PROTEOGLYCAN 4: A DYNAMIC REGULATOR OF PARATHYROID HORMONE BIOLOGIC ACTIONS by Chad M. Novince A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Oral Health Sciences) in The University of Michigan 2011 Doctoral Committee: Professor Laurie K. McCauley, Chair Professor Jan C. Hu Professor Russell S. Taichman Associate Professor Hedwig S. Murphy DEDICATION To my parents, Loretta and William To my bothers, Ryan and Zachary ii ACKNOWLEDGEMENTS I express sincere gratitude to all parties that provided me support throughout my doctoral studies in the Oral Health Sciences Ph.D. program. I am deeply appreciative of the time and effort that Dr. Laurie McCauley, my Ph.D. mentor and the chair of my dissertation committee, devoted to my scientific training. I am thankful for her invaluable guidance and support, which empowered me to have a successful Ph.D. training experience. Her sincere passion for the advancement of science and commitment to mentoring is inspiring to developing sc ie ntis t. I acknowledge and thank the members of my dissertation committee; Dr. Laurie McCauley, Dr. Russell Taichman, Dr. Jan Hu, and Dr. Hedwig Murphy for providing me with the direction and support that enabled me to make continual progress throughout my doctoral studies. I am grateful to the members of my committee for unselfishly sharing their expertise and individually meeting with me on a regular basis during my Ph.D. training. The enthusiasm and encouragement conveyed by my committee members towards my doctoral research and training was deeply motivational. I individually acknowledge Dr. Russell Taichman and Dr. Thomas Rosol for their informal mentorship. Scientific interactions with Drs. Taichman and Rosol were formative experiences during my doctoral training. Moreover, their willingness to iii collaborate on scientific projects broadened the scope and impact of my Ph.D research stud ies. I a m ever indebted to the members of Dr. Laurie McCauley’s research laboratory for sharing their vast knowledge and expertise in carrying out scientific research. The enthusiasm for research and discovery that is conveyed by the McCauley lab members provided for an exciting and motivational Ph.D. training experience. I thank Amy Koh, Jan Berry, Glenda Pettway, Xin Li, Jinhui Liao, Flavia Pirih, Serkin Park, Zhengyan Wang, Sun Wook Cho, Fabiana Soki, Megan Michalski, Matt Eber, Payam Entezami, Mallory Van P utten, Naomi Lin, and David Sadler for their friendship and support during my doctoral research training. Individually, I extend sincere thanks to Amy, Megan, and Payam for playing an integral ro le in the progress and success of my Ph.D. research studies. Other individuals I acknowledge for their contributions include Junro Yamashita, Julie Marchesan, Chris Wilson, Ben S inder, Ken Kozloff, C hris Strayhorn, Chris Jung, Taocong Jin, Holly Brown, Ramiro Toribio, Thomas Wronski, and Matthew Warman. Special thanks go to Dr. Charlotte Mistretta, director of the Oral Health Sciences Ph.D. program, for her sincere commitment to the well being and success of the Ph.D. students. Her guidance and enthusiastic support throughout the P h.D. training experience was a source of inspiration that was very meaningful to me. I thank Dr. Robert Bradley fo r his pre-candidate mentors hip. I am grateful to Dean Peter Polverini for taking a sincere interest in my Ph.D. training and professional development. I also extend thanks to Dr. Darnell Kaigler for his positive encouragement and generous advice. iv I acknowledge and thank Patricia Schultz, Janet Riggs, and Diane Lafferty for their guidance and assistance in applying for grants and managing the administrative aspects of my Ph.D. training. Concerning the clinical component of my dual D.D.S./Ph.D. studies I recognize and thank Dr. Marilyn Woolfolk and Dr. Don Heys for facilitating the structuring o f the clinical component of D.D.S./Ph.D. program. Their understanding and willingness to accommodate the demands of the P h.D. curriculum, while never compromising the quality of the D.D.S. curriculum, allowed me to have an optimal undergraduate dental training experience. I extend thanks to Dr. Don Heys, Dr. Philip Richards, and Dr. Bryan Williams for being influential clinical mentors during my D.D.S. training. I also thank Dr. Peter Yaman, Dr. Gisele Neiva, and Dr. John Heys for acting as clinical mentors throughout my graduate dental training in the Graduate Restorative Dentistry Clinic. I acknowledge and extend tremendous gratitude to my brothers Ryan and Zachary, who insp ir ed me to apply to the Oral Health Sciences Ph.D. program. Both Ryan and Zach played formative roles in my scientific development and the progression of my doctoral studies. I relied on them to discuss scientific