ABSTRACT:CYP2C9 and CYP2C19 are clinically important drug-metabolizing enzymes. The expression level of CYP2C9 is much higher than that of CYP2C19, although the factor(s) responsible for the difference between the expression levels of these genes is still unclear. It has been reported that hepatocyte nuclear factor 4␣ (HNF4␣) plays an important role in regulation of the expression of liver-enriched genes, including P450 genes. Thus, we hypothesized that HNF4␣ contributes to the difference between the expression levels of these genes. Two direct repeat 1 (DR1) elements were located in both the CYP2C9 and CYP2C19 promoters. The upstream and downstream elements in these promoters had the same sequences, and HNF4␣ could bind to both elements in vitro. The transactivation levels of constructs containing two DR1 elements of the CYP2C9 promoter were increased by HNF4␣, whereas those of the CYP2C19 promoter were not increased. The introduction of mutations into either the upstream or downstream element in the CYP2C9 gene abolished the responsiveness to HNF4␣. We also examined whether HNF4␣ could bind to the promoter regions of the CYP2C9 and the CYP2C19 genes in vivo. The results of chromatin immunoprecipitation assays showed that HNF4␣ could bind to the promoter region of the CYP2C9 gene but not to that of the CYP2C19 promoter in the human liver. Taken together, our results suggest that HNF4␣ is a factor responsible for the difference between the expression levels of CYP2C9 and CYP2C19 in the human liver.

Pregnane X receptor (PXR; NR1I2), a key transcriptional factor that regulates genes encoding drug-metabolizing enzymes and drug transporters, is abundantly expressed in the human liver. However, studies on the molecular mechanism of human PXR gene regulation are limited. In this study, we examined the involvement of hepatocyte nuclear factor 4alpha (HNF4alpha; NR2A1) in the transcriptional regulation of the human PXR gene in the human liver. The activities of the human PXR promoter containing the direct repeat 1 (DR1) element located at -88/-76 of the promoter were significantly increased by co-expression of HNF4alpha in the human hepatocellular carcinoma cell line. In addition, introduction of mutation into the DR1 element abolished the transcriptional activation of the human PXR promoter by exogenous HNF4alpha. The results of gel mobility shift assays and chromatin immunoprecipitation assays showed that HNF4alpha was bound to the promoter region containing the DR1 element. A knock-down of HNF4alpha by siRNA significantly decreased expression levels of endogenous PXR mRNA in HepG2 cells. Furthermore, expression levels of PXR mRNA positively correlated with those of HNF4alpha mRNA in 18 human liver samples. These results suggested that HNF4alpha transactivated the human PXR gene by binding to the DR1 element located at -88/-76 of the promoter and was involved in the expression of PXR in the human liver.

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