Human Metabolism Section, Laboratory of Pharmacology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA.
Drug Metab Dispos. 2010 Apr;38(4):591-9. doi: 10.1124/dmd.109.030387. Epub 2010 Jan 19.
CYP2C enzymes are expressed constitutively and comprise approximately 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 HNF4alpha in the pregnane X receptor (PXR)/rifampicin-mediated up-regulation of CYP2C8, CYP2C9, and CYP2C19 gene expression. We first identified new proximal cis-acting HNF4alpha 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 HNF4alpha in gel shift assays. Thus, these and recent studies identified a total of three HNF4alpha sites in the CYP2C9 promoter and two in the CYP2C8 promoter. Mutational studies showed that the HNF4alpha sites are needed for up-regulation of the CYP2C8 and CYP2C9 promoters by rifampicin. Furthermore, silencing of HNF4alpha abolished transactivation of the CYP2C8 and CYP2C9 promoters by rifampicin. Constitutive promoter activity was also decreased. Quantitative polymerase chain reaction analysis demonstrated that silencing HNF4alpha 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 HNF4alpha contributes to the constitutive expression of the human CYP2C genes and is also important for up-regulation by the PXR agonist rifampicin.
CYP2C 酶是组成型表达的，约占人类肝脏总细胞色素 P450 的 20%。然而，影响 CYP2C 亚家族转录调控的因素最近才被阐明。在本研究中，我们使用原代人肝细胞培养物来研究 HNF4alpha 在 pregnane X 受体 (PXR)/利福平介导的 CYP2C8、CYP2C9 和 CYP2C19 基因表达上调中的作用。我们首先在 CYP2C8 启动子的近端 [距翻译起始位点 -181 个碱基 (bp)] 和 CYP2C9 启动子中鉴定出新的近端 HNF4alpha 结合位点 (-211 bp)。凝胶迁移分析显示这两个位点均能与 HNF4alpha 结合。因此，这些和最近的研究总共在 CYP2C9 启动子中鉴定出三个 HNF4alpha 位点，在 CYP2C8 启动子中鉴定出两个。突变研究表明，HNF4alpha 结合位点是利福平上调 CYP2C8 和 CYP2C9 启动子所必需的。此外，HNF4alpha 的沉默消除了利福平对 CYP2C8 和 CYP2C9 启动子的转录激活作用。组成性启动子活性也降低。定量聚合酶链反应分析表明，沉默 HNF4alpha 降低了 CYP2C8 (53%)、CYP2C9 (55%) 和 CYP2C19 (43%) mRNA 的组成型表达，并显著降低了利福平介导的 CYP2C8 (6.6-与 2.7 倍)、CYP2C9 (3-与 1.5 倍)和 CYP2C19 (1.8-与 1.1 倍)诱导的幅度。这些结果提供了明确的证据，表明 HNF4alpha 有助于人类 CYP2C 基因的组成型表达，并且对于 PXR 激动剂利福平的上调也很重要。
