Studies exposure of human liver cell lines and primary human hepatocytes to clinical drugs, herbal remedies and other xenobiotics have 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 the fact that in vivo studies in humans previous exposure to drugs and herbal remedies decreases the half-life to many drugs. 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 and in human hepatocytes (1). Our last report identified NCOA6 as a new HNF4alpha binding protein, and published data indicating that the coactivator NCOA6 is involved in forming a bridge between the distal CAR site and the proximal HNF4 site in the CYP2C9 promoter (Surapureddi et al., 2010). Our present studies show that NCOA6 also bridges between PXR and HNF4 sites, producing synergistic activation in the presence of PXR ligand (2). Recently collaborative studies have shown shown that coactivator NCOA6 is responsible for the recruitment of ASCOM complex to the promoter bound FXR, responsible for epigenetic modification of the regulatory region (H3K4me3 epigenetic mark) which was found to be essential and prerequiste for the activation/induction of bile salt export pump (BSEP), multi drug resistance associated protein 2 (MRP2) and sodium taurcholate cotransporting polypeptide (NTCP) genes which belong to the transporter family of genes regulated by nuclear receptors (3). In contrast, collaborative studies have shown that PXR represses human sulfotransfease SULT1E1 by altering chromatin structure (4). We have shown in cultured primary human hepatocytes (a surrogate for human liver) that mutation of binding sites for the liver-enriched receptor HNF4 alpha also abolishes induction by CAR and PXR agonists (1). 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 (5). 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 Med 25, a member of the Mediator Complex, as a new HNF4 interacting protein(6). We over expressed CAR and HNF4 in adenoviral vectors and performed pull downs with GST-HNF4. Coactivators and members of the of the mediator complex were identified as part of a complex associated with GST-HNF4 from nuclear extracts of HepG2 cells by Western blotting and by mass spectrometric analysis, but when Med 25 was silenced repressor proteins were identified in the complex. The Mediator Complex is important in recruiting PolI to gene promoters and initiating transcription. We showed that Med 25 protein interacts with HNF4alpha in GST pull downs through an LXXLL motif. Nuclear cofactors such as NCOA6 and PGC-1 were also identified in the complex. Using CYP2C9 promoter assays we could show that adenoviral constructs to shRNAs for Med 25 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 in HepG2 cells and primary human hepatocytes, antibodies toMed 25 and polymerase II pulled down the HNF4 binding site of the CYP2C9 extracts of primary human hepatocytes bound to the HNF4 site, and binding was shifted by antibodies to HNF4. Antibody to flag peptide-tagged Med 25 shifted the nuclear complex from hepatocytes infected with FLAG-Mediator protein. Antibody to polymerase II also shifted the nuclear complex in all nuclear extracts except those silenced by shRNA to Med25. Using primary human hepatocytes and shRNA for HNF4, over expression of HNF4 and shRNA for the Med25 we have shown with qPCR and microarray analysis that silencing the Med25 decreases upregulation of a number of CYP mRNAs and proteins by HNF4 alpha. These include upregulation of various CYP genes including drug metabolizing enzymes such as CYP2C9, CYP3A4, CYP2B6 and also enzymes involved in fatty acid metabolism but not those involved in glucose regulation. Thus Med 25 selectively initiates transcription of some but not all pathways controlled by HNF4 alpha.
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