The immunomodulator 2-amino-2-[2-(4-octylphenyl)ethyl]-<em>1</em>,3-propanediol (FTY720) has promising therapeutic effects in multiple sclerosis (MS), a degenerative disease in which demyelination of the central nervous system is accompanied by death of oligodendrocytes (OLGs), the myelin-producing cells. In vivo phosphorylation of FTY720 generates an agonist for G protein-coupled receptors for <em>sphingosine</em>-<em>1</em>-<em>phosphate</em>, a lipid mediator that plays a crucial role in the stimulation of OLG survival by neurotrophin-3 (NT-3). The mechanisms underlying the action of FTY720 in MS are not clearly understood, although the effects of this drug in autoimmune diseases are thought to stem from its ability to reduce lymphocyte infiltration and inflammation. Interestingly, we now found that FTY720 also has a direct effect on OLG progenitors. Treatment of these cells with FTY720 causes activation of extracellular signal-regulated kinase <em>1</em>/2 and Akt, accompanied by protection from apoptosis. However, FTY720 also arrested OLG differentiation. Importantly, this effect was counteracted by NT-3, which not only enhanced the survival of OLG progenitors induced by FTY720 but also stimulated their maturation. Altogether, these observations suggest that in addition to its immunosuppressive functions, FTY720 could also have a beneficial effect in MS by direct action on OLG progenitors. However, the finding that FTY720 blocks the differentiation of these cells raises the question of whether MS therapies with FTY720 should include the use of differentiation-enhancing factors such as NT-3. This approach would ensure both protection of existing OLG progenitor pools against immune-mediated insults as well as stimulation of remyelination by enhancing the maturation of these cells.

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) concentration in plasma and serum has been estimated to be within 200-900 nM. Among plasma and serum components, S<em>1</em>P is concentrated in lipoprotein fractions with a rank order of high-density lipoprotein (HDL>>low-density lipoprotein (LDL>>very low-density lipoprotein (VLDL>>lipoprotein-deficient plasma (LPDP) when expressed as the per unit amount of protein. It is well known that LDL, especially oxidized LDL, is closely correlated and HDL is inversely correlated, with the risk of cardiovascular disease, such as atherosclerosis. Evidence was presented that a part of HDL-induced actions previously reported are mediated by the lipoprotein-associated S<em>1</em>P. Furthermore, S<em>1</em>P content in LDL was markedly decreased during its oxidation. This paper will discuss whether S<em>1</em>P is an atherogenic mediator or an anti-atherogenic mediator.

BACKGROUND

Numerous in vitro studies suggest that <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P), a bioactive lysosphingolipid associated with high-density lipoproteins, accounts at least partly for the potent antiinflammatory properties of high-density lipoprotein and, thereby, contributes to the antiatherogenic potential attributed to high-density lipoproteins. The present study was undertaken to investigate whether modulation of S<em>1</em>P signaling would affect atherosclerosis in a murine model of disease.

RESULTS

Low-density lipoprotein receptor-deficient mice on a cholesterol-rich diet were given FTY720, a synthetic S<em>1</em>P analogue, at low (0.04 mg/kg per day) or high (0.4 mg/kg per day) doses for <em>1</em>6 weeks. FTY720 dose-dependently reduced atherosclerotic lesion formation, both in the aortic root and brachiocephalic artery, and almost completely blunted necrotic core formation. Plasma lipids remained unchanged during the course of FTY720 treatment. However, FTY720 lowered blood lymphocyte count (at a high dose) and significantly interfered with lymphocyte function, as evidenced by reduced splenocyte proliferation and interferon-gamma levels in plasma. Plasma concentrations of proinflammatory cytokines such as tumor necrosis factor-alpha, interleukin (IL)-6, IL-<em>1</em>2, and regulated on activation normal T cell expressed and secreted were reduced by FTY720 administration. Moreover, lipopolysaccharide-elicited generation of nitrite/nitrate and IL-6--two markers of classical (M<em>1</em>) macrophage activation--was inhibited, whereas IL-4-induced production of IL-<em>1</em>-receptor antagonist, a marker of alternative (M2) macrophage activation, was augmented in peritoneal macrophages from FTY720-treated low-density lipoprotein receptor-deficient mice.

