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Hydroxylase Gene Transcription and Bile Acid Synthesis In VITAMIN D RECEPTOR REGULATION OF CHOLESTEROL 7α- HYDROXYLASE GENE TRANSCRIPTION AND BILE ACID SYNTHESIS IN HUMAN HEPATOCYTES A dissertation submitted to Kent State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy by Shuxin Han December, 2009 Dissertation written by Shuxin Han B.S., Anhui Agriculture University, 1998 M.A., Temple University, 2004 Ph.D., Kent State University, 2009 Approved by Director, Doctoral Dissertation Committee John Y. L. Chiang , John Y. L. Chiang, Professor Members, Doctoral Dissertation Committee John R. D. Stalvey , John R. D. Stalvey, Professor James P. Hardwick , James P. Hardwick, Associate Professor Daniel L. Ely , Daniel L. Ely, Professor Yong Lu , Yong Lu, Assistant Professor Yijing Chen , Yijing Chen, Assistant Professor Accepted by Robert V. Dorman , Director, School of Biomedical Sciences John R. D. Stalvey , Dean, College of Arts and Sciences ii TABLE OF CONTENTS LIST OF TABLES............................................................................................................vi LIST OF FIGURES.........................................................................................................vii ACKNOWLEDGEMENTS..............................................................................................x CHAPTER I. INTRODUCTION.....................................................................................1 A. Bile acid synthesis and physiological functions.............................................................2 1. Bile acid synthesis...........................................................................................................2 1.1 The classical or neutral pathways..................................................................................2 1.2 The alternative or acidic pathways................................................................................6 2. Physiological functions..................................................................................................10 3. Bile acid transport..........................................................................................................11 3.1. Bile acid excretion of hepatocytes..............................................................................11 3.2. Bile acid transport in cholangiocytes..........................................................................17 3.3. Intestinal absorption and transport of bile aicds.........................................................18 3.4. Hepatic uptake of bile acids........................................................................................20 4. Cholestasis.....................................................................................................................22 4.1. Hereditary cholestatic diseases...................................................................................22 4.2. Acquired cholestatic diseases.....................................................................................24 4.2.1. Sepsis-associated cholestasis...................................................................................24 4.2.2. Drug-induced cholestasis.........................................................................................25 4.2.3. Intrahepatic cholestasis of pregnancy......................................................................25 B. Regulation of bile acid biosynthesis.............................................................................26 1. Nuclear hormone receptor regulation of bile acid synthesis.........................................27 1.1. Nuclear hormone receptor..........................................................................................28 1.1.1. FXR (NR1H4).........................................................................................................32 1.1.2. PXR (NR1I2)...........................................................................................................36 1.1.3. VDR (NR1I1)...........................................................................................................38 1.1.3.1. VDR expression....................................................................................................38 1.1.3.2. Physiological functions of VDR...........................................................................39 1.1.3.3. VDR transcription regulation................................................................................40 2. Bile acid-activated membrane signaling regulation of bile acid synthesis....................43 2.1. MAPK signaling pathways.........................................................................................44 2.1.1. The ERK1/2 pathway...............................................................................................47 2.1.2. The JNK pathway....................................................................................................50 2.1.3. The p38 pathway......................................................................................................51 2.1.4. Specificity of MAPK signaling…............................................................................52 iii 2.2. Fibroblast growth factor 15/19 (FGF15/19) signaling................................................53 2.3. Cell signaling pathways directly activated by bile acids............................................55 2.4. Bile acid-induced cytokine released by nonparechymal cells....................................57 2.5. Insulin and glucagon signals.......................................................................................59 3. Cell signaling pathways involved in VDR regulation of gene expression....................60 4. Bile acid feedforward and feedback regulation of bile acid synthesis..........................64 4.1. Feedforward regulation of CYP7A1...........................................................................64 4.2. Feedback inhibition of CYP7A1.................................................................................65 4.3. VDR and bile acid metabolism...................................................................................66 C. Hypothesis….................................................................................................................67 CHAPTER II. EXPERIMENTAL MATERIALS AND PROCEDURES..................68 A. Materials........................................................................................................................68 1. Cell culture.....................................................................................................................68 2. Reporter and expression plasmids.................................................................................69 3. Other materials...............................................................................................................70 B. Methods.........................................................................................................................71 1. Site-directed mutagensis................................................................................................71 2. Luciferase reporter assay...............................................................................................72 2.1. Calcium-phosphate-DNA co-precipitation transfection.………................................73 2.2. Lipofectamin2000 transfection……….......................................................................74 2.3. Tfx-20 transfection………..........................................................................................74 2.4. Luciferase and β-gal assays........................................................................................75 3. Mammalian one and two-hybrid assays........................................................................76 4. Quantitative real-time PCR...........................................................................................76 4.1. RNA isolation.............................................................................................................81 4.2. Reverse transcription PCR..........................................................................................82 4.3. Real-time PCR............................................................................................................82 5. Electrophoretic mobility shift assay (EMSA)................................................................84 6. Chromatin immunoprecipitation (ChIP) assay..............................................................85 7. Protein extraction and immunoblotting analysis............................................................88 8. Immunoprecipitation (IP) assay.....................................................................................89 9. Co-immunoprecipitation (Co-IP) assay.........................................................................90 10. In vitro ERK1/2 kinase assay.......................................................................................91 11. GST pull-down assay...................................................................................................94 11.1. GST fusion protein expression..................................................................................97 11.2. GST pull-down experiment.......................................................................................97 12. Bile acid assay..............................................................................................................98 13. Small RNA interference assay.....................................................................................99
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