca hyperabsorption, increased bone resorption, and decreased renal tubule Ca reabsorption, all of which can be accounted for by the elevated serum 1,25(OH)2D3 found in about 50% of patients. There is evidence for a defect in the regulation of the 1-hydroxylase, but the nature of the dysregulation remains unknown. In those patients with normal serum 1,25(OH)2D3 levels, the possibility of vitamin D receptor excess as found in GHS rats, offers a testable hypothesis.


8. 9. 10. 11. 12. 13. 14. 15. 16.

17.

18. 19. 20. 21. 22.

References 1. Flocks RH 1939 Calcium and phosphorus excretion in the urine of patients with renal or ureteral calculi. JAMA 13:1466–1471. 2. Albright F, Henneman P, Benedict PH, Forbes HR 1953 Idiopathic hypercalciuria. A preliminary report. Proc R Soc Med Lond (Biol) 46:1077. 3. Henneman PH, Benedict PH, Forbes AP Dudley HR 1958 Idiopathic hypercalciuria. N Engl J Med 259:802–807. 4. Hodgkinson A, Pyrah LN 1958 The urinary excretion of calcium and inorganic phosphate in 344 patients with calcium stones of renal origin. Br J Surg 46:10–18. 5. Robertson WG, Morgan DB 1972 The distribution of urinary calcium excretions in normal persons and stone-formers. Clin Chim Acta 37:503–508. 6. Coe FL, Parks JH, Asplin JR 1992 The pathogenesis and treatment of kidney stones. N Engl J Med 327:1141–1152. 7. Coe FL 1977 Treated and untreated recurrent calcium nephrolithiasis in patients with idiopathic hypercalciuria,

23. 24.

25. 26. 27. 28.

hyperuricosuria, or no metabolic disorder. Ann Intern Med 87:404–410. McGeown MG 1960 Heredity in renal stone disease. Clin Sci 19:465–471. Resnick M, Pridgen DB, Goodman HO 1968 Genetic predisposition of calcium oxalate renal calculi. N Engl J Med 278:1313–1318. Coe FL, Parks JH, Moore ES 1979 Familial idiopathic hypercalciuria. N Engl J Med 300:337–340. Pak CYC, McGuire J, Peterson R, Britton F, Harrod MJ 1981 Familial absorptive hypercalciuria in a large kindred. J Urol 126:717–719. Mehes K, Szelid Z 1980 Autosomal dominant inheritance of hypercalciuria. Eur J Pediatr 133:239–242. Moore ES, Coe FL, McMann BJ, Favus MJ 1978 Idiopathic hypercalciuria in children: Prevalence and metabolic characteristics. J Pediatr 92:906–910. Coe FL, Favus MJ 1981 Hypercalciuric states. Miner Electrolyte Metab 5:183–200. Bushinsky DA, Favus MJ 1988 Mechanism of hypercalciuria in genetic hypercalciuric rats. Inherited defect in intestinal calcium transport. J Clin Invest 82:1585–1591. Li X-Q, Tembe V, Horwitz GM, Bushinsky DA, Favus MJ 1993 Increased intestinal vitamin D receptor in genetic hypercalciuric rats: A cause of intestinal calcium hyperabsorption. J Clin Invest 91:661–667. Broadus AE, Insogna KL, Lang R, Mallette LE, Oren DA, Gertner JM, Kliger AS, Ellison AF 1984 A consideration of the hormonal basis and phosphate leak hypothesis of absorptive hypercalciuria. J Clin Endocrinol Metab 58:161–169. Buckalew VM, Purvis ML, Shulman MG, Herndon CN, Rudman D 1974 Hereditary renal tubular acidosis. Medicine 53:229–254. Frymoyer PA, Scheinman SJ, Dunham PB, Jones DB, Hueber P, Schroeder ET 1991 X-linked recessive nephrolithiasis with renal failure. N Engl J Med 325:681–686. Low RK, Stoller ML 1997 Endoscopic mapping of renal papillae for Randall’s plaques in patients with urinary stone disease. J Urol 158:2062–2064. Randall A 1937 The origin and growth of renal calculi. Ann Surg 105:1009–1027. Evan AP, Lingeman JE, Coe FL, Parks JH, Bledsoe SB, Shao Y, Sommer AJ, Paterson RF, Kuo RL, Grynpas M 2003 Randall’s plaque of patients with nephrolithiasis begins in basement membranes of thin loops of Henle. J Clin Invest 111:607–616. Bushinsky DA 2003 Nephrolithiasis: site of the initial solid phase. J Clin Invest 111:602–605. Klugman VA, Favus MJ 1996 Intestinal absorption of calcium, magnesium, and phosphorus. In: Coe FL, Favus MJ, Pak CYC, Parks JH, Preminger GM (eds) Kidney Stones: Medical and Surgical Management, 1st Ed. Lippincott-Raven: Philadelphia, Pennsylvania, pp. 210–221. Bronner F, Pansu D, Stein WD 1986 An analysis of intestinal calcium transport across the rat intestine. Am J Physiol 250:G561–G569. Favus MJ 1985 Factors that influence absorption and secretion of calcium in the small intestine and colon. Am J Physiol 248:G147–G157. Caniggia A, Gennari C, Cesari L 1965 Intestinal absorption of 45Ca in stone-forming patients. Br Med J 1:427–429. Birge SJ, Peck WA, Berman M, Wheadon GD 1969 Study of calcium absorption in man: A kinetic analysis and physiologic model. J Clin Invest 48:1705–1713.


29. Wills MR, Zisman E, Wortsman J, Evens RG, Pak CYC, Bartter FC 1970 The measurement of intestinal calcium absorption in nephrolithiasis. Clin Sci 39:95–106. 30. Pak CYC, East DA, Sanzenbacher LJ, Delea CS, Bartter FC 1972 Gastrointestinal calcium absorption in nephrolithiasis. J Clin Endocrinol Metab 35:261–270. 31. Ehrig U, Harrison JE, Wilson DR 1974 Effect of long-term thiazide therapy on intestinal calcium absorption in patients with recurrent renal calculi. Metabolism 23:139–149. 32. Pak CYC, Ohata M, Lawrence EC, Snyder W 1974 The hypercalciurias: Causes, parathyroid functions, and diagnostic criteria. J Clin Invest 54:387–400. 33. Kaplan RA, Haussler MR, Deftos LJ, Bone H, Pak CYC 1977 The role of 1,25-dihydroxyvitamin D in the mediation of intestinal hyperabsorption of calcium in primary hyperparathyroidism and absorptive hypercalciuria. J Clin Invest 59:756–760. 34. Shen FH, Baylink DJ, Nielsen RL, Sherrard DJ, Ivey JL, Haussler MR 1977 Increased serum 1,25-dihydroxyvitamin D in idiopathic hypercalciuria. J Lab Clin Med 90:955–962. 35. Barilla DE, Tolentino R, Kaplan RA, Pak CYC 1978 Selective effects of thiazide on intestinal absorption of calcium in absorptive and renal hypercalciurias. Metabolism 27:125–131. 36. Lemann J Jr 1992 Pathogenesis of idiopathic hypercalciuria and nephrolithiasis. In: Coe FL, Favus MJ (eds) Disorders of Bone and Mineral Metabolism, 1st Ed. Raven: New York, pp. 685–706. 37. Duncombe VM, Watts RWE, Peters TJ 1984 Studies on intestinal calcium absorption in patients with idiopathic hypercalciuria. Q J Med 209:69–79. 38. Insogna KL, Broadus AE, Dreyer BE, Ellison AF, Gertner JM 1985 Elevated production rate of 1,25-dihydroxyvitamin D in patients with absorptive hypercalciuria. J Clin Endocrinol Metab 61:490–495. 39. Bataille P, Bouillon R, Fournier A, Renaud H, Gueris J, Idrissi A 1987 Increased plasma concentrations of total and free 1,25(OH)2D3 in calcium stone formers with idiopathic hypercalciuria. Contrib Nephrol 58:137–142. 40. Coe FL, Favus MJ, Crockett T, Strauss LM, Parks JH, Porat A, Gantt CL, Sherwood LM 1982 Effects of low-calcium diet on urine calcium excretion, parathyroid function, and serum 1,25(OH)2D3 levels in patients with idiopathic hypercalciuria and in normal subjects. Am J Med 72:25–32. 41. Broadus AE, Insogna KL, Lang R, Ellison AF, Dreyer BE 1984 Evidence for disordered control of 1,25-dihydroxyvitamin D production in absorptive hypercalciuria. N Engl J Med 311:73–80. 42. Gray RW, Wilz DR, Caldas AE, Lemann J Jr 1977 The importance of phosphate in regulating plasma l,25(OH)2 vitamin D levels in humans: Studies in healthy subjects, in calcium stone formers, and in patients with primary hyperparathyroidism. J Clin Endocrinol Metab 45:299–306. 43. Reed BY, Gitomer WL, Heller HJ, Hsu MC, Lemke M, Padalino P, Pak CYC 2002 Identification and characterization of a gene with base substitutions associated with the absorptive hypercalciuria phenotype and low spinal bone density. J Clin Endocrinol Metab 87:1476–1485. 44. Econs MJ, Foroud T 2002 Editorial: The genetics of absorptive hypercalciuria—A note of caution. J Clin Endocrinol Metab 87:1473–1475. 45. Edwards NA, Hodgkinson A 1965 Metabolic studies in patients with idiopathic hypercalciuria. Clin Sci 29:143–157. 46. Peacock M, Nordin BEC 1968 Tubular reabsorption of calcium in normal and hypercalciuric subjects. J Clin Pathol 21:353–358.

1353 47. Sutton RAL, Walker VR 1980 Responses to hydrochlorothiazide and acetazolamide in patients with calcium stones. N Engl J Med 302:709–713. 48. Bianchi G, Vezzoli G, Cusi D, Cova T, Elli A, Soldati L, Tripodi G, Surian M, Ottaviano E, Rigatti P 1988 Abnormal red-cell calcium pump in patients with idiopathic hypercalciuria. N Engl J Med 319:897–901. 49. Alhava EM, Juuti M, Karjalainen P 1976 Bone mineral density in patients with urolithiasis. Scand J Urol Nephrol 10:154–156. 50. Lawoyin S, Sismilich S, Browne R, Pak CYC 1979 Bone mineral content in patients with calcium urolithiasis. Metabolism 28:1250–1254. 51. Borgi L, Meschi T, Guerra A, Maninetti L, Pedrazzoni M, Macato A, Vescovi P, Novarini A 1991 Vertebral mineral content in diet-dependent and diet-independent hypercalciuria. J Urol 146:1334–1338. 52. Bataille P, Achard JM, Fournier A, Boudailliez B, Westeel PF, Laval Jeantet MAL, Bouillon R, Sebert JL 1991 Diet, vitamin D, and vertebral mineral density in hypercalciuric calcium stone formers. Kidney Int 39:1193–1205. 53. Pietschmann F, Breslau NA, Pak CYC 1992 Reduced vertebral bone density in hypercalciuric nephrolithiasis. J Bone Miner Res 7:1383–1388. 54. Barkin J, Wilson DR, Manuel MA, Arnold B, Murray T, Harrison J 1985 Bone mineral content in idiopathic calcium nephrolithiasis. Miner Electrolyte Metab 11:19–24. 55. Liberman UA, Sperling O, Atsmon A, Frank M, Modan M, de-Vries A 1968 Metabolic and calcium kinetic studies in idiopathic hypercalciuria. J Clin Invest 47:2580–2590. 56. Malluche HH, Tschoepe W, Ritz E, Meyer-Sabelle W, Massry SG 1980 Abnormal bone histology in idiopathic hypercalciuria. J Clin Endocrinol Metab 50:654–658. 57. Steiniche T, Mosekilde L, Christensen MS, Melsen F 1989 A histomorphometric determination of iliac bone remodeling in patients with recurrent renal stone formation and idiopathic hypercalciuria. APMIS 97:309–316. 58. Sutton RAL, Walker VR 1986 Bone resorption and hypercalciuria in calcium stone formers. Metabolism 35:465–488. 59. Urivetzky M, Anna PS, Smith AD 1988 Plasma osteocalcin levels in stone disease. A potential aid in the differential diagnosis of calcium nephrolithiasis. J Urol 139:12–14. 60. Asplin JR, Bauer KA, Kinder J, Muller G, Coe BJ, Parks JH, Coe FL 2003 Bone mineral density and urine calcium excretion among subjects with and without nephrolithiasis. Kidney Internat 63:662–669. 61. Melton LJ III, Crowson CS, Khosla S, Wilson DM, O’Fallon WM 1998 Fracture risk among patients with urolithiasis: A population-based cohort study. Kidney Int 53: 459–464. 62. Pak CYC, Kaplan R, Bone H, Townsend J, Waters O 1975 A simple test for the diagnosis of absorptive, resorptive, and renal hypercalciurias. N Engl J Med 292:497–500. 63. Van Den Berg CJ, Kumar R, Wilson DM, Heath III H, Smith LH 1980 Orthophosphate therapy decreases urinary calcium excretion and serum 1,25-dihydroxyvitamin D concentrations in idiopathic hypercalciuria. J Clin Endocrinol Metab 51:998–1001. 64. Breslau NA, Preminger GM, Adams BV, Otey J, Pak CYC 1992 Use of ketoconazole to probe the pathogenetic importance of 1,25-dihydroxyvitamin D in absorptive hypercalciuria. J Clin Endocrinol Metab 75:1446–1452. 65. Pak CYC, Britton F, Peterson R, Ward D, Northcutt C, Breslau NA, McGuire J, Sakahee K, Bush S, Nicar M, Norman DA,

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83. Krieger NS, Stathopoulos VM, Bushinsky DA 1996 Increased sensitivity to 1,25(OH)2D3 in bone from genetic hypercalciuric rats. Am J Physiol 271:C130–C135. 84. Kirn M, Sessler NE, Tembe V, Favus MJ, Bushinsky DA 1993 Response of genetic hypercalciuric rats to a low calcium diet. Kidney Int 43:189–196. 85. Parks JH, Coe FL 1996 Pathogenesis and treatment of calcium stones. Semin Nephrol 16:398–411. 86. Wilson DR, Strauss AL, Manuel MA 1984 Comparison of medical treatments for the prevention of recurrent calcium nephrolithiasis. Urol Res 12:39–40. 87. Edwards BR, Baer PG, Sutton RA, Dirks JH 1973 Micropuncture study of diuretic effects on sodium and calcium reabsorption in the dog nephron. J Clin Invest 52:2418–2427. 88. Costanzo LS, Windhager EE 1978 Calcium and sodium transport by the distal convoluted tubule of the rat. Am J Physiol 235:F492–F506. 89. Zerwekh JE, Pak CYC 1980 Selective effects of thiazide therapy on serum l-alpha,25-dihydroxyvitamin D and intestinal calcium absorption in renal and absorptive hypercalciurias. Metabolism 29:13–17. 90. Coe FL, Parks JP, Bushinsky DA, Langman CB, Favus MJ 1988 Chlorthalidone promotes mineral retention in patients with idiopathic hypercalciuria. Kidney Int 33:1140–1146. 91. Wasnich RD, Benfante RJ, Yano K, Heilbrun L, Vogel JM 1983 Thiazide effect on the mineral content of bone. N Engl J Med 309:344–347. 92. LaCroix AZ, Wienpahl J, White LR, Wallace RB, Scherr PA, George LK 1990 Thiazide diuretic agents and the incidence of hip fracture. N Engl J Med 322:286–290. 93. Asplin JR, Favus MJ, Coe FL 1996 Nephrolithiasis. In: Brenner BR (ed) The Kidney, 5th Ed. Saunders: Philadelphia, Pennsylvania, pp. 1893–1935. 94. Ohkawa M, Tokunga S, Nakashima T, Orito M, Hisazumi H 1992 Thiazide treatment for calcium nephrolithiasis in patients with idiopathic hypercalciuria. Br J Urol 69:571–576. 95. Cheskis B, Lemon BD, Uskokovic M, Lomedico PT, Freedman LP 1995 Vitamin D3 retinoid X receptor dimerization, DNA binding, and transactivation are differentially affected by analogs of 1,25-dihydroxyvitamin D3. Mol Endocrinol 9:1814–1824. 96. Skowronski RJ, Peehl DM, Feldman D 1995 Actions of vitamin D3, analogs on human prostate cancer cell lines: Comparison with 1,25-dihydroxyvitamin D3. Endocrinology 136:20–26. 97. Fleet JC, Bradley J, Reddy GS, Ray R, Wood RJ 1996 1 alpha,25-(OH)2 vitamin D analogs with minimal in vivo calcemic activity can stimulate significant transepithelial calcium transport and mRNA expression in vitro. Arch Biochem Biophys 329:228–234. 98. Naveh-Many T, Silver J 1993 Effects of calcitriol, 22-oxacalcitriol, and calcipotriol on serum calcium and parathyroid hormone gene expression. Endocrinology 133:2724–2728. 99. Brown AJ, Finch J, Grieff M, Ritter C, Kubodera N, Nishii Y, Slatopolsky E 1993 The mechanism for the disparate actions of calcitriol and 22-oxacaltriol in the intestine. Endocrinology 133:1158–1164.

CHAPTER 78

Hypercalcemia Due to Vitamin D Toxicity MISHAELA R. RUBIN AND SUSAN THYS-JACOBS Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, New York

FREDRIECH K. W. CHAN Department of Medicine, Queen Elizabeth Hospital, Hong Kong

LILIA M. C. KOBERLE Health Sciences Department, Federal University, Sao Carlos, Brazil

JOHN P. BILEZIKIAN Department of Medicine and Department of Pharmacology, College of Physicians and Surgeons, Columbia University, New York, New York

I. II. III. IV. V.

Introduction Forms of Exogenous Vitamin D Toxicity Forms of Endogenous Vitamin D Toxicity Mechanisms of Vitamin D Toxicity Clinical Manifestations

I. INTRODUCTION Vitamin D toxicity is not a common cause of hypercalcemia. In the differential diagnosis of hypercalcemia, it is often buried amid a long list of other more and less common causes (Table I). Among the more common causes, primary hyperparathyroidism and hypercalcemia of malignancy are the principal etiologies. Together, primary hyperparathyroidism and hypercalcemia of malignancy constitute the overwhelming majority of causes of hypercalcemia. They are in fact so common that the practical issue in the diagnosis of a hypercalcemic individual is to distinguish between these two etiologies first and not to consider other etiologies until these two have been ruled out. Patients with primary hyperparathyroidism tend to be asymptomatic, whereas patients with hypercalcemia of malignancy tend to be ill. The diagnosis of primary hyperparathyroidism is established by an elevated concentration of parathyroid hormone (PTH), an association that is made in over 90% of patients with primary hyperparathyroidism. In contrast, patients with hypercalcemia of malignancy, including those whose hypercalcemia is due to the elaboration of parathyroid hormone-related protein (PTHrP), show levels of PTH that are typically suppressed. If the PTH level is suppressed, the diagnosis of primary hyperparathyroidism is ruled out. The diagnosis of malignancy, however, is VITAMIN D, 2ND EDITION FELDMAN, PIKE, AND GLORIEUX

VI. VII. VIII. IX.

Diagnosis of Vitamin D Toxicity Treatment of Vitamin D Toxicity Evidence for Benefits of Higher Vitamin D Levels Summary and Conclusions References

not necessarily ruled in. Certainly, if a malignancy is detected that is classically associated with hypercalcemia, such as squamous cell carcinoma of the lung, the etiology becomes clear. However, the longer list of other causes of hypercalcemia is also associated, with rare exceptions, with reduced levels of PTH. This situation, namely, elevated serum calcium concentration with reduced or undetectable levels of PTH, is seen in the various forms of vitamin D toxicity. If primary hyperparathyroidism is ruled out and malignancy is not apparent, the likelihood of vitamin D toxicity looms as an important possible etiology of hypercalcemia. In that long list of other causes, vitamin D toxicity now becomes a major diagnostic consideration (Table I). This chapter reviews the various forms of vitamin D toxicity, mechanisms of hypercalcemia due to vitamin D toxicity, clinical manifestations, diagnosis, and management.

II. FORMS OF EXOGENOUS VITAMIN D TOXICITY Vitamin D toxicity can be life threatening and associated with high morbidity, if not identified quickly. Hypervitaminosis D with hypercalcemia may be secondary to excessive intake of parent vitamin D, its metabolites 25-hydroxyvitamin D (25OHD), 1,25dihydroxyvitamin D [1,25(OH)2D], or vitamin D Copyright © 2005, Elsevier, Inc. All rights reserved.

1356 TABLE I Differential Diagnosis of Hypercalcemia Primary hyperparathyroidism Sporadic (adenoma, hyperplasia, or carcinoma) Familial Isolated Cystic Multiple endocrine neoplasia type I or II Malignancy Parathyroid hormone-related protein Excess production of 1,25-dihydroxyvitamin D Other factors (cytokines, growth factors) Disorders of vitamin D Exogenous vitamin D toxicity—parent D compound, 25OHD,1,25(OH)2D Endogenous production of 25-hydroxyvitamin D (Williams syndrome) Endogenous production of 1,25-dihydroxyvitamin D Granulomatous diseases a. Sarcoidosis b. Tuberculosis c. Histoplasmosis d. Coccidioidomycosis e. Leprosy f. Others Lymphoma Nonparathyroid endocrine disorders Thyrotoxicosis Pheochromocytoma Acute adrenal insufficiency Vasoactive intestinal polypeptide hormone-producing tumor (VIPoma) Medications Thiazide diuretics Lithium Estrogens/antiestrogens, testosterone in breast cancer Milk-alkali syndrome Vitamin A toxicity Familial hypocalciuric hypercalcemia Immobilization Parenteral nutrition Aluminum excess Acute and chronic renal disease

analogs; to increased production of 25OHD or 1,25(OH)2D from exogenous substrate; and even to topical applications of potent vitamin D analogs.

A. Vitamin D and 25-Hydroxyvitamin D Toxicity The most common etiology of vitamin D toxicity is inadvertent or improper oral use of pharmaceutical preparations. Excessive ingestion of vitamin D (usually greater than 10,000 IU daily) can cause vitamin D intoxication that is recognized by markedly elevated levels of 25OHD (usually >150 ng/ml) in association with levels of 1,25(OH)2D that are only slightly

MISHAELA R. RUBIN

ET AL .

elevated. Hyperphosphatemia typically accompanies the hypercalcemia [1–3]. The hyperphosphatemia can be a clue to the etiology of the hypercalcemia as due to vitamin D toxicity. The usual setting of vitamin D toxicity is in its use as a therapy for the hypophosphatemic disorders: hypoparathyroidism, pseudohypoparathyroidism, osteomalacia, renal failure, or osteoporosis. Ingestion of excessive quantities of 25OHD, 1-alpha-hydroxyvitamin D, 1,25(OH)2D, dihydrotachysterol, or exuberant use of the topical calcipotriene (Dovonex) for psoriasis can cause vitamin D intoxication [4]. Health conscious adults have been reported to ingest large doses of megavitamins from over the counter supplements, in amounts that may exceed 2 million IU of vitamin D daily [5]. Cancer patients, in particular, have been observed to consume excess nutritional supplements such as calcium, vitamin D, and shark cartilage [6]. Excessive sunlight exposure can raise serum concentrations of 25OHD to as high as 79 ng/ml (normal range 9–52 ng/ml), but there is no evidence that sunlight exposure alone can result in vitamin D toxicity and hypercalcemia in normal individuals [7]. Hypercalcemia associated with granulomatous diseases, such as sarcoidosis, can be worsened by excessive sunlight exposure. Natural foods, in general, other than fatty fish, eggs, milk, and liver do not contain much vitamin D. Hypervitaminosis D has been associated with drinking milk when erroneously fortified with massive concentrations of vitamin D. One investigation of eight patients manifesting symptoms of nausea, vomiting, weight loss, hyperirritability, or failure-to-thrive revealed markedly elevated mean concentrations of 25OHD of 293 ± 174 ng/ml (nl: 9–52 ng/ml) [3]. Analysis of the milk production facility at the local dairy revealed excessive vitamin D fortification of milk with up to 245,840 IU per liter (232,565 IU of vitamin D3 per quart). Usual fortification of milk in the United States is 400 IU per quart. Milk is not fortified with vitamin D in other parts of the world. Generally, milk is the only dairy product that is fortified with vitamin D in the United States. In addition to milk, vitamin D fortification of natural foods includes certain breakfast cereals, pasta, baked goods, fats, and recently orange juice oils [8]. There is no documentation that excessive ingestion of any of these other fortified foods has ever resulted in vitamin D toxicity. However, industrial contamination of table sugar with vitamin D3 and consequent severe vitamin D toxicity (25OHD 1555 nmol/L, nl: 20–80 nmol/l) has been reported [9]. Vitamin D2 and vitamin D3, although used interchangeably in the treatment of metabolic bone diseases, may differ in toxic potential at higher doses. In general, vitamin D3 appears to be somewhat more toxic than D2. Investigations in rats, sheep, pigs, horses, and primates support differences in metabolic clearance

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CHAPTER 78 Hypercalcemia Due to Vitamin D Toxicity

rates and in toxicity between the two vitamin D compounds [10]. In horses, vitamin D2 has a lower toxicity compared to vitamin D3 [11]. Massive doses of vitamin D3 administered to Old World primates can cause toxicity and death, whereas equivalent doses of vitamin D2 are better tolerated [12]. In human subjects, it has been shown that vitamin D3 increases 25OHD levels 1.7 times more than the equivalent dose of vitamin D2 [13]. The current, officially recommended dietary allowance (RDA) for vitamin D is 400 IU per day, but many authoritative bodies are calling for increases in the requirements to 600 units in individuals over the age of 70 [14]. Although this chapter concerns itself with vitamin D toxicity, the reason for the trend to increase recommendations for vitamin D intake is the large numbers of free-living adults who are being shown worldwide to have vitamin D deficiency (see Chapters 61–62) [15,16]. The smallest dose of parent vitamin D in adults that can produce toxicity and hypercalcemia is not known, but is clearly much higher than the RDA [17]. The threshold for vitamin D toxicity was evaluated in a study in which 61 subjects were randomized to 1000 or 4000 IU vitamin D3 daily for 2–5 months. Increases in 25OHD were greater in the higher dose than lower dose group (96.4 ± 14.6 vs. 68.7 ± 16.9 nmol/L), but remained within the physiologic range, leading the authors to conclude that as much as 4000 IU daily was a safe dose [18]. On the other hand, in infants, daily dosages of 2000 IU or less have been associated with hypercalcemia and nephrocalcinosis [19]. Intermittent oral dosages of 15 mg or 600,000 IU to infants to prevent vitamin deficiency have been shown to be excessive during the first year of life, resulting in transient hypercalcemia and vitamin D overload [20,21,22]. Lower amounts of 5 mg (200,000 IU) every 6 months or 2.5 mg (100,000 IU) every 3 months appear to be safer and to provide better protection in high risk infants. In adults, doses of greater than 40,000–60,000 IU per day, as commonly used in the treatment of hypoparathyroidism, can be associated with significant toxicity. Individuals manifest wide variations both in their response to hypercalcemic doses of vitamin D and in the duration of the effect. This variation in individual responsiveness might reflect differences in intestinal absorption and vitamin D metabolism, in the concentration of free vitamin D metabolites, in the rate of degradation of the metabolites and conversion to inactive metabolites, and in the capacity of storage sites for 25OHD [17]. Factors that enhance susceptibility to vitamin D toxicity and hypercalcemia include increased dietary calcium intake, reduced renal function, co-administration of vitamin A, and granulomatous disorders such as sarcoidosis that render subjects more sensitive to vitamin D (see Chapter 79) [2]. Hypercalciuria in hypervitaminosis D usually presents much earlier than

hypercalcemia, but it is easily missed for the obvious reason that hypercalciuria is not routinely measured in the absence of renal symptomatology.

B. 1,25-Dihydroxyvitamin D Toxicity The greater potency of 1,25(OH)2D3 and its direct actions on target tissues have resulted in its increased use for a variety of metabolic bone diseases [23]. Its ability to inhibit PTH synthesis and secretion has also made 1,25(OH)2D3 and its analogs useful agents in patients with renal osteodystrophy and secondary hyperparathyroidism. Most recently, 1,25(OH)2D3 has been found to inhibit the growth of human cancer cells in vitro [24] (see below, and Section VIII of this book). As 1,25(OH)2D3 is increasingly recognized for its antiproliferative, prodifferentiating, and immunomodulatory actions, its potential therapeutic use is expanding [25–27]. Thus, considerable attention has focused on possible toxic effects of 1,25(OH)2D3 not usually associated with the parent vitamin D compound. The incidence of hypercalcemia and hypercalciuria with 1,25(OH)2D3 use has been reported as very high, with one review citing complications in two-thirds of treated patients [28]. The mechanisms of the hypercalcemia are increased intestinal absorption and potentiation of osteoclastic activity. Dosages of 1,25(OH)2D3 above 0.75 µg/day have been associated with toxicity, whereas dosages at or below 0.5 µg/day rarely result in toxicity. One investigation showed that over 90% of patients on doses of 1,25(OH)2D3 between 1.0 and 2.0 µg/day became hypercalcemic, and all had hypercalciuria when calcium intake was set at 1000 mg per day [29]. Accelerated deterioration of renal function was recorded in a number of reports in patients with renal insufficiency receiving 1,25(OH)2D3 therapy [30]. Compared to oral therapy, intravenous administration of 1,25(OH)2D3 to renal dialysis patients induces hypercalcemia less frequently, with a smaller increment in the serum calcium concentration and a more effective reduction of PTH levels [31]. Other studies, however, suggest that intermittent oral pulse administration of 1,25(OH)2D3 may be effective, though not as effective as intravenous 1,25(OH)2D3, in suppressing PTH in uremic patients with secondary hyperparathyroidism [32–34] (see Chapter 76 for further discussion).

C. Toxicity Due to Synthetic Analogs In one investigation, oral pulse therapy with 1α-hydroxyvitamin D3 (1αOHD3) resulted in a rapid control of secondary hyperparathyroidism without causing hypercalcemia or hyperphosphatemia [35].

1358 However, 1α-OHD3 may harbor potential calcemic effects similar to 1,25(OH)2D3 in the treatment of renal osteodystrophy. Crocker et al. [36] investigated the comparative toxicity of vitamin D, 1α-OHD3, and 1,25(OH)2D3 in weanling male mice at three different doses over a four-week period. 1α-OHD3 appeared to be more toxic in the high dose group only, with significantly higher serum calcium levels, higher urinary calcium excretion, and severe nephrocalcinosis [36]. 1α-OHD3 has been described as less potent than 1,25(OH)2D3 at low doses but equipotent at doses greater than 2.0 µg/day. At the higher doses, there is a delayed onset of action and a prolonged half-life, suggesting a potential for cumulative toxicity in renal insufficiency [37,38]. The potential for hypercalcemia, hypercalciuria, and soft tissue calcifications limits the clinical usefulness of 1α-OHD3. Mortensen and colleagues compared the toxicity of both 1α-OHD3 and 1,25(OH)2D3 in rats fed standard or low calcium diets. High doses of either compound resulted in severe hypercalcemia, with retarded growth, nephrosis, and structural bone changes in the rats fed the standard diet. On the low calcium diet, however, slight hypercalcemia occurred, but without growth retardation or bone changes. There was minimal effect on the kidney. Calcium restriction again proved effective in protecting the animals against the toxic effects of the vitamin D analogs. Animals fed the low calcium diet tolerated 1α-OHD3 at dose levels up to 10 times higher than rats on the standard diets [39]. In human subjects, 1α-OHD3 causes toxic effects at doses above 1.0 µg/day, but doses of 0.5 to 1.0 µg/day appear to be safe. Because of the relatively narrow therapeutic window of vitamin D3 compounds, a synthetic analog of vitamin D2, 1α-OHD2 (doxercalciferol) was developed with the concept that the window of therapeutic efficacy to toxicity would be wider. In postmenopausal osteopenic women, doses of doxercalciferol ranging from 1.0 to 5.0 µg/day were administered to 15 subjects. There was no evidence of vitamin D toxicity manifesting as either hypercalciuria or hypercalcemia, whereas significant therapeutic effects on osteoblastic activity were demonstrated [40]. Similar to the 1α-OHD3, doxercalciferol requires obligatory hepatic 25-hydroxylation for activation. However, doxercalciferol is able to activate its catabolic pathway via hepatic 24-hydroxylation with a lower potential for toxicity [41]. These investigations on synthetic analogs seem to confirm earlier reports that vitamin D2 compounds, in general, are as efficacious and somewhat better tolerated than D3 compounds. Because of our understanding of the nonclassic target tissue effects of vitamin D in the modulation of hormones and cytokines, and in the regulation of cellular differentiation and proliferation, newer clinical uses have been developed (see Section IX of this book).

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The clinical applications of these newer properties of vitamin D, however, have also been tempered by the potential for complications, such as hypercalcemia and hypercalciuria, prompting the development of other analogs to distinguish even better calcemic from antiproliferative effects [42] (see also Section VIII of this book). Depending on the chemical modification of the basic structure of vitamin D, some analogs do demonstrate reduced calcemic activity, but others have been developed with increased calcemic activity owing to enhanced intestinal calcium absorption and bone mineral mobilization. Fluorination of C-24, C-26, or C-27 apparently results in markedly increased calcemic activity resulting from reduced enzymatic degradation of the side chain. Calcemic potency of 1,25(OH)2D3 and its analogs can be also enhanced at least two- to fivefold by epimerization at the C-20 site [43]. The vitamin D analogs in use for secondary hyperparathyroidism in the United States include doxercalciferol, 22-oxacalcitriol (1,25-dihydroxy22-oxavitamin D3) and paricalcitol (19-nor-1,25dihydroxyvitamin D2). Each analog retains suppressive action on PTH and parathyroid gland growth, but has less calcemic and phosphatemic activity than calcitriol. It is unclear how the analogs compare to each other, although in rats, paricalcitol is less calcemic and phosphatemic than doxercalciferol [44]. Overall, the effect of vitamin D analogs to minimize the calcium-phosphate product might reduce vascular calcification [45] and mortality in the renal failure population [46]. Of additional potential importance is the decreased likelihood of low bone turnover, or adynamic bone disease, with the use of these agents [47–48]. The mechanism for the differential actions of vitamin D analogs is not completely understood. Oxacalcitriol, for example, has a low affinity for vitamin D–binding protein, so more of the drug circulates in the free form, allowing it to be more rapidly metabolized than calcitriol [49]. This leads to a shorter half-life, which could explain the small and transient stimulation of intestinal calcium absorption. It does not, however, seem to account for the prolonged inhibition of PTH release (see Chapter 86). Other vitamin D analogs, such as topical calcipotriol (MC903), have proved very effective in the treatment of psoriasis (see Chapter 101). Because of its low absorption rate and rapid degradation, calcipotriol is believed to have negligible effects on systemic calcium homeostasis when administered topically. However, isolated cases of hypercalcemia and hypercalciuria have been reported, even in patients taking recommended doses [50]. In one investigation, Bourke and colleagues noted suppression of serum PTH concentrations in all patients within two weeks of treatment with calcipotriol. Mean serum and urine calcium


levels increased during treatment and fell following withdrawal [51]. The authors concluded that although this particular synthetic analog alters serum and urinary calcium with a dose-dependent effect on systemic calcium homeostasis, it is well tolerated and effective for mild to moderate chronic plaque psoriasis. However, it is potentially hazardous in extensive, unstable, exfoliative disease [52].

III. FORMS OF ENDOGENOUS VITAMIN D TOXICITY A. Endogenous Production of 25-Hydroxyvitamin D Hypervitaminosis D with hypercalcemia is rarely due to endogenous dysregulation of vitamin D metabolites as seen in Williams syndrome [53]. Williams syndrome, an idiopathic infantile form of hypercalcemia, is associated with late psychomotor development, selective mental deficiency, and supravalvular aortic stenosis [54]. The hypercalcemia has been reported to range widely from 12 to 19 mg/dl but usually subsides by 4 years of age. One report suggests an exaggerated production of 25OHD with small doses of vitamin D as a possible etiology of the hypervitaminosis D [53].

B. Production of 1,25-Dihydroxyvitamin D 1. GRANULOMATOUS DISEASES

In contrast to the megadosages of vitamin D that are usually required to produce vitamin D toxicity, patients with granulomatous diseases can develop hypercalcemia rather easily without excessive intake of exogenous vitamin D. They are said to be hypersensitive to vitamin D. The etiology of the vitamin D toxicity in this syndrome is due to poorly regulated extrarenal synthesis of 1,25(OH)2D by the granulomatous tissue itself (as described in detail in Chapter 79). In contrast to the various presentations of vitamin D toxicity described earlier, the responsible metabolite in granulomatous disease is quite different. In the case of vitamin D toxicity due to overdosage of vitamin D or 25OHD, 25OHD is the active metabolite; renal production of 1,25(OH)2D in this setting is highly regulated and not excessively high. In granulomatous tissue, however, 1,25(OH)2D formation is not subject to control by any recognized regulators, such as PTH, phosphorus, or calcium. Thus, this syndrome is due to ectopic production of 1,25(OH)2D by the granulomatous tissue itself. The mechanisms by which hypercalcemia occurs, however, are similar to all other vitamin D toxic

1359 states, namely, increased intestinal calcium absorption and enhanced osteoclastic bone resorption [55,56]. Many studies have led to greater understanding of the pathophysiology and immunological features associated with this syndrome. a. Sarcoidosis Abnormalities in calcium metabolism have long been noted in patients with sarcoidosis [57]. Sarcoidosis is also the most common granulomatous disease associated with hypercalcemia. Approximately 10% of patients with sarcoidosis will develop hypercalcemia, and as many as 50% will experience hypercalciuria at some time during the course of the disease [58]. Hypercalciuria is invariably present when patients develop hypercalcemia. In the 1950s, studies had already revealed similarities between hypercalcemia of sarcoidosis and the hypercalcemia of vitamin D toxicity, namely, increased intestinal absorption of calcium, hypercalciuria, and therapeutic efficacy of glucocorticoids [59,60]. The major distinguishing feature was in the amount of vitamin D associated with the hypercalcemia and/or hypercalciuria. Seasonal variation of the serum calcium level in sarcoidosis was correlated with availability of sunlight as a source of vitamin D [61]. In the late 1970s, two independent groups showed that the vitamin D-like principle that appeared to be responsible in sarcoidosis was, in fact, the active metabolite of vitamin D, 1,25(OH)2D3 [56,62]. Ectopic production of 1,25(OH)2D3 was confirmed by demonstrating high circulating concentrations of 1,25(OH)2D3 in anephric patients with sarcoidosis on hemodialysis during hypercalcemic episodes [63,64]. This observation showed unequivocally that the kidney, usually the sole source of 1,25(OH)2D3 in nonpregnant individuals, could not be the source of 1,25(OH)2D3 in these patients. The serum calcium and 1,25(OH)2D3 levels were positively correlated with indices of disease activity [65–67], namely, the extent of granuloma formation and the angiotensin-converting enzyme level. It was subsequently shown that the granulomatous tissue was, in fact, the site of 1,25(OH)2D3 production. The lα-hydroxylase enzyme responsible for formation of 1,25(OH)2D3 was present in lymph node homogenates [68]. Moreover, pulmonary alveolar macrophages [69] could be shown to catalyze the formation of an 3H-labeled 25OHD3 metabolite. This metabolite was definitively identified as 1,25(OH)2D3 by high-performance liquid chromatography (HPLC), by the chick intestinal receptor assay for 1,25(OH)2D3, by UV spectroscopy, and by mass spectrometry [70]. The production of mRNA for 1α-hydroxyalse is markedly increased in alveolar macrophages isolated from hypercalcemic patients with sarcoid [71]. Importantly, control of the macrophage 1α-hydroxylase enzyme differs

1360 from that of the renal 1α-hydroxylase. The renal 1α-hydroxylase is regulated at the level of transcription by calciotropic hormones, and is exquisitely autoregulated by 1,25(OH)2D3 itself [72]. In contrast, the macrophage 1α-hydroxylase mRNA expression is potently stimulated by inflammatory agents, such as γ-interferon [73], and shows no feedback control in response to 1,25(OH)2D3 [74]. Communication between signaling pathways of γ-interferon and the vitamin D receptor has recently been reported [75]. These mechanisms account for the uncontrolled synthesis of 1,25(OH)2D3 and the characteristic finding of increased sensitivity to vitamin D in these patients [76], so that patients even without major increases in 1,25(OH)2D3 can become hypercalemic. Conversely, abnormal 1,25(OH)2D3 metabolism has been described in some patients with sarcoidosis who are normocalciuric and normocalcemic [77]. Another property of the macrophage 1α-hydroxylase enzyme is that it is inhibited in a dose-dependent fashion by dexamethasone and chloroquine that do not influence the renal 1α-hydroxylase enzyme that catalyzes synthesis of 1,25(OH)2D3 [78]. These in vitro observations have direct clinical relevance. There are several mechanisms by which calcium metabolism is disturbed in sarcoidosis [79]. First, 1,25(OH)2D3 causes hypercalcemia, in part, by stimulating intestinal calcium absorption. A low calcium diet [80,81], alone or in association with cellulose phosphate [82], was found to normalize the calcium level in some patients with sarcoidosis. Second, 1,25(OH)2D3 directly stimulates osteoclastic-mediated bone resorption; skeletal granulomas are not required for this effect [83–85]. The increased flux of calcium into the extracellular space by these gastrointestinal and skeletal mechanisms, aided by suppression of PTH [62–64], leads to hypercalciuria. Chronic hypercalciuria favors nephrocalcinosis and renal stone formation [86]. When the kidneys are unable to excrete the calcium presented to them, because of either declining renal function, enhanced bone resorption, a sudden influx of dietary calcium, dehydration, or any combination of these events, hypercalcemia ensues [87]. Granulomatous production of PTHrP may also play a role in abnormal calcium metabolism [88], where TNFα and interleukin-6, produced by macrophages, increase PTHrP gene expression. PTHrP was reported in one series to be present in 85% of biopsies of granulomatous tissue from patients with sarcoidosis [88]. b. Tuberculosis Longitudinal studies from the United States [89] and India [90] suggested that 16 to 28% of patients with tuberculosis develop hypercalcemia. However, in these early studies, vitamin D supplements were employed, increasing the risk and severity

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of hypercalcemia. A similar study from Greece [91] reported a figure as high as 48% when serum calcium was corrected to a normal albumin level. Other studies from the United Kingdom [92], Belgium [93], Hong Kong [94], and Malaysia [95] have shown a much lower prevalence of hypercalcemia, in the range of 0 to 2.3%. It is likely that hypercalcemia is not as common in tuberculosis as was previously thought [96]. This discrepancy might be attributable to regional differences in calcium and vitamin D intake, which can unmask hypercalcemia [97], along with increased sun exposure. Reports of high circulating levels of 1,25(OH)2D3 in three anephric patients with tuberculosis support an extrarenal source of the active vitamin D metabolite [98,99]. Positive correlation of the albumin-adjusted calcium level with the radiographic extent of the disease has been shown [94]. Hypercalcemia in tuberculosis may occur weeks to months after starting antituberculosis chemotherapy [89,90]. Thus, the hypercalcemia is not related to the presence of viable acid-fast bacilli, but rather to the granulomatous process and associated reactions. As with sarcoidosis, hypercalcemia in tuberculosis can be controlled by administration of glucocorticoids [100]. In patients with tuberculous pleuritis, the mean free 1,25(OH)2D3 concentration in pleural fluid was selectively concentrated by 5.3-fold over that in serum [101]. Positive correlation between the concentrations of substrate (25OHD3) and product [1,25(OH)2D3] in pleural fluid supported the idea that 1,25(OH)2D3 was produced locally by activated inflammatory cells in or adjacent to the pleural space. The pleural fluid was found to have high concentrations of γ-interferon, a cytokine known to stimulate activated macrophages in vitro to synthesize 1,25(OH)2D3 [102]. Cells obtained from bronchoalveolar lavage in patients with tuberculosis were also found to synthesize 1,25(OH)2D3 in vitro. An important source of the active vitamin D metabolite appears to be the CD8 + T lymphocytes at the granulomatous sites [103]. If one wonders etiologically about the production of 1,25(OH)2D3 under these circumstances, the immunomodulatory functions of 1,25(OH)2D3 acting as a beneficial local paracrine factor could be pertinent (see Chapter 79). Viewed in this context, hypercalcemia occurs when 1,25(OH)2D3 is produced in such quantities that it gains entry into the circulation. Hypercalcemia in tuberculosis is usually mild and asymptomatic. Besides glucocorticoids, ketoconazole administration has been associated with a rapid decline in 1,25(OH)2D3 and normalization of serum calcium levels [104]. Long-term antituberculosis therapy with isoniazid and rifampin can also be effective in treating the hypercalcemia by controlling the disease.


c. Other Granulomatous Diseases Besides the more detailed studies of hypercalcemia in tuberculosis, hypercalcemia has also been reported in other infectious diseases, including leprosy [105], coccidioidomycosis [106], histoplasmosis [107], candidiasis [108], catscratch disease [109], and pneumocystis carinii pneumonia [110]. Noninfectious associations, aside from sarcoidosis, have been reported with eosinophilic granuloma [111], berylliosis [112], silicone-induced granuloma [113], paraffin-induced granulomatosis [114], Wegener’s granulomatosis [115], Langerhans’ cell granulomatosis [116], Crohn’s disease [117], and infantile fat necrosis [118]. The mechanism of increased production of the active vitamin D metabolite is believed to be shared by all of these granulomatous disorders. Spontaneous idiopathic excess production of calcitriol in the absence of granulomatous disease has also been reported in patients with elevated angiotensin converting enzyme levels [119], presumably with increased calcitriol production by macrophages. A possible role for 1,25(OH)2D3 in these granulomatous disorders as noted above includes immunomodulatory features, which are discussed in Chapters 36 and 98. 2. LYMPHOMA

Hypercalcemia has been reported to occur in 5% [120] and 15% [121] of patients with Hodgkin’s disease and non-Hodgkin’s lymphoma (NHL), respectively. Up to 80% of patients with human T-cell leukemia virus type 1 (HTLV-l)-associated adult T-cell lymphoma/ leukemia (ATLL) will develop hypercalcemia [122]. As is the case with other malignancies, hypercalcemia is a poor prognostic feature in lymphoma [123], adding substantially to morbidity and mortality. The humoral mediators of hypercalcemia in lymphoma are multiple and heterogeneous. However, evidence has shown 1,25(OH)2D3 to be an important factor in many cases. Hodgkin’s disease is most consistently associated with 1,25(OH)2D3 when hypercalcemia develops. Since the first report of hypercalcemia complicating Hodgkin’s disease in 1956 [124], more than 60 cases have been described. In a retrospective review of the literature [125], 84% of patients had a peak serum calcium above 12 mg/dl, 74% of the patients had Ann Arbor stage III or IV disease, and 68% were symptomatic with night sweats, fever, and weight loss. Only 3 of 23 patients had radiological evidence of lytic bone lesions. In 17 hypercalcemic patients, all but one patient had an elevated 1,25(OH)2D3 level. There is no evidence to implicate parathyroid hormone-related peptide (PTHrP) as a mediator of hypercalcemia in Hodgkin’s disease. Two patients with Hodgkin’s disease [126,127] were reported to have intermittent hypercalcemia

1361 during two consecutive summers or on vitamin D challenge. There was a close association between hypercalcemia and the abnormally raised 1,25(OH)2D3 level, but serum 25OHD3 was within the normal range. These observations support the idea that the mechanism of the hypercalcemia in Hodgkin’s disease is similar to that of the granulomatous diseases, namely, production by the lymphomatous tissue of 1,25(OH)2D3. A number of cases of 1,25(OH)2D3-induced hypercalcemia in non-Hodgkin’s lymphoma have been described. Most patients had bulky or advanced stage disease, but no clinically or radiographically evident bone lesions. In one case, the 1,25(OH)2D3-mediated hypercalcemia was associated with transformation from a chronic lymphocytic leukemia to an aggressive high-grade non-Hodgkin’s lymphoma [128]. Data supporting extrarenal synthesis of 1,25(OH)2D3 are the presence of severe renal failure in a number of instances [129,130]; the demonstration of in vitro conversion of 25OHD3 to 1,25(OH)2D3 by excised lymph node homogenates [131]; the prompt decline of 1,25(OH)2D3 levels to normal after excision of an isolated splenic lymphoma [132] and a primary ovarian lymphoma [133]; and sensitivity to glucocorticoid suppression [130]. Five of ten patients with either AIDS or non-AIDS associated non-Hodgkin’s lymphoma and hypercalcemia had frankly elevated serum 1,25(OH)2D3 concentrations [134]. Other malignant lymphoproliferative diseases associated with 1,25(OH)2D3–mediated hypercalcemia include lymphomatoid granulomatosis [135], dysgerminoma [136], and an inflammatory myofibroblastic tumor [137]. In a prospective study by Seymour et al. [138], a control group was composed of 16 patients with hypercalcemia and multiple myeloma. Using the mean serum 1,25(OH)2D3 level of the control patients plus 3 standard deviations, the investigators defined the upper limit of expected serum 1,25(OH)2D3 during hypercalcemia as 42 pg/ml, well below the upper limit of 76 pg/ml for the normocalcemic reference range. Thus, the typical hypercalcemic patient, if represented by this cohort of patients with multiple myeloma, shows a lower range of normal for 1,25(OH)2D3 concentration. Of the 22 hypercalcemic patients with non-Hodgkin’s lymphoma, 12 (55%) had elevated serum 1,25(OH)2D3 levels. Moreover, the serum levels of corrected calcium and 1,25(OH)2D3 were strongly correlated with one another. Even in the normocalcemic group with non-Hodgkin’s lymphoma, 71% were hypercalciuric and 18% had elevated serum 1,25(OH)2D3 levels. The precise cell type responsible for the extrarenal synthesis of 1,25(OH)2D3 in lymphoma remains to be established. There are two possibilities. One is the tumor-infiltrating reactive macrophage, recognized by

1362 a “starry-sky” appearance [139] in intermediate and high-grade lymphomas, in which hypercalcemia is also most common. Alternatively, it may be that a particular clone of the malignant lymphoma cell synthesizes 1,25(OH)2D3 [140]. Recent immunohistochemical analysis of the enzyme 1α-hydroxylase in a B-cell lymphoma associated with hypercalcemia and raised circulating levels of 1,25(OH)2D3 suggests that the tumor itself is not a source of the steroid hormone [141]. Rather, macrophages adjacent to the tumor are likely to be the major site of ectopic 1,25(OH)2D3 synthesis [141]. 1,25-Dihydroxyvitamin D is only one cause of hypercalcemia in lymphoma. About half of the patients with non-Hodgkin’s lymphoma and hypercalcemia have suppressed 1,25(OH)2D3 levels. Additional circulating or local osteolytic factors are likely to be involved. Two of 22 patients in the study by Seymour et al. had elevated PTHrP levels. A few other cases of hypercalcemia in non-Hodgkin’s lymphoma associated with elevated levels of PTHrP have been reported [142–144]. Cytokines such as interleukin-1, tumor necrosis factor-α (TNFα), and transforming growth factor (TGFβ) may also play a role in the pathogenesis of lymphomaassociated hypercalcemia. Although HTLV-1-transformed lymphocytes were shown in vitro to possess the capacity to convert 25OHD3 to 1,25(OH)2D3 [145], most studies have shown reduced 1,25(OH)2D3 levels in hypercalcemia associated with HTLV-1-related adult T-cell leukemia/ lymphoma [146,147]. PTHrP is most strongly implicated as the major mediator in this syndrome [148]. PTHrP messenger RNA has been demonstrated in HTLV-1-infected T cells [149] and tumor cells from adult T-cell lymphoma/leukemia (ATLL) patients with hypercalcemia [150]. Nevertheless, there are two welldocumented instances of elevated 1,25(OH)2D3 levels in ATLL [122,130]. In the first case, a PTHrP level was not available. In the second case, concomitant elevation of 1,25(OH)2D3 and PTHrP was shown, suggesting the possibility of increased renal 1α-hydroxylase activity secondary to PTHrP. Alternatively, the tissue could be the site of both PTHrP and 1,25(OH)2D3 formation. Most patients with hypercalcemia due to classic squamous cell carcinoma have elevated PTHrP levels and either suppressed or normal 1,25(OH)2D3 levels.

IV. MECHANISMS OF VITAMIN D TOXICITY A. General Mechanisms Vitamin D toxicity may occur in patients due to any one of the three forms of vitamin D, namely, the vitamin D parent compound, 25OHD, or 1,25(OH)2D.

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Multiple factors may influence susceptibility to vitamin D toxicity and include the concentration of the vitamin D metabolite itself, vitamin D receptor (VDR) number, activity of lα-hydroxylase, the metabolic degradation pathway, and the capacity of the vitamin D–binding protein (DBP). Vitamin D2 or D3 toxicity is more difficult to manage than toxicity due to its metabolites 25OHD or 1,25(OH)2D. In part, this is due to the extensive lipid solubility of the parent compound in liver, muscle, and fat tissues and corresponding large storage capacity. As a result, the half-life of vitamin D ranges from 20 days to months. In contrast, the biological half-life of the less lipophilic compound 25OHD is shorter, approximately 15 days [151]. The biological half-life of the least lipophilic compound 1,25(OH)2D, is much shorter, approximately 15 hr [152]. In general, duration of toxicity is related to the half-life of the vitamin D compound. Thus, the hypercalcemia of parent vitamin D overdose can last for as long as 18 months, long after dosing is discontinued, because of its slow release from fat deposits. Over-dosage of 25OHD can persist for weeks also, but excessive 1,25(OH)2D toxicity is more rapidly reversed because 1,25(OH)2D is not stored in appreciable amounts in the body [66]. The toxicity of either parent vitamin D or 25OHD is due to 25OHD. In an investigation examining the concentrations of vitamin D3 and its metabolites in the rat as influenced by various intakes of vitamin D3 or 25OHD, Shepard and DeLuca found that large intakes of vitamin D3, ranging from 0.65 to 6500 nmol/day, resulted in excessive concentrations of vitamin D3 and 25OHD3 but not in 1,25(OH)2D3 (Table II) [153]. Similarly, increased dosages of 25OHD3 ranging from 0.46 to 4600 nmol/day resulted in excessive amounts of 25OHD3, but not of vitamin D3 or 1,25(OH)2D3 (Table III). In the setting of toxicity due to overadministration of 1,25(OH)2D3, the active metabolite itself is responsible for the hypercalcemia [154]. Unlike 1,25(OH)2D whose production is tightly regulated in the kidney, the production of 25OHD is not tightly controlled by the liver. The high capacity for 25-hydroxylation of vitamin D in the liver as well as poor regulation at this site allows for massive amounts of 25OHD to be generated from large amounts of vitamin D. Thus, excessive concentrations of 25OHD are typically measured in vitamin D toxicity. As would be expected, PTH levels are suppressed in this form of hypercalcemia.

B. Role of Vitamin D Receptor (VDR) in Vitamin D Toxicity Various investigations have helped to shed light on the interrelationship among vitamin D metabolites, the

1363


TABLE II Plasma Concentrations of Vitamin D3 and Metabolites in Rats Given Various Amounts of Vitamin D3a Amount (nmol/day)

Vitamin D3 (ng/ml)

25OHD3 (ng/ml)

0.65 6.5 65 650 6500

11.3 ± 6.1 110 ± 43 368 ± 121 1339 ± 329c 3108

2.3 ± 1.9 14.7 ± 8.6 74.2 ± 14.5 643 ± 93b 1111

Lactone (ng/ml) < 0.06 0.35 ± 0.12 10.3 ± 3.9 64.5 ± 19.1c 43.6

24,25(OH)2D3 (ng/ml)

25,26(OH)2D3 (ng/ml)

1,25(OH)2D3 (pg/ml)

Plasma calcium (mg/100 ml)

0.56 ± 0.13 3.98 ± 1.90 25.5 ± 5.2 73.5 ± 29.6d 86.5

< 0.2 0.20 ± 0.36 7.60 ± 2.78 16.4 ± 4.7c 8.4

80 ± 60 77 ± 64 88 ± 9 51 ± 11c 37

9.0 ± 0.1 9.4 ± 0.4 9.7 ± 0.3 12.4 ± l.06 13.8

a Rats were orally dosed daily for 14 days with indicated amounts of vitamin D . Data are means of 5 rats ± SD. Reprinted with permission from 3 Shepard RM, DeLuca HF 1980. Arch Biochem Biophys 202:43–50. b Differs from control group (0.65 nmol/day) and from group receiving 65 nmol/day at p < 0.001. c Differs from group receiving 65 nmol/day at p < 0.001. d Differs from control group (0.65 nmol/day) at p < 0.001 and from group receiving 65 nmol/day at p < 0.010.

VDR, and PTH in vitamin D toxicity. The biologically active form of vitamin D, 1,25(OH)2D, as is typical of other steroid hormones, binds to a specific intracellular receptor protein (VDR) within its target tissues. The hormone-VDR complex then triggers subsequent transcriptional events by binding to DNA elements. Regulation of cellular VDR numbers is believed to be an important mechanism by which cellular responsiveness to 1,25(OH)2D is modulated, because the biological activity of 1,25(OH)2D is proportional both to tissue VDR number and concentration of 1,25(OH)2D (see Section II of this book for a detailed discussion). Increased VDR concentrations imply enhanced tissue responsiveness to 1,25-dihydroxyvitamin D, whereas decreased receptor numbers indicate reduced tissue responsiveness. Several investigations have suggested that exogenous 1,25(OH)2D3 can lead to homologous

TABLE III Amount (nmol/day) 0.46 4.6 46 460 4600

up-regulation of VDR in vitro and in vivo, in contrast to endogenous production of 1,25(OH)2D3. In vitro and in vivo administration of 1,25(OH)2D3 to rats has been shown to increase VDR content. In vitro exposure of human skin fibroblasts and osteosarcoma cells to 1,25(OH)2D3 has been shown to result in a three- to fivefold increase in VDR number [155]. Similarly, in vivo studies have shown increased VDR with exogenous administration of 1,25(OH)2D3. Costa and Feldman administered 1500 pmol/kg of 1,25(OH)2D3 daily to rats and found a 30% increase in intestinal VDR and a threefold increase in renal VDR concentration [156]. Reinhart et al. infused rats with 250 pmol/kg of 1,25(OH)2D3 daily for six days and noted a 22% increase in VDR levels in the intestine and a 37% increase in bone [157]. Goff and colleagues infused 36 ng of 1,25(OH)2D3 to rats over seven days and found

Plasma Concentrations of Vitamin D3 and Metabolites in Rats Given Various Amounts of 25OHD3a Vitamin D3 (ng/ml) < 0.5 < 0.5 < 0.5 < 0.5 < 0.5

25OHD3 (ng/ml) 6.2 ± 2.3 56.3 ± 11.4 199 ± 24 436 ± 58 688 ± 145b

Lactone (ng/ml) 0.31 ± 0.05 3.02 ± 0.63 32.5 ± 8.5 118 ± 26 110 ± 38c

24,25(OH)2D3 (ng/ml)

25,26(OH)2D3 (ng/ml)

2.29 ± 0.54 11.7 ± 3.3 57.3 ± 19.5 170 ± 22 214 ± 117d

< 0.2 < 0.2 1.19 ± 0.49 4.02 ± 1.02 6.31 ± 1.79e

1,25(OH)2D3 (pg/ml) 187 ± 72 192 ± 65 82 ± 29 33 ± 8 22 ± 1f

Plasma calcium (mg/100 ml) 9.8 ± 0.5 9.3 ± 0.5 10.2 ± 0.4 9.7 ± 0.4 14.0 ± 0.5*

a Rats were orally dosed daily for 14 days with indicated amounts of 25OHD . Data are means of 5 rats ± SD. Reprinted with permission from Shepard RM, 3 DeLuca HF 1980 Arch Biochem Biophys 202:43–50. b Differs from control group (0.46 nmol/day) at p < 0.001 and from group receiving 460 nmol/day at p < 0.010. c Differs from control group (0.46 nmol/day) at p < 0.001. d Differs from control group (0.46 nmol/day) at p < 0.005. e Differs from group receiving 460 nmol/day at p < 0.005. f Differs from control group (0.46 nmol/day) at p < 0.001 and from group receiving 460 nmol/day at p < 0.050. *Differs from control group (0.46 nmol/day) and from group receiving 460 nmol/day at p < 0.001.

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600

Control

p < 0.01a

Vitamin-D2-treated Vitamin-D3-treated

500

p < 0.05 Unoccupied receptor (fmol/mg protein)

a 1.5-fold increase in duodenal VDR content and a three-fold increase in renal VDR content [158]. Goff et al. [158] also demonstrated that endogenously produced 1,25(OH)2D3 has a different effect than exogenous administration of 1,25(OH)2D3 on tissue VDR content. Rats fed a calcium-restricted diet resulting in “nutritional” hyperparathyroidism achieved a similar increase in endogenous 1,25(OH)2D3 concentration as rats administered exogenous 1,25(OH)2D3. However, calcium-restricted rats failed to up-regulate VDR content in the duodenum or kidney, presumably a consequence of the negative control of VDR by PTH [159]. This point has at least conceptual relevance in the case of vitamin D toxicity. Rather than downregulation occurring during hypervitaminosis D, which is a more typical regulatory and protective event to limit tissue responsiveness, exposure of cells to exogenous 1,25(OH)2D results in enhanced responsiveness by virtue of up-regulation. Such a mechanism would be of particular clinical relevance if the toxicity were due to overexposure of 1,25(OH)2D. Moreover, in this setting, the associated suppression of PTH would prevent the regulatory mechanism from being operative. Evidence suggests that in parent vitamin D toxicity, target tissues are responding to high concentrations of 25OHD, not 1,25(OH)2D. Concentrations of 1,25(OH)2D are typically only slightly increased, if at all. The hypercalcemia is due to the effects of pharmacologically high levels of 25OHD, even though in physiological settings, 25OHD has little potency. At high concentrations, 25OHD can compete for binding at VDR sites, and thereby produce biological effects similar to those of 1,25(OH)2D on intestine and bone [160]. Beckman and colleagues [161] suggested, furthermore, that hypervitaminosis D, like excessive 1,25(OH)2D, is associated with homologous up-regulation of intestinal VDR. Their investigation demonstrated that supraphysiological amounts of vitamin D2 or vitamin D3 administered to rats at doses of 25,000 IU

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400

300

200

100

0

Day 6 of treatment

FIGURE 1 Intestinal VDR in rats treated six days with 25,000 IU/ day of either vitamin D2 or vitamin D3 relative to the response in age-matched controls. aResponse in vitamin D3-treated rats significantly different from that in vitamin D2-treated rats (p < 0.05). Reprinted with permission from Beckman MJ, et al. [161].

daily for six days resulted in increasing plasma 25OHD concentrations with significant up-regulation of intestinal VDR concentration and hypercalcemia. Plasma 1,25(OH)2D levels were not altered substantially (see Table IV and Fig. 1). A comparison between hypervitaminosis D3 and D2 was also made [161]. No differences in 25OHD and plasma calcium concentrations were noted between either preparations. Concentrations of 25OHD in each case were markedly higher than the control group. The concentration of 1,25(OH)2D was observed to be only slightly greater in the vitamin D3-treated group than the vitamin D2-treated group. Because the 25OHD

TABLE IV Changes in Body Weight, Plasma Calcium, and Plasma Vitamin D Metabolites in Rats Treated for Six Days with Either 25,000 IU/day of Vitamin D2 or Vitamin D3a Group Control Vitamin D2-treated Vitamin D3-treated

Body weight (g) 251 ± 5 230 ± 17 201 ± 18*

Plasma calcium (mg/dl) 9.5 ± 0.7 11.8 ± 0.6c 12.0 ± 0.9c

25OHD (ng/ml)

1,25(OH)2D (pg/ml)

20 ± 2 466 ± 36a 506 ± 67b

112 ± 11 123 ± 12 150 ± 8c,d

represent means ± SE. Reprinted with permission from Beckman MJ et al,1990. Biochem Biophys Res Commun 169:910–915. difference at p < 0.01 of the treated groups relative to the control group. c Significant difference at p < 0.05 of the treated group relative to the control group. d Statistical significance between the D - and D -treated groups (n = 6). 2 3 a Data

b Significant

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concentrations were elevated twenty- to twenty-five-fold, whereas 1,25(OH)2D showed only minimal increases, the biochemical and clinical changes associated with parent vitamin D toxicity were attributed to 25OHD. The data provided further support for the importance of 25OHD as the major toxic metabolite in vitamin D–associated hypercalcemia, as well as for the importance of increased intestinal VDR in the pathophysiological process that leads to enhanced effects of this metabolite.

C. Control of Renal 1α-Hydroxylase in Vitamin D Toxicity Some investigators have suggested that toxic effects of excessive concentrations of 25OHD may result from PTH suppression and down-regulation of 1α-hydroxylase with increased concentrations of 25OHD. PTH and 1,25(OH)2D have known reciprocal actions on 1α-hydroxylase and 24-hydroxylase activities. PTH stimulates 1α-hydroxylase activity and down-regulates 24-hydroxylase activity; 1,25(OH)2D, on the other hand, down-regulates 1α-hydroxylase activity and stimulates 24-hydroxylase activity. Beckman and colleagues [162] studied the effects of an excess of vitamin D3 and dietary calcium restriction on tissue 1α-hydroxylase and 24-hydroxylase activity in rats. Four groups of rats with different dietary calcium and vitamin D3 concentrations were studied (normal calcium, NC; low calcium, LC; and the excess vitamin D groups with normal or low calcium, NCT and LCT). The data showed that in the setting of a calciumrestricted diet, a nutritional hyperparathyroidism ensued (Table V). Under conditions of excess vitamin D3 at doses of 75,000 IU per week and on a calcium-restricted diet, elevations in PTH facilitated the elimination of

25OHD3 through its metabolism to 1,25(OH)2D3 and or degradation to 24,25(OH)2D3. The elevation in PTH was accompanied by increased activation of renal 1α-hydroxylase activity, lower concentrations of 25OHD3, increased activation of intestinal 24-hydroxylase activity, and lower renal VDR content compared to the normal calcium group (Table VI). In contrast, the normal calcium diet in the vitamin D3 excess group contributed to the toxicity by virtue of suppressed PTH concentrations resulting in down-regulation of renal 1α-hydroxylase and decreased 24-hydroxylase activity, and, thus, higher 25OHD3 concentrations. On the other hand, dietary calcium restriction in the setting of vitamin D3 excess seemed to be protective, providing less biological stimulation due to higher PTH concentrations with reduced VDR, increased activation of both 1α-hydroxylase and 24-hydroxylase activities, greater reductions in 25OHD3 concentrations, and lower concentrations of total calcium resulting in a less toxic state. So the low calcium diet protects, not only by contributing to less hypercalcemia, but also by facilitating metabolic pathways of vitamin D inactivation.

D. Inhibition of the Catabolic Pathway of 24-Hydroxylase Others have proposed that inhibition of the enzymes that degrade the vitamin D metabolites may have a role in the pathogenesis of hypervitaminosis D. 1,25(OH)2D is a known regulator of its own catabolism and an inhibitor of its synthesis. In the kidney, intestine and other targets 1,25(OH)2D induces the enzyme 24-hydroxylase. This enzyme initiates a catabolic cascade that ultimately causes side chain oxidation, cleavage, and metabolic elimination of both 1,25(OH)2D and 25OHD, and it accounts for 35–40% of the

TABLE V Changes in Plasma Calcium, Phosphorus, PTH, and 1,25(OH)2D3 Concentrations in Response to Dietary Calcium Restriction and Vitamin D3 Excessa Treatmentb NC NCT LC LCT

Calcium (mg/dl)

Phosphorus (mg/dl)

11.2 ± 0.1 14.6 ± 0.3c 9.1 ± 0.1c 9.7 ± 0.4d,e

9.3 ± 0.7 9.5 ± 0.5 8.9 ± 0.5 9.0 ± 0.7

25OHD3 (ng/ml) 15.2 ± 1.7 443 ± 43c 24,25(OH)2D > 1,25(OH)2D [168]. Of note is the fact that the potent metabolite 1,25(OH)2D has the least affinity for DBP, but the highest affinity for the intracellular VDR that triggers subsequent transcriptional events. Therefore, freeing

bound 1,25(OH)2D metabolite from DBP could promote its entry into various tissues and promote biological activity [169]. In states of vitamin D toxicity, the presence of elevated free 1,25(OH)2D levels despite normal total 1,25(OH)2D levels suggests that 1,25(OH)2D is displaced from DBP by 25OHD, resulting in a rise of serum free calcitriol [170]. Evidence indicates that the biologically active form of the vitamin D steroid hormone is the free hormone that is accessible to cells [171]. Because of technical difficulties in measuring the free hormone, the determination of vitamin D status involves a measurement combining free vitamin D and DBP concentrations. In normal individuals, 85% of the total 1,25(OH)2D is bound to DBP, 15% is bound to albumin, and 0.4% is free [172]. However, under conditions of altered or reduced albumin and DBP concentrations, as in liver or kidney disease, the free hormone may provide different information compared to the total measured concentration of vitamin D. Theoretically, total hormone concentration in such settings may erroneously suggest deficiency of vitamin D with needless institution of replacement therapy. Bikle and colleagues noted that subjects with liver disease have reduced DBP concentrations with low total 1,25(OH)2D and 25OHD levels, whereas free forms are normal [173,174]. Similarly, in certain forms of renal disease, the concentrations of DBP and vitamin D metabolites are reduced, thus measurements of total hormone may provide an inaccurate reflection of vitamin D status. Koenig et al. [175] investigated free and total 1,25(OH)2D concentrations in subjects with renal disease. Patients with nephrotic syndrome, with varying degrees of renal failure, and on chronic hemodialysis and peritoneal dialysis were studied. The serum concentrations of total and free 1,25(OH)2D correlated well with one another in the patients with renal failure

1368 and those undergoing hemodialysis. The concentrations of DBP and 25OHD, thus, were unaffected by renal function. The concentrations of total 1,25(OH)2D accurately reflected free 1,25(OH)2D in patients with varying degrees of renal failure when DBP levels remained normal. However, this did not hold true for the subjects with nephrotic syndrome or those on chronic peritoneal dialysis, who lost DBP and bound vitamin D metabolites into the urine or peritoneal fluid, respectively, with a rise in the percentage free 1,25(OH)2D (also megalin may play a role as discussed in Chapter 10). Measurement of free metabolite in these particular patients may be important to avoid vitamin D toxicity when supplementation is instituted. Thus, in this context, the binding proteins of the vitamin D metabolites not only serve a transport function, but also may provide a buffering mechanism to protect against toxicity [176].

V. CLINICAL MANIFESTATIONS The clinical manifestations of vitamin D toxicity resulting from hypercalcemia reflect the essential role of calcium in many tissues and targets, including bone, the cardiovascular system, nerves, and cellular enzymes. Initial signs and symptoms of hypervitaminosis D may be similar to other hypercalcemic states and include generalized weakness and fatigue. Central nervous system features may include confusion, difficulty in concentration, drowsiness, apathy, and coma [177]. Neuropsychiatric symptoms include depression and psychosis, which resolve following improvement of the hypercalcemia. Hypercalcemia can affect the gastrointestinal tract and cause anorexia, nausea, vomiting, and constipation. It can induce hypergastrinemia, but only in men does it appear to be associated with peptic ulcer disease. There is no evidence that peptic ulcers are more common in any other form of hypercalcemia. Rarely, pancreatitis may be a presentation of either acute or chronic hypercalcemia. In the heart, hypercalcemia may shorten the repolarization phase of conduction reducing the Q-T interval on the electrocardiogram (EKG). EKG changes in vitamin D toxicity have been mistaken for myocardial ischemia [178]. A more accurate EKG indication of the level of hypercalcemia is the Q-T interval corrected for rate. Bradyarrhythmias and first degree heart block have been described, but are rare. Hypercalcemia may potentiate the action of digitalis on the heart [179]. Kidney function is affected because high concentrations of calcium alter the action of vasopressin on the renal tubules. The net result is reduced urinary

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concentrating ability and a form of nephrogenic diabetes insipidus. This usually presents as polyuria, but rarely is the volume as high as that associated with central diabetes insipidus. Symptoms may include polydipsia, which is an expected consequence of polyuria. Hypercalciuria is one of the earliest signs of vitamin D toxicity and precedes the occurrence of hypercalcemia. The initial hypercalciuria may be ameliorated as renal failure progresses because of reduced calcium clearance. The pathophysiology of hypercalcemia can be rapidly worsened when dehydration develops. When reduced renal blood flow occurs, less calcium is presented to the renal glomerulus, and hypercalcemia can rapidly progress. Renal impairment from the hypercalcemia is reversible if of short duration. Chronic, uncontrolled hypercalcemia can lead to deposition of calcium phosphate salts in the kidney and permanent damage with eventual nephrocalcinosis. In an investigation of vitamin D–induced nephrocalcinosis, Scarpelli and colleagues [180] noted that cell damage, specifically in mitochondria, preceded intracellular calcium deposition. The hypercalcemia induced in rats by excessive vitamin D administration caused mitochondrial swelling, cell injury, and subsequent calcification. Ectopic soft tissue calcification can be a particular problem in hypervitaminosis D. The tendency towards soft tissue calcification is compounded by the combination of hypercalcemia and hyperphosphatemia, often exceeding the solubility product of the two ions [181–183]. In rats exposed to excessive vitamin D, Hass and colleagues demonstrated that the pathological processes of vitamin D toxicity were related to dosage, length of time between doses, and duration of exposure [184]. For rats subjected to sublethal doses, generalized calcinosis was seen after only eight days, when a total of 300,000 units of ergosterol was administered. Pathologically, bones appeared more brittle than normal, with increased cortical bone resorption, increased numbers of osteoclasts, and reduced numbers of osteoblasts. Abnormal calcium deposits were noted in the aorta and its major branches, heart, kidney, muscle, and respiratory tract. The earliest evidence of hypervitaminosis D was in the proximal aorta. Muscle tissue was the least resistant to calcification, with the order of decreasing susceptibility being smooth muscle > cardiac muscle > skeletal muscle [185]. The liver, brain, and pituitary were not affected by high doses of vitamin D. Permanent dental changes have also been reported with hypervitaminosis D, including enamel hypoplasia and focal pulp calcification [186]. Bone mineral density can be decreased due to excessive bone resorption [182,187], changes which can be reversed when vitamin D levels return to normal [188].

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VI. Diagnosis of Vitamin D Toxicity With modern assays for calciotropic hormones, PTH, 25OHD, and 1,25(OH)2D (see Chapter 58), one can readily differentiate vitamin D metabolite-mediated hypercalcemia from other causes of hypercalcemia. The circulating intact PTH level, preferably measured by the two-site immunoradiometric assay (IRMA) or immunochemiluminometric assay (ICMA), should be suppressed in virtually all hypercalcemic disorders with the exception of primary hyperparathyroidism, familial hypocalciuric hypercalcemia, administration of lithium or thiazides, and renal failure. Although patients with malignancy-associated hypercalcemia tend to have a higher serum calcium concentration than those with other causes of hypercalcemia, diminished glomerular filtration rate and subsequent reduction in renal calcium excretion can dramatically increase the serum calcium level in any hypercalcemic patient. In contrast to the low serum phosphorus level in patients with hypercalcemia due to PTH or PTHrP, the serum phosphorus level is at the upper limit of normal or frankly elevated in patients with vitamin D metabolite-mediated hypercalcemia. This is due to increased intestinal absorption and reduced renal clearance of phosphate. An elevated 25OHD concentration with normal 1,25(OH)2D level is indicative of toxicity with exogenously administered vitamin D or 25OHD. The serum 1,25(OH)2D level may be normally increased in patients with primary hyperparathyroidism due to the induction of renal 1α-hydroxylase by PTH. Abnormally high 1,25(OH)2D levels, in the setting of suppressed PTH and hypercalcemia, indicate dysregulated production of 1,25(OH)2D due to either granulomatous diseases, lymphoma, or toxicity with exogenous 1,25(OH)2D or lα-OHD. In cases of hypercalcemia due to PTHrP or local osteolytic factors, the serum 1,25(OH)2D concentration is usually suppressed. In patients with hypercalcemia due to toxicity with other vitamin D analogs such as dihydrotachysterol (DHT) [189] and calcipotriol, the active metabolites may not be recognized by the conventional competitive protein binding assays for 1,25(OH)2D. The diagnosis of vitamin D toxicity can be made on clinical grounds. Detailed clinical and drug history are of paramount importance in order to make an early diagnosis. Most patients who are suffering from vitamin D toxicity are taking vitamin D for osteoporosis, hypoparathyroidism, pseudohypoparathyroidism, hypophosphatemia, osteomalacia, or renal osteodystrophy in excessive dosages or at too frequent dosing intervals. Therefore, one should have a high index of suspicion in patients who are being treated with pharmacological dosages of vitamin D or its metabolites.

Patients with granulomatous diseases or lymphoma usually have widespread active disease when hypercalcemia develops. In such cases, the diagnosis is obvious at the time of presentation. However, exceptions do exist. In patients with unexplained hypercalcemia, if the 1,25(OH)2D level is elevated and other more easily identifiable causes for this elevation such as primary hyperparathyroidism, pregnancy, and exogenous toxicity (by history) are excluded, measurement of angiotensin converting enzyme level and a systemic search for lymph node enlargement, pulmonary, renal, hepatosplenic, ocular, central nervous system, and bone marrow granulomas or lymphoma should be made.

VII. Treatment of Vitamin D Toxicity Dietary calcium and vitamin D restriction and avoidance of exposure to sunlight and other ultraviolet light sources should be advised to patients at high risk to develop vitamin D metabolite-mediated hypercalcemia. Those at risk include patients with granulomatous diseases and lymphoma whose disease is widespread and active and patients who are already hypercalciuric. Daily dietary calcium intake should be minimized to approximately 400 mg or less in these patients. Any use of vitamin D supplements should be discontinued. The patient should be encouraged to use sunscreen [sun protection factor (SPF) >15] as much as possible when out of doors. The calcium level should be monitored closely in patients who have a previous history of hypercalcemia or hypercalciuria, or who have recently taken diets enriched in vitamin D and calcium, or who have a recent history of excessive sunlight exposure. A reduction in oxalate intake may also be advisable, so as to prevent an increase in oxalate absorption and hyperoxaluria, which may increase the risk of kidney stone formation, despite a reduction in urinary calcium excretion [190]. When hypercalcemia develops, the aforementioned preventive measures will help to ameliorate the severity of hypercalcemia. General measures in those who are symptomatic include hydration with normal saline and the judicious use of a loop diuretic, like furosemide. Specific inhibitors of bone resorption, such as bisphosphonates [182,187] and calcitonin, can be helpful. Recently, a 3-month-old infant with vitamin D intoxication due to oversupplementation (serum calcium 18.5 mg/dl and 25OHD 360 ng/ml) was treated with alendronate (5–10 mg/d) for 18 days with resolution of the hypercalcemia [22]. Glucocorticoids have proved to be particularly effective in vitamin D intoxication, granulomatous diseases, and lymphoma (see also Chapter 73). The precise

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VIII. EVIDENCE FOR BENEFITS OF HIGHER VITAMIN D LEVELS A. Bone Health Recent evidence indicates that the accepted threshold of vitamin D sufficiency might not be satisfactory, a shift in paradigm which could conceivably lead towards more zealous vitamin D replacement. Most of the daily vitamin D requirements in a healthy individual (3000–5000 IU) are met with cutaneously synthesized accumulations from solar sources during the preceding summer [199]. However, summer sun exposure is probably not sufficient. In 26 healthy men,

Cumulative probability of fracture

mechanism of action of glucocorticoids in calcium homeostasis is not known. Nonetheless, they are useful because they (1) directly inhibit gastrointestinal absorption of calcium by decreasing the synthesis of calcium-binding protein (calbindin-D) and decreasing active transcellular transport [191], (2) increase urinary excretion of calcium [192], and (3) may alter hepatic vitamin D metabolism to favor the production of inactive vitamin D metabolites, resulting in lower concentrations of 25OHD [193]. Evidence also suggests that they may increase the degradation of 1,25(OH)2D at the receptor sites [194]. Glucocorticoids may also limit osteoclastic bone resorption [195]. Institution of glucocorticoid therapy results in prompt decline of the circulating 1,25(OH)2D concentrations within 3 to 4 days [66]. Patients with nonhematological malignancies and those with primary hyperparathyroidism do not usually respond to glucocorticoids. Aminoquinolones (chloroquine and hydroxychloroquine) are also capable of reducing the 1,25(OH)2D and calcium concentrations in patients with sarcoidosis [196]. The theoretical advantage of aminoquinolones over glucocorticoids is that correction of the 1,25(OH)2D should result in rapid recovery of at least some of the bone density lost to the disease [188]. In lymphoma cells, however, aminoquinolones do not have the same regulatory effects on the excess 1,25(OH)2D as they do in granulomatous disease. In the presence of lymphoma, it is preferable to use steroid-containing antitumor regimens [198]. Owing to the limited experience with aminoquinolone drugs as antihypercalcemic agents and their potential side effects, they should be reserved for patients in whom steroid therapy is unsuccessful or specifically contraindicated. Ketoconazole, an antifungal agent, in high dosages can inhibit the mitochondrial cytochrome P450-linked 25OHD 1α-hydroxylase irrespective of whether it is renal [189] or extrarenal as in sarcoidosis [197] and tuberculosis [104].

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0.14 Vitamin D 0.12 Placebo 0.10 0.08 0.06 0.04 0.02 0

0

10

20

30

40

50

60

70 Months

FIGURE 2 Cumulative probability of any first fracture in a randomized controlled trial according to treatment with vitamin D (n =1345) or placebo (n = 1341), based on Cox regression; difference between two groups, p = 0.04. Reproduced with permission from Trivedi, et al. [201].

25OHD levels went from 122 nmol/L in late summer to 74 nmol/L in late winter [200]. Furthermore, 25OHD levels that are well above the bottom end of the conventional reference range are probably, in fact, not optimal. Heaney and colleagues recently demonstrated that 25OHD needs to be at least 80 nmol/L (32.4 ng/ml) to maximize intestinal calcium absorption [14]. This finding was validated by the subsequent results of a large randomized controlled trial (n = 2686), in which treatment with vitamin D in a dose sufficient to raise serum 25OHD from 53 to 74 nmol/L decreased fracture risk at hip, forearm, or spine by 33% ( p = 0.02) (see Fig. 2) [201]. Precisely how much more vitamin D supplementation might be necessary has not been determined, although some estimates can be made. It is known that an eight week course of additional vitamin D at 400 IU daily will raise 25OHD by 11 nmol/L [1] and that maintenance of a normal PTH in the absence of sun exposure requires 1000 IU of daily vitamin D [202]. It thus remains to be seen whether the recommended daily allowance (RDA) for vitamin D will be increased and whether this will enhance the potential for vitamin D toxicity (see also Chapters 61 and 62).

B. Cellular Health Once thought to exert its effects solely on bone, kidney, and intestine, 1,25(OH)2D and its synthetic analogs are increasingly recognized to possess a wider variety of noncalcemic roles, including antiproliferative, prodifferentiative, and immunomodulatory actions. It has now been ascertained that prostate, colon, skin, and

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osteoblasts can express 1α-hydroxylase and synthesize 1,25(OH)2D locally [24, 203]. An example of the clinical ramifications of the nonclassic actions of 1,25(OH)2D is the efficacy of calcipotriene in treating psoriasis [4]. Another novel potential action of vitamin D might be to increase bone formation. A highly potent 1,25(OH)2D analog (2MD) was recently shown to have an anabolic effect in the rodent skeleton [204] (Chapter 87). The noncalcemic actions of 1,25(OH)2D, like the calcemic ones, appear to be mediated through the VDR. One constraint in the use of vitamin D for cellular health has been the dose-limiting hypercalcemia, although this has been partially circumvented with the use of the newer synthetic vitamin D analogs. For example, paricalcitol, a less-calcemic vitamin D analog, has been found in vitro to inhibit proliferation of myeloid leukemia, myeloma, and colon cancer cells [27]. As with 1,25(OH)2D, the VDR controls most of the effects of the synthetic analogs on proliferation, apoptosis, differentiation, and angiogenesis [205]. The antiproliferative effects of 1,25(OH)2D have been demonstrated directly in the prostate and colon and indirectly in the parathyroid. Human prostate cancer cells contain receptors for 1,25(OH)2D and respond to vitamin D in vitro with increases in differentiation and apoptosis and decreases in proliferation, invasiveness, and metastases [26]. Epidemiologically, an association has been observed between decreased sun exposure or vitamin D deficiency and an increased risk of prostate cancer at an earlier age [26]. In a small clinical trial, 22 patients with prostate cancer recurrence were treated with calcitriol 0.5 µg/kg once weekly for 10 months with only transient hypercalcemia. This strategy of intermittent dosing apparently allows very high doses of calcitriol to be administered without hypercalcemic side-effects, although the primary efficacy endpoint of a 50% reduction in PSA was not achieved [206]. In the colon, similar in vitro evidence indicates that cultured transformed colon cancer cells can convert 25OHD to 1,25(OH)2D [24]. mRNA for 1αhydroxylase has been identified in normal colon tissue and in malignant and adjacent normal colon tissue [24]. As in prostate cancer, epidemiologic data suggest that the risk of dying from colorectal cancer is highest in areas with the least amount of sunlight (see Chapter 90). Finally, recent evidence suggests that local production of 1,25(OH)2D regulates parathyroid cell growth and differentiation. The production of 1αhydroxylase has been detected in parathyroid tissue, but at higher levels in adenomas and hyperplastic tissue. This implies that in addition to feedback control by circulating 1,25(OH)2D levels, parathyroid cells may also be influenced by local 1α-hydroxylase activity with possible growth controlling effects [207].

1,25(OH)2D is known to exert a potent immunomodulatory effect on activated human lymphocytes in vitro. In fact, it has been proposed that 1,25(OH)2D produced by macrophages in granulomatous disease exerts a paracrine immunoinhibitory effect on neighboring, activated lymphocytes to slow an otherwise overly exuberant immune response that may be detrimental to the host [208]. The physiological significance of this has been highlighted by the recent development of 1α-hydroxylase knockout mouse models [209,210], which present with multiple enlarged lymph nodes (see Chapter 67). An additional immunomodulatory action of vitamin D is inhibition of the autoimmune reaction targeted towards the β cells of the pancreas. In nonobese diabetic (NOD) mice, a murine model of human type I diabetes mellitus, 1,25(OH)2D pretreatment decreased the incidence of type 1 diabetes [211] (see Chapter 99). More recently, a vitamin D–sufficient status alone was shown to confer partial protection against the development of type I diabetes mellitus in NOD mice [212]. These observations appear to have direct clinical relevance. The risk of type 1 diabetes mellitus was reduced by 80% in children treated with 2000 IU vitamin D daily after age 1 [25]. These noncalcemic actions of vitamin D thus have many potential pharmacologic applications; whether this will enhance the potential for vitamin D toxicity remains to be seen.

VIII. SUMMARY AND CONCLUSIONS Vitamin D toxicity is not a common cause of hypercalcemia, but it can be life threatening if not identified quickly. The major causes of hypercalcemia are primary hyperparathyroidism and malignancy. If these two etiologies are excluded, vitamin D toxicity becomes an important diagnostic consideration. There are many forms of exogenous and endogenous vitamin D toxicity. Inadvertent excessive use of pharmaceutical preparations is the most common etiology of exogenous toxicity. Excessive amounts of the parent compound, vitamin D, can be most difficult to manage as compared to toxicity due to the metabolites 25OHD or 1,25(OH)2D. Extensive lipid solubility of vitamin D accounts for its extraordinary half-life and tendency for prolonged hypercalcemia. New clinical applications of 1,25(OH)2D and its synthetic analogs have been accompanied by the increased potential for toxicity. Endogenous etiologies may result from ectopic production of 1,25(OH)2D in granulomatous diseases, such as sarcoidosis and tuberculosis, or in lymphoma. Many different mechanisms have been proposed to account for vitamin D toxicity, including the vitamin D metabolite itself, VDR number, activity of 1α-hydroxylase,

1372 inhibition of vitamin D metabolism, and the capacity of DBP. Mounting evidence that higher levels of vitamin D may have beneficial effects on bone and cellular health may predispose to enhanced administration of vitamin D in the future and thereby increased frequency of vitamin D toxicity.

Acknowledgment This review was facilitated, in part, by support from a grant from the National Institutes of Health (DK 32333).

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184. Hass GM, Trueheart RE, Taylor CB, Stumpe M 1958 An experimental histologic study of hypervitaminosis D. Am J Pathol 34(3):395–431. 185. Swierczynski, J, Nagel G, Zydowo MM 1987 Calcium content in some organs of rats treated with a toxic calciol dosis. Pharmacology 34(1):57–60. 186. Giunta JL, Dental changes in hypervitaminosis D 1998 Oral Surg Oral Med Oral Pathol Oral Radiol Endod 85(4):410–413. 187. Selby PL, Davies M, Marks JS, Mawer EB 1995 Vitamin D intoxication causes hypercalcemia by increased bone resorption which responds to pamidronate. Clin Endocrinol (Oxf) 43(5):531–536. 188. Adams JS, Lee G 1997 Gains in bone mineral density with resolution of vitamin D intoxication. Ann Intern Med 127(3):203–206. 189. Glass AR, Eil C 1988 Ketoconazole-induced reduction in serum 1,25-dihydroxyvitamin D and total serum calcium in hypercalcemic patients. J Clin Endocrinol Metab 66(5): 934–938. 190. Kogan BA, Konnak JW, Lau K 1982 Marked hyperoxaluria in sarcoidosis during orthophosphate therapy. J Urol 127(2): 339–340. 191. Feher JJ, Wasserman RH 1979 Intestinal calcium-binding protein and calcium absorption in cortisol-treated chicks: effects of vitamin D3 and 1,25-dihydroxyvitamin D3. Endocrinology 104(2):547–551. 192. Suzuki, Y, Ichikawa Y, Saito E, Homma M 1983 Importance of increased urinary calcium excretion in the development of secondary hyperparathyroidism of patients under glucocorticoid therapy. Metabolism 32(2):151–156. 193. Lukert BP, Raisz LG 1990 Glucocorticoid-induced osteoporosis: pathogenesis and management. Ann Intern Med 112(5):352–364. 194. Carre, M, Ayigbede O, Miravet L, Rasmussen H 1974 The effect of prednisolone upon the metabolism and action of 25-hydroxy- and 1,25-dihydroxyvitamin D3. Proc Natl Acad Sci USA 71(8):2996–3000. 195. Bilezikian JP, 1989 Etiologies and therapy of hypercalcemia. Endocrinol Metab Clin North Am 18(2):389–414. 196. O’Leary TJ, Jones G, Yip A, Lohnes D, Cohanim M, Yendt ER 1986 The effects of chloroquine on serum 1,25dihydroxyvitamin D and calcium metabolism in sarcoidosis. N Engl J Med 315(12):727–730. 197. Adams JS, Sharma OP, Diz MM, Endres DB 1990 Ketoconazole decreases the serum 1,25-dihydroxyvitamin D and calcium concentration in sarcoidosis-associated hypercalcemia. J Clin Endocrinol Metab 70(4):1090–5. 198. Adams JS, Kantorovich V 1999 Inability of short-term, lowdose hydroxychloroquine to resolve vitamin D–mediated hypercalcemia in patients with B-cell lymphoma. J Clin Endocrinol Metab 84(2):799–801. 199. Heaney RP, Davies KM, Chen TC, Holick MF, Barger-Lux MJ 2003 Human serum 25-hydroxycholecalciferol response

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CHAPTER 79

Extra-renal 1α-Hydroxylase Activity and Human Disease MARTIN HEWISON JOHN S. ADAMS

Division of Medical Sciences, The University of Birmingham, Queen Elizabeth Medical Centre, Birmingham B15 2TH, UK Division of Endocrinology, Metabolism and Diabetes, Cedars-Sinai Medical Center, Los Angeles, USA

I. Introduction II. Vitamin D and Granuloma-Forming Disease: Historical Perspective III. Pathophysiology of Disordered Calcium Balance in Sarcoidosis: A Model for the Extra-renal Production of an Active Vitamin D Metabolite in Human Disease IV. Local Immunoregulatory Effects of Active Vitamin D Metabolites

V. Human Diseases Associated with the Extra-renal Overproduction of Active Vitamin D Metabolites VI. Diagnosis, Prevention, and Treatment of the Patient with Endogenous Vitamin D Intoxication References

I. INTRODUCTION

immunological targets for active vitamin D metabolites and propose a model in which macrophage-derived vitamin D metabolites play a role in the modulation of the local immune responses. The fifth section will provide a comprehensive review of the various human diseases proposed to be associated with the overproduction of active vitamin D metabolites from an extra-renal source. The sixth and final section of this chapter will address the clinical aspects of disordered extra-renal 1α-hydroxylase; this will include a discussion of the diagnosis, treatment, and prevention of hypercalcemia and hypercalciuria in the patient with endogenous vitamin D intoxication.

