title: Cytokines induce tight junction disassembly in airway cells via an EGFR-dependent MAPK/ERK1/2-pathway.
abstract: 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-a, IL-4 and IFN-g 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 mitogenactivated 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-a (47±3%), IL-4 (43±1%) and INF-g (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. G(T) values were, respectively, 1.030±0.0, 1.300±0.04, 1.260±0.020 and 2.220±0.015 (mS/cm²)1000 in control cultures and in those exposed to TNF-a, IFN-g 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 μM) or a MAP kinase inhibitor (U0126, 25 μM). In conclusion, cytokine-induced epithelial injury includes TJ disassembly and epithelial barrier permeability alteration and involves the EGFR-dependent MAPK/ERK1/2 signaling pathway.
authors.affiliation: Pulmonary Disease Unit, G. Gaslini Institute, Genoa, Italy. loredanapetecchia@gmail.com
authors.forename: Loredana
authors.initials: L
authors.lastname: Petecchia
authors.forename: Federica
authors.initials: F
authors.lastname: Sabatini
authors.forename: Cesare
authors.initials: C
authors.lastname: Usai
authors.forename: Emanuela
authors.initials: E
authors.lastname: Caci
authors.forename: Luigi
authors.initials: L
authors.lastname: Varesio
authors.forename: Giovanni A
authors.initials: GA
authors.lastname: Rossi
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D016207:Cytokines / Q000378:metabolism*; D004249:DNA Damage; D017097:Electric Impedance; D004847:Epithelial Cells / Q000166:cytology; D066246:ErbB Receptors / Q000378:metabolism*; D006801:Humans; D020287:In Situ Nick-End Labeling; D020935:MAP Kinase Signaling System; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D008856:Microscopy, Fluorescence; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D062793:Occludin; D010750:Phosphoproteins / Q000378:metabolism; D011817:Rabbits; D020545:Respiratory Mucosa / Q000166:cytology / Q000201:enzymology / Q000378:metabolism; D019108:Tight Junctions / Q000201:enzymology / Q000378:metabolism*; D062826:Zonula Occludens-1 Protein
chemical_list: D016207:Cytokines; D008565:Membrane Proteins; C568985:OCLN protein, human; D062793:Occludin; C568986:Ocln protein, mouse; D010750:Phosphoproteins; C569042:TJP1 protein, human; C569043:Tjp1 protein, mouse; D062826:Zonula Occludens-1 Protein; C512478:EGFR protein, human; D066246:ErbB Receptors; D020929:Mitogen-Activated Protein Kinase Kinases