Regulation of transient receptor potential ankyrin-1 agonist-induced airway epithelial cell injury and repair by protein kinase C and p38 mitogen-activated kinase.

The airway epithelium must constantly adapt to maintain function, as disrupted epithelial homeostasis contributes to asthma and other respiratory diseases. Toxic electrophilic aldehydes, quinones, and substituted phenols are transient receptor potential ankyrin-1 (TRPA1) channel agonists found in common air pollutants, including wood and biomass smoke, cigarette smoke, and vehicle exhaust. TRPA1 agonists damage airway epithelial cells in part by disrupting calcium homeostasis and triggering endoplasmic reticulum stress. A detailed understanding of how inhaled toxicants affect epithelial homeostasis is critical for understanding their effects and managing adverse effects. We integrated results from kinase-targeted phenotypic screening, unbiased mass spectrometry-based global proteomics and phosphoproteomics, live-cell imaging, calcium flux assays, gene expression assays, and Western blot analyses to decipher systematic changes in kinase signaling that control TRPA1 agonist-induced injury. We show that injury coincides with changes in cell structure and motility overlaid with epidermal growth factor receptor (EGFR) transactivation, and that inhibiting kinase pathways known to favor survival and wound repair, including EGFR, extracellular signal-regulated kinase 1/2, phosphoinositide 3-kinase/protein kinase B, and c-Jun N-terminal kinases, exacerbated injury, while inhibiting protein kinase C and p38 mitogen-activated kinase, and others, reduced injury. We attributed the protective effects of protein kinase C and p38 mitogen-activated kinase inhibition to an attenuation of TRPA1 activity, balanced by enhanced transient receptor potential vanilloid-3 expression, resulting in a net shift toward proadaptive kinase-driven signaling downstream of EGFR. Our results support a model in which repair depends on kinase-regulated transitions through a transient injury state that could potentially be targeted prophylactically to reduce injury caused by selected inhaled toxicants. SIGNIFICANCE STATEMENT: This study shows that inhibiting protein kinase C and p38 mitogen-activated kinase reduces transient receptor potential ankyrin-1 activity and enhances transient receptor potential vanilloid-3 expression and prosurvival kinase signaling, favoring cell adaptation and repair following acute injury, offering insight into potential therapeutic approaches to limit transient receptor potential ankyrin-1 agonist-induced injury through a vital network of transient receptor potential channels.