Hepatocyte Nuclear Factor 4α Regulates Rifampicin-Mediated Induction of CYP2C Genes in Primary Cultures of Human Hepatocytes

CYP2C enzymes are expressed constitutively and comprise similar to 20% of the total cytochrome P450 in human liver. However, the factors influencing the transcriptional regulation of the CYP2C subfamily have only been addressed recently. In the present study, we used primary cultures of human hepatocytes to investigate the role of HNF4 alpha in the pregnane X receptor (PXR)/rifampicin-mediated upregulation of CYP2C8, CYP2C9, and CYP2C19 gene expression. We first identified new proximal cis-acting HNF4 alpha sites in the proximal CYP2C8 promoter [at -181 base pairs (bp) from the translation start site] and the CYP2C9 promoter (at -211 bp). Both sites bound HNF4 alpha in gel shift assays. Thus, these and recent studies identified a total of three HNF4 alpha sites in the CYP2C9 promoter and two in the CYP2C8 promoter. Mutational studies showed that the HNF4 alpha sites are needed for up-regulation of the CYP2C8 and CYP2C9 promoters by rifampicin. Furthermore, silencing of HNF4 alpha abolished transactivation of the CYP2C8 and CYP2C9 promoters by rifampicin. Constitutive promoter activity was also decreased. Quantitative polymerase chain reaction analysis demonstrated that silencing HNF4 alpha reduced the constitutive expression of CYP2C8 (53%), CYP2C9 (55%), and CYP2C19 (43%) mRNAs and significantly decreased the magnitude of the rifampicin-mediated induction of CYP2C8 (6.6-versus 2.7-fold), CYP2C9 (3-versus 1.5-fold), and CYP2C19 (1.8-versus 1.1-fold). These results provide clear evidence that HNF4 alpha contributes to the constitutive expression of the human CYP2C genes and is also important for upregulation by the PXR agonist rifampicin.