<em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> is a bioactive lipid that is mitogenic for human glioma cell lines by signaling through its G protein-coupled receptors. We investigated the role of <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> receptors and the enzymes that form <em>sphingosine</em>-<em>1</em>-<em>phosphate</em>, <em>sphingosine</em> kinase (SphK)-<em>1</em>, and -2 in human astrocytomas. Astrocytomas of various histologic grades expressed three types of <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> receptors, S<em>1</em>P<em>1</em>, S<em>1</em>P2, and S<em>1</em>P3; however, no significant correlation with histologic grade or patient survival was detected. Expression of SphK<em>1</em>, but not SphK2, in human astrocytoma grade 4 (glioblastoma multiforme) tissue correlated with short patient survival. Patients whose tumors had low SphK<em>1</em> expression survived a median 357 days, whereas those with high levels of SphK<em>1</em> survived a median <em>1</em>02 days. Decreasing SphK<em>1</em> expression using RNA interference or pharmacologic inhibition of SphK significantly decreased the rate of proliferation of U-<em>1</em>242 MG and U-87 MG glioblastoma cell lines. Surprisingly, RNA interference to knockdown SphK2 expression inhibited glioblastoma cell proliferation more potently than did SphK<em>1</em> knockdown. SphK knockdown also prevented cells from exiting G<em>1</em> phase of the cell cycle and marginally increased apoptosis. Thus, SphK isoforms may be major contributors to growth of glioblastoma cells in vitro and to aggressive behavior of glioblastoma multiforme.

The factors directing marginal zone B cells to the splenic marginal zone are not well understood. Here we report that FTY720, a drug that targets <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) receptors, induced marginal zone B cell migration into follicles. Marginal zone B cells expressed S<em>1</em>P receptors <em>1</em> and 3 (S<em>1</em>P(<em>1</em>) and S<em>1</em>P(3), respectively). Using gene-targeted mice, we show that S<em>1</em>P(<em>1</em>) but not S<em>1</em>P(3) was required for localization in the marginal zone. In mice lacking the chemokine CXCL<em>1</em>3, S<em>1</em>P(<em>1</em>)-deficient marginal zone B cells reacquired a marginal zone distribution. Exposure to lipopolysaccharide or antigen caused marginal zone B cells to downregulate S<em>1</em>P(<em>1</em>) and S<em>1</em>P(3) and to migrate into the splenic white pulp. These data suggest that marginal zone B cell localization to the marginal zone depends on responsiveness to the blood lysophospholipid S<em>1</em>P, with S<em>1</em>P(<em>1</em>) signaling overcoming the recruiting activity of CXCL<em>1</em>3.

The time at which ovarian failure (menopause) occurs in females is determined by the size of the oocyte reserve provided at birth, as well as by the rate at which this endowment is depleted throughout post-natal life. Here we show that disruption of the gene for acid sphingomyelinase in female mice suppressed the normal apoptotic deletion of fetal oocytes, leading to neonatal ovarian hyperplasia. Ex vivo, oocytes lacking the gene for acid sphingomyelinase or wild-type oocytes treated with <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> resisted developmental apoptosis and apoptosis induced by anti-cancer therapy, confirming cell autonomy of the death defect. Moreover, radiation-induced oocyte loss in adult wild-type female mice, the event that drives premature ovarian failure and infertility in female cancer patients, was completely prevented by in vivo therapy with <em>sphingosine</em>-<em>1</em>-<em>phosphate</em>. Thus, the sphingomyelin pathway regulates developmental death of oocytes, and <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> provides a new approach to preserve ovarian function in vivo.

Defining critical points of modulation across heterogeneous clinical syndromes may provide insight into new therapeutic approaches. Coagulation initiated by the cytokine-receptor family member known as tissue factor is a hallmark of systemic inflammatory response syndromes in bacterial sepsis and viral haemorrhagic fevers, and anticoagulants can be effective in severe sepsis with disseminated intravascular coagulation. The precise mechanism coupling coagulation and inflammation remains unresolved. Here we show that protease-activated receptor <em>1</em> (PAR<em>1</em>) signalling sustains a lethal inflammatory response that can be interrupted by inhibition of either thrombin or PAR<em>1</em> signalling. The <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) axis is a downstream component of PAR<em>1</em> signalling, and by combining chemical and genetic probes for S<em>1</em>P receptor 3 (S<em>1</em>P3) we show a critical role for dendritic cell PAR<em>1</em>-S<em>1</em>P3 cross-talk in regulating amplification of inflammation in sepsis syndrome. Conversely, dendritic cells sustain escalated systemic coagulation and are the primary hub at which coagulation and inflammation intersect within the lymphatic compartment. Loss of dendritic cell PAR<em>1</em>-S<em>1</em>P3 signalling sequesters dendritic cells and inflammation into draining lymph nodes, and attenuates dissemination of interleukin-<em>1</em>beta to the lungs. Thus, activation of dendritic cells by coagulation in the lymphatics emerges as a previously unknown mechanism that promotes systemic inflammation and lethality in decompensated innate immune responses.