The period of time following the publication of the first edition of this book has witnessed some remarkable advances in our understanding of the enzyme 25-hydroxyvitamin D-1α-hydroxylase (1α-hydroxylase), including the cloning of the gene for this enzyme (CYP1α or CYP27b), the development of knockout animal models, and vastly improved tools for analysis of tissue-specific expression of CYP1α. In this chapter we have incorporated these new developments into the framework of the original chapter on the pathophysiology of dysregulated vitamin D metabolism associated with granuloma-forming and malignant lymphoproliferative disorders. We have placed the seminal observations of extra-renal 1α-hydroxylase activity in diseases, such as sarcoidosis, alongside the current studies that have highlighted a much wider tissue distribution of the enzyme, including epithelial cells. The fundamental structure of the chapter has been retained, but additional sections have been added. After this brief introduction (first section), the second section of the chapter will review the historical aspects of extra-renal synthesis of 1,25-dihydroxyvitamin D (1,25(OH)2D) associated with inflammatory disease, including recent studies that have expanded the pathological relevance of extra-renal 1α-hydroxylase. The third section of the chapter will describe what we know about the mechanics and regulation of the vitamin D metabolizing enzymes present in inflammatory cells. The fourth section will recapitulate the potential VITAMIN D, 2ND EDITION FELDMAN, PIKE, AND GLORIEUX

II. VITAMIN D AND GRANULOMAFORMING DISEASE: HISTORICAL PERSPECTIVE A. Evidence of Endogenous Vitamin D Intoxication Associated with Sarcoidosis A pathophysiological relationship between vitamin D and sarcoidosis was first recognized by Harrell and Fisher in 1939 [1]. Among the six hypercalcemic patients in their initial report, one was observed to experience a steep rise in the serum calcium concentration following ingestion of cod liver oil known to be enriched in vitamin D. Almost two decades passed before Henneman et al. [2] demonstrated in 1956 that the hypercalcemic syndrome of sarcoidosis, characterized Copyright © 2005, Elsevier, Inc. All rights reserved.

1380 by increased intestinal calcium absorption and bone resorption, was remarkably similar to that of exogenous vitamin D intoxication and was treatable by the administration of glucocorticoids. This is summarized in Section IV of this chapter and is documented in greater detail in Chapters 5, 7, and 93–96. In 1963, Taylor and coworkers [3] performed the first, large-scale seasonal evaluation of serum calcium levels in patients with sarcoidosis. They found that there was a significant increase in the mean serum calcium concentration in 345 patients with sarcoidosis from winter to summer, but no such change in over 12,000 control subjects. This was the first evidence that there was an association between enhanced vitamin D synthesis, known to occur principally during the summer months, and the blood level of calcium in patients with sarcoidosis. This observation was prospectively confirmed by Dent [4], who was able to increase the serum calcium concentration in patients with active sarcoidosis upon exposure to whole body ultraviolet radiation. The Dent study also helped validate the earlier work of Hendrix [5] who achieved resolution of hypercalcemia and hypercalciuria in two patients with sarcoidosis by institution of vitamin D–deficient diets and environmental sunlight deprivation.

B. Evidence for the Extra-renal Overproduction of an Active Vitamin D Metabolite The above mentioned studies led Bell et al. [6] to propose in 1964 that development of a clinical abnormality in calcium balance in patients with active sarcoidosis resulted from an increase in target organ responsiveness to vitamin D. This view persisted for more than a decade. However, after the discovery of 1,25(OH)2D as the active vitamin D hormone [7–9] and the development of sensitive and specific assays for the hormone in blood [10–12], investigators were quick to determine that the hypercalcemia of sarcoidosis was the result of an increase in the circulating concentrations of a vitamin D metabolite that interacted with the vitamin D receptor (VDR) [13–16]. The fact that a vitamin D hormone was made outside the kidney in hypercalcemic patients with sarcoidosis was first discovered by Barbour and colleagues in 1981 [17]. These investigators reported high concentrations of a vitamin D metabolite detected as 1,25(OH)2D in the circulation of a hypercalcemic, anephric patient with active sarcoidosis. Two years later, Adams et al. [18] determined the macrophage to be the extra-renal source of the active vitamin D metabolite. Unequivocal structural characterization of the metabolite as 1,25(OH)2D was obtained by these same investigators in 1985 [19].

MARTIN HEWISON AND JOHN S. ADAMS

C. Cloning of the CYP1α Gene Provides New Perspectives for Extra-renal Synthesis of 1,25(OH)2D The original studies describing synthesis of 1,25(OH)2D by activated macrophages and the potential consequences of this with respect to inflammatory diseases such as sarcoidosis has stimulated a much broader appreciation of extra-renal activation of vitamin D. This is summarized in section IV of this chapter and is documented in greater detail in Chapters 7, 93, 94, and 95. Further investigation of macrophage 1α-hydroxylase activity also highlighted several crucial differences between the activity of the enzyme in these cells when compared to its classical renal counterpart. For example, the macrophage 1α-hydroxylase is not subject to the exquisite autoregulation characteristic of its kidney counterpart, raising the possibility that renal and extra-renal synthesis of 1,25(OH)2D is catalyzed by distinct enzymes. This and other mechanistic features of extra-renal 1α-hydroxylase are discussed in section III of this chapter. The most significant contributing factor to our current understanding of extra-renal 1,25(OH)2D production has been the cloning of the gene for 1α-hydroxylase (CYP1α). After initial isolation of the mouse gene (Cyp1α) from renal tissue [20], it is notable that the human homolog (CYP1α) was cloned from keratinocytes, a well-established extra-renal site for 1,25(OH)2D production [21]. That this gene was identical to that in the kidney strongly supported the notion of a single but differentially regulated 1α-hydroxylase protein in renal and extra-renal tissues. Further support for this postulate was provided by Mawer and colleagues who showed that macrophages from patients harboring mutations in the CYP1α gene had impaired levels of 1,25(OH)2D production similar to that observed in the renal enzyme [22]. The availability of sequence information has also facilitated the development of specific antisera and probes for 1α-hydroxylase. This has further emphasized the widespread tissue distribution of 1α-hydroxylase, but has also helped to confirm the identity between the renal and extra-renal enzymes [23,24]. Advances in our understanding of extra-renal 1α-hydroxylase have also led to its implication in diseases beyond the original observation of abnormal synthesis of 1,25(OH)2D in some patients with sarcoidosis. A key development has been the expression and function of 1α-hydroxylase in breast, prostate, and colon cancer, and this is discussed in greater detail in Chapters 93–95. In the remainder of this chapter, we will focus on the established link between extra-renal 1α-hydroxylase and granulomatous diseases.

CHAPTER 79 Extra-renal 1α-Hydroxylase Activity and Human Disease

III. PATHOPHYSIOLOGY OF DISORDERED CALCIUM BALANCE IN SARCOIDOSIS: A MODEL FOR THE EXTRA-RENAL PRODUCTION OF AN ACTIVE VITAMIN D METABOLITE IN HUMAN DISEASE A. Clinical Evidence for Dysregulated Overproduction of the Vitamin D Hormone As has been described in detail in earlier chapters, the synthesis of 1,25(OH)2D by the renal 1α-hydroxylase is normally strictly regulated with levels of the hormone product being some 1000-fold less plentiful in the circulation than that of the principal substrate for the enzyme, 25-hydroxyvitamin D (25OHD). Hormone synthesis in the kidney is stimulated by an increase in the serum parathyroid hormone (PTH) concentration, a decrease in the serum phosphate concentration, and a decrease in the activity of the competing vitamin D 24-hydroxylase (24-hydroxylase). Synthesis of 1,25(OH)2D is inhibited by a decrease in the circulating PTH levels, increased serum phosphate, and increased 24-hydroxylase activity. There are now at least four clear lines of clinical evidence to indicate that endogenous 1,25(OH)2D production in hypercalcemic/ hypercalciuric patients with sarcoidosis is dysregulated and not bound by the same set of endocrine factors known to regulate 1,25(OH)2D synthesis in the kidney [25]. First, hypercalcemic patients with sarcoidosis possess a frankly high or inappropriately elevated serum 1,25(OH)2D concentration, although their serum PTH level is suppressed and their serum phosphate concentration is relatively elevated [26,27]. If 1,25(OH)2D synthesis were under the regulation of PTH, phosphate, and 1,25(OH)2D itself, then 1,25(OH)2D concentrations in such patients should be low. Second, the serum 1,25(OH)2D concentration in patients with active sarcoidosis is very sensitive to an increase in available substrate [28], while the serum 1,25(OH)2D level in normal individuals is not influenced by small or even moderate increments in the circulating 25OHD concentration. Clinically, this aspect of dysregulation is manifest by the long-recognized association of the appearance of hypercalciuria and/or hypercalciuria in sarcoidosis patients in the summer months or following holidays to geographic locations at lower latitudes than those at which the patient normally resides [28,29]. This link between an increase in cutaneous vitamin D synthesis and the development of a clinical abnormality in calcium balance can be replicated by the oral administration of vitamin D [15,27,30] to such patients.

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It can also be substantiated on a biochemical basis by demonstration of a positive correlation between the serum 25OHD and 1,25(OH)2D concentrations in patients with active sarcoidosis, but not in normal human subjects [31]. Third, the rate of endogenous 1,25(OH)2D production, which is significantly increased in patients with sarcoidosis [32], is unusually sensitive to inhibition by factors (i.e. drugs) that do not influence the renal 1α-hydroxylase at the same doses. Anti-inflammatory concentrations of glucocorticoids have long been recognized as effective combatants of sarcoidosis-associated hypercalcemia and have also been shown to dramatically lower elevated 1,25(OH)2D levels [30,31,33]. On the other hand, administration of the same glucocorticoid doses to patients without sarcoidosis is not associated with a clinically relevant reduction in the serum 1,25(OH)2D or calcium concentration. Chloroquine and its hydroxylated analog, hydroxychloroquine, are other examples of pharmaceutical agents that appear to act preferentially on the extra-renal vitamin D-1α-hydroxylase reaction, which is active in patients with sarcoidosis [34–36]. Fourth, the serum calcium and 1,25(OH)2D concentrations are positively correlated to indices of disease activity in patients with sarcoidosis [37–39]; patients with widespread disease and high angiotensinconverting enzyme (ACE) activity are more likely to be hypercalciuric or frankly hypercalcemic.

B. Correlates In Vitro for Dysregulated 1,25(OH)2D Production In Vivo Investigators have now generated a substantial body of experimental data from cells, including inflammatory cells harvested directly from patients with sarcoidosis, to indicate that the dysregulated vitamin D hormone synthesis in sarcoidosis is probably not due to expression of a 1α-hydroxylase that is different from the renal enzyme, but rather to expression of the authentic 1αhydroxylase in a macrophage, not a kidney cell [22]. In fact, each of the above mentioned pieces of clinical evidence for dysregulated vitamin D hormone production in this disease can be borne out in vitro in cells from patients with this disease [40]. 1. SUBCELLULAR LOCALIZATION, SUBSTRATE SELECTIVITY, AND KINETICS OF THE MACROPHAGE VITAMIN D-1-HYDROXYLASE

As is the case with the 1α-hydroxylase of renal origin, the macrophage enzyme is a mitochondrial mixed function oxidase with detectable cytochrome P450 activity [41] (see Fig. 1). Like the renal 1α-hydroxylase reconstituted from mitochondrial extracts, the presence

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Mitochondrial membrane

NADPH

FP

e−

Fdxred

1,25-D

CYP1α

1α-hydroxylase

e−

NADP+

e−

FPred

Fdx

e−

CYP1α red

_ O2

25-D

+ NO

FIGURE 1

Mitochondrial electron transport associated with the vitamin D-1α-hydroxylase reaction: a model for interaction with nitric oxide (NO). NADPH supplies the electron transport chain of accessory proteins associated with 1α-hydroxylase, consisting of a flavoprotein reductase (FP), a ferredoxin (Fdx), and the 1α-hydroxylase cytochrome P450 (CYP1α). A stimulatory effect on the enzyme may also be mediated by relatively low intracellular NO levels. An electron (e−) generated from NO is donated to oxidized NADP, thus forming NADPH. On the other hand, the inhibitory effect on the enzyme which occurs at relatively high NO levels in the cell results from competition with O2 binding to the P450 heme group, inhibiting the enzyme.

of a flavoprotein, ferredoxin reductase, an electron source, and molecular oxygen (O2) are all required for electron transfer to the cytochrome P450 and for the insertion of an oxygen atom in the substrate [41]. Also, like the renal 1α-hydroxylase, we now know the macrophage 1α-hydroxylase is inhibited by the napthoquinones, molecules which compete with reductase for donated electrons, and by the imidazoles, compounds which compete with the enzyme for receipt of O2 [42]. Similar to the 1α-hydroxylase isolated from the mitochondria of proximal renal tubular epithelial cells, the macrophage enzyme requires a secosterol (vitamin D sterol molecule with an open B-ring) as substrate [43]. Also similar to the renal 1α-hydroxylase, the macrophage enzyme has a particular affinity for secosterols bearing a carbon-25 hydroxy group as is encountered in the two preferred substrates for this enzyme, 25OHD and 24,25-dihydroxyvitamin D (24,25(OH)2D) [43,44]; the calculated Km (affinity) of the 1α-hydroxylase in pulmonary alveolar macrophages derived directly from patients with active sarcoidosis is in the range of 50–100 nM for these two substrates [43,44]. The availability of cDNA sequences for 1α-hydroxylase expression studies has shed more light on the catalytic properties of the enzyme (20,21,45,46) but, as yet, has failed to provide a clear mechanism for the differential regulation of 1,25(OH)2D production in renal and extra-renal tissues. Some of the potential explanations for this are discussed in the following sections. 2. MACROPHAGES LACK RESPONSIVENESS TO PTH, CALCIUM, PHOSPHATE

In vivo there appear to be three major regulators of the renal 1α-hydroxylase—the serum concentration

of calcium, parathyroid hormone, and phosphate [47] (left panel, Fig. 2). Hypocalcemia enhances the activity of the renal 1α-hydroxylase, but much of this stimulatory effect may be indirectly mediated through parathyroid hormone (PTH). Any decrease in the serum calcium concentration below normal is a stimulus for increased secretion of PTH [48] which, in turn, is a direct stimulator of the renal 1α-hydroxylase [49]. Recent promoter-reporter analyses have shown that both PTH and calcitonin stimulate transactivation of 1α-hydroxylase [50,51], although other studies have suggested that PTH can also effect changes in 1,25(OH)2D production via transient alteration in the phosphorylation status of the ferredoxin, which contributes electrons to 1α-hydroxylase [52]. A change in the serum phosphate concentration is the other major regulator of renal 1,25(OH)2D production; in adult humans, dietary phosphorus restriction causes an increase in circulating concentrations of 1,25(OH)2D to 80% above control values, an increase not due to accelerated metabolic clearance of this hormone [53]. Dietary phosphorous supplementation will have the opposite effect. Although the mechanism by which a drop in the serum phosphate level will increase renal 1,25(OH)2D production remains uncertain [54], there is no doubt that there exists a concerted, cooperative attempt of the calcium-phosphorous-PTH axis in man to regulate the conversion of 25OHD to 1,25(OH)2D in the kidney. For example, a drop in the serum calcium concentration will be immediately registered by the parathyroid cell calcium receptor, which will release its inhibition on PTH production and secretion. An increase in the circulating PTH will directly stimulate the renal 1α-hydroxylase, while a PTH-mediated phosphaturic response and a subsequent decrement in

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Kidney

Macrophage

24,25-D & 1,24,25-D

+

+

25-D

PTH

X X

_

1,25-D

[P]

[Ca]

1,25-D

25-D

X

X

PTH

[P]

[Ca]

FIGURE 2

Model distinguishing the regulation of 1α-hydroxylase in the proximal renal tubular epithelial cell of the kidney (left panel) and in the granuloma-forming disease-activated macrophage (right panel). In the kidney, the enzymatic conversion of substrate 25-hydroxyvitamin D (25-D) to product 1,25-dihydroxyvitamin D (1,25-D) is subject to negative feedback control with down-regulation of enzyme activity under the influence of 1) a calcium-mediated decrease in the circulating parathyroid (PTH); 2) a 1,25-D-mediated increase in the serum phosphate [P] level; and 3) a 1,25-D–mediated increase in vitamin D 24-hyroxylase activity (24,25-dihydroxyvitamin D (24,25-D) and 1,24,25-trihydroxyvitamin D (1,24,25-D) production). The macrophage lacks responsiveness to changes in the extracellular PTH and [P] and harbors little or no detectable vitamin D-24-hydroxylase (lack of regulation designated X).

the serum phosphate level will indirectly promote 1,25(OH)2D production. The macrophage 1α-hyroxylase, on the other hand, is immune to the stimulatory effects of PTH and phosphate [42,55] (right panel, Fig. 2). The macrophage plasma membrane is not enriched with PTH receptors [56], and there is no evidence that any PTH receptors which are resident in the macrophage membrane are responsive to PTH or PTHrP in terms of stimulating the protein kinase signaling pathways that are associated with stimulation of the renal 1α-hydroxylase. Similarly, the macrophage enzyme appears to be uninfluenced by changes in the extracellular phosphate concentration [42]. Moreover, exposure of activated macrophages expressing 1α-hydroxylase to a calcium ionophore stimulates the hydroxylation reaction [57], while increasing the extracellular calcium concentration has the opposite, inhibitory effect on the renal 1α-hydroxylase [58]. These observations appear to confirm the fact that the three most important extracellular signaling systems for the renal 1α-hydroxylase are not heeded by the macrophage enzyme and provide an explanation for why 1,25(OH)2D production by the macrophage in diseases like sarcoidosis is not subject to negative feedback control as reflected by a drop in the serum PTH concentration and an increase in the circulating calcium and phosphate level. Furthermore, with

the possible exception of insulin-like growth factor-1 (IGF-1) [59], there is no evidence that the macrophage 1α-hydroxylation reaction is influenced by any of the other endocrine factors, including estrogen, prolactin, and growth hormone, purported to increase the renal production of 1,25(OH)2D [60–63]. By contrast, macrophage 1α-hydroxylase activity is potently inhibited by anti-inflammatory agents, such as glucocorticoids, which have little or no effect on the renal enzyme. In vivo, this is likely to be due in part to the effects of glucocorticoids on macrophage differentiation and apoptosis. However, studies in vitro suggest that there is also direct inhibition of macrophage 1α-hydroxylase activity by glucocorticoids [40].

3. Macrophages Lack 1,25(OH)2D-directed 24-hydroxylase Activity The other major contributor to the circulating 1,25(OH)2D level is the activity of 24-hydroxylase. Like the 1α-hydroxylase, 24-hydroxylase is a hemebinding mitochondrial enzyme requiring NADPH, molecular oxygen, and magnesium ions [52,64]. The cDNA and gene sequences for human, rat, and chicken 24-hydroxylase, now referred to as CYP24/Cyp24, were cloned several years prior to CYP1α [65–67].

1384 As depicted in Fig. 2 (left panel), expression of CYP24 is stimulated in kidney cells by 1,25(OH)2D, especially if the protein kinase C (PKC) pathway is also upregulated [68–70]. PTH appears to exert an opposite, inhibitory effect on CYP24 gene transcription and 24,25(OH)2D synthesis [71]. There is dual impact of this mitochondrial, cytochrome P450-linked enzyme system on vitamin D and calcium balance in adult animals, including man. Because it is coexpressed in the kidney along with the 1α-hydroxylase, the first point of impact is on regulation of substrate 25OHD available to the 1α-hydroxylase. Like the 1α-hydroxylase, the 24-hydroxylase exhibits a preference for 25hydroxylated secosterol substrates [72]. Although its affinity for 25OHD is reported to be somewhat less than that of renal 1α-hydroxylase, its capacity for substrate is substantially greater [44]. Hence, when up-regulated under the influence of circulating or locallyproduced 1,25(OH)2D or diminished serum PTH levels, the 24-hydroxylase has the capacity to compete with 1α-hydroxylase for substrate 25OHD. Under physiological conditions, this state of competitive substrate deprivation for the 1α-hydroxylase will persist until the serum calcium and PTH concentration are normalized. The second point of impact of the vitamin D-24hydroxylase on the circulating 1,25(OH)2D concentration is at the level of catabolism of 1,25(OH)2D itself. Although both 25OHD and 1,25(OH)2D are metabolized by 24-hydroxylase [73], current data strongly suggest that the latter is the preferred substrate [64]. Considering the fact that the 24-hydroxylase is the initial step in the conversion of 1,25(OH)2D to nonbiologically-active, water-soluble, excretable metabolites of the hormone, up-regulation of this enzyme will contribute to the lowering of 1,25(OH)2D hormone levels. In contrast to precursor monocytic cells, the macrophage lacks detectable 24-hydroxylase activity (Fig. 2, right panel) [43]. Therefore, unlike the renal tubular epithelial cells and indeed other epithelial cells [74], macrophages do not possess the capability of shunting substrate 25OHD or the 1α-hydroxylase product 1,25(OH)2D down the catabolic 24-hydroxylase pathway. The net result is dysregulated overproduction of 1,25(OH)2D by the macrophage, escape of the hormone into the general circulation, and the eventual development of hypercalcemia. 4. MACROPHAGE 1α-HYDROXYLASE EXHIBITS RESPONSIVENESS TO IMMUNE CELL REGULATORS

The lack of negative feedback control on the 1α-hydroxylase expressed in the macrophage as just described can account for the failure to appropriately inhibit 1,25(OH)2D synthesis in inflammatory diseases like sarcoidosis. However, this does not adequately


explain the fact that 1,25(OH)2D production rates are increased well above normal in patients with these diseases at a time when the renal 1α-hydroxylase is inhibited. This observation suggests that there must be an alternative, “nonclassical” set of factors that stimulate the synthesis of 1,25(OH)2D by the macrophage but not by the kidney. Clinical observations from a number of investigative groups around the world indicate that sarcoidosis patients with diffuse, infiltrative pulmonary disease are at greater risk to develop dysregulated vitamin D metabolism. Cultured pulmonary alveolar macrophages (PAM) from such patients were more likely to synthesize more 1,25(OH)2D in vitro on a per cell basis than PAM from a host with less intense or no alveolitis [75,76]. These results led to the conclusion that the specific activity of the 1α-hydroxylase reaction in macrophages from patients with active pulmonary sarcoidosis was regulated by endogenously-synthesized factors, which also modulated the intensity of the host immune response. Of the various bioactive cytokines concentrated in the alveolar space of patients with active sarcoidosis [77,78], IFNγ was found to be the principal cytokine stimulator of the sarcoid macrophage 1α-hydroxylation reaction [77]; by itself at maximally effective concentrations in vitro, IFNγ increased basal hydroxylase activity over four-fold. However, it is now clear that other immunomodulators are also able to stimulate macrophage 1α-hydroxylase, including other cytokines such as tumor necrosis factor α (TNFα) [43,79] and interleukin-2 (IL-2) [55], as well as pathogen-associated peptides such as bacterial lipopolysaccharide (LPS) [42,55]. As all of these factors have been implicated in the maturation of macrophage responsiveness within the innate immune system, it seems likely that up-regulated 1α-hydroxylase activity is a common feature of activated macrophages. However, two key questions remain: first, is there a specific mechanism involved in up-regulation of macrophage 1α-hydroxylase activity; second, why is macrophage 1α-hydroxylase activity pathologically elevated in patients with inflammatory diseases, such as sarcoidosis? These issues are addressed in the following sections. a. Cytokines Despite the advent of gene sequence information for CYP1α and in particular promoter analyses for classical 1α-hydroxylase regulators such as PTH, our current understanding of the molecular mechanisms involved in regulating macrophage 1α-hydroxylase activity is still poor. It seems likely that several pathways are involved—for example, IFNγ signals via Janus Kinase 1 (JAK1) and JAK2 with subsequent phosphorylation of signal transducers and activators of transcription alpha (STAT alpha) and subsequent transregulation of target genes via cis-acting

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promoter elements [80]. However, the JAK/STAT pathway is essential for the effects of many cytokines and growth factors, including some members of the interleukin family (e.g. IL-2 and IL-6) [81]. The JAK/ STAT system may also interact with other signaling pathways, including p38 mitogen-activated protein kinase (MAP kinase) and nuclear factor-κB (NF-κB) [82,83]. The net effect of this extensive cross-talk means that a variety of factors, including the cytokines outlined above together with appropriate growth factors such as granulocyte-macrophage colonystimulating factor (GM-CSF), may be able to stimulate 1α-hydroxylase in macrophages. Disappointingly, the availability of CYP1α gene sequence information and promoter-reporter constructs has shed no further light on the nuclear transactivation factors that mediate cytokine-induced synthesis of 1,25(OH)2D. Nevertheless, the CYP1α promoter region includes putative AP-1 and NF-κB binding sites, which are potential targets for cytokine-regulation of 1α-hydroxylase [50,51]. Alternatively signaling via cytokines such as IFNγ may lead to the activation of other, calcium-dependent pathways in the macrophage, specifically the protein kinase C (PKC) [84] and phospholipase A2 (PLA2) pathways [85,86]. Because the macrophage 1α-hydroxylase was not influenced by attempts to directly stimulate or inhibit PKC, attention has focused on the PLA2 pathway and the endogenous arachidonic acid metabolic cascade as the signal transduction pathway of most influence over the macrophage enzyme. Further dissection of the intracellular arachidonate metabolic pathway in this cell demonstrated that signal transduction through the 5-lipoxygenase pathway, specifically with the generation of leukotriene C4 (LTC4), was most critical to an increase in 1,25(OH)2D synthesis [87]. These studies were extended to investigate another compound with potential actions in the PLA2arachidonic acid pathway, the 4-amino quinoline derivative chloroquine. 1,25(OH)2D synthesis by macrophages was completely inhibited by exposure to 10−6 M chloroquine in vitro [36]. Furthermore, this effect is independent of chloroquine’s apparent ability to alter the pH of intracellular organelles. When given orally to a hypercalcemic patient with sarcoidosis, chloroquine [34,36] or its analog hydroxychloroquine [35] can effectively reduce the serum 1,25(OH)2D and calcium concentration within a matter of 36 hours. b. Lipopolysaccharide (LPS) LPS is a bioactive lipid extractable from the cell wall of infectious microorganisms, including the mycobacterium. On macrophages, LPS interacts with a complex that includes the cell surface CD14 receptor and toll-like receptor 4 (TLR4), together with the accessory proteins MD-2 and MyD88 [88]. TLR4 is one of ten TLR proteins,

similar to the TNF receptor family, that function as pathogen-recognition receptors and which signal via NF-κB and p38 MAP kinase [89]. TLR4/CD14 is strongly expressed on cells from the immune system, including macrophages and dendritic cells (DCs), and LPS is a potent inducer of 1α-hydroxylase in human monocyte/macrophage-like cells [42,55] (Fig. 3). TLRs are also expressed by epithelial cells at “barrier” sites, including the skin, lungs, gastrointestinal tract, and distal nephron. Here, as with macrophages, TLR expression is able to support LPS inducibility of 1,25(OH)2D production. In recent studies, Hewison and co-workers demonstrated the presence of CD14 and TLR4 on cortical collecting duct HCD cells, but not proximal tubule HKC-8 cells [90]. As a consequence, HCD cells showed potent induction of 1α-hydroxylase activity in response to both PTH and LPS, while HKC-8 cells responded to PTH alone. Thus, it seems likely that TLR expression and signaling acts as a pivotal mechanism in regulating extra-renal 1α-hydroxylase activity. In fact, the most reproducibly effective stimulation of the macrophage 1α-hydroxylase

Macrophage CD14 TNFα IL-1β MD-2 TLR4 LPS hsp70 IFNγ

CD14 IFNr

VDR

iNOS CYP1α

IDBP

1,25-D

NO + e− O2 P450 1α 25-D IDBP 25-D

FIGURE 3 Proposed mechanism for the amplification of macrophage 1α-hydroxylase. Macrophage stimulatory agents lipopolysaccharide (LPS), interferon-γ (IFNγ), and heat-shock proteins (hsps) with their respective cell-surface receptor molecules (LPS and hsps: CD14, toll-like receptor 4 (TLR4), MD-2 complex. IFNγ: IFN receptor (IFNr). This leads to: 1) up-regulation of CYP1α mRNA expression and increased levels of 1α-hydroxylase protein (P4501α); 2) up-regulation of iNOS expression. Nitric oxide (NO) is synthesized from an extracellular source of molecular oxygen (O2) and L-arginine (L-arg). NO can serve as an electrondonating source for enzymatic conversion of 25-hydroxyvitamin D (25-D) to 1,25-dihydroxyvitamin D (1,25-D). 3) increased conversion of 25-D to 1,25-D. The transport of 25-D to mitochondrial 1αhydroxylase and 1,25-D to nucleus is facilitated by hsp70-like intracellular vitamin D–binding proteins (IDBP). NO and 1,25-D act in an intracrine fashion to up-regulate expression of the cytokines interleukin-1β (IL-1ß), tumor necrosis factor α (TNFα), and the LPS receptor molecules; TNF and CD14 promote intracrine stimulation of NO and 1,25-D synthesis.

1386 in vitro is achieved by coexposure of macrophages to IFNγ and LPS [55]. LPS and IFNγ commonly activate different signal transduction pathways, but as outlined previously, there is potential for cross-talk between these pathways, which may have a significant impact on transactivation of CYP1α. Notably, IFNγ and LPS are also the two most effective stimulators of nitric oxide (NO) synthesis in macrophages, and this led Adams and co-workers to hypothesize that production of NO and 1,25(OH)2D in macrophage-like cells may be functionally linked [91–93]. c. Nitric Oxide (NO) The generation of NO in the macrophage is under the control of the enzyme inducible nitric oxide synthase (iNOS) [94]. In contrast to the more stringently regulated, constitutively expressed isoforms of the enzyme (cNOS) that are localized to endothelial cells and neurons, are regulated by calcium, and are capable of producing only modest amounts of NO, the calcium-independent iNOS remains tonically active when “induced” and is capable of generating large quantities of NO in and around the cell [95]. It is therefore interesting that two of the major stimulators of the human macrophage 1α-hydroxylase, IFNγ and LPS, are also key transcriptional regulators of the iNOS gene [96,97], which is itself a cytochrome P450-linked oxidase [98]. These observations coupled with the fact that NO has established inhibitory effects on other cytochrome P450s [99,100] suggested a possible link with the enzymes involved in vitamin D metabolism. Data indicate that NO may act as an alternative to NADPH as a source of unpaired electrons for the 1α-hydroxylase reaction in macrophages [91,92]. However, as the amount of NO generated inside the macrophage continues to increase, there is a reflex decrease in 1,25(OH)2D production [93], suggesting that there is a built-in limit on the ability of the cell to produce active vitamin D. This inhibitory effect of NO on the macrophage 1α-hydroxylase is almost certainly due to competition of NO with oxygen for binding to the heme center of the enzyme. A similar effect has been very recently demonstrated for a number of heme-containing enzymes [99,100], including those involved in steroid hormone metabolism [101]. d. Other Potential Regulators of the Macrophage 1α-hydroxylase Another potential autoregulator of macrophage 1,25(OH)2D synthesis is the stressinduced heat shock-70 (hsp70) family of proteins [102,103]. These proteins are ubiquitously distributed in the nuclear, cytoplasmic, mitochondrial, and endoplasm reticular compartments of eukaryotic cells. Hsp70s were first recognized as heat-shock-responsive ATP-binding proteins with ATPase activity [104]. Stress-induced proteins have a well-established link


with immune responses and inflammatory disease [104,105]. This stems first from the fact that peptides bound or linked to HSPs can elicit potent antigenspecific immunity [106]. In addition, proteins such as hsp70 are able to stimulate cells of the innate immune system directly, and thus act as “danger”-signaling molecules in a similar fashion to LPS [107]. These observations are supported by studies which have shown that TLRs act as exogenous and enodogenous signal transduction pathways for hsp responses [108,109]. A specific link between hsp/TLR signaling and vitamin D metabolism has yet to be studied, but it seems likely that hsps will act as important regulators of the macrophage 1α-hydroxylase. This may be due in part to TLR/NF-κB/p38 MAP kinase-mediated transcriptional regulation as detailed in section 4.b. However, hsps are also characterized functionally by their ability to bind and release hydrophobic segments of an unfolded polypeptide chain in an ATP-hydrolytic reaction cycle [103]. This so-called “chaperone” function of hsp70 is critical for a number of intracellular protein-protein interactions [105], and in the targeting and translocation of molecules across the endoplasmic and mitochondrial membrane [105,110–112]. In macrophage-like cells, hsp70 expression is known to be induced by both physical (i.e. heat) and cytokine (i.e. IFN) stimuli, but its expression is also dramatically enhanced by 1,25(OH)2D [113]. Furthermore, in a series of studies we have shown that proteins from the hsp70 family have a high capacity for intracellular binding of 25-hydroxylated vitamin D metabolites [114]. These hsp70 homologs now termed intracellular vitamin D–binding proteins (IDBPs) were originally identified as vitamin D–resistant cells from New World primates [115]. Initially, these proteins were thought to be the functional basis for the end-organ resistance to 1,25(OH)2D in New World primates. However, subsequent data have shown that the IDBPs are in fact potent activators of 1,25(OH)2D–induced gene transactivation [116]. Rather, the underlying cause of vitamin D resistance in New World primates has now been shown to be due to constitutive overexpression of a vitamin D response element-binding protein (VDRE-BP) from the heterogeneous nuclear ribonucleoprotein in the A family (see Chapter 21), with the IDBPs acting as a putative compensatory mechanism by acting as intracellular chaperones for 1,25(OH)2D [117]. Crucially, cells overexpressing IDBPs also showed increased synthesis of 1,25(OH)2D, indicating that their chaperone activity was not restricted to increased VDR-mediated transactivation [116]. Thus, it is possible that up-regulated IDBP expression in disease-activated macrophages is another key factor in the increased synthesis of 1,25(OH)2D by these cells. Because of its

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capacity to bind 25OHD as well as 1,25(OH)2D, hsp70/IDBP may serve to concentrate, on a relatively low-affinity, high-capacity binding protein, substrate for the 1α-hydroxylase from the general circulation. In fact, by virtue of their organelle-targeting sequences, hsp70s or related molecules may be critical in the directed translocation of 25OHD to the inner mitochondrial membrane where the 1α-hydroxylase actually resides. In this respect, IDBPs may provide a crucial link between the intracellular organelles and endocytic proteins, such as megalin, which are known to act as an interface with serum-bound steroid hormones [118]. Megalin is strongly expressed in the proximal renal tubules where it plays a pivotal role in transporting 25OHD [119], but its role in directing macrophage metabolism of vitamin D remains unclear. The actions of megalin are discussed in greater detail in Chapter 10.

IV. LOCAL IMMUNOREGULATORY EFFECTS OF ACTIVE VITAMIN D METABOLITES A. Intracrine/Autocrine Action on the Monocyte/Macrophage The immunomodulatory properties of 1,25(OH)2D and synthetic analogs of vitamin D are discussed extensively in Chapter 36, which also outlines potential therapeutic applications for auto-immune disease and host-graft rejection. However, a key question remains as to the normal physiological function of locally synthesized 1,25(OH)2D. Furthermore, what is the

Paracrine Action of 1,25-D

+ 1-OHase

25-D

Endocrine Action of 1,25-D Hypercalcemia =“tip of the iceburg”

+ 1,25-D

MACROPHAGE

1-OHase

25-D

1,25-D

1,25-D

antigen

+ LYMPHOCYTES lymphokines

Effect endocrine

MACROPHAGE

+ IL-1, TNF

Target Tissues bone, gut

IL-1, TNF

+

lymphocyte

paracrine

macrophage

intracrine

antigen − LYMPHOCYTES

increasing 1,25-D production

Autocrine Action of 1,25-D

purpose of 1,25(OH)2D production in patients with granuloma-forming disease like sarcoidosis? Is local synthesis of the hormone by macrophages beneficial or detrimental to the host? These are important questions that investigators in a number of centers around the world have been addressing since the 1980s when it became known that the activated, circulating monocytes and tissue macrophages expressed the VDR [120,121]. Expression of the receptor for the active vitamin D hormone indicated that the macrophage could actually be a target for the 1,25(OH)2D that the cell itself was making. Indeed, investigators have suggested that 1,25(OH)2D has the potential to interact with the monocyte/macrophage in either an intracrine or autocrine mode [122,123] (left panel, Fig. 4). For example, incubation with a VDR-saturating concentration of 1,25(OH)2D increases IL-1ß expression by eightfold and decreases by 1000-fold the concentration of stimulator lipopolysaccharide (LPS) required to achieve maximal IL-1ß gene expression [124]. This extraordinary priming effect of the 1,25(OH)2D for LPS stimulation of the IL-1 gene can also be observed for another monokine gene product, TNF [125] and is due to 1,25(OH)2D–mediated induction of the gene for CD14, which acts as an accessory receptor with TLR4. The multiplicity and complexity of actions of 1,25(OH)2D on the macrophage are detailed in Chapter 36. Suffice it to say that it is now widely accepted that the actions of hormone are directed toward stimulation of macrophage function. For example, 1,25(OH)2D is known to enhance giant cell formation [126], monokine production [127–129], and cytotoxic function [130,131]. Conversely, 1,25(OH)2D acts as a

lymphokines

FIGURE 4 Schemes for local production and action of macrophage-derived 1,25-dihydroxyvitamin D (1,25-D). In an autocrine or intracrine mode (left panel), 1,25-D promotes antigen handling and monokine production. In a paracrine mode (middle panel), 1,25-D acts in a negative feedback fashion to “brake” what may turn out to be an overexuberant lymphocyte response to presented antigen and local monokines. If the immune response and 1,25-D production are persistent, then the hormone can escape the local inflammatory microenvironment and act in an endocrine mode (right panel) to alter host calcium balance.

1388 potent suppressor of antigen presentation by both macrophages [132] and other professional antigen presenting cells (APCs), such as dendritic cells (DCs) [133]. The latter has attracted considerable recent attention because of the link between vitamin D responsiveness in DCs and macrophages. Specifically, the ability of 1,25(OH)2D and vitamin D analogs to inhibit DC function appears to be dependent on the suppression of DC differentiation, thereby maintaining DCs in an immature, immune tolerant state [134]. Importantly, studies in vitro indicate that this is accompanied by a reinduction of the macrophage marker CD14 [135,136], suggesting that the suppressive effects of 1,25(OH)2D on DCs are counterbalanced by enhanced macrophage development/function. In this way, 1,25(OH)2D may play an important role in modulating the balance between innate (macrophage/phagocytic) and acquired (DC/APC) immune responses. The significance of this has been further underlined through recent studies by us and others who have shown that monocyte-derived DCs express the same 1α-hydroxylase as renal cells and macrophages and are able to synthesize significant levels of 1,25(OH)2D [137,138]. As a result, DC differentiation and function was potently suppressed by 25OHD, as well as 1,25(OH)2D, suggesting that this may be the pivotal mechanism linking vitamin D status with normal immune function. This process would also be consistent with the overproduction of 1,25(OH)2D observed in diseases such as sarcoidosis. Specifically, the enhanced localized synthesis of 1,25(OH)2D by DCs following an immune challenge would further stimulate tissue macrophage population. This, in turn, may lead to even higher levels of 1,25(OH)2D production and in some cases potential spill-over into systemic levels of the hormone. These functional consequences of hormone action indicate that elaboration of 1,25(OH)2D by activated macrophages and DCs in human diseases like sarcoidosis is important in modulation of the local cellular immune response to the granuloma-causing antigen, promoting antigen processing, containment, and destruction. The system is designed for maximal efficiency in that the hormone interacts with the VDR in the same cell in which the hormone is made. Thus, under normal circumstances, relatively high intracellular and local concentrations of hormone can be achieved to modulate macrophage/DC action without having a generalized, endocrine effect on the host (see Fig. 4).

B. Paracrine Action on Lymphocyte As outlined above, vitamin D is a potent autocrine/ intracrine regulator of both innate and acquired


immune reponses. The effects of 1,25(OH)2D on acquired, lymphocyte-directed immune reponses are due, at least in part, to indirect actions via the suppression of antigen presentation by macrophages and DCs. However, there is also a breadth of evidence for direct effects of 1,25(OH)2D on lymphocytes [139–142]. Specifically, if lymphokine stimulation of macrophage 1,25(OH)2D synthesis were persistent because of difficulty in macrophage-mediated elimination of the offending antigen, then one might conceive of a situation in which the lipid soluble vitamin D hormone escapes the confines of the macrophage (middle panel, Fig. 4). Once outside of the macrophage, 1,25(OH)2D would be free to interact in a paracrine fashion with antigen-activated T- and B-lymphocytes in the local inflammatory microenvironment. The many reported actions of active vitamin D metabolites on cells of the T-lymphocyte (T-cell) lineage are also described in Chapter 36. In general and in contrast to stimulatory effects of the hormone on monocyte-macrophage cells, 1,25(OH)2D and most of the nonhypercalcemic analogs of vitamin D will inhibit T-cell responsiveness to mitogen or antigen challenge. Interaction of the VDR with its cognate ligand in activated lymphocytes and natural killer (NK) cells inhibits cellular proliferation [143,144], generally decreases lymphokine production [143,145], and inhibits T-cell-directed B-cell immunoglobulin synthesis [146] and delayed-type hypersensitivity reactions [147]. More recent studies have shown that 1,25(OH)2D and vitamin D analogs also influence the nature of T-cell responses by promoting specific T-cell subgroups. Initial studies indicated that 1,25(OH)2D preferentially enhanced a shift from potentially damaging cellular-based T-cell responses involving type 1 helper T-cells (Th1) to a more benign humoral-based immunity via Th2 cells [148–150]. However, more recent work has highlighted an exciting alternative role for 1,25(OH)2D as a potent stimulator of suppressor T-cells also termed T-regulatory cells [151–153]. In view of the importance of T-regulatory cells in directing immune tolerance, the implications for the effects of 1,25(OH)2D on these cells are considerable both in terms of its therapeutic potential and as a basis for a clearer role for vitamin D in normal immune responses. These and other issues are discussed in much greater detail in Chapter 36. In summary it is now clear that immunomodulatory actions of either exogenously added or locally synthesized 1,25(OH)2D can be broadly divided into either autocrine/intracrine activation of macrophage function or paracrine suppression of lymphocyte function. We have postulated that this apparent paradox in immunoactions of the hormone, to dampen lymphocyte activity


while stimulating monocyte/macrophage function, is designed to maximize the ability of the host to combat and contain the granuloma-causing antigen, while controlling the potentially self-destructive lymphocytic response to that offending antigen. In other words, in order to prevent “overstimulation” of lymphocytes by monokines elaborated at the site of inflammation, some hormone produced by the macrophage will escape the confines of the cell in which it was made, will interact with neighboring, VDR-expressing, activated lymphocytes, and will tend to restrain what might be an otherwise overzealous, self-destructive T-cell and B-cell response to the offending antigen. As depicted in the right panel of Fig. 4, only at times of heightened immunoreactivity (i.e. extraordinary disease activity) does monokine production escape the confines of the site of inflammation and spill over into the general circulation causing elevated 1,25(OH)2D concentrations. This model would indicate that the endocrine actions of a locally produced vitamin D metabolite that escapes the inflammatory microenvironment is the exception rather than the rule. It also suggests that 1,25(OH)2D is by design an immunomodulatory cytokine in these lymphoproliferative diseases and not a hormone meant to modulate calcium homeostasis in the host. The overall importance of vitamin D as part of the normal immunomodulatory machinery is still open to some debate. Studies in vivo using animals [154–157] and humans [158,159] have highlighted significant immune abnormalities associated with vitamin D deficiency. Likewise, vitamin D supplementation has been shown to have positive effects on immune responses [160]. The extent to which this is mediated via macrophage 1α-hydroxylase and the contribution of this mechanism to the effects of UV exposure on diseases such as TB [161] are still under scrutiny and are discussed in much greater detail in Chapter 36. However, further light has been shed on this issue following the recent development of VDR [162] and 1α-hydroxylase [163] knockout mice. Although initial analysis of the VDR knockout mouse revealed minimal changes in the immune function [164], subsequent studies have documented dysregulated T-helper cell function in these animals [165]. In a similar fashion both VDR and 1α-hydroxylase knockout mice have characteristic lymph nodes dysplasia that is consistent with abnormal DC function [134,163]. These observations have underlined the potential importance of vitamin D metabolism and signaling as a regulator of immune responses and in future studies it will be interesting to assess the way these animals respond to specific inflammatory diseases.

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C. Accumulation of 1,25(OH)2D at Sites of Inflammation 1. GRANULOMA-FORMING DISEASES

Although much effort has been expended to ascertain the immunomodulatory potential of active vitamin D metabolites in human disease, considerably less is known about the immunoactions of these endogenously synthesized molecules in vivo in man and animals. If 1,25(OH)2D is truly a naturally occurring “cytokine,” then one should be able to document accumulation of the metabolite at sites of inflammation and show that the inflammatory cells at this site are under the influence of the locally-produced vitamin D metabolite. This was first accomplished by Barnes et al. [166], not in sarcoidosis, but in the infectious granuloma-forming disease, tuberculosis. They determined that the pleural space in nonhypercalcemic/calciuric patients infected with mycobacterium tuberculosis was one such site of 1,25(OH)2D accumulation. They detected a steep gradient for free, biologically-active 1,25(OH)2D across the visceral pleura in patients’ tuberculous effusions (but not in patients with nontuberculous effusions), showed that PPD-reacting T-cell clones from these patients expressed the VDR, and determined that the stimulated proliferation of these T-cell clones was susceptible to 1,25(OH)2D–mediated inhibition. They also showed that the pleural fluid of these patients contained an IFN-like peptide that stimulated the synthesis of 1,25(OH)2D by heterologous sarcoid macrophages [167]. Collectively, these data supported the idea put forward by Rook and colleagues [168] that there exists in the pleural microenvironment of patients with active pulmonary tuberculosis a system whereby: 1) mycobacterium-activated macrophages are stimulated to make 1,25(OH)2D; 2) this synthetic reaction is supported by proliferating and lymphokine- (i.e. IFNγ) producing lymphocytes in the local site of inflammation; and 3) the local accumulation of lymphokines, in turn, acts to further augment the local production of 1,25(OH)2D by the macrophage (see Fig. 4). Investigators [169] have viewed this sort of positive feedback effect of IFNγ on macrophage 1,25(OH)2D production in vivo as an efficient mechanism for dealing with antigens, like myco-bacteria, the “sarcoid antigen,” or certain viruses that are difficult for the host to irradicate. 2. OTHER INFLAMMATORY DISEASE STATES

Mawer and colleagues [170,171] have demonstrated substrate-dependent accumulation of 1,25(OH)2D in the synovial fluid of patients with “inflammatory arthritis,” including subjects with rheumatoid arthritis. These investigators speculate that the local increase in 1,25(OH)2D synthesis may contribute to periarticular

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bone loss in such individuals. A positive tissue-toserum gradient for 1,25(OH)2D has also been suggested in peritoneal dialysis patients, particularly when afflicted with peritonitis [164,165]; peritoneal macrophages from such patients have been shown to metabolize 25OHD to 1,25(OH)2D in vitro [172,173].

V. HUMAN DISEASES ASSOCIATED WITH THE EXTRA-RENAL OVERPRODUCTION OF ACTIVE VITAMIN D METABOLITES (see Table I) A. Granuloma-Forming Diseases 1. SARCOIDOSIS

Sarcoidosis is the human disease most commonly complicated by endogenous vitamin D intoxication [1–6,14–19,174–177]. In the most expansive studies published on the topic, roughly 10% of patients with sarcoidosis will develop hypercalcemia [177], and up to 50% will suffer from hypercalciuria [177] at some time during the course of their disease. In their retrospective, worldwide review of serum calcium concentrations in 3,676 patients with sarcoidosis, James et al. [177] recorded an 11% incidence of hypercalcemia (serum calcium ≥ 10.5 mg/dL). Studdy et al. [176]

TABLE I Human Disease Associated with 1,25-Dihydroxyvitamin D-mediated Hypercalcemia/Hypercalciuria Granuloma-forming diseases Infectious Tuberculosis Leprosy Candidiasis Crytococcosis Histoplasmosis Coccidioidomycosis AIDS-related pneumocystis Noninfectious Sarcoidosis Silicone-induced granulomatosis Eosinophilic granuloma Wegener’s granulomatosis Langerhans cell histiocytosis Berylliosis Infantile fat necrosis Crohn’s disease Malignant lymphoproliferative disease Hodgkin’s disease Non-Hodgkin’s lymphoma Dysgerminoma/seminoma

[182–191] [192] [193] [194] [195] [196] [197] [14–19] [198] [199] [200] [201] [202] [203] [204,205] [209–211, 213] [212, 214–216] [217]

studied 547 patients with biopsy-proven sarcoidosis in Great Britain and found hypercalcemia to be 38% more frequent in men than women and more common among Caucasians than individuals of West Indian descent. Although not systematically studied, the frequency of hypercalcemia among patients with sarcoidosis tends to be consistently higher in North America than in Northern Europe [178]. This is perhaps due to the lower latitude and more direct sunlight exposure in the United States. Although fractional intestinal calcium absorption may be increased under the influence of 1,25-(OH)2D and fractional urinary calcium excretion may be decreased in patients with renal insufficiency [38], the principal source of calcium which accumulates in the circulation in this disease is the skeleton. This fact is perhaps most strongly confirmed by the observations of Rizatto et al. [179] who documented in serial fashion a significant decrease in bone mineral density in a group of patients with chronic active sarcoidosis in whom anti-inflammatory agents, including glucocorticoids, were not used in management compared to age- and sex-matched control subjects. This fact is confirmed by the long-standing observations that hypercalcemia persists in patients with active sarcoidosis in absence of ingested calcium and may be contributed to by increased bone resorption [33]. The proximal cause of bone loss is increased osteoclastmediated bone resorption [180] and does not require the presence of extensive granulomata in the bone [181]. These observations suggest that an osteoclast activating factor (OAF) exists in this disease. One such bonafide OAF is of course already known, namely 1,25(OH)2D. 2. TUBERCULOSIS

Of the other human granuloma-forming diseases reported to be associated with vitamin D metabolitemediated hypercalcemia, tuberculosis is the most commonly reported aside from sarcoidosis. Hypercalcemia has been recognized as a complication of infection with mycobacterium tuberculosis for over eight decades [182]. That this disturbance in calcium balance is caused by the extra-renal overproduction of an active vitamin D metabolite was confirmed by investigators in the mid 1980s [183,184]. As is the case with sarcoidosis, the circulating vitamin D metabolite causing hypercalcemia 1) appears to be 1,25(OH)2D [185,186]; 2) is synthesized by disease-activated macrophages [187,188], 3) is abnormally responsive to small changes in the serum concentration of substrate 25OHD [189], and 4) is reducible under the influence of glucocorticoid in vivo [190,191]. The prevalence of hypercalcemia


in patients may be as high as 26% [182] and may be even higher, particularly in the era of AIDS, because of frequent association of hyopalbuminemia (i.e. from malnutrition) in patients with tuberculosis. The source of 1,25(OH)2D in this disease is, as it is in all of the other granuloma-forming diseases, extra-renal [183] most likely arising from the macrophage [187]. 3. OTHER INFECTIOUS DISEASES

Hypercalciuria or overt hypercalcemia has also been observed in a number of infectious diseases, most characterized by widespread granuloma formation and macrophage proliferation in infected tissue. Included among these diseases are leprosy [192], disseminated candidiasis [193], crytococcosis [194], histoplasmosis [195], and coccidioidomycosis [196]. Hypercalcemia in most of these conditions has been documented to be associated with inappropriately elevated serum concentrations of 1,25(OH)2D. The true prevalence and incidence of hypercalcemia and hypercalciuria in patients with these diseases in unknown. However, it is likely that this complication of dysregulated vitamin D metabolism and action associated with these diseases will increase in frequency as the number of immunocompromised patients, especially those with AIDS, increases worldwide. For example, hypercalcemia in association with elevated circulating levels of 1,25(OH)2D has been reported in an AIDS patient with pneumocystis [197]; both serum calcium and 1,25(OH)2D concentrations dropped in this patient with successful treatment of his opportunistic infection. 4. NONINFECTIOUS GRANULOMA-FORMING DISEASES

The syndrome of extra-renal overproduction of 1,25(OH)2D has also been documented in adult patients with widespread silicone-induced granulomata [198], eosinophilic granuloma [199], Wegener’s granulomatosis [200], and Langerhans cell histiocytosis [201]. Although the active vitamin D metabolite was not measured, dysregulated calcium balance in the granuloma-forming pulmonary disease berylliosis is also attributed to the extra-renal production of 1,25(OH)2D [202]. In addition, 1,25(OH)2D–mediated hypercalcemia has been observed in newborn infants suffering from massive subcutaneous fat necrosis [203]; this is a transient disorder associated with birth trauma and characterized histopathologically by the proliferation of “foreign body-type” giant cells around cholesterolshaped crystals in necrotizing, subcutaneous adipose tissue. Finally, there are also reports of elevated serum levels of 1,25(OH)2D and associated hypercalcemia in patients with inflammatory bowel disease [204]. The possible impact of extra-renal 1α-hydroxylase in this

1391

clinical situation is an exciting new development, in part because of the prevalence of Crohn’s disease particularly in developed countries [205], but also because of several recent reports which have documented expression of 1α-hydroxylase along the gastrointestinal tract [24,206–208].

B. Malignant Lymphoproliferative Disorders By the 1980s, data was accumulating to suggest that a vitamin D–mediated disturbance in calcium metabolism was not confined to patients with granuloma-forming diseases and could also be observed in patients with lymphoproliferative neoplasms [209–212]. More recent reports [213,214] indicate that the extra-renal overproduction of 1,25(OH)2D is the most common cause of hypercalciuria and hypercalcemia in patients with non-Hodgkin and Hodgkin lymphoma, especially in patients with B-cell neoplasms, whether or not the tumor is associated with AIDS in the patient [212]. In fact, in the Seymour study [206] 71% of normocalcemic patients with non-Hodgkin lymphoma had hypercalciuria (fractional urinary calcium excretion >0.15 mg/dL glomerular filtrate) and most of these had serum 1,25(OH)2D levels that were above the mid range of normal or frankly elevated. As is the situation with hypercalciuric/calcemic patients with sarcoidosis or other granuloma-forming disease and elevated circulating 1,25(OH)2D levels, the serum concentrations of PTH are suppressed and PTHrP normal (i.e. not elevated) in lymphoma patients, indicative of the state of dysregulated overproduction of the active vitamin D hormone. Results of clinical studies of hypercalcemic patients with lymphoma pre and post successful anti-tumor therapy [212–215] are most compatible with either the tumor being an immediate source of an active vitamin D metabolite or the source of a soluble factor (i.e. peptide), which stimulates the production of 1,25(OH)2D in the kidney or in other inflammatory cells. Recent data from our group suggest that the latter is the case. Specifically, we carried out extensive analysis of a patient with hypercalcemia and raised circulating levels of 1,25(OH)2D associated with a splenic B-cell lymphoma [216]. The abnormalities in serum 1,25(OH)2D and calcium were corrected following resection of the spleen, and subsequent immunonhistochemical analysis of this tissue revealed increased expression of 1α-hydroxylase in macrophages adjacent to the tumor, but not in the tumor itself. This raises further potentially important questions: 1) what is the nature of the tumor-derived factor that is able to stimulate macrophage 1α-hydroxylase?

1392 2) Is macrophage-derived 1,25(OH)2D a contributing factor to the hypercalcemia associated with other types of tumors? In seeking to answer the latter question, current studies within our group have focused on the expression and function of macrophage 1α-hydroxylase in dysgerminomas, gonadal tumors that are associated with granulomata and which have shown previously to be linked to hypercalcemia [217].

C. Non-Granuloma-forming Conditions As outlined in section II.C, cloning of the gene for 1α-hydroxylase (CYP1α) has enabled a much more comprehensive appraisal of the tissue distribution of this enzyme than was previously available. Indeed, the human cDNA for CYP1α was cloned from keratinocytes, a well-established extra-renal source of 1,25(OH)2D [218,219]. Studies have defined both the renal and extra-renal tissues that express 1α-hydroxylase [23,24]. Specific sites of interest include skin [24,218,219], prostate [220], placenta [24,221,222] (one of the first tissues to show extra-renal synthesis of 1,25(OH)2D [223]), parathyroids [224], vasculature [225,226], gut [24,206–208], brain [24], and pancreas [24]. The precise function of 1α-hydroxylase expression at these sites is currently the focus of considerable attention. In tissues such as the prostate [220,227], colon, [206–208], and parathyroids [224,228], the enzyme has been postulated to fulfill an antitumor function by increasing local concentrations of antiproliferative 1,25(OH)2D, and this is discussed in greater detail in Chapters 93–96. However, in many extrarenal tissues particularly the pancreas, gut, vasculature, and brain, it is probable that 1α-hydroxylase will fulfill a function which is more akin to the macrophage, namely as a generator of immunomodulatory 1,25(OH)2D. For example, the most potent activator of 1α-hydroxylase in cultured human endothelial cells [225], as well as epithelial cells from the distal nephron [90], is LPS, with both cell types, like macrophages, showing strong expression of CD14/TLR4. Thus, the function of extra-renal 1α-hydroxylase may be far more diverse than originally thought: putative roles in preclampsia [221], implantation [222], and vascular disease [225] have been proposed in addition to its link with common cancers [206–208,220,227]. Nevertheless, at the present moment in time the most well-documented pathological paradigm for extra-renal 1α-hydroxylase remains the overproduction of 1,25(OH)2D associated with granulomatous disease. The clinical management of this is therefore discussed in greater detail in the following sections.


VI. DIAGNOSIS, PREVENTION, AND TREATMENT OF THE PATIENT WITH ENDOGENOUS VITAMIN D INTOXICATION A. Diagnosis The diagnosis of so-called “endogenous” vitamin D intoxication is made when the following three criteria are met. First is the presence of hypercalciuria and/or hypercalcemia in a patient with an inappropriately elevated serum 1,25(OH)2D (i.e. the serum 1,25(OH)2D concentration is not suppressed below 20 pg/ml). Second is the presence in the serum of an appropriately suppressed PTH level if the patient’s free (ionized) serum calcium concentration is high; this is evidence that the calcium sensing receptor in the plasma membrane of the host’s parathyroid cell is normally operative. This distinguishes the patient with primary hyperparathyroidism and elevated 1,25(OH)2D levels, in whom the calcium-sensing receptor signal transduction pathway to control PTH synthesis and release is disrupted, and from the individual with endogenous vitamin D intoxication and elevated 1,25(OH)2D levels. The other major exception here is the patient with absorptive hypercalciuria who possesses, as a primary or secondary abnormality, an inappropriately elevated circulating 1,25(OH)2D concentration [229]. Third is the exclusion of exogenous vitamin D intoxication arising from the oral or parental administration of an active vitamin D metabolite or the substrate for endogenous synthesis of an active vitamin D metabolite. The most common cause of exogenous vitamin D intoxication occurs with the ingestion or injection of large doses of vitamin D2 (ergocalciferol) or vitamin D3 (cholecalciferol), and can usually be detected by measuring a frankly elevated serum 25OHD level; most, if not all, currently available serum assays for 25OHD do not distinguish 25OHD2 from 25OHD3 [230]. Exogenous vitamin D intoxication may occur in patients taking too much 1-α-OHD, dihydrotachysterol (DHT), or 1,25(OH)2D itself; the two former compounds undergo 25-hydroxylation in the host hepatocyte. In these instances, the 1,25(OH)2D, not the 25OHD, concentration will be elevated, making the distinction from endogenous vitamin D intoxication from the extra-renal overproduction of 1,25(OH)2D impossible on strictly biochemical grounds. In these situations, a complete knowledge of the medications to which the patient has access is critical for making the correct diagnosis. Examples of such patients would be those receiving relatively large amounts of vitamin D, a vitamin D metabolite, or a vitamin D analog (i.e. patients with hypoparathyroidism, renal failure, and


psoriasis, respectively). Most of the newer vitamin D analogs currently in clinical use [231] will not be measured efficiently in the serum 1,25(OH)2D assay, so awareness of the use of these kinds of topically- and orally-administered agents is of particular importance to the diagnosing clinician.

B. Early Detection and Prevention of Hypercalciuria/Hypercalcemia 1. IDENTIFYING PATIENTS AT RISK

Considering the fact that the means of specifically inhibiting the production of active metabolites of vitamin D or of blocking the response of cells to active vitamin D derivatives is not yet available, the best way to treat vitamin D–mediated abnormalities in calcium balance is to prevent their occurrence. The first step is to identify patients at risk. This encompasses primarily patients with granuloma-forming disease as well as patients with malignant lymphoproliferative disorders, especially B-cell and Hodgkin’s lymphoma. Disordered calcium balance in these groups of patients results from the endogenous and dysregulated overproduction of 1,25(OH)2D by inflammatory cells. Production of the offending vitamin D metabolite is, in turn, directly related to the amount of substrate 25OHD available to the macrophage 1α-hydroxylase (see Fig. 2) as well as to the severity and activity of the underlying disease. In terms of sarcoidosis, for example, patients at risk would be those with: 1) widespread, active disease; 2) a previous history of hypercalciuria or hypercalcemia; 3) a diet enriched in vitamin D and/or calcium; 4) a recent history of sunlight exposure or treatment with vitamin D; and 5) an intercurrent condition, or medicinal treatment of an intercurrent condition, that increases bone resorption or decreases the glomerular filtration rate. 2. SCREENING PATIENTS AT RISK

Since hypercalciuria almost always precedes the development of overt hypercalcemia in this set of disorders, patients at risk should be checked for the presence of occult hypercalciuria. This is best accomplished by a fasting two-hour urine collection for calcium and creatinine. If the calcium:creatinine ratio (gm:gm) is not abnormally high ( 2.8 2.2 >4 0.2 1.6 2.1 1.8 0.7 0.4 0.5

t1/2 (hr) 7.0 2.5

Pharmacokinetic Data on Vitamin D Analogs

AUC∞ (ng/ml × hr) 9596 7355 13,228 27 1216 255 267 142 46 40

Serum clearance (ml/hr/kg)

Binding affinity for DBP (M)

Relative binding affinity for DBPb

Rate of metabolismc

21 27 15 7407 167 784 693 1408 4348 5000

9 × 10−9 1.5–6.0 × 10−7 1.7 × 10−8 1.7 × 10−6 5.2 × 10−6 7.9 × 10−6 3.2 × 10−5 6.5 × 10−5 n.b. n.b.

33 1 17 0.1 0.1 0.03 0.02 0.007 0 0

Very slow Fast – Very fast Fast Slow – – Fast –

Metabolic clearance rate (ml/min)

Relative binding affinity for DBPe

Rate of metabolismf

1 266

Fast Very fast

5.0 48.2

Baseline (pg/ml)

AUC∞ t1/2 (hr)

67.0 10−6 3 × 10−9 1 × 10−9 3 × 10−9 3 × 10−9

> 10−6 5 × 10−12 1 × 10−12 1 × 10−10 5 × 10−11

Hybrid analogs 1β-20-epi-24a,26a,27a-tri-homo-25(OH)2D3 1β-(hydroxymethyl)-3α-20-epi-22-oxa-24a,26a,27a-tri-homo-25-OH-D3

1 × 10−7 >10−6

2 × 10−9 1 × 10−8

Analogs with double bond between C16 and C17 1α,25(OH)2-16-ene-D3 1α,25(OH)2-16-ene-23-yne-D3

1 × 10−9 0.8 × 10−9

1 × 10−10 6 × 10−10

a ED 3 50 (in M), ligand concentration required to reach 50% displacement of [ H]1,25(OH)2D3 binding to recombinant human VDR from transfected COS-1 cells. b ED (in M), effective dose required to produce 50% of maximal transcription activation of a reporter gene containing the osteocalcin VDRE in ROS 50 17/2.8 cells in serum-free culture medium. The transcriptional activities of 1,25(OH)2D3 and the last two analogs in the table (16-ene analogs) were also examined in cells grown in 10% serum. Under these conditions, the ED50 of the three compounds were 5 ×10−10 M, 6 ×10−12 M, and 2 × 10−11 M, respectively.

CHAPTER 83 Molecular Basis for Differential Action of Vitamin D Analogs

their transcriptional activity increases manyfold without a change in their affinity for the VDR [56,57].

C. The D-ring The crystal structure of VDR-1,25(OH)2D3 complex show that the D-ring forms several hydrophobic interactions in the ligand-binding pocket, although these interactions are less critical than those contributed by the A-ring and the side chain [43]. A modification that enhances VDR-mediated transcriptional activity of the hormone is insertion of a double bond between carbons 16 and 17 in the D-ring [58]. Compounds containing this modification, with or without additional chemical modifications in the side chain, have transcriptional activity significantly greater than that of 1,25(OH)2D3 without a significant increase in their affinity for VDR. In addition, this group of analogs exhibits diminished calcium regulating activity. The unique properties of these compounds include lower affinity for vitamin D– binding protein and slower catabolism [59,60], both of which may increase potency (Table I). It is not known what the unsaturation at C16-C17 is doing to the mode of ligand interaction with the VDR, but dot maps suggest that this modification restricts the flexibility of the side chain, and therefore has the potential to force it into contact points that are distinguishable from those used by the side chain of the natural hormone [61]. In conclusion, the A-ring regulates high affinity for VDR, the D-ring controls ligand uptake and metabolism and possibly contributes to the flexibility of the side chain, and the side chain regulates both affinity for VDR and its transactivation (Fig. 1).

III. DIFFERENTIAL ACTIVATION OF THE VDR BY SYNTHETIC ANALOGS A large amount of information on the structure of the LBDs of various nuclear receptors bound to natural or synthetic ligands has accumulated in the past few years. These studies provide evidence for the structural flexibility of nuclear receptors and a partial explanation for the mechanism of action of agonists, antagonist, and selective receptor modulators. Additional studies, using biochemical, molecular, and cellular biology approaches, provide ample evidence to the many ways by which nuclear receptor actions can be manipulated and refined by synthetic ligands. Interestingly, seven years ago we had little evidence for similar flexibility and heterogeneity in transcriptional activation of the VDR. However, today we are able to distinguish at least three groups of ligands that modulate the VDR

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differently from the natural hormone. These include superaognists (such as the 20-epi analogs mentioned above), antagonists, and tissue- or gene-selective agonists. Unfortunately, structural studies provide only partial explanation for the mechanism of action of superagonist, and structural data is not yet available for VDR’s LBD bound to antagonists or to tissue-selective agonists. Therefore, the text below will describe primarily biological and biochemical experiments that provide evidence for the differential activation of the VDR by these three groups of compounds.

A. Superagonists 1. DEFINITION AND ASSESSMENT OF SUPERAGONISTS

Superagonists are analogs that are significantly more transcriptionally potent or efficacious than 1,25(OH)2D3. Earlier studies of analogs’ actions used primarily ligand-binding assays (to determine affinity for the VDR) and cellular assays (to determine antiproliferative activities) [41]. More recently, the identification and assessment of their activities is accomplished by using common molecular endocrinology methodologies to assess transcriptional activation of genes by steroid hormone receptors. When several DNA binding sites for VDR-RXR complexes in the promoters of vitamin D responsive genes were characterized, the most common tool to assess transcriptional activity induced by analogs became reporter gene assays, in which a fusion gene containing a vitamin D response element attached to a heterologous promoter and a reporter gene is transfected into eukaryotic cells that express the VDR [62–64]. A typical dose-response curve of reporter gene expression in cells treated with the natural hormone shows a gradual induction of reporter gene expression that may reach plateau at 10 nM, with an effective dose for 50% of maximal activity (ED50) of 1–5 nM. A superagonist by definition is an analog that induces transcription with an ED50 significantly lower than that of the natural hormone, or that induces severalfold greater maximal transcription [Fig. 4 and ref. 21]. The advantages of this assay are that, unlike the growthinhibitory assay, it directly measures a single VDRmediated transcriptional activity, it is not subject to other vitamin D–mediated signaling that may take place in cell growth assays, and it is not dependent on vitamin D–mediated effects on RNA stability. It is also faster and more reproducible than the growth inhibitory assays. Its disadvantage is that, in eukaryotic cells (especially mammalian cells), transcriptional activity may still depend on analog uptake (which may vary with differences in binding to DBP in the serum) [65]

1476

SARA PELEG

A

B

Transcription

Dimerization

100

20E-1,25D3

80 60

1,25D3

40 20 0

C

120

% Maximal activity

% Maximal activity

120

D

100 80

1,25D3

60 40

20E-1,25D3

20 0

1,25D3

60 40 20

0 .001 .01 .1 1 10 100 1000 10000 Ligand concentration (nM)

GRIP-1 binding 120

1 10 100 1000 10000 0 .001 .01 .1 Ligand concentration (nM)

% Maximal activity

% Maximal activity

120

20E-1,25D3

80

0

0 .001 .01 .1 1 10 100 1000 10000 Ligand concentration (nM)

SRC-1 binding

100

100 80

20E-1,25D3

60 40

1,25D3

20 0

1 10 100 1000 10000 0 .001 .01 .1 Ligand concentration (nM)

FIGURE 4 Transcriptional potency of the superagonist 20-epi-1,25(OH)2D3 is correlated with an increase in dimerization potency but not with an increase in binding to the p160 coactivators SRC-1 and GRIP. Transcription (A) was assessed by co-transfecting CV-1 cells with the hVDR and a reporter containing the osteocalcin VDRE attached to the minimal thymidine kinase promoter and the growth hormone gene. Dimerization (B) binding to SRC (C) and binding to GRIP (D) were assessed by pull-down assays, using GST-fusion protein to bind the 35S-labeled in vitro synthesized hVDR. Bound VDR was eluted, separated by SDS-PAGE, visualized by autoradiography, and quantified by densitometry scanning. These experiments suggest that the dimerization interface rather than the coactivator binding interface is distinct in the VDR-hormone and VDR-20-epi analog complexes [69].

and on the cellular metabolism of the analog [66,67]. For these reasons, it is necessary to confirm differential activation of the VDR by testing analogs in eukaryotic systems that do not contain vitamin D metabolic enzymes (such as yeast) [68], by cell-free transcriptional assays, or by other in vitro assays that examine individual ligand-dependent events that are essential for transcriptional activity in vivo (binding to DNA or interactions with dimerization partners and with other partners of transcription) (Fig. 4) [21,69]. 2. THE 20-NATURAL SUPERAGONISTS

Using these approaches, several analogs have been identified that have greater transcriptional potency than 1,25(OH)2D3. The common feature of these compounds is that they have side-chain modifications but an unmodified A-ring. These analogs’ modifications, such as unsaturation at C-23 or fluorine atoms at C-24, limit their availability to 24-hydroxylase [41].

Additional modifications, such as substitution of hydrogen for fluorine atoms at positions 26 and 27 or homologation at these positions, alter access of these compounds to the 26-hydroxylase in the kidney [70]. However, these modifications also have enormous effect on antiproliferative activity and transcriptional response as determined by reporter gene assays in cultured cells. As mentioned above, another modification, unsaturation at positions 16–17 of the D ring, also increases the transcriptional potency of these compounds. This modification alone, or in combination with the aforementioned side-chain modifications, has been shown to slow down 24-hydroxylation and cause cellular accumulation of active catabolic intermediates [71]. Therefore, it appears that almost all of the increases in the potencies of compounds from this group are due to altered pharmacokinetics, including a decrease in binding to DBP that in vitro causes the cells to take up the analogs faster than 1,25(OH)2D3 [65]. However, several studies


have provided in vitro evidence for a true change in their ability to induce receptor-mediated activity. First, Uskokovic et al. have shown that the analogs containing the 16-ene modification plus hexafluorine substitutions at the 26 and 27 positions have a twofold greater affinity for the VDR than the natural hormone [71], although this affinity is not proportional to the increase in transcriptional potency (100- to 1000-fold) [72]. Another study has shown that several fluorinated compounds increase the affinity of the VDR-analog complex for its dimerization partner RXR or increase the affinity of the VDR-RXR complex for DNA [72]. Again, these changes are not proportional to the observed increases in transcriptional activities of these compounds in culture, suggesting that these modifications may affect additional parameters of transcriptional activation of the VDR. 3. THE 20-EPI SUPERAGONISTS

The best-characterized modulators of the VDR are the 20-epi analogs, including 20-epi-1α,25(OH)2D3 (MC-1288) and 20-epi-22-oxa-24a,26a,27a-tri-homo1α,25(OH)2D3 (KH-1060) (Table I) [55]. These compounds, synthesized by Leo Pharmaceuticals, share one distinct modification, a stereochemical change at carbon 20. Dot maps revealed that the energy-minimized side chain conformers of these analogs have mostly northwest orientations, instead of the northeast orientations of side chains of the natural hormone and 20-natural analogs [73]. Without chemical modification of the 20-epi side chain, the analog 20-epi 1,25(OH)2D3 (MC-1288) has a hundredfold greater growth-inhibitory and transcriptional activities than 1,25D3 does. The addition of chemical modifications to the 20-epi side chains in the analogs KH-1060 and MC-1301 (20-epi24a,26a,27a-tri-homo-1α,25(OH)2D3) increased their activities up to 3,000-fold compared with that of 1,25D3 [21,55,74]. These phenomenal improvements in activities are not associated with any increase in affinity for the VDR [21,55]. These compounds act directly by altering VDR actions, as they induce reporter gene expression more effectively than 1,25D3 in many cell lines and through several types of vitamin D–response elements [21]. These results occurred in both the presence and the absence of DBP, thus reducing the probability that altered DBP-modulated delivery of these ligands contributes to their potency in culture. It has been shown that 24-hydroxylase does not effectively catabolize several 20-epi analogs [67,75]. However, that they are still significantly more potent than 1,25(OH)2D3 in cells lacking 24-hydroxylase activity (ROS 17/2.8) [21] excludes differential catabolism as a primary explanation for their potency. The most compelling evidence for the

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molecular basis of the enhanced activity of these compounds came from a series of studies demonstrating, in a yeast two-hybrid system, that the 20-epi analog KH-1060 induces dimerization more effectively than 1,25(OH)2D3 does [68]. More importantly, by using cell-free assay systems, it was shown that binding of 20-epi analogs to the VDR induces a conformation distinct from that induced by the natural hormone [21]. This distinct conformation is associated with the enhanced ability of VDR-20-epi analog complexes to dimerize with RXR [69,76]; to bind to specific DNA sequences (VDREs) [21]; to interact with a key component of the transcription apparatus, DRIP 205; and to induce transcription in cell-free systems [77] (Fig. 4). These biochemical findings strongly suggest that the contact points of 20-epi analogs with the VDR are different from those used by the natural hormone and that these differences cause conformational changes that affect the properties or availability of VDR binding domains for transcription partners (Fig. 4). Biochemical evidence that the modes of interaction of the natural hormone and the analogs are different came from studies of site-directed mutagenesis of the VDR and from comparing the effects of mutations on binding of the hormone and the 20-epi analogs [78]. These experiments showed that the ability of the 20-epi analogs to interact with a VDR that lacked contact sites essential for binding of 1,25D3 is not impaired. The difference in binding requirement appears to involve residues in the C-terminal region of the VDR, including the AF-2 core/helix 12. This domain, which is essential for coordinating the interaction of coactivators with the VDR, also has contact points for the side chain of 1,25D3 but appears to be less important for the binding of the 20-epi analogs to VDR. Studies demonstrating that the half-lives of the VDR-20-epi analog complexes were significantly greater than the half-lives of VDR complexes with their 20-natural counterparts suggested that the 20-epi analogs may be buried more deeply in the binding pocket than the 20-natural compounds are [78]. Interestingly, structural analysis of the VDR-20-epi analog complexes by X-ray crystallography did not support the biochemical data, because it did not show that the 20-epi analogs use contact points different from those used by 1,25D3, unless the analogs had chemical modifications in their side chains. Furthermore, the structural studies did not provide evidence for a significant change in the functional surface of the VDR-20-epi analog complexes that would explain the modified interactions of these complexes with dimerization partners and with the bridging factor DRIP 205 in vitro [79]. An explanation for this discrepancy could be that 20-epi analogs shift their position in the ligand-binding pocket when the VDR is associated with transcription

1478 A

SARA PELEG

VDR-20-epi 1,25D3 monomers Trypsin

20-epi 1,25D3 (-LOG M)

NL NL 11 10 9

8 7

VDR

34 kDa 32 kDa 28 kDa

B

VDR-20-epi 1,25D3 heterodimers Trypsin

20-epi 1,25D3 (-LOG M)

NL 11 10 9

8

7 34 kDa 32 kDa 28 kDa

FIGURE 5 The effect of VDR interaction with transcriptional partners on the mode of analog binding. (A) In vitro translated VDR was incubated with 20-epi analog (20-epi-1,25(OH)2D3) and then subjected to trypsin digestion. (B) In vitro translated VDR was incubated with the 20-epi analog, with GST-RXR and gluatathione-Sepharose beads. VDR-RXR complexes were separated from the unbound VDR and then subjected to trypsin digestion. Note the differences in the conformation of VDR-analog complexes with RXR and VDR-analog monomers. Also, note the 100-fold increase in the ability of the analog to stabilize the conformation of the heterodimerized VDR [69].

partners [83] (Fig. 5), and these changes are not reflected in the monomer structure that was analyzed by X-ray crystallography. Additional explanation could be that VDR bound to 20-epi analogs in the cells undergoes modifications that enhance its abilities to bind DNA and partners of transcription, and these modifications do not occur in the cell-free systems [80,81].

B. Low-calcemic Analogs/Selective Agonists 1. ASSESSMENT OF NONCALCEMIC SELECTIVE AGONISTS

Surprisingly, a major misconception exists in the vitamin D field because analogs have been defined as noncalcemic or low-calcemic if they have significant receptor binding activity in vitro and growth-inhibitory activity in culture but when administered to animals do not induce hypercalcemia/hypercalciurea at the concentration range at which 1,25(OH)2D3 does [41]. However, low calcemic activity in vivo may simply be due to pharmacokinetic properties such as rapid clearance

rate and short terminal half-life, which would lead to very poor overall biological activities [70]. Therefore, a more accurate definition for low-calcemic analog is a compound that has a wider safety window than 1,25(OH)2D3 to induce a biological response without inducing hypercalcemia [82]. We define analogs that have these desirable qualities in vivo as “selective” agonists because, by definition, the ability to induce biological response without changing calcium homeostasis requires that the analog has a preference for a given target tissue (e.g., tumor cells, the parathyroid gland, and skin) over calcium-regulating organs such as intestine, kidney, and bone. In this review, we will discuss only those low-calcemic selective agonists that have shown evidence of regulating VDR functions differently from 1,25(OH)2D3 in culture and in cell-free systems. 2. MECHANISM OF ACTION OF SELECTIVE AGONISTS

The best-characterized analogs that can be defined as low-calcemic but biologically active at a reasonable concentration range in vivo and as selective modulators of the VDR in vitro are three structurally unrelated compounds (Fig. 6). One is Leo Pharmaceuticals’ EB-1089, a side chain-modified analog (22,24-diene26,27-bishomo-1,25-dihydroxyvitamin D3) that inhibits tumor growth in vivo with half the calcemic activity of 1,25(OH)2D3. Another of these analogs is Chugai’s OCT (22-oxa-1,25-dihydroxyvitamin D3), also a side chain modified analog that inhibits parathyroid hormone secretion without inducing hypercalcemia. The third analog is Roche’s Ro-26-9228 (1αF-16-ene-20epi-23-ene-26,27-bishomo-25-hydroxyvitamin D3), a hybrid analog that restores bone loss without inducing hypercalcemia at a wide concentration range. Examination of the mechanism of action of these apparent selective agonists has raised three questions: (1) Is there evidence that their mode of interaction with and transcription activation of the VDR is significantly different from that of the natural hormone? (2) Is there evidence that gene expression in cells or tissues that regulate calcium homeostasis is modulated differently by the natural hormone and by these analogs? (3) Is there compelling evidence that the analog has tissue or gene preference different from that of 1,25(OH)2D3? The compounds described below each have several of these features. However, additional studies are required to further evaluate and substantiate the mechanisms for their apparent selective actions. Leo Pharmaceutical developed EB-1089 primarily for use in chemotherapy of malignancies, with a focus on breast cancer [83–85]. In vivo, EB-1089 has a reasonable safety window for inhibiting tumor growth without hypercalcemia, but how that occurs is not

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22

24

22 O

OH

H

H

20 23

OH

H

H

OH

H

1 HO 3β

1αOH 1

EB-1089

OH

HO OCT

HO

F Ro-26-9228

FIGURE 6 Structural formulas of selective agonists. EB-1089 was synthesized by Leo Pharmaceuticals; OCT was synthesized by Chugai, and Ro-26-9228 was synthesized by Roche Bioscience.

known [84,86]. There have been few attempts to distinguish EB-1089’s mechanism of VDR activation from VDR-mediated actions of 1,25(OH)2D3. In vitro assays demonstrated that the electrophoretic mobilities of VDR-EB-1089-DNA complexes and VDR1,25(OH)2D3-DNA complexes are different, suggesting that the conformations of the complexes or their compositions are different [87]. Another study showed that EB-1089-VDR complexes have an apparent binding preference for IP9 (inverted repeat with nine intervening nucleotides) VDRE instead of the classic DR3 (direct repeats with three intervening nucleotides) VDREs, whereas 1,25(OH)2D3-VDR complexes do not. Cell culture studies also revealed that EB-1089, but not 1,25(OH)2D3, has some preference for inducing transcription through the IP9 VDRE of a transfected reporter gene [87,88]. Because the response elements used were synthetic and not present in natural genes, the relevance of these findings is not clear, except that they showed differences in the molecular properties of EB-1089-VDR and 1,25(OH)2D3-VDR complexes. Unfortunately, these studies were not extended to test cause and effect relationship between these phenomena and the pharmacological actions of EB-1089 in vitro and in vivo. The results of cell-culture and in vivo studies to determine the mechanism for the low calcemic activity of EB-1089 have been somewhat equivocal and provide only a partial explanation for its selective activities. Cell culture studies have shown that EB-1089 is as good or better than 1,25(OH)2D3 at inducing bone resorption [89]. In vivo studies demonstrated that EB-1089 is somewhat less effective than 1,25(OH)2D3 at inducing 24-hydroxylase and calbindin D9K mRNAs in the duodenum [90]. On the other hand, EB-1089 and 1,25(OH)2D3 have similar abilities to induce these genes in the kidney. These results suggest that EB-1089 has different preference for the duodenum than

1,25(OH)2D3 and that the lower calcemic activity of EB-1089 may be associated with a lower ability to induce gene expression at a site that regulates calcium absorption [90]. It may also suggest that EB-1089 has a different tissue preference in vivo. In conclusion, these in vitro and cell culture studies provide some evidence that EB-1089 is a selective modulator of the VDR, and that its selectivity may be either at the level of target genes or target tissues, but additional studies in vivo and in vitro are necessary to substantiate these findings. Another side chain modified analog, OCT, has been shown to be potent in animals without inducing hypercalcemia [41]. The pharmacokinetic properties of this analog are significantly different from those of 1,25(OH)2D3. First and foremost, OCT binds very poorly to DBP [91], which may contribute to its short half-life in animals. However, there is strong evidence that this analog has a selective action in vivo, as it induces only brief intestinal calcium absorption but prolonged inhibition of parathyroid hormone secretion. The brief intestinal calcium absorption correlates with transient induction of calbindin D9K, a vitamin D receptor-modulated gene. The brief induction of calbindin D9K by OCT is significantly different from the longer 1,25(OH)2D3-dependent induction of the gene [91–93]. Two interesting features about these differences in gene expression between the two compounds are that the analog is retained in the intestine longer and that its maximal binding to the intestinal VDR is higher than that of 1,25(OH)2D3. These findings suggest that the brief period of gene expression is not due to a short half-life of the intestinal VDR-OCT complexes, but perhaps to transcriptional activation events downstream of the formation of VDR-ligand complexes. That OCT is indeed a selective agonist has been supported by in vitro assays that showed differences in

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C. Antagonists 1. ASSESSMENT OF ANTAGONISTS

Antagonists are receptor-binding compounds that inhibit the actions of the respective natural hormone and, on their own, are unable to elicit a transcriptional response through the receptor. Therapeutically, these compounds are exceedingly valuable, as they are used to prevent growth of hormone-responsive malignancies (estrogen antagonists) [96–98] and to regulate reproductive processes (progesterone antagonists) [99]. Only a few of these compounds are pure antagonists, but in many cases they may act as antagonists on certain target genes or in certain tissues and as moderate

% Maximal reporter gene expression

A

Transcription in Caco-2 cells 120

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ED50 1,25D3 = 2 nM Ro-26-9228 = 120 nM

80

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.1

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recruitment of transcriptional coactivators to VDR-OCT and VDR-1,25(OH)2D3 complexes [94]. However, the assays did not provide direct proof that the coactivator selectivity of VDR-OCT complexes in vitro leads to cell-type selective or gene-selective actions in vivo. Another example of an analog with convincing tissue- and cell-selective properties is the Roche compound Ro-26-9228 [82]. Biochemically it is a “hybrid” analog because it contains modifications in both the A-ring and the side chain. Its affinity for VDR is 8- to 10-fold lower than the affinity of the natural hormone, as would be expected from replacement of the 1α-OH group with a fluorine atom. However, examination of its transcriptional activities in cell culture showed that Ro-26-9228 is equipotent to the natural hormone in osteoblasts, whereas in intestinal cells it is 60 times less potent (Fig. 7 and [82]). Further studies of the VDR from the two cell types has revealed that when it binds the analog in intestinal cells, it does not acquire the ability to interact with dimerization partners and transcription coactivators, whereas when the analog binds the VDR from osteoblasts or synthetic VDR in vitro, it does have these abilities. In contrast, the natural hormone 1,25(OH)2D3 has these abilities in both cell types [95]. This apparent cell selectivity in vitro mimics the tissue preference in vivo: the administration of Ro-26-9228 to rats induces VDR–dependent gene expression in the bone but not in the duodenum (Fig. 8 and [82]). These properties of the analog in vivo are associated with prevention of bone loss in osteopenic rats without induction of hypercalcemia over a very wide concentration range. This suggests that poor recognition of the analog by the VDR in the duodenum spares the animals from hypercalcemia induced by enhanced calcium absorption, whereas the analog’s preference for bone (probably for osteoblasts) promotes bone-remodeling activities that lead to a net bone gain [82].

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Transcription in MG-63 cells 120

100

ED50 1,25D3 = 3 nM Ro-26-9228 = 5 nM

80

60

1,25D3

40 Ro-26-9228 20

0 0 .0001 .001 .01

.1

1

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FIGURE 7

A selective modulation of VDR-mediated transcription in cultured cells. Human colon carcinoma cells (Caco-2) or human osteosarcoma cells, MG-63 were transfected with a reporter gene containing a minimal thymidine kinase promoter and a vitamin D response element. The cells were treated with the indicated doses of 1,25(OH)2D3 or the analog Ro-26-9228, and reporter gene expression was assessed 48 h later. Note that the analog is equipotent to 1,25(OH)2D3 in the osteoblast-like cells but is 60 times less potent than 1,25(OH)2D3 in the intestinal-like cells [82].

agonists in others. These properties opened numerous therapeutic possibilities that are best represented by the synthetic estrogen receptor-binding ligands termed selective estrogen receptor modulators (SERMs) that include tamoxifen and raloxifene [96,97]. Their antagonistic properties are used to inhibit the growth of estrogen-dependent breast cancer cells, while their agonist activities are used to maintain bone integrity in

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TGF-β2 (Bone)

24-hydroxylase (Duodenum) 8 * 8

6

4

2

0 Sham

OVX

1,25D3 Ro-26-9228 (0.2 µg/kg) (5 µg/kg)

Densitometry units (normalized)

Densitometry units (normalized)

10

*

6

*

4

2

0 Sham

OVX

1,25D3 Ro-26-9228 (0.2 µg/kg) (5 µg/kg)

FIGURE 8

A selective modulation of gene expression in target tissues for vitamin D action by the analog Ro-26-9228, in vivo. Female rats were either sham-operated or ovariectomized for three weeks and then given vehicle, or the indicated dose of the vitamin D compound. Total RNA was isolated from the duodenum and the tibia of these animals 7 h after treatment. The mRNAs were quantified by northern blots (24-hydroxylase) or by semiquantitative RT-PCR (TGF-β2). Note that Ro-26-9228 did not induce a significant gene expression in the duodenum whereas it did so in the bone. In contrast 1,25(OH)2D3 induced expression of these genes in both tissues [82].

postmenopausal women. At the molecular level, binding of antagonists to their respective receptors induces a conformational change that is not permissive for recruitment of coactivators. In fact, antagonist binding prevents coactivator binding in vitro by causing a conformational change in the position of the C-terminal AF-2 core [46]. The cellular conditions that render these complexes transcriptionally active are debated but include changing ratios of coactivators and corepressors, and interaction of another transcription activation domain (the N-terminal AF-1) with the transcription apparatus under conditions that are not permissive for this interaction through the AF-2 domain of the antagonist-bound receptor (Fig. 3 and [98,99]). Numerous vitamin D analogs have been synthesized, but until five years ago none exhibited clear antagonistic activities. One reason could be the remarkable structural flexibility of vitamin D derivatives (unlike the relatively rigid four-ring steroid hormones) [100]. This flexibility may also allow these ligands to adapt their conformations to the binding pocket of the VDR despite many structural modifications, and therefore vitamin D analogs may be less likely to disrupt formation of a functional VDR surface. Another possibility is that, for practical reasons, vitamin D antagonists are not as obviously useful clinically as selective agonists might be, and therefore have not been synthesized strategically or have not been investigated. Despite these limitations, there are two groups of vitamin D analogs with significant

antagonistic activities. The best representative of the first group is the 26,23-lactone TEI 9647, synthesized at the Teijin Institute for Biomedical Research. The other group of antagonists is the carboxylic esters, the most potent representative of which is ZK 159222, synthesized at Schering AG (Fig. 9). 2. THE LACTONES

The lactone analog TEI 9647 (23S-25-dehydro1α-hydroxyvitamin D3-26,23-lactone) has an affinity for VDR which is one-tenth that of 1,25(OH)2D3 [101]. Its mode of interaction with the VDR is different from that of 1,25(OH)2D3, because the analog stabilizes a conformation that is significantly different from that stabilized by 1,25(OH)2D3 [102]. This distinct Antagonists OH O 23

H

O

22

OH TEI 9647

26 O

O

H

HO

24

H H

HO

OH

ZK 159222

FIGURE 9 Structural formulas of antagonists: TEI 9647 was synthesized by Teijin, and ZK 159222 was synthesized by Schering.

1482 conformation is evident in the VDR monomer and in the VDR complexes with RXR and with DNA [103,104]. It is possible that the antagonist activities are due to disruption of the conformation of helix 12 by the bulky lactone group at the side chain of this compound. This disruption causes the inactivation of the AF-2 domain (Fig. 3) and a loss of transcriptional activity of the antagonist-bound VDR. However, X-ray crystallography of the VDR bound to this ligand confirms that hypothesis has not yet been performed. Despite the lack of structural information, this analog can effectively inhibit 1,25(OH)2D3-mediated transcriptional activity in COS-7 cells cotransfected with the human VDR and a reporter gene containing the 24-hydroxylase VDREs [103]. It also inhibits 1,25(OH)2D3-mediated transcription in Saso-2 cells transfected with the same reporter but with the endogenous VDR, and in MCF-7 cells transfected with a reporter gene containing a single DR3-type VDRE [103]. The antagonistic activity of TEI 9647 seems to involve two LBD-mediated actions: the analog appears to inhibit 1,25(OH)2D3-induced VDR-RXR dimerization and to inhibit interaction of VDR with the p160 coactivator SRC-1 [103,104]. Using HL60 cells as a model for the growth inhibitory and differentiating actions of 1,25(OH)2D3, cellular responses that are largely considered to be VDRmediated, it was shown that TEI 9647 did not induce any of the differentiation markers induced by 1,25(OH)2D3 and prevented all aspects of the growth-inhibitory and differentiating actions of 1,25(OH)2D3 [101]. These results underscore the notion that growth inhibition and differentiation induced by 1,25(OH)2D3 are indeed mediated through the nuclear VDR, although some aspects of these cellular processes are thought to be mediated through a distinct membrane receptor for 1,25D3 (see Chapter 23). TEI 9647 was examined for antagonistic and agonistic activities in normocalcemic rats and in vitamin D–and calcium-depleted rats [105]. TEI 9647 has a moderate ability to inhibit the increase in serum calcium in normocalcemic rats injected with pharmacological amounts of 1,25(OH)2D3, but this analog does not inhibit the normal physiological activities of 1,25(OH)2D3. In vitamin D–deficient and calcium-deficient rats, TEI 9647 acts as a poor agonist of intestinal calcium absorption and a somewhat better agonist of bone resorption, and it very effectively inhibits parathyroid hormone secretion. Interestingly, when given together with 1,25(OH)2D3 to the vitamin D–deficient rats, TEI 9647 can inhibit the calcium-absorbing and bone-resorbing activities of 1,25(OH)2D3 as well as the 1,25(OH)2D3-mediated inhibition of parathyroid hormone secretion [105]. These results suggest that under normal physiological conditions in vivo, TEI 9647 is a poor antagonist.

