Cerebellar granule cells from <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) lyase-deficient mice were used to study the toxicity of this potent sphingolipid metabolite in terminally differentiated postmitotic neurons. Based on earlier findings with the lyase-stable, semi-synthetic, cis-4-methyl<em>sphingosine</em> <em>phosphate</em>, we hypothesized that accumulation of S<em>1</em>P above a certain threshold induces neuronal apoptosis. The present studies confirmed this conclusion and further revealed that for S<em>1</em>P to induce apoptosis in lyase-deficient neurons it must also be produced by <em>sphingosine</em>-kinase2 (SK2). These conclusions are based on the finding that incubation of lyase-deficient neurons with either <em>sphingosine</em> or S<em>1</em>P results in a similar elevation in cellular S<em>1</em>P; however, only S<em>1</em>P addition to the culture medium induces apoptosis. This was not due to S<em>1</em>P acting on the S<em>1</em>P receptor but to hydrolysis of S<em>1</em>P to <em>sphingosine</em> that was phosphorylated by the cells, as described before for cis-4-methyl<em>sphingosine</em>. Although the cells produced S<em>1</em>P from both exogenously added <em>sphingosine</em> as well as <em>sphingosine</em> derived from exogenous S<em>1</em>P, the S<em>1</em>P from these two sources were not equivalent, because the former was primarily produced by SK<em>1</em>, whereas the latter was mainly formed by SK2 (as also was cis-4-methyl<em>sphingosine</em> <em>phosphate</em>), based on studies in neurons lacking SK<em>1</em> or SK2 activity. Thus, these investigations show that, due to the existence of at least two functionally distinct intracellular origins for S<em>1</em>P, exogenous S<em>1</em>P can be neurotoxic. In this model, S<em>1</em>P accumulated due to a defective lyase, however, this cause of toxicity might also be important in other cases, as illustrated by the neurotoxicity of cis-4-methyl<em>sphingosine</em> <em>phosphate</em>.

<em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) is a bioactive sphingolipid that acts as both an extracellular ligand for the endothelial differentiation gene-<em>1</em> (EDG-<em>1</em>) G-protein coupled receptor (GPCR) family and as an intracellular messenger. Cellular levels of S<em>1</em>P are low and tightly regulated in a spatial-temporal manner not only by <em>sphingosine</em> kinase (SPHK) but also by degradation catalyzed by S<em>1</em>P lyase, specific S<em>1</em>P phosphohydrolases, and by general lipid <em>phosphate</em> phosphohydrolases (LPPs). LPPs are characterized as magnesium-independent, insensitive to inhibition by N-ethylmaleimide (NEM) and possessing broad substrate specificity with a variety of phosphorylated lipids, including S<em>1</em>P, phosphatidic acid (PA), and lysophosphatidic acid (LPA). LPPs contain three highly conserved domains that define a phosphohydrolase superfamily. Recently, several specific S<em>1</em>P phosphohydrolases have been identified in yeast and mammalian cells. Phylogenetic and biochemical analyses indicate that these enzymes constitute a new subset of the LPP family. As further evidence, S<em>1</em>P phosphohydrolases exhibit high specificity for phosphorylated sphingoid bases. Enforced expression of S<em>1</em>P phosphohydrolase alters the cellular levels of sphingolipid metabolites in yeast and mammalian cells, increasing <em>sphingosine</em> and ceramide, bioactive sphingolipids that often have opposing biological actions to S<em>1</em>P. By regulating the cellular ratio between ceramide/<em>sphingosine</em> and S<em>1</em>P, S<em>1</em>P phosphohydrolase is poised to be a critical factor in cell survival/cell death decisions. Indeed, expression of S<em>1</em>P phosphohydrolase in mammalian cells increases apoptosis, whereas deletion of S<em>1</em>P phosphohydrolases in yeast correlates with resistance to heat stress. In this review, we discuss the role of phosphohydrolases in the metabolism of S<em>1</em>P and how turnover of S<em>1</em>P can regulate sphingolipid metabolites signaling.

