NADPH oxidase subunit p22phox: A marker of oxidase-dependent oxidative stress and target for stress suppression in nonphagocytic cells

Reactive oxygen species (ROS)-producing NADPH oxidase (Nox) family proteins are involved in host defense. The overproduction of ROS leads to oxidative stress, which is associated with a myriad of diseases. The Nox subunit p22^phox is essential for Nox1–4 activity, and p22^phox and Nox2 proteins are mutually stabilized in phagocytic cells. This study investigated the suitability of p22^phox protein as a marker of Nox activity. To avoid contamination by phagocytic p22^phox, we developed global Cybb (encoding Nox2)-knockout mice and analyzed p22^phox stability and the expression profiles of Nox proteins in lysates of various tissues. We found that consistent with Nox2 in phagocytic cells, p22^phox protein was detected when Nox1–4 were coexpressed in nonphagocytic cells. Furthermore, p22^phox protein degradation was suppressed by Nox1–4, suggesting that p22^phox is a suitable marker of Nox family-dependent oxidative stress. Thus, we examined p22^phox protein levels in tissue lysates prepared from Cybb-knockout mice to avoid the contamination of phagocytic p22^phox. Cybb-knockout mice show moderately reduced p22^phox protein levels in lung tissue, suggesting that Nox2 and other Nox family members stabilized p22^phox protein. Paradoxically, this result implied that p22^phox knockdown concurrently suppressed various Nox family-dependent oxidative stress mechanisms, and this was confirmed by the suppression of Nox family-dependent directed migration in p22^phox-knockdown A549 human lung epithelial cells. Therefore, p22^phox not only served as a marker of Nox-dependent oxidative stress but also as a target to suppress this stress in various tissues and cells.