The novel immunomodulator FTY720 is effective in experimental models of transplantation and autoimmunity, and is currently undergoing Phase III clinical trials for prevention of kidney graft rejection. In contrast to conventional immunosuppressants, FTY720 does not impair T- and B-cell activation, proliferation and effector function, but interferes with cell traffic between lymphoid organs and blood. The molecular basis for the mode of action of the drug has only recently been established. FTY720, after phosphorylation, acts as a high-affinity agonist at the G protein-coupled <em>sphingosine</em> <em>1</em>-<em>phosphate</em> receptor-<em>1</em> (S<em>1</em>P(<em>1</em>)) on thymocytes and lymphocytes, thereby inducing aberrant internalization of the receptor. This renders the cells unresponsive to the serum lipid <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P), depriving them from an obligatory signal to egress from lymphoid organs. As a consequence, lymphocytes are unable to recirculate to peripheral inflammatory tissues and graft sites but remain functional in the lymphoid compartment. In addition to the effects on lymphocyte recirculation, the drug acts on endothelial cells and preserves vascular integrity by enhancing adherens junction assembly and endothelial barrier function. The available data establish S<em>1</em>P(<em>1</em>) as a key target for FTY720, and further point to therapeutically relevant effects of the drug on lymphocytes and vascular endothelium.

<em>Sphingosine</em>, a metabolite of membrane sphingolipids, regulates proliferation of quiescent Swiss 3T3 fibroblasts (Zhang, H., N. E. Buckley, K. Gibson. and S. Spiegel. <em>1</em>990. J. Biol. Chem. 265:76-8<em>1</em>). The present study provides new insights into the formation and function of a unique phospholipid, a metabolite of <em>sphingosine</em>, which was unequivocally identified as <em>sphingosine</em>-<em>1</em>-<em>phosphate</em>. The rapid increase in 32P-labeled <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> levels induced by <em>sphingosine</em> was concentration dependent and correlated with its effect on DNA synthesis. Similar to the mitogenic effects of <em>sphingosine</em>, low concentrations of <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> stimulated DNA synthesis and induced pronounced morphological alterations. Both <em>sphingosine</em> and <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> stimulated DNA synthesis in cells made protein kinase C deficient by prolonged treatment with phorbol ester and <em>sphingosine</em> still elicited similar increases in <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> levels in these cells. Although both <em>sphingosine</em> and <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> acted synergistically with a wide variety of growth factors, there was no additive or synergistic effect in response to a combination of <em>sphingosine</em> and <em>sphingosine</em>-<em>1</em>-<em>phosphate</em>. Using a digital imaging system for measurement of calcium changes, we observed that both <em>sphingosine</em> and <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> are potent calcium-mobilizing agonists in viable 3T3 fibroblasts. The rapid rise in cytosolic free calcium was independent of the presence of calcium in the external medium, indicating that the response is due to the mobilization of calcium from internal store. Our results suggest that <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> may be a component of the intracellular second messenger system that is involved in calcium release and the regulation of cell growth induced by <em>sphingosine</em>.

Many enzymes of sphingolipid metabolism are regulated in response to extra- and intracellular stimuli and in turn serve as regulators of levels of bioactive lipids (such as <em>sphingosine</em>, ceramide, <em>sphingosine</em> <em>1</em>-<em>phosphate</em>, and diacylglycerol), and as such, they serve a prototypical modular function in cell regulation. However, lipid metabolism is also closely interconnected in that a product of one enzyme serves as a substrate for another. Moreover, many cell stimuli regulate more than one of these enzymes, thus adding to the complexity of regulation of lipid metabolism. In this paper, we review the status of enzymes of sphingolipid metabolism in cell regulation and propose a role for these enzymes in integration of cell responses, a role that builds on the modular organization while also taking advantage of the complexity and interconnectedness of lipid metabolism, thus providing for a combinatorial mechanism of generating diversity in cell responses. This may be a general prototype for the involvement of metabolic pathways in cell regulation.

