Ventilator-induced lung injury syndromes are characterized by profound increases in vascular leakiness and activation of inflammatory processes. To explore whether excessive cyclic stretch (CS) directly causes vascular barrier disruption or enhances endothelial cell sensitivity to edemagenic agents, human pulmonary artery endothelial cells (HPAEC) were exposed to physiologically (5% elongation) or pathologically (<em>1</em>8% elongation) relevant levels of strain. CS produced rapid (<em>1</em>0 min) increases in myosin light chain (MLC) phosphorylation, activation of p38 and extracellular signal-related kinase <em>1</em>/2 MAP kinases, and actomyosin remodeling. Acute (<em>1</em>5 min) and chronic (48 h) CS markedly enhanced thrombin-induced MLC phosphorylation (2.<em>1</em>-fold and 3.2-fold for <em>1</em>5-min CS at 5 and <em>1</em>8% elongation and 2.<em>1</em>-fold and 3.<em>1</em>-fold for 48-h CS at 5 and <em>1</em>8% elongation, respectively). HPAEC preconditioned at <em>1</em>8% CS, but not at 5% CS, exhibited significantly enhanced thrombin-induced reduction in transendothelial electrical resistance but did not affect barrier protective effect of <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (0.5 microM). Finally, expression profiling analysis revealed a number of genes, including small GTPase rho, apoptosis mediator ZIP kinase, and proteinase activated receptor-2, to be regulated by CS in an amplitude-dependent manner. Thus our study demonstrates a critical role for the magnitude of CS in regulation of agonist-mediated pulmonary endothelial cell permeability and strongly suggests phenotypic regulation of HPAEC barrier properties by CS.

The bioactive lysophospholipid mediator <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) promotes the egress of newly formed T cells from the thymus and the release of immature B cells from the bone marrow. It has remained unclear, however, where and how S<em>1</em>P is released. Here, we show that in mice, the S<em>1</em>P transporter spinster homolog 2 (Spns2) is responsible for the egress of mature T cells and immature B cells from the thymus and bone marrow, respectively. Global Spns2-KO mice exhibited marked accumulation of mature T cells in thymi and decreased numbers of peripheral T cells in blood and secondary lymphoid organs. Mature recirculating B cells were reduced in frequency in the bone marrow as well as in blood and secondary lymphoid organs. Bone marrow reconstitution studies revealed that Spns2 was not involved in S<em>1</em>P release from blood cells and suggested a role for Spns2 in other cells. Consistent with these data, endothelia-specific deletion of Spns2 resulted in defects of lymphocyte egress similar to those observed in the global Spns2-KO mice. These data suggest that Spns2 functions in ECs to establish the S<em>1</em>P gradient required for T and B cells to egress from their respective primary lymphoid organs. Furthermore, Spns2 could be a therapeutic target for a broad array of inflammatory and autoimmune diseases.

FTY720, a potent immunomodulator, becomes phosphorylated in vivo (FTY-P) and interacts with <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) receptors. Recent studies showed that FTY-P affects vascular endothelial growth factor (VEGF)-induced vascular permeability, an important aspect of angiogenesis. We show here that FTY720 has antiangiogenic activity, potently abrogating VEGF- and S<em>1</em>P-induced angiogenesis in vivo in growth factor implant and corneal models. FTY720 administration tended to inhibit primary and significantly inhibited metastatic tumor growth in a mouse model of melanoma growth. In combination with a VEGFR tyrosine kinase inhibitor PTK787/ZK222584, FTY720 showed some additional benefit. FTY720 markedly inhibited tumor-associated angiogenesis, and this was accompanied by decreased tumor cell proliferation and increased apoptosis. In transfected HEK293 cells, FTY-P internalized S<em>1</em>P<em>1</em> receptors, inhibited their recycling to the cell surface, and desensitized S<em>1</em>P receptor function. Both FTY720 and FTY-P apparently failed to impede VEGF-produced increases in mitogen-activated protein kinase activity in human umbilical vascular endothelial cells (HUVEC), and unlike its activity in causing S<em>1</em>PR internalization, FTY-P did not result in a decrease of surface VEGFR2 levels in HUVEC cells. Pretreatment with FTY720 or FTY-P prevented S<em>1</em>P-induced Ca2+ mobilization and migration in vascular endothelial cells. These data show that functional antagonism of vascular S<em>1</em>P receptors by FTY720 potently inhibits angiogenesis; therefore, this may provide a novel therapeutic approach for pathologic conditions with dysregulated angiogenesis.

