Multiple Mechanisms of Hepatoprotective Mrp3 Induction

该项目的目标是确定MRP3在肝脏保护反应中转录激活的机制(S)。MRP3定位于肝细胞的窦状膜，从肝脏向血液中输出广泛的I-有机阴离子。因此，肝脏MRP3的作用是降低肝脏中潜在有毒分子的浓度。MRP3在肝脏中的表达通常很低，但在暴露于某些诱导剂后以及在胆汁淤积期间显著增加，进一步证明了MRP3介导的输出作为一种肝脏保护手段的重要性。胆汁淤积性肝损伤造成巨大的临床负担，每年估计导致100,000人死亡，通常被列为多器官功能障碍。目前对胆汁淤积症的临床治疗包括苯巴比妥，它可以减少胆汁淤积的副作用对肝脏的毒性。我之前已经演示过 用多个CYP2B1/2的还原剂(由CAR转录激活)，如苯巴比妥，以及NADP(H)的诱导剂：苯醌氧化还原酶(通过Nrf2激活)也能够诱导MRP3，显示出对I期药物代谢基因和III期异源转运体的协调调节。研究还表明，胆汁淤积导致MRP3水平升高，但降低了CYP2B1/2水平，这表明参与MRP3调控的机制不同。此外，胆汁淤积症的其他结果包括氧化应激增加、高胆红素血症、炎症和细胞因子释放，所有这些单独影响异生转运体或I期药物代谢基因的调节，其方式与胆汁淤积症的效果一致。因此， 为了验证这一假说，即在化学伤害和胆汁淤积应激期间，MRP3都受到不同的调控：1)确定构成雄激素受体在微粒体酶诱导剂激活MRP3表达的转录激活中的作用。2)确定胆汁淤积过程中MRP3的诱导是否由NRF2的促氧化剂或抗氧化剂激活所介导 在胆汁淤积过程中，MRP3的释放、信号转导和核因子-kB的激活参与了MRP3的不同调控。4)确定在化学损伤和胆汁淤积应激过程中，负责转录激活的mrp3启动子元件。了解控制mrp3介导的有机阴离子排泄的机制可能会为科学界创造安全和生物活性的药物提供潜在的服务，从而减轻 特定的运输缺陷或上调患者或接触者的化学物质的排泄。

The goal of this project is to determine the mechanism(s) by which Mrp3 is transcriptionally activated in a hepatoprotective response. Mrp3, which is localized to the sinusoidal membrane of hepatocytes, exports a wide range of I organic anions from the liver, into the blood. Thus, the role of hepatic Mrp3 is to decrease the concentration of potentially toxic molecules in the liver. Mrp3 expression is normally low in liver but is significantly increased after exposure to certain inducers and also during cholestasis, further demonstrating the importance of Mrp3-mediated export as a means of hepatoprotection. Cholestatic liver injury results in a substantial clinical burden leading to an estimated 100,000 deaths per year often listed as multiple organ dysfunction. The current clinical management of chotestasis includes phenobarbital, which decreases hepatotoxicity as a side effect of cholestasis. I have previously demonstrated that treatment with multiple reducers of CYP2B 1/2, (transcriptionally activated by CAR) such as phenobarbital, as well as inducers of NADP(H):quinone oxidoreductase (activated through Nrf2) are also capable of inducing Mrp3, showing coordinate regulation of both the Phase I drug-metabolizing genes and Phase III xenobiotic transporters. It has also been demonstrated that cholestasis results in increased Mrp3 but decreased CYP2B 1/2 levels suggesting distinct mechanisms involved in the regulation of Mrp3. Additionally, other outcomes of cholestasis include an increase in oxidative stress, hyperbilirubinemia, inflammation and cytokine releasc, all of which, individually, effect the regulation of xenobiotic transporters or Phase I drug metabolizing genes in a manner consistent with the effects of cholestasis. Therefore, the following aims have been designed to test the hypothesis that Mrp3 is differentially regulated during periods of both chemical insult and cholestatic stress: 1) Determine the role of the Constitutive Androstane Receptor in transcriptional activation by microsomal enzyme inducers that activate Mrp3 expression. 2) Determine whether the induction of Mrp3 during cholestasis is mediated by either prooxidant or antioxidant activation of Nrf2.3) Determine whether cytokine release, signaling, and NF-kB activation are responsible for the differential regulation of Mrp3 during cholestasis. 4) Define the Mrp3 promoter elements that are responsible for the transcriptional activation during both chemical insult and cholestatic stress. Understanding the mechanisms that control Mrp3-mediated excretion of organic anions can potentially serve the scientific community in our objective to create safe and biologically active drugs that alleviate specific transport deficiencies or up-regulate the excretion of chemicals in patients or exposed individuals.