There are two isoforms of <em>sphingosine</em> kinase (SphK) that catalyze the formation of <em>sphingosine</em> <em>1</em>-<em>phosphate</em>, a potent sphingolipid mediator. Whereas SphK<em>1</em> stimulates growth and survival, here we show that SphK2 enhanced apoptosis in diverse cell types and also suppressed cellular proliferation. Apoptosis was preceded by cytochrome c release and activation of caspase-3. SphK2-induced apoptosis was independent of activation of <em>sphingosine</em> <em>1</em>-<em>phosphate</em> receptors. Sequence analysis revealed that SphK2 contains a 9-amino acid motif similar to that present in BH3-only proteins, a pro-apoptotic subgroup of the Bcl-2 family. As with other BH3-only proteins, co-immunoprecipitation demonstrated that SphK2 interacted with Bcl-xL. Moreover, site-directed mutation of Leu-2<em>1</em>9, the conserved leucine residue present in all BH3 domains, markedly suppressed SphK2-induced apoptosis. Hence, the apoptotic effect of SphK2 might be because of its putative BH3 domain.

Lysophosphatidic acid (LPA) exerts a variety of biological responses through specific receptors: three subtypes of the EDG-family receptors, LPA<em>1</em>, LPA2, and LPA3 (formerly known as EDG-2, EDG-4, and EDG-7, respectively), and LPA4/GPR23, structurally distinct from the EDG-family receptors, have so far been identified. In the present study, we characterized the action mechanisms of 3-(4-[4-([<em>1</em>-(2-chlorophenyl)ethoxy]carbonyl amino)-3-methyl-5-isoxazolyl] benzylsulfanyl) propanoic acid (Ki<em>1</em>6425) on the EDG-family LPA receptors. Ki<em>1</em>6425 inhibited several responses specific to LPA, depending on the cell types, without any appreciable effect on the responses to other related lipid receptor agonists, including <em>sphingosine</em> <em>1</em>-<em>phosphate</em>. With the cells overexpressing LPA<em>1</em>, LPA2, or LPA3, we examined the selectivity and mode of inhibition by Ki<em>1</em>6425 against the LPA-induced actions and compared them with those of dioctyl glycerol pyro<em>phosphate</em> (DGPP 8:0), a recently identified antagonist for LPA receptors. Ki<em>1</em>6425 inhibited the LPA-induced response in the decreasing order of LPA<em>1</em>>>/= LPA3>>) LPA2, whereas DGPP 8:0 preferentially inhibited the LPA3-induced actions. Ki<em>1</em>6425 inhibited LPA-induced guanosine 5'-O-(3-thio)tri<em>phosphate</em> binding as well as LPA receptor binding to membrane fractions with a same pharmacological specificity as in intact cells. The difference in the inhibition profile of Ki<em>1</em>6425 and DGPP 8:0 was exploited for the evaluation of receptor subtypes involved in responses to LPA in A43<em>1</em> cells. Finally, Ki<em>1</em>6425 also inhibited LPA-induced long-term responses, including DNA synthesis and cell migration. In conclusion, Ki<em>1</em>6425 selectively inhibits LPA receptor-mediated actions, especially through LPA<em>1</em> and LPA3; therefore, it may be useful in evaluating the role of LPA and its receptor subtypes involved in biological actions.

GPR55 is an orphan G protein-coupled receptor. In this study, we explored a possible endogenous ligand for GPR55 using HEK293 cells which expressed GPR55. We found that lysophosphatidylinositol induced rapid phosphorylation of the extracellular signal-regulated kinase in transiently or stably GPR55-expressing cells. On the other hand, lysophosphatidylinositol did not induce phosphorylation of the extracellular signal-regulated kinase in vector-transfected cells. Lysophosphatidic acid and <em>sphingosine</em> <em>1</em>-<em>phosphate</em> also induced phosphorylation of the extracellular signal-regulated kinase in GPR55-expressing cells. However, these lipid phosphoric acids elicited similar responses in vector-transfected cells. Various types of other lysolipids as well as the cannabinoid receptor ligands did not induce phosphorylation of the extracellular signal-regulated kinase. We also found that lysophosphatidylinositol elicited a rapid Ca2+ transient in GPR55-expressing cells. Lysophosphatidylinositol also stimulated the binding of GTPgammaS to the GPR55-expressing cell membranes. These results strongly suggest that GPR55 is a specific and functional receptor for lysophosphatidylinositol.

