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(Cyp8b1) Gene; an Alternative Mechanism for Bile Acid

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(Cyp8b1) Gene; an Alternative Mechanism for Bile Acid BHATT, ASMEEN, Ph.D., May, 2006 BIOMEDICAL SCIENCES CYTOKINE REPRESSION OF THE HUMAN STEROL 12α-HYDROXYLASE (CYP8B1) GENE; AN ALTERNATIVE MECHANISM FOR BILE ACID SUPPRESSION OF CYP8B1 (228 pp.) Director of Dissertation: John Y. L. Chiang, Ph.D. Bile acids, synthesized from precursor cholesterol molecules, serve as an important mechanism for elimination of excess cholesterol from the body. Bile acid synthesis is tightly regulated since excess bile acids are toxic and can cause liver damage. Bile acids inhibit their own synthesis by inhibiting the bile acid biosynthetic genes. Cholesterol 7α- hydroxylase (CYP7A1) is the rate-limiting enzyme of classic bile acid biosynthesis, which synthesizes two primary bile acids, cholic acid (CA) and chenodeoxycholic acid (CDCA) in the liver. Sterol 12α-hydroxylase (CYP8B1) catalyzes CA synthesis in the liver and is feedback inhibited by bile acids. In addition to activating farnesoid X receptor (FXR, nuclear receptor subfamily 1H4, NR1H4), bile acids also induce the release of inflammatory cytokines, like interleukin 1β (IL-1β) from Kupffer cells in the liver. The objective of this study was to investigate the mechanism by which inflammatory cytokines inhibit human CYP8B1 gene transcription. Real time PCR assays revealed that both CDCA and IL-1β markedly reduced CYP8B1, CYP7A1, and nuclear receptor hepatocyte nuclear factor 4α (HNF4α, NR2A1) mRNA expression levels in human primary hepatocytes. However, CDCA induced, but IL-1β reduced the negative nuclear receptor, small heterodimer partner (SHP, NR0B2) mRNA expression. IL-1β inhibited human CYP8B1 reporter activity only in liver cells, and a c-Jun N-terminus kinase (JNK)-specific inhibitor blocked IL-1β inhibition. Activated JNK1 or c-Jun inhibited, whereas their dominant negative forms blocked IL-1β inhibition of CYP8B1 transcription. Mutagenesis analyses mapped an IL-1β response element to a previously identified bile acid response element, which contains an HNF4α binding site. Furthermore, IL-1β inhibited HNF4α gene transcription, protein expression and binding to the CYP8B1 gene. JNK1 phosphorylated HNF4α and a JNK-specific inhibitor blocked the IL-1β inhibition of HNF4α expression. Expression of c-Jun, a downstream target of the JNK pathway, was induced by both IL-1β and CDCA in primary human hepatocytes. c-Jun inhibited the HNF4α and coactivator peroxisome proliferator-activated receptor γ co-activator-1α (PGC-1α) mediated trans-activation of CYP8B1 reporter activity. Co- immunoprecipitation revealed an interaction between c-Jun and HNF4α, which was confirmed by GST pull down assay. IL-1β increased the ratio of c-Jun or phosphorylated c-Jun bound to HNF4α in HepG2 cells. Chromatin immunoprecipitation (ChIP) assay revealed that c-Jun did not affect HNF4α binding but blocked HNF4α recruitment of PGC-1α to the CYP8B1 chromatin. This study also showed that HNF4α stimulated the gene expression of SHP by binding to the promoter of the latter. The results suggest that IL-1β inhibits CYP8B1 gene transcription, in a SHP-independent manner via the JNK pathway that inhibits HNF4α gene expression and its DNA-binding ability. IL-1β also induces c-Jun, which blocks HNF4α recruitment of PGC-1α to CYP8B1 chromatin. This mechanism may play an important role in the adaptive response to inflammatory cytokines and in the protection of the liver during cholestasis. CYTOKINE REPRESSION OF THE HUMAN STEROL 12α-HYDROXYLASE (CYP8B1) GENE; AN ALTERNATIVE MECHANISM FOR BILE ACID SUPPRESSION OF CYP8B1 A dissertation submitted to Kent State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy By Asmeen Bhatt May, 2006 Dissertation written by Asmeen Bhatt M.B.B.S., University of Mumbai, 2000 Ph.D. Kent State University, 2006 Approved by Chair, Doctoral Dissertation Committee John Chiang John Y. L. Chiang, Professor Members, Doctoral Dissertation Committee Philip Westerman Philip W. Westerman, Professor James Hardwick James P. Hardwick, Associate Professor Hans Folkesson Hans G. Folkesson, Associate Professor Kathleen Doane Kathleen J. Doane, Associate Professor Accepted by Robert Dorman Director, School of Biomedical Sciences John Stalvey Dean, College of Arts and Sciences ii TABLE OF CONTENTS LIST OF FIGURES.......................................................................................................... v LIST OF TABLES.......................................................................................................... vii ACKNOWLEDGEMENTS .......................................................................................... viii CHAPTER I: INTRODUCTION.................................................................................... 