The Effects Of Moesin Phosphorylation In Sphingosine-1-Phosphate Induced Responses In Endothelialcells

The vascular endothelium serves as a semi-selective barrier between circulating blood and surrounding tissues and regulates many biological processes such as protein and fluid transport, inflammation and angiogenesis. Endothelial barrier dysfunction induced by inflammatory agonists is the direct underlying cause of vascular leak and pulmonary edema in sepsis and an essential component of angiogenesis, tumor metastasis, and atherosclerosis. Therefore, the preservation of vascular endothelial cell (EC) barrier integrity has the potential for profound clinical impact. Previous studies described potent EC barrier enhancement induced by the platelet-derived phospholipid sphingosine-1-phosphate (SIP), which involves Rac GTPase-dependent cortical actin rearrangement as an integral step. Sphingosine-1-phosphate (SIP) is a bioactive sphingolipid in plasma. SIP is mainly synthesized and secreted by platelets. And other cells such as erythrocytes, neutrophils, macrophages, monocytes, mast cells and endothelial cells are also able to produce and release SIP. Concentration of SIP can be adjusted through a complex metabolic process, and its change in plasma concentration directly regulates its biological effects. Intracellular SIP can interact with regulatory molecules such as enzymes, channel proteins and transcription factors as a second messenger. SIP can also serve as the extracellular ligand binding to SIP membrane receptors existing in almost all cell types. SIP affects cell proliferation, survival, differentiation, migration and morphogenesis through different signal transduction pathways. There are five cognate G-protein-coupled receptors, to which SIP specifically binds, designated S1PR1, S1PR2, S1PR3, S1PR4and S1PR5, respectively. The SIP receptors most abundantly expressed in endothelial cells are S1PR1, S1PR2and S1PR3with S1PR1>S1PR2=S1PR3. Different subtypes of SIP receptor binding different G protein, S1PR1is coupled to Gi; S1PR2/3is associated to Gi, Gq, G12/13. Further, S1PR1and S1PR2/3have different affinities to SIP. An appropriate or physiological level of SIP (0.5-1.0μmol/L) causes the activation of S1PR1, resulting in the strengthening of the barrier integrity of endothelial cells by inducing Rac signaling pathway, while the lack of SIP will be harmful in endothelial barrier function. While over-dose SIP (5-10.0μmol/L) will bind to receptor2and3and the activation of S1PR2/R3would disrupt the inter-endothelial junctions by evoking the RhoA and ROCK pathway. The balances in the expression and activation of S1PR1, R2and R3help to maintain the physiological functions of ECs. Our preliminary research confirmed that lipopolysaccharide (LPS) or tumor necrosis factor-a (TNF-a) significantly upregulated S1PR2mRNA and protein levels. Physiological level of SIP (0.1-1.0μmol/L) resulting in the strengthening of the barrier integrity of endothelial cells is supposed to play a critical role in clinic treatment, it may provide therapy approaches for pathological conditions such as infection, injury of tissue inflammation, especially for the rise of vascular permeability caused by the tissue edema. therefor, Further study of S1P on regulation of endothelial cell function would play a critical role in the application of SIP as an endogenous vascular permeability stabilizing mediator in the treatment of inflammatory diseases in future.Previous studies have proposed a working model of EC barrier regulation in which the vascular barrier is regulated by a balance between competing EC contractile forces, which generate centripetal tension, and adhesive cell-cell and cell-matrix tethering forces, imposed by focal adhesion and adherens junctions (AJ), which together regulate cell shape change. EC barrier enhancement induced by SIP and other barrier protective factors, such as oxidized phospholipids, human growth factor (HGF), ATP or simvastatin requires actomyosin remodeling, including formation of a prominent cortical actin rim, peripheral accumulation of phosphorylated regulatory myosin light chains (MLC), and disappearance of central stress fibers, which is regulated by Rac-dependent mechanisms. However, the downstream targets of these signaling pathways leading to the cytoskeletal changes remain incompletely defined.The widely distributed