Growth signalling networks that use glycerophospholipid metabolites as second messengers have been well characterized, but less is known of the second messengers derived from sphingolipids, another major class of membrane lipids. A tantalizing link between sphingolipids and cellular proliferation has emerged from the discovery that the sphingolipid metabolites sphingosine and sphingosine-1-phosphate stimulate growth of quiescent Swiss 3T3 fibroblasts by a pathway that is independent of protein kinase C. Sphingosine-1-phosphate is rapidly produced from sphingosine and may mediate its biological effects. Furthermore, sphingosine-1-phosphate triggers the dual signal transduction pathways of calcium mobilization and activation of phospholipase D, prominent events in the control of cellular proliferation. Here we report that activation of sphingosine kinase and the formation of sphingosine-1-phosphate are important in the signal transduction pathways activated by the potent mitogens platelet-derived growth factor (PDGF) and fetal calf serum (FCS).

Sphingosine-1-phosphate (S1P), an important sphingolipid metabolite, regulates diverse cellular processes, including cell survival, growth, and differentiation. Here we show that S1P signaling is critical for neural and vascular development. Sphingosine kinase-null mice exhibited a deficiency of S1P which severely disturbed neurogenesis, including neural tube closure, and angiogenesis and caused embryonic lethality. A dramatic increase in apoptosis and a decrease in mitosis were seen in the developing nervous system. S1P 1 receptor-null mice also showed severe defects in neurogenesis, indicating that the mechanism by which S1P promotes neurogenesis is, in part, signaling from the S1P 1 receptor. Thus, S1P joins a growing list of signaling molecules, such as vascular endothelial growth factor, which regulate the functionally intertwined pathways of angiogenesis and neurogenesis. Our findings also suggest that exploitation of this potent neuronal survival pathway could lead to the development of novel therapeutic approaches for neurological diseases.Sphingosine-1-phosphate (S1P) is a signaling molecule that is crucial for the regulation of several diverse cellular events, including cell survival, growth, differentiation, motility, and calcium mobilization (29). Recently, numerous studies on S1P have demonstrated its importance in the development of the vascular system (1,14,16,18), the heart (15), and immunity (2, 5, 21) by signaling through a family of G protein-coupled receptors designated S1P 1 to S1P 5 . The S1P signaling pathway through S1P receptors induces activation of the protein kinase Akt, phosphatidylinositol 3-kinase, the small GTPase Rac, phospholipase C, and extracellular signal-regulated kinase, leading to cell survival and proliferation (11). In higher plants, S1P also plays an important role in calcium regulation and the control of guard cells (7).Sphingosine kinase (SphK) is an enzyme that catalyzes the phosphorylation of sphingosine to form S1P. Two isoforms of mammalian SphK (SphK1 and SphK2) have been cloned and characterized (13,17). Recent studies revealed that overexpression of SphK2 suppressed cell growth and also markedly enhanced apoptosis in cultured cells (12,19), in sharp contrast to findings for SphK1, which generally promoted cell survival and growth (26,27).Recently, we generated SphK1 knockout mice to study the enzyme's physiological functions (3). The SphK1 Ϫ/Ϫ mice were viable and fertile and lacked any obvious abnormalities, although total SphK activity was substantially, but not completely, reduced. These results raise the possibility that SphK1 and SphK2 might have redundant functions in mice and that SphK2 could compensate for a deficiency in SphK1 activity. To investigate the physiological functions of both isoforms, we generated SphK2 knockout mice, as well as SphK1/SphK2 double-knockout mice. Simultaneous deletion of both enzymes resulted in mice with undetectable levels of S1P. The studies on S1P-deficient mice described herein reveal a novel function for S1P signaling ...

Sphingosine-1-phosphate (S1P) is a biologically active metabolite of plasma-membrane sphingolipids that is essential for immune-cell trafficking. Its concentration is increased in many inflammatory conditions, such as in asthma and autoimmunity. Much of the immune function of S1P results from engagement of a family of G-protein-coupled receptors (S1PR1-S1PR5). Recent findings on the role of S1P in immunosurveillance, the discovery of regulatory mechanisms in S1P-mediated immune-cell trafficking, and new advances in understanding how S1P affects immune-cell function indicate that the alliance between S1P and its receptors has a fundamental role in immunity.Sphingolipids are essential plasma-membrane lipids that concentrate in liquid-ordered domains (commonly known as lipid rafts or cholesterol-enriched membrane microdomains). These lipids can be rapidly metabolized upon stimulation of various plasma-membrane receptors through the activation of an enzymatic cascade (FIG. 1), which converts sphingolipids, such as sphingomyelin or complex glycosphingolipids, to ceramide and subsequently to sphingosine. Two sphingosine kinases (SPHK1 and SPHK2) then phosphorylate sphingosine to generate the lysosphingolipid sphingosine-1-phosphate (S1P) 1 . This sphingolipid metabolite has both cell intrinsic and extrinsic activity, affecting cell homeostasis and function 2 . Here, we focus on its cell-extrinsic function in the immune system as a ligand for a family of five G-protein-coupled receptors (GPCRs), known as S1PR1-S1PR5 3 .The fundamental physiological role of the interaction between S1P and S1PRs in immune-cell function was recognized through studies of the immunosuppressant FTY720. This compound rapidly induces lymphopenia through the sequestration of lymphocytes in lymph nodes and by blocking the egress of mature thymocytes from the thymus. A breakthrough in the understanding of its mechanism, together with a link to S1PR signalling, came with the realization that FTY720 is a sphingosine analogue that could be phosphorylated by SPHKs to produce a S1PR ligand 4 with potent effects including S1PR agonism and downregulation of S1PR expression 5-7 .In this article, we discuss the recent advances towards understanding how S1P and its receptors regulate immune-cell trafficking and function. New data have emerged demonstrating a role for S1P-S1PR in immunosurveillance, immune cell-differentiation and immune responses. The recent flurry of research activity in this area has shown that the interplay between S1P metabolism and receptor function has broad effects on the immune system. The evolving paradigm is that the alliance of S1P-S1PR is an essential regulatory circuit in immunity.

