Abstract
Tumor necrosis factor-alpha (TNF-alpha) signaling is central to the transmission of the innate immune response and subsequent activation of the adaptive immune system. The functioning of both systems is required for optimal clearance of pathogens from the airways. In cystic fibrosis (CF), dysfunction of the CF transmembrane conductance regulator (CFTR) is associated with recurrent pulmonary infections despite an intense inflammatory and immune response. We reported recently that TNF-alpha decreased gap junction connectivity in non-CF airway cells, a mechanism that was absent in CF cells expressing the DeltaPhe-508 mutant of CFTR. We have now identified the tyrosine kinase c-Src as a possible pathway between the mediators of inflammation and the gap junction protein connexin43 (Cx43). Indeed, TNF-alpha increased the proportion of activated c-Src in non-CF airway cells. Moreover, pharmacological antagonists and expression in non-CF cells of a dominant negative construct of c-Src prevented Cx43 channel closure by TNF-alpha. Finally, gap junction channel closure was prevented by expression of a Cx43 mutant lacking tyrosine phosphorylation sites for c-Src. Additional experiments showed that activation of c-Src was defective in CF airway cells but rescued in CFTR-corrected CF cells. These data suggest that CFTR dysfunction is associated with altered TNF-alpha signaling, resulting in the persistence of gap junction connectivity in CF airway cells. We propose that altered regulation of c-Src may contribute to the dysregulated inflammatory response that is characteristic of the CF phenotype.
Highlights
Tumor necrosis factor-␣ (TNF-␣) signaling is central to the transmission of the innate immune response and subsequent activation of the adaptive immune system
We reported recently that TNF-␣ decreased gap junction connectivity in non-cystic fibrosis (CF) airway cells, a mechanism that was absent in CF cells expressing the ⌬Phe-508 mutant of cystic fibrosis transmembrane regulator (CFTR)
Effects of TNF-␣ on Gap Junction Channel Activity in Airway Cells—We have reported previously that 100 units/ml TNF-␣, a concentration that maximally stimulates the release of IL-8, decreased gap junction connectivity in non-CF but not CF airway cells within 15 min by a yet undefined mechanism [26]
Summary
Cell Culture—The normal human bronchial epithelial Beas2B cell line was purchased from the American Type Culture Collection (Manassas, VA); the human nasal epithelial CF15 cell line, which was derived from a patient homozygous for the ⌬Phe-508 mutation of CFTR, was previously characterized by Jefferson and colleagues [39]. Dye Coupling—Dye coupling studies were performed on subconfluent monolayers of cells incubated in a solution (external solution) containing (in mM): 136 NaCl, 4 KCl, 1 CaCl2, 1 MgCl2, and 2.5 glucose and was buffered to pH 7.4 with 10 mM HEPES-NaOH. For dye coupling experiments performed in cell clusters expressing EGFP, a filter specific for fluorescein (excitation range 465– 490 nm) was used, and images of the fluorescent clusters were acquired before the injection of Lucifer Yellow. Because of the brighter intensity of the Lucifer Yellow signal, the image acquisition time was typically 10 times smaller than that used for EGFP, so that the green fluorescence of the cells was not detected by the camera. Electrical Coupling—For electrical coupling studies, the dual wholecell patch clamp approach was applied on pairs of cells incubated in the external solution. All data are shown as mean Ϯ S.E. and compared using unpaired t tests
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