Drug Metabolizing Enzymes In Humans And Animal Models

我们使用人肝细胞系和原代人肝细胞的研究来证明临床药物、环境化学品和其他外源性物质转录上调药物代谢酶CYP 2C亚家族（CYP 2C 8、2C 9和2C 19）的表达，从而增加许多临床和非处方药物以及杀虫剂和除草剂的代谢。这一数据与人体体内研究一致，这些研究表明，接触某些药物和草药会缩短许多药物的半衰期。这可能会产生药物耐受性或使患者治疗复杂化的药物-药物相互作用。在肝脏和肠中，通过预先给药，CYP 2C可增加>2-8倍。我们已经表明，人CYP 2C基因的启动子区受结合异种感应核受体CAR（组成型雄烷受体）、PXR（雄烷X受体）和肝脏富集受体（如HNF 4 α（HNF 4a））的元件调节。药物如利福平、圣约翰草、苯巴比妥和其他异生物质与CAR和PXR相互作用，CAR和PXR结合启动子中的元件，增加转录。新的研究（3）使用酵母双杂交筛选PXR作为诱饵发现E3泛素连接酶RBCK 1（与蛋白激酶C-1相互作用的环B盒卷曲蛋白）是人PXR相互作用蛋白。 用抗PXR和RBCK 1抗体的免疫共沉淀试验证实，RBCK 1和hPXR相互作用在AD-293细胞中共表达。 GST-pulldown实验证实了这两种蛋白之间存在相互作用。 泛素化研究证实，RBCK 1泛素化hPXR，推测其靶向蛋白酶体降解。 RBCK 1过表达降低HepG 2细胞内源性PXR水平。 原代人肝细胞中RBCK 1的过表达和沉默分别导致内源性PXR蛋白的减少和增加。 沉默RBCK 1增加PXR配体利福平对PXR靶基因的诱导。这些结果表明RBCK 1参与PXR的蛋白酶体降解。 其次，我们发现microRNAS 103和107对乳腺癌药物紫杉醇代谢酶CYP 2C 8有选择性的调节作用。(2)在人类肝细胞中。 此外，这些microRNA的水平与人类肝脏中的蛋白质呈负相关。 在一个正在进行的新项目中，双杂交筛选显示Med 25是介体复合物的成员，是一种HNF 4a结合蛋白。 我们已经证明Med 25对于RNA聚合酶II的募集是重要的，以选择HNF 4a激活的启动子集，例如重要的药物代谢基因细胞色素P450 2C 9（CYP 2C 9）。 我们假设这涉及Med 25和HNF 4之间的直接相互作用，以改变CYP 2C 9基因的染色质构象，使其处于转录活性状态。 构象变化需要被募集到靶基因的酶修饰组蛋白。 组蛋白修饰包括组蛋白N-末端尾部上赖氨酸和精氨酸氨基酸的甲基化或乙酰化。 例如，组蛋白3赖氨酸4二甲基化（H3 K4 me 2）与基因激活相关，而组蛋白3赖氨酸27三甲基化（H3 K27 me 3）是基因沉默的标志物。 在这项研究中，我们使用HepG 2细胞来确定Med 25在HNF 4a依赖性CYP 2C 9表达的表观遗传调控中的作用。 我们进行了染色质免疫沉淀，以确定组蛋白修饰在HNF 4a结合位点的Med 25蛋白水平。 我们的研究结果表明，改变Med 25的表达改变乙酰化和甲基化的某些组蛋白。. 然而，当用小发夹环RNAi沉默Med 25时，发生了作为原型基因沉默标记的修饰。 这些结果表明，Med 25在CYP 2C 9近端HNF 4结合位点诱导容许染色质状态。 类似地，共聚焦显微镜显示Med 25与关键组蛋白修饰标记共定位。 我们还确定了开放的CYP 2C 9染色质水平激活条件下使用甲醛辅助分离的调控元件（FAIRE）。 FAIRE数据表明，在激活核受体CAR和HNF 4a的存在下，CYP 2C 9近端启动子的HNF 4位点周围的染色质是开放的。 最近我们发现，亲电体和氧化应激通过AP-1位点激活CYP 2C 9。 已知许多药物被激活为亲电体。 这是CYP 2C 9激活的一种新机制。

We use studies in human liver cell lines and primary human hepatocytes to whow that clinical drugs, environmental chemicals and other xenobiotics transcriptionally upregulate expression of the CYP2C subfamily of drug-metabolizing enzymes (CYP2C8, 2C9, and 2C19) which then increases the metabolism of many clinical and over-the counter drugs as well as pesticides and herbicides. This data is consistent with in vivo studies in humans which have shown that exposure to certain drugs and herbal remedies decrease the half-life to many drugs. This can 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 shown that the promoter regions of the human CYP2C genes are regulated by elements which bind xenosensing nuclear receptors CAR (constitutive androstane receptor), PXR (pregnane X receptor), and liver-enriched receptors such as HNF4 alpha (HNF4a). Drugs such as rifampicin, St Johns Wort, phenobarbital and other xenobiotics interact with CAR and PXR which bind to elements in the promoter increasing transcription. New studies (3) using yeast two-hybrid screens with PXR as bait found an E3 ubiquitin Ligase, RBCK1 (Ring-B-box coiled-col proteini interactining with protein kinase C-1) to be a human PXR-interacting protein. Co-immunoprecipitation assays with antibodies to PXR and RBCK1 confirmed that RBCK1 and hPXR interacted were co-expressed in AD-293 cells. GST-pulldown