CONCLUSIONS

The present results demonstrate that an S<em>1</em>P analogue inhibits atherosclerosis by modulating lymphocyte and macrophage function, and these results are consistent with the notion that S<em>1</em>P contributes to the antiatherogenic potential of high-density lipoprotein.

The mitochondrial enzyme monoamine oxidase (MAO), its isoform MAO-A, plays a major role in reactive oxygen species-dependent cardiomyocyte apoptosis and postischemic cardiac damage. In the current study, we investigated whether sphingolipid metabolism can account for mediating MAO-A- and reactive oxygen species-dependent cardiomyocyte apoptosis. In H9c2 cardiomyoblasts, MAO-A-dependent reactive oxygen species generation led to mitochondria-mediated apoptosis, along with <em>sphingosine</em> kinase-<em>1</em> (SphK<em>1</em>) inhibition. These phenomena were associated with generation of proapoptotic ceramide and decrease in prosurvival <em>sphingosine</em> <em>1</em>-<em>phosphate</em>. These events were mimicked by inhibition of SphK<em>1</em> with either pharmacological inhibitor or small interfering RNA, as well as by extracellular addition of C(2)-ceramide or H(2)O(2). In contrast, enforced expression of SphK<em>1</em> protected H9c2 cells from serotonin- or H(2)O(2)-induced apoptosis. Analysis of cardiac tissues from wild-type mice subjected to ischemia/reperfusion revealed significant upregulation of ceramide and inhibition of SphK<em>1</em>. It is noteworthy that SphK<em>1</em> inhibition, ceramide accumulation, and concomitantly infarct size and cardiomyocyte apoptosis were significantly decreased in MAO-A-deficient animals. In conclusion, we show for the first time that the upregulation of ceramide/<em>sphingosine</em> <em>1</em>-<em>phosphate</em> ratio is a critical event in MAO-A-mediated cardiac cell apoptosis. In addition, we provide the first evidence linking generation of reactive oxygen species with SphK<em>1</em> inhibition. Finally, we propose sphingolipid metabolites as key mediators of postischemic/reperfusion cardiac injury.

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) is a lipid agonist that regulates smooth muscle cell (SMC) and endothelial cell functions by activating several members of the S<em>1</em>P subfamily of G-protein-coupled Edg receptors. We have shown previously that SMC differentiation is regulated by RhoA-dependent activation of serum response factor (SRF). Because S<em>1</em>P is a strong activator of RhoA, we hypothesized that S<em>1</em>P would stimulate SMC differentiation. Treatment of primary rat aortic SMC cells with S<em>1</em>P activated RhoA as measured by precipitation with a glutathione S-transferase-rhotekin fusion protein. In SMC and <em>1</em>0T<em>1</em>/2 cells, S<em>1</em>P treatment up-regulated the activities of several transiently transfected SMC-specific promoters, and these effects were inhibited by the Rho-kinase inhibitor, Y-27632. S<em>1</em>P also increased smooth muscle alpha-actin protein levels in SMC but had no effect on SRF binding to the smooth muscle alpha-actin CArG B element. Quantitative reverse transcriptase-PCR showed that S<em>1</em>P treatment of SMC or <em>1</em>0T<em>1</em>/2 cells did not increase the mRNA level of either of the recently identified SRF co-factors, myocardin or myocardin-related transcription factor-A (MRTF-A). MRTF-A protein was expressed highly in SMC and <em>1</em>0T<em>1</em>/2 cultures, and importantly the effects of S<em>1</em>P were inhibited by a dominant negative form of MRTF-A indicating that S<em>1</em>P may regulate the transcriptional activity of MRTF-A. Indeed, S<em>1</em>P treatment increased the nuclear localization of FLAG-MRTF-A, and the effect of MRTF-A overexpression on smooth muscle alpha-actin promoter activity was inhibited by dominant negative RhoA. S<em>1</em>P also stimulated SMC growth by activating the early growth response gene, c-fos. This effect was not attenuated by Y-27632 but could be inhibited by the MEK inhibitor, UO<em>1</em>26. S<em>1</em>P enhanced SMC growth through ERK-mediated phosphorylation of the SRF co-factor, Elk-<em>1</em>, as measured by gel shift and Elk-<em>1</em> activation assays. Taken together these results demonstrate that S<em>1</em>P activates multiple signaling pathways in SMC and regulates proliferation by ERK-dependent activation of Elk-<em>1</em> and differentiation by RhoA-dependent activation of MRTF-A.