ideas, trouble shoot problems at the bench, and when necessary to travel to Ann Arbor to assist me in performing scientific experiments. I am ever grateful to my parents for providing unconditional love and support. The ir commitment to professionalism and helping others was influential in my decision to pursue a career in science and healthcare. The unselfish love, support, and positive encouragement provided by my family members played a critical role in the progression and success of my D.D.S./Ph.D. s tud ies. v TABLE OF CONTEN TS DEDICATION…………………………………………………………………………...ii ACKNOWLEDGEMENTS…………………………………………………………….iii LIST OF FIGURES……………………………………………………………………viii LIST OF TABLES……………………………………………………………………….x ABSTRACT……………………………………………………………………………...xi CHAPTER 1 INTRODUCTION……………………………………………………………….1 Problem statement…………………………………………………………1 General hypothesis………………………………………………………...3 Specific aims………………………………………………………………3 Background and significance……………………………………………...4 References………………………………………………………………..12 CHAPTER 2 PROTEOGLYCAN 4, A NOVEL IMMUNOMODULATORY FACTOR, REGULATES PARATHYROID HORMONE ACTIONS ON HEMATOPOIETIC CELLS…………………………………………………..20 Abstract…………………………………………………………………..20 Introduction………………………………………………………………21 Materials and methods…………………………………………………...23 Results……………………………………………………………………29 Disc uss io n………………………………………………………………..35 References………………………………………………………………..50 CHAPTER 3 PROTEOGLYCAN 4: A DYNAMIC REGULATOR OF SKELETOGENESIS AND PTH SKELETAL ANABOLISM………………56 Abstract…………………………………………………………………..56 vi Introduction………………………………………………………………57 Materials and methods…………………………………………………...59 Results……………………………………………………………………66 Discussion………………………………………………………………..75 References………………………………………………………………..93 CHAPTER 4 THE IMPACT OF PROTEOGLYCAN 4 AND PARATHYROID HORMONE ON ARTICULAR CAR TILAGE DEGENERATION………...99 Abstract…………………………………………………………………..99 Introduction……………………………………………………………..100 Materials and methods………………………………………………….102 Results…………………………………………………………………..105 Discussion………………………………………………………………107 References………………………………………………………………119 CHAPTER 5 CONCLUSIONS………………………………………………………………124 Proteoglycan 4, a novel immunomodulatory factor, regulates parathyroid hormone actions on hematopoietic cells………………………………..125 Proteoglycan 4: a dynamic regulator of skeletogenesis and PTH skeletal anabolism……………………………………………………………….127 The impact of proteoglycan 4 and parathyroid hormone on articular cartilage degeneration…………………………………………………..129 Summary………………………………………………………………..130 References………………………………………………………………133 vii LIST OF FIGURES Fig ure 2.1. PTH re gula tio n o f Prg4 mRN A………………………………………………….40 2.2. Histological assessment of bone marrow, liver, and spleen……………………..41 2.3. PTH re gula tio n o f gene expression………………..……………………………..42 2.4. Per ip hera l b lood cells…………………………………………………………….43 2.5. Bone marrow immune cells……………………………………………………...44 2.6. Bone marrow hematopoietic progenitor cells……………………………………45 2.7. Bone marrow megakaryopoietic cells……………………………………………46 2.8. Bone marrow IL6 and SDF-1 express io n………………………………………..47 3.1. PTH re gula tio n o f Prg4 mRN A………………………………………….………82 3.2. Trabecular bone area and volume analysis………………………………………83 3.3 Tibial growth plate, femur length, and bone turnover serum biochemical analysis in young mice…………………………………………………………………….84 3.4. Bone marrow stromal cell (BMSC) in vitro osteoblastogenesis assays…………85 3.5. Proximal tibia bone cell numbers and activity…………………………………...86 3.6. Ad ult Prg4 mice - serum biochemical analysis of bone turnover and mineral homeostasis……………………………………………………………….……...87 3.7. Joint range of motion and animal mobility………………………………………88 3.8. PTH regulation of bone marrow and liver gene expression……………………..89 viii 4.1. Knee joint histopathology…………....................................................................114 4.2. Subchondral bone morphology…………………………………………………115 4.3. Articular cartilage degradation…………………………………………………116 4.4 Chondrocyte apoptosis………………………………………………………….117 4.5. SDF-1 expression in the synovium……………………………………………..118 5.1. Proteoglycan 4 indirectly supports PTH anabolic actions.……...……………...132 ix LIST OF TABLES Table 2.1 Prg4 protein products……………………………………………………………48 2.2 Peripheral blood cells………………………………………………………….....49 3.1 Femur micro-CT analysis………………………………………………………..90 3.2 Bone marrow and liver gene expression…………………………………………91 3.3 Serum biochemical analysis – IGF-I and F GF-2………………………………...92 x ABSTRACT PROTEOGLYCAN 4: A DYNAMIC REGULATOR OF PARATHYROID HORMONE BIOLOGIC ACTIONS By Chad M. Novince Chair: Laurie K. McCauley Intermittent parathyroid hormone (PTH) (1-34) is the only anabolic agent currently approved for the treatment of osteoporo s is in the U.S. PTH (1-34) is under clinical