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

1.细胞与细胞之间形成紧密接触后，血管内皮细胞停止生长。在接触诱导的生长停滞过程中，细胞周期调节因子p27^&lt；Kip1&gt；的表达水平上调。这种上调是由于转录上调。2.我们建立了一种检测系统，以确定在培养的内皮细胞中与细胞-细胞接触形成反应的转录调控元件。3.我们获得了一个包含全长p27^；lt；Kip1&gt；基因的猪基因组克隆，并利用1500个核苷酸的上游区域检测了启动子的活性。-333~-247nT区域对细胞-细胞亲和性接触的形成有反应，而对HeLa细胞的接触无反应。因此，该启动子区域在接触诱导生长停滞期间上调p27^&lt；Kip1&gt；的表达起着重要作用。4.我们开发了一种新的方法，利用在人类免疫缺陷病毒转录因子Tat蛋白中发现的穿透性多肽，将蛋白质导入质膜完整的细胞中。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的活性需要在细胞周期的晚期G_1期才能使内皮细胞进入S期。5.我们发现共转染叉头转录因子afx激活了p27^lt；Kip1&gt；启动子。

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.