151 TRPA1/V3-Dependent Regulation of Airway Epithelial Cell Damage and Repair Triggered by wood/Biomass Smoke Particulate

Abstract

Abstract Wood/biomass smoke particulate material (WBSPM) is a pervasive environmental pollutant. Acute high-dose inhalation exposure can cause airway epithelial cell (AEC) damage and respiratory dysfunction. We hypothesized that AEC damage is limited by compensatory signaling to allow epithelial repair. RNA sequencing, proteomics, targeted interventions, and cultured human AECs treated with WBSPM and Transient Receptor Potential Ankyrin-1 (TRPA1) agonists revealed TRPA1-driven endoplasmic reticulum stress (ERS) and oxidative stress (OS) as contributory pathways to cytotoxicity. Alternatively, TRP vanilloid-3 (TRPV3) counteracted these events despite also being activated by WBSPM. Using the TRPA1 agonist AITC (and others) as a surrogate for WBSPM, morphological distortion indicative of AEC damage was observed 2-4h post-treatment; at >6h cells recovered. Strikingly, at 2h, the abundance of only 6 proteins changed including loss of fibronectin and tubulin and increased heme oxygenase 1. At 6h, 36 proteins changed, including ER chaperones and transcriptional/cell cycle regulatory proteins, OS-response, and structural/matrix proteins. Analysis of the phosphoproteome revealed changes in 2672 and 1886 unique phosphosites at 2 and 6h, attributable to PKC, p38 MAPK, EGFR, Akt and other kinase activities based on “phosphosite-GSEA” analysis. A kinase inhibitor screen linked PKC, p38 MAPK, GSK3b and ERK1/2 with cytotoxicity, while EGFR, MEK1/2, JNK1/2/3 and Akt were protective. Considering that TRPV3 expression is regulated by EGFR following WBSPM injury and TRPV3 enhanced adhesion and slowed proliferation via attenuated EGFR signaling, we propose a mechanism whereby the balance of TRPA1, TRPV3, and calcium/growth factor receptor-dependent kinase signaling coordinates WBSPM injury, compensation and repair.