The importance of <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (Sph-<em>1</em>-P) as an intercellular sphingolipid mediator has been established in various systems, and this is especially true in the areas of vascular biology and immunology. Blood platelets store Sph-<em>1</em>-P abundantly and release this bioactive lysophospholipid extracellularly upon stimulation, while vascular endothelial cells and smooth muscle cells respond dramatically to this platelet-derived bioactive lipid. Most of the responses elicited by extracellular Sph-<em>1</em>-P are believed to be mediated by G protein-coupled cell surface receptors, i.e., S<em>1</em>Ps. It is likely that regulation of Sph-<em>1</em>-P biological activity could be important for therapeutics, including but not limited to control of vascular disorders. Furthermore, elucidation of the mechanisms by which the levels of Sph-<em>1</em>-P in the blood are regulated seems important. Accordingly, the application of Sph-<em>1</em>-P analysis to laboratory medicine may be an important task in clinical medicine. In this review, Sph-<em>1</em>-P-related metabolism in the plasma will be summarized. Briefly, the levels and bioactivities of plasma Sph-<em>1</em>-P in vivo may be regulated by various factors, including Sph-<em>1</em>-P release from platelets (and red blood cells, based upon the recent reports), Sph-<em>1</em>-P distribution between albumin and lipoproteins, and S<em>1</em>P expression and lipid <em>phosphate</em> phosphatase activity on the cell surface. Then, application of Sph-<em>1</em>-P analysis to laboratory medicine will be discussed.

BACKGROUND

Platelets release the immune-modulating lipid <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P). However, the mechanisms of platelet S<em>1</em>P secretion are not fully understood.

OBJECTIVE

The present study investigates the function of thromboxane (TX) for platelet S<em>1</em>P secretion during platelet activation and the consequences for monocyte chemotaxis.

METHODS

S<em>1</em>P was detected using thin-layer chromatography in [(3)H]<em>sphingosine</em>-labeled platelets and by mass spectrometry. Monocyte migration was measured in modified Boyden chamber chemotaxis assays.

RESULTS

Release of S<em>1</em>P from platelets was stimulated with protease-activated receptor-<em>1</em>-activating peptide (PAR-<em>1</em>-AP, <em>1</em>00 μM). Acetylsalicylic acid (ASA) and two structurally unrelated reversible cyclooxygenase inhibitors diclofenac and ibuprofen suppressed S<em>1</em>P release. Oral ASA (500-mg single dose or <em>1</em>00 mg over 3 days) attenuated S<em>1</em>P release from platelets in healthy human volunteers ex vivo. This was paralleled by inhibition of TX formation. S<em>1</em>P release was increased by the TX receptor (TP) agonist U-466<em>1</em>9, and inhibited by the TP antagonist ramatroban and by inhibitors of ABC-transport. Furthermore, thrombin-induced release of S<em>1</em>P was attenuated in platelets from TP-deficient mice. Supernatants from PAR-<em>1</em>-AP-stimulated human platelets increased the chemotactic capacity of human peripheral monocytes in a S<em>1</em>P-dependent manner via S<em>1</em>P receptors-<em>1</em> and -3. These effects were inhibited by ASA-pretreatment of platelets.

CONCLUSIONS

TX synthesis and TP activation mediate S<em>1</em>P release after thrombin receptor activation. Inhibition of this pathway may contribute to the anti-inflammatory actions of ASA, for example by affecting activity of monocytes at sites of vascular injury.

OBJECTIVE

Mouse models are useful for glaucoma research, but it is unclear whether intraocular pressure (IOP) regulation in mice operates through mechanisms similar to those in humans. Our goal was to determine whether pharmacologic compounds that affect conventional outflow facility in human eyes exert similar effects in C57BL/6 mice.

METHODS

A computerized perfusion system was used to measure conventional outflow facility in enucleated mouse eyes ex vivo. Paired eyes were perfused sequentially, either immediately after enucleation or after 3 hours storage at 4°C. Three groups of experiments examined <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P), S<em>1</em>P with antagonists to S<em>1</em>P(<em>1</em>) and S<em>1</em>P(2) receptors, and the prostanoid EP(4) receptor agonist 3,7-dithia PGE(<em>1</em>). We also examined whether a 24-hour postmortem delay affected the response to 3,7-dithia prostaglandin E(<em>1</em>) (PGE(<em>1</em>)).

RESULTS

S<em>1</em>P decreased facility by 39%, and was blocked almost completely by an S<em>1</em>P(2), but not S<em>1</em>P(<em>1</em>), receptor antagonist. The S<em>1</em>P(2) receptor antagonist alone increased facility nearly 2-fold. 3,7-dithia PGE(<em>1</em>) increased facility by <em>1</em>06% within 3 hours postmortem. By 24 hours postmortem, the facility increase caused by 3,7-dithia PGE(<em>1</em>) was reduced 3-fold, yet remained statistically detectable.