In this study we addressed the role of sphingolipid metabolism in the inflammatory response. In a L929 fibroblast model, tumor necrosis factor-alpha (TNF) induced prostaglandin E2 (PGE2) production by 4 h and cyclooxygenase-2 (COX-2) induction as early as 2 h. This TNF-induced PGE2 production was inhibited by NS398, a COX-2 selective inhibitor. GC-MS analysis revealed that only COX-2-generated prostanoids were produced in response to TNF, thus providing further evidence of COX-2 selectivity. As sphingolipids have been implicated in mediating several actions of TNF, their role in COX-2 induction and PGE2 production was evaluated. <em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) induced both COX-2 and PGE2 in a dose-responsive manner with an apparent ED50 of <em>1</em>00-300 nM. The related sphingolipid <em>sphingosine</em> also induced PGE2, though with much less efficacy. TNF induced a 3.5-fold increase in <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> levels at <em>1</em>0 min that rapidly returned to baseline by 40 min. Small interfering RNAs (siRNAs) directed against mouse SK<em>1</em> decreased (typically by 80%) SK<em>1</em> protein and inhibited TNF-induced SK activity. Treatment of cells with RNAi to SK<em>1</em> but not SK2 almost completely abolished the ability of TNF to induce COX-2 or generate PGE2. By contrast, cells treated with RNAi to S<em>1</em>P lyase or S<em>1</em>P phosphatase enhanced COX-2 induction leading to enhanced generation of PGE2. Treatment with SK<em>1</em> RNAi also abolished the effects of exogenous <em>sphingosine</em> and ceramide on PGE2, revealing that the action of <em>sphingosine</em> and ceramide are due to intracellular metabolism into S<em>1</em>P. Collectively, these results provide novel evidence that SK<em>1</em> and S<em>1</em>P are necessary for TNF to induce COX-2 and PGE2 production. Based on these findings, this study indicates that SK<em>1</em> and S<em>1</em>P could be implicated in pathological inflammatory disorders and cancer.

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) is a membrane-derived lysophospholipid that acts primarily as an ex-tracellular signaling molecule. Signals initiated by S<em>1</em>P are transduced by five G protein-coupled receptors, named S<em>1</em>P<em>1</em>-5 Cellular and temporal expression of the S<em>1</em>P receptors (S<em>1</em>PRs) determine their specific roles in various organ systems, but they are particularly critical for regulation of the cardiovascular, immune, and nervous systems, with the most well-known contributions of S<em>1</em>PR signaling being modulation of vascular barrier function, vascular tone, and regulation of lymphocyte trafficking. However, our knowledge of S<em>1</em>PR biology is rapidly increasing as they become attractive therapeutic targets in several diseases, such as chronic inflammatory pathologies, autoimmunity, and cancer. Understanding how the S<em>1</em>PRs regulate interactions between biological systems will allow for greater efficacy in this novel therapeutic strategy as well as characterization of complex physiological networks. Because of the rapidly expanding body of research, this review will focus on the most recent advances in S<em>1</em>PRs.

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P), a potent sphingolipid mediator produced by <em>sphingosine</em> kinase isoenzymes (SphK<em>1</em> and SphK2), regulates diverse cellular processes important for breast cancer progression acting in an autocrine and/or paracrine manner. Here we show that SphK<em>1</em>, but not SphK2, increased S<em>1</em>P export from MCF-7 cells. Whereas for both estradiol (E(2)) and epidermal growth factor-activated SphK<em>1</em> and production of S<em>1</em>P, only E(2) stimulated rapid release of S<em>1</em>P and dihydro-S<em>1</em>P from MCF-7 cells. E(2)-induced S<em>1</em>P and dihydro-S<em>1</em>P export required estrogen receptor-alpha, not GPR30, and was suppressed either by pharmacological inhibitors or gene silencing of ABCC<em>1</em> (multidrug resistant protein <em>1</em>) or ABCG2 (breast cancer resistance protein). Inhibiting these transporters also blocked E(2)-induced activation of ERK<em>1</em>/2, indicating that E(2) activates ERK via downstream signaling of S<em>1</em>P. Taken together, our findings suggest that E(2)-induced export of S<em>1</em>P mediated by ABCC<em>1</em> and ABCG2 transporters and consequent activation of S<em>1</em>P receptors may contribute to nongenomic signaling of E(2) important for breast cancer pathophysiology.

