Nuclear Receptor Coactivator 6 Mediates the Synergistic Activation of Human Cytochrome P-450 2C9 by the Constitutive Androstane Receptor and Hepatic Nuclear Factor-4α

Abstract

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-

Deciphering the roles of the constitutive androstane receptor in energy metabolism

“…Nevertheless, with the help of additional factors, crosstalk between CAR and other transcriptional factors will be facilitated and will exhibit synergistic effects [67,[69][70][71] . However, several CAR inhibitory factors, such as sterol regulatory elementbinding protein 1 (SREBP1), liver X receptor (LXR), farnesoid X receptor (FXR), estrogen receptor (ER), and histone deacetylase 1 (HDAC1), also exist [72][73][74][75][76] .…”

Interaction of PER2 with the Constitutive Androstane Receptor Possibly Links Circadian Rhythms to Metabolism

“…A similar arrangement with a serine residue in front of a LXXLL motif can also be observed in NCoA6, another CAR co-activator. 28,30 As both PER2 and PGC1α are co-activators of PPARγ, a NR involved in lipid and carbohydrate metabolism, attention was focused on the homology of the nuclear receptor. Most receptor residues that are in contact with LXXLL motifs have no charge pointing towards the co-activator motifs, besides two very distinct lysins at both the CAR and PPARγ at homologous positions.…”

“…The cells were held at 5% CO2 and 37 °C and transfered to 96-well microplates 24 hours prior to transfections with 5 × 10 6 cells in DMEM and 10% FBS. For transfections X-tremeGENE HP DNA (Roche) was 28, 30 The letters h and m before the protein name designate species Homo sapiens and Mus musculus, with the sequential number of the noted LXXLL and UniProt/Swiss-Prot entry number following the protein name. The predicted LXXLL motifs with residues marked as 1-5 are shown in red, as are serines located just before LXXLL.…”

Potential role of estradiol and progesterone in insulin resistance through constitutive androstane receptor

“…Recruitment analysis of CAR, FOXO1 and HNF4a on the CAR responsive element and HNF4a and FOXO1 on their responsive element ChIP assays were performed on the chromatin extracts from the HepG2 cells to examine the effect of CAR ligand and sex steroids, E 2 and progesterone, on the recruitment of CAR, FOXO1 and HNF4a on the CAR responsive element in CYP2C9 promoter (Surapureddi et al 2008), HNF4a on the HNF4a responsive element in the PEPCK promoter (Bhalla et al 2004), and FOXO1 on the FOXO1 binding site in the G6Pase promoter (Vander Kooi et al 2003). CAR ligand CITCO enhanced the recruitment of CAR, FOXO1, HNF4a, and coactivator SRC-1 compared with ethanol treatment, but E 2 and/or progesterone decreased these recruitments on the CAR responsive element (Fig.…”

“…After reversing the cross-links, DNA was purified and used as a template in PCR. PCR was performed by primer sets specific for the CAR responsive element in the CYP2C9 promoter (Surapureddi et al 2008), the HNF4a responsive element in the PEPCK promoter (Bhalla et al 2004), and FOXO1 binding site in the G6Pase promoter (Vander Kooi et al 2003). (nZ6 per group).…”