Quantitative proteomics analysis of human endothelial cell membrane rafts - Evidence of MARCKS and MRP regulation in the sphingosine 1-phosphate-induced barrier enhancement

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.