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2D3-Induced Genes in Osteoblasts THE REGULATION AND FUNCTION OF 1,25-DIHYDROXYVITAMIN D3- INDUCED GENES IN OSTEOBLASTS by AMELIA LOUISE MAPLE SUTTON Submitted in partial fulfillment of the requirements for the Degree of Doctor of Philosophy Advisor: Dr. Paul N. MacDonald Department of Pharmacology CASE WESTERN RESERVE UNIVERSITY August 2005 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of ______________________________________________________ candidate for the ________________________________degree *. (signed)_______________________________________________ (chair of the committee) ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ (date) _______________________ *We also certify that written approval has been obtained for any proprietary material contained therein. I would like to dedicate this dissertation to my amazing mother. This is the very least I can do to say thank you to the woman who has dedicated her entire life to me. TABLE OF CONTENTS Dedication ii Table of Contents iii List of Tables iv List of Figures v Acknowledgments vii Abstract xiv Chapter I Introduction 1 Chapter II The 1,25(OH)2D3-regulated transcription factor MN1 64 stimulates VDR-medicated transcription and inhibits osteoblast proliferation Chapter III The 1,25(OH)2D3-induced transcription factor 98 CCAAT/enhancer-binding protein-β cooperates with VDR to regulate 24-hydroxylase expression and may be required for osteoblast differentiation and bone mineralization Chapter IV Targeted overexpression of the 1,25(OH)2D3- 120 regulated gene semaphorin3B in osteoblasts causes increased bone resorption and osteopenia Chapter V Investigating the function of thrombomodulin, a 160 1,25(OH)2D3-regulated anticoagulant protein, in osteoblasts and in bone Chapter VI Summary and future directions 183 References 201 iii LIST OF TABLES Table I-1 Gene Expression Profiling Reveals Decreased Expression 43 of 92 Genes in 1,25(OH)2D3-Treated Osteoblastic Cells Table I-2 Gene Expression Profiling Reveals Increased Expression of 46 33 Genes in 1,25(OH)2D3-Treated Osteoblastic Cells iv LIST OF FIGURES Figure I-1 Metabolism and mineral homeostatic functions of the 48 vitamin D endocrine system Figure I-2 Domain structure of VDR 50 Figure I-3 The vitamin D receptor undergoes a conformational 52 change upon binding hormone Figure I-4 Schematic of the conserved domains in SRC family 54 members Figure I-5 Model of the DRIP complex interacting with liganded VDR 56 Figure I-6 Domain structure of NCoA-62/SKIP 58 Figure I-7 Model of temporal association of coactivators during VDR- 60 mediated transcription Figure I-8 Long bones are formed by endochondral ossification 62 Figure II-1 1,25(OH)2D3 induces MN1 expression in a time- and dose- 84 dependent manner Figure II-2 1,25(OH)2D3 induction of MN1 requires de novo RNA 86 synthesis but not protein synthesis Figure II-3 MN1 augments 1,25(OH)2D3-dependent transcription 88 through the VDR ligand-binding domain Figure II-4 Effect of MN1 on other nuclear receptors 90 Figure II-5 MN1 synergizes with SRC coactivators 92 Figure II-6 MN1 inhibits proliferation in osteoblastic cells 94 Figure II-7 MN1 decreases S-phase entry in osteoblastic cells 96 Figure III-1 1,25(OH)2D3 increases C/EBPβ expression in primary 112 osteblasts Figure III-2 C/EBPβ is required for maximal 1,25(OH)2D3-mediated 114 induction of 24OHase expression v Figure III-3 Targeted deletion of C/EBPβ disrupts osteoblast 116 mineralization in vitro Figure III-4 Targeted deletion of both C/EBPβ and VDR causes 118 undermineralized bones in vivo Figure IV-1 1,25(OH)2D3 induces SEMA3B expression 140 Figure IV-2 SEMA3B is expressed in the long bones of mice 142 Figure IV-3 Establishment of transgenic mice overexpressing SEMA3B 144 in osteoblasts Figure IV-4 Transgenic mice have reduced body weight and shorter 146 bones Figure IV-5 Altered cranial morphology in some transgenic mice 148 Figure IV-6 Transgenic mice have decreased bone mineral density 150 Figure IV-7 Transgenic mice have diminished trabecular bone 152 Figure IV-8 Normal osteoblast function but potentially increased bone 154 resorption in transgenic mice Figure IV-9 Transgenic osteoblasts show increased differentiation and 156 mineralization in vitro Figure IV-10 Transgenic osteoblasts stimulate increased 158 osteoclastogenesis Figure V-1 1,25(OH)2D3 induces thrombomodulin expression in a 173 time- and dose-dependent manner Figure V-2 1,25(OH)2D3 induction of thrombomodulin requires active 175 transcription but does not require protein synthesis Figure V-3 1,25(OH)2D3 increases protein levels of thrombomodulin 177 Figure V-4 Stable MG-63 cell lines overexpressing thrombomodulin 179 display an increased proliferation rate Figure V-5 Mice with an osteoblast-selective knockout of 181 thrombomodulin weigh the same as control