CONCLUSIONS

C57BL/6 mice showed opposing effects of S<em>1</em>P(2) and EP(4) receptor activation on conventional outflow facility, as observed in human eyes. Pharmacologic effects on facility were detectable up to 24 hours postmortem in enucleated mouse eyes. Mice are suitable models to examine the pharmacology of S<em>1</em>P and EP(4) receptor stimulation on IOP regulation as occurs within the conventional outflow pathway of human eyes, and are promising for studying other aspects of aqueous outflow dynamics.

Activated platelets release large lipid-protein complexes termed microparticles. These platelet microparticles (PMP) are composed of vesicular fragments of the plasma membrane and alpha-granules. PMP facilitate coagulation, promote platelet and leukocyte adhesion to the subendothelial matrix, support angiogenesis and stimulate vascular smooth muscle proliferation.

OBJECTIVE

PMP were separated into 4 size classes to facilitate identification of active protein and lipid components. PMP were obtained from activated human platelets and separated into 4 size classes by gel filtration chromatography. Proteins were identified using 2-dimensional, liquid chromatography tandem mass spectrometry. Functional effects on platelets were determined using the PFA-<em>1</em>00 and on endothelial cells by measuring transendothelial cell electrical resistance. PMP size classes differed significantly in their contents of plasma membrane receptors and adhesion molecules, chemokines, growth factors and protease inhibitors. The two smallest size classes (3 and 4) inhibited collagen/adenosine-di<em>phosphate</em>-mediated platelet thrombus formation, while fractions 2 and 4 stimulated barrier formation by endothelial cells. Heat denaturation blocked the effect of fraction 4 on endothelial cell function, but not fraction 2 implying that the active component in fraction 4 is a protein and in fraction 2 is a heat-stable protein or lipid but not <em>sphingosine</em>-<em>1</em>-<em>phosphate</em>. Proteomic and functional analysis of PMP size fractions has shown that PMP can be separated into different size classes that differ in protein components, protein/lipid ratio, and functional effects on platelets and endothelial cells. This analysis will facilitate identification of active components in the PMP and clarify their involvement in diseases such as atherosclerosis and cancer.

BACKGROUND

Lysophosphatidic acid (LPA) and <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) are two prominent signaling lysophospholipids (LPs) exerting their functions through a group of G protein-coupled receptors (GPCRs). This review covers current knowledge of the LP signaling in the function and pathology of the reproductive system.

METHODS

PubMed was searched up to May 2008 for papers on lysophospholipids/LPA/S<em>1</em>P/LPC/SPC in combination with each part of the reproductive system, such as testis/ovary/uterus.

RESULTS

LPA and SIP are found in significant amounts in serum and other biological fluids. To date, <em>1</em>0 LP receptors have been identified, including LPA(<em>1</em>-5) and S<em>1</em>P(<em>1</em>-5). In vitro and in vivo studies from the past three decades have demonstrated or suggested the physiological functions of LP signaling in reproduction, such as spermatogenesis, male sexual function, ovarian function, fertilization, early embryo development, embryo spacing, implantation, decidualization, pregnancy maintenance and parturition, as well as pathological roles in ovary, cervix, mammary gland and prostate cancers.

CONCLUSIONS

Receptor knock-out and other studies indicate tissue-specific and receptor-specific functions of LP signaling in reproduction. More comprehensive studies are required to define mechanisms of LP signaling and explore the potential use as a therapeutic target.

OBJECTIVE

The present review considers recent reports that identify the roles of key intermediate signaling components and mediators during and after mast cell activation and degranulation leading to anaphylaxis.

RESULTS

Mechanisms of anaphylaxis are becoming better understood as the interaction of several regulatory systems in the mast cell activation and degranulation signaling cascade. Multiple tyrosine kinases, activated after immunoglobulin E binding to the high-affinity receptors for immunoglobulin E (FcepsilonRI), exert both positive and negative regulation on the signaling cascade, which may vary with genetic background or mutations in signaling proteins. Calcium influx, the essential, proximal intracellular event leading to mast cell degranulation, is controlled also by both negative and positive regulation through calcium channels. <em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> is emerging as a newly realized mediator of anaphylaxis, acting as a signaling component within the mast cell and as a circulating mediator.

