We use studies in human liver cell lines and primary human hepatocytes to whow that clinical drugs, environmental chemicals and other xenobiotics transcriptionally upregulate expression of the CYP2C subfamily of drug-metabolizing enzymes (CYP2C8, 2C9, and 2C19) which then increases the metabolism of many clinical and over-the counter drugs as well as pesticides and herbicides. This data is consistent with in vivo studies in humans which have shown that exposure to certain drugs and herbal remedies decrease the half-life to many drugs. This can 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 shown that the promoter regions of the human CYP2C genes are regulated by elements which bind xenosensing nuclear receptors CAR (constitutive androstane receptor), PXR (pregnane X receptor), and liver-enriched receptors such as HNF4 alpha (HNF4a). Drugs such as rifampicin, St Johns Wort, phenobarbital and other xenobiotics interact with CAR and PXR which bind to elements in the promoter increasing transcription. New studies (3) using yeast two-hybrid screens with PXR as bait found an E3 ubiquitin Ligase, RBCK1 (Ring-B-box coiled-col proteini interactining with protein kinase C-1) to be a human PXR-interacting protein. Co-immunoprecipitation assays with antibodies to PXR and RBCK1 confirmed that RBCK1 and hPXR interacted were co-expressed in AD-293 cells. GST-pulldown assays confirmed that there was an interaction between the two proteins. Ubiquination studies confirmed that RBCK1 ubiquinates hPXR, presumably targeting it for proteasomal degradation. Overexpression of RBCK1 decreased endogenous levels of PXR in HepG2 CELLS. Overexpression and silencing of RBCK1 in primary human hepatocytes resulted in decreases and increases respectively of endogenous PXR protein. Silencing RBCK1 increases in induction of PXR target genes by the PXR ligand rifampicin. These results indicate RBCK1 is involved in the proteasomal degradation of PXR. Secondly we have tound that microRNAS 103 and 107 regulate CYP2C8 the enzyme which metabolizes the breast anticancer drug taxol translationally.(2) in human liver cells. Moreover the levels of these microRNAs is inversely related to protein in human liver. In an ongoing new project, two-hybrid screens showed that Med25, a member of the mediator complex, is an HNF4a-binding protein. We have shown that Med25 is important for the recruitment of RNA Polymerase II to select sets of HNF4a-activated promoters such as the important drug-metabolizing gene cytochrome P450 2C9 (CYP2C9). We hypothesized that this involves a direct interaction between Med25 and HNF4 to alter chromatin conformation of the CYP2C9 gene to a transcriptionally active state. Conformational change requires the modification of histones by enzymes that are recruited to target genes. Histone modifications include methylation or acetylation of lysine and arginine amino acids on histone N-terminal tails. For example, histone 3 lysine 4 dimethylation (H3K4me2) is associated with gene activation, while histone 3 lysine 27 trimethylation (H3K27me3) is a marker of gene silencing. In this study, we used HepG2 cells to determine the role of Med25 in the epigenetic regulation of HNF4a-dependent CYP2C9 expression. We performed chromatin immunoprecipitation to identify histone modifications at the HNF4a binding site in relation to Med25 protein levels. Our results indicate that altering Med25 expression modified acetylation and methylation of certain histones. . However, when Med25 was silenced with small-hairpin looped RNAi, the modifications occurred which were a prototypical gene-silencing marker. These results indicate that Med25 induces a permissive chromatin state at the CYP2C9 proximal HNF4 binding site. Similarly, confocal microscopy revealed that Med25 colocalized with key histone modification markers. We have also determined levels of open CYP2C9 chromatin under activating conditions using formaldehyde-assisted isolation of regulatory elements (FAIRE). FAIRE data indicated that the chromatin around the HNF4 sites of the CYP2C9 proximal promoter was open in the presence of activating nuclear receptors CAR and HNF4a. Recently we found that electrophiles and oxidative stress activate CYP2C9 through AP-1 sites. Many drugs are known to be activated to electrophiles. This is a new mechanism of activation of CYP2C9.
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