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In contrast, in vitro it can antagonize a wide range of 1,25(OH)2D3-mediated VDR actions without a target gene or target tissue preferences. Therefore, it is not likely that TEI 9647 has mixed agonist-antagonist activities that would make it useful in a clinical setting. However, all of the in vitro experiments on TEI 9647 were performed with human cells or recombinant human VDR, and the in vivo experiments were performed with rats. One might speculate that the analog might not be an effective antagonist of the rat VDR as it is of the human VDR, and therefore its actions in the rat may differ from those in humans. 3. THE 26-CARBOXYLIC ESTERS

The other group of antagonists is the carboxylic esters ZK 159222 and ZK 168281 [106]. These compounds appear to act as typical antagonists of nuclear receptors in vitro and in cultured cells, and in very high doses they act as poor agonists. In the presence of 1,25D3, they inhibit VDR-mediated transcriptional activities. In vitro they induce a VDR conformation different from that induced by 1,25(OH)2D3, a clear indication of a difference in the mode of interaction with the receptor. These analogs’ binding to VDR does not prevent dimerization and binding to DNA (as with the lactone compounds), but they do not induce interaction with transcription coactivators of the p160 family and they partially inhibit the 1,25(OH)2D3-mediated interaction of VDR with these factors [106,107]. These results suggest that the step in VDR activation that is disrupted by the 26-carboxylic esters is the induction of a VDR conformation that allows interaction with the coactivators. Since this is an AF-2-dependent function, it suggests that the long side chain of these analogs disrupts the agonist conformation of the helix 12/AF2 core in the VDR [108]. Without X-ray crystallography data, however, it is not possible to determine whether the AF-2 core in these VDR-analog complexes assumes an antagonist conformation such as that seen in the AF-2 core in estrogen receptor-tamoxifen complexes (e.g., the coactivators binding site is masked) or simply is not able to properly interact with coactivators because its conformation is similar to that of the AF-2 core in the unoccupied receptor (Fig. 3). So far, tissue- or genespecific activities of these antagonists have not been identified, either in culture or in animal studies.

IV. CLINICAL SIGNIFICANCE FOR SELECTIVE MODULATION OF THE VDR BY VITAMIN D ANALOGS Vitamin D metabolites (Calderol of Organon, Rocaltrol of Hoffmann-LaRoche) and analogs that are


in fact prehormones of 1,25(OH)2D3 (One-alpha of Leo Pharmaceuticals, Hectoral of Bone Care International) have been used for years for treatment of senile osteoporosis, postmenopausal osteoporosis, secondary hyperparathyroidism, and the skin disease psoriasis [109–116]. These usages suggest that new analogs of vitamin D will be developed and used first and foremost for treatment of these conditions. For instance, the relatively new analog Dovonex (synthesized at Leo Pharmaceuticals) has been used in the past few years for treatment of psoriasis [117–119] and Zemplar (19nor-1,25(OH)2D3, Abbott Laboratories) and maxacalcitol (OCT, synthesized at Chugai) have recently been approved for use in secondary hyperparathyroidism [120–122]. Of these compounds, however, only OCT has been established as a selective modulator of the VDR. Other interesting analogs are in clinical trials for osteoporosis, including ED-71 of Chugai [123,124] and Roche’s selective modulator Ro-26-9228. Both analogs have been carried to phase II clinical trials in postmenopausal osteoporosis and appear to be well tolerated and effective [82]. The analog Ro-26-9228 (also named BXL628) is currently being tested in clinical trials for treatment of benign prostate hyperplasia (BPH), and it will be used in clinical trials for posttransplantation immunosuppression through the next three years (Dr. L. Adorini, personal communication, and unpublished results). Because the side effects common to drugs used for treatment of these two conditions include bone loss, this bone-protecting vitamin D analog may have a dual beneficial effect for BPH and transplantation patients. Until recently, there did not seem to be a specific clinical use for vitamin D antagonists. However, recent studies on Paget’s disease have suggested a specific increase in osteoclasts’ sensitivity to the differentiating action of 1,25(OH)2D3 as the principal underlying mechanism for abnormal bone formation in patients with this disease. These findings suggest that potent vitamin D antagonists might be useful drugs to inhibit the abnormal activation of osteoclasts in this disease [125,126]. Another clinical condition that has not yet been explored is the osteolytic form of metastatic bone disease. This form of bone metastases is common in multiple myeloma and renal carcinoma, and is associated with massive activation of osteoclast actions. Osteoclast activation in osteolytic metastatic bone disease may be coupled to osteoblasts’ production of cytokines that promote osteoclast differentiation and function (primarily RANKL) [127]. Therefore, it remains to be examined whether 1,25(OH)2D3-regulated cytokine production by osteoblasts can be blocked in this form of metastatic bone disease by vitamin D antagonists, without adverse effects on calcium homeostasis and bone turnover.

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One of the most attractive pharmacological features of vitamin D analogs is their ability to inhibit malignant cell growth in vitro and in animal models. These effects depend on the ability of these compounds to induce cell differentiation or apoptosis and to inhibit angiogenesis in vivo [85,128,129]. One of the compounds most thoroughly studied in that respect is the selective modulator EB-1089 (secocalcitol of Leo Pharmaceuticals). In preclinical studies, it inhibited various malignancies including breast, colon, and prostate cancers [84,86,130,131]. EB-1089 even has a significant effect on progression of breast cancer cells into bone in nude mice [132]. In culture and in vivo, EB-1089 appears to have significant differentiating and apoptotic effects and its inhibition of breast cancer cells metastasis into bone suggests that it is also effective on angiogenesis, an important parameter of tumor progression. Interestingly, EB-1089 has been successful in clinical trials of advanced liver cancer and is also being tested in advanced pancreatic and breast cancer [133,134]. Although EB-1089 is efficacious in animal models of malignancies and also has a significant effect on several human malignancies, it still exhibits significant calcemic. Consequently activity, its therapeutic window is somewhat limited [130,133]. Therefore, the future development of additional low-calcemic analogs that are potent inhibitors of tumor cell growth and have a wider therapeutic window will be necessary to further establish vitamin D analogs in cancer therapy.

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CHAPTER 84

Development of New Vitamin D Analogs LISE BINDERUP, ERNST BINDERUP, WAGN O. GODTFREDSEN, AND ANNE-MARIE KISSMEYER Medicinal Chemistry and Biological Research, LEO Pharma, Ballerup, Denmark

I. Introduction II. Strategy for Development of New Vitamin D Analogs III. Structure-Activity Relationships

IV. Biological Activities V. Clinical Development of LEO Analogs References

I. INTRODUCTION

reviews our main efforts and achievements in this field since the mid-1980s.

The involvement of LEO Pharma in the synthesis and evaluation of new vitamin D analogs and metabolites dates back to the early 1970s, with the development of 1α-hydroxycholecalciferol [1α(OH)D3] for the treatment of renal osteodystrophy and hyperparathyroidism. Our interest was further stimulated in the early 1980s with the appearance of reports describing receptors for 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3] in nonclassic target tissues and the demonstration of the role of 1,25(OH)2D3 in regulating growth and differentiation of various cancer cell lines, in vitro and in vivo [1,2]. At the same time, a number of reports suggested that 1,25(OH)2D3 might also influence various functions of activated lymphocytes and thereby play a role as a physiological regulator of the immune system [3,4]. These findings suggested new therapeutic possibilities for 1α(OH)D3 and 1,25(OH)2D3, especially in neoplastic and immune-mediated diseases. In 1983, LEO took steps to initiate clinical trials with 1α(OH)D3 in leukemia and non-Hodgkin’s lymphomas [5,6]. The therapeutic usefulness of 1α(OH)D3 and 1,25(OH)2D3 was, however, likely to be limited by their potent effects on calcium metabolism, leading to side effects such as hypercalcemia and soft tissue calcifications. It was therefore decided to try to develop new analogs with a more favorable therapeutic profile. In 1985, the preliminary testing of a small series of new synthetic analogs led to the discovery of a promising candidate, MC903, later named calcipotriol. At the same time, clinical observations suggested that 1α(OH)D3 and 1,25(OH)2D3 might exert antipsoriatic effects [7,8]. It was therefore decided to test calcipotriol in patients with psoriasis and to further expand our engagement in vitamin D chemistry. This chapter VITAMIN D, 2ND EDITION FELDMAN, PIKE, AND GLORIEUX

II. STRATEGY FOR DEVELOPMENT OF NEW VITAMIN D ANALOGS A. Basic Screening Strategy At the start of the program for synthesis and evaluation of new vitamin D analogs, the primary aim was to identify compounds that were potent regulators of cell proliferation and differentiation, but which had a reduced ability to exert the classic effects of 1,25(OH)2D3 on calcium homeostasis. Structure-activity relationships of a relatively large number of analogs (more than 1000) have been studied in detail over the last 15 years. Initially, the effects of all newly synthesized vitamin D analogs were tested in the human histiocytic lymphoma cell line U 937, which expresses the vitamin D receptor (VDR) and responds to 1,25(OH)2D3 with a decrease in proliferation and induction of differentiation along the monocyte-macrophage pathway [9,10]. The analogs were tested in the cell culture system for 4 days, at concentrations ranging from 10−12 to 10−7M, and their effects were compared to those of 1,25(OH)2D3. More recently, the U 937 cell cultures were supplemented with other cell types, notably mammary cancer cells (MCF-7, human breast carcinoma cell line) and skin cells (HaCaT, human keratinocyte cell line). The binding of the vitamin D analogs to the VDR was assessed by displacement of bound 3H-1,25(OH)2D3 from receptor protein obtained from the intestinal epithelium of rachitic chickens [11]. As the binding affinity of many analogs did not show a direct correlation Copyright © 2005, Elsevier, Inc. All rights reserved.

1490 with their biological activities (see also Section IV.A), the VDR binding studies were extended to include studies of the transcriptional activity of the analogs, using the vitamin D responsive element in the promoter region of the osteocalcin gene. To assess the effects of new vitamin D analogs on calcium metabolism, an in vivo model was chosen [10]. The analogs were administered orally to rats, daily for 7 days. Urine was collected daily, and blood was collected by cardiac puncture at the end of the experiment. Metaphyseal bone was prepared from tibiae. Calcium levels were determined in urine and serum samples, and the calcium content in bone was assessed after ashing. To assure detection of even small differences in potency between various analogs, the rats were given a vitamin D–replete diet with a high calcium content (1%), in contrast to many of the older studies in rats and chickens given low calcium and/or low vitamin D diets [12,13]. As a follow-up to the screening system described above, the metabolic stability (serum half-life) of selected analogs was initially tested in vivo after intravenous administration to rats [14]. At a later stage, an in vitro test using the rat liver post-mitochondrial fraction (S9) was introduced. The analogs were tested at a single concentration, and the percentage of intact analog after 1 hour of incubation was assessed. The analogs were classified as unstable (35% intact analog). Analogs with a low metabolic stability were considered as candidates for topical use, and analogs with a high metabolic stability as candidates for systemic use.

B. Synthesis Strategy From the beginning of the project it was decided to concentrate our efforts on the synthesis of analogs of 1,25(OH)2D3 (I) in which the C-17 side chain was modified while the seco-steroid ring system was kept intact. This decision was partly dictated by the fact that this part of the molecule is more easily accessible to chemical manipulation than the ring system, but it was also decisive that the side chain is known to play a crucial role in the binding of 1,25(OH)2D3 to its receptor [15]. It is beyond the scope of this chapter to detail the synthesis of the more than 1000 analogs that have been made in the LEO laboratories, but some general pathways are outlined in the following sections. Our starting material has been the readily available ergocalciferol (II) (Scheme 1), which, according to a method originally devised by Hesse [16,17], can be converted to a 1α-hydroxylated trans vitamin derivative

LISE BINDERUP

20 17

ET AL .

25 OH

H H

3

1

HO

OH (I)

(IIa), conveniently isolated as the pure, crystalline, bis-silyl ether (III) [18]. After protection of the conjugated triene system as the sulfur dioxide adduct (IV), the side chain can be cleaved selectively by ozonolysis with formation of the aldehyde (V). By heating (V) in the presence of NaHCO3, SO2 is expelled, and the key intermediate (VI) can be isolated as a crystalline compound [18]. Compound (VI) has been a cornerstone in our synthetic work. By means of the Wittig reaction, new side chains could be introduced. Scheme 2 illustrates the synthesis of the anticancer drug EB 1089 (IX) (seocalcitol, see Table II), which contains two conjugated double bonds in the side chain [19]. The product of the Wittig reaction (VII) is reacted with ethyl lithium to give (VIII), which is then isomerized to the cis form by ultraviolet irradiation in the presence of the photosensitizer anthracene [20]. Finally, the hydroxyl groups are deprotected with tetrabutylammonium fluoride in tetrahydrofuran to give (IX). A Wittig reaction with (VI) is also a step in the synthesis of the antipsoriatic drug MC 903 (calcipotriol, see Table III) [18]. Another route to new analogs involves NaBH4 reduction of the aldehyde (VI), followed by tosylation of the resulting alcohol to give (X), which is subsequently reacted with a Grignard reagent to form (XI). Finally, (XI) is isomerized and deprotected to provide the analog CB 966 (XII) (see Table II) [21]. Analogs containing an oxygen atom in the 22 position (22-oxa analogs) can be synthesized as shown in Scheme 3, which depicts the synthesis of the C-20 epimeric compounds KH 1139 and KH 1060 (see Table IV) [22]. Oxidation of the aldehyde (VI) with air in the presence of a copper catalyst yields the methylketone (XIII), which on reduction with NaBH4 forms a mixture of the two epimeric alcohols (XIVa and XIVb), where the 20-epi isomer (XIVb) dominates. Alkylation of the two alcohols, followed by isomerization

1491

CHAPTER 84 Development of New Vitamin D Analogs

H

H

H

H

HO

HO (II)

O

SO2

H

H

Si

Si

O

(IIa)

O

Si

(III)

CHO H O

O H

H

O

H

H O3

S Si O

CHO

O

O H S

Si O

Si

H

NaHCO3 O

(IV)

∆

H

Si

Si

O

(V)

O

Si

(VI)

SCHEME 1

and deprotection as described above, yields the cis analogs (XVa) (KH 1139) and (XVb) (KH 1060), respectively. As described in Section III.D, epimerization at C-20 has a profound influence on the biological properties of the analogs. Another route to 20-epi compounds (Scheme 3) starts with an epimerization of the aldehyde (VI) to the 20-epi aldehyde (XVI), which then, by the same

COOCH3

CHO (C6H5)3P

N

sequence of reactions used in the 20-normal series (Scheme 2), can be converted to 20-epi analogs, such as 20-epi-1,25(OH)2D3 (XVII) (MC 1288) (see Table IV) [23]. The tosylate (XVIII), used in the synthesis of MC 1288, has also been used to synthesize the 20-epi23-thia analog (XIX) (GS 1790) (see Table V) [24]. The reactions depicted in Schemes 1–3 are typical of the pathways used in the synthesis of a wide variety

COOCH3 N

(VI)

N=

H

(VII) H

1. NaHBH4 2.TsCl - Pyridine

2 EtLi Si

O

O

Si

OH OTs N (X)

N (VIII) 1. Anthracene/UV-light 2. TBA+ F− OR

H M=

H

OH A (XI) R = Si(Ch3)3 ; A = N (XII) CB 966 (R = H ; A = M)

M (IX) EB 1089

SCHEME 2

HO

OH

1492

LISE BINDERUP

CHO

ET AL .

CHO

OH−

N

N

(VI)

(XVI)

OH M (XVII) MC 1288

2+

O2, Cu , DABCO 2,2′-Bipyridyl OH O

OTs

N

N

(XIII)

(XVIII)

S M (XIX) GS 1790

NaBH4 OH N (XIVa)

+

N (XIVb)

OH

OH

O

OH

O

+

M

M

(XVa) KH 1139

(XVb) KH 1060

SCHEME 3 N and M have the same meaning as in Scheme 2.

of side chain analogs, but it is obvious that many variations have been necessary.

III. STRUCTURE-ACTIVITY RELATIONSHIPS In this section, the effect of systematic chemical modifications of the 1,25(OH)2D3 side chain on various biological parameters is presented. All the analogs discussed here have been tested for calcemic activity, antiproliferative activity, and ability to induce cell differentiation, as described in Section II.A. However, because the antiproliferative and differentiation-inducing properties run parallel, only the antiproliferative potencies are shown in the tables. All values are given in relation to 1,25(OH)2D3.

A. Variation of Chain Length In Table I the biological activities of a number of analogs which differ from 1,25(OH)2D3 with respect to the length of the C-17 side chain are listed [21]. It is seen that if the chain length is increased with one methylene group as in MC 1127, the ability to inhibit proliferation is increased, whereas the calcemic activity is reduced to about one-third of that of 1,25(OH)2D3. This compound has also been described by Ostrem et al. [25]. If the two terminal methyl groups in MC 1127 are replaced by ethyl groups as in CB 966, the antiproliferative potency is increased

and the calcemic activity further reduced. On the other hand, if propyl groups are substituted for the methyl groups, the antiproliferative potency is reduced to about the same level as that of 1,25(OH)2D3. An increase of the 1,25(OH)2D3 side chain with two methylene groups (MC 1147), also described by Kutner et al. [26], causes a further reduction of the calcemic activity, whereas the antiproliferative potency remains the same as in MC 1127. However, with the introduction of one more methylene group (MC 1179), the antiproliferative activity is reduced. In other words, the optimal number of methylene groups between C-20 and the tertiary hydroxyl group seems to be four or five.

B. Introduction of Double and Triple Bonds The effect of introducing one or two double bonds in the C-17 side chain is illustrated in Table II. Whereas the introduction of a ∆22 double or triple bond in CB 966 decreases the antiproliferative activity [23,27], the introduction of a further ∆24 double bond leads to EB 1089 [19], which, with respect to cell proliferation, is the most active in the series, being 100 times more potent than 1,25(OH)2D3. Because the calcemic activity of EB 1089 is three times lower, a substantial separation of the effects has been achieved. EB 1089 has been chosen as candidate for clinical testing in cancer patients (see Section V.B.2). Another analog, CB 1093, is characterized by several modifications in the side chain. In addition to the triple bond, CB 1093 has altered stereochemistry at

1493


TABLE I Variation of Chain Length Compound 1,25(OH)2D3

Inhibition of U937 cell proliferation IC50 (M)

Side chain structure OH

Calcemic activity relative to 1,25(OH)2D3 (%)

3 × 10−8 [1X]a

100

5 × 10−9 [6X]

38

1 × 10−9 [30X]

17

5 × 10−8 [0.6X]

n.d.b

OH

MC 1127

OH

CB 966

OH

CB 973

MC 1147

OH

5 × 10−9 [6X]

4

CB 953

OH

2 × 10−8 [1X]

2

4 × 10−8 [0.7X]

2

OH

MC 1179 aBoldface bNot

figures indicate activity relative to 1,25(OH)2D3. determined.

C-20 and an ethoxy group at C-22. These modifications increase the antiproliferative activity of the compound to the same level as that of EB 1089. CB 1093 has recently been shown to be a potent inducer of apoptosis (see Section IV.C). The last compound in Table II, HEP 187, is a 20-epi-vitamin D analog, in which the terminal side chain hydroxy group has been replaced by a fluorine atom. This compound has a relatively low antiproliferative effect, compared to the other 20-epi-analogs, but it has been shown to exert interesting effects on bone mineral metabolism (see Section IV.E.3).

C. Calcipotriol and Related Analogs One of the first 1,25(OH)2D3 analogs synthesized at LEO for which a clear separation between the calcemic

activity and the effects on cell regulation was achieved was MC 903 [10,18] (Table III), which later received the United States Adopted Name (USAN) calcipotriene and the International Nonproprietary Name (INN) calcipotriol. In this compound, a ∆22 double bond is introduced in the side chain, the 25-hydroxyl is moved to the 24 position (with the indicated stereochemistry), and a cyclopropane ring is substituted for the isopropyl group in 1,25(OH)2D3. Table III shows that calcipotriol has retained the cell-regulating potency of 1,25(OH)2D3, whereas its calcemic activity has been reduced by a factor of 200. As described in Sections IV.E.2 and V.B.1 of this chapter and in Chapter 101, calcipotriol has become an important antipsoriatic drug. Its 24-epimer MC 900 [18] has a considerably lower antiproliferative potency, and the same holds true for MC 1046 and MC 1080, the two main metabolites of calcipotriol [28].

1494

LISE BINDERUP

TABLE II Compound

Double and Triple Bonds Inhibition of U937 cell proliferation IC50 (M)

Side chain structure

1,25(OH)2D3

ET AL .

OH


3 × 10−8 [1X]a

100

1 × 10−9 [30X]

17

2 × 10−8 [1.5X]

24

3 × 10−8 [1X]

n.d.b

3 × 10−10 [100X]

31

3 × 10−10 [100X]

24

5 × 10−9 [6X]

34

OH

CB 966

OH

MC 1473 OH

CB 1309

OH

EB 1089 (seocalcitol) O OH

CB 1093 F

HEP 187 aBoldface bNot

figures indicate activity relative to 1,25(OH)2D3. determined.

The effect of the size of the terminal ring was studied by Calverley [29], who synthesized three pairs of 24-epimeric analogs in which the cyclopropyl group in calcipotriol is replaced by cyclobutyl, cyclopentyl, and cyclohexyl groups, respectively. Although the stereochemistry of the 24-hydroxyl group in these compounds has not been rigorously established, their polarities suggest that in MC 1070, MC 1052, and MC 1048 the 24-hydroxyl group has the same stereochemistry as in calcipotriol, whereas in MC 1069, MC 1050, and MC 1033 the stereochemistry of the 24-hydroxyl group is as in MC 900. It was found that the antiproliferative potencies of the cyclobutyl and cyclopentyl analogs MC 1070 and MC 1050 were similar to that of calcipotriol, whereas the cyclohexyl analog MC 1048 was less potent. All these compounds were, however, significantly more calcemic than calcipotriol.

D. Epimerization at C-20 A seemingly minor modification of the side chain in 1,25(OH)2D3 that has a dramatic effect on its biological activities is epimerization at C-20. As Table IV shows, the 20-epimer of 1,25(OH)2D3 (MC 1288) [23] is about 100 times more potent than the natural hormone as an inhibitor of cell proliferation, whereas its calcemic activity has increased by a factor of only 2. Even more pronounced is the effect of 20-epimerization on immunosuppressive properties [30]. In view of these results, we found it mandatory to investigate the effect of 20-epimerization more broadly [22,23]. Table IV shows the activities of pairs of 20epimers. Both the antiproliferative potency and the calcemic activity are generally higher in the 20-epi than in the 20-normal series. A particularly noteworthy compound is the 22-oxa analog KH 1060 (lexacalcitol),

1495


TABLE III Compound

Calcipotriol and Analogs Inhibition of U937 cell proliferation IC50 (M)

Side chain structure

1,25(OH)2D3


3 × 10−8 [1X]a

100

2 × 10−8 [1.5X]

0.5

>1 × 10−7 [ 50% reduction) levels. Patients with bone pain at study entry have had pain relief. As discussed in Section VI.F, cell culture and animal studies indicate that calcitriol and its analogs can enhance the cytotoxic effects of conventional chemotherapeutic drugs, and investigators have begun testing these combinations in phase I and phase II trials. Trump, Johnson, and co-workers are conducting two phase I trials testing 1,25(OH)2D3 in combination with carboplatin or paclitaxel. In the study testing calcitriol and carboplatin, no dose-limiting toxicity has been seen up to calcitriol doses of 13 µg/day orally for three days every four weeks with carboplatin, and the study

continues [230]. In the second trial, paclitaxel is given with escalating doses of calcitriol every day for three days per week for a period of six weeks, and so far no dose limiting toxicity has been encountered [231]. Beer et al. [232] are conducting a phase II study to evaluate weekly oral administration of very high dose calcitriol (0.5 µg/kg) on day 1 followed by intravenous administration of docetaxel on day 2 in patients with AIPC, repeated for six consecutive weeks of each eightweek cycle. In a recent report Beer et al. [233] showed that the combination of a once weekly oral high-dose calcitriol and weekly docetaxel was a well-tolerated regimen for patients with AIPC. Thus far 22 out of 37 patients exhibit a > 75% reduction in serum PSA at the end of an eight-week treatment cycle, confirmed four weeks later. The results of this study indicate that PSA and measurable disease response rates, as well as time to progression and survival, are more promising with the combination approach when compared to phase II studies of the single agent docetaxel in AIPC. To confirm these encouraging findings these investigators have started a placebo-controlled, double-blinded randomized comparison of docetaxel plus calcitriol to docetaxel alone. The result of the various clinical investigations so far support the potential promise of exploiting vitamin D compounds alone or in combination with other agents to control PCa progression and further studies are clearly warranted.

VIII. SUMMARY AND CONCLUSIONS A number of studies have established the role of 1,25(OH)2D3 as an antiproliferative agent in normal and malignant prostate cells. Investigations using animal models have also demonstrated the anti-tumor effects of 1,25(OH)2D3 and its less hypercalcemic analogs. The mechanisms underlying the anti-cancer effects of vitamin D compounds in PCa cells are varied and cell-specific and include growth arrest, apoptosis, pro-differentiation effects, and modification of growth factor activity. Some studies suggest additional effects to inhibit invasiveness and anti-angiogenesis, but there are less data on these issues. 1,25(OH)2D3 interacts with androgen signaling in PCa cells possibly enhancing its prodifferentiating activity. Investigators are making progress in identifying 1,25(OH)2D3-regulated genes and understanding their role in the mediation of the above-mentioned effects. Such studies would unveil novel 1,25(OH)2D3–responsive genes and provide new therapeutic targets. Administration of pharmacological doses of calcitriol to men with PCa results in hypercalcemia and hypercalciuria, which limits the concentration of

1700 calcitriol that can safely be administered to patients. Consequently, structural analogs of calcitriol that effectively activate the VDR but exhibit less hypercalcemic effects are being developed and evaluated as potential anti-cancer agents. A challenging area of research involves defining the mechanisms by which various vitamin D analogs maintain potent growth inhibitory effects and yet are less able to induce hypercalcemia. Intermittent dosing regimens with very high boluses of calcitriol are an interesting approach that apparently inhibits PCa while not causing persistent hypercalcemia. Combinations of calcitriol or its analogs with inhibitors of 24-hydroxylase or other growth-inhibitory molecules such as retinoids, glucocorticoids, or cytotoxic chemotherapeutic drugs permit the use of vitamin D and the other agents at relatively lower doses, thereby avoiding toxic side effects while achieving synergistic antitumor effects. Several investigators are carrying out clinical trials to determine whether calcitriol or analogs singly or in combination with other agents can prevent the progression of PCa. Investigators are also examining the role of vitamin D compounds as chemopreventive agents. We believe that progress in these areas of research will result in rational drug design and lead to the development of more potent and safer vitamin D analogs. It remains to be determined what will be the most effective use of calcitriol and its analogs in terms of dosage regimen, combination with other agents, and phase of the disease in which it will be most useful in the treatment and/or prevention of PCa.

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1701

CHAPTER 94 Vitamin D and Prostate Cancer

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227.

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vitamin D3 (calcitriol) [published erratum appears in J Urol 1998 Sep; 160(3 Pt 1):840]. J Urol 159:2035–2039; discussion 2039–2040. Smith DC, Johnson CS, Freeman CC, Muindi J, Wilson JW, Trump DL 1999 A phase I trial of calcitriol (1,25-dihydroxycholecalciferol) in patients with advanced malignancy. Clin Cancer Res 5:1339–1345. Beer TM, Lemmon D, Lowe BA, Henner WD 2003 Highdose weekly oral calcitriol in patients with a rising PSA after prostatectomy or radiation for prostate carcinoma. Cancer 97:1217–1224. Liu G, Oettel K, Ripple G, Staab MJ, Horvath D, Alberti D, Arzoomanian R, Marnocha R, Bruskewitz R, Mazess R, Bishop C, Bhattacharya A, Bailey H, Wilding G 2002 Phase I trial of 1α-hydroxyvitamin D2 in patients with hormone refractory prostate cancer. Clin Cancer Res 8: 2820–2827. Johnson CS, Hershberger PA, Bernardi RJ, McGuire TF, Trump DL 2002 Vitamin D receptor: a potential target for intervention. Urology 60:123–130; discussion 130–121. Muindi JR, Peng Y, Potter DM, Hershberger PA, Tauch JS, Capozzoli MJ, Egorin MJ, Johnson CS, Trump DL 2002 Pharmacokinetics of high-dose oral calcitriol: results from a phase 1 trial of calcitriol and paclitaxel. Clin Pharmacol Ther 72:648–659. Beer TM, Hough KM, Garzotto M, Lowe BA, Henner WD 2001 Weekly high-dose calcitriol and docetaxel in advanced prostate cancer. Semin Oncol 28:49–55. Beer TM, Eilers KM, Garzotto M, Egorin MJ, Lowe BA, Henner WD 2003 Weekly high-dose calcitriol and docetaxel in metastatic androgen-independent prostate cancer. J Clin Oncol 21: 123–128.

CHAPTER 95

Vitamin D and Colon Cancer HEIDE S. CROSS Department of Pathophysiology, Medical University of Vienna, Austria I. Introduction II. Molecular Basis of Vitamin D Action on Neoplastic Colonocytes III. Vitamin D Metabolism in Normal and Neoplastic Colon Cells

IV. Nutritional Regulation of CYP27B1 and CYP24 V. Conclusion References

I. INTRODUCTION

than 3.75 micrograms vitamin D/day was associated with a 50% reduction in the incidence of colorectal cancer [11]. The same nested case-control study based on a cohort of more than 25,000 individuals demonstrated that a moderately elevated serum concentration of 25-(OH)-D3 (65–100 nmol/l) was associated with a highly significant reduction in colorectal cancer incidence [11]. Several later studies, however, provided ambiguous results: a large 10–17 year retrospective study of Washington County residents did not provide any link between colorectal cancer incidence and serum levels of 25-(OH)-D3, or of 1,25-dihydroxyvitamin D3 (1,25-(OH)2-D3) evaluated prior to disease occurrence [12]. When serum 25-(OH)-D3 concentrations in patients with colonic neoplasia were compared with those of noncancer patients, no correlation with the disease was found either [13]. Tangrea et al. [14], however, did find that the estimated relative risk of large bowel cancer decreased with increasing serum 25-(OH)-D3 levels, and that the association was most pronounced for rectal cancer in a nested case-control study within a Finnish clinical trial cohort. It is highly interesting that in this large study levels of 1,25-(OH)2-D3 again did not correlate at all with colon cancer risk. One of the very few studies demonstrating a correlation between low circulating levels of 1,25-(OH)2-D3 (below 26 pg/ml, a level considered to be below normal) and enhanced risk was found in the prospective Nurses’ Health Study. A higher risk of distal colorectal adenomas was found in individuals with lower than 26 pg 1,25-(OH)2-D3/ml serum [15]. In another study by Niv et al. [16], which was marred by the small number of patients involved, a steady reduction of serum 1,25-(OH)2-D3 was observed in parallel with advancing tumor stages, but not with the biological tumor grade. A plethora of studies has been based on semiquantitative food frequency questionnaires, which often involved not only vitamin D but dairy food intake as well. Therefore, calcium ingestion may be a confounding factor in these evaluations. The consensus is that,

A. Colonic Tumor Prevention by Vitamin D 1. EPIDEMIOLOGICAL EVIDENCE

As early as 1980 Garland et al. [1] proposed that vitamin D may be a protective factor against colorectal cancer. This hypothesis was based on the observation that the geographic distribution of colon cancer mortality in the U.S.A. was highest in regions where the population was least exposed to solar radiation. UV-B is responsible for vitamin D production in the skin, and serum levels of 25-hydroxyvitamin D3 (25-(OH)-D3) are a direct reflection of sunlight exposure, of the use of sun blockers, and of skin pigmentation [2]. Thus, low serum 25-(OH)-D3 levels generally found in African-Americans probably are a reflection of reduced vitamin D synthesis due to high melanin concentrations in the skin [3]. This population segment also has enhanced incidence of colorectal, breast, and prostate cancer [4]. The link between colorectal cancer incidence and solar radiation was later confirmed by Freedman et al. [5] and by several large studies comparing southern and northern parts of the U.S.A. [6]. Recently, Grant [7] suggested that actually 20–30% of colorectal cancer incidence is due to insufficient exposure to sunlight (see Chapters 90 and 91). A follow-up study by Garland et al. [8] in almost 2000 males demonstrated that risk of colorectal cancer correlated inversely with dietary vitamin D and calcium intake. Further studies on dietary vitamin D and calcium (see [9]) appeared to confirm these data. When Garland et al. [10] demonstrated in blood samples from a Washington County population that a serum 25-(OH)-D3 concentration of 20 ng/ml or more decreased threefold the risk of colon cancer, a direct connection between serum vitamin D levels and colon cancer incidence appeared to be established. However, in this study, cancer incidence was evaluated only 1–8 years after blood sampling. In a 19-years prospective study, it was subsequently established that a dietary intake of more VITAMIN D, 2ND EDITION FELDMAN, PIKE, AND GLORIEUX


1710 if vitamin D consumption has any preventive effect on colorectal cancer incidence, it is a very modest one. However, this protective effect might be augmented by intake of multivitamin supplements (see [17–23]). In this respect, the argument was raised that solar radiation or nutritional vitamin D intake, for instance by consuming fatty fish, would not be sufficient to effectively prevent the occurrence of colorectal tumors. Indeed, in a later study Garland et al. [24] suggested that intake of at least 800 IU (20 micrograms) of vitamin D3 together with 1000 mg calcium would be needed to significantly reduce the incidence and mortality rates of human colon cancer. 2. CONCLUSION

Thus, cumulative epidemiological evidence suggests that there is a direct correlation between reduced colorectal cancer incidence and sunlight exposure, low skin pigmentation, nutritional vitamin D intake, and high serum levels of 25-(OH)-D3. This association could be strengthened by vitamin D supplementation. In a recent human pilot study, Holt et al. [25] demonstrated for the first time that rectal crypt proliferation was inversely correlated with 25-(OH)-D3 levels in serum. This indicates, though in an indirect way, that low 25-(OH)-D3 levels may be indeed associated with colorectal cancer incidence. However, no relationship between serum 1,25-(OH)2-D3 and disease occurrence was convincingly apparent in any of the cited studies, except at very low serum 25-OH-D concentrations.

II. MOLECULAR BASIS OF VITAMIN D ACTION ON NEOPLASTIC COLONOCYTES Since the 1980s, 1,25-(OH)2-D3 has been recognized as a potent cellular antiproliferative and prodifferentiating agent in the colon. More recently, there has been intense interest in its effects on apoptosis, malignant cell invasion, and metastasis. The classical signaling pathway is via a nuclear vitamin D receptor (VDR), which is a transcription factor (see [26]). The existence of a separate “membrane” receptor has also been suggested [27]; however, recent data from a VDR knockout mouse provide good evidence that this purported receptor is of minor importance in the intestine [28].

A. The Vitamin D Receptor in Normal and Malignant Colon Cells In 1982, Frampton et al. [29] provided evidence that the VDR was present in many human cancer cell lines

HEIDE S. CROSS

and also in all colonic cell lines they investigated. Since then, the presence of the VDR has been extensively studied in a variety of colon cancer cell lines. Giuliano et al. [30] showed in Caco-2 cells that the VDR was functional in these colon cancer cells and that expression was increased when Caco-2 cells became confluent and differentiated in culture. Interestingly, Zhao and Feldman [31] demonstrated convincingly that, at least in HT-29 cells which were differentiated by chemical means, VDR abundance was actually decreased with decreased proliferation and increased differentiation. Brehier and Thomasset [32] found no specific binding of 1,25-(OH)2-D3 in differentiated HT-29 cells. Harper et al. [33] also found smaller amounts of the receptor in galactose-grown, i.e. differentiated, HT-29 cells when compared with undifferentiated (glucosegrown) HT-29 cells. In Caco-2 cells, however, there was strong expression of the VDR upon differentiation. Conversely, activation of proliferation in these cells by epidermal growth factor (EGF) resulted in down-regulation of the expression of the high affinity receptor [34]. The question of whether the VDR was more highly expressed in proliferating or differentiated colon cancer cells was further studied by Shabahang et al. [35]. Their conclusion was that the more differentiated the colonic cell lines were, the higher was their VDR expression. They also evaluated presence of the VDR in human malignant colonic tissue. In the majority of these tumors, they found lower expression of VDR than in tumor-adjacent normal mucosa from the same patient; however, the number of cases analyzed was small (12 patients). Lointier et al. [36] investigated VDR expression in 23 human tumor tissue specimens and in adjacent normal-appearing mucosa. They did not observe any difference in VDR distribution between normal right and left colon, or the rectum. However, they did find the receptor in most of the normal tissue specimen, but only in very few of the adenocarcinomas. Notably, all positive adenocarcinomas were of the welldifferentiated (low grade) type. Vandewalle et al. [37] demonstrated significantly higher VDR expression in transformed colon than in normal tissue in the proximal and distal colon, but not in the rectum. These data were accrued by binding studies with tissue homogenates. Cross et al. [38] were the first to demonstrate by immunoblotting that VDR protein expression was increasing during the transition from normal mucosa to polyps and during progression into malignancy. In rather advanced tumor stages, however, expression was diminished or disappeared completely. This suggests that colon cancer cells express the VDR as long as they retain a certain level of differentiation. This could explain why their data differed from those obtained by

1711

CHAPTER 95 Vitamin D and Colon Cancer

TABLE I Semi-quantitative Evaluation of VDR and EGFR mRNA Expression in Epithelial Cells Adenocarcinoma Normal adjacent mucosa VDR EGFR

17.5 ±1.4 58.2 ± 5.5

Low grade

High grade

125.0 ± 19.7 * 122.0 ± 18.1 *

46.3 ± 5.5 142.5 ± 27*

In situ hybridization (IHS) reactivity scores were calculated by multiplying the percentage of receptor positive cells by the average signal intensity. Data are mean ± SEM. * indicates statistically significant difference from respective IHS score in normal adjacent mucosa at p < 0.01 (using Student’s unpaired t test).

some other groups, who did not take into account tumor staging and grading. Further studies by Sheinin et al. [39], on a larger number of human colon tissues demonstrated convincingly, by in situ techniques, that in normal colon VDR expression is weak and positivity is found mainly in luminal cells, i.e. differentiated crypt cells. During tumor progression, the number of VDR-positive colonocytes increased dramatically in parallel with epidermal growth factor receptor (EGFR) expression, and it reached its maximum in low-grade (well to moderately differentiated) tumor tissue, whereas in high grade cancers (G3/4, low differentiation or undifferentiated tissue), VDR expression was very low. In contrast, EGFR expression rose even further in undifferentiated tumors (Table I). When establishing primary cultures from human premalignant and malignant colonic tissue at diverse

stages of tumor progression, Tong et al. [40] have shown a mosaic pattern of VDR expression in colon cells. This demonstrated that a large fraction of cells isolated from human colon tumors, but not all, expressed the VDR and thus could respond to the genomic action of vitamin D compounds. Cross et al. [41] and Kállay et al. [42] demonstrated by immunoblotting and RT-PCR in colon tissue derived from 61 patients that there was indeed little VDR present in normal tissue, and that expression rose during colon tumor progression. In Fig. 1, VDR expression is shown in parallel with a proliferation marker, Ki-67. Barely any VDR expression is found in a (G3/4) tumor with cells at a low differentiation level (Fig. 1C), when it is was compared with expression in normal colon mucosa (Fig. 1A) or in a moderately-differentiated (G2) tumor (Fig. 1B). Low VDR expression in normal mucosa (Fig. 1A) is paralleled by low Ki-67 positivity mainly in the lower crypt area (Fig. 1D). With enhanced VDR expression in low grade cancer (Fig. 1B) there is also markedly increased Ki-67 positivity (Fig. 1E), whereas in high grade tumors with strong proliferation, there is almost no more VDR positivity apparent (Fig. 1C and F). These, and data from other laboratories, led to the suggestion that vitamin D receptor expression could be used as a predictive marker of biological behavior in human colorectal cancer [43]. The importance of the VDR for prevention of colonic hyperproliferation and of potential tumorigenesis was demonstrated by Kállay et al. [44] in the VDR knockout mouse model established by the group of Kato [45]. Complete loss of the VDR resulted in colonic hyperproliferation, cyclin D1 elevation, and

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FIGURE 1 Evaluation of vitamin D receptor (A, B, C) and Ki-67 (D, E, F) expression in normal and malignant human colon. A, D: Normal colon mucosa, B, E: Moderately differentiated (G2) adenocarcinoma, and C, F: Adenocarcinoma of the colon at low differentiation (G3/4). (Cross and co-workers, unpublished data).

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a dramatic increase of DNA damage mainly in the distal colon (as measured by 8-hydroxy-2′-deoxyguanosine accumulation). This suggests that, at least in this animal model, growth control by 1,25-(OH)2-D3 is highly effective in the distal [44], but not in the proximal colon, and may be essential for maintenance of normal growth conditions of mucosal cells (see also Fig. 2). Recently, data from a population-based case-control study of colon cancer suggested that normal molecular variants of the VDR gene, i.e. polymorphisms of the VDR, might be related to the development of colon cancer [46]. The different variants could have less or more transcriptional activity upon binding of the hormonal ligand (see Chapter 68). Epidemiological studies to

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B. Effect of 1,25-(OH)2-D3 and of Vitamin D Analogs on Proliferation, Differentiation, and Apoptosis of Colonic Cells The vitamin D receptor is known to exert its antimitotic and prodifferentiating effect by binding vitamin D metabolites and analogs. This not only maintains normal growth of the colonic crypt, but potentially can prevent the progression into premalignancy (see [42]). Interestingly, it has been observed recently [47] that there may be other paths of colon cancer prevention following VDR activation: the VDR can also bind the enteric carcinogen lithocholic acid, and can activate its detoxification via transcriptional induction of cytochrome P450 enzymes (see Chapter 53). The following sections will present, with some selectivity, experimental evidence for the effects of various vitamin D compounds on proliferation, differentiation, and apoptosis of colon cells in in vitro systems, in animal models, and in human studies. 1. IN VITRO MODELS

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evaluate the association of these variants with diet and lifestyle factors are needed.

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A: Expression of Cyclin D1 protein by immunoblotting in the ascending (CA) and descending (CD) colon of wild-type (VDR+/+), heterozygous (VDR+/−), and VDR knockout (VDR−/−) mice. Values are expressed as mean ± SD, n = 5 animals per genotype. Statistically significant differences compared to the VDR+/+ group are indicated as * ( p < 0.05) (Student’s t test). B: Immunohistochemical evaluation of 8-OHdG expression in ascending (CA) and descending (CD) colon of wild-type (VDR+/+), heterozygous (VDR +/−), and VDR knockout (VDR−/−), mice. For quantification three 35 mm photo slides were taken randomly from each sample and were analyzed by NIH Image freeware. Values are expressed as mean ± SD, n = 5 animals of each genotype. Statistical significance was indicated as **(p< 0.01). (Student’s t test). 8-OHdG, 8-hydroxy2′-deoxyguanosine.

a. Action of 1,25-(OH)2-D3 In 1987 Lointier et al. [48] demonstrated that 10 nM 1,25-(OH)2-D3 inhibited growth of the LoVo colon cancer cell line under serumfree conditions. Brehier et al. [32], by evaluating induction of brush border hydrolase activity, demonstrated the differentiating effect of 1,25-(OH)2-D3 in HT-29 cells. Harper et al. [33] observed a decrease in the growth rate of HT-29 cells at the low concentration of 10 pM 1,25(OH)2-D3. Cross et al. [49–51] provided extensive evidence that 1–10 nM 1,25-(OH)2-D3 decreased growth and increased activity of the differentiation marker alkaline phosphatase in the human colon adenocarcinoma-derived cell line Caco-2. Induction of hyperproliferation of Caco-2 cells resulted in 100-fold higher sensitivity to the antimitotic prodifferentiating action of the secosteroid hormone [49]. Evidence for a dosedependent reduction of proliferation and increased alkaline phosphatase activity in Caco-2 cells was provided also by Halline et al. [52]. Responsiveness of primary cultures obtained from human normal colon, colon adenomas, and carcinomas to 1,25-(OH)2-D3 was demonstrated for the first time by Tong et al. in 1998 [40]. The proliferative rate of adenoma cells was initially twice that of cells obtained from normal mucosa. When adenoma cells were treated with 10 nM of the secosteroid, their mitotic rate was reduced to that of normal colonocytes in culture. Carcinoma-derived primary cultures responded to 1,25-(OH)2-D3 and vitamin D analogs in

1713


a concentration-dependent manner with respect to proliferation and differentiation. Since colorectal tumors are frequently under mitotic control by EGF or TGF-α, and during human colon tumor progression EGFR expression is up-regulated in parallel to that of the VDR [39] (see also Table I), Tong et al. [53] evaluated the interaction of EGF with the 1,25-(OH)2-D3. Their data demonstrated a mutual modulation of the VDR by the hormones, which resulted in enhanced activity of 1,25-(OH)2-D3 in EGFtreated Caco-2 cells. Bareis et al. [54] pointed out that only well-differentiated colonic cell lines or primary cultures were also able to respond to 10 nM 1,25-(OH)2-D3 by growth reduction. Their data showed that EGF treatment of a differentiated Caco-2 clone, but not of an undifferentiated one, increased VDR expression (see also Fig. 3). This suggested that VDR mediated growth inhibition by 1,25-(OH)2-D3 would be effective only in differentiated human colorectal carcinomas. Franceschi et al. [55] found that 1,25-(OH)2-D3 was able to stimulate fibronectin synthesis in colon cancer

A 0.0 nM

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A: Evaluation of [3H]-thymidine incorporation into DNA in Caco-2/AQ and Caco-2/15 clones. Cells were treated for 48 hours with 10 nM 1,25-(OH)2-D3. Data are accumulated from two separate experiments and are presented in percent of ethanol control (mean ± SD; n = 6 per group; **: p < 0.01). B: Western blot analysis of VDR expression in Caco-2/AQ and Caco-2/15 cells with and without exposure to 10 ng EGF for 48 hours.

cell lines, indicating that the secosteroid may also play a restrictive role in tumor metastasis. Recently, Palmer et al. [56] demonstrated that only VDR–positive clones derived from the human colon carcinoma cell line SW480 were able to respond to the differentiating action of 1,25-(OH)2-D3. Conversely those clones that lacked the VDR did not respond. This vitamin D– induced differentiation process resulted in induction of E-cadherin and of other adhesion proteins, and promoted the translocation of β-catenin from the nucleus to the plasma membrane. 1,25-(OH)2-D3 also repressed β-catenin/TCF-4 transcriptional activity and modulated target genes in a manner opposite to that of βcatenin. Wilson et al. [57] demonstrated in HT-29 cells the significance of this effect with respect to regulation of the c-myc oncogene: elevated β-catenin/TCF signaling due to mutations in the adenomatous polyposis coli (APC) gene resulted in increased transcriptional activity of c-myc, one of the early immediate genes initiating cell cycle traverse. 1,25-(OH)2-D3 induced transcriptional blockage that resulted in decreased c-myc expression. TGF-β, a well recognized growth inhibitor of normal colonocytes, is no longer active in human colon cancer cells. However, 1,25-(OH)2-D3 treatment activated TGF-β signaling in Caco-2 cells, and enhanced abundance of the type 1 TGF-β receptor [58]. Thus, the secosteroid sensitizes Caco-2 cells to the growth-inhibitory action of TGF-β1. According to very recent data by Thompson et al. [59], transcriptional activity of the liganded VDR may result also in induction of cytochrome P450 detoxification enzymes, which may contribute to colonic chemoprotective mechanisms by detoxification of enteric carcinogens. Though several reports have been published on rapid and membrane based signal transduction mechanisms following exposure of colon cancer cells to 1,25-(OH)2-D3 (see [60–62]), none is of specific relevance yet for colon cancer prevention or therapy by 1,25-(OH)2-D3. b. Vitamin D Analogs It is becoming increasingly evident that adjuvant treatment of colorectal cancer patients with 1,25-(OH)2-D3 for its antimitotic prodifferentiating activity would necessitate a pharmacological dose, which would have the classical adverse consequences, namely hypercalcemia, soft tissue calcification, and nephrocalcinosis. Therefore, over the past decades, more than 400 analogs of vitamin D have been chemically synthesized and their biological properties have been systematically explored, calcemic effects have been quantified, and cell-differentiating and antimitotic potential have been evaluated (for reviews see [63,64] and Chapters 80–88). Action of some of these analogs on colon cancer cells will be reviewed here.

1714 Cross et al. [51] evaluated concentration-dependent growth inhibition in relation to hypercalcemic potential of two side chain-modified synthetic vitamin D analogs (Ro23-4319, Ro23-7553). Ro23-7553, a 16ene23yne side chain-modified vitamin D analog, was tenfold more effective than the 1,25-(OH)2-D3 in suppressing growth of Caco-2 cells; however, it was also tenfold more potent in stimulating calcium release from cultured mouse calvariae. With respect to intestinal calcium absorption the analogs were rather less effective than the parent hormone. Several other studies [65–69] evaluated only differential growth inhibition of colon cancer cells by a variety of analogs, and the main conclusion was that side chain-modified analogs improve activity of the secosteroid maximally by a factor 10, while their hypercalcemic activity is still moderately high. Only the 1β-(hydroxymethyl) congeners of the natural hormone, though still possessing significant antimitotic activity in spite of their low affinity for the VDR [70], did not promote osteoclast differentiation in vitro. This suggests that at nanomolar doses they would not cause hypercalcemia in human studies [71]. Oh et al. [72] implied that the antiproliferative activity of EB1089 in HT-29 cells was, at least in part, due to decreased secretion of IGF-II and increased concentrations of the IGF-II binding protein IGFBP-6. Tanaka et al. [73,74] observed enhanced differentiation of HT-29 cells upon treatment with a combination of sodium butyrate and vitamin D analogs and proposed this combination as a differentiation-based therapy for the clinical management of human colon cancer. Evaluation of Ro25-6760 action on HT-29 cells demonstrated significant growth inhibition, apoptosis induction, with enhanced expression of p21Waf1 and G1/Go cell cycle arrest [75]. An increase of the proapoptotic protein BAK induced by the vitamin D analog EB1089 in colon cell lines also suggested a mechanism of action involving apoptosis [76]. Another vitamin D analog was shown to increase expression of the cdk inhibitors p21Waf1 and of p27Kip1 independent of changes in pRB [77]. 2. ANIMAL MODELS FOR COLORECTAL TUMOR PREVENTION BY VITAMIN D COMPOUNDS

As early as 1988, Pence and Buddingh [78] evaluated the effect of 2000 IU vitamin D3/kg diet on 1,2-dimethylhydrazine-induced colon carcinogenesis in male rats. Development of cancer was promoted with 20% corn oil in the diet. Their data suggested that only in animals on a high fat diet (i.e. the promoter) did vitamin D3 significantly reduce tumor incidence. Subsequent work by Kawaura et al. [79], who instilled intrarectally lithocholic acid in rats with N-methyl-N-nitrosourea-induced colonic tumors, demonstrated that 1α-(OH)-D3 as well

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was inhibiting promotion of tumorigenesis. Apparently, without exogenous promoters, vitamin D compounds did not affect colonic carcinogenesis and did not interfere with formation of bile acid profiles [80]. Comer et al. [81] also provided evidence that dietary levels between 250–10,000 IU vitamin D3/kg diet did not alter carcinogen-induced tumor incidence without prior promoter treatment, while work by Belleli et al. [82] using 400 ng 1,25-(OH)2-D3 per rat (an exceedingly high dose) suggested a protective effect of the secosteroid if it was delivered before the mutagen. When the protective action of vitamin D in combination with dietary calcium on carcinogen-induced colon tumors in rodents was investigated [83,84], it became apparent that both substances affected cellular kinetic indices, i.e., tumor size and not tumor incidence, and that their mode of action appeared to be a cooperative one. Newmark and Lipkin [85] introduced the concept of a nutritional stress diet for mice designed to represent the human Western diet, which is deficient in calcium and vitamin D, and rich in phosphate and fat. This diet led to hyperproliferation and hyperplasia in the rodent colon and, when fed long enough, to functional and structural modifications in the colon mucosa similar to those found in humans at increased risk for colonic neoplasia [86]. Mokady et al. [87] demonstrated that rats fed the Western stress diet had an enhanced response to tumor induction by a carcinogen, whereas supplementary vitamin D3 abrogated this tumor induction. (See also Chapter 91.) Several studies addressed the question of the efficacy of vitamin D analogs in rodent colon tumor models [88–91]. In these it was claimed that blood calcium levels generally were not raised by the analogs and that the development of aberrant crypt foci, a putative neoplastic lesion, or of carcinogen-induced tumors was significantly reduced by vitamin D compounds. The most pronounced inhibition was found if analogs were administered after carcinogen treatment. The wellknown vitamin D analog EB1089 was used in a human colon cancer LoVo cell xenograft study in a nude mouse model and led to 50% inhibition of tumor growth [92]. Another xenograft study with human colorectal cancer cells differing in VDR content demonstrated that tumor growth of VDR-positive cells was reduced in a concentration-dependent manner by 1,25-(OH)2-D3 and the hexafluorinated analog Ro25-6760, whereas growth of VDR-negative xenografts was not [93]. Tanaka et al. [94] used a novel analog, DD-003, to inhibit HT-29 human colon cancer cell growth under the renal capsule of immunodeficient mice. Interestingly, PKC isoform expression was significantly altered in precancerous lesions in rat colon after treatment with Ro24-5531 [95].

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While some of these vitamin D–derived compounds appear to be quite effective in reducing tumor size and tumor burden, hypercalcemia is still sometimes detected. The general consensus is clear: vitamin D analogs do inhibit colon carcinogenesis specifically when administered in the postinitiation phase by reducing colonocyte proliferation. A possible additional mechanism of action for these substances in rodent models might be inhibition of angiogenesis, as was demonstrated by Iseki et al. [96], and by inhibition of metastasis [97]. Recently, Wali et al. [98] demonstrated in rat colon that azoxymethane-induced aberrant crypt foci and tumors expressed enhanced levels of cyclin D1, of cyclooxygenase-2, and of inducible nitric oxide synthase, as well as reduced E-cadherin levels. These changes were significantly inhibited by a fluorinated vitamin D analog. Studies on tumor prevention by 1,25-(OH)2-D3 and its analogs were recently extended to the Apc(min) mouse by Huerta et al. [99]. They observed a significant decrease in total tumor burden while serum calcium in the group treated with analogs was only moderately elevated. These results suggest that such analogs may ultimately have utility as in humans chemopreventive agents at least in population groups at high risk for colon cancer, if serum calcium concentration is constantly monitored.

C. Clinical Studies In 1992, Thomas et al. [100,101] evaluated in vitro crypt cell production rate (CCPR) in rectal tissue obtained from familial adenomatous polyposis (FAP) patients. 1 µM–100 pM 1,25-(OH)2-D3, as well as the synthetic vitamin D analogs MC-903 and EB-1089, reduced CCPR in explant cultures by approximately 50%, whereas EGF increased CCPR by 100% [100]. This indicated for the first time that human colorectal tissue would indeed respond to 1,25-(OH)2-D3 and its analogs in a similar manner to cell and animal experiments. Accumulating experimental evidence from in vitro and from animal studies suggested that the analog EB-1089 was potentially useful for human treatment due to weaker calcemic side-effects while still maintaining high antimitotic, prodifferentiating, and apoptotic activity. In this respect, a promising phase I study with EB-1089 in patients with advanced colon and breast cancer was initiated [102]. An initial evaluation of this study [103] showed that hypercalcemia was still seen in patients receiving 17 microgram/day EB-1089 for 10 to 234 days. Hypercalcemia was reversible by discontinuing administration of the substance or by reducing the amount, and a tolerable dose for most patients was established at 7 microgram/day. There were no complete or partial responses, but 6 out

of 21 patients on treatment for more than 90 days showed stabilization of their disease. The only other human trial was performed on FAP patients who had previously undergone colectomy but had upper gastrointestinal polyps. In this double-blind randomized crossover trial, the effectiveness of sulindac, a specific inhibitor of cyclooxygenase-2, was compared with that of calcium in combination with calciferol. While sulindac treatment resulted in reduction of the crypt cell proliferation index in gastric epithelium but not in duodenal mucosa, calcium and calciferol had no effect whatsoever [104].

D. Conclusion Experimental results show quite clearly that, while 1,25-(OH)2-D3 and also some of its analogs indeed have antimitotic and prodifferentiating activity in colon cancer cells in vitro, their use in vivo, especially in the human patient, has not yet been well explored. While administration at low doses does not appear to be very effective, the high nanomolar concentrations needed to inhibit growth frequently are prohibitively hypercalcemic. Epidemiological data have not reliably supported the hypothesis that in humans serum 1,25-(OH)2-D3 at the highest physiological range showed a negative correlation with colorectal tumor incidence (see Section I.A.1). However, there is apparently a negative correlation between high levels of 25-(OH)-D3, the proliferative index of crypt cells [25], and colorectal cancer incidence. While this obviously favors the population group living at latitudes with high incident sunshine, also those consuming vitamin supplements could have higher levels of the precursor of the active metabolite. This precursor could conceivably be used by colon cells for extrarenal synthesis of 1,25-(OH)2-D3, which may function as an autocrine/paracrine cell cycle regulator in the colon. Evidence for this new concept of organlocalized accumulation of 1,25-(OH)2-D3, which would not influence its serum levels, will be discussed in the following sections.

III. VITAMIN D METABOLISM IN NORMAL AND NEOPLASTIC COLON CELLS A. Human Colon Cancer Cell Lines and Primary Cultures In 1990, Tomon et al. [105] were the first to demonstrate vitamin D catabolism in an in vitro intestinal cell

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model, the human colon adenocarcinoma-derived cell line Caco-2. These cells did not exhibit constitutive 25-(OH)-D3-24-hydroxylase (CYP24) activity. Catabolic activity was inducible upon treatment with 100 nM 1,25-(OH)2-D3. While, at this early date, only conjectures about extrarenal synthesis and degradation of 1,25-(OH)2-D3 existed, its role in growth control in the intestine was already well established. Early data by Birge et al. [106] demonstrated, that in rats dosed with vitamin D, mucosal cell turnover was accelerated and there was an approximately 20% increase in villus height, whereas in vitamin D–deficient rats, villus height was blunted. Thus, there is a trophic as well as a differentiating influence on intestinal morphology, which, at the time, was attributed to serum 1,25-(OH)2-D3 that had been synthesized in the kidney from the precursor 25-(OH)-D3. However, in retrospect this growth regulation could also have been attributed to extrarenal intestinal synthesis of 1,25-(OH)2-D3. It was only much later in 1997 when Cross et al. [107] demonstrated in Caco-2 cells the conversion of the precursor 25 OH D3 into 1,25-(OH)2-D3. They found constitutive expression of the 25-D3-1α-hydroxylase (CYP27B1) in almost any growth phase of this cell

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line, and the sequential metabolism/catabolism of the secosteroid along the C-24 and C-23 oxidative pathways. It is therefore conceivable that human colon cells can control their growth via 1,25-(OH)2-D3 in an autocrine/ paracrine manner dependent upon presence of the vitamin D receptor. Bischof et al. [108] provided evidence that distinct oxidation pathways for 1,25-(OH)2-D3 catabolism were used by two Caco-2 clones differing in their level of differentiation. Colonic metabolism of vitamin D was subsequently evaluated in a variety of primary cultures, which were established from human colon tumors at different grades and stages. Such cultures are more closely related to human physiology and more appropriate to verify evidence obtained from cell lines. Figure 4 compares 25-(OH)-D3 metabolism in two different human colonic cancer cell types. From high performance liquid chromatography (HPLC) analysis, it was obvious that COGA-13 cells, which were isolated from a G2/3 human adenocarcinoma of the right colon, had no innate 1α-hydroxylase activity, which, however, was pronounced in Caco-2 cells (Fig. 4A and 4B). Caco-2 cells responded with reduced 1α-hydroxylation and enhanced 24-hydroxylation to 1,25-(OH)2-D3 treatment (Fig. 4 C), B

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FIGURE 4 Comparison of HPLC profiles of 25-(OH)-D3 metabolism in human colonic cancer cells. Cells were incubated with the precursor 25-(OH)-[26,27-methyl-3H]-D3. The 1α-hydroxylated compounds appear after 20–25 minutes elution time, whereas the 24-hydroxylated metabolites are seen much earlier between 5–10 minutes elution time, just after the precursor peak. A: Untreated Caco-2 cells have mainly 1α-hydroxylated metabolite production and almost no 24-hydroxylase activity. B: Untreated COGA-13 cells have constitutively high 24-hydroxylase activity and no 1α-hydroxylation. C: In Caco-2 cells treatment with 10 nM 1,25-(OH)2-D3 results in significant down-regulation of 1α-hydroxylase activity and significant increase of 24-hydroxylation. D: In COGA-13 cells, the same treatment yields no detectable effects on 25-(OH)-D3 metabolism (Cross and co-workers, unpublished data).

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whereas COGA-13 cells were insensitive, probably because their maximal 24-hydroxylation capacity had already been reached (Fig. 4D) (see also Section III.A.1). These results support the hypothesis that it could be the degree of differentiation of cells that determines their ability for 1,25-(OH)2-D3 synthesis or catabolism (see also [54]). Even more direct evidence for extrarenal vitamin D metabolism present in cancer patient-derived colon tissue was also provided. Cells were isolated from freshly excised human colon tumors, as well as from the adjacent normal mucosa outside the tumor border. After testing cells for general metabolic activity, they were immediately processed for HPLC assays. Bareis et al. [109] demonstrated unequivocally with this method that in human colon cancer cells there is very active vitamin D metabolism including both 24-hydroxylase and 1α-hydroxylase activity, whereas in the adjacent normal mucosa there is very little of either. Bareis and co-workers also demonstrated by RT-PCR analysis in human colon tumor-derived primary cultures low CYP27B1 mRNA and high CYP24 mRNA expression. The latter, however, was not only present as the wild-type transcript, but also as a splice variant. The relationship between splice variants and enzymatic activity is as yet unknown [109]. There is accumulating evidence that human colon cancer cells express varying levels of the metabolic and catabolic vitamin D hydroxylases, supporting the hypothesis that the human colon tissue has differential capacity to accumulate the active hormonal metabolite. In addition, there are apparently unique regulatory processes that depend upon difficult to define levels of differentiation (see also Chapter 79). 1. REGULATION OF VITAMIN D METABOLISM CATABOLISM BY 1,25-(OH)2-D3 AND EPIDERMAL GROWTH FACTOR IN VITRO

of their 24-hydroxylation pattern after treatment with the active vitamin D metabolite (Fig. 4D). Thus, there is, on the one hand, constitutive expression of this catabolic enzyme in cells derived from a colon tumor at a low differentiation level. On the other hand, in Caco-2 cells even under vitamin D treatment, there is still 1α-hydroxylation though reduced. However, there is also 24-hydroxylating activity. This clearly demonstrates the potential problem of hormone degradation, which might exist in colon tissue following localized synthesis of 1,25-(OH)2-D3. Two different clones derived from the Caco-2 cell line were analyzed (Caco-2/AQ with high proliferation, lower differentiation; Caco-2/15 with low proliferation, high differentiation). When their CYP27B1 expression levels after treatment with 10 nM 1,25-(OH)2-D3 were compared, it became obvious that in Caco-2/AQ cells not only activity but also protein (and mRNA) level of CYP27B1 was down-regulated, whereas in Caco-2/15 cells it was increased [54]. Such up-regulated expression of CYP27B1 appeared to be typical for well-differentiated cell lines, and again suggests the primary importance of the biological grade of cells, which would determine synthesis and degradation of 1,25(OH)2-D3 in the human cancerous colon mucosa under physiological conditions. Regulation of vitamin D metabolic and catabolic enzymes was also seen with EGF [54]. This latter observation may be of some physiological importance for colonic synthesis of vitamin D, since expression of the EGFR is well recognized to increase during colon tumor progression (see Table I and [39]) and transforming growth factor α (TGF-α), also a ligand for the EGFR, is an autocrine growth factor during tumorigenesis. Moreover, as described above in Section II.A.1.a, there is mutual regulation between the VDR and the EGFR in colon cancer cell lines (see also [110]).

AND

If cells in the human colon synthesize and catabolize 1,25-(OH)2-D3 to different extents, this may be due to regulatory factors present in the cellular environment. The active hormone 1,25-(OH)2-D3 could itself be present due to local synthesis, and this could result in regulation similar to the well known pathway in the kidney, i.e. down-regulation of 1α-hydroxylase activity and up-regulation of 24-hydroxylase activity. Consequently, treatment of Caco-2 cells for 48 hours with 10 nM 1,25-(OH)2-D3 resulted in induced 24-hydroxylation and in considerable reduction of the 1α-hydroxylated peak, as measured by HPLC analysis (Fig. 4C). COGA-13 cells isolated from a high-grade colon adenocarcinoma, while expressing CYP24 strongly in the control, did not show any modulation

B. Expression of CYP27B1 and CYP24 in Human Intestine There is increasing evidence that during tumor progression differential cellular regulation of vitamin D metabolism and catabolism occurs possibly similar to the regulation demonstrated by Cross et al. [41] in colon cancer patients with respect to vitamin D receptor expression: VDR is elevated at the mRNA level very early during progression, i.e. in differentiated tumors, while late during progression VDR expression is significantly reduced [38,41]. Such a regulatory pattern points towards a physiological defense mechanism against tumor progression, which may fail during late stages.

1718 1. EVALUATION OF MRNA EXPRESSION CYP27B1 AND CYP24

FOR

Evaluation of tissue specimens from 50 colorectal adenocarcinomas by RT-PCR demonstrated convincingly that CYP27B1 mRNA was elevated in G1 and G2 tumors when compared with adjacent normal mucosa from the same patient, and also in comparison with colon mucosa from noncancer patients. In G3 tumors expression dropped to low levels [41]. This seemed to be true only for early stages of the disease (pT1-pT3), during late stage (pT4) disease both G2 and G3 tumors had low CYP27B1 mRNA expression [109]. Tangpricha et al. [111] studied CYP27B1 mRNA expression by real-time PCR in normal colon and colon tumors, though a quantitative evaluation was not possible due to the small number of patients. Recently, Ogunkolade et al. [112] showed CYP27B1 expression in colonic tissue also by real-time RT-PCR in a larger number of individuals. They did not confirm the increase of CYP27B1 in tumors compared with healthy colon samples from noncancer individuals described by Cross et al. [41] and Bareis et al. [109]. It has to be pointed out, however, that Ogunkolade et al. did not examine colonic tumors with respect to the biological grade of cells as was done in other studies [41,109]. Cross et al. [113] provided very recently the evaluation of CYP27B1 mRNA by real-time PCR in colon tumors and adjacent mucosa from 18 cancer patients, as well as in colon mucosa from 5 noncancer patients. All tumor patients had high to medium differentiated (G1, G2) primary adenocarcinomas. The authors clearly demonstrated that the normal mucosa from tumor as well as from nontumor patients had similarly low levels of CYP27B1 mRNA, whereas this was consistently increased in tumor tissue. The discrepancies in results between different laboratories [41,109,113], and [111,112], are most likely caused by the fact, that (1) G3 tumors frequently have very low expression of CYP27B1 mRNA, and (2) that sometimes the adjacent “normal” mucosa of a G3 tumor displays high expression of CYP27B1 mRNA similar to that found in early colon tumors [42]. Bareis et al. [109] detected a transcript of CYP24 in human cancerous colon lesions as well as in the adjacent mucosa of the same patient. While CYP24 was consistently higher in tumor tissue than in adjacent normal tissue, they also found at least two transcripts of differing size, where the larger one contained an additional sequence with homology to intron 1. Expression levels of the smaller transcript appeared to be highest in late-stage high-grade tumors, whereas the larger one was present in low-grade highly-differentiated tumor tissue.

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2. CYP27B1 PROTEIN EXPRESSION IN HUMAN COLON

THE

Zehnder et al. [114] were the first to demonstrate by immunohistochemistry and immunoblotting extrarenal expression of the CYP27B1. Among the tissues investigated was also the human colon where staining was found in epithelial cells and apparently also in parasympathetic ganglia. They suggested that the discrete pattern of staining found in various organs in the human body emphasized a possible role for the hydroxylase as an intracrine modulator of vitamin D function in peripheral tissues. When Cross and co-workers evaluated CYP27B1 protein expression in colon tissue samples (normal mucosa from noncancer patients, adenomas, low- and high-grade tumors and adjacent mucosa from the same patient) from 38 patients by immunofluorescence, they found that at least 50% of tumor patients were positive. This positivity depended completely upon histology of the tissue: As long as there were glandular differentiated structures present, these were positive for CYP27B1, even if the rest of the tissue consisted of cells of low differentiation and was negative for CYP27B1. Fig. 5A shows normal human colon mucosa with barely any positivity. Fig. 5B exemplifies strongly enhanced expression of CYP27B1 in a colon adenoma, while Fig. 5C shows strong expression in the small intestine (ileum). a. Coexpression of CYP27B1, VDR, and Ki-67 Proteins in Human Colon Further evaluation of human colon tissue sections from cancer patients by immunofluorescence showed that many cells positive for the VDR were also positive for CYP27B1 as recently shown by Bises et al. [115]. However, there were also many cells only positive for the VDR, especially in normal and premalignant tissues. This again points to an autocrine/paracrine mode of action for the secosteroid. While the VDR is obviously present also in normal mucosa where it may regulate mucosal function and growth with 1,25-(OH)2-D3 as ligand, it is only during onset of malignant progression that CYP27B1 expression is strongly induced in more than 50% of patients. While the VDR frequently is found also in proliferating cells (there is coexpression with the Ki-67 antigen, see Fig. 1), CYP27B1 positivity is rarely found in Ki-67positive cells (Cross et al., unpublished observations). While CYP27B1 positivity is barely apparent by immunofluorescence in the normal colonic mucosa, it is interesting to observe that in small intestine (ileum) much more positivity is present (Fig. 5C). This observation suggests that in the small intestine there is an innate defense against hyperproliferation due to high

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B

A

C

FIGURE 5

Immunofluorescence analysis of CYP27B1 protein in normal human colon mucosa (A), colon adenoma (B), and small intestine (C). (Cross and co-workers, unpublished data).

local synthesis of the 1,25-(OH)2-D3. This could, at least partially, be responsible for the low tumor incidence occurring in the human small intestine.

C. CYP27B1, CYP24, and VDR Expression in a Mouse Model Kállay et al. [44] demonstrated in a mouse model that VDR expression was higher in the proximal than in the distal colon. Their results from a VDR-knockout (VDR-KO) mouse showed that it was mainly the distal colon that was negatively affected by the lack of genomic action of 1,25-(OH)2-D3 [44]. However, CYP27B1 mRNA expression in proximal and distal mouse colon seemed quite similar, whereas CYP24 expression was much higher in the proximal than in the distal colon [116]. This implies that in the proximal colon less active vitamin D metabolite would be potentially available. Recently Kállay et al. [117] demonstrated for the first time by RT-PCR that the regulation of CYP27B1 and CYP24 mRNA in the mouse colon is completely different from that in the kidney. In the VDR-KO mouse kidney, CYP27B1 mRNA expression is highly induced due to lack of the VDR and consequent ineffectiveness of 1,25-(OH)2-D3 action, while, in the colon CYP27B1 expression is significantly downregulated in parallel with enhanced proliferation (as shown by increased PCNA expression) (Fig. 6A, B). These very recent results demonstrate again the unique aspects of extrarenal 1,25-(OH)2-D3 synthesis, the completely different regulation in the colon in contrast to the one in the kidney, and also the potential importance this could have for colorectal cancer prevention.

IV. NUTRITIONAL REGULATION OF CYP27B1 AND CYP24 This new concept of extrarenal synthesis and catabolism of vitamin D in colonocytes should gain increasing importance for tumor prevention as well as for therapy, since 1,25-(OH)2-D3 could act locally and prevent hyperproliferation and dedifferentiation without causing generalized hypercalcemia. However, some cells could respond to local accumulation of 1,25-(OH)2-D3 with increased activity of CYP24 and decreased activity A Kidney

Colon

B

VDR +/+

FIGURE 6

VDR −/−

A: Comparison of the CYP27B1 status in the kidney and colon of wild-type (VDR+/+) and knockout (VDR−/−) mice. B: Expression of the proliferation marker PCNA in the colon of wild-type (VDR+/+) and knockout (VDR−/−) mice.

1720 of CYP27B1. Therefore, the regulation of these colonic enzymes by, for instance, nutritional means to improve localized accumulation of 1,25-(OH)2-D3 might be of some importance for cancer prevention and also for therapy of low-grade, early-stage tumors. Considerable physiological evidence is accumulating for a protective effect of estrogenic substances against colorectal cancer incidence. At all ages, women are less likely than men to develop colon cancer, and postmenopausal hormone replacement therapy even further reduces colon cancer risk by up to 25%. Potter et al. [118] demonstrated lower risk of adenomatous polyps of the large bowel with hormone replacement therapy. In addition, in several colon cancer animal models, male rodents were shown to have higher tumor burden and increased aberrant crypt formation rates (see [119]), the latter being a typical precursor lesion of colorectal cancer. Very recently a large comprehensive study by the Women’s Health Initiative Investigators [120] on physiological effects of hormone replacement therapy (HRT) was stopped, since most parameters that were assumed to be beneficially affected by HRT, were either negatively (for instance, breast cancer) or not at all affected. The only highly significant exceptions were reduction in incidence of colorectal cancer and of osteoporosis. Thus, the reluctance to use HRT for a longer period and for minor problems seems to be well founded. However, the observed reduction of colorectal cancer incidence needs to be explored further. The mechanism of action could conceivably involve modulation of the vitamin D system. In the clinical situation, for instance, plasma 1,25(OH)2-D3 levels are elevated during human pregnancy and remain high postpartum in lactating women [120, 121] even beyond that component explained by increased DBP (see Chapter 51). In animal studies, female rats treated with estradiol benzoate daily for eight days had increased 1,25-(OH)2-D3 concentrations in plasma, gut mucosa, and kidneys [123]. Interactions between vitamin D and estrogen have also been observed in murine colon carcinoma [124]. However, evidence that estrogenic substances indeed regulate vitamin D metabolism was still missing.

A. Regulation of 1,25-(OH)2-D3 Synthesis by Phytoestrogens Dramatically reduced incidence, especially of hormone-related cancers such as mammary and prostate tumors, has been linked to the consumption of a typical Asian diet, which contains high amounts of soy products and thus is rich in phytoestrogens. It is conceivable that these substances, through their potential to

HEIDE S. CROSS

act as selective estrogen receptor modulators, could have an effect on vitamin D–related inhibition of tumor growth in the mammary and prostate gland. Interestingly, isoflavones belonging to the group of phytoestrogens have been shown to down-regulate estrogen receptor (ER) expression, which could lead to reduced estrogenic responses, i.e. protection against deleterious sex hormone effects in hormone-responsive tissues [125]. Foley and co-workers [126] suggested that malignant transformation of the human colon is associated with a marked diminution of ER-β expression, which is widely regarded to be the predominant ER-subtype in normal colonic tissue [127]. Phytoestrogens bind with high affinity to ER-β. This could indicate a possible protective mode of action for phytoestrogens in the colon, even though this requires further experimental evaluation. Kállay et al. [128] demonstrated in a mouse model that soy feeding elevated CYP27B1 mRNA expression both in the proximal and distal sections of the colon. In contrast, CYP24 mRNA expression was considerably reduced by soy consumption. When they administered genistein, a well-known isoflavone, by gavage to mice, a similar effect was observed (Table II). This suggests that genistein, a major constituent of soy, is one of the nutritional substances that could be used to modulate vitamin D metabolism and catabolism. These results imply potentially enhanced colonic synthesis of 1,25-(OH)2-D3 in populations on high nutritional soy consumption, and conceivably enhanced protection against colorectal cancer. While, in breast and prostate tissue, phytoestrogens contained in soy may very well interfere with the action of estrogen itself, they could also stimulate extrarenal vitamin D synthesis

TABLE II Expression of CYP27B1 and CYP24 mRNA in the Murine Colon CYP27B1 Right colon AIN 76A diet Phytoestrogen diet

242 ± 176 468 ± 135*

Left colon

CYP24 Right colon

Left colon

148 ± 76 111 ± 22 33 ± 9.4 460 ± 182* 23 ± 11* 27 ± 6.7

Multiplex RT-PCR, i.e. simultaneous amplification of transcripts specific either for CYP27B1 or for CYP24, and a transcript specific for the epithelial cell marker cytokeratin 8, was carried out for semi-quantitative evaluation of respective mRNA expression levels. PCR products were analyzed on agarose gels. The level of CYP27B1 and CYP24 expression was correlated with that of the epithelial cell marker CK 8. Values are expressed as mean ± SD, n = 8 animals in each group. Statistical significance is indicated as * ( p < 0.05).

1721


in such tissues, enabling the steroid hormone to exert its well recognized antimitotic, prodifferentiating action [129].

Acknowledgment I thank Professor Meinrad Peterlik, PhD., MD, for invaluable suggestions and criticisms, and Dr. Enikö Kállay for critically reviewing the manuscript.

V. CONCLUSION While epidemiology has provided strong support for the concept, that vitamin D could be a prevention factor during colon tumorigenesis, it was only the serum level of the precursor 25-(OH)-D3 and not 1,25-(OH)2-D3 itself that correlated convincingly with human colorectal tumor incidence. While 1,25-(OH)2-D3 is known to prevent proliferation and to induce differentiation and apoptosis in colonocytes, pharmacological doses are necessary, regardless whether it is tested in an in vitro or an in vivo system. Such high concentrations, however, are prohibitively hypercalcemic. While some analogs may maintain their effectiveness as cell cycle regulators at 10–100-fold lower doses than the parent compound, even these concentrations could result in hypercalcemia in patients. It therefore appears unlikely that any of these compounds will ever be used for preventive purposes. However, in late stage colon cancers, they may very well prove to stabilize the disease. Recent data have demonstrated extrarenal synthesis of the secosteroid in the colon. Physiological regulation of vitamin D metabolic and catabolic hydroxylases in normal and malignant human colonic tissue suggests a role for the locally accumulated hormone in prevention of tumor progression. In addition, during low-grade earlystage malignancy, colonic synthesis of 1,25-(OH)2-D3 could potentially provide a block to progression, if 1,25-(OH)2-D3 catabolism could be inhibited. Renal or colonic 1,25-(OH)2-D3 synthesis and catabolism is differentially regulated. While lack of vitamin D action due to absence of the VDR results in elevated expression of CYP27B1 in the kidney, the same enzyme is down-regulated in the colon, probably due to enhanced proliferation of the tissue. This implies that there may exist substances that could enhance extrarenal 1,25-(OH)2-D3 accumulation without affecting renal synthesis. Recent results allude to such action mechanisms for phytoestrogens. Genistein, a phytoestrogen and major component of soy, can induce expression of CYP27B1 and reduce that of CYP24 in the mouse colon. The involvement of phytoestrogens in the regulation of the vitamin D system could conceivably explain the observation that women have less colorectal cancer incidence than men, probably due to their higher estrogenic background. Consumption of phytoestrogens, however, could also be acceptable for men to protect against colorectal cancer incidence.

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1724 74. Tanaka Y, Bush KK, Klauck TM, Higgins PJ 1989 Enhancement of butyrate-induced differentiation of HT-29 human colon carcinoma cells by 1,25-dihydroxyvitamin D3. Biochem Pharmacol 38:3859–3865. 75. Evans SR, Soldatenkov V, Shchepotin EB, Bogrash E, Shchepotin IB 1999 Novel 19-nor-hexafluoride vitamin D3 analog (Ro 25-6760) inhibits human colon cancer in vitro via apoptosis. Int J Oncol 14:979–985. 76. Diaz GD, Paraskeva C, Thomas MG, Binderup L, Hague A 2000 Apoptosis is induced by the active metabolite of vitamin D3 and its analogue EB1089 in colorectal adenoma and carcinoma cells: possible implications for prevention and therapy. Cancer Res 60:2304–2312. 77. Scaglione-Sewell BA, Bissonnette M, Skarosi S, Abraham C, Brasitus TA 2000 A vitamin D3 analog induces a G1-phase arrest in CaCo-2 cells by inhibiting cdk2 and cdk6: roles of cyclin E, p21Waf1, and p27Kip1. Endocrinology 141: 3931–3939. 78. Pence BC, Buddingh F 1988 Inhibition of dietary fat-promoted colon carcinogenesis in rats by supplemental calcium or vitamin D3. Carcinogenesis 9:187–190. 79. Kawaura A, Tanida N, Sawada K, Oda M, Shimoyama T 1989 Supplemental administration of 1α-hydroxyvitamin D3 inhibits promotion by intrarectal instillation of lithocholic acid in N-methyl-N-nitrosourea-induced colonic tumorigenesis in rats. Carcinogenesis 10:647–649. 80. Oda M, Kawaura A, Tanida N, Sawada K, Maekawa S, Kano M, Shimoyama T 1990 Effects of 1α-hydroxyvitamin D3 on N-methyl-N-nitrosourea-induced colonic tumorigenesis, and on fecal bile acid profiles with respect to soluble and precipitated phases in rats. Tokushima J Exp Med 37:75–81. 81. Comer PF, Clark TD, Glauert HP 1993 Effect of dietary vitamin D3 (cholecalciferol) on colon carcinogenesis induced by 1,2-dimethylhydrazine in male Fischer 344 rats. Nutr Cancer 19:113–124. 82. Belleli A, Shany S, Levy J, Guberman R, Lamprecht SA 1992 A protective role of 1,25-dihydroxyvitamin D3 in chemicallyinduced rat colon carcinogenesis. Carcinogenesis 13: 2293–2298. 83. Sitrin MD, Halline AG, Abrahams C, Brasitus TA 1991 Dietary calcium and vitamin D modulate 1,2-dimethylhydrazineinduced colonic carcinogenesis in the rat. Cancer Res 51: 5608–5613. 84. Beaty MM, Lee EY, Glauert HP 1993 Influence of dietary calcium and vitamin D on colon epithelial cell proliferation and 1,2-dimethylhydrazine-induced colon carcinogenesis in rats fed high fat diets. J Nutr 123:144–152. 85. Newmark HL, Lipkin M 1992 Calcium, vitamin D, and colon cancer. Cancer Res 52:2067s–2070s. 86. Risio M, Lipkin M, Newmark H, Yang K, Rossini FP, Steele VE, Boone CW, Kelloff GJ 1996 Apoptosis, cell replication, and Western-style diet-induced tumorigenesis in mouse colon. Cancer Res 56:4910–4916. 87. Mokady E, Schwartz B, Shany S, Lamprecht SA 2000 A protective role of dietary vitamin D3 in rat colon carcinogenesis. Nutr Cancer 38:65–73. 88. Otoshi T, Iwata H, Kitano M, Nishizawa Y, Morii H, Yano Y, Otani S, Fukushima S 1995 Inhibition of intestinal tumor development in rat multi-organ carcinogenesis and aberrant crypt foci in rat colon carcinogenesis by 22-oxa-calcitriol, a synthetic analogue of 1α,25-dihydroxyvitamin D3. Carcinogenesis 16:2091–2097. 89. Wali RK, Bissonnette M, Khare S, Hart J, Sitrin MD, Brasitus TA 1995 1α,25-dihydroxy-16-ene-23-yne-26,27hexafluorocholecalciferol, a noncalcemic analogue of

HEIDE S. CROSS

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1α,25-dihydroxyvitamin D3, inhibits azoxymethane-induced colonic tumorigenesis. Cancer Res 55:3050–3054. Salim EI, Wanibuchi H, Taniyama T, Yano Y, Morimura K, Yamamoto S, Otani S, Nishizawa Y, Morii H, Fukushima S 1997 Inhibition of development of N,N′-dimethylhydrazineinduced rat colonic aberrant crypt foci by pre, post, and simultaneous treatments with 24R,25-dihydroxyvitamin D3. Jpn J Cancer Res 88:1052–1062. Taniyama T, Wanibuchi H, Salim EI, Yano Y, Otani S, Nishizawa Y, Morii H, Fukushima S 2000 Chemopreventive effect of 24R,25-dihydroxyvitamin D3 in N,N′-dimethylhydrazine-induced rat colon carcinogenesis. Carcinogenesis 21:173–178. Akhter J, Chen X, Bowrey P, Bolton EJ, Morris DL 1997 Vitamin D3 analog, EB1089, inhibits growth of subcutaneous xenografts of the human colon cancer cell line, LoVo, in a nude mouse model. Dis Colon Rectum 40:317–321. Evans SR, Schwartz AM, Shchepotin EI, Uskokovic M, Shchepotin IB 1998 Growth inhibitory effects of 1,25-dihydroxyvitamin D3 and its synthetic analog, 1α,25dihydroxy-16-ene-23yne-26,27-hexafluoro-19-nor-cholecalciferol (Ro 25-6760), on a human colon cancer xenograft. Clin Cancer Res 4:2869–2876. Tanaka Y, Wu AY, Ikekawa N, Iseki K, Kawai M, Kobayashi Y 1994 Inhibition of HT-29 human colon cancer growth under the renal capsule of severe combined immunodeficient mice by an analog of 1,25-dihydroxyvitamin D3, DD-003. Cancer Res 54:5148–5153. Wali RK, Bissonnette M, Khare S, Aquino B, Niedziela S, Sitrin M, Brasitus TA 1996 Protein kinase C isoforms in the chemopreventive effects of a novel vitamin D3 analog in rat colonic tumorigenesis. Gastroenterology 111:118–126. Iseki K, Tatsuta M, Uehara H, Iishi H, Yano H, Sakai N, Ishiguro S 1999 Inhibition of angiogenesis as a mechanism for inhibition by 1α-hydroxyvitamin D3 and 1,25-dihydroxyvitamin D3 of colon carcinogenesis induced by azoxymethane in Wistar rats. Int J Cancer 81:730–733. Evans SR, Shchepotin EI, Young H, Rochon J, Uskokovic M, Shchepotin IB 2000 1,25-dihydroxyvitamin D3 synthetic analogs inhibit spontaneous metastases in a 1,2-dimethylhydrazine-induced colon carcinogenesis model. Int J Oncol 16:1249–1254. Wali RK, Khare S, Tretiakova M, Cohen G, Nguyen L, Hart J, Wang J, Wen M, Ramaswamy A, Joseph L, Sitrin M, Brasitus T, Bissonnette M 2002 Ursodeoxycholic acid and F(6)-D3 inhibit aberrant crypt proliferation in the rat azoxymethane model of colon cancer: roles of cyclin D1 and E-cadherin. Cancer Epidemiol Biomarkers Prev 11:1653–1662. Huerta S, Irwin RW, Heber D, Go VL, Koeffler HP, Uskokovic MR, Harris DM 2002 1α,25-(OH)2-D3 and its synthetic analog decrease tumor load in the Apc(min) Mouse. Cancer Res 62:741–746. Thomas MG 1995 Luminal and humoral influences on human rectal epithelial cytokinetics. Ann R Coll Surg Engl 77:85–89. Thomas MG, Tebbutt S, Williamson RC 1992 Vitamin D and its metabolites inhibit cell proliferation in human rectal mucosa and a colon cancer cell line. Gut 33:1660–1663. Hansen CM, Maenpaa PH 1997 EB 1089, a novel vitamin D analog with strong antiproliferative and differentiation-inducing effects on target cells. Biochem Pharmacol 54:1173–1179. Gulliford T, English J, Colston KW, Menday P, Moller S, Coombes RC 1998 A phase I study of the vitamin D analog EB 1089 in patients with advanced breast and colorectal cancer. Br J Cancer 78:6–13.


104. Seow-Choen F, Vijayan V, Keng V 1996 Prospective randomized study of sulindac versus calcium and calciferol for upper gastrointestinal polyps in familial adenomatous polyposis. Br J Surg 83:1763–1766. 105. Tomon M, Tenenhouse HS, Jones G 1990 Expression of 25-hydroxyvitamin D3-24-hydroxylase activity in Caco-2 cells. An in vitro model of intestinal vitamin D catabolism. Endocrinology 126:2868–2875. 106. Birge SJ, Alpers DH 1973 Stimulation of intestinal mucosal proliferation by vitamin D. Gastroenterology 64:977–982. 107. Cross HS, Peterlik M, Reddy GS, Schuster I 1997 Vitamin D metabolism in human colon adenocarcinoma-derived Caco-2 cells: expression of 25-hydroxyvitamin D3-1alpha-hydroxylase activity and regulation of side-chain metabolism. J Steroid Biochem Mol Biol 62:21–28. 108. Bischof MG, Siu-Caldera ML, Weiskopf A, Vouros P, Cross HS, Peterlik M, Reddy GS 1998 Differentiationrelated pathways of 1 alpha,25-dihydroxycholecalciferol metabolism in human colon adenocarcinoma-derived Caco-2 cells: production of 1 alpha,25-dihydroxy-3epi-cholecalciferol. Exp Cell Res 241:194–201. 109. Bareis P, Bises G, Bischof MG, Cross HS, Peterlik M 2001 25-hydroxyvitamin D metabolism in human colon cancer cells during tumor progression. Biochem Biophys Res Commun 285:1012–1017. 110. Tong WM, Hofer H, Ellinger A, Peterlik M, Cross HS 1999 Mechanism of antimitogenic action of vitamin D in human colon carcinoma cells: relevance for suppression of epidermal growth factor-stimulated cell growth. Oncol Res 11:77–84. 111. Tangpricha V, Flanagan JN, Whitlatch LW, Tseng CC, Chen TC, Holt PR, Lipkin MS, Holick MF 2001 25-hydroxyvitamin D-1alpha-hydroxylase in normal and malignant colon tissue. Lancet 357:1673–1674. 112. Ogunkolade BW, Boucher BJ, Fairclough PD, Hitman GA, Dorudi S, Jenkins PJ, Bustin SA 2002 Expression of 25-hydroxyvitamin D-1-alpha-hydroxylase mRNA in individuals with colorectal cancer. Lancet 359:1831–1832. 113. Cross HS, Kallay E, Farhan H, Weiland T, Manhardt T 2003 Regulation of extrarenal vitamin D metabolism as a tool for colon and prostate cancer prevention. Recent Results Cancer Res 164:413–425. 114. Zehnder D, Bland R, Williams MC, McNinch RW, Howie AJ, Stewart PM, Hewison M 2001 Extrarenal expression of 25-hydroxyvitamin d(3)-1 alpha-hydroxylase. J Clin Endocrinol Metab 86:888–894. 115. Bises G, Kállay E, Weiland T, Wrba F, Wenzl E, Bonner E, Kriwanek S, Obrist P, Cross HS 2004 25-hydroxyvitamin D31α-hydroxylase expression in normal and malignant human colon. J Histochem Cytochem 52(7):985–989. 116. Cross HS, Kállay E, Korchide M, Lechner 2003 Regulation of extrarenal synthesis of 1,25-dihydroxyvitamin D3— relevance for colonic cancer prevention and therapy. Molecular Aspects of Medicine. 24(6):459–465.

1725 117. Kállay E, Bajna E, Cross HS 2002 Gender- and sitespecific expression of 25-hydroxyvitamin D3-1α-hydroxylase in the mouse large intestine: relevance for colonocyte hyperproliferation. Proceedings of the 93rd Anual Meeting of the American Association for Cancer Research. San Francisco. 43:127. 118. Potter JD, Bostick RM, Grandits GA, Fosdick L, Elmer P, Wood J, Grambsch P, Louis TA 1996 Hormone replacement therapy is associated with lower risk of adenomatous polyps of the large bowel: the Minnesota Cancer Prevention Research Unit Case-Control Study. Cancer Epidemiol Biomarkers Prev 5:779–784. 119. Ochiai M, Watanabe M, Kushida H, Wakabayashi K, Sugimura T, Nagao M 1996 DNA adduct formation, cell proliferation, and aberrant crypt focus formation induced by PhIP in male and female rat colon with relevance to carcinogenesis. Carcinogenesis 17:95–98. 120. Design of the Women’s Health Initiative clinical trial and observational study. The Women’s Health Initiative Study Group. Control Clin Trials 19:61–109. 121. Kumar R, Cohen WR, Silva P, Epstein FH 1979 Elevated 1,25-dihydroxyvitamin D plasma levels in normal human pregnancy and lactation. J Clin Invest 63:342–344. 122. Lund B, Selnes A 1979 Plasma 1,25-dihydroxyvitamin D levels in pregnancy and lactation. Acta Endocrinol (Copenh) 92:330–335. 123. Baksi SN, Kenny AD 1978 Does estradiol stimulate in vivo production of 1,25-dihydroxyvitamin D3 in the rat? Life Sci 22:787–792. 124. Smirnoff P, Liel Y, Gnainsky J, Shany S, Schwartz B 1999 The protective effect of estrogen against chemically-induced murine colon carcinogenesis is associated with decreased CpG island methylation and increased mRNA and protein expression of the colonic vitamin D receptor. Oncol Res 11:255–264. 125. Sathyamoorthy N, Wang TT 1997 Differential effects of dietary phyto-oestrogens daidzein and equol on human breast cancer MCF-7 cells. Eur J Cancer 33:2384–2389. 126. Foley EF, Jazaeri AA, Shupnik MA, Jazaeri O, Rice LW 2000 Selective loss of estrogen receptor beta in malignant human colon. Cancer Res 60:245–248. 127. Campbell-Thompson M, Lynch IJ, Bhardwaj B 2001 Expression of estrogen receptor (ER) subtypes and ERbeta isoforms in colon cancer. Cancer Res 61:632–640. 128. Kallay E, Adlercreutz H, Farhan H, Lechner D, Bajna E, Gerdenitsch W, Campbell M, Cross HS 2002 Phytoestrogens regulate vitamin D metabolism in the mouse colon: relevance for colon tumor prevention and therapy. J Nutr 132: 3490S–3493S. 129. Farhan H, Wahala K, Cross HS 2003 Genistein inhibits vitamin D hydroxylases CYP24 and CYP27B1 expression in prostate cells. J Steroid Biochem Mol Biol 84: 423–429.