The adipose tissue has become a central focus in the pathogenesis of obesity-mediated cardiovascular and metabolic disease. Here we demonstrate that adipose sphingolipid metabolism is altered in genetically obese (ob/ob) mice. Expression of enzymes involved in ceramide generation (neutral sphingomyelinase [NSMase], acid sphingomyelinase [ASMase], and serine-palmitoyl-transferase [SPT]) and ceramide hydrolysis (ceramidase) are elevated in obese adipose tissues. Our data also suggest that hyperinsulinemia and elevated tumor necrosis factor (TNF)-alpha associated with obesity may contribute to the observed increase in adipose NSMase, ASMase, and SPT mRNA in this murine model of obesity. Liquid chromatography/mass spectroscopy revealed a decrease in total adipose sphingomyelin and ceramide levels but an increase in <em>sphingosine</em> in ob/ob mice compared with lean mice. In contrast to the adipose tissue, plasma levels of total sphingomyelin, ceramide, <em>sphingosine</em>, and <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) were elevated in ob/ob mice. In cultured adipocytes, ceramide, <em>sphingosine</em>, and S<em>1</em>P induced gene expression of plasminogen activator inhibitor-<em>1</em>, TNF-alpha, monocyte chemoattractant protein-<em>1</em>, interleukin-6, and keratinocyte-derived chemokine. Collectively, our results identify a novel role for sphingolipids in contributing to the prothrombotic and proinflammatory phenotype of the obese adipose tissue currently believed to play a major role in the pathogenesis of obesity-mediated cardiovascular and metabolic disease.

The mechanisms involved in renal ischemia-reperfusion injury (IRI) are complex and appear to involve the early participation of bone marrow-derived cells. T lymphocytes participate in the pathogenesis of IRI. <em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) induces peripheral T cell depletion. Therefore, we hypothesized that S<em>1</em>P<em>1</em> receptor activation protects kidney from IRI. FTY-720, a non-receptor-selective <em>sphingosine</em> analog, was given intraperitoneally to C57BL/6 mice, and animals were subjected to ischemia for 32 min followed by reperfusion for 24 h. Plasma creatinine, blood count, myeloperoxidase (MPO) activity, and renal histology were determined. IRI led to a marked increase in plasma creatinine, MPO activity, leukocyte infiltration, and vascular permeability. FTY-720 significantly decreased plasma creatinine in a dose-response manner with a maximal reduction of approximately 73 and approximately 69% with doses of 240 and 48 microg/kg, respectively. MPO, leukocyte infiltration, vascular permeability, and peripheral blood lymphocyte counts were markedly decreased with FTY-720 treatment. The protective effect of FTY-720 was reversed with VPC-44<em>1</em><em>1</em>6, a selective S<em>1</em>P<em>1</em> receptor antagonist. Furthermore, SEW-287<em>1</em>, a selective S<em>1</em>P<em>1</em> agonist, significantly decreased plasma creatinine in a dose-response manner with a maximal reduction of approximately 70% with a dose of <em>1</em>0 mg/kg. Analysis of kidneys by light microscopy revealed minimal histological signs of ischemic injury with FTY-720 or SEW-287<em>1</em> treatment compared with the vehicle group. Using RT-PCR, we found a time-dependent increase in the S<em>1</em>P<em>1</em> mRNA expression following IRI that begins after 2 h with the maximum expression at approximately 4 h. We conclude that the protective effect of FTY-720 is due primarily to activation of S<em>1</em>P<em>1</em> receptors. The mechanism of protection is not known but may be related to peripheral lymphocyte depletion or direct effects on kidney cells expressing S<em>1</em>P<em>1</em> receptor.