CONCLUSIONS

Anaphylaxis is a systemic reaction involving multiple organ systems, but it is believed that it may be influenced by cellular events in mast cells and basophils resulting in the release of mediators. Therefore, understanding the mechanisms of mast cell activation and degranulation is critical to understanding the mechanisms of anaphylaxis. Recent reports have identified important regulatory components of the signaling cascade and, consequently, potential targets for therapeutic intervention.

Characterizing the metabolic changes pertaining to hepatocellular carcinoma (HCC) in patients with liver cirrhosis is believed to contribute towards early detection, treatment, and understanding of the molecular mechanisms of HCC. In this study, we compare metabolite levels in sera of 78 HCC cases with <em>1</em>84 cirrhotic controls by using ultra performance liquid chromatography coupled with a hybrid quadrupole time-of-flight mass spectrometry (UPLC-QTOF MS). Following data preprocessing, the most relevant ions in distinguishing HCC cases from patients with cirrhosis are selected by parametric and non-parametric statistical methods. Putative metabolite identifications for these ions are obtained through mass-based database search. Verification of the identities of selected metabolites is conducted by comparing their MS/MS fragmentation patterns and retention time with those from authentic compounds. Quantitation of these metabolites is performed in a subset of the serum samples (<em>1</em>0 HCC and <em>1</em>0 cirrhosis) using isotope dilution by selected reaction monitoring (SRM) on triple quadrupole linear ion trap (QqQLIT) and triple quadrupole (QqQ) mass spectrometers. The results of this analysis confirm that metabolites involved in sphingolipid metabolism and phospholipid catabolism such as <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S-<em>1</em>-P) and lysophosphatidylcholine (lysoPC <em>1</em>7:0) are up-regulated in sera of HCC vs. those with liver cirrhosis. Down-regulated metabolites include those involved in bile acid biosynthesis (specifically cholesterol metabolism) such as glycochenodeoxycholic acid 3-sulfate (3-sulfo-GCDCA), glycocholic acid (GCA), glycodeoxycholic acid (GDCA), taurocholic acid (TCA), and taurochenodeoxycholate (TCDCA). These results provide useful insights into HCC biomarker discovery utilizing metabolomics as an efficient and cost-effective platform. Our work shows that metabolomic profiling is a promising tool to identify candidate metabolic biomarkers for early detection of HCC cases in high risk population of cirrhotic patients.

Activated protein C (APC) has both anticoagulant activity and direct cell-signaling properties. APC has been reported to promote cancer cell migration/invasion and to inhibit apoptosis and therefore may exacerbate metastasis. Opposing these activities, APC signaling protects the vascular endothelial barrier through <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> receptor-<em>1</em> (S(<em>1</em>)P(<em>1</em>))activation, which may counteract cancer cell extravasation. Here, we provide evidence that endogenous APC limits cancer cell extravasation, with in vivo use of monoclonal antibodies against APC. The protective effect of endogenous APC depends on its signaling properties. The MAPC<em>1</em>59<em>1</em> antibody that only blocks anticoagulant activity of APC does not affect cancer cell extravasation as opposed to MPC<em>1</em>609 that blocks anticoagulant and signaling properties of APC. Combined administration of anti-APC antibodies and S(<em>1</em>)P(<em>1</em>) agonist (SEW287<em>1</em>) resulted in a similar number of pulmonary foci in mice in presence and absence of APC, indicating that the protective effect of APC depends on the S(<em>1</em>)P(<em>1</em>) pathway. Moreover, endogenous APC prevents cancer cell-induced vascular leakage as assessed by the Evans Blue Dye assay, and SEW287<em>1</em> treatment reversed MPC<em>1</em>609-dependent vascular leakage. Finally, we show that cancer cells combined with MPC<em>1</em>609 treatment diminished endothelial VE-cadherin expression. In conclusion, endogenous APC limits cancer cell extravasation because of S(<em>1</em>)P(<em>1</em>)-mediated VE-cadherin-dependent vascular barrier enhancement.

