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Srebp2 and Reverb-Alpha Regulation of Human Cyp8b1 SREBP2 AND REVERB-ALPHA REGULATION OF HUMAN CYP8B1 A thesis submitted to Kent State University in partial fulfillment of the requirements for the Degree of Master of Sciences By Hailin Feng December, 2009 Thesis written by Hailin Feng B.S. Guangzhou University, 1992 M.S. Guangzhou Medical College, 1995 M.S. Kent State University, 2009 Approved by ________________________________, Advisor Dr. John Y. Chiang ________________________________, Director, School of Biomedical Science Dr. Robert V. Dorman ________________________________, Dean, College of Arts and Sciences Dr. John R.D. Stalvey ii TABLE OF CONTENTS LIST OF FIGURES………………………………………………………………….vi ACKNOWLEGEMENTS……………………………………………………………viii CHAPTER I INTRODUCTION…………………………………………………………….1 1. Bile Acids………………………………………………………………..…2 1.1 Structure and function……………………………………………………..2 1.2 Synthesis pathways and enzymes………………………………………….5 1.2.1 Classic pathway………………………………………………………….5 1.2.2 Acidic pathway………………………………………………………….6 1.3 Regulation of bile acid synthesis…………………………………………..7 1.3.1 Cholesterol homeostasis…………………………………………………7 1.3.2 Enterohepatic circulation of bile………………………………………..10 2. Nuclear Hormone Receptor…………………………………………….…..10 2.1 Structure………………………………………………………………......10 2.2 Response elements………………………………………………………..15 2.3 Ligands……………………………………………………………….…...16 3. Hormone Receptors Related to Bile Acid Synthesis………………….……16 3.1 Hepatocyte nuclear factor 4α (HNF4α, NR2A1)……………………..…..16 3.2 Farnesoid receptor (FXR, NR1H4)……………………………………….17 iii 3.3 Human α-fetoprotein transcription factor (CPF, NR5A2)………………..18 3.4 Reverb-α (NR1D1) …………………………………………………….....19 3.5 Small heterodimer partner (SHP, NR2A1)……………………………..…21 4. Transcription factors, co-activator and corepressor related to bile acid synthesis……………………………………………………………………....22 4.1 Sterol regulatory element binding proteins (SREBPs)……………………22 4.2 Peroxisome proliferator-activated receptor-γ co-activator-1α (PGC-1α)………………………………………………………………….…..24 4.3 Member of PAS super family 3 (BMAL, or MOP3)……………………...27 4.4 N-COR/histone deacetylase 3 corepressor (N-COR3)…………………....28 5. Regulation of CYP8B1 Expression…………………………………………28 6. Hypothesis, Specific aims, Approaches and Significance…………………..33 II Materials and Methods…………………………………………………….…..37 1. Cell Culture………………………………………………………………....37 1.1 Human hepatoma cell line culture…………………………………………37 1.2 Primary human hepatocyte culture………………………………………..38 2. Plasmids DNA Preparation…………………………………………………38 2.1 Large scale DNA preparation………………………………………….….38 2.2 Small scale DNA preparation……………………………………………..39 3. Preparation of Competent Cells…………………………………………….40 4. Bacterial Cell Transformation…………………………………………..….41 5. Transient Transfection Assay……………………………………...………..41 iv 5.1) Luciferase assay…………………………………………………………..42 5.2) β-Galactosidase activity assay………………………………………..…..42 6. Mammalian One-Hybrid Assay………………………………………….…43 7. Site-Directed Mutagenesis………………………………………………….44 8. Quantitative Teal-Time PCR……………………………………………….44 8.1) RNA isolation…………………………………………………………….44 8.2) Reverse transcription PCR……………………………………………….45 8.3 Rea-Time PCR……………………………………………………...……..46 III Results…………………………………………………………………….…..48 IV Discussion………………………………………………………………….…74 APPENDIX ABBREVIATIONS……………………………………………….……79 REFERENCES………………………………………………………………….……84 v LIST OF FIGURES Figure 1. Chemical structure of cholesterol and bile acids………………………..……..3 Figure 2.Major pathways of bile acid synthesis…………………………………….…...9 Figure 3. Enter hepatic circulation of bile acid salts……………………………….……11 Figure 4. General structure of nuclear hormone receptors and types of hormone response element (HRE)………………………………………………………………….….……13 Figure 5. Model of SREBPs regulating genes related to cholesterol homeostasis….…..25 Figure 6. The partial nucleotide sequences of 5’-flanking region of the human CYP8B1…………………………………………………………………………..…….31 Figure7. Effects of SREBP2 on human CYP8B1 reporter activity………………...…..50 Figure 8. Mapping the response element of SREBP2 on the human CYP8B1 gene…..52 Figure9. Effect of HNF4α and CPF binding site mutation on the SREBP2 inhibitory effect on human CYP8B1 luciferase reporter activity…………………………...……..54 Figure10. Mammalian one-hybrid assay………………………………………..………56 Figure11 SREBP2 influences the co-activation effect of HNF4α and PGC-1α on human CYP8B1 reporter activity……………………………………………………..………...60 Figure.12 Dose-dependent effects of REVERB-α on human CYP8B1 reporter………62 Figure. 13 Mapping the response element……………………………………..….……64 vi Figure. 14 Effect of Reverb-α binding site mutation on human CYP8B1 luciferase reporter activity………………………………………………………..…………….…..66 Figure. 15. Effects of hemin on human CYP8B1 reporter activity in HepG2 cells….....68 Figure. 