In T cells, the PI3K pathway promotes proliferation and survival induced by Ag or growth factors, in part by inactivating the FOXO transcription factor <em>1</em>. We now report that FOXO<em>1</em> controls the expression of L-selectin, an essential homing molecule, in human T lymphocytes. This control is already operational in unprimed T cells and involves a transcriptional regulation process that requires the FOXO<em>1</em> DNA-binding domain. Using transcriptional profiling, we demonstrate that FOXO<em>1</em> also increases transcripts of EDG<em>1</em> and EDG6, two <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> receptors that regulate lymphocyte trafficking. Additionally, FOXO<em>1</em> binds the promoter of the cell quiescence and homing regulator Krüppel-like factor 2 and regulates its expression. Together, these results reveal a new function of FOXO<em>1</em> in the immune system and suggest that PI3K controls a coordinated network of transcription factors regulating both cell quiescence and homing of human T lymphocytes.

<em>Sphingosine</em> kinase (SK) is an oncogenic sphingolipid-metabolizing enzyme that catalyzes the formation of the mitogenic second messenger <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) at the expense of proapoptotic ceramide. Thus, SK is an attractive target for cancer therapy because blockage of S<em>1</em>P formation leads to inhibition of proliferation, as well as the induction of apoptosis in cancer cells. We have recently identified novel SK inhibitors with nanomolar to low micromolar potencies toward recombinant human SK. This study describes the continuing analysis of these inhibitors through in vitro and in vivo experiments. All three structurally diverse SK inhibitors tested showed antitumor activity in mice without exhibiting toxicity. Blood and tumor inhibitor concentrations exceeded in vitro potency levels. Cell signaling analyses in vitro revealed mixed inhibition of mitogen-activated protein kinase kinase and Akt phosphorylation by the SK inhibitors. Importantly, 4-[4-(4-chloro-phenyl)-thiazol-2-ylamino]-phenol (SKI-II) is orally bioavailable, detected in the blood for at least 8 h, and showed a significant inhibition of tumor growth in mice. These compounds are the first examples of nonlipid selective inhibitors of SK with in vivo antitumor activity and provide leads for further development of inhibitors of this important molecular target.

Sphingolipids and glycosphingolipids are emerging as major players in many facets of cell physiology and pathophysiology. We now present an overview of sphingolipid biochemistry and physiology, followed by a brief presentation of recent advances in translational research related to sphingolipids. In discussing sphingolipid biochemistry, we focus on the structure of sphingolipids, and their biosynthetic pathways--the recent identification of most of the enzymes in this pathway has led to significant advances and better characterization of a number of the biosynthetic steps, and the relationship between them. We then discuss some roles of sphingolipids in cell physiology, particularly those of ceramide and <em>sphingosine</em>-<em>1</em>-<em>phosphate</em>, and mention current views about how these lipids act in signal transduction pathways. We end with a discussion of sphingolipids and glycosphingolipids in the etiology and pathology of a number of diseases, such as cancer, immunity, cystic fibrosis, emphysema, diabetes, and sepsis, areas in which sphingolipids are beginning to take a central position, even though many of the details remain to be elucidated.

Angiogenesis, the process by which new blood vessels arise from preexisting ones, is critical for embryonic development and is an integral part of many disease processes. Recent studies have provided detailed information on how angiogenic sprouts initiate, elongate, and branch, but less is known about how these processes cease. Here, we show that S<em>1</em>PR<em>1</em>, a receptor for the blood-borne bioactive lipid <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P), is critical for inhibition of angiogenesis and acquisition of vascular stability. Loss of S<em>1</em>PR<em>1</em> leads to increased endothelial cell sprouting and the formation of ectopic vessel branches. Conversely, S<em>1</em>PR<em>1</em> signaling inhibits angiogenic sprouting and enhances cell-to-cell adhesion. This correlates with inhibition of vascular endothelial growth factor-A (VEGF-A)-induced signaling and stabilization of vascular endothelial (VE)-cadherin localization at endothelial junctions. Our data suggest that S<em>1</em>PR<em>1</em> signaling acts as a vascular-intrinsic stabilization mechanism, protecting developing blood vessels against aberrant angiogenic responses.

