Regulation of TRPA1 Agonist-Induced Airway Epithelial Cell Injury and Repair by PKC and p38 MAPK

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 (MS)-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 overlayed with EGFR transactivation, and that inhibiting kinase pathways known to favor survival and wound repair, including EGFR, ERK1/2, PI3K/AKT, and JNK kinases, exacerbated injury, while inhibiting protein kinase C (PKC) and p38 mitogen-activated kinase (p38 MAPK), and others, reduced injury. We attributed the protective effects of PKC and p38 MAPK inhibition to an attenuation of TRPA1 activity, balanced by enhanced TRPV3 expression, resulting in a net shift toward pro-adaptive 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.