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Molecular Mechanisms Study of WNT3A Promoting Osteoblasts Proliferation and Differentiation by Binding and Stabilizing FZD2 and Activating Wnt Signaling Pathway |
CUI Yongjian, LI Yan, WANG Qiaomei, et al |
The Sixth Affiliated Hospital of Xinjiang Medical University, Xinjiang Urumqi 830002, China |
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Abstract Objective: To investigate the effects and the molecular mechanisms of ligand WNT3A on osteoblasts' 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 levels 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. Adenovirus-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-Catenin, 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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