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Degree

PHD, Kent State University, School of Biomedical Sciences, 2006.

Abstract

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 7alpha 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 HNF4alpha, the key activator of human CYP7A1, and blocked HNF4alpha interaction with co-activator PGC-1alpha, 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 HNF4alpha, SRC-1 and PGC-1alpha. SHP, a negative nuclear receptor, reduced PXR recruitment of HNF4alpha 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 LXRalpha ligand and the PXR/CYP27A1/LXRalpha 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.

Keywords

PXR; BILE; BILE ACID; HNF4α; CYP7A1; CHOLESTEROL; rifampicin

Advisor

John Y.L. Chiang

Document number: kent1132160196
Permalink: http://rave.ohiolink.edu/etdc/view?acc_num=kent1132160196

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