Drug Metabolizing Enzymes In Humans And Animal Models

在一项大型前瞻性流行病学研究中，我们研究了CYP2C9基因多态性（一种重要的药物代谢酶（CYP2C9））和维生素K酰化酶复合物1基因多态性对控制心血管（血栓溶栓）疾病所需的华法林剂量和危及生命的副作用风险的影响，该研究对302名白种美国人和273名非裔美国人进行了为期2年的华法林治疗。与Limdi等人（阿拉巴马大学）合作。我们开发了一种新的快速焦磷酸测序基因分型检测方法，用于检测白种人和非裔美国人CYP2C9等位基因（*2、*3、*5、*6、10和11变体）中已知或疑似缺陷的等位基因。具有异常CYP2C9等位基因的个体需要较低剂量的华法林。CYP2C9和VKORC1分别占美国白种人华法林所需剂量变异的30%和18%，非裔美国人占10%。我们还在一位因危及生命的出血住院的非裔美国人身上发现了CYP2C9 （Asp360Glu）的一个新的编码等位基因(1)。在其他研究中，人类肝细胞系和原代人肝细胞暴露于临床药物、草药和其他外源药物中，已被证明可以转录上调CYP2C亚家族（CYP2C8、2C9和2C19）的酶，从而增加这些酶对药物的代谢。这一数据与人类体内研究一致。这可能会产生对药物的耐受性或使患者治疗复杂化的药物-药物相互作用。在肝脏和肠道中，CYP2Cs可通过事先给药增加2-8倍。我们已经证明，人类CYP2C基因的启动子区域是由结合核受体CAR（组成型雄烷受体）、PXR（孕烷X受体）、类视黄酮相关孤儿受体（RORs）和肝脏富集受体（如HNF4 α）的元件调节的。药物如利福平、圣约翰草、苯巴比妥和其他外源药物与CAR和PXR相互作用，增加激活。此外，启动子中的HNF4 α位点增强了CAR或PXR的诱导作用，并且HNF4和CAR以及PXR协同作用增加了肝细胞系中CYP2C9的表达。我们发表的数据表明，共激活子NCOA6可能参与在CYP2C9启动子的远端CAR位点和近端HNF4位点之间形成桥梁。我们最近回顾了所有这些关于各种核受体（包括CAR、PXR、HNF4alpha和RORs）上调CYP2C酶的研究，其中大部分是在我们的实验室完成的(3)。我们目前发现NCOA6也在PXR和HNF4之间架起桥梁，在PXR配体存在的情况下产生协同激活。最近，我们在培养的原代人肝细胞（人类肝脏的替代品）中发现，肝脏富集受体HNF4 α的结合位点对于cyp2c基因的CYP2C8和CYP2C9启动子的上调是必需的。这些位点的突变阻止了PXR激动剂利福平的诱导。PXR位点或HNF4位点的突变均可消除诱导作用。siRNA to HNF4 α几乎可以消除原代人肝细胞中CYP2C8、2C9或2C19 mRNA的诱导作用，而原代人肝细胞是目前人类肝脏的最佳体外模型(4)。在其他研究中，发现小鼠Cyp2c Cyp2c55可将内源性物质花生四烯酮代谢为19-羟基二十碳四烯酸。Cyp2c55是由CAR和PXR激动剂苯巴比妥和pcn（一种特异性小鼠PXR激动剂）诱导的。这两种激动剂的这种诱导作用发生在CAR+/+和PXR+/+中，但不发生在敲除小鼠中，可能在肿瘤促进中很重要(6)。由于肝脏富集受体HNF4 α在诱导药物代谢和各种生理疾病过程（如糖尿病和脂质代谢）中的重要性，我们使用酵母双杂交筛选来鉴定HNF4 α相互作用蛋白。最近，酵母两种杂交筛选的结果确定了一种与中介复合物相关的蛋白，作为一种新的HNF4相互作用蛋白。我们在腺病毒载体上过表达CAR和HNF4，并使用GST-HNF4进行拉下。通过Western blotting和质谱分析，从HepG2细胞的核提取物中鉴定出与GST-HNF4相关的复合物的两个成员。中介复合物在招募PolI到基因启动子和启动转录中起着重要作用。我们发现新的中介相关蛋白在GST下拉过程中与HNF4alpha相互作用。核辅因子如NCOA6和PGC-1也在该复合体中被鉴定。通过Western blotting和质谱分析在HNF4alpha核复合体中鉴定了中介复合物的成员和相关蛋白。通过CYP2C9启动子实验，我们可以发现腺病毒构建的介导相关蛋白sinas下调了CYP2C9基因的hnf4α激活以及核受体CAR和HNF4的协同激活。这些数据也反映在CYP2C9 mRNA的研究中。在染色质免疫沉淀（ChIP）实验中，针对中介蛋白和聚合酶II的抗体拉低了CYP2C9启动子的HNF4结合位点，表明这些因子在体内与启动子结合。凝胶位移显示，原代人肝细胞的核提取物与HNF4位点结合，并被抗体HNF4转移。对标记的中介蛋白的抗体将核复合物从感染了flag-中介蛋白的肝细胞转移。除了siRNA沉默的核提取物外，抗聚合酶ii抗体也将所有核提取物中的核复合体转移到中介蛋白上。利用原代人肝细胞和HNF4的siRNA，过表达HNF4和中介蛋白的siRNA，我们通过微阵列分析表明，沉默中介相关蛋白会沉默一些被HNF4上调的CYP mrna的表达。这些代表了各种药物代谢酶，如CYP2C9、CYP3A4、CYP2B6以及参与脂肪酸代谢的酶，但不包括参与葡萄糖调节的酶。

We examined the effects of genetic polymorphisms of CYP2C9 (an important drug metabolizing enzyme (CYP2C9) and polymorphisms in the vitamin K epoxide reductase complex 1 on the dose of warfarin required to control cardiovascular (thromboembolytic) disease and risk for life-threatening side effects in a large prospective epidemiology study of 302 Caucasian Americans and 273 African-American patients treated with warfarin in over a 2 year period, in a collaboration with Limdi et al (University of Alabama). We developed new rapid pyrosequencing genotyping tests for known or suspected defective alleles of human CYP2C9 alleles (*2, *3, *5, *6, 10, and 11 variants) which occur in Caucasians and African Americans. Individuals with aberrant CYP2C9 alleles required lower doses of warfarin. CYP2C9 and VKORC1 accounted for 30% and 18% of the variability of required warfarin dose in American Caucasians and 10% in African-Americans. We also identified a new coding allele of CYP2C9 (Asp360Glu) in an African-American hospitalized for life-threatening hemorrhage (1). In other studies exposure of human liver cell lines and primary human hepatocytes to clinical drugs, herbal remedies and other xenobiotics has been shown to transcriptionally upregulate the CYP2C subfamily (CYP2C8, 2C9, and 2C19) of enzymes increasing metabolism of drugs by these enzymes. This data is consistent with invivo studies in humans. This could produce tolerance to drugs or drug-drug interactions complicating patient therapy. In liver and intestine the CYP2Cs can be increased >2-8 fold by prior administration of drugs. We have then shown that the promoter regions of the human CYP2C genes are regulated by elements which bind the nuclear receptors CAR (constitutive androstane receptor), PXR (pregnane X receptor), retinoid related orphan receptors (RORs) and liver-enriched receptors such as HNF4 alpha. Drugs such as rifampicin, St Johns Wort, phenobarbital and other xenobiotics interact with CAR and