Mast cells secrete various substances that initiate and perpetuate allergic responses. Cross-linking of the high-affinity receptor for IgE (FcepsilonRI) in RBL-2H3 and bone marrow-derived mast cells activates <em>sphingosine</em> kinase (SphK), which leads to generation and secretion of the potent sphingolipid mediator, <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P). In turn, S<em>1</em>P activates its receptors S<em>1</em>P<em>1</em> and S<em>1</em>P2 that are present in mast cells. Moreover, inhibition of SphK blocks FcepsilonRI-mediated internalization of these receptors and markedly reduces degranulation and chemotaxis. Although transactivation of S<em>1</em>P<em>1</em> and Gi signaling are important for cytoskeletal rearrangements and migration of mast cells toward antigen, they are dispensable for FcepsilonRI-triggered degranulation. However, S<em>1</em>P2, whose expression is up-regulated by FcepsilonRI cross-linking, was required for degranulation and inhibited migration toward antigen. Together, our results suggest that activation of SphKs and consequently S<em>1</em>PRs by FcepsilonRI triggering plays a crucial role in mast cell functions and might be involved in the movement of mast cells to sites of inflammation.

The phosphorylated sphingolipid metabolites <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) and ceramide <em>1</em>-<em>phosphate</em> (C<em>1</em>P) have emerged as potent bioactive agents. Recent studies have begun to define new biological functions for these lipids. Generated by <em>sphingosine</em> kinases and ceramide kinase, they control numerous aspects of cell physiology, including cell survival and mammalian inflammatory responses. Interestingly, S<em>1</em>P is involved in cyclooxygenase-2 induction and C<em>1</em>P is required for the activation and translocation of cPLA2. This suggests that these two sphingolipid metabolites may act in concert to regulate production of eicosanoids, important inflammatory mediators. Whereas S<em>1</em>P functions mainly via G-protein-coupled receptors, C<em>1</em>P appears to bind directly to targets such as cPLA2 and protein phosphatase <em>1</em>/2A. S<em>1</em>P probably also has intracellular targets, and in plants it appears to directly regulate the G protein alpha subunit GPA<em>1</em>.

Sphingolipids comprise a complex family of naturally occurring molecules that are enriched in lipid rafts and contribute to their unique biochemical properties. Membrane sphingolipids also serve as a reservoir for bioactive metabolites including <em>sphingosine</em>, ceramide, <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> and ceramide-<em>1</em>-<em>phosphate</em>. Among these, <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> has emerged as a central regulator of mammalian biology. <em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> is essential for mammalian brain and cardiac development and for maturation of the systemic circulatory system and lymphatics. In addition, <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> contributes to trafficking and effector functions of lymphocytes and other hematopoietic cells and protects against various forms of tissue injury. However, <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> is also an oncogenic lipid that promotes tumor growth and progression. Recent preclinical and clinical investigations using pharmacological agents that target <em>sphingosine</em>-<em>1</em>-<em>phosphate</em>, its receptors and the enzymes required for its biosynthesis and degradation demonstrate the promise and potential risks of modulating <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> signaling in treatment strategies for autoimmunity, cancer, cardiovascular disease and other pathological conditions.

