We examined the effects of genetic polymorphisms of CYP2C9 (an important drug metabolizing enzyme (CYP2C9) and polymorphisms in the vitamin K epoxide reductase complex 1 on the dose of warfarin required to control cardiovascular (thromboembolytic) disease and risk for life-threatening side effects in a large prospective epidemiology study of 302 Caucasian Americans and 273 African-American patients treated with warfarin in over a 2 year period, in a collaboration with Limdi et al (University of Alabama). We developed new rapid pyrosequencing genotyping tests for known or suspected defective alleles of human CYP2C9 alleles (*2, *3, *5, *6, 10, and 11 variants) which occur in Caucasians and African Americans. Individuals with aberrant CYP2C9 alleles required lower doses of warfarin. CYP2C9 and VKORC1 accounted for 30% and 18% of the variability of required warfarin dose in American Caucasians and 10% in African-Americans. We also identified a new coding allele of CYP2C9 (Asp360Glu) in an African-American hospitalized for life-threatening hemorrhage (1). In other studies exposure of human liver cell lines and primary human hepatocytes to clinical drugs, herbal remedies and other xenobiotics has been shown to transcriptionally upregulate the CYP2C subfamily (CYP2C8, 2C9, and 2C19) of enzymes increasing metabolism of drugs by these enzymes. This data is consistent with invivo studies in humans. This could produce tolerance to drugs or drug-drug interactions complicating patient therapy. In liver and intestine the CYP2Cs can be increased >2-8 fold by prior administration of drugs. We have then shown that the promoter regions of the human CYP2C genes are regulated by elements which bind the nuclear receptors CAR (constitutive androstane receptor), PXR (pregnane X receptor), retinoid related orphan receptors (RORs) and liver-enriched receptors such as HNF4 alpha. Drugs such as rifampicin, St Johns Wort, phenobarbital and other xenobiotics interact with CAR and PXR increasing activation. Moreover HNF4 alpha sites in the promoter enhance inducibility by CAR or PXR, and HNF4 and CAR as well as PXR act synergistically to increase CYP2C9 in liver cell lines. We have published data indicating that the coactivator NCOA6 may be involved in forming a bridge between the distal CAR site and the proximal HNF4 site in the CYP2C9 promoter. We recently reviewed all these studies available on up regulation of the CYP2C enzymes by various nuclear receptors including CAR, PXR, HNF4alpha, and RORs, most of which was done in our laboratory (3). We are presently finding that NCOA6 also bridges between PXR and HNF4, producing synergistic activation in the presence of PXR ligand. Recently we have shown in cultured primary human hepatocytes (a surrogate for human liver) that binding sites for the liver-enriched receptor HNF4 alpha are necessary for the up regulation of the CYP2C8 and CYP2C9 promoter of the CYP2Cs genes. Mutation of these sites prevents induction by the PXR agonist rifampicin. Mutation of either the PXR sites or HNF4 sites abolishes induction. siRNA to HNF4 alpha almost abolishes induction of CYP2C8, 2C9 or 2C19 mRNA in primary human hepatocytes which are the best in vitro model we have for human liver (4). In other studies, Cyp2c55, a murine Cyp2c, has been found to metabolize the endogenous substance arachidonic to 19-hydroxyeicosatranenoic acid. Cyp2c55 is induced by the CAR and PXR agonists phenobarbital and PCN-a specific murine PXR agonist. This induction by these two agonists occurs in CAR+/+ and PXR+/+, but not knockout mice and could be important in tumor promotion (6). Because of the importance of the liver enriched receptor HNF4alpha in induction of drug metabolism and various physiological disease processes such as diabetes and lipid metabolism, we have used yeast two-hybrid screens to identify HNF4 alpha interacting proteins. Very recently, results of yeast two hybrid screens identified a protein associated with the the Mediator Complex, as a new HNF4 interacting protein. We over expressed CAR and HNF4 in adenoviral vectors and performed pull downs with GST-HNF4. Two members of the of the mediator complex) were identified as part of a complex associated with with GST-HNF4 from nuclear extracts of HepG2 cells by Western blotting and by mass spectrometric analysis. The Mediator Complex is important in recruiting PolI to gene promoters and initiating transcription. We showed that the new mediator associated protein interacts with HNF4alpha in GST pull downs. Nuclear cofactors such as NCOA6 and PGC-1 were also identified in the complex. Members of the mediator complex and the associated protein were identified in the HNF4alpha nuclear complex by Western blotting and mass spectrometry. Using CYP2C9 promoter assays we could show that adenoviral constructs to siRNAs for the mediator associated protein down-regulated HNF4alpha activation of CYP2C9 gene and the synergistic activation by the nuclear receptors CAR and HNF4. These data were also reflected in studies of mRNA for CYP2C9. In chromatin immunoprecipitation (ChIP) assays antibodies to the mediator protein and polymerase II pulled down the HNF4 binding site of the CYP2C9 promoter showing these factors bind to the promoter in vivo. Gel shifts showed that nuclear extracts of primary human hepatocytes bound to the HNF4 site, and binding was shifted by antibodies to HNF4. Antibody to flag-tagged mediator protein shifted the nuclear complex from hepatocytes infected with FLAG-Mediator protein. Antibody to polymerase IIalso shifted the nuclear complex in all nuclear extracts except those silenced by siRNA to the Mediator protein. Using primary human hepatocytes and siRNA for HNF4, over expression of HNF4 and siRNA for the Mediator protein we have shown with microarray analysis that silencing the Mediator associated protein silences expression of a number of CYP mRNAs that are upregulated by HNF4. These represent various drug metabolizing enzymes such as CYP2C9, CYP3A4, CYP2B6 and also enzymes involved in fatty acid metabolism but not those involved in glucose regulation.
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