Zhang Bo, Luo Chenglin, Xiao Wenjin
Department of Radiology, The Second Affiliated Hospital of Soochow University, Suzhou, China.
Department of Gastroenterology, Suzhou Xiangcheng People's Hospital, Suzhou, China.
Ann Transl Med. 2021 Sep;9(18):1474. doi: 10.21037/atm-21-4474.
Lysyl oxidase (LOX) has been identified to modulate osteoclast activity, so we explored the role of LOX, the highest frequency single nucleotide polymorphism in LOX, in osteoclast formation and its potential relationship to autophagy.
The ability of the LOX mutant, LOX, 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-AMPKα and p-mTOR proteins, were further evaluated by western blot and qPCR assays.
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-AMPKα 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 LOX mutation, indicating that promoting autophagy could enhance the ability of LOX to induce osteoclast formation.
LOX mutant (LOX) might promote osteoclast formation for RAW264.7 by enhancing autophagy via the AMPK/mTOR pathway, which is a new direction for bone disease research.
赖氨酰氧化酶（LOX）已被证实可调节破骨细胞活性，因此我们探究了LOX及其最高频率单核苷酸多态性在破骨细胞形成中的作用及其与自噬的潜在关系。
在破骨前体细胞系RAW264.7中评估LOX突变体（LOX）促进自噬和破骨细胞形成的能力。此外，分别通过蛋白质免疫印迹法和电子显微镜检测自噬相关蛋白表达和自噬体。同时，使用抗酒石酸酸性磷酸酶（TRAP）染色试验和骨吸收试验检测破骨细胞形成和吸收能力。另外，通过蛋白质免疫印迹法和定量聚合酶链反应（qPCR）试验进一步评估破骨细胞相关蛋白和信使核糖核酸（mRNA），以及磷酸化腺苷酸活化蛋白激酶α（p-AMPKα）和磷酸化哺乳动物雷帕霉素靶蛋白（p-mTOR）。
自噬抑制剂3-甲基腺嘌呤（3-MA）抑制了转染MUT质粒的RAW264.7中Beclin-1和自噬相关蛋白5（ATG5）的蛋白水平、微管相关蛋白轻链3-II（LC3-II）与微管相关蛋白轻链3-I（LC3-I）的比率，以及自噬体形成，并增强了p62蛋白表达。同时，3-MA还减少了破骨细胞形成和吸收，以及破骨细胞的丝状肌动蛋白环水平。此外，3-MA抑制了破骨细胞相关蛋白和mRNA表达，包括活化T细胞核因子1（NFATC1）、抗酒石酸酸性磷酸酶5（ACP5）、组织蛋白酶K（CTSK）。并且，3-MA处理使自噬相关途径蛋白p-AMPKα增加而p-mTOR减少。然而，自噬激动剂雷帕霉素（RAPA）逆转了3-MA对具有LOX突变的RAW264.7的作用，表明促进自噬可增强LOX诱导破骨细胞形成的能力。
LOX突变体（LOX）可能通过AMPK/mTOR途径增强自噬，从而促进RAW264.7破骨细胞形成，这是骨病研究的一个新方向。
