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Local inflammatory reactions affect the integrity of intestinal epithelial cells, such as E-cadherin-mediated cell-cell interactions. To elucidate this event, we investigated the effects of an inflammatory mediator, leukotriene D(4 )(LTD(4)), on the phosphorylation status and properties of vinculin, a multi-binding protein known to interact with both the E-cadherin-catenin complex and the cytoskeleton. Treatment of an intestinal epithelial cell line with LTD(4 )induced rapid tyrosine phosphorylation of vinculin, which was blocked by the Src family tyrosine kinase inhibitor PP1. Simultaneously, LTD(4) caused an increased association between vinculin and actin, and that association was decreased by PP1. LTD(4) also induced dissociation of vinculin from alpha-catenin without affecting the catenin complex itself. This dissociation was not blocked by PP1 but was mimicked by the protein kinase C (PKC) activator 12-O-tetradecanoylphorbol 13-acetate (TPA). Also, the PKC inhibitor GF109203X abolished both the LTD(4)- and the TPA-induced dissociation of vinculin from alpha-catenin. Furthermore, LTD(4) caused a colocalisation of vinculin with PKC-alpha in focal adhesions. This accumulation of vinculin was blocked by transfection with a dominant negative inhibitor of PKC (PKC regulatory domain) and also by preincubation with either GF109203X or PP1. Thus, various LTD(4)-induced phosphorylations of vinculin affect the release of this protein from catenin complexes and its association with actin, two events that are necessary for accumulation of vinculin in focal adhesions. Functionally this LTD(4)-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.
Massoumi et al. (Tue,) studied this question.