Although binge alcohol-induced liver injury has been studied extensively in the context of endotoxins and alcohol itself, it was recently revealed that circulating protein and RNA factors play an essential role as well. Ethanol- inducible cytochrome P450-2E1 (CYP2E1), a key enzyme in ethanol metabolism to generate Reactive Oxygen Species (ROS), promotes alcohol-induced hepatic steatosis and inflammatory liver disease, at least in part by mediating changes in intestinal permeability to endotoxins and release of extracellular proteins and RNAs into the blood stream. Specific miRNAs, including let-7 family miRNAs, have been shown to be amplified in serum from alcohol use disorder (AUD) cohorts having decreased liver function, suggesting that these miRNAs contribute to ethanol-induced injury of organs such as the liver. In my published studies and preliminary investigations, I made two key observations that contribute to my scientific premise that let-7b is released from the intestine through exosomes upon binge alcohol: 1) I demonstrated that the RNA-binding protein AUF1 binds mature let-7b miRNA, the ligand of Toll-like Receptor 7 (TLR7) in liver, and that this binding modulates its existence in cytosol and exosome; 2) I showed that the Serine/Threonine kinase MST1 mediates oxidative stress-induced phosphorylation of RNA-binding proteins such as AUF1 and is required for let-7b enrichment in blood exosomes after alcohol binging. This latter finding suggests that ROS-mediated signaling modulates interaction of AUF1 and miRNA through MST1-mediated phosphorylation. Based on these observations, I hypothesize that ethanol-induced activation of MST1 kinase results in the AUF1-dependent exosomal release of let-7b targeting hepatic TLR7, resulting in transcriptional activation of pro-inflammatory cytokines that cause liver injury through hepatic senescence. For this project, we will focus on elucidating the critical functions of AUF1 phosphorylation by MST1 in the dissociation of let-7b from AUF1, the release of let-7b from the intestine to the blood stream via cargo proteins QKI and hnRNP K, and subsequent hepatic injury.
Alcohol-induced liver injury occurs at multiple molecular levels such as penetration of endotoxins, circulation of cytokines, release of pro-inflammatory RNAs, and even directly by ethanol in the blood stream. We propose to study MST1-AUF1-let-7b signaling pathway that mediates alcohol's damaging effect on distant organs by regulating gut exosomal release in intestinal epithelial cells. The proposed studies will provide critical new insights into fundamental molecular mechanisms that mediate alcohol-induced injury in the liver as well as direct clinical relevance by targeting hepatic inflammatory receptors to develop new therapeutics.
