Abstract
The redox-active pyocyanin (PCN) secreted by the respiratory pathogen Pseudomonas aeruginosa generates reactive oxygen species (ROS) and causes oxidative stress to pulmonary epithelial cells. Nuclear factor (erythroid-derived 2)-like 2 (NRF2) confers protection against ROS-mediated cell death by inducing the expression of detoxifying enzymes and proteins via its binding to the cis-acting antioxidant response element (ARE). However, a clear relationship between NRF2 and PCN-mediated oxidative stress has not been established experimentally. In this study, we investigated the induction of NRF2-ARE response by PCN in the pulmonary epithelial cells. We analyzed the effect of PCN on NRF2 expression and nuclear translocation in cultured human airway epithelial cells, and in a mouse model of chronic PCN exposure. NRF2-dependent transcription of antioxidative enzymes was also assessed. Furthermore, we used inhibitors to examine the involvement of EGFR and its downstream signaling components that mediate NRF2-ARE-activation in response to PCN. PCN enhances the nuclear NRF2 accumulation and activates the transcription of ARE-mediated antioxidant genes. Furthermore, PCN activates NRF2 by inducing the EGFR-phosphoinositide-3-kinase (PI3K) signaling pathway and its main downstream effectors, AKT and MEK1/2-ERK1/2 MAP kinases. Inhibition of the EGFR-PI3K signaling markedly attenuates PCN-stimulated NRF2 accumulation in the nucleus. We demonstrate for the first time that PCN-mediated oxidative stress activates the EGFR-PI3K-AKT/MEK1/2-ERK1/2 MAP kinase signaling pathway, leading to nuclear NRF2 translocation and ARE responsiveness in pulmonary epithelial cells.
Highlights
Pseudomonas aeruginosa (PA) is an important bacterial pathogen causing acute nosocomial infections in immunocompromised patients, and chronic recurring lung infections in patients with cystic fibrosis (CF) or non-CF bronchiectasis [1,2]
We demonstrate that PCN activates the EGFR-PI3K-AKT/MEK1/ 2-ERK1/2 signaling pathway, leading to increased nuclear translocation of Nuclear factor (erythroid-derived 2)-like 2 (NRF2), with corresponding increase in the transcription of antioxidant response element (ARE)-containing c-GCSh and NQO1 genes in the lung epithelial cells
Three lines of evidence support these conclusions: (i) Western blotting and immunofluorescence analyses show that clinically-relevant concentrations of PCN induce the translocation and accumulation of NRF2 from cytoplasm to nuclei of lung epithelial cells at both concentration and time-dependent manners; (ii) Chronic exposure to PCN enhances nuclear translocation of NRF2 in airway epithelial cells developing goblet cell hyperplasia and metaplasia (GCHM) and mucus hypersecretion; (iii) Increased levels of nuclear NRF2 are correlated with enhanced expression of two NRF2-ARE controlled antioxidant enzymes, NQO1 and c-GCSh
Summary
Pseudomonas aeruginosa (PA) is an important bacterial pathogen causing acute nosocomial infections in immunocompromised patients, and chronic recurring lung infections in patients with cystic fibrosis (CF) or non-CF bronchiectasis [1,2]. Pyocyanin (PCN), a blue redox-active secondary metabolite, is an important virulence factor in the pathogenesis of pseudomonal lung diseases. Chronic instillation of PCN causes goblet cell hyperplasia and metaplasia (GCHM), fibrosis, destruction of alveolar airspace and influx of neutrophils in mouse lungs, pathophysiological features that resemble those observed in the CF and non-CF bronchiectatic lungs [10,11]. The virulence effects of PCN are mediated by its ability to induce the formation of ROS, which oxidatively damage pulmonary epithelial cells [12,13,14,15]. Upon entry into host cells, PCN undergoes a non-enzymatic reduction by NADPH or other reducing agents and rapidly reacts with molecular oxygen to produce superoxide [16] and, by dismutation, hydrogen peroxide [17]. Superoxide could react with nitric oxide to form highly toxic reactive nitrogen species that damage DNA, proteins and phospholipids and modulate host immune response [18]
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