ERK1/2 activation modulates pyocyanin-induced toxicity in A549 respiratory epithelial cells

Abstract

Pyocyanin (PCN), a virulence factor produced by Pseudomonas aeruginosa, has many damaging effects on mammalian cells. Several lines of evidence suggest that this damage is primarily mediated by its ability to generate oxidative stress. However mechanisms underlying PCN-induced oxidative injury remain unclear. Although oxidative stress and subsequent MAPK signaling has been shown to modulate cell death in other models, its role in PCN-induced cytotoxicity remains unknown. Therefore the aim of this study was to investigate the role of redox-sensitive MAPK in PCN-induced toxicity in A549 cells. Here we show that PCN (50μM) rapidly increased ERK1/2 phosphorylation after 5min. Pre-treatment of A549 cells with the MEK1/2 inhibitor U0126 (10μM) decreased PCN-induced ERK1/2 phosphorylation and protected cells against apoptosis and cell injury suggesting a role for ERK signalling. In contrast, JNK and p38 MAPK phosphorylation remained unchanged following exposure to PCN and pretreatment with either the JNK or p38 MAPK inhibitors (10μM SP600125 and 10μM SB203580, respectively) did not afford protection against PCN toxicity. This would suggest that PCN-induced cytotoxicity appears to occur independently of JNK and p38 MAPK signaling pathways. Finally, although we confirm that oxidative stress contributes to PCN-induced toxicity, our data suggest the contribution of oxidative stress is independent of ERK1/2 signaling. These findings may provide insight for novel targeted therapies to reduce PCN-mediated lung injury in patients with chronic P. aeruginosa respiratory infections.

Keywords: A549 cells; CF; DMEM; Dulbecco’s modified eagle medium; ERK; Extracellular signal-regulated kinase (ERK); GSH; GSSG; MAPK; Mitogen-activated protein kinases (MAPK); N-acetylcyteine; NAC; Oxidative stress; PCN; Pyocyanin; ROS; cystic fibrosis; extracellular signal-regulated kinase; mitogen-activated protein kinases; oxidised glutathione; pyocyanin; reactive oxygen species; reduced glutathione; siRNA; small interfering RNA.