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Abstract
Interferon gamma (IFNγ) 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 IFNγ 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. IFNγ (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 IFNγ 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 IFNγ treatment and siRNA studies showed this was dependent upon the Src-kinase isoform Fyn. E-cadherin ubiquitinylation and subsequent proteasomal degradation stimulated by IFNγ 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, IFNγ 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 IFNγ regulates E-cadherin stability and suggest a novel mechanism of disruption of epithelial cell contact, which could contribute to perturbed epithelial barrier function.
Highlights
The integrity of the intestinal epithelial monolayer constitutes an important regulated barrier that controls access of the gut microflora, an abundant population of commensal and potentially pathogenic microbes, to the mucosa and is a key modulator of immune-mediated inflammatory activity within the intestinal submucosa [1]
Given our data identifying a key role for the Src kinase Fyn in IFNc-evoked increases in epithelial barrier function [8], we sought to determine if Src activity was required for IFNc-evoked changes in E-cadherin expression and localization in T84 epithelia
As a comparison with tight junction structure, a 48 h treatment with IFNc resulted in the expected focal discontinuities in zonula occludens (ZO)-1 [21], that was unaffected by PP1 co-treatment (Figure 1)
Summary
The integrity of the intestinal epithelial monolayer constitutes an important regulated barrier that controls access of the gut microflora, an abundant population of commensal and potentially pathogenic microbes, to the mucosa and is a key modulator of immune-mediated inflammatory activity within the intestinal submucosa [1]. As the association between microbial-driven inflammation and cancer becomes more apparent, a greater awareness of mechanisms of altered gut epithelial function during inflammatory responses may lead to additional or improved treatment strategies. Interferon-gamma (IFNc) is a key inflammatory cytokine primarily secreted by T cells and natural killer (NK) cells that has a well-described role during intestinal inflammation [3,4]. IFNc may serve to exacerbate inflammatory responses via its effects on the epithelial barrier
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