Cytokines induce tight junction disassembly in airway cells via an EGFR-dependent MAPK/ERK1/2-pathway

Epithelial barrier permeability is altered in inflammatory respiratory disorders by a variety of noxious agents through modifications of the epithelial cell structure that possibly involve tight junction (TJ) organization. To evaluate in vitro whether pro-inflammatory cytokines involved in the pathogenesis of respiratory disorders could alter TJ organization and epithelial barrier integrity, and to characterize the signal transduction pathway involved Calu-3 airway epithelial cells were exposed to TNF-alpha, IL-4 and IFN-gamma to assess changes in: (a) TJ assembly, that is, occludin and zonula occludens (ZO)-1 expression and localization, evaluated by confocal microscopy; (b) apoptotic activity, quantified using terminal transferase deoxyuridine triphosphate nick-end labeling staining; (c) epithelial barrier integrity, detected as transmembrane electrical resistance and expressed as G(T) values; (d) epidermal growth factor receptor (EGFR)-dependent mitogen-activated protein (MAP) kinase (MAPK)/extracellular signal-regulated kinases (ERK)1/2 phosphorylation, assessed by western blotting. Exposure to cytokines for 48 h induced a noticeable downregulation of the TJ transmembrane proteins. The degree ZO-1 and occludin colocalization was 62 +/- 2% in control cultures and significantly decreased in the presence of TNF-alpha (47 +/- 3%), IL-4 (43 +/- 1%) and INF-gamma (35 +/- 3%). Although no apoptosis induction was detected following exposure to cytokines, changes in the epithelial barrier integrity were observed, with a significant enhancement in paracellular conductance. GT values were, respectively, 1.030 +/- 0.0, 1.300 +/- 0.04, 1.260 +/- 0.020 and 2.220 +/- 0.015 (mS/cm(2)) x 1000 in control cultures and in those exposed to TNF-alpha, IFN-gamma and IL-4. The involvement of EGFR-dependent MAPK/ERK1/2 signaling pathway in cytokine-induced damage was demonstrated by a significant increase in threonine/tyrosine phosphorylation of ERK1/2, already detectable after 5 min incubation. All these cytokine-induced changes were markedly prevented when Calu-3 cells were cultured in the presence of an EGFR inhibitor (AG1478, 1 mu M) or a MAP kinase inhibitor (U0126, 25 mu M). In conclusion, cytokine-induced epithelial injury includes TJ disassembly and epithelial barrier permeability alteration and involves the EGFR-dependent MAPK/ERK1/2 signaling pathway. Laboratory Investigation (2012) 92, 1140-1148; doi:10.1038/labinvest.2012.67; published online 14 May 2012