TNF-α通过JNK和AP-1途径调节乳腺癌MCF-7细胞VEGF的表达

Tumor necrosis factor-α regulates expression of vascular endothelial growth factor via JNK and AP-1 pathway in MCF-7 cells

目的:探讨肿瘤坏死因子(tumor necrosis factor-α,TNF-α)诱导血管内皮生长因子(vascular endothelial growth fac-tor,VEGF)表达的机制。方法:以20 ng/ml TNF-α处理MCF-7细胞,用Western blotting检测MAPK(JNK,p38,ERK)信号通路中蛋白磷酸化水平的变化以及AP-1家族(c-Jun,Jun-B,c-Fos,Fra-1,Fra-2,JunD)的蛋白表达及磷酸化水平的变化;以免疫共沉淀法检测激活后的AP-1存在形式;以RT-PCR以及Western blotting检测VEGF mRNA和蛋白表达水平;以MAPK抑制剂预处理后,检测VEGF蛋白表达水平;运用ChIP的方法验证p-c-Jun结合在VEGF启动子区。结果:TNF-α通过激活JNK信号转导通路活化AP-1;被TNF-α激活后AP-1以p-c-Jun-c-Jun和p-c-Jun-JunB同源二聚体形式存在;TNF-α通过激活转录因子AP-1促进VEGF的转录,并增强VEGF的蛋白表达水平;p-c-jun通过与VEGF启动子AP-1结合参与对VEGF转录的调控。结论:在TNF-α作用下,AP-1通过p-c-jun同源二聚体结合在VEGF启动子的AP-1结合位点上,直接对VEGF转录进行调控。

Objective：To investigate the mechanism by which tumor necrosis factor-α （TNF-α） induces expression of vascular endothelial growth factor（VEGF） in MCF-7 cells. Methods： MCF-7 cells were treated with TNF-α （20 ng,/ml） for different time periods. Western blotting was used to detect the phosphorylation of MAPK pathway proteins （ JNK, P38, and ERK） and the expression and phosphorylation of AP-1 family members（ c-Jun, Jun-B, c-Fos, Fra-1, Fra-2, JunD）. The activation of AP-1 was detected by immunoprecipitation（IP）. RT-PCR and Western blotting were used to detect the VEGF mRNA and protein expression. The VEGF protein expression was also detected after pre-treatment with inhibitor of MAPK. ChIP method was used to verify the binding of p-c-Jun to the promoter region of VEGF. Results： TNF--α activated AP-1 through JNK signal pathway. AP-1 existed in the form of p-c-Jun-c-Jun and p-c-Jun-JunB homodimer after activa- tion. TNF-ot increased VEGF transcription by activating transcriptional factor AP-1 and result in increased expression of VEGF protein. P-c-jun regulated the transcription of VEGF through binding to the AP-1 site of VEGF. Conclusion ： TNF--α can mediate the transcription of VEGF through a AP-1-dependent pathway.