Abstract

CYP2C enzymes are expressed constitutively and comprise ∼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 HNF4α in the pregnane X receptor (PXR)/rifampicin-mediated up-regulation of CYP2C8, CYP2C9, and CYP2C19 gene expression. We first identified new proximal cis-acting HNF4α 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 HNF4α in gel shift assays. Thus, these and recent studies identified a total of three HNF4α sites in the CYP2C9 promoter and two in the CYP2C8 promoter. Mutational studies showed that the HNF4α sites are needed for up-regulation of the CYP2C8 and CYP2C9 promoters by rifampicin. Furthermore, silencing of HNF4α abolished transactivation of the CYP2C8 and CYP2C9 promoters by rifampicin. Constitutive promoter activity was also decreased. Quantitative polymerase chain reaction analysis demonstrated that silencing HNF4α 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 HNF4α contributes to the constitutive expression of the human CYP2C genes and is also important for up-regulation by the PXR agonist rifampicin.