Syntenin上调FAK磷酸化水平促进人脑胶质瘤细胞迁移的分子机制

目的探讨Syntenin促进人脑胶质瘤细胞迁移的分子机制。方法采用划痕实验和Western blot检测CHG-5、稳定表达人源性Syntenin的CHG-hS细胞在纤维连接蛋白(fibronectin,FN)和多聚赖氨酸(Poly-l-lysine,PL)两种基质表面迁移能力、FAK磷酸化位点及相关信号分子的变化情况;分别在P38MAPK特异性抑制剂SB239063和PI3K特异性抑制剂LY294002处理后,FN基质上CHG-5、CHG-hS细胞迁移能力和下游信号分子JNK和AKT磷酸化水平的改变。结果细胞划痕实验结果显示,CHG-hS细胞在FN表面的运动能力显著高于CHG-5细胞(P<0.05);而在多聚赖氨酸包被的培养板上,CHG-hS细胞的运动能力与CHG-5组细胞表面无显著性差异(P>0.05)。Western blot检测结果显示,FN作用下CHG-hS细胞中FAK Tyr397、FAK Tyr576、FAK Tyr925位点的磷酸化水平随时间延长而逐渐升高(P<0.05),而FAK FAK Tyr861位点的磷酸化水平没有变化(P>0.05);相关信号Src、JNK、AKT的磷酸化水平也随时间延长而升高(P<0.05)。分别采用SB239063和LY294002处理后,伴随着p-JNK和p-AKT的磷酸化水平减弱,CHG-hS迁移能力下降。结论 Syntenin通过与p-Src结合,随后触发FAK Tyr397、FAK Tyr925、FAK Tyr576位点的磷酸化作用,最大程度的激活FAK并上调JNK、AKT等下游信号分子的磷酸化水平,最终通过Syntenin-Src/FAK/MAPK和Syntenin-Src/FAK/PI3K两条通路增强FN相关胶质瘤细胞的迁移能力。

Molecular mechanism of Syntenin promoting human glioma cell migration though upregulating FAK phosphorylation

Objective To investigate the molecular mechanism of Syntenin promoting human glioma cell migration.Methods The migration ability tests of CHG-5 cells(human glioma cell line) and CHG-hS cells(CHG-5 stably expression human Syntenin) on fibronectin(FN) and poly-L-lysine(PL) were performed by scratch wound assay.FAK phosphorylation and activation of its relative signal molecules JNK,AKT and Src were detected by Western blotting.After CHG-hS and CHG-5 cells were treated with P38 MAPK specific inhibitor SB239063 and PI3K specific inhibitor LY294002,respectively,the migration ability of the cells on FN was investigated by scratch wound assay and the phosphorylation levels of JNK and AKT were detected by Western blotting.Results The result of scratch wound assay showed that the migration ability of the CHG-hS cells on FN-coated plates was significantly higher than that of the CHG-5 cells(P<0.05),while the migration ability of the CHG-hS cells on PL-coated plates showed no significant difference compared with that of the CHG-5 cells(P>0.05).Western blot analysis showed that the phosphorylation levels of FAK phosphorylation sites(Tyr397,Tyr576,Tyr925,but not Tyr861) and FAK-related signal molecules(phosphor-Src,phosphor-JNK and phosphor-AKT) in the CHG-hS cells on FN increased in a time-dependent manner(P<0.05).The migration ability of the CHG-hS cells decreased along with the reduced levels of phosphor-JNK and phosphor-AKT after treated with SB239063 and LY294002,respectively.Conclusion Syntenin specifically interacting with p-Src promotes the phosphorylation of FAK Tyr397,Tyr925 and Tyr576,which activates FAK to a maximum extent and upregulates the phosphorylation levels of downstream signal molecules such as JNK and AKT,and finally promotes FN-related glioma cell migration though the Syntenin-Src/FAK/MAPK and Syntenin-Src/FAK/PI3K pathways.