The immunomodulatory drug FTY720 is phosphorylated in vivo, and the resulting FTY720 <em>phosphate</em> as a ligand for <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> receptors is responsible for the unique biological effects of the compound. So far, phosphorylation of FTY720 by murine <em>sphingosine</em> kinase (SPHK) <em>1</em>a had been documented. We found that, while FTY720 is also phosphorylated by human SPHK<em>1</em>, the human type 2 isoform phosphorylates the drug 30-fold more efficiently, because of a lower Km of FTY720 for SPHK2. Similarly, murine SPHK2 was more efficient than SPHK<em>1</em>a. Among splice variants of the human SPHKs, an N-terminally extended SPHK2 isoform was even more active than SPHK2 itself. Further SPHK superfamily members, namely ceramide kinase and a "SPHK-like" protein, failed to phosphorylate <em>sphingosine</em> and FTY720. Thus, only SPHK<em>1</em> and 2 appear to be capable of phosphorylating FTY720. Using selective assay conditions, SPHK<em>1</em> and 2 activities in murine tissues were measured. While activity of SPHK2 toward <em>sphingosine</em> was generally lower than of SPHK<em>1</em>, FTY720 phosphorylation was higher under conditions favoring SPHK2. In human endothelial cells, while activity of SPHK<em>1</em> toward <em>sphingosine</em> was 2-fold higher than of SPHK2, FTY720 phosphorylation was 7-fold faster under SPHK2 assay conditions. Finally, FTY720 was poorly phosphorylated in human blood as compared with rodent blood, in line with the low activity of SPHK<em>1</em> and in particular of SPHK2 in human blood. To conclude, both SPHK<em>1</em> and 2 are capable of phosphorylating FTY720, but SPHK2 is quantitatively more important than SPHK<em>1</em>.

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P), 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 S<em>1</em>P. In mammals, S<em>1</em>P 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, S<em>1</em>P may participate in various pathological conditions. For example, S<em>1</em>P 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 S<em>1</em>P. The ubiquity of the mediator and its receptors, as well as the evolutionary conservation of S<em>1</em>P 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.

The bioactive lipid <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) is involved in multiple cellular signalling systems and has a pivotal role in the control of immune cell trafficking. As such, S<em>1</em>P has been implicated in disorders such as cancer and inflammatory diseases. This Review discusses the ways in which S<em>1</em>P might be therapeutically targeted - for example, via the development of chemical inhibitors that target the generation, transport and degradation of S<em>1</em>P and via the development of specific S<em>1</em>P receptor agonists. We also highlight recent conflicting results observed in preclinical studies targeting S<em>1</em>P and discuss ongoing clinical trials in this field.

We recently reported the critical importance of Rac GTPase-dependent cortical actin rearrangement in the augmentation of pulmonary endothelial cell (EC) barrier function by <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P). We now describe functional roles for the actin-binding proteins cortactin and EC myosin light chain kinase (MLCK) in mediating this response. Antisense down-regulation of cortactin protein expression significantly inhibits S<em>1</em>P-induced barrier enhancement in cultured human pulmonary artery EC as measured by transendothelial electrical resistance (TER). Immunofluorescence studies reveal rapid, Rac-dependent translocation of cortactin to the expanded cortical actin band following S<em>1</em>P challenge, where colocalization with EC MLCK occurs within 5 min. Adenoviral overexpression of a Rac dominant negative mutant attenuates TER elevation by S<em>1</em>P. S<em>1</em>P also induces a rapid increase in cortactin tyrosine phosphorylation (within 30 s) critical to subsequent barrier enhancement, since EC transfected with a tyrosine-deficient mutant cortactin exhibit a blunted TER response. Direct binding of EC MLCK to the cortactin Src homology 3 domain appears essential to S<em>1</em>P barrier regulation, since cortactin blocking peptide inhibits both S<em>1</em>P-induced MLC phosphorylation and peak S<em>1</em>P-induced TER values. These data support novel roles for the cytoskeletal proteins cortactin and EC MLCK in mediating lung vascular barrier augmentation evoked by S<em>1</em>P.