CHAPTER 96

Vitamin D and Hematological Malignancy JAMES O’KELLY ROBERTA MOROSETTI H. PHILLIP KOEFFLER

I. II. III. IV.

Division of Hematology/Oncology, Department of Medicine, Cedars-Sinai Medical Center, UCLA School of Medicine, Los Angeles, California Pediatric Oncology Division, Catholic University of Rome, Rome, Italy Division of Hematology/Oncology, Department of Medicine, Cedars-Sinai Medical Center, UCLA School of Medicine, Los Angeles, California

Overview of Hematopoiesis Vitamin D Receptors in Blood Cells Effects of Vitamin D Compounds on Normal Hematopoiesis Effects of Vitamin D Compounds on Leukemic Cell Lines

I. OVERVIEW OF HEMATOPOIESIS Hematopoiesis is the process that leads to the formation of the highly specialized circulating blood cells from bone marrow pluripotent progenitor stem cells. These stem cells are the most primitive blood cells, and they have the ability to either self-replicate or differentiate. They are regulated by a feedback system and are affected by various stimuli such as bone marrow depletion, hemorrhage, infection, and stress. They produce more mature “committed” cells that are able to proliferate and differentiate into cells of different lineages, acquiring specific functional properties (Fig. 1). The pluripotent stem cell common to granulocytes, erythrocytes, monocytes, and megakaryocytes is called the colony-forming unit-GEMM (CFU-GEMM), and the committed cells giving rise to the lineage specific cells are assayed in vitro as erythroid burst-forming units (BFU-E), megakaryocyte colony-forming units (CFUMK), and granulocyte-monocyte colony-forming units (CFU-GM). Each of these stem cells has cell surface receptors for specific cytokines. Binding of cytokines to these receptors stimulates secondary intracellular signals that deliver a message to the nucleus to stimulate proliferation, differentiation, and/or activation. The CFU-GMs, in the presence of cytokines, undergo a differentiation program progressing to granulocytes and monocytes. The growth factors acting primarily on the granulocyte-macrophage pathway are granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), and macrophage colony-stimulating factor (M-CSF). The GM-CSF also stimulates VITAMIN D, 2ND EDITION FELDMAN, PIKE, AND GLORIEUX

V. Vitamin D Analogs Effective Against Leukemic Cells VI. Summary and Conclusions References

eosinophils, enhances megakaryocytic colony formation, and increases erythroid colony formation in the presence of erythropoietin (Epo). In vivo, it causes an increase in granulocytes, monocytes, and eosinophils. It can activate these cells to efficiently fight microbes. The G-CSF stimulates the formation of granulocyte colonies in vitro. It is able to act synergistically with interleukin-3 (IL-3), GM-CSF, and M-CSF. This cytokine is active in vivo, stimulating an increase of peripheral blood granulocytes. The M-CSF stimulates the formation of macrophage colonies in vitro. It maintains the survival of differentiated macrophages and increases their anti-tumor activities and secretion of oxygen reduction products and plasminogen activators. This cytokine binds to a receptor that is the product of the protooncogene c-fms. Interleukin-3 has multilineage stimulating activity and acts directly on the granulocyte-macrophage pathway, but also enhances the development of erythroid, megakaryocytic, and mast cells, and possibly T lymphocytes. In synergy with Epo, IL-3 stimulates the formation of early erythroid stem cells, promoting the formation of colonies of red cells in soft gel culture known as BFU-E. In addition, it supports the formation of early multilineage blast cells in vitro. IL-3 also induces leukemic blasts to enter the cell cycle and induces, either alone or in combination with other growth factors, the production of all the myeloid cells in vivo. Stem cell factor (SCF) promotes survival, proliferation and differentiation of hematopoeitic progenitor cells. It synergizes with other growth factors such as IL-3, GM-CSF, G-CSF, and Epo to support the colony growth of BFU-E, CFU-GM, and CFU-GEMM in vitro. Copyright © 2005, Elsevier, Inc. All rights reserved.

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JAMES O’KELLY, ROBERTA MOROSETTI, AND H. PHILLIP KOEFFLER

SCF EPO IL-3

BFU-E

EPO

SCF IL -3 BFU-MK

CFU-Meg

SCF IL-3

SCF CFU-GEMM

SCF IL-3 GM-CSF TPO

CFU-M IL-3 GM-CSF CFU-GM G-CSF CFU-G

STEM CELL

Macrophage

Neutrophil

Eosinophil

CFU-Eu CFU-Blast

Platelets

GM-CSF M-CSF Monocyte

IL-3 GM-CSF G-CSF

IL-3 GM-CSF IL-5 SCF

IL -6 TPO Megakaryocyte

IL-3 GM-CSF G-MSF

IL -3 GM-CSF -

Red Cell

Reticulocyte

CFU-E

IL -3 IL -4 CFU-Bas

Basophil IL-1 IL IL-2 IL IL-6 IL IL-7 IL

Pre-T

T lymphocyte

SCF Lymphoid stem cell

SCF IL-7

IL-6 IL-4

Pre-B B lymphocyte

FIGURE 1 Scheme of hematopoiesis. The key progenitor cells and their growth factors are shown. CFUBlast, colony-forming unit-blast; CFU-GEMM, CFU-granulocyte, erythrocyte, megakaryocyte, macrophage; BFU-E, burst-forming unit-erythroid; CFU-E, CFU-erythroid; BFU-MK, BFU-megakaryocyte; CFU-Meg, CFU-megakaryocyte; CFU-GM, CFU-granulocyte-monocyte; CFU-Eo, CFU-eosinophil; CFU-Bas, CFUbasophil; SCF, stem cell factor; IL-3, interleukin-3; GM-CSF, granulocyte-monocyte colony-stimulating factor; EPO, erythropoietin; TPO, thrombopoietin.

Although SCF alone has a modest effect on colony growth, in the presence of other cytokines SCF increases both the size and the number of these colonies. It is a ligand for the c-kit receptor, a tyrosine kinase receptor that is expressed in hematopoeitic progenitor cells. The growth factor Epo stimulates the formation of erythroid colonies (CFU-E) in vitro and is the primary hormone of erythropoiesis in animals and humans in vivo. It binds to a specific receptor (Epo-R). Production of erythroblasts is hormonally regulated by a feedback mechanism mediated by the linear correlation between tissue oxygenation of Epo-producing cells in the kidney mediated by oxygen-carrying hemoglobin in red blood cells. Anemia causes tissue hypoxia, resulting in an increase of serum Epo levels.

II. VITAMIN D RECEPTORS IN BLOOD CELLS The genomic actions of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] are mediated by the intracellular vitamin D receptor (VDR), which belongs to a large

family of nuclear receptors [1]. VDR forms a heterodimer with the retinoid X receptor (RXR); this complex regulates expression of target genes by binding to vitamin D responsive elements (VDREs) in the promoter regions of their target genes [2]. The mechanism of action of 1,25(OH)2D3 via the VDR is discussed in Chapters 11 and 13. Expression of VDR has been detected in various normal and leukemic hematopoeitic cells. It is expressed constitutively in monocytes, in certain subsets of thymocytes, and after in vitro activation of B and T lymphocytes [3–5]. Expression of VDR is induced in the lymphocytes of patients with rheumatoid arthritis, in human tonsillar lymphocytes, and in pulmonary lymphocytes of patients with tuberculosis and sarcoidosis [6–8]. In addition, lymphocytes of patients with hereditary vitamin D–resistant rickets type II (HVDRR) have various alterations of the VDR [9]. Also, fewer receptors have been detected in the peripheral blood mononuclear cells of patients with X-linked hypophosphatemic rickets [10]. Examination of a large array of myeloid leukemia cell lines blocked at various stages of maturation showed that they all

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CHAPTER 96 Vitamin D and Hematological Malignancy

expressed VDR, albeit at different levels [3]. Bone marrow-derived stromal cells express VDR and show a reduction of their proliferation that occurs after their exposure to 1,25(OH)2D3. Both T-helper and T-suppressor lymphocytes express similar concentrations of VDR. In particular, T lymphocytes express high levels of VDR mRNA, whereas resting B lymphocytes express either very low or nondetectable levels of VDR transcripts [3]. Nevertheless, 1,25(OH)2D3 inhibits the synthesis of immunoglobulins (Ig) by B lymphocytes in vitro [11,12]. This suppression, however, could be the result of the inhibition of T-helper activity [12]. Production of lymphokines, including IL-2, is markedly decreased by 1,25(OH)2D3 in activated T lymphocytes, and this could cause the suppression of Ig synthesis [13–16]. The effects of vitamin D on the immune system are discussed in Chapter 36. Studies by us in VDR knockout (KO) mice indicated that expression of VDR is dispensible for normal myeloid development [17]. No difference in the numbers and percentages of red and white cells were found between VDR KO and wild-type (WT) mice. Committed myeloid stem cells from the bone marrow cultured in methylcellulose formed similar numbers of colonies when grown in the presence of either GM-CSF, G-CSF, M-CSF alone or in combination with IL-3. Furthermore, bone marrow cells from VDR KO and WT mice formed a similar number and percentage of granulocyte, macrophage, and granulocyte/macrophage mixed colonies when cultured in methylcellulose with GMCSF and IL-3. Under these conditions, treatment with 1,25(OH)2D3 dramatically increased the percentage of macrophage colonies derived from WT but not VDR KO bone marrow cultures. This observation demonstrates the requirement of VDR expression for 1,25(OH)2D3–induction of bone marrow progenitors into monocytes/macrophages. The proportion of T- and B-cells were normal in the VDR KO mice. However, the antigen-stimulated spleen cells from VDR KO mice produced less IFNγ and more IL-4 than those from WT mice, indicating impaired Th1 differentiation. Additionally, IL-12 stimulation induced a weaker proliferative response in VDR KO splenocytes as compared to WT, and expression of STAT4 was reduced. These results suggest that VDR plays an important role in the Th1-type immune response. The HL-60 myeloblastic cell line cultured in the presence of 1,25(OH)2D3 (10−7 M) has a 50% decrease of VDR protein levels at about 24 hr, which returned to normal levels after 72 hr; no change of VDR mRNA expression occurred [3]. These data suggested that one of the major sites of regulation of VDR expression occurs at the posttranscriptional level. The same cell line exposed to a lower dose of 1,25(OH)2D3 for 12 hr

appeared to have an increased number of VDRs, as determined by immunoprecipitation, which returned to normal levels after 72 hr [18]. The HL-60 myeloblasts cultured with retinoic acid (RA) and dimethyl sulfoxide (DMSO) or 12-Otetra-decanoylphorbol-13-acetate (TPA) terminally differentiate into granulocytes or macrophages, respectively. The differentiation is associated with induction of high expression of VDR mRNA. Also, normal human nondividing macrophages express VDR mRNA, and these levels do not change after exposure to activating factors such as tumor necrosis factor α (TNFα). The expression of VDR mRNA was not detectable in nonproliferating lymphocytes harvested from normal human peripheral blood, but VDR mRNA expression increased in proliferating lymphocytes after a 24 hr exposure to the lectin phytohemagglutinin-A (PHA), suggesting that in lymphocytes a major site of regulation of VDR expression is at the transcriptional level [3, 19]. Moreover, low levels of VDR expression were detected in low-grade non-Hodgkin’s lymphoma (NHL) tumor samples and in the follicular lymphoma B-cell lines SU-DHL4 and SU-DHL5 [20]. The VDR can bind to the osteocalcin response element along with the activator protein-1 (AP1) complexes [21]. In addition, Jun and Fos proto-oncogenes are upregulated by 1,25(OH)2D3 [22]. Jun-D DNA binding activity is increased during cell cycle arrest in the human chronic myelogenous leukemia RWLeu-4 cultured with 1,25(OH)2D3, suggesting that Jun D binding activity may play a role in the regulation of cell proliferation by 1,25(OH)2D3 [21].

III. EFFECTS OF VITAMIN D COMPOUNDS ON NORMAL HEMATOPOIESIS The role of 1,25(OH)2D3 in cell differentiation was first described by Abe et al. [23] in the murine leukemia cell line Ml, which was induced to differentiate into more mature cells by 1,25(OH)2D3. Normal human bone marrow committed stem cells cultured in either soft agar with 1,25(OH)2D3 (10−7 M) or in liquid culture with 1,25(OH)2D3 (5 × 10−9 M for 5 days) and monocytes cultured in serum-free medium with 1,25(OH)2D3 (5 × 10−8 M for 7 days) differentiate into macrophages [24,25]. In further studies, these macrophages were functionally competent and able to release large amounts of TNFα and IL-6 [26]. Furthermore, the terminal differentiation of monocytes into mature macrophages can be obtained in vitro by culturing these cells in the presence of serum or in a serum-free medium with the addition of vitamin D3 compounds [4,26,27].

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As mentioned earlier, 1,25(OH)2D3 is able to inhibit IL-2 synthesis and the proliferation of peripheral blood lymphocytes [12–15]. Indeed, 1,25(OH)2D3 appears to be able to regulate many lymphokines. For example, Tobler et al. [28] showed that expression of the lymphokine GM-CSF is regulated by 1,25(OH)2D3 through VDR by a process independent of IL-2 production. In particular, 1,25(OH)2D3 was able to inhibit both GMCSF mRNA and protein expression in PHA-activated normal human peripheral blood lymphocytes (PBL). The former occurred at least in part by destabilizing and shortening the half-life of the GM-CSF mRNA [28]. The down-regulation of GM-CSF was obtained at 1,25(OH)2D3 concentrations similar to those reached in vivo, with a 50% reduction of GM-CSF activity occurring at 10−10 M 1,25(OH)2D3. In addition, IL-2 did not affect the modulation of GM-CSF production by 1,25(OH)2D3 in the PBL cocultured with 1,25(OH)2D3 (10−10−10−7 M) and high concentrations of IL-2.

IV. EFFECTS OF VITAMIN D COMPOUNDS ON LEUKEMIC CELL LINES All of the studies conducted so far with 1,25(OH)2D3 emphasize the need for new vitamin D3 analogs with greater anti-leukemic effects and less toxicity. In spite of the promising data obtained from in vitro studies, results of clinical trials in leukemia

with 1,25(OH)2D3 are limited in scope and thus far have exhibited only mediocre results. For example, the myelodysplastic syndrome (MDS) is associated with anemia, thrombocytopenia, and leukopenia and an increased number of myeloid progenitor cells in the bone marrow. Some patients with MDS go on to develop acute myeloid leukemia. We treated 18 MDS patients with increasing doses of 1,25(OH)2D3 up to a maximum of 2 µg/day for 12 weeks. Although an improvement of at least one hematologic parameter occurred in 8 patients after more than 4 weeks, the response was not durable and not detectable at the end of the study at 12 weeks [24]. Nine patients developed hypercalcemia, which was the dose-limiting toxicity. In another study, seven MDS patients were treated with 1,25(OH)2D3 (2.5 µg/day, for at least 8 weeks), with no beneficial effects [29]. A major drawback in using 1,25(OH)2D3 is its calcemic effect, which prevents pharmacological doses of the compound from being given. Vitamin D analogs have been synthesized that have enhanced potency to inhibit proliferation and promote differentiation of cancer cells, with less calcemic activity as compared to 1,25(OH)2D3 (see Chapters 80–88). Many of these analogs in vitro are between 10- and a 1000-fold more active than the parental 1,25(OH)2D3 in their growth suppressive activity. A comparison of the relative anti-leukemic potencies of vitamin D compounds is provided in Table I. These analogs could provide

TABLE I Effect of Vitamin D Compounds on Clonal Proliferation of HL-60 Cells in Soft Agar and Calcium Levels in Mice Compound 1,25(OH)2D3 1,25(OH)2-16-ene-D3 1,25(OH)2-16-ene-23-yne-D3 1,25(OH)2-16-ene-5,6-trans-D3 1,25(OH)2-16-ene-24-oxo-D3 1,25(OH)2-16-ene-19-nor-D3 1,25(OH)2-16-ene-24-oxo-19-nor-D3 1,25(OH)2-20-epi-D3 1,25(OH)2-20-epi-22-oxa-24,26,27-trishomo-D3d 1,25(OH)2-diene-24,26,27-trihomo-D3e 19-nor-1,25(OH)2D2f

ED50a (×10−9 mol/l) 4–18 0.015 3 0.03 0.2 0.8 0.1 0.006 0.001 0.23 2.4

aED represents the effective dose achieving 50% growth inhibition of HL-60 cells. 50 bMTD, Maximally tolerated dose; highest dose reported that did not produce hypercalcemia

toneally, three times per week. cND, not done. dLeo Pharmaceutical code name is KH 1060. e Leo Pharmaceutical code name is EB 1089. f Abbott Laboratories code name is Paricalcitol.

MTDb (µg) 0.0625 0.125 2 4 NDc 0.5 6 0.00125 0.0125 0.25 0.1

Reference [102–109] [102] [102, 104] [103] [107] [107] [106] [104, 108, 109] [108] [104] [31]

or other noticeable toxicities in mice when injected intraperi-

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a larger therapeutic window for the treatment of hematologic malignancies, retaining the useful properties of 1,25(OH)2D3 [30].

A. Celluar Effects of Vitamin D Compounds on Leukemic Cells The various vitamin D compounds have similar effects on inducing differentiation and inhibiting proliferation of several acute myeloid leukemia cell lines such as HL-60, U937, THP-1, HEL, and NB4. In contrast, more immature myeloid leukemia cell lines such as HL-60 blasts, KG1, KGla, and K562 do not respond to the hormone. Vitamin D analogs inhibit cell growth mainly by inducing cell cycle arrest. Many studies have shown that treated leukemic cell lines accumulate in the G0/G1 and G2/M phase of the cell cycle, with a concomitant decrease in the proportion of cells in S-phase [31–33]. These effects of vitamin D compounds on the cell cycle are discussed in Chapter 92. HL-60 cells treated with 1,25(OH)2D3 acquire the morphology and functional characteristics of macrophages. Expression of the cell-surface markers CD14 and CD11b are up-regulated. The cells become adherent to charged surfaces, develop pseudopodia, stain positively for nonspecific esterase (NSE) with a reduction of nitroblue tetrazolium (NET), and acquire the ability to phagocytose yeast during incubation with 1,25(OH)2D3 (10−10−10−7 M for 7 days) [25,34]. In addition, the treated cells acquired the ability to degrade bone marrow matrix in vitro, raising the possibility that the cells may have acquired some osteoclast-like characteristics. Leukemic cells from acute myelogenous leukemia (AML) patients respond to vitamin D compounds when cultured in vitro; however, they are often less sensitive than the cell lines. They are often still able to undergo partial monocytic differentiation as assessed by NBT reduction, morphology, and phagocytic ability. Furthermore, their clonal growth is often inhibited [25,34]. The molecular targets of vitamin D3 compounds in leukemic cells are described in the following section, and are summarized in Table II.

B. Molecular Mechanisms of Action of Vitamin D Compounds Against Leukemic Cells Vitamin D compounds can exert their anti-cancer effects by activating the VDR and modulating the transcription of various target genes. Some of these target genes are associated with inhibition of growth and

TABLE II Molecular Targets of Vitamin D Compounds in Leukemic Cellsa Cell Cycle/Apoptosis Cyclin A ↑ Cyclin D1 ↑ Cyclin E ↑ p15 ↑ p21Waf1 ↑ p27Kip1 ↑ Bcl-2 ↓ Differentiation Markers CD11b ↑ CD14 ↑

Oncogenes c-myc ↓ Dek ↓ Fli ↓ Tumor Suppressors PTEN ↑ BTG ↑ Kinases PI3-K ↑activity p38 MAPK ↑activity ERK 1/2 ↑activity PKC ↑levels

a Regulation of expression or activity may occur either directly or as a consequence of differentiation. See text for details.

induction of differentiation, but this modulation may not be a direct effect, as it may simply reflect the entire process of differentiation. Myeloid leukemic cell lines treated with 1,25(OH)2D3 undergo an initial proliferative burst, which is followed by growth inhibition, terminal differentiation, and subsequent apoptosis [36,37]. Levels of cyclin A, D1, and E increased in U937 cells within 24 hours of 1,25(OH)2D3–treatment, decreasing after 48 hours, although cyclin dependent kinase (CDK) levels did not change [37]. The CDK inhibitors p21 and p27, important regulators of the cell cycle, were elevated during the periods of both proliferation and growth inhibition. A strong correlation appears to exist between early induction of p21 and the beginning of the differentiation program. The marked increase of p21 protein expression in response to 1,25(OH)2D3 may be due to enhanced posttranscriptional stabilization of p21 mRNA [38]. The up-regulation of p21 mRNA occurred independently of de novo protein synthesis, further supporting the hypothesis that p21 is an early response gene. Indeed, the p21 promoter contains a vitamin D response element, and induction requires the presence of VDR. Also, experiments using a variety of cell lines showed that 1,25(OH)2D3 and other differentiating agents could mediate their induction of p21-independent of an intact p53 gene [38]. Using differential hybridization, Liu et al. [39] showed that p21 is differentially expressed in response to 1,25(OH)2D3 in the myelomonocytic cell line U937. Transient overexpression of p21 and p27 in U937 cells

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promoted the appearance of the cell surface differentiation molecules CD14 and CD11b. One series of experiments showed that the p15, p16, p18, p21, and p27 CDKIs were up-regulated in a time-dependent manner after the addition of 1,25(OH)2D3 [39]. This induction occurred within 4 hr of the addition of 1,25(OH)2D3 in the presence of cycloheximide (CHX), suggesting a direct transcriptional activation by VDR. In another study, the protein expression of different G1-phase regulators has been examined in HL-60 cells exposed to different concentrations of 1,25(OH)2D3. A strong up-regulation of p27 protein expression was evident after 72 hr of exposure to the compound, and it was dependent on 1,25(OH)2D3 concentration. This up-regulation was also associated with increased levels of D- and E-cyclins, coinciding with the G1 arrest. These results suggested a prominent role of p27 in mediating the antiproliferative activity of 1,25(OH)2D3 in this cell line [40]. Activation of the protooncogene c-myc by retroviral insertion or chromosomal rearrangement is a typical feature of human leukemias. The HL-60 leukemia cell line is characterized by high levels of expression of cmyc due to gene amplification [41, 42]. Treatment of this cell line with 1,25(OH)2D3 results in a down-regulation of expression of this oncogene related to cell differentiation [43]. Supression of c-myc by 1,25(OH)2D3 and its noncalcemic analogs has been demonstrated to occur at the transcriptional level in HL-60 cells [30,44]. 1,25(OH)2D3 is thought to up-regulate proteins such as the homeobox gene, HoxB4, that binds to the first exon/intron border of c-myc to prevent transcriptional elongation, a process dependent on activation of PKCβ [45,46]. Another homeobox gene, HoxA10, was found by differential display to be a gene transcriptionally induced by 1,25(OH)2D3 during differentiation of U937 cells [47]. 1,25(OH)2D3 has a protective effect against apoptosis in HL-60 cells [48,49]. This effect lends support to the observation that monocytic differentiation interferes with programs leading to apoptotic death. In other cell types, inhibition of apoptosis correlates with elevated levels of Bcl-2, but this does not appear to be the case with myeloid cells. In fact, after culture with 1,25(OH)2D3 a down-regulation of Bcl-2 was observed both at the mRNA and protein levels [49]. Exposure of HL-60 cells to 1,25(OH)2D3 induces the expression of the protooncogene c-fms, which occurs in parallel with the induction of CD14 expression and a block of their cell cycle in G0/G1 phase [50]. In the chronic myelogenous leukemia (CML) cell line RWLeu-4, an inhibition of proliferation was observed after 1,25(OH)2D3 treatment. Moreover, the binding activity of the protooncogene junD was enhanced by 1,25(OH)2D3 in these cells

during their cell cycle arrest, whereas it was not decreased in a 1,25(OH)2D3–resistant variant cell line [21]. Fusion proteins involving the retinoic acid receptor alpha (RAR α) with either the PML or PLZF nuclear proteins are the genetic markers of acute promyelocytic leukemias (APLs). Although APLs with PMLRARα are more sensitive to retinoic acid, expression of either PML-RARα or PLZF-RARα in U937 and HL-60 cells blocks terminal differentiation induced by 1,25(OH)2D3 [51]. Both PML-RARα or PLZF-RARα can bind to VDR in U937 cells and sequester VDR away from its normal sites of localization [52]. Overexpression of VDR overcomes the block in 1,25(OH)2D3–stimulated differentiation caused by the fusion proteins. The cell lines HL-60 and U937 have been used to attempt to identify early response genes directly regulated by VDR. Bories et al. [53] identified a serine protease, myeloblastin, that was down-regulated by phorbol esters in promyelocytic cells, causing growth arrest and cell differentiation. They also isolated cDNAs coding for fructose 1,6-biphosphatase, whose expression is up-regulated by 1,25(OH)2D3 in HL-60 cells and peripheral blood monocytes. Genes regulated during the course of 1,25(OH)2D3–mediated HL-60 cell differentiation have been analyzed using cDNA array analysis [54]. Among the genes shown to be down-regulated were the putative oncogenes Dek and Fli-1, and up-regulated genes included the antiproliferative BTG1. Increasing evidence suggests that both the antiproliferative and differentiation-inducing effects of vitamin D compounds require their modulation of the intracellular kinase pathways, p38 MAPK, ERK, and PI3-K. Activation of PI3-K has been shown to be required for 1,25(OH)2D3–stimulated myeloid differentiation, as determined by induction of CD14 expression [55]. PI3-K was activated by 1,25(OH)2D3 in THP-1 cells within 20 minutes. Pretreatment with the PI3-K inhibitors, LY 294004 and wortmanin, inhibited CD14 induction in response to 1,25(OH)2D3 in THP-1 cells and peripheral blood monocytes. Furthermore, antisense oligonucleotides against PI3-K blocked induction of CD14 expression in THP-1 and U937 cells. Expression of the VDR was required for activation of PI3-K; and interestingly, VDR was found to associate with the active form of the kinase. Inhibitors of PI3-K have also been shown to block the differentiation induced by 1,25(OH)2D3 in HL-60 cells [56]. Exposure of either HL-60 or NB-4 cells to differentiation-inducing concentrations of vitamin D compounds causes activation and nuclear translocation of MAPK [57–59]. In addition, the vitamin D3 analog EB1089 was recently demonstrated to induce apoptosis of B-cell chronic lymphocytic leukemia cells from


patients, an event preceded by stimulation of p38 MAPK and suppression of ERK activity [60]. Furthermore, 1,25(OH)2D3 was found to stimulate the transient [24–48h] phosphorylation of ERK1/2, which was followed by growth arrest and differentiation of HL-60 cells [61]. In another study, PD98059, an ERK1/2 inhibitor, blocked the 1,25(OH)2D3–stimulated differentiation of HL-60 cells [62]. Activation of PKC by the phorbol diesters such as TPA, promotes monocyte differentiation of leukemic cell lines [63,64]. Differentiation of HL-60 cells in response to 1,25(OH)2D3 is accompanied by increased levels of PKCβ, and this differentiation can be inhibited by the specific PKC inhibitor, chelerythrine chloride [65]. Other vitamin D analogs have been shown to stimulate expression and translocation of PKCα and delta during NB-4 monocytic differentiation [66]. Some of the effects of vitamin D compounds on the signaling pathways occur within seconds. For example, rapid changes in the phosphorylation status of MAPK (within 30 seconds) have been demonstrated in response to 1,25(OH)2D3 in NB-4 cells [58]. These effects occur too quickly to be attributed to the genomic actions of vitamin D–mediated activated transcription of target genes by VDR. Nonetheless, 1,25(OH)2D3–activated intracellular signaling pathways require the presence of VDR to stimulate monocyte/macrophage differentiation, as demonstrated by studies on bone marrow cells from VDR KO mice [17] and cells from patients with vitamin D–dependent rickets type II [67,68]. The rapid nongenomic activities of vitamin D are described in detail in Chapter 23.

C. Vitamin D Compounds in Combination with Other Agents Because of the potential toxicity of 1,25(OH)2D3 and its analogs at the concentrations required in vivo, various attempts have been made to use them with other compounds that might act synergistically to achieve an anti-leukemic effect capable of promoting cell differentiation, yet with an acceptable toxicity. A range of compounds with different mechanisms of action have been studied. Vitamin D compounds may cooperate with other differentiating agents such as retinoids, tissue plasminogen activator, and interferon (IFN). For example, 1,25(OH)2D3 can potentiate IFN-γ action to induce the expression of CD11b and CD14. We and others have shown that the combination of vitamin D analogs and either all-trans-retinoic acid (ATRA) or 9-cis-retinoic acid (9-cis-RA) can potentiate the terminal differentiation process of HL-60 cells down the monocyte-macrophage pathway [69,70]. These findings have also been demonstrated in other studies [71,72].

1733 Cells cultured in the presence of the combination of 1,25(OH)2D3 and ATRA developed atypically, having a neutrophilic morphology, but in other properties were typical of monocytes (e.g., CD14 expression, ability to bind to bacterial LPS, and ability to develop sodium fluoride-inhibited NSE) [69,70]. The combination of ATRA (10−9 M) and the vitamin D3 analogs l,25(OH)216-ene-23-yne D3 or 1,25(OH)2-23-yne D3 (10−9 to 10−10 M) showed a synergistic effect on the induction of differentiation and inhibition of proliferation of HL-60 cells [73]. A decrease of c-myc expression was also observed in the presence of ATRA and l,25(OH)2-16ene-23-yne D3. This down-regulation of c-myc was stronger than that observed using single agents and correlated with the initiation of differentiation. A synergistic antineoplastic effect of l,25(OH)2-16-ene-23yne D3 and ATRA has been shown in HL-60 cells [73]. A HL-60 clone resistant to ATRA was much more sensitive to inhibition of proliferation by l,25(OH)2-16ene-23-yne D3 as compared with 1,25(OH)2D3. In addition, the induction of differentiation of these cells by l,25(OH)2-16-ene-23-yne D3 was much stronger in these cells in contrast to wild-type HL-60 cells. Another retinoid-resistant acute promyelocyte leukemia cell line (UF-1) was induced towards granulocye differentiation by 1,25(OH)2D3, in association with stimulation of p21Waf1 and p27Kip1 expression [74]. These effects were enhanced by the addition of ATRA. In the promyelocytic cell line NB4, carrying the translocation t(15;17) typical of APL, vitamin D compounds can act as weak inducers of monocytic differentiation [75,76]. Bathia et al. [77] showed that the combination of 1,25(OH)2D3 and TPA resulted in a synergistic response in NB4 cells, causing a complete differentiation to fully functional adherent macrophages with a rapid arrest of cell growth in the first 24 hr. Remarkable inhibition of proliferation and induction of differentiation occurred when NB4 cells were cultured with both 9-cis-RA and KH1060 (a 20-epi-vitamin D3 analog) [76]. ATRA and 1,25(OH)2D3 also synergistically induce monocytic differentiation in the promonocytic cell line U937 [78]. The same group observed that U937 cells exposed to a moderate thermal stress responded with increased differentiation after the addition of 1,25(OH)2D3 and ATRA [79]. Nonsteroidal anti-inflammatory drugs (NSAIDs) have been shown to enhance the differentiation of HL-60 cells in respnse to 1,25(OH)2D3 and its analogs [80,81]. This effect may occur because of the ability of NSAIDs to inhibit an aldoketoreductase; this enzyme supresses the production of natural PPAR γ ligands by blocking the conversion of prostaglandin D2 to prostaglandin J2 [82].

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Vitamin D compounds have also been combined successfully with naturally occurring plant products. One of these is carnosic acid, a plant-derived polyphenol antioxidant recently shown to potentiate the prodifferentiative effects of 1,25(OH)2D3 [83,84]. These studies demonstrated that carnosic acid enhanced the growth arrest and CD11b and CD14 expression induced by 1,25(OH)2D3 in a HL-60 subline, and combining these compounds produced greater stimulation of VDR expression and activity as determined by gelshift analysis. Differentiation was correlated with antioxidant activity, and was associated with activation of the RAF-ERK pathway and increased binding of the AP-1 transcription factor to the promoter of VDR. Furthermore, potentiation of differentiation by carnosic acid with 1,25(OH)2D3 (10−9 M) did not lead to the increase in intracellular calcium concentrations as compared to when the cells were treated with 10−7 M 1,25(OH)2D3 alone, although differentiation levels were equivalent. Combining vitamin D compounds with traditional chemotherapy agents such as cisplatin, etoposide, and doxorubicin has been shown to reduce the concentrations required for their anti-leukemic activities [85, 86]. Studies in other cancers have also shown that a vitamin D compound combined with a chemotherapeutic agent was more effective than either agent alone [87–89]. A recent phase I clinical trial has demonstrated the therapeutic potential of this method, as very high doses of 1,25(OH)2D3 could be given orally with paclitaxel [90]. Another novel approach to circumvent the calcemic side effects of vitamin D compounds has been to give high doses of 1,25(OH)2D3 subcutaneously every other day [91]. In a phase I clinical trial, the maximum tolerated dose administered in this fashion was five times greater than the daily oral dose that had previously been shown to cause hypercalcemia [91].

V. VITAMIN D ANALOGS EFFECTIVE AGAINST LEUKEMIC CELLS The first attempts using analogs focused on the compound 1α-hydroxyvitamin D3 (1αOHD3), a vitamin D3 analog that is efficiently converted to 1,25(OH)2D3 in vivo by D3-25-hydroxylase. This compound was administered to mice previously inoculated with the Ml leukemia cell line and showed greater activity than 1,25(OH)2D3 [92]. Its conversion to the active form resulted in a more prolonged elevation of plasma levels of 1,25(OH)2D3, and the dose (25 pmol, every other day) produced only a slight and not significant elevation of serum calcium. In addition, survival of the leukemic mice was increased by 50–60%; however, the more effective doses produced hypercalcemia. Also, the

administration of 1αOHD3 produced tumor regression in follicular NHLs in rats, but hypercalcemia was the dose-limiting factor [20]. In one study, six patients with MDS were treated with 1αOHD3 at 1 µg/day for a minimum of three months, but neither a good clinical response nor toxicity was observed in these cases [93]. In another clinical study, thirty MDS patients were included in two different groups: one group received 1α-OHD3 at 4–6 µg/day and the other group received placebo; the patients were treated for a median of 17 weeks [94]. An improvement of hematologic parameters was detected in only one patient, but the authors felt the treated group had a greater proportion of patients who did not progress to leukemia as compared to the control group. Hypercalcemia and increased serum creatinine were observed in two patients, and these abnormal measurements regressed with reduction of the dose [94]. A case has been reported of an individual with chronic myelomonocytic leukemia (subtype of MDS) who achieved complete remission with 25-hydroxyvitamin D3 therapy for 15 months; this remission was sustained for 15 months after the end of the treatment [95]. These results are surprising because 25OHD3 has low activity by itself and in vitro has little anti-leukemic activity. However, as substrate for 1α-hydroxylase it may lead to local production of 1,25(OH)2D3 (see Chapter 79). Calcipotriol (MC903) has a cyclopropyl group at the end of the side chain formed by the fusion of C-26 and C-27, a hydroxyl group at C-24, and a double bond at C-22. This compound was equipotent to 1,25(OH)2D3 in inhibiting the proliferation and inducing the differentiation of the monoblastic cell line U937 [96,97]. In bone marrow cultures, the analog promoted the formation of multinucleated osteoclastlike cells, a vitamin D–mediated function. The effects of this compound on the immune system were very similar to those induced by 1,25(OH)2D3. By interfering with T-helper cell activity, calcipotriol reduced immunoglobulin production and blocked the proliferation of thymocytes induced by IL-1 [98,99]. Exposure of the follicular NHL B-cell lines SU-DUL4 and SUDUL5, carrying the t(14;18) translocation characteristic of the disease, to MC903 inhibited of proliferation only at high concentrations of the compound (10 −7 M) [20]. At the same time, calcipotriol was 100-fold less active than 1,25(OH)2D3 in inducing hypercalcemia and mobilizing bone calcium in rats [100]. However, the analog is rapidly inactivated in the intact animal, and therefore has been developed as a topical agent (see Chapter 101). Introduction of a double bond at carbon 16 has proved to be an effective modification of 1,25(OH)2D3 [101].


When combined with other motifs the 16 ene modification has led to a series of analogs with potent antiproliferative and differentiation-promoting activities with decreased calcemic effects. Prior studies by us have shown that vitamin D3 analogs having the C-16-ene motif were almost 100-fold more potent than 1,25(OH)2D3 in inhibiting growth of HL-60 leukemia cells, while the calcemic activity was the same or markedly less than 1,25(OH)2D3 [102,103]. Combination of the C-16-double bond and the C-23-triple bond [1,25(OH)2-16-ene-23yne-D3] produces a compound that is a more potent inducer of growth inhibition and differentiation in HL-60 cells than 1,25(OH)2D3, and is 15 times less hypercalcemic in mice [102]. The analog l,25(OH)2-16-ene-23yne D3 has potent antiproliferative and differentiating effects on leukemic cells in vitro [104]. In blocking HL60 clonal growth, l,25(OH)2-16-ene-23-yne D3 has a potency about four times higher than 1,25(OH)2D3. This compound administered to vitamin D–deficient chicks is about 30 times less effective than 1,25(OH)2D3 in stimulating intestinal calcium absorption and about 50 times less effective in inducing bone calcium mobilization [97]. Further experiments have demonstrated the therapeutic potential of l,25(OH)2-16-ene-23-yne D3 by its ability to prolong markedly the survival of mice inoculated with the myeloid leukemic cell line WEHI 3BD+ when treated with a high dose (1.6 µg every other day) of the compound [105]. The 1,25(OH)2-16-ene-19-nor-24-oxo-D3 was synthesized as a result of previous studies that isolated 24-oxo metabolites of potent vitamin D3 analogs, which were formed in a rat kidney perfusion system [106]. We found that these 24-oxo-metabolites had markedly reduced calcemic activity, but possessed at least an equal ability as the unmetabolized analogs to inhibit the clonal growth of breast and prostate cancer cells and myeloid leukemia cells in vitro. Taken together, these findings prompted the chemical synthesis of a series of vitamin D3 analogs with 1,25(OH)2-16ene-19-nor-24-oxo-D3 being one of the more exciting compounds, having the ability to inhibit acute myeloid leukemia cells in the concentration range of 10−10 M [107]. Remarkably, this compound had very little calcemic activity even when 6 µg was administered intraperitoneally to the mice three times a week [107]. The compound l,25(OH)2-20-epi D3 is characterized by an inverted stoichiometry at C-20 of the side chain. The monoblastic cell line U937 cultured with this compound showed a strong induction of differentiation [108]. It was also a potent modulator of cytokinemediated T-lymphocyte activation and exerted calcemic effects comparable to 1,25(OH)2D3 in rats. A study by us suggested that l,25(OH)2-20-epi D3 is the most potent vitamin D3 compound at inhibiting the clonal

1735 growth of HL-60 cells and at inducing cell differentiation. In fact, it was about 2600-fold more potent than 1,25(OH)2D3 in inhibiting the clonal growth of HL-60 cells and about 5000-fold more effective in preventing clonal growth of fresh human leukemic myeloid cells [109]. 1,25(OH)2-20-epi D3 exerts its effects by binding directly to VDR as shown by a T-lymphocytic cell line established from a patient with vitamin D–dependent rickets type II (HVDRR) lacking a functional VDR. Clonal growth was not inhibited after treatment of these cells with high doses of either 1,25(OH)2-20epi D3 or 1,25(OH)2D3 (10−7 M). In contrast, control experiments showed that these compounds [1,25(OH)220-epi D3 > 1,25(OH)2D3] were powerful inhibitors of proliferation of a human T-cell leukemia virus type I (HTLV-I) transformed T-cell line that possessed VDR. KH1060 is a potent vitamin D3 20-epi analog with an oxygen in place of C-22 and three additional carbons in the side chain. It is about 14,000-fold more potent than 1,25(OH)2D3 in inhibiting the clonal growth of the monoblastic cell line U937 [108]. It also has a powerful effect on other leukemic cells [70,108, 109]. However, it has the same hypercalcemic activity and the same receptor binding affinity as 1,25(OH)2D3. One promising new vitamin D analog is paricalcitol (19-nor-1,25-dihydroxyvitamin D2), which has been approved by the Food and Drug Administration for the clinical treatment of secondary hyperparathyroidism. Clinical trials have demonstrated that it possesses very low calcemic activity [110,111]. Studies by us and another group have demonstrated that paricalcitol has antiproliferative, prodifferentiation activities against myeloid leukemia and myeloma cell lines [31,112]. Paricalcitol activity was dependent on the presence of VDR, as it was unable to induce differentiation of mononuclear bone marrow cells from VDR knockout mice, whereas cells from wild-type mice were differentiated towards monocytes/macrophages [31]. Furthermore, paracalcitol was able to inhibit tumor growth without causing hypercalcemia in nude mice. These observations have prompted us to begin a clinical trial of paricalcitol aimed at treating patients with MDS. Potential mechanisms by which vitamin D analogs may have increased biological activity compared to 1,25(OH)2D3 are: reduced affinity to the serum vitamin D–binding protein; decreased catabolism by 24-hydroxylase; retention of biological activities by metabolic products of vitamin D analogs; increased stability of the ligand-VDR complex; increased VDR DNA–binding and dimerization with RXR; and enhanced recruitment of the DRIP coactivator complex. These topics are covered in detail in Chapters 81–83. In conclusion, new vitamin D analogs have potent anti-leukemic activity and lower hypercalcemic

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effects than 1,25(OH)2D3, and should be considered for the treatment of hematologic malignancies either alone or in combination with other differentiating agents. However, more phase I, II, and III trials are still necessary to assess the safety and effectiveness of these treatments.

VI. SUMMARY AND CONCLUSIONS The hormone 1,25(OH)2D3 plays a role in normal hematopoiesis, enhancing the activity of monocytesmacrophages and inhibiting cytokine production by T lymphocytes. It can also inhibit proliferation and induce differentiation of various myeloid leukemia cell lines. Its activity is mediated by vitamin D receptors that belong to the superfamily of steroid-thyroid receptors. However, the anti-leukemic activity of 1,25(OH)2D3 in vivo is associated with high toxicity and the onset of hypercalcemia as the dose-limiting effect. Limited clinical trials have been performed for the treatment of preleukemia with differentiating agents including 1,25(OH)2D3, but the in vitro effective dose caused hypercalcemia in vivo. Since the mid-1980s, many vitamin D analogs have been identified with reduced hypercalcemic activity and high potential to induce cell differentiation and to inhibit proliferation of leukemic cells. Further studies have been performed in vitro and in vivo using these analogs with other differentiating agents such as retinoids, in the hopes that the combination of agents working through different pathways could lead to synergistic activity. Proof of principle that 1,25(OH)2D3 and its analogs are beneficial in cancer has occurred in experiments conducted in vitro and in laboratory animals; however, the results of currently ongoing and future clinical trials in patients using vitamin D analogs will determine their ultimate therapeutic value.

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81. Sokoloski J, Sartorelli A 1998 Induction of the differentiation of HL-60 promyelocytic leukemia cells by nonsteroidal anti-inflammatory agents in combination with low levels of vitamin D3. Leuk Res 22:153–161. 82. Desmond J, Mountford J, Drayson M, Walker E, Hewison M, Ride J, Luong Q, Hayden R, Vanin E, Bunce C 2003 The aldo-keto reductase AKR1C3 is a novel suppressor of cell differentiation that provides a plausible target for the noncyclooxygenase-dependent antineoplastic actions of nonsteroidal anti-inflammatory drugs. Cancer Res 63: 505–512. 83. Danilenko M, Wang X, Studzinski G 2001 Carnosic acid and promotion of monocytic differentiation of HL60-G cells initiated by other agents. J Natl Cancer Inst 93: 1224–1233. 84. Danilenko M, Wang Q, Wang X, Levy J, Sharoni Y, Studzinski G 2003 Carnosic acid potentiates the antioxidant and prodifferentiation effects of 1α,25-dihydroxyvitamin D3 in leukemia cells but does not promote elevation of basal levels of intracellular calcium. Cancer Res 63: 1325–1332. 85. Torres R, Calle C, Aller P, Mata F 2000 Etoposide stimulates 1,25-dihydroxyvitamin D3 differentiation activity, hormone binding and hormone receptor expression in HL-60 human promyelocytic cells. Mol Cell Biochem 208:157–162. 86. Siwinska A, Opolski A, Chrobak A, Wietrzyk J, Wojdat E, Kutner A, Szelejewski W, Radzikowski C 2001 Potentiation of the antiproliferative effect in vitro of doxorubicin, cisplatin, and genistein by new analogs of vitamin D. Anticancer Res 21:1925–1929. 87. Koshizuka K, Koike M, Kubota T, Said J, Binderup L, Koeffler H 1998 Novel vitamin D3 analog (CB1093) when combined with paclitaxel and cisplatin inhibit growth of MCF-7 human breast cancer cells in vivo. Int J Oncol 13:421–428. 88. Koshizuka K, Koike M, Asou H, Cho S, Stephen T, Rude R, Binderup L, Uskokovic M, Koeffler H 1999 Combined effect of vitamin D3 analogs and paclitaxel on the growth of MCF7 breast cancer cells in vivo. Breast Cancer Res Treat 53:113–120. 89. Yu W, McElwain M, Modzelewski R, Russell D, Smith D, Trump D, Johnson C 1998 Enhancement of 1,25-dihydroxyvitamin D3–mediated anti-tumor activity with dexamethasone. J Natl Cancer Inst. 90:134–141. 90. Muindi J, Peng Y, Potter D, Hershberger P, Tauch J, Capozzoli M, Egorin M, Johnson C, Trump D 2002 Pharmacokinetics of high-dose oral calcitriol: results from a phase 1 trial of calcitriol and paclitaxel. Clin Pharmacol Ther 72:648–659. 91. Smith D, Johnson C, Freeman C, Muindi J, Wilson J, Trump D 1999 A phase I trial of calcitriol (1,25-dihydroxycholecalciferol) in patients with advanced malignancy. Clin Cancer Res 5:1339–1345. 92. Honma Y, Hozumi M, Abe E, Konno K, Fuku S, Hima M, Hata S, Nishii Y, DeLuca H, Suda T 1983 1,25-dihydroxyvitamin D3 prolong survival time mice inoculated with myeloid leukemia cells. Proc Natl Acad Sci USA 80: 201–204. 93. Metha A, Kumaran T, Marsh G 1984 Treatment of myelodysplastic syndrome with alfacalcidol. Lancet 2:761. 94. Motomura S, Kanamori H, Maruta A, Kodama F, Ohkubo T 1991 The effect of 1-hydroxyvitamin D3 for prolongation of leukemic transformation-free survival in myelodysplastic syndromes. Am J Hematol 38:67–68.

1739 95. Mellibovsky L, Diez A, Aubia J, Nogues X, Perez-Vila E, Serrano S, Recker R 1993 Long-standing remission after 25-OH-D3 treatment in a case of chronic myelomonocytic leukemia. Br J Haematol 85:811–812. 96. Binderup L, Bramm E 1988 Effects of a novel vitamin D analogue MC903 on cell proliferation and differentiation in vitro and on calcium metabolism in vivo. Biochem Pharmacol 37:889–895. 97. Brown A, Dusso A, Slatopolsky E 1994 Selective vitamin D analogs and their therapeutic applications. Semin Nephrol 14:156–174. 98. Muller K, Svenson M, Bendtzen K 1988 1,25Dihydroxyvitamin D3 and a novel vitamin D analog MC903 are potent inhibitors of human interleukin 1 in vitro. Immunol Lett 17:361–366. 99. Muller K, Heilmann C, Poulsen L, Barington T, Bendtzenk K 1991 The role of monocytes and T-cells in 1,25-dihydroxyvitamin D3–mediated inhibition of B-cell function in vitro. Immunopharmacology 21:121–128. 100. Rebel V, Ossenkoppele G, van de Loosdrecht A, Wijermans P, Beelen R, Langenhuijsen M 1992 Monocytic differentiation induction of HL-60 cells by MC 903, a novel vitamin D analog. Leuk Res 16:443–451. 101. Uskokovic M, Stuzinski G, Gardner J, Reddy S, Campbell M, Koeffler H 1997 The 16-ene vitamin D analogs. Curr Pharm Design 3:99–123. 102. Jung S, Lee Y, Pakkala S, de Vos S, Elsner E, Norman A, Green J, Uskokovic M, Koeffler H 1996 1,25-(OH)2-16-enevitamin D3 is a potent antileukemic agent with low potential to cause hypercalcemia. Leuk Res 18:453–463. 103. Hisatake J, Kubota T, Hisatake Y, Uskokovic M, Tomoyasu S, Koeffler H 1999 5,6-trans-16-ene-vitamin D3: a new class of potent inhibitors of proliferation of prostate, breast, and myeloid leukemic cells. Cancer Res 59:4023–4029. 104. Pakkala S, de Vos S, Elstner E, Ruder K, Uskokovic M, Binderup L, Koeffler H 1995 Vitamin D3 analogs: Effect on leukemic clonal growth and differentiation, and on serum calcium levels. Leuk Res 19:65–72. 105. Zhou J, Norman A, Chen D, Sun G, Uskokovic M, Koeffler H 1990 1,25(OH)2-16ene-23yne vitamin D3 prolongs survival time of leukemic mice. Proc Natl Acad Sci USA 87:3929–3932. 106. Campbell M, Reddy G, Koeffler H 1997 Vitamin D3 analogs and their 24-oxo metabolites equally inhibit clonal proliferation of a variety of cancer cells but have different molecular effects. J Cell Biochem 66:413–425. 107. Shiohara M, Uskokovic M, Hisitake J, Hisatake Y, Koike K, Komiyama A, Koeffler H 2001 24-oxo metabolites of vitamin D3 analogs: Disassociation of their prominent antileukemic effects from their lack of calcium modulation. Cancer Res 61:3361–3368. 108. Binderup L, Latini S, Binderup E, Bretting C, Calverley M, Hansen K 1991 20-epi-vitamin D3 analogs: A novel class of potent regulators of cell growth and immune response. Biochem Pharmacol 42:1569–1575. 109. Elstner E, Lee Y, Hashiya M, Pakkala S, Binderup L, Norman A, Okamura W, Koeffler H 1994 l,25-dihydroxy-20epi-vitamin D3: An extraordinarily potent inhibitor of leukemic cell growth in vitro. Blood 84:1960–1967. 110. Llach F, Keshav G, Goldblat M, Lindberg J, Sadler R, Delmez J, Arruda J, Lau A, Slatopolsky E 1998 Suppression of parathyroid hormone secretion in hemodialysis patients by a novel vitamin D analog: 19-nor-1,25-dihydroxyvitamin D2. Am J Kidney Dis 32:S48–54.

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111. Martin K, Gonzalez E, Gellens M, Hamm L, Abboud H, Lindberg J 1998 19-nor-1-α-25-dihydroxyvitamin D2 (Paricalcitol) safely and effectively reduces the levels of intact parathyroid hormone in patients on hemodialysis. J Am Soc Nephrol 9:1427–1432.

112. Molnar I, Kute T, Willingham M, Powell B, Dodge W, Schwartz G 2003 19-nor-1α,25-dihydroxyvitamin D2 (paricalcitol): effects on clonal proliferation, differentiation, and apoptosis in human leukemic cell lines. J Cancer Res Clin Oncol 129:35–42.

CHAPTER 97

Clinical Development of Calcitriol and Calcitriol Analogs in Oncology: Progress and Considerations for Future Development* DONALD L. TRUMP JOSEPHIA MUINDI CANDACE S. JOHNSON

PAMELA A. HERSHBERGER I. II. III. IV.

Department of Medicine, Roswell Park Cancer Institute, Buffalo, NY Department of Medicine, Roswell Park Cancer Institute, Buffalo, NY Department of Pharmacology & Therapeutics, Roswell Park Cancer Institute, Buffalo, NY Department of Pharmaceutical Sciences, State University at Buffalo, Buffalo, NY Department of Pharmacology, University of Pittsburgh, Pittsburgh, PA

Introduction Clinical Trials Laboratory-Clinical Extrapolations of Calcitriol Exposure High Dose Intermittent Calcitriol

I. INTRODUCTION 1,25 dihydroxyvitamin D3 (calcitriol), a central factor in bone and mineral metabolism, is a potent antiproliferative agent in a wide variety of malignant cell types [1–12]. As noted in preceding chapters, calcitriol and calcitriol analogs have significant anti-tumor activity in vitro and in vivo in animal and human hematopoietic and epithelial cancer models. Calcitriol enhances the in vitro and in vivo anti-tumor effects of platinum and taxane analogs, as well as antimetabolites (cytosine arabinoside, gemcitabine), topoisomerase inhibitors (etoposide, irinotecan), and alkylating agents [12–14]. Calcitriol as a single agent induces G0/G1 arrest, modulates p27Kipl and p21Waf1/Cipl (the cyclin-dependent kinase (cdk) inhibitors implicated in G1 arrest), induces cleavage of caspase 3, PARP, and the growth-promoting/ prosurvival signaling molecule mitogen-activated protein kinase (MEK) in a caspase-dependent manner [4,9,11,12,15]. In association with these effects, full

*This work is supported by grants from the NCI (CA95045, CA67267, and CA85142) and CaPCURE/The Prostate Cancer Research Foundation. VITAMIN D, 2ND EDITION FELDMAN, PIKE, AND GLORIEUX

V. Calcitriol + Cytotoxic Agent Combinations VI. Calcitriol Analogs VII. The Future References

length MEK, phospho-Erk (P-Erk), and phospho-Akt (P-Akt) are lost. The phosphorylation and expression of Akt, a kinase regulating a second cell survival pathway, is also inhibited after treatment with calcitriol. In contrast to changes that occur during cytotoxic drug-induced apoptosis, the pro-apoptotic signaling molecule MEKK-1 is significantly up-regulated by calcitriol [9]. Enhancement of cytotoxic agent-mediated apoptosis by calcitriol is associated with an increase in PARP-, MEK-, MEKK-1, and caspase-cleavage; P-Erk and P-Akt decrease. In addition, the expression of the p53 homolog, p73, is strongly induced by calcitriol, and p73 can sensitize tumor cells to the cytotoxic effects of platinum and taxanes [20]. Glucocorticoids (GC) potentiate the anti-tumor effect of calcitriol and decrease calcitriol-induced hypercalcemia [16,17]. Both in vitro and in vivo, GC significantly increase vitamin D receptor (VDR) ligand-binding in the tumor while decreasing binding in intestinal mucosa [16], the site of calcium absorption [17]. P-Erk and P-Akt are decreased with calcitriol/GC, compared to either agent alone [16]. These preclinical data support the development of calcitriol-based approaches to cancer therapy. Historically, a limited number of trials in cancer patients have been completed testing vitamin D-based approaches. Copyright © 2005, Elsevier, Inc. All rights reserved.

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DONALD L. TRUMP, JOSEPHIA MUINDI, CANDACE S. JOHNSON, AND PAMELA A. HERSHBERGER

While early studies were largely negative, the improved understanding of the molecular changes that occur following calcitriol treatment and new approaches to the use of calcitriol and analogs provide encouraging data on the potential to develop calcitriol-based anti-tumor approaches. This chapter will review the current status of the clinical development of vitamin D analogs in cancer therapeutics, emphasizing important new pharmacokinetic and combination therapy approaches.

II. CLINICAL TRIALS A. Early Trials Oral calcitriol and analogs such as EB 1089 and 1α(OH)D3 have been used in a number of clinical trials. Hematopoietic disorders (myelodysplasia, acute leukemia) have been the most commonly studied diseases. These studies were largely negative and have suffered from a number of limitations: small numbers of patients, lack of control groups, and use of a calcitriol analog in combination with cytotoxic agents in single arm trials, such that conclusions regarding the contribution of the calcitriol analog are difficult [21–26]. Gross and colleagues and Osborn and co-workers conducted straightforward trials of oral daily calcitriol administration in prostate cancer [27,28]. Gross et al. studied patients in whom the prostate-specific antigen was rising following local therapy, and Osborn et al. studied men with androgen-independent prostate cancer. As noted in Chapter 94, both studies were carefully designed, with sufficient power to delineate positive effects of calcitriol. While both studies provided evidence suggesting some positive benefit of calcitriol, both studies illustrate the major shortcomings of all previous studies of calcitriol-based therapeutics: doses of calcitriol and the schedule employed were not reflective of calcitriol exposures achieved in preclinical models in which substantial anti-tumor effects have been demonstrated. Both Gross et al. and Osborn et al. utilized the daily doses of calcitriol employed in the management of benign disease (1.5–2.0 µg). In each study, perturbations of calcium metabolism occurred that led the investigators to limit dose escalation—hypercalciuria (Gross) and hypercalcemia (Osborn). These are the predicted toxicities of vitamin D–based therapies and might have led to investigators abandoning attempts to administer calcitriol in either epithelial or hematopoietic malignancies. Most approaches to overcoming the hypercalcemic effects of calcitriol-based therapies have focused on the development of “nonhypercalcemic” vitamin D analogs. Thousands of such analogs have been synthesized and as will be noted below, some are now entering clinical trials. However, two groups

of investigators have evaluated a different approach to averting the hypercalcemic effects of vitamin D. Reflecting on the fact that in vivo and in vitro experiments utilize high exposure, limited duration treatment, Johnson and Trump as well as Beer and colleagues have evaluated the feasibility of administering high-dose, intermittent regimens of calcitriol.

B. High Dose, Intermittent Calcitriol Regimens Smith and colleagues explored a higher dose subcutaneous regimen of calcitriol, hypothesizing that an every-other-day (QOD) schedule combined with a subcutaneous route of administration might permit safe dose escalation of calcitriol [29]. These investigators were able to administer 8 µg QOD calcitriol safely— this represents a >twofold dose escalation compared to the oral, daily schedule. Beer and colleagues studied oral weekly administration and showed that doses as high as 2.6 µg/kg (approximately 180 µg weekly) could be administered without toxicity. These workers also demonstrated that at doses of >0.5 µg/kg, there appeared to be loss of dose-proportional increase in systemic exposure as doses of oral calcitriol were increased. Importantly, no limiting toxicity was noted in the patients treated by Beer and colleagues. Muindi and co-workers further evaluated these findings of apparent “saturable absorption” during the conduct of a trial of paclitaxel (intravenous, weekly × 6) + dose escalation of oral calcitriol daily for three consecutive days each week (QD × 3, weekly). Calcitriol was administered safely at doses as high as 38 µg QD×3 weekly in combination with paclitaxel [31]. These workers confirmed the loss of dose-proportional increase in calcitriol exposure with increasing dose. Loss of doseproportional increase in exposure appeared to occur at 16–18 µg of calcitriol (Fig. 1). In this figure, baselinesubtracted serum calcitriol AUC0→24hr (area under the concentration-time curve for the 24-hour-period after calcitriol administration) is plotted against dose. A fit to the Michaelis Menten function (AUC = a × dose/ (1 + b × dose) indicates that AUC0→24hr is not proportional to dose (a = 540 ± 140 pg.hr/ml.µg); if AUC were proportional to dose, b would equal 0. The effect of this nonlinearity over the range of doses studied is large; the value of AUC0→24hr at 38 µg is only 4 times that at 4 µg, instead of the 9.5 times expected for a proportional relationship. No deviation from linearity can be detected up to a dose of 17 µg (p =0.4). In this trial, patients with advanced cancer received paclitaxel (80 mg/m2 weekly × 6) + escalating doses of calcitriol, QD×3 weekly ×6. The starting dose of calcitriol was 4 µg po QD × 3 weekly, and patients were entered

1743

CHAPTER 97 Clinical Development of Calcitriol and Calcitriol Analogs in Oncology

A

B Calcitriol AUC0–>24hr (pg.hr/ml)

1400

Cmax (pg/ml)

1200 1000 800 600 400 200

8000

4000

0 0

10

20

30

40

0

10

Calcitriol dose (µg)

20

30

40

Calcitriol dose (µg)

FIGURE 1 (A) Scatter plot of the maximum serum calcitriol concentration (Cmax) vs. calcitriol doses. Closed symbols represent mean values at each dose level. (B) Baseline-subtracted serum calcitriol AUC0 →24hr (area under the concentration-time curve for the 24-hour period after calcitriol administration) plotted against dose, a fit of the Michaelis-Menten function.

Calcitriol AUC0–>24hr (pg.hr/ml)

through the 38 µg dose level. No dose-limiting toxicity was encountered. In this study, the effect of calcitriol on paclitaxel pharmacokinetics was evaluated. No changes in peak concentration, AUC, or t1/2 were noted, indicating the lack of drug-drug interactions between calcitriol and paclitaxel. In these studies as well as those of Beer and colleagues, the commercially available formulation of calcitriol (Rocaltrol®) was used—a formulation available only as 0.25 µg and 0.5 µg caplets. Hence, in these studies, patients were asked to take up to 75–100 caplets at one dose. To investigate whether this apparent limited absorption of calcitriol was related to pharmaceutical limitations posed by multiple caplet ingestion, Muindi and colleagues evaluated patients receiving escalating doses of calcitriol at 14 µg and higher using a liquid formulation of calcitriol (Fig. 2). No change in the curvilinear relationship between dose and AUC was noted.

Taken together these studies clearly indicate that high dose, intermittent administration of calcitriol is safe. Hypercalcemia was transient, and calcium returned to the normal range within approximately 24 hours after dosing. Table I summarizes the doses, concomitant drugs, and limiting toxicities seen in the series of trials conducted by these two groups of investigators. Dose escalation more than tenfold above that achieved with the daily doses of calcitriol is possible— without any apparent toxicity. It is also clear that pharmacokinetic considerations will complicate the use of the current standard formulations of calcitriol. Dose escalation in the studies of Trump and Johnson was ceased when it became clear that dose proportional increase in exposure was not feasible and substantial interpatient variation in exposure was noted (see Fig. 1).

TABLE I Clinical Experience with High Dose Oral Calcitriol Route

Agent1

Schedule

5000

5000

0 14

16 18 Calcitriol dose (µg)

20

Oral, oral SQ, SQ Oral Oral Oral Oral Oral Oral

MDA2

Hypercalcemia

QD

0

2 µg

Yes

QOD QD×3 QD×3 QD× 3 QD× 3 Weekly × 1 Weekly × 6

0 Dex Carboplatin Paclitaxel 0 0 Docetaxel

10 µg 12 µg 24 µg 38 µg 24 µg 2.6 µg/kg 0.5 µg/kg

Yes No No No No No No

FIGURE 2

AUC from patients treated with calcitriol from the phase I trial of calcitriol where pk was determined on day one following calcitriol administration. Patients received either the capsule form (cross symbol) or the liquid form (open symbol).

1 Agents

administered with calcitriol. maximum daily dose administered. QD, every day; QOD, every other day; QD × 3, each day for 3 days/week. 2 MDA,

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III. LABORATORY-CLINICAL EXTRAPOLATIONS OF CALCITRIOL EXPOSURE

100

Among the first questions to arise in attempting to duplicate in the clinic the favorable anti-tumor in vivo effects of calcitriol that can be demonstrated in the laboratory is whether the systemic exposures obtained in the laboratory can be achieved in the clinic. While the initial bias of many has been that administration of high doses of calcitriol is not feasible, the studies of Beer and colleagues and Trump, Johnson, and Muindi clearly show that large doses of calcitriol can be administered safely—the limitation to date is pharmacokinetic, not toxicologic. While realizing that apparently saturable processes of absorption must still be overcome, Muindi and colleagues have characterized the systemic exposure achieved in mice at doses that exhibit anti-tumor and drug potentiating effects in several tumor models. These studies provide a rough “target concentration” that may be necessary to achieve in humans if these anti-tumor effects are to be realized. Figure 3 depicts the plasma concentration-time curve achieved in mice following intraperitoneal administration of “effective doses.” As shown in Table II, at 0.125 µg (the lowest dose to consistently produce significant anti-tumor effects in mice), the AUC0→24hr was 37.3 ng.hr/ml; this compares in man at a 38 µg dose to 7.5 ng.hr/ml. Similarly, in mice the Cmax was 9.2 ng/ml compared to 1.4 ng/ml in man. At the 0.042 µg dose in mice, an anti-tumor effect could be seen but was not consistently observed. Therefore, effective serum calcitriol levels are 5–7 times higher in mice than those achieved at the highest oral dose administered in man (38 µg) in the studies of Muindi and colleagues. The highest AUC0→48hr reported by Beer and colleagues was 47 ng.hr/ml. Beer studied a relatively small number of patients at these high doses. Both groups’ results are in agreement that intermittent high doses of calcitriol can be given safely and that attaining exposure in humans comparable to those required in mice to achieve optimal anti-tumor effects will be difficult with current formulations because they are inconvenient, highly

TABLE II

Plasma calcitriol (ng/ml)

0.125 µg/mouse 0.5 µg/mouse

10

1

0.1

0.01 0

0.042 0.125 0.5

6

9 12 15 Sampling times (hr)

18

21

24

FIGURE 3

Plots of plasma 1,25-D3 I concentration-time curves of normal C3H/HeJ mice; groups of 5–9 mice treated with a single 1,25-D3 i.p. injection of either 0.125 or 0.5 mg dose/mouse. Plasma 1,25-D3 concentrations were measured by RIA. Results are presented as mean ± SD.

variable in absorption, and at the highest doses display apparent saturable absorption characteristics.

IV. HIGH DOSE INTERMITTENT CALCITRIOL Several other studies have confirmed that high-dose intermittent calcitriol is safe and well tolerated.

A. Calcitriol + Dexamethasone Trump and colleagues [33] have completed a 43-patient study of calcitriol in escalating doses to a maximum of 12 µg calcitriol QD ×3 each week together with dexamethasone (4 mg QD ×4, weekly). While it was anticipated that this would be an aggressive calcitriol regimen that would be difficult to administer, this was not the case in practice. No patient ceased therapy because of hypercalcemia. Trimonthly urinary tract radiographs were completed to monitor for urinary

Calcitriol Pharmacokinetic Parameters Mice and Man

Mouse (IP) Dose (µg)

3

Man (PO)

AUC (0→24) (ng.hr/ml)

Cmax (ng/ml)

Dose (µg)

AUC (0→24) (ng.hr/ml)

Cmax (ng/ml)

3.6 37.3 123.9

0.7 9.2 43.4

13 17 38

3.9 ± 1.4 5.4 ± 2.1 7.5 ± 2.1

0.5 ± 0.3 0.5 ± 2.2 1.4 ± 0.9


tract stones. Two patients developed stones; one symptomatic and one asymptomatic. Symptomatic improvement and PSA responses (>50% decline) were seen in 28% of patients, but this response frequency is not clearly greater than one might expect with dexamethasone alone.

B. Single Agent Calcitriol in Androgen-independent Prostate Cancer (AIPC) Following this trial Trump and colleagues undertook a phase I dose escalation trial of calcitriol alone in AIPC to define the maximum tolerated dose and determine response. Dose escalation up to 36 µg QD ×3 weekly was possible without hypercalcemia or urinary tract stones. This study was terminated when it became clear that there was not dose-proportional increase in exposure as drug dose was increased.

C. Single Agent Calcitriol in Androgen-dependent Prostate Cancer Beer and colleagues administered weekly oral calcitriol, 0.5 µg/kg, to 22 men with PSA rising after local therapy (prostatectomy or irradiation) [34]. No toxicity was encountered. No men met the criteria for response established by these investigators. These data clearly indicate that very high intermittent oral doses of calcitriol can be administered safely. Single-agent oral calcitriol therapy (Rocaltrol) appears to have limited activity, at least in prostate cancer, and optimal systemic exposure is limited by variable and incomplete oral absorption. It is important to emphasize that formal bioavailability studies of oral calcitriol have not been conducted to evaluate the exact mechanism of the loss of dose proportional increase in AUC with increasing dose. It is by inference and circumstantial evidence that this observation has been attributed to “decreased absorption.” However, increased rate of metabolism consequent to increased calcitriolinduced 24-hydroxylase activity may also play a role.

V. CALCITRIOL CYTOTOXIC AGENT COMBINATIONS A. Carboplatin Trump and colleagues initiated a phase I trial of carboplatin + calcitriol, based on the considerable data that

1745

platinum analogs are potentiated by calcitriol [35–37]. Patients with advanced cancer were treated with carboplatin (AUC = 5) every 28 days + escalating doses of calcitriol QD × 3 every 28 days. Calcitriol starting dose was 4 µg QD × 3. Studies were designed such that in each patient, carboplatin was given on day 1 before calcitriol in one of the first two cycles of treatment and on day 3 after two days of high dose calcitriol on the other. This permitted comparison of AUC of carboplatin in the same patient before and after pretreatment with calcitriol. Dose-limiting toxicity was not encountered in this trial. The AUC of carboplatin was higher in patients who received carboplatin following 3 days of calcitriol than in patients in whom carboplatin was administered before calcitriol (mean AUC = 7.6 µg/ml.hr ± 1.8, carboplatin day 3 [DDDC] vs. AUC = 6.6 µg/ml.hr ± 1.4, carboplatin day 1 [CDDD], p = 0.04) (Fig. 4). While no-dose-limiting toxicity has been seen, myelosuppression (% change in platelet count) following the sequence carboplatin →calcitriol (CDDD) was less than that following calcitriol → carboplatin (DDDC), consistent with the change in AUC. No clinically detectable renal impairment was seen with either sequence. These data indicate that potentiation of carboplatin by calcitriol may in part be related to reduced carboplatin clearance. No patients became hypercalcemic. This trial was halted when the concerns regarding predictable and dose proportional exposure became evident in this and other studies of oral calcitriol (Rocaltrol).

B. Taxanes We have discussed the trial of Trump and colleagues evaluating the combination of high dose oral calcitriol + the taxane, paclitaxel. No dose-limiting toxicity was noted at calcitriol doses up to 38 µg QD ×3 weekly. Recently, Beer and colleagues reported a phase I trial demonstrating that patients can tolerate weekly oral dosing of calcitriol at 0.5 µg/kg + docetaxel (36 mg/m2 weekly × 6) without significant toxicity [38]. This group has reported the results of a phase II trial of this regimen in men with androgen-independent prostate cancer. Among 37 men treated with weekly calcitriol (Rocaltrol) + docetaxel, 81% (95% confidence interval [CI] 68–94%) achieved a PSA response rate as measured by a greater than 50% reduction in PSA [39]. This response rate appears greater than response rates of 38–46% reported in phase II studies of single-agent weekly docetaxel. Among 15 patients with measurable tumor masses 8 (53%, Ci 43%–75%) achieved a tumor mass response defined by standard criteria. These are quite encouraging data with respect to anti-tumor effects of calcitriol-based therapy and has led to an

1746


12 DDDC CDDD 10

Carboplatin AUC

8

6

4

2

0 1

2 4 µg

3

4

5 6 µg

6

7

8 8 µg

9

10

11

11 µg

12

13

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14 µg

16

16 µg

17 24 µg

Patients and calcitriol dose

FIGURE 4 Carboplatin AUC from patients treated at selected doses of calcitriol with either carboplatin on day 1, calcitriol day 1, 2, and 3 (CDDD) (open bars), or calcitriol day 1, 2, and 3 followed by carboplatin on day 3 (DDDC) (closed bars). Carboplatin AUC is significantly greater with the DDDC sequence than the sequence CDDD ( p < 0.04).

ongoing phase III trial to more rigorously evaluate this regimen (see below).

C. Reformulation of Calcitriol In view of the fact that high-dose intermittent calcitriol is safe, feasible—but inconvenient and not dependably absorbed using the currently available and tested formulation Rocaltrol—efforts have been undertaken to develop a more “bioavailable” and more convenient preparation of calcitriol. The express purpose of this effort is to develop a preparation that might allow full testing of calcitriol in high dose alone and with cytotoxic agents. A new formulation, DN 101 (Novocea, Inc.) has completed phase 1 testing; initial results indicate a more linear relationship between dose and exposure. No unusual toxicity or effects have been noted [40]. DN 101 is the calcitriol preparation being used in a randomized trial of docetaxel +/− calcitriol in AIPC. This trial is testing whether the increased PSA response rate seen in the trial of Beer et al. can be confirmed in a randomized trial.

VI. CALCITRIOL ANALOGS While considerable work has been done to demonstrate that high-dose intermittent calcitriol administration is feasible and that blood levels in the range of those found to be effective in animal models have not yet been achieved, great interest remains in the development of vitamin D analogs that retain the antiproliferative and/or prodifferentiative properties of calcitriol with less propensity to cause hypercalcemia. Two analogs have been tested in substantial numbers of cancer patients: EB 1089 or seocalcitol and 1α(OH)D2.

A. EB 1089 (seocalcitol) Phase 1 trials of seocalcitol [(1(S),3(R)-dihydroxy20(R)-(5′-ethyl-5′-hydroxy-hepta-1′(E),3′(E)-dien-1′-yl)9,10-secopregna-5(Z),7(E),10(19)-triene] (see Chapter 84) have been conducted using a daily oral schedule of administration [41]. 7–15 µg/m2m is estimated to be tolerable and all patients who received 17 µg/sqm per day developed hypercalcemia. Preclinical data indicate


seocalcitol is 50–200 times more potent than calcitriol in terms of antiproliferative activity; these phase I data indicate that seocalcitol is 1/7–1/10 as potent in inducing hypercalcemia. As discussed elsewhere in this text, phase II studies have been conducted in breast, pancreatic, colorectal hepatocellular carcinomas (HCC), as well as leukemia [42,43] (Chapter 84). Anti-tumor responses have been seen in HCC, but not in the other diseases.

B. 1(OH)D2 This analog has been developed as one potentially more active and less prone to cause hypercalcemia. This agent is converted to 1,25(OH)2D2 and 1,24(OH)2D2, both of which activate VDR-mediated biologic effects; the 1,24(OH)2D2 metabolite is substantially less potent than 1,25(OH)2D2 or 1,25(OH)2D3 [44,45]. Liu and colleagues conducted a phase I trial of 1α(OH)D2 in prostate cancer patients [46]. Daily dosing from 5 to 15 µg per day was employed. Hypercalcemia with dehydration and azotemia was noted at 15 µg QD; 12.5 µg QD was well tolerated in 3 of 3 patients. Two of 25 patients treated demonstrated evidence of antitumor effect; interestingly both responses were seen at “low” doses (5 µg and 7.5µg). It would appear that 1α(OH)D2 is approximately 1/10 as prone to induce hypercalcemia as a similar dose of calcitriol on a QD-dosing schedule. The anti-tumor effects of seocalcitol and 1α(OH)D2 are modest in the studies conducted. The activity of seocalcitol in HCC is of considerable interest in view of the importance of this disease worldwide.

VII. THE FUTURE These data, in combination with the considerable preclinical information indicating the potential role of vitamin D–based therapies in cancer, continue to stimulate the interest of several research groups. The ongoing study of the new calcitriol formulation + docetaxel in AIPC will be very important in establishing the potential for calcitriol, on this dose and schedule, in the management of prostate cancer. Among the unanswered questions are: 1. What is the proper dose and schedule? Preclinical data indicate that all studies have been conducted at 1/5 – 1/10 the drug exposure that is effective in animal models; and clinical evidence suggests that administration of the exposures effective in preclinical models may be safe and feasible. Trials of intravenous calcitriol and new formulations of calcitriol to establish the maximum possible dose on an intermittent schedule are underway. 2. What is the

1747

“best analog”? While preclinical data suggest advantages for a number of analogs, substantial clinical work remains to evaluate the analogs currently in clinical trials. The greatest preclinical and clinical work has been done with calcitriol. 3. Are there additional novel approaches that may capitalize on modulation of the vitamin D system? Potentiation of the anti-tumor activity of growth factor receptor antagonists (gefitinib), as well as many other cytotoxics, has been well described in preclinical models [47–54]. Vitamin D analogs have been described to have antiangiogenic effects, as well as direct anti-tumor activities; this suggests that combinations and applications in settings where tumor blood vessels are the target may have merit [56–58]. Regional administration has merit. Regional arterial infusions (e.g. hepatic artery) or topical (e.g. cutaneous or bronchial) therapy are being investigated. Regional approaches have the great advantage of permitting the administration of high doses, locally with limited systemic effects. While definitive data regarding the use of vitamin D in the management of cancer remains elusive, preclinical data are persuasive and considerable progress has been made in developing clinical strategies utilizing vitamin D in the treatment of epithelial and hematopoietic cancers.

References 1. Bikle DD, Pillai S 1993 Vitamin D, calcium, and epidermal differentiation. Endocr Rev 14(1):3–19. 2. Reichel H, Koeffler HP, Norman AW 1989 The role of vitamin D endocrine system in health and disease. N Engl J Med 320(15):980–991. 3. McElwain MC, Dettlebach MA, Modezelewski RA, Russell DM, Uskokovic MR, Smith DC, Trump DL, Johnson CS 1995 Antiproliferative effects in vitro and in vivo of 1,25-dihydroxyvitamin D3 and a vitamin D3 analog in a squamous cell carcinoma model system. Mol Cell Diff 3(1):31–50. 4. Getzenberg RH, Light BW, Lapco PE, Konety BR, Nangia AK, Acierno JS, Dhir R, Shurin Z, Day RS, Trump DL, Johnson CS 1997 Vitamin D inhibition of prostate adenocarcinoma growth and metastasis in the Dunning rat prostate model system. Urology 50:999–1006. 5. Mangelsdorf DJ, Koeffler HP, Donaldson CA, Pike JW, Haussler MR 1984 1,25-dihydroxyvitamin D3 induced differentiation in a human promyelocytic leukemia cell line HL-60: receptor mediated maturation to macrophage-like cells. J Cell Biol 98:391–398. 6. Colston KW, Chander SK, Mackay AG, Coombes RC 1992 Effects of synthetic vitamin D analog on breast cancer cell proliferation in vivo and in vitro. Biochem Pharmacol 44:693–702. 7. Shabahang M, Buras RR, Davoodi F, Schumaker LM, Nauta RJ, Uskokovic MR, Brenner RV, Evans SR 1994 Growth inhibition of HT-29 human colon cancer cells by analogs of 1,25dihyroxyvitamin D3. Cancer Res 54:4057–4064. 8. Peehl DM, Skowronski RJ, Leung GK, Wong ST, Stamey TA, Feldman D 1994 Antiproliferative effects of 1,25-dihydroxyvitamin D3 on primary cultures of human prostatic cells. Cancer Res 54(3):805–810.

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9. McGuire TF, Trump DL, Johnson CS 2001 Vitamin D3–induced apoptosis of murine squamous cell carcinoma cells: Selective induction of caspase-dependent MEK cleavage and up-regulation of MEKK-1. J Biol Chem 276(28):26365–26373. 10. Zhou JY, Norman AW, Chen DL, Sun GW, Uskokovic M, Koeffler HP 1990 1,25-dihydroxy-16-ene-23-yne-vitamin D3 prolongs survival time of leukemic mice. Proc Natl Acad Sci USA 87(10):3929–3932. 11. Modzelewski RA 1999 Apoptotic effects of paclitaxel and calcitriol in rat dunning MLL and human PC-3 prostate tumor cells in vitro. Proc Amer Assoc Cancer Res 40:580. 12. Hershberger PA, Yu WD, Modzelewski RA, Rueger RM, Johnson CS, Trump DL 2001 Enhancement of paclitaxel anti-tumor activity in squamous cell carcinoma and prostatic adenocarcinoma by 1,25-dihydroxycholecaciferol (1,25-D3). Clin Cancer Res 7:1043–1051. 13. Light BW, Yu W-D, McElwain MC, Russell DM, Trump DL, Johnson CS 1997 Potentiation of cisplatin anti-tumor activity using a vitamin D analog in a murine squamous cell carcinoma model system. Cancer Res 57(17):3759–3764. 14. Christakos S, Raval-Pandya M, Wernyj RP, Yang W 1996 Genomic mechanisms involved in the pleiotropic actions of 1,25-dihydroxivitamin D3. Biochem J 316(Pt 2):361–371. 15. Hershberger PA, Modzelewski RA, Shurin ZR, Rueger RM, Trump DL, Johnson CS 1999 In vitro and in vivo modulation of p21Wafl/Cip1 and p27Kip1 in squamous cell carcinoma I response to 1,25-dihydroxycholecalciferol (calcitriol). Cancer Res 59:2644–2649. 16. Yu W-D, McElwain MC, Modzelewski RA, Russell DM, Smith DC, Trump DL, Johnson CS 1998 Enhancement of 1,25-dihydroxyvitamin D3–mediated anti-tumor activity with dexamethasone. J Natl Cancer Inst 90(2):134–141. 17. Bernardi RJ, Trump DL, Yu W-D, McGuire TF, Hersherber PA, Johnson CS 2001 Combination of 1α,25-dihydroxyvitamin D3 with dexamethasone enhances cell cycle arrest and apoptosis: Role of nuclear receptor cross-talk and Erk/Akt signaling. Clin Cancer Res 7:4165–4173. 18. Evans RM 1998 The steroid and thyroid hormone receptor superfamily. Science 240:889. 19. Darwish HM, DeLuca HF 1996 Recent advances in the molecular biology of vitamin D action. In: Progress in Nucleic Acid Research and Molecular Biology. Academic Press Inc, Vol 53, pp. 321 . 20. Darwish H, DeLuca HF 1993 Vitamin D–regulated gene expression. Critical Reviews in Eukaryotic Gene Expression 3(2):89–116. 21. Rustin GJ, Quinnell TG, Johnson J, Clarke H, Nelstrop AE, Bollag W 1996 Br J. Trial of isotretinoin and calcitriol monitored by CA 125 in patients with ovarian cancer. Cancer 74(9):1479–1481. 22. Slapak CA, Desforges JF, Fogaren T, Miller KB 1992 Treatment of acute myeloid leukemia in the elderly with lowdose cytarabine, hydroxyurea, and calcitriol. Am J Hematol 41(3):178–183. 23. Petrini M, Caracciolo F, Corini M, Valentini P, Sabbatini AR, Grassi B 1991 Low-dose ARA-C and 1(OH)D3 administration in acute nonlymphoid leukemia: pilot study. Haematologica 76(3):200–203. 24. Hellstrom E, Robert KH, Samuelsson J, Lindemalm C, Grimfors G, Kimby E, Oberg G, Winqvist I, Billstrom R, Carneskog J 1990 Treatment of myelodysplastic syndromes with retinoic acid and 1 alpha-hydroxy-vitamin D3 in combination with low-dose ara-C is not superior to ara-C alone.

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metastatic androgen-independent prostate cancer. J Clin Oncol 21(1):123–128. Beer TM (personal communication). Gulliford T, English J, Colston KW, Menday P, Moller S, Coombes RC 1998 A phase I study of the vitamin D analog EB 1089 in patients with advanced breast and colorectal cancer. Br J Cancer 78(1):6–13. Dalhoff K, Dancey J, Astrup L, Skovsgaard T, Hamberg KJ, Lofts FJ, Rosmorduc O, Erlinger S, Bach Hansen J, Steward WP, Skov T, Burcharth F, Evans TR 2003 A phase II study of the vitamin D analog seocalcitol in patients with inoperable hepatocellular carcinoma. Br J Cancer 89(2):252–257. Evans TR, Colston KW, Lofts FJ, Cunningham D, Anthoney DA, Gogas H, de Bono JS, Hamberg KJ, Skov T, Mansi JL 2002 A phase II trial of the vitamin D analog seocalcitol (EB1089) in patients with inoperable pancreatic cancer. Br J Cancer 86(5):680–685. Sjoden G, Lindgren JU, DeLuca HF 1984 Antirachitic activity of 1alpha-hydroxyergocalciferol and 1alpha-hydroxycholecalciferol in rats. J Nutr 114(11):2043–2046. Sjoden G, Smith C, Lindgren U, DeLuca HF 1985 1alphahydroxyvitamin D2 is less toxic than 1 alpha-hydroxyvitamin D3 in the rat. Proc Soc Exp Biol Med 178(3):432–436. Liu G, Oettel K, Ripple G, Staab MJ, Horvath D, Alberti D, Arzoomanian R, Marnocha R, Bruskewitz R, Mazess R, Bishop C, Bhattacharya A, Bailey H, Wilding G 2002 Phase I trial of 1alpha-hydroxyvitamin D2 in patients with hormone refractory prostate cancer. Clin Cancer Res 8(9):2820–2827. Elstner E, Linker-Israeli M, Umiel T, Le J, Grillier I, Said J, Shintaku IP, Krajewski S, Reed JC, Binderup L, Loeffler HP 1996 Combination of a potent 20-epi-vitamin D3 analog (KH 1060) with 9-cis-retinoic acid irreversibly inhibits clonal growth, decreases bc1–2 expression, and induces apoptosis in HL-60 leukemic cells. Cancer Res 56:3570–3576. Tong WM, Kallay E, Hofer H, Hulla W, Manhardt T, Peterlik M, Cross HS 1998 Growth regulation of human colon cancer cells by epidermal growth factor and 1,25-dihydroxyvitamin D3 is mediated by mutual modulation of receptor expression. Euro J of Can 34:2119–2125.

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49. Koga M, Eisman JA, Sutherland RL 1988 Regulation of epidermal growth factor receptor levels by 1,25-dihydroxyvitamin D3 in human breast cancer cells. Can Res 48:2734–2739. 50. Gonzales EA, Disthabanchong S, Kowalewski R, Martin KJ 2002 Mechanisms of the regulation of EGF receptor gene expression by calcitriol and parathyroid hormone in UMR 106-01 cells. Kidney Int 61:1627–1634. 51. Boyle BJ, Zhao XY, Cohen P, Feldman D 2001 Insulin-like growth factor binding protein-3 mediates 1 alpha,25-dihydroxyvitamin D3 growth inhibition in the LNCaP prostate cancer cell line through p21/WAF1. J Urol 165:1319–1324. 52. Rozen F, Yang XF, Huynh H, Pollak M 1997 Antiproliferative action of vitamin D–related compounds and insulin-like growth factor-binding protein 5 accumulation. J Natl Cancer Inst 89:652–656. 53. Rozen F, Pollak M 1999 Inihibition of insulin-like growth factor I receptor signaling by the vitamin D analog EB 1089 in MCF-7 breast cancer cells: A role for insulin-like growth factor binding proteins. Int J Oncol 15:589–594. 54. Danielpour D 1996 Induction of transforming growth factorbeta autocrine activity by all-transretinoic acid and 1 alpha,25dihydroxyvitamin D3 in NRP-152 rat prostatic epithelial cells. J Cell Physiol 166:231–239. 55. Yang L, Yang J, Venkateswarly S, Ko T, Brattain MG 2001 Autocrine TGF beta signaling mediates vitamin D3 analoginduced growth inhibition in breast cells. J Cell Physiol 188:383–393. 56. Mantell DJ, Owens PE, Bundred NJ, Mawer EB, Canfield AE 2000 1 alpha,25-dihydroxyvitamin D3 inhibits angiogenesis in vitro and in vivo. Circ Res 87(3):214–220. 57. Bernardi RJ, Johnson CS, Modzelewski RA, Trump DL 2002 Antiproliferative effects of 1alpha,25-dihydroxyvitamin D3 and vitamin D analogs on tumor-derived endothelial cells. Endocrinology 143(7):2508–2514. 58. Iseki K, Tatsuta M, Uehara H, Iishi H, Yano H, Sakai N, Ishiguro S 1999 Inhibition of angiogenesis as a mechanism for inhibition by 1alpha-hydroxyvitamin D3 and 1,25-dihydroxyvitamin D3 of colon carcinogenesis induced by azoxymethane in Wistar rats. Int J Cancer 81(5):730–733.

CHAPTER 98

Vitamin D3: Autoimmunity and Immunosuppression JACQUES LEMIRE

Division of Pediatric Nephrology, Department of Pediatrics, University of California, San Diego, La Jolla, California

I. Introduction II. Autoimmunity

I. INTRODUCTION Since the mid-1980s, a variety of new properties and applications have been discovered for the hormone 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. Almost simultaneously the antiproliferative, prodifferentiating, and immunosuppressive activities of this metabolite of vitamin D were defined. It became obvious from the early investigations that in order to achieve maximal immunosuppressive activity in vitro, 1,25(OH)2D3 was required at a concentration higher than that needed to obtain antiproliferative activity. This observation explains in part the early success of the hormone when used for the treatment of psoriasis, whereas, currently, the inherent hypercalcemic properties of 1,25(OH)2D3 still prevent its clinical use for immunosuppression in humans. However, the development of analogs of the active metabolite has now broadened the potential clinical applications of the hormone. The optimal compound would be one that exerts maximal immunosuppressive activity while sparing the recipient of hypercalcemic complications. To achieve that goal, a variety of animal models of autoimmunity have been studied using 1,25(OH)2D3 and related analogs. This work has led to a potential application for the hormone in human autoimmune diseases. This review will concentrate on the mechanisms of action and effectiveness of 1,25(OH)2D3 in animal models of autoimmunity (excluding psoriasis, which is discussed in Chapter 101, and diabetes, which is discussed in Chapter 99) and describe the practical application of the hormone in humans for autoimmunity. Vitamin D regulation of the immune response is covered in Chapter 36.

II. AUTOIMMUNITY A. Immune Mechanisms Operational in Autoimmunity It is beyond the scope of this section to provide an extensive review of mechanisms involved in VITAMIN D, 2ND EDITION FELDMAN, PIKE, AND GLORIEUX

References

autoimmunity. However, by outlining some key factors described in the dysregulation of the immune system leading to the process, a better understanding of some targets of 1,25(OH)2D3 can be achieved. The description of helper T-cells with different cytokine secretion patterns has opened a new understanding of cell–cell interaction and regulation. Three different T-helper cell subsets have been well described: Th0, Th1, and Th2 cells. The first subset, Th0, represents what appears to be an early precursor or transitory cell that subsequently differentiates into Th1 or Th2 cells on the appropriate stimulus, which is most likely provided by antigen-presenting cells (monocytes, macrophages, B-cells, or dendritic cells). One of these stimuli is interleukin-12 (IL-12), which can be produced by both monocytes, macrophages, and B-cells [1] and promotes Th0 cells to differentiate along the Th1 pathway [2]. The helper T-cell subsets Th1 and Th2 are determined by their cytokine secretion patterns. The Th0 cells produce an unrestricted pattern of cytokines. Th1 cells produce IL-2 and interferon-γ (IFN-γ), whereas Th2 cells produce IL-4, -5, -6, -10, and -13 [3]. Furthermore, Th1 cells can transfer delayed-type hypersensitivity (DTH) [4] and provide help to B-cells to produce the antibody isotype immunoglobulin G2a (IgG2a), whereas Th2 cells help B-cells for IgGi and IgE secretion [5]. Because of their IFN-γ production, Th1 cells can also interact with macrophages to increase bactericidal properties [6]. These Th subsets also cross-regulate one another: IFN-γ produced by Th1 cells can down-modulate Th2 cells, and IL-4 and IL-10 produced by Th2 cells inhibits Th1 cells [7]. Interestingly, IL-10 produced by Th2 cells can indirectly inhibit Th1 cell responses by acting on monocytes that are required by Th1 cells for antigen-specific proliferation and lymphokine secretion [8], most likely by inhibiting IL-12 secretion [9]. The lymphokine IFN-γ produced by Th1 cells also enhances class II antigen expression [10]. This dichotomy between Th1 and Th2 cells has been confirmed in humans. A similar pattern of Copyright © 2005, Elsevier, Inc. All rights reserved.

1754 cytokine secretion for both Th subsets is present [11]. Th1 cells express cytolytic activity against antigenpresenting cells and provide helper function for IgM, IgG, and IgA synthesis at low T-cell/B-cell ratios. At T/B ratios higher than 1:1, a decline in B-cell help is observed, related to the lytic activity of Th1 cells against autologous antigen-presenting B-cells [12]. This downregulation of antibody responses could be operational in vivo. In contrast, Th2 cells develop in response to allergens or parasites, provide help for all immunoglobulin classes including IgE, and lack cytolytic potential [13]. The absence of lytic activity of Th2 cells may account for the long-term IgE responses of patients with atopy or parasitic infections [13]. High efficiency cloning of peripheral blood CD4+ T cells from healthy individuals generates the Th1, Th0, and Th2 cytokine profiles roughly distributed according to a 2:4:1 ratio [13]. The heterogeneity of the cytokine profile in humans is not restricted to CD4+ cells; CD8+ cells, which have the phenotype of cytotoxic and suppressor cells, can also be further defined by analysis of their lymphokine profile [14]. The recognition of the presence of regulatory T-cells leading to peripheral tolerance to extrinsic antigens or autoantigens has helped to understand some of the mechanisms involved in autoimmunity and to provide potential tools for new therapeutic agents [15]. Among those regulatory cells, CD25+T-cells appear to provide a protective effect in the prevention of the autoimmune process [16]. The role of Th2 cells as regulators of physiologic autoimmunity remains unclear. However, a protective effect of Th2 has been shown in situations of induction of autoimmunity such as antigen administration [15]. Further evidence of such a down-regulating role of Th2 cells has been provided by animal models: abrogation of tolerance in IL-4 deficient mice [17] or prevention of diabetes of non-obese diabetic (NOD) mice with administration of Th2 cytokines such as IL-4, IL-10 or IL-13 [18]. Induction of regulatory cells by T-cell vaccination such as T-cell receptor (TCR) peptide vaccination has been accomplished in experimental animal models and provides promising possibilities [19].

