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Chapter I Introduction LI, TIANGANG, Ph.D., May, 2006 BIOMEDICAL SCIENCES PREGNANE X RECEPTOR REGULATION OF BILE ACID METABOLISM AND CHOLESTEROL HOMEOSTASIS (189 PP.) Director of Dissertation: John Y. L. Chiang, Ph.D The nuclear receptor pregnane X receptor (PXR) is activated by bile acids, steroids and drugs and regulates a network of genes in lipid and drug mechanisms. The goal of this study is to investigate the role of PXR in the coordinate regulation of bile acid synthetic and detoxification genes and its implications in cholestatic liver diseases and treatments. Cholesterol 7α hydroxylase (CYP7A1) catalyzes the rate-limiting step in the classic bile acids synthetic pathway and plays a key role in controlling bile acids homeostasis. Quantitative real-time PCR (Q-PCR) showed human PXR agonist rifampicin inhibited CYP7A1 mRNA expression in primary human hepatocytes. Mammalian two-hybrid assays, co-immunoprecipitation (co-IP) assays and chromatin immunoprecipitation (ChIP) assay revealed that ligand-activated PXR strongly interacted with HNF4α, the key activator of human CYP7A1, and blocked HNF4α interaction with co-activator PGC-1α, thus resulted in inhibition of CYP7A1. CYP3A4 is the most abundant cytochrome P450 monooxygenase expressed in human liver and intestine. Bile acids and drugs-activated PXR induces CYP3A4, which converts toxic bile acids to non-toxic metabolites for excretion. Studies using Q-PCR, reporter assays, GST pull-down assays and ChIP assays revealed that PXR strongly induced CYP3A4 gene transcription by interacting with HNF4α, SRC-1 and PGC-1α. SHP, a negative nuclear receptor, reduced PXR recruitment of HNF4α and SRC-1 to the CYP3A4 chromatin and inhibited CYP3A4. Interestingly, PXR concomitantly inhibited SHP gene transcription and maximized the PXR induction of CYP3A4. Taken together, PXR inhibits CYP7A1 to reduce bile acid synthesis and induces CYP3A4 to detoxify bile acid. Thus, PXR may play a protective role against cholestasis. Drugs targeted to PXR may be developed for treating cholestatic liver diseases induced by bile acids and drugs. Mitochondrial sterol 27-hydroxylase (CYP27A1) catalyzes the side-chain cleavage reaction in bile acid synthetic pathways and 27-hydroxylation of cholesterol mainly in the peripheral tissues. Q-PCR revealed that rifampicin induced CYP27A1 mRNA expression in the intestine-derived Caco2 cells, but not in primary human hepatocytes and HepG2 cells. Rifampicin stimulated CYP27A1 gene transcription in cholesterol laden Caco2 cells and increased intracellular 27HOC, which stimulates cholesterol efflux by induction of cholesterol efflux transporters ABCA1 and ABCG1. Mutagenesis analysis, electrophoretic mobility shift assay and ChIP assays identified a functional PXR binding site in the human CYP27A1 gene. These data suggest that 27-hydroxycholesterol is an endogenous LXRα ligand and the PXR/CYP27A1/LXRα signaling pathway regulate cholesterol efflux in intestine cells. Results revealed an intestine-specific regulation of human CYP27A1 gene by PXR and suggested a novel role for PXR in cholesterol metabolism and detoxification in the intestine. PREGNANE X RECEPTOR REGULATION OF BILE ACID METABOLISM AND CHOLESTEROL HOMEOSTASIS A dissertation submitted to Kent State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy by Tiangang Li May, 2006 Dissertation written by Tiangang Li B.S., Jilin University, 1999 Ph.D., Kent State University, 2006 Approved by Director, Doctoral Dissertation Committee , John Y. L. Chiang, Professor Members, Doctoral Dissertation Committee ________________________________, Amy Milsted, Professor ________________________________, Dean E. Dluzen, Professor ________________________________, James P. Hardwick, Associate Professor ________________________________, John R. D. Stalvey, Professor ________________________________, Philip W. Westerman, Professor Accepted by ________________________________, Director, School of Biomedical Sciences ________________________________, Dean, College of Arts and Sciences TABLE OF CONTENTS LIST OF FIGURES..........................................................................................................v ACKNOWLEDGEMENTS ...........................................................................................vii CHAPTER I. INTRODUCTION .................................................................................... 1 A. BILE ACID BIOSYNTHESIS ............................................................................................ 1 1. Chemistry................................................................................................................ 1 2. Physiological function ............................................................................................ 5 3. Bile acid biosynthesis pathways ............................................................................. 5 3.1. The classic or neutral pathway......................................................................... 8 3.2. Acidic or alternative pathway .......................................................................... 8 4. Bile acid transport................................................................................................... 9 4.1. Enterohepatic circulation ................................................................................. 9 4.2. Hepatic bile acid transport ............................................................................. 12 4.2.1. hepatic bile acid uptake........................................................................... 12 4.2.2. canalicular bile acid excretion ................................................................ 13 4.3. Intestinal bile acid transport........................................................................... 14 4.3.1. apical bile acid uptake into the enterocytes ............................................ 14 4.3.2. basolateral efflux of bile acids from enterocytes .................................... 15 5. Cholestasis ............................................................................................................ 16 B. CHOLESTEROL HOMEOSTASIS .................................................................................... 18 C. REGULATION OF BILE ACID BIOSYNTHESIS AND CHOLESTEROL HOMEOSTASIS........... 22 1. Nuclear hormone receptor..................................................................................... 22 2. Regulation of bile acid synthesis .......................................................................... 28 2.1. Feedforward regulation of CYP7A1............................................................... 29 2.2. Feedback inhibition of CYP7A1..................................................................... 32 2.2.1. FXR/SHP dependent pathway ................................................................ 32 2.2.2. FXR/SHP independent pathways............................................................ 35 3. PXR and bile acid metabolism.............................................................................. 37 3.1. The nuclear receptor PXR.............................................................................. 37 3.2. PXR and xenobiotic metabolism ................................................................... 38 3.3. PXR and bile acid metabolism....................................................................... 42 4. LXR and cholesterol homeostasis......................................................................... 43 D. HYPOTHESIS.............................................................................................................. 45 CHAPTER II. EXPERIMENTAL PROCEDURES.................................................... 49 A. CELL CULTURE .......................................................................................................... 49 B. LUCIFERASE REPORTER ASSAY .................................................................................. 49 1. Calcium-phosphate-DNA co-precipitation transfection method .......................... 50 2. Lipofectamin2000 transfection method ................................................................ 51 3. Luciferase and β-gal assays .................................................................................. 52 C. MAMMALIAN HYBRID ASSAYS................................................................................... 52 D. SITE-DIRECTED MUTAGENESIS................................................................................... 55 E. QUANTITATIVE REAL-TIME PCR................................................................................ 56 1. RNA isolation ....................................................................................................... 56 2. Reverse transcription PCR.................................................................................... 57 3. Real time PCR....................................................................................................... 57 F. ELECTROPHORETIC MOBILITY SHIFT ASSAY (EMSA)................................................. 58 G. CHROMATIN IMMUNOPRECIPITATION (CHIP) ASSAY ................................................. 60 H. WESTERN IMMUNOBLOTTING ANALYSIS ................................................................... 63 I. CO-IMMUNOPRECIPITATION ASSAY............................................................................. 64 J. GST PULL-DOWN ASSAY............................................................................................. 65 1. GST fusion protein expression.............................................................................
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