Recent data have revealed that soluble oligomeric amyloid-beta peptide (Abeta) may be the proximate effectors of neuronal injuries and death in Alzheimer's disease (AD) by unknown mechanisms. Consistently, we recently demonstrated the critical role of a redox-sensitive cytosolic calcium-dependent phospholipase A2 (cPLA2)-arachidonic acid (AA) pathway in Abeta oligomer-induced cell death. According to the involvement of oxidative stress and polyunsaturated fatty acids like AA in the regulation of sphingomyelinase (SMase) activity, the present study underlines the role of SMases in soluble Abeta-induced apoptosis. Soluble Abeta oligomers induced the activation of both neutral and acidic SMases, as demonstrated by the direct measurement of their enzymatic activities, by the inhibitory effects of both specific neutral and acidic SMase inhibitors, and by gene knockdown using antisense oligonucleotides. Furthermore, soluble Abeta-mediated activation of SMases and subsequent cell death were found to be inhibited by antioxidant molecules and a cPLA2-specific inhibitor or antisense oligonucleotide. We also demonstrate that <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> is a potent neuroprotective factor against soluble Abeta oligomer-induced cell death and apoptosis by inhibiting soluble Abeta-induced activation of acidic sphingomyelinase. These results suggest that Abeta oligomers induce neuronal death by activating neutral and acidic SMases in a redox-sensitive cPLA2-AA pathway.

<em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P), the bioactive product of <em>sphingosine</em> kinase (SK) activation, is a survival factor for endothelial cells. The mechanism of SK-mediated survival was investigated in endothelial cells with moderately raised intracellular SK activity. Overexpression of SK mediated survival primarily through the activation of the phosphatidyl inositol 3-kinase (PI-3K)/protein kinase B (Akt/PKB) pathway and an associated up-regulation of the antiapoptotic protein B cell lymphoma gene 2 (Bcl-2) and down-regulation of the proapoptotic protein bisindolylmaleimide (Bcl-2 interacting mediator of cell death; Bim). In addition there was an up-regulation and dephosphorylation of the junctional molecule platelet endothelial cell adhesion molecule-<em>1</em> (PECAM-<em>1</em>), which was obligatory for activation of the PI-3K/Akt pathway, for SK-induced cell survival, and for the changes in the apoptosis-related proteins. Thus, raised intracellular SK activity induced a molecule involved in cell-cell interactions to augment cell survival through a PI-3K/Akt-dependent pathway. This is distinct from the activation of both PI-3K/Akt and mitogen-activated protein kinase (MAPK) pathways seen with exogenously added S<em>1</em>P. Cells overexpressing SK showed enhanced survival under conditions of serum deprivation and absence of attachment to extracellular matrix, suggesting a role for SK in the regulation of vascular phenomena that occur under conditions of stress, such as angiogenesis and survival in unattached states, as would be required for a circulating endothelial cell.

Bile acids have been shown to be important regulatory molecules for cells in the liver and gastrointestinal tract. They can activate various cell signaling pathways including extracellular regulated kinase (ERK)<em>1</em>/2 and protein kinase B (AKT) as well as the G-protein-coupled receptor (GPCR) membrane-type bile acid receptor (TGR5/M-BAR). Activation of the ERK<em>1</em>/2 and AKT signaling pathways by conjugated bile acids has been reported to be sensitive to pertussis toxin (PTX) and dominant-negative Gα(i) in primary rodent hepatocytes. However, the GPCRs responsible for activation of these pathways have not been identified. Screening GPCRs in the lipid-activated phylogenetic family (expressed in HEK293 cells) identified <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> receptor 2 (S<em>1</em>P(2) ) as being activated by taurocholate (TCA). TCA, taurodeoxycholic acid (TDCA), tauroursodeoxycholic acid (TUDCA), glycocholic acid (GCA), glycodeoxycholic acid (GDCA), and S<em>1</em>P-induced activation of ERK<em>1</em>/2 and AKT were significantly inhibited by JTE-0<em>1</em>3, a S<em>1</em>P(2) antagonist, in primary rat hepatocytes. JTE-0<em>1</em>3 significantly inhibited hepatic ERK<em>1</em>/2 and AKT activation as well as short heterodimeric partner (SHP) mRNA induction by TCA in the chronic bile fistula rat. Knockdown of the expression of S<em>1</em>P(2) by a recombinant lentivirus encoding S<em>1</em>P(2) shRNA markedly inhibited the activation of ERK<em>1</em>/2 and AKT by TCA and S<em>1</em>P in rat primary hepatocytes. Primary hepatocytes prepared from S<em>1</em>P(2) knock out (S<em>1</em>P(2) (-/-) ) mice were significantly blunted in the activation of the ERK<em>1</em>/2 and AKT pathways by TCA. Structural modeling of the S<em>1</em>P receptors indicated that only S<em>1</em>P(2) can accommodate TCA binding. In summary, all these data support the hypothesis that conjugated bile acids activate the ERK<em>1</em>/2 and AKT signaling pathways primarily through S<em>1</em>P(2) in primary rodent hepatocytes.

