Vascular endothelial cells undergo morphogenesis into capillary networks in response to angiogenic factors. We show here that sphingosine-1-phosphate (SPP), a platelet-derived bioactive lipid, activates the EDG-1 and -3 subtypes of G protein-coupled receptors on endothelial cells to regulate angiogenesis. SPP induces the Gi/mitogen-activated protein kinase/cell survival pathway and the small GTPase Rho- and Raccoupled adherens junction assembly. Both EDG-1-and EDG-3-regulated signaling pathways are required for endothelial cell morphogenesis into capillary-like networks. Indeed, SPP synergized with polypeptide angiogenic growth factors in the formation of mature neovessels in vivo. These data define SPP as a novel regulator of angiogenesis.

The sphingolipid metabolite sphingosine-1-phosphate (SPP) has been implicated as a second messenger in cell proliferation and survival. However, many of its biological effects are due to binding to unidentified receptors on the cell surface. SPP activated the heterotrimeric guanine nucleotide binding protein (G protein)-coupled orphan receptor EDG-1, originally cloned as Endothelial Differentiation Gene-1. EDG-1 bound SPP with high affinity (dissociation constant = 8.1 nM) and high specificity. Overexpression of EDG-1 induced exaggerated cell-cell aggregation, enhanced expression of cadherins, and formation of well-developed adherens junctions in a manner dependent on SPP and the small guanine nucleotide binding protein Rho.

Sphingosine-1-phosphate (SPP), a bioactive lipid, acts both intracellularly and extracellularly to cause pleiotropic biological responses. Recently, we identified SPP as a ligand for the G protein–coupled receptor Edg-1 (Lee, M.-J., J.R. Van Brocklyn, S. Thangada, C.H. Liu, A.R. Hand, R. Menzeleev, S. Spiegel, and T. Hla. 1998. Science. 279:1552–1555). Edg-1 binds SPP with remarkable specificity as only sphinganine-1-phosphate displaced radiolabeled SPP, while other sphingolipids did not. Binding of SPP to Edg-1 resulted in inhibition of forskolin-stimulated cAMP accumulation, in a pertussis toxin–sensitive manner. In contrast, two well-characterized biological responses of SPP, mitogenesis and prevention of apoptosis, were clearly unrelated to binding to Edg-1 and correlated with intracellular uptake. SPP also stimulated signal transduction pathways, including calcium mobilization, activation of phospholipase D, and tyrosine phosphorylation of p125FAK, independently of edg-1 expression. Moreover, DNA synthesis in Swiss 3T3 fibroblasts was significantly and specifically increased by microinjection of SPP. Finally, SPP suppresses apoptosis of HL-60 and pheochromocytoma PC12 cells, which do not have specific SPP binding or expression of Edg-1 mRNA. Conversely, sphinganine-1-phosphate, which binds to and signals via Edg-1, does not have any significant cytoprotective effect. Thus, SPP is a prototype for a novel class of lipid mediators that act both extracellularly as ligands for cell surface receptors and intracellularly as second messengers.

