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Hedgehog Signaling Modulates Cholesterol Homeostasis in Chondrocytes and in Osteoarthritis

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Hedgehog Signaling Modulates Cholesterol Homeostasis in Chondrocytes and in Osteoarthritis HEDGEHOG SIGNALING MODULATES CHOLESTEROL HOMEOSTASIS IN CHONDROCYTES AND IN OSTEOARTHRITIS by Shabana Amanda Ali A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Institute of Medical Science University of Toronto © Copyright by Shabana Amanda Ali 2014 HEDGEHOG SIGNALING MODULATES CHOLESTEROL HOMEOSTASIS IN CHONDROCYTES AND IN OSTEOARTHRITIS Shabana Amanda Ali Doctor of Philosophy Institute of Medical Science University of Toronto 2014 Abstract Osteoarthritis (OA) is a common degenerative disease of the joint that is characterized by degradation and calcification of articular cartilage, and subchondral bone changes. Hedgehog (Hh) signaling is known to be activated in human and murine OA. Since Hh signaling regulates Gli‐mediated gene expression, we identified Hh target genes that are expressed in chondrocytes. Microarray analyses were performed to detect changes in gene expression when the Hh pathway was modulated in human OA cartilage samples. Results from the Affymetrix Human Gene 1.0 ST microarray were analyzed for differentially expressed genes from three patient samples. Using Ingenuity® Pathway analysis, several genes known to be involved in sterol homeostasis were found to be modulated with Hh inhibition. We hypothesized that Hh signaling regulates cholesterol biosynthesis in chondrocytes, and that modulating cholesterol homeostasis impacts the severity of OA. To investigate the function of cholesterol in the cartilage, mice with chondrocyte‐specific cholesterol accumulation were generated. This was achieved by excising Insig1 and Insig2, major negative regulators of cholesterol homeostasis. Over time, mice with chondrocyte‐specific cholesterol accumulation exhibited impaired growth of the long bones. With aging or surgically induced joint instability, these mice ii developed more severe OA than control littermates. They expressed typical OA markers, such as type X collagen, indicating chondrocyte hypertrophy in the articular cartilage. Genetic manipulation of Hh signaling in these mice suggests that Hh signaling is modulating the phenotype by regulating sterol homeostasis. Hh reduction and statin treatment both lowered cholesterol production and rescued the phenotype, reducing the severity of OA. Here we identified novel Hh target genes in chondrocytes which regulate intracellular cholesterol levels, and found that cholesterol dysregulation in the chondrocytes predisposes to OA. These data suggest that pharmacologic correction of intra‐articular sterol imbalance can be used as a treatment for osteoarthritis. iii Acknowledgements For taking a chance on me, I thank my supervisor Dr. Benjamin Alman. His vision saw beyond the limited scientific experience I brought to the lab, and his support afforded me the training environment to flourish as a scientist. To my past and present lab mates, I am forever indebted for the patience, assistance, and inspiration provided to me. For their scientific guidance and/or experimental assistance I thank Dr. Louisa Ho, Dr. Claire Hsu, Dr. ZhuJuan (Sue) Li, Dr. Jason Rockel, Dr. Saeid Amini‐Nik, Dr. Farasat Zaman, Dr. Alvin Lin, Mushriq Al‐Jazrawe, Heather Whetstone, Raymond Poon, Henry Ma, and Puviindran Nadesan. For his contributions, both significant (help with mathematical calculations) and trivial (changing my outlook on life), I thank my desk‐ mate Dr. Gurpreet Baht. I am thankful for the scientific expertise offered by my supervisory committee, Dr. Christopher McCulloch, Dr. Khosrow Adeli, and Dr. Jane Aubin. These individuals were critical in shaping the direction of this project. The participation of my esteemed thesis examination committee, Dr. Frank Beier, Dr. Mohit Kapoor, and Dr. Dominic Ng, is greatly appreciated. For his participation in both my reclassification exam and my final committee meeting, I thank Dr. Chi‐Chung Hui. His advice, both scientific and personal, was instrumental to my graduate experience. He is a dedicated mentor and I am grateful for his investment in me. Without collaboration this work would not be possible. I am thankful to Dr. Khosrow Adeli, Dr. Philip Connelly, Dr. Carolyn Cummins and their respective laboratories for providing experimental guidance, and to Dr. David Backstein at Mount Sinai Hospital (Toronto, Canada) for granting access to human cartilage specimens. iv For making me the person I am today, I thank my grandparents, my parents, my brother, and all of my good friends along the way. My grandmother who raised me during the early years taught me my first, and arguably most important, life lessons. Her struggle with osteoarthritis motivated me to pursue this field of research. If my small