Transcriptional activation of CYP2C9, CYP1A1, and CYP1A2 by hepatocyte nuclear factor 4α requires coactivators peroxisomal proliferator activated receptor-γ coactivator 1α and steroid receptor coactivator 1

Hepatocyte nuclear factor 4 alpha ( HNF4 alpha) 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 HNF4 alpha but express only marginal P450 levels. We found that the HNF4 alpha-mediated P450 transcription in HepG2 is impaired by the low level of coactivators peroxisomal proliferator activated receptor-gamma coactivator 1 alpha ( PGC1 alpha) 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 HNF4 alpha. A deletion mutant lacking the two proximal HNF4 alpha binding sites in the CYP2C9 promoter did not respond to PGC1 alpha or SRC1, demonstrating that coactivators were acting through HNF4 alpha response elements. Adenovirus-mediated transfection of PGC1 alpha in human hepatoma cells caused a significant dose-dependent increase in CYP2C9, CYP1A1, and CYP1A2 and in the positive control CYP7A1. PGC1 alpha 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 HNF4 alpha and PGC1 alpha to HNF4 alpha 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 PGC1 alpha and a concomitant down-regulation of P450s. Our results establish the importance of coactivators PGC1 alpha and SRC1 for the hepatic expression of human P450s and uncover a new HNF4 alpha-dependent regulatory mechanism to constitutively control the CYP1A1/2 cluster.