Reduced surface expression of epithelial E-cadherin evoked by interferon-gamma is Fyn kinase-dependent.
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.
Gastrointestinal Research Group, Department of Physiology & Pharmacology, Calvin, Phoebe and Joan Snyder Institute for Chronic Diseases, University of Calgary, Calgary, Alberta, Canada.
David
D
Smyth
Gabriella
G
Leung
Maria
M
Fernando
Derek M
DM
McKay
D015820:Cadherins / Q000096:biosynthesis* / Q000235:genetics; D002460:Cell Line; D003106:Colon / Q000166:cytology / Q000378:metabolism*; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000502:physiology*; D006801:Humans; D007371:Interferon-gamma / Q000235:genetics / Q000378:metabolism*; D007413:Intestinal Mucosa / Q000166:cytology / Q000378:metabolism*; D055550:Protein Stability; D051076:Proto-Oncogene Proteins c-fyn / Q000235:genetics / Q000378:metabolism*
D015820:Cadherins; C573415:IFNG protein, human; D007371:Interferon-gamma; C494957:FYN protein, human; D051076:Proto-Oncogene Proteins c-fyn