B. Mechanisms of Action of 1,25(OH)2D3 in Autoimmunity A significant body of evidence suggests that while the resulting effects of 1,25(OH)2D3 on the immune response are suppressive, its actions are complex. The final response results in part from the interaction of the hormone with both antigen-presenting cells (APC) and T-cells leading to a dual response: suppression of

JACQUES LEMIRE

enhancers and amplification of down-regulators of the immune response. 1. HELPER T-CELLS

From the early discovery of the action of 1,25(OH)2D3 on the immune system, an interest sparked by the discovery of VDR in lymphocytes, a direct antiproliferative effect of the hormone on T-cells has been described [20]. However, its specificity of action resulted from the analysis of cytokine production by T-cells. The sterol inhibits the production of the Th1mediated cytokines IL-2 and IFN-γ, both at transcriptional levels [21,22]. The pro-Th2 cytokine pathway appears to be spared by the sterol, but controversy has recently risen as to the net effect of the hormone on IL4 production. First, 1,25(OH)2D3 leads to an increased production of IL-10 by helper T-cells [23]. By studying human helper T-cell clones, already committed to a specific antigen, the following observations were made after exposure of the cloned T-cells to the sterol. Helper T-cell clones were isolated from atopic patients sensitive to the rye grass antigen Lol pI and characterized as Th0, Th1, and Th2 based on their lymphokine secretion pattern [24]. The Th subsets were activated with Lol pI and antigen-presenting cells in the presence or absence of 1,25(OH)2D3 or the analog 1,25(OH)2-16-ene D3, and the effect of the vitamin D compounds on the lymphokine production was analyzed (Fig. 1). Both 1,25(OH)2D3 and l,25(OH)2-16-ene D3 suppressed the production of IFN-γ by Th1 cells in a dose-dependent manner, but these compounds had minimal effect on IL-4 production by Th2 cells and only at the highest concentrations tested. Interestingly, Th0 cells, producer of both cytokines, showed a profound reduction in IFN-γ in the presence of the vitamin D compounds, whereas IL-4 secretion was less inhibited, suggesting once again a pro-Th1 effect of the hormone and its analog [25]. Further evidence for an immunosuppressive effect of 1,25(OH)2D3 on a Th1-mediated biological activity was provided by the passive transfer of myelin basic protein (MBP)-specific Th1 clones. A characteristic of Th1 cells is their ability to transfer delayed-type hypersensitivity (DTH) [4]. MBP-reactive T-cell clones were activated with syngeneic spleen cells and antigen (MBP) in the presence or absence of 1,25(OH)2D3 before being washed and transferred to the footpads of naive mice. Swelling, as an index of DTH, was measured before and 18 hr after cell transfer using a pressure-sensitive caliper. A complete inhibition of the passive transfer of DTH was observed with 10 nM 1,25(OH)2D3 (Fig. 2) [26]. These results suggested that the hormone could directly interfere with functional activity of Th1 cells.

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Th1 and Th2 100 80 60 40 20 0

10−10 10−9 10−8 10−7

% Suppression of IFN (−) and IL-4 (- -)

% Suppression of IFN (−) and IL-4 (- -)

CHAPTER 98 Vitamin D3: Autoimmunity and Immunosuppression

Th0 100 80 60 40 20 0

10−10 10−9 10−8 10−7

FIGURE 1 Effect of 1,25(OH)2D3 ( , ●) and analog 1,25(OH)2-16-ene D3 ( , ■) on lymphokine production by Th1 (left, open symbols), Th2 (left, filled symbols), and Th0 (right) cell clones. Th clones were activated with antigen, APC, and the lymphokines IL-4 and IFN-γ and assayed by radioimmunoassay. The data are expressed as percentage suppression.

More recently, it was shown that 1,25(OH)2D3 could directly affect naïve CD4+ T cells to enhance the development of Th2 cells [27]. When given in vivo, in IL-4–deficient mice, 1,25(OH)2D3 was less effective in its ability to suppress experimental autoimmune encephalitis (EAE) induction [28]. However, other studies have also shown a reduction of [29] or no effect

DTH response (mm×10−10)

12 10 8 6 4 2 1 0

10−10 10−9 10−8

10−7

1,25-Dihydroxyvitamin D3 (M)

FIGURE 2 Delayed-type hypersensitivity (DTH) response in histocompatible recipients of cocultures of MBP-activated, cloned T cells and syngeneic spleen cells exposed to 20 µg/ml MBP. Cocultures were incubated for 5 days in the presence or absence of varying mean ± SD of the DTH response value in five individual mice. An asterisk signifies a significant (p < 0.005) decrease in the DTH response compared to cells not exposed to hormone. From Lemire and Adams [29] with permission.

on [30,31] IL-4 secretion by T-cells, questioning the 1,25(OH)2D3 “switch” to a pro-Th2 response. So far, these studies would suggest a primarily Th1–mediated inhibitory effect of 1,25(OH)2D3 with a resulting enhanced Th2 functional ability. Whether or not IL-4 is affected remains to be clarified in an in vivo situation. 2. SUPPRESSOR, CYTOXIC, AND REGULATORY T-CELLS

The modulation of suppressor/cytotoxic T-cells in the context of autoimmunity by 1,25(OH)2D3 is unclear. Studies in transplantation immunology have suggested the following. In the context of the mixed lymphocyte reaction [MLR] using human cells, when added at the initiation of the MLR, 1,25(OH)2D3 induced suppressor cell activity, reduced the generation of cytotoxic T-cells and the expression of class II antigen [32]. The steroid hormone could also inhibit natural killer cell activity [33–35] and reduce the activity of cytotoxic T cell lines [36]. Further alternative regulatory mechanisms can be attributed to the steroid hormone. 1,25(OH)2D3 could instead promote or allow for the development of regulatory T-cells and to induce tolerance. Naïve human and mouse CD4+T-cells can be induced into regulatory cells producing IL-10 by treatment with 1,25(OH)2D3 [23]. Transplantation tolerance through the induction of CD4+CD25+ regulatory T-cells can be achieved with 1,25(OH)2D3 [37]. Finally, the steroid hormone can interfere with apoptosis by the down-regulation of CD95L, a cell surface molecule not only activating apoptosis but also promoting Th1 activation through antigen-presenting cell maturation [38].

1756 3. ANTIGEN-PRESENTING CELLS

The first evidence of a direct effect of 1,25(OH)2D3 on antigen-presenting cells was provided by the dosedependent inhibition of IL-12, a pro-Th1 cytokine, by the steroid hormone [39]. Recent studies suggest that the resulting in vitro effect of the drug, when administered early, is to interfere with the differentiation and maturation of APC, thereby leading to altered T-cell responsiveness. In vitro treatment with 1,25(OH)2D3 and analogs leads to down-regulated expression of the co-stimulatory molecules CD40, CD80, CD86 and to decreased IL-12 and enhanced IL-10 production, resulting in decreased T-cell activation, and with APC with tolerogenic properties [40]. Some in vivo evidence of such tolerogenic properties of APC induced by 1,25(OH)2D3 and analogs has been shown in allograft rejection models with the sterol given orally [37] or following the passive transfer of 1,25(OH)2D3 treated APC [41].

C. Effects of 1,25(OH)2D3 on Animal Models of Autoimmunity Table I illustrates the most representative studies of 1,25(OH)2D3 and analogs in animal models of autoimmunity (excluding diabetes, Chapter 99). The first one, a long-standing useful model for the study of autoimmune diseases, has been the animal model of multiple sclerosis, EAE. In this model, immunization of susceptible mice or rats with central nervous system proteins will induce a progressive paralysis in the recipients within two weeks. Developments in peptide technology have led to a higher rate of disease induction in the susceptible recipients [42]. There is strong evidence that EAE is a Th1-mediated disease since antigen-specific Th1 cells can transfer disease [43]. Moreover, at the peak of the disease, there is a predominance of Th1 cytokines (IL-2 and IFN-γ) in the central nervous system of the mice; during remission, IL-10 prevails, suggesting a Th2 predominance [44]. In this model, 1,25(OH)2D3 and analogs can prevent the induction and the relapses of the disease [31]. While 1,25(OH)2D3 clearly exerts a Th1 inhibitory effect in favor of a pro-Th2 cytokine secreting effect in vitro, this dichotomy is harder to demonstrate in vivo [45–47]. A particularly interesting model is the experimental lupus of MRL/l mice. Lupus is an autoimmune disorder that leads to the formation and deposition of immune complexes throughout the body. Sites of predilection include the kidneys, causing nephritis, often with renal failure, and the skin, causing rash and inflammation. A potential role of Th1-mediated IgG2a in the pathogenesis of the disease was suggested by

JACQUES LEMIRE

treatment of MRL/1 mice with anti-IgM anti-sera from birth. This resulted in a depletion of IgG2a antibodies and prevented the development of skin, but not glomerular lesions [48]. A significant increase (eightfold) in IgG2a-producing cells is observed in MRL/1 mice between two to five months of age [49]. In response to thymus-dependent antigens, the IgG subclass profiles in all systemic lupus erythematosus (SLE) mice differ from those of normals, with a predominance of IgG2b and IgG2a rather than IgG1. In addition, sera of the majority of MRL/1 mice contain rheumatoid factors that react most strongly with IgG2a [50], The dependence of IgG2a secretion on Th1 cells [5], as well as class II expression secondary to IFN-γ secretion by Th1 cells, would suggest an important role for the Th1 cell subset in the pathogenesis of experimental SLE. The administration 1,25(OH)2D3 from an early age in these mice could completely inhibit the development of skin lesions, characteristics of this animal model [51]. The development of nephritis and resulting proteinuria was not prevented with the same treatment. However, a subsequent study suggested that a diet with a normal to high calcium content (0.87%) administered to MRL/l mice undergoing similar therapy with 1,25(OH)2D3 could even prevent the development of nephritis (proteinuria) [52]. The animal model of inflammatory bowel disease, experimental murine inflammatory bowel disease, provides additional information about the potential mechanisms of action of the sterol besides reducing the severity of the disease. IL-10 knockout mice, made vitamin D–deficient, develop a severe wasting syndrome; treatment with 1,25(OH)2D3 improved symptoms and prevented the progression of existing disease [61]. These observations suggest once again that the primary target of 1,25(OH)2D3 is through inhibition of Th1 cell activity rather than direct stimulation of the Th2 pathway.

D. Effects of 1,25(OH)2D3 in Autoimmunity in Humans The first and most studied application for 1,25(OH)2D3 in an autoimmune disease in humans is psoriasis and the experience is reviewed in Chapter 101. Limited application of the hormone for other autoimmune disease in humans results from the intrinsic hypercalcemic properties of the hormone, restricting the therapeutic potential of 1,25(OH)2D3. Two significant advances have held promising perspectives for the use of the steroid hormone in humans: the synergistic properties of 1,25(OH)2D3 with known immunosuppressive agents, such as corticosteroids [67]

1757

CHAPTER 98 Vitamin D3: Autoimmunity and Immunosuppression

TABLE I 1,25 (OH)2D3 and Analogs in Autoimmunity

Organ

Dose (µg/kg)a

Outcome, treated/ controls (measure)

Serum calcium (mg/dl)

1,25(OH)2D3

5/2d

80%/5% (survival)

9.7

Lemire [53]

5/2d 7.5/2d

1/4 (disease activity) 1/4 (disease activity)

11.2 9.7

Lemire et al. [54]

2.5/2d

3.3/5 (disease activity)

8.7

Lemire et al. [55]

EAE/mouse

1,25(OH)2D3-16-ene D3 1,25(OH)2-24-oxo16-ene D3 1,25(OH)2D3-16-ene-23ene-26,27hexafluroro D3 MC1288

0.2/2d

10.2

Lemire et al. [56]

EAE/mouse

Ro 63-2023

240/2d

10.0

Mattner et al. [31]

Experimental autoimmune thyroiditis mouse Adjuvant arthritis/rat Murine Lyme arthritis Collagen-induced arthritis Collagen-induced arthritis Experimental Murine Inflammatory Bowel Disease Heymann nephritis/ rat

1,25(OH)2D3

0.2/d

25%/92.8% (disease incidence) 100%/50% (survival) 1.4/3.1 (relapses) 50%/85.7% (histologic incidence)

N/Ab

Fournier et al. [57]

1,25(OH)2D3

0.2/d

12.0

Boissier et al. [58]

1,25(OH)2D3

1/d

11.2

Cantorna et al. [59]

1,25(OH)2D3

2.5/d

7.9

Cantorna et al. [59]

MC 1288

0.1/d

2.6 mmol

Larsson [60]

1,25(OH)2D3

0.25/d

11.9/16.9 (arthritic score) 0.2/0.23 cm (ankle size) 0%/100% (disease incidence) 50%/100% (disease incidence) 1.7/3.0 (histology score)

3 mmol

Cantorna [61]

1,25(OH)2D3

0.5/d

11.8

Branisteau et al. [62]

KH1060

0.5/d

Mercury chlorideinduced nephritis/rat

1,25(OH)2D3

0.1/d

KH1060

0.3/d

Nephrotoxic serum nephritis/rat Lupus nephritis/ mouse Lupus nephritis/ mouse Lupus nephritis/ mouse Lupus nephritis/ mouse Lupus/MRL mouse

1,25(OH)2D3

0.5/d

1,24R(OH)2D3

0.1/d

OCT

0.002–0.1/d 10–40%/80% (proteinuria incidence) 5/2d < 4/>6 (urinary protein/ creatinine ratio) 5–10/d 0–0.2/2–2.5 (severity score) 5/2d None/present (skin lesions)

Model species

Nervous EAE/mouse system EAE/mouse EAE/mouse EAE/mouse

Thyroid Joint

Bowel

Kidney

Kidney

Skin

ad,

daily; 2d, every second day. not available.

bN/A:

Vitamin D3 or analog

1,25(OH)2D3 1,25(OH)2D3 1,25(OH)2D3

80/210 (mg urinary protein/day) 210/210 (mg urinary protein/day) 180/780 (mg urinary protein/day) 14 11

Hattori [64]

N/A

Koizumi et al. [65]

10.4

Abe et al. [66]

8

Lemire et al. [51]

N/A

Deluca [52]

8

Lemire et al. [51]

1758

Oral calcitrol and total skin score Patients with systemis scleroderma

40

Total skin score [mean & SE]

and cyclosporine [57,58,68,69], and the development of 1,25(OH)2D3-analogs with reduced hypercalcemic activity. These exciting possibilities have yet to be applied in the treatment of autoimmune conditions. The ideal application for 1,25(OH)2D3 in humans is a disease in which a significant autoimmune component plays a role and no efficacious treatment is available. Systemic scleroderma might represent such a disease. While the cause of the disease is unknown, the pathogenesis is multifactorial [70]. An early immunologic trigger may lead to expansion of fibrogenic clones of tissue fibroblasts accompanying clinical expansion with early migration of CD4+ and CD8+ cells with a preponderance of CD4+ cells in the skin of affected patients [71,72]. These activated T-cells express HLA-DR molecules, IL-2 receptors, and increased CD4+ markers [73,74]. By precisely suppressing those immune mechanisms, 1,25(OH)2D3 could play a role in modulating the disease. A tolerability and feasibility study of calcitriol in the treatment of systemic sclerosis was done in 10 patients with a diagnosis of systemic sclerosis [75]. One patient withdrew within two weeks of the study secondary to drug intolerance. The 9 patients (M/F ratio of 2/7; age: 44 ± 14 years), with a disease duration of 6.9 ± 4.6 years, completed the six-month trial. After initial assessment and instructions to patients to follow a low calcium diet (800 mg/day), calcitriol was started at low dosage (0.25 or 0.5 mcg/day) and increased on a monthly basis to the maximum tolerable dosage, i.e. urinary calcium excretion of 70 years exhibit wintertime levels of 25OHD < 30 nmol/l [84]. Serum levels of 25OHD have been shown by Chapuy et al. [85] to correlate with latitude (r = −0.7, p < 0.01). More pronounced seasonal variations in 25OHD at higher latitudes might be a contributing factor to the increase in hip fracture incidence during wintertime at high latitude. Exposure to sunlight is quite essential in maintenance of normal vitamin D status and in the prophylaxis against HDM. Normally the skin supplies the body with 80–100% of its requirements of vitamin D [86]. If sunlight exposure of the skin is limited, however, 25OHD will decrease and HDM may develop. A study of veiled Caucasian women living in Denmark showed that lack of direct sunlight exposure required a daily supply of 800 IU vitamin D3 in order to maintain normal vitamin D status [65]. This is in agreement with the study of Holick [86], who investigated vitamin D levels in submariners. A supply of more than 600 IU vitamin D3 was necessary to maintain normal levels of vitamin D during the absence of sunlight for three months. In elderly fallers Dhesi et al. [87] described a relationship between 25OHD levels and the number of times per week the patient went outside. Thus, homebound frail old people have a high risk for development of HDM making them even more immobile. They get less sunlight exposure, and moreover their age-dependent skin atrophy further reduces the ability to produce vitamin D, despite ample sunlight exposure [86]. These data lead to the following conclusion: all individuals

1814

HENNING GLERUP AND ERIK FINK ERIKSEN

at risk of HDM, especially the elderly (> 65 years) who are not regularly exposed to sunlight, should be given a daily supplement of at least 800 IU vitamin D3 in order to avoid HDM.

33% of elderly people experience at least one fall per year [72–74]. Mowe et al. [58] found lower levels of 25OHD among fallers compared to nonfallers with an inverse correlation between serum levels of 25OHD and the risk of falls (r = −0.27, p < 0.001). This is in agreement with the findings of Stein et al. [56], who described a correlation between secondary hyperparathyroidism and the risk of falls. In a study of 4251 elderly Australian women living in residential care (age > 84 years), Flicker et al. [88] recently identified low serum levels of 25OHD as a major risk factor for falls (hazard risk ratio 0.64, p < 0.004). In a “falls clinic” in London, to which patients with at least one fall within the last 8 weeks, were referred, Dhesi et al. [87,89] described severe hypovitaminosis D (25OHD < 30 nmol/l) in 31.8% and moderate hypovitaminosis (25OHD < 50 nmol/l) in 72.8%. Patients with low levels of 25OHD (< 30 nmol/l) displayed significantly impaired psychomotor function measured by a performance test (AFPT), postural sway, choice reaction time (CRT), and isometric quadriceps strength measurement. In a multivariate analysis, 25OHD was identified as an independent variable for AFPT, CRT, and body sway. For quadriceps strength, PTH was found to be an independent variable. Pfeifer et al. [90] investigated the effect on body sway and fall incidence in 148 women (aged 74 ± 1 year) treated with either a daily dose of 1200 mg calcium in combination with 800 IU cholecalciferol or 1200 mg of calcium alone. After one year, a 9% decrease in body sway was seen in the vitamin D–treated group compared to the group treated with calcium only (p = 0.043). The number of falls per subject per year was 0.45 in the calcium-only group compared to 0.24 in the vitamin D–treated group (p = 0.034). Bischoff et al. [9] treated 122 elderly women (mean age 83.3 years) with either 1200 mg calcium in combination with 800 IU cholecalciferol (Cal + D) or calcium only (Cal). Musculoskeletal function was measured by knee extension and flexion strength, grip strength, and timed “up & go” test. After three months, a significant improvement in musculoskeletal function was found in the Cal + D group (p < 0.0094). The risk of falling was reduced by 49% when comparing the Cal + D to the Cal group (p < 0.01) (see Fig. 8). The data presented above lend strong support to the hypothesis, that it is vitamin D supplementation more

Probability

B. Vitamin D and Risk of Falls

0.8 0.7

Cal + D

0.6

Cal

0.5 0.4 0.3 0.2 0.1 0

0

1

2 3 Number of falls

>=4

FIGURE 8 Number of falls and the effect of treatment with 800 IU cholecalciferol + 1200 mg calcium daily (Cal + D) versus calcium alone (Cal). Adjusted probabilities and SEs for having zero, one, or multiple falls for subjects in both treatment groups. SEs are calculated by taking 1 SD above and below the mean rate of falls and calculating the resulting Poisson probabilities. Adjustments have been performed for length of observation in the treatment period, previous falls in pretreatment period, a person who fell in the pretreatment period, age, and baseline 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D. Reproduced from Bischoff HA, et al. J Bone Miner Res 2003;18:343–351 with permission of the American Society for Bone and Mineral Research.

than calcium supplementation that protects against falls. The analysis of these studies suggests that a daily dose of at least 800 IU vitamin D3 should be given in order to ensure vitamin D sufficiency and significant improvement in muscle function and reduction in the incidence of falls.

VII. IS HDM CAUSED BY LOW LEVELS OF 25OHD, 1,25(OH)2D OR ELEVATED PTH? From a theoretical point of view, the most likely effector associated with HDM should be 1,25(OH)2D. VDR has been identified in muscle [35–38] and 1,25(OH)2D shows the highest affinity for the receptor. In agreement with this theory, experimental research has almost exclusively focused on the muscular effects of 1,25(OH)2D. Clinical experience, however, indicates that complaints associated with HDM correlate to serum levels of 25OHD and not to 1,25(OH)2D. The lack of correlation to 1,25(OH)2D levels might be due to increased renal 1-α-hydroxylase activity caused by secondary hyperparathyroidism. PTH, on the other hand, might also exert direct effects on muscle. In the following sections the three “candidates” will be discussed separately.

1815

CHAPTER 102 Muscles and Falls

A. PTH

B. 1,25(OH)2D

It has been suggested that HDM is caused by raised levels of PTH associated with secondary hyperparathyroidism, rather vitamin D deficiency itself [91]. Fatigue and muscular weakness are classically associated with primary hyperparathyroidism [92–95], and successful parathyroid surgery results in significant improvement of these muscular symptoms [92,94,96]. In addition, muscle biopsies obtained from patients with primary hyperparathyroidism reveal type II fiber atrophy, very much like the changes seen in HDM. Treatment with PTH reduces the intracellular content of inorganic phosphate, creatine phosphate, and CaATPase [97], which are exactly the findings we reported after analysis of muscle biopsies and 31P-MR-spectroscopy in patients with HDM [23,66]. Furthermore, mitochondrial oxygen consumption and the activity of creatine phosphokinase and CaATPase are reduced and oxidation of long-chain fatty acids impaired by PTH [98]. Thus, many parallels exist between myopathy caused by primary hyperparathyroidism and HDM. However, muscle weakness is not always present in patients with primary hyperparathyroidism [3,99], and HDM can be present despite normal PTH levels [9]. Further, improvements in muscle strength after surgery for primary hyperparathyroidism do not correlate to postoperative decreases in PTH or calcium [94,100]. If PTH was the only effector on skeletal muscle, reversal of secondary hyperparathyroidism by a high intake of calcium should reverse the muscular symptoms. However, in their rat study Rodman and Baker [45] detected severe perturbations of muscle function in vitamin D-deficient rats despite high serum levels of calcium and phosphate (Fig. 4). Furthermore, in a study on type II fiber size in hip fracture patients, Sato et al. [54] reported a significant correlation between serum levels of 25OHD and fiber size—no correlation to PTH was reported. Generally, clinical studies [8,56,89] report inverse correlations between PTH and muscle symptoms, and positive correlations to 25OHD [8,56,58,59,89]. In a comment accompanying the study of Stein et al. [56], Birge [83] suggested that PTH might be a better biological marker for vitamin D deficiency at the tissue level than serum levels of 25OHD, and this could be the reason why PTH and not 25OHD come out as significant determinants in multiple regression analyses. In this context, however, the significant interaction between 25OHD and PTH has to be taken into account. In conclusion: There is strong evidence for a direct effect of vitamin D on muscle both in clinical and experimental studies. It is possible, however, that secondary hyperparathyroidism may exert additive or synergistic effects on HDM development.

Theoretically, 1,25(OH)2D concentrations should be the most likely effector of vitamin D effects on muscle— and indeed, a large amount of experimental data support this notion (see Chapter 55). In clinical studies, however, this expected relationship finds less support [59]. Glerup et al. [8,23] found no correlation between muscle function and 1,25(OH)2D (r = −0.14, NS), whereas maximal knee extension strength correlated significantly to 25OHD (r = 0.34, p < 0.01). In fact, it is common to see severe symptoms of HDM with normal or even elevated values of 1,25(OH)2D. Furthermore, hypovitaminosis D–related symptoms (diffuse muscle pain, deep bone pain, paresthesia, fatigue, muscle cramps, joint pain) all correlated to 25OHD (KruskalWallis ANOVA: p < 0.001) but not to 1,25(OH)2D (NS). The absence of correlations to 1,25(OH)2D is probably explained by several factors. First, renal 1-αhydroxylase activity is under tight control by PTH levels, resulting in normal or even elevated levels of 1,25(OH)2D, despite very low levels of 25OHD. Second, serum levels of 1,25(OH)2D don’t necessarily tell anything about the intracellular levels of the hormone in muscle cells. Third, Geusens et al. [67] have shown clinical importance of VDR-genotypes, which support an in vivo effect of VDR-mediated effects. Thus, 1,25(OH)2D seems to be involved in the pathogenesis of HDM. One hypothesis may reconcile the inconsistencies outlined above, namely the purported presence of intracellular, autocrine production of 1,25(OH)2D from 25OHD in muscle cells [68]. There is an increasing amount of evidence suggesting the clinical importance of extrarenal 1,25(OH)2D synthesis [101–107] (see Chapter 79). Two features distinguish extrarenal from renal synthesis of 1,25(OH)2D: 1) it is not under control of PTH, but is dependent on the availability of the substrate 25OHD; 2) local 1,25(OH)2D synthesis has been shown to take place in the mitochondria [101]. Muscle has a very high content of mitochondria, which makes muscle a very likely site of extrarenal 1,25(OH)2D synthesis. Intracellular production of 1,25(OH)2D could explain the correlation between 25OHD and the muscular effects of vitamin D. Further, this pathway still requires 1,25(OH)2D to be the final effector of vitamin D’s muscular effects. It has been argued that local 1,25(OH)2D synthesis in muscle should result in increased serum levels of 1,25(OH)2D. Significant local 1,25OH2D production has been identified in other tissues (endothelium [102], prostate [106], bone cells, liver cells, skin, etc. [103]), but these sites do not result in increased serum levels of 1,25(OH)2D. The absence of increased 1,25(OH)2D in these instances is most likely explained by the presence of highly-induced intracellular 24-hydroxylase

1816 activity, ensuring degradation of 1,25OH2D before it reaches the circulation. Also, failure of release of 1,25(OH)2D into the circulation may be an additional factor.

C. 25OHD 25OHD is considered to be the storage and circulating form of vitamin D, and measurement of serum levels of 25OHD best reflect the vitamin D status of the body. 25OHD has been presumed to be biologically inert, but recent data challenge this notion. As already mentioned above, serum levels of 25OHD correlate to the biological effects of vitamin D in vivo. The effects of 25OHD on muscle cells could be mediated in several ways. 25OHD has some affinity for VDR, but the affinity of 1,25(OH)2D for VDR is approximately 1000-fold higher than 25OHD. The serum concentration of 25OHD is about 500–1000 times higher than 1,25(OH)2D, but most is bound to vitamin D binding protein (DBP) (see Chapter 8). Competitive binding of the two vitamin D metabolites to VDR might be possible under some circumstances [108–110]. No specific receptor for 25OHD has been identified. As mentioned in the paragraph above, a more likely explanation is the local synthesis of 1,25(OH)2D from 25OHD as substrate. Finally, it is possible that 25OHD could exert direct effects on muscle via an effector-mechanism, which is still under investigation. Recently, Nykjaer et al. [111–113] (see Chapter 10) identified the cubilinmegalin receptor system as being responsible for renal reuptake of vitamin D metabolites bound to DBP. Muscle tissue possesses receptors of the LDL receptor family, which potentially could be involved in tissue specific uptake of 25OHD, but this still needs to be investigated (personal communication A. Nykjaer).

VIII. OTHER POSSIBLE MUSCULAR EFFECTS OF VITAMIN D A. Insulin Resistance in Vitamin D Deficiency — Due To HDM? Vitamin D deficiency has been reported to increase the risk of developing insulin resistance and abnormal oral glucose tolerance tests (OGTT) [114–117]. Striated muscle is central in the pathogenesis of Type 2 diabetes. GLUT4 is the most important glucose transporter in muscle [118–120]. The GLUT4 content of muscles declines with age [118,120], especially in the fast type II muscle fibers. Furthermore, GLUT4


is reduced in type 2 diabetes [119]. More research is necessary to establish a possible effect of vitamin D on the GLUT4 content of the muscle. In type 2 diabetes, serum levels of free fatty acids are elevated [121,122]. Significant perturbations in the energy metabolism of mitochondria in muscle has been described in hypovitaminosis D [66], as well as in the presence of increased levels of PTH [97]. Additional research is warranted on the possible effects of vitamin D and PTH on fatty oxidation in striated muscle.

B. Possible Effects of Vitamin D on Muscle Regeneration During exercise, serum levels of 1,25(OH)2D have been reported to increase temporarily [123–127]. Exercise damages the muscle fibers and induces regeneration and growth of the muscle through enhanced satellite cell proliferation [128,129]. It could be speculated that 1,25OH2D might be of importance in the regeneration process of muscle. Furthermore, reduced IGF-I levels seem to play a role in age-related muscle degeneration. A possible interrelationship between IGF-I levels and vitamin D levels should be investigated [130].

IX. SUMMARY In this chapter we have reviewed the increasing evidence pointing to direct effects of vitamin D on striated muscle, making striated muscle an important target organ for vitamin D. Hypovitaminosis D myopathy (HDM) is a reversible disease that can recover completely, usually with significant improvement within a few weeks to a month after beginning vitamin D treatment [7,131,132]. Full restoration of severe HDM, however, may take 6 to 12 months of treatment with vitamin D [10]. Moreover, there is strong evidence for the prophylactic effects of vitamin D to reduce the risk of falls through improved muscular function and thereby to decrease the incidence of fractures. A daily dose of at least 800 IU (20 µg) cholecalciferol preferably in combination with 1000–1200 mg calcium seems to be the most effective treatment. Consequently combined vitamin D and calcium prophylaxis should be considered to combat hip fractures in the elderly. All patients at risk for vitamin D deficiency (i.e., lack of sunlight exposure) should be suspected to suffer from HDM. Those patients suspected of having HDM should have a blood test performed for measurement of 25OHD and PTH. In severe cases of HDM, treatment


should be initiated with a higher dose of vitamin D in order to speed up recovery. 300,000 IU cholecalciferol or ergocalciferol can be given either as an oral dose or intramuscular injection. This can be given as a single dose or repeated every month for three months. The high dose vitamin D should be combined with a daily supply of calcium. In order to avoid HDM, the serum levels of 25OHD should be kept above 50 nmol/l and PTH levels should be suppressed to the normal range. Maintenance of normal 25OHD levels in the elderly should have a high priority, as hip fractures and disability carry a high cost for society as well as for the individual patients. Treatment of HDM results in significant improvement in quality of life. However, vitamin D is not the solution to every musculoskeletal problem in the aging population. The age-related loss of muscle power (approximately 1.5% per year [32]) seems to be obligatory and unrelated to vitamin D deficiency. The data summarized in this review, lead to new questions, of which the ones, we consider most important are listed below: 1. Do muscle cells have the capacity to synthesize 1,25(OH)2D from 25OHD? 2. Is hydroxylation of 25OHD to 1,25(OH)2D necessary in order to mediate its effect on muscle, or does 25OHD have an effect of its own? 3. How do elevated PTH levels interact with vitamin D in muscle? 4. Finally, is the uptake of 25OHD and 1,25(OH)2D in muscle a matter of simple diffusion, or do muscle cells possess a system for facilitated uptake of the compounds?

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1819 85. Chapuy MC, Preziosi P, Maamer M, Arnaud S, Galan P, Hercberg S, Meunier PJ 1997 Prevalence of vitamin D insufficiency in an adult normal population. Osteoporos Int 7:439–443. 86. Holick MF 1994 McCollum Award Lecture, 1994: vitamin D— new horizons for the 21st century. Am J Clin Nutr 60: 619–630. 87. Dhesi JK, Moniz C, Close JC, Jackson SH, Allain TJ 2002 A rationale for vitamin D prescribing in a falls clinic population. Age Ageing 31:267–271. 88. Flicker L, Mead K, Nowson CA, Scherer SC, Stein MS, Thomas J, Hopper JL, Wark JD 2001 Risk factors for falls in older women in residential care in Australia. J Bone Miner Res 16(Suppl 1):S170. 89. Dhesi JK, Bearne LM, Moniz C, Hurley MV, Jackson SH, Swift CG, Allain TJ 2002 Neuromuscular and psychomotor function in elderly subjects who fall and the relationship with vitamin D status. J Bone Miner Res 17:891–897. 90. Pfeifer M, Begerow B, Minne HW, Abrams C, Nachtigall D, Hansen, C 2000 Effects of a short-term vitamin D and calcium supplementation on body sway and secondary hyperparathyroidism in elderly women. J Bone Miner Res 15: 1113–1118. 91. McCarty MF 2002 Vitamin D status and muscle strength. Am J Clin Nutr 76:1454–1455. 92. Colliander EB, Strigard K, Westblad P, Rolf C, Nordenstrom J 1998 Muscle strength and endurance after surgery for primary hyperparathyroidism. Eur J Surg 164:489–494. 93. Turken SA, Cafferty M, Silverberg SJ, De La Cruz L, Cimino C, Lange DJ, Lovelace RE, Bilezikian JP 1989 Neuromuscular involvement in mild, asymptomatic primary hyperparathyroidism. Am J Med 87:553–557. 94. Kristoffersson A, Bjerle P, Stjernberg N, Järhult J 1988 Preand postoperative respiratory muscle strength in primary hyperparathyroidism. Acta Chir Scand 154:415–418. 95. Patten MP, Mallette LE, Prince A, Aurbach GD, Belezikian JP, King Engel W 1974 Neuromuscular disease in primary hyperparathyroidism. Ann Intern Med 80:182–193. 96. Deutch SR, Jensen MB, Christiansen PM, Hessov I 2000 Muscular performance and fatigue in primary hyperparathyroidism. World J Surg 24:102–107. 97. Baczynski R, Massry SG, Magott M, el Belbessi S, Kohan R, Brautbar N 1985 Effect of parathyroid hormone on energy metabolism of skeletal muscle. Kidney Int 28: 722–727. 98. Smogorzewski M, Piskorska G, Borum PR, Massry SG 1988 Chronic renal failure, parathyroid hormone, and fatty acids oxidation in skeletal muscle. Kidney Int 33: 555–560. 99. Jansson S, Grimby G, Hagne I, Hedman I, Tisell LE 1991 Muscle structure and function before and after surgery for primary hyperparathyroidism. Eur J Surg 157:13–16. 100. Chou FF, Sheen Chen SM, Leong CP 1995 Neuromuscular recovery after parathyroidectomy in primary hyperparathyroidism. Surgery 117:18–25. 101. Zehnder D, Bland R, Walker EA, Bradwell AR, Howie AJ, Hewison M, Stewart PM 1999 Expression of 25-hydroxyvitamin D3-1alpha-hydroxylase in the human kidney. J Am Soc Nephrol 10:2465–2473. 102. Zehnder D, Bland R, Chana RS, Wheeler DC, Howie AJ, Williams MC, Stewart PM, Hewison M 2002 Synthesis of 1,25-dihydroxyvitamin D3 by human endothelial cells is regulated by inflammatory cytokines: a novel autocrine determinant of vascular cell adhesion. J Am Soc Nephrol 13:621–629.

1820 103. Dusso A, Brown A, Slatopolsky E 1994 Extrarenal production of calcitriol. Semin Nephrol 14:144–155. 104. Dusso A, Lopez-Hilker S, Rapp N, Slatopolsky E 1988 Extrarenal production of calcitriol in chronic renal failure. Kidney Int 34:368–375. 105. Dusso AS, Finch J, Brown A, Ritter C, Delmez J, Schreiner G, Slatopolsky E 1991 Extrarenal production of calcitriol in normal and uremic humans. J Clin Endocrinol Metab 72: 157–164. 106. Schwartz GG, Whitlatch LW, Chen TC, Lokeshwar BL, Holick MF 1998 Human prostate cells synthesize 1,25-dihydroxyvitamin D3 from 25-hydroxyvitamin D3. Cancer Epidemiol Biomarkers Prev 7:391–395. 107. Barreto AM, Schwartz GG, Woodruff R, Cramer SD 2000 25-hydroxyvitamin D3, the prohormone of 1,25-dihydroxyvitamin D3, inhibits the proliferation of primary prostatic epithelial cells. Cancer Epidemiol Biomarkers Prev 9:265–270. 108. Reichel H, Koeffler HP, Norman AW 1989 The role of the vitamin D endocrine system in health and disease. N Engl J Med 320:980–991. 109. Barger-Lux MJ, Heaney RP, Lanspa SJ, Healy JC, DeLuca HF 1995 An investigation of sources of variation in calcium absorption efficiency. J Clin Endocrinol Metab 80:406–411. 110. Barger-Lux MJ, Heaney RP, Dowell S, Bierman J 1996 Relative molar potency of 25-hydroxyvitamin D indicates a major role in calcium absorption. J Bone Miner Res 11:S423. 111. Nykjaer A, Dragun D, Walther D, Vorum H, Jacobsen C, Herz J, Melsen F, Christensen EI, Willnow TE 1999 An endocytic pathway essential for renal uptake and activation of the steroid 25-(OH) vitamin D3. Cell 96:507–515. 112. Nykjaer A, Fyfe JC, Kozyraki R, Leheste JR, Jacobsen C, Nielsen MS, Verroust PJ, Aminoff M, de la CA, Moestrup SK, Ray R, Gliemann J, Willnow TE, Christensen EI 2001 Cubilin dysfunction causes abnormal metabolism of the steroid hormone 25(OH) vitamin D3. Proc Natl Acad Sci USA 20:98, 13895–13900. 113. Leheste JR, Melsen F, Wellner M, Jansen P, Schlichting U, Renner-Muller I, Andreassen TT, Wolf E, Bachmann S, Nykjaer A, Willnow TE 2002 Hypocalcemia and osteopathy in mice with kidney-specific megalin gene defect. Faseb J 114. Boucher BJ 1998 Inadequate vitamin D status: does it contribute to the disorders comprising syndrome ‘X’? Br J Nutr 79:315–327. 115. Boucher BJ, Mannan N, Noonan K, Hales CN, Evans SJ 1995 Glucose intolerance and impairment of insulin secretion in relation to vitamin D deficiency in east London Asians. Diabetologia 38:1239–1245. 116. Baynes KCR, Boucher BJ, Feskens EJM, Kromhout D 1999 Vitamin D, glucose tolerance, and insulinanemia in elderly men. Diabetologia 40:344–347. 117. Rudnicki PM, Molsted-Pedersen L 1997 Effect of 1,25-dihydroxycholecalciferol on glucose metabolism in gestational diabetes mellitus. Diabetologia 40:40–44.


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CHAPTER 103

Renal Failure and Secondary Hyperparathyroidism MASAFUMI FUKAGAWA KIYOSHI KUROKAWA

Division of Nephrology & Dialysis Center, Kobe University School of Medicine, Kobe, Japan Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo, Japan

I. Role of Vitamin D in the Development of Hyperparathyroidism in Renal Failure II. Resistance to 1,25(OH)2D as a Cause of Severe Secondary Hyperparathyroidism in Chronic Renal Failure

I. ROLE OF VITAMIN D IN THE DEVELOPMENT OF HYPERPARATHYROIDISM IN RENAL FAILURE The kidney is the main organ for the production of active vitamin D, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) [1], a process that is catalyzed by 25-hydroxyvitamin D3 1alpha-hydroxylase (1α-hydroxylase) in the proximal tubule cells [2–4]. Activity of this enzyme is attenuated in chronic renal failure due to phosphate load [5,6] as well as to the decreased numbers of viable nephrons [1]. Furthermore, it has recently been shown that fibroblast growth factor-23 (FGF-23) may suppress activation of vitamin D [7]. FGF-23 is a newly discovered phosphaturic factor (see Chapters 26 and 29) and increased serum levels have been reported in patients with renal dysfunction [8]. In addition to the decreased production of 1,25(OH)2D3 in the kidney, the importance of vitamin D deficiency has been recognized again, especially in chronic kidney disease (CKD) stages 3 and 4 [9]. Vitamin D deficiency is reflected by decreased serum concentrations of 25(OH)D [10]. Such decrease of 25(OH)D level may result from the loss of vitamin D-binding protein into the urine [11] as well as malnutrition [12]. In addition, a decrease of megalin on the brush border of proximal tubules has been reported [13], which results in diminished reuptake of filtered 25(OH)D [14] (see Chapter 10). In chronic renal failure, the secretion of parathyroid hormone (PTH) is stimulated by several factors, primarily hypocalcemia and reduced production of 1,25(OH)2D3 [1]. In addition, direct stimulatory VITAMIN D, 2ND EDITION FELDMAN, PIKE, AND GLORIEUX

III. Management of Severe Hyperparathyroidism Refractory to Medical Therapy IV. Future Roles of Vitamin D Analogs in Chronic Renal Failure References

action of phosphate on parathyroid has recently been demonstrated [15–17]. Thus, secondary hyperparathyroidism develops almost inevitably in patients with chronic renal failure without appropriate therapy [1]. Excess PTH accelerates bone turnover and results in a typical bone abnormality known as osteitis fibrosa [18]. Vitamin D metabolites suppress the secretion of PTH by correcting hypocalcemia and also by direct action on parathyroid cells in patients of chronic renal failure [19,20]. However, it is still difficult to suppress PTH secretion in substantial numbers of patients by vitamin D treatment. Such patients usually have marked parathyroid hyperplasia [21]. Since conventional uses of vitamin D in mild and advanced renal failure, including 1,25(OH)2D3 pulse therapy, are discussed in the Chapter 76 by Dusso, Brown, and Slatopolsky, we will focus on patients with severe disease that are refractory to medical therapy and summarize the new therapeutic uses of vitamin D metabolites.

II. RESISTANCE TO 1,25(OH)2D AS A CAUSE OF SEVERE SECONDARY HYPERPARATHYROIDISM IN CHRONIC RENAL FAILURE A. Resistance to 1,25(OH)2D in Chronic Renal Failure Despite physiological plasma concentrations of 1,25(OH)2D, as well as those of calcium ion obtained by routine treatment, there are still many patients with elevated plasma PTH levels. Some of these patients respond to supraphysiological concentration of 1,25(OH)2D3 Copyright © 2005, Elsevier, Inc. All rights reserved.

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MASAFUMI FUKAGAWA AND KIYOSHI KUROKAWA

achieved either by intravenous or oral intermittent high doses of 1,25(OH)2D3, which is also refered to as “1,25(OH)2D3 pulse therapy” [22–24]. These observations suggest that the resistance of parathyroid cells to 1,25(OH)2D may play a major role in the pathogenesis of severe secondary hyperparathyroidism in chronic renal failure [25]. Resistance to physiological concentrations of 1,25(OH)2D may develop during the early phase of chronic renal failure. In rat models of mild renal failure, PTH secretion, synthesis, and parathyroid cell proliferation were all enhanced even in the presence of a normal plasma concentration of calcium and 1,25(OH)2D [26]. Hyperparathyroidism returned to normal with pharmacological doses of 1,25(OH)2D3 without the induction of hypercalcemia. In these rats, 1,25(OH)2D receptor (VDR) density in parathyroid glands, detected by Western blot, was decreased compared to levels seen in normal rats. Such reduction of VDR density in parathyroid glands also has been demonstrated in enlarged parathyroid glands of chronic dialysis patients [27], as well as in animal models of chronic uremia [28,29]. This abnormality, reduced VDR concentration, is currently considered the central feature responsible for the resistance of parathyroid glands to 1,25(OH)2D in chronic renal failure [30]. In addition to the decreased density of VDR, several mechanisms have been proposed (Fig. 1). Decreased density of retinoid receptor X (RXR), which forms heterodimers with VDR, has been suspected [31]; however, the significance of this observation still remains unclear. Hsu and associates have been focused

on the possible inhibition of 1,25(OH)2D action by uremic toxins [32]. They have shown that serum from uremic patients inhibited the interaction between the 1,25(OH)2D-VDR complex and DNA [33], possibly through the formation of a Schiff base [33]. Although they have examined the effects of glyoxylate [35], other uremic toxins responsible for this inhibition still remain to be identified [36]. In addition, calreticulin has been shown to inhibit the binding of the 1,25(OH)2D-VDR complex to the vitamin D responsive element (VDRE) in the PTH gene promoter [37]. Hypocalcemia was found to induce increased concentrations of calreticulin exclusively in parathyroid glands. It is possible that this molecule plays some role in the regulation of VDR function by extracellular calcium [38,39]. Decreased action of 1,25(OH)2D due to these mechanisms finally results in disturbed up-regulation of VDR, which further increases resistance to 1,25(OH)2D in chronic renal failure (Fig. 1) [40–42]. Such a vicious cycle may be prevented by early vitamin D treatment as suggested by the animal models [43].

B. Advantages and Limitations of Intravenous Calcitriol Therapy Considering that these abnormalities cause vitamin D resistance, it is quite reasonable that supraphysiological concentrations of 1,25(OH)2D have been shown effective in suppressing PTH secretion in chronic dialysis patients resistant to conventional oral calcitriol [22–24]. Since the peak concentration of 1,25(OH)2D is more important

Calcium ion 1,25(OH)2D RXR 1,25(OH)2D receptor

1,25 D-receptor complex

Uremic toxins Up-regulation Calreticulin

DNA Nucleus AAAAAA mRNA Up-regulation Calcium-sensing receptor

FIGURE 1 Mechanisms of resistance to 1,25(OH)2D in chronic renal failure. Several steps of 1,25(OH)2D action are disturbed in chronic renal failure, leading to further reduction of VDR.

CHAPTER 103 Renal Failure and Secondary Hyperparathyroidism

for the suppression of PTH secretion than the total dose of calcitriol, as shown in dialysis patients [22] and in experimental animals [44], higher doses of calcitriol theoretically should be more effective. However, high doses of calcitriol often cause hypercalcemia and hyperphosphatemia, resulting in reduction or disconcentration of therapy. High Ca × Pi product leads to metastatic calcification including within blood vessels, which may result in a higher mortality risk [45]. This has been the main reason why less calcemic vitamin D analogs, such as 19-nor-1,25(OH)2D2 [46] and 22-oxa1,25(OH)2D3 [47], have been developed (see Section VIII of this book). Even with these less calcemic vitamin D analogs, PTH secretion is still difficult to control in some patients.

C. Parathyroid Size as a Marker for the Prognosis of Vitamin D Therapy In order to avoid unnecessary vitamin D treatment and metastatic calcification, it is certainly necessary to have a good predictor of the prognosis of vitamin D therapy for parathyroid suppression in these patients. Although recent data suggest that serum FGF-23 levels may be a marker for the future prognosis of hyperparathyroidism [48], parathyroid gland size assessed by ultrasonography is the most simple and useful marker at this time. Marked parathyroid gland hyperplasia is a unique feature of secondary hyperparathyroidism in chronic dialysis patients [49]. Although the size of each of the four glands is usually different, even in the same patient, it has been recognized by experience that the size of the largest gland roughly correlates with the length and severity of uremia and with the degree of prevailing plasma and stimulated peak PTH levels [50,51]. The size also correlates with the degree of abnormal control of PTH secretion [52,53], which may be normalized by calcitriol pulse therapy [54]. Clinical observations of dialysis patients suggest that the size of the largest gland is the critical marker for the long-term prognosis of vitamin D therapy [55]. If the largest gland is larger than 1 cm in diameter or about 0.5 cm3 in volume, it is usually difficult to suppress PTH secretion by calcitriol pulse therapy. In such patients, secondary hyperparathyroidism always persists or relapses even if it initially responded to calcitriol pulse therapy. By contrast, patients with only smaller glands usually respond well to calcitriol pulse therapy, and parathyroid gland function can be controlled then with oral active vitamin D sterols. Thus, the size of the parathyroid gland may have more relevance than plasma PTH levels in assessment of calcitriol pulse

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therapy [37]. Furthermore, Tominaga et al. demonstrated in patients treated by surgical parathyroidectomy that autoimplantation of tissue fragments from glands heavier than 0.5 g resulted in frequent relapse of hyperparathyroidism [56]. Thus, the critical size for the management strategy for hyperparathyroidism in chronic dialysis patients seems to be less than 0.5 cm3 in volume. The correlation between the gland size and the resistance to calcitriol can be explained by the degree of decrease of VDR density. VDR density is inversely correlated with the weight of enlarged glands [57]. Large parathyroid glands are usually composed of nodular hyperplasia, a more advanced type of pathology than diffuse hyperplasia seen in small glands [58]. It has been reported that cells in nodular hyperplasia glands have higher proliferative potentials [59,60,62] and more abnormal regulation of PTH secretion [63] than cells in diffuse hyperplasia glands. We and others have clearly shown that the VDR number was decreased more in nodular hyperplasia than in diffuse hyperplasia [57,64]. Since 90% of the glands heavier than 0.5 g were composed of nodular hyperplasia as shown by Tominaga and Takagi [61], the difference in the response to calcitriol that is dependent upon gland size can be explained by the difference in the type of hyperplasia in the larger glands. In nodular hyperplasia, decreased density of the calcium-sensing receptor also has been demonstrated [65,66]. Although it is still controversial whether this decrease is the cause or the result of secondary hyperparathyroidism, a direct correlation between cell proliferation and decrease of calcium-sensing receptor has been suggested [67,68]. Thus, glands with nodular hyperplasia are less responsive to the suppressive effect of ambient calcium. This may partially explain the empirical finding of high PTH levels in the presence of hypercalcemia in patients with nodular hyperplasia. The progression of parathyroid hyperplasia is summarized in Fig. 2. It is of note that some enlarged glands smaller than 0.5 cm3 may be composed of nodular hyperplasia [61]. Nodule formation may be recognized by the shape of the glands detected by the latest models of ultrasonography devices [70]. Furthermore, Onoda et al. recently reported that positive blood supply detected inside the gland was highly suggestive of nodular hyperplasia [71].

III. MANAGEMENT OF SEVERE HYPERPARATHYROIDISM REFRACTORY TO MEDICAL THERAPY Prevention of parathyroid hyperplasia from the early phase of chronic renal failure is the most important

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Normal parathyroid

Diffuse hyperplasia

Responsive to medical therapy

Early nodularity in diffuse hyperplasia

Point of no-return?

Refractory to medical therapy Nodular hyperplasia

Indication for parathyroid intervention

FIGURE 2 Progression of parathyroid hyperplasia in chronic renal failure. Cells with more severe reduction of VDR receptor and calcium-sensing receptor within diffuse hyperplasia form small nodules leading to nodular hyperplasia.

strategy for the management of secondary hyperparathyroidism in chronic renal failure. This can be achieved by dietary phosphate restriction and the early use of phosphate binders and cautious use of active vitamin D sterols as described in Chapter 76. Recent data suggest that the direct effects of phosphate on parathyroid cells is especially important in the early phase of chronic renal failure [72].

A. Selective Percutaneous Ethanol Injection Therapy (PEIT) Although the introduction of new vitamin D analogs and calcimimetics may be promising in the treatment of secondary hyperparathyroidism, what can be done for patients with nodular hyperplasia? Do they have any choice other than surgical parathyroidectomy [73,74].

The ongoing discussion indicates that small glands composed of diffuse hyperplasia should still be responsive to calcitriol even in such patients. However, what about the patients that have already progressed to nodular hyperplasia? For patients with nodular hyperplasia, two new techniques have been established. The first technique is the selective percutaneous ethanol injection therapy (PEIT) [75–77]. The second technique is direct vitamin D injection therapy (see below). In PEIT, glands with nodular hyperplasia are “selectively” destroyed by ethanol injection under ultrasonographic guidance. Other glands with diffuse hyperplasia are then controlled by medical therapy (Fig. 3). Recently, this technique has become more powerful and safer than ever and has become widely used, especially in Japan. According to the guideline by Japanese Society for Parathyroid Intervention [78],


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before PEIT in patients with more than three glands of critical size. Recurrent nerve palsy due to leakage of ehanol is one of the most important complications of PEIT [77]. It is also suspected that adhesions in the tissue surrounding the parathyroids may be caused by leakage of ethanol. This could be a major problem if surgical parathyroidectomy will be needed in the future. With the routine use of color Doppler flow mapping by ultrasonography, the volume of ethanol used for PEIT has become minimal, leading to the lower rate of such complications [79]. Nevertheless, skilled operators and appropriate equipments are certainly required for successful and safe PEIT [78]. Ethanol vitamin D

B. Direct Vitamin D Injection Therapy

Diffuse hyperplasia Nodular hyperplasia

FIGURE 3 Parathyroid intervention under ultrasonographic guidance. Glands with nodular hyperplasia (hatched circle) are treated by intervention using percutaneous ethanol injection therapy (PEIT). Other glands with diffuse hyperplasia (open circle) are then controlled by medical therapy.

patients having one or two glands with nodular hyperplasia are the best candidates for PEIT. After the initial ethanol injection, PTH levels and recurrence should be monitored carefully. More importantly, after the successful destruction of nodular hyperplasia, residual glands with diffuse hyperplasia should be managed by appropriate medical therapy, including dietary phosphorus restriction. Thus, good compliance of the patients to medical therapy and regular check-ups after PEIT is essential. In the largest study by Kakuta et al., 46 patients were treated by selective PEIT on an outpatient basis followed by appropriate medical therapy. PTH levels in 80% of the patients remained within the target range at one year after initial treatment [79]. Long-term follow-up (three years) after PEIT has been also reported by this group [80]. Failure of PTH suppression despite successful ablation of glands with nodular hyperplasia suggests the existence of another gland containing nodular hyperplasia beyond the reach of ultrasonography [81]. If ectopic glands are recognized before the procedure is performed, initial surgical parathyroidectomy is indicated. Thus, it may be reasonable to search for ectopic glands

The second technique for treating nodular hyperplasia is direct calcitriol injection therapy under ultrasonographic guidance (PCIT) [82]. In this therapy, very high local concentration of 1,25(OH)2D3 or other vitamin D metabolites is achieved exclusively in injected parathyroid glands. As already reported, direct calcitriol injection therapy not only suppressed PTH secretion, but also restored the responsiveness to medical therapy. Such effects have been confirmed by other studies with different protocols [83–85]. Furthermore, direct injections of 22-oxacalcitriol have also been tried with favorable results [86]. In contrast to PEIT, the risk of recurrent nerve palsy is extremely low with PCIT. However, a recent report suggested that direct injection of 22-oxacalcitriol might evoke inflammation, resulting in adhesions of the surrounding tissue [87]. The use of vitamin D analogs is promising and such risks should be avoidable with improvements in technique. Future development may allow direct injection of new calcimimetics and even adenovirus-mediated gene transfer to treat parathyroid hyperplasia, as has been shown recently in animal models [88].

C. Regression of Parathyroid Hyperplasia: Is It Really Possible? The cell cycle of parathyroid cells is usually very slow, even in the hyperplastic glands [89]. Although prevention of parathyroid hyperplasia by several therapeutic modalities has been demonstrated in rat models of chronic kidney disease [90], suppression of PTH secretion may not lead to the complete normalization of parathyroid cell function including proliferation site in patients with secondary hyperparathyroidism [91,92].

1826 Thus, it is still an unsettled issue whether hyperplastic parathyroid glands do regress after proper medical therapy or after kidney transplantation. Regression of parathyroid hyperplasia has been reported in chronic dialysis patients treated by oral calcitriol pulse therapy [93–95], although controversial data have been also reported [96]. Such a regression was observed not only in cases with successful PTH suppression, but also in small glands even in cases without significant suppression of PTH [55,97]. Thus, in our opinion it is reasonable to conclude that glands with diffuse hyperplasia regress after effective medical therapy. In contrast, as discussed above, glands with nodular hyperplasia do not regress except for a few cases in which spontaneous apoplexy of the gland was suspected [98,99]. Due to the lack of established parathyroid cell lines for in vitro studies, mechanisms of regression have not yet been satisfactorily elucidated [100]. In order to achieve regression of hyperplastic glands, suppression of cell proliferation may not be sufficient. Negative cell balance by increased apoptosis may be needed. However, in rats, it has been very hard to demonstrate the apoptosis of parathyroid cells, which takes place in a limited number of cells during cell turnover [101–104]. Moreover, interpretation of apoptotic cells demonstrated in surgically removed parathyroid glands in dialysis patients has been controversial [100]. In a 1977 report by Henry et al. [105], reduction of parathyroid cell number was clearly demonstrated in three-month-old vitamin D–deficient chickens treated with vitamin D replacement. In contrast, 1,25(OH)2D3 treatment suppressed parathyroid cell proliferation, but did not reverse hyperplasia in experimental uremia, as demonstrated by Szabo et al. [106]. In recent animal studies by Lewin et al. [107,108], hyperparathyroidism induced by long-term uremia returned to normal following kidney transplantation. However, parathyroid hyperplasia was persistent. Such a suppression of PTH secretion with persistent hyperplasia has also been demonstrated in rat models of secondary hyperparathyroidism induced by high phosphorus diet, by switching to low phosphorus diet. In these animal models, reversal of reduced VDR or calcium-sensing receptor has not been confirmed at least in the short term. As discussed above, it has been suggested recently that regression of nodular hyperplasia may be induced by direct vitamin D injection therapy, originally performed with calcitriol [82]. By injecting directly into enlarged glands under ultrasonography, very high local concentration of vitamin D or analog can be achieved transiently. Shiizaki et al. recently reported that direct injection of 22-oxa-calcitriol solution into enlarged glands in patients leads to the regression of hyperplasia [84].


By repeated parathyroid biopsy before and after the therapy, they clearly demonstrated the induction of apoptosis of parathyroid cells in the injected glands. They also suggested that such a regression was associated with up-regulation of VDR and the calcium-sensing receptor. These data suggest that direct vitamin D injection therapy not only induces apoptosis of parathyroid cells, but also restores the responsiveness of residual parathyroid cells to medical therapy, leading to normalization of parathyroid hyperplasia. It may be possible that such specific effects of vitamin D on parathyroid cells may also be achieved by oral or intravenous preparations, if vitamin D analogs with these specific actions can be designed in the future. It is also of note that increased 25-hydroxyvitamin D3 1α-hydroxylase and reduced 25-hydroxyvitamin D3 24-hydroxylase expression have been reported in parathyroid tumors [109]. Thus, parathyroid is not only a target organ of vitamin D, but it also metabolizes vitamin D. Since parathyroid glands possess 1α-hydroxylase, it may become possible to develop new vitamin D metabolites that use this system to be activated only in parathyroid.

IV. FUTURE ROLES OF VITAMIN D ANALOGS IN CHRONIC RENAL FAILURE A. Design of Vitamin D Analogs with Specific Actions on Specific Tissues in Chronic Renal Failure The parathyroid glands are not the only target organ of vitamin D therapy in patients with chronic renal failure. The skin and the immune system are other examples; however, the role of vitamin D treatment on these systems, as well as other organs, has not been fully clarified yet [110,111]. A recent report suggests that paricalcitol treatment leads to better survival than calcitriol treatment in chronic dialysis patients [112]. It is still unclear whether such a difference is due to the less calcemic effect of paricalcitol or to its effects on other organ systems. Bone is a representative classic target organ of vitamin D. Thus, different effects of 22-oxacalcitriol versus calcitriol on bone turnover have been noted in animal models of chronic renal failure [113] (see Chapter 86). As recently suggested, the different effects of vitamin D analogs on bone, seen in in vitro and in vivo experiments, might be clues that help in the future elucidation of the mechanism for the differential actions of analogs in various tissues [114]. 22-oxacalcitriol was originally developed as an analog

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with potent activity on the differentiation of leukemic cell lines [115]. Such an activity has also been examined with paricalcitol [116]. Recently, it has been demonstrated that different vitamin D analogs utilize specific cofactors for target gene regulation [117]. Different cofactors bind to different genes, evoking different actions on the same cell. Thus, it is expected that design of vitamin D analogs with differential actions in specific organs will become possible [118]. For example, it may be possible to design analogs that specifically induce apoptosis of parathyroid cells as well as less calcemic analogs.

B. Possible Treatment of Chronic Kidney Disease by Vitamin D Analogs The kidney is not only the site of active vitamin D production, but also is its target organ. As intensively discussed in several previous chapters, vitamin D metabolites modulate the activity of the enzymes involved in vitamin D synthesis and degradation. Calcium-binding proteins [119] are also induced by vitamin D in the distal tubules of the kidney. VDRs have been identified in various parts of the kidney and no doubt are regulated by vitamin D metabolites [120]. Inhibition of renal cell proliferation by vitamin D was initially demonstrated in renal cell carcinoma lines [121]. It has also been shown that 1,25(OH)2D3 diminished 3H-thymidine incorporation, cell counts, and TGF-β secretion into the supernatant of cultured proximal tubular cell lines [122–124] and in cultured human mesangial cells [125]. Regulation of mesangial cell smooth muscle phenotype has also been suggested [126]. In vivo, 1,25(OH)2D3 reduced renal weight, protein content, DNA content, and the number of mitoses in the remnant kidney with compensatory hypertrophy after uninephrectomy [127]. On the contrary, 1,25(OH)2D3 may induce type IV collagen synthesis, possibly through up-regulation of TGF-β type II receptor [126] and upregulation of protein-1 [128]. It may be possible that vitamin D ameliorates glomerular injury seen in chronic kidney disease, although the effects may depend on the phase of renal injury. It has recently been shown that 1,25(OH)2D3 inhibited progressive glomerulosclerosis in subtotally nephrectomized rats [129] and reduced proteinuria, glomerular hyper-cellularity and inflammatory infiltration in anti-Thy-1.1 nephritis [130]. Similar suppressive effects have been also demonstrated with retinoic acids [131,132]. These studies suggest the possibility that vitamin D may alter the rate of progression of CKD. In contrast, there has been a concern that oral vitamin D treatment

in CKD patients may increase the risk of accelerating the progression of renal dysfunction by increasing urinary calcium excretion. In this respect, the lesscalcemic vitamin D analogs may be more suitable for this purpose [133]. 22-oxa-calcitriol is one such less-calcemic vitamin D compound used for the treatment of severe secondary hyperparathyroidism in chronic dialysis patients [134], as extensively reviewed in Chapter 86. It has recently been shown that 22-oxa-calcitriol also effectively ameliorated glomerular sclerosis in two rat models of chronic kidney disease without affecting calcium and phosphorus levels [135,136]. Although further studies are needed, a vitamin D analog with such properties may be a promising agent for the treatment of chronic kidney disease in the near future.

Acknowledgements This work was partly supported by grants from Renal Osteodystrophy Foundation and from Renal Anemia Foundation, Japan. Authors are grateful to Ms Tomoko Nii-Kono for her secretarial assistance and art work.

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43. Denda M, Finch J, Brown AJ, Nishii Y, Kubodera N, Slatopolsky E 1996 1,25-dihydroxyvitamin D3 and 22-oxacalcitriol prevent the decrease in vitamin D receptor content in the parathyroid glands of uremic rats. Kidney Int 50:34–39. 44. Reichel H, Szabo A, Uhl J, Pesian S, Schmultz A, SchmidtGayk H, Ritz E 1993 Intermittent versus continuous administration of 1,25-dihydroxyvitamin D3 in experimental renal hyperparathyroidism. Kidney Int 44:1259–1265. 45. Block GA, Hulbert-Shearon TE, Levin NW, Port FK 1998 Association of serum phosphorus and calcium × phosphate product with mortality risk in chronic hemodialysis patients: a national study. Am J Kidney Dis 31:607–617. 46. Martin KJ, Gonzalez EA, Gellens M, Hamm LL, Abboud H, Lindberg J 1998 19-Nor-1-alpha-25-dihydroxyvitamin D2 (Paricalcitol) safely and effectively reduces the levels of intact parathyroid hormone in patients on hemodialysis. J Am Soc Nephrol 9:1427–1432. 47. Brown AJ, Ritter CR, Finch JL, Morrissey J, Martin KJ, Murayama E, Nishii Y, Slatopolsky E 1989 The noncalcemic analog of vitamin D, 22-oxacalcitriol, suppresses parathyroid hormone synthesis and secretion. J Clin Invest 84:728–732. 48. Kazama JJ, Gejyo F, Shigematsu T, Fukumoto S, Yamashita T, Nakanishi S, Fukagawa M 2003 Circulating fibroblast growth factor-23 (FGF-23) levels predict the development of secondary hyperparathyroidism in long-term dialysis patients (abstract). J Am Soc Nephrol, 14:209A. 49. Drüeke TB 2000 Cell biology of parathyroid gland hyperplasia in chronic renal failure. J Am Soc Nephrol 11: 1141–1152. 50. Malmaeus J, Glimelius L, Johansson G, Akerstrom G, Ljunghall S 1984 Parathyroid pathology in hyperparathyroidism secondary to chronic renal failure. Scand J Urol Nephrol 18:157–166. 51. McCarron DA, Muther RS, Lenfesty B, Bennett WM 1982 Parathyroid function in persistent hyperparathyroidism: Relationship to gland size. Kidney Int 22:662–670. 52. Wallfelt CH, Larsson R, Gylfe E, Ljunghall S, Rasted J, Akerstrom G 1988 Secretory disturbance in hyperplastic parathyroid nodules of uremic hyperparathyroidism: Implication for parathyroid autoplantation. World J Surg 12:431–438. 53. Felsenfeld AJ, Llach F 1993 Parathyroid function in chronic renal failure. Kidney Int 43:771–789. 54. Delmez JA, Tindra C, Grooms P, Dusso A, Windus DW, Slatopolsky E 1989 Parathyroid hormone suppression by intravenous 1,25(OH)2 vitamin D. A role for increased sensitivity to calcium. J Clin Invest 83:1349–1355. 55. Fukagawa M, Kitaoka M, Yi H, Fukuda N, Matsumoto T, Ogata E, Kurokawa K 1994 Serial evaluation of parathyroid size by ultrasonography is another useful marker for the long-term prognosis of calcitriol pulse therapy in chronic dialysis patients. Nephron 68:221–228. 56. Tominaga Y, Sato K, Tanaka Y, Numano M, Uchida K, Takagi H 1995 Histopathology and pathophysiology of secondary hyperparathyroidism due to chronic renal failure. Clin Nephrol 44:S42–S47. 57. Fukuda N, Tanaka H, Tominaga Y, Fukagawa M, Kurokawa K, Seino Y 1993 Decreased 1,25-dihydroxyvitamin D3 receptor density is associated with a more severe form of parathyroid hyperplasia in chronic uremic patients. J Clin Invest 92:1436–1443. 58. Åkerstrom G, Malmaeus J, Grimelius L, Ljunghall S, Bergstrom R 1984 Histological changes in parathyroid

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1830 75. Kitaoka M, Fukagawa M, Ogata E, Kurokawa K 1994 Reduction of functioning parathyroid cell mass by ethanol injection in chronic dialysis patients. Kidney Int 46:1110–1117. 76. Giangrande A, Castiglioni A, Sorbiati L, Allaria P 1992 Ultrasound guided percutaneous fine needle ethanol injection into parathyroid glands in secondary hyperparathyroidism. Nephrol Dial Transplant 7:412–421. 77. Kitaoka M 2003 Ultrasonographic diagnosis of parathyroid glands and percutaneous ethanol injection therapy. Nephrol Dial Transplant 18(Suppl 3):iii27–iii30. 78. Fukagawa M, Kitaoka M, Tominaga Y, Akizawa T, Kurokawa K, for Japanese Society for Parathyroid Intervention 2003 Guidelines for percutaneous ethanol injection therapy (PEIT) of the parathyroid glands in chronic dialysis patients. Nephrol Dial Transplant 18(Suppl 3):31–33. 79. Kakuta T, Fukagawa M, Fujisaki T, Hida M, Suzuki H, Sakai H, Kurokawa K, Saito A 1999 Prognosis of parathyroid function after successful percutaneous ethanol injection therapy (PEIT) guided by color Doppler flow mapping in chronic dialysis patients. Am J Kidney Dis 33:1091–1099. 80. Tanaka R, Kakuta T, Fujisaki T, Tanaka S, Sakai H, Kurokawa K, Saito A 2003 Long-term (three years) prognosis of parathyroid function in chronic dialysis patients after percutaneous ethanol injection therapy guided by color Doppler ultrasonography. Nephrol Dial Transplant 18: (Suppl 3):iii58–iii61. 81. Fukagawa M, Nakanishi S 2003 Role of parathyroid intervention in the management of secondary hyperparathyroidism in chronic renal failure. Nephrol Dial Transplant 18 (Suppl 3):23–26. 82. Kitaoka M, Fukagawa M, Kurokawa K 1995 Direct injection of calcitriol into parathyroid hyperplasia in chronic dialysis patients with severe parathyroid hyperfunction. Nephrology 1:563–568. 83. Kitaoka M, Onoda N, Kitamura H, Koiwa M, Tanaka M, Fukagawa M 2003 Percutaneous calcitriol injection therapy (PCIT) for secondary hyperparathyroidism: multicentre trial. Nephrol Dial Transplant 18(Suppl 3):iii38–iii41. 84. Shiizaki K, Negi S, Mizobuchi M, Hatamura I, Narukawa N, Sakaguchi T, Kitabata Y, Sumikado S, Akizawa T 2003 Effect of percutaneous calcitriol injection therapy on secondary hyperparathyroidism in uremic patients. Nephrol Dial Transplant 18(Suppl 3):iii42–iii46. 85. Nakanishi S, Yano S, Nomura R, Tsukamoto T, Shimizu Y, Shin J, Fukagawa M 2003 Efficacy of direct injection of calcitriol into the parathyroid glands in uraemic patients with moderate to severe secondary hyperparathyroidism. Nephrol Dial Transplant 18(Suppl 3):iii47–iii49. 86. Shiizaki K, Hatamura I, Negi S, Narukawa N, Mizobuchi M, Sakaguchi T, Ooshima A, Akizawa T 2003 Percutaneous maxacalcitol injection therapy regresses hyperplasia of parathyroid and induces apoptosis in uremia. Kidney Int 64:992–1003. 87. Yamamoto H, Katoh N, Takeyama H, Ikeda M, Yokoyama K, Shigematsu T, Kawaguchi Y, Hosoya T 2003 Surgical verification of percutaneous maxacalcitriol injection therapy on enlarged parathyroid glands in chronic dialysis patients. Nephrol Dial Transplant 18(Suppl 3):iii50–iii52. 88. Iwasaki Y, Kakuta T, Haruguchi H, Fukuda N, Kurokawa K, Fukagawa M 2003 Adenovirus-mediated functional gene transfer into parathyroid cells in vivo and in vitro. Nephrol Dial Transplant 18(Suppl 3):iii18–iii22. 89. Parfitt AM 1982 Hypercalcemic hyperparathyroidism following renal transplantation: Differential diagnosis, management, and implications for cell population control in the parathyroid gland. Mineral Electrolyte Metab 8:92–112.


90. Wada M, Nagano N, Furuya Y, Chin J, Nemeth EF, Fox J 2000 Calcimimetic NPS R-568 prevents parathyroid hyperplasia in rats with severe secondary hyperparathyroidism. Kidney Int 57:50–58. 91. Takahashi F, Denda M, Finch JL, Brown AJ, Slatopolsky E 2002 Hyperplasia of the parathyroid gland without secondary hyperparathyroidism. Kidney Int 6:1332–1338. 92. Ritter CS, Martin DR, Lu Y, Slatopolsky E, Brown AJ 2002 Reversal of secondary hyperparathyroidism by phosphate restriction restores parathyroid calcium-sensing receptor expression and function. J Bone Miner Res 17: 2206–2213. 93. Fukagawa M, Okazaki R, Takano K, Kaname S, Ogata E, Kitaoka M, Harada S, Sekine N, Matsumoto T, Kurokawa K 1990 Regression of parathyroid hyperplasia by calcitriolpulse therapy in patients on long-term dialysis. N Engl J Med 323:421–422. 94. Hyodo T, Koumi T, Ueda M, Miyagawa I, Kodani K, Doi S, Ishibashi M, Takemoto M 1991 Can oral 1,25(OH)2D3 therapy reduce parathyroid hyperplasia? Nephron 59:171–172. 95. Cannella G, Bonucci E, Rolla D, Ballanti P, Moriero E, De Grandi R, Augeri C, Claudiani F, Di Maio G 1994 Evidence of healing of secondary hyperparathyroidism in chronically hemodialyzed uremic patients treated with long-term intravenous calcitriol. Kidney Int 46:1124–1132. 96. Quarles LD, Yohay DA, Carroll BA, Spritzer CE, Minda SA, Bartholomay D, Lobaugh BA 1994 Prospective trial of pulse oral versus intravenous calcitriol treatment of hyperparathyroidism in ESRD. Kidney Int 45:1710–1721. 97. Fukagawa M, Fukuda N, Yi H, Kitaoka M, Kurokawa K 1996 Derangement of parathyroid function in renal failure: Biological and clinical aspects. J Nephrol 9:219–224. 98. Nylen E, Shah A, Hall J 1996 Spontaneous remission of primary hyperparathyroidism from parathyroid apoplexy. J Clin Endocrinol Metab 81:1326–1328. 99. Chaffanjon PC, Chavanis N, Chabre O, Brichon PY 2003 Extracapsular hematoma of the parathyroid glands. World J Surg 27:14–17. 100. Drueke TB, Zhang P, Gogusev J 1997 Apoptosis: background and possible role in secondary hyperparathyroidism. Nephrol Dial Transplant 12:2228–2233. 101. Naveh-Many T, Rahamimov R, Livni N, Silver J 1995 Parathyroid cell proliferation in normal and chronic renal failure rats. The effects of calcium, phosphate, and vitamin D. J Clin Invest 96:1786–1793. 102. Zhang P, Duchambon P, Gogusev J, Nabarra B, Sarfati E, Bourdeau A, Drueke TB 2000 Apoptosis in parathyroid hyperplasia of patients with primary or secondary uremic hyperparathyroidism. Kidney Int 57:437–445. 103. Canalejo A, Almaden Y, Torregrosa V, Gomez-Villamandos JC, Ramos B, Campistol JM, Felsenfeld AJ, Rodriguez M 2000 The in vitro effect of calcitriol on parathyroid cell proliferation and apoptosis. J Am Soc Nephrol 11:1865–1872. 104. Jara A, Gonzalez S, Felsenfeld AJ, Chacon C, Valdivieso A, Jalil R, Chuaqui B 2001 Failure of high doses of calcitriol and hypercalcemia to induce apoptosis in hyperplastic parathyroid glands of azotaemic rats. Nephrol Dial Transplant 16:506–512. 105. Henry HL, Taylor AN, Norman AW 1977 Response of chick parathyroid glands to the vitamin D metabolites, 1,25-dihydroxycholecalciferol and 24,25-dihydroxycholecalciferol. J Nutr 107:1918–1926. 106. Szabo A, Merke J, Beier E, Mall G, Ritz E 1989 1,25(OH)2 vitamin D3 inhibits parathyroid cell proliferation in experimental uremia. Kidney Int 35:1049–1056.


107. Lewin E, Wang W, Olgaard K 1997 Reversibility of experimental secondary hyperparathyroidism. Kidney Int 52:1232–1241. 108. Lewin E, Garfia B, Recio FL, Rodriguez M, Olgaard K 2003 Persistent down-regulation of calcium-sensing receptor mRNA in rat parathyroids when severe secondary hyperparathyroidism is reversed by an isogenic kidney transplantation. J Am Soc Nephrol 13:2110–2116. 109. Correa P, Segersten U, Hellman P, Akerstrom G, Westin G 2002 Increased 25-hydroxyvitamin D3 1alpha-hydroxylase and reduced 25-hydroxyvitamin D3 24-hydroxylase expression in parathyroid tumors—new perspects for treatment of hyperparathyroidism with vitamin D. J Clin Endocrinol Metab 87:5826–5829. 110. Mathieu C, Van Etten E, Gysemans C, Decallonne B, Kato S, Laureys J, Depovere J, Valckx D, Verstuyf A, Bouillon R 2001 In vitro and in vivo analysis of the immune system of vitamin D receptor knockout mice. J Bone Miner Res 16:2057–2065. 111. Kira M, Kobayashi T, Yoshikawa K 2003 Vitamin D and the skin. J Dermatol 30:429–437. 112. Teng M, Wolf M, Lowrie E, Ofsthun N, Lazarus JM, Thadhani R 2003 Survival of patients undergoing hemodialysis with paricalcitol or calcitriol therapy. N Engl J Med 349: 446–456. 113. Monier-Faugere MC, Geng Z, Friedler RM, Qi Q, Kubodera N, Slatopolsky E, Malluche HH 1999 22-oxacalcitriol suppresses secondary hyperparathyroidism without inducing low bone turnover in dogs with renal failure. Kidney Int 55:821–832. 114. Shibata T, Shira-Ishi A, Sato T, Masaki T, Masuda A, Hishiya A, Ishikura N, Higashi S, Uchida Y, Saito MO, Ito M, Ogata E, Watanabe K, Ikeda K 2002 Vitamin D hormone inhibits osteoclastogenesis in vivo by decreasing the pool of osteoclast precursors in bone marrow. J Bone Miner Res 17:622–629. 115. Abe J, Morikawa M, Miyamoto K, Kaiho S, Fukushima M, Miyaura C, Abe E, Nishii Y 1987 Synthetic analogs of vitamin D3 with an oxygen atom in the side chain skeleton. FEBS Lett 226:58–62. 116. Molnar I, Kute T, Willingham MC, Powell BL, Dodge WH, Schwartz GG 2003 19-nor-1alpha,25-dihydroxyvitamin D2 (paricalcitol): effects on clonal proliferation, differentiation, and apoptosis in human leukemic cell lines. J Cancer Res Clin Oncol 129:35–42. 117. Takeyama K, Masuhiro Y, Fuse H, Endoh H, Murayama A, Kitanaka S, Suzawa M, Yanagisawa J, Kato S 1999 Selective interaction of vitamin D receptor with transcriptional coactivators by a vitamin D analog. Mol Cell Biol 19:1049–1055. 118. Fujishima T, Kittaka A, Yamaoka K, Takeyama K, Kato S, Takayama H 2003 Synthesis of 2,2-dimethyl-1,25-dihydroxyvitamin D3: A-ring structural motif that modulates interactions of vitamin D receptor with transcriptional coactivators. Org Biomol Chem 1:1863–1869. 119. Christakos S, Barletta F, Huening M, Dhawan P, Liu Y, Porta A, Peng X 2003 Vitamin D target proteins: function and regulation. J Cell Biochem 88:238–244. 120. Iida K, Shinki T, Yamaguchi A, DeLuca HF, Kurokawa K, Suda T 1995 A possible role of vitamin D receptors in regulating vitamin D activation in the kidney. Proc Natl Acad Sci USA 92:6112–6116. 121. Nagakura K, Abe E, Suda T, Hayakawa M, Nakamura H, Tazaki H 1986 Inhibitory effect of 1alpha,25-dihydroxyvitamin D3 on the growth of the renal carcinoma cell line. Kidney Int 29:834–840.

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122. Weih M, Orth S, Weinreich T, Reichel H, Ritz E 1994 Inhibition of growth by calcitriol in a proximal tubular cell line (OK). Nephrol Dial Transplant 9:1390–1394. 123. Weinreich T, Landolt M, Booy C, Wuthrich R, Binswanger U 1999 1,25-dihydroxyvitamin D3 stimulates transforming growth factor-beta1 synthesis by mouse renal proximal tubular cells. Kidney Blood Press Res 22:99–105. 124. Weinreich T, Muller A, Wuthrich RP, Booy C, Binswanger U 1996 1,25-dihydroxyvitamin D3 and the synthetic vitamin D analog, KH 1060, modulate the growth of mouse proximal tubular cells. Kidney Blood Press Res 19:325–331. 125. Weinreich T, Merke J, Schonermark M, Reichel H, Diebold M, Hansch GM, Ritz E 1991 Actions of 1,25dihydroxyvitamin D3 on human mesangial cells. Am J Kidney Dis 18:359–366. 126. Abe H, Iehara N, Utsunomiya K, Kita T, Doi T 1999 A vitamin D analog regulates mesangial cell smooth muscle phenotypes in a transforming growth factor beta type II receptor-mediated manner. J Biol Chem 274: 20874–20878. 127. Matthias S, Busch R, Merke J, Mall G, Thomasset M, Ritz E 1991 Effects of 1,25(OH)2D3 on compensatory renal growth in the growing rat. Kidney Int 40:212–218. 128. Kobayashi T, Uehara S, Ikeda T, Itadani H, Kotani H 2003 Vitamin D3 up-regulated protein-1 regulates collagen expression in mesangial cells. Kidney Int 64:1632–1642. 129. Schwarz U, Amann K, Orth SR, Simonaviciene A, Wessels S, Ritz E 1998 Effect of 1,25(OH)2 vitamin D3 on glomerulosclerosis in subtotally nephrectomized rats. Kidney Int 53:1696–1705. 130. Panichi V, Migliori M, Taccola D, Filippi C, De Nisco L, Giovannini L, Palla R, Tetta C, Camussi G 2001 Effects of 1,25(OH)2D3 in experimental mesangial proliferative nephritis in rats. Kidney Int 60:87–95. 131. Datta PK, Lianos EA 1999 Retinoic acids inhibit inducible nitric oxide synthase expression in mesangial cells. Kidney Int 56:486–493. 132. Xu Q, Konta T, Kitamura M 2002 Retinoic acid regulation of mesangial cell apoptosis. Exp Nephrol 10:171–175. 133. Hirata M, Katsumata K, Endo K, Fukushima N, Ohkawa H, Fukagawa M 2003 In subtotally nephrectomized rats 22-oxacalcitriol suppresses parathyroid hormone with less risk of cardiovascular calcification or deterioration of residual renal function than 1,25(OH)2 vitamin D3. Nephrol Dial Trasplant 18:1770–1776. 134. Akizawa T, Suzuki M, Akiba T, Nishizawa Y, Ohashi Y, Ogata E, Slatopolsky E, Kurokawa K 2002 Long-term effect of 1,25-dihydroxy-22-oxavitamin D3 on secondary hyperparathyroidism in hemodialysis patients. One-year administration study. Nephrol Dial Transplant 17(Suppl) 10:28–36. 135. Makibayashi K, Tatematsu M, Hirata M, Fukushima N, Kusano K, Ohashi S, Abe H, Kuze K, Fukatsu A, Kita T, Doi T 2001 A vitamin D analog ameliorates glomerular injury on rat glomerulonephritis. Am J Pathol 158:1733–1741. 136. Hirata M, Makibayashi K, Katsumata K, Kusano K, Watanabe T, Fukushima N, Doi T 2002 22-oxacalcitriol prevents progressive glomerulosclerosis without adversely affecting calcium and phosphorus metabolism in subtotally nephrectomized rats. Nephrol Dial Transplant 17: 2132–2137.

CHAPTER 104

Inhibition of Benign Prostatic Hyperplasia by Vitamin D Receptor Ligands

I. II. III. IV.

MARIO MAGGI CLARA CRESCIOLI

Andrology Unit, Department of Clinical Physiopathology, University of Florence, Florence, Italy

LUCIANO ADORINI

BioXell, Milan, Italy

Endocrinology Unit, Department of Clinical Physiopathology, University of Florence, Florence, Italy

Introduction Pathogenesis of BPH Effects of Androgens and Growth Factors on Human BPH Cells Vitamin D Receptor Expression in Prostate Cells

I. INTRODUCTION The prostate is a gland surrounding the male urethra below the neck of the bladder and producing the prostatic fluid, a secretion which contributes 30% to the total ejaculate. The prostatic fluid is rich in fibrinolytic enzymes, such as prostatic-specific antigen (PSA), acid phosphatase, citric acid, and zinc. In humans, the prostate gland is composed of 40 to 50 ducts distributed essentially in three distinct zones: peripheral, central, and transitional or periurethral. While cell transformation in the peripheral zone gives rise to prostate cancer, cell growth in the periurethral zone leads to the most common age-related disease of the male: benign prostatic hyperplasia (BPH). The prostate weight is only a few grams at birth, and it increases during puberty, reaching approximately 20 g in the young adult. In contrast to the pubertal growth phase, which involves the entire gland, during the fifth decade of life, in the majority of men, there is a second growth phase selectively involving the periurethral zone leading to BPH [1]. The prevalence of BPH increases with age so that by age 80, about 90% of men have histological evidence of BPH [1]. In a subset of elderly men (27–35%), BPH can cause lower urinary tract symptoms (LUTS), which may require medical or surgical treatment [2] due to the compression by the enlarged prostate of the prostatic urethra, which decreases bladder outflow. In the earliest stages, this obstruction is compensated by an increased activity of the bladder detrusor muscular system but eventually complete voiding of the bladder is prevented, due to the slackening of the neck musculature. Urinary obstruction VITAMIN D, 2ND EDITION FELDMAN, PIKE, AND GLORIEUX

V. Antiproliferative Effects of BXL-353 on Human BPH Cells VI. Inhibition of in Vivo Prostate Growth by BXL-353 VII. Conclusions References

and even renal insufficiency might follow. This may lead to emergency surgery for acute urinary retention with an increased risk of morbidity and even death when compared to elective surgery [3].

II. PATHOGENESIS OF BPH Periurethral prostate overgrowth involves both epithelial and stromal components, including both fibroblasts and smooth muscle cells, in various combinations. One of the earliest events in BPH development is the reduction of the epithelium/stroma ratio, most probably due to an imbalance between growth and death programs. Indeed, in the hyperplastic prostate, the epithelium regularly undergoes apoptosis, whereas stromal cells escape it [4], with a consequent increase in the stromal volume [5]. In addition, stromal growth factors (GFs) induce epithelial overgrowth and glandular hyperplasia [6,7]. All these events are clearly androgen-dependent, as shown by the observation that BPH does not develop in hypogonadal men, and that either surgical or pharmacological castration results in a decrease gland size [8–10]. However, BPH develops mainly in older men, when circulating testosterone, and in particular free testosterone, is progressively decreasing. Therefore, it is possible that sensitivity to androgens, rather than circulating androgen levels, are involved in BPH pathogenesis. A higher transcriptional activity of the androgen receptor (AR) due to a decreased number of CAG repeats in exon 1 has been reported in the majority [11–13], although not in all studies [14,15]. Interestingly, in hypogonadal Copyright © 2005, Elsevier, Inc. All rights reserved.

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MARIO MAGGI, CLARA CRESCIOLI, AND LUCIANO ADORINI

patients the effect of androgen substitution on prostate growth was inversely related to the extent of CAG residues [16]. In addition, a recent study has indicated a decreased expression of the AR co-repressor DAX-1 in BPH [17]. These studies support the view that AR activity is up-regulated in the prostate of BPH patients. Therefore, blocking AR activity represents a promising approach in the treatment of BPH. This could be achieved by reducing androgen levels, for example, blocking their formation with GnRH analogs or by antagonizing androgen activity at the receptor level using AR antagonists. Although both these strategies might be indeed effective, in clinical practice they are unacceptable because of the major side effects caused by complete androgen ablation in otherwise healthy individuals. Wilson 1972 [18], first hypothesized that the main androgen inducing prostate hyperplasia was not testosterone (T), but its highly biologically active metabolite dihydrotestosterone (DHT), which is formed locally by two 5α-reducing iso-enzymes (5α-reductase type 1 and 2, the latter being predominant, see [19] for review). Interestingly, intra-prostatic DHT content is not decreased as a function of age [20–22]. According to Wilson’s original hypothesis, blocking DHT formation with a type 2 selective (finasteride) or with a dual (dutasteride) inhibitor of 5α-reductase isoforms is, indeed, an effective treatment for BPH [23,24]. However, prostate size reduction obtained with this strategy is relatively limited (about 25%). Also some men experience sexual side effects related to partial androgen deficiency (decreased libido and impotence) that are not well tolerated, in particular in the ageing male [3,25]. It is possible that the limited clinical response to 5α-reductase inhibitors is due to a compensatory increase in intra-prostatic growth factor (GF) receptors, which follows androgen deprivation [26,27]. Therefore, an alternative strategy to reduce age-related prostate overgrowth is to decrease the activity of androgen-induced prostatic GFs, which are considered to mediate, at least partially, the proliferative activity of sex steroids in the gland [7,28,29]. It is interesting to note that the prostate gland is one of the few androgen targets retaining a proliferative responsiveness to androgens in adulthood. Therefore disrupting androgeninduced, intra-prostatic GF signaling is an attractive option to obtain a selective, and sexual side-effect free, therapy for BPH.

mutual interactions between the two compartments (reviewed in [30]). However, as discussed above, stromal rather than epithelial cells are thought to be primarily involved in the pathogenesis of BPH. As shown in Fig. 1, BPH-derived stromal cells (BPH cells) express the AR gene and protein, with a high affinity for the ligand (Kd = 72 ± 34 pM), as well as both isoforms of 5α-reductase [31]. In addition, they respond to androgens with an increased growth (EC50 = 380 ± 200 pM, [31]). An increase in BPH cell proliferation was also obtained with addition of specific GFs, such as epidermal growth factor (EGF [32]), keratinocyte-GF (KGF [32,33]), and insulin-like growth factor-I (IGF-I [34]). Data in Fig. 2 show the maximal stimulatory activity of KGF (10 ng/ml), Des (1–3) IGF-I (an IGF-I analog which does not bind to binding proteins, 10 ng/ml) and T (10 nM) on BPH cell proliferation. KGF- and Des [1–3] IGF-I-induced proliferation was completely blocked only by specific antibodies, but not by unrelated antibodies or immunoglobulins (Fig. 2). Conversely, testosterone-induced cell growth was completely abolished not only by an AR antagonist (cyproterone acetate) or by a type 2 5α-reductase inhibitor (finasteride), but also by antibodies against the receptors for KGF (KGFR) and IGF-I (IGFR1) (Fig. 2). This indicates that T-induced proliferative activity in BPH cells is at least partially mediated by KGFR and IGFR1. This finding is consistent with data from organ cultures of neonatal rat ventral prostates, in which exogenous administration of KGF completely replaces the requirement of T for prostate growth and branching morphogenesis [35]. In addition, KGF has been also shown to replace androgen in eliciting growth and differentiation of seminal vesicles [36]. Hence, KGF, the predominant fibroblast GF (FGF) in human prostate [37], is considered one of the main prostatic andromedins, that is mediators of androgen-induced growth [38]. Also, the IGF system has been implicated in the pathogenesis of BPH. IGFR1 and IGF-II expression were higher in the periurethral zone of the human prostate than in other zones, and IGF-II levels were strictly correlated with the intra-prostatic androgen level [39]. Patients with the highest circulating levels of IGF-I have an elevated risk of BPH [40] and transgenic mice overexpressing IGF-I protein in the prostate show sign of hyperplasia in the ventral lobe, the most androgen-dependent zone [41].

III. EFFECTS OF ANDROGENS AND GROWTH FACTORS ON HUMAN BPH CELLS

IV. VITAMIN D RECEPTOR EXPRESSION IN PROSTATE CELLS

In the human prostate, the AR is expressed in both the epithelial and the stromal compartments and regulates

The aforementioned experimental and clinical studies indicate that an ideal medical treatment for BPH might be an agent able to disrupt the intra-prostatic

NC

hfPSMC

CHO 1827

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FIGURE 1 Expression of AR, 5α-reductase and VDR in human BPH cells. Panel A: homologous competition curve for [3H]R1881 binding. R1881 binds with high affinity (Kd = 72 ± 34 pM) and low capacity (Bmax = 2.64 ± 0.5 fM) to a single class of sites. Ordinate: B/T, Bound to total ratio for [3H]R1881; Abscissa: Total concentration (molar) of labeled and unlabeled R1881. Inset: RT-PCR detection of the AR gene. Products are derived from total RNA using specific primers for AR (upper panel) and GAPDH (lower panel). Panel B: RT-PCR amplimers from total RNA of BPH cells, prostate tissue, CHO 1827 (transfected with 5α-reductase 1, 5α-R1, gene) or CHO 1829 (transfected with 5α-reductase 2, 5α-R2 gene), CHO cells, human fetal penile smooth muscle cells (hfPSMC), using specific primers for 5α-R1 (upper panel), 5α-R2 (second panel), VDR (third panel) and GAPDH (bottom panel). CHO 1827 or 1829, hfPSMC and human prostate were used as positive controls for 5α-R1, 5α-R2 and AR. GAPDH mRNA amplification was performed to verify the integrity and loading of the extracted total RNA. MW, molecular weight markers; NC, negative control. The blots are representative of three separate experiments. *

160 *

* *

*

*

*

* 120

*

100

Des IGF-I (10 ng/ml)

IgG

Anti-IGFR1

Anti-KGFR

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Anti-KGFR

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60 40

*

*

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Cell number (% of control)

140

T (10 nM)

FIGURE 2 Antiproliferative effect of BXL-353 on BPH cell growth induced by GFs or testosterone (T). Incubation for 48 h with KGF (10 ng/ml), Des [1–3] IGF-I (10 ng/ml) or T (10 nM) significantly induced BPH cell proliferation. Anti-androgens such as the 5α-R2 inhibitor, finasteride (F, 1 nM), or the AR antagonist cyproterone acetate (Cyp, 100 nM) completely reverted T-induced stimulation of BPH cell growth, but they did not exert any effect on basal cell growth. Specific antibodies against KGFR (Anti-KGFR, 1 µg/ml) and IGFR1 (AntiIGFR1, 1 µg/ml) blocked cell growth stimulated by their cognate GFs. T-induced cell proliferation was blunted by both types of anti-GF receptor antibodies. Immunoglobulin controls (IgG 1 µg/ml) failed to block either GFor T-stimulated proliferation. BXL-353 (10 nM) was able to block BPH cell growth both in basal condition and in the presence of T or GFs. Results are expressed as percent increase (mean ± SEM) over their relative controls in 4 different experiments performed in quadruplicate (* P< 0.01 vs control; ° P < 0.01 vs GF- or T- treated cells by one-way ANOVA and paired or unpaired Student’s t tests). The data are derived from Crescioli et al., 2003.

