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.

Basuroy S, Dunagan M, Sheth P, Seth A, Rao RK. Hydrogen peroxide activates focal adhesion kinase and c-Src by a phosphatidylinositol 3 kinase-dependent mechanism and promotes cell migration in Caco-2 cell monolayers. Am J Physiol Gastrointest Liver Physiol 299: G186-G195, 2010. First published April 8, 2010 doi:10.1152/ajpgi.00368.2009.-Recent studies showed that c-Src and phosphatidylinositol 3 (PI3) kinase mediate the oxidative stress-induced disruption of tight junctions in Caco-2 cell monolayers. The present study evaluated the roles of PI3 kinase and Src kinase in the oxidative stress-induced activation of focal adhesion kinase (FAK) and acceleration of cell migration. Oxidative stress, induced by xanthine and xanthine oxidase system, rapidly increased phosphorylation of FAK on Y397, Y925, and Y577 in the detergent-insoluble and soluble fractions and increased its tyrosine kinase activity. The PI3 kinase inhibitors, wortmannin and LY294002, and the Src kinase inhibitor, 4-amino-5[chlorophyll]-7-[tbutyl]pyrazolo [3-4-d]pyrimidine, attenuated tyrosine phosphorylation of FAK. Oxidative stress induced phosphorylation of c-Src on Y418 by a PI3 kinase-dependent mechanism, whereas oxidative stress-induced activation of PI3 kinase was independent of Src kinase activity. Hydrogen peroxide accelerated Caco-2 cell migration in a concentration-dependent manner. Promotion of cell migration by hydrogen peroxide was attenuated by LY294002 and PP2. Reduced expression of FAK by siRNA attenuated hydrogen peroxide-induced acceleration of cell migration. The expression of constitutively active c-Src Y527F enhanced cell migration, whereas the expression of dominant negative c-Src K296R/Y528F attenuated hydrogen peroxide-induced stimulation of cell migration. Oxidative stress-induced activation of c-Src and FAK was associated with a rapid increase in the tyrosine phosphorylation and the levels of paxillin and p130 CAS in actin-rich, detergent-insoluble fractions. This study shows that oxidative stress activates FAK and accelerates cell migration in an intestinal epithelium by a PI3 kinase-and Src kinase-dependent mechanism. epithelium; barrier function; cell motility; protein phosphorylation A SIGNIFICANT BODY OF EVIDENCE indicates that oxidative stress disrupts epithelial tight junctions and adherens junctions, leading to an increase in the paracellular permeability in Caco-2 and Madin-Darby canine kidney (MDCK) cell monolayers (16 -19). Oxidative stress induces tyrosine phosphorylation of a wide spectrum of proteins, including occludin, zonula occludens-1, E-cadherin, and ␤-catenin and tyrosine kinase inhibitors attenuate the oxidative stress-induced increase in paracellular permeability (1,16,19). Previous studies demonstrated that oxidative stress activates c-Src and phosphatidylinositol (PI3) kinase, and these activities are involved in the oxidative stress-induced disruption of tight junctions and increase in paracellular permeability in Caco-2 and MDCK cell monolayers (2, 23).Focal adhesion kinase (FAK) plays an importan...

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Oxidative stress plays a crucial role in carcinogenesis and tumor metastasis. Numerous studies demonstrated that H2O2 destabilizes intercellular junctions, a process that is involved in cell migration. In the present study, we evaluated the effect of H2O2 on epithelial differentiation and cell migration in Caco‐2 cells. Epithelial differentiation was induced by 3D growth of Caco‐2 cells on matrigel and cell migration analyzed by scratch wound method. H2O2 (100‐250 μM) was administered to the medium. Src kinase inhibitor, PP2 (3 μM) and PI3‐kinase inhibitor, LY294002 (25 μM) were added 30 min prior to H2O2. The role of c‐Src was determined by stable expression of constitutively active c‐Src(pY418). Cell morphology recorded by phase contrast microscopy and the intercellular junctions assessed by confocal microscopy for occludin and ZO‐1. Results show that H2O2 dose‐dependently accelerated cell migration, which was attenuated by PP2 or LY294002, suggesting that H2O2 promotes cell migration by a c‐Src and PI3‐kinase‐dependent mechanism. H2O2‐induced cell migration was associated with redistribution of occludin and ZO‐1 from the intercellular junctions, which was also attenuated by PP2 and LY294002. Expression of c‐Src(pY418) significantly accelerated cell migration. Caco‐2 cells on Matrigel grew to form glandular structure enclosing a cavity. However, in the presence of H2O2 Caco‐2 cells grew into an irregular clump of cells with no cavity; this was associated with irregular distribution of occludin and ZO‐1. These results suggest that oxidative stress may prevent epithelial differentiation and promote cell migration.

helped me with various aspects of the project. Chris Waters allowed me to use his microscope, even on weekends. Anjaparavanda Naren provided use of equipment and advice. Susan Senogles and Gabor Tigyi offered valuable advice and criticism as my committee members. Larry Tague and Clark Blatteis offered valuable career guidance, lunch debate, and experimental assistance. The Physiology department of the University of Tennessee Health Science Center has offered a supportive working environment, and I appreciate the assistance and training from all.