assays confirmed that there was an interaction between the two proteins. Ubiquination studies confirmed that RBCK1 ubiquinates hPXR, presumably targeting it for proteasomal degradation. Overexpression of RBCK1 decreased endogenous levels of PXR in HepG2 CELLS. Overexpression and silencing of RBCK1 in primary human hepatocytes resulted in decreases and increases respectively of endogenous PXR protein. Silencing RBCK1 increases in induction of PXR target genes by the PXR ligand rifampicin. These results indicate RBCK1 is involved in the proteasomal degradation of PXR. Secondly we have tound that microRNAS 103 and 107 regulate CYP2C8 the enzyme which metabolizes the breast anticancer drug taxol translationally.(2) in human liver cells. Moreover the levels of these microRNAs is inversely related to protein in human liver. In an ongoing new project, two-hybrid screens showed that Med25, a member of the mediator complex, is an HNF4a-binding protein. We have shown that Med25 is important for the recruitment of RNA Polymerase II to select sets of HNF4a-activated promoters such as the important drug-metabolizing gene cytochrome P450 2C9 (CYP2C9). We hypothesized that this involves a direct interaction between Med25 and HNF4 to alter chromatin conformation of the CYP2C9 gene to a transcriptionally active state. Conformational change requires the modification of histones by enzymes that are recruited to target genes. Histone modifications include methylation or acetylation of lysine and arginine amino acids on histone N-terminal tails. For example, histone 3 lysine 4 dimethylation (H3K4me2) is associated with gene activation, while histone 3 lysine 27 trimethylation (H3K27me3) is a marker of gene silencing. In this study, we used HepG2 cells to determine the role of Med25 in the epigenetic regulation of HNF4a-dependent CYP2C9 expression. We performed chromatin immunoprecipitation to identify histone modifications at the HNF4a binding site in relation to Med25 protein levels. Our results indicate that altering Med25 expression modified acetylation and methylation of certain histones. . However, when Med25 was silenced with small-hairpin looped RNAi, the modifications occurred which were a prototypical gene-silencing marker. These results indicate that Med25 induces a permissive chromatin state at the CYP2C9 proximal HNF4 binding site. Similarly, confocal microscopy revealed that Med25 colocalized with key histone modification markers. We have also determined levels of open CYP2C9 chromatin under activating conditions using formaldehyde-assisted isolation of regulatory elements (FAIRE). FAIRE data indicated that the chromatin around the HNF4 sites of the CYP2C9 proximal promoter was open in the presence of activating nuclear receptors CAR and HNF4a. Recently we found that electrophiles and oxidative stress activate CYP2C9 through AP-1 sites. Many drugs are known to be activated to electrophiles. This is a new mechanism of activation of CYP2C9.