Estrogen receptor alpha-activated adenosine monophosphate-activated protein kinase signaling pathway promotes proliferation and differentiation of osteoblasts

doi:10.12307/2025.731

Abstract

Abstract: BACKGROUND: Estrogen receptor α can act as an upstream protein to regulate the expression and phosphorylation level of adenosine monophosphate-activated protein kinase (AMPK). Activation of the estrogen receptor α-AMPK signaling pathway promotes osteogenic proliferation and differentiation.
OBJECTIVE: To explore the molecular mechanism of estrogen receptor α regulating AMPK and its effect on osteoblast proliferation and differentiation at osteoblast cell line and molecular biology levels.
METHODS: (1) The passaged MC3T3-E1 mouse embryonic osteoblasts were divided into three groups: blank control group, mock group (transfected with pCDNA3.1 control plasmid), and estrogen receptor α group (transfected with pCDNA3.1-estrogen receptor α overexpression plasmid), and RT-qPCR and western blot methods were used to detect the hepatic kinase B1, CaMKKβ, and AMPKα1 mRNA, protein and phosphorylation levels. (2) ChIP-qPCR was used to demonstrate that estrogen receptor α interacts with the hepatic kinase B1 promoter. Dual luciferase assay was used to demonstrate that estrogen receptor α interacts with the hepatic kinase B1 promoter region to activate its transcriptional expression. (3) The cells were divided into three groups: mock+shNC group, estrogen receptor α+shNC group, and estrogen receptor α+shLKB1 group. Changes in the expression levels of hepatic kinase B1, phosphorylated hepatic kinase B1, and phosphorylated AMPKα1 proteins in the cells were detected by western blot. (4) The cells were divided into four groups: mock group, estrogen receptor α group, estrogen receptor α+5 μmol/L Compound C (AMPK inhibitor) group, and estrogen receptor α+10 μmol/L Compound C group. The expression of proteins related to the AMPK signaling pathway and related to osteogenesis and osteoinductivity were detected by western blot method. Cells were transfected for 24 hours and then subjected to osteogenic induction for 14 days. Alkaline phosphatase staining was performed and cell viability in each group was detected. Mineralized nodule formation was detected by alizarin red staining at 21 days of osteogenic induction. (5) The cells were transfected and pretreated with different concentrations of AMPK inhibitor in corresponding groups, and cell viability was detected by cell counting kit 8.
RESULTS AND CONCLUSION: (1) Estrogen receptor α activates the AMPK signaling pathway in MC3T3-E1 cells. (2) Estrogen receptor α promotes liver kinase B1 transcription and mediates AMPK signaling pathway activation. (3) Estrogen receptor α promotes the proliferation and differentiation of MC3T3-E1 cells by activating the AMPK signaling pathway, and the expression of AMPKα1, p-AMPKα, osteoprotegerin, osteopontin, and Runx2 proteins was down-regulated under the intervention of AMPK inhibitor, and the viability of osteoblasts was decreased. (4) To conclude, estrogen receptor α activates the AMPK signaling pathway by acting on liver kinase B1 promoter, promotes osteoblast proliferation and osteogenic differentiation, and prevents osteoporosis.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

Key words: postmenopausal osteoporosis, estrogen receptor α overexpression, AMPK, signaling pathway, liver kinase B1, engineered tissue construction

CLC Number:

Yang Feng, Xu Jinfan, Long Huan, Yang Fengchun, Zhang Guixin, Jiang Tao, Chen Qingzhen, Shao Min. Estrogen receptor alpha-activated adenosine monophosphate-activated protein kinase signaling pathway promotes proliferation and differentiation of osteoblasts[J]. Chinese Journal of Tissue Engineering Research, 2025, 29(24): 5061-5070.

