Quantitative Proteomics Analysis of Human Endothelial Cell Membrane Rafts Evidence of MARCKS and MRP Regulation in the Sphingosine 1-Phosphate-induced Barrier Enhancement

The pulmonary endothelium serves as a semipermeable cellular barrier between blood and the interstitium and airspaces of the lung. Endothelial cell (EC)1 barrier dysfunction results in increased vascular permeability, which is a cardinal feature of inflammation and an essential component of tumor metastasis, angiogenesis, and atherosclerosis. Proteins and lipids released after platelet activation have long been appreciated as enhancing the integrity of the microcirculation (1, 2). Sphingosine 1-phosphate (S1P), a biologically active phosphorylated lipid growth factor released from platelet, has multiple EC effects, including promoting barrier integrity in vivo and in vitro across both human and bovine pulmonary artery and lung microvascular ECs (3, 4). Our previous studies demonstrated S1P mediated barrier enhancement to be dependent on S1P binding to its major surface receptor, S1P1; activation of the small GTPase, Rac1; and rearrangement of the cortical actin cytoskeleton (3, 5, 6). Very recently, we further detailed the essential involvement of PI 3-kinase, Tiam1, and α-actinin in specialized membrane domains (i.e. membrane rafts) in S1P-treated EC barrier regulation (7). However, the underlying signaling mechanisms by which S1P increases vascular integrity via signaling to the endothelial cytoskeleton remain poorly understood. As defined by the recent Keystone Symposium on lipid rafts and cell function (March 23–28, 2006, in Steamboat Springs, CO) (8), “Membrane rafts are small (10–200 nm), heterogeneous, highly dynamic, sterol- and sphingolipid-enriched domains that compartmentalize cellular processes. Small rafts can sometimes be stabilized to form larger platforms through protein-protein and protein-lipid interactions.” Many biophysical, biochemical, and microscopy studies suggest that membrane rafts truly exist and are implicated in diverse cellular processes including signal transduction (9–12). Currently, two commonly used methods to isolate membrane rafts, resistant to either high pH or nonionic detergents, involve separation of membrane rafts from other proteins by density gradient centrifugation, with detergent resistance the more widely utilized method. 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 µm, 5 min) using quantitative proteomics analysis. The time period and concentration for S1P treatment were based on previous studies (3, 4). Because of the extremely hydrophobic nature of membrane rafts, we chose a solution-based proteomics approach, isobaric tagging for relative and absolute quantitation (iTRAQ™) method. The protein changes in membrane rafts were confirmed by Western blot analysis. Linking these proteins to EC barrier enhancement was then explored using siRNA transfection and measurement of transendothelial electrical resistance (TER).