1,25-DIHYDROXYVITAMIN D3-INDUCED GENES IN OSTEOBLASTS: UNCOVERING NEW FUNCTIONS FOR MENINGIOMA 1 AND SEMAPHORIN 3B IN SKELETAL PHYSIOLOGY by XIAOXUE ZHANG Submitted in partial fulfillment of the requirements for the Degree of Doctor of Philosophy Thesis advisor: Paul N. MacDonald Department of Pharmacology CASE WESTERN RESERVE UNIVERSITY May 2009 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of _____________________________________________________ candidate for the ______________________degree *. 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I dedicate this thesis to my mother and father for their lifelong love, encouragement and sacrifice TABLE OF CONTENTS Table of Contents ii List of Tables iii List of Figures iv Acknowledgements vii Abbreviations x Abstract xiii Chapter I Introduction 1 Chapter II Meningioma 1 (MN1) is a 1,25-dihydroxyvitamin D3- 44 induced transcription coactivator that promotes osteoblast proliferation, motility, differentiation, and function Chapter III Semaphorin 3B (SEMA3B) is a 1,25- 108 dihydroxyvitamin D3-induced gene in osteoblasts that promotes osteoclastogenesis and induces osteopenia in mice Chapter IV Summary and future directions 155 Reference List 175 ii LIST OF TABLES Table II-1 Amplification primers used to generate cDNA probes for 77 Northern blot analysis iii LIST OF FIGURES Figure I-1 The metabolism of vitamin D 32 Figure I-2 The structure and activation of VDR 34 Figure I-3 The model of VDR mediated transcription 36 Figure I-4 The scheme of bone remodeling 38 Figure I-5 MN1 knockout mice have severe defects in cranial skeletal 40 development Figure I-6 The scheme of vertebrate semaphorins and SEMA3B 42 signaling Figure II-1 1,25(OH)2D3 induces MN1 expression in MG-63 78 osteoblastic cells Figure II-2 The mechanism of MN1 regulation in MG-63 cells 80 Figure II-3 MN1 mRNA transcripts are induced by 1,25(OH)2D3 and 82 by osteoblastic cell differentiation Figure II-4 MN1 augments 1,25(OH)2D3/VDR mediated transcription 84 Figure II-5 MN1 shows selectivity on coactivating nuclear receptors 86 Figure II-6 MN1 cooperates with SRC coactivators to stimulate VDR- 88 mediated transcription Figure II-7 MN1 knockout osteoblasts have diminished VDR-mediated 90 transcriptional responses Figure II-8 MN1 knockout osteoblasts have reduced growth rate in 92 vitro Figure II-9 MN1 knockout calvarial cells have reduced proliferation in 94 vivo iv Figure II-10 MN1 knockout osteoblasts have altered morphology and 96 reduced motility Figure II-11 MN1 knockout osteoblasts have reduced alkaline 98 phosphatase activity Figure II-12 MN1 knockout osteoblasts show decreased mineralization 100 Figure II-13 MN1 knockout osteoblasts show enhanced adipogenesis 102 Figure II-14 MN1 knockout osteoblasts have reduced ability to support 104 1,25(OH)2D3-stimulated osteoclastogenesis Figure II-15 MN1 stimulates RANKL promoter activity 106 Figure III-1 1,25(OH)2D3 induces SEMA3B expression in MG-63 133 osteoblastic cells Figure III-2 The mechanism of SEMA3B regulation in MG-63 cells 135 Figure III-3 SEMA3B is induced by 1,25(OH)2D3 and by osteoblastic 137 differentiation in ST-2 bone marrow stromal cells Figure III-4 SEMA3B is induced by 1,25(OH)2D3 and by differentiation 139 in MC3T3-E1 cells and primary osteoblasts Figure III-5 SEMA3B is expressed in the long bones of mice 141 Figure III-6 Transgenic mice with targeted osteoblast-selective 143 expression of SEMA3B have reduced body weight and shorter bones Figure III-7 SEMA3B-expressing transgenic mice have decreased 145 bone mineral density and diminished trabecular bone Figure III-8 Transgenic bones have normal osteoblasts but increased 147 osteoclastogenesis Figure III-9 SEMA3B transgenic osteoblasts have increased ability to 149 support 1,25(OH)2D3-stimulated osteoclastogenesis v Figure III-10 SEMA3B transgenic osteoblasts have increased ability to 151 support 1,25(OH)2D3-stimulated osteoclastogenesis Figure III-11 SEMA3B promotes osteoclast differentiation in RAW 264.7 153 cells vi Acknowledgements I would like to acknowledge my advisor, Dr. Paul MacDonald, for the continuous support and encouragement during the graduate study. Being patient and knowledgeable, he gave me step-by-step instruction and guidance on science. I would like to thank Dr. Diane Dowd, our lab member and my thesis committee member, who provided a lot of expertise on experiments and assistance on scientific writing. I would like to acknowledge my thesis committee members, Dr. Ruth Keri, Dr. David Danielpour, and Dr. Yu-Chung Yang, for their valuable