Multiple Mechanisms of Hepatoprotective Mrp3 Induction

本项目的目标是确定Mrp 3在肝保护反应中转录激活的机制。mrp 3定位于肝细胞的窦状膜，从肝脏输出广泛的I有机阴离子进入血液。因此，肝脏Mrp 3的作用是降低肝脏中潜在毒性分子的浓度。Mrp 3的表达通常在肝脏中较低，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Mrp 3介导的输出作为肝脏保护手段的重要性。胆汁淤积性肝损伤导致大量的临床负担，导致每年估计100，000例死亡，通常被列为多器官功能障碍。目前临床上对胆汁淤积的治疗包括苯巴比妥，它可以降低胆汁淤积的副作用肝毒性。我之前已经证明了 用CYP 2B 1/2的多种还原剂（通过CAR转录激活）如苯巴比妥以及NADP（H）的诱导剂：醌氧化还原酶（通过Nrf 2激活）处理也能够诱导Mrp 3，显示I相药物代谢基因和III相外源性转运蛋白的协调调节。还已经证明，胆汁淤积导致Mrp 3增加，但CYP 2B 1/2水平降低，表明参与Mrp 3调节的不同机制。此外，胆汁淤积的其他结果包括氧化应激、高胆红素血症、炎症和细胞因子释放的增加，所有这些单独地以与胆汁淤积的作用一致的方式影响异生物质转运蛋白或I相药物代谢基因的调节。因此 已经设计了以下目的来检验Mrp 3在化学损伤和胆汁淤积性应激期间受到差异调节的假设：1）确定组成型雄甾烷受体在激活Mrp 3表达的微粒体酶诱导剂的转录激活中的作用。2)确定胆汁淤积期间Mrp 3的诱导是否由Nrf 2的促氧化剂或抗氧化剂活化介导。 释放、信号传导和NF-kB活化负责胆汁淤积期间Mrp 3的差异调节。4)定义在化学损伤和胆汁淤积应激期间负责转录激活的Mrp 3启动子元件。了解控制Mrp 3介导的有机阴离子排泄的机制可能有助于科学界实现我们的目标，即创造安全且具有生物活性的药物， 特定的运输缺陷或上调患者或暴露个体的化学品排泄。

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.