RANKL drives autophagy and osteoclast differentiation via ROS-mediated HIF-1α down-regulation

Abstract

Objective‍ ‍To investigate the molecular mechanism by which receptor activator of nuclear factor-κB ligand (RANKL) regulates autophagy and osteoclast differentiation through the ROS-HIF-1α signaling axis. Methods‍ ‍Mouse monocytic/macrophage RAW264.7 cells were randomly divided into control group, RANKL group, and 3 combined intervention groups, that is, RANKL+CQ group (intervention with autophagy inhibitor, CQ), RANKL+FG-4592 group (intervention with HIF-1α stabilizer, FG-4592), and RANKL+NAC group (intervention with ROS scavenger, NAC). The number of mature multinucleated osteoclasts was quantitatively detected with tartrate-resistant acid phosphatase (TRAP) staining; the protein levels of HIF-1α and LC3 were detected by Western blotting and the mRNA levels of osteoclast differentiation-related genes (TRAP, Cath K and MMP-9) were analyzed by qRT-PCR; autophagy flux and activity were observed by adenovirus mRFP-GFP-LC3 labeling system combined with confocal laser microscopy; and the production of intracellular reactive oxygen species (ROS) was measured by DCFH-DA probe. Results‍ ‍Compared with the control group, the RANKL group obtained significantly increased number of TRAP-positive multinucleated osteoclasts (P<0.05), up-regulated mRNA levels of osteoclast differentiation-related genes, activated autophagy pathway, as evidenced by the increased LC3-Ⅱ/LC3-Ⅰ ratio (P<0.05) and number of autophagic lysosomes (P<0.05), elevated intracellular ROS generation (P<0.05), and down-regulated HIF-1α protein level (P<0.05). NAC intervention significantly inhibited intracellular ROS accumulation (P<0.05), reversed the lowered expression of HIF-1α protein (P<0.05), decreased the LC3-Ⅱ/LC3-Ⅰ ratio (P<0.05) and autophagy flux and activity (P<0.05), and reduced expression levels of osteoclast differentiation-related genes and the number of mature osteoclasts (P<0.05). In the RANKL+FG-4592 group, HIF-1α protein level was significantly increased (P<0.05), autophagy activation was inhibited [with decreased LC3-Ⅱ/LC3-Ⅰ ratio (P<0.05) and autophagy flux and activity (P<0.05)], and the expression of osteoclast differentiation-related genes and number of mature osteoclasts were decreased (P<0.05) when compared with the RANKL group. While CQ treatment resulted in inhibited autophagy flux and activity (P<0.05), declined expression levels of osteoclast differentiation-related genes, and reduced number of mature osteoclasts (P<0.05). Conclusion‍ ‍RANKL activates autophagy through ROS-mediated down-regulation of HIF-1α, and thereby promotes osteoclast differentiation.

Keywords

• hif-1α
• autophagy
• osteoclast differentiation
• reactive oxygen species