脑血管内皮细胞中转化生长因子β以Smad4依赖方式上调Notch4

摘要

目的 研究内皮细胞中NOTCH受体是否作为转化生长因子β(transforming growth factor β,TGF-β)/Smad4信号通路下游靶分子而被TGF-β/Smad4直接调节.方法 利用实时荧光定量PCR、Western印迹实验研究TGF-β对notch1、notch4基因表达的调节,利用荧光素酶报告基因实验研究TGF-β对notch1、notch4基因启动子活性的调节,利用染色质免疫共沉淀实验研究notch1、notch4基因启动子与SMAD蛋白之间的相互作用.结果 TGF-β1和骨形态发生蛋白4(bone morphogenetic protein 4, BMP4)的刺激在转录水平上促进notch1和notch4表达.它们还促进SMAD4与notch4基因启动子上SMAD结合位点的结合.在敲低SMAD4或notch4基因启动子SMAD结合位点突变的情况下,notch4的表达失去对TGF-β1和BMP4的反应性.结论 脑血管内皮细胞中SMAD4介导的TGF-β/BMP信号直接上调NOTCH受体表达.%Objective To find out whether NOTCH receptors can serve as direct downstream targets of transforming growth factor β(TGF-β)/Smad4 signaling in endothelial cells.Methods Real-time PCR and Western blotting were performed to verify whether the expression of notch1 and notch4 was regulated by TGF-β pathway.Luciferase reporter assay was employed to investigate how the promoter of notch1 and notch4 was regulated by TGF-β.Then, ChIP assay was used confirm whether the promoter of notch1 and notch4 physically interacted with SMAD protein.Results TGF-β1 and bone morphogenetic protein 4 (BMP4) treatment increased the expression of both notch1 and notch4 at the transcriptional level.In addition, SMAD4 was physically associated with the SMAD binding sites on the notch4 promoter, which was largely enhanced under the treatment of TGF-β1 and BMP4.Importantly, TGF-β1 and BMP4 failed to transactivate notch4 in the absence of endogenous SMAD4 or the SMAD binding regions on the notch4 promoter.Conclusion The expression of NOTCH receptor can be directly up-regulated by SMAD4-mediated TGF-β/BMP signaling in cerebrovascular endothelial cells.