Post-translational modifications to hemidesmosomes in human airway epithelial cells following diacetyl exposure

Abstract

Abstract Diacetyl (DA; 2,3-butanedione) is a highly reactive alpha (α)-diketone. Inhalation exposure to DA can cause significant airway epithelial cell injury, however, the mechanisms of toxicity remain poorly understood. The purpose of these experiments was to assess for changes in abundance and distribution of hemidesmosome-associated proteins following DA exposure that contribute to DA-induced epithelial toxicity. Human bronchial epithelial cells were grown in submerged cultures and exposed to three occupationally-relevant concentrations of DA (5.7, 8.6, or 11.4 mM) for 1 h. Following DA exposure, epithelial cells were cultured for 4 days to monitor for cell viability by MTT and WST-1 assays as well as for changes in cellular distribution and relative abundance of multiple hemidesmosome-associated proteins, including keratin 5 (KRT5), plectin (PLEC), integrin alpha 6 (ITGα6) and integrin beta 4 (ITGβ4). Significant toxicity developed in airway epithelial cells exposed to DA at concentrations ≥ 8.6 mM. DA exposure resulted in post-translational modifications to hemidesmosome-associated proteins with KRT5 crosslinking and ITGβ4 cleavage. Following DA exposure at 5.7 mM, these post-translational modifications to KRT5 resolved with time. Conversely, at DA concentrations ≥ 8.6 mM, modifications to KRT5 persisted in culture with decreased total abundance and perinuclear aggregation of hemidesmosome-associated proteins. Significant post-translational modifications to hemidesmosome-associated proteins develop in airway epithelial cells exposed to DA. At DA concentrations ≥ 8.6 mM, these hemidesmosome modifications persist in culture. Future work targeting hemidesmosome-associated protein modifications may prevent the development of lung disease following DA exposure.