In this report, sphingosine-1-phosphate (S1P), a serum-borne bioactive lipid, is shown to activate tight-junction-associated protein Zonula Occludens-1 (ZO-1), which in turn plays a critical role in regulating endothelial chemotaxis and barrier integrity. After S1P stimulation, ZO-1 was redistributed to the lamellipodia and cell-cell junctions via the S1P1/G i /Akt/Rac pathway. Similarly, both endothelial barrier integrity and cell motility were significantly enhanced in S1P-treated cells through the G i /Akt/Rac pathway. Importantly, S1P-enhanced barrier integrity and cell migration were abrogated in ZO-1 knockdown cells, indicating ZO-1 is functionally indispensable for these processes. To investigate the underlying mechanisms, we demonstrated that cortactin plays a critical role in S1P-induced ZO-1 redistribution to the lamellipodia. In addition, S1P significantly induced the formation of endothelial tight junctions. ZO-1 and ␣-catenin polypeptides were colocalized in S1P-induced junctional structures; whereas, cortactin was not observed in these regions. Together, these results suggest that S1P induces the formation of two distinct ZO-1 complexes to regulate two different endothelial functions: ZO-1/cortactin complexes to regulate chemotactic response and ZO-1/␣-catenin complexes to regulate endothelial barrier integrity. The concerted operation of these two ZO-1 complexes may coordinate two important S1P-mediated functions, i.e. migration and barrier integrity, in vascular endothelial cells.Endothelial barrier integrity is an important physiological function of the endothelium in vivo. Dysregulated barrier integrity is implicated in a variety of pathological conditions, such as stroke, inflammation, various immune responses, etc. (1). To elucidate the function and regulation of endothelial barrier integrity, cultured brain microvascular endothelial cells have been widely employed as an in vitro model system to study the blood-brain barrier (BBB) 2 (2). Evidence from these studies indicates that BBB plays a critical role in regulating the homeostatic environment of the brain and the transportation of plasma constituents into brain. Furthermore, it has been shown that severely impaired blood-brain barrier integrity is attributed to the pathological states of various neurological disorders, such as multiple sclerosis (3, 4), Alzheimer disease (5, 6), and human immunodeficiency virus-1-associated encephalitis or dementia (7,8).Sphingosine 1-phosphate (S1P), a serum-borne bioactive lipid mediator secreted by activated platelets (9), enhances barrier formation in cultured pulmonary endothelial cells (ECs) (10). However, the molecular details for the formation and maintenance of endothelial barrier integrity are poorly understood. It was recently reported that the association of cortactin, an F-actin cross-linking polypeptide, and myosin light chain kinase is crucial in S1P-enhanced endothelial barrier integrity (11). Furthermore, it is well documented that tight junctions are important in regulating BBB formation...

Lee JF, Gordon S, Estrada R, Wang L, Siow DL, Wattenberg BW, Lominadze D, Lee MJ. Balance of S1P 1 and S1P2 signaling regulates peripheral microvascular permeability in rat cremaster muscle vasculature. Am J Physiol Heart Circ Physiol 296: H33-H42, 2009. First published November 14, 2008 doi:10.1152/ajpheart.00097.2008.-Sphingosine-1-phosphate (S1P) regulates various molecular and cellular events in cultured endothelial cells, such as cytoskeletal restructuring, cell-extracellular matrix interactions, and intercellular junction interactions. We utilized the venular leakage model of the cremaster muscle vascular bed in Sprague-Dawley rats to investigate the role of S1P signaling in regulation of microvascular permeability. S1P signaling is mediated by the S1P family of G protein-coupled receptors (S1P 1-5 receptors). S1P1 and S1P2 receptors, which transduce stimulatory and inhibitory signaling, respectively, are expressed in the endothelium of the cremaster muscle vasculature. S1P administration alone via the carotid artery was unable to protect against histamineinduced venular leakage of the cremaster muscle vascular bed in Sprague-Dawley rats. However, activation of S1P 1-mediated signaling by SEW2871 and FTY720, two agonists of S1P 1, significantly inhibited histamine-induced microvascular leakage. Treatment with VPC 23019 to antagonize S1P 1-regulated signaling greatly potentiated histamine-induced venular leakage. After inhibition of S1P2 signaling by JTE-013, a specific antagonist of S1P2, S1P was able to protect microvascular permeability in vivo. Moreover, endothelial tight junctions and barrier function were regulated by S1P 1-and S1P 2-mediated signaling in a concerted manner in cultured endothelial cells. These data suggest that the balance between S1P 1 and S1P2 signaling regulates the homeostasis of microvascular permeability in the peripheral circulation and, thus, may affect total peripheral vascular resistance.spingosine-1-phosphate receptor subtypes; vascular integrity; signal transduction SPINGOSINE-1-PHOSPHATE (S1P), a serum-borne bioactive lipid mediator, regulates an array of biological activities in various cell types (13,14,28,42). Most, if not all, S1P-regulated functions are mediated by the S1P family of G protein-coupled receptors (1,20,48). Five members of the S1P receptor family have been identified: S1P 1 , S1P 2 , S1P 3 , S1P 4 , and S1P 5 , previously known as endothelial differentiation gene (EDG)-1, -5, -3, -6, and -8, respectively (6). It was demonstrated that S1P receptor subtypes couple to different G␣ polypeptides to regulate specific signaling pathways (2, 16, 46a). S1P receptor subtypes are expressed in distinct combinations in different cell types to produce an appropriate biological effect. For example, S1P 1 and S1P 3 are expressed in cultured endothelial cells (ECs) (18). The signaling pathways regulated by the S1P 1 and S1P 3 receptors in ECs are required for chemotaxis, adherens junction assembly, morphogenesis, and angiogenic response in vitro and in vivo (18 -20). H...

