The reactive oxygen species (ROS)-producing NADPH oxidase (Nox) family plays important roles in host defense (Song et al., 2023; Paclet et al., 2022; Dumas and Knaus, 2021; Panday et al., 2015). Nox-derived ROS serve as microbicidal agents (Nauseef, 2019) and redox signaling molecules in host defense (Aviello and Knaus, 2018). Additionally, redox signaling processes are involved in angiogenesis (Craige et al., 2011), lung wound healing (Bernard et al., 2014), and mucosal wound healing (Fu et al., 2014) during the later stages of injury. However, ROS overproduction leads to oxidative stress, which is associated with a myriad of diseases (Schröder, 2024; Bernard et al., 2014).

In phagocytic cells, Nox2 produces ROS that act as powerful microbicidal agents. The heterodimerization of Nox2 with p22phox (Yu et al., 1997; DeLeo et al., 2000; Kawai et al., 2023) results in enzymatic activation (Zhu et al., 2006), protein stabilization (Kawai et al., 2023; Parkos et al., 1989; Porter et al., 1994; Miyano et al., 2021a), and cellular localization of enzymatically active Nox2 (De Leo et al., 2000). Nox2 or p22phox gene deficiency leads to chronic granulomatous disease (CGD), and affected individuals are highly susceptible to severe, recurrent staphylococcal infections (Roos, 2019). Interestingly, p22phox protein is absent in phagocytes from patients with Nox2-deficient CGD (Zhen et al., 1993; Bu-Ghanim et al., 1995; Yu et al., 1999), indicating that p22phox forms a mutually stabilizing complex with Nox2.

Phagocytic oxidase Nox2 is responsible for phagocyte-mediated host defense (Paclet et al., 2022; Song et al., 2023). In nonphagocytic cells, Nox family proteins, including Nox2, are involved in various biological processes. ROS produced by the Nox family act as signal molecules in oxidative stress/redox signaling during tissue homeostasis and disease (Nazari et al., 2023; Dumas and Knaus, 2021; Buvelot et al., 2019). Additionally, Nox-dependent oxidative stress regulates the migration of endothelial (Craige et al., 2011; Miyano et al., 2020a) and epithelial cells (Kwon et al., 2016; Miyano et al., 2020b), playing a key role in wound healing (Lopez et al., 2022). It is also involved in disease progression in tissues and physiological processes (Jiang et al., 2017; Begum et al., 2022; Meitzler et al., 2014). Thus, determination of Nox-derived ROS levels in healthy tissues is important because Nox family activity is either enhanced or suppressed during disease progression in tissues (Jiang et al., 2017; Begum et al., 2022). The expression level of Nox protein is directly related to the level of ROS production. However, the measurement of Nox-derived ROS is challenging because of its rapid metabolization and low production levels in nonphagocytic cells. Furthermore, except for Nox2 (Kawai et al., 2022; Kawai et al., 2018), few commercially available antibodies detect the Nox family with sufficiently high sensitivity for analysis via immunoblotting.

Nox1, Nox3, and Nox4 (but not Nox5) heterodimerize with p22phox in the same manner as Nox2, and this is necessary for Nox enzymatic activity (Miyano et al., 2021a; Ambasta et al., 2004; Kawahara et al., 2005; Nakano et al., 2007; von Löhneysen et al., 2008; Zana et al., 2018; Miyanoet al., 2022). Thus, if p22phox forms a mutually stabilizing complex with Nox1, Nox3, and Nox4, as is the case with Nox2, it would be valuable to determine the expression pattern of the Nox subunit p22phox in different types of healthy tissues because its presence would indicate the presence of Nox family proteins, which are difficult to detect at the protein level. Highly sensitive antibodies against p22phox are commercially available, including CS9 monoclonal antibodies (Taylor et al., 2004) and polyclonal antibodies (Miyano et al., 2021a; Miyano et al., 2022). However, measurement is difficult because the background of p22phox protein is high due to contamination by phagocytic cells like neutrophils and macrophages during tissue lysate preparation.

This study aimed to investigate p22phox protein as a marker of Nox activity by analyzing its stability and the tissue expression profiles of Nox family proteins in global Cybb (encoding Nox2)-knockout mice in which the Cybb gene was deleted to prevent contamination by phagocytic p22phox. We also investigated the migration capacity of A549 human lung epithelial cells in the presence and absence of Nox-dependent redox signaling.

We concluded that (i) the measurement of p22phox protein is useful to confirm Nox1–4-dependent oxidative stress in various tissues and (ii) p22phox suppression is effective in inhibiting Nox1–4-dependent oxidative stress in response to biological events.