The cloning and analysis of the first identified lysophosphatidic acid (LPA) receptor gene, lpA<em>1</em> (also referred to as vzg-<em>1</em> or edg-2), led us to identify homologous murine genes that might also encode receptors for related lysophospholipid ligands. Three murine genomic clones (designated lpB<em>1</em>, lpB2, and lpB3) were isolated, corresponding to human/rat Edg-<em>1</em>, rat H2<em>1</em>8/AGR<em>1</em>6, and human edg-3, respectively. Based on the amino acid similarities of their predicted proteins (44-52% identical), the three lpB genes could be grouped into a separate G-protein coupled receptor subfamily, distinct from that containing the LPA receptor genes lpA<em>1</em> and lpA2. Unlike lpA<em>1</em> and lpA2, which contain multiple coding exons, all lpB members contained a single coding exon. Heterologous expression of individual lpB members in a hepatoma cell line (RH7777), followed by 35S-GTPgammaS incorporation assays demonstrated that each of the three LPB receptors conferred <em>sphingosine</em>-<em>1</em>-<em>phosphate</em>-dependent, but not lysophosphatidic acid-dependent, G-protein activation. Northern blot and in situ hybridization analyses revealed overlapping as well as distinct expression patterns in both embryonic and adult tissues. This comparative characterization of multiple <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> receptor genes and their spatiotemporal expression patterns will aid in understanding the biological roles of this enlarging lysophospholipid receptor family.

<em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> is a sphingolipid metabolite that regulates cell proliferation, migration and apoptosis through specific signaling pathways. <em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> lyase catalyzes the conversion of <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> to ethanolamine <em>phosphate</em> and a fatty aldehyde. We report the cloning of the Drosophila <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> lyase gene (Sply) and demonstrate its importance for adult muscle development and integrity, reproduction and larval viability. Sply expression is temporally regulated, with onset of expression during mid-embryogenesis. Sply null mutants accumulate both phosphorylated and unphosphorylated sphingoid bases and exhibit semi-lethality, increased apoptosis in developing embryos, diminished egg-laying, and gross pattern abnormalities in dorsal longitudinal flight muscles. These defects are corrected by restoring Sply expression or by introduction of a suppressor mutation that diminishes sphingolipid synthesis and accumulation of sphingolipid intermediates. This is the first demonstration of novel and complex developmental pathologies directly linked to a disruption of sphingolipid catabolism in metazoans.

Studies in skeletal muscle demonstrate that elevation of plasma FFAs increases the sphingolipid ceramide. We aimed to determine the impact of FFA oversupply on total sphingolipid profiles in a skeletal muscle model. C2C<em>1</em>2 myotubes were treated with palmitate (PAL). Lipidomics analysis revealed pleiotropic effects of PAL on cell sphingolipids not limited to ceramides. (<em>1</em>3)C labeling demonstrated that PAL activated several branches of sphingolipid synthesis by distinct mechanisms. Intriguingly, PAL increased <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> independently of de novo synthesis. Quantitative real-time PCR demonstrated that PAL increased <em>sphingosine</em> kinase <em>1</em> (SK<em>1</em>) mRNA by approximately 4-fold. This was accompanied by a 2.3-fold increase in <em>sphingosine</em> kinase enzyme activity. This upregulation did not occur upon treatment with oleate, suggesting some level of specificity for PAL. These findings were recapitulated in the diet-induced obesity mouse model, in which high-fat feeding increased SK<em>1</em> message in skeletal muscle over 2.3-fold. These data suggest that the impact of elevated FFA on sphingolipids reaches beyond ceramides and de novo sphingolipid synthesis. Moreover, these findings identify PAL as a novel regulatory stimulus for SK<em>1</em>.