16. Time course of hemin effects on human CYP8B1 mRNA level…………...70 Figure.17. Human CYP8B1 mRNA level after depleting heme with succinylacetone…72 vii ACKNOWLEGEMENTS I would like to thank my advisor Dr. John Y. Chiang, for his great guiding, valuable advice and strong support. I also thank my committee members: Dr. Hardwick and Dr. Lee for their generous and critical comments on my thesis. I am grateful to all the members in Dr. Chiang’s lab for their help. viii CHARPTER I INTRODUCTION Cholesterol is the precursor molecule of primary bile acids including chenodeoxycholic acid (CDCA) and cholic acid (CA). Several enzymes are involved in the synthesis of primary bile acids. For example, cholesterol 7alpha-hydroxylase (CYP7A1) is the rate- limiting enzyme in classic bile acid biosynthesis. Sterol 12-hydroxylase (CYP8B1) is a liver specific enzyme that catalyzes the synthesis of CA. Importantly, CYP8B1 determines the ratio of CA to CDCA in bile. Since CA is more hydrophilic than CDCA, CYP8B1 may determine the hydrophobicity of the bile acid pool, which in turn regulates bile acid synthesis [1]. In addition, CYP8B1 is crucial for cholesterol absorption in the intestine; therefore, this enzyme is important for cholesterol homeostasis [2, 3]. The expression of CYP8B1 is regulated by bile acids, cholesterol, insulin and diurnal rhythm, mainly at the gene transcriptional levels [3-6]. In animal models, transcription factors, such as FXR, HNF4α, SREBPs, FTF, clock gene DBP, have been identified to be involved in the transcriptional regulation of CYP8B1 gene expression [7-10]. But in human liver cell, it is not known whether CYP8B1 is transcriptionally regulated by SREBPs and clock gene Reverb-α. The objective of this study is to identify whether human CYP8B1 promoter activity can be regulated by SREBP2 and Reverb-α, and to reveal the possible mechanisms underlying the transcriptional regulation of human 1 2 CYP8B1. The significance of this project is to provide important clues for the treatment of metabolism disease related to CYP8B1 activity, especially in cholesterol metabolism disease in human. 1. Bile Acids 1.1 Structure and function Bile acids are the end products of cholesterol catabolism in the liver. Their common structure includes a saturated sterol nucleus and an aliphatic side chain. Bile acids are planar amphipaths. They are rigid molecules with a hydrophilic side and hydrophobic side (Fig. 1). In human, bile acids usually are classified into primary and secondary bile acids. In the human liver, cholesterol is converted into primary bile acid: cholic acid (CA) and chenodeoxycholic acid (CDCA). While CA has 3 hydroxyl groups (3α, 7α and 12α), CDCA only has two hydroxyl groups (3α, 7α). Accordingly, CA is more hydrophilic than CDCA. In the intestine, bacteria enzymes remove the hydroxyl group from the position 7 of the sterol, which converts the CA and CDCA into deoxycholic acid (DCA) and lithocholic acid (LCA), respectively. The amphipathic structure of bile acids makes them as excellent detergents for lipid absorption and transportation. Therefore, bile acids act as physiological detergent to solubilize many lipids. In physiology conditions, bile acids are present as sodium salts, and are conjugated with taurine or glycine [11-13, 126-127]. 3 Fig. 1 4 Fig. 1. Chemical structure of cholesterol and bile acids. In the human liver, cholesterol is converted into two major primary bile acids: cholic acid and chenodeoxycholic acid; in intestine, cholic acid and chenodeoxycholic acid are converted into secondary bile acids: deoxycholica acid and lithocholic acid, respectively. 5 1.2 Synthesis pathways and enzymes In the liver, there are two major bile acid synthesis pathways. One is the “classic or neutral pathway”; another is “acidic or alternative pathway” [11-14]. The conversion of cholesterol to bile acids includes about 15 steps (Fig. 2). Briefly, the ring structure of cholesterol is hydroxylated and oxidized by multiple steps, and the side chain is shortened. These complicate chemical reactions involve different enzymes that are located in endoplasmic reticulum, mitochondria, cytosol and peroxisome of the cell. In the classic pathway, cholesterol 7α-hydroxylase (CYP7A1) is the rate-limiting enzyme located in the microsome of the liver cell. However, the acidic pathway is initiated by mitochondrial sterol 27-hydroxylase (CYP27A1). The classic pathway produces about 80% of bile acids in human body. In contrast, less than 18% of bile acids in the liver are produced through the acidic pathways. 1.2.1 Classic pathway As illustrated in Fig. 2, rate-limiting enzyme CYP7A1 converts cholesterol into 7α hydroxycholesterol. Then, 3β-hydroxy-27-steroid dehydrogenase (3β-HSD), an isomerase in microsome, catalyzes 7α-hydroxycholesterol into
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