Reduced Surface Expression of Epithelial E-Cadherin Evoked by Interferon-Gamma Is Fyn Kinase-Dependent

Interferon gamma (IFN gamma) is an important regulatory cytokine that can exert a pro-inflammatory effect in the gut, where it has been shown to increase epithelial permeability via disruption of the tight junctions. Here we investigated the potential for IFN gamma to regulate the adherens junction protein E-cadherin, an important mediator of normal epithelial tissue function, using the model T84 human colonic epithelial cell line. IFN gamma (10 ng/ml) stimulated increased internalization of E-cadherin as assessed by immunofluorescence microscopy; internalization was reversed when cells were treated with PP1 (125 nM), a Src kinase-selective inhibitor. Immunoprecipitation studies demonstrated loss of E-cadherin from membrane fractions following IFN gamma treatment and a corresponding increase in cytosolic E-cadherin and its binding partners, p120-catenin and beta-catenin: effects that were Src-kinase dependent. E-cadherin and p120-catenin phosphorylation was increased by IFN gamma treatment and siRNA studies showed this was dependent upon the Src-kinase isoform Fyn. E-cadherin ubiquitinylation and subsequent proteasomal degradation stimulated by IFN gamma was found to be dependent upon Fyn and the E-cadherin-selective ubiquitin ligase, Hakai. Use of Fyn and Hakai siRNA inhibited the internalization of E-cadherin as shown by immunoblotting and confocal fluorescence microscopy. Finally, IFN gamma treatment resulted in a more fragile T84 cell monolayer with increased cell detachment in response to physical stress, which was prevented by PP1 and siRNA targeting Fyn or Hakai. Collectively, these results demonstrate a Fyn kinase-dependent mechanism through which IFN gamma regulates E-cadherin stability and suggest a novel mechanism of disruption of epithelial cell contact, which could contribute to perturbed epithelial barrier function.