The five known members of the <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) receptor family exhibit diverse tissue expression profiles and couple to distinct G-protein-mediated signalling pathways. S<em>1</em>P<em>1</em>, S<em>1</em>P2, and S<em>1</em>P3 receptors are all present in the heart, but the ratio of these subtypes differs for various cardiac cells. The goal of this review is to summarize data concerning which S<em>1</em>P receptor subtypes regulate cardiac physiology and pathophysiology, which G-proteins and signalling pathways they couple to, and in which cell types they are expressed. The available information is based on studies using a lamentably limited set of pharmacological agonists/antagonists, but is complemented by work with S<em>1</em>P receptor subtype-specific knockout mice and <em>sphingosine</em> kinase knockout mice. In cardiac myocytes, the S<em>1</em>P<em>1</em> receptor subtype is the predominant subtype expressed, and the activation of this receptor inhibits cAMP formation and antagonizes adrenergic receptor-mediated contractility. The S<em>1</em>P3 receptor, while expressed at lower levels, mediates the bradycardic effect of S<em>1</em>P agonists. Studies using knockout mice indicate that S<em>1</em>P2 and S<em>1</em>P3 receptors play a major role in mediating cardioprotection from ischaemia/reperfusion injury in vivo. S<em>1</em>P receptors are also involved in remodelling, proliferation, and differentiation of cardiac fibroblasts, a cell type in which the S<em>1</em>P3 receptor predominates. Receptors for S<em>1</em>P are also present in endothelial and smooth muscle cells where they mediate peripheral vascular tone and endothelial responses, but the role of this regulatory system in the cardiac vasculature is unknown. Further understanding of the contributions of each cell and receptor subtype to cardiac function and pathophysiology should expedite consideration of the endogenous S<em>1</em>P signalling pathway as a therapeutic target for cardiovascular disease.

Regulation of endothelial cell (EC) permeability by bioactive molecules is associated with specific patterns of cytoskeletal and cell contact remodeling. A role for mechanical factors such as shear stress (SS) and cyclic stretch (CS) in cytoskeletal rearrangements and regulation of EC permeability becomes increasingly recognized. This paper examined redistribution of focal adhesion (FA) proteins, site-specific focal adhesion kinase (FAK) phosphorylation, small GTPase activation and barrier regulation in human pulmonary EC exposed to laminar shear stress (<em>1</em>5 dyn/cm2) or cyclic stretch (<em>1</em>8% elongation) in vitro. SS caused peripheral accumulation of FAs, whereas CS induced randomly distributed FAs attached to the ends of newly formed stress fibers. SS activated small GTPase Rac without effects on Rho, whereas <em>1</em>8% CS activated without effect on Rac. SS increased transendothelial electrical resistance (TER) in EC monolayers, which was further elevated by barrier-protective phospholipid <em>sphingosine</em> <em>1</em>-<em>phosphate</em>. Finally, SS induced FAK phosphorylation at Y576, whereas CS induced FAK phosphorylation at Y397 and Y576. These results demonstrate for the first time differential effects of SS and CS on Rho and Rac activation, FA redistribution, site-specific FAK phosphorylation, and link them with SS-mediated barrier enhancement. Thus, our results suggest common signaling and cytoskeletal mechanisms shared by mechanical and chemical factors involved in EC barrier regulation.

