GSK-3β通过调节Snail参与Fas诱导的EMT过程

GSK-3β regulates Snail expression during Fas-induced EMT in gastrointestinal cancer

目的探讨ERK、GSK3β和Snail在Fas诱导的EMT中的作用。方法选用结肠癌SW480细胞及胃癌AGS细胞作为研究对象,使用U0126以阻断其ERK/MAPK通路的激活,稳定转染GSK3βS9A以抑制其GSK3βSer9位点的磷酸化及信号转导,稳定转染Snail shRNA以抑制其Snail活性,并对接受上述处理的细胞分别给予低剂量FasL刺激后,再利用侵袭试验、免疫荧光、免疫印迹、RT-PCR、qRTPCR、免疫共沉淀及荧光素酶报告基因等方式检测细胞的形态、功能变化。结果 Fas信号通路的激活可抑制E-cadherin的转录表达,且这一过程依赖于ERK/MAPK通路。通过转染稳定敲除Snail表达后,Fas对E-cadherin的转录抑制作用显著减弱。在细胞内过表达突变型GSK3βS9A可显著降低Fas通路激活后Snail的表达上调水平、E-cadherin的转录抑制水平及细胞的侵袭能力增加程度。免疫共沉淀提示,GSK3β与ERK、Snail在细胞核存在相互作用。结论在消化道肿瘤细胞中,Fas诱导EMT的调控机制包括ERK/MAPK通路的激活、ERK对GSK3β的磷酸化(Ser9位点)失活、GSK3β失活导致的Snail表达上调及核易位,以及Snail作用下的E-cadherin转录水平下调。

Aims To explore the role of ERK, GSK3βand Snail in Fas-induced EMT process. Methods Fas-signaling-induced changes of cellular morphology and functions in SW480 or AGS cells with blockage of ERK/MAPK pathway, inhibition of GSK3βtranscriptional activity or knock-down of Snail were detected by invasion assay, immunofluorescence, Western blot, RT-PCR , qRT-PCR, immunoprecipitation and luciferase reporter gene assay. Results The activation of the Fas signaling pathway could inhibit the transcription of E-cadherin in an ERK/MAPK pathway dependent manner. The inhibition of E-cadherin transcription by Fas signaling was significantly reduced after stable transfection of Snail shRNA. The overexpression of mutant GSK3βS9A significantly reduced Fas-induced Snail upregulation, E-cadherin downregulation, and cellular motility enhancement. The result of coimmunoprecipitation demonstrated the interaction between GSK3β, ERK and Snail in nucleus. Conclusion In gastrointestinal cancer cells, Fas signaling can induce EMT through the activation of ERK/MAPK pathway, the phosphorylation and inactivation of GSK3βby ERK, the upregula-tion and nuclear translocation of Snail resulting from GSK3βinactivation, as well as Snail-induced downregu-lation of E-cadherin.