Induction of osteoclast formation by LOX mutant (LOXG473A) through regulation of autophagy

Background: Lysyl oxidase (LOX) has been identified to modulate osteoclast activity, so we explored the role of LOXG473A, the highest frequency single nucleotide polymorphism in LOX, in osteoclast formation and its potential relationship to autophagy. Methods: The ability of the LOX mutant, LOXG473A, to promote autophagy and osteoclast formation was evaluated in the pre-osteoclast cell line RAW264.7. Furthermore, autophagy-related protein expression and autophagosomes were detected by western blot and electron microscopy, respectively. Simultaneously, osteoclast formation and resorption ability were also detected using TRAP staining assay and bone resorption assay. In addition, the osteoclast-related proteins and mRNAs, as well as p-AMPK alpha and p-mTOR proteins, were further evaluated by western blot and qPCR assays. Results: Autophagy inhibitor 3-MA suppressed the Beclin-1 and ATG5 protein levels and the ratio of LC3-II to LC3-I, as well as autophagosome formation in RAW264.7 transfected with the MUT plasmid and enhanced p62 protein expression. Simultaneously, 3-MA also reduced osteoclast formation and resorption, as well as the F-actin ring level of osteoclasts. In addition, 3-MA inhibited osteoclast-related protein and mRNA expression, including NFATC1, ACP5, CTSK. And the autophagy-related pathway protein p-AMPK alpha was increased and p-mTOR was reduced by 3-MA treatment. However, autophagy agonist RAPA reversed the effect of 3-MA on RAW264.7 with LOXG473A mutation, indicating that promoting autophagy could enhance the ability of LOXG473A to induce osteoclast formation. Conclusions: LOX mutant (LOXG473A) might promote osteoclast formation for RAW264.7 by enhancing autophagy via the AMPK/mTOR pathway, which is a new direction for bone disease research.

Automated summary

This study showed that the LOX mutant LOXG473A may promote osteoclast formation by enhancing autophagy through the AMPK/mTOR pathway. The autophagy inhibitor 3-MA reduced osteoclast formation and resorption, while the autophagy agonist RAPA reversed these effects, suggesting that promoting autophagy can enhance the ability of LOXG473A to induce osteoclast formation.