Benzo(a)pyrene induces MUC5AC expression through the AhR/mitochondrial ROS/ERK pathway in airway epithelial cells

Abstract

Objectives: Benzo(a)pyrene (BaP) is a ubiquitous air pollutants, and BaP exposure leads to a risk of respiratory diseases. The oversecretion of airway mucus and high expression of mucin 5AC (MUC5AC) are associated with common respiratory disorders caused by air pollution. We aimed to investigate the effect of BaP on MUC5AC expression, especially the mechanisms by which BaP induces MUC5AC gene expression.

Methods: The human airway epithelial cell NCI-H292 was used to test the effects of BaP on the expression of MUC5AC in vitro. MUC5AC mRNA and protein expression were assessed with real-time quantitative PCR, immunochemistry, and western blotting. A luciferase assay was conducted to detect the activity of the promoter. The total cellular ROS and mitochondrial ROS were measured by corresponding probes. Small-interfering RNAs were used for gene silencing. AhR-overexpressing cell lines were constructed by transfection with AhR overexpression lentivirus.

Results: We found that BaP stimulation upregulated the MUC5AC mRNA and protein levels and activated the ERK pathway. Suppressing ERK with U0126 (an ERK inhibitor) or knocking down ERK with siRNA decreased BaP-induced MUC5AC expression. The luciferase activity transfected with the MUC5AC promoter and cAMP-response element (CRE) was increased after BaP treatment, whereas CREB siRNA suppressed the BaP-induced overexpression of MUC5AC. In addition, BaP increased mitochondrial ROS production, and Mito-TEMP, a mitochondrial ROS inhibitor, inhibited BaP-induced MUC5AC expression and ERK activation. BaP increased the mRNA levels of CYP1A1 and CYP1B1, while Alizarin, a CYP1s inhibitor, suppressed the effects of BaP, including the MUC5AC overexpression, ERK activation and mitochondrial ROS generation. BaP induced the translocation of aryl hydrocarbon receptor (AhR) from the cytoplasm to the nucleus. SiRNA-mediated knockdown or chemical inhibition of AhR decreased the BaP-induced expression of MUC5AC, while the overexpression of AhR significantly enhanced the BaP-induced expression of MUC5AC. ITE, an endogenous AhR ligand, also upregulated the mRNA and protein expression of MUC5AC. Furthermore, resveratrol treatment inhibited the BaP-induced MUC5AC overexpression, AhR translocation, mitochondrial ROS production and ERK pathway activation.

Conclusion: Here, we highlighted the crucial role of AhR/mitochondrial ROS/ERK pathway activation in BaP-induced MUC5AC overexpression and identified resveratrol as a promising drug to reduce BaP-induced MUC5AC overexpression.

Keywords: Aryl hydrocarbon receptor; Benzo(a)Pyrene; Mucin 5AC; Reactive oxygen species; Resveratrol.