Figures/Tables 5

2.1 雌激素受体α过表达对MC3T3-E1细胞中AMPK通路相关 mRNA表达的影响雌激素受体α过表达促进MC3T3-E1细胞中AMPKα1和肝激酶B1的mRNA增加(P < 0.05)，而对CaMKKβ mRNA无影响(P > 0.05)，见图1。 2.2 雌激素受体α过表达对MC3T3-E1细胞中AMPK通路相关蛋白表达的影响雌激素受体α过表达促进MC3T3-E1细胞中p-AMPKα1蛋白表达(P < 0.05)，提示雌激素受体α激活MC3T3-E1细胞中AMPKα1信号通路。同时，雌激素受体α过表达可使AMPKα1信号通路上游的蛋白肝激酶B1和磷酸化肝激酶B1蛋白增加(P < 0.05)，而对CaMKKβ和p-CaMKKβ蛋白表达无影响(P > 0.05)，见图2。 2.3 ChIP-qPCR证明雌激素受体α与肝激酶B1启动子互作 JASPAR预测结合位点示意图见图3。ChIP-PCR结果显示：与IgG比较，雌激素受体α上富集的肝激酶B1启动子显著增加(P < 0.05)，提示雌激素受体α与肝激酶B1启动子结合，见图4。 2.4 双荧光素酶实验证明雌激素受体α与肝激酶B1启动子区域互作激活其转录表达见图5。 2.5 雌激素受体α过表达或与shLKB1共同转染对MC3T3-E1细胞AMPK通路相关蛋白表达的影响首先，将shNC和3条shLKB1分别转染MC3T3-E1细胞中24 h后，采用RT-PCR检测肝激酶B1的表达水平变化，见图6。结果表明，3条shLKB1均可以抑制MC3T3-E1细胞中肝激酶B1表达，其中shLKB1抑制效果最佳，用于后续实验研究。其次，雌激素受体α过表达单独或与shLKB1共同转染MC3T3-E1细胞后，采用Western blot检测细胞中肝激酶B1、磷酸化肝激酶B1和p-AMPKα1蛋白的表达变化。结果表明，雌激素受体α过表达促进MC3T3-E1细胞中肝激酶B1、磷酸化肝激酶B1和p-AMPKα1蛋白表达，而转染shLKB1可以显著抑制以上作用，提示雌激素受体α通过调控肝激酶B1表达激活AMPK信号通路，见图7。 2.6 不同浓度AMPK抑制剂干预AMPK信号通路相关蛋白和成骨分化相关蛋白的表达 Western blot检测结果显示，雌激素受体α过表达促进MC3T3-E1细胞中AMPKα1、p-AMPKα1、骨保护素、骨桥蛋白、Runx2蛋白表达，而给予5 μmol/L Compound C具有逆转以上现象的趋势，但无显著差异，但是给予10 μmol/L Compound C可以显著逆转以上现象(P < 0.05)，提示雌激素受体α通过激活AMPK信号通路促进MC3T3-E1细胞分化，见图8。 2.7 各组细胞碱性磷酸酶染色及活性检测结果雌激素受体α过表达促进MC3T3-E1细胞碱性磷酸酶活性及染色阳性，而给予AMPK抑制可以显著逆转以上现象，提示雌激素受体α通过激活AMPK信号通路促进MC3T3-E1细胞碱性磷酸酶染色阳性和活力，见图9，10。 2.8 茜素红染色检测矿化结节形成情况雌激素受体α过表达促进MC3T3-E1细胞茜素红染色阳性，而给予AMPK抑制可以显著逆转以上现象，提示雌激素受体α通过激活AMPK信号通路促进MC3T3-E1细胞钙化，见图11。 2.9 AMPK抑制剂对过表达雌激素受体α后MC3T3-E1细胞增殖的影响 CCK8法结果显示，雌激素受体α过表达促进MC3T3-E1细胞增殖，而给予AMPK抑制可以显著逆转以上现象，提示雌激素受体α通过激活AMPK信号通路促进MC3T3-E1细胞增殖，见图12。"

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