Transcriptional activation of CYP2C9, CYP1A1, and CYP1A2 by hepatocyte nuclear factor 4alpha requires coactivators peroxisomal proliferator activated receptor-gamma coactivator 1alpha and steroid receptor coactivator 1.
Hepatocyte nuclear factor 4alpha (HNF4alpha) is a key transcription factor for the constitutive expression of cytochromes P450 (P450s) in the liver. However, human hepatoma HepG2 cells show a high level of HNF4alpha but express only marginal P450 levels. We found that the HNF4alpha-mediated P450 transcription in HepG2 is impaired by the low level of coactivators peroxisomal proliferator activated receptor-gamma coactivator 1alpha (PGC1alpha) and steroid receptor coactivator 1 (SRC1). Reporter assays with a chimeric CYP2C9-LUC construct demonstrated that the sole transfection of coactivators induced luciferase activity in HepG2 cells. In HeLa cells however, CYP2C9-LUC activity only significantly increased when coactivators were cotransfected with HNF4alpha. A deletion mutant lacking the two proximal HNF4alpha binding sites in the CYP2C9 promoter did not respond to PGC1alpha or SRC1, demonstrating that coactivators were acting through HNF4alpha response elements. Adenovirus-mediated transfection of PGC1alpha in human hepatoma cells caused a significant dose-dependent increase in CYP2C9, CYP1A1, and CYP1A2 and in the positive control CYP7A1. PGC1alpha also showed a moderate activating effect on CYP3A4, CYP3A5, and CYP2D6. Adenoviral transfection of SRC1 had a lessened effect on P450 genes. Chromatin immunoprecipitation assay demonstrated in vivo binding of HNF4alpha and PGC1alpha to HNF4alpha response sequences in the CYP2C9 promoter and to three new regulatory regions in the common 23.3 kilobase spacer sequence of the CYP1A1/2 cluster. Insulin treatment of HepG2 and human hepatocytes caused repression of PGC1alpha and a concomitant down-regulation of P450s. Our results establish the importance of coactivators PGC1alpha and SRC1 for the hepatic expression of human P450s and uncover a new HNF4alpha-dependent regulatory mechanism to constitutively control the CYP1A1/2 cluster.
Martínez-Jiménez
Celia P
CP
Unidad de Hepatología Experimental, Centro de Investigación, Hospital La Fe, Avenida de Campanar, 21, 46009 Valencia, Spain.
Castell
José V
JV
Gómez-Lechón
M José
MJ
Jover
Ramiro
R
D000256:Adenoviridae / Q000378:metabolism; D001189:Aryl Hydrocarbon Hydroxylases / Q000235:genetics*; D002478:Cells, Cultured; D019363:Cytochrome P-450 CYP1A1 / Q000235:genetics*; D019388:Cytochrome P-450 CYP1A2 / Q000235:genetics*; D065729:Cytochrome P-450 CYP2C9; D005260:Female; D006367:HeLa Cells; D006360:Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism*; D051557:Hepatocyte Nuclear Factor 4 / Q000235:genetics / Q000378:metabolism*; D022781:Hepatocytes / Q000187:drug effects; D051548:Histone Acetyltransferases / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007328:Insulin / Q000494:pharmacology; D008297:Male; D008875:Middle Aged; D056920:Nuclear Receptor Coactivator 1; D000071248:Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha; D011485:Protein Binding / Q000187:drug effects; D020218:Response Elements / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation / Q000187:drug effects; D014162:Transfection
D006360:Heat-Shock Proteins; D051557:Hepatocyte Nuclear Factor 4; D007328:Insulin; C486497:PPARGC1A protein, human; D000071248:Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha; D014157:Transcription Factors; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; D001189:Aryl Hydrocarbon Hydroxylases; D019363:Cytochrome P-450 CYP1A1; D019388:Cytochrome P-450 CYP1A2; D051548:Histone Acetyltransferases; C538859:NCOA1 protein, human; D056920:Nuclear Receptor Coactivator 1