inputs on my projects, especially when I was in difficult times and needed discussions and suggestions. I would like to thank my previous and current lab members for their help on experiments, classes, and presentation practice. Dr. Amelia Sutton identified MN1 and SEMA3B and started excellent studies about these two genes. She generated SEMA3B transgenic animals, performed bone imaging analysis, and set up protocols for animal handling and bone cell analysis. Ms. Meika Moore gave plenty of assistance on animal experiments, including the maintenance, breeding, and genotyping of experimental mice. Dr. Tara Ellison made nuclear receptor expression constructs, gave valuable suggestions on molecular biology, and provided useful discussions on troubleshooting. Dr. Chi Zhang taught me a lot about graduate studies and assisted me to settle down in Cleveland. I am also grateful to other laboratories for generously sharing various equipments and vii providing valuable experimental instructions, especially to the laboratories of Drs. Ruth Keri, Ruth Siegel, Monica Montano, and Yu-Chung Yang. I would like to acknowledge the previous and current departmental members, including Dr. Erin Milliken, Dr. Yu Chen, Dr. Hui Wang, Dr. Yiping Rong, Dr. Gang Zheng, for their help on classes, experiments, presentations, preliminary exams, and career choice. I would also like to thank the Pharmacology Department for providing this great training program. I would like to acknowledge Dr. Ellen C. Zwarthoff (Erasmus Medical Center, Rotterdam, The Netherlands) and Dr. Gerard Grosveld (St. Jude Children’s Research Hospital, Memphis, TN) for providing MN1 knockout mice breeding pairs. I would also like to thank Dr. Benoit de Crombrugghe for providing the 2300lacZ plasmid, Dr. John D. Minna for providing the pcDNA3- SEMA3B plasmid, Dr. Edward Greenfield for the assistance with in vitro osteoblast and osteoclast assays, and Ms. Patty Lott and the staff at the University of Alabama at Birmingham, Center for Metabolic Bone Disease, Histomorphometry and Molecular Analysis Core Laboratory, for performing the mineralized bone histology and histomorphometry. Finally, I would like to acknowledge Dr. Bernard Tandler for helping me edit the thesis. I would like to thank my undergraduate teacher, Dr. Zihe Rao, for his continuous care and unconditional support during the past ten years. I would like to thank all the friends in my everyday life. It is their care and encouragement that made me able to accomplish the study abroad. I would like viii to thank all my family members, who are so far away, but trying their best to support me to finish my graduate study. ix Abbreviations 1,25(OH)2D3 1,25-dihydroxyvitamin D3 1α-hydroxylase 25-hydroxyvitamin D-1α-hydroxylase 24-hydroxylase 25-hydroxyvitamin D3-24-hydroxylase 25-hydroxylase vitamin D 25-hydroxylase 25(OH)D3 25-hydroxycholecalciferol 9-cis RA 9-cis retinoic acid AF-2 Activation function-2 ALP Alkaline phosphatase AML Acute myeloid leukemia ATP Adenosine triphosphate ATRA All trans retinoic acid BCA Bicinchoninic acid BMP Bone morphogenetic protein BrdU 5-bromo-2-deoxyuridine C/EBP CCAAT/enhancer-binding protein cAMP Cyclic adenosine monophosphate CaSR Calcium-sensing receptor Cbfa Core binding factor CBP CREB-binding protein CBS Bovine calf serum CCD Cleidocranial dysplasia CDK Cyclin dependent kinase CDKI CDK inhibitor CM Conditional media CT Computed tomography CREB cAMP response element binding protein CTD carboxyl terminal domain DAPI 4',6-diamidino-2-phenylindole DBD DNA-binding domain DEXA Dual-energy X-ray absorptiometry Dlx Distal-less homeobox DNA Deoxyribonucleic acid DR Direct repeats DRIP Vitamin D receptor interacting protein E2 17β-estradiol EGFR Epidermal growth factor receptor EMSA Electrophoretic mobility shift assay ER Estrogen receptor FABP4 Fatty acid binding protein 4 FBS Fetal bovine serum FGF Fibroblast growth factor FRP-4 Fizzled-related protein 4 GAPDH Glyceraldehyde-3-phosphate dehydrogenase GDF Growth/differentiation factor x GR Glucocorticoid receptor GRE Glucocorticoid response element GRIP Glucocorticoid receptor interacting protein HAT Histone acetyltransferase HC Hypertrophic chondrocyte zone HDAC Histone deacetylase Hr Hairless IGF Insulin-like growth factor IGFBP IGF binding protein Ihh Indian hedgehog IL Interleukin KO Knockout LBD Ligand-binding domain LOH Loss of heterozygosity LPL Lipoprotein lipase LRP Low-density lipoprotein receptor-related protein MAP Mitogen-activated protein MAR Mineral apposition rate MARRS Membrane-associated, rapid