BACKGROUND

S<em>1</em>PL is an aldehyde-lyase that irreversibly cleaves <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) in the terminal step of sphingolipid catabolism. Because S<em>1</em>P modulates a wide range of physiological processes, its concentration must be tightly regulated within both intracellular and extracellular environments.

METHODS

In order to better understand the function of S<em>1</em>PL in this regulatory pathway, we assessed the in vivo effects of different levels of S<em>1</em>PL activity using knockout (KO) and humanized mouse models.

RESULTS

Our analysis showed that all S<em>1</em>PL-deficient genetic models in this study displayed lymphopenia, with sequestration of mature T cells in the thymus and lymph nodes. In addition to the lymphoid phenotypes, S<em>1</em>PL KO mice (S<em>1</em>PL(-/-)) also developed myeloid cell hyperplasia and significant lesions in the lung, heart, urinary tract, and bone, and had a markedly reduced life span. The humanized knock-in mice harboring one allele (S<em>1</em>PL(H/-)) or two alleles (S<em>1</em>PL(H/H)) of human S<em>1</em>PL expressed less than <em>1</em>0 and 20% of normal S<em>1</em>PL activity, respectively. This partial restoration of S<em>1</em>PL activity was sufficient to fully protect both humanized mouse lines from the lethal non-lymphoid lesions that developed in S<em>1</em>PL(-/-) mice, but failed to restore normal T-cell development and trafficking. Detailed analysis of T-cell compartments indicated that complete absence of S<em>1</em>PL affected both maturation/development and egress of mature T cells from the thymus, whereas low level S<em>1</em>PL activity affected T-cell egress more than differentiation.

CONCLUSIONS

These findings demonstrate that lymphocyte trafficking is particularly sensitive to variations in S<em>1</em>PL activity and suggest that there is a window in which partial inhibition of S<em>1</em>PL could produce therapeutic levels of immunosuppression without causing clinically significant S<em>1</em>P-related lesions in non-lymphoid target organs.

The splenic marginal zone is a unique microenvironment where resident immune cells are exposed to the open blood circulation. Even though it has an important role in responses against blood-borne antigens, lymphocyte migration in the marginal zone has not been intravitally visualized due to challenges associated with achieving adequate imaging depth in this abdominal organ. Here we develop a two-photon microscopy procedure to study marginal zone and follicular B-cell movement in the live mouse spleen. We show that marginal zone B cells are highly motile and exhibit long membrane extensions. Marginal zone B cells shuttle between the marginal zone and follicles with at least one-fifth of the cells exchanging between compartments per hour, a behaviour that explains their ability to deliver antigens rapidly from the open blood circulation to the secluded follicles. Follicular B cells also transit from follicles to the marginal zone, but unlike marginal zone B cells, they fail to undergo integrin-mediated adhesion, become caught in fluid flow and are carried into the red pulp. Follicular B-cell egress via the marginal zone is <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> receptor-<em>1</em> (S<em>1</em>PR<em>1</em>)-dependent. This study shows that marginal zone B cells migrate continually between marginal zone and follicles and establishes the marginal zone as a site of S<em>1</em>PR<em>1</em>-dependent B-cell exit from follicles. The results also show how adhesive differences of similar cells critically influence their behaviour in the same microenvironment.

<em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) is a lipid mediator that exerts multiple cellular functions through activation of G-protein-coupled receptors. Although the role of S<em>1</em>P on angiogenesis is well established, its role in neurogenesis is unknown. We examined the effects of S<em>1</em>P on G-protein activation in brain sections of rat embryo and on neural progenitor cells in culture. Intense S<em>1</em>P-stimulated [35S]GTPgammaS labeling was observed as early as E<em>1</em>5 in the neuroepithelium and differentiating fields throughout the brain, suggesting that functional S<em>1</em>P receptors are expressed in brain areas with active neurogenesis. mRNA transcripts for several S<em>1</em>P receptor subtypes (S<em>1</em>P<em>1</em>, S<em>1</em>P2, S<em>1</em>P3 and S<em>1</em>P5) were expressed in neural progenitor cells prepared from embryonic rat hippocampus. S<em>1</em>P induced phosphorylation of extracellular signal-regulated kinase (ERK) and proliferation of neural progenitor cells as determined by BrdU incorporation in a pertussis toxin-sensitive manner. These effects were prevented by the ERK signaling inhibitor U0<em>1</em>26. S<em>1</em>P augmented telomerase activity in neural progenitor cells with similar potency as that of FGF-2. Furthermore, S<em>1</em>P induced cell-cell aggregation. This morphological change was transient and prevented by Y-27632, an inhibitor of Rho-associated kinase. These results suggest that S<em>1</em>P plays a pleiotropic role in neurogenesis via pathways involving S<em>1</em>P receptors, MAP kinases and Rho kinase.

