Lee O H, Lee D J, Kim Y M, Kim Y S, Kwon H J, Kim K W, Kwon Y G
Institute of Environment and Life Science, Hallym Academy of Science, Hallym University, Kangwon-Do, Chunchon, 200-702, Korea.
Biochem Biophys Res Commun. 2000 Feb 5;268(1):47-53. doi: 10.1006/bbrc.2000.2087.
We have previously shown that sphingosine 1-phosphate (S1P) stimulates motility of human umbilical vein endothelial cells (HUVECs) (O.-H. Lee et al., Biochem. Biophys. Res. Commun. 264, 743-750, 1999). To investigate the molecular mechanisms by which S1P stimulates HUVEC motility, we examined tyrosine phosphorylation of p125 focal adhesion kinase (p125(FAK)) which is important for cell migration. S1P induces a rapid increase in tyrosine phosphorylation of p125(FAK). Compared with other structurally related lipid metabolites such as sphingosine, C2-ceramide, and lysophosphatidic acid, S1P uniquely stimulated p125(FAK) tyrosine phosphorylation and migration of HUVECs. The effect of S1P on p125(FAK) tyrosine phosphorylation was markedly reduced by treatment with pertussis toxin or U73122, a phospholipase C (PLC) inhibitor. As a downstream signal of PLC, p125(FAK) tyrosine phosphorylation in response to S1P was totally blocked by depletion of the intracellular calcium pool. However, protein kinase C (PKC) inhibitor had no effect on the response to S1P. Finally, chemotaxis assays revealed that inhibition of PLC but not PKC significantly abrogated S1P-stimulated HUVEC migration. These results suggest that the G(i)-coupled receptor-mediated PLC-Ca(2+) signaling pathway may be importantly involved in S1P-stimulated focal adhesion formation and migration of endothelial cells.
我们之前已经表明，1-磷酸鞘氨醇（S1P）可刺激人脐静脉内皮细胞（HUVECs）的运动（O.-H. Lee等人，《生物化学与生物物理研究通讯》264，743 - 750，1999年）。为了研究S1P刺激HUVEC运动的分子机制，我们检测了对细胞迁移很重要的p125粘着斑激酶（p125(FAK)）的酪氨酸磷酸化。S1P可诱导p125(FAK)的酪氨酸磷酸化迅速增加。与其他结构相关的脂质代谢产物如鞘氨醇、C2 - 神经酰胺和溶血磷脂酸相比，S1P独特地刺激了p125(FAK)的酪氨酸磷酸化以及HUVECs的迁移。用百日咳毒素或磷脂酶C（PLC）抑制剂U73122处理可显著降低S1P对p125(FAK)酪氨酸磷酸化的作用。作为PLC的下游信号，细胞内钙库耗竭可完全阻断S1P诱导的p125(FAK)酪氨酸磷酸化。然而，蛋白激酶C（PKC）抑制剂对S1P的反应没有影响。最后，趋化性分析表明，抑制PLC而非PKC可显著消除S1P刺激的HUVEC迁移。这些结果表明，G(i)偶联受体介导的PLC - Ca(2+)信号通路可能在S1P刺激的内皮细胞粘着斑形成和迁移中起重要作用。
