8-prenylgenistein exerts osteogenic effects via ER alpha and Wnt-dependent signaling pathway
8-prenylgenistein exerts osteogenic effects via ER alpha and Wnt-dependent signaling pathway
复制标题
8-异戊二烯金雀异黄酮通过 ER α 和 Wnt 依赖性信号通路发挥成骨作用
DOI：
10.1016/j.yexcr.2020.112186
复制
发表时间：
2020
影响因子：
3.7
通讯作者：
Wong Man-sau
中科院分区：
文献类型：
--
作者：
Qiu Zuo-cheng;Zhang Yan;Xiao Hui-hui;Poon Christina-chui-wa;Li Xiao-li;Cui Jian-fang;Wong Man-kin;Yao Xin-sheng;Wong Man-sau
8-异戊二烯基染料木黄酮（8 PG）先前被报道比染料木黄酮（一种众所周知的大豆植物雌激素）发挥更强的成骨活性。然而，8 PG对成骨细胞作用的分子机制还远不清楚。在本研究中，8 PG和染料木素的成骨作用和机制进行了研究，使用人骨髓基质细胞和小鼠前成骨细胞MC 3 T3-E1细胞。我们的结果表明，与MPP（10− 6 M，ERα拮抗剂）共孵育可消除8 PG和染料木素对成骨细胞分化的刺激作用，但与PHTPP（10− 6 M，ERβ拮抗剂）共孵育则无此作用。分子对接结果表明，8 PG与ERα的结合方式与genistein相似，不能解释二者的成骨作用差异，而计算机靶点分析结果表明，Wnt/β-catenin通路的关键介导因子糖原合成酶激酶-3 β（glycogen synthase kinase-3β，GSK-3β）参与了8 PG的作用。然而，在成骨细胞MC 3 T3-E1中，发现8 PG和染料木素不是直接抑制GSK-3β酶活性，而是诱导GSK-3β在丝氨酸-9处磷酸化。8 PG对MC 3 T3-E1细胞中LRP 5、β-catenin、Runx 2、骨钙素、alp、opg、Wnt信号通路中主要蛋白和基因标志物的表达的促进作用强于genistein。此外，DKK 1对Wnt信号通路的抑制作用可被8 PG和genistein恢复。然而，8 PG，而不是genistein，刺激ERα依赖的β-catenin蛋白在MC 3 T3-E1细胞中的表达。此外，通过与LY 294002（10− 5 M，PI 3 K通路抑制剂）共同处理，8 PG和染料木素引起的ALP活性、LRP 5和磷酸化Akt/Akt表达的增加被消除。综上所述，我们的研究结果表明，8 PG的成骨活性是通过GSK-3β磷酸化介导的，其途径是通过Wnt/β-catenin和ERα相关的PI 3 K/Akt信号通路。
8-prenylgenistein (8PG) was previously reported to exert stronger osteogenic activity than genistein, a well-known soy phytoestrogen. However, the molecular mechanism underlying the actions of 8PG on osteoblasts was far from clear. In the present study, the osteogenic effects and mechanisms of 8PG and genistein were studied using human BMSC and murine pre-osteoblast MC3T3-E1 cells. Our results indicated that the stimulatory effects of 8PG and genistein on osteoblast differentiation were abolished by co-incubation with MPP (10−6M, an ERα antagonist), but not PHTPP (10−6M, an ERβ antagonist). Molecular docking indicated that the binding mode of 8PG toward ERα was similar to that of genistein and therefore could not account for their differential osteogenic actions.In silicotarget profiling identified the involvement of glycogen synthase kinase-3β (GSK-3β), a key mediator of Wnt/β-catenin pathway, in the actions of 8PG. However, instead of directly inhibiting GSK-3β enzymatic activities, 8PG and genistein were found to induce GSK-3β phosphorylation at Serine-9 in osteoblastic MC3T3-E1 cells. 8PG exerted more potent effects than genistein in stimulating expressions of LRP5, β-catenin, Runx2, osteocalcin, alp, opg, major protein and gene markers involved in Wnt signaling pathway in MC3T3-E1 cells. Moreover, the inhibition of Wnt signaling by DKK1 could be restored by treatment with 8PG and genistein. However, 8PG, but not genistein, stimulated ERα-dependent β-catenin protein expression in MC3T3-E1 cells. Furthermore, the increase in ALP activity, LRP5 and phospho-Akt/Akt expression by 8PG and genistein were abolished by co-treatment with LY294002 (10−5M, a PI3K pathway inhibitor). Collectively, our results suggested that the osteogenic activities of 8PG was mediated by GSK-3β phosphorylation through the induction of Wnt/β-catenin and ERα-associated PI3K/Akt signaling.