Nox4 Promotes RANKL-Induced Autophagy and Osteoclastogenesis via Activating ROS/PERK/eIF-2α/ATF4 Pathway

Receptor activator of nuclear factor-κB ligand (RANKL) has been found to induce osteoclastogenesis and bone resorption. However, the underlying molecular mechanisms remain unclear. Via conducting a series of biochemical experiments with in vitro cell lines, this study investigated the role and mechanism of NADPH Oxidase 4 (NOX4) in RANKL-induced Autophagy and osteoclastogenesis. In the current study, we found that RANKL dramatically induced Autophagy and osteoclastogenesis, inhibition of Autophagy with chloroquine (CQ) markedly attenuates RANKL-induced osteoclastogenesis. Interestingly, we found that the protein level of NOX4 was remarkably upregulated by RANKL treatment. Inhibition of NOX4 by 5-O-methyl quercetin or knockdown of NOX4 with specific shRNA markedly attenuated RANKL-induced Autophagy and osteoclastogenesis. Furthermore, we found that NOX4 stimulated the production of nonmitochondrial Reactive Oxygen Species (ROS), activating the critical unfolded protein response (UPR)-related signaling pathway PERK/eIF-2α/ATF4, leading to RANKL-induced Autophagy and osteoclastogenesis. Blocking the activation of PERK/eIF-2α/ATF4 signaling pathway either by NOX4 shRNA, ROS scavenger (NAC) or PERK Inhibitor (GSK2606414) significantly inhibited Autophagy during RANKL-induced osteoclastogenesis. Collectively, this study reveals that NOX4 promotes RANKL-induced Autophagy and osteoclastogenesis via activating ROS/PERK/eIF-2α/ATF4 pathway, suggesting that the pathway may be a novel potential therapeutic target for osteoclastogenesis-related disease.