1836 cross-talk between AR and GFs but devoid of antiandrogenic properties. Calcitriol analogs might comply with such criteria. The strict inter-relationships between vitamin D and prostate have been extensively described. Vitamin D deficiency has been proposed to be a risk factor for prostate cancer [42,43], because prostate cancer mortality in the USA increases as the availability of ultraviolet light exposure, and therefore of vitamin D formation, decreases [44] (see Chapter 90). Polymorphisms in the VDR gene have also been associated with increased risk of prostate cancer in some studies [45–47] (see Chapter 68). Malignant prostate cells express the VDR, and treatments with calcitriol, or lesshypercalcemic analogs, can inhibit prostate cancer proliferation and invasiveness (see Chapter 94 and ref. [48,49] for review). Interestingly, also epithelial and stromal cells of both human [50,51] (see also Figs. 1 and 2) and rat [51] normal prostate cells express the VDR, and addition of 1,25 (OH)2D3 inhibits cell growth [50].

V. ANTIPROLIFERATIVE EFFECTS OF BXL-353 ON HUMAN BPH CELLS We have extensively studied the antiproliferative effects of VDR ligands, and in particular of 1,25-dihydroxy-16-ene-23-yne D3 (BXL-353 or analog V), a compound 30-fold less calcemic than calcitriol, on human stromal prostate cells (BPH cells). BPH cells were obtained from prostate tissues derived from patients, who underwent suprapubic adenomectomy for BPH and did not receive any pharmacological treatment in the three months preceding surgery [33]. BPH cells showed positive staining for smooth-muscle actin, vimentin, and desmin, suggesting fibromuscular morphological features. Conversely, they were negative for epithelial and endothelial markers such as cytokeratin and factor VIII [33]. As shown in Fig. 2 BXL-353 completely inhibited GF- or T-induced BPH cell proliferation and also decreased the growth of unstimulated cells [31,33,34]. To better understand the antiproliferative effect of BXL-353, we have studied its effects on the cell cycle distribution of partially synchronized BPH cells after a 24 h culture with medium or KGF (10 ng/ml). As shown in Fig. 3, after serum starvation, more than 75% of the cells were in G0/G1-early S phase, as indicated by fluorescence emission of propidium iodide-stained nuclei. Treatment with KGF allowed the cells to progress through the cell cycle with a statistically significant decrease in the proportion of cells accumulated in G0/G1-early S and an increase in cells traversing the G2/M phase. The simultaneous addition of BXL-353 completely antagonized the KGF-induced effects on cell-cycle progression.


In BPH cells GFs and steroids not only stimulated DNA synthesis and cell proliferation but also prolonged cell survival, via induction of the anti-apoptotic protein Bcl-2 [31,33,34] (see also Fig. 4). Members of the Bcl-2 family are essential mediators of cell survival and apoptosis, and include both anti- and pro-apoptotic intracellular proteins residing at the mitochondrial outer membrane [52–55]. Their classification is based on the presence or absence of Bcl-2 homology (BH) domains: BH1, BH2, BH3, and BH4 [56]. In particular, Bcl-2 and Bcl-XL members, both containing all four BH domains, inhibit apoptosis and promote cell survival [57]. Bcl-2 activity, derived by integrating signals from survival and death stimuli, seems to be regulated by several different mechanisms, like homo- and heterodimerization with other family members, or posttranslational modifications such as phosphorylation and proteolysis [52,58]. BXL-353 not only dramatically reduced GF- or T-induced Bcl-2 overexpression and survival, but also in the presence of these anti-apoptotic factors was able to stimulate a sustained death program (Fig. 4). Hence, in BPH cells, BXL-353 induced a decrease in the progression through the cell cycle and an increase in the rate of programmed cell death. Similar results were observed with calcitriol in breast cancer cells [59], in the androgen-dependent prostate cancer cell line LNCaP [60,61] as well as in metastatic Dunning rat prostate carcinoma [62]. Interestingly, in LNCaP cells, overexpression of Bcl-2 completely blocked calcitriol-induced apoptosis but only partially affected cell cycle arrest [61], indicating that partially independent pathways mediate the effects of calcitriol on cell proliferation and cell death. The inhibitory effect of BXL-353 on prostate growth and survival is at least partially explained by the inhibition of GF-induced receptor activation. We have found that a rapid incubation of both benign [33] and malignant [63] prostate cells with BXL-353 dramatically reduces agonist-induced KGF-R auto-phosphorylation, one of the earliest event of KGF signaling. Because this effect was rapid, induced in a few minutes, and accompanied by an increase in intracellular calcium concentrations [33], we speculated the involvement of a nontranscriptional mechanism which, in agreement with recent results in chick myoblast [64], might involve the same VDR translocated from the nucleus to the microsomal fraction. Other studies have shown that rapid effects of calcitriol are mediated by a binding protein different from the VDR [65–68]. It has also been shown [69] that the VDR, upon ligand binding, physically interacts with the catalytic subunit of protein phosphatases PP1 and PP2Ac, thereby promoting their enzymatic activities with the consequent inactivation of p70S6k, a kinase essential

1837

CHAPTER 104 Inhibition of Benign Prostatic Hyperplasia by Vitamin D Receptor Ligands

A

G0/G1

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Phase percentage

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G2/M

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64

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FIGURE 3 Effect of BXL-353 on cell cycle distribution of BPH cells. Panel A shows the effect of 24 h treatment with KGF (10 ng/ml) with or without BXL-353 (10 nM) on partially synchronized (24 h serum starvation) BPH cells. KGF significantly reduced the number of BPH cell accumulated in the G0/G1 phase, thereby increasing the percentage of nuclei in G2/M. Simultaneous treatment with BXL-353 completely blocked the mitogenic activity of KGF. Results are expressed as cell cycle phase percentage. * P < 0.01 vs. control by one-way ANOVA and paired or unpaired Student’s t tests. For assay method see ref. [86]. Panel B shows results from a typical experiment.

in G0/G1 transition. VDR ligands not only induced a prompt decrease in phosphorylated KGFR [33,63], but also in phospho-Erk and phospho-Akt [70,71]. Hence, it is possible that calcitriol and related analogs might activate the catalytic subunit of distinct families of phosphatases, leading to antiproliferative effects by targeting GF signaling. Interestingly, in the human epidermoid A431 cells, overexpressing an autocrine growth loop for EGF, calcitriol not only induced a rapid alteration of EGFR auto-phosphorylation (as previously observed by us on KGFR), but also impaired EGFR membrane trafficking and signaling via the classic VDR-dependent mechanism [72]. In conclusion, it is possible that genomic (nuclear VDR-dependent) as well as rapid or nongenomic (cytoplasmic VDR-dependent?) mechanisms simultaneously contribute to the growthsuppressing activity of VDR ligands on prostate cells.

VI. INHIBITION OF IN VIVO PROSTATE GROWTH BY BXL-353 To investigate whether calcitriol analogs might represent a new opportunity to decrease prostrate cell

overgrowth and, therefore, to treat BPH, we carried out a series of studies using the rat as an experimental model. Taking advantage of the high sensitivity to androgens of the ventral prostate, castrated rats were supplemented with T with or without increasing doses of BXL-353 for various time periods [31]. Changes in ventral prostate volume and morphology, along with measurements of calcemia and hormonal values were studied. BXL-353 has a maximum tolerated dose of 30 µg/Kg, and at any dose tested never caused hypercalcemia. One week treatment with BXL-353 was sufficient to decrease significantly and dose-dependently ventral prostate weight, with an IC50 = 1.5 ± 1 µg/Kg (Fig. 5, panel A). Similar results were obtained with a two-week treatment of BXL-353 (Fig. 5, panel B). A 30% reduction of ventral prostate weight was induced by one month treatment with BXL-353 to intact adult rats (Fig. 5, panel C). It is interesting to note that prostate weight reduction induced by BXL-353 (∼50% decrease) is similar to that obtained in similar experimental models with 10 mg/Kg finasteride [31]. Because we observed that human BPH stromal cells underwent apoptosis even after short-term in vitro exposure to BXL-353, we investigated the fate of rat

A 60 * 50

Apoptotic index %

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35

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FIGURE 4

+Des (1–3) IGF (10 ng/ml)

+T (10 nm)

Effect of BXL-353 on apoptosis and Bcl-2 expression in BPH cells. The effect of 48 h treatment with BXL-353 (10 nM) alone or in combination with GFs (KGF and Des [1–3] IGF-I, 10 ng/ml) or T (10 nM) is reported on apoptosis (panel A) and Bcl-2 expression (panel B). Apoptotic index was obtained from in situ end labeling (ISEL) experiments and represents the number of stained nuclei over total cells in each of at least 5 separate fields per slide. Percentage of Bcl-2 stained cells was calculated by counting the number of immunopositive BPH cells over total cells in each of at least 5 separate fields per slide. Treatment with GFs or T significantly decreased the number of apoptotic cells (panel A), while it increased Bcl-2 positivity (panel B). Simultaneous treatment with GFs or T and BXL-353 significantly blunted the effect of both GFs and T on the number of ISEL (panel A) and Bcl-2 (panel B) positive cells. A 48 h exposure to BXL-353 induced a massive increase in apoptotic index (panel A), while Bcl-2 expression decreased (panel B). *P < 0.01 vs. control; °P < 0.01 vs. GF- or T-treated cells by one-way ANOVA and paired or unpaired Student’s t tests. The data are partially derived from Crescioli et al., 2000; 2002; 2003.


A

B

IC50 = 1.52 ± 1.05 µg/Kg

IC50 = 7.4361 ± 1.25 µg/Kg

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1839

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IC50 = 4.2041 ± 2.1 µg/Kg

Ventral prostate weight (% of vehicle)

100 95 90 85 80 75 70 65

1000 10000 BXL-353 (µg/Kg)

FIGURE 5 Effect of BXL-353 on rat ventral prostate weight. Panel A: Castrated rats were supplemented with a single injection of T enanthate (30 mg/Kg/week) and orally treated for 4 days with vehicle or increasing concentrations of BXL-353 (1–30 µg/Kg). Ventral prostate weight is expressed as % variation (mean ± SEM) of the weight of T-replaced castrated rats, in two separate experiments. ^P < 0.05 and *P < 0.01 vs T-supplemented vehicle-treated rats by one-way ANOVA and paired or unpaired Student’s t tests. Panel B: Castrated rats were injected with T enanthate (15 mg/Kg/week) and orally treated for 5 day/week for two consecutive weeks with vehicle or increasing concentrations of BXL-353 (3–30 µg/Kg). Ventral prostate weight is expressed as % variation (mean ± SEM) of the weight of T-replaced castrated rats (n = 4). *P < 0.01 vs. T-supplemented vehicle-treated rats by one-way ANOVA and paired or unpaired Student’s t tests. Panel C: Intact adult rats were orally treated for over one month (5 times/week for a total of 27 administrations) with vehicle (control) or increasing concentrations of BXL-353 (3–30 µg/Kg). Ventral prostate weight is expressed as % variation (mean ± SEM) of the weight of control rats (n = 4) (*P < 0.01 vs control rats). Data are derived from Crescioli et al., 2003.

prostate cells after sub-acute (7–14 days, castrated rats) or prolonged (1 month, intact rats) treatment with the analog. By using terminal deoxynucleotidyl transferase (TdT) mediated dUTP nick end-labeling (TUNEL), we observed the typical hallmark of nuclear fragmentation in both the epithelial and stromal cells of the BXL-353-treated prostate in all the experimental protocols studied [31]. In addition, we found that BXL353 treatments induced a dose- and time-dependent up-regulation of clusterin gene and protein. Clusterin (CLU), or testosterone-repressed message 2 (TRPM-2), is an ubiquitous, puzzling protein expressed also in the rat prostate [73–75], which is down-regulated by androgens and up-regulated by growth arrest and cell

death (see in [76]). CLU has different intracellular and extracellular functions. As an extracellular, secretory glycoprotein, it has a chaperone-like role, binding a wide range of unrelated molecules and probably clearing cellular debris. Conversely, the intracellular ∼49 kDa protein, after appropriate stimuli, is transported from the cytoplasm to the nucleus, where it binds DNA helicases, as Ku70/Ku86, thereby reducing DNA repair and allowing cell death [77]. Hence, CLU is generally considered an androgen-regulated, pro-apoptotic protein. We confirmed that in the rat prostate, CLU expression was increased by castration and finasteride administration [73,75], and we found that BXL-353 induced a sustained increase in CLU gene and protein

1840


expression [31]. Interestingly, CLU-positive cells were more apparent in the glands of BXL-353 treated rats showing the more pronounced features of involution and atrophy. In conclusion, studies in the rat strongly support findings in human BPH cells: BXL-353, similar to finasteride, counteracts the growth promoting effect of T, by inducing growth cell arrest and apoptosis. However, BXL-353, at variance with finasteride, is not an antiandrogen. It does not bind to the AR, as demonstrated by competition studies using the synthetic androgen [3H] R1881on BPH homogenates, and it does not inhibit 5α-reductase activity, as shown by the failure to interfere with DHT formation in CHO cells transfected with type 1 or type 2 5α-reductase iso-enzymes [31]. In addition, BXL-353 did not affect the gonadal or pituitary secretion of testosterone or gonadotrophin [31]. Hence it should act downstream of the AR receptor ligand interaction. The activated AR is a multiple phosphorylated protein and some of its phosphorylation sites (as Ser 650) are required for full transcriptional activity (see ref. [78]). Hence, it is possible that BXL353 might activate the catalytic subunit of distinct families of phosphatases, therefore exerting its antiproliferative effects acting on AR-dependent signaling. Alternatively, BXL-353 may disrupt androgendependent GF-mediated survival pathways, thus hampering T-induced BPH cell growth.

patients using α-blockers [81]. A possible mechanism of action underlying the risk reduction of BPH-related surgery by finasteride is that 5α-reductase inhibitors, by blocking DHT formation, shrink the prostate volume, which, in turn, is shown to be itself an important risk factor for BPH progression and, consequently, BPH-related surgery [80,82]. In fact, although many variables make it difficult to predict an individual’s clinical course, prostatic size is reported to be one of the most important risk factors along with age and prostatic specific antigen (PSA) value [83]. Hence, the ideal treatment for BPH should include a medication that reduces prostate volume without interfering with androgen activity. Actually, patient compliance for finasteride may be limited by consistent sexual side effects, such as decreased libido, altered sexual potency, or ejaculatory dysfunction [84], especially in men with borderline erectile function [85]. Well-tolerated calcitriol analogs, such as BXL-353, might represent such a new class of drugs, because they decrease AR-mediated prostate growth, acting downstream of the AR on the GF-mediated proliferation pathways. Based on the data reviewed here, a double-blind, placebo-controlled phase II study is currently ongoing in Italy to evaluate the effects of a nonhypercalcemic calcitriol analog in patients with BPH.

VII. CONCLUSIONS

1. Berry SJ, Coffey DS, Walsh PC, Ewing LL 1984 The development of human benign prostatic hyperplasia with age. J Urol 132:474–479. 2. Jacobsen SJ, Girman CJ, Guess HA, Panser LA, Chute CG, Oesterling JE, Lieber MM 1995 Do prostate size and urinary flow rates predict health care-seeking behavior for urinary symptoms in men? Urology 45:64–69. 3. Thorpe A, Neal D 2003 Benign prostatic hyperplasia. Lancet 361:1359–1367. 4. Claus S, Berges R, Senge T, Schulze H 1997 Cell kinetic in epithelium and stroma of benign prostatic hyperplasia. J Urol 158:217–221. 5. Shapiro E, Becich MJ, Hartanto V, Lepor H 1992 The relative proportion of stromal and epithelial hyperplasia is related to the development of symptomatic benign prostate hyperplasia. J Urol 147:1293–1297. 6. McNeal JE 1978 Origin and evolution of benign prostatic enlargement. Invest Urol 15:340–345. 7. Serio M, Fiorelli G 1991 Dual control by androgens and peptide growth factors of prostatic growth in human benign prostatic hyperplasia. Mol Cell Endocrinol 78:C77–C81. 8. Peters CA, Walsh PC 1987 The effect of nafarelin acetate, a luteinizing-hormone-releasing hormone agonist, on benign prostatic hyperplasia. N Engl J Med 317:599–604. 9. McConnell JD 1990 Androgen ablation and blockade in the treatment of benign prostatic hyperplasia. Urol Clin North Am 17:661–670. 10. Schroder FH 1994 5 alpha-reductase inhibitors and prostatic disease. Clin Endocrinol 41:139–147.

A large proportion of aging males develop BPH and, until recently, the only options for treatment were surgical intervention or watchful waiting. During the last 10 years, progress in medical therapy of BPH has resulted in effective treatments patterns leading to a significant improvement in the quality of life of affected patients. At present, two different classes of agents are available for BPH treatment: α-blockers and 5α-reductase inhibitors. Although sexual related side effects are more often reported with 5α-reductase inhibitors than with α-blockers [79], the reverse is true for reduction in risk of BPH-related surgeries [80]. A population-based cohort study, conducted in more than 5000 patients, receiving either α-blockers or 5α-reductase inhibitors (finasteride), showed that the incidence of BPH-related surgery was higher in α-blocker-treated patients than in 5α-reductase inhibitor-treated ones [80]. Similar results, obtained from a retrospective analysis of patients’ data, demonstrated that the risk of experiencing serious complication related to BPH progression (catheterization, acute urinary retention, surgery) was significantly lower in finasteride-treated patients compared to

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28. 29. 30. 31.

32.

33.

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35.

36.

37. 38. 39.

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41.

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Index

A AC. See Adenylyl cyclase ACE. See Angiotensin-converting enzyme Acidemia, rickets/osteomalacia features evident in, 984 Acidosis, 1α-hydroxylase inhibited by, 1316 Actin, 142 DBP sequestering, 147–148, 148f Actin-scavenger system, 143 DBP-actin complex implications for, 148–149 Activating domain (AD), AF-2, 279, 280f Activation function (AF-2) domain D analogs changing conformation of, 1452f, 1453 mutations v. D analogs, 1456 NCoA62/SKIP/VDR interaction independent of, 296 in NR LBDs, 279, 280f in 1,25(OH)2D3 transactivation of VDR, 1472 VDR LBD coactivator binding site v., 1473, 1473f WSTF coactivating ligand-induced, 309, 310f Activator protein (AP-1), in 1,25(OH)2D3-induced differentiation, 1640 AD. See Activating domain; Addison’s disease; Alzheimer’s disease Adaptor proteins, receptors requiring, 159 Addison’s disease (AD), VDR polymorphisms v., 1148 Adenylyl cyclase (AC), in 1α,25(OH)2D3/Ca2+ mechanism, 890f, 891 ADH. See Antidiuretic hormone ADHR. See Autosomal dominant hypophosphatemic rickets Adjusted apposition rate (Aj.AR) FP v., 1034 MAR v. osteoid surface in, 956–957 O.Th v., 1035, 1035f Adults acute hypocalcemia therapy for, 1058 D3 dosage considerations for, 1003–1005, 1009t D insufficiency in, 1091–1092, 1091f, 1096–1097 elderly D insufficiency in, 825–826, 1092–1094, 1092f D supplementation v. bone loss in, 825–826 insufficiency v. hip fracture risk in, 1089 intestinal D absorption reduced in, 1087–1088 infant D3 dosage v., 1003 minuscule D3 intake recommendations for, 1008 AF-2. See Activation function domain AFM. See Atomic force microscopy

Africans. See also specific nationalities VDR polymorphism allele frequencies in, 1128t, 1129 VDR polymorphisms v. PCa in, 1139t, 1145 Aging cutaneous D production v., 823–824 D metabolism v., 823–833 D tissue responsiveness in, 831–833 dietary D/intestinal absorption v., 824–826 FCA in response to free 1,25(OH)2D v., 832, 832f 25OHD serum concentration v., 826–827 1,25(OH)2D synthesis/metabolism v., 826–831 25OHD synthesis v., 826 PCa incidence v., 1679, 1680 as PCa risk factor, 1601, 1601t PTH/1,25(OH)2D v., 828, 828f Agonists. See also specific agonists action mechanism of selective, 1478–1480, 1479f assessment of noncalcemic selective, 1478 differential VDR activation by noncalcemic selective, 1478–1480 nonsecosteroid VDR, 1557–1567 AIDS, granulomatous disease incidence increasing with, 1391 AIPC. See Androgen-independent prostate cancer Aj.AR. See Adjusted apposition rate Al. See Aluminum Alaskans, D deficiency in child/infant, 795 Alcohol. See Ethanol Alcoholic liver disease bone disorders associated with, 1306 clinical features of, 1306 D metabolism impaired in, 1306, 1306f management, 1306 Alcoholics, inadequate dietary D/sunlight exposure in, 1266 Alcoholism hypophosphatemia in, 1177 magnesemia in, 1177 Alendronate bone loss prevented by, 1248 Ca absorption/bone formation suppression v., 1244 Alfacalcidol. See 1α-Hydroxyvitamin D3 Algerians, sunlight exposure v. rickets in, 1066–1067 Alien, NR co-repressor, 299 Alkaline phosphatase Ca entry v., 416 mineral properties in KO animals, 479t

1846 Alkaline phosphatase (Continued) in mineralization, 479t in mineralization v. 1α,25(OH)2D3, 716 1α(OH)ase-null mice demonstrating elevated, 438 1,25(OH)2D3 elevating matrix vesicle, 590–591 tissue’s ability to calcify associated with, 580 Alkalosis, serum phosphate depressed by, 1177 All-trans-retinoic acid (ATRA), in leukemia combination therapy, 1733 Allograft rejection Geminis inhibiting, 1519–1521 immunoregulatory mechanisms inhibiting, 642 19-nor Gemini v. vascularized heart, 1520–1521, 1520t, 1521f 1,25(OH)2D3/analogs inhibiting acute, 640–641, 641t orally active VDR ligand treatments for, 643 VDR ligands/1,25(OH)2D3 analogs inhibiting chronic, 642 VDR ligands v., 640–642 Alopecia in HVDRR, 1208, 1208f, 1210 HVDRR associated with, 1229–1230 in HVDRR phenotype, 621 HVDRR severity correlating with, 1210 in VDR-ablated mice with normal mineral ions, 542 VDR locus mutation/expression in, 1404 VDR-null mice having progressive, 346–347 VDR RXRα heteropartner role supported by, 233 Aluminum (Al) D metabolism influenced by, 1255t, 1270–1271 in osteomalacia, 1270 toxicity from chronic renal disease, 979–981 Alzheimer’s disease (AD), 1779–1780 D analogs treating, 1785 Ameloblasts calbindin-D9K/D28K in, 724–725, 730t D-dependent molecules in, 602–604, 603f enamel elaborated by, 599 American Indians, metabolic bone disease v. lactose intolerance in, 917t Analogs. See also Agonists; Antagonists; Deltanoids; Geminis; Superagonists 2-carbon-modified 19-Nor-1α,25-dihydroxyvitamin D3, 1543–1554 aromatic rings in, 1495, 1497t autoimmunity v. 1,25(OH)2D3, animal models of, 1756, 1757t bone diseases v., 1501–1502 breast cancer cell apoptosis induced by 1,25(OH)2D3, 1665–1666, 1666f, 1672f breast cancer cells/tumors v., 1668–1669 breast cancer cells v. 1,25(OH)2D3, 1663–1667, 1664t breast cancer v. 1,25(OH)2D3, 1663–1672 breast cancer v. preclinical/clinical trials of, 1668–1669 C-ring, 1562, 1563f structures/activities of, 1562, 1564t calcemic v. antiproliferative effect of 1,25(OH)2D3, 1573, 1575t cancer models v., 1499–1500 catabolism v. selectivity of, 1459–1461, 1460f cell-specific catabolism of, 1459–1460 cell-surface receptor v. nongenomic activity of, 1461–1462 chronic kidney disease treated with, 1827 chronic renal failure v. action/tissue specificity of, 1826–1827 chronic renal failure v. future roles of, 1826–1827 clinical development of LEO, 1502–1505 clinical studies of cancer v., 1574 colon cancer cells v. antiproliferative activity by, 1521–1522, 1522f, 1523f, 1523t colon cell proliferation/differentiation/apoptosis v.

INDEX

Analogs (Continued) clinical studies of, 1715 in vitro, 1713–1714 in vivo animal models for, 1714–1715 D-ring, 1562, 1563f structures/activities of, 1562, 1565t DBP affinity v. side chain modifications in, 1457 dermal inflammation/epidermal proliferation/differentiation v., 1781 developing new, 1489–1505 basic screening strategy for, 1489–1490 strategy for, 1489–1492 synthesis strategy for, 1490–1492 development of OCT/ED-71, 1525–1539 dialysis patient PTH secretion controlled by, 1822–1823 differential initiation complex recruitment by, 1455–1456 differential VDR activation by, 1475–1482, 1476f differentiating/antiproliferative v. calcemic activities in, 1449 DNA binding v. selectivity of, 1455 E-ring, 1562–1565, 1563f structures/activities of, 1562–1564, 1566t EAE treated with, 1784 efficacy/safety in clinical development of, 1504–1505 20-epi 1,25(OH)2D3, 1494–1495, 1496t, 1511–1522 transcriptional activity of, 1511 epidermal proliferation marker modulation v. topical, 1781, 1783f in future HVDRR therapy, 1229 hyperproliferative skin disorders v. less calcemic, 1788–1789 hypertension v. low calcemic, 1514–1516 IDBP interactions v. selectivity of, 1462–1463 identifying selective, 1449–2900 immunological diseases v., 1500–1501 immunosuppressive agents synergistically influencing 1,25(OH)2D3, 1520 implications for action mechanism of, 1441–1442 kidney stone formation v., 1351–1352 leukemia v., 1734–1736 leukemia v. 20-epi, 1730t, 1735 leukemia v. C-16-ene, 1730t, 1734–1735 leukemic cell lines v., 1730t level of selectivity in, 1451–1452 ligand binding determining selectivity of, 1452f, 1453 mechanisms for selective actions by, 1449–2911 metabolism of, 1423–1443 biological systems in studying, 1428–1429, 1428t drug design implications in, 1442 examples for, 1429–1440 general considerations in, 1423–1429 implications from studying, 1440–1443 invalid comparisons of in vivo/in vitro, 1442 non-D-related enzymes in, 1428–1429 pharmacokinetic information correlating with, 1440–1441, 1441t questionably valid assumptions regarding, 1442 radioactive analogs in studying, 1428–1429 specific analogs exemplifying, 1429–1440 metabolism of 20-epi-/20-methyl, 1433–1434, 1433f, 1435f metabolism of cyclopropane-ring containing, 1431–1433, 1432f metabolism of homologated, 1434–1436 metabolism of oxa-group containing, 1438 metabolism of unsaturated, 1436–1437 molecular basis for differential action of, 1471–1483 non-psoriatic skin diseases v., 1787–1788 nonsecosteroid CD ring modifications in, 1562–1565 nonsteroidal, 1557–1567

INDEX

Analogs (Continued) in normal/psoriatic skin, 1781–1784 1,25(OH)2D3, 1423–1424, 1425t–1426t animal model diabetes prevented by, 1768–1772 biological activities of, 1495–1502 bisphenols v., 1565 clinical trials of, 1742–1743 diabetes/auto-immune diabetes v., 1772–1773 diabetes early intervention using, 1768–1770, 1768t diabetes v. immune modulators combined with, 1771–1772, 1771t early trials of, 1742 oncology using, 1741–1747 secondary hyperparathyroidism in chronic renal failure v., 1331–1332, 1332f tumors v., 1741–1742 VDR binding/gene expression v., 1495–1498, 1511, 1512f 1,25(OH)2D3 side chain, 1490–1492, 1490f, 1491f, 1492f chain length v. structure-activity of, 1492, 1493t diabetes v., 1768t, 1770–1771 double/triple bonds in, 1492–1493 structure-activity relationships in, 1492–1495 1,25(OH)2D3 structure in secosteroid, 1558, 1558f overview of, 1403–1404 PCa treatment clinical trials of, 1698–1699 PCa v., 1689–1690 PD/AD treated with, 1785 pharmacokinetic data describing, 1440–1441, 1441t pharmacokinetics/metabolism in clinical development of, 1502–1503 preclinical experience with, 1499–1502 prodrug, 1423–1424, 1424t prodrug/1α,25(OH)2D3 analog classification of, 1423 protein interactions v. in vivo selectivity of, 1452–1463, 1452f psoriasis treated with, 1450 psoriasis v. biological effects of, 1781, 1781f psoriasis v. clinical use of 1,25(OH)2D3, 1784–1787 psoriasis v. topical 1,25(OH)2D3, 1784–1785 PTH-suppressing/noncalcemic, 1449–2898 quantifying selectivity of, 1451 rat kidney perfusion in studying D3, 1513–1514, 1516f selection criteria for, 1543–1544 serum DBP interacting with, 1456–1459 stabilization/increased VDR v. activity of, 1456 strong/weak calcemic/noncalcemic, 1440–1441 synthesizing 20-epi, 1491, 1491f, 1492f, 1496t, 1497t synthesizing 22-oxa, 1490–1491, 1492f, 1496t target cell enzymes activating/deactivating, 1441–1442 toxicity due to synthetic, 1357–1359 VDR/coactivator interaction enhanced by 20-epi, 1497–1498 VDR conformational change v. selectivity in, 1452f, 1453 VDR interactions v. binding of 20-epi, 1477–1478, 1478f VDR modulated by, 1482–1483 VDR phosphorylation possibly influenced by, 1454 VDR stabilized by, 1456 VDR transcriptional activity v. 1α,25(OH)2D3, 1474–1475, 1474t Androgen human BPH cells v., 1834 in PCa treatment, 1679–1680 PCa v. D interaction with, 1687–1688 Androgen-independent prostate cancer (AIPC) high dose intermittent 1,25(OH)2D3 v., 1745 1,25(OH)2D3/taxane combinations v., 1745–1746 1,25(OH)2D3 v., 1742 Androgen receptor (AR) in human BPH cells, 1834, 1835f in PCa, 1679–1680

1847 Anemia, in hemodialysis patients associated with VDR polymorphism, 246 Angiogenesis, D compounds v., 1574–1576, 1693 Angiotensin-converting enzyme (ACE), in RAS cascade, 871, 871f Anonymous polymorphisms, VDR gene analysis v., 1138 Antagonists assessment of, 1480–1481 26-carboxylic ester, 1481f, 1482 differential VDR activation by, 1480–1482 lactone, 1481–1482, 1481f structure of representative, 1481, 1481f Anthropometry, VDR gene polymorphisms v., 1143 Anticonvulsants, D metabolism influenced by, 1255t, 1264–1265 Antidiuretic hormone (ADH) IMCD water reabsorption increased by, 558 in RAS cascade, 871–872, 871f Antigen presenting cells (APCs) immunosuppressive treatments not targeting, 1519 1,25(OH)2D3/analog autoimmunity v., 1756 Antimycotics, kidney 1α-hydroxylase activity regulated by, 78 Antioxidant, 1,25(OH)2D3 as, 764t, 765–767 Antituberculous agents, D metabolism influenced by, 1255t, 1271 AP-1. See Activator protein Apa polymorphisms, 1131t–1132t, 1135–1137 BMD v., 1142–1143 Apatite hydroxyapatite v., 477, 478–480, 478f vertebrate tissues with largest/smallest crystals of, 477 APCs. See Antigen presenting cells APLs. See Promyelocyticleukemias Apoptosis BXL-353/GFs v. BPH cell, 1836–1837, 1838f calbindin-D28K regulating, 727 calbindin-D28K v. bone cell, 725, 725f 20-epi D analogs inducing, 1498 hypertrophic chondrocyte, 577 1,25(OH)2D3 inhibiting tumor cells through, 1580 1,25(OH)2D3 protecting epidermal keratinocytes from, 766–767 1,25(OH)2D3 protecting HL-60 cells from, 1732 1,25(OH)2D3 v. PCa cell, 1691 oncogenes/tumor suppressor genes in, 1577–1580 AR. See Androgen receptor Arabs D metabolism in United Arab Emirates/non-Gulf, 794 MVC v. D in veiled Danish, 1812, 1812f Area under the curve (AUC), high dose intermittent regimen v. 1,25(OH)2D3, 1742–1743, 1743f Aromatase, 858 D v. expression of, 859 Asian Indians bone histomorphometry of, 793 bone mass in, 793 D metabolism in, 790t, 792–793 low 25OHD in immigrant, 1026 sunlight exposure v. rickets in, 1066–1067 VDR polymorphisms v. diabetes in, 1146 Asians high hVDR gene frequency in, 244–245 metabolic bone disease v. lactose intolerance in, 917t PCa risk in, 1601, 1601t rickets in immigrant Southeast, 968–971 VDR polymorphism allele frequencies in, 1128t, 1129 Astrocytes, in brain antioxidative/detoxification processes, 766 Atomic force microscopy (AFM), bone crystals imaged by, 482 ATP, muscle cell energy from, 1808 ATRA. See All-trans-retinoic acid Australians, D deficiency in, 795

1848

INDEX

Autoimmune disorders calbindin-D28K Ca buffering v., 726 as D analog therapeutic target, 1451 D analogs treating, 1451 orally active VDR ligand treatments for, 643 VDR ligand immunomodulation mechanisms in models of, 636–640, 637t VDR polymorphisms v., 1146–1148 VDR-RXR heterodimer-activating ligands v., 241 Autoimmune polyglandular syndrome type 1, hypocalcemia v., 1053 Autoimmunity, 1753–1759 D3 in, 1753–1759 immune mechanisms in, 1753–1754 1,25(OH)2D3 action mechanisms in, 1754–1756 regulatory T cells in, 1754 Autosomal dominant hypophosphatemic rickets (ADHR), 463t, 467–468 clinical features, 1189–1190 as disorder of phosphate metabolism, 1189–1190 as hypophosphatemic disease, 1163

B B lymphocytes, antigens recognized by, 631 BAF. See Brahma/SWI2-related gene 1 associated factor Basal cell carcinoma, VDR polymorphisms v., 1146 Basic multicellular units (BMUs) conditions for progress by, 504–505 as instrument of bone remodeling, 501–506, 502f, 503f, 504f originations/progressions/terminations in, 503 posttarget progression by, 503, 505f Bedouins HHRH linkage analysis in, 469 osteomalacia/Ca deficiency in, 1077 serum 25OHD in dark-skinned, 794–795 vitamin D deficiency in, 41 Benign prostatic hyperplasia (BPH) 1α-hydroxylase in cells derived from, 1608–1609, 1610f pathogenesis, 1833–1834 VDR ligands inhibiting, 1833–1840 Beri-beri, nutrition v., 3 β cells clinical trials of D metabolites v., 1765–1767 D deficiency v. in vivo, 1764 D influencing characteristics of, 1767 D metabolites v. in vitro, 1764–1765 D v., 1764–1767 D’s metabolic influences on, 1764–1767 BFR. See Bone formation rate Bile, inactive polar D derivatives in, 1295 Bile acids enterohepatic circulation reclaiming, 863–864 fate of, 863 in lipid digestion/absorption, 863 NRs regulating metabolism of, 865–866 production of secondary, 864–865, 864f VDR as sensor for carcinogenic, 863–869 Binding proteins, intracellular VDRE, 351–361 Binding sites proposed for regulatory trans-proteins, 94–95, 95f rat CYP24A1 proximal promoter region, 93–97, 94f Biopsy, bone, 951–952 Bisphenols, 1557–1561 antiproliferative potency of nonsecosteroid, 1559, 1559t

Bisphenols (Continued) identification/structure/synthesis of nonsecosteroid, 1557–1558 metabolic properties altered in, 1567 mutant VDR v. nonsecosteroid, 1561–1562 1,25(OH)2D3 template v., 1565 in vitro characterization of nonsecosteroid, 1558–1560, 1559t in vivo nonsecosteroid, 1560–1561 Bisphosphonates, D metabolism influenced by, 1255t, 1268 Blacks. See also Africans age-adjusted PCa mortality in, 1600, 1600f bone histomorphometry of, 792 bone mass in, 791–792 colon cancer v. skin pigmentation in, 1709–1710 D metabolism in, 790–791, 790t dietary calcium reduction response by, 778 hypertension v. D deficiency in, 899–900 metabolic bone disease v. lactose intolerance in, 917t 25OHD concentration/vitamin D deficiency in, 38–39 osteoporosis/atraumatic fractures in, 792 osteoporosis/Ca absorption in, 816 PCa in Caucasians/Nigerians/American, 1680–1681 PCa mortality/incidence in American, 1625 PCa risk in, 1601, 1601t PTH-stimulated bone resorption in, 778 Blood cells, VDR in, 1728–1729 Blood pressure. See also Hypertension D endocrine system regulating, 291, 873 RAS regulating, 871 regulation v. D3, 999t sunlight/D v., 873–874, 874f in VDR-null mice, 345 BMAR. See Bone mineral apposition rate BMC. See Bone mineral content BMD. See Bone mineral density 2BMD. See 2β-Methyl-19-nor-(20S)-1,25-dihydroxyvitamin D3 BMUs. See Basic multicellular units Bone, 565–768 activation frequency v. turnover, 959, 1034 age-related loss of, 506–508 age v. thickness of trabecular, 500 aging v. D responsiveness by, 833, 833t apposition rates influenced by 1,25(OH)2D3, 656–657 biopsy, 951–952 black/Caucasian formation rate of, 792 Ca in formation of, 411 calbindin-D9K/D28K in, 724–725 cancellous, marrow composition for, 500–501, 501t CaR in, 558 CKD with low 1,25(OH)2D3 or 1,25(OH)2D3/VDR resistance in, 1325 collagen synthesis regulation in, 704–705 D action redundant on, 429 D actions on mineralization of, 565–566 D analog nongenomic pathway in, 1462 D/D metabolites acting on, 565–571 D deficiency impairing resorption of, 776, 777, 777f D-deficient, 487–488 D direct/indirect effects on, 1042–1044 D insufficiency impact on, 1089–1090 D intake v. winter loss of, 1090t D mobilizing mineral in, 665–666, 666f D modulating formation of, 327–328 deformities caused by D deficiency, 972f–973f, 973, 974f density correlated with VDR gene polymorphisms, 184, 200 density influenced by exercise, 796 density seasonal decline v. D/Ca, 998

INDEX

Bone (Continued) dento-alveolar complex of, 599–605 development v. perturbed D metabolism, 107–108 disease in gastrointestinal disorders, 1297–1299 disorders v. gastrointestinal/hepatobiliary disease, 1293–1307 driving transfers of Ca2+ in mineralization of, 774, 775f estrogen/PTH/D metabolism v. age-related loss of, 1107–1109 evolution of D-related disease of, 1035–1039 formation rates, 958 formation selectively induced by deltanoid, 1415f, 1416 fracture aluminum toxicity associated with, 980–981 D3 dose preventing, 997, 998f in Looser’s zones, 973 manifesting osteoporosis, 1101 normal 25OHD v., 1022–1023 rate v. D supplementation, 1094–1096, 1095f, 1096f repair influenced by 24,25(OH)2D, 108 risk v. aging/activity level, 508 risk v. D, 1813–1814 risk v. serum 25OHD, 1370, 1370f VDR polymorphisms v., 1143–1144 in vertebrae v. 12523, 1111–1112 glucocorticoid therapy causing loss of, 1239 growth in central skeleton, 499, 500f health supporting higher D levels, 1370 hydroxide-deficient apatite crystals in, 477 24-hydroxylase enzyme regulation in, 92 irreversible loss of, 506, 507f, 508f length, 499–500 loss influenced by age-related PTH increase, 1107, 1108f partitioned into horizontal/vertical components, 506–507, 509f VDR polymorphisms associated with alveolar, 246 marrow rejection inhibited by 1,25(OH)2D3/analogs, 641t matrix proteins influenced by D, 479t, 484–487 matrix synthesis gene expression v. 1α,25(OH)2D3, 715 metabolism v. ED-71/17β-estradiol, 1536, 1536f mineral content/density v. pregnancy, 840–841 mineral metabolism influenced by HEP 187, 1493, 1494t mineralization assessed by histomorphometry, 954–957 mineralization mechanisms in, 480 mineralization v. vitamin D, 5–6 modeling v. remodeling, 497–498, 498t OCT influence on, 1527–1529, 1529t OCT v. formation rate of cortical, 1531, 1533f 1,25(OH)2D3 regulating type I collagen expression in, 703–705 1,25(OH)2D3 v. mineral homeostasis in, 220–221, 221f 1,25(OH)2D3 v. 24,25(OH)2D3 in endochondral formation of, 583 1α,25(OH)2D3 VDRnuc in, 385t osteocalcin gene expression modulation during, 328–329 osteomalacia v. properties of, 489–490, 490f Pi flux in human, 453–455, 454f Pi transport in, 465–467 progenitor cell commitment v. 1,25(OH)2D3, 650f, 655–656 PTHrP expression in, 739t remodeling, 497–511 activation frequency, 504–505 balance assessment, 959–961 BMUs, 501–506, 502f, 503f, 504f bone age/mineral density v., 503f, 506 cycle in cancellous bone, 506, 507f cycles v. bone loss, 507, 509f focal imbalance, 506, 508f influences mediated by strain, 508 initiated by fatigue microdamage, 503

1849 Bone (Continued) osteonal/hemiosteonal, 502–503 purpose, 500–501 in structural/metabolic bone, 501, 501t remodeling markers, 921t remodeling periods, 959 renal osteodystrophy v. periosteal formation of, 979 resorption/formation v. ED-71, 1537, 1537f, 1538 resorption in GHS rats, 1349 resorption in vitro v. 1,25(OH)2D3, 568–569, 569f resorption in vivo v. D, 569–570, 570f resorption induced by 1,25(OH)2D3, 680–681, 681f resorption influenced by D, 568–570 resorption rates, 958 resorption signal transduced by RANKL/ODF, 672–673 RUNX2∆C-homozygous mice not forming, 335 steroid receptors/actions in, 1239–1241 strength structural determinants, 961 structural/cellular basis for growth of, 498–500 structure assessed by histomorphometry, 961–963 target genes v. D, 566–568 turnover, 497 assessment, 958–959 dissimilarly affected by PTH/1,25(OH)2D3, 510 turnover/density v. D insufficiency in elderly, 825 two types mineralization in, 1030–1031 VDR in growth/maturation/remodeling of, 430f, 433 VDR overexpression strengthening, 488–489, 489f volume v. VDR ablation, 343, 344t Bone cells. See also Osteoblasts; Osteoclasts in bone modeling/remodeling, 497–511 calbindin-D28K v. degeneration of, 725 coordinated activities of, 497, 498t Bone disease. See also specific bone diseases approaching patients with metabolic, 913–928 D analogs v., 1501–1502 gastrointestinal conditions associated with, 1299–1303 gastrointestinal disease with metabolic, 1298–1299 non-Ca/D factors in gastrointestinal disease-associated, 1298 Bone formation rate (BFR) in HVO, 1035–1036 OCT v., 1528, 1529t Bone histomorphometry, 951–963 biopsy, 951–952 adverse effects, 952 indications, 952 procedure, 951–952, 951f bone remodeling indices in derived, 953, 953t primary, 953, 953t bone structure assessment by, 961–963 three-dimensional, 962–963 two-dimensional, 961–962 dynamic indices from mineralizing perimeter/surface, 958 future developments in, 963 limitations, 954 methodology, 952 remodeling balance assessment by, 959–961 bone formation v., 959–960, 959f bone resorption v., 960–961, 960f terminology, 952–954 referents/abbreviations, 952–953, 953t theoretical considerations influencing, 952 in vivo tetracycline labeling in, 954, 955, 955f Bone mass D in GIO treatment v., 1245–1247

1850 Bone mass (Continued) ED-71 v. ovariectomized rat, 1537, 1537f IH patients having low, 1342–1343 loss reduced by D supplementation, 1094 2MD v. ovariectomized rat, 1547, 1548f Bone matrix proteins, 712, 712f 1α,25(OH)2D3 regulating, 712, 713t target genes in, 711–717 VDREs identified in, 712, 713t Bone mineral apposition rate (BMAR), D metabolites influencing, 656–657 Bone mineral content (BMC) maternal D status v. infant, 842–843 1,25(OH)2D3 restoring PDDR, 1202, 1202f Bone mineral densitometry, in evaluating bone metabolic disease, 922–924 Bone mineral density (BMD) alendronate v., 1248 Cdx2 polymorphism v., 1130 corticosteroid/Ca/D v., 1245–1247, 1247f D metabolites v., 1245, 1246t in defining normal serum 25OHD, 1022, 1022f in ED-71 clinical trials, 1538–1539, 1538f intestinal CA absorption/VDR polymorphisms v., 1141–1142 1,25(OH)2D v., 1107 1αOHD3 v., 1247–1248 in osteoporosis/fracture, 1141, 1143–1144 prednisone/Ca/D v., 1245, 1246f VDR gene polymorphism v. lumbar spine, 243 VDR gene polymorphism v. women’s, 243 VDR polymorphisms v., 1142–1143 Bone scintigraphy, skeletal abnormalities uncovered with, 922 Bone sialoprotein (BSP) expression suppressed by 1,25(OH)2D3, 486 1α,25(OH)2D3 suppressing, 715 production v. D, 567–568 BPH. See Benign prostatic hyperplasia BPH cells AR/5α-reductase/VDR in, 1834, 1835f BXL-353/GFs v. apoptosis/Bcl-2 expression in, 1836–1837, 1838f BXL-353 v., 1836–1837, 1837f KGF/IGF-I/T v., 1834, 1835f Brahma/SWI2-related gene 1 associated factor (BAF) components in WINAC, 308, 308f in 1,25(OH)2D3-liganded VDR-RXR heterodimer transactivation, 238, 239f Brain detoxification v. 1,25(OH)2D3, 1783 development influenced by D3, 999t homeostasis v. 12523, 1784, 1785f 1,25(OH)2D3 antioxidant activities in, 764t 1,25(OH)2D3 in development/disorders of, 1779–1785, 1785f 1,25(OH)2D3 v. tumors in, 1783–1784 PTHrP expression in, 739t Breast cancer angiogenesis/invasion/metastasis v. 1,25(OH)2D3, 1666–1667 cell proliferation v. D analogs, 1451 D3 protecting against, 999t D sensitivity, 1667–1668 EB1089 v., 1499 epidemiology of D v., 1671 natural ligands v. synthetic analogs treating, 1668 1,25(OH)2D3/analog antiproliferative effects v., 1459, 1460f 1,25(OH)2D3/analogs v., 1663–1672 1,25(OH)2D3 prooxidant activities in, 764–765, 764t

INDEX

Breast cancer (Continued) 1,25(OH)2D3/VDR modulating cell proliferation/apoptosis in, 857–858 preclinical studies of D preventing, 1670 prevention and D, 1669–1672, 1670t prognosis v. tumor VDR expression, 1668 PTHrP expression in, 739t PTHrP production v., 742 PTHrP v. osteolytic bone metastasis in, 744 risk, for nationality, 245 risk v. VDR polymorphisms, 1671–1672 VDR polymorphisms v., 1145 Breast cancer cells apoptosis-independent 1,25(OH)2D3 inhibition of, 1580 apoptosis induced by 1,25(OH)2D3/analogs in, 1665–1666, 1666f, 1672f D compounds v. oncogenes/tumor suppressor genes in, 1664–1665 D resistance in, 1667–1668 EB1089 inhibiting IGF-I-stimulated, 1498 EB1089 v., 1669 1,25(OH)2D3/analog actions on, 1663–1667, 1664t 1,25(OH)2D3/deltanoid-induced differentiation modeled in, 1637t 1,25(OH)2D3/EB1089 arresting progression of, 1663–1664, 1672f 1,25(OH)2D3/EB1089 v. estrogen action in MCF-7, 1665 1,25(OH)2D3 v. proliferation of, 1663–1665, 1672f VDR expression/regulation in, 1667 Breast milk Ca absorption fraction from, 813, 814t maternal D/Ca intake v. D/Ca in, 846–847 neonatal hypocalcemia incidence v., 841–842, 842f substitutes v. rickets risk, 1066 Brown tumors, in hyperparathyroidism, 978f, 979 Brush border membrane lipids v. Ca entry, 415–416 membrane Na/Pi transport reduced in Npt2a KO mice, 462 membrane Pi transport, 455 permeability increased by D, 414 Bsm polymorphisms, 1131t–1132t, 1135–1137 BMD v., 1142–1143 BSP. See Bone sialoprotein Burns, hypophosphatemia associated with, 1177 BXL-353. See 1,25-Dihydroxy-16-ene-23-yne D3

C C-3 epimerization, 1,25(OH)2D3, 24, 25f C-24 oxidation pathway, to calcitroic acid, 1424–1427, 1426f C-cells, CaR stimulating CT secretion by, 556 c-Fos, osteoclastogenesis v. RANKL-induced expression of, 677–678 c-Src family proteins, osteoclast differentiation v., 677, 677f C27 sterol 27-hydroxylase, 54–55 mitochondrial D3 25-hydroxylase is, 55 C-terminal extension (CTE), hVDR DBD/VDRE alternative positions, 231f, 232 Ca. See Calcium Caffeine, D metabolism influenced by, 1255t, 1271–1272 Calbindin, 417–419, 721–730 Ca absorption v., 418 Ca entry process initiation v., 418–419 in cytosolic Ca diffusion, 419 distribution, 730t EF-hand HLH structural motif characterizing, 722, 722f

INDEX

Calbindin (Continued) functions of, 418–419 gene expression regulation, 728–730 localization/functional significance, 722–728 1,25(OH)2D3 v. in brain, 1781 synthesis, 418 VDRnuc responsible for production of, 384–385 Calbindin-D9K, 523–525, 721–730 in placenta/yolk sac/uterus, 726, 730t structure, 524 Calbindin-D28K, 523–525, 721–730 EF hands contained in, 722, 723f in egg shell gland, 726 enzymes activated by, 727–728 in nervous system, 726–727 PMCa pump regulated by, 524 regulatory function v. apoptosis, 727 structure, 524 Calbindin-D9K gene genomic organization, 729 1,25(OH)2D3 regulating, 729 steroids/other factors regulating, 729–730 Calbindin-D28K gene genomic organization, 728 1,25(OH)2D3 regulating, 728 regulation, 728–729 steroids/other factors regulating, 728–729 Calcidiol. See 25-Hydroxycholecalciferol Calcimimetics, CaR activated by, 554 Calcipotriol breast cancer cells/tumors v., 1668 discovery, 1489 face/skin flexures treated with, 1786 leukemic cells v., 1734 metabolism, 1431–1433, 1432f monotherapy/combination therapy v. psoriasis, 1504, 1504t as noncalcemic analog, 1440, 1441t pharmacokinetics/metabolism in clinical development of, 1495t, 1502–1503 psoriasis treated with OCT v., 1533, 1533f psoriasis treated with topical, 1784 psoriatic lesions v. topical, 1450 scalp psoriasis treated with, 1785–1786 skin irritation in psoriasis treatment with, 1785 structure-activity relationships, 1493–1494, 1495t toxicity, 1358–1359 Wittig reaction in synthesis of, 1490, 1491f, 1495t Calcitonin (CT) C cells producing CGRP and, 689 CaR stimulating C-cell secretion of, 556 CYP24A1 expression regulated by, 93, 94f CYP24A1 promoter expression increased by, 100 D metabolism influenced by, 1254t, 1256–1257 hypocalcemia from overzealous use of, 1057 induction during CYP24A1 promoter mutational analysis, 96–97, 97f intestinal CYP24 expression v., 21 kidney 1α-hydroxylase activity regulated by, 77 levels regulated by D3, 689–691 PTHrP expression/production stimulated by, 741t renal Pi excretion decreased by, 516t Calcitonin (CT) gene D influencing thyroid C cell, 687–697 model system for 1,25(OH)2D3 v. transcription of, 690–691 transcription down-regulated by D, 693–696 transcriptional regulation, 689, 690f, 691–693

1851 α-Calcitonin gene-related peptide (CGRP), C cells producing CT and, 689 Calcitriol. See 1,25-Dihydroxyvitamin D3 Calcitroic acid C-24 oxidation pathway to, 1424–1427, 1426f in calcipotriol metabolism, 1432, 1432f 1,25(OH)2D2 metabolism to, 25–26 Calcium (CA), 1,25(OH)2D3 v. intestinal transport of, 679f Calcium (Ca). See also Calciuria; Eucalcemia; Familial hypocalciuric hypercalcemia; Hereditary hypophosphatemic rickets with hypercalciuria; Hypercalcemia; Hypercalciuria; Hypocalcemia; Normocalcemia; Transcaltachia; Vascular calcification; Williams syndrome absorption by paracellular path, 421–422 absorption by VDR-null mice functional aspects of, 433–435, 434f molecular aspects of, 435–437 absorption enhancers in food, 818 absorption in infancy/childhood, 811–818 absorption in Npt2-null/Hyp mice, 440 absorption in postmenopausal/age-related osteoporosis, 1110 absorption increased by alendronate, 1244 absorption influenced by 1,25(OH)2D3/VDR binding, 220–221, 221f absorption location/timing in gut, 778–779, 779f absorption v. IH, 1341, 1341f absorption v. intake, 779–781, 779f, 780f absorption v. prednisone/glucocorticoids, 1244–1245, 1245f absorption v. serum 1,25(OH)2D in IH, 1342, 1343f absorption v. VDR polymorphism/BMD relationship, 1141–1142 absorption v. vitamin D3, 5 absorptive efficiency v. transit time, 778 absorptive input, 778–782 active/passive absorption of, 781–782 adolescents v. inadequate intake of, 816 adult human body, 773 aging v. intestinal absorption of, 831–832 aging v. total/ionized, 829, 829t balance disordered in sarcoidosis, 1381–1387 balance v. lactation, 4 binding proteins in brain v. 1,25(OH)2D3, 1781 biological importance of, 411 in cancer risk epidemiology, 1617–1629 case-control/cohort studies of colorectal cancer v., 1622 CKD impairing 1α-hydroxylase induction by, 1315–1316 colorectal adenoma v., 1622–1623 colorectal cancer v., 1621–1624 cytosolic transfer of, 417–419 calbindins in, 419 D deprivation/repletion v. muscle, 1810f, 1811 D enhancing paracellular absorption of, 412f, 421–422 D3 improving absorption of, 999t D/25OHD3/1,25(OH)2D3 influencing renal handling of, 518–519 in dairy products v. PCa, 1627–1629 demand response of ECR, 776–778 dietary deficiency of, 1074–1076 duodenal/ileal transcellular/paracellular absorption of, 422–424, 423f ECaC1/2 expression in fine-tuning transport of, 432 in eggshells/embryos, 851 entry step v. absorption, 416–417, 417f entry step v. calbindins, 418–419 extrusion across basolateral membrane, 419–421, 420f falls v. D3 and, 1814, 1814f fetuses/neonates v. low maternal intake of, 843, 843f food fortification with, 817–818

1852 Calcium (Continued) fractional absorption from human milk/formula, 813, 814t GHS rat response to low dietary, 1349, 1350f homeostasis in muscle cells, 1807-1808, 1808f homeostasis v. caffeine, 1271–1272 homeostasis v. cardiovascular disease, 901 in HVDRR therapy, 1227–1228 IH decreasing renal reabsorption of, 1342 IH v. restricted dietary, 1351 intake/absorption v. age, 1103–1106 intake v. absorption, 412–413, 412f, 413f, 782, 782f, 783–784, 784f intervention studies of colorectal adenoma v., 1623–1624 evidence interpretation in, 1623 implications of, 1624 mechanism in, 1623–1624 intestinal absorption of estrogens’ genomic effects v., 444–445 1,25(OH)2D3 v., 411–424 VDR WT/KO mouse gestation and, 440–442, 441f, 441t, 442f VDR WT/KO mouse lactation and, 441f, 441t, 442–444, 442f, 443f jejuno-ileal bypasses reducing serum, 1303 in keratinocyte differentiation, 615–619, 615f keratinocyte differentiation v. intracellular, 616 kidney/parathyroid VDR regulated by, 522–523, 525t KO mice/human intestinal absorption of, 429–437, 446t long-term OCT administration v., 1530–1531, 1530f low Ca intake v. total body, 781 macrophages unresponsive to, 1382–1383, 1383f malabsorption in gastrointestinal disease, 1297 maternal Ca intake v. breast milk, 847, 847f mechanisms of age-related changes in, 1104–1106 metabolism during lactation/weaning, 843–846 metabolism during pregnancy, 839–840 metabolism v. IH therapeutics, 1351 molecular bases for entry of, 415–416 2MP/2MbisP/2Mpregna v. serum, 1552–1553, 1553f nuclear import regulated by, 371–374 obligatory losses of, 774–776 OCT v. renal failure rat, 1527, 1528f 1,25(OH)2D3 promoting absorption of, 291 1α,25(OH)2D3 regulating muscle, 886–887 1,25(OH)2D synthesis influenced by, 828 1,25(OH)2D3 v. intestinal transport of, 678–680 oral supplements, 1059t PTH defending serum, 1050 PTH/1,25(OH)2D3 in maternal regulation of, 859 PTH v., 552, 553f PTHrP expression/production stimulated by, 741t reabsorption by kidney, 515–516 regulation in ECF, 774–778, 775f renal reabsorption, 6 reproduction v. active absorption of, 440–445 restriction/24-hydroxylase catabolism in D toxicity, 1365–1367, 1366t restriction v. renal 1α-hydroxylase in D toxicity, 1365, 1365t serum 25OHD values v. intake of, 1023–1024 supplementation in metabolic bone disease, 927 systemic/intracellular homeostasis of, 751 transcellular absorption of, 414–421 CA entry v., 415–417, 417f calbindin v., 418 cytosolic transfer, 417–419 D in initiating, 414 extrusion across basolateral membrane in, 419–421, 420f

INDEX

Calcium (Continued) in intestine, 414 model for, 423f, 424 thermodynamic parameters, 414 transport in GHS rats, 1348, 1348f, 1348t VDR ablation v., 341 VDR expression affected by, 202t, 204–205 vesicular transport of, 422 VSCCs inactivated by, 755–756, 756f Calcium channel blockers, D metabolism influenced by, 1255t, 1269 Calcium deficiency, hypocalcemia due to dietary, 1056 Calcium economy, 773–778 body calcium compartments in, 773–774 D in, 773–785 fetal bone mineral accretion v., 841, 841f HVDRR Ca absorption efficiency v., 782 Calcium-sensing receptor (CaR), 551–559 amino acids allosterically activating, 554 as “calciostat,” 552–554 fetal role of, 854 intracellular signaling, 551–554 pathways modulated by, 554 isolation, 551 keratinocytes’ alternately spliced variant of, 616 in keratinocytes’ Ca response, 615f, 616–617, 620f in kidney, 556–558 1,25(OH)2D3 regulating, 543 parathyroid cell proliferation v., 545 in parathyroid gland, 554–556 parathyroid VDR interacting with, 556, 556t in placental Ca transport, 853–854 predicted structure, 551–552, 552f PTH secretion and, 537–538 Calcium transport ECaC1/2 expression in fine-tuning, 432, 432f, 432t genes, VDR-null mouse Ca absorption v. expression of, 435–437, 436t, 437t in GHS rats, 1348 1,25(OH)2D3 v. intestinal, 678–680, 679f vesicular, 422 Calciuria, calbindin-D28K in, 724 Calcospherulites, 1030 Calmodulin Ca entry v., 416 in keratinocyte growth/differentiation, 619 Calreticulin, 543 increased in hypocalcemic rats, 544 1,25(OH)2D3 PTH gene regulation v., 543–544 VDR DBD mutations in binding site of, 1220 Cancer. See also specific types of cancer clinical studies of D analogs v., 1574 clinical studies of D compounds v., 1573–1574 D analog therapy v., 1450–1451 D v., 1571–1577 epidemiology of, 1571–1573 diet/Ca intake/D relationship with, 1572 EB1089 v., 1499–1500 efficacy/safety of D analogs treating, 1504–1505 growth/development v. 1,25(OH)2D3, 1573, 1574t, 1575t Hopkins QW-1624F2-2 hybrid deltanoid v., 1417, 1417f hVDR gene polymorphisms v., 245 models v. D analogs, 1499–1500 nonsecosteroidal D mimics v., 1565 1,25(OH)2D3/analog therapeutic potential v., 169 1,25(OH)2D3/carboplatin combinations v. advanced, 1745, 1746f

INDEX

Cancer (Continued) risk epidemiology v. D/Ca, 1617–1629 soy consumption/CYP24A1 expression v., 98 sunlight/vitamin D v., 42 VDR in, 1571, 1572t VDR polymorphisms v., 1145–1146 Cancer cells D resistance/metabolism in, 1583–1584 EB1089 v. PTHrP production in, 744 24-hydroxylase enzyme regulation in, 93 1α-hydroxylase v. 1,25(OH)2D3 PTHrP inhibition in, 744, 745f 1,25(OH)2D3 inducing apoptosis of, 1580 PTHrP expressed by normal/cancer, 739–740, 739t PTHrP stimulators/inhibitors in normal/cancer, 740–742, 741f CaP. See Prostate cancer CaR. See Calcium-sensing receptor CaR gene, abnormalities v. hypocalcemia, 1053 Carbonate, in bone apatite, 480 Carboplatin, 1,25(OH)2D3 in combination with, 1745, 1746f Cardiomyocytes, D signaling in, 903 Cardiovascular disease, VDR polymorphisms v., 1148 Cardiovascular medicine, 899–905 D dose/response curve, 905, 905f Cardiovascular system D signaling in health of, 899–901 epidemiology of, 899–900 functions v. D, 874–875 indirect D actions on, 901–902 VDR genetics v. disease of, 900–901 Cartilage. See also Growth plates bone forming from calcified, 575 Ca/Phosphate supply v. D metabolites in, 581 calbindin-D9K/D28K in, 730t changes during maturation, 577–578 embryonic variations in, 575 genomic/nongenomic regulation by 1,25(OH)2D3/24,25(OH)2D3, 575–591 metabolism regulated by D, 579–582, 581t mineralization mechanisms in, 480 1,25(OH)2D3 v. 24,25(OH)2D3 in, 582–583 Cartilage oligomeric matrix protein (COMP), D metabolites bound by, 579 Catabolism, 1,25(OH)2D3, 1424–1427 C-26 hydroxylation/26,23-lactone formation in, 1427, 1427f Caucasians. See also Whites; specific nationalities age-adjusted PCa mortality in, 1600, 1600f bone mass in, 793 Cdx2 polymorphism in, 1125f, 1126 D deficiency in American juvenile, 1067 graphical LD display across VDR gene in, 1127, 1128f low hVDR gene frequency in, 244–245 osteoporosis/atraumatic fractures in, 792 osteoporosis/Ca absorption in, 816 UV v. PCa in American, 1601–1602, 1602f, 1603f VDR gene association studies in, 1141 VDR polymorphism allele frequencies in, 1128t, 1129 VDR polymorphisms v. breast cancer in, 1145 VDR polymorphisms v. colon cancer in, 1145–1146 VDR polymorphisms v. diabetes in, 1146 VDR polymorphisms v. PCa in, 1139t, 1145 VDR polymorphisms v. psoriasis in, 1146 CB1093, metastasis v., 1576 CB 966, synthesis, 1490, 1491f, 1494t CC. See Chief complaint Cdk5, in monocytic differentiation marker expression, 1639

1853 Cdk inhibitory (CDKI) proteins Cdk regulated by, 1643–1644, 1643f 1,25(OH)2D3/deltanoids up-regulating, 1646–1649, 1647t–1648t Cdk5/p35 pathway, in 1,25(OH)2D3 differentiation signal propagation, 1639 CDKIs. See Cdk inhibitory proteins Cdks. See Cyclin-dependent kinases Cdx2 polymorphism, 1130, 1131t bone fracture risk v., 1144 in Caucasians, 1125f, 1126, 1130 in Japanese, 1126, 1130 polymorphism functionality parameters associated with, 1135f, 1137 Celiac disease Ca malabsorption in, 1297 clinical features of, 1300–1301 D deficiency development in, 1301 management, 1301 Cell cycle apoptosis v. differentiation at block in, 1580 compartments/checkpoints, 1640–1641, 1641f D influencing, 1577–1580, 1578f–1579f deltanoid-induced differentiation v., 1640–1650 deltanoids modulating events in, 1646–1650 differentiation v., 1635–1651 20-epi D analogs regulating, 1498 G1 arrest v. c-Myc expression down-regulation, 1649–1650 G1 block controlled by retinoblastoma protein, 1649 G2 retardation v. polyploidization, 1650 G1/S block, 1646–1650, 1646f machinery features, 1640–1642 1,25(OH)2D3/EB1089 in arrest of breast cancer, 1663–1664, 1672f oncogenes/tumor suppressor genes in, 1577–1580 Cell cycle progression G2/M phase transition in, 1645, 1645f G1/S phase transition in, 1643–1644, 1643f mechanisms driving, 1642, 1642f regulation of, 1643–1645 S phase DNA licensing in, 1644–1645 Cell cycle traverse Cdks controlling, 1642, 1642f checkpoints controlling, 1641, 1641f 1,25(OH)2D3 inhibiting, 1646–1649, 1646f Cells. See also specific types of cells antioxidant mechanisms of, 762 D in oxidative stress response of, 761–768 DBP associated with, 123 higher D levels in health of, 1370–1371 HVDRR studies using various, 1218 1,25(OH)2D3 v. growth/differentiation of cells, 696 PTHrP expressed by normal/cancer, 739–740, 739t PTHrP stimulators/inhibitors in normal/cancer, 740–742, 741f redox state of, 761–762 ROS causing death of, 761 cementoblasts, cementum elaborated by, 599 Cementum bone sharing matrix components with, 602 D bioinactivation causing hypomineralization in, 602 in tooth root/periodontium, 601–602 Central nervous system (CNS) neurodegenerative disease etiology/physiopathology in, 1779–1780 1,25(OH)2D3 actions in, 1781–1783 1,25(OH)2D3 v. tumors in, 1783–1784 VDR/1,25(OH)2D3 targets in, 1780–1781

1854 Cerebrotendinous xanthomatosis (CTX), CYP27A1 expression v., 58–59 CGRP. See α-Calcitonin gene-related peptide Checkpoints, cell cycle traverse controlled by, 1641, 1641f CHF. See Congestive heart failure Chickens D deficiency v. fertility in, 854–855 embryonic development/egg hatchability v. D in, 851–852 Chief complaint (CC), in metabolic bone disease diagnosis, 915 Children. See also Infants; Puberty adolescent Ca absorption in, 815–817 factors influencing, 815–816 early hypocalcemia in, 1036t, 1037 inadequate Ca/D intake in, 816 nutritional rickets in, 968, 969f soda v. Ca intake by, 817 Ca absorption in prepubertal, 815 Ca/D-fortified foods for, 817–818 CA intake by developing countries’, 817 D deficiency and nutritional rickets in, 1065–1077 D deficiency/Ca absorption in, 811–818 deficiency in breast-fed, 1067 PTH resistance in rachitic, 1069 Chinese bone mass in, 793 Bsml RFLP v. nephrolithiasis in, 1142 Buddhist vegetarians v. metabolic bone disease, 917t Ca v. colorectal cancer in, 1622 D metabolism in, 793 PCa mortality/incidence in, 1624–1625 sunlight exposure v. rickets in, 1066–1067 VDR polymorphism v. PCa risk in, 1682 VDR polymorphisms v. colon cancer in, 1145 Chloride channel (ClC)-5, in Ca homeostasis, 159 Chloride channels, 1α,25(OH)2D3 opening ROS 17/2.8 cell, 393, 394f Cholecalciferol. See Vitamin D3 Cholesterol bile acids converted from, 863, 864, 864f D3 derived from, 931 D endocrine system having low concentration of, 1001, 1001f Chondrocytes in bone growth, 498–499, 499f cartilage produced by, 575 cell maturation specific Ca2+ ion kinetics in, 585 chickens/rats modeling growth plate, 577 D metabolite action mechanisms in, 587–588, 587f, 588f lineage of, 575–577 maturation of growth plate, 108 maturation rates of, 575–576 membrane signaling in, 586–587 mineralization v. hypertrophic, 576–577, 576f 1,25(OH)2D3-deficient matrix vesicles produced by, 580 1,25(OH)2D hormone v. differentiation/function of, 112–113 phospholipids altering membrane fluidity in, 585 stereospecific membrane receptors in, 586 zone of maturation v. D3 metabolite response of, 582 Chondrodysplasias, rickets v. differential diagnoses for, 984, 987f Chromatin architecture in nucleus, 328, 329f, 330f ATP-dependent remodeling complexes for, 267–268 binding v. NR co-repressors, 292, 293f in comodulator activity integrated model, 300, 300f Mediator-D HAT activity v. remodeling, 267, 295, 299–300 osteocalcin gene organized by, 330–332

INDEX

Chromatin (Continued) PBAF repressing, 238–240, 239f remodeling facilitating promoter accessibility/regulatory integration, 329–332 remodeling in VDR promoter/VDR targeting, 305–312, 306f remodeling in VDR transcription model, 268–269, 269f remodeling v. DNA repair/replication, 307 RUNX elements in remodeling, 332 WINAC in reorganizing, 310, 311f WINAC inducing ATP-dependent remodeling of, 308–309, 309f Chromatin immunoprecipitation (ChIP) coactivator/transcription factor assembly details from, 299–300 NCoA62SKIP/VDR-activated transcription influence shown by, 297 OC gene promoter organization remodeling v., 332 VDR/WINAC association shown by, 309 Chronic kidney disease (CKD). See also Chronic renal failure abnormal 1,25(OH)2D3/VDR activity in, 1320–1322, 1320f bone loss in, 1325 D bioactivation to 1,25(OH)2D in, 1313–1317 decreased renal mass/GFR v., 1313–1314, 1314f PTH/Ca induction of renal 1α-hydroxylase v., 1315–1316 renal 1α-hydroxylase substrate availability v., 1315 defective homologous VDR up-regulation in, 1317–1318 mechanisms impairing 1,25(OH)2D3/VDR transcriptional activity, 1320–1321, 1320f megalin expression/1,25(OH)2D3 resistance in, 1319 1,25(OH)2D/VDR action altered in, 1317–1322 TGFα/EGFR expression v. parathyroid hyperplasia in, 1323–1324, 1323f, 1324f uremia v. 1,25(OH)2D3/VDR transcriptional activity in, 1320–1321, 1321f VDR polymorphisms v. expression/function in, 1318–1319 Chronic renal failure D therapy in, 1327–1332 1,25(OH)2D3 v. secondary hyperparathyroidism in, 1327–1331, 1330f, 1331–1332, 1332f Chugai OCT cell-specific catabolism of, 1459–1460 DBP affinity v. plasma levels, 1457–1458, 1457f hypercalcemia of malignancy v., 1450–1451 mouse primary immune response v., 1451 PTH suppression/low calcemic activity by, 1449–2898, 1450f SAR in design of, 1412, 1413f Cimetidine, D metabolism influenced by, 1255t, 1270 Cirrhosis, hypocalcemia/secondary hyperparathyroidism in primary biliary, 1056 CKD. See Chronic kidney disease ClC-5. See Chloride channel-5 CNS. See Central nervous system Coagulation cascade, cardiovascular disease v. D regulating blood, 902 Cod liver oil historical importance of, 70 rickets v., 4 Collagen in bone matrix scaffolding, 485 D influencing, 485–486, 486f factors modulating synthesis of, 703–704 molecular mechanisms regulating, 705–706 1,25(OH)2D3 inhibiting organ culture synthesis of, 704 1,25(OH)2D3 regulating expression of type I, 703–705 1,25(OH)2D3 regulating matrix proteins in type I, 703 synthesis regulation in bone, 704–705 Colon dietary Ca absorption in, 424

INDEX

Colon (Continued) 1α-hydroxylase expression in human, 1718–1719, 1719f 1α-hydroxylase/VDR/Ki-67 coexpression in, 1718–1719, 1719f 1α,25(OH)2D3-mediated rapid response in, 386t 1α,25(OH)2D3 VDRnuc in, 385t VDR’s clinical relevance to, 246–249 Colon cancer D supplementation v. fat-promoted, 246 D v., 1709–1721 D/VDR v., 866–867 EB1089 v., 1500 20-epi D analogs v. IGF-II in, 1498–1499 high-fat diet v., 866–867, 1572 rationale for Gemini analogs treating, 1521–1522 sunlight/latitude v. death rate from, 1571–1572 sunlight v. incidence of, 246 VDR/EGFR expression in, 1710–1711, 1711f, 1711t VDR gene polymorphisms v., 1619–1620 VDR polymorphisms v., 1145–1146 VDR polymorphisms v. development of, 1712 Colon cancer cells D metabolism in lines/primary cultures of, 1715–1717, 1716f metabolic/catabolic D hydroxylases expressed in, 1717 1,25(OH)2D3/analog antiproliferative activity v., 1521–1522, 1522f, 1523f, 1523t 1,25(OH)2D3/deltanoid-induced differentiation modeled in, 1637t 1,25(OH)2D3/Ro-26–9228 v., 1480, 1480f Colon cells D metabolism in normal/neoplastic, 1715–1719 1,25(OH)2D3 action on neoplastic, 1710–1715 1,25(OH)2D3/D analogs v. proliferation/differentiation/apoptosis of, 1712–1715 clinical studies of, 1715 in vitro, 1712–1714 in vivo animal models for, 1714–1715 VDR in normal/malignant, 1710–1712 Colorectal adenoma Ca v., 1622–1623 D plasma markers v., 1619 dietary D v., 1619 intervention studies of Ca v., 1623–1624 evidence interpretation in, 1623 implications of, 1624 mechanism in, 1623–1624 VDR gene polymorphisms v., 1619–1620 Colorectal cancer, 1618–1619 Ca v., 1621–1624 case-control/cohort studies of Ca v., 1622 circulating D v., 1618–1619 D dietary/supplementary intake v., 1618 dietary Ca v., 1621–1622 risk epidemiology v. D/Ca, 1617–1629 sunlight exposure v., 1618 Colorectal neoplasms, 1618–1624 Combination therapy advanced cancer v. 1,25(OH)2D3/carboplatin, 1745, 1746f in animal PCa models, 1689 calcipotriol in psoriasis, 1504, 1504t cancer cell differentiation v., 1582–1583 falls v. D3/Ca, 1814, 1814f leukemia v. D compounds in, 1733–1734 PCa, 1695–1698 PCa v. 1,25(OH)2D3/taxane, 1745–1746 psoriasis, 1504, 1504t COMP. See Cartilage oligomeric matrix protein

1855 Competitive chemiluminescence immunoassay (CLIA), 25OHD quantitation using 125I-RIA v. automated, 939, 939f, 940f, 940t Competitive protein binding assays (CPBAs) for D/25OHD, 931–932 25OHD assay consistency v., 1023 Computerized tomography (CT), tumors causing TIO v., 988 Congestive heart failure (CHF), VDR polymorphisms v., 1148 Cortical thick ascending limbs (CTAL), CaR controlling Ca/Mg reabsorption in, 557, 557f Corticosteroids active intestinal Ca absorption v., 445 D metabolism influenced by, 1255t, 1265–1266 CPBAs. See Competitive protein binding assays Crohn’s disease bone disease in, 1302 Ca malabsorption in, 1297 D/25OHD/Ca malabsorption in, 1302 hypercalcemia/D hypersensitivity in, 1361 management of, 1302 VDR polymorphisms v., 1147 CT. See Calcitonin; Computerized tomography CT/CGRP gene alternative RNA processing of, 689, 689f cAMP-induced enhancer, 691 cAMP/neuroendocrine enhancers in 1,25(OH)2D3-inhibited transcription of, 693, 695f cAMP responsive enhancer, 690f mapping negative VDRE of, 694-696, 695f neuroendocrine-specific enhancer, 690f neuroendocrine-specific HLH enhancer, 691–692 MAP kinase stimulation/sumatriptan repression, 692, 692f 1,25(OH)2D3 transcription repression mechanism v., 696, 696f, 696t CT gene. See Calcitonin gene CTAL. See Cortical thick ascending limbs CTE. See C-terminal extension CTX. See Cerebrotendinous xanthomatosis Cubilin, 158–159, 158f Cyclin-dependent kinases (Cdks), cell cycle restriction points v., 1642, 1642f CYP24. See 24-Hydroxylase CYP27. See 25-Hydroxylase CYP1α mRNA v. 1α,25(OH)2D3, 75, 75t mRNA v. PTH, 76, 76t promoter activity v. 1α,25(OH)2D3, 76, 76t promoter activity v. PTH, 76–77, 77f CYP24A1. See 24-Hydroxylase CYP24A1 human, 91 rat, 91 cyp24A1, 24-hydroxylation v., 107–109 CYP27A1. See 25-Hydroxylase CYP27A1 intraacinar localization, 58–59 1,25(OH)2D3 administration v., 57–58, 58f, 59f regulation/enzyme activity, 56–58 transcription v. bile acids, 57 cyp27A1, hepatic 25-hydroxylation v., 105–106 CYP1α gene, extrarenal 1,25(OH)2D synthesis v., 1380 CYP24A1 promoter, rat, 93–97 mutational analysis v., 95–97, 96f, 97f CYP27B1. See 1α-Hydroxylase CYP27B1, structure, 74, 74f cyp27B1, 1α-hydroxylation v., 109–113 CYP2C11. See 25-Hydroxylase

1856

INDEX

CYP2D25. See 25-Hydroxylase CYP2R1. See 25-Hydroxylase Cytochrome P4501α, 1442 cloning/gene structure, 73–75, 74f Cytochromes P450 function of, 88 isoform modeling studies of, 1443 molecular modeling of, 88–89, 89f nomenclature for, 105, 106t in 25OHD3 1α-hydroxylation, 71–72, 71f ROS generation implicating, 762 Cytokines in keratinocyte growth/differentiation, 614 in macrophage 1α-hydroxylase regulation, 1384–1385 Cytoplasm, protein import retarded by docking in, 374 Cytotoxic T cells, 1,25(OH)2D3 autoimmunity v., 1755 Cytotoxic therapy, hyperphosphatemia in, 1178

D D. See Vitamin D D2. See Vitamin D2 D3. See Vitamin D3 Daivobet, in combination therapy of psoriasis, 1504, 1504t DBDs. See DNA binding domains DBP. See Vitamin D binding protein DBP-A, DBP-B structure compared with, 135–136 DBP-actin complexes, 122t, 123, 142–149 other actin complexes compared with, 147 structure of, 143–147, 143f β/γ-actin v., 145t, 146–147 hydrophobic DBP residue interaction v., 144t–145t, 146, 146f structures reported for, 149 tissue necrosis/cell disruption increasing, 126, 142 DBP-B, DBP-A structure compared with, 135–136 DBP-vitamin D complex, D binding site structure in, 137–142, 139f–140f biological implications of, 142 VDR D pocket structure v., 142 DCs. See Dendritic cells DCT. See Distal convoluted tubule Deficiency, 968–974 abnormal fetal organ development v., 852–853 adolescent, 816 age-dependent signs/symptoms of, 916t animal fertility reduced by, 854–855, 854t bisphosphonate antiresorptive effect v., 1114 in breast-fed children, 1067 Ca deprivation increasing susceptibility to, 1051–1052, 1052f cartilage v., 579 resorption of, 581 in celiac disease, 1301 classifying states of, 1024–1026 correction in osteoporosis treatment, 1110–1111 D absorption/input v. risk of, 784 development of acquired, 1296, 1296f development of depletion and, 1293–1296 diet and contemporary, 777–778 ECF [Ca2+] demand response mediated by, 776–777, 777f in elderly v. D synthesis/lifestyle, 823–824 epidemic in industrialized countries, 42 epidemiology of nutritional rickets/D, 1066–1067 HDM v. insulin resistance in, 1816 immune abnormalities associated with, 1389 in infancy/childhood, 811–818

Deficiency (Continued) insufficiency v., 1085–1086 intrinsic/extrinsic, 1029 in last trimester of pregnancy, 803–804 many diseases causing, 968 mild/moderate/severe stages of, 1024, 1025t mineralization v., 487–488 muscle myopathies in, 893 myopathy in, 1805 neonatal Ca metabolism v. maternal, 841 nutritional rickets in children v., 1065–1077 radiographic abnormalities of rickets v. treating, 970f, 971, 974f, 982f RAS v. blood pressure/cardiovascular function in, 875 risk groups, 1026 spiral of developing, 1294, 1295f ubiquity of, 796, 1024–1026, 1025f in vivo β cells v., 1764 7-Dehydrocholesterol (7-DHC) D3 biosynthesis from, 1405, 1408f decreased in elderly subjects, 823 keratinocytes producing D3 from, 609, 610f Dehydroepiandrosterone (DHEA) OCT production using, 1526 as steroid precursor in deltanoid synthesis, 1412, 1413f Delayed-type hypersensitivity (DTH), 1,25(OH)2D3 v. passive transfer of, 1754, 1755f Deltanoids c-Myc v. differentiation/G1 arrest induced by, 1649–1650 catabolism-inhibiting, 1406–1408 CDKIs up-regulated by, 1646–1649, 1647t–1648t cell cycle events modulated by, 1646–1650 cell cycle v. differentiation induced by, 1640–1650 cell type v. proliferation inhibition by, 1650 cellular models of differentiation induced by, 1635–1636, 1637t differentiation induced by, 1635–1640 generalized structure of, 1405, 1407f metabolic rationale guiding development of, 1405–1408 molecular biology rationale in development of, 1408–1411, 1410f multistep synthesis of hybrid, 1416–1418, 1416f, 1417f, 1418f, 1419f nearby structural changes v. catabolism of, 1407–1408, 1407f, 1410f neoplastic cell proliferative quiescence induced by, 1635 1,25(OH)2D3 hypercalcemia/calcification v., 1405 organic chemistry rationale in development of, 1411–1418 physiologically active metabolites of, 1408 potency of 22-ethyl, 1409, 1411f prodrugs/indications in, 1405, 1406f rational design of, 1405–1418 remote structural changes v. catabolism of, 1407, 1410f 6-s-cis-locked, 1412, 1412f SAR in design of 22-oxa, 1412 steroid precursors in constructing, 1412–1416, 1413f, 1414f, 1415f Dendritic cells (DCs) in acute allograft rejection, 1519 adaptive immune responses mediated by, 631 in generating effector/regulatory T cells, 634, 634f immunointervention targeting, 632–633, 632t 1,25(OH)2D3 inducing tolerogenic, 1519 VDR ligand immunoregulation of, 633–635 Dengue fever, VDR polymorphism v. hemorrhagic form of, 246 Dentin characteristics, 600–601 odontocytes elaborating, 599 in tooth root/periodontium, 601–602

INDEX

Dentin phosphoproteins (DPPs), high degree of phosphorylation in, 600–601 Dentin sialoprotein (DSP), in dentin, 601 Dento-alveolar bone complex, 599–605 formation/functions, 599–602 Depression, D3 preventing, 999t DEXA. See Dual energy x-ray absorptiometry Dexamethasone high dose intermittent 1,25(OH)2D3 with, 1744–1745 WT mouse intestinal Ca absorption v., 445, 447f 7-DHC. See 7-Dehydrocholesterol DHEA. See Dehydroepiandrosterone Diabetes in animal models v. 1,25(OH)2D3/analogs, 1768–1772 calbindin-D28K Ca buffering v. type I, 726 calbindin-D28K v. apoptotic cell death in, 727 D/immune system in type 1, 1767–1773 D signaling in regulating, 899 D v., 1763–1774 early intervention with 1,25(OH)2D3/analogs, 1768–1770, 1768t incidence v. sunlight exposure, 1766 insulin v. 1α-hydroxylation in, 1259 late 1,25(OH)2D3 intervention v., 1770 1,25(OH)2D3 analogs v., 1772–1773 1,25(OH)2D3/analogs with immune modulators v., 1771–1772, 1771t 1,25(OH)2D3 v. NOD mouse, 1404 risk v. D3, 999t risk v. VDR polymorphism, 1773 VDR ligand treatment v. type 1, 637–638, 637t VDR polymorphism associated with, 246 VDR polymorphism v., 1146–1147 Diabetic ketoacidosis, hypophosphatemia in, 1177 Diet age-related 25OHD decrease v., 1101 bone mass v. macrobiotic/vegetarian, 795 Ca deficient, 1074–1076 Ca in paleolithic/contemporary, 777–778 celiac disease v., 1300–1301 colon cancer v. high-fat, 866–867, 1572 colorectal cancer v. Ca from, 1621–1622 D hormonal system dependent on high Ca, 780–781, 781f D insufficiency v., 1087–1088, 1087f D3 intake from, 1026 D metabolism influenced by, 789–796 D metabolism v. vegetarian/omnivorous, 795 D negligible in Western, 1599 D status v. normal, 1066 D supplied by, 1294, 1294f, 1617 D toxicity from, 1356 ECF [Ca2+] demand response mediated by, 777–778 elderly v. D intake from, 824 groups/sects/ethnicities with vegetarian, 917t 25OHD half-life reduced by high fiber, 1076 osteoporosis v. Ca/D supplements to, 1112–1113, 1113t as PCa risk factor, 1681 PCa v. Ca/D from dairy products in, 1627–1629 PCa v. D from, 1606, 1625 rickets v. macrobiotic/vegetarian, 1067, 1077 VDR expression affected by, 204-205 vegetarian, 795, 917t, 1067, 1077 Differentiation cell cycle v., 1635–1651 cell cycle v. deltanoid-induced, 1640–1650 cellular models of 1,25(OH)2D3/deltanoids-induced, 1635–1636, 1637t

1857 Differentiation (Continued) 1,25(OH)2D3/deltanoids inducing, 1635–1640 1,25(OH)2D3 influencing tumor cell, 1580–1581 1,25(OH)2D3 signal propagation pathways v., 1636–1639 1,25(OH)2D3 synthesis/catabolism v., 1717 1,25(OH)2D3 v. PCa cell, 1691–1692 signals for 1,25(OH)2D3 influences on, 1636 TF role in 1,25(OH)2D3-induced differentiation, 1639–1640 DiGeorge sequence, hypocalcemia v., 1052–1053 Digital rectal exam (DRE), in PCa diagnosis, 1679 Dihydrotachysterol, metabolism, 1429–1430, 1430f 1,25-Dihydroxy–16-ene-23-yne D3 (BXL-353) BPH cell apoptosis/Bcl-2 expression v., 1836–1837, 1838f BPH cells v. antiproliferative influence of, 1836–1837, 1837f in vivo prostate growth v., 1837–1840, 1839f 10,19-dihydro–1α,25-dihydroxyvitamin D3, activity, 1544 19-Nor-1,25-dihydroxyvitamin D3, early 2-carbon analogs of, 1545 19-Nor-1α,25-dihydroxyvitamin D3 2-carbon-modified analogs of, 1543–1554 synthesis, 1544 23(S),25(R)1,25-Dihydroxyvitamin D3-26,23-lactone, in C-23 oxidative pathway discovery, 23 2α-Methyl-19-nor-(20S)-1,25-dihydroxyvitamin D3 (2αMD), 1545–1546 bone selectivity by, 1546–1547 structure, 1545f 1α,24R-Dihydroxyvitamin D3 [1α,24R(OH)2D3], 1425t–1426t, 1439 C24 oxidation steps in catabolism of, 1426f, 1439 metabolism, 1439 psoriasis treatment using OCT v., 1533 skin irritation in psoriasis treatment with, 1785 2β-Methyl-19-nor-(20S)-1,25-dihydroxyvitamin D3 (2BMD), 1545–1546 structure, 1545f 2-Methylene-19-nor-(20S)-1,25-dihydroxyvitamin D3 (2MD), 1545–1546 bone anabolic activity of, 1546–1547, 1547f bone selectivity by, 1546–1547 1,25(OH)2D3 v., 1546, 1546f ovariectomized rat bone mass v., 1547, 1548f structure, 1545f tissue selectivity/enhanced potency, 1548–1551 VDR binding to promoters stimulated by, 1549–1550, 1549f VDR conformation induced by, 1550, 1550f VDR interactions promoted by, 1550–1551, 1551f 1,25-Dihydroxyvitamin D [1,25(OH)2D], 1389–1390 age v., 1102–1103 as autocrine hormone in prostate, 1607–1610 Ca absorption v. age-related decrease in, 1104 Ca absorption v. age-related resistance to, 1104–1105, 1105f CKD altering VDR-mediated actions of, 1317–1322 CKD v. D bioactivation to, 1379–1383 clearance v. age, 1103 clinical evidence for dysregulated overproduction of, 1381 concentration having clinical relevance, 948t, 949 cutaneous production of, 609–613 CYP1α gene cloning v. extrarenal synthesis of, 1380 detecting, 942–947 competing approaches, 946–947, 946f, 947f issues/improvements v., 946–947 diseases v. hypercalcemia/hypercalciuria mediated by, 1390–1392, 1390t estimating human serum, 942, 943t Geminis as analogs of, 1511–1522 gene expression regulated by, 1212, 1213f in GHS rats, 1348, 1348f

1858 1,25-Dihydroxyvitamin D [1,25(OH)2D] (Continued) granulomatous disease inflammation sites accumulating, 1389 as HDM cause, 1815–1816 health v. changed concentration of, 1102 heightened immunoreactivity elevating, 1387f, 1389 hormonal regulation of, 610–611 hypercalcemia in lymphoma v., 1362 hypocalcemia due to hereditary resistance to, 1056 IH elevating, 1341–1342, 1342f inflammatory arthritis/RA inflammation sites accumulating, 1389–1390 inhibiting its own production, 20 in keratinocytes, 611 intracrine/autocrine action on monocytes/macrophages, 1387–1388, 1387f intravenous therapy v. renal failure, 1822–1823 levels v. Ca absorption in IH, 1342, 1343f macrophages lacking 24-hydroxylase activity directed by, 1383–1384 mechanism of action, 1210–1212 mechanisms of age-related changes in, 1102–1103 metabolism kinetics, 828 in muscle regeneration, 1816 25OHD hydroxylation producing, 1599 25OHD plasma half-life v. concentration of, 1104, 1104f 25OHD seasonality tracked by, 1019–1020, 1020f paracrine suppression of lymphocytes, 1387f, 1388–1389 PCa v., 1610–1611, 1611f PCa v. serum, 1602–1604 in PDDR, 1198–1199, 1199f plasma concentration, 28t production v. diseases, 1359–1362 psoriasis v. serum, 1784 resistance causing secondary hyperparathyroidism in renal failure, 1821–1823 resistance mechanisms in chronic renal failure, 1822, 1822f RIA methodology for detecting, 945–947, 945f assay calibrator preparation for, 945 sample/calibrator extraction/pretreatment in, 945 solid-phase extraction/silica purification chromatography in, 945 RRA methodology for detecting, 943–945, 944f calf thymus VDR preparation for, 943 RRA in, 944f sample extraction for, 943 solid-phase extraction/purification chromatography in, 943, 944f SCC keratinocytes producing, 612 serum concentration/PR/MCR v. aging, 828, 828f substrate-dependent synthesis of, 1019–1020, 1020f trophic factors influencing synthesis of, 828–831 in vitro correlates for dysregulated overproduction of, 1381–1387 1,25-Dihydroxyvitamin D2 [1,25(OH)2D2], isolation/identification, 19 1,25-Dihydroxyvitamin D3 [1,25(OH)2D3], 167–184, 412 A-ring in VDR transactivation, 1472f, 1473–1474 action mechanisms in autoimmunity, 1754–1756 actions in CNS, 1781–1783 actions in VSMCs, 902–903 as active form of vitamin D, 19, 782 in analog synthesis, 1490, 1490f analogs in oncology, 1746–1747 angiogenesis v., 1574–1576, 1693 in animal cancer models, 1573, 1574t animal model diabetes prevented by, 1768–1772 as antioxidant, 764t, 765–767 AUC for lowest anti-tumor dose in mice, 1744, 1744t

