The unfolded protein an integrated stress response in cigarette smoke-induced interference in Wnt/β-catenin signaling

Abstract

Background: Failure of endogenous repair mechanisms, such as the canonical Wnt/β-catenin pathway, may contribute to COPD pathogenesis. ER stress may interfere with Wnt/β-catenin signaling, whereas COPD and cigarette smoke (CS) exposure are associated with ER stress. Furthermore, the unfolded protein response (UPR) and the integrated stress response (ISR) are activated by oxidative stress and infection. Therefore, our aim was to determine the effect of CS and inducers of the UPR /ISR on Wnt/β-catenin signaling. Methods: MRC-5 fibroblasts were exposed to thapsigargin (Tg; 100 nM), tunicamycin (Tm; 5 µg/ml) or CS extract (CSE; expressed as arbitrary units; AU/ml) and stimulated for 6 or 24 hours with the Wnt/β-catenin activator CHIR99021. Expression of markers for the UPR/ISR, oxidative stress and Wnt/β-catenin signaling was measured by qRT-PCR. Results: Stimulation with Tm, Tg and CSE induced expression of the ISR markers GADD34 and CHOP at 6 and 24h in MRC-5 fibroblasts. Expression of the UPR markers BiP and spliced XBP1 was increased by Tg and Tm, but not by CSE stimulation, suggesting that CSE causes an ER stress-independent activation of the ISR. Exposure to CSE induced the expression of the oxidative stress marker HMOX-1. Expression of the Wnt/β-catenin target Axin-2 was decreased by Tg, Tm, exposure to CSE at 6 and 24h. Conclusion: We conclude that CSE causes a dose-dependent increase in oxidative stress and in expression of the ISR markers GADD34 and CHOP and this was accompanied by disturbed Wnt/β-catenin signaling. In the future, we will investigate which eIF2α kinase is responsible for CS-induced ISR activation and disturbed Wnt signaling.