littermates vi ACKNOWLEDGEMENTS There are so many people that have made this dissertation possible that I would need to write an entire other dissertation just to thank them properly. In the next few pages, I hope I can capture just a glimpse of the contributions of so many wonderful people that have touched my life in the last several years. First, I would like to thank my advisor Dr. Paul MacDonald for guiding me through this process. He gave me the freedom I needed to think and work independently, but always stepped in to offer advice, expertise, or motivation when my work (or I) was stalled. I will always be deeply indebted to him for all of his encouragement and support while I have been his laboratory. I would also like to thank Dr. Diane Dowd for sharing her technical expertise and continual support both as a member of my thesis committee and in the laboratory. I am very grateful to the other members of my thesis committee, Dr. Hung-Ying Kao, Dr. Clark Distelhorst, and Dr. John Nilson, for their encouragement, time, and thoughtful scientific discussions throughout this process. I would like to express a special thank you to John for his support throughout my career as an M.D./Ph.D. student. John’s enthusiasm about science and philosophy of education was one of the things that attracted me to pursuing my Ph.D. in addition to my M.D., and I know that I would not have made it this far without his help. vii I would like to thank all the past and present members of the MacDonald laboratory. Dr. Chi Zhang provided much-needed company during many late nights in the laboratory. I am grateful to Dr. Brenda Altose for her friendship, kindness, and advice throughout my education. I would like to thank Tara Ellison for providing endless dietary, intellectual, and emotional nourishment throughout the past several years. I am indebted to her for her friendship and amazing culinary prowess. I am grateful to Meika Moore for her excellent technical assistance in maintaining the mouse breeding and performing flawless genotyping for me, sometimes on a moment’s notice and always without complaint. I am very thankful to Xiaoxue Zhang for her hard work in performing the studies regarding coactivator activity of MN1. I would also like to thank Fusong Chen for his efforts in continuing the C/EBPβ project. One of the best aspects of my experience in the Pharmacology department has been interacting with so many talented, enthusiastic, helpful, entertaining, and supportive graduate students. Whether it has been sharing a protocol or lending an understanding ear, I would have never been able to complete this dissertation without them. In particular, I would like to thank Dr. Erin Milliken for commiserating with me throughout this rather rocky road. I am grateful to both Erin and Jonathan Mosley for leading the “Science, Non-Science” discussion group, which has proven to be a very engaging distraction from pipetting. I would also like to thank Jonathan for his continuous statistical advice and his unique perspective on just about everything. I am indebted to Mike Davis for his patient viii assistance with several flow cytometry experiments. Even though these experiments were not fruitful for my project, I learned a great deal from him and really appreciate the time and effort he put into helping me. Through this department and the Medical Scientist Training Program, I have met three lifelong friends that have been an endless source of love and support. First, I would like to thank Dr. Michelle Kahlenberg for her kindness, encouragement, many late nights of ice cream therapy and for forging ahead in medical school so that I may try to follow in her path of excellence. I am indebted to Dr. Nicole Bianco for her support, insightful advice, and hours of comic relief. I would like to express a very special thank you to Dr. Helai Mohammad who has always been there when I needed her the most, both scientifically and personally, and has provided much entertaining diversions from the laboratory. I am grateful to all members of the Nilson and Keri laboratories for generously sharing their technical expertise, equipment, and reagents and for letting me crash all their parties. I would like to give a special thank you to Dave Peck and Kristen Lozada, who taught me more than I ever wanted to know about mouse breeding. I would also like to thank Kristen for her support and friendship, and taking me out when I needed to get away from the lab. I am indebted to Dr. Ruth Keri for always being available to discuss science and numerous unrelated topics, and for always being there with a kind word when I really needed it. Ruth has also been very open with sharing her laboratory’s reagents and equipment, and I would not have been able to complete these studies without her generosity.
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