INDEX

1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] (Continued) autoimmunity v., 1753–1759 animal models of, 1756, 1757t human, 1756–1759 avian parathyroid PTH/VDR gene expression regulated by, 541–542 β cells v., 1764–1767 basis for 25OHD interaction with, 783 biological effects in psoriasis, 1781, 1781f biphasic effects on osteoblasts, 650 blood pressure inversely associated with, 874, 874f bone apposition rates influenced by, 656–657 brain calbindins/Ca-binding proteins v., 1781 in brain detoxification, 1783 in brain homeostasis control, 1784, 1785f in breast cancer angiogenesis/invasion/metastasis, 1666–1667 breast cancer cell apoptosis induced by, 1665–1666, 1666f, 1672f breast cancer cell proliferation v., 1663–1665, 1672f breast cancer cells acted on by, 1663–1667, 1664t breast cancer sensitivity to, 1667–1668 breast cancer v., 1663–1672 C-/D-/E-ring modifications of, 1562–1565, 1563f C-3 epimerization, 24, 25f Ca/P demand regulating, 566 calbindin-D9K gene expression regulated by, 729 calbindin-D28K gene expression regulated by, 728 calcium channels regulated by, 1782–1783 in cancer combination therapy, 1582–1583 cancer growth/development v., 1573, 1574t, 1575t 20-carbon epimerization, 1544 cartilage differentiation requiring, 582 cartilage regulated by, 575–591 catabolism, 1406, 1409f, 1424–1427 C-26 hydroxylation/26,23-lactone formation, 1427, 1427f CDKIs up-regulated by, 1646–1649, 1647t–1648t cell cycle influenced by, 1577–1580, 1578f–1579f cell growth/differentiation v., 696 cell type v. proliferation inhibition by, 1650 cellular effects on leukemic cells, 1731, 1731t cellular models of differentiation induced by, 1635–1636, 1637t CKD v. catabolism of, 1316–1317 clinical use in psoriasis, 1784–1787 CNS activity v. in vitro investigations, 1781–1783 CNS tumors v., 1783–1784 colon cancer cells v. antiproliferative activity by, 1521–1522, 1522f, 1523f, 1523t colon cancer v., 1709–1721 epidemiology of, 1709–1710 colon cell proliferation/differentiation/apoptosis v. clinical studies of, 1715 in vitro, 1712–1714 in vivo animal models for, 1714–1715 colon tumors prevented by, 1709–1710 concentration v. immunosuppression/hypercalcemia, 1753 conformationally flexible seco-B-ring of, 1408f, 1412 CT/CGRP gene transcription repression mechanism of, 696, 696f, 696t CT levels regulated by, 689–691 CYP24A1 expression induced by, 93–97 cytotoxic agents combined with, 1745–1746 D endocrine system VSCCs influenced by, 751–757 D mediation v., 1040–1042 D-ring in VDR transactivation, 1475 in diabetes early intervention, 1768–1770, 1768t in diabetes late intervention, 1770 diabetes v., 1763–1774

INDEX

1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] (Continued) diabetes v. immune modulators combined with, 1771–1772, 1771t differential cell response to, 651 differentiation induced by, 1635–1640 differentiation signal propagation pathways, 1636–1639 DNA motifs mediating stimulatory effects of, 705–706 dose escalation using QOD schedule, 1742–1743, 1743t early clinical trials of, 1742 epidermal keratinocytes/dermal inflammation v., 1781, 1782f 20-epimerization, 1494–1495, 1496t factors influencing concentration of, 517t fat/bone progenitor cell commitment v., 650f, 655–656 formation, 516–517 G1 block v. transient p21Cip1 up-regulation by, 1646–1648, 1646f GADD45 expression stimulated by, 1577, 1578f–1579f genomic actions’ molecular basis, 313–314 GFR v. serum, 1314, 1314f growth inhibition effect requiring 1α-hydroxylase, 744, 745f high dose intermittent, 1743f, 1743t, 1744–1745 high dose intermittent regimens of, 1742–1743 hormone/GF influence on osteoblasts v., 654–655 human PCa cell line proliferation v., 1683, 1683f hypercalcemic effect limiting use, 542 hyperparathyroidism v. intravenous, 1330–1331 IGF system interacting with, 1581–1582 immunosuppressive agents synergistically influencing, 1520 insulin secretion influenced by, 1764–1765 in intestinal Ca absorption, 411–424 intestinal calbindin increased by, 722–723 isolation/identification, 291 keratinocyte growth/VDR expression regulation linked in, 1781 kidney/parathyroid VDR regulated by, 522–523, 525t laboratory/clinical extrapolations of exposure to, 1744, 1744f, 1744t leukemic cell lines v., 1730–1731, 1730t ligand availability v. breast cancer sensitivity, 1666–1667 low-affinity neurotrophin receptor regulated by, 1781 2MD v., 1546, 1546f membrane-initiated Ca2+ responses to, 753–754 metabolically influencing β cells, 1764–1767 metastasis v., 1576 2-methylene/2α-methyl/2β-methyl derivatives of, 1545–1546, 1545f molecular mechanisms for leukemic cell influence by, 1731–1733 in MS pathophysiology, 1784 multiple membrane receptors for, 1462 as negative endocrine regulator of RAS, 875–878 as neuroactive hormone, 1779–1785, 1785f normal hematopoiesis influenced by, 1729–1730 NOS II v., 1783 novel target genes of, 1693–1695, 1694t oncogene expression regulated by, 1577–1579 oncology using 1,25(OH)2D3 analogs and, 1741–1747 in osteoblast differentiation/activity, 649–658 osteoblast differentiation pathway/status in vitro v., 649–653, 650f osteoblast heterogeneity not caused by, 652 osteoblast proliferation/differentation-associated genes v., 653–654 in osteoporosis treatment, 1111 parathyroid glands influenced by low, 1324–1325, 1325f parathyroid size correlating with resistance to, 1823 PCa cell apoptosis v., 1691 PCa cell growth arrested by, 1690–1691 PCa cell growth inhibition mechanisms, 1690–1695

1859 1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] (Continued) PCa cell invasion/metastasis inhibited by, 1693 PCa cell resistance to antiproliferative effect of, 1684–1685 PCa growth inhibited in vitro by, 1683–1685 PCa treatment clinical trials, 1698–1699, 1698t PCa v. androgen interaction with, 1687–1688 phytoestrogens regulating synthesis of, 1720–1721, 1720t pleiotropism, 1403 in postmenopausal osteoporosis treatment, 1111–1112 potency/toxicity in PCa treatment, 1689–1690 pre-pro PTH mRNA v., 1527, 1528f primary PCa cell cultures v., 1683–1684, 1684f as prooxidant, 763–765, 764t prooxidant/antioxidant activities, 764t, 768 proposed hormonal mechanism, 167, 168f prostate cells synthesizing, 1607, 1608f prostate cells v. antiproliferative influence of, 1607–1608, 1609f psoriasis v. oral, 1785, 1785f psoriasis v. topical, 1784–1785 PTH gene expression regulated by, 539–543 PTH in chronic renal failure v. intravenous, 1329–1330, 1330f PTH-induced Ca2+ influx primed by, 755, 755f PTHrP production inhibited by, 741t, 743, 1576–1577 PTHrP production v. EB1089 analog of, 744 rapid action on growth-zone chondrocytes, 587–588, 587f reformulation for high dose intermittent regimens, 1746 renal Ca/P handling influenced by, 518–519 renin activity inversely associated with, 874, 874f resistance in breast cancer cells, 1667–1668 resistance mechanisms besides VDR mutation, 1226–1227 resistance v. uremia in CKD, 1320–1321, 1321f retinoids/thyroid hormones effects interdependent with, 655 ROS levels/redox-associated molecules v., 767, 767t Runx2/Cbfal TF modulated by, 566–567 19-nor/10,19-saturated derivatives of, 1544 secondary hyperparathyroidism in chronic renal failure v., 1327–1331, 1330f self-induced metabolism, 22 sex steroids synergistically stimulating synthesis of, 1260 side chain in VDR transactivation, 1474–1475 side effects v. medical applications, 142 signals of differentiation influences by, 1636 in skin, 1780 structural requirements for VDR transactivation by, 1472–1475, 1472f structure, 1472f, 1490f, 1558f supplementation safety, 1112–1113 targets in CNS, 1780–1781 TFs in differentiation induced by, 1639–1640 Th subset lymphokine production v., 1754, 1755f therapeutic use v. calcemic/phosphatemic activities, 1449 therapy in chronic renal failure, 1327–1332 tissue v. abnormal VDR function/low, 1322–1327 toxicity, 1357 tumor cell differentiation v., 1580–1581 tumor growth factors/receptors v., 1581–1582 tumor suppressor gene expression regulated by, 1579 VDR expression affected by, 201–204, 202t VDR mediated growth inhibition v. differentiation, 1713, 1713f virally transformed prostate cells v., 1684 in vitro D metabolism/catabolism v., 1717 in vitro osteoblasts influenced by, 649–656 VSCCs Ca2+/transcriptional responses to, 756–757 VSCCs v., 753–754 1α,25-Dihydroxyvitamin D3 [1α,25(OH)2D3], 381 biological actions mediated by VDRnuc, 384–385, 386t

1860 1α,25-Dihydroxyvitamin D3 [1α,25(OH)2D3] (Continued) biological response generation pathways, 383, 385f biosynthesis, 1408f bonds in 16-ene, 23-yne analog of, 1425t–1426t, 1436 bone matrix protein target genes, 711–717 conformational flexibility, 382f, 383, 388, 391, 399–400, 399f deltanoids emulating conformational flexibility of, 1411–1412, 1412f dual role in osteoblast differentiation, 713–714, 714f 20-epi-1α,25(OH)2D3 biological activity advantages over, 1433–1434, 1433f 20-epi compound analogs v., 285–287, 286f Geminis v., 287–288, 878 growth plate effects of 24R,25(OH)2D3 v., 582–583 kidney 1α-hydroxylase activity regulated by, 75–76, 75t, 76t muscle Ca homeostasis regulated by, 886–887 muscle cell proliferation/differentiation influenced by, 887–889 osteoblasts as source of, 717 production of extrarenal, 69–70 renal, 69, 70f rapid response v. 6-s-cis-shaped, 391, 398f rapid responses mediated by, 381–400 molecular tools for study of, 392, 393f schematic model of, 399–400, 399f structure-function evaluation of, 392–397 rational design of analogs for, 1405–1418 side chain modifications v. VDR transcription, 1474–1475, 1474t skeletal homeostasis v., 711–712 structure, 381–383, 382f VDR binding to, 313 24,25-Dihydroxyvitamin D3 [24,25(OH)2D3] bone mineralization stimulated by, 16 cartilage regulated by, 575–591 rapid action on resting-zone chondrocytes, 588–589, 588f 24,25-Dihydroxyvitamin D [24,25(OH)2D] concentration lacking clinical relevance, 948t, 949 detecting, 940–942, 941t preparative normal-phase HPLC in, 941 RIA for, 941–942, 942f sample extraction for, 941 solid-phase extraction chromatography in, 941 25,26-Dihydroxyvitamin D3 [25,26(OH)2D3], R/S isomer mixture in natural, 24 24R,25-Dihydroxyvitamin D3 [24R,25(OH)2D3] growth plate effects of 1α,25(OH)2D3 v., 582–583 in quail/chicken embryonic development/hatchability, 851 Disabled-2, megalin function in PCT requiring, 159 Diseases extrarenal 1α-hydroxylase activity in human, 1379–1394 extrarenal D metabolite overproduction v. human, 1390–1392 VDR gene association analysis v. phenotypes of, 1138 VDR polymorphism association analysis v. states of, 1137–1148 VDR polymorphisms v. hyperproliferative, 1145–1146 VDR polymorphisms v. immune-related, 1146–1148 VDR polymorphisms v. risk of, 1121–1149 Distal convoluted tubule (DCT), transcellular Ca/Mg reabsorption in, 557–558 DNA chromatin packaging genomic, 329, 329f VDR binding, 171 DNA binding domains (DBDs) dimerizing in presence of DNA, 314 DNA binding role v point mutations in, 233, 234f as homodimers bound to mouse osteopontin VDRE, 223t, 230, 231f

INDEX

DNA binding domains (Continued) hVDR nuclear translocation mediated by, 231f, 232–233 hVDR zinc finger, 229–233, 231f HVDRR-related mutations in VDR, 1218–1220 Arg50Gln, 1219, 1219f Arg80Gln, 1219, 1219f description of, 1218–1220, 1219f Gly46Asp, 1219–1220, 1219f His35Gln, 1219, 1219f Lys45Glu, 1219, 1219f structural analysis of, 1220 sequence similarity of hVDR, 229–230, 231f VDR, 1210–1211, 1211f DNA licensing, control of, 1644–1645 DPPs. See Dentin phosphoproteins DRE. See Digital rectal exam DRIP coactivator complex. See Mediator-D coactivator complex Drugs D analog metabolism v. design of, 1442–1443 D metabolism influenced by, 1253, 1255t, 1263–1274 deltanoids as, 1405, 1406f deltanoids as candidate, 1405, 1407f leukemia combination therapy with chemotherapeutic, 1734 mineral/skeletal homeostasis influenced by, 915t, 917 PCa combination therapy with chemotherapeutic, 1697–1698 DSP. See Dentin sialoprotein DTH. See Delayed-type hypersensitivity Dual energy x-ray absorptiometry (DEXA), BMD assessment with, 922–924 Duodenum Ca absorption by VDR-null mouse, 435 high Ca absorption rate in, 778 transcellular/paracellular Ca absorption in, 422–424, 423f Dutch dietary Ca intake of French v., 1096 hypovitaminosis D in institutionalized elderly, 1094

E EAE. See Experimental allergic encephalomyelitis Early neonatal hypocalcemia (ENH), premature infants affected by, 805–806 EB1089 antiproliferative activity v. IGF-II, 1714 bone mass loss in microgravity v., 1501–1502 breast cancer cell progression arrested by, 1663–1664, 1672f breast cancer cells/tumors v., 1669 cancer models v., 1499–1500 cell cycle regulated by, 1498 double bond system, 1425t–1426t, 1436 early clinical trials of, 1742 efficacy/safety of D analogs treating, 1504–1505 GADD45 expression stimulated by, 1577, 1578f–1579f hypercalcemia of malignancy v., 1450–1451 IGF system interacting with, 1581–1582 LNCaP xenograft growth inhibited by, 1688–1689, 1688f mechanism of action, 1478–1479 metastasis v., 1576 as 1,25(OH)2D3 analog in cancer/tumor treatment, 1746–1747 1,25(OH)2D3 v., 744 PCa v., 1690 pharmacokinetics/metabolism in clinical development of, 1503, 1503f, 1503t PTHrP expression/production inhibited by, 741t structure, 1479f, 1560, 1560f

INDEX

EB1089 (Continued) as superagonist, 1477–1478 VDR transcriptional activity v., 1474t in vitro metabolism, 1436–1437, 1437f in vivo colon cells influenced by, 1714 as weak calcemic analog, 1440, 1441t Wittig reaction introducing side chains to, 1490, 1491f, 1494t EBT. See Electron beam tomography ECaC. See Epithelial calcium channel ECaC1, functional properties, 431–432 ECaC2 expression in WT/VDR KO mice, 434f, 436t, 438 fast initial phase in Ca-dependent inactivation of, 432 functional properties, 431–432 tissue distribution, 431 ECD. See Extracellular domain ecologic fallacy, PCa risk v., 1602 ECs. See Endothelial cells ED-71 action mechanism, 1535–1537, 1537f convergent synthesis, 1534, 1534f development, 1525, 1534–1539 17β-estradiol bone metabolism influence v., 1536, 1536f linear synthesis, 1534, 1534f metabolism, 1439–1440 osteoporosis treatment using 1αOHD3 v., 1534–1535, 1535f ovariectomized rat bone mass v., 1537, 1537f as strong calcemic analog, 1440, 1441t structure, 1425t–1426t, 1439, 1525, 1526f EDAX. See Energy dispersive X-ray analysis EGF. See Epidermal growth factor EGF receptor (EGFR), 1710–1712 D compounds v. signaling through, 1499 expression in colon cancer, 1710–1711, 1711t EGFR. See EGF receptor EKG. See Electrocardiogram Electrocardiogram (EKG), changes in D toxicity v. myocardial ischemia, 1368 Electron beam tomography (EBT), coronary artery calcification v., 979 Electron microscopy, crystal size/shape shown by, 481 Enamel ameloblasts producing, 599, 601 apatite crystal size/impurities in, 480 D bioinactivation causing dysplasia/hypomaturation/hypomineralization in, 602 Endothelial cells (ECs), 1,25(OH)2D3 responses by, 903–904 Endothelin-1 (ET-1), parathyroid cell proliferation v., 545 Energy dispersive X-ray analysis (EDAX) growth plate ion concentrations monitored by, 487–488 mineral composition information from, 481 English, winter hypovitaminosis D in adult, 1091, 1091f ENH. See Early neonatal hypocalcemia Enterocyte Ca extrusion across basolateral membrane of, 419–421, 420f structure v. Ca flux, 413–414 vesicular CA transport in, 422 Enterohepatic circulation bile acids reclaimed through, 863–864 D/D metabolites in, 1295–1296 Enzymes D analog selectivity v. metabolic, 1460–1461 metabolic future study directions for, 112–113 mutant mouse models of, 105–114 microsomal, 51t–52t

1861 Enzymes (Continued) mitochondrial, 54–59 sex hormones regulating, 61 Epidermal differentiation, 1,25(OH)2D hormone likely to influence, 112 Epidermal growth factor (EGF) 20-epi D analogs interfering with, 1498–1499 keratinocyte proliferation stimulated by, 614 mitogenic activity inhibited by D compounds, 1665 PTHrP expression/production stimulated by, 741t VDR expression/cell differentiation v., 1713, 1713f in vitro D metabolism/catabolism v., 1717 Epidermis microanatomy of, 613, 613f 1,25(OH)2D3 antioxidant activities in, 764t regulatory Ca gradient in, 609, 613, 615 vitamin A influencing development of, 614 Epithelial calcium channel (ECaC), 527–528 architecture, 430, 431f in CA absorption, 415–416 Ca influx v., 430–432 molecular architecture, 431f, 432 nomenclature, 415n 1,25(OH)2D3 regulating expression of, 558 S100A10-annexin 2 required for functional expression of, 432 TRP superfamily/TRPV family, 430 VDREs in human, 527 Epithelial cells, mouse/human gastrointestinal ECaC2 expression restricted to, 431 ER. See Estrogen receptor EREs. See Estrogen response elements Ergocalciferol. See Vitamin D2 ERKOα hull mice, reduced duodenal ECaC2 mRNA expression in, 441f, 445 ERKOβ hull mice, duodenal gene expression unchanged in, 441f, 445 Estradiol, D metabolism influenced by, 1254t, 1259–1261 Estrogen. See also Estradiol Ca absorption v. deficiency of, 1105 deficiency increasing PTH with age, 1108–1109, 1108f intestinal Ca absorption v., 441f, 441t, 444–445, 444f 1,25(OH)2D v. deficiency of, 1103 PTHrP expression/production stimulated by, 741t serum PTH influenced by, 1024 VDR expression affected by, 202t, 206 Estrogen receptor (ER) localization in plasma membrane caveolae, 400 signaling down-regulated by 1,25(OH)2D3/EB1089, 1665 signaling v. breast cancer cell proliferation, 1663, 1672f Estrogen response elements (EREs) hVDR promoter, 200 optimal VDREs resembling half-sites of, 222 ET-1. See Endothelin-1 Ethanol D metabolism influenced by, 1255t, 1266 hypocalcemia/hypomagnesemia v., 1057 hypomagnesemia associated with abuse of, 1054 Ethiopians climate v. D in, 1026 low serum 25OHD in, 1026 sunlight exposure v. rickets in, 1066–1067 Eucalcemia Ca/D supplements for stable, 1059, 1059t, 1060t D in long-term maintenance of, 1050–1051 Europeans. See also specific nationalities D deficiency in, 1025–1026

1862

INDEX

Europeans (Continued) hypovitaminosis D in institutionalized elderly, 1093, 1093f PCa mortality in northern, 1600, 1600f PCa mortality/incidence in, 1625 Every-other-day (QOD) schedule, 1,25(OH)2D3 dose escalation permitted by, 1742–1743, 1743t Exercise bone density influenced by, 796 D metabolism influenced by, 789–796 Exosome, hnRNP role in, 359 Experimental allergic encephalomyelitis (EAE) D analogs treating, 1784 1,25(OH)2D3 down-regulating NOS II in, 1783 1,25(OH)2D3 v., 1755, 1784 VDR ligand treatment v., 637t, 638–639 Export receptors, nuclear export mediated by, 366–368, 367f Extracellular domain (ECD), CaRs’ large amino-terminal, 551, 552f Extracellular fluid (ECF) [Ca2+] demand response, 776–778 Ca2+ homeostasis in, 553f, 554–555, 555f Ca2+ obligatory loss from, 774–776 [Ca2+] regulation in, 774–778, 775f driving transfers of Ca2+ in, 774–776

F 26,27-F6-1α,25-(OH)2D3. See 26,26,26,27,27,27-Hexafluoro1α,25-(OH)2D3 Falls, D status in risk of, 1814, 1814f Familial hypocalciuric hypercalcemia (FHH), heterozygous inactivating CaR mutations causing, 552–553 Family history (FH), in approaching metabolic bone disease, 918 Famine, 777 Fanconi syndrome (FS) phosphate homeostasis in, 1174–1175 treatment, 1175 type I, 1174 type II, 1174–1175 Farnesoid X receptor (FXR), bile acid synthesis/flow regulated by, 865 Fat, progenitor cell commitment v. 1,25(OH)2D3, 650f, 655–656 Fatty acids, VDR expression affected by, 202t, 205 FCA. See Fractional calcium absorption Ferredoxin mitochondrial characteristics of, 72 hormones regulating, 72–73 in 25OHD3 1α-hydroxylation, 71f, 72–73 Fertility D in, 854–855 in mice with poor D function, 852 Fetus Ca homeostasis in mother and, 853–854 CaR in, 854 D synthesis v. development of, 852–853 low maternal D/Ca intake v., 841–843 maternal Ca economy v. bone mineralization by, 841, 841f FFAs. See Free fatty acids FGF-23. See Fibroblast growth factor-23 FGF-23 gene ADHR caused by, 1190 FLAG-tagged native/R176Q mutant, 1190, 1191f FH. See Family history FHH. See Familial hypocalciuric hypercalcemia

Fibroblast growth factor (FGF)-23 in ADHR, 468, 1164–1165 biological activity, 1165 in OHO pathogenesis, 468 as PHEX substrate, 1166–1167 as phosphatonin, 1164–1166 in XLH, 1193 Fibroblasts, in HVDRR studies, 1212–1217 Fibromyalgia, D3 mitigating, 999t Fibrous dysplasia, pathogenesis of hypophosphatemia in, 1192 FISH. See Fluorescent in situ hybridization Fish D3 in fatty, 1026 in diet v. PCa, 1601, 1606 Fluorescence resonance energy transfer (FRET) cytoplasmic VDR-RXR heterodimer shown by, 364–365, 365f GFP-VDR/RXR-BFP focal binding v., 374–375, 375f Fluorescent in situ hybridization (FISH), VDR gene mapping with, 1122, 1124f Fluoride, D metabolism influenced by, 1255t, 1273 FokI RFLP, 1130–1135, 1131t, 1133t, 1134t BMD v., 1142–1143 bone fracture risk v., 1143–1144 Japanese young adult height v., 1143 polymorphism functionality parameters associated with, 1135f, 1137 Foods Ca absorption enhancers in, 818 Ca/D fortification of, 817–818 rationale for, 817–818 D content of selected, 996t–997t D fortification of, 1065–1066 D toxicity v. natural/D-fortified, 1356 targeted/untargeted fortification of, 1073–1074 Formation period (FP) in osteoid indices, 1034 prolonging, 1034–1035 Fos family proteins, in osteoclastogenesis, 677–678 Founder effect PDDR, 1200 Tyr295stop mutation v., 1221 FP. See Formation period Fractal analysis, in bone structure assessment, 962 Fractional calcium absorption (FCA), response to 1,25(OH)2D v. aging, 832, 832f Free fatty acids (FFAs), DBP-A/B binding sites for, 136 French dietary Ca intake of Dutch v., 1096 hypovitaminosis D in adult urban, 1091, 1092t hypovitaminosis D in elderly, 1092–1093 hypovitaminosis D in institutionalized elderly, 1094 FRET. See Fluorescence resonance energy transfer FS. See Fanconi syndrome FXR. See Farnesoid X receptor

G G protein-couple receptor (GCPR) superfamily CaR as member of, 551–552 large ECDs in, 551–552 Gastrointestinal disease acquired bone disease in, 1297–1299 metabolic bone disease development in, 1298–1299 metabolic disturbances in, 1293–1297

INDEX

Gastrointestinal disease (Continued) non-Ca/D bone disease factors in, 1298 secondary hyperparathyroidism accompanying, 1297 type of acquired bone disease in, 1297–1298 Gc-globulin. See Vitamin D binding protein GDNF. See Glial cell-derived neurotrophic factor Gelsolin in actin-scavenger system, 143 growth nucleation v. DBP, 147 Geminis, 1511–1522 colon cancer treatment with, 1521–1522 20-epimeric-24R-hydroxy/19-nor, 1513, 1513f 23-yne-26,27-hexafluoro, 1514, 1514f, 1516f, 1517f 24R-hydroxy metabolite of, 1513–1514 configuration of, 1513, 1514f hypertension v., 1518 1α,25(OH)2D3 v., 287–288, 878 renin expression suppressed by 19-nor, 1518, 1518t as renin inhibitors, 1514–1518 synthesis of, 1511–1513, 1512f vascularized heart allograft rejection v. 19-nor, 1520–1521, 1520t, 1521f Gene expression 20-epi D analogs v., 1495–1498 VDR-mediated control mechanisms influencing, 235–243 Gene inactivation, tissue-specific, Cre/lox strategy for, 112, 113f Genetic hypercalciuric stone-forming (GHS) rats bone resorption in, 1349 establishment of colony of, 1347 as IH animal model, 1346–1350 intestinal Ca transport in, 1348, 1348t low Ca diet response by, 1349, 1350f mineral balance in, 1348 pathogenesis in, 1350, 1350f serum 1,25(OH)2D in, 1348, 1348f serum/urine chemistries in, 1347–1348 VDR in, 1348–1349, 1349f Geography adult D insufficiency v., 1091–1094, 1091f, 1092f D metabolism influenced by, 790–795 D nutrition/acquired bone disease v., 1297–1298 diabetes incidence v., 1766 hip fracture risk v., 1813 hypertension/stroke v., 873 low D intake v., 1087–1088, 1087f as PCa risk factor, 1601, 1601t rickets/colon cancer similarity in, 866 serum 25OHD v. Argentine, 795 Gestation, VDR WT/KO mouse intestinal Ca absorption during, 440–442, 441f, 441t, 442f GFR. See Glomerular filtration rate GFs. See Growth factors GH. See Growth hormone GHS rats. See Genetic hypercalciuric stone-forming rats GIO. See Glucocorticoid-induced osteoporosis Glass, vitamin D photosynthesis v., 41, 42f Glial cell-derived neurotrophic factor (GDNF) in C cell migration, 687–688 RET/GDNFR-α signaling system, 688, 688f Glomerular filtration rate (GFR), 1,25(OH)2D3 v., 1380, 1380f Glucocorticoid-induced osteoporosis (GIO) D as treatment for, 1243–1248 D metabolism v., 1239–1248 D preparations’ efficacy v., 1247–1248 studies of serum D metabolites in, 1241, 1242t

1863 Glucocorticoid receptor (GR), 1239–1240 actions in bone, 1240 Glucocorticoid response elements (GREs), hVDR promoter, 200 Glucocorticoids Ca absorption v., 1244–1245 CYP27A1 regulated by, 57 D metabolism influenced by, 1241–1243 genomic/nongenomic effects of, 1239 GIO v. effects mediated by, 1239 mineral/skeletal metabolic indices v., 1243, 1243f non-GR TFs mediating, 1239–1240 1,25(OH)2D3 influenced by, 1741 osteoblasts/osteoblast progenitors influenced by, 1240 in PCa combination therapy, 1696 PKC activity/CYP24A1 expression v., 99 proximal tubular Pi transport v., 461t PTHrP expression/production inhibited by, 741t renal Pi excretion decreased by, 516t type I collagen synthesis v., 704 VDR expression affected by, 202t, 205–206 Glucose, serum phosphate depressed by insulin and, 1177 Gluten, celiac disease v., 1300–1301 Goats, Ca balance v. sunlight in lactating, 4 Granulomatous diseases extrarenal D metabolite overproduction v., 1390–1391, 1390t 1,25(OH)2D accumulation in, 1389 1,25(OH)2D production v., 1359–1361 Grave’s disease D deficiency v. subtotal thyroidectomy for, 1262 VDR polymorphisms v., 1148 GREs. See Glucocorticoid response elements Growth, VDR ablation v., 341–345, 342f Growth factor receptors, 1,25(OH)2D3 influencing tumor, 1581–1582 Growth factors (GFs) 20-epi D analogs interfering with, 1498–1499 human BPH cells v., 1834 1,25(OH)2D3 influencing tumor, 1581–1582 1,25(OH)2D3 v. signaling by, 1665 PCa v. actions of, 1692–1693 PTHrP expression/production stimulated by, 741t PTHrP inhibition v. signaling interference in, 743 VDR expression affected by, 207–208 Growth hormone (GH) acromegalic/normal 1,25(OH)2D3 v., 1257–1258 D metabolism influenced by, 1254t, 1257 D metabolism influenced by IGF-I and, 1254t, 1258 renal Pi excretion decreased by, 516t VDR expression affected by, 208 Growth plates, 108 calbindin-D9K/D28K in, 724, 730t chondrocyte differentiation in, 576, 576f chondrocytes changing extracellular matrix composition of, 576f, 577–578 cyp24A1-null mouse, 108–109, 108f D actions influencing, 581–582, 581t dietary Ca increasing thickness of, 112 formation of, 575 in growing skeleton, 1031 horizontal/vertical organization of, 498, 499f 24,25(OH)2D3 targeting cartilage in, 582–583 24R,25(OH)2D3 v. 1α,25(OH)2D3 in, 582–583 rickets v. disorganization of, 1071, 1071f subperiosteal erosions near, 976f, 977 VDR ablation affecting, 343, 343f GS 1500, VDR DNA-binding complex induced by, 1496, 1497t

1864

INDEX

GS 1790, bone loss/strength v., 1497t, 1501, 1502t Gy mice, XLH modeled by, 463, 463t

H Habitus bone histomorphometry v., 795–796 bone mass v., 795 D metabolism influenced by, 789–796, 795 Hair cycle D endocrine system in, 291 regulation of, 621–622 factors implicated in, 621 VDR’s different role in, 622 VDR mediating, 234–235 VDR-RXR triggering mechanism unknown, 249 VDR triggering telogen/anagen transition in, 247f, 248–249 Hair follicles development of mouse, 347 1α,25(OH)2D3 VDRnuc in, 385t VDR ablation effect mouse on, 346–348, 346f VDR in function of, 224–225 VDR’s clinical relevance to, 246–249 Hairless gene (hr), as NR co-repressor, 299 Haplotypes polymorphic variation/disease risk v., 1126–1127 polymorphic variation encapsulated by, 1125f, 1127, 1128f VDR gene v. gene-wide, 1135–1137, 1136f Harrison’s sulcus, 1067–1068, 1068f HAT coactivator complexes. See Histone acetyltransferase coactivator complex HD. See Hemodialysis; Huntington’s disease HDM. See Hypovitaminosis D myopathy Health claiming D influence on, 999–1000 D dose/response curve for cardiovascular, 905, 905f D3 nutrition effects anticipated on, 1009t D signaling in cardiovascular, 899–901 D synthesis/absorption v., 1294 FGF-23 in, 1193–1194 serum 25OHD directly related to, 825 sunlight/dietary vitamin D affecting, 42 VDR-mediated signaling implications for, 243–249 Heart D supplementation benefiting, 874 19-nor Gemini v. allograft rejection in, 1520–1521, 1520t, 1521f 1,25(OH)2D3/analog immunoregulatory properties in, 1519 Heat shock-70 proteins (hsp70s), in macrophage 1α-hydroxylase regulation, 1386–1387 Helix-loop-helix (HLH) proteins, in CT gene transcription, 691 Helper T cells interaction/regulation by Th subsets, 1753–1754 1,25(OH)2D3 in autoimmunity v., 1754–1755 1,25(OH)2D3 v. lymphokine production by Th, 1754, 1755f Hematological malignancy, D v., 1727–1736 Hematopoiesis, 1727–1728, 1728f bone remodeling resembling, 505–506 D compounds influencing, 1729–1730 Hemochromatosis, hypoparathyroidism/hypocalcemia v., 1053 Hemodialysis (HD), long-term OCT dose in, 1530–1531, 1531f HEP 187 bone loss/strength v., 1494t, 1501 bone mineral metabolism influenced by, 1493, 1494t Heparin, D metabolism influenced by, 1255t, 1269–1270 Hepatic osteodystrophy, 1303–1304

Hereditary 1,25-dihydroxyvitamin D-resistant rickets. See Hereditary vitamin D-resistant rickets Hereditary hypophosphatemic rickets with hypercalciuria (HHRH), 469 genetic defect underlying, 1173–1174 pathophysiology, 1173 phosphate homeostasis in, 1169t, 1173–1174 treatment, 1174 XLH/ADHR/OHO v., 469 Hereditary vitamin D-resistant rickets (HVDRR), 224, 1207–1231 alopecia associated with, 1229–1230 arginine altered to leucine in, 233, 234f bisphenol analogs v., 1561–1562, 1561f Ca absorption efficiency in, 782 Ca/P/lactose v., 111–112, 111f cases described, 1212–1213, 1214t–1216t cell lines suppressing RXR-VDR transactivation/VDRE binding, 354f, 355–356 cellular basis of, 1212–1218 clinical/biochemical findings in, 1208–1209 clinical features, 1208–1210 cultured fibroblasts in initial studies of, 1212–1217 D-resistant human with signs of, 355 D v. Ca/phosphate absorption in, 566 defective mineralization/rickets in, 1208, 1208f environmental causes v. disease resembling, 1226–1227 first recognition of, 1212 first report of, 1208 functional domains, 1197, 1198f glutamate 420 altered to lysine in, 233, 234f ligand contact point disruption as basis for, 1224 molecular basis for, 1218–1227 non-VDR protein mutations causing, 1226–1227 25OHD 1α-hydroxylase enzyme activity v. PDDR, 1199f 1,25(OH)2D/1,25(OH)2D3-regulated processes in, 1210 pathophysiology of, 1209 prenatal diagnosis of, 1228 R391C mutant hVDR-RXR heterodimerization impaired in, 233, 234f recognition/semantics of, 1197 serum biochemistry levels in, 1208, 1209t severity correlating with alopecia, 1210 spontaneous healing of Tyr295stop/Arg73Gln, 1228 terminology, 1207 therapy for, 1227–1229 Ca in, 1227–1228 D analogs in future, 1229 D in, 1227 VDR/1α(OH)ase ablation causing symptoms of, 429 VDR arginine/cysteine mutation in, 233, 234f VDR gene defects causing, 224, 429, 1207 Herodotus, sunlight effects v. bone recognized by, 565 Heterogeneous nuclear ribonucleoproteins (hnRNPs), 357–359 classical view of, 357 HVDRR v. abnormal expression of, 1226 as multifunctional proteins, 357–359 26,26,26,27,27,27-Hexafluoro–1α,25-(OH)2D3 [26,27-F61α,25-(OH)2D3], potency/metabolism, 1440 HHM. See Humoral hypercalcemia of malignancy HHRH. See Hereditary hypophosphatemic rickets with hypercalciuria High-performance liquid chromatography (HPLC) D/25OHD analyzed with, 932 D v. quantitative reversed-phase, 934, 934f deficiency/depletion evaluated by, 1086

INDEX

High-performance liquid chromatography (Continued) 24R-monohydroxylated Gemini metabolite configuration determined by, 1513–1514, 1514f, 1515f 25OHD assay consistency v., 1023 Hispanics bone mass in, 794 D metabolism in, 793–794 metabolic bone disease v. lactose intolerance in, 917t Histone acetyltransferase (HAT) coactivator complex enzymatic activity in SRC/p160 family, 264–265 VDR linkage to, 176, 264–265 in VDR transcription model, 268–270, 269f History of present illness (HPI) in approaching metabolic bone disease, 916 nutritional factors in, 916, 917t HLH proteins. See Helix-loop-helix proteins hnRNP A (hnRNPA) family direct repeat half-site specificity in, 357 New World primate VDRE-BPs in, 354 REBiP similar to proteins in, 355 hnRNPs. See Heterogeneous nuclear ribonucleoproteins Hodgkin’s disease, extrarenal D metabolite overproduction in, 1390t, 1391–1392 Homer, rickets described by, 1065 Hopkins QW-1624F2-2, 1410f 24F2-1,25(OH)2D3 v., 1407–1408, 1410f large-scale synthesis/availability of, 1408 Hormone resistance, concept evolution, 1207 Hormones. See also specific hormones D metabolism influenced by, 1253–1263, 1254t proximal tubular Pi transport regulated by, 461–462, 461t HPI. See History of present illness HPLC. See High-performance liquid chromatography HPLCs. See Human periodontal ligament cells hr. See Hairless gene HSA. See Human serum albumin hsp70s. See Heat shock-70 proteins Human periodontal ligament cells (HPLCs), osteoblast-like differentiation in, 602 Human physiology, 773–905 Human serum albumin (HSA), DBP structural similarity to, 135–136, 136–137, 136f, 137f, 138f Human VDR (hVDR) ablated in rodents/humans, 224–225 HVDRR v. point mutations in, 233–235, 234f putative NLSs in, 368–369, 368f structure of superagonist ligands complexed to, 285–287, 286f Humans D requirements from evolution of, 995 D resistance in, 355–356 D supply in genomic selection of, 1006 intestinal Ca absorption in, 429–437 Humoral hypercalcemia of malignancy (HHM), Ca reabsorption in, 1256 Huntington’s disease (HD), 1779 hVDR∆ biological properties, 280–281 LBD topology, 281, 283f ligand-binding pocket, 281–282, 283f radii of gyration, 281, 282f solution studies, 281 hVDR∆−1α,25(OH)2D3 complex, crystal structure, 281–282 HVDRR. See Hereditary vitamin D-resistant rickets HVO. See Hypovitaminosis D osteopathy Hydroxyapatite apatite v. natural, 477, 478–480, 478f

1865 Hydroxyapatite (Continued) chondrocyte matrix supporting, 577 crystal formation in matrix vesicles, 578 crystal growth in dentin, 600 crystal growth in enamel, 601 lattice structure in bone mineral, 1030 2-Methylene-19-nor-(20S)-1α-hydroxybishomopregnacalciferol (2MbisP), 1551–1552, 1552f tissue selectivity, 1552–1553, 1553f 25-Hydroxycholecalciferol, in HVO evolution, 1038t, 1041–1042 2-Methylene-19-nor-1α-hydroxyhomopregnacalciferol (2MP), 1551–1552, 1552f tissue selectivity, 1552–1553, 1553f 25-Hydroxylase, 47–62 activity v. sex, 61 D molecule interaction model, 56 human/mouse, 54 identifying novel types of, 106–107 microsomal, 50t–51t expression sites for, 48, 49t porcine, 53–54, 54f sex differences in, 52–53 mitochondrial, 51t–52t expression sites for, 48, 49t ontogeny, 60 specificity, 55–56 vitamin D activated by, 17–19 26-Hydroxylase, 24 24-Hydroxylase (CYP24), 20–21, 85–100 activity in kidney, 517–518 catabolic pathway/Ca restriction in D toxicity, 1365–1367, 1366t cellular expression, 86–87 in CYP24A1 KO/transgenic animals, 90–91 cellular metabolism by, 1459–1461, 1460f distribution in kidney, 528 enzyme functional in PDDR patients, 1199 kinetic analysis, 89–90 metabolic analysis, 89 spectral analysis, 90 structure/function, 88–90 enzyme pathways, 87–88, 88f enzyme structure/function, 88–90 expression regulators, 93, 94f function/regulated expression, 85–86, 86f human intestine expressing, 1717–1719 in 23-hydroxylation, 23 inhibitors in PCa combination therapy, 1695–1696 keratinocyte differentiation v. activity of, 611–612, 611f macrophages lacking 1,25(OH)2D-directed activity of, 1383–1384 molecular aspects, 93–100 mouse proximal/distal colon expressing, 1719 mRNA expression evaluation for, 1718 nutritional regulation, 1719–1721 1,25(OH)2D actions terminated by, 22 25OHD3/1,25(OH)2D3-, 528 1,25(OH)2D3 regulating Ca deficiency v., 21 intestinal., 21 PCa growth-inhibitory responses influenced by, 1685–1686, 1686f physiological role, 21–22 properties, 87 responsiveness v. VDREs, 316t, 320 terminal reaction, 87, 87f ubiquity, 20–21

1866 1α-Hydroxylase (CYP27B1), 69–78 age v. activity of renal, 1103, 1109 in breast cancer, 1667 CKD directly inhibiting, 1316 CKD v. PTH/Ca induction of, 1315–1316 cytokines stimulating macrophage, 1384–1385 in D toxicity v. restricted Ca diet, 1365, 1365t, 1366t deficiency v. HVDRR, 1208, 1209t enzyme v. 24-hydroxylase enzyme, 1199 factors influencing activity of, 517t functional diversity, 1392 hsp70s regulating macrophage, 1386–1387 human disease/extrarenal activity of, 1379–1394 human intestine expressing, 1717–1719 immune cell regulators v. macrophage, 1384–1387 importance of phosphorus in regulating, 1161 keratinocyte differentiation v. activity of, 611–612, 611f levels in PCas/noncancerous prostates, 1608–1609 LPS amplifying macrophage, 1385–1386, 1385f in macrophages, 1381–1382, 1382f mouse proximal/distal colon expressing, 1719, 1719f mRNA expression evaluation for, 1718 new developments in disease v., 1379 NO regulating macrophage, 1386 nutritional regulation, 1719–1721 1,25(OH)2D3 growth inhibition requiring, 744, 745f PCa growth-inhibitory responses influenced by, 1686–1687, 1687f PCa v., 1626–1627 in prostate, 1607 PTH/Ca not regulating prostatic, 1609–1610 renal phosphate transport/phosphorus v., 1161, 1163f species distribution, 70–71 substrate availability/1,25(OH)2D bioactivation in CKD, 1315 TLR expression/signaling v. extrarenal, 1385 in vitamin D metabolism, 19–20 23-Hydroxylation, vitamin D3, 23 2-Methylene-19-nor-1α-hydroxypregnacalciferol (2Mpregna), 1551–1552, 1552f tissue selectivity, 1552–1553, 1553f 1,25-Hydroxyvitamin D3, urinary loss in megalin KO mice, 154, 155t 23(S),25(R)25-Hydroxyvitamin D3-26,23-lactone, in C-23 oxidative pathway discovery, 23 1α-Hydroxyvitamin D2 [1αOHD2] as 1,25(OH)2D3 analog in cancer/tumor treatment, 1747 toxicity, 1358 1α-Hydroxyvitamin D3 [1αOHD3] early clinical trials of, 1742 in GIO treatment, 1247–1248 leukemic cells v., 1734 osteoporosis treatment using ED-71 v., 1534–1535, 1535f PDDR treatment with, 1201t, 1202 toxicity, 1357–1358 25-Hydroxyvitamin D3/DBP complexes cubilin as endocytic receptor for, 158–159, 158f glomerular filtration of, 153–154, 154f megalin as endocytic receptor for, 154–156, 157–158, 157f, 158f megalin influencing cellular uptake of, 156–157, 156f receptors/co-receptors for, 157–159 two-receptor model for, 159, 159f 25-Hydroxyvitamin D3 [25OHD3] basis for 1,25(OH)2D3 interaction with, 783 biological static/dynamic methods v. normal serum, 1020t BMD v. normal, 1022, 1022f Bone fracture risk defining normal, 1022–1023

INDEX

25-Hydroxyvitamin D3 [25OHD3] (Continued) Ca intake v. serum, 1023–1024 colon cancer v., 1709–1710 CYP27A1 disruption v., 60 D3/metabolite plasma concentrations v., 1362, 1363t desirable target plasma concentration of, 1009t dose-response relationship with D3, 1003, 1005f early 2-carbon analogs of, 1545 endocytic pathways, 153–160, 160f as hormonally active form of vitamin D3, 16 interlaboratory assay variability v., 1023, 1023f, 1023t, 1025 intestinal Ca absorption efficiency correlated with, 783 metabolism in human colon cancer cells, 1716–1717, 1716f neoplastic colonocytes acted on by, 1710–1715 nutritional status improvement v., 1003–1005, 1004t PDDR treatment with, 1201–1203, 1201t, 1202f, 1203f proteins in 1α-hydroxylation of, 71–73 renal endocytosis, 153–159 renal handling of Ca/P influenced by, 518–519 secondary hyperparathyroidism in defining normal, 1020–1022 serum concentration indicating nutritional status, 1003, 1004t serum concentration of D status clinical outcomes v. normal, 1019 defining normal, 1019–1026, 1020t sampling biases v. reference value, 1019 strategies increasing concentration of, 1003, 1004t urinary loss in megalin KO mice, 154, 155t variables influencing normal values of, 1023–1024 25-Hydroxyvitamin D [25OHD]. See also 25-Hydroxyvitamin D3 age v., 1101–1102, 1102f age v. synthesis of, 826, 1617 automated instrumentation CLIA methodology for detecting, 939–940, 939f, 940f, 940t bone fracture risk v. serum, 1370, 1370f Ca absorption role of, 1106 clearance v. age, 1102 clinical implications of keratinocytes producing, 612–613 concentration having clinical relevance, 948t, 949 cytokines regulating, 610f, 612 deficiency caused by defective CYP27A1, 61–62 deficiency v. age, 1103, 1109 detecting, 935–939 endogenous production in hypervitaminosis D, 1359 as HDM cause, 1816 HPLC methodology for detecting, 936 quantitative normal-phase HPLC in, 936, 937f sample extraction for, 936 silica cartridge chromatography in, 933f, 936 solid-phase extraction chromatography in, 936 hydroxylation, 1599 keratinocyte differentiation regulating, 611–612 kidney in metabolism of, 516–518 liver production, 17–19 mechanisms of age-related decrease in, 1101–1102 metabolism/identification of products, 609–610 muscle power/MVC v., 1812–1813, 1812f 1,25(OH)2D concentration v. plasma half-life of, 1296, 1296f parent D/25OHD toxicity due to, 1362 PCa v., 1610–1611, 1611f PCa v. serum, 1602–1604 plasma concentration, 28t poor assay performance v., 939–940, 940t prostate cells v. antiproliferative influence of, 1607–1608, 1609f psoriasis v. serum, 1796 RIA methodology for detecting, 936–938 assay calibrator preparation for, 936

INDEX

25-Hydroxyvitamin D [25OHD] (Continued) other D compounds v., 937–938, 938f, 938t, 939t radioimmunoassay in, 937, 938f sample/calibrator extraction for, 936 seasonal variation, 1019–1020, 1020f, 1069 serum concentration influenced by aging, 826–827 Hyp mice abnormal 24-hydroxylase regulation in, 465 XLH modeled by, 463, 463t, 464f, 465 Hypercalcemia. See also Humoral hypercalcemia of malignancy calcipotriol/related analogs v., 1493–1494 causes of, 1355, 1356t clinical manifestations of D toxicity from, 1368 D-induced, 568–569, 986, 988f D metabolite use limited by, 1403 D single dose rickets therapy v., 1072, 1073 D toxicity causing, 1355–1372 dehydration worsening pathophysiology of, 1368 early detection/prevention of, 1393 EB1089 v., 744 20-epi 1,25(OH)2D3 analogs v., 1494–1495, 1496t excessively fortified milk causing, 1065 identifying patients at risk for, 1393 lowest D3 dose causing, 1005f, 1008–1009 nonsecosteroid analogs v., 1565–1567 in Npt2a KO mice, 462 1,25(OH)2D3 allograft rejection therapy v., 1518 1,25(OH)2D3/analogs influencing, 542–543 24F2-1,25(OH)2D3 and Hopkins QW-1624F2-2 v., 1407–1408, 1410f 1,25(OH)2D3 causing, 744 preventing, 1393 SCCs associated with, 612 screening patients at risk for, 1393 stanniocalcin v., 462 therapeutic 1,25(OH)2D3 inducing, 1449 treating, 1393–1394 Hypercalciuria. See also Idiopathic hypercalciuria D metabolite use limited by, 1403 early detection/prevention of, 1393 as early sign of D toxicity, 1368 identifying patients at risk for, 1393 IH diagnosis v. causes of normocalcemic, 1340, 1340t in Npt2a KO mice, 462 1,25(OH)2D3 causing, 744 screening patients at risk for, 1393 treating, 1393–1394 Hyperesthesia, hypovitaminosis D associated with, 1090 Hyperkalemia, hypocalcemia v., 1049 Hypermagnesemia CaR mutation causing, 553–554 hypocalcemia masked by, 1049 Hyperparathyroidism. See also Secondary hyperparathyroidism D insufficiency in elderly v. mild, 825 D supplementation v., 1094 high-Ca diet preventing KO mouse, 429 intracortical bone resorption in, 975f, 977 intravenous 1,25(OH)2D3 v., 1330–1331 management of severe/refractory, 1823–1826 in megalin KO mice, 154–156, 155t models, 544 in renal osteodystrophy, 976–979 resorbed distal phalangeal tufts v. treated, 975f, 977 subperiosteal erosions in, 975f, 976–977 VDR ablation v., 342, 343f in VDR KO mice, 433, 488 VDR polymorphisms v., 1146

1867 Hyperphosphatemia in altered phosphate load disorders, 1179–1180 dystrophic calcification associated with, 478 1α-hydroxylase inhibited by, 1316 hypocalcemia induced by, 1056–1057 1,25(OH)2D inhibited by, 1161 Pi vs., 454–455 therapeutic 1,25(OH)2D3 inducing, 1449 Hyperproliferative skin disorders, VDR-RXR heterodimeractivating ligands v., 241 Hypertension D analogs v., 878 D signaling in regulating, 899 Gemini compounds v., 1518 inappropriate RAS activation leading to, 871 low calcemic D analogs v., 1514–1516 Hypervitaminosis D2, hypervitaminosis D3 v., 1364–1365 Hypervitaminosis D3 cartilage/bone v., 582 23-hydroxylation in, 23 hypervitaminosis D2 v., 1364–1365 Hypocalcemia. See also Early neonatal hypocalcemia; Late neonatal hypocalcemia biochemical changes induced by, 1051–1052 breast milk/formula v. neonatal, 841–842, 842f CYP27B1 gene mutation v., 109 in CYP27B1-null mice, 703 dermopathy in chronic, 920, 920f differential diagnosis/D therapy v., 1049–1060 differential diagnosis of, 1052–1058 early/late neonatal, 1055 fasting/famine/drought causing, 777 of IDM, 807 long-term treatment of, 1058–1060 management of acute, 1058 in megalin KO mice, 154–156, 155t in 1α(OH)ase-null mice, 438, 489 1,25(OH)2D3 production increased by, 19–20 1,25(OH)2D3 v. HVDRR patient’s, 357 parathyroid cell proliferation stimulated by, 545 physiology of, 1049–1050 PTH gene expression increased by, 544 PTH increasing 1,25(OH)2D3 production in, 703 rickets/osteomalacia v. chronic, 1049 signs/symptoms, 919–920, 919t skeletal homeostasis/D metabolism/illness causing, 1054–1058 therapy for, 1058–1060 uncalcified osteoid v. 1,25(OH)2D3 treatment of, 1041, 1041f in VDR KO mice, 433, 488 Hypokalemia, hypocalcemia masked by, 1049 Hypolipidemics, D metabolism influenced by, 1255t, 1267–1268 Hypomagnesemia in alcoholics, 1177 CaR mutation causing, 553–554 hypocalcemia v., 1049, 1053–1054, 1054 Hypoparathyroidism hypocalcemia v. idiopathic, 1053 in Npt2a KO mice, 462 Hypophosphatemia in altered phosphate load disorders, 1178–1179 bone mineralization abnormalities in, 488 causes of chronic, 924, 924t correction v. therapeutic objectives, 928, 928f in CYP27B1-null mice, 703 dental defects associated with familial, 602 GH regulating 1,25(OH)2D3 in, 1258

1868

INDEX

Hypophosphatemia (Continued) in Npt2a KO mice, 462 in 1α(OH)ase-null mice, 438 1,25(OH)2D increased by, 1161 pathogenesis in fibrous dysplasia, 1192 Pi vs., 454 radiology of renal tubular defect, 981–984 in stage II rickets, 1070 TIO/XLH/ADHR characterized by, 1162–1163 transcellular shift in, 1176–1177 Hypophosphatemic bone disease, FGF-23 gene mutation detection in, 1190 Hypophosphatemic diseases, 1162–1176 common pathway of pathogenesis in, 1163–1168 FGF-23 activity v. phenotypic abnormalities of, 1166 Hypovitaminosis D, urinary loss of 25OHD3 causing, 153 Hypovitaminosis D myopathy (HDM), 1805–1817 clinical studies of, 1811–1813 biopsies in, 1811 muscle function in, 1811–1813 D deficiency insulin resistance v., 1816 misdiagnosis, 1805–1806 muscle physiology v., 1806–1808, 1806f, 1807f 25OHD/1,25(OH)2D/PTH causing, 1814–1816 sunlight v., 1813–1814 symptoms/signs, 1805–1806 Hypovitaminosis D osteopathy (HVO), 1035 biochemical evolution of, 1036–1039, 1038t D metabolism in, 1039–1040 pathogenesis of, 1029–1044 stages of, 1036, 1036t

I IBD. See Inflammatory bowel disease Ichthyosis, D therapy v., 1787 IDBPs. See Intracellular vitamin D binding proteins Idiopathic hypercalciuria (IH), 1339–1346 Ca absorption v. 1,25(OH)2D in, 1342, 1343f diagnosis v. causes of normocalcemic hypercalciuria, 1340, 1340t dietary Ca restriction v., 1351 elevated 1,25(OH)2D in, 1341–1342, 1342f genetic hypercalciuric rats as animal model of, 1346–1350 human genetic, 1346f, 1350–1351, 1351t inheritance, 1340–1341, 1340f intestinal Ca absorption in, 1341, 1341f low bone mass associated with, 1342–1343 nephrolithiasis in, 1142, 1339–1352 overview, 1339–1341 pathogenesis of human, 1341–1346 pathogenetic models of, 1343–1346, 1344f external Ca balance v., 1344–1345, 1345f fasting PTH/urine Ca v., 1344 1,25(OH)2D excess v., 1344f, 1345–1346, 1347f tests of, 1344–1346 urine Ca/Ca balance/low Ca diet v., 1345, 1345f, 1346f renal Ca reabsorption decreased by, 1342 therapeutics v. Ca metabolism, 1351 thiazides v., 1351 IDM. See Infants of diabetic mothers IFNγ. See Interferon-γ IGF. See Insulin-like growth factor IGF-I D metabolism influenced by GH and, 1254t, 1258 20-epi D analogs interfering with, 1498–1499

IGF-II in EB1089 antiproliferative activity, 1714 20-epi D analogs interfering with, 1498–1499 IH. See Idiopathic hypercalciuria Ileum high Ca absorption quantity in, 778 transcellular/paracellular Ca absorption in, 422–424, 423f Ilium, fracture suggesting D deficiency, 973 IMCal. See Intestinal membrane calcium-binding protein IMCD. See Inner medullary collecting duct Immune diseases D analogs v., 1500–1501 VDR polymorphisms v., 1146–1148 Immune responses cardiovascular disease v. D modulating, 902 innate/adaptive layers of, 631 selective intervention in, 631–633, 632t VDR ligands mediating, 631–643 mechanisms involved in, 643, 643f Immune system CKD/1,25(OH)2D3 deficiency/resistance in, 1326–1327 D-deficiency-associated abnormalities in, 1389 D/diabetes v., 1767–1773 D endocrine system involved in, 291 D metabolite local regulatory effects on, 1387–1390 DBP role in, 126–127 VDR ablation effect on, 346 Immunosuppressants D analogs combined with, 1500 D metabolism influenced by, 1255t, 1272–1273 Import receptors, nuclear import mediated by, 366, 367f Importins. See Import receptors Infants acute hypocalcemia therapy for, 1058 Ca absorption in human milk/formula-fed, 813, 814t D actions in perinatal, 803–808 D deficiency/Ca absorption in, 811–818 D3 dosage considerations for, 1002–1003, 1009t D supplementation for formula-fed, 808 D supplementation for low-birth-weight, 808 early/late neonatal hypocalcemia in, 1055 infant/maternal D supplementation v. 25OHD in, 846–847, 846f low maternal D/Ca intake v., 841–843 maternal Ca intake v. BMC in, 843, 843f normal term, 804–805 Ca absorption in, 813–814 Ca intake recommendations for, 813, 813t D deficiency/Ca absorption in, 813–814 D in, 814 supplemental D sources for, 814, 814t premature, 805–807 D deficiency/Ca absorption in, 812–813 D in, 806, 806f early neonatal hypocalcemia v., 805–806 nutritional rickets in, 968 osteopenia risk criteria for, 812, 812t postnatal D supplementation for, 806–807 rickets and Chinese, 793 term growth-retarded, 805 VDDR v. black, 791 Infants of diabetic mothers (IDM), pathogenic factors in, 807 Infections children with rickets v., 1068 VDR polymorphisms v., 1148 Inflammation, 126 DBP role in, 126–127

INDEX

Inflammatory bowel disease (IBD). See also Crohn’s disease clinical/biochemical features of, 1302 VDR ligand treatment v., 637t Infrared spectroscopy, tissue mineralization quantified with, 481, 482–483, 482f Inner medullary collecting duct (IMCD), vasopressin-stimulated water reabsorption v. CaR, 558 Inorganic phosphate (Pi) aging v. serum concentration of, 455 chemistry, 453 circadian rhythm in serum concentration of, 455 disorders associated with renal wasting of, 467–470 common metabolic pathway hypothesized in, 469–470 distribution in body, 453 extracellular homeostasis of, 453–455, 454f homeostasis, 453–469 regulation, 1159–1161 v. NPT2a-related signaling, 1161 1α-hydroxylase activity v. restricted, 464, 464f intestinal absorption of, 455–457 cellular aspects in, 455 molecular mechanisms in, 456, 456t NPT2b gene expression v., 455–456, 456t regulation of, 455–456 kidney excreting, 516, 516t kidney reabsorbing, 516 NPT2a regulating reabsorption of, 459, 461t renal transport of, 457–463 cellular aspects of, 457–458, 458f molecular aspects of, 456t, 458–459 physiology/tubular localization in, 457 supplementation in metabolic bone disease, 927 transport in bone, 465–467 type II cotransporter in handling, 1160 Insufficiency in adults/elderly, 1085–1097 consequences of, 1088–1090 deficiency v., 1085–1086 determinants, 1086–1088 elderly v. consequences of, 825–826 medical causes of, 1088 muscle weakness in, 1090 prevalence, 1090–1094 preventing, 1094–1096, 1097 Insulin D metabolism influenced by, 1254t, 1259 EGF keratinocyte proliferation stimulation enhanced by, 614 proximal tubular Pi transport v., 461t renal Pi excretion decreased by, 516t resistance in D deficiency v. HDM, 1816 secretion influenced by 1,25(OH)2D3, 1764–1765 secretion stimulated by 1α,25(OH)2D3, 394–395, 395f serum phosphate depressed by glucose and, 1177 synthesis/secretion in NOD mice, 1765, 1766f synthesis/secretion in VDR KO mice, 1765, 1765f type I collagen synthesis increased by, 704 Insulin-like growth factor (IGF) mitogenic activity inhibited by D compounds, 1665 in 1,25(OH)2D3 actions on prostate cells, 1692–1693, 1692f system interacting with 1,25(OH)2D3/EB1089, 1581–1582 Integument D’s role in, 609–622 VDR ablation effect on, 346–348, 346f Interferon-γ (IFNγ) CYP24A1 up-regulation inhibition v., 100 D metabolism influenced by, 1254t, 1263

1869 Intestinal membrane calcium-binding protein (IMCal), Ca entry v., 416 Intestine. See also Colon; Duodenum; Ileum active Ca transport in, 414 age v. 1,25(OH)2D resistance/Ca absorption by, 1104–1105, 1105f calbindin-D9K/D28K in, 722–724, 730t CaR in, 559 CKD with low 1,25(OH)2D3 or 1,25(OH)2D3/VDR resistance in, 1322 corticosteroids influencing Ca absorption in, 445 CYP27B1/CYP24 expression in, 1717–1719 D analogs v. induced tumors in, 1451 D deficiency/depletion v., 1294 D-dependent Ca absorption mechanisms in, 433–440 dexamethasone influencing Ca absorption in, 445, 447f ECaC2/calbindin-D9K mediating CA absorption by, 430f, 440 estrogens v. Ca absorption in, 444–445 gestation v. Ca absorption in, 440–442, 441f, 441t, 442f 24-hydroxylase enzyme regulation in, 92 lactation v. Ca absorption in, 441f, 441t, 442–444, 442f, 443f LCA detoxification in, 867–868, 868f 1,25(OH)2D3 and Ca absorption by, 411–424 1,25(OH)2D3 antioxidant activities/CA absorption in, 764t, 767 25OHD correlated with Ca absorption efficiency in, 783 paracellular path in Ca absorption, 421–422 Pi absorption in, 455–457 cellular aspects of, 455 molecular mechanisms of, 456, 456t NPT2b gene expression v., 456–457, 457t regulation of, 455–456 segments v. Ca absorption, 413, 422–424, 423f transcellular/paracellular Ca absorption in, 429, 430f Intoxication Ca/24-hydroxylase catabolic pathway in, 1365–1367, 1366t Ca v. renal 1α-hydroxylase in, 1365, 1365t, 1366t clinical manifestations of, 1368 D absorption/input v. risk of, 784 D2 causing most cases of, 1008 DBP/free metabolite level in, 1367–1368 diagnosis of, 1369 diagnosis of endogenous D, 1392–1393 diagnosis/prevention/treatment v. endogenous, 1392–1394 excessively fortified milk causing, 1009t–1010, 1066 forms of exogenous, 1355–1359 hypercalcemia due to, 1355–1372 metabolic bone disease patient SH v., 917 myocardial ischemia v. EKG changes in, 1368 sarcoidosis associated with endogenous D, 1379–1380 target tissue response to 25OHD in, 1364 TPTX rat 24-hydroxylase activity v., 1366–1367, 1367t treatment of, 1369–1370 VDR in, 1362–1365 Intracellular receptor gene family, VDR in, 172–173 Intracellular vitamin D binding proteins (IDBPs) D analog selectivity influenced by, 1462–1463 in D-resistant New World primates, 359–360 hsp70 family members homologous to, 359, 360f in intracellular D trafficking model, 360 in 25OHD3 1α-hydroxylation, 73 “sink”/“swim” hypotheses for, 359–360, 360f Intragenic interaction, VDR gene analysis v., 1136f, 1138 Irish, winter hypovitaminosis D in adult, 1091, 1091f Isoflavones, PCa combination therapy with soy, 1697 Italians Ca v. colorectal cancer in, 1622

1870

INDEX

Italians (Continued) climate v. D in, 1026 hypovitaminosis D in elderly, 1092–1093 winter hypovitaminosis D in adult, 1091, 1091f

J Japanese Buddhist vegetarians v. metabolic bone disease, 917t Cdx2 polymorphism in, 1126 CKD/VDR polymorphisms/expression in, 1319 FokI RFLP v. height in young adult, 1143 hypovitaminosis D in adult, 1091 PCa risk for indigenous, 1681 PCa risk in, 1601 VDR gene polymorphism v. BMD in, 243 VDR polymorphism v. PCa risk in, 1682 VDR polymorphisms v. diabetes in, 1146 VDR polymorphisms v. MS in, 1147 VDR polymorphisms v. PCa in, 1139t, 1145 VDR polymorphisms v. psoriasis in, 1146 VDR polymorphisms v. renal cell carcinoma in, 1146 westernization v. PCa in, 1600, 1600f Jejuno-ileal bypass bone disorders associated with, 1302–1303 clinical features of, 1302–1303 management, 1303 Jejunum, high Ca absorption quantity in, 778 Jews metabolic bone disease v. lactose intolerance in, 917t serum 25OHD in light-skinned, 794–795 JG cells. See Juxtaglomerular cells JNK. See Jun-N-terminal kinase Jun-N-terminal kinase (JNK), in RANKL-induced osteoclastogenesis, 678 Juxtaglomerular (JG) cells, renin synthesized/secreted by, 871, 872f

K Kellgren score, OA diagnosed with, 1144 Keratinocyte-GF (KGF) BPH cells v. BXL–353 and, 1836–1837, 1837f human BPH cells v., 1834, 1835f Keratinocytes Ca regulating proliferation/differentiation of, 609 Ca sensing mechanism of, 615–616, 615f Ca switch inducing changes in, 616 Ca switch stimulating phosphoinositide metabolism in, 617–618 clinical implications of 1,25(OH)2D production by, 612–613 DRIP205/p160 recruitment in differentiation of, 273 epidermal layers of, 613, 613f growth/differentiation regulators of, 613–615 24-hydroxylase enzyme regulation in, 93 1,25(OH)2D3/deltanoid-induced differentiation modeled in, 1637t 1,25(OH)2D3 inhibiting differentiation of, 1781 1,25(OH)2D3 inhibiting PTHrP production in, 741t 1,25(OH)2D production by transformed, 612 1,25(OH)2D production regulated by differentiation of, 611–612 1,25(OH)2D3 protecting epidermal, 764t, 766–767 1,25(OH)2D regulating differentiation of, 619–621, 620f regulation of differentiation by, 613–621 retinoic acid receptors identified in, 614 VDRs in mammalian/lamprey, 228

Ketoconazole D metabolism influenced by, 1255t, 1266–1267 kidney 1α-hydroxylase activity regulated by, 78 KGF. See Keratinocyte-GF KH1060 analog-VDR complex stabilized by, 1496 autoimmune type I diabetes prevented by, 1500 breast cancer cells/tumors v., 1669 in leukemia combination therapy, 1733 leukemic cells v., 1735 metabolism, 1438, 1439f metastasis v., 1576 as noncalcemic analog, 1440, 1441t structure v. 1α,25(OH)2D3, 285–287, 286f synthesis, 1490–1491, 1492f, 1496t Kidney aging v. D responsiveness by, 833 Ca handling by, 515–516 calbindin-D9K/D28K in, 724, 730t CaR in, 556–558 CKD with low 1,25(OH)2D3 or 1,25(OH)2D3/VDR resistance in, 1325–1326 D analogs treating chronic disease of, 1827 D-dependent protein distribution/regulation in, 519–528 D-responsive proteins in, 520t D v., 515–528 DBP uptake in megalin-deficient, 154, 154f, 155f dystrophic mineral deposits in, 477–478, 478t ECaC1 expression restricted to mouse, 431 function deteriorating with aging, 827 glucocorticoids directly affecting, 1243, 1243f 1α-hydroxylase/24-hydroxylase expression in fetal, 852–853, 853f 24-hydroxylase distribution in, 528 24-hydroxylase enzyme regulation in, 91–92 24-hydroxylase expression in, 86–87 24-hydroxylation in, 21–22 JG apparatus producing renin in, 871 keratinocyte 1,25(OH)2D negative feedback loop v., 611 mass/CKD v. 1,25(OH)2D bioactivation, 1379–1380 mouse models with defective phosphate transport by, 462–463 1,25(OH)2D3/analog immunoregulatory properties in, 1519 1α,25(OH)2D3-mediated rapid response in, 386t in 25OHD metabolism, 516–518 1,25(OH)2D3 produced by, 782 1α,25(OH)2D3 produced in, 69, 70f 1,25(OH)2D v. phosphate transport in, 1161–1162, 1162f 1,25(OH)2D3/VDR-mediated signal termination in, 221f, 222 1α,25(OH)2D3 VDRnuc in, 385t Pi flux in human, 453–455, 454f Pi handling by, 516, 516t Pi homeostasis arbitrated by, 1159 Pi transport in, 457–463 cellular aspects of, 457–458, 458f molecular aspects of, 456t, 458–459 physiology/tubular localization of, 457 PTHrP expression in, 739t rejection inhibited by 1,25(OH)2D3/analogs, 641t tubular defects and hypophosphatemia, 981–984 VDR in, 520–523 polyclonal antibodies detecting, 521, 521f Kidney disease 1,25(OH)2D secretion v., 1313 secretory control/hormonal interactions v., 1313, 1314f Klotho gene, kidney 1α-hydroxylase activity v., 78

1871

INDEX

Knockout (KO) mice alopecia in, 1229–1230 c-src, osteopetrosis in, 677 calbindin D28K, urinary Ca/creatinine ratio in, 525 cyp24A1 growth plate architecture in, 108–109, 108f 24-hydroxylation in, 107–109 phenotype of, 107–109, 107t cyp27A1 hepatic 25-hydroxylation in, 105–106 phenotype of, 106 cyp27B1 D-dependent gene expression in, 110 1α-hydroxylation in, 109–113 hypocalcemia/hypophosphatemia/secondary hyperparathyroidism in, 703 phenotype of, 109–110 phenotype rescue in, 110–112 DBP, 149 serum D metabolites v. bone abnormalities in, 489f viable immune function in, 127 ERα/ERβ, 441f, 445 FGF-23, phosphate homeostasis in, 1193–1194 full-length CaR, 616–617 intestinal Ca absorption in, 429–437, 446t JNK1, RANKL-induced osteoclastogenesis in, 678 megalin, 154–156 Npt2 Ca absorption in, 440 generation/characteristics of, 439–440 phosphate homeostasis in, 439–440 Npt2a intrinsic osteoclast defect in, 467 Npt2c expression in NHERF and, 1162 renal phosphate transport defects in, 462 renal Pi reabsorption v. P450c1α gene expression in, 464f, 465 1α(OH)ase ECaC1 expression reduced in, 438 generating, 438 hypocalcemia/rickets/osteomalacia in, 489 lower calbindins-D expression in, 525 mineral homeostasis in, 438–439 PDDR in, 438–439, 489 prehypertrophic chondrocytes showcased by, 578 RXRα conditional, alopecia in, 233 SRC coactivator family models in, 294–295 VDR, 224–225, 341–348 alopecia in, 620, 621, 665 Ca absorption in, 433–437, 434f Ca absorption v. Ca transporter gene expression in, 435–437, 436t, 437t Ca entry v. Ca transport in, 417 Ca/P ameliorating skeletal abnormalities in, 1403–1404 calbindin D28K expression v. age in, 525 D-dependent active intestinal Ca absorption in, 433–438 dietary intervention v. Ca absorption in, 434f, 436t, 437–438 estrogen deficiency in, 441f, 445 generating, 433 gestation/intestinal Ca absorption in, 440–442, 441f, 441t, 442f HVDRR/alopecia link in, 621 hypocalcemia in, 665 infertility in, 665 intraperitoneal glucose tolerance test in, 1765, 1765f lactation/intestinal Ca absorption in, 441f, 441t, 442–444, 442f, 443f renin expression/Ang II production in, 875–877, 876f

KO mice. See Knockout mice Koreans VDR polymorphisms v. psoriasis in, 1146 VDR polymorphisms v. RA in, 1147 Kuwaitis, sunlight exposure v. rickets in, 1066–1067

L Lactation BMC/BMD during, 843f, 844–845 Ca homeostasis v., 204 D/Ca metabolism during, 843–844 D metabolism in, 839, 843–847 maternal Ca economy v., 845, 845f PRL v. D metabolism during, 1258 VDR WT/KO mouse intestinal Ca absorption during, 441f, 441t, 442–444, 442f, 443f Lactose intolerance, Ca intake v., 1026 Lampreys, VDRs in, 227–228, 279 Late neonatal hypocalcemia (LNH), 807–808 Latinas, hVDR polymorphisms v. breast cancer risk in U.S., 245 LBDs. See Ligand binding domains LC/MS. See Liquid chromatography/mass spectrometry LCA. See Lithocholic acid LD. See Linkage disequilibrium Lebanese climate v. D in, 1026 D metabolism in, 794 hypovitaminosis D in, 1087 low serum 25OHD in, 1026 Leo KH-1060, SAR in design of, 1412, 1413f Leprosy extrarenal D metabolite overproduction v., 1390t, 1391 hypercalcemia/D hypersensitivity in, 1361 Leukemia D analogs effective against, 1734–1736 D compound combination therapy v., 1733–1734 RUNX TF localization in acute myelogenous, 335 Leukemia cells bisphenol compounds v. differentiation of, 1559, 1559f calcipotriol v., 1734 D compounds v., 1730–1734, 1730t, 1731, 1731t molecular mechanisms of D compounds v., 1731–1733 1,25(OH)2D3 v. myeloid, 1731 1αOHD3 v., 1734 Leukocytes, antigenic stimulants activating phagocytic function in, 127 Libyans, rickets in infant, 795 Ligand binding domains (LBDs) coactivator binding v. HVDRR-related mutations in VDR, 1225–1226 Glu420Lys, 1226 connecting region poorly conserved in, 279–280 crystal structure of D NR, 279–288, 280f D analogs inducing conformational change in VDR, 1452f, 1453, 1550, 1550f deltanoids v. nuclear VDR, 1408–1411, 1410f 16-ene-24-sulfone deltanoid v. rickets mutant VDR, 1411, 1411f human/rat VDR, 174–176, 175f HVDRR/alopecia from Glu329Lys/366delC mutations in VDR, 1221f, 1226 HVDRR-related mutations in VDR, 1222–1226 hypothetical conformations of VDR, 1473, 1473f ligand binding changing conformation of VDR, 176, 279, 280f, 293, 294f, 313, 321

1872 Ligand binding domains (Continued) NRs dimerizing via, 314 1,25(OH)2D3 binding v. HVDRR-related mutations in VDR, 1222–1224, 1223f Arg274Leu, 1222–1223, 1223f Cys190Trp, 1223f, 1224 His305Gln, 1223, 1223f Ile314Ser, 1223, 1223f Ile268Thr, 1223f, 1224 structural analysis of, 1224 Trp286Arg, 1223f, 1224 1,25(OH)2D3 stabilizing VDR, 313 proteins with 1α,25(OH)2D3, 387–392 putative alternative VDR, 400 “squelching” in NR, 291–292 structures in hVDR∆/zVDR, 284–285, 284f topology of hVDR∆, 281, 283f VDR, 1211–1212, 1211f in VDR/DNA binding, 178 VDR-RXR heterodimerization v. HVDRR-related mutations in VDR, 1224–1225 Arg391Cys, 1221f, 1224 Gln259Pro, 1221f, 1225 Phe251Cys, 1221f, 1225 structural analysis of, 1225 VDRnuc v. DBP, 387, 387f zVDR, 284, 284f zVDR-Gemini channel showing adaptability in, 287–288 Ligands allograft tolerance induced using VDR, 1519 BPH inhibited by VDR, 1833–1840 classes of nonsecosteroid D, 1565 genomic/nongenomic action specificity of, 1461 hVDR∆ binding, 280–281 identification of ODF, 670–673 immune responses regulated by VDR, 631–643 immunoregulation by VDR, 633, 643, 643f immunosuppressive agents combining with VDR, 642 nonsecosteroid VDR, 1557, 1558f novel mammalian VDR, 225–227, 227f potential uncharacterized novel VDR, 234f, 235 regulatory T cells enhanced by, 636 structure of hVDR complexed to superagonist, 285–287, 286f tissue selectivity in VDR, 271 VDR immunomodulatory mechanisms in autoimmune disease models, 636–640, 637t VDR signaling activated by, 235–237, 236f Linkage disequilibrium (LD) FokI RFLP surrounded by small, 1128f, 1130 polymorphisms predicted by, 1126–1127 Lipids, bile acids in digesting/absorbing, 863 Lipopolysaccharide (LPS) in macrophage 1α-hydroxylase amplification, 1385–1386, 1385f 1,25(OH)2D3 down-regulating NOS II in injection of, 1783 Liquid chromatography/mass spectrometry (LC/MS), 20-methyl-1α,25(OH)2D3 metabolite, 1434, 1435f Lithium, D metabolism influenced by, 1255t, 1274 Lithocholic acid (LCA) detoxification amplified by supplemental D, 246, 247f in ED-71 synthesis, 1534, 1534f intestinal detoxification of, 867–868, 868f as nonsecosteroid VDR agonist, 1557, 1558f VDR regulating CYP3A-dependent detoxification of, 867 Liver D 25-hydroxylation in, 17–19 in D metabolism, 1294–1296

INDEX

Liver (Continued) D metabolites secreted in, 1295 D3 uptake by, 47–48 DBP production in, 121 enterohepatic circulation in, 863–864, 1295–1296 1,25(OH)2D3/analog immunoregulatory properties in, 1519 1α,25(OH)2D3-mediated rapid response in, 386t PTHrP expression in, 739t rejection inhibited by 1,25(OH)2D3/analogs, 641t Liver disease. See also specific liver diseases bone disorders associated with, 1303–1306 Liver X receptor (LXR), cholesterol/bile acid levels controlled by, 865 LNH. See Late neonatal hypocalcemia Locke, John, rickets reported by, 967 Looser’s zones impaired mineralization ambiguously shown by, 1043 in osteomalacia, 971–973, 972f–973f radionuclide bone scans detecting, 986–988, 989f in severe rickets, 1071 in XLH patients, 981, 982f, 984f LPS. See Lipopolysaccharide LXR. See Liver X receptor Lymphocytes, paracrine 1,25(OH)2D suppression of, 1387f, 1388–1389 Lymphoma, hypercalcemia in, 1361–1362 Lysosomes, DBP degradation in, 157, 157f Lythgoe coupling, in steroid precursors, 1414–1416, 1414f, 1415f

M Macrophages 24-hydroxylase enzyme regulation in, 92 D-1α-hydroxylase in, 1381–1382, 1382f 1α-hydroxylase v. immune cell regulators in, 1384–1387 intracrine/autocrine 1,25(OH)2D activation of, 1387–1388, 1387f 1,25(OH)2D-directed 24-hydroxylase activity in, 1383–1384 PTH/Ca/Phosphate responsiveness lacking in, 1382–1383, 1383f Magnesium (Mg). See also Hypermagnesemia; Hypomagnesemia CaR v., 553–554 jejuno-ileal bypasses reducing serum, 1303 in PTH secretion, 1053 supplementation in acute hypocalcemia therapy, 1058 Magnetic resonance imaging (MRI) acid phosphate distribution/crystal structure from, 483 in evaluating bone metabolic disease, 924 tumors causing TIO v. whole body, 988 Malignant hyperthermia, hyperphosphatemia in, 1178 Malnutrition hypocalcemia due to, 1052f, 1055–1056 in last trimester of pregnancy, 803 Mammary gland D in, 857–858 development role of D endocrine system, 291 development v. VDR, 345–346 VDR expression/role in normal, 1669–1670, 1670t MAP kinase. See Mitogen-activated protein kinase MAR. See Mineral apposition rate MARRS. See Membrane-associated rapid response steroid binding protein Matrix attachment regions (MARs) chromatin units between, 314, 315f genomic domain/nuclear scaffold association mediated by, 329f Matrix extracellular phosphoglycoprotein (MEPE), as phosphatonin/minhibin, 1164, 1166

INDEX

Matrix Gla protein (MGP), in mineralization v. 1α,25(OH)2D3, 716 Matrix metalloproteinase–9 (MMP–9) gene, 1,25(OH)2D3 targeting, 566, 567 Matrix vesicles D metabolites modulating PKC in, 586 extracellular matrix growth factors activated by, 589–590 genomically controlled production of, 589 in matrix calcification, 589 nongenomic regulation of, 589–591 proposed mechanism for, 590–591, 590f 1,25(OH)2D3 accelerating crystal formation in, 591 1,25(OH)2D3/24,25(OH)2D3 affecting matrix calcification through, 590–591 Pi transport in, 466 Maturation (mitosis) promoting factor (MPF), cell cycle G2/M transition regulated by, 1645, 1645f Maxacalcitol. See 22-Oxa-calcitriol Maximum voluntary contraction (MVC) D v. veiled Danish Arabs’, 1812, 1812f 25OHD v., 1812 2MbisP. See 2-Methylene-19-nor-(20S)-1αhydroxybishomopregnacalciferol MC1288 analog-VDR complex stabilized by, 1496 chronic graft rejection inhibited by, 642 structure v. 1α,25(OH)2D3, 285–287, 286f as superagonist, 1477–1478 VDR transcriptional activity v., 1474t MC–903 cellular differentiation assay v., 1543 psoriasis v., 1543 McCollum, E. V., in vitamin A/B/D discoveries, 4 McCune-Albright syndrome, fibrous dysplasia as component of, 1192 MCM proteins. See Mini-chromosome maintenance proteins MCR. See Metabolic clearance rate 2MD. See 2-Methylene-19-nor-(20S)-1,25-dihydroxyvitamin D3 2αMD. See 2α-Methyl-19-nor-(20S)-1,25-dihydroxyvitamin D3 Mediator-D coactivator complex, 265–267 in comodulator activity integrated model, 300, 300f D analogs recruiting, 1455 functionality, 266–267 HAT activity absent in, 267, 295 identification, 265–266 MC1288 enhancing recruitment of, 272–273 multiple binding motifs in, 269 other complexes closely related to, 266 subunit composition, 265t transcription factors interacting with, 269–270 unified nomenclature v. DRIP, 295 VDR linkage to, 176 VDR-RXR heterodimers interacting with, 296, 296f in VDR transcription model, 268–270, 269f Medullary thick ascending limbs (MTAL), CaR inhibiting NaCl reabsorption in, 557 Medullary thyroid carcinoma (MTC) 5-HT1 receptor activation v. Ca in, 693, 694f C cells v., 688 D analogs v., 696 hereditary/sporadic, 688 Megalin, 157–158, 157f, 158f expression v. 1,25(OH)2D3 resistance in CKD, 1319 in 25OHD3 1α-hydroxylation, 73 as 25OHD3/DBP complex endocytic receptor, 154–156 Megalin-DBP complexes, in 25OHD3/1,25(OH)2D3 conversion, 125 Melanin, vitamin D photosynthesis v., 38–39, 40–41 Melanoma, VDR polymorphisms v., 1146

1873 Membrane-associated rapid response steroid binding protein (MARRS), odontocytes expressing, 599, 600f Membrane VDR (VDRmem), 391–392 membrane-initiated responses v. 1α,25(OH)2D3 interacting with, 383 rapid responses v. 1α,25(OH)2D3 interacting with, 399–400, 399f Men. See also Prostate cancer D intake v. PCa risk in American, 1681 normative histomorphometric data for, 956t 1,25(OH)2D concentration/MCR/PR in young/elderly, 827, 827f PCa in American, 1679 testosterone v. 1,25(OH)2D3 in hypogonadal, 1261 Metabolic bone disease adverse influences on mineral/skeletal homeostasis in, 915, 915t approaching patients with, 913–928 biochemical investigation of, 924–926 diagnostic evaluation of, 913–926 epidemic, 913 histopathological assessment of, 926 HPI in addressing, 916 laboratory testing for, 921, 921t medical history in evaluating, 914, 915–918, 915t patient complaints in addressing, 915, 916t physical examination for, 916t, 919–921, 919t radiological examination for, 921–924 review of systems in addressing, 918 treatment, 926–928 Metabolic clearance rate (MCR), young/elderly men’s 1,25(OH)2D, 827, 827f Metabolism, 15–29, 1000f, 1423–1424 aging v., 823–833 aging v. 1,25(OH)2D, 826–831 Al v., 1255t, 1270–1271 analog, 1423–1443 biological systems in studying, 1428–1429, 1428t drug design implications of, 1442–1443 examples of, 1429–1440 general considerations in, 1423–1429 implications from studying, 1440–1443 invalid in vivo/in vitro comparisons in, 1442 non-D-related, 1428–1429 pharmacokinetic information correlating with, 1440–1441, 1441t questionably valid assumptions in, 1442 radioactive analogs in studying, 1428–1429 anticonvulsants v., 1255t, 1264–1265 antituberculous agents v., 1255t, 1271 bisphosphonates v., 1255t, 1268 Ca channel blockers v., 1255t, 1269 caffeine v., 1255t, 1271–1272 calcitonin v., 1254t, 1256–1257 cimetidine v., 1255t, 1270 corticosteroids v., 1255t, 1265–1266 cyclopropane ring-containing analogs’, 1431–1433, 1432f D, 15–29, 16–17 mutant mouse metabolic enzyme models v., 105 overview, 15–17 species variation in, 27–29, 28t D2 D3 metabolism v., 16–17 pathways of, 18f unique aspects in, 25–26 D3, 69–70, 70f, 85–86, 86f, 1000–1002, 1000f pathways of, 16–17, 17f D3 abundance v., 1000f, 1001 D2 derivative, 1430–1431

1874 Metabolism (Continued) dihydrotachysterol, 1429–1430, 1430f drugs influencing, 1255t, 1263–1274 20-epi-/20-methyl analog, 1433–1434, 1433f, 1435f estradiol v., 1254t, 1259–1261 ethanol v., 1255t, 1266 exogenous/stimulated PTH v., 1253–1255, 1254t fluoride v., 1255t, 1273 gastrointestinal disease v., 1293–1297 GH/IGF-I v., 1254t, 1258 GH v., 1254t, 1257 GIO v., 1239–1248 heparin v., 1255t, 1269–1270 homologated analog, 1434–1436 hormones influencing, 1254t, 1263–1274 in HVO, 1039–1040 D-24-hydroxylase in, 1459–1461, 1460f hypolipidemics v., 1255t, 1267–1268 IFNγ v., 1254t, 1263 immunosuppressants v., 1255t, 1272–1273 insulin v., 1254t, 1259 ketoconazole v., 1255t, 1266–1267 kinetics of 1,25(OH)2D, 828 lithium v., 1255t, 1274 liver’s roles in, 1294–1296 in normal/neoplastic colon cells, 1715–1719 olestra v., 1255t, 1273 orlistat v., 1255t, 1274 oxa-group containing analog, 1438 phosphate homeostatic disorders v. paradoxical regulation of, 1161 Pi-dependent modulation of, 1161–1162 progesterone v., 1254t, 1261–1262 prolactin v., 1254t, 1258 prostaglandins v., 1254t, 1262–1263 PTHrP v., 1254t, 1255–1256 reciprocal regulation of phosphate homeostasis, D, 1159 sex steroids v., 1254t, 1259–1262 testosterone v., 1254t, 1261 theophylline v., 1255t, 1272 thiazide diuretics v., 1255t, 1268–1269 thyroid hormone v., 1254t, 1262 TNFα v., 1254t, 1263 unsaturated analog, 1436–1437 in vitro regulation by 1,25(OH)2D3/EGF, 1717 Metabolites, early evidence of converted, 6–7 Metastasis, D compounds v., 1576 Metastatic calcification in arteries/around joints, 979, 980f chronic renal failure causing, 979 treating, 979, 980f Mg. See Magnesium MGP. See Matrix Gla protein MI. See Mineralization index; Myocardial infarction Mice. See also specific types of mice in coculture system recruiting osteoclasts, 666–667, 667f 1,25(OH)2D3 v. diabetes/MS in NOD, 1404 Michaelis-Menten equation Ca flux described by, 412 high dose intermittent 1,25(OH)2D3 fitted to, 1742, 1743f Miconazole, kidney 1α-hydroxylase activity regulated by, 78 Microcomputerized tomography (µCT), bone structural data provided by, 481 Microvillar membrane. See Brush border Middle Easterners low 25OHD in immigrant, 1026 rickets in immigrant, 968–971

INDEX

Migraine, 1,25(OH)2D3 repression of CT/CGRP expression v., 690 Mineral apposition rate (MAR) bone formation indices derived from, 955–956 ED-71 v. ovariectomized rat, 1537, 1537f in Mlt, 1033 OCT v., 1528, 1529t Mineral homeostasis, 411–559, 453–469 adverse influences on, 915, 915t clinical disorders of phosphate, 1159–1180, 1164t ECaCs v. Ca, 430–432 factors causing disorders in phosphate, 1162, 1164t fetal/maternal Ca, 853–854 kidney in, 515–528 maternal D status v. neonatal Ca, 843 metabolic bone disease diagnosis v., 914 1,25(OH)2D3 regulating Ca/P, 1403 Pi, 453–469 extracellular, 453–455, 454f PTHrP in fetal C9, 3–4 regulation of Pi, 1159–1161 systemic/intracellular Ca2+, 751 VDR ablation v., 341–345 VDR-null mice skin change v., 346f, 347 VDR-null mice with normal, 344 Mineralization, 477–490 bone histomorphometry assessing, 954–957 complex mineral transport in, 1044 D/D metabolites influencing, 478–480, 565–571 D-deficiency decreasing, 484, 485f D-deficiency v., 487–488 D influencing, 483–487, 565–566 D v. cell/matrix molecule, 479t, 484–487 D v. pathogenesis of impaired, 1040–1044 definitions/terminology, 477–478 dynamic indices of, 955–957 epitaxial/heterogeneous crystal formation in, 477 gene expression v. 1α,25(OH)2D3 during, 715–716 histology, 1036t hypocalcemia due to accelerated skeletal, 1057 maternal D status v. infant, 842–843 microscopic examination of in situ, 1029–1030 in model systems with D alterations, 487–490 variations/dependencies, 487 morphologic/biochemical aspects of, 1029–1031 normocalcemic/D-deficient, 1043 1α,25(OH)2D3 v. available Ca in, 711 osteoblasts influencing, 1044 osteoid indices in recognizing impaired, 1034–1035 as phase transformation, 1030 phosphatonins/minhibins regulating, 1163 physical chemistry of, 478, 479t possible D targets influencing, 509–510 precipation in, 483–484 primary/secondary, 1032 quantifying tissue, 480–483 ash weight/density in, 481 mineral characterization in, 481–483, 482f questions in, 480 radiographic methods showing, 480–481 two types of bone, 1030–1031 type I collagen required for bone matrix, 703 unexpected/dystrophic, 477–478 VDR alteration v., 488–489, 489f in vitro, 483 Mineralization index (MI), osteomalacia diagnosed with, 1035–1036

1875

INDEX

Mineralization lag time (Mlt), 957 in osteoid accumulation/osteomalacia pathogenesis, 1033–1034, 1033f osteomalacia defined using, 1035–1036 Mini-chromosome maintenance (MCM) proteins, in DNA licensing/replication, 1644–1645 Mitogen-activated protein (MAP) kinase cascade up-regulation, 888 CGRP transcription controlled by, 692–693, 692f pathways in 1,25(OH)2D3 differentiation signaling, 1638–1639, 1638f Mitosis, MPF regulating cell cycle transition to, 1645, 1645f Mixed-function oxidases, in 25OHD3 1α-hydroxylation, 71–73, 71f Mlt. See Mineralization lag time MMP–9 gene. See Matrix metalloproteinase–9 gene Monocytes 24-hydroxylase enzyme regulation in, 92 intracrine/autocrine 1,25(OH)2D interaction with, 1387–1388, 1387f Moroccans, serum 25OHD/PTH in dark-skinned Dutch, 794 2MP. See 2-Methylene-19-nor-1α-hydroxyhomopregnacalciferol MPF. See Maturation (mitosis) promoting factor 2Mpregna. See 2-Methylene-19-nor-1α-hydroxypregnacalciferol MRI. See Magnetic resonance imaging MS. See Multiple sclerosis MTAL. See Medullary thick ascending limbs MTC. See Medullary thyroid carcinoma µCT. See Microcomputerized tomography Multiple sclerosis (MS), 1780 D3 v., 999t EAE role supporting 1,25(OH)2D3 involvement in, 1784 1,25(OH)2D3 v. NOD mouse, 1404 VDR polymorphisms v., 1147 Muscle biopsies in HDM clinical studies, 1811 Ca v. D deprivation/repletion, 1810f, 1811 cell culture/animal studies of D, 1809–1811 contraction, 1807, 1807f, 1808f contraction/relaxation v. D, 1809, 1809f D influencing, 883–894 D influencing striated, 1809–1811 D3 metabolites v. phosphate uptake by, 887 dystrophic mineral deposits in, 478t falls v. D’s influence on, 1805–1817 fiber types, 1808 filament components, 1807, 1807f function in HDM clinical studies, 1811–1813 function v. VDR polymorphisms, 1813 genomic/nongenomic D influence on striated, 1809 1α,25(OH)2D3 mechanisms in, 889–893 genomic, 889–890 nongenomic, 890–893, 890f 1α,25(OH)2D3 regulating Ca homeostasis in, 886–887 1α,25(OH)2D3-stimulated signaling in growth of, 888–889, 889 physiology v. HDM, 1806–1808, 1806f, 1807f power v. serum 25OHD, 1812–1813, 1812f proliferation/differentiation influenced by 1α,25(OH)2D3, 887–889 regeneration v. 1,25(OH)2D, 1816 tyrosine phosphorylation mediating 1α,25(OH)2D3 in growth of, 888 VDR in, 885–886 VDR polymorphisms v. strength of, 1143 weakness with D insufficiency, 1090

Muscle cells ATP supplying energy to, 1808 Ca homeostasis in, 1807–1808, 1808f 1,25(OH)2D3/deltanoid-induced differentiation modeled in, 1637t MVC. See Maximum voluntary contraction Myelodysplasia, 1α-(OH)D3 v., 1573 Myocardial infarction (MI), VDR polymorphisms v., 1148 Myopathies animal model studies of, 884–885 clinical background of, 883–884 D-dependent, 883–885, 893