FTY720 (2-amino-2[2-(-4-octylphenyl)ethyl]propane-<em>1</em>,3-diol hydrochloride) is a new <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> receptor agonist being developed for multiple sclerosis and prevention of solid organ transplant rejection. A physiologically based pharmacokinetic model was developed to predict the concentration of FTY720 in various organs of the body. Single oral and intravenous doses of FTY720 were administered to male Wistar rats, with blood and tissue sampling over 360 h analyzed by liquid chromatography/tandem mass spectrometry. A well stirred model (perfusion rate-limited) described FTY720 kinetics in heart, lungs, spleen, muscle, kidneys, bone, and liver, with a permeability rate-limited model being required for brain, thymus, and lymph nodes. Tissue-to-blood partition coefficients (RT) ranged from 4.69 (muscle) to 4<em>1</em>.4 (lungs). In lymph nodes and spleen, major sites for FTY720-induced changes in sequestration of lymphocytes, RT values were 22.9 and 34.7, respectively. Permeability-surface area products for brain, thymus, and lymph nodes were 39.3, <em>1</em>22, and <em>1</em>76 ml/min. Intrinsic hepatic clearance was 23,<em>1</em>45 l/h/kg for the free drug in blood (f(ub) 0.000333); systemic clearance was 0.748 l/h/kg and terminal half-life was 23.4 h. The fraction orally absorbed was 7<em>1</em>%. The model characterized well FTY720 disposition for this extensive dosing and tissue collection study in the rat. On scaling the model to dogs and humans, good agreement was found between the actual and predicted blood concentration-time profiles. More importantly, brain concentrations in dogs were well predicted from those of the rat. In absolute terms, the predictions were slightly lower than observed values, just under a <em>1</em>.5-fold deviation, but the model accurately predicted the terminal elimination of FTY720 from the brain.

The humoral immune response to protein antigens is composed of a rapid low-affinity IgM antibody response followed by an IgG response exhibiting higher affinity. Here, we demonstrate that Lsc, a protein that regulates G protein-coupled-receptor signaling and RhoA activation, is required by B lymphocytes for the antigen-specific IgM antibody response to a protein antigen. We further show that in lsc(-/-) mice, MZB cells are selectively affected such that naive and in vivo-activated MZB cells migrate toward <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> at increased proportions but release inefficiently from integrin ligands. Consequently, lsc(-/-) MZB cells do not traffick appropriately in an immune response and do not contribute to the TD antibody response. These data demonstrate that Lsc regulates the migration and adhesion of MZB cells, and this regulation appears to be required for these cells to contribute to the antibody response to TD antigens.

Sphingomyelin or the products derived from its metabolism may constitute a signaling system involved in a variety of cellular processes. The activation of a plasma membrane neutral sphingomyelinase, which catalyzes the first step in sphingomyelin turnover, has been suggested to play an important role in cellular differentiation. We have studied the effect of exogenous staphylococcal sphingomyelinase on DNA synthesis and on the composition of membrane sphingolipids in quiescent Swiss 3T3 fibroblasts. Sphingomyelinase stimulated proliferation of Swiss 3T3 cells and potentiated the mitogenic action of other growth factors, such as insulin, epidermal growth factor, and bombesin. Treatment with sphingomyelinase produced a significant decrease in sphingomyelin accompanied by a corresponding increase in ceramide levels. No significant increases were detected in the levels of products derived from ceramide, i.e. ceramide <em>1</em>-<em>phosphate</em>, <em>sphingosine</em>, or <em>sphingosine</em> <em>1</em>-<em>phosphate</em>. To further investigate the role of ceramide in cellular proliferation, we studied the effect of cell-permeable analogs of ceramide on DNA synthesis in quiescent Swiss 3T3 cells. Both N-hexanoyl<em>sphingosine</em> and N-acetyl<em>sphingosine</em> at low concentrations stimulated [3H]thymidine incorporation and acted synergistically with a wide variety of growth factors known to induce proliferation of quiescent Swiss 3T3 fibroblasts. Similar effects were observed with bovine brain ceramides. These results suggest that ceramide may be involved in the regulation of cellular proliferation.

The regulation of glioma cell proliferation by <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) was studied using the human glioblastoma cell line U-373 MG. U-373 MG cells responded mitogenically to nanomolar concentrations of S<em>1</em>P, and express mRNA encoding the S<em>1</em>P receptors S<em>1</em>P<em>1</em>/endothelial differentiation gene (EDG)-<em>1</em>, S<em>1</em>P3/EDG-3 and S<em>1</em>P2/EDG-5. S<em>1</em>P-induced proliferation required extracellular signal-regulated kinase activation and was partially sensitive to pertussis toxin and wortmannin, indicating involvement of a Gi-coupled receptor and phosphatidylinositol 3-kinase. Moreover, S<em>1</em>P<em>1</em>, S<em>1</em>P3 and S<em>1</em>P2 receptors are expressed in the majority of human glioblastomas as determined by reverse transcriptase-polymerase chain reaction analysis. Thus, S<em>1</em>P signaling through EDG receptors may contribute to glioblastoma growth in vivo.

