Cigarette smoke extract induces cytosolic phospholipase A2 expression via NADPH oxidase, MAPKs, AP-1, and NF-κB in human tracheal smooth muscle cells

Up-regulation of cytosolic phospholipase A(2) (cPLA(2)) by cigarette smoke extract (CSE) may play a critical role in airway inflammatory diseases. However, the mechanisms underlying CSE-induced cPLA(2) expression in human tracheal smooth muscle cells (HTSMCs) remain unknown. CSE induced cPLA(2) protein and mRNA expression, and ROS generation was attenuated by pretreatment with a reactive oxygen species (ROS) scavenger (N-acetylcysteine), or inhibitors of NADPH oxidase (diphenyleneiodoniun chloride, apocynin) and transfection with p47(phox) siRNA, suggesting that CSE-induced cPLA(2) expression was mediated through NADPH oxidase activation and ROS production in HTSMCs. Furthermore, CSE-induced cPLA2 expression was attenuated by pretreatment with the inhibitors of MEK1/2 (U0126), p38 MAPK (SB202190), and JNK (SP600125), which were further confirmed by transfection with siRNAs of JNK1, p42, and p38 to down-regulate the expression Of respective proteins and reduce cPLA(2) expression. Induction of cPLA(2) by CSE was attenuated by selective inhibitors of NF-kappa B (helenalin) and AP-1 (curcumin). Moreover, promoter assays revealed that increases of cPLA(2), NF-kappa B, and AP-1 luciferase activities Stimulated by CSE were attenuated by these inhibitors. These results suggest that in HTSMCs, CSE induced NADPH oxidase activation leading to phosphorylation of p42/p44 MAPK, p38 MAPK, and JNK. These reactions induced nuclear transcription NF-kappa B and AP-1 activities which were essential for CSE-induced cPLA(2) gene expression. (C) 2009 Elsevier Inc. All rights reserved.