Nuclear receptor coactivator 6 mediates the synergistic activation of human cytochrome P-450 2C9 by the constitutive androstane receptor and hepatic nuclear factor-4alpha.
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.
Laboratory of Pharmacology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA.
Sailesh
S
Surapureddi
Ritu
R
Rana
Janardan K
JK
Reddy
Joyce A
JA
Goldstein
D000595:Amino Acid Sequence; D001189:Aryl Hydrocarbon Hydroxylases / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D000090702:Constitutive Androstane Receptor; D065729:Cytochrome P-450 CYP2C9; D015971:Gene Expression Regulation, Enzymologic; D020868:Gene Silencing; D051557:Hepatocyte Nuclear Factor 4 / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D008024:Ligands; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D056918:Nuclear Receptor Coactivators; D011485:Protein Binding; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020218:Response Elements / Q000235:genetics; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
D000090702:Constitutive Androstane Receptor; D051557:Hepatocyte Nuclear Factor 4; D047908:Intracellular Signaling Peptides and Proteins; D008024:Ligands; C486559:NCOA6 protein, human; D056918:Nuclear Receptor Coactivators; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D018160:Receptors, Cytoplasmic and Nuclear; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; D001189:Aryl Hydrocarbon Hydroxylases