Regulation Of The Human Cyp2c Enzymes

目的：提出以下问题：1)哪些孤儿受体(如CAR（组成型雄甾烷受体）和PXR（妊娠X受体）（SXR）（类固醇-异种结合元件）参与了人类CYP2Cs的调节？2)人类CAR与小鼠CAR有何不同？3) CYP2Cs是如何调控的，涉及哪些核因子？临床使用的药物和环境诱导自身的代谢，并通过增加其代谢而引起相互作用。化学物质。苯巴比妥（PB）是这些药物的原型。现在已知药物和环境化学物质可以激活许多基因启动子区域的应答元件，包括细胞色素P450，如人类CYP2B6。此外，核受体CAR和PXR可以与这些元件结合，并组成性和诱导性地激活转录。我们实验室正在研究人类p450的CYP2C亚家族的调控，包括CYP2C8、CYP2C9和CYP2C19。这是临床上重要的一组p450，它们代谢了人类接触到的20%的药物。研究表明，由人类CYP2Cs代谢的药物在与PB或抗生素利福平共同使用时，会显示出药代动力学特征的改变。用稳定转染CAR的HepG2细胞进行的实验表明，通过RT-PCR监测，CAR可组成性地激活内源性CYP2C8和CYP2C9转录物水平。这种激活是由pb型诱导剂诱导的。CYP2C9似乎至少部分受到PB反应受体CAR的调节。然而，人类CAR（hCAR）的作用似乎与小鼠CAR（mCAR）不同。此外，不同化合物如苯巴比妥和利福平对CYP2C8和CYP2C9的诱导作用不同，表明其控制机制存在差异。用含有前3kb CYP2C9的荧光素酶报告基因构建体转染研究表明，CAR可以组成性和诱导性地激活转录。我们已经在CYP2C9中分离出一个CAR结合元件，它似乎调节构成性表达。然而，这种反应并不能完全解释对诱导剂的反应，表明其他区域也可能含有元素。对该CYP2C9启动子区域的缺失分析定位了可能参与CAR介导激活的CYP2C9近端区域。我们还发现了CYP2C9启动子的远端元件，该元件与hCAR>hPXR>mCAR结合在电泳迁移率转移试验中。转染该元件驱动胸苷激酶启动子的研究表明，hCAR>hPXR>mCAR组成性地激活了HepG-2细胞中该元件的调控。

Accomplishments: AIMS: To ask the questions :1)Which orphan receptors such as CAR (constitutive androstane receptor) and PXR(pregnane X receptor)(SXR)(Steroid-Xenobiotic binding Element) are involved in the regulation of the human CYP2Cs?" 2)How does human CAR differ from mouse CAR. 3)How are the CYP2Cs regulated and what nuclear factors are involved? Clinically used drugs and environmentals induce their own metabolism and cause interactions by increasing their metabolism. chemicals. Phenobarbital (PB) is a prototype of these drugs. It is now known that drugs and environmental chemicals can activate response elements in the promoter regions of many genes, including cytochromes P450 such as human CYP2B6. In addition, the nuclear receptor CAR and PXR can bind to these elements, and constitutively and inducibly activate transcription. Our laboratory is studying the regulation of the CYP2C subfamily of P450s in humans, which includes CYP2C8, CYP2C9, and CYP2C19. These are a clinically important group of P450s which metabolize about 20% of all drugs to which humans are exposed It has been shown that drugs metabolized by the human CYP2Cs show altered pharmacokinetic profiles when co-administered with PB or the antibiotic rifampicin. Experiments using HepG2 cells stably transfected with CAR demonstrated that CAR constitutively activates endogenous levels of CYP2C8 and CYP2C9 transcripts monitored by RT-PCR. This activation is inducible by PB-type inducers. CYP2C9 appears to be at least partly regulated by the PB responsive receptor CAR. However, human CAR (hCAR) appears to act differently than mouse CAR(mCAR). Moreover , CYP2C8 and CYP2C9 are differentially induced by different compounds such as phenobarbital and rifampicin indicating differences in control mechanisms. Transfection studies with luciferase reporter constructs containing the first 3 kb of of CYP2C9 have shown that CAR can activate transcription constitutively and inducibily . We have isolated one CAR binding element in CYP2C9 which appears to regulate constituitive expression. This response does not, however, totally account for the response to inducers indicating other regions may also contain elements. Deletional analysis of this CYP2C9 promoter region has localized a possible proximal region of CYP2C9 which is involved in CAR mediated activation. We have also identified a distal element of the CYP2C9 promotor that binds to hCAR>hPXR>mCAR in electrophoretic mobility shift assays. Transfection studies with this element driving a thymidine kinase promoter show that hCAR>hPXR>mCAR constitutively activates regulation of this element in HepG-2 cells.