Interferon-γ-induced increases in intestinal epithelial macromolecular permeability requires the Src kinase Fyn

Interferon-gamma (IFN gamma) is an important immunoregulatory cytokine that can also decrease intestinal epithelial barrier function. Little is known about the intracellular signalling events immediately subsequent to IFN gamma/IFN gamma receptor interaction that mediate increases in epithelial permeability; data that could be used to ablate this effect of IFN gamma while leaving its immunostimulatory effects intact. This study assessed the potential involvement of Src family kinases in IFN gamma-induced increases in epithelial permeability using confluent filter-grown monolayers of the human colon-derived T84 epithelial cell line. Inhibition of Src kinase with the pharmacologic PP1 and use of Fyn kinase-specific siRNA significantly reduced IFN gamma-induced increases in epithelial permeability as gauged by translocation of noninvasive E. coli (HB101 strain) and flux of horseradish peroxidase (HRP) across monolayers of T84 cells. However, the drop in transepithelial resistance elicited by IFN gamma was not affected by either treatment. Immunoblotting revealed that IFN gamma activated the transcription factor STAT5 in T84 cells, and immunoprecipitation studies identified an IFN gamma-inducible interaction between STAT5b and the PI3K regulatory subunit p85 alpha through formation of a complex requiring the adaptor molecule Gab2. siRNA targeting STAT5b and Gab2 reduced IFN gamma-induced increases in epithelial permeability and phosphorylation of PI3K(p85 alpha). PP1 and Fyn siRNA reduced IFN gamma-induced PI3K activity (indicated by decreased phospho-Akt) and the formation of the STAT5b/PI3K(p85 alpha) complex. Collectively, the results suggest the formation of a Fyn-dependent STAT5b/Gab2/PI3K complex that links IFN gamma to PI3K signalling and the regulation of macromolecular permeability in a model enteric epithelium. Laboratory Investigation (2011) 91, 764-777; doi:10.1038/labinvest.2010.208; published online 14 February 2011