Vascular stabilization, a process by which nascent vessels are invested with mural cells, is important in angiogenesis. Here we describe the molecular basis of vascular stabilization regulated by <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P), a platelet-derived lipid mediator. S<em>1</em>P<em>1</em> receptor-dependent cell-surface trafficking and activation of the cell-cell adhesion molecule N-cadherin is essential for interactions between endothelial and mural cells. Endothelial cell S<em>1</em>P<em>1</em>/Gi/Rac pathway induces microtubule polymerization, resulting in trafficking of N-cadherin to polarized plasma membrane domains. S<em>1</em>P treatment modulated the phosphorylation of N-cadherin as well as p<em>1</em>20-catenin and induced the formation of cadherin/catenin/actin complexes containing novel regulatory and trafficking factors. The net result of endothelial cell S<em>1</em>P<em>1</em> receptor activation is the proper trafficking and strengthening of N-cadherin-dependent cell-cell adhesion with mural cells. Perturbation of N-cadherin expression with small interfering RNA profoundly attenuated vascular stabilization in vitro and in vivo. S<em>1</em>P-induced trafficking and activation of N-cadherin provides a novel mechanism for the stabilization of nascent blood vessels by mural cells and may be exploited to control angiogenesis and vascular diseases.

Numerous cell surface receptors, including tyrosine kinase and G protein-coupled receptors, play critical roles in endothelial cell function and blood vessel development. These receptors share the ability of stimulating an intricate network of intracellular signaling pathways, including the activation of members of the Ras and Rho family of small GTPases. However, the contribution of these signaling molecules to the numerous biological activities performed by endothelial cells is still not fully understood. Here, we have used a conditional Cre/Flox approach, enabling the deletion of the Rac<em>1</em> gene in endothelial cells, to examine the role of the Rho-related GTPase Rac<em>1</em> in endothelial cell function and vascular development. Rac<em>1</em> excision in primary endothelial cells in vitro revealed that Rac<em>1</em> plays a central role in endothelial cell migration, tubulogenesis, adhesion, and permeability in response to vascular endothelial growth factor (VEGF) and <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P), which is likely due to the inability of Rac<em>1</em>-deficient endothelial cells to form lamellipodial structures and focal adhesions, and to remodel their cell-cell contacts. Importantly, endothelial-specific excision of Rac<em>1</em> results in embryonic lethality in midgestation (around E9.5), and defective development of major vessels and complete lack of small branched vessels was readily observed in these endothelial Rac<em>1</em>-deficient embryos and their yolk sacs. These findings provide direct evidence that the activity of Rac<em>1</em> in endothelial cells is essential for vascular development and suggest that Rac<em>1</em> and its downstream targets may represent promising therapeutic targets for the treatment of numerous human diseases that involve aberrant neovascularization.

Sphingolipids play a very important role in cell membrane formation, signal transduction, and plasma lipoprotein metabolism, all of which may well have an impact on the development of atherosclerosis. To investigate the relationship between sphingolipid metabolism and atherosclerosis, we utilized myriocin to inhibit mouse serine palmitoyl-CoA transferase (SPT), the key enzyme for sphingolipid biosynthesis. We injected 8-week-old apoE-deficient mice with myriocin (0.3 mg/kg/every other day, intraperitoneal) for 60 days. On a chow diet, myriocin treatment caused a significant decrease (50%) in liver SPT activity (p < 0.00<em>1</em>), significant decreases in plasma sphingomyelin, ceramide, and <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> levels (54, 32, and 73%, respectively) (p < 0.000<em>1</em>), and a significant increase in plasma phosphatidylcholine levels (9<em>1</em>%) (p < 0.000<em>1</em>). Plasma total cholesterol and triglyceride levels demonstrated no significant changes, but there was a significant decrease in atherosclerotic lesion area (42% in root and 36% in en face assays) (p < 0.0<em>1</em>). On a high fat diet, myriocin treatment caused marked decreases in plasma sphingomyelin, ceramide, and <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> levels (59, 66, and 8<em>1</em>%, respectively) (p < 0.000<em>1</em>), and a marked increase in plasma phosphatidylcholine levels (<em>1</em>00%) (p < 0.000<em>1</em>). Total cholesterol and triglyceride demonstrated no significant changes, but there was a significant decrease in atherosclerotic lesion area (39% in root and 37% in en face assays) (p < 0.0<em>1</em>). These results indicate that, apart from cholesterol levels, sphingolipids have an effect on atherosclerotic development and that SPT has proatherogenic properties. Thus, inhibition of SPT activity could be an alternative treatment for atherosclerosis.

