摘要
目的:探讨配体WNT3A通过稳定和激活FZD2(Frizzled2)增加成骨细胞骨形成的活性及其分子机制.方法:24 只雌性6～8 周龄C57BL/6J小鼠随机分为4 组,对照(Control)组,假手术(Sham)组,双侧卵巢摘除术(ovariectomy,OVX)诱导骨质疏松症(Osteoporosis,OP)小鼠模型组(OVX 组),OVX +雌二醇治疗组(OVX+E2 Treatment组),每组6 只小鼠.建立OVX小鼠模型.Western blot法测定小鼠后肢胫骨组织中WNT3A、FZD2、Active-β-Catenin、β-Catenin、ALP 和 Runx2 以及磷酸化(p-)STAT3、STAT3、p-JAK2 和JAK2 的表达水平.CCK-8 测定小鼠胚胎成骨细胞MC3T3-E1 的增殖能力.腺病毒-shRNA-FZD2 介导敲低MC3T3-E1 细胞中的FZD2.免疫共沉淀(co-Immunoprecipitation,co-IP)法测定WNT3A处理MC3T3-E1 细胞前后FZD2 与泛素(ubiquitin,Ub)的直接结合情况.结果:与OVX组相比,OVX+E2 Treatment组小鼠胫骨组织中WNT3A、FZD2、Active-β-Catenin、β-Catenin、ALP 和Runx2 的表达水平均被上调(P<0.05).与Control组相比,WNT3A Treatment组MC3T3-E1 细胞中 FZD2、Active-β-Catenin、β-Catenin、ALP 和Runx2 的表达水平均升高(P<0.05);细胞增殖能力增强(P<0.05).与WNT3A Treatment组相比,WNT3A Treatment+Adv-shRNA-FZD2 组FZD2、Active-β-Catenin、β-Catenin、ALP 和Runx2 的表达水平均降低(P<0.05);p-STAT3 和p-JAK2 的磷酸化水平均降低(P<0.05);增殖能力降低(P<0.05);而2 组细胞中STAT3 和JAK2 的表达水平无统计学差异(P>0.05).在 IP:Ub 组中,与WNT3A 处理(-)的细胞相比,WNT3A 处理(+)的细胞中 FZD2 的表达水平升高(P<0.05).结论:WNT3A的促骨合成代谢活性是通过结合并稳定FZD2 激活Wnt/β-Catenin信号通路实现的.
Abstract
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.
基金项目
新疆维吾尔自治区自然科学基金(2022D01C417)
维普
万方数据