Mouse SPNS2 functions as a sphingosine-1-phosphate transporter in vascular endothelial cells

Sphingosine-1-phosphate (S1P), a sphingolipid metabolite that is produced inside the cells, regulates a variety of physiological and pathological responses via S1P receptors (S1P1-5). Signal transduction between cells consists of three steps; the synthesis of signaling molecules, their export to the extracellular space and their recognition by receptors. An S1P concentration gradient is essential for the migration of various cell types that express S1P receptors, such as lymphocytes, pre-osteoclasts, cancer cells and endothelial cells. To maintain this concentration gradient, plasma S1P concentration must be at a higher level. However, little is known about the molecular mechanism by which S1P is supplied to extracellular environments such as blood plasma. Here, we show that SPNS2 functions as an S1P transporter in vascular endothelial cells but not in erythrocytes and platelets. Moreover, the plasma S1P concentration of SPNS2-deficient mice was reduced to approximately 60% of wild-type, and SPNS2-deficient mice were lymphopenic. Our results demonstrate that SPNS2 is the first physiological S1P transporter in mammals and is a key determinant of lymphocyte egress from the thymus.     

To stay or to leave: Stem cells and progenitor cells navigating the S1P gradient

Most hematopoietic stem progenitor cells (HSPCs) reside in bone marrow (BM), but a small amount of HSPCs have been found to circulate between BM and tissues through blood and lymph. Several lines of evidence suggest that sphingosine-1-phosphate (S1P) gradient triggers HSPC egression to blood circulation after mobilization from BM stem cell niches. Stem cells also visit certain tissues. After a temporary 36 h short stay in local tissues, HSPCs go to lymph in response to S1P gradient between lymph and tissue and eventually enter the blood circulation. S1P also has a role in the guidance of the primitive HSPCs homing to BM in vivo, as S1P analogue FTY720 treatment can improve HSPC BM homing and engraftment. In stress conditions, various stem cells or progenitor cells can be attracted to local injured tissues and participate in local tissue cell differentiation and tissue rebuilding through modulation the expression level of S1P1, S1P2 or S1P3 receptors. Hence, S1P is important for stem cells circulation in blood system to accomplish its role in body surveillance and injury recovery.     

Sphingosine 1-phosphate signaling: providing cells with a sense of direction

Sphingosine 1-phosphate (S1P) is a sphingolipid metabolite that regulates diverse biological functions. S1P has been identified as a high-affinity ligand for a family of five G-protein-coupled receptors, known as the S1P receptors. The physiological role of the S1P receptor S1P(1) in vascular maturation was recently revealed by gene disruption in mice. In addition to other cellular processes, the binding of S1P to its receptors regulates motility and directional migration of a variety of cell types, including endothelial cells and vascular smooth muscle cells. This review focuses on the important role of S1P and its receptors in cell migration and describes a new paradigm for receptor cross-communication in which transactivation of S1P(1) by a receptor tyrosine kinase (PDGFR) is crucial for cell motility.     

S1P and eNOS regulation

In the mammalian cardiovascular system, nitric oxide (NO), a small diffusible gaseous signal mediator, plays pivotal roles in the maintenance of vascular homeostasis. The endothelial isoform of nitric oxide synthase (eNOS) is activated by diverse agonist-modulated cell surface receptors, and eNOS-derived NO is a key determinant of blood pressure, platelet activation, angiogenesis and other fundamental responses in the vascular wall. Sphingosine 1-phosphate (S1P) has recently been identified as an important activator of eNOS. This review summarizes the roles of sphingosine 1-phosphate and S1P receptors in eNOS activation, and analyzes the eNOS regulatory processes evoked by S1P. The implications of S1P activation of eNOS in cardiovascular (patho)physiology will be also discussed.     

