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

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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.