While binge alcohol-induced gut leakage has been studied extensively in the context of reactive oxygen species (ROS)-mediated signaling, it was recently revealed that post-transcriptional regulation plays an essential role as well. Ethanol-inducible cytochrome P450-2E1 (CYP2E1) is a key enzyme in ethanol metabolism, and promotes alcohol-induced hepatic steatosis and inflammatory liver disease at least in part by mediating changes in intestinal permeability. For instance, gut leakage and elevated intestinal permeability to endotoxins have been shown to be regulated by enhancing CYP2E1 mRNA and CYP2E1 protein levels. Although it is understood that ethanol promotes CYP2E1 induction and activation, the mechanisms by which CYP2E1 expression is regulated in the context of intestinal damage remain poorly defined. Specific miRNAs, including miR-132, miR-212, miR- 378, and miR-552, have been shown to repress the expression of CYP2E1, suggesting that these miRNAs contribute to ethanol-induced intestinal injury. In my published studies and preliminary investigations, I made two key observations that contribute to my scientific premise that CYP2E1 expression is regulated post- transcriptionally through miRNA-mediated degradation: 1) I demonstrated that the RNA-binding protein AUF1 binds mature miRNAs, including CYP2E1-targeting miRNAs, and that this binding modulates the degradation of corresponding target mRNAs; 2) I showed that the Serine/Threonine kinase MST1 mediates oxidative stress- induced phosphorylation of RNA-binding proteins such as AUF1. This finding suggests that ROS-mediated signaling modulates AUF1/miRNA interaction through MST1-mediated phosphorylation. Based on these observations, I hypothesize that ethanol-induced activation of the MST1 kinase results in the AUF1-dependent destabilization of miRNAs targeting CYP2E1 mRNA, resulting in transcript stabilization and increased levels of CYP2E1 protein which cause intestinal injury through damage to the epithelial barrier. For this project in the COBRE for Digestive & Liver Disease, we will focus on elucidating the critical functions of AUF1 phosphorylation by MST1 in the decay of miRNAs targeting CYP2E1, the stabilization of CYP2E1 mRNA in the presence of ethanol, and subsequent intestinal injury.
