自噬在OPG抑制破骨细胞分化和骨吸收过程中的作用及分子机制

The differentiation and bone resorption of osteoclasts play a central role in bone reconstruction, during which Osteoprotegerin (OPG) is one of the key regulating factors. Autophagy is involved in many biological processes including skeletal turnover. We have found that among OPG-induced inhibited effects on differentiation and bone resorption of osteoclasts, the level of autophagy were significantly decreased. But the effects and mechanisms of autophagy in this inhibition process have not been illuminated. In this item, M-CSF and RANKL are used to induce RAW264.7 and murine bone marrow cells differenciated to osteoclasts and osteoclast precursor cells (OPC), with OPG treated for different time periods. Firstly, PathScan technology, QRT-PCR, Western blotting, immunofluorescence and transmission electron microscopy (TEM) are used to explore the expression of genes and proteins related to autophagy signal and select the key proteins participated in autophagy regulation. Then overexpress/silence the selected genes to detect their effects on autophagy, cytoskeleton, the expression of cytoskeleton related proteins, autophagy-lysosome positioning, demonstrating the role of autophagy participated in OPG-induced inhibition of differentiation and bone resorption in osteoclasts, and figuring out the targets of autophagy in this process. Thereafter, the regulating factors of cytoskeleton are silenced or overexpressed to detect the effects of autophagy on cytoskeleton. Our study will shed light on the effects and mechanisms of autophagy on OPG induced inhibition of differentiation and of bone resorption in osteoclasts.

破骨细胞分化和骨吸收是骨重建的关键，OPG是其调控因子。自噬是骨重建等多种生命活动的重要参与者。申请者发现在OPG抑制破骨细胞分化和骨吸收中，自噬显著降低。目前关于自噬在此过程中的作用及机制仍不清楚。本项目通过M-CSF和RANKL诱导小鼠RAW264.7和骨髓细胞产生破骨细胞前体细胞和成熟细胞，OPG处理不同时间。利用PathScan、QRT-PCR、免疫印迹、免疫荧光和电镜等研究自噬通路基因或蛋白表达，并筛选出与调控自噬相关的关键蛋白。再将上述蛋白沉默或过表达，检测对自噬、细胞骨架和骨架调节因子表达及其与自噬溶酶体共定位的影响，确定自噬调控OPG抑制破骨细胞分化和骨吸收中的信号通路和自噬靶标。然后沉默或过表达骨架调控因子，研究OPG处理前后细胞骨架对自噬的影响，从而阐明自噬在OPG抑制破骨细胞分化和骨吸收中的作用及分子机制。

自噬在破骨细胞（OC）主导的骨重建中至关重要。本研究表明OPG活化AMPKα诱导自噬，抑制OC分化。阐明AMPK/mTOR/p70S6K通路介导自噬在OPG抑制OC中的机制。.1.AMPK激活剂AICAR抑制OC形成，降低CTSK、c-Fos、NFATc1、p62、p-mTOR/mTOR、Rheb及p-p70S6K/p70S6K表达，增加LC3聚点，上调Beclin1、LC3Ⅱ、p-AMPKα/AMPKα及TSC2表达。OPG降低OC数量，对自噬影响与AICAR相似，对ROS和GβL无影响。表明OPG经增加自噬及AMPK通路表达抑制OC分化。.2.氯喹(QC)和雷帕霉素(RAPA)减少OC数量，增加LC3聚点；QC+OPG组比QC组c-Fos下调，p62、Rheb及p-p70S6K/p70S6K上调，对p-AMPKα/AMPKα和TSC2无影响；RAPA+OPG组比RAPA组c-Fos、p62、Rheb及p-p70S6K/p70S6K下调，Beclin1、p-AMPKα/AMPKα及TSC2上调。自噬抑制剂3-MA减弱OPG抑制OC分化，上调OC数量和LC3Ⅱ、Atg7、mTORC1、Rheb及p-p70S6K/p70S6K表达，下调CTSK、c-Fos、NFATc1、Beclin1、Atg（5,12）、p-AMPKα/AMPKα及TSC2。表明3-MA经AMPK/mTOR/p70S6K减弱OPG抑制OC分化中自噬。.3.溶酶体抑制剂BAF+OPG组比OPG组CTSK、c-Fos、NFATc1、LC3Ⅱ、p62、p-mTOR/mTOR、Raptor、Rheb及p-p70S6K/p70S6K表达上调，Beclin1、Atg（5,7,12）表达下调，GβL、p-AMPKα/AMPKα及TSC2表达无影响，LC3聚点增多。表明BAF在OPG抑制OC分化中经mTOR及 p70S6K磷酸化抑制自噬。.4.AMPK抑制剂Com C组、Com C+OPG组和siAMPK+OPG组OC数量增加，CTSK、c-Fos、LC3Ⅱ、p62、p-mTOR/TOR、Raptor、Rheb和p-p70S6K/p70S6K表达上升，NFATc1、Beclin1、Atg（5,7,12）、p-AMPKα/AMPKα和TSC2表达下降，LC3聚点增多。表明AMPK通路介导自噬参与OPG抑制OC分化。