mTOR磷酸化水平对成骨MC3T3E1细胞增殖、自噬和分化的作用及其机制

Effect of mTOR phosphorylation level on proliferation,autophagy,and differentiation of MC3T3-E1 osteoblasts and its mechanism

目的:通过雷帕霉素(RAPA)和MHY1485抑制和激活哺乳动物雷帕霉素靶蛋白(mTOR)的磷酸化,探讨磷酸化mTOR(p-mTOR)水平变化对成骨细胞增殖、自噬和分化的作用及其机制。方法:小鼠成骨MC3T3-E1细胞分为对照组、20 nmol·L^(-1)RAPA组、60 nmol·L^(-1)RAPA组、160 nmol·L^(-1)RAPA组和2.0μmol·L^(-1)MHY1485组。流式细胞术检测各组不同细胞周期MC3T3-E1细胞百分率,Western blotting和实时荧光定量PCR(RT-qPCR)法检测各组细胞中mTOR及下游真核翻译起始因子4E结合蛋白1(4E-BP1)和核糖体S6激酶1(S6K1)、B淋巴细胞瘤2(Bcl-2)、Bcl-2相关X蛋白(Bax)和Caspase-3、碱性磷酸酶(ALP)、Runt相关转录因子2(Runx2)和成骨相关转录因子Osterix的表达水平,茜素红染色观察各组成骨MC3T3-E1细胞矿化能力。结果:与对照组比较,20和60 nmol·L^(-1)RAPA组细胞增殖活性升高(P<0.05或P<0.01),G1期细胞百分率降低(P<0.05或P<0.01),S期细胞百分率升高(P<0.05或P<0.01),细胞中p-mTOR及其下游通路磷酸化4E-BP1(p-4E-BP1)、磷酸化S6K1(p-S6K1)水平和p62、Bax、Cleaved-Caspase-3表达水平降低(P<0.05或P<0.01)、LC3-Ⅱ、Bcl-2、ALP和Runx2表达水平升高(P<0.05或P<0.01);与对照组比较,160 nmol·L^(-1)RAPA组细胞增殖活性降低(P<0.01)、细胞中p-mTOR、p-4E-BP1和p-S6K1水平明显降低(P<0.05或P<0.01),2.0μmol·L^(-1)MHY1485组细胞增殖活性明显降低(P<0.01),细胞中p-mTOR、p-4E-BP1和p-S6K1水平明显升高(P<0.01),160 nmol·L^(-1)RAPA和2μmol·L^(-1)MHY1485组细胞中Bax和Cleaved-Caspase-3表达水平升高(P<0.05或P<0.01),Bcl-2、ALP、Runx2和Osterix表达水平降低(P<0.05或P<0.01);作用12 d后,20和60 nmol·L^(-1)RAPA组细胞中可见明显矿化结节,160 nmol·L^(-1)RAPA和2.0μmol·L^(-1)MHY1485组成骨细胞中见极少数钙盐结晶。结论:轻中度抑制mTOR的磷酸化可促进成骨细胞增殖、自噬和分化,而过度抑制和增强mTOR磷酸化则具有相反作用。

Objective:To inhibit and activate the phosphorylation of mammalian target protein of rapamycin(mTOR)by rapamycin(RAPA)and MHY1485,and to discuss the effect of level of phosphorylated mTOR(p-mTOR)on the proliferation,autophagy and differentiation of osteoblasts and its mechanism.Methods:The mouse MC3T3-E1 osteoblasts were divided into control group,20 nmol·L^(-1) RAPA group,60 nmol·L^(-1) RAPA group,160 nmol·L^(-1)RAPA group and 2.0μmol·L^(-1) MHY1485group.The percentages MC3T3E1 osteoblasts at different cell cycles in various groups were detected by flow cytometry;the expression levels of mTOR and downstream signals—IF4E-binding protein 1(4E-BP1)and ribosome protein subunit 6 kinase 1(S6K1),B cell lymphoma-2(Bcl-2),Bcl-2 related X protein(Bax),and Caspase-3,alkaline phosphatase(ALP),Runt-related transcription factor 2(Runx2),and osteoblast-related transcription factor Osterix were detected by Western blotting and Real-time fluorescence quantitative PCR(RT-qPCR)methods;the mineralization ability of MC3T3-E1 osteoblasts in various groups was observed by Alizarin red staining.Results:Compared with control group,the proliferation activities of MC3T3-E1 osteoblasts in 20 and 60 nmol·L^(-1)RAPA groups were significantly increased(P<0.05 or P<0.01),the percentages of MC3T3-E1 osteoblasts at G1 phase were increased(P<0.05 or P<0.01),the percentages of the MCET3-E1 osteoblasts at S phase were increased(P<0.05 or P<0.01),the expression levels of p-mTOR and its downstream pathway of p-4E-BP1 and p-S6K1,as well as p62,Bax,and Cleaved-Caspase-3 were decreased(P<0.05 or P<0.01),and the expression levels of LC3-Ⅱ,Bcl-2,ALP and Runx2 were increased(P<0.05 or P<0.01).Compared with control group,the proliferation activity of MC3T3-E1 osteoblasts in 160 nmol·L^(-1) RAPA group was significantly decreased(P<0.01),and the expression levels of p-mTOR,p-4E-BP1,and p-S6K1 were decreased(P<0.05 or P<0.01);the proliferation activity of MC3T3-E1 osteoblasts in 2.0μmol·L^(-1)MHY1485 group was decreased(P<0.01),and the expression levels of p-mTOR,p-4E-BP1,and p-S6K1 were decreased(P<0.01);however,the expression levels of Bax and Cleaved-Caspase-3 in both160 nmol·L^(-1) RAPA and 2.0μmol·L^(-1) MHY1485 groups were increased(P<0.05 or P<0.01),and the expression levels of Bcl-2,ALP,Runx2,and Osterix were decreased(P<0.05 or P<0.01).Twelve days after treatment,the mineralized nodules were found in the MC3T3-E1 osteoblasts in 20 and60 nmol·L^(-1) RAPA groups,and only a very small amount of calcium salt were found in the MC3T3-E1 osteoblasts in 160 nmol·L^(-1) RAPA and 2.0μmol·L^(-1) MHY1485 groups.Conclusion:Mild to moderate inhibition of mTOR phosphorylation can promote the proliferation,autophagy and differentiation of the MC3T3-E1 osteoblasts,while excessive inhibition and enhancement of mTOR phosphorylation have the opposite effect.