Sphingosine-1-phosphate (SPP) is a bioactive lipid that has recently been identified as the ligand for the EDG family of G protein–coupled cell surface receptors. However, the mitogenic and survival effects of exogenous SPP may not correlate with binding to cell-surface receptors (Van Brocklyn, J.R., M.J. Lee, R. Menzeleev, A. Olivera, L. Edsall, O. Cuvillier, D.M. Thomas, P.J.P. Coopman, S. Thangada, T. Hla, and S. Spiegel. 1998. J. Cell Biol. 142:229–240). The recent cloning of sphingosine kinase, a unique lipid kinase responsible for the formation of SPP, has provided a new tool to investigate the role of intracellular SPP. Expression of sphingosine kinase markedly increased SPP levels in NIH 3T3 fibroblasts and HEK293 cells, but no detectable secretion of SPP into the medium was observed. The increased sphingosine kinase activity in NIH 3T3 fibroblasts was sufficient to promote growth in low- serum media, expedite the G1/S transition, and increase DNA synthesis and the proportion of cells in the S phase of the cell cycle with a concomitant increase in cell numbers. Transient or stable overexpression of sphingosine kinase in NIH 3T3 fibroblasts or HEK293 cells protected against apoptosis induced by serum deprivation or ceramide elevation. N,N-Dimethylsphingosine, a competitive inhibitor of sphingosine kinase, blocked the effects of sphingosine kinase overexpression on cell proliferation and suppression of apoptosis. In contrast, pertussis toxin did not abrogate these biological responses. In Jurkat T cells, overexpression of sphingosine kinase also suppressed serum deprivation- and ceramide-induced apoptosis and, to a lesser extent, Fas-induced apoptosis, which correlated with inhibition of DEVDase activity, as well as inhibition of the executionary caspase-3. Taken together with ample evidence showing that growth and survival factors activate sphingosine kinase, our results indicate that SPP functions as a second messenger important for growth and survival of cells. Hence, SPP belongs to a novel class of lipid mediators that can function inside and outside cells.

Sphingosine-1-phosphate (SPP) is a novel lipid messenger that has dual function. Intracellularly it regulates proliferation and survival, and extracellularly, it is a ligand for the G protein-coupled receptor Edg-1. Based on peptide sequences obtained from purified rat kidney sphingosine kinase, the enzyme that regulates SPP levels, we report here the cloning, identification, and characterization of the first mammalian sphingosine kinases (murine SPHK1a and SPHK1b). Sequence analysis indicates that these are novel kinases, which are not similar to other known kinases, and that they are evolutionarily conserved. Comparison with Saccharomyces cerevisiae and Caenorhabditis elegans sphingosine kinase sequences shows that several blocks are highly conserved in all of these sequences. One of these blocks contains an invariant, positively charged motif, GGKGK, which may be part of the ATP binding site. From Northern blot analysis of multiple mouse tissues, we observed that expression was highest in adult lung and spleen, with barely detectable levels in skeletal muscle and liver. Human embryonic kidney cells and NIH 3T3 fibroblasts transiently transfected with either sphingosine kinase expression vectors had marked increases (more than 100-fold) in sphingosine kinase activity. The enzyme specifically phosphorylated D-erythro-sphingosine and did not catalyze the phosphorylation of phosphatidylinositol, diacylglycerol, ceramide, D,L-threo-dihydrosphingosine or N,N-dimethylsphingosine. The latter two sphingolipids were competitive inhibitors of sphingosine kinase in the transfected cells as was previously found with the purified rat kidney enzyme. Transfected cells also had a marked increase in mass levels of SPP with a concomitant decrease in levels of sphingosine and, to a lesser extent, in ceramide levels. Our data suggest that sphingosine kinase is a prototypical member of a new class of lipid kinases. Cloning of sphingosine kinase is an important step in corroborating the intracellular role of SPP as a second messenger.The sphingolipid metabolite, sphingosine-1-phosphate (SPP), 1 is emerging as a prototype of a new class of lipid second messengers, which has both intracellular and extracellular actions (1-4). Ample evidence indicates that SPP can serve as an intracellular second messenger; SPP modulates intracellular pathways important for diverse biological processes including cell growth, survival, motility, and cytoskeletal changes (reviewed in Ref. 5). Moreover, because SPP antagonizes apoptosis mediated by ceramide, a stress-induced sphingolipid metabolite (3, 6), we have proposed that the relative intracellular levels of these two sphingolipid metabolites is an important factor that determines whether cells will survive or die (3). In support of this idea, it has recently been shown that unfertilized mouse oocytes exposed to the anticancer drug doxorubicin undergo ceramide-mediated apoptosis that is inhibited by SPP (7). In addition, it seems that this ceramide/SPP rheostat is an evolutionarily conserved stress...