Hippo signaling represents a tumor suppressor pathway that regulates organ size and tumorigenesis through phosphorylation and inhibition of the transcription coactivator YAP. Here, we show that serum deprivation dramatically induces YAP Ser<em>1</em>27 phosphorylation and cytoplasmic retention, independent of cell-cell contact. Through chemical isolation and activity profiling, we identified serum-derived <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) and lysophosphatidic acid (LPA) as small molecule activators of YAP. S<em>1</em>P induces YAP nuclear localization through S<em>1</em>P(2) receptor, Rho GTPase activation, and F-actin polymerization, independent of the core Hippo pathway kinases. Bioinformatics studies also showed that S<em>1</em>P stimulation induces YAP target gene expression in mouse liver and human embryonic stem cells. These results revealed potent small molecule regulators of YAP and suggest that S<em>1</em>P and LPA might modulate cell proliferation and tumorigenesis through YAP activation.

There is mounting evidence linking Abeta42 generation in Alzheimer's disease (AD) with sphingomyelin catabolism. Using microarray technology to study <em>1</em>7 brain regions from subjects with varying severity of AD and dementia we detected multiple gene expression abnormalities of the key enzymes that control sphingolipid metabolism. These changes were correlated with the progression of clinical dementia. The upregulation of gene expression of the enzymes controlling synthesis de novo of Cer and the downregulation of the enzymes involved in glycosphingolipid synthesis was evident as early in disease progression as in mild dementia. Together these changes suggest a shift in sphingolipid metabolism towards accumulation of Cer, depletion of glycosphingolipids and the reduction of synthesis of the anti-apoptosis signaling lipid-<em>sphingosine</em> <em>1</em>-<em>phosphate</em> as a function of disease progression. This disrupted balance within the sphingolipid metabolism may trigger signaling events promoting neurodegeneration across cortical regions. This potential mechanism may provide a link between lipid metabolism disturbance and AD.

The cellular dynamics of the egress of lymphocytes from lymph nodes are poorly defined. Here we visualized the branched organization of lymph node cortical sinuses and found that after entry, some T cells were retained, whereas others returned to the parenchyma. T cells deficient in <em>sphingosine</em> <em>1</em>-<em>phosphate</em> receptor type <em>1</em> probed the sinus surface but failed to enter the sinuses. In some sinuses, T cells became rounded and moved unidirectionally. T cells traveled from cortical sinuses into macrophage-rich sinus areas. Many T cells flowed from medullary sinuses into the subcapsular space. We propose a multistep model of lymph node egress in which cortical sinus probing is followed by entry dependent on <em>sphingosine</em> <em>1</em>-<em>phosphate</em> receptor type <em>1</em>, capture of cells in a sinus region with flow, and transport to medullary sinuses and the efferent lymph.

The two lysophospholipids (LPs) lysophosphatidic acid and <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) regulate diverse biological processes. Over the past decade, it has become clear that medically relevant LP activities are mediated by specific G protein-coupled receptors, implicating them in the etiology of a growing number of disorders. A new class of LP agonists shows promise for drug therapy: the experimental drug FTY720 is phosphorylated in vivo to produce a potent S<em>1</em>P receptor agonist (FTY720-P) and is currently in Phase III clinical trials for kidney transplantation and Phase II for multiple sclerosis. Recent genetic and pharmacological studies on LP signaling in animal disease models have identified new areas in which interventions in LP signaling might provide novel therapeutic approaches for the treatment of human diseases.

Programmed cell death is an important physiological response to many forms of cellular stress. The signaling cascades that result in programmed cell death are as elaborate as those that promote cell survival, and it is clear that coordination of both protein- and lipid-mediated signals is crucial for proper cell execution. Sphingolipids are a large class of lipids whose diverse members share the common feature of a long-chain sphingoid base, e.g., <em>sphingosine</em>. Many sphingolipids have been shown to play essential roles in both death signaling and survival. Ceramide, an N-acyl<em>sphingosine</em>, has been implicated in cell death following a myriad of cellular stresses. <em>Sphingosine</em> itself can induce cell death but via pathways both similar and dissimilar to those of ceramide. <em>Sphingosine</em>-<em>1</em>-<em>phosphate</em>, on the other hand, is an anti-apoptotic molecule that mediates a host of cellular effects antagonistic to those of its pro-apoptotic sphingolipid siblings. Extraordinarily, these lipid mediators are metabolically juxtaposed, suggesting that the regulation of their metabolism is of the utmost importance in determining cell fate. In this review, we briefly examine the role of ceramide, <em>sphingosine</em>, and <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> in programmed cell death and highlight the potential roles that these lipids play in the pathway to apoptosis.

