鞘氨醇 1-磷酸鹽引發人類內皮細胞表面之血小板/內皮細胞附著因子-1磷酸化機制之研究

Abstract

Sphingosine 1-phosphate (S1P) is a multifunctional bioactive lipid. Through a family of specific G protein-coupled receptors encoded by endothelial differentiation gene (edg), S1P regulates various cellular functions in different cell types. In endothelial cells, S1P has been demonstrated to modulate cell proliferation, adhesion, migration, wound healing, matix remodeling, tube formation, adhesion molecule expression, cytokine and chemokine releasing. Platelet/endothelial cell adhesion molecule-1 (PECAM-1) is a 130-kDa transmembrane glycoprotein which forms trans-homophilic bindings at cell-cell border. PECAM-1 performs both structural and signaling mediatory functions. Its cytoplasmic immunoreceptor tyrosine inhibitory motifs (ITIM) and tyrosine residues, when being phosphorylated, provide docking sites for various signaling molecules and modulate following cellular functions, such as cell migration, cell survival, tube formation, and transendothelial migration. However, the effect of S1P on endothelial PECAM-1 is still unknown, and the identification of the kinases responsible for PECAM-1 phosphorylation varies in different cell types upon different stimulations. In this study, we confirmed that S1P treatment induced PECAM-1 phosphorylation in human umbilical cord vein cells (HUVECs) by immunoprecipitation and following immunoblotting. The induction occurred immediately in less than three minutes. The induction could be block by pertussis toxin (PTx) and PP2 pretreatment, indicating the participation of Gi and Src family kinases. Results from immunoblotting and confocal microscopy proved that S1P also triggered the activation of Src family kinases at cell border. Finally, to identify the Src member responsible for PECAM-1 phosphorylation, specific siRNA was transfected to HUVECs. The obtained PECAM-1 phosphorylation ratio revealed that both cSrc and Fyn participated in this S1P-induced PECAM-1 phosphorylation.