Wang Hongbing, Faucette Stephanie, Moore Rick, Sueyoshi Tatsuya, Negishi Masahiko, LeCluyse Edward
Division of Drug Delivery and Disposition, School of Pharmacy, University of North Carolina at Chapel Hill, North Carolina 27599, USA.
J Biol Chem. 2004 Jul 9;279(28):29295-301. doi: 10.1074/jbc.M400580200. Epub 2004 Apr 28.
Compared with its rodent orthologs, little is known about the chemical specificity of human constitutive androstane receptor (hCAR) and its regulation of hepatic enzyme expression. Phenytoin (PHY), a widely used antiepileptic drug, is a potent inducer of CYP2B6 in primary human hepatocytes, but does not activate human pregnane X receptor (PXR) significantly in cell-based transfection assays at the same concentrations associated with potent induction of CYP2B6. Based on this observation, we hypothesized that PHY may be a selective activator of hCAR. In primary human hepatocytes, expression of CYP2B6 reporter genes containing phenobarbital-responsive enhancer module (PBREM) or PBREM/xenobiotic-responsive enhancer module (XREM) response elements were activated up to 14- and 28-fold, respectively, by 50 microm PHY. By contrast, parallel experiments in HepG2 cell lines co-transfected with an hPXR expression vector did not show increased reporter activity. These results indicated that a PXR-independent pathway, which is retained in primary hepatocytes, is responsible for PHY induction of CYP2B6. Further experiments revealed that PHY effectively translocates hCAR from the cytoplasm into the nucleus in both primary human hepatocytes and CAR(-/-) mice. Compared with vehicle controls, PHY administration significantly increased CYP2B6 reporter gene expression, when this reporter construct was delivered together with hCAR expression vector into CAR(-/-) mice. However, PHY did not increase reporter gene expression in CAR(-/-) mice in the absence of hCAR vector, implying that CAR is essential for mediating PHY induction of CYP2B6 gene expression. Taken together, these observations demonstrate that, in contrast to most of the known CYP2B6 inducers, PHY is a selective activator of CAR in humans.
与啮齿动物的直系同源物相比，人们对人类组成型雄甾烷受体（hCAR）的化学特异性及其对肝脏酶表达的调控了解甚少。苯妥英（PHY）是一种广泛使用的抗癫痫药物，是原代人肝细胞中CYP2B6的强效诱导剂，但在基于细胞的转染实验中，在与强效诱导CYP2B6相关的相同浓度下，它对人类孕烷X受体（PXR）的激活作用并不显著。基于这一观察结果，我们推测PHY可能是hCAR的选择性激活剂。在原代人肝细胞中，含有苯巴比妥反应增强子模块（PBREM）或PBREM/异生素反应增强子模块（XREM）反应元件的CYP2B6报告基因的表达分别被50微摩尔的PHY激活了14倍和28倍。相比之下，在与hPXR表达载体共转染的HepG2细胞系中进行的平行实验并未显示报告基因活性增加。这些结果表明，原代肝细胞中保留的一条不依赖PXR的途径负责PHY对CYP2B6的诱导。进一步的实验表明，PHY在原代人肝细胞和CAR（-/-）小鼠中均能有效地将hCAR从细胞质转运到细胞核中。与载体对照组相比，当将该报告构建体与hCAR表达载体一起导入CAR（-/-）小鼠时，给予PHY可显著增加CYP2B6报告基因的表达。然而，在没有hCAR载体的情况下，PHY并未增加CAR（-/-）小鼠中的报告基因表达，这意味着CAR对于介导PHY诱导CYP2B6基因表达至关重要。综上所述，这些观察结果表明，与大多数已知的CYP2B6诱导剂不同，PHY是人类中CAR的选择性激活剂。