The Edg family G protein-coupled receptors for lysophospholipids: their signaling properties and biological activities

Sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) are blood-borne lysophospholipids with a wide spectrum of biological activities, which include stimulation of cell growth, prevention of apoptosis, regulation of actin cytoskeleton, and modulation of cell shape, cell migration, and invasion. Activated platelets appear to be a major source of both S1P and LPA in blood. Despite the diversity of their biosynthetic origins, they are considered to share substantial structural similarity. Indeed, recent investigation has revealed that S1P and LPA act via a single family of G protein-coupled receptors designated as Edg. Thus, the Edg isoforms, Edg1 (also called S1P(1)), Edg5 (S1P(2)), Edg3 (S1P(3)), Edg6 (S1P(4)), and Edg8 (S1P(5)), are specific receptors for S1P (and SPC with a lower affinity), whereas Edg2 (LPA(1)), Edg4 (LPA(2)), and Edg7 (LPA(3)) serve as receptors specific for LPA. Each receptor isoform displays a unique tissue expression pattern and coupling to a distinct set of heterotrimeric G proteins, leading to the activation of an isoform-specific panel of multiple intracellular signaling pathways. Recent studies on knockout mice have unveiled non-redundant Edg receptor functions that are essential for normal development and vascular maturation. In addition, the Edg lysophospholipid signaling system may play a role in modulating cell motility under such pathological conditions as inflammation, tumor cell dissemination and vascular remodeling.     

Sphingosine 1-Phosphate Receptor Signaling Regulates Proper Embryonic Vascular Patterning

Sphingosine 1-phosphate (S1P) binds G-protein-coupled receptors (S1P_1–5) to regulate a multitude of physiological effects, especially those in the vascular and immune systems. S1P receptors in the vascular system have been characterized primarily in mammals. Here, we report that the S1P receptors and metabolic enzymes are conserved in the genome of zebrafish Danio rerio. Bioinformatic analysis identified seven S1P receptor-like sequences in the zebrafish genome, including duplicated orthologs of receptors 3 and 5. Sphingolipidomic analysis detected erythrocyte and plasma S1P as well as high plasma ceramides and sphingosine. Morpholino-mediated knockdown of s1pr1 causes global and pericardial edema, loss of blood circulation, and vascular defects characterized by both reduced vascularization in intersegmental vessels, decreased proliferation of intersegmental and axial vessels, and hypersprouting in the caudal vein plexus. The s1pr2 gene was previously characterized as a regulator of cell migration and heart development, but its role in angiogenesis is not known. However, when expression of both s1pr1 and s1pr2 is suppressed, severely reduced vascular development of the intersegmental vessels was observed with doses of the s1pr1 morpholino that alone did not cause any discernible vascular defects, suggesting that s1pr1 and s1pr2 function cooperatively to regulate vascular development in zebrafish. Similarly, the S1P transporter, spns2, also cooperated with s1pr1. We propose that extracellular S1P acts through vascular S1P receptors to regulate vascular development.     

Flow-Regulated Endothelial S1P Receptor-1 Signaling Sustains Vascular Development

During angiogenesis, nascent vascular sprouts fuse to form vascular networks, enabling efficient circulation. Mechanisms that stabilize the vascular plexus are not well understood. Sphingosine 1-phosphate (S1P) is a blood-borne lipid mediator implicated in the regulation of vascular and immune systems. Here we describe a mechanism by which the G protein-coupled S1P receptor-1 (S1P(1)) stabilizes the primary vascular network. A gradient of S1P(1) expression from the mature regions of the vascular network to the growing vascular front was observed. In the absence of endothelial S1P(1), adherens junctions are destabilized, barrier function is breached, and flow is perturbed, resulting in abnormal vascular hypersprouting. Interestingly, S1P(1) responds to S1P?as well as laminar shear stress to transduce flow-mediated signaling in endothelial cells both in?vitro and in?vivo. These data demonstrate that blood flow and circulating S1P activate endothelial S1P(1) to stabilize blood vessels in development and homeostasis.     

