脑血管内皮细胞中转化生长因子β以Smad4依赖方式上调Notch4

发布时间：2018-08-05 12:17

【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 factorβ,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。
[Abstract]:Aim to investigate whether the NOTCH receptor is directly regulated by TGF- 尾 / Smad4 as a downstream target of TGF- 尾 / Smad4 signaling pathway in endothelial cells. Methods the regulation of notch1notch4 gene expression by TGF- 尾 and the activity of notch1notch4 gene promoter by luciferase reporter gene were studied by real-time quantitative PCR- 尾 Western blotting and luciferase reporter gene assay respectively, and TGF- 尾 was used to study the effect of TGF- 尾 on the activity of notch1nnotch4 gene promoter. The interaction between notch1notch4 gene promoter and SMAD protein was studied by chromatin immunoprecipitation assay. Results TGF- 尾 1 and bone morphogenetic protein 4 (BMP4) stimulated the expression of notch1 and notch4 at the transcriptional level. They also promote the binding of SMAD4 to SMAD binding sites on the promoter of notch4 gene. The expression of notch4 lost its responsiveness to TGF- 尾 1 and BMP4 under the condition of knockout of SMAD binding site mutation of SMAD4 or notch4 promoter. Conclusion SMAD4 mediated TGF- 尾 / BMP signal directly up-regulates the expression of NOTCH receptor in cerebrovascular endothelial cells.
【作者单位】：军事医学科学院生物工程研究所;
【基金】：国家自然科学基金资助项目(31301203,81370596,91439128)
【分类号】：R743

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