Study On The Role And Meehanism Of OPG-RANKL-WNT Signaling Pathway In Bone Metabolism Of Osteoporosis

Background information:Nuclear factor-?B receptor activator(RANK)and its ligands(RANKL)and osteoprotegerin(OPG)are cytokines discovered in recent years to regulate osteoclast differentiation and activation.It plays an important role in osteoporosis-related bone metabolism.RANKL combined with RANK induced osteoclast differentiation and activation and inhibited osteoclast apoptosis,while OPG blocked this process and reduced bone destruction.?-catenin,as a key molecule in the classical Wnt/?-catenin signaling pathway,is involved in osteoblast differentiation,maturation and apoptosis.OPG/RANKL-Wnt/?-catenin signaling system is involved in the regulation of a variety of bone metabolism-related diseases,and is a promising therapeutic target.This study focused on the role and potential mechanism of OPG/RANKL-Wnt/P-catenin signaling pathway in bone metabolism of osteoporosis.Methods:1.Primary human osteoblasts were obtained and cultured from healthy controls and osteoporosis patients,respectively.The expression of DKK1,DKK2,phosphorylated ?-catenin and RANKL/OPG at mRNA and protein levels,osteocalcin and alkaline phosphatase synthesis and cell proliferation were detected under basic condition and 1,25-dihydroxyvitamin D 3 stimulation.2.The expression of conditional knockout ?-catenin protein in mature osteoclasts of mice was compared with that of wild-type mice:the changes of bone mass and the morphological changes of osteoclasts were analyzed by osteoclast specific TRAP immunohistochemical staining and HE staining.Primary bone marrow-derived osteoclasts were cultured in vitro and stimulated by RANKL and M-CSF to evaluate the effects of RANKL-Wnt signaling pathway on the maturation,differentiation and number of osteoclasts.The expression of OPG and RANKL mRNA in tibia of the two groups was detected by real-time PCR.Results:1.Compared with the healthy control group,the ratio of RANKL/OPG in osteoporosis group was significantly higher(P<0.05).Exogenous vitamin D3 treatment increased the expression of RANKL/OPG and DKK2,but decreased the expression of DKK1.However,there was no significant effect on the expression of ?-catenin.Osteocalcin and alkaline phosphatase production and osteoblast proliferation were decreased in osteoporosis group.2.We successfully achieved specific knockout of ?-catenin gene in mouse osteoblasts,and found that after knockout of ?-catenin gene in osteoclasts,the mice maintained normal bone development before the age of 4 weeks.However,severe bone mass loss and an increase in the number of osteoclasts occurred after 4 weeks.Further in vitro experiments confirmed that the activation of Wnt/?-catenin signaling pathway could promote the apoptosis of osteoclasts,reduce the number of osteoclasts,and knockout ?-catenin gene could also lead to the decrease of OPG expression in non-osteoclasts.Which in turn leads to a decrease in bone mass throughout the body.Conclusion:1.Osteoblasts in the pathological state of osteoporosis may affect the synthesis and secretion of DKK family members through autocrine or paracrine mechanism,and then inhibit Wnt/?-catenin signaling pathway leads to the decrease of osteocalcin synthesis,cell proliferation and osteogenic activity of osteoblasts,and stimulates bone resorption by increasing the expression of RANKL.2.Osteoclasts in the pathological state of osteoporosis affect Wnt/?-catenin signaling pathway in a cell independent way to regulate the life span of osteoclasts and reduce the number of osteoclasts.At the same time,by regulating OPG,the imbalance of RANKL/OPG can promote the maturation and differentiation of osteoclasts,resulting in the loss of total bone mass,and eventually lead to the formation of osteoporosis.