Vascular endothelial cells (ECs) have a finite lifespan when cultured in vitro and eventually enter an irreversible growth arrest state called "cellular senescence." It has been shown that sphingolipids may be involved in senescence; however, the molecular links involved are poorly understood. In this study, we investigated the signaling and functions of sphingosine 1-phosphate (S1P), a serum-borne bioactive sphingolipid, in ECs of different in vitro ages. We observed that S1P-regulated responses are significantly inhibited and the S1P 1-3 receptor subtypes are markedly increased in senescent ECs. Increased expression of S1P 1 and S1P 2 was also observed in the lesion regions of atherosclerotic endothelium, where senescent ECs have been identified in vivo. S1P-induced Akt and ERK1/2 activation were comparable between ECs of different in vitro ages; however, PTEN (phosphatase and tensin homolog deleted on chromosome 10) activity was significantly elevated and Rac activation was inhibited in senescent ECs. Rac activation and senescent-associated impairments were restored in senescent ECs by the expression of dominant-negative PTEN and by knocking down S1P 2 receptors. Furthermore, the senescent-associated impairments were induced in young ECs by the expression of S1P 2 to a level similar to that of in vitro senescence. These results indicate that the impairment of function in senescent ECs in culture is mediated by an increase in S1P signaling through S1P 2 -mediated activation of the lipid phosphatase PTEN.Spingosine 1-phosphate (S1P), 2 a serum-borne bioactive lipid mediator, regulates an array of biological activities in various cell types (1-4). Most, if not all, of S1P-regulated functions are mediated by the S1P family of G-protein-coupled receptors (GPCRs) (5-7). There are five identified members of the S1P receptor family: S1P 1 , S1P 2 , S1P 3 , S1P 4 , and S1P 5 (old nomenclature: EDG-1, -5, -3, -6, and -8, respectively) (8). It was demonstrated that S1P receptor subtypes couple to different G ␣ polypeptides to regulate distinct signaling pathways (9 -11). The S1P receptor subtypes were expressed in distinct combinations in different cell types to produce an appropriate biological effect. For example, S1P 1 and S1P 3 are expressed in endothelial cells (ECs) (12). The signaling pathways regulated by the S1P 1 and S1P 3 receptors are required for the chemotaxis of endothelial cells, adherens junction assembly, endothelial morphogenesis, and angiogenic response in vitro and in vivo (7,(12)(13). However, the functional outcomes resulting from the concerted effects of the signaling pathways mediated by the distinct S1P receptor subtypes are currently unknown in a physiological environment.In contrast to S1P 1 -stimulating chemotaxis, S1P 2 -mediated signaling was shown to inhibit cell migration (14 -16). For example, embryonic fibroblasts isolated from S1P 2 null mice exhibited an enhanced chemotaxis toward S1P, serum, and platelet-derived growth factor; this enhancement was reversed by the reintroduction of S...

Sphingosine-1-phosphate (S1P), a serum-borne lipid mediator, was demonstrated to be a potent chemoattractant of endothelial cells. It was recently shown that the colocalization of cortactin and actin related protein 2/3 (Arp2/3) in the lamellipodia is critical to S1P-induced endothelial chemotaxis. In this report, we describe that S1P-stimulated cortactin translocation to the cell periphery to form lamellipodia is specifically mediated by the endothelial S1P1 G-protein coupled receptor, and is regulated by G(i)-mediated Akt-dependent S1P1 receptor phosphorylation and Cdc42/Rac activation pathways. In contrast to Src-dependent fibroblast growth factor-induced cortactin translocation, tyrosine phosphorylation cascades are not required for S1P-mediated lamellipodia formation and chemotaxis. Furthermore, we also demonstrate that S1P signaling, via the G(i)/Akt/S1P1 phosphorylation/Rac pathway, regulates the cortactin-Arp2/3 complex formation, which ultimately results in membrane ruffling, formation of the lamellipodia and endothelial migration.

Sphingosine-1-phosphate (S1P) regulates a wide array of biological functions in endothelial cells. We previously showed that S1P receptor subtype 2 (S1P2) is significantly up-regulated in the atherosclerotic endothelium (J. Biol. Chem. 283:30363, 2008). In this study, we investigated the roles of S1P2-mediated signaling in the proinflammatory responses of endothelial cells. Treatment with tumor necrosis factor-α (TNFα), a proinflammatory cytokine, increased the expression of S1P2 receptors in endothelial cells. TNFα treatment also enhanced sphingosine kinase 1 expression and increased S1P production. Pharmacological inhibition or knockdown of S1P2 receptors completely abrogated the TNFα-induced VCAM-1 (vascular cell adhesion molecule 1) and ICAM-1 (intercellular adhesion molecule 1) expression in endothelial cells. In contrast, pharmacological inhibition or knockdown of other S1P receptor subtypes had no effect on the TNFα-stimulated ICAM-1 and VCAM-1 expression. Moreover, ectopic expression of S1P2 receptors increased VCAM-1 and ICAM-1 expression in endothelial cells in response to S1P stimulation. Mechanistically, we show that antagonizing S1P2 signaling markedly inhibited the TNFα-stimulated NFκB activation. Utilizing the NFκB reporter luciferase assay, the S1P/S1P2 signaling was shown to stimulate NFκB activation. Moreover, the S1P/S1P2-stimulated VCAM-1/ICAM-1 expression was completely abolished by the pharmacological inhibitor of NFκB. Collectively, our data suggest that TNFα treatment activates autocrine S1P/S1P2 signaling, which subsequently activates NFκB and leads to the proinflammatory responses in endothelial cells.