Acetaldehyde disrupts tight junctions in Caco-2 cell monolayers by a protein phosphatase 2A-dependent mechanism

Abstract

Acetaldehyde is accumulated at high concentrations in the colonic lumen following ethanol administration. Previous studies demonstrated that acetaldehyde disrupts intestinal epithelial tight junctions and increases paracellular permeability. In the present study, we investigated the role of PP2A in the acetaldehyde-induced disruption of intestinal epithelial tight junctions. Caco-2 cell monolayers were exposed to 200-600 μM acetaldehyde for varying times, and the epithelial barrier function was evaluated by measuring transepithelial electrical resistance and inulin permeability. Acetaldehyde treatment resulted in a time-dependent increase in inulin permeability and redistribution of occludin and ZO-1 from the intercellular junctions. Treatment of cells with fostriecin (a PP2A-selective inhibitor) or knockdown of PP2A by siRNA blocked acetaldehyde-induced increase in inulin permeability and redistribution of occludin and ZO-1. The effects of fostriecin and acetaldehyde were confirmed in mouse intestine ex vivo. Acetaldehyde-induced tight junction disruption and barrier dysfunction were also attenuated by a PP2A-specific inhibitory peptide, TPDYFL. Coimmunoprecipitation studies showed that acetaldehyde increased the interaction of PP2A with occludin and induced dephosphorylation of occludin on threonine residues. Fostriecin and TPDYFL significantly reduced acetaldehyde-induced threonine dephosphorylation of occludin. Acetaldehyde failed to change the level of the methylated form of PP2A-C subunit. However, genistein (a tyrosine kinase inhibitor) blocked acetaldehyde-induced association of PP2A with occludin and threonine dephosphorylation of occludin. These results demonstrate that acetaldehyde-induced disruption of tight junctions is mediated by PP2A translocation to tight junctions and dephosphorylation of occludin on threonine residues.

Role of defective methylation reactions in ethanol-induced dysregulation of intestinal barrier integrity

“…Despite the important role that intact intestinal barrier integrity plays in preventing organ injury, the molecular mechanism responsible for ethanol-induced gut leakiness remains an ongoing area of research [12,[26][27][28][38][39][40][41][42][43][44][45][46][47][48][49][50][51]. It has been established that the loss of the molecular architecture of TJ which is a complex interaction of transmembrane proteins occludin, claudins with cytoplasmic adapter protein ZO-1 and a cytoskeletal network [13][14][15]52], regulates barrier function [12,[26][27][28][38][39][40][41][42][43][44][45][46][47][48][49][50][51].…”

“…It has been established that the loss of the molecular architecture of TJ which is a complex interaction of transmembrane proteins occludin, claudins with cytoplasmic adapter protein ZO-1 and a cytoskeletal network [13][14][15]52], regulates barrier function [12,[26][27][28][38][39][40][41][42][43][44][45][46][47][48][49][50][51]. Using Caco-2 cells that exhibits this complex protein barrier, we show that ethanol exposure severely compromises the function of this barrier by affecting the localization of the major tight junction protein, occludin-1.…”

“…TEER was recorded using Endohm EVOM epithelial tissue voltohmmeter (World Precision Instruments) on cell monolayers in 6.5 mm diameter transwell inserts before and after 24 h of treatment as described before [16,[26][27][28]. Percent increase and decrease in TEER was calculated by subtracting the TEER value obtained after treatment from the TEER value obtained before treatment.…”

“…These cells spontaneously differentiate into a monolayer of cells that morphologically resemble normal small intestinal enterocytes with a defined apical brush border and a highly organized TJ network [25]. This cell model has been extensively used for studying structural barrier integrity by conducting microscopic and Western blot analysis of TJ proteins and determining the functional barrier integrity using transepithelial electrical resistance (TEER) and paracellular solute flux measurements [16,[26][27][28][29].…”

Abnormal ultrastructure of intestinal epithelial barrier in mice with alcoholic steatohepatitis

“…Alcohol could induce redistribution of tight junction and adherens junction proteins from the intercellular junctions and disrupt AJC by altering phosphorylation status, including regulation to protein tyrosine/PKC/RhoA (Atkinson & Rao, 2001;Basuroy, Sheth, Mansbach, & Rao, 2005;Dunagan, Chaudhry, Samak, & Rao, 2012;Keshavarzian et al, 2009;Li et al, 2013;Tong, Wang, Chang, Zhang, Liu, et al, 2013;. Increased expression of inducible nitric oxide synthase (iNOS) by alcohol disrupts intestinal barrier function because of increased levels of disassembled tubulin, which subsequently damage the microtubule cytoskeleton and disrupt barrier function in alcohol-treated CaCo 2 cells (Banan, Fields, Decker, Zhang, & Keshavarzian, 2000;Tang, Forsyth, Farhadi, et al, 2009).…”