Abstract-Sphingosine 1-phosphate (S1P), an abundant lipid mediator in plasma, regulates vascular and immune cells by activating S1P receptors. In this report, we investigated the mechanisms by which high plasma S1P levels are maintained in mice. We found that plasma S1P turns over rapidly with a half-life of Ϸ15 minutes, suggesting the existence of a high-capacity biosynthetic source(s). Transplantation of bone marrow from wild-type to Sphk1 Ϫ/Ϫ Sphk2 ϩ/Ϫ mice restored plasma S1P levels, suggesting that hematopoietic cells are capable of secreting S1P into plasma. However, plasma S1P levels were not appreciably altered in mice that were thrombocytopenic, anemic, or leukopenic. Surprisingly, reconstitution of Sphk1 Ϫ/Ϫ Sphk2 ϩ/Ϫ bone marrow cells into wild-type hosts failed to reduce plasma S1P, suggesting the existence of an additional, nonhematopoietic source for plasma S1P. Adenoviral expression of Sphk1 in the liver of Sphk1 Ϫ/Ϫ mice restored plasma S1P levels. In vitro, vascular endothelial cells, but not hepatocytes, secreted S1P in a constitutive manner. Interestingly, laminar shear stress downregulated the expression of S1P lyase (Sgpl) and S1P phosphatase-1 (Sgpp1) while concomitantly stimulating S1P release from endothelial cells in vitro. Modulation of expression of endothelial S1P lyase with small interfering RNA and adenoviral expression altered S1P secretion, suggesting an important role played by this enzyme. These data suggest that the vascular endothelium, in addition to the hematopoietic system, is a major contributor of plasma S1P. Key Words: sphingosine 1-phosphate (S1P) Ⅲ sphingosine kinase (Sphk) Ⅲ S1P lyase (Sgpl) Ⅲ plasma S1P gradient Ⅲ Shear stress T he bioactive lipid sphingosine 1-phosphate (S1P) is a potent regulator of numerous biological responses, the most well characterized being cardiovascular and immune effects. S1P binds to and activates a widely expressed family of G protein-coupled receptors, termed S1PRs. Intracellular signaling of these receptors are thought to mediate most of the effects of S1P. [1][2][3][4] S1P is abundant (0.1 to 1.2 mol/L) in plasma, where it is mainly bound to albumin and high-density lipoprotein (HDL). 5,6 Thus, S1P receptors on blood-borne cells are likely to be constitutively activated. In contrast, S1P levels in tissues are considerably lower (0.5 to 75 pmol/mg wet weight), although tissues with high blood content, such as spleen, are exceptions. This concentration difference of S1P between plasma and tissues has been termed the vascular S1P gradient, which was shown to be functionally important in lymphocyte egress from the lymphoid tissues and the thymus. 7,8 The regulation of S1P production and release is not well understood. Secretion of S1P is observed in a variety of cells including platelets, 9 -11 erythrocytes, 9,12,13 mononuclear cells, neutrophils, 9 mast cells, 14,15 and endothelial cells. 16 The concentration of S1P in the cell is determined by the activity of biosynthetic enzymes (sphingosine kinase [Sphk]-1 and -2) and the degradative ...

Sphingosine 1-phosphate (S1P), a multifunctional lipid mediator, regulates lymphocyte trafficking, vascular permeability, and angiogenesis by activation of the S1P 1 receptor. This receptor is activated by FTY720-P, a phosphorylated derivative of the immunosuppressant and vasoactive compound FTY720. However, in contrast to the natural ligand S1P, FTY720-P appears to act as a functional antagonist, even though the mechanisms involved are poorly understood. In this study, we investigated the fate of endogenously expressed S1P 1 receptor in agonist-activated human umbilical vein endothelial cells and human embryonic kidney 293 cells expressing green fluorescent protein-tagged S1P 1 . We show that FTY720-P is more potent than S1P at inducing receptor degradation. Pretreatment with an antagonist of S1P 1 , VPC 44116, prevented receptor internalization and degradation. FTY720-P did not induce degradation of internalization-deficient S1P 1 receptor mutants. Further, small interfering RNA-mediated down-regulation of G protein-coupled receptor kinase-2 and ␤-arrestins abolished FTY720-P-induced S1P 1 receptor degradation. These data suggest that agonistinduced phosphorylation of S1P 1 and subsequent endocytosis are required for FTY720-P-induced degradation of the receptor. S1P 1 degradation is blocked by MG132, a proteasomal inhibitor. Indeed, FTY720-P strongly induced polyubiquitinylation of S1P 1 receptor, whereas S1P at concentrations that induced complete internalization was not as efficient, suggesting that receptor internalization is required but not sufficient for ubiquitinylation and degradation. We propose that the ability of FTY720-P to target the S1P 1 receptor to the ubiquitinylation and proteasomal degradation pathway may at least in part underlie its immunosuppressive and anti-angiogenic properties.Sphingosine 1-phosphate (S1P) 2 is recognized as a multifunctional bioactive lipid mediator involved in immune cell trafficking, regulation of vascular permeability, and angiogenesis (1, 2). It acts via a family of G protein-coupled receptors referred to as S1P n receptors (3). The prototypical receptor, S1P 1 was originally isolated as an inducible gene from vascular endothelial cells (4). Knock out of S1P 1 resulted in embryonic lethality due to a vascular maturation defect (5). We recently showed that S1P 1 function in endothelial cells is needed for proper endothelial-pericyte interaction, a critical event in vascular maturation (6). In addition, we demonstrated previously that S1P 1 is needed for the assembly of vascular endothelialcadherin-based adherens junctions on vascular endothelial cells (7). This event is needed for regulation of paracellular permeability, a model system vascular leak syndrome (8). In the immune system, selective deletion of S1P 1 in T-cells led to inhibition of lymphocyte egress from lymph nodes and the thymus (9, 10). However, function of S1P 1 in efferent lymphatics may also be important for the regulation of lymphocyte egress, as activation of this receptor may lead to "gate clos...

