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

Loredana Petecchia, Federica Sabatini, Cesare Usai, Emanuela Caci, Luigi Varesio, Giovanni A. Rossi

Research output: Contribution to journalArticlepeer-review


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-α, IL-4 and IFN-γ 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-α (47±3%), IL-4 (43±1%) and INF-γ (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/cm 2) × 1000 in control cultures and in those exposed to TNF-α, IFN-γ 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.

Original languageEnglish
Pages (from-to)1140-1148
Number of pages9
JournalLaboratory Investigation
Issue number8
Publication statusPublished - Aug 2012


  • airway inflammation
  • IL-4
  • INF-γ
  • occludin
  • TNF-α
  • zonula occludens family

ASJC Scopus subject areas

  • Pathology and Forensic Medicine
  • Cell Biology
  • Molecular Biology


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