N Na. See Sodium Na/Pi cotransporters, type I/II/III, 1159–1160 NaCl. See Sodium chloride NADPH-ferredoxin reductase, in 25OHD3 1α-hydroxylation, 71f, 73 Native Americans. See American Indians NCoA62/SKIP coactivator complex, 296, 297f in comodulator activity integrated model, 300, 300f transcription/mRNA slicing coupled by, 298 VDR gene expression comodulated by, 296–297, 297f VDR interaction independent of AF-2 domain, 296 NCoR, NR co-repressor, 298–299 NDOs. See Non-digestible oligosaccharides Necrosis, as inflammatory process component, 126 Negative VDRE (nVDRE) as complex VDR binding site, 321–322 positive VDREs v., 321–322 VDR-RXR heterodimer repressing transcription through, 309–310, 310f Neonatal severe hyperparathyroidism (NSHPT), homozygous inactivating CaR mutations causing, 553 Nephrolithiasis in IH, 1142, 1339–1352 renal histopathology in Ca oxalate, 1341 Nephron Ca reabsorption in, 519, 520f 25OHD3 in, 519 Nerve growth factor (NGF), 1,25(OH)2D3 stimulating, 1781–1782 Nervous system calbindin-D28K in, 726–727, 730t CKD/1,25(OH)2D3 deficiency/resistance in, 1327 Neurotrophic factors, 1,25(OH)2D3 influencing, 1781–1782 Neurotrophin receptor, 1,25(OH)2D3 regulating low-affinity, 1782 New World primates biochemical phenotype of rachitic, 352–353, 353f D hormone resistance in, 352, 354–355 early evolution of, 351, 352f high 1,25(OH)2D3 levels in, 353, 353f IDBP function in, 359–360, 360f IDBPs over-expressed by, 359 VDP carrying 25OHD in, 352 NF-κB, activation in osteoclast differentiation, 678 NGF. See Nerve growth factor Nigerians, sunlight exposure v. rickets in, 1066–1067 Nitric oxide (NO), in macrophage 1α-hydroxylase regulation, 1386 NLSs. See Nuclear localization sequences NMR. See Nuclear magnetic resonance NO. See Nitric oxide Non-digestible oligosaccharides (NDOs), enhancing Ca absorption from foods, 818

1876 Non-Hodgkin’s lymphoma extrarenal D metabolite overproduction in, 1390t, 1391–1392 1α-(OH)D3 v., 1573 PTHrP associated with end-stage, 740 Nongenomic response, steroid hormones in, 98–99 Normocalcemia, CaR in restoring, 551 NOS II. See Type II nitric oxide synthase NPC. See Nuclear pore complex NPT2a, not responsible for HHRH, 469 NPT2a gene, expression/protein production regulation in kidney, 1160–1161, 1160f NPT2a protein mediating Na/Pi cotransport, 459 Na/Pi transport changes v., 1160–1161 PTH/AKAP79/RAP retrieving, 461 regulation, 459–462, 461t signaling mechanisms in insertion/retrieval of, 1161 structure-function, 459, 460f NPT2b protein, molecular mechanisms regulating, 456–457, 457t NRs. See Nuclear receptors NSHPT. See Neonatal severe hyperparathyroidism Nuclear localization sequences (NLSs) calcitriol-induced transcription v. mutant, 372f hVDR putative, 368–369, 368f RXR, 369, 370f Nuclear magnetic resonance (NMR), acid phosphate distribution/crystal structure from, 483 Nuclear pore complex (NPC) nucleocytoplasmic macromolecule transport through, 365–374, 367f VDR/RXR exiting nucleus through, 366f, 369 Nuclear receptors (NRs), 279 apo/holo forms of, 279 bile acid metabolism regulated by, 865–866 co-repressors in, 292, 293f, 298–299 Alien, 299 hr, 299 SMRT/NCoR class of, 298–299 comodulators linking VDR/PIC, 292, 293f crystal structures of LBDs in, 279–288, 280f DNA response element recognition by, 230, 231f everted repeats in, 317–319, 318f export of, 368 first described defect in superfamily of, 1218 human, 225, 226f–227f interaction domains/coactivators in, 235–237 N-terminal variants of, 195 1,25(OH)2D3 inducing focal accumulation of, 374–375, 375f protein interactions facilitating export of, 374 RNA processing linked to coactivators in, 297–298 RXR as heterodimeric partner for, 291, 292f VDRs in superfamily of, 225–228 Nuclear VDR (VDRnuc) 1α,25(OH)2D3 gene transcription mediated by, 383 deltanoids selectively modulating, 1408–1411, 1410f LBD of human, 387–388, 387f, 389f, 390f–391f, 398f LBD v. DBP LBD, 387, 387f 1α,25(OH)2D3 biological actions mediated by, 384–385, 386t structure v. DBP structure, 391, 392t tissue distribution of 1α,25(OH)2D3, 384–385, 385t Nucleosome, hnRNP insertion in, 358 Nucleus acceptor sites in, 334 architectural parameters in, 335–336 gene expression in architecture of, 328–329, 329f, 330f gene expression regulatory component scaffolding in, 332–333

INDEX

Nucleus (Continued) import/export mechanisms in, 365–368, 367f protein export retarded by docking in, 374 signal pathway targeting/integration in compartments of, 335 skeletal gene expression regulatory machinery organization in, 327–336 skeletal regulatory factor trafficking in, 334–335 transcription machinery/factor complexes compartmentalized in, 332 transcriptionally active compartments in, 334–335 VDR translocation to, 1452f, 1454 Nutrition, early research in, 3 Nutritional recovery syndrome, hypophosphatemia in, 1177 nVDRE. See Negative VDRE

O OA. See Osteoarthritis Obesity bone histomorphometry in, 795–796 bone mass v., 795 D metabolism v., 795 OC. See Osteocalcin OC gene chromatin organization of, 330–332 nuclear/gene expression relationship represented by, 328–329, 331f OCIF. See Osteoclastogenesis inhibitory factor OCIF/OPG characterization, 668–670 discovery, 668–670 OCT. See 22-Oxa-calcitriol ODF. See Osteoclast differentiation factor Odontoblasts D-dependent molecules in, 602–604, 603f dentin elaborated by, 599 PHEX expressed in, 467 VDR gene/MARRS expressed in, 599, 600f Odontogenesis, process/functions, 599–602 1,25(OH)2D. See 1,25-Dihydroxyvitamin D 1,25(OH)2D2. See 1,25-Dihydroxyvitamin D2 1,25(OH)2D3. See 1,25-Dihydroxyvitamin D3 1α,25(OH)2D3. See 1α,25-Dihydroxyvitamin D3 25OHD. See 25-Hydroxyvitamin D 25OHD3. See 25-Hydroxyvitamin D3 25,26(OH)2D3. See 25,26-Dihydroxyvitamin D3 OHO. See Tumor-induced osteomalacia Olestra, D metabolism influenced by, 1255t, 1273 Omt. See Osteoid maturation time Oncogenes in cell cycle/apoptosis control, 1577–1580 1,25(OH)2D3/EB1089 regulating breast cancer cell, 1664–1665 1,25(OH)2D3 regulating expression of, 1577–1579 Oncogenic hypophosphatemic osteomalacia (OHO). See Tumor-induced osteomalacia Oncology, 1,25(OH)2D3/1,25(OH)2D3 analogs in, 1741–1747 OPG. See Osteoprotegerin OPN. See Osteopontin ORC proteins. See Origin recognition complex proteins Origin recognition complex (ORC) proteins, in DNA licensing/replication, 1644–1645 Orlistat, D metabolism influenced by, 1255t, 1274 OS/BS. See Osteoid surface per unit of bone surface Osteoarthritis (OA) VDR polymorphisms v., 1144–1145 in XLH patients, 983

INDEX

Osteoblast genes, D in vivo response in, 566 Osteoblasts in BMU termination, 503 BMUs assembling teams of, 501–502 bone formation by, 498 calbindin-D9K/D28K in, 724–725, 730t as D analog therapy target, 1501 D compounds v. apoptosis of, 1498 D-dependent molecules in, 602–604, 603f D influencing differentiation/activity of, 649–658 death of, 506 different dexamethasone/1,25(OH)2D3 influences on, 652 differentiation of, 712–713 dual role of 1α,25(OH)2D3 in, 713–714, 714f gene expression sequence v., 328 nuclear architecture v. transcription during, 335 1α,25(OH)2D3 v., 712–714, 712f factors stimulating Na/Pi transport in, 466, 466t glucocorticoids increasing differentiation of, 1240 L-type VSCC v. Ca2+ influx into, 754–755, 755f 2MD inducing mineralization in, 1546–1547, 1547f Mlt v. age of, 1033–1034, 1033f Na/Pi transport in renal proximal tubular cells v., 461t, 466, 466t nonadherent marrow cells in 1,25(OH)2D3 influencing, 652–653 1,25(OH)2D3 causing gene up-regulation in mature, 650f, 652–653 1,25(OH)2D3 deficiency v. mineralization by, 1043–1044 1,25(OH)2D3 directly affecting, 703 1,25(OH)2D3 having biphasic effects on, 650 1,25(OH)2D3 influencing, 1240–1241 1,25(OH)2D3 influencing in vitro, 649–656 1,25(OH)2D3 influencing proliferation of precursors to, 653–654 1,25(OH)2D3 modifying hormone/GF responsiveness of, 654–655 1,25(OH)2D3 “pushing” maturity of, 651–652 1,25(OH)2D3 regulating genes influencing proliferation/ differentiation of, 653–654 1,25(OH)2D3 regulating products of, 654 as 1α,25(OH)2D3 source, 717 1,25(OH)2D3 target proteins with in vitro, 566 1,25(OH)2D3 v. heterogeneity of, 652 1,25(OH)2D3 v. in vitro differentiation pathway/status of, 649–653, 650f 1α,25(OH)2D3 v. gene expression in proliferating, 714–715 osteocalcin promoter remodeling v. phenotype development in, 331f osteoclast differentiation/activation regulated by, 677, 677f PHEX expressed in, 467 site-dependent differential activity of, 599 VSCC/VICC interactions in, 755, 755f Osteocalcin (OC), 176 D deficiency rickets v., 1070 expression stimulated by 1,25(OH)2D3, 486–487 human gene promoter induced by 1,25(OH)2D3, 176–177 human/rat 1,25(OH)2D3/PTH v., 567, 567f mineral properties in KO animals, 479t in mineralization, 479t in mineralization v. 1α,25(OH)2D3, 715–716 1,25(OH)2D3 inducing/glucocorticoids suppressing, 1240 1,25(OH)2D3 regulating matrix protein expression in, 703 regulation as VDR-regulated transcription model, 176–178 VDR target gene polymorphism associated with, 246 Osteoclast differentiation factor (ODF) ligand identification, 670–673 molecular cloning, 670–672, 671f OPG-binding protein as candidate, 670 in osteoclastogenesis hypothesis, 667–668, 668f

1877 Osteoclastogenesis, 665–681 factors in molecular mechanism of, 668–676 RANK as signaling receptor in in vitro, 673–674, 673f RANKL-RANK signal transduction pathways in, 676–678 Osteoclastogenesis inhibitory factor (OCIF), isolation/cloning, 668 Osteoclasts in BMU origination, 503 bone resorption by, 498 molecular mechanisms forming/activating, 674, 676f mouse coculture system recruiting, 666–668, 667f Na/Pi transport in, 466–467 OC recruiting, 487 1,25(OH)2D3 v bone resorption by, 678–680, 679f RANKL/ODF differentiating osteoclast progenitors to, 672 Osteocytes, PHEX expressed in, 467 Osteoid accumulation indices, 1034–1035 accumulation of unmineralized, 1031–1035 in growing skeleton, 1031 histomorphometric measurement of, 954–955, 955f kinetics, 1031 Mlt in understanding accumulation of, 1033–1034, 1033f Osteoid-bone interface, 1031–1032 Osteoid maturation time (Omt), 957 human Mlt v., 1032f, 1033 Osteoid seams, 1032 in adult skeleton, 1031–1034 in bone formation, 1032–1033, 1032f D metabolites’ influence on BMAR v., 657 width measurement of, 955f Osteoid surface per unit of bone surface (OS/BS) osteoid seam life span/FP determining, 1034 O.Th v., 1035, 1035f Osteoid thickness (O.Th) in Mlt, 1033 osteomalacia defined using, 1035–1036 Osteoid volume per unit of bone volume (OV/BV), aging/osteoporosis increasing, 1034 Osteomalacia, 967. See also Tumor-induced osteomalacia Al toxicity in, 980, 1270 biochemical evolution of, 1036–1039 causes of, 925–926, 925t–926t characteristics in CYP27B1-null mice, 703 D3 curing, 999t D deficiency causing, 566 D/intestinal Ca absorption v., 703 D metabolism in, 1039–1040 focal, 958 generalized, 957–958, 957f histological diagnosis of, 957–958 histological evolution/kinetic definition of, 1035–1036 histomorphometric diagnosis of, 955 hypovitaminosis D causing, 154, 156t Looser’s zones in, 971–973, 972f–973f, 981, 982f, 984f mean Mlt/mean O.Th in defining, 1035–1036 mineralization index in diagnosing, 1035–1036 mineralization v. bone properties in, 489–490, 490f Mlt in understanding pathogenesis of, 1033–1034, 1033f in 1α(OH)ase-null mice, 489 osteoid seam width/Mlt in, 957 pathogenesis, 1029–1044 in postgastrectomy bone disease, 1299, 1300 radiology of, 967–990 radiology of D-deficiency, 971–973 rickets defined v., 1031 risk v. race/population, 789, 790t

1878 Osteomalacia (Continued) secondary hyperparathyroidism in, 973, 975f VDR ablation v., 342–343 Osteopenia atrophic rickets characterized by, 1071 D deficiency v., 510 premature infant risk criteria for, 812, 812t renal disease/bone resorption causing, 979 Osteopetrosis bone formation/resorption imbalance causing, 665 in c-src KO mice, 677 Osteophytosis, VDR in, 1144–1145 Osteopontin gene, VDR homodimer-mediated activation v., 179–180 Osteopontin (OPN) mineral properties in KO animals, 479t in mineralization, 479t 1,25(OH)2D3 increasing synthesis of, 567–568 1,25(OH)2D3 regulating matrix protein expression in, 703 1,25(OH)2D3 up-regulating, 487 Osteoporosis, 1101–1114. See also Glucocorticoid-induced osteoporosis in African-Americans/Caucasians, 816 bone formation/resorption imbalance causing, 665 calbindin-D28K v. apoptotic cell death in, 727 D in treatment of established, 1109–1114 D supplementation in treatment of, 1113–1114 D v. mineralization in, 489–490 D3 v., 999t ED-71 v. clinical results using, 1537–1539, 1538f development of, 1525, 1534–1539 planned studies of, 1539 preclinical results using, 1534–1537 safe dosage for, 1537–1538 increased net bone resorption in type I, 1109, 1109f 1,25(OH)2D3 in treating, 1111 1,25(OH)2D safety margin v., 998 PTH/25OHD in type II, 1109 rationale/principles of D treatment of, 1109–1111 renal disease/bone resorption causing, 979 Ro 26–9228 hybrid deltanoid v., 1416–1417, 1417f VDR gene association studies in, 1141–1144 VDR gene polymorphisms predicting, 184 VDR target gene polymorphism associated with, 246 Osteoprotegerin (OPG) bone-resorbing factors regulating, 675–676, 676t clinical trials, 676 discovery, 668 as RANKL decoy receptor, 568 regulating expression of, 674–676 structure of human, 668, 669f in TNF receptor family, 668 Osteosclerosis radiology of, 981, 982f in renal disease/secondary hyperparathyroidism, 977, 977f O.Th. See Osteoid thickness OV/BV. See Osteoid volume per unit of bone volume Ovarian cancer, VDR in, 856 Ovary D in, 856 VDR ablation reducing aromatase activity in, 345 22-Oxa-calcitriol (OCT) adverse reactions to topical, 1533–1534 angiogenesis v., 1574 BFR v., 1528, 1529t

INDEX

22-Oxa-calcitriol (Continued) bone influenced by, 1527–1529, 1529t bone metabolism marker changes v., 1531, 1532f breast cancer cells/tumors v., 1669 cortical bone formation rate v., 1531, 1533f development, 1525–1534 MAR v., 1528, 1529t mechanism of action, 1479–1480 metabolism, 1438 as noncalcemic analog, 1440, 1441t PCa v., 1690 psoriasis vulgaris v., 1531–1534, 1533f PTH v., 1527, 1527f, 1528f PTHrP gene expression/secretion inhibited by, 1576 reduced parathyroid VDR v., 1317, 1318f secondary hyperparathyroidism v., 1527–1531 structure, 1479f, 1525, 1526f synthesis for large-scale production, 1525–1526, 1526f Oxidation-reduction (redox) homeostasis D/cellular response to, 761–768 ROS v., 761–763

P P. See Phosphorus PAD. See Peripheral arterial vascular disease Paget’s disease of bone, calcitonin treating, 1257 Pakistanis, D metabolism in, 790t, 792–793 Pancreas calbindin-D28K in, 725–726, 730t CKD/1,25(OH)2D3 deficiency/resistance in, 1326 Pancreatic disease, metabolic bone disease v., 1301 Pancreatitis, hypocalcemia/tetany in, 1057 Paracellular path, CA absorption by, 421–422 D-dependency of, 412f, 421–422 thermodynamic parameters of, 421 Parathyroid gland. See also Hyperparathyroidism; Hypoparathyroidism; Neonatal severe hyperparathyroidism Ca2+ elevation v., 553f, 554–555, 555f CaR activity v. D effect on, 543 CaR in, 554–556 cell proliferation v. secondary hyperparathyroidism, 544–547 CKD with low 1,25(OH)2D3 or 1,25(OH)2D3/VDR resistance in, 1322–1325, 1323f, 1324f, 1325f D in, 537–547 hyperplasia v. TGFα/EGFR expression in CKD, 1323–1324, 1323f, 1324f low 1,25(OH)2D3 v., 1324–1325, 1325f model of factors regulating, 546f 1,25(OH)2D3 deficiency v. VDR content in, 1317, 1318f as 1,25(OH)2D3 target organ, 539, 540f physiological insights from homozygous null mice, 545–547 PTHrP expression in, 739t removal v. rat 1,25(OH)2D synthesis, 463–464 VDR/CaR interactions in, 556, 556t Parathyroid hormone (PTH) age/glomerular filtration rate v. serum, 1024 age v., 1106–1107 biosynthesis, 537 blacks showing decreased skeletal sensitivity to, 791 bone loss/D deficiency v., 509–510 bone loss v. age-related increase in, 1107, 1108f Ca channel blockers v., 1269 Ca reabsorption in D-replete/deficient rats v., 518–519, 519f Ca v., 552, 553f

INDEX

Parathyroid hormone (Continued) CaR mutations v., 555 CaR v., 537–538 CKD impairing 1α-hydroxylase induction by, 1315–1316 CYP24A1 expression regulated by, 93, 94f CYP24A1 promoter expression/CYP24A1 activity v., 99–100 D bone resorption inhibition v., 570 as D deficiency/insufficiency/repletion index, 1085–1086 D insufficiency increasing serum, 1088–1089 D metabolism influenced by exogenous/stimulated, 1253–1255, 1254t degradation in PTH control, 544 24,25-dihydroxyvitamin D3 suppressing secretion of, 16 diuretics v. serum, 1024 early D deficiency associated with normal, 1069 ECF [Ca2+] demand response mediated by, 776 effector mechanism independence/redundancy, 778 estrogen deficiency v. age-related increase in, 1108–1109, 1108f estrogen influencing serum, 1024 function v. age, 1106 as HDM cause, 1815 hypocalcemia due to abnormal availability of, 1052–1054 hypocalcemia due to resistance to, 1054 hypocalcemia v. defective secretion of, 1053–1054 hypocalcemia v. postsurgery reduction in, 1053 intravenous 1,25(OH)2D3 in renal failure v., 1329–1330, 1330f kidney 1α-hydroxylase activity regulated by, 76–77, 76t, 77t kidney/parathyroid VDR regulated by, 522–523 long-term OCT administration v., 1530–1531, 1530f macrophages unresponsive to, 1382–1383, 1383f mechanisms of age-related increases in, 1106–1107 2MP/2MbisP/2Mpregna v., 1552–1553 noncalcemic D analogs suppressing, 1449–2898 OCT v., 1527, 1527f in 1,25(OH)2D3 feedback loop, 539 1,25(OH)2D3 production increased by, 19–20 1,25(OH)2D synthesis influenced by, 830–831, 830f 25OHD v., 1088, 1088f osteoblast Ca2+ entry stimulated by, 755, 755f osteoblast Na/Pi transport stimulated by, 466, 466t during pregnancy, 839 renal Pi excretion decreased by, 516t renal Pi reabsorption regulated by, 460–461, 461t resistance in rachitic children, 1069 secretion control in dialysis patients, 1822–1823 secretion suppression set-point v. age, 1107 secretion v. cardiovascular disease, 901–902 serum Ca defended by, 1050 serum 25OHD v. serum, 1020–1022, 1021f type I collagen synthesis inhibited by, 704 VDR expression affected by, 208 VDR level 1,25(OH)2D3 regulation of, 540f, 541–542 Parathyroid hormone-related peptide (PTHrP), 737–746 antisense RNA technology inhibiting production of, 743 D compounds inhibiting expression/secretion of, 1576–1577 D therapy v. production of, 741t, 743–746 gene/gene products, 737–738 hypercalcemic cancer patient survival v., 740–742, 740f immunotherapy neutralizing, 742–743 mechanism, 738–739 normal/cancer cells/tissues expressing, 739–740, 739t production regulation, 739–742 SCCs elaborating, 612 stimulators/inhibitors in normal/cancer cells/tissues, 740–742, 741f therapeutic strategies inhibiting production of, 742–746, 742f

1879 Parathyroid hormone-related protein (PTHrP) D metabolism influenced by exogenous/stimulated, 1254t, 1255–1256 in placental Ca transport, 853 synthetic molecules’ actions v., 1256 Parathyroid hyperplasia in D therapy prognosis, 1823, 1824f regression of, 1825–1826 Parkinson’s disease (PD), 765, 1779 D analogs treating, 1785 1,25(OH)2D3 antioxidant activities in, 764t, 766–767 Past medical history (PMH), in approaching metabolic bone disease, 917 PBAF. See Polybromo- and BAF-containing complex PBC. See Primary biliary cirrhosis p450c24. See 24-Hydroxylase PCa. See Prostate cancer PCIT. See Percutaneous calcitriol injection therapy PCR. See Polymerase chain reaction PCTs. See Proximal convoluted tubules PD. See Parkinson’s disease PDDR. See Pseudo-vitamin D-deficiency rickets PEIT. See Percutaneous ethanol injection therapy PENK. See Proenkephalin Peptide growth factors, in keratinocyte growth/differentiation, 614–615 Peptide hormones, VDR expression affected by, 207–208 Percutaneous calcitriol injection therapy (PCIT), nodule hyperplasia v., 1825 Percutaneous ethanol injection therapy (PEIT) nodule hyperplasia v. selective, 1824–1825 severe hyperparathyroidism v. selective, 1825f Perinatal period, 803 D actions, 803–808 recommended D intake in, 808 Periodontium cementum in, 601–602 D influencing, 604–605 dentin in, 601–602 Peripheral arterial vascular disease (PAD), D deficiency associated with, 900 Peripheral quantitative computerized tomography (pQCT), peripheral cortical/trabecular density measured with, 481 Peroxisome proliferator-activated receptor (PPAR), ligands in PCa combination therapy, 1696–1697 Phagocytic cells DBP allele activation v. recruiting, 127 tissue damage causing recruitment/activation of, 126–127 Pharmacology, 995–1010 D3 dosage considerations in, 1002–1006, 1009t D3 nutrition questions in, 1009t DBP in, 1002 pharmacokinetic principles in, 1006–1007 PHEX, in XLH/TIO/ADHR pathogenesis model, 1165–1166 PHEX gene, responsible for XLH, 463t, 467 Phorbol ester PKC activity/CYP24A1 expression v., 99 PTHrP expression/production stimulated by, 741t Phosphate. See also Autosomal dominant hypophosphatemic rickets; Hereditary hypophosphatemic rickets with hypercalciuria; Hyperphosphatemia; Hypophosphatemia; Inorganic phosphate (Pi); Phosphaturia; Tumor-induced osteomalacia; X-linked hypophosphatemia absorption v. 1,25(OH)2D3/VDR binding, 220–221, 221f D3 metabolites modifying muscle uptake of, 887 deficiency-related disorders, 467–470

1880 Phosphate (Continued) common metabolic pathway in, 469–470 deprivation, 1176 disorders in homeostasis of, 1159–1180, 1164t disorders in metabolism of, 1189–1194 disorders in renal transport of, 1162–1175 disorders of decreased, 1176 disorders of increased, 1177–1178 disorders related to altered, 1176–1180 gastrointestinal malabsorption of, 1176 homeostasis FGF-23 in maintaining, 1193–1194 in Npt2-null/Hyp mice, 439–440 homeostasis v. cardiovascular disease, 901 macrophages unresponsive to, 1382–1383, 1383f mineralization v. low plasma, 1042 mouse models with defective transport of, 462–463 1,25(OH)2D3 production v. plasma, 20 1,25(OH)2D3 promoting absorption of, 291 1,25(OH)2D v. renal transport of, 1161–1162, 1162f phosphatonins/minhibins regulating renal transport of, 1163 renal D metabolism regulation v., 463–465, 464f retention from chronic renal failure, 979 Phosphatidylinositol 3-kinase (PI3-K), in 1,25(OH)2D3 differentiation signal propagation, 1638 Phosphatonin/minhibin protein candidate biological activity, 1167–1168, 1167t sFRP–4 as, 1164 Phosphatonin/minhibin proteins FGF-23 as, 1164–1166 MEPE as, 1164, 1166 postulated, 1163 Phosphaturia in 1α(OH)ase-null mice, 438 1,25(OH)2D3 v., 519 Phosphorus (P) absorption v. vitamin D3, 5 abundance/ubiquity in tissues, 1159 aging v., 829, 829t altered phosphate load disorders causing abnormal, 1178–1180 combined mechanisms decreasing serum, 1177 D/25OHD3/1,25(OH)2D3 influencing renal handling of, 518–519 disorders related to transcellular shift of, 1176–1177 kidney 1α-hydroxylase activity regulated by dietary, 77 1,25(OH)2D synthesis influenced by, 828–830, 829f PTH infusion/recovery v., 830, 831f renal reabsorption of, 6 treatment of abnormal serum, 1180 VDR ablation v., 342 VDR expression affected by, 202t, 205 Photobiology, 37–43 history, 37–38 Photosynthesis, 38–41, 38f latitude/season/time v., 39, 40f ultraviolet B-absorbing materials v., 40–41, 41f, 42f Phytoestrogens, 1,25(OH)2D3 synthesis regulated by, 1720–1721, 1720t Pi. See Inorganic phosphate PI3-K. See Phosphatidylinositol 3-kinase PIC. See Preinitiation complex PKA. See Protein kinase A PKC. See Protein kinase C PKCα, 1α,25(OH)2D3 muscle stimulation translocating, 890f, 891 Placenta Ca transport using PTHrP/CaR in, 853–854 calbindin-D9K in, 726, 730t

INDEX

Placenta (Continued) 1,25(OH)2D3 production in, 859 PTHrP expression in, 739t Plants, vitamin D2 synthesized by, 15 Plasma membrane calcium (PMCa) pump, 419–420, 525–527 activity modulators, 420–421 CA transported across basolateral membrane by, 419–420 calbindins stimulating, 420 distribution, 527, 527t in duodenal/ileal Ca absorption, 422–424, 423f epitopes in distal tubular cell basolateral membrane, 525–527, 526f Plasma membrane sodium-calcium exchanger, CA transported across basolateral membrane by, 421 Plasma membranes Ca2+ response to 1,25(OH)2D3 initiated by, 754–755 resting potential “left-shifting” in, 754–755, 755f PMCa pump. See Plasma membrane calcium pump PMH. See Past medical history Pneumonia hypercalcemia/D hypersensitivity in pneumocystis carinii, 1361 resistance influenced by D3, 999t Polybromo- and BAF-containing complex (PBAF) in 1,25(OH)2D3-liganded VDR-RXR heterodimer transactivation, 238–240, 239f in VDR transcriptional function, 268 Polymerase chain reaction (PCR), in HVDRR studies, 1218 Polynesians bone mass in, 794 D metabolism in, 794 Postgastrectomy bone disease, 1299–1300 biochemistry, 1300 bone turnover in, 1299 clinical features, 1299–1300 D metabolism of, 1299 management, 1300 osteomalacia in, 1299 pattern of, 1299 PPAR. See Peroxisome proliferator-activated receptor pQCT. See Peripheral quantitative computerized tomography PR. See Production rate Prednisone, Ca absorption v., 1244–1245, 1245f Pregnancy bone mineral content/density during, 840–841 Ca homeostasis v., 204 D/Ca metabolic adaptations during, 839–841 D metabolism in, 839–843 last trimester, 803–804 D deficiency in, 803–804 D supplementation in, 804, 808 malnutrition in, 803 1,25(OH)2D v. Ca absorption during, 839–840, 840f Pregnane X receptor (PXR) accumulated toxins v., 865–866 VDR ligand binding/heterodimerization/transactivation domains compared with, 233–235, 234f Preinitiation complex (PIC) in comodulator activity integrated model, 300, 300f VDR contacts with, 291–292 Previtamin D3, vitamin D3 produced from, 38, 38f, 39f Primary biliary cirrhosis (PBC) bone disorders associated with, 1304–1306 clinical features of, 1304 D metabolism in, 1305, 1305f management of, 1305–1306 Primates. See New World primates PRL. See Prolactin

INDEX

Production rate (PR), young/elderly men’s 1,25(OH)2D, 827, 827f Proenkephalin (PENK), 1,25(OH)2D3/other osteotropic hormones regulating, 655 Progesterone, D metabolism influenced by, 1254t, 1261–1262 Prolactin (PRL), D metabolism influenced by, 1254t, 1258 Promyelocyticleukemias (APLs), RAR α fusion proteins marking, 1732 Prooxidant, 1,25(OH)2D3 as, 763–765, 764t Prostaglandins, D metabolism influenced by, 1254t, 1262–1263 Prostate D endocrine/autocrine systems in, 1608 as D target, 1682–1683 1α-hydroxylase in, 1607 1,25(OH)2D as autocrine hormone in, 1607–1610 1,25(OH)2D3 synthesized from 25OHD3 in, 1607, 1608f 1,25(OH)2D3 v. virally transformed cells of, 1684 VDR in, 1682–1683 Prostate cancer cells D analogs v. growth of, 1451 enzymes in D metabolite response by, 1685–1687 growth factor actions in regulating, 1692–1693 mechanisms of D-mediated growth inhibition v., 1690–1695 1,25(OH)2D3 arresting growth of, 1690–1691 1,25(OH)2D3/deltanoid-induced differentiation modeled in, 1637t 1,25(OH)2D3 inhibiting invasion/metastasis by, 15 1,25(OH)2D3 v. apoptosis in, 1691 1,25(OH)2D3 v. differentiation in, 1691–1692 1,25(OH)2D3 v. primary cultured, 1683–1684, 1684f 1,25(OH)2D3 v. proliferation of human, 1683, 1683f resistance to 1,25(OH)2D3 antiproliferative effect in, 1684–1685 Prostate cancer (PCa) animal models/in vivo studies of, 1688–1689 Ca/dairy products v., 1627–1629 circulating D v., 1625–1626 clinical trials evaluating 1,25(OH)2D3/analogs, 1698–1699 combination therapy v., 1695–1698 D analogs v., 1689–1690 D3 protecting against, 999t D/sunlight/natural history v., 1599–1611 D v., 1679–1700 dietary D v., 1625 D’s antiproliferative/prodifferentation actions in, 1682 EB1089 v., 1499 epidemiology, 1680–1681 epidemiology v. age/race/place, 1599–1600 etiology/treatment/hormonal factors, 1679–1680 genetic factors v., 1681–1682 high dose intermittent 1,25(OH)2D3 v. androgen-dependent, 1745 high incidence of incidental, 1600 mortality/incidence, 1624–1625 1,25(OH)2D3 inhibiting in vitro growth of, 1683–1685 1,25(OH)2D3 v., 1742 25OHD v. risk of, 1000f, 1002 risk epidemiology v. D/Ca, 1617–1629 risk factors v. vitamin D hypothesis, 1601, 1601t risk v. D, 1600–1601 sunlight v., 1617, 1625 VDR gene polymorphisms/1α-hydroxylase v., 1626–1627 VDR genetic polymorphisms associated with, 1261, 1681–1682 VDR polymorphisms v., 1139t, 1145 vitamin D hypothesis, 1600–1602, 1610–1611, 1611f dietary D studies, 1606 observational studies, 1602–1606 seroepidemiological studies, 1602–1604 sunlight exposure studies, 1605–1606 VDR polymorphism studies, 1604–1605

1881 Prostate cells BXL–353 v. in vivo growth of, 1837–1840, 1839f VDR expression in, 1834–1836 Prostate specific antigen (PSA) doubling time v. 1,25(OH)2D3 PCa treatment, 1698, 1698t in early PCa diagnosis, 1679 Protein kinase A (PKA) PKC interaction v. VDR regulation by, 209–210 VDR expression affected by, 209 Protein kinase C (PKC) activity v. 1α,25(OH)2D3 muscle stimulation, 890f, 891 D metabolites modulating matrix vesicle, 586 glucocorticoid/CYP24A1 expression v., 99 hVDR phosphorylation v. nuclear localization/DNA binding, 231f, 232–233 keratinocyte differentiation mechanism unclear in, 618–619 in keratinocyte growth/differentiation, 618–619, 620f in 1,25(OH)2D3 differentiation signal propagation, 1637–1638 in 1α,25(OH)2D3-mediated rapid responses, 386t phorbol ester/CYP24A1 expression v., 99 PKA interaction v. VDR regulation by, 209–210 VDR expression affected by, 209 Protein kinases, 1,25(OH)2D3/mitogen-activated, 97–98 Protein sulfhydryl (-SH) groups, Ca entry v., 416 Protusio acetabulae, 972f–973f, 973 Provitamin D3, photolysis, 38, 39f Proximal convoluted tubules (PCTs), in 25OHD3/1,25(OH)2D3 conversion, 153 PSA. See Prostate specific antigen Pseudo-vitamin D-deficiency rickets (PDDR), 1197–1204 biochemical findings in, 1198–1199 clinical manifestations, 1197–1198 CYP27B1 gene causing, 109 D/1,25(OH)2D3 v. 1,25(OH)2D3 in, 1199, 1199f defective 25OHD3/1,25(OH)2D3 conversion in, 1197, 1198f founder effect in Northeastern Quebec, 1200 genetic studies, 1199–1200 HVDRR v., 1208, 1209t 1α-hydroxylase gene defects in, 1200–1201, 1201f hypocalcemia in, 1056 in 1α(OH)ase-null mice, 438–439, 489 1,25(OH)2D in, 16 1,25(OH)2D replacement therapy v., 110 1,α(OH)D replacement therapy v., 111 1,25(OH)2D3 therapy v. rickets phenotype in, 1202, 1203f placenta studies, 1199, 1200f recognition/semantics, 1197 tooth enamel dysplasia in, 1202, 1203f treatment, 1201–1203 Pseudohypoparathyroidism, hypocalcemia v., 1054 Psoriasis, 1791–1801 calcipotriol/betamethasone dipropionate treating, 1500 calcipotriol treating scalp, 1785–1786 clinical use of 1,25(OH)2D3/analogs v., 1784–1787 D/analog biological effects in, 1781, 1781f D analogs treating, 1450 D analogs v., 744 efficacy/safety of D analogs treating, 1504, 1504t MC–903 v., 1543 OCT development for, 1525–1534 OCT v., 1531–1534, 1533f pathogenesis of, 1791–1792 safe VDR ligand treatments for, 643 serum 1,25(OH)2D3/25OHD in, 1784 skin lesion histology in, 1791–1792 specific therapies for, 1785–1787

1882

INDEX

Psoriasis (Continued) Théramex hybrid deltanoid v., 1416, 1416f therapies increasing topical D analog efficacy in, 1786–1787 topical 1,25(OH)2D3/analogs v., 1784–1785 topical 1,25(OH)2D3 v., 1785, 1785f treatment response v. VDR genotypes, 1784 VDR ligand treatment v., 637t, 640 VDR polymorphisms v., 1146 PTH. See Parathyroid hormone PTH gene, 538–539 abnormalities v. hypocalcemia, 1053 expression v. VDREs, 539 1,25(OH)2D3 regulating, 539 calreticulin v., 543–544 in vivo studies of, 539, 540f promoter sequences, 538–539 regulation, 538 structure, 538 PTHrP. See Parathyroid hormone-related peptide; Parathyroid hormone-related protein PTHrP gene, 737–738 organization/structure-function relationships, 737–738, 738f Puberty, Ca absorption efficiency/utilization during, 815–816, 815f, 816f Purdah, rickets incidence v., 1067 PXR. See Pregnane X receptor

Q qBEI. See Quantitative backscattered electron imaging QCT. See Quantitative computed tomography QOD schedule. See Every-other-day schedule Quantitative backscattered electron imaging (qBEI) localized mineral measurement with, 483 old bone mineralization measured with, 489 Quantitative computed tomography (QCT), BMD assessment with, 922–924 Québecois, PDDR in, 1199–1200

R RA. See Retinoic acid; Rheumatoid arthritis 9-cis-RA, in leukemia combination therapy, 1733 RAA axis. See Renin-angiotensin-aldosterone axis Race. See also Blacks; Whites; specific nationalities D metabolism influenced by, 790–795, 790t as PCa risk factor, 1601, 1601t rickets/osteomalacia risk v., 789, 790t Radiation, hypocalcemia v., 1053 Radioimmunoassays (RIAs) 125I-labeled tracers coupled with, 932 improved antibody for, 946, 946t metabolites assayed as 1,25(OH)2D by, 946–947, 947f 25OHD assay consistency v., 1023 1,25(OH)2D measured by RRA v., 946, 946f 25OHD/1,25(OH)2D quantitated with, 932 Radiology imaging, 986–990 nuclear medicine in, 986–988 plain radiographs in, 986 rickets/osteomalacia v., 967–990 Radioreceptor assays (RRAs), for 1,25(OH)2D, 932, 942–947, 943t Raman spectroscopy, tissue mineralization quantified with, 481, 482–483, 482f

RANK. See Receptor activator of NFκB RANKL. See Receptor activator of NFκB ligand RANKL/ODF bone resorption signal transduced by, 672–673 cloning/structure, 670–672, 671f osteoclast progenitors/osteoclasts differentiated by, 672 receptor identified as RANK, 673–674 as sole in vivo RANKL receptor, 674 RANKL-RANK signaling, alternations v. skeletal phenotype, 678, 679t Rapid response, 381 6-s-cis-shaped 1α,25(OH)2D3 optimal agonist for, 391, 398f case studies, 392–397, 397t 1α,25(OH)2D3-mediated, 381–400, 386t molecular tools for study of, 392, 393f structure-function evaluation of, 392–397 in ROS 17/2.8 cells, 392–394, 394f schematic model, 399–400, 399f steroid hormones generating, 386 VSMC v. 1α,25(OH)2D3-mediated, 395–397, 396f RAREs. See Retinoic acid response elements RAS. See Renin-angiotensin system Rat calvaria cells biphasic 1,25(OH)2D3/osteoblast relationship confirmed in, 651 1,25(OH)2D3/dexamethasone stimulating adipogenesis in, 655–656 1,25(OH)2D3/osteoblast relationship modeled by, 649–650, 650f Rat osteoblastic osteosarcoma (ROS), 1,25(OH)2D3 inhibiting collagen synthesis in, 704 Rats. See also Genetic hypercalciuric stone-forming rats D deficiency v. fertility of male/female, 855 vitamin D3 discriminated against by, 29 RDA. See Recommended dietary allowance Reactive oxygen species (ROS), 761 generation/degradation of, 762 Janus face of, 761 in neurodegenerative diseases, 1779–1780 redox homeostasis v., 761–763 as signaling cascade messengers, 762–763 REBiP. See Response element binding protein Receptor activator of NFκB ligand (RANKL), 568 bone-resorbing factors regulating, 675–676, 676t mRNA/serum Ca v. 1,25(OH)2D3, 680, 680f 1α,25(OH)2D3 inducing expression of, 713 osteoblasts regulating osteoclasts through inducing, 568 RANK as sole in vivo receptor for, 674 regulating expression of, 674–676 Receptor activator of NFκB (RANK) RANKL/ODF receptor identified as, 673–674 as RANKL/ODF signaling receptor in in vitro osteoclastogenesis, 673–674, 673f Receptor interacting domain (RID), NCoA62/SKIP, 296, 297f Recommended dietary allowance (RDA), 1357 calls for increasing, 1357 Redox homeostasis. See Oxidation-reduction homeostasis Redox state, cellular, 761–762 Regulator of G protein signaling (RGS)–2, 1,25(OH)2D3 inversely modulating, 654 Regulatory T cells in autoimmunity, 1754 1,25(OH)2D3 autoimmunity v., 1755 Renal cell carcinoma, VDR polymorphisms v., 1146 Renal endocytosis, 25OHD3, 153–159 cell biology of, 156–157 molecular biology of, 157–159 physiology of, 153–156

1883

INDEX

Renal failure D analog action/tissue specificity v. chronic, 1826–1827 D in, 1313–1333 future D analog roles in chronic, 1826–1827 hyperparathyroidism v. D in, 1821 intravenous 1,25(OH)2D therapy v., 1822–1823 mechanisms of 1,25(OH)2D resistance in chronic, 1822, 1822f OCT in dog model of chronic, 1527 1,25(OH)2D resistance causing secondary hyperparathyroidism in, 1821–1823 1,25(OH)2D resistance in chronic, 1821–1822 parathyroid hyperplasia in chronic, 1823, 1824f secondary hyperparathyroidism v., 1821–1827 Renal osteodystrophy, 974 aluminum toxicity in, 979–981 metastatic calcification in, 979 1,25(OH)2D3/VDR action in, 1322–1326 periosteal new bone formation in, 979 radiology of, 974–981, 975f Renin 19-nor Gemini suppressing expression of, 1518, 1518t D suppression v. other mechanisms regulating, 877 Gemini analogs inhibiting, 1514–1518 RAS cascade rate limited by, 871 synthesis/secretion control, 872–873 Renin-angiotensin-aldosterone (RAA) axis, D regulating, 901 Renin-angiotensin system (RAS), 871–879 CKD/1,25(OH)2D3 deficiency/resistance in, 1326 components in hypertension treatment, 1514 D endocrine system interaction with, 877–878, 878f Gemini compounds inhibiting, 1516–1518, 1518t 1,25(OH)2D3 as negative endocrine regulator of, 875–878 animal studies evaluating, 875–877 hypothesis of, 875 physiological implications of, 877–878 overview, 871–872, 871f Renin gene expression regulation, 873 expression suppressed by D, 877 Reproduction active Ca absorption during, 440–445 VDR ablation affecting, 345–346 Reproductive organs, D’s role in, 851–860 Resistance humans having New World primate-like, 355–356 hypocalcemia due to hereditary, 1056 index case in humans, 355 in New World primates, 352 biochemical nature of, 354–355 VDRE-BP-2 causing, 355 Response element binding protein (REBiP) binding in cis, 356, 356f D-resistant human patient over-expressing, 355 human, 356–357 overexpression v. HVDRR patient 1,25(OH)2D3 resistance, 356–357, 356f Restriction fragment length polymorphisms (RFLPs) HVDRR-related VDR gene mutation v., 1220 in prenatal VDR gene mutation diagnosis, 1228 VDR gene, 1124, 1125f Retinoblastoma protein, deltanoid-induced G1 block controlled by, 1649 Retinoic acid (RA), VDR expression affected by, 202t, 206–207 Retinoic acid response elements (RAREs), human VDR promoter, 200

Retinoid X Receptor (RXR) import/export receptors interacting with, 368–371 intranuclear trafficking of, 374–376 as NR/VDR heterodimeric partner, 291, 292f nucleocytoplasmic trafficking regulation, 371–374 “piggyback” nuclear import of, 369, 371f putative NLSs in, 369, 370f shuttling v. transcription, 371 subcellular trafficking, 363–376 in VDR/DNA binding, 178–180, 179f, 220, 220f Retinoids in PCa combination therapy, 1696 VDR expression affected by, 206–207 RFLPs. See Restriction fragment length polymorphisms RGS-2. See Regulator of G protein signaling–2 Rhabdomyolysis, hyperphosphatemia in, 1178 Rheumatoid arthritis (RA) 1,25(OH)2D accumulation in, 1389–1390 subperiosteal erosions simulating, 975f, 976 VDR ligand treatment v., 637, 637t VDR polymorphisms v., 1147 RIAs. See Radioimmunoassays Rickets, 967. See also Osteopenia; specific types of rickets Al toxicity in, 980 biochemical abnormalities in nutritional, 1069–1070 biochemical evolution of, 1036–1039 bone turnover markers elevated in nutritional, 1069–1070 Ca2+ availability v. mineralization in, 579 Ca v. D deficiency in, 1077, 1077f cartilage prehypertrophic/hypertrophic zones increased in, 579, 580f causes of, 925–926, 925t–926t Chinese incidence of infantile, 793 in cities, 967, 1065 classical features of, 1067–1068, 1068f clinical presentation of nutritional, 1067–1069 D3 curing, 999t D deficiency and children’s nutritional, 1065–1077 D deficiency causing, 566 D deficiency impairing bone resorption in, 777, 777f D/intestinal Ca absorption v., 703 D metabolism in, 1039–1040 D single dose therapy v., 1072 deltanoids v. VDR mutants associated with, 1409, 1411f dental phenotype of, 602, 602f dietary Ca deficiency causing, 1074–1075, 1075f epidemiology of D deficiency/nutritional, 1066–1067 in exclusively breast-fed children, 777 geographical distribution of colon cancer and, 866 high-Ca diet preventing KO mouse, 429 history, 4, 37–38, 967, 1065–1066 HVOii/HVOiii v. infantile, 1038 in Los Angeles Zoo New World primates, 352–353, 353f mineralization in healing, 1071–1072, 1071f oncogenic, 983–984 pathogenesis, 1029–1044, 1076–1077, 1077f pathophysiological progression of D-deficiency, 1070 PDDR v. nutritional, 1197 premature infants v. nutritional, 812, 812t prevention of nutritional, 1072–1074 radiology of, 967–990, 981, 982f, 1070–1072 radiology of D-deficiency, 968–971, 969f, 970f, 971f restricted definition of, 1031 risk v. race/population, 789, 790t sunlight v. incidence of, 246 symptoms caused by defective CYP27A1, 61–62

1884

INDEX

Rickets (Continued) treatment of nutritional, 1072 ultraviolet/sunlight v., 4–5 VDR ablation v., 342–343 in VDR-null mice v. mineral homeostasis, 433 vitamin D3 activity v. vitamin D-dependency, 7 RID. See Receptor interacting domain Ro-26–9228 mechanism of action, 1480 osteoporosis v., 1451 potency in intestinal cells, 1480, 1480f structure, 1479f target tissue gene expression v., 1480, 1481f ROS. See Rat osteoblastic osteosarcoma; Reactive oxygen species RRAs. See Radioreceptor assays RUNX proteins chromatin remodeling due to, 332 gene expression suppressed by, 333 intranuclear targeting signal v. function of, 335 localization, 334 1,25(OH)2D3 regulation dependent on osteoblast maturity, 651 promoter element/co-regulatory protein interactions by, 332 promoter regulatory complex organization determined by, 333

S SAGE. See Serial analysis of gene expression SAR. See Structure-activity relationship Sarcoidosis active D metabolite produced extrarenally in, 1380 disordered Ca balance pathophysiology in, 1381–1387 dysregulated overproduction of 1,25(OH)2D in, 1381 endogenous D intoxication associated with, 1379–1380 extrarenal D metabolite overproduction v., 1390, 1390t hypercalcemia/D hypersensitivity in, 1359–1360 VDR polymorphisms v., 1147 Saudi Arabians bone mass in, 794 D metabolism in, 794 low serum 25OHD in, 1026 sunlight exposure v. rickets in, 1066–1067 winter hypovitaminosis D in adult, 1091 Scandinavians hypovitaminosis D in adult, 1091, 1091f hypovitaminosis D in healthy elderly, 1092, 1092f hypovitaminosis D in institutionalized elderly, 1093, 1093f Scanning small-angle X-ray scattering (scanning-SAXS), mineral particle thickness/alignment from, 481–482 SCCs. See Squamous cell carcinomas Scleroderma D analog therapy v., 1787 1,25(OH)2D3 treating, 1758–1759, 1758f, 1758t, 1759t SCP. See Start codon polymorphism Scurvy, nutrition v., 3 Secondary hyperparathyroidism bone turnover due to, 509–510 CaR expression v. primary/uremic, 556 in chronic renal failure v. 1,25(OH)2D3 analogs, 1331–1332, 1332f CYP27B1 gene mutation v., 109 CYP27B1-null mice developing, 703 D insufficiency v. senile, 1088, 1088f gastrointestinal diseases associated with, 1297 normal serum 25OHD v., 1020–1022 OCT ameliorating osteopathy in, 1531, 1533f

Secondary hyperparathyroidism (Continued) OCT development for, 1525–1534 OCT v., 1527–1531 clinical results of, 1529–1531 preclinical results of, 1527–1529 1α(OH)ase-null mice demonstrating severe, 438 1,25(OH)2D3 deficiency/CKD v., 1324–1325, 1325f parathyroid cell proliferation v., 544–547 parathyroid gland hyperplasia in, 1823 radiology of D deficiency, 973–974, 975f, 980f renal disease stimulating, 974–976 renal failure and, 1821–1827 treatment in chronic renal failure before dialysis, 1327–1328 during hemodialysis, 1328–1331, 1330f Secreted frizzle-related protein (sFRP)–4, as phosphatonin/ minhibin, 1164 Selective estrogen receptor modulators (SERMs), in VDR ligand tissue selectivity, 270–271 Selective progesterone receptor modulators (SPRMs), in VDR ligand tissue selectivity, 270–271 Seocalcitol. See EB1089 Sepsis, hypocalcemia in acute, 1057 Serial analysis of gene expression (SAGE), TIO tumors v., 1192 SERMs. See Selective estrogen receptor modulators Sex steroids D metabolism influenced by, 1254t, 1259–1262 1,25(OH)2D synthesis influenced by, 828 sFRP–4. See Secreted frizzle-related protein–4 SH. See Social history Shwachman-Diamond syndrome, rickets v. differential diagnoses for, 984–985, 987f Side-chain cleavage, vitamin D2/D3, 25–26 Side-chain oxidation vitamin D2, 25 vitamin D3, 17f Simian bone disease, 351–352 D deficiency v., 351–352 New World primates susceptible to, 351–352 Single dose therapy, 1066 hypercalcemia v., 1072, 1073 patient compliance problem avoided by, 1072 Skeletal genes controlling in vivo expression of, 327–328 intranuclear organization of D-mediated regulatory machinery for, 327–336 Skeletal muscle, as D target tissue, 883–894 Skin D analog actions in normal/psoriatic, 1781–1784 D system in normal/psoriatic, 1792–1793 dystrophic mineral deposits in, 478t 1,25(OH)2D3/analog immunoregulatory properties in, 1519 psoriasis/other diseases of, 1791–1801 rejection inhibited by 1,25(OH)2D3/analogs, 641t structure/function deteriorating with age, 823 VDR in human, 1792f, 1793 Skin cancer D analog therapy v., 1787 D photosynthesis v. recommendations for reducing, 1087 vitamin D photosynthesis v., 42–43 Skin lesions in children v. 1,25(OH)2D3 ointment, 1786 in HIV patients v. oral 1,25(OH)2D3, 1786, 1786f Skull, subperiosteal erosions causing “pepper pot,” 975f, 977 SLE. See Systemic lupus erythematosus SMRT, NR co-repressor, 298–299

1885

INDEX

Social history (SH) in approaching metabolic bone disease, 917–918 vegetarianism in, 917t, 918 vertebral crush deformity correction guided by, 918, 918f Soda, v. Ca intake by adolescents, 817 Sodium chloride (NaCl), CaR inhibiting MTAL reabsorption of, 557 Sodium (Na), excretion v. 1,25(OH)2D3 in TPTX dogs, 518 Software, VDRE screening, 322 Sp1 transcription factor, in 1,25(OH)2D3-induced differentiation, 1640 Spanish, hypovitaminosis D in elderly, 1092–1093 Spermatogenesis, SRC-2 deletion resulting in, 295 Spliceosome, hnRNPs associated with, 358–359 SPRMs. See Selective progesterone receptor modulators Squamous cell carcinomas (SCCs), 1,25(OH)2D produced by keratinocytes from, 612 SRC-1 See Steroid receptor coactivator 1 Stanniocalcin hypercalcemia v., 462 proximal tubular Pi transport regulated by, 461t Star volume, in bone structure assessment, 961–962, 962f Start codon polymorphism (SCP), 1124 Steroid hormones calbindin-D28K/D in producing, 726 calbindin-D9K regulated by, 729–730 calbindin-D28K regulated by, 728–729 CYP24A1 transcription regulated by, 97 D structure v., 381, 382f kidney 1α-hydroxylase activity regulated by, 77–78 nongenomic actions reported for, 98–99 VDR expression affected by, 205–206 Steroid precursors, in deltanoids, 1412–1416, 1413f, 1414f, 1415f Steroid receptor coactivator 1 (SRC-1), VDR stabilization v. interaction with, 1472–1473, 1472f Sterols. See also Steroid hormones equilibrium of bound/free, 124 interpretation/relevance of measurements of antirachitic, 947–949 plasma proteins v. transport/function of, 124–125 Stomach, Ca absorption v. aging, 1105–1106 Store-operated Ca2+ (SOC) anti-INAD antibody v. 1α,25(OH)2D3-dependent influx from, 893, 893f INAD-based signaling complexes in 1α,25(OH)2D3-modulated influx from, 893, 894f muscle influx mediated by TRPC3 proteins/VDR, 892–893, 892f Stosstherapie. See Single dose therapy Structure-activity relationship (SAR), in deltanoid design, 1412 Strut analysis, in bone structure assessment, 961, 962f Sunlight alcoholics lacking exposure to, 1266 blood pressure v. D and, 873–874, 874f colon cancer death rate v., 1571–1572 colon cancer v., 1709–1710 D from UVB component of, 1006–1007 D intake from food v., 995, 996t–997t D nutrition/acquired bone disease v., 1297–1298 D supplementation required by high latitude, 784–785 deprivation determining D insufficiency, 1086–1087 diabetes incidence v., 1766 exposure increasing D3 status, 1009t exposure v. age, 1102, 1109 exposure v. colorectal cancer, 1618 fatal breast/prostate cancer v., 1572 glass v. D synthesis induction by, 823

Sunlight (Continued) HDM v., 1813–1814 high rickets incidence despite, 1066–1067 25OHD v. excessive, 1356 PCa v., 1599–1611, 1617, 1625 PCa v. exposure to, 1680–1681 rickets/colon cancer incidence v., 246 rickets v., 777 vitamin D photosynthesis regulated by, 38–39 Sunscreen, vitamin D photosynthesis v., 40–41, 41f Superagonists, 1475–1476, 1476f differential VDR activation by, 1475–1478, 1476f 20-epi, 1477–1478 20-natural, 1476–1477 Suppressor T cells, 1,25(OH)2D3 autoimmunity v., 1755 Swiss, hypovitaminosis D in adult, 1091 Systemic lupus erythematosus (SLE), VDR ligand treatment v., 637t, 639–640

T T cell response, VDR KO mice showing abnormal, 246 T cells. See also specific types of T cells in acute allograft rejection, 1519 immunosuppressive therapy v. pathogenic, 631–632, 632t VDR ligand immunoregulation of, 635–636, 635t VDR ligands enhancing regulatory, 636 T-cells, in psoriasis, 1791–1792 T lymphocytes, antigens recognized by, 631 Tacalcitol. See 1α,24R-Dihydroxyvitamin D3 Taq polymorphisms, 1131t–1132t, 1135–1137 BMD v., 1142–1143 Taxanes, 1,25(OH)2D3 in combination with, 1745–1746 TC. See Tumoral calcinosis TEI 9647, 1481–1482, 1481f Testis calbindin-D28K in, 726 D in, 855–856 1α,25(OH)2D3 VDRnuc in, 385t SRC-2 deletion causing defects in, 295 VDR ablation reducing aromatase activity in, 345 Testosterone, D metabolism influenced by, 1254t, 1261 Tetany in hypocalcemia, 920, 1049 in PDDR, 1197 TFs. See Transcription factors TGF. See Transforming growth factor TGFα, keratinocytes producing, 614 Theophylline, D metabolism influenced by, 1255t, 1272 Thiazide diuretics D metabolism influenced by, 1255t, 1268–1269 as IH therapeutics, 1351 Thyroid C cells, 687 D controlling CT gene in, 687–697 embryonic development of, 687–688 neoplasia of, 688 origin/function of, 687–688 Thyroid gland C cells in normal adult, 688 PTHrP expression in, 739t Thyroid hormone, D metabolism influenced by, 1254t, 1262 Thyroid receptor-associated proteins (TRAP) coactivator complex. See Mediator-D coactivator complex Thyroparathyroidectomized (TPTX) dogs, 1,25(OH)2D3 v. phosphate/Ca/Na excretion in, 518

1886 Thyroparathyroidectomized (TPTX) rats 24-hydroxylase activity v. D toxicity in, 1366–1367, 1367t 1,25(OH)2D3 v. Ca excretion in, 518, 518f TIO. See Tumor-induced osteomalacia TLRs. See Toll-like receptors TNF. See Tumor necrosis factor TNF receptor-associated factor (TRAF) family proteins, in osteoclastogenesis, 676–677 TNF receptor family. See Tumor necrosis factor receptor family TNFα, D metabolism influenced by, 1254t, 1263 Toddlers, Ca absorption in, 815 Toll-like receptors (TLRs) LPS-induced 1,25(OH)2D production supported by, 1385 pathogens recognized by, 631 Tooth crown influenced by D, 604 D in formation/mineralization of, 601–602 dentin/cementum in root of, 601–602 eruption delayed by rickets, 1068 eruption delayed in PDDR, 1198 Tooth enamel hypoplasia in HVDRR, 1208 1,25(OH)2D3 partially correcting PDDR, 1202, 1203f Total parenteral nutrition (TPN) bone disease caused by Al in, 1270, 1303 bone disorders associated with, 1303 clinical features of, 1303 management, 1303 Toxicity, 26–27. See also Intoxication adipose tissue loss v., 1007, 1010 arterial dystrophic calcification induced by, 478 biological markers for monitoring, 1009t Ca/D supplementation v., 1059 cardiovascular, 899, 904–905 D2/D3, 26–27, 1356–1357 D effects on bone v. hypercalcemia in, 510 D/25OHD, 1356–1357 D osteolytic response/hypercalcemic effects in, 569 EAE v. 1,25(OH)2D3, 1784 factors affecting, 27 high dose intermittent 1,25(OH)2D3, 1743, 1743t increased serum phosphorus associated with, 1177–1178 mechanisms of D, 1362–1368 1,25(OH)2D3, 1357 1,25(OH)2D3 concentration v., 1008 overview, 26 PCa treatment v. 1,25(OH)2D3, 1689–1690 pharmacological issues of safety and, 1007–1010 psoriasis treatment v., 1791 radiology of, 986, 988f synthetic analog, 1357–1359 thresholds in adults/infants, 1357 topically applied/systemically administered D compound, 1450 TPN. See Total parenteral nutrition TPTX dogs. See Thyroparathyroidectomized dogs Trabecular bone pattern factor, in bone structure assessment, 962 TRAF family proteins. See TNF receptor-associated factor family proteins Transactivation coregulators in VDR/RXR, 180–181 HVDRR cell lines suppressing RXR-VDR-mediated, 354f, 355–356 1,25(OH)2D3-liganded VDR-RXR, 237–243, 239f REBiP squelching hormone-directed, 358, 358f RXR’s direct involvement in, 181 squelching VDR-directed VDRE-reporter-driven, 355, 355f

INDEX

Transcaltachia cell-surface receptor in analog-stimulated, 1461 structure-function summary analysis, 397, 397t Transcription apparatus, REBiP in, 358, 358f Transcription factors (TFs) intranuclear pathways directing, 335–336 in 1,25(OH)2D3-induced differentiation, 1639–1640 osteoblast differentiation status varying, 653 as regulatory component scaffolding, 332–333 Transcription start sites (TSSs), 1,25(OH)2D3 target gene VDREs near, 314, 315f, 316t Transforming growth factor (TGF), in 1,25(OH)2D3 actions on prostate cells, 1692–1693 Transient Receptor Potential (TRP) channel superfamily, Ca2+ influx v., 430 muscle, 892–893, 892f Transient Receptor Potential Vanilloid (TRPV) family, Ca influx v. TRPV5/TRPV6 members of, 430 Translation, hnRNPs as ribosome recognition proteins in, 359 Transport receptors in nucleocytoplasmic VDR/RXR trafficking, 365–376 Ran-GTPase regulating, 365–366, 367f TRAP coactivator complex. See Mediator-D coactivator complex TRP channel superfamily. See Transient Receptor Potential channel superfamily TRPV5. See ECaC1 TRPV6. See ECaC2 TRPV family. See Transient Receptor Potential Vanilloid family TSSs. See Transcription start sites Tuberculosis D3 preventing, 999t extrarenal D metabolite overproduction v., 1390–1391, 1390t hypercalcemia/D hypersensitivity in, 1360 VDR polymorphisms v., 1148 Tumor cells D influences on, 1577–1582 D influencing, 1577–1582 D resistance/metabolism in, 1583–1584 1,25(OH)2D3 stimulating proliferation of, 1574t, 1584–1585, 1585f Tumor-induced osteomalacia (TIO), 463t, 468, 983–984 benign/malignant tumors in, 983, 986f as disorder of phosphate metabolism, 1190–1192 genes overexpressed in, 1192 phosphatonin secretion resulting in, 1190 tumors eluding detection in, 983–984, 989f Tumor necrosis factor (TNF) causing apoptosis v. calbindin-D28K/osteoblasts, 725, 725f type I collagen synthesis inhibited by, 704 Tumor necrosis factor (TNF) receptor family nomenclature, 674, 675f OPG in, 668 Tumor suppressor genes in cell cycle/apoptosis control, 1577–1580 1,25(OH)2D3/EB1089 regulating breast cancer cell, 1664–1665 1,25(OH)2D3 regulating expression of, 1579 Tumoral calcinosis (TC), hyperphosphatemic, 1175–1176 Tumors D/analogs differentially influencing canine/human, 1577 D preventing colon, 1709–1710 1,25(OH)2D3/1,25(OH)2D3 analogs v., 1741 1,25(OH)2D3 stimulating development of, 1574t, 1584–1585, 1585f prognosis v. VDR expression in breast cancer, 1668 VDR in, 1572t VDR-RXR heterodimer-activating ligands v., 241

1887

INDEX

Turks serum 25OHD/PTH in dark-skinned Dutch, 794 sunlight exposure v. rickets in, 1066–1067 sunlight exposure v. serum 25OHD in, 794 Type II nitric oxide synthase (NOS II), 1,25(OH)2D3 v., 1783

U UL. See Upper limit Ultraviolet (UV) light age/serum D v., 823, 824f PCa mortality v., 1601–1602, 1602f, 1603f PCa v., 1605–1606 rickets v., 565 serum 25OHD directly related to, 825 Upper limit (UL), conservative safety margin of, 1008 Uremia, 1,25(OH)2D3/VDR-mediated transcription v., 1320–1321, 1321f Uterus calbindin-D9K/D28K in, 726, 730t D in, 856–857 PTHrP expression in, 739t 3’UTR polymorphisms, 1131t–1132t, 1135–1137 BAt/baT haplotype expression in, 1131t–1132t, 1135 UV light. See Ultraviolet light UVB in combination psoriasis therapy, 1504, 1504t as D3 dose, 1006–1007 D status in elderly improved by, 1087

V Vascular calcification, 899 calcitropic hormones v., 904–905 D regulating, 904–905 overview, 904 regression v. treatment, 979 Vascular endothelial growth factor (VEGF) gene, 1,25(OH)2D3 targeting, 566, 567 Vascular inflammation, D signaling in regulating, 899 Vascular smooth muscle cells (VSMCs) 1,25(OH)2D3 actions in, 902–903 1α,25(OH)2D3 promoting migration of, 395–397, 396f Vasculature, direct D actions in, 902–904 Vasopressin. See Antidiuretic hormone VDCCs. See Voltage-dependent calcium channels VDR. See Vitamin D receptor VDR gene, 182–184 arrangement, 1210, 1211f complexity v. polymorphism identification, 1125f, 1129 defects causing HVDRR, 1207 exon-intron structure/polymorphism position, 1124, 1125f gene deletion prematurely terminating, 1222 genomic mapping to chromosome 12q13.1, 1122, 1123f haplotype importance in, 1135–1137, 1136f HVDRR caused by mutated, 111 LD measured across, 1125f, 1127 LD strength display across Caucasian, 1127, 1128f locus, 194–201 structure, 194–195, 195f nephrolithiasis v., 1142 odontoblasts expressing, 599 organization, 182–183, 183f polymorphism association analysis in disease states, 1137–1148

VDR gene (Continued) polymorphism in human, 172, 183–184, 200–201, 200f, 244f disease risk/functional consequences v., 243–246 ethnic variation v., 1127–1129, 1128t undiscovered/functionally significant, 245 polymorphisms/1α-hydroxylase v. PCa, 1626–1627 polymorphisms associated with cancer, 1572–1573 polymorphisms v. colorectal cancer/adenoma, 1619–1620 polymorphisms v. disease risk, 1121–1149 polymorphisms v. sequence comparisons, 1124–1126, 1125f premature termination v. HVDRR-related mutations, 1220–1222 promoters v. D analog selectivity, 1452f, 1453 12q13 locus, genomic structure, 1122, 1123f, 1124f RFLPs, 1124, 1125f sequence variations near anonymous markers, 1125f, 1135 splice site mutations prematurely terminating, 1222 Glu92fs, 1221f, 1222 Leu233fs, 1221f, 1222 stop mutations prematurely terminating, 1220–1222, 1221f Arg30stop, 1221–1222, 1221f Arg73stop, 1221, 1221f Gln152stop, 1221, 1221f Gln317stop, 1221f, 1222 Tyr295stop, 1220, 1221f Tyr295stop ochre, 1214t–1216t, 1220–1221 structural complexity, 193 structure, 194–196 structure/polymorphisms, 1122–1137 study size v. analysis of, 1137–1138 VDR homodimers D analog selectivity v., 1454–1455 RXR-independent 1,25(OH)2D3 signaling v., 319 VDR promoters, 196–200 human, 197–200, 198f, 199f nonhuman, 197, 198f targeting VDR through chromatin remodeling complex, 305–312, 306f VDR-RXR heterodimers 9-cis RA stimulating, 241 allosteric model of, 235–237, 236f cyclic model for transactivation by 1,25(OH)2D3-liganded, 237–243, 239f limits of, 241 cytoplasmic dimerization of, 369, 371f D ligands influencing DNA interaction with, 1455 DNA binding v. hexameric core binding motifs, 319 DNA complex formation of, 318f everted repeats, 317–319, 318f FRET experiments showing cytoplasmic, 364–365, 365f hVDR∆ in, 281 intestinal CYP enzymes regulated by, 246–248, 247f Mediator-D complex interacting with, 296, 296f 1,25(OH)2D3 signaling mediated by, 319 in 1,25(OH)2D3/VDR control of D responsive genes, 1320, 1320f structure/function of, 230–236, 231f, 234f therapeutic potential of ligands activating, 241 VDR signaling v. 1,25(OH)2D3/9-cis RA binding in, 237 VDR superagonist ligand v. enhanced formation of, 271–272, 272f VDRE binding sites for, 222–224, 223t VDRE interacting with, 539–541 VDRM-like activity-originating mechanisms in, 273 VDRE. See Vitamin D response element VDRE-BP. See Vitamin D response element binding protein VDRmem. See Membrane VDR VDRnuc. See Nuclear VDR

1888 Vegetarians diet for bone mass v. macrobiotic/vegetarian, 795 D metabolism v. vegetarian/omnivorous, 795 groups/sects/ethnicities, 917t rickets v., 1067, 1077 metabolic bone disease v. Chinese Buddhist, 917t VEGF gene. See Vascular endothelial growth factor gene Vertebrates, vitamin D3 synthesized by, 15 VICCs. See Voltage-independent calcium channels Vitamin A deficiency inducing squamous metaplasia, 614 in keratinocyte growth/differentiation, 614 McCollum/Davis discovering, 3–4 Vitamin B, McCollum/Osborne/Mendel discovering, 4 Vitamin D autocrine system, in prostate, 1608 Vitamin D binding protein (DBP), 117–128 actin-binding property conserved in vertebrates, 146 analog interaction with, 1456–1459 asymmetric unit having DBP-A/B molecules, 135 binding v. D compound structure, 1457 C-/D-/E-ring analogs binding poorly with, 1563–1565, 1567 changes with age, 1102–1103 concentration v. ecological factors, 126 D analogs’ affinity for, 1440–1441, 1441–1442, 1441t D3 metabolite/analog affinity for, 138, 141t in D3 pharmacology, 1002 D toxicity/free metabolite level v., 1367–1368 20-epi-1,25(OH)2D3/1,25(OH)2D3-induced hGH reporter gene expression v., 1433–1434, 1433f functional features, 121–127 gene, 117–121 allele distribution, 120–121 chromosomal location/linkages, 117–119 evolution within gene family, 118f, 119–120, 119f polymorphisms, 120–121 structural features, 117, 118f transcriptional orientation, 119, 119f hepatic failure/multiple trauma risk v., 148–149 interactions v. free hormone theory, 125–126 isoforms v. disease susceptibility, 121 LBD v. VDRnuc LBD, 387, 387f molecular interactions, 122–123, 122t as multifunctional protein, 124 overview, 117 in PCa etiology, 1681 physiological roles, 124–127 polymorphisms, 120–121 primate D hormone movement from, 354–355 proteolysis after receptor/ligand scavenging, 125 species variation in, 29 structural features, 117, 388–391, 390f–391f, 398f structure explaining unique functions, 149, 150f structure v. VDRnuc structure, 391, 392t synthesis/turnover, 121–122 three-dimensional structure, 135–149, 136f in vertebrate evolution, 120 Vitamin D (D). See also Hypovitaminosis D absorption v. age, 1101–1102 adolescents v. inadequate intake of, 816 analog development, 1489–1505 basic screening strategy, 1489–1492 strategy, 1489–1492 synthesis strategy, 1490–1492 analog selectivity mechanisms, 1449–2911 animal/cell culture muscle influenced by, 1809–1811

INDEX

Vitamin D (Continued) aromatase expression v., 859 β cell characteristics influenced by, 1767 β cells v., 1764–1767 benefits of higher levels of, 1370 in bone fracture clinical trials, 1112, 1113t bone fracture risk v., 1813–1814 bone mass influenced by, 1245–1247 cancer/differentiation v., 1571–1586 in cancer risk epidemiology, 1617–1629 cancer v., epidemiology of, 1571–1573 in cardiovascular medicine, 899–905 cell cycle influenced by, 1577–1580, 1578f–1579f chemistry/metabolism/circulation, 15–160 chicken embryonic development/egg hatchability v., 851–852 colon cancer v., 1709–1721 epidemiology of, 1709–1710 colon cancer v. protective role of, 866 colorectal adenoma v. dietary, 1619 colorectal adenoma v. plasma, 1619 colorectal cancer v. dietary/supplementary, 1618 colorectal cancer v. plasma, 1618–1619 compounds v. leukemic cell lines, 1730–1734, 1730t concentration lacking clinical relevance, 947–948, 948t controlling factors in supply of, 1293–1294 cutaneous production v. age, 823–824 definitions/models for studying rapid actions of, 583–585 detecting, 933–935 methodology for, 933–934, 935f sample extraction for, 933 silica cartridge chromatography in, 933–934, 934f detecting D metabolites and, 931–949 diabetes v., 1763–1774 dietary intake by elderly, 824 discovery, 4, 291 disease states v. D metabolites and, 948t diseases/conditions prevented by, 998–1000, 999t drug-oriented perspective on, 995–997 ED-71 as “long-lived,” 1440 epidemiology of breast cancer v., 1671 estimated requirement v. recommended intake, 785 estimating human serum, 933, 933t factors influencing metabolism of, 789–796 in fertility, 854–855 fetuses/neonates v. low maternal intake of, 841–843 FGF-23 in homeostasis of, 1193 food fortification with, 817–818 genomic/nongenomic influence on striated muscle, 1809 as GIO treatment, 1243–1248 in granuloma-forming disease, 1379–1380 hematological malignancy v., 1727–1736 high latitude summer generation of, 1087 historical perspective on, 3–8 in human physiology, 773–905 in HVDRR therapy, 1227 hydroxylation, 1599 25-hydroxylation, 17–19 in hyperparathyroidism development in renal failure, 1821 hypocalcemia due to malabsorption of, 1056 immune system in type 1 diabetes v., 1767–1773 insufficiency v. low intake of, 1087–1088, 1087f intestinal absorption by elderly, 824–825 intracellular trafficking in IDBP model, 360 ligands, interacting with DBP, 122–123 in mammary gland, 857–858 maternal D intake v. breast milk, 846–847

INDEX

Vitamin D (Continued) mechanism, 167–400 metabolically influencing β cells, 1764–1767 metabolism, 789–790 during pregnancy, 839–840 metabolism during lactation/weaning, 843–846 metabolism in pregnancy/lactation, 839–847 metabolism v. race/geography, 790–795 metabolites influencing β cells in vitro, 1764–1765 metabolites v. β cells in clinical trials, 1765–1767 mineralization influenced by, 478–480 molecular mechanisms in leukemia v., 1731–1733 mouse models lacking function of, 852 muscle contraction/relaxation v., 1809, 1809f muscle influenced by, 883–894 naming conventions, 15 non-bone effects of, 998–1000 not a vitamin, 4–5 optimal status of, 782–785, 784f osteoblast differentiation/activity influenced by, 649–658 in osteoporosis, 1101–1114 in ovary, 856 in parathyroid gland, 537–547 pathogenesis of impaired mineralization v., 1040–1044 in PCa, 1599–1611 PCa risk v. circulating, 1625–1626 PCa v., 1610–1611, 1611f, 1679–1700 PCa v. dietary, 1625 PDDR treatment with, 1201t perinatal actions, 803–808 pharmacology, 995–1010 photobiology, 37–43 physiological sources for activity of, 782–783 plasma half-life, 27 preparations v. GIO, 1247–1248 prostate targeted by, 1682–1683 rapid actions/nongenomic mechanisms, 583–589 in renal failure, 1313–1333 renal handling of Ca/P influenced by, 518–519, 518f replete/deficient state classification, 1024–1026 in reproductive organs, 851–860 resistance in breast cancer cells, 1667–1668 in rickets management, 1072 striated muscle influenced by, 1809–1811 supplementation/fortification, 784–785 supplements for related agents and, 1060t synthesis, 1599 synthesis v. fetal development, 852–853 target organs/actions, 565–768 in testis, 855–856 therapy in chronic renal failure, 1327–1332 tissue responsiveness/role in aging, 831–833 tumor cells influenced by, 1577–1582 in uterus, 856–857 winter oral intake v. photosynthesis of, 1087 Vitamin D2 (D2), 5f, 16f assays not monitoring therapeutic, 939–940, 940t clinical use, 11, 12t D intoxication v., 1108 derivatives’ metabolism, 1430–1431 in diet, 1599 isolation/identification, 5 molecular structure, 931 1,25(OH)2D3 analog synthesis from, 1490, 1491f 25OHD/VDR concentration/hypercalcemia v. supraphysiological, 1364, 1364f, 1364t

1889 Vitamin D2 (Continued) PDDR treated with, 1199f, 1201 skin diseases treated with, 1791 Vitamin D3 (D3), 5f, 16f. See also Hypervitaminosis D3 activation, 7, 8f in autoimmunity, 1753–1759 benefits overlooked, 1000 C-25 hydroxylation, monooxygenase activity, 48 clinical use of D2 v., 1005, 1006t D3/metabolite plasma concentrations v., 1362, 1363t deltanoids v. interconverting 6-s-cis/trans, 1408f, 1412, 1412f derivatives functionalized at C-24, 22–23 dietary/photosynthesized, 1599 differentiation/proliferation regulation pathways dissociated, 1581 distribution, 1006–1007 dose-response relationship with 25OHD3, 1003, 1005f dose v. tissue stores, 1007 falls v. Ca and, 1814, 1814f half-life, 1007, 1009t indications/clinical use, 997–1000, 1009t isolation/identification, 5, 7 metabolic transformations, 931, 932f metabolism in vertebrates, 220–222, 221f metabolism/regulation, 1000–1002, 1000f metabolite/analog formulas/DBP affinity, 141t metabolites, 8 molecular structure, 931 as new drug, 995–997 “normal” disease prevalence v. increased, 999t, 1001 24R,25(OH)2D3 as inactive metabolite of, 851–852 25OHD/VDR concentration/hypercalcemia v. supraphysiological, 1364, 1364f, 1364t in osteoclastogenesis, 665–681 as prosteroid hormone, 15 recommendations v. public health, 1026 serum 25OHD levels ensured by, 1003, 1004t skin diseases treated with, 1791 storage/25OHD conversion, 1007 supplementation safety margin, 1010 Vitamin D-dependent rickets type II. See Hereditary vitamin Dresistant rickets Vitamin D endocrine system, 383–386 adapting to 1,25(OH)2D3 concentration, 1000f, 1001 calbindin-D28K/VDR colocalization in understanding, 721 description of, 383, 384f discovery, 7 implicated in OA, 1144 osteoporosis/fracture v., 1141–1144 physiological process role, 291 pleiotropic effects v. VDR gene association analysis, 1138–1141, 1139t–1140t in prostate, 1608 RAS interaction with, 877–878, 878f VSCCs v. 1,25(OH)2D3, 751–757 Vitamin D hormone receptors, new functions indicated by, 6 Vitamin D hypothesis colorectal cancer v., 1618 experimental studies, 1606–1607 observational studies v., 1602–1606 PCa risk factors in, 1601, 1601t PCa v., 1599–1611, 1611f studies v., 1626 Vitamin D pseudodeficiency. See Pseudo-vitamin D-deficiency rickets (PDDR) Vitamin D-receptor interacting protein coactivator complex. See Mediator-D coactivator complex

1890 Vitamin D receptor (VDR), 167–184, 219–250, 1210–1212. See also Human VDR (hVDR); VDR gene; specific types of VDR ablated in rodents/humans, 224–225 ablation in mice, 341–348 absent in tibial dyschondroplasia, 579 activation functions, 176 affinity v. D analog activity, 1452f, 1453 allosteric model of signaling activation in, 235–237, 236f analog activity v. increased, 1456 analog selectivity in ligand-dependent regulation of, 1452f, 1456 analog selectivity v. RXR heterodimerization of, 1452f, 1454 analogs modulating, 1482–1483 antagonist/partial agonist response paradigms, 271 basal gene transcription apparatus contacting, 263 baT haplotype allele v. nephrolithiasis, 1142 as bile acid receptor, 866–867 binding sites in classical, 314–319 complex, 319–322 binding v. 20-epi D analogs, 1495–1498 binding v. deltanoid A-ring conformation, 1412, 1412f binding v. 1,25(OH)2D3 20-carbon epimerization, 1544 biochemical properties, 170–171, 170t bisphenol analogs as agonists of mutant, 1561–1562, 1561f in blood cells, 1728–1729 in cancer, 1571, 1572t in cancer cell growth regulatory response, 1583–1584 cardiovascular disease and genetics of, 900–901 cellular/tissue distribution, 168–170, 169t characterization, 168–171 chromatin remodeling v. transcriptional control by, 305–307, 306f CKD altering 1,25(OH)2D3 mediation by, 1317–1322 cloning, 171–172 in CNS, 1780–1781 coactivators associating with, 242–243, 264–268, 293–298 different means of recruitment for, 273 Ets-1, 298 interaction mechanism for, 293, 294f ligand-induced/tissue-selective recruitment of, 270–271 Mediator-D, 265–267, 265t multifunctional HAT activity assemblies of, 264–265 NCoA62/SKIP, 265–267 Smad 3, 298 SRC family of, 293–295, 295f SRC/p160 family of, 264 TFIIA/TFIIB/TFIID, 298 WSTF, 309–310, 310f cofactor complexes affecting, 263–274 ATP-dependent remodeling, 267–268 colonic hyperproliferation/tumorigenesis v., 1711–1712, 1712f comodulators, 242–243, 291–300 integrated model for activity by, 299–300, 300f in cultured metanephros, 521–522, 524f cyclic dynamics, 181–182, 181f, 182f D analog selectivity determined by interaction with, 1452–1456, 1452f D endocrine system v. calbindin colocalizing with, 721 D pocket structure, 142 defects causing hypocalcemia, 1056 deltanoids v. rickets-associated mutants of, 1409, 1411f in developing rodent kidney, 521–522, 522f, 523f differential activation by analogs, 1475–1482, 1476f differential activation by antagonists, 1480–1482 differential activation by noncalcemic selective agonists, 1478–1480 differential activation by superagonists, 1475–1478, 1476f

INDEX

Vitamin D receptor (Continued) discovered, 8–9, 167–168, 219–220 DNA binding, 171, 313–314 capacity in normal/renal failure rats, 543 endogenous gene promoters binding to, 177–178, 178f ethnic variation in polymorphisms of, 1127–1129, 1128t evolutionary insights from, 227–228 expression/abundance regulation, 193–210, 202t heterologous, 204–210 homologous, 201–204 expression in colon cancer, 1710–1711, 1711f, 1711t expression in prostate cells, 1834–1836 expression/regulation in breast cancer cells, 1667 expression/role in normal mammary gland, 1669–1670, 1670t functional analysis, 176–182 Gemini/1α,25(OH)2D3 binding efficiency, 287 gene targets/biological actions, 220–225 genomic structure surrounding, 1122, 1123f genotype responses as serum marker differences, 1133t, 1135–1136 genotypes v. psoriasis treatment response, 1784 in GHS rats, 1348–1349, 1349f GR sharing coactivators/corepressors with, 1240 group 1I/1H cholesterol derivatives recognized by, 227f, 229 similarities in, 229, 229f growth inhibition v. differentiation, 1713, 1713f helix 12’s intramolecular interactions in, 281, 283f homologous up-regulation defective in CKD, 1317–1318 in human BPH cells, 1834, 1835f import/export receptors interacting with, 368–371 import v. coactivators, 369, 370f interactions v. 20-epi analog binding, 1477–1478, 1478f intranuclear trafficking of, 374–376 in kidney, 520–523 lamprey, 227–228, 279 LBD hypothetical conformation, 1473, 1473f ligand immunomodulation v. graft rejection, 1519–1520 ligand-triggered protein-protein interactions, 313 ligands as immunoregulatory agents, 633 ligands enhancing regulatory T cells, 636 ligands inhibiting BPH, 1833–1840 localization, 363–365, 364f models, 363–365, 364f in shuttle model, 364–365, 364f localization in plasma membrane caveolae, 400 2MD inducing unique conformation of, 1550, 1550f 2MD promoting interactions by, 1550–1551, 1551f 2MD stimulating promoter binding by, 1549–1550, 1549f mineralization v. altered, 488–489, 489f mouse proximal/distal colon expressing, 1719 muscle, 885–886 muscle SOC influx v. TRPC3 proteins and, 892–893, 892f myeloid development v. expression of, 1729 neocytoplasmic trafficking, 365–374 nonsecosteroidal D mimics differentially regulating, 1565 nonsteroidal analogs inducing unique conformation of, 1567 normal/leukemic hematopoeitic cells expressing, 1728–1729 in normal/malignant colon cells, 1710–1712 novel co-regulatory complexes interacting with, 307–308, 307f nuclear export, 369–370, 372f nuclear export v. transcription, 371, 372f nucleocytoplasmic trafficking regulation, 371–374 1,25(OH)2D3 A-ring in transactivation of, 1472f, 1473–1474 1,25(OH)2D3 D-ring in transactivation of, 1475 1,25(OH)2D3 deficiency reducing parathyroid, 1317, 1318f

1891

INDEX

Vitamin D receptor (Continued) 1,25(OH)2D3 inducing focal accumulation of, 374–375, 375f 1,25(OH)2D3 ligand binding by v. hair cycle regulation, 234–235 in 1α,25(OH)2D3-modulated SOC influx, 893, 894f 1,25(OH)2D3 regulating PTH gene at, 540f, 541–542 1,25(OH)2D3 side chain in transactivation of, 1474–1475 1,25(OH)2D3 up-regulation, 201–204, 202t parathyroid CaR interacting with, 556, 556t phenotype v. fracture risk, 1143–1144 phosphorylation possibly regulating activity, 1454 PIC linked to, 291–292 “piggyback” nuclear import of, 369, 371f polymorphism association analysis in disease states, 1137–1148 polymorphism functionality, 1129–1137, 1131t–1134t polymorphism testing levels, 1129, 1135f polymorphism v. diabetes risk, 1773 polymorphisms, 1122–1126 polymorphisms influencing breast cancer risk, 1671–1672 polymorphisms influencing muscle function, 1813 polymorphisms/intestinal Ca absorption v. BMD, 1141–1142 polymorphisms v. cancer/hyperproliferative disease, 1145–1146 polymorphisms v. colon cancer development, 1712 polymorphisms v. disease risk, 1121–1149 polymorphisms v. PCa, 1604–1605 polymorphisms v. PCa risk, 1681–1682 polymorphisms v. structure/function in CKD, 1318–1319 in prostate, 1682–1683 shuttling v. transcription, 371 skeletal homeostasis not requiring, 344 species having characterized, 279–280 stabilization v. SRC–1 interaction, 1472–1473, 1472f structural domains, 173–176, 173f DNA binding, 174 Ligand binding, 174–176, 175f PXR v. ligand binding/heterodimerization/transactivation, 233–235, 234f zinc finger DNA binding, 229–233, 231f structural gene, 171–176 structural organization, 171 structural requirements for 1,25(OH)2D3 transactivation of, 1472–1475, 1472f structure/function, 229–236 subcellular distribution, 168f, 170 subcellular trafficking, 363–376 superagonist/selective coactivator recruitment paradigms, 271–273 target gene diversity, 313–322 tissue distribution, 193–194, 228 tissue effects of low 1,25(OH)2D3/abnormal, 1322–1327 tissue selective ligands in, 270–273 established paradigms for, 270–271 tissue source/species conservation, 172 as toxic bile acid sensor, 863–869 in toxicity, 1362–1365 transcription regulated by integrated pathways, 268–270, 269f transcription v. 1α,25(OH)2D3/analog side chain modification, 1474–1475, 1474t translocation mechanisms determining analog selectivity, 1452f, 1454 variants, 195–196, 195f, 196f VDR promoter targeting, 305–312, 306f VDRE interaction v. interference footprinting protocols, 542 Vitamin D response element binding protein (VDRE-BP) dominant negative action, 354, 354f compensation for, 359–360 New World primate, 354–355

Vitamin D response element (VDRE) analogy selectivity v. genes with, 1454–1455 binding proteins in intracellular, 351–361 in bone proteins, 712, 713t clusters, 320–321 complex, 316t as complex/multiple TF binding site structures, 320 direct 1,25(OH)2D3 osteoblast modulation v., 654 DNA bend induced by VDR-RXR bonding to, 541 DNA binding polarity, 180, 317–318 DR6/DR3-type, 316t, 317 DR3-type, 314–315 classical VDRE structure in, 314 DBD-DBD distance in, 318, 318f multiple signaling pathways v., 316–317 strongest VDR-RXR heterodimer binding in, 314–315 DR4-type, 316t, 317 ER9-type, 316t DBD-DBD distance in, 318, 318f in human ECaC promoter region, 527 human VDR promoter, 200 known natural types of, 316t location/sequence of positive natural, 177, 177t mapping CT/CGRP gene, 694–696, 695f neuroendocrine-specific/cAMP enhancers in, 693–696 nonhuman VDR promoter, 197 in 1,25(OH)2D3 regulation of PTH gene expression, 539–541 1,25(OH)2D3 target gene promoter regions having, 314 positive/negative/optimal, 222–224, 223t rat CYP24A1 proximal promoter region, 93–97, 94f, 95f simple v. complex, 319–320 software v. identifying complex, 322 VDR interaction v. interference footprinting protocols, 542 VDR-regulated genes originating, 222–224, 223t VDR/RXR recognition by PTHrP, 743–744 VDR structure supporting specific binding to, 230–232, 231f whole-genome screening for putative, 322 Vitamins, discovery of, 3–4 Voltage-dependent calcium channels (VDCCs), in nongenomic 1α,25(OH)2D3 actions in muscle, 890–893, 890f Voltage-independent calcium channels (VICCs), Osteoblast VSCCs interacting with, 755, 755f Voltage-sensitive calcium channels (VSCCs), 751–753 α1 subunit types/functions of, 752, 752t L-type Ca2+ inactivating, 755–756, 756f 1,25(OH)2D3 v. open time in, 754–755, 755f pore-forming α1 subunit transmembrane organization in, 752, 752f subunit structure of, 752, 752f membrane/nuclear action cross-talk in, 757 1,25(OH)2D3 Ca2+/transcriptional responses by, 756–757 1,25(OH)2D3 regulating, 1782–1783 1,25(OH)2D3 v., 753–754 1,25(OH)2D3 v. D endocrine system, 751–757 VSCCs. See Voltage-sensitive calcium channels VSMCs. See Vascular smooth muscle cells

W Weaning BMC/BMD after, 845–846, 845f D/Ca metabolism after, 845 maternal Ca economy after, 846, 846f

1892 Weight, serum 25OHD v., 1007 Whites, dietary calcium reduction response by, 778 Wild-type (WT) mice dexamethasone influencing Ca absorption in, 445, 447f dietary intervention v. Ca absorption in, 434f, 436t, 437–438 intraperitoneal glucose tolerance test in, 1765, 1765f low-Pi diet v. Hyp and, 464f, 465 Williams syndrome defective chromatin remodeling complex WINAC in, 238–240 hypervitaminosis D/hypercalcemia in, 1359 1,25(OH)2D3 repression of CT/CGRP expression v., 690 WSTF gene deleted in patients with, 267–268 Williams syndrome transcription factor (WSTF) as VDR interactant, 307–308, 307f VDR ligand-induced transactivation coactivated by, 309–310, 310f WINAC/VDR interaction through, 268 Wilson’s disease, hypoparathyroidism/hypocalcemia v., 1053 WINAC coactivator complex components, 308, 308f cooperative function with co-regulator complexes, 311–312 nucleosome arrays disrupted by, 309, 309f, 310, 311f purification/identification, 308, 308f VDR promoter targeting mechanism, 310–311, 310f, 311f in VDR transcription model, 268–270 Williams syndrome associated with defective, 238–240 WSTF in VDR interaction with, 268 Women. See also Latinas BMD/insufficiency/secondary hyperparathyroidism in, 1089 bone remodeling markers in young/elderly, 1089–1090, 1090t Ca absorption v. intake in, 779–780, 779f, 780f Ca absorption v. load in, 779–780, 780f Ca v. age-related 1,25(OH)2D resistance in, 1104–1105, 1105f cervical cell carcinoma common in, 857 D deficiency in black, 791 D metabolism in Iranian, 794 D supplementation reducing bone mass loss in, 1094 D v. risk of falls in postmenopausal, 1114 estrogen v. 1,25(OH)2D3 in postmenopausal, 1260 estrogen v. PTH/bone resorption in, 1108–1109, 1108f normative histomorphometric data for, 956t 1,25(OH)2D3 v. postmenopausal osteoporosis in, 1111–1112 25OHD v. age in, 1101, 1102f 25OHD v. PTH in French post-menopausal, 1021–1022, 1021f OPG v. breast carcinoma-related bone metastases in, 676 postmenopausal OPG clinical trials in, 676 VDR target gene associated with osteoporosis in, 246 rickets prevention strategies v., 1072 serum 1,25(OH)2D in, 827 winter hypovitaminosis D in young/elderly, 1093, 1093t

INDEX

WSTF. See Williams syndrome transcription factor WT mice. See Wild-type mice

X X-linked hypophosphatemia (XLH), 467, 981 bone modeling abnormalities in, 982, 983f extraskeletal ossification in, 982–983, 984f, 985f FGF-23 in, 1193 genetic defect underlying, 1170–1172 Hyp/Gy mouse models for, 463, 463t Hyp mouse models for, 464f, 465 in original D-resistant rickets case, 1207 osteoarthritis in, 983 pathogenesis, 1172 pathophysiology, 1168–1170 phosphate homeostasis in, 1168–1173, 1169t radiography of rickets v., 922, 923f, 924f reinforcing FGF-23 as phosphatonin, 1165 treatment, 467, 1172–1173 X-linked recessive hypophosphatemia (XLRH), phosphate homeostasis in, 1175 X-ray diffraction, bone mineral presence determined by, 481, 482f X-rays D-deficient osteomalacia revealed by, 481 discovery of, 967 metabolic bone disease evaluated with, 914, 921–922, 923f, 924f Xenobiotic detoxification, VDR in, 228, 229f XLH. See X-linked hypophosphatemia XLRH. See X-linked recessive hypophosphatemia

Y Yeast higher eukaryote complex diversity v., 266 Mediator-D/related complex function studied in, 266

Z Zebrafish VDR (zVDR) bound to 1α,25(OH)2D3, 284–285, 284f characterized, 279 ZK 159222, 1481f, 1482 zVDR. See Zebrafish VDR zVDR-1α,25(OH)2D3 complex, crystal structure, 284–285, 284f zVDR-Gemini complex channel extending original pocket in, 287–288 structure, 287–288 zVDR-1α,25(OH)2D3 structure v., 287

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