Vitamin D Receptor (VDR) and Cholesterol Homeostasis: Interplay of VDR Enzyme Targets and Vitamin D Deficiency by Holly P. Quach A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Department of Pharmaceutical Sciences University of Toronto © Copyright by Holly P. Quach, 2016 Vitamin D Receptor (VDR) and Cholesterol Homeostasis: Interplay of VDR Enzyme Targets and Vitamin D Deficiency Holly P. Quach Doctor of Philosophy Department of Pharmaceutical Sciences University of Toronto 2016 Abstract Vitamin D deficiency is speculated to play a role in hypercholesterolemia. However, there has been little molecular evidence to link the two until recent evidence identified the vitamin D receptor (VDR) and its natural ligand, 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3], as key regulators of cholesterol metabolism. In the liver, 1,25(OH)2D3-liganded VDR directly inhibited the small heterodimer partner (Shp) to increase expression of cholesterol 7α-hydroxylase (Cyp7a1), the rate-limiting enzyme for cholesterol metabolism to bile acids, a mechanism independent of the farnesoid X receptor. Vitamin D deficiency was established in mice after 8 weeks of the D-deficient diet, which resulted in decreased levels of plasma and liver 1,25(OH)2D3, downregulation of hepatic Vdr and Cyp7a1, and elevation of Shp. Consequently, higher plasma and liver cholesterol levels were observed. Intervention with 1,25(OH)2D3 or vitamin D3 reversed the altered expression of these cholesterol-regulating genes and lowered cholesterol levels back to baseline levels. The correlations between liver cholesterol vs. liver 1,25(OH)2D3 and Cyp7a1 expression in mice were also found in human liver tissue, suggesting that the VDR could be a ii potential therapeutic target for cholesterol lowering. However, the therapeutic utility of 1,25(OH)2D3 is limited by hypercalcemia and by its feedback control mechanisms. When given exogenously, 1,25(OH)2D3 triggered inhibition of its synthesis enzyme (Cyp27b1) but induction of its degradation enzyme (Cyp24a1) in a dose-dependent manner, events that are predicted by compartmental and physiologically-based pharmacokinetic/pharmacodynamic modeling. These models provide a foundation for predicting 1,25(OH)2D3 disposition for inter-species scaling and for exploration of alternative dosing schemes and routes of administration to describe the dynamics of 1,25(OH)2D3 in its new therapeutic roles. Overall, these findings highlight the importance of vitamin D status and the VDR as regulators of hypercholesterolemia. iii Acknowledgments First, I would like to express my gratitude and respect for my mentor, Dr. K. Sandy Pang. I am grateful for her guidance, support, and infinite contributions to this work. Thank you for always demonstrating what determination, enthusiasm, and commitment can accomplish. Your work ethic is unparalleled and truly inspirational. I am thankful to my advisory committee members, Drs. Ping Lee, Micheline Piquette-Miller, and Reinhold Vieth, for their efforts and words of wisdom. I would also like to thank our collaborators, Drs. Donald Mager, Carolyn Cummins, Geny Groothuis, and Albert Li for sharing their expertise. I am further grateful to Dr. Geny Groothuis for her hospitality and providing me with the opportunity to work in her laboratory at the University of Groningen in The Netherlands. I would like to thank all of the past and present members of the Pang lab. This work could not have been accomplished without all of your efforts, especially Edwin, Matthew, Paola, Joy, and Stacie. I am so appreciative of the support and friendships that you have provided me with both inside and outside of the lab. I would also like to acknowledge the financial support that I have received throughout the years from the Natural Sciences and Engineering Research Council of Canada, Ontario Graduate Student Scholarship, and the University of Toronto Fellowship. Finally, I would like to thank my parents for their love, patience, and support throughout the years. I am eternally grateful for the sacrifices that you have made for me. Andrew, thank you for your endless support, encouragement, and laughter. I could not have done it without you by my side every step (or running stride) of the way. iv Table of Contents Acknowledgments.......................................................................................................................... iv Table of Contents .............................................................................................................................v Abbreviations and Terms .................................................................................................................x List of Tables ............................................................................................................................... xiii List of Figures ................................................................................................................................xv List of Appendices ....................................................................................................................... xix 1 Introduction ...............................................................................................................................1 1.1 The Vitamin D Receptor (VDR) ............................................................................................1 1.1.1 Role of the VDR .....................................................................................................2 1.1.1.1 Physiological roles ...................................................................................2 1.1.1.2 VDR tissue distribution and species differences .....................................4 1.1.1.3 VDR regulation of enzymes, transporters, and nuclear receptors ...........4 1.1.1.4 Polymorphisms ........................................................................................5 1.1.2 VDR ligands............................................................................................................6 1.1.2.1 VDR ligand binding pocket ....................................................................7 1.1.2.2 1α,25-Dihydroxyvitamin D3 or 1,25(OH)2D3 ........................................7 1.1.2.3 Vitamin D analogs and alternate ligands of the VDR ............................8 1.1.3 Pharmacokinetics/pharmacodynamics of 1,25(OH)2D3 ........................................10 1.1.3.1 Pharmacokinetics of 1,25(OH)2D3 .......................................................10 1.1.3.2 Modeling ..............................................................................................11 1.2 Cholesterol and Bile Acid Homeostasis ..............................................................................13 1.2.1 Metabolic pathway and regulation ........................................................................15 1.2.2 Hypercholesterolemia ...........................................................................................16 1.2.3 Role of the VDR in cholesterol .............................................................................17 1.3 Vitamin D Deficiency ..........................................................................................................18 1.3.1 Pathogenesis and clinical associations ..................................................................19 1.3.2 Vitamin D deficiency and hypercholesterolemia ..................................................20 1.3.3 Experimental models of vitamin D deficiency and hypercholesterolemia ...........23 1.4 Significance of the VDR in cholesterol and deficiency .......................................................27 2 Statement of Purpose ..............................................................................................................28 2.1 Purpose of investigation .......................................................................................................28 2.2 Hypotheses ...........................................................................................................................29 2.3 Thesis outline .......................................................................................................................30 3 Temporal Gene Changes in Tissue 1α,25-Dihydroxyvitamin D3, Vitamin D Receptor Target Genes, and Calcium and PTH Levels After 1,25(OH)2D3 Treatment in Mice .......................31 3.1 Abstract .................................................................................................................................32 3.2 Introduction ...........................................................................................................................32 3.3 Materials and methods ..........................................................................................................35 3.3.1 Materials ...............................................................................................................35 3.3.2 Pharmacokinetic study of 1,25(OH)2D3 in mice ...................................................35 3.3.3 Plasma calcium and phosphorus analyses and PTH assay ....................................36 3.3.4 Tissue 1,25(OH)2D3 extraction and 1,25(OH)2D3 EIA .........................................36 3.3.5 Preparation of subcellular protein fractions of kidney and intestinal tissues ........37 3.3.6 Western blotting ....................................................................................................38 v 3.3.7 Quantitative real-time PCR ...................................................................................38