FTY720 (fingolimod) is a first-in-class <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) receptor modulator that was highly effective in Phase II clinical trials for Multiple Sclerosis (MS). FTY720 is phosphorylated in vivo by <em>sphingosine</em> kinase-2 to form the active moiety FTY720-<em>phosphate</em> that binds to four of the five G protein-coupled S<em>1</em>P receptor subtypes. Studies using conditional S<em>1</em>P<em>1</em> receptor-deficient and <em>sphingosine</em> kinase-deficient mice showed that the egress of lymphocytes from lymph nodes requires signalling of lymphocytic S<em>1</em>P<em>1</em> receptors by the endogenous ligand S<em>1</em>P. The S<em>1</em>P mimetic FTY720-<em>phosphate</em> causes internalization and degradation of cell membrane-expressed S<em>1</em>P<em>1</em>, thereby antagonizing S<em>1</em>P action at the receptor. In models of human MS and demyelinating polyneuropathies, functional antagonism of lymphocytic S<em>1</em>P<em>1</em> slows S<em>1</em>P-driven egress of lymphocytes from lymph nodes, thereby reducing the numbers of autoaggressive TH<em>1</em>7 cells that recirculate via lymph and blood to the central nervous system and the sciatic/ischiatic nerves. Based on its lipophilic nature, FTY720 crosses the blood-brain barrier, and ongoing experiments suggest that the drug also down-modulates S<em>1</em>P<em>1</em> in neural cells/astrocytes to reduce astrogliosis, a phenomenon associated with neurodegeneration in MS. This may help restore gap-junctional communication of astrocytes with neurons and cells of the blood-brain barrier. Additional effects may result from (down-) modulation of S<em>1</em>P3 in astrocytes and of S<em>1</em>P<em>1</em> and S<em>1</em>P5 in oligodendrocytes. In conclusion, FTY720 may act through immune-based and central mechanisms to reduce inflammation and support structural restoration of the central nervous system parenchyma. Beyond the autoimmune indications, very recent studies suggest that short-term, low-dose administration of FTY720 could help treat chronic (viral) infections. Differential effects of the drug on the trafficking of naïve, central memory and effector memory T cell subsets are discussed.

<em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) stimulates signaling pathways via G-protein-coupled receptors and triggers diverse cellular processes, including growth, survival, and migration. In S<em>1</em>P<em>1</em> receptor-deficient embryos, blood vessels were incompletely covered by vascular smooth muscle cells (VSMCs), indicating the S<em>1</em>P<em>1</em> receptor regulates vascular maturation. Because S<em>1</em>P<em>1</em> receptor expression is not restricted to a particular cell type, it was not known whether the S<em>1</em>P<em>1</em> receptor controlled VSMC coverage of vessels in a cell-autonomous fashion by functioning directly in VSMCs or indirectly through its activity in endothelial cells (ECs). By using the Cre/loxP system, we disrupted the S<em>1</em>P<em>1</em> gene solely in ECs. The phenotype of the conditional mutant embryos mimicked the one obtained in the embryos globally deficient in S<em>1</em>P<em>1</em>. Thus, vessel coverage by VSMCs is directed by the activity of the S<em>1</em>P<em>1</em> receptor in ECs.

Sphingolipids have been implicated in the regulation of cell growth, differentiation, and programmed cell death. The current paradigm for their action is that complex sphingolipids such as gangliosides interact with growth factor receptors, the extracellular matrix, and neighboring cells, whereas the backbones--<em>sphingosine</em> and other long-chain or "sphingoid" bases, ceramides, and <em>sphingosine</em> <em>1</em>-<em>phosphate</em>--activate or inhibit protein kinases and phosphatases, ion transporters, and other regulatory machinery. Tumor necrosis factor-alpha, interleukin <em>1</em>beta, and nerve growth factor, for example, induce sphingomyelin hydrolysis to ceramide. Other agonists, such as platelet-derived growth factor, trigger further hydrolysis of ceramide to <em>sphingosine</em> and activate <em>sphingosine</em> kinase to form <em>sphingosine</em> <em>1</em>-<em>phosphate</em>. These metabolites either stimulate or inhibit growth and may be cytotoxic (in some cases via induction of apoptosis), depending on which products are formed (or added exogenously), the cellular levels (and possibly intracellular localization), and the cell type. In Swiss 3T3 cells, for example, <em>sphingosine</em> and <em>sphingosine</em> <em>1</em>-<em>phosphate</em> are growth stimulatory at low concentrations via calcium mobilization from intracellular stores and activation of the mitogen-activated protein kinase (MAP kinase) pathway and transcription factors (AP-<em>1</em>), but are toxic at high concentrations. High levels of endogenous sphingoid bases are also produced by inhibition of ceramide synthase by fumonisins, mycotoxins produced by Fusarium moniliforme, resulting in growth stimulation or toxicity. Thus, sphingolipid metabolites appear to serve as second messengers for growth factors, cytokines, and other "physiological" agonists and, when elevated abnormally, to lead to disease.