EDG-<em>1</em> is a heterotrimeric guanine nucleotide binding protein-coupled receptor (GPCR) for <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (SPP). Cell migration toward platelet-derived growth factor (PDGF), which stimulates <em>sphingosine</em> kinase and increases intracellular SPP, was dependent on expression of EDG-<em>1</em>. Deletion of edg-<em>1</em> or inhibition of <em>sphingosine</em> kinase suppressed chemotaxis toward PDGF and also activation of the small guanosine triphosphatase Rac, which is essential for protrusion of lamellipodia and forward movement. Moreover, PDGF activated EDG-<em>1</em>, as measured by translocation of beta-arrestin and phosphorylation of EDG-<em>1</em>. Our results reveal a role for receptor cross-communication in which activation of a GPCR by a receptor tyrosine kinase is critical for cell motility.

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (SPP) has been shown to inhibit chemotaxis of a variety of cells, in some cases through intracellular actions, while in others through receptor-mediated effects. Surprisingly, we found that low concentrations of SPP (<em>1</em>0-<em>1</em>00 nM) increased chemotaxis of HEK293 cells overexpressing the G protein-coupled SPP receptor EDG-<em>1</em>. In agreement with previous findings in human breast cancer cells (Wang, F., Nohara, K., Olivera, O., Thompson, E. W., and Spiegel, S. (<em>1</em>999) Exp. Cell Res. 247, <em>1</em>7-28), SPP, at micromolar concentrations, inhibited chemotaxis of both vector- and EDG-<em>1</em>-overexpressing HEK293 cells. Nanomolar concentrations of SPP also induced a marked increase in chemotaxis of human umbilical vein endothelial cells (HUVEC) and bovine aortic endothelial cells (BAEC), which express the SPP receptors EDG-<em>1</em> and EDG-3, while higher concentrations of SPP were less effective. Treatment with pertussis toxin, which ADP-ribosylates and inactivates G(i)-coupled receptors, blocked SPP-induced chemotaxis. Checkerboard analysis indicated that SPP stimulates both chemotaxis and chemokinesis. Taken together, these data suggest that SPP stimulates cell migration by binding to EDG-<em>1</em>. Similar to SPP, sphinganine <em>1</em>-<em>phosphate</em> (dihydro-SPP), which also binds to this family of SPP receptors, enhanced chemotaxis; whereas, another structurally related lysophospholipid, lysophosphatidic acid, did not compete with SPP for binding nor did it have significant effects on chemotaxis of endothelial cells. Furthermore, SPP increased proliferation of HUVEC and BAEC in a pertussis toxin-sensitive manner. SPP and dihydro-SPP also stimulated tube formation of BAEC grown on collagen gels (in vitro angiogenesis), and potentiated tube formation induced by basic fibroblast growth factor. Pertussis toxin treatment blocked SPP-, but not bFGF-stimulated in vitro angiogenesis. Our results suggest that SPP may play a role in angiogenesis through binding to endothelial cell G(i)-coupled SPP receptors.

The essential role of the <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) receptor S<em>1</em>P(<em>1</em>) in regulating lymphocyte trafficking was demonstrated with the S<em>1</em>P(<em>1</em>)-selective nanomolar agonist, SEW287<em>1</em>. Despite its lack of charged headgroup, the tetraaromatic compound SEW287<em>1</em> binds and activates S<em>1</em>P(<em>1</em>) through a combination of hydrophobic and ion-dipole interactions. Both S<em>1</em>P and SEW287<em>1</em> activated ERK, Akt, and Rac signaling pathways and induced S<em>1</em>P(<em>1</em>) internalization and recycling, unlike FTY720-<em>phosphate</em>, which induces receptor degradation. Agonism with receptor recycling is sufficient for alteration of lymphocyte trafficking by S<em>1</em>P and SEW287<em>1</em>. S<em>1</em>P(<em>1</em>) modeling and mutagenesis studies revealed that residues binding the S<em>1</em>P headgroup are required for kinase activation by both S<em>1</em>P and SEW287<em>1</em>. Therefore, SEW287<em>1</em> recapitulates the action of S<em>1</em>P in all the signaling pathways examined and overlaps in interactions with key headgroup binding receptor residues, presumably replacing salt-bridge interactions with ion-dipole interactions.