Abstract
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.
Highlights
Bone homeostasis is maintained through elaborate bone remodeling via coordinated bone formation and bone resorption (Upadhyay et al, 2015)
Our study is focus on the molecular mechanism of RANKL-induced osteoclastogenesis and we found that RANKL upregulates the protein level of NADPH oxidase 4 (Nox4), enhancing the level of reactive oxygen species (ROS), activating PERK/eIF-2α/ATF4 pathway, leading to autophagy and osteoclastogenesis
We found that RANKL induced osteoclastogenesis via autophagy
Summary
Bone homeostasis is maintained through elaborate bone remodeling via coordinated bone formation and bone resorption (Upadhyay et al, 2015). Osteoclasts are the principal cells responsible for bone resorption (Chung et al, 2014). RANKL, one of the tumor necrosis factor superfamily (Pacifici, 2012), has been demonstrated to interact with the RANK receptor, expressed on osteoclast precursors, to activate multiple osteoclastogenesis-related signaling pathways (NF-κB, Src, MAPK, etc.), releasing nuclear transcription factors (NFATc1, AP-1, etc.) and regulating the expression of osteoclastogenesis-related genes, which induce the differentiation and maturation of osteoclasts in the bone microenvironment (Feng and Teitelbaum, 2013; Kimura et al, 2014). Denosumab, a monoclonal antibody with activity against RANKL, has been demonstrated to be effective in the prevention and treatment of osteolytic disease (Dahiya et al, 2015). The detailed mechanisms of osteoclastogenesis induced by RANKL remain unclear
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