ERM family of membrane-associated proteins (ezrin, radixin, moesin) regulates the structure and function of specific domains of the cell cortex. The ERM proteins are actin-binding linkers that connect filamentous F-actin and the plasma membrane, either directly via binding to transmembrane proteins or indirectly via scaffolding proteins attached to transmembrane proteins. This linker function makes ERM proteins essential for many fundamental cellular processes including cell adhesion, determination of cell shape, motility, cytokinesis and integration of membrane transport with signaling pathways. The activation state of ERM proteins is tightly regulated by phosphorylation events. Binding of the protein to membrane lipid phosphatidylinositol4,5-bisphosphate (PIP2) and subsequent phosphorylation of a conserved C-terminal threonine (T567in ezrin, T564in radixin, T558in moesin) are believed to disrupt the intramolecular association, thus unmasking sites for interactions with other proteins. In addition, phosphorylation of ezrin on other residues may also be required to direct specific targeted effects in cells. Several kinases have been implicated in regulating ERM protein function through phosphorylation of the C-terminal threonine residue. However, the identity of kinases that directly phosphorylate ERM in many cells remains to be clearly defined. ERM proteins also associate with cytoplasmic signaling molecules in cellular processes that require membrane cytoskeletal reorganization. ERM proteins appear to act both downstream and upstream of the Rho family of GTPases, which regulates remodeling of the actin cytoskeleton. However, information is limited concerning the possible role of ERM proteins in the remodeling of endothelial cytoskeleton in response to different agonists. Koss and coworkers demonstrated that ERM proteins are phosphorylated on C-terminal threonine residues by TNF-α-induced signaling events and likely play important roles in modulating the cytoskeletal changes and permeability increases in human pulmonary microvascular EC. Previous study suggest that the potential involvement of ERM proteins in the remodeling of the endothelial cytoskeleton that is essential to the SIP barrier-enhancing response. ERM proteins are phosphorylated on this critical C-terminal threonine residue by SIP-induced signaling events and, despite their structural similarities and reported functional redundancy, ERM proteins differentially modulate SIP-induced changes in lung EC cytoskeleton and permeability. These results advance our mechanistic understanding of EC barrier regulation and identify the ERM family as potential clinically important targets for therapeutic manipulation during high permeability processes. Despite structural similarities and reported functional redundancy, the ERM proteins differentially modulate SIP-induced alterations in lung EC cytoskeleton and permeability. These results suggest that ERM activation is an important regulatory event in EC barrier responses to SIP. Due to the wide range of ezrin, radixin and moesin cytophysiological features, detailed exploration of the ERM biochemistry will provide a series of answers to questions about ambiguous functions in many intracellular signal transduction pathways. ERM are important effective proteins in regulating the plastically of plasma membrane downstream signal transduction pathways in the living cells, but its function still under determined. Studies have confirmed that the endothelial cells mainly express moesin. This study aims to explore the effects of moesin phosphorylation in sphingosine-1-phosphate induced responses in endothelial cells.Objective:Studies have found that an appropriate or physiological level of SIP (0.5-1.0μmol/L) causes the activation of S1PR1, resulting in the strengthening of the barrier integrity of endothelial cells by inducing Rac signaling pathway, while the lack of SIP will be harmful in endothelial barrier function. While over-dose SIP (5-10.0μmol/L) will bind to receptor2and3and the activation of S1PR2/R3would disrupt the inter-endothelial junctions by evoking the RhoA and ROCK pathway. Our preliminary studies had proved that1.AGE-HSA could change the morphological and functional of HUVECs then damage the vascular permeability by increase moesin phosphorylation.2. S1PR2mediated high doseSIP disrupt endothelial barrier function. This