The sphingosine-1-phosphate receptors S1P1, S1P2, and S1P3 function coordinately during embryonic angiogenesis

Sphingosine-1-phosphate (S1P) elicits diverse cellular responses through a family of G-protein-coupled receptors. We have shown previously that genetic disruption of the S1P(1) receptor, the most widely expressed of the family, results in embryonic lethality because of its key role within endothelial cells in regulating the coverage of blood vessels by vascular smooth muscle cells. To understand the physiologic functions of the two other widely expressed S1P receptors, we generated S1P(2) and S1P(3) null mice. Neither the S1P(2) null mice nor the S1P(3) null mice exhibited significant embryonic lethality or obvious phenotypic abnormalities. To unmask possible overlapping or collaborative functions between the S1P(1), S1P(2), and S1P(3) receptors, we examined embryos with multiple S1P receptor mutations. We found that S1P(1) S1P(2) double null and S1P(1) S1P(2) S1P(3) triple null embryos displayed a substantially more severe vascular phenotype than did embryos with only S1P(1) deleted. We also found partial embryonic lethality and vascular abnormalities in S1P(2) S1P(3) double null embryos. Our results indicate that the S1P(1), S1P(2) and S1P(3) receptors have redundant or cooperative functions for the development of a stable and mature vascular system during embryonic development.     

Physiological and pathological actions of sphingosine 1-phosphate

Sphingosine 1-phosphate (S1P), a product of sphingomyelin (SM) metabolism, occurs widely in nature. Although, originally described as an intracellular second messenger, its role as an extracellular lipid mediator in higher organisms has recently been shown with the discovery of the G protein-coupled receptors (GRCR) for S1P. In mammals, S1P receptors are widely expressed and are thought to regulate important physiological actions, such as immune cell trafficking, vascular development, vascular tone control, cardiac function, and vascular permeability, among others. In addition, S1P may participate in various pathological conditions. For example, S1P has been implicated as an important mediator in autoimmunity, transplant rejection, cancer, angiogenesis, vascular permeability, female infertility, and myocardial infarction. It is important to emphasize that these findings represent an early understanding of the physiological and pathological roles of S1P. The ubiquity of the mediator and its receptors, as well as the evolutionary conservation of S1P metabolism and action, argues that it is a potent and ubiquitous physiological factor in many contexts, and warrant a fuller understanding of its actions at the molecular, cellular and organismal levels.     

Relevance and potential of sphingosine-1-phosphate in vascular inflammatory disease

The typical pathological feature of atherosclerosis is inflammation. In the last years, it has become evident that inhibition of inflammation is one important therapeutic option in atherosclerosis. Recently, sphingolipid sphingosine-1-phosphate (S1P) was identified as a crucial molecule with potent anti-inflammatory properties. Indeed, S1P activates various G protein-coupled receptors, namely S1P1-S1P5. In the vasculature, mainly S1P1-3 receptors are present. FTY720, after phosphorylation to FTY720-P, is an orally active S1P mimetic. FTY720 has been developed for therapy in the field of autoimmune diseases and organ transplantation. In analogy to S1P, FTY720 shows potent anti-inflammatory effects and several groups have tested the in vivo effects of FTY720 on the progression of inflammatory vascular diseases. They could show that S1P receptor activation might lead to a partial inhibition of the progression of atherosclerotic lesions. S1P receptor activation therefore might be a concept for anti-inflammatory drug treatment. However, it is not clear how S1P and FTY720 exactly act on vascular inflammation. This review article gives a brief overview over the known actions of S1P in vascular inflammatory disease.     

