Multiple Mechanisms of Hepatoprotective Mrp3 Induction

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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.