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Transcriptional Activation of CYP2C9, CYP1A1, and CYP1A2 by Hepatocyte Nuclear Factor 4 Requires Coactivators Peroxisomal Proliferator Activated Receptor- Coactivator 1 and Steroid Receptor Coactivator 1

Unidad de Hepatología Experimental, Centro de Investigación, Hospital Universitario La Fe, Valencia, Spain (C.P.M.-J., J.V.C., M.J.G.-L., R.J.); and Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Universidad de Valencia, Spain (J.V.C., R.J.)

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

Address correspondence to: Dr. Ramiro Jover Atienza, Unidad de Hepatología Experimental, Centro de Investigación, Hospital La Fe, Avenida de Campanar, 21, 46009 Valencia, Spain. E-mail: ramiro.jover{at}uv.es

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