Two mammalian <em>sphingosine</em> kinase (SphK) isoforms, SphK<em>1</em> and SphK2, possess identical kinase domains but have distinct kinetic properties and subcellular localizations, suggesting each has one or more specific roles in <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) generation. Although both kinases use <em>sphingosine</em> as a substrate to generate S<em>1</em>P, the mechanisms controlling SphK activation and subsequent S<em>1</em>P generation during lung injury are not fully understood. In this study, we established a murine lung injury model to investigate LPS-induced lung injury in SphK<em>1</em> knockout (SphK<em>1</em>(-/-)) and wild-type (WT) mice. We found that SphK<em>1</em>(-/-) mice were much more susceptible to LPS-induced lung injury compared with their WT counterparts, quantified by multiple parameters including cytokine induction. Intriguingly, overexpression of WT SphK<em>1</em> delivered by adenoviral vector to the lungs protected SphK<em>1</em>(-/-) mice from lung injury and attenuated the severity of the response to LPS. However, adenoviral overexpression of a SphK<em>1</em> kinase-dead mutant (SphKKD) in SphK<em>1</em>(-/-) mouse lungs further exacerbated the response to LPS as well as the extent of lung injury. WT SphK2 adenoviral overexpression also failed to provide protection and, in fact, augmented the degree of LPS-induced lung injury. This suggested that, in vascular injury, S<em>1</em>P generated by SphK2 activation plays a distinctly separate role compared with SphK<em>1</em>-dependent S<em>1</em>P generation and survival signaling. Microarray and real-time RT-PCR analysis of SphK<em>1</em> and SphK2 expression levels during lung injury revealed that, in WT mice, LPS treatment caused significantly enhanced SphK<em>1</em> expression ( approximately 5x) levels within 6 h, which declined back to baseline levels by 24 h posttreatment. In contrast, expression of SphK2 was gradually induced following LPS treatment and was elevated within 24 h. Collectively, our results for the first time demonstrate distinct functional roles of the two SphK isoforms in the regulation of LPS-induced lung injury.

Lysophosphatidic acid (LPA) and <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) are both low-molecular-weight lysophospholipid (LPL) ligands which are recognized by the Edg family of G protein-coupled receptors (GPCRs). In endothelial cells, these two ligands activate Edg receptors resulting in cell proliferation and cell migration. Interleukin-8 (IL-8) is a C-X-C chemokine and acts as a chemoattractant of neutrophils, whereas monocyte chemoattractant protein-<em>1</em> (MCP-<em>1</em>) is a C-C chemokine and functions mainly as a chemoattractant of monocytes/macrophages. Both factors are secreted from endothelial cells and have been implicated in the processes leading to atherosclerosis. We examined the effects of LPLs on the expression of IL-8 and MCP-<em>1</em>, key regulators of leukocyte recruitment in human umbilical cord vein endothelial cells (HUVECs). Work illustrated in this article showed that LPA and S<em>1</em>P enhanced IL-8 and MCP-<em>1</em> mRNA expressions, and protein secretions in dose- and time-dependent fashions. Maximal mRNA expression appeared at <em>1</em>6 hr post-ligand treatment. Using prior treatments with chemical inhibitors, LPLs enhanced IL-8 and MCP-<em>1</em> expressions through a Gi-, Rho-, and NFkappaB-dependent mechanism. In a chemotaxis assay system, LPL treatments of endothelial cells enhanced monocyte recruitment through upregulating IL-8 and MCP-<em>1</em> protein secretions. Pre-incubation with AF<em>1</em>2<em>1</em>98, an IL-<em>1</em> receptor antagonist or IL-<em>1</em> functional blocking antibody both suppressed the enhanced effects elicited by LPLs of IL-8 and MCP-<em>1</em> mRNA expressions in HUVECs. These results suggest that LPLs released by activated platelets might enhance the IL-8- and MCP-<em>1</em>-dependent chemoattraction of monocytes toward the endothelium through an IL-<em>1</em>-dependent mechanism, which may play an important role in facilitating wound-healing and inflammation processes.