Lysophospholipids and the cardiovascular system

The lysophospholipids sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) have varied effects on the cardiovascular system. S1P is necessary for normal vascular development and may play an important role in angiogenesis. These molecules may exert potentially detrimental effects. Both S1P and LPA are released from activated platelets and can in turn stimulate platelet aggregation. These thrombogenic effects would further enhance ischemia in acute coronary syndromes and myocardial infarction. LPA is a major component of the lipid core of human atherosclerotic plaques and can stimulate vascular smooth muscle proliferation. Both LPA and S1P cause cardiac myocyte hypertrophy in vitro. Beneficial effects include cardioprotection both in vitro and during ischemia/reperfusion in an ex vivo whole heart mouse model. Understanding both the acute and the chronic physiologic and pathophysiologic roles of the lysophospholipids and their cognate receptors and signaling pathways in the cardiovascular system, which are likely to be species-, tissue-, and cell-specific, may allow the development of molecules that can be targeted to stimulate or inhibit a specific function.     

The carboxyl tail of protease-activated receptor-1 is required for chemotaxis. Correlation of signal termination and directional migration.

The G protein-coupled thrombin receptor, protease-activated receptor 1 (PAR1), mediates many of the actions of thrombin on cells including chemotaxis. In contrast to the reversible agonist binding that regulates signaling by most G protein-coupled receptors (GPCRs), PAR1 is activated by an irreversible proteolytic mechanism. Although activated PAR1 is phosphorylated, uncoupled, and internalized like typical GPCRs, signal termination is additionally dependent on lysosomal degradation of cleaved and activated receptors. In the present study we exploit two PAR1 mutants to examine the link between chemotaxis and receptor shutoff. One, a carboxyl tail deletion mutant (Y397Z), is defective in phosphorylation and internalization. The other, a carboxyl tail chimeric receptor (P/S), is phosphorylated and internalized upon activation but recycles to the plasma membrane like reversibly activated GPCRs. Expression of these receptors in a hematopoietic cell line disrupted cell migration along thrombin gradients. Thrombin activation of cells expressing P/S or Y397Z resulted in persistent signaling independent of the continued presence of thrombin. Signaling in response to the soluble agonist peptide SFLLRN was reversible for P/S but persisted for Y397Z. Strikingly, cells expressing P/S responded chemokinetically to thrombin but chemotactically to SFLLRN. In contrast, Y397Z-mediated migration was largely chemokinetic to both agonists. These studies suggest that termination of PAR1 signaling at the level of the receptor is necessary for gradient detection and directional migration.     

S1P activates store-operated calcium entry via receptor- and non-receptor-mediated pathways in vascular smooth muscle cells

Sphingosine-1-phosphate (S1P) has been shown to modulate intracellular Ca(2+) through both G protein-coupled receptors and intracellular second messenger pathways. The precise mechanism by which S1P activates store-operated calcium entry (SOCE) in vascular smooth muscle cells (VSMCs) has not been fully characterized. Because sphingolipids and Ca(2+) modulate proliferation and constriction in VSMCs, characterizing the connection between S1P and SOCE may provide novel therapeutic targets for vascular diseases. We found that S1P triggered STIM1 puncta formation and SOCE in VSMCs. S1P-activated SOCE was inhibited by 2-aminoethoxydiphenyl borate (2-APB), diethylstilbestrol (DES), and gadolinium (Gd(3+)). SOCE was observed in VSMCs lacking either S1P(2) or S1P(3) receptors, suggesting that S1P acts via multiple signaling pathways. Indeed, both extracellular and intracellular S1P application increased the total internal reflection fluorescence signal in VSMCs cells transfected with STIM1-yellow fluorescent protein in a 2-APB-sensitive manner. These data, and the fact that 2-APB, DES, and Gd(3+) all inhibited S1P-induced cerebral artery constriction, suggest that SOCE modulates S1P-induced vasoconstriction in vivo. Finally, S1P-induced SOCE was larger in proliferative than in contractile VSMCs, correlating with increases in STIM1, Orai1, S1P(1), and S1P(3) receptor mRNA. These data demonstrate that S1P can act through both receptors and a novel intracellular pathway to activate SOCE. Because S1P-induced SOCE contributes to vessel constriction and is increased in proliferative VSMCs, it is likely that S1P/SOCE signaling in proliferative VSMCs may play a role in vascular dysfunction such as atherosclerosis and diabetes.     

