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Elucidation of the Intracellular Signal Networks Involved in the Growth Arrest induced by Cell-cell Contact in Vascular Endothelial Cells

阐明血管内皮细胞细胞间接触引起的生长停滞所涉及的细胞内信号网络

基本信息

批准号：
14570675
负责人：
HIRANO Mayumi
金额：
$ 2.24万
依托单位：
Kyushu University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2003
项目状态：
已结题

项目摘要

1.The vascular endothelial cells ceased their growth upon formation of the tight cell-cell contact. The expression level of the cell cycle regulator p27^<Kip1> was upregulated during the contact-induced growth arrest. This up-regulation was found to be due to transcriptional up -regulation.2.We have developed an assay system to determine the transcriptional regulatory element that responds to the formation of cell-cell contact in the cultured endothelia cells.3.We obtained a porcine genomic clone containing a full-length p27^<Kip1> gene, and examined the promoter activity by using a 1500-nt up-stream region. The region -333to -247 nt was found to respond to the formation of homophilic cell-cell contact but not to the contact to HeLa cells. This promoter region is thus suggested to play an important role in up-regulating p27^<Kip1> expression during the contact-induced growth arrest.4.We have developed a novel method to introduce protein into the cells with intact plasma membrane with a help of cell-penetrating peptide found in human immunodeficiency viral transcription factor Tat protein. By this method, proteins can be introduced in a quantitative and reversible manner. The time-specific transduction of the inhibitory peptide of RhoA revealed that the activity of-RhoA is required during the late G_1 phase of the cell cycle for the endothelial cells to progress to the S phase.5.We found that co-transfection of the forkhead transcription factor AFX activated the p27^<Kip1> promoter.

1.细胞与细胞形成紧密接触后，血管内皮细胞停止生长。在接触诱导的生长停滞期间，细胞周期调节因子p27 α的表达水平<Kip1>上调。2.建立了一种检测内皮细胞中细胞间接触形成的转录调控元件的方法; 3.获得了猪p27^基因的全长克隆<Kip1>，并对其上游1500个核苷酸的启动子活性进行了检测。-333 ~-247nt区域对嗜同性细胞间接触的形成有反应，但对HeLa细胞的接触无反应。因此，该启动子区在接触诱导生长停滞过程中上调p27^表达中起重要作用<Kip1>。4.我们开发了一种新的方法，利用人免疫缺陷病毒转录因子达特蛋白中的细胞穿透肽将蛋白质导入具有完整质膜的细胞中。通过这种方法，可以以定量和可逆的方式引入蛋白质。RhoA抑制肽的时间特异性转导表明，RhoA活性在G_1期晚期是内皮细胞进入S期所必需的。5.我们发现叉头转录因子AFX共转染激活了p27^<Kip1>启动子。