contribution makes a difference, it is for her. My parents worked tirelessly to provide me with support of all kinds. My mother showed me the meaning of strength, of hard work, of unconditional love. My father encouraged my curiosity, gave me a sense of worth, and instilled in me the importance of education. Together, my parents created a life for me that they themselves could not have. Despite the room for improvement that remains (of which you’re always reminding me), I hope I have made you proud. My big little brother Jordan is constantly pushing me to be better, leading me with his own incredible example. I know that he will always be there for me, because he has always been there for me. I did the thing which I thought I could not do; time to set new goals. My best friend Natalie is a perpetual source of inspiration, leading me with her own incredible example. She sees the best in me, and firmly reminds me when I forget. These two give me the perfect balance of tough and unconditional love, adjusted accordingly on a daily basis, at their wise and trusted discretion. To them I owe my sanity (a state that is constantly challenged in graduate school). These words only begin to express my gratitude. I share this accomplishment with each of you. v Contributions Under the supervision of Dr. Benjamin Alman, I designed experiments, interpreted results, and wrote the manuscripts resulting from the work described here. The results described in Chapter Two, as partly published in Analytical Biochemistry (Ali and Alman 2012), were acquired independently. The results described in Chapters Three and Four, the manuscript for which is being prepared for publication, were acquired with the assistance of the following individuals. Mushriq Al‐Jazrawe assisted with histological grading, protein blots, and statistical analyses. Heather Whetstone assisted with histological sectioning and staining. Raymond Poon and Henry Ma assisted with primary human chondrocyte culture and reporter construct experiments. Sarah Farr and Mark Naples from the laboratory of Dr. Khosrow Adeli performed radiotracer experiments to assess cholesterol biosynthesis. The contributions made by these individuals are also described in‐text. vi Table of Contents Abstract ii Acknowledgements iv Contributions vi List of Figures x List of Tables xii Chapter One: Introduction 1 Background Osteoarthritis: Prevalence, Pathophysiology, Etiology, Burden of disease 2 Experimental models of osteoarthritis 7 Articular joints: Articular cartilage, Subchondral bone 9 Articular chondrocytes and growth plate chondrocytes 12 Hedgehog signaling and osteoarthritis 14 Gene expression analyses in osteoarthritis 17 Systemic influences in osteoarthritis 19 Cholesterol homeostasis 21 Cholesterol homeostasis and Hedgehog signaling 25 Cholesterol homeostasis and osteoarthritis 27 vii Current treatment strategies in osteoarthritis: Pharmacologics, Lifestyle changes, Surgery, Cartilage restoration 30 Rationale 35 Hypothesis 36 Objectives 36 Chapter Two: Hh signaling regulates expression of cholesterol biosynthetic genes in chondrocytes 37 Summary 38 Introduction 39 Results Optimized RNA extraction from human osteoarthritic cartilage 41 Gene expression profiling in osteoarthritic cartilage: microarray analyses 47 Identifying Hh targets: cholesterol biosynthetic genes 52 Discussion 58 Materials and Methods 62 Chapter Three: Hh signaling modulates cholesterol accumulation in chondrocytes 67 Summary 68 Introduction 69 Results Cholesterol accumulation in chondrocytes: InsigDKO mice 72 viii Hedgehog signaling regulates cholesterol biosynthesis in chondrocytes 78 Cholesterol accumulation in chondrocytes impairs growth of the long bones 81 Discussion 88 Materials and Methods 92 Chapter Four: Cholesterol modulation can alter the severity of osteoarthritis 95 Summary 96 Introduction 97 Results Cholesterol accumulation in chondrocytes predisposes to osteoarthritis 99 Cholesterol inhibition protects against osteoarthritis in mice 105 Statin treatment reduces OA markers in human cartilage 115 Discussion 118 Materials and Methods 122 Chapter Five: Conclusions and future research 127 Future Research 130 Relevance 139 References 140 Appendix: Microarray gene list 155 ix List of Figures Figure 1. Representative total knee arthroplasty sample showing features of osteoarthritis. Figure 2. Schematic representation of articular chondrocytes and growth plate chondrocytes. Figure 3. Schematic representation of the Hedgehog (Hh) signaling pathway. Figure 4. Schematic representation of critical regulators of cholesterol homeostasis. Figure 5. Representative electropherograms comparing RNA quality. Figure 6. Hh inhibition in human articular cartilage. Figure 7. Microarray analysis: gene filtering. Figure 8. Heatmap of cholesterol homeostatic genes. Figure 9. Real‐time PCR validation of cholesterol homeostatic
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