Quantitative proteomics analysis of human endothelial cell membrane rafts - Evidence of MARCKS and MRP regulation in the sphingosine 1-phosphate-induced barrier enhancement
Quantitative proteomics analysis of human endothelial cell membrane rafts - Evidence of MARCKS and MRP regulation in the sphingosine 1-phosphate-induced barrier enhancement
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DOI：
10.1074/mcp.m600398-mcp200
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发表时间：
2007-04-01
影响因子：
7
通讯作者：
Garcia, Joe G. N.
中科院分区：
文献类型：
--
作者：
Guo, Yurong;Singleton, Patrick A.;Garcia, Joe G. N.
内皮细胞屏障功能障碍导致在炎症、肿瘤转移、血管生成和动脉粥样硬化中观察到的血管通透性增加。1-磷酸鞘氨醇（S1 P）是一种具有生物活性的磷酸化脂质生长因子，从活化的血小板中释放出来，在体外和体内增强内皮细胞屏障的完整性。为了开始鉴定介导S1 P诱导的内皮屏障增强的分子机制，在不存在或存在S1 P刺激的情况下，对从人肺动脉内皮细胞分离的膜筏进行定量蛋白质组学分析（iTRAQTM）。我们的研究结果表明，S1 P介导的快速和特异性招聘（1 μ m，5分钟）的豆蔻酰丙氨酸丰富的蛋白激酶C底物（MARCKS）和MARCKS相关蛋白（MRP）的膜筏。Western印迹实验证实了MARCKS和MRP的这些发现。最后，小干扰RNA介导的MARCKS或MRP或两者的沉默减弱S1 P介导的内皮细胞屏障增强。这些数据表明，通过MARCKS和MRP的细胞特异性定位的S1 P介导的内皮细胞屏障增强的调节，并验证了蛋白质组学方法在鉴定新的分子靶点中的实用性。
Endothelial cell barrier dysfunction results in the increased vascular permeability observed in inflammation, tumor metastasis, angiogenesis, and atherosclerosis. Sphingosine 1-phosphate (S1P), a biologically active phosphorylated lipid growth factor released from activated platelets, enhances the endothelial cell barrier integrity in vitro and in vivo. To begin to identify the molecular mechanisms mediating S1P induced endothelial barrier enhancement, quantitative proteomics analysis (iTRAQ(TM)) was performed on membrane rafts isolated from human pulmonary artery endothelial cells in the absence or presence of S1P stimulation. Our results demonstrated that S1P mediates rapid and specific recruitment (1 mu m, 5 min) of myristoylated alanine-rich protein kinase C substrate (MARCKS) and MARCKS-related protein (MRP) to membrane rafts. Western blot experiments confirmed these findings with both MARCKS and MRP. Finally, small interfering RNA-mediated silencing of MARCKS or MRP or both attenuates S1P-mediated endothelial cell barrier enhancement. These data suggest the regulation of S1P-mediated endothelial cell barrier enhancement via the cell specific localization of MARCKS and MRP and validate the utility of proteomics approaches in the identification of novel molecular targets.