CDLD Alumni Research Project: Cyp2e1 regulation of ethanol-induced miRNAs in intestinal epithelial cells

Je-Hyun Yoon – Department of Biochemistry, Graduated CDLD 2020

Alcohol (EtOH), in particular acute EtOH exposure, is known to lead to gut leakage of bacteria leading to intestinal injury and hepatic inflammation. The mechanism(s) by which EtOH increases the permeability of the intestinal barrier and allows penetration of pathogens (“EtOH-induced gut leakage”) centers around P450-2E1 (CYP2E1), a prominent enzyme that converts EtOH to acetaldehyde, generates reactive oxygen species (ROS), and promotes intestinal cell injury (Figure 1). Importantly, and critical to the premise of this project, is that CYP2E1 is upregulated in intestinal epithelial cells after binge EtOH exposure. The overarching goal of this application is to elucidate the CYP2E1-dependent molecular mechanism(s) mediating EtOH-induced intestinal injury.

 

EtOH-related regulation of CYP2E1 function occurs at multiple molecular levels, including rapid mRNA degradation, post-transcriptional silencing, CYP2E1 release from the intestine. While specific miRNAs that repress expression of CYP2E1 transcripts have previously been described, their involvement in binge EtOH-induced gut injury has not been investigated. Recently, it has been shown that binge EtOH feeding activates the serine/threonine protein kinase, Mammalian Ste20-Like Kinase 1 (MST1; encoded by STK4), a critical regulator of mRNA decay and translation.

In preliminary studies, we have shown that CYP2E1-targeting miRNAs are down-regulated in response to binge EtOH-induced phosphorylation of RNA-binding protein AUF1 (AU-binding factor 1; encoded by HNRNPD), and that AUF1 phosphorylation is mediated by the MST1 Ser/Thr kinase. We have specifically demonstrated that EtOH reduces miR-132, miR-212, miR-378, and miR-552 expression, and that lower levels of these miRNAs lead to stabilization of CYP2E1 mRNA. The mechanism underlying their reduced expression is through MST1 phosphorylation of AUF1; in its non-phosphorylated state, it binds and stabilizes the expression of these miRNAs, preventing their degradation. Thus, EtOH-mediated phosphorylation of AUF1 by MST1 results in AUF1’s dissociation from the miRNAs, thereby promoting their turnover.

These findings support the existence of an MST1-AUF1-miRNA-CYP2E1 signaling axis that mediates EtOH’s damaging effect on gut permeability by regulating CYP2E1 levels in intestinal epithelial cells (Figure 1). We therefore hypothesize that EtOH-induced activation of MST1 results in phosphorylation of AUF1, causing the release and destabilization of miRNAs targeting CYP2E1. This in turn stabilizes the expression of CYP2E1 mRNA and promotes EtOH-induced intestinal injury.