The Role Of Autophagy In The Regulation Of Osteoclast Resorption By Osteoprotegerin Via AKT/mTOR/ULK1 Signaling Pathway

Osteoclasts(osteoclast,OC)and osteoblasts(osteoblast,OB)are a pair of tightly coordinated mechanism to ensure the health of bone tissue.Macrophage colony stimulating factor(M-CSF)and nuclear factor kappa B receptor activating factor(RANKL)are main cytokines that promote osteoclast differentiation and resorption.Osteoprotegerin(osteoprotegerin,OPG)increases bone mineral density by inhibiting osteoclast formation,activation and bone resorption.It has been found that the process of bone resorption is closely related to autophagy,and the mechanism of autophagy in the process of bone resorption regulated by OPG has been understood poorly.In this study,BALB/c mouse bone marrow-derived macrophages(BMMs)differentiation of osteoclast formation as the research object,after treatment with autophagy inhibitor CQ,agonist RAP and OPG,to clarify the role and mechanism of autophagy mediated by AKT/mTOR/ULK1 signaling pathway in osteoclast absorption regulated by OPG.In three aspects,will we elucidate the mechanism:the effect of OPG on autophagy in osteoclasts,the role of autophagy in osteoclast bone resorption regulated by OPG,and the mechanism of autophagy mediated by AKT/mTOR/ULK1 signaling pathway in osteoclast bone resorption regulated by OPG.The results of our study will provide a theoretical basis for clinical application of OPG in the treatment of bone metabolic diseases.1.Effects of OPG on autophagy in osteoclastsIn order to reveal the effect of OPG on autophagy in osteoclasts cells were treated with different concentrations of OPG(0,20,40,80 ng/mL)for different time gaps(0,3,6,12h),besides CQ and RAP were also used to study the changes of autophagy flux.The fluorescence intensity of MDC and LC3 was detected by immunofluorescence.The results showed that 40ng/mLOPG significantly increased the level of autophagy at 3 h.OPG weakened the inhibitory effect of CQ on osteoclast autophagy,while RAP and OPG can significantly enhance the level of autophagy in osteoclasts;OPG significantly enhanced MDC and LC3 fluorescence intensity and the number of autophagosomes,while CQ inhibited the degradation of osteoclast autolysosome,enhanced MDC and LC3 fluorescence intensity,the number of autophagic bodies decreased significantly.Addition of RAP promoted the autophagy of osteoclasts,and OPG could enhance the effect of RAP on autophagy.The results showed that 40 ng/mL OPG had a significant promotion effect on the differentiation of osteoclasts to the first of the 3 days,and promoted autophagy flux cells in osteoclasts.2.The role of autophagy in OPG-induced inhibition of osteclast bone resorptionIn order to clarify he role of autophagy played in OPG-induced inhibition of osteclast bone resorption,we used autophagy inhibitor CQ and agonist RAP to treat osteoclasts,and used xCelligence system to monitor real-time cell index,resorption pit assay to observe the change of osteoclastic bone resorption ability,fluorescence quantitative PCR and western blotting to detect gene and protein expression levels of cathepsin CathepsinK and tartrate resistant acid phosphatase TRAP.The results showed that the treatment of OPG and RAP significantly inhibited the survival rate of osteoclasts at 80 h,the RAP group maintained at a low level in the late stage compared with the control group,the bone resorption ability of osteoclasts in OPG and RAP treated group was significantly lower,while CQ only partly attenuated the inhibition of OPG on bone resorption of osteoclasts;RAP significantly increased the transcription and protein expression levels of CathepsinK and TRAP,while CQ partially counteracted the inhibitory effect of OPG on the expression of CathepsinK and TRAP protein.This indicated that OPG significantly increased the bone resorption capacity while enhancing the autophagy of osteoclasts.3.The role of AKT/mTOR/ULK1 signaling pathway in the regulation process of autophagy by OPGIn order to investigate the role of AKT/mTOR/ULK1 signaling pathway in the regulation process of osteoclast autophagy by OPG,Western blotting were applied to detect the phosphorylation levels of AKT,mTOR and ULK1 protein,and immunofluorescence to detect LC3 and LAMP2,transmission electron microscopy to detect the autophagy in bilayer membrane.The results showed that OPG decreased the expression of P-AKT and P-mTOR protein and increase the expression of P-ULK1 protein in a dose-dependent manner.OPG enhanced the inhibitory effect of PI3K/AKT blocker LY294002 on P-AKT and P-mTOR,and enhanced the inhibition of RAP on P-AKT,and promoted the autophagy of osteoclasts,the results suggested that OPG could enhance the level of autophagy in osteoclasts by inhibiting the AKT/mTOR signaling pathway;OPG significantly enhanced the fluorescence intensity of LC3 and LAMP2,and showed significant colocalization,autophagy inhibitor 3-MA significantly reduce the intensity of fluorescence,mTOR inhibitor RAP can significantly enhanced the level of autophagy,showing an increase in fluorescence intensity;showed an increase in fluorescence intensity;OPG attenuated the inhibitory effect of 3-MA on the autophagy of osteoclasts,and the number of OPG and 3-MA coprocessing group increased significantly.40 ng/mL OPG significantly enhanced the autophagy in osteoclast differentiated for 3 days through AKT/mTOR/ULKl signaling pathway...