Azithromycin-mediated CHIP regulation promotes proteasomal degradation of NOX4

Introduction: Transforming growth factor (TGF)-b is one of the major profibrotic cytokines orchestrating lung fibrogenesis. NADPH oxidase 4 (NOX4) play a pivotal role in TGF-b signaling via reactive oxygen species (ROS) production in association with lung fibrosis development. We hypothesized that anti-fibrotic properties of azithromycin (AZM) may be attributed to regulating NOX4 protein levels. Methods: Using human lung fibroblasts (LFB), western blotting (WB) of α-smooth muscle actin (SMA) and typeⅠcollagen was performed to evaluate TGF-b-induced myofibroblast differentiation. Si RNA-mediated knockdown experiments were performed to elucidate the role of C terminus of HSC70-Interacting Protein (CHIP), a known E3 ubiquitin ligase for NOX4. 20S proteasome activity was measured by adding the fluorogenic substrates for proteasome. Bleomycin (BLM)-induced lung fibrosis models were used to evaluate anti-fibrotic properties of AZM. Results: AZM suppressed TGF-b-induced NOX4 protein expression and myofibroblast differentiation in LFB. AZM significantly increased CHIP protein levels with concomitantly enhanced proteasome activity. CHIP knockdown attenuated AZM-induced colocalization of NOX4 and ubiquitin, resulting in restoration of NOX4 reduction during AZM treatment. BLM-induced NOX4 expression and lung fibrosis were efficiently attenuated by AZM treatment. Conclusion: AZM-mediated NOX4 degradation is governed through NOX4 ubiquitination by CHIP and enhanced proteasomal degradation. AZM-mediated NOX4 regulation is responsible for attenuating TGF-b-induced myofibroblast differentiation and lung fibrosis development by BLM. AZM may also be used for the treatment of the fibrotic lung disease, IPF.