We investigated mechanisms for inhibition of B<em>1</em>6 melanoma cell migration and invasion by <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P), which is the ligand for the Edg family G protein-coupled receptors and also implicated as an intracellular second messenger. S<em>1</em>P, dihydro-S<em>1</em>P, and sphingosylphosphorylcholine inhibited B<em>1</em>6 cell migration and invasion with the relative potencies expected as S<em>1</em>P2 receptor agonists. The S<em>1</em>P2-selective antagonist JTE0<em>1</em>3 completely abolished the responses to these agonists. In addition, JTE0<em>1</em>3 abrogated the inhibition by <em>sphingosine</em>, which is the S<em>1</em>P precursor but not an agonist for S<em>1</em>P receptors, indicating that the <em>sphingosine</em> effects were mediated via S<em>1</em>P2 stimulation, most likely by S<em>1</em>P that was converted from <em>sphingosine</em>. S<em>1</em>P induced inhibition and activation, respectively, of Rac and RhoA in B<em>1</em>6 cells, which were abrogated by JTE0<em>1</em>3. Adenovirus-mediated expression of N<em>1</em>7Rac mimicked S<em>1</em>P inhibition of migration, whereas C3 toxin pretreatment, but not Rho kinase inhibitors, reversed the S<em>1</em>P inhibition. Overexpression of S<em>1</em>P2 sensitized, and that of either S<em>1</em>P<em>1</em> or S<em>1</em>P3 desensitized, B<em>1</em>6 cells to S<em>1</em>P inhibition of Rac and migration. In JTE0<em>1</em>3-pretreated, S<em>1</em>P3-overexpressing B<em>1</em>6 cells, S<em>1</em>P stimulated cellular RhoA but failed to inhibit either Rac or migration, indicating that RhoA stimulation itself is not sufficient for inhibition of migration. These results provide compelling evidence that endogenously expressed S<em>1</em>P2 negatively regulates cell motility and invasion through ligand-dependent reciprocal regulation of cellular Rac and RhoA activities. In the presence of JTE0<em>1</em>3, S<em>1</em>P instead stimulated Rac and migration in B<em>1</em>6 cells that overexpress either S<em>1</em>P<em>1</em> or S<em>1</em>P3, unveiling counteractions between S<em>1</em>P2 and S<em>1</em>P<em>1</em> or S<em>1</em>P3 chemotactic receptor.

Neural stem/progenitor cells (NSPCs) migrate toward a damaged area of the central nervous system (CNS) for the purpose of limiting and/or repairing the damage. Although this migratory property of NSPCs could theoretically be exploited for cell-based therapeutics of CNS diseases, little is known of the mechanisms responsible for migratory responses of NSPCs. Here, we found that <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (Sph-<em>1</em>-P), a physiological lysophospholipid mediator, had a potent chemoattractant activity for NSPCs, in which, of Sph-<em>1</em>-P receptors, S<em>1</em>P(<em>1</em>) was abundantly expressed. Sph-<em>1</em>-P-induced NSPC migration was inhibited by the pretreatment with pertussis toxin, Y-27632 (a Rho kinase inhibitor), and VPC230<em>1</em>9 (a competitive inhibitor of S<em>1</em>P(<em>1</em>) and S<em>1</em>P(3)). Sph-<em>1</em>-P does not act as intracellular mediator or in an autocrine manner, because [(3)H]<em>sphingosine</em>, incorporated into NSPCs, was mainly converted to ceramide and sphingomyeline intracellularly, and the stimulation-dependent formation and extracellular release of Sph-<em>1</em>-P were not observed. Further, Sph-<em>1</em>-P concentration in the spinal cord was significantly increased at 7 days after a contusion injury, due to accumulation of microglia and reactive astrocytes in the injured area. This locally increased Sph-<em>1</em>-P concentration contributed to the migration of in vivo transplanted NSPCs through its receptor S<em>1</em>P(<em>1</em>), given that lentiviral transduction of NSPCs with a short hairpin RNA interference for S<em>1</em>P(<em>1</em>) abolished in vivo NSPC migration toward the injured area. This is the first report to identify a physiological role for a lipid mediator in NSPC migration toward a pathological area of the CNS and further indicates that the Sph-<em>1</em>-P/S<em>1</em>P(<em>1</em>) pathway may have therapeutic potential for CNS injuries.