Structural and functional characteristics of S1P receptors

The sphingosine-1-phosphate (S1P) family of G protein-coupled receptors (GPCR) regulates essential cellular processes such as proliferation, migration, cytoskeletal organization, adherens junction assembly, and morphogenesis. S1P, a product from the breakdown of sphingomyelin, binds to the five members of this receptor family, S1P(1), S1P(2), S1P(3), S1P(4), and S1P(5), previously referred to as endothelial differentiation gene (EDG)-1, -5, -3, -6, and -8. S1P receptors are widely expressed in different tissues, so it is not surprising that the S1P receptor family regulates many physiological processes, such as vascular maturation, cardiac development, lymphocyte trafficking, and vascular permeability. FTY720, a new S1P receptor agonist, is undergoing clinical trials as an immunosuppressor. Understanding the physiological role of these receptors and the basics of the ligand-receptor interaction will potentially provide new therapies to control a variety of diseases.     

Roles of sphingosine-1-phosphate signaling in angiogenesis

Sphingosine-1-phosphate (S1P) is a blood-borne lipid mediator with pleiotropic biological activities. S1P acts via the specific cell surface G-protein-coupled receptors, S1P(1-5). S1P(1) and S1P(2) were originally identified from vascular endothelial cells (ECs) and smooth muscle cells, respectively. Emerging evidence shows that S1P plays crucial roles in the regulation of vascular functions, including vascular formation, barrier protection and vascular tone via S1P(1), S1P(2) and S1P(3). In particular, S1P regulates vascular formation through multiple mechanisms; S1P exerts both positive and negative effects on angiogenesis and vascular maturation. The positive and negative effects of S1P are mediated by S1P(1) and S1P(2), respectively. These effects of S1P(1) and S1P(2) are probably mediated by the S1P receptors expressed in multiple cell types including ECs and bone-marrow-derived cells. The receptor-subtype-specific, distinct effects of S1P favor the development of novel therapeutic tactics for antitumor angiogenesis in cancer and therapeutic angiogenesis in ischemic diseases.     

Sphingosine 1-phosphate and its receptors: an autocrine and paracrine network

Sphingosine 1-phosphate (S1P) is a biologically active lysophospholipid that transmits signals through a family of G-protein-coupled receptors to control cellular differentiation and survival, as well as the vital functions of several types of immune cell. In this Review article, we discuss recent results that indicate that S1P and its receptors are required for the emigration of thymocytes from the thymus, the trafficking of lymphocytes in secondary lymphoid organs and the migration of B cells into splenic follicles. In an autocrine manner, through interactions with different G-protein-coupled receptors, S1P also enhances optimal mast-cell migration and release of pro-inflammatory mediators in allergic reactions. S1P-S1P-receptor regulatory systems might therefore be novel targets for the therapy of diverse immunological diseases.     

Sphingosine-1-phosphate signaling and biological activities in the cardiovascular system

The plasma lysophospholipid mediator sphingosine-1-phosphate (S1P) is produced exclusively by sphingosine kinase (SPHK) 1 and SPHK2 in vivo, and plays diverse biological and pathophysiological roles by acting largely through three members of the G protein-coupled S1P receptors, S1P(1), S1P(2) and S1P(3). S1P(1) expressed on endothelial cells mediates embryonic vascular maturation and maintains vascular integrity by contributing to eNOS activation, inhibiting vascular permeability and inducing endothelial cell chemotaxis via Gi-coupled mechanisms. By contrast, S1P(2), is expressed in high levels on vascular smooth muscle cells (VSMCs) and certain types of tumor cells, inhibiting Rac and cell migration via a G(12/13)-and Rho-dependent mechanism. In rat neointimal VSMCs, S1P(1) is upregulated to mediate local production of platelet-derived growth factor, which is a key player in vascular remodeling. S1P(3) expressed on endothelial cells also mediates chemotaxis toward S1P and vasorelaxation via NO production in certain vascular bed, playing protective roles for vascular integrity. S1P(3) expressed on VSMCs and cardiac sinoatrial node cells mediates vasopressor and negative chronotropic effect, respectively. In addition, S1P(3), together with S1P(2) and SPHK1, is suggested to play a protective role against acute myocardial ischemia. However, our recent work indicates that overexpressed SPHK1 is involved in cardiomyocyte degeneration and fibrosis in vivo, in part through S1P activation of the S1P(3) signaling. We also demonstrated that exogenously administered S1P accelerates neovascularization and blood flow recovery in ischemic limbs, suggesting its usefulness for angiogenic therapy. These results provide evidence for S1P receptor subtype-specific pharmacological intervention as a novel therapeutic approach to cardiovascular diseases and cancer.     

Unraveling the complexities of sphingosine-1-phosphate function: the mast cell model

Sphingosine-1-phosphate (S1P) is a lipid mediator involved in diverse biological processes, from vascular and neural development to the regulation of lymphocyte trafficking. Many of its functions are regulated by five widely expressed S1P G-protein-coupled receptors (S1P(1-5)). S1P is produced mostly intracellularly, thus, much of its potential as an autocrine and paracrine mediator depends on how, when, and where it is generated or secreted out of the cells. However, S1P can also have intracellular activity independent of its receptors, adding to the complexity of S1P function. The mast cell, a major effector cell during an allergic response, has proven instrumental towards understanding the complex regulation and function of S1P. Antigen (Ag) engagement of the IgE receptor in mast cells stimulates sphingosine kinases, which generate S1P and are involved in the activation of calcium fluxes critical for mast cell responses. In addition, mast cells secrete considerable amounts of S1P upon activation, thus affecting the surrounding tissues and recruiting inflammatory cells. Export of S1P is also involved in the autocrine transactivation of S1P receptors present in mast cells. The in vivo response of mast cells, however, is not strictly dependent on their ability to generate S1P, but they are also affected by changes in S1P in the environment previous to Ag challenge. This review will discuss the recent advances towards understanding the intricacies of S1P generation, secretion and regulation in mast cells. In addition, how S1P receptors are activated and their involvement in mast cell functions will also be covered, including new insights on the role of S1P in the mast cell-mediated allergic response of systemic anaphylaxis.     

Athero- and thrombogenic actions of lysophosphatidic acid and sphingosine-1-phosphate

Lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) are potent bioactive phospholipids with specific and multiple effects on blood cells and cells of the vessel wall. Released by activated platelets, LPA and S1P mediate physiological wound healing processes such as vascular repair. Evidence is accumulating that these lipid mediators can, however, under certain conditions become athero- and thrombogenic molecules that might aggravate cardiovascular disease. For example, LPA present in minimally modified LDL and within the intima of atherosclerotic lesions may play a role in the early phase of atherosclerosis by inducing barrier dysfunction and increased monocyte adhesion of the endothelium, as well as in the late phase by triggering platelet activation and intra-arterial thrombus formation upon rupture of the atherosclerotic plaque. Moreover, LPA and S1P, by stimulating the proliferation of fibroblasts and by enhancing the survival of inflammatory cells are likely to play a central role in the excessive fibroproliferative and inflammatory response to vascular injury that characterizes the progression of atherosclerosis. Furthermore, LPA can cause the phenotypic dedifferentiation of medial vascular smooth muscle cells, and S1P is able to stimulate the migration and proliferation of intimal vascular smooth muscle cells; both processes ultimately lead to the formation of the neointima. Most importantly, as LPA and S1P bind to and activate multiple G-protein receptors, it emerges that the beneficial or harmful action of LPA and S1P are critically dependent on the expression profile of their receptor subtypes and their coupling to different signal transduction pathways in the target cells. By targeting specific subtypes of LPA and S1P receptors in selective cells of the vascular wall and blood, new strategies for the prevention and therapy of cardiovascular diseases can be envisioned.