research is designed to investigate whether moesin phosphorylation is involved in high-dose SIP induced endothelial barrier dysfunction, and to clarify the SIP receptors participating in moesin phosphorylation.MethodsHuman umbilical vein endothelial cell line (HUVECs) were cultured in35mm dish, petri dish, and transwell, respectively, to90%confluent and starved for8hours before being stimulated with SIP in indicated doses and times. In the case of receptor antagonist (S1PR1with W146, S1PR2with JTE-013, or S1PR3with CAY10444) treatment, HUVECs were pretreated with10μmol/L for receptor antagonists for30min, then cultured in fresh complete medium with10μmol/L SIP for20min. Then immunoblotting was used to detect the phosphorylation of moesin. HUVECs were stimulated with SIP (10μmol/L) for5min,10min,20min,40min,60min, and90min in time-dependent experiment, or with SIP (0μmol/L,1μmol/L,5μmol/L,10μmol/L) for20min in dose-dependent experiment. Moesin siRNA was applied to transfect endothelial cell for48h and subculture24h. HUVECs were then starved for8hours before being stimulated with SIP in indicated doses and times. Trans endothelial electric resistance (TEER) was detected by resistance meter to clarify the alteration of endothelial barrier function,the formation of stress fiber in HUVECs were observed with immunocytochemistry under confocal microscope of Zeiss LSM780.Results:1. SIP induced time-dependent and does-dependent moesin phosphorylation in HUVECs.The Exposure of HUVECs to10μmol/L SIP resulted in time-dependent increase of moesin phosphorylation and this increase reached the peak at20min and then sustained at relatively stable level for90min. The stimulation of HUVECs with SIP (0μmol/L,0.5μmol/L,1.0μmol/L,5.0μmol/L and10.0μmol/L) for20min induced does-dependent increase of moesin phosphorylation (n=3, P<0.05). These results demonstrated that SIP induced time-dependent and does-dependent moesin phosphorylation in HUVECs.2. High dose S1P (10μmol/L) induced moesin phosphorylation by S1PR2.S1PR1antagonist W146, S1PR2antagonist (JTE-013), or S1PR3antagonist (CAY10444) was used to precultured with HUVECs for30min, and then SIP (10μmol/L) was applied for20min. Results showed that there were no difference between S1PR1/3antagonist W146, CAY10444pretreated group and SIP (10μmol/L) stimulated-group in moesin phosphorylation levels, while the increase of moesin phosphorylation in JTE-013-pretreated group was significantly suppressed compared with that of with S1P (10μmol/L) stimulation alone.3. Knockdown of moesin using moesin siRNA suppressed SIP (10μmol/L) induced endothelial barrier dysfunction.Human umbilical vein endothelial cell line (HUVECs) were cultured in35mm, the expression of moesin in HUVECs were down-regulated by moesin siRNA transfection. Normal HUVECs, moesin siRNA or control siRNA transfected HUVECs were stimulate with SIP (10μmol/L). The phosphorylation of moesin was subsequently decreased.In functional research, HUVECs of different groups were then cultured in transwell inserts, trans-endothelial electric resistance (TEER) was detected by resistance meter to clarify the alteration of endothelial barrier function in90min. In SIP (10μmol/L) stimulated-group and negative siRNA control group plus SIP (10μmol/L) stimulated-group, TEER was significantly reduced in20min. Transfection of moesin siRNA inhibited the reduction of TEER induced by SIP (10μmol/L) stimulation. Those results indicated that knockdown of moesin using moesin siRNA suppressed SIP (10μmol/L)-induced endothelial barrier dysfunction.In morphological study, HUVECs of different groups were then cultured in Petri dish. The formation of stress fiber was detected by F-actin and nuclear staining. In SIP (10μmol/L) stimulated group or negative siRNA control plus SIP (10μmol/L) stimulated-group, the formation of stress fiber were obvious and the transfection of moesin siRNA inhibited the formation of stress fiber induced by SIP (10μmol/L) stimulation. Those results indicated that knockdown of moesin using moesin siRNA suppressed SIP (10μmol/L)-induced endothelial morphological alteration.Conclusion1. SIP induced time-dependent and does-dependent phosphorylation of moesin in HUVECs.2. Moesin phosphorylation induced endothelial barrier dysfunction via S1PR2.3. Knockdown of moesin using moesin siRNA suppressed SIP (10μmol/L) induced endothelial barrier dysfunction.