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.

Mast cells secrete various substances that initiate and perpetuate allergic responses. Cross-linking of the high-affinity receptor for IgE (FcɛRI) in RBL-2H3 and bone marrow–derived mast cells activates sphingosine kinase (SphK), which leads to generation and secretion of the potent sphingolipid mediator, sphingosine-1–phosphate (S1P). In turn, S1P activates its receptors S1P1 and S1P2 that are present in mast cells. Moreover, inhibition of SphK blocks FcɛRI-mediated internalization of these receptors and markedly reduces degranulation and chemotaxis. Although transactivation of S1P1 and Gi signaling are important for cytoskeletal rearrangements and migration of mast cells toward antigen, they are dispensable for FcɛRI-triggered degranulation. However, S1P2, whose expression is up-regulated by FcɛRI cross-linking, was required for degranulation and inhibited migration toward antigen. Together, our results suggest that activation of SphKs and consequently S1PRs by FcɛRI triggering plays a crucial role in mast cell functions and might be involved in the movement of mast cells to sites of inflammation.

Sphingosine-1-phosphate (S1P), a lipid signaling molecule that regulates many cellular functions, is synthesized from sphingosine and ATP by the action of sphingosine kinase. Two such kinases have been identified, SPHK1 and SPHK2. To begin to investigate the physiological functions of sphingosine kinase and S1P signaling, we generated mice deficient in SPHK1. Sphk1 null mice were viable, fertile, and without any obvious abnormalities. Total SPHK activity in most Sphk1؊/؊ tissues was substantially, but not completely, reduced indicating the presence of multiple sphingosine kinases. S1P levels in most tissues from the Sphk1؊/؊ mice were not markedly decreased. In serum, however, there was a significant decrease in the S1P level. Although S1P signaling regulates lymphocyte trafficking, lymphocyte distribution was unaffected in lymphoid organs of Sphk1؊/؊ mice. The immunosuppressant FTY720 was phosphorylated and elicited lymphopenia in the Sphk1 null mice showing that SPHK1 is not required for the functional activation of this sphingosine analogue prodrug. The results with these Sphk1 null mice reveal that some key physiologic processes that require S1P receptor signaling, such as vascular development and proper lymphocyte distribution, can occur in the absence of SPHK1.Sphingosine-1-phosphate (S1P) 1 is a signaling molecule that influences cellular functions including proliferation, survival, migration, adhesion molecule expression, and morphogenesis (1-4). S1P binds to members of the S1P receptor family (also known as EDG receptors) and, via G proteins, triggers multiple signaling pathways (5, 6). S1P has also been shown to function intracellularly mediating calcium homeostasis, cell growth, and suppression of apoptosis (7,8). In mammals, vascular development and lymphocyte trafficking are dependent on S1P receptor signaling (9 -13).Sphingosine kinase (SPHK) catalyzes the synthesis of S1P via the phosphorylation of sphingosine. SPHK activity is elevated by several stimuli, including platelet-derived growth factor, vascular endothelial growth factor, tumor necrosis factor-␣, and phorbol ester, which trigger an increase in cellular S1P levels (14). Sphk genes have been identified in mammals (15-18), insects (19), plants (20), yeast (21), worm (22), and slime mold (23, 24). Mammals carry two known SphK genes, which in mice are encoded by Sphk1 and Sphk2. The two enzymes contain five highly conserved regions (C1-C5) and an ATP binding site within a conserved lipid kinase catalytic domain (15, 16). SPHK1 has a predominantly cytoplasm localization but can be induced to localize to the inner leaflet of the plasma membrane. Interestingly in endothelial cells SPHK1 is secreted and is capable of producing S1P extracellularly (25). Sphk1 shows a tissue distribution and developmental expression pattern different from Sphk2, although both enzymes are widely expressed (16,26).The importance of S1P receptor signaling in lymphocyte trafficking was first illuminated by the activities of FTY720, a potent immunosuppressive agent. FT...