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

Sphingolipids and their metabolites, ceramide, <em>sphingosine</em> and <em>sphingosine</em>-<em>1</em>-<em>phosphate</em>, are involved in a variety of cellular processes including differentiation, cellular senescence, apoptosis and proliferation. Ceramide is the main second messenger, and is produced by sphingomyelinase-induced hydrolysis of sphingomyelin and by de novo synthesis. Many stimuli, e. g. growth factors, cytokines, G protein-coupled receptor agonists and stress (UV irradiation) increase cellular ceramide levels. Sphingomyelin in the plasma membrane is located primarily in the outer (extracellular) leaflet of the bilayer, whilst sphingomyelinases are found at the inner (cytosolic) face and within lysosomes/endosomes. Such cellular compartmentalisation restricts the site of ceramide production and subsequent interaction with target proteins. Glycosphingolipids and sphingomyelin together with cholesterol are major components of specialised membrane microdomains known as lipid rafts, which are involved in receptor aggregation and immune responses. Many signalling molecules, for example Src family tyrosine kinases and glycosylinositol<em>phosphate</em>-anchored proteins, are associated with rafts, and disruption of these domains affects cellular responses such as apoptosis. <em>Sphingosine</em> and <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> derived from ceramide are also signalling molecules. In particular, <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> is involved in proliferation, differentiation and apoptosis. <em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> can act both extracellularly through endothelial-differentiating gene (EDG) family G protein-coupled receptors and intracellularly through direct interactions with target proteins. The importance of sphingolipid signalling in cardiovascular development has been reinforced by recent reports implicating EDG receptors in the regulation of embryonic cardiac and vascular morphogenesis.

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P), a novel lipid mediator, is concentrated in the fraction of lipoproteins that include high density lipoprotein (HDL) and low density lipoprotein (LDL) in human plasma. Here, we show that oxidation of LDL resulted in a marked reduction in the S<em>1</em>P level in association with a marked accumulation of lysophosphatidylcholine (LPC). We therefore investigated the role of the lipoprotein-associated lipids especially S<em>1</em>P in the lipoprotein-induced cytoprotective or cytotoxic actions in human umbilical vein endothelial cells. The viability of the cells gradually decreased in the absence of serum or growth factors in the culture medium. The addition of oxidized LDL (ox-LDL) accelerated the decrease in the cell viability. LPC and 7-ketocholesterol mimicked ox-LDL actions. On the other hand, HDL and LDL almost completely reversed the serum deprivation- or ox-LDL-induced cytotoxicity. Exogenous S<em>1</em>P mimicked cytoprotective actions. Moreover, the S<em>1</em>P-rich fraction and chromatographically purified S<em>1</em>P from HDL exerted cytoprotective actions, but the rest of the fractions did not. The cytoprotective actions of HDL and S<em>1</em>P were associated with extracellular signal-regulated kinase (ERK) activation and were almost completely inhibited by pertussis toxin and PD98059, an ERK kinase inhibitor. The HDL-induced action was specifically desensitized in the S<em>1</em>P-pretreated cells. Taken together, these results indicate that the lipoprotein-associated S<em>1</em>P and the lipid receptor-mediated signal pathways may be responsible for the lipoprotein-induced cytoprotective actions. Furthermore, the decrease in the S<em>1</em>P content, in addition to the accumulation of cytotoxic substances such as LPC, may be important for the acquisition of the cytotoxic property to ox-LDL.

We have observed that conditioning for hematopoietic transplantation by lethal irradiation induces a proteolytic microenvironment in the bone marrow (BM) that activates the complement cascade (CC). As a result, BM is enriched for proteolytic enzymes and the soluble form of the terminal product of CC activation, the membrane attack complex C5b-C9 (MAC). At the same time, proteolytic enzymes induced in irradiated BM impