Nonphagocytic oxidase 1 causes death in lung epithelial cells via a TNF-RI-INK signaling axis

Airway epithelial cells are simultaneously exposed to and produce cytokines and reactive oxygen species (ROS) in inflammatory settings. The signaling events and the physiologic outcomes of exposure to these inflammatory mediators remain to be elucidated. Previously we demonstrated that in cultured mouse lung epithelial cells exposed to bolus administration of H2O2, TNF-alpha-induced NF-kappa B activity was inhibited, whereas c-Jun-N-terminal kinase (INK) activation was enhanced via a mechanism involving TNF receptor-1 (TNF-RI). In this study we used the nonphagocytic NADPH oxidase (Nox1) to study the effects of endogenously produced ROS on a line of mouse alveolar type II epithelial cells. Nox1 expression and activation inhibited TNF-alpha-induced inhibitor of kappa B kinase (IKK), and NF-kappa B while promoting JNK activation and cell death. Nox1-induced INK activation and cell death were attenuated through expression of a dominant-negative TNF-RI construct, implicating a role for TNF-RI in Nox1 signaling. Furthermore, Nox1 used the TNF-RI adaptor protein TNF-receptor-associated factor-2 (TRAF2), and the redox-regulated INK MAP3K, apoptosis signal kinase-1 (ASK1), to activate INK. In addition, ASK1 siRNA attenuated both Nox1-induced INK activity and cell death. Collectively, these studies suggest a mechanism by which ROS produced in lung epithelial cells activate INK and cause cell death using TNF-RI and the TRAF2-ASK1 signaling axis.