Abstract
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) 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 microM, 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.
Highlights
Endothelial cell barrier dysfunction results in the increased vascular permeability observed in inflammation, tumor metastasis, angiogenesis, and atherosclerosis
Human pulmonary artery Endothelial cell (EC) were cultured in T150 flasks to confluence, either left untreated or treated with Sphingosine 1-phosphate (S1P) (1 M, 5 min)
Representative MS/MS spectra for peptides identified from myristoylated alanine-rich protein kinase C substrate (MARCKS), MARCKS-related protein (MRP), and caveolin-1 are shown in Fig. 3, with the peaks for the signature ions shown in the insets
Summary
EVIDENCE OF MARCKS AND MRP REGULATION IN THE SPHINGOSINE 1-PHOSPHATE-INDUCED BARRIER ENHANCEMENT*□S. To begin to identify the molecular mechanisms mediating S1P induced endothelial barrier enhancement, quantitative proteomics analysis (iTRAQTM) was performed on membrane rafts isolated from human pulmonary artery endothelial cells in the absence or presence of S1P stimulation. To begin to identify the underlying signaling mechanisms by which S1P increases vascular integrity, we chose to identify protein changes in membrane rafts isolated from human pulmonary artery ECs in the presence or absence of S1P treatment (1 M, 5 min) using quantitative proteomics analysis. Linking these proteins to EC barrier enhancement was explored using siRNA transfection and measurement of transendothelial electrical resistance (TER)
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