The Adaptor Protein p62 Is Involved in RANKL-induced Autophagy and Osteoclastogenesis
The Adaptor Protein p62 Is Involved in RANKL-induced Autophagy and Osteoclastogenesis
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接头蛋白 p62 参与 RANKL 诱导的自噬和破骨细胞生成
DOI：
10.1369/0022155414551367
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发表时间：
2014-12-01
影响因子：
3.2
通讯作者：
Zhao, Yi-Fang
中科院分区：
文献类型：
--
作者：
Li, Rui-Fang;Chen, Gang;Zhao, Yi-Fang
以前的研究表明，自噬与破骨细胞分化有关。本研究的目的是研究p62在RANKL诱导的破骨细胞形成中的潜在作用。实时定量聚合酶链式反应和免疫印迹分析检测RANKL诱导小鼠巨噬细胞样RAW264.7细胞破骨分化过程中自噬相关标志物的表达水平。同时，用双标记免疫荧光法检测p62/LC3定位与F-肌动蛋白环形成之间的关系。然后用小干扰RNA(SiRNA)下调RAW264.7细胞中p62的表达，检测其对RANKL诱导的自噬激活、破骨细胞分化和F-肌动蛋白环形成的影响。数据显示，在RANKL诱导的破骨细胞分化过程中，包括p62在内的几个关键的自噬相关标记发生了显着变化。此外，免疫印迹和免疫荧光分析显示，p62的表达和定位分别与Lc3积累量和F-肌动蛋白环的形成呈负相关。重要的是，p62基因的敲除明显减弱了RANKL诱导的自噬和破骨细胞相关基因的表达、TRAP阳性多核细胞的形成、Lc3的积聚以及F-肌动蛋白环的形成。我们的研究表明，p62可能在RANKL诱导的自噬和破骨细胞生成中发挥重要作用，这可能有助于开发一种治疗破骨细胞生成相关疾病的新策略。
Previous studies have implicated autophagy in osteoclast differentiation. The aim of this study was to investigate the potential role of p62, a characterized adaptor protein for autophagy, in RANKL-induced osteoclastogenesis. Real-time quantitative PCR and western blot analyses were used to evaluate the expression levels of autophagy-related markers during RANKL-induced osteoclastogenesis in mouse macrophage-like RAW264.7 cells. Meanwhile, the potential relationship between p62/LC3 localization and F-actin ring formation was tested using double-labeling immunofluorescence. Then, the expression of p62 in RAW264.7 cells was knocked down using small-interfering RNA (siRNA), followed by detecting its influence on RANKL-induced autophagy activation, osteoclast differentiation, and F-actin ring formation. The data showed that several key autophagy-related markers including p62 were significantly altered during RANKL-induced osteoclast differentiation. In addition, the expression and localization of p62 showed negative correlation with LC3 accumulation and F-actin ring formation, as demonstrated by western blot and immunofluorescence analyses, respectively. Importantly, the knockdown of p62 obviously attenuated RANKL-induced expression of autophagy- and osteoclastogenesis-related genes, formation of TRAP-positive multinuclear cells, accumulation of LC3, as well as formation of F-actin ring. Our study indicates that p62 may play essential roles in RANKL-induced autophagy and osteoclastogenesis, which may help to develop a novel therapeutic strategy against osteoclastogenesis-related diseases.