Sustained expression of NADPH oxidase 4 by p38 MAPK-Akt signaling potentiates radiation-induced differentiation of lung fibroblasts

Radiation-induced fibrosis (RIF) is a long-term adverse effect of curative radiotherapy; however, the distinct molecular mechanisms of RIF in neighboring normal tissue are not fully understood. We investigated the mechanisms underlying radiation-induced fibroblast differentiation into myofibroblasts. Lung fibroblasts produced reactive oxygen species (ROS) immediately after irradiation, the level of which remained increased for 24 h. The NADPH oxidase inhibitor, diphenyleneiodonium (DPI), suppressed ROS production and significantly decreased the radiation-induced expression of α-smooth muscle actin (α-SMA) and fibronectin (FN). The mRNA and protein expression of Nox4 was increased by radiation, and siRNA knockdown of Nox4 reduced α-SMA and FN levels. Increased phosphorylation of p38MAPK, Erk, and PI3k/Akt was observed after irradiation. Inhibitors of p38 MAPK and Akt, but not of Erk, reduced radiation-induced fibroblast differentiation and Nox4 expression. Notably, DPI partially decreased phosphorylation of p38MAPK and Akt, suggesting that p38MAPK, Akt, and Nox4 may cooperate in a positive feedback loop. Nox4 expression was also increased during bleomycin-induced fibroblast differentiation, and downregulation of Nox4 reduced α-SMA levels and extracellular matrix (ECM) accumulation. These results demonstrate that interfering Nox4 activation can be a potential strategy to disrupt fibrotic process.