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

Laboratory of Pharmacology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA.
Molecular pharmacology (Impact Factor: 4.13). 07/2008; 74(3):913-23. DOI: 10.1124/mol.108.048983
Source: PubMed


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 4alpha (HNF4alpha)-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 HNF4alpha transcriptionally up-regulates CYP2C9 via proximal sites. We demonstrate ligand-enhanced synergistic cross-talk between CAR and HNF4alpha. We identify NCOA6 as crucial to the underlying mechanism of this cross-talk. NCOA6 was identified as an HNF4alpha-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 HNF4alpha 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 HNF4alpha. Silencing of NCOA6 abrogated the synergistic activation of the CYP2C9 promoter and the synergistic induction of the CYP2C9 gene by CAR-HNF4alpha. Chromatin immunoprecipitation analysis revealed that NCOA6 can pull down both the proximal HNF4alpha 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 HNF4alpha.

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Available from: Ritu Rana, Oct 05, 2015
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    • "The PXR protein was translated in vitro in rabbit reticulocyte lysate (Promega, Madison, WI), using a TNT Quick coupled transcription and translation system with radiolabeled [ 35 S]methionine (MP Biomedicals, Solon, OH). Expression and purification of recombinant GST fusion proteins and interaction assays were as described previously (Surapureddi et al., 2008). Immunoblotting. "
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    ABSTRACT: The pregnane X receptor (PXR, NR1I2) plays a pivotal role in the disposition and detoxification of numerous foreign and endogenous chemicals by increasing transcription of numerous target genes including phase I and II drug-metabolizing enzymes and transporters. In the present study, yeast two-hybrid screening identified an E3 ubiquitin ligase, RBCK1 (RBCC (RING-B-box-Coiled-coil) protein interacting with PKC-1), as a hPXR-interacting protein. Coimmunoprecipitation studies confirmed the interaction between RBCK1 and hPXR when both were ectopically expressed in AD-293 cells. Domain mapping studies showed that the interaction between RBCK1 and hPXR requires different RBCK1 domains. We further demonstrate that RBCK1 ubiquitinates human PXR (hPXR), and this may target hPXR for degradation by the ubiquitin-proteasome pathway. Simultaneous ectopic overexpression of RBCK1 and PXR decreased PXR levels in AD-293 cells and this decrease was inhibited by the proteasomal inhibitor MG-132. Furthermore, overexpression of RBCK1 decreased endogenous levels of PXR in HepG2 cells. Importantly, ectopic overexpression and silencing of endogenous RBCK1 in primary human hepatocytes resulted in a decrease and increase, respectively, in endogenous PXR protein levels and in the induction of PXR target genes by rifampicin. These results suggest that RBCK1 is important for the ubiquitination of PXR and may play a role in its proteasomal degradation.
    Drug metabolism and disposition: the biological fate of chemicals 11/2012; 41(2). DOI:10.1124/dmd.112.048728 · 3.25 Impact Factor
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    • "Key transcriptional regulators of CYP2C basal expression and xenobiotic-mediated induction are xenoreceptors CAR and PXR [286] [287] [288]. The expression of CYP2C genes is also regulated by several transcription factors including HNF4 [271] [289] [290] [291] [292] [293] [294] [295], C/EBP [270], HNF3 [296], transcription factor GATA-4 [297] [298], nuclear receptor co-activator 6 (NCoA-6) [299], PGC-1 and SRC-1 [292]. VDR binds and transactivates those xenobiotic-responsive elements (ER6, DR3, and DR4) previously identified in CYP2C9 promoter and shown to be targeted by PXR and/or CAR [201]. "
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    ABSTRACT: Retinoic acid receptors (RARs), retinoid X receptors (RXRs) and thyroid hormone receptors (TRs) are nuclear receptors that are crucial transcriptional regulators of many cellular processes such as differentiation, development, apoptosis, carbohydrate and lipid metabolism, homeostasis etc. In addition, RXRs are common heterodimerization partners for several receptors including vitamin D receptor, pregnane X receptor (PXR), constitutive androstane receptor (CAR) etc. In the course of 90s', PXR and CAR were discovered as key xenosensors regulating drug-metabolizing enzymes. Since there exist various cross-talks between cell signaling pathways, this was not surprising that RXRs, RARs and TRs were identified as regulators of human drug-metabolizing cytochromes P450 and cytochromes P450 involved in metabolism of endogenous compounds. Hence, a link between regulation of xenobiotic metabolizing enzymes and regulatory pathways of intermediary metabolism was established. Additionally, several drug-metabolizing enzymes are involved in metabolism of retinoids, rexinoids and thyroid hormones. In the current paper, we summarize the knowledge on the role of RARs, RXRs and TRs in the regulation of drug metabolizing cytochromes P450, and vice versa on the role of P450s in homeostasis of retinoids, rexinoids and thyroid hormone.
    Current Drug Metabolism 03/2011; 12(2):71-88. DOI:10.2174/138920011795016881 · 2.98 Impact Factor
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    • "Li and Chiang (2006) concluded that the competition between PXR and HNF4␣ for their coactivators PGC-1␣ and SRC-1 contributes to an interaction between these receptors. Our laboratory recently reported cross-talk between the proximal HNF4␣ and the upstream CAR/PXR sites of the CYP2C9 promoter (Chen et al., 2005; Surapureddi et al., 2008). HNF4␣ and CAR/PXR synergistically activate the CYP2C9 promoter in HepG2 cells in the presence of the CAR agonist 6-(4-chlorophenyl)imidazo[2,1-b][1] [3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime or the PXR agonist rifampicin. "
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    Drug metabolism and disposition: the biological fate of chemicals 04/2010; 38(4):591-9. DOI:10.1124/dmd.109.030387 · 3.25 Impact Factor
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