Naive CD4(+) T cells differentiate into diverse effector and regulatory lineages to orchestrate immunity and tolerance. Here we found that the differentiation of proinflammatory T helper type <em>1</em> (T(H)<em>1</em>) cells and anti-inflammatory Foxp3(+) regulatory T cells (T(reg) cells) was reciprocally regulated by S<em>1</em>P(<em>1</em>), a receptor for the bioactive lipid <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P). S<em>1</em>P(<em>1</em>) inhibited the generation of extrathymic and natural T(reg) cells while driving T(H)<em>1</em> development in a reciprocal manner and disrupted immune homeostasis. S<em>1</em>P(<em>1</em>) signaled through the kinase mTOR and antagonized the function of transforming growth factor-β mainly by attenuating sustained activity of the signal transducer Smad3. S<em>1</em>P(<em>1</em>) function was dependent on endogenous <em>sphingosine</em> kinase activity. Notably, two seemingly unrelated immunosuppressants, FTY720 and rapamycin, targeted the same S<em>1</em>P(<em>1</em>) and mTOR pathway to regulate the dichotomy between T(H)<em>1</em> cells and T(reg) cells. Our studies establish an S<em>1</em>P(<em>1</em>)-mTOR axis that controls T cell lineage specification.

Ceramidases catalyze hydrolysis of ceramides to generate <em>sphingosine</em> (SPH), which is phosphorylated to form <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P). Ceramide, SPH, and S<em>1</em>P are bioactive lipids that mediate cell proliferation, differentiation, apoptosis, adhesion, and migration. Presently, 5 human ceramidases encoded by 5 distinct genes have been cloned: acid ceramidase (AC), neutral ceramidase (NC), alkaline ceramidase <em>1</em> (ACER<em>1</em>), alkaline ceramidase 2 (ACER2), and alkaline ceramidase 3 (ACER3). Each human ceramidase has a mouse counterpart. AC, NC, and ACER<em>1</em>-3 have maximal activities in acidic, neutral, and alkaline environments, respectively. ACER<em>1</em>-3 have similar protein sequences but no homology to AC and NC. AC and NC also have distinct protein sequences. The human AC (hAC) was implicated in Farber disease, and hAC may be important for cell survival. The mouse AC (mAC) is needed for early embryo survival. NC is protective against inflammatory cytokines, and the mouse NC (mNC) is required for the catabolism of ceramides in the digestive tract. ACER<em>1</em> is critical in mediating cell differentiation by controlling the generation of SPH and S<em>1</em>P and that ACER2's role in cell proliferation and survival depends on its expression or the cell type in which it is found. Here, we discuss the role of each ceramidase in regulating cellular responses mediated by ceramides, SPH, and S<em>1</em>P.

<em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (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-<em>1</em> (Lee, M.-J., J.R. Van Brocklyn, S. Thangada, C.H. Liu, A.R. Hand, R. Menzeleev, S. Spiegel, and T. Hla. <em>1</em>998. Science. 279:<em>1</em>552-<em>1</em>555). Edg-<em>1</em> binds SPP with remarkable specificity as only sphinganine-<em>1</em>-<em>phosphate</em> displaced radiolabeled SPP, while other sphingolipids did not. Binding of SPP to Edg-<em>1</em> 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-<em>1</em> and correlated with intracellular uptake. SPP also stimulated signal transduction pathways, including calcium mobilization, activation of phospholipase D, and tyrosine phosphorylation of p<em>1</em>25(FAK), independently of edg-<em>1</em> 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 PC<em>1</em>2 cells, which do not have specific SPP binding or expression of Edg-<em>1</em> mRNA. Conversely, sphinganine-<em>1</em>-<em>phosphate</em>, which binds to and signals via Edg-<em>1</em>, 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.

Consistent with their function in immune surveillance, natural killer (NK) cells are distributed throughout lymphoid and nonlymphoid tissues. However, the mechanisms governing the steady-state trafficking of NK cells remain unknown. The lysophospholipid <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P), by binding to its receptor S<em>1</em>P<em>1</em>, regulates the recirculation of T and B lymphocytes. In contrast, S<em>1</em>P5 is detected in the brain and regulates oligodendrocyte migration and survival in vitro. Here we show that S<em>1</em>P5 was also expressed in NK cells in mice and humans and that S<em>1</em>P5-deficient mice had aberrant NK cell homing during steady-state conditions. In addition, we found that S<em>1</em>P5 was required for the mobilization of NK cells to inflamed organs. Our data emphasize distinct mechanisms regulating the circulation of various lymphocyte subsets and raise the possibility that NK cell trafficking may be manipulated by therapies specifically targeting S<em>1</em>P5.