Molecular Mechanisms Study of WNT3A Promoting Osteoblasts Proliferation and Differentiation by Binding and Stabilizing FZD2 and Activating Wnt Signaling Pathway
Objective:To investigate the effects and the molecular mechanisms of ligand WNT3A on os-teoblasts'bone formation by stabilizing and activating FZD2(Frizzled2).Methods:Twenty-four female C57BL/6J mice aged 6～8 week were randomly divided into 4 groups:control group,sham group,ovariectomy-induced osteoporosis(OP)mouse model group(OVX group),and OVX+estradiol treatment group(OVX+ E2 treatment group),with 6 mice in each group.The OVX mouse model was established.The expression lev-els of WNT3A,FZD2,Active-β-Catenin,β-Catenin,ALP and Runx2,and phosphorylated(p-)STAT3,STAT3,P-JAK2 and JAK2 in the tibia tissues of mouse hind limbs were determined by Western blot.The proliferation ability of mouse embryonic osteoblast MC3T3-E1 cells were determined by CCK-8 assay.Adeno-virus-shRNA-FZD2 was used to knockdown FZD2 in MC3T3-E1 cells.Co-Immunoprecipitation(co-IP)was used for detecting the direct binding of FZD2 to ubiquitin(Ub)before and after MC3T3-E1 cells treated with WNT3A.Results:Compared with the OVX group,the expression levels of WNT3A,FZD2,Active-β-Catenin,β-Catenin,ALP,and Runx2 in the tibia tissue of mice in the OVX+E2 treatment group were up-regulated(P<0.05).Compared with Control group,in the WNT3A treatment group the expression levels of FZD2,Active-β-Catenin,β-Catenin,ALP,and Runx2 in MC3T3-E1 cells were increased(P<0.05);and the cell proliferation ability was increased(P<0.05).Compared with WNT3A treatment group,in the WNT3A treatment+Adv-shRNA-FZD2 group,the expression levels of FZD2,Active-β-Catenin,β-Cate-nin,ALP,and Runx2 were decreased(P<0.05);the phosphorylation levels of p-STAT3 and p-JAK2 were both decreased(P<0.05);the proliferation ability was decreased(P<0.05).There were no significant differences in the expression levels of STAT3 and JAK2 between the two groups(P>0.05).In the IP:Ub group,compared with WNT3A-treated(-)cells,the expression level of FZD2 was increased in WNT3A-treated(+)cells(P<0.05).Conclusion:The bone anabolic activity of WNT3A is mediated by binding and stabilizing FZD2 to activate the Wnt/β-Catenin signaling pathway.
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