Acetaldehyde and Epithelial Junctions: Role of PP2A and Occludin Phosphorylation

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DESCRIPTION (provided by applicant): Acetaldehyde is a toxic metabolite of ethanol consumption. It can disrupt tight junctions between intestinal epithelial cells, allowing toxins into the body. This raises the risk of alcoholic liver disease and certain cancers. This disruption involves dephosphorylation of occludin and other tight junction proteins. PP2A, a serine/threonine phosphatase, is known to play a role in regulating tight junctional integrity. My preliminary data suggests that acetaldehyde destabilizes tight junctions and adherens junctions and promotes epithelial- to-mesenchymal transition by a PP2A-dependent mechanism. The long-range goal of this project is to elucidate the mechanisms involved in the ethanol-induced increase in risk for colon cancer. My first specific aim is to determine the role of PP2A and Thr-dephosphorylation of occludin, a tight junction protein, in acetaldehyde-induced disruption of tight junctions. This will be achieved by examining acetaldehyde-induced disruption of tight junctions with transepithelial resistance, inulin flux, and immunofluorescence confocal microscopy. Protection by fostriecin, a PP2A inhibitor, and by siRNA to knock down PP2A expression, will be used to examine the role of PP2A in disruption of tight junctions and in promotion of cell migration by acetaldehyde. My second specific aim is to determine that acetaldehyde induces epithelial-to-mesenchymal transition(EMT) by a PP2A-dependent mechanism. We will examine acetaldehyde-induced EMT by phase- contrast and immunofluorescence confocal microscopy as well as RT-PCR and Western blot to reveal changes in gene transcription and protein expression. We will use fostriecin and the generation and growth of occludin threonine mutants under 2D and 3D culture conditions to examine the roles of PP2A and threonine dephosphorylation in acetaldehyde-induced EMT. PUBLIC HEALTH RELEVANCE: Acetaldehyde is a toxin produced in the colon when a person drinks alcoholic beverages. This toxin opens the tight junction, a barrier between intestinal cells that protects the body from substances in the colon. Disruption of the barrier increases the risk of liver disease, pancreatitis, and cancer, so I am studying how acetaldehyde breaks connections between tight junction proteins and how to keep these connections intact.