University of Kentucky UKnowledge Theses and Dissertations--Pharmacy College of Pharmacy 2013 Regulation of 7-Dehydrocholesterol Reductase by Vitamin D3 Ling Zou University of Kentucky, [email protected] Right click to open a feedback form in a new tab to let us know how this document benefits ou.y Recommended Citation Zou, Ling, "Regulation of 7-Dehydrocholesterol Reductase by Vitamin D3" (2013). Theses and Dissertations--Pharmacy. 28. https://uknowledge.uky.edu/pharmacy_etds/28 This Doctoral Dissertation is brought to you for free and open access by the College of Pharmacy at UKnowledge. It has been accepted for inclusion in Theses and Dissertations--Pharmacy by an authorized administrator of UKnowledge. For more information, please contact [email protected]. STUDENT AGREEMENT: I represent that my thesis or dissertation and abstract are my original work. Proper attribution has been given to all outside sources. 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Ling Zou, Student Dr. Todd Porter, Major Professor Dr. Jim Pauly, Director of Graduate Studies REGULATION OF 7-DEHYDROCHOLESTEROL REDUCTASE BY VITAMIN D3 DISSERTATION A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the College of Pharmacy at the University of Kentucky By Ling Zou Lexington, Kentucky Director: Dr. Todd D Porter, Professor of Pharmaceutical Sciences Lexington, Kentucky 2013 Copyright © Ling Zou 2013 ABSTRACT OF DISSERTATION Regulation of 7-Dehydrocholesterol Reductase by Vitamin D3 7-Dehydrocholesterol (7-DHC) is the substrate of 7-dehydrocholesterol reductase (DHCR7) in the cholesterol synthesis pathway. Keratinocytes in human skin possess the enzymes necessary for cholesterol synthesis but are also responsible for vitamin D3 synthesis from 7-DHC by exposure to UVB irradiation. It has been well established that DHCR7 is regulated by the SREBP pathway in the regulation of cholesterol synthesis, but little is known about the regulation of DHCR7 by the vitamin D pathway. In this study, the regulation of DHCR7 activity by vitamin D was explored. Treatment of adult human epidermal keratinocyte (HEKa) cells with vitamin D3 resulted in a rapid decrease in DHCR7 activity which was not due to changes in the amount of enzyme present. This suppression of activity was observed only in HEKa cells, a primary cell line cultured from normal human skin, and not in an immortalized skin cell line (HaCaT cells) nor in two liver-derived hepatoma cell lines. Because vitamin D3 treatment of HEKa cells did not change the content of lanosterol nor 7-DHC, these results suggest that vitamin D3 rapidly down-regulates the entire cholesterolgenesis pathway, presumably at a very early step in the pathway. 25-Hydroxyvitamin D3, the first metabolite and circulating form of vitamin D3, had a lesser effect on DHCR7 activity, while 1,25-dihydroxyvitamin D3, the activated form of the vitamin, had no effect on DHCR7, indicating that the vitamin D receptor is not involved. The decrease in DHCR7 activity was due neither to the dephosphorylation of the enzyme, an established mechanism of inactivation, nor to direct inhibition by vitamin D3. Vitamin D3 markedly inhibited proliferation and induced differentiation of HEKa cells, suggesting a possible role for hedgehog signaling in the decrease in DHCR7 activity. KEYWORDS: Cholesterol, 7-Dehydrocholesterol reductase, Vitamin D3, HEKa, Hedgehog signaling Ling Zou Student’s Signature December 2, 2013 Date REGULATION OF 7-DEHYDROCHOLESTEROL REDUCTASE BY VITAMIN D3 By Ling Zou Todd Porter Director of Dissertation Jim Pauly Director of Graduate Studies December 2, 2013 Date To Wenlong and Lucy ACKNOWLEDGMENTS The following dissertation benefited from the direction of several people. First of all, I would like to thank my mentor, Dr. Todd Porter, for his guidance, inspiration, and understanding throughout my graduate studies. I am deeply grateful to him for giving me this precious opportunity in research and encouraging me all through these several years. I would like to acknowledge Dr. Charles Loftin, Dr. Gregory Graf, Dr. Hollie Swanson for all the valuable advices that helped me overcoming difficulties and making progress in my research. I would also like to thank Dr. Austin Cantor for agreeing to my outside examiner for my dissertation defense. In addition, I would like to thank my colleagues for their technical assistance and support. Finally, I would like to thank my family and friends for their generous support and great joy during my doctoral studies. iii TABLE OF CONTENTS Acknowledgments……………………………………………………………………....iii List of Tables…………………………………………………………………...