Hepatocyte nuclear factor 4␣ (HNF4␣) controls the expression of many critical metabolic pathways, and the Mediator complex occupies a central role in recruiting RNA polymerase II (Pol II) to these gene promoters. An impaired transcriptional HNF4␣ network in human liver is responsible for many pathological conditions, such as altered drug metabolism, fatty liver, and diabetes. Here, we report that Med25, an associated member of the Mediator complex, is required for the association of HNF4␣ with Mediator, its several cofactors, and RNA Pol II. Further, increases and decreases in endogenous Med25 levels are reflected in the composition of the transcriptional complex, Pol II recruitment, and the expression of HNF4␣-bound target genes. A novel feature of Med25 is that it imparts "selectivity." Med25 affects only a significant subset of HNF4␣ target genes that selectively regulate drug and lipid metabolism. These results define a role for Med25 and the Mediator complex in the regulation of xenobiotic metabolism and lipid homeostasis.The liver is the central organ that controls/regulates such metabolic processes as lipid metabolism (lipogenesis and fatty acid ␤-oxidation [FAO]), energy homeostasis (glycolysis and gluconeogenesis), bile acid synthesis, and drug metabolism (4,48,49,57). Metabolism in the liver is governed by a highly dynamic transcriptional regulatory network whose key receptors may be classical (glucocorticoid), adopted (peroxisome proliferator activated receptor, farnesoid X receptor, liver specific X receptor, and constitutively active receptor/pregnane X receptor), or orphan (hepatocyte nuclear factor 4␣ [HNF4␣]) (6). HNF4␣, also known as NR2A1, is an important member of the orphan nuclear receptor superfamily (51, 53). When HNF4␣ binds to DNA, it regulates the expression of both constitutive genes, such as apolipoprotein genes (37), CYP7A1 and CYP8B1 (25, 27), the FXII and XIIIB genes (26), the proline oxidase gene (30), CYP2D6 (5), CYP2A6 (42), UGT1A9 (1), SULTA1 (31), and the glucose-6-phosphatase (G6PC) and Pck1 genes (47), and also xenobiotic inducible genes, such as CYP3A4 (56), CYP2C9 (8), and CYP2B6 (24). Malfunctions in the regulation of genes by HNF4␣ are responsible for alterations in drug metabolism and metabolic disorders, such as atherosclerosis, diabetes, hemophilia, hypoxia, medium-chain acyl-coenzyme A (CoA) dehydrogenase (MCAD) deficiency, and ornithine transcarbamylase (OTC) deficiency (16,22).Like most nuclear receptors, HNF4␣ displays a typical DNA binding domain (DBD), including a double zinc finger motif, a ligand binding domain (LBD), and two activation domains, AF1 and AF2, located in the N-and C-terminal regions, respectively (21, 53). The DNA binding domain binds to a cisacting element which optimally consists of a direct repeat of AGGTCA with a 1-or 2-nucleotide spacer (DR1/DR2) to which HNF4␣ binds as a homodimer (52). The ligand binding domain is structurally homologous to other receptors of the retinoid receptor family, with a well-defined hydrophobic pocket capable of ...

While some studies show that carbon tetrachloride-mediated metabolic oxidative stress exacerbates steatohepatitic-like lesions in obese mice, the redox mechanisms that trigger the innate immune system and accentuate the inflammatory cascade remain unclear. Here we have explored the role of the purinergic receptor P2X7-NADPH oxidase axis as a primary event in recognizing the heightened release of extracellular ATP from CCl4-treated hepatocytes and generating redoxmediated Kupffer cell activation in obese mice. We found that an underlying condition of obesity led to the formation of protein radicals and post-translational nitration, primarily in Kupffer cells, at 24 h post-CCl4 administration. The free radical-mediated oxidation of cellular macromolecules, which was NADPH oxidase- and P2X7 receptor-dependent, correlated well with the release of TNF- α and MCP-2 from Kupffer cells. The Kupffer cells in CCl4-treated mice exhibited increased expression of MHC Class II proteins and showed an activated phenotype. Increased expression of MHC Class II was inhibited by the NADPH oxidase inhibitor apocynin , P2X7 receptor antagonist A438709 hydrochloride, and genetic deletions of the NADPH oxidase p47 phox subunit or the P2X7 receptor. The P2X7 receptor acted upstream of NADPH oxidase activation by up-regulating the expression of the p47 phox subunit and p47 phox binding to the membrane subunit, gp91 phox. We conclude that the P2X7 receptor is a primary mediator of oxidative stress-induced exacerbation of inflammatory liver injury in obese mice via NADPH oxidase-dependent mechanisms.

Nuclear receptor coactivator 6 (NCOA6) also known as PRIP/ RAP250/ASC-2 anchors a steady-state complex of cofactors and function as a transcriptional coactivator for certain nuclear receptors. This is the first study to identify NCOA6 as a hepatic nuclear factor 4␣ (HNF4␣)-interacting protein. CYP2C9 is an important enzyme that metabolizes both commonly used therapeutic drugs and important endogenous compounds. We have shown previously that constitutive androstane receptor (CAR) (a xenobiotic-sensing receptor) up-regulates the CYP2C9 promoter through binding to a distal site, whereas HNF4␣ transcriptionally up-regulates CYP2C9 via proximal sites. We demonstrate ligand-enhanced synergistic cross-talk between CAR and HNF4␣. We identify NCOA6 as crucial to the underlying mechanism of this cross-talk. NCOA6 was identified as an HNF4␣-interacting protein in this study using a yeast two-hybrid screen and GST pull-down assays. Furthermore, we identified NCOA6, CAR, and other coactivators as part of a mega complex of cofactors associated with HNF4␣ in HepG2 cells. Although the interaction of NCOA6 with CAR is specifically through the first LXXLL motif of NCOA6, both LXXLL motifs are involved in its interaction with HNF4␣. Silencing of NCOA6 abrogated the synergistic activation of the CYP2C9 promoter and the synergistic induction of the CYP2C9 gene by CAR-HNF4␣. Chromatin immunoprecipitation analysis revealed that NCOA6 can pull down both the proximal HNF4␣ and distal CAR binding sites of the CYP2C9 promoter and provides the basis for the recruitment of other cofactors. We conclude that the coactivator NCOA6 mediates the mechanism of the synergistic activation of the CYP2C9 gene by CAR and HNF4␣.Cytochrome P450 2C9 (CYP2C9) is a major member of the cytochrome P450 superfamily in human liver, metabolizing numerous therapeutically used drugs and physiologically important endogenous compounds (Goldstein, 2001). Hepatic expression of CYP2C9 exhibits considerable interindividual variability in humans. Some of this interindividual variability is due to the up-regulation of CYP2C9 levels by prior exposure to drugs and xenobiotics such as rifampicin, hyperforin, phenobarbital, and paclitaxel (Taxol) (Raucy et al., 2002;Madan et al., 2003;Komoroski et al., 2004). Studies in primary hepatocytes and clinical studies in vivo in humans have confirmed that CYP2C9 levels and the clearances of CYP2C9 substrates are increased after the administration of drugs (Williamson et al., 1998;Henderson et al., 2002).Recent studies have shown that the constitutive androstane receptor (CAR) and the pregnane X receptor (PXR) -(3,4-dichlorobenzyl)oxime; PXR, pregnane X receptor; CREB, cAMP response element-binding pro-