The role of the protein kinase Akt in cell migration is incompletely understood. Here we show that sphingosine-1-phosphate (S1P)-induced endothelial cell migration requires the Akt-mediated phosphorylation of the G protein-coupled receptor (GPCR) EDG-1. Activated Akt binds to EDG-1 and phosphorylates the third intracellular loop at the T(236) residue. Transactivation of EDG-1 by Akt is not required for G(i)-dependent signaling but is indispensable for Rac activation, cortical actin assembly, and chemotaxis. Indeed, T236AEDG-1 mutant sequestered Akt and acted as a dominant-negative GPCR to inhibit S1P-induced Rac activation, chemotaxis, and angiogenesis. Transactivation of GPCRs by Akt may constitute a specificity switch to integrate rapid G protein-dependent signals into long-term cellular phenomena such as cell migration.

Sphingosine 1-phosphate (SPP), a platelet-derived bioactive lysophospholipid, is a regulator of angiogenesis. However, molecular mechanisms involved in SPPinduced angiogenic responses are not fully defined. Here we report the molecular mechanisms involved in SPP-induced human umbilical vein endothelial cell (HUVEC) adhesion and migration. SPP-induced HUVEC migration is potently inhibited by antisense phosphothioate oligonucleotides against EDG-1 as well as EDG-3 receptors. In addition, C3 exotoxin blocked SPP-induced cell attachment, spreading and migration on fibronectin-, vitronectin-and Matrigel-coated surfaces, suggesting that endothelial differentiation gene receptor signaling via the Rho pathway is critical for SPPinduced cell migration. Indeed, SPP induced Rho activation in an adherence-independent manner, whereas Rac activation was dispensible for cell attachment and focal contact formation. Interestingly, both EDG-1 and -3 receptors were required for Rho activation. Since integrins are critical for cell adhesion, migration, and angiogenesis, we examined the effects of blocking antibodies against ␣ v ␤ 3 , ␤ 1 , or ␤ 3 integrins. SPP induced Rho-dependent integrin clustering into focal contact sites, which was essential for cell adhesion, spreading and migration. Blockage of ␣ v ␤ 3 -or ␤ 1 -containing integrins inhibited SPP-induced HUVEC migration. Together our results suggest that endothelial differentiation gene receptor-mediated Rho signaling is required for the activation of integrin ␣ v ␤ 3 as well as ␤ 1 -containing integrins, leading to the formation of initial focal contacts and endothelial cell migration. Sphingosine 1-phosphate (SPP)1 is a bioactive lipid produced by metabolism of the membrane phospholipid, sphingomyelin.Activation of the sphingomyelinase enzyme followed by sequential catalysis by ceramidase and sphingosine kinase results in the formation of SPP (1). Although the site of synthesis and mechanisms involved in secretion of SPP are not well understood, it is clear that one mechanism by which SPP acts is via the interaction with plasma membrane-localized G-proteincoupled receptors (GPCR) of the EDG family (2, 3). To date, EDG-1, -3, -5, -6, and -8 were shown to bind to SPP and transduce various intracellular signals (4 -9). Signal transduction mechanisms of EDG-1, -3, and -5 have been defined. These GPCRs are stimulated by nanomolar concentrations of SPP

Sphingosine 1-phosphate (S1P), produced by Sphks (sphingosine kinases), is a multifunctional lipid mediator that regulates immune cell trafficking and vascular development. Mammals maintain a large concentration gradient of S1P between vascular and extravascular compartments. Mechanisms by which S1P is released from cells and concentrated in the plasma are poorly understood. We recently demonstrated [Ancellin, Colmont, Su, Li, Mittereder, Chae, Stefansson, Liau and Hla (2002) J. Biol. Chem. 277, 6667-6675] that Sphk1 activity is constitutively secreted by vascular endothelial cells. In the present study, we show that among the five Sphk isoforms expressed in endothelial cells, the Sphk-1a isoform is selectively secreted in HEK-293 cells (human embryonic kidney cells) and human umbilical-vein endothelial cells. In sharp contrast, Sphk2 is not secreted. The exported Sphk-1a isoform is enzymatically active and produced sufficient S1P to induce S1P receptor internalization. Wild-type mouse plasma contains significant Sphk activity (179 pmol x min(-1) x g(-1)). In contrast, Sphk1-/- mouse plasma has undetectable Sphk activity and approx. 65% reduction in S1P levels. Moreover, human plasma contains enzymatically active Sphk1 (46 pmol x min(-1) x g(-1)). These results suggest that export of Sphk-1a occurs under physiological conditions and may contribute to the establishment of the vascular S1P gradient.