<em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) is a lipid second messenger that signals via five G protein-coupled receptors (S<em>1</em>P<em>1</em>-5 ). S<em>1</em>P receptor (S<em>1</em>PR) signalling is associated with a wide variety of physiological processes including lymphocyte biology, their recirculation and determination of T-cell phenotypes. The effect of FTY720 (Fingolimod, Gilenya™) to regulate lymphocyte egress and to ameliorate paralysis in experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis led to the use of FTY720 as a first-line oral agent for treatment of relapsing-remitting multiple sclerosis. However, a significant body of research suggests that S<em>1</em>P signalling may participate in diverse immune regulatory functions other than lymphocyte trafficking. This review article discusses the current knowledge of S<em>1</em>P signalling in the fate and function of T regulatory, T helper type <em>1</em>7 and memory T cells in health and disease.

EDG-6 is a recently cloned member of the endothelial differentiation gene (EDG) G protein-coupled receptor family that is expressed in lymphoid and hematopoietic tissue and in the lung. Homology of EDG-6 to the known <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (SPP) receptors EDG-<em>1</em>, EDG-3, and EDG-5 and lysophosphatidic acid (LPA) receptors EDG-2 and EDG-4 suggested that its ligand may be a lysophospholipid or lysosphingolipid. We examined the binding of [(32)P]SPP to HEK293 cells, transiently transfected with cDNA encoding EDG-6. Binding of [(32)P]SPP was saturable, demonstrating high affinity (K(D) = 63 nmol/L). Binding was also specific for SPP, as only unlabeled SPP and sphinganine-<em>1</em>-<em>phosphate</em>, which lacks the trans double bond at the 4 position, potently displaced radiolabeled SPP. LPA did not compete for binding of SPP at any concentration tested, whereas sphingosylphosphorylcholine competed for binding to EDG-6, but only at very high concentrations. In addition, SPP activated extracellular signal-regulated kinase (Erk) in EDG-6 transfected cells in a pertussis toxin-sensitive manner. These results indicate that EDG-6 is a high affinity receptor for SPP, which couples to a G(i/o) protein, resulting in the activation of growth-related signaling pathways. (Blood. 2000;95:2624-2629)

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) receptors (S<em>1</em>PRs) belong to the class A family of G protein-coupled receptors (GPCRs). S<em>1</em>PRs are widely expressed on many cell types, including those of the immune, cardiovascular, and central nervous systems. The S<em>1</em>PR family is rapidly gaining attention as an important mediator of many cellular processes, including cell differentiation, migration, survival, angiogenesis, calcium homeostasis, inflammation and immunity. Importantly, S<em>1</em>PRs are known drug targets for multiple sclerosis (MS), for which the newly developed oral therapy fingolimod, an S<em>1</em>PR modulator, has recently been approved for clinical use. Much progress has also recently been made in the field of structural biology and in the modeling of heterotrimeric GPCRs allowing the crystal structure of the S<em>1</em>PR<em>1</em> subtype to be elucidated and key interactions defined. Here, we outline the structure and function of S<em>1</em>PR<em>1</em>, highlighting the key residues involved in receptor activation, signaling, transmembrane interactions, ligand binding, post-translational modification, and protein-protein interactions.

During development, natural killer (NK) cells exit the BM to reach the blood. CXCR4 retains NK cells in the BM, whereas the <em>sphingosine</em>-<em>1</em> <em>phosphate</em> receptor 5 (S<em>1</em>P5) promotes their exit from this organ. However, how the action of these receptors is coordinated to preserve NK-cell development in the BM parenchyma while providing mature NK cells at the periphery is unclear. The role of CXCR4 and S<em>1</em>P5 in NK-cell recirculation at the periphery is also unknown. In the present study, we show that, during NK-cell differentiation, CXCR4 expression decreases whereas S<em>1</em>P5 expression increases, thus favoring the exit of mature NK cells via BM sinusoids. Using S<em>1</em>P5(-/-) mice and a new knockin mouse model in which CXCR4 cannot be desensitized (a mouse model of warts, hypogammaglobulinemia, infections, and myelokathexis [WHIM] syndrome), we demonstrate that NK-cell exit from the BM requires both CXCR4 desensitization and S<em>1</em>P5 engagement. These 2 signals occur independently of each other: CXCR4 desensitization is not induced by S<em>1</em>P5 engagement and vice versa. Once in the blood, the S<em>1</em>P concentration increases and S<em>1</em>P5 responsiveness decreases. This responsiveness is recovered in the lymph nodes to allow NK-cell exit via lymphatics in a CXCR4-independent manner. Therefore, coordinated changes in CXCR4 and S<em>1</em>P5 responsiveness govern NK-cell trafficking.