Leukotriene D-4 affects localisation of vinculin in intestinal epithelial cells via distinct tyrosine kinase and protein kinase C controlled events

Local inflammatory reactions affect the integrity of intestinal epithelial cells, such as E-cadherin-mediated cell-cell interactions. To elucidate thi s event, we investigated the effects of an inflammatory mediator, leukotrie ne D-4 (LTD4), on the phosphorylation status and properties of vinculin, a multi-binding protein known to interact with both the E-cadherin-catenin co mplex and the cytoskeleton, Treatment of an intestinal epithelial cell line with LTD4 induced rapid tyrosine phosphorylation of vinculin, which was bl ocked by the Src family tyrosine kinase inhibitor PP1, Simultaneously, LTD4 caused an increased association between vinculin and actin, and that assoc iation was decreased by PP1, LTD4 also induced dissociation of vinculin fro m alpha -catenin without affecting the catenin complex itself. This dissoci ation was not blocked by PP1 but was mimicked by the protein kinase C (PKC) activator 12-O-tetradecanoylphorbol 13-acetate (TPA), Also, the PKC inhibi tor GF109203X abolished both the LTD4- and the TPA-induced dissociation of vinculin from alpha -catenin, Furthermore, LTD4 caused a colocalisation of vinculin with PKC-alpha in focal adhesions. This accumulation of vinculin w as blocked by transfection with a dominant negative inhibitor of PKC (PKC r egulatory domain) and also by preincubation with either GF109203X or PP1, T hus, various LTDs-induced phosphorylations of vinculin affect the release o f this protein from catenin complexes and its association with actin, two e vents that are necessary for accumulation of vinculin in focal adhesions. F unctionally this LTD4-induced redistribution of vinculin was accompanied by a PKC-dependent upregulation of active beta1 integrins on the cell surface and an enhanced beta1 integrin-dependent adhesion of the cells to collagen IV.