Abstract
The respiratory epithelium is subject to continuous environmental stress and its responses to injury or infection are largely mediated by transactivation of the epidermal growth factor receptor (EGFR) and downstream signaling cascades. Based on previous studies indicating involvement of ATP-dependent activation of the NADPH oxidase homolog DUOX1 in epithelial wound responses, the present studies were performed to elucidate the mechanisms by which DUOX1-derived H2O2 participates in ATP-dependent redox signaling and EGFR transactivation. ATP-mediated EGFR transactivation in airway epithelial cells was found to involve purinergic P2Y2 receptor stimulation, and both ligand-dependent mechanisms as well as ligand-independent EGFR activation by the non-receptor tyrosine kinase Src. Activation of Src was also essential for ATP-dependent activation of the sheddase ADAM17, which is responsible for liberation and activation of EGFR ligands. Activation of P2Y2R results in recruitment of Src and DUOX1 into a signaling complex, and transient siRNA silencing or stable shRNA transfection established a critical role for DUOX1 in ATP-dependent activation of Src, ADAM17, EGFR, and downstream wound responses. Using thiol-specific biotin labeling strategies, we determined that ATP-dependent EGFR transactivation was associated with DUOX1-dependent oxidation of cysteine residues within Src as well as ADAM17. In aggregate, our findings demonstrate that DUOX1 plays a central role in overall epithelial defense responses to infection or injury, by mediating oxidative activation of Src and ADAM17 in response to ATP-dependent P2Y2R activation as a proximal step in EGFR transactivation and downstream signaling.
Highlights
The respiratory epithelium forms a first line defense against inhaled pathogens and pollutants, and has developed intricate innate response mechanisms against diverse environmental challenges to provide important initial host defense and to protect airway structure and function
epidermal growth factor (EGFR) phosphorylation at Y845 implies the involvement of Src, which was confirmed by assessing tyrosine auto-phosphorylation at Y416, which indicated that ATP dosedependently enhanced Src phosphorylation at Y416 (Fig. 1D), whereas phosphorylation at Y527, which is associated with inhibition of Src, was not significantly affected by ATP
The present studies highlight the central involvement of the NADPH oxidase DUOX1 in EGFR transactivation in tracheobronchial epithelial cells in response to exogenous ATP, and thereby further establish DUOX1 as a critical factor in innate defense mechanisms within the respiratory epithelium
Summary
The respiratory epithelium forms a first line defense against inhaled pathogens and pollutants, and has developed intricate innate response mechanisms against diverse environmental challenges to provide important initial host defense and to protect airway structure and function. EGFR activation in response various diverse environmental or microbial stresses typically involves the initial stimulation of various G-protein-coupled receptors (GPCR), which promotes EGFR transactivation by as yet incompletely understood mechanisms involving ligand-independent intracellular mechanisms as well as activation of EGFR ligands by ADAM (a disintegrin and metalloproteinase) family sheddases [4,5,6,7]. One GPCR family of particular interest in the context of epithelial injury and wound responses includes purinergic receptors, which are activated by epithelial release of ATP in response to both mechanical and molecular stresses [8,9], and are critical in epithelial responses to injury or infection promoting mucociliary clearance and stimulating cellular repair mechanisms [8,10,11,12], and transactivation of EGFR has been implicated in these ATP-mediated wound responses in various cell systems [13,14,15]
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