Stable knock-down of the sphingosine 1-phosphate receptor S1P1 influences multiple functions of human endothelial cells
Stable knock-down of the sphingosine 1-phosphate receptor S1P1 influences multiple functions of human endothelial cells
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DOI：
10.1161/01.atv.0000154360.36106.d9
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发表时间：
2005-03-01
影响因子：
8.7
通讯作者：
Siess, W
中科院分区：
文献类型：
--
作者：
Krump-Konvalinkova, V;Yasuda, S;Siess, W
目的 - 1 - 磷酸鞘氨醇（S1P）是一种具有生物活性的磷脂，它既作为G蛋白偶联受体S1P（1 - 5）的配体，又作为一种第二信使。由于S1P（1）基因敲除在转基因小鼠中是致死的，所以需要一种替代方法来研究S1P（1）在内皮细胞中的功能。
方法与结果 - 所分析的所有人类内皮细胞都表达大量的S1P（1）转录本。我们（通过RNA干扰）永久性地沉默了人类内皮细胞系AS - M.5和ISO - HAS.1中S1P（1）的表达。S1P（1）敲低细胞呈现出独特的形态，对S1P既没有肌动蛋白 ruffles反应，也没有血管生成反应。此外，这些细胞对氧化应激介导的损伤更敏感。在人类内皮细胞中确定了新的依赖于S1P（1）的基因靶点。S1P（1）沉默降低了血小板 - 内皮细胞黏附分子 - 1和血管内皮钙黏蛋白的表达，并消除了脂多糖或肿瘤坏死因子 - α刺激细胞后E - 选择素的诱导。微阵列分析显示S1P（1）沉默后更多内皮特异性转录本下调。
结论 - S1P（1）的长期沉默使我们首次能够证明S1P（1）参与内皮细胞的关键功能，并确定新的依赖于S1P（1）的基因靶点。
Objectives - Sphingosine 1-phosphate (S1P) is a bioactive phospholipid acting both as a ligand for the G protein - coupled receptors S1P(1-5) and as a second messenger. Because S1P(1) knockout is lethal in the transgenic mouse, an alternative approach to study the function of S1P(1) in endothelial cells is needed.Methods and Results - All human endothelial cells analyzed expressed abundant S1P(1) transcripts. We permanently silenced ( by RNA interference) the expression of S1P(1) in the human endothelial cell lines AS-M. 5 and ISO-HAS.1. The S1P(1) knock-down cells manifested a distinct morphology and showed neither actin ruffles in response to S1P nor an angiogenic reaction. In addition, these cells were more sensitive to oxidant stress - mediated injury. New S1P(1)-dependent gene targets were identified in human endothelial cells. S1P(1) silencing decreased the expression of platelet - endothelial cell adhesion molecule-1 and VE-cadherin and abolished the induction of E-selectin after cell stimulation with lipopolysaccharide or tumor necrosis factor-alpha. Microarray analysis revealed downregulation of further endothelial specific transcripts after S1P(1) silencing.Conclusions - Long-term silencing of S1P(1) enabled us for the first time to demonstrate the involvement of S1P(1) in key functions of endothelial cells and to identify new S1P(1)-dependent gene targets.