title: Expression of kinase-inactive c-Src delays oxidative stress-induced disassembly and accelerates calcium-mediated reassembly of tight junctions in the Caco-2 cell monolayer.
abstract: The activity of Src kinases appears to play a role in both assembly and disassembly of tight junction. However, the role of a specific isoform of Src kinase in regulation of tight junction is not known. In the present study the role of c-Src in regulation of epithelial tight junction was investigated in Caco-2 cell monolayers. Oxidative stress (xanthine oxidase + xanthine) induced an activation and membrane translocation of c-Src. The oxidative stress-induced decrease in transepithelial electrical resistance, increase in inulin permeability, and redistribution of occludin and ZO-1 from the intercellular junctions were prevented by PP2. The rates of oxidative stress-induced activation of c-Src, tyrosine phosphorylation of ZO-1 and beta-catenin, decrease in resistance, increase in permeability to inulin, and redistribution of occludin and ZO-1 were significantly greater in cells transfected with wild type c-Src, whereas it was low in cells transfected with kinase-inactive c-SrcK297R mutant, when compared with those in empty vector-transfected cells. The rates of recovery of resistance, increase in barrier to inulin, and reorganization of occludin and ZO-1 into the intercellular junctions during the calcium-induced reassembly of tight junction were much greater in Caco-2 cells transfected with c-SrcK297R as compared with those in cells transfected with empty vector or wild type c-Src. These results show that the dominant-negative expression of kinase-inactive c-Src delays the oxidative stress-induced disruption of tight junction and accelerates calcium-induced assembly of tight junction in Caco-2 cells and demonstrate that oxidative stress-induced disruption of tight junction is mediated by the activation of c-Src.
authors.affiliation: Department of Physiology, University of Tennessee Health Sciences Center, Memphis, Tennessee 38163, USA.
authors.forename: Shyamali
authors.initials: S
authors.lastname: Basuroy
authors.forename: Parimal
authors.initials: P
authors.lastname: Sheth
authors.forename: Dhan
authors.initials: D
authors.lastname: Kuppuswamy
authors.forename: Sundar
authors.initials: S
authors.lastname: Balasubramanian
authors.forename: Ramesh M
authors.initials: RM
authors.lastname: Ray
authors.forename: Radhakrishna K
authors.initials: RK
authors.lastname: Rao
mesh_terms: D000818:Animals; D000081247:CSK Tyrosine-Protein Kinase; D018938:Caco-2 Cells; D002118:Calcium / Q000378:metabolism*; D002463:Cell Membrane Permeability / Q000502:physiology; D003598:Cytoskeletal Proteins / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D007444:Inulin / Q000493:pharmacokinetics; D008565:Membrane Proteins / Q000378:metabolism; D062793:Occludin; D018384:Oxidative Stress / Q000502:physiology*; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000235:genetics* / Q000378:metabolism*; D019108:Tight Junctions / Q000201:enzymology*; D015534:Trans-Activators / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism; D062826:Zonula Occludens-1 Protein; D051176:beta Catenin; D019061:src-Family Kinases
chemical_list: C495270:CTNNB1 protein, human; D003598:Cytoskeletal Proteins; D008565:Membrane Proteins; C568985:OCLN protein, human; D062793:Occludin; D010750:Phosphoproteins; C569042:TJP1 protein, human; D015534:Trans-Activators; D062826:Zonula Occludens-1 Protein; D051176:beta Catenin; D014443:Tyrosine; D007444:Inulin; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases; C000631346:CSK protein, human; D002118:Calcium