Transcriptional Regulation of Human CYP3A4 Basal Expression by CCAAT Enhancer-Binding Protein α and Hepatocyte Nuclear Factor-3γ

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Abstract
Cytochrome P450 3A4 (CYP3A4) is involved in the metabolism of more than 50% of currently used therapeutic drugs, yet the mechanisms that control CYP3A4 basal expression in liver are poorly understood. Several putative binding sites for CCAAT/enhancer-binding protein (C/EBP) and hepatic nuclear factor 3 (HNF-3) were found by computer analysis in CYP3A4 promoter. The use of reporter gene assays, electrophoretic mobility shift assays, and site-directed mutagenesis revealed that one proximal and two distal C/EBPα binding sites are essential sites for thetrans-activation of CYP3A4 promoter. Notrans-activation was found in similar reporter gene experiments with a HNF-3γ expression vector. The relevance of these findings was further explored in the more complex DNA/chromatin structure within endogenous CYP3A4 gene. Using appropriate adenoviral expression vectors, we found that both hepatic and nonhepatic cells overexpressing C/EBPα had increasedCYP3A4 mRNA levels, but no effect was observed when HNF-3γ was overexpressed. In contrast, overexpression of HNF-3γ simultaneously with C/EBPα resulted in a greater activation of theCYP3A4 gene. This cooperative effect was hepatic-specific and also occurred in CYP3A5 andCYP3A7 genes. To investigate the mechanism for HNF-3γ action, we studied its binding to CYP3A4 promoter and the effect of the deacetylase inhibitor trichostatin A. HNF-3γ was able to bind CYP3A4 promoter at a distal position, near the most distal C/EBPα binding site. Trichostatin A increased C/EBPα effect but abolished HNF-3γ cooperative action. These findings revealed that C/EBPα and HNF-3γ cooperatively regulateCYP3A4 expression in hepatic cells by a mechanism that probably involves chromatin remodeling.