1 1. Bile Acids.................................................................................................................... 1 a. Structure .................................................................................................................. 2 b. Function .................................................................................................................. 5 c. Synthesis ................................................................................................................. 5 d. Mutations and deficiencies of important enzymes in bile acid biosynthesis.......... 9 e. Enterohepatic circulation of bile ........................................................................... 13 2. Cholesterol Homeostasis in the liver ........................................................................ 19 3. Nuclear Hormone Receptors..................................................................................... 23 a. Structure ................................................................................................................ 26 b. Response Elements ............................................................................................... 26 c. Ligands.................................................................................................................. 27 4. Important Nuclear Receptors involved in regulation of bile acid biosynthesis........ 28 a. Farnesoid X Receptor (FXR, NR1H4).................................................................. 28 b. Pregnane X Receptor (PXR, NR1I2).................................................................... 29 c. Vitamin D3 Receptor (VDR, NR1I1) ................................................................... 30 d. Hepatocyte nuclear factor 4α (HNF4α, NR2Α1) ................................................. 31 e. Human α-fetoprotein transcription factor (hFTF, NR5A2) .................................. 32 f. Small Heterodimer Partner (SHP, NR0B2)........................................................... 33 g. Peroxisome Proliferator Activated Receptor α (PPARα, NR1C1) ...................... 34 h. Liver X Receptor (LXR, NR1H3) ........................................................................ 35 5. Important Transcription factors and Coactivators involved in regulation of bile acid biosynthesis................................................................................................................... 36 a. Sterol Regulatory Element Binding Proteins (SREBPs)....................................... 36 b. c-Jun...................................................................................................................... 38 c. Peroxisome Proliferator-Activated Receptor-γ Coactivator 1α (PGC-1α):.......... 39 6. Regulation of Bile Acid Biosynthesis....................................................................... 40 a. Feedforward Regulation by Cholesterol. .............................................................. 41 b. Feedback Regulation by Bile Acids...................................................................... 41 7. Regulation of CYP8B1 ............................................................................................. 47 8. Mitogen Activated Protein Kinase (MAPK) Pathways ............................................ 50 9. Cholestatic liver disease............................................................................................ 54 10. Proinflammatory cytokine mediated gene regulation in the liver........................... 56 iii 11. Hypothesis, Specific aims, Approaches and Significance...................................... 60 Chapter II: MATERIALS AND METHODS .............................................................. 64 1. Cell Culture............................................................................................................... 64 Cell line culture:........................................................................................................ 64 Primary cell culture:.................................................................................................. 65 2. Plasmids .................................................................................................................... 65 Reporters:.................................................................................................................. 66 Expression Vectors: .................................................................................................. 66 3. Preparation of Competent Cells................................................................................ 67 4. Bacterial Cell Transformation................................................................................... 68 5. Preparation of Plasmid DNA ...................................................................................
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