………viii List of Figures…………………………………………………………………….……..ix Chapter One: Introduction…………………………………………………………...…1 1.1 Structure and function of human skin………………………………………...1 1.1.1 Cholesterol function……………………………………………..………1 1.1.2 Cholesterol homeostasis………………………………………….……2 1.1.2.1 Cholesterol biosynthesis, uptake, storage and metabolism..3 1.1.2.2 Regulation of cholesterol homeostasis………………….……5 1.2 Cholesterol function and homeostasis………………………………………10 1.2.1 Function of mammalian skin………………………………………….10 1.2.2 Structure of mammalian skin…………………………………………10 1.2.3 Regulation of epidermal lipogenesis…………………………………12 1.3 Vitamin D production, metabolism and function in human skin…………..15 1.3.1 Function of vitamin D in calcium homeostasis……………………...15 1.3.2 Regulation of vitamin D production and metabolism in mammalian skin………………………………………………………………………16 1.3.2.1 Regulation of vitamin D production in mammalian skin……16 1.3.2.2 Metabolism of vitamin D3 to its biologically active products.17 1.3.3 The role of vitamin D in skin cancer…………………………………18 iv 1.3.3.1 Vitamin D inhibits proliferation and promotes differentiation of keratinocytes………………………………………………...19 1.3.3.2 Vitamin D inhibits hedgehog signaling pathway in keratinocytes…………………………………………………...20 1.3.3.2.1 Hedgehog signaling pathway…………………………….20 1.3.3.2.2 Hedgehog signaling in skin cancer………………………21 1.3.3.2.3 Potential roles of vitamin D3 and 7-dehydrocholesterol reductase in hedgehog signaling………………………...22 1.4 The role of 7-dehydrocholesterol reductase in vitamin D deficiency…….24 1.4.1 The biochemical characteristics of human 7-dehydrocholesterol reductase……………………………………………………………….24 1.4.2 The genetic variations of 7-dehydrocholesterol reductase in vitamin D deficiency…………………………………………………………….26 Chapter Two: Experimental Rationale.................................................................27 Experimental rationale………………………………………………………...…..27 Specific aims………………………………………………………………….……29 Chapter Three: Materials and Methods...............................................................31 List of Chemicals, Buffers, and Reagents………………………………………31 Cell Culture…………………………………………………………………………34 Activity Assays……………………………………………………………………..35 GC/MS Sample Analysis by Wsearch32………………………………………..36 v UVB Irradiation……………………………………………………………………..37 Gel Electrophoresis and immunoblotting………………………………………..38 RT-PCR……………………………………………………………………………..38 Chapter Four: Results……………………………………………………………… 4.1 Vitamin D3 decreases DHCR7 activity in adult human epidermal keratinocytes……………………………………………………………………….41 4.2 Vitamin D3 has no effect on DHCR7 activities in McA-RH7777, HepG2, and HaCaT cells…………………………………………………………………...45 4.3 Effects of 25-hydroxyvitamin D3 and 1α,25-dihydroxyvitamin D3 on DHCR7 activity in HEKa and HaCaT cells………………………………………………………………………………….48 4.4 Vitamin D3 does not change lanosterol and 7-dehydrocholesterol contents in HEKa cells……………………………………………………………………….52 4.5 No effect of UVB irradiation on DHCR7 activity……………………………………………………………………………….54 4.6 Regulation of DHCR7 activity by vitamin D3 is not due to phosphorylation/dephosphorylation…………………………………………..….55 4.7 Regulation of DHCR7 activity by vitamin D3 is not due to direct binding of vitamin D3 to DHCR7 in HEKa cells……………………………………………..58 vi 4.8 Vitamin D3 inhibits proliferation and induces differentiation of HEKa cells………………………………………………………………………………….60 4.9 Modest effect of cyclopamine on DHCR7 activity………………………….64 Chapter Five: Summary of Results…………………………………………………..66 Chapter Six: Discussion………………………………………………………………71 References……………………………………………………………………………..77 Vita………………………………………………………………………………………82