Abnormally high epithelial permeability and oxidant-induced epithelial injury have been implicated in the pathogenesis of a wide spectrum of intestinal inflammatory disorders, including necrotizing enterocolitis and inflammatory bowel disease. We have recently demonstrated that oxidants at physiologically relevant concentrations increase paracellular permeability in Caco-2 and T84 cell monolayers. The oxidant induced increase in permeability was dependent on protein tyrosine phosphorylation, and it was inhibited by the presence of epidermal growth factor (EGF), a well recognized mucosal protective factor. On the basis of preliminary studies it is hypothesized that, a) oxidant induced increase in paracellular permeability is associated with an alteration of the tyrosine phosphorylation of occludin and E- cadherin (junctional proteins) at specific sites, b) oxidant induced alterations of occludin and E-cadherin phosphorylation is caused by the activation and membrane translocation of pp125FAK and pp60-c-src, and c) EGF delays oxidant-induced activation of pp125FAK and pp60-c-src alteration of tyrosine phosphorylation of occludin and E-cadherin, and dissociation of junctional complexes. Using the above mentioned model of oxidant injury, we propose to determine, 1) the specific site of tyrosine phosphorylation in occludin which undergoes dephosphorylation during oxidant treatment, 2) whether oxidants induce activation and translocation of pp125FAK and pp60-c-src from the cytosol to the membrane, 3) if modulation of pp60-c-src gene expression in Caco-2 cells alter the ability of oxidants to disrupt the barrier function, and 4) whether the mechanisms of EGF-mediated epithelial protection include a prevention of oxidant-induced activation and translocation of pp125FAK and pp60-c-src, alteration of the tyrosine phosphorylation status of occludin and E-cadherin, and dissociation of occludin/ZO-l and E- cadherin/beta-catenin complexes. Information derived from these studies has the potential to expand our understanding of oxidant-induced injury in intestinal epithelium, by identifying some of the mechanisms of oxidant-induced disruption of paracellular junctional complexes and epithelial protection by EGF.
Showing the most recent 10 out of 48 publications