PXR increasing activation. Moreover HNF4 alpha sites in the promoter enhance inducibility by CAR or PXR, and HNF4 and CAR as well as PXR act synergistically to increase CYP2C9 in liver cell lines. We have published data indicating that the coactivator NCOA6 may be involved in forming a bridge between the distal CAR site and the proximal HNF4 site in the CYP2C9 promoter. We recently reviewed all these studies available on up regulation of the CYP2C enzymes by various nuclear receptors including CAR, PXR, HNF4alpha, and RORs, most of which was done in our laboratory (3). We are presently finding that NCOA6 also bridges between PXR and HNF4, producing synergistic activation in the presence of PXR ligand. Recently we have shown in cultured primary human hepatocytes (a surrogate for human liver) that binding sites for the liver-enriched receptor HNF4 alpha are necessary for the up regulation of the CYP2C8 and CYP2C9 promoter of the CYP2Cs genes. Mutation of these sites prevents induction by the PXR agonist rifampicin. Mutation of either the PXR sites or HNF4 sites abolishes induction. siRNA to HNF4 alpha almost abolishes induction of CYP2C8, 2C9 or 2C19 mRNA in primary human hepatocytes which are the best in vitro model we have for human liver (4). In other studies, Cyp2c55, a murine Cyp2c, has been found to metabolize the endogenous substance arachidonic to 19-hydroxyeicosatranenoic acid. Cyp2c55 is induced by the CAR and PXR agonists phenobarbital and PCN-a specific murine PXR agonist. This induction by these two agonists occurs in CAR+/+ and PXR+/+, but not knockout mice and could be important in tumor promotion (6). Because of the importance of the liver enriched receptor HNF4alpha in induction of drug metabolism and various physiological disease processes such as diabetes and lipid metabolism, we have used yeast two-hybrid screens to identify HNF4 alpha interacting proteins. Very recently, results of yeast two hybrid screens identified a protein associated with the the Mediator Complex, as a new HNF4 interacting protein. We over expressed CAR and HNF4 in adenoviral vectors and performed pull downs with GST-HNF4. Two members of the of the mediator complex) were identified as part of a complex associated with with GST-HNF4 from nuclear extracts of HepG2 cells by Western blotting and by mass spectrometric analysis. The Mediator Complex is important in recruiting PolI to gene promoters and initiating transcription. We showed that the new mediator associated protein interacts with HNF4alpha in GST pull downs. Nuclear cofactors such as NCOA6 and PGC-1 were also identified in the complex. Members of the mediator complex and the associated protein were identified in the HNF4alpha nuclear complex by Western blotting and mass spectrometry. Using CYP2C9 promoter assays we could show that adenoviral constructs to siRNAs for the mediator associated protein down-regulated HNF4alpha activation of CYP2C9 gene and the synergistic activation by the nuclear receptors CAR and HNF4. These data were also reflected in studies of mRNA for CYP2C9. In chromatin immunoprecipitation (ChIP) assays antibodies to the mediator protein and polymerase II pulled down the HNF4 binding site of the CYP2C9 promoter showing these factors bind to the promoter in vivo. Gel shifts showed that nuclear extracts of primary human hepatocytes bound to the HNF4 site, and binding was shifted by antibodies to HNF4. Antibody to flag-tagged mediator protein shifted the nuclear complex from hepatocytes infected with FLAG-Mediator protein. Antibody to polymerase IIalso shifted the nuclear complex in all nuclear extracts except those silenced by siRNA to the Mediator protein. Using primary human hepatocytes and siRNA for HNF4, over expression of HNF4 and siRNA for the Mediator protein we have shown with microarray analysis that silencing the Mediator associated protein silences expression of a number of CYP mRNAs that are upregulated by HNF4. These represent various drug metabolizing enzymes such as CYP2C9, CYP3A4, CYP2B6 and also enzymes involved in fatty acid metabolism but not those involved in glucose regulation.