| Osteoclasts(OCs)are highly differentiated terminal multinuclear cells.Together with osteoblasts(OBs)and osteocytes(OSTs),OCs maintain the balance of bone metabolism.Osteoprotegerin(OPG)is a cytokine secreted by osteoblasts which inhibits the differentiation and bone resorption activity of osteoclast.Besides,It has been recently reported that OPG can stimulate autophagy in OCs and reduce osteoclastic bone resorption.Mitochondria are important organelles for cell energy metabolism and mantaining the homeostasis of osteoclasts.However,the mechanism of OPG on mitophagy in osteoclasts has been poorly understood.In this study,Osteoclasts were differentiated from bone marrow mononclear macrophages of BALB/c mouse.Cells were treated with autophagy inhibitor CQ,autophagy agonist RAP,mitochondrial fission inhibitor Mdivi-1 and OPG.Then,technologies of molecular biology were applied to obseve the mitochondrial function,morphology,fission and dectect the expression of mitophagy related proteins.The study aims to clarify the effects of OPG on mitophagy in osteoclasts via PINK1/Parkin signaling pathway suggesting a scientific theory for the clinical application of OPG in the treatment of bone metabolic diseases.1.The effects of OPG on mitochondrial morphology and function in OCsTo reveal the effects of OPG on the function and morphology of mitochondria in osteoclasts,the cells were treated with OPG(0,20,40,80 ng·mL-1)for 3 h,or pretreated with either CQ for 30 min or RAP for 1 h,following the treatment with 40 ng·mL-1 OPG for another 3 h.Reactive oxygen species(ROS)and mitochondrial membrane potential(MMP)of osteoclasts were detected by flow cytometry.ATP was detected by ELISA and read by microplate reader,the ultrastructure of mitochondria was observed by transmission electron microscope(TEM).The results showed that compared with Control group OPG reduced ROS level(P<0.05).Compared with the OPG group,CQ significantly rescued OPG-induced ROS decrease(P<0.01).Compared with Control group,OPG reduced MMP of osteoclasts(P<0.05).Compared with the OPG group,the MMP level was inhibited obviously by the combination use of RAP and OPG(P<0.01).Compared with Control group,OPG increased significantly ATP contents in osteoclasts(P<0.01).While compared with the OPG group,RAP obviously attenuated OPG-induced the increase of ATP contents(P<0.01).OPG treatment induced the swelling of mitochondria and mitophagy.RAP could enhance the effect of OPG on mitophagy.The results suggested that OPG could cause mitochondrial dysfunction of osteoclasts and promote the formation of mitophagy bodies in osteoclasts.2.The effects of OPG on mitochondrial fission in OCsIn order to elucidate the effect of OPG on mitochondrial fission in osteoclasts,osteoclasts were pretreated with mitochondrial fission inhibitor Mdivi-1 and then treated with OPG,Transmission electron microscopy(TEM)was used to observe mitochondrial fission.Immunofluorescence was applied to observe the colocalization of p-Drpls616 and Mito tracker Red(a mitochondrial probe).Western blot was used to detect the expression of mitochondrial fission-fusion related proteins(Drpl,Fis1,MFF and Mfn2).The results showed that OPG induced mitochondrial swelling and mitochondrial fragmentation.Compared with the OPG group,the Mdivi-1+OPG enlongated the mitochondria.Compared with the Control group,OPG enhanced the fluorescence intensity and colocalization of p-Drpls616 and mitochondria.Compared with the Control group,the expression levels of MFF,Fisl,mito-Drpl and cyto-Drpl in OPG group significantly increased(P<0.01),and Drpl shifted to mitochondria.While OPG inhibited the expression level of Mfn2(P<0.05).Mdivi-1 obviously weakened the effects of OPG on the expression of mitochondrial fission-related proteins except for Mfn2(P<0.01).The results denmostrated that OPG promoted mitochondrial fission in osteoclasts.3.The role of PINKl/Parkin signaling pathway in OPG-induced mitophagy of OCsIn order to explore the role of PINK1/Parkin pathway in OPG-induced mitophagy of OCs,Western blot was applied to detect the expression of proteins(PINK1,Parkin,Mfn2,VDAC1 and TOMM20).Immunofluorescence was used to observe the flourescence intensity of Parkin,TOMM20 and ubiquitin(Ub)and the colocalization of LC3,Mito tracker Red and LAMP2.Interaction between Mfn2 and Ub was detected by immunoprecipitation.The results showed that OPG up-regulated the expression levels of PINK1,Parkin,VDAC1 and TOMM20 proteins(P<0.05),and down-regulated Mfn2 expression(P<0.05).OPG enhanced the inflourescence intensity of Parkin and TOMM20 as well as Parkin and Ub,and colocalization of LC3,mitochondria and LAMP2.The ratio of interaction between Mfn2 and Ub is closely related to the dose of OPG In conclusion,The results indicated that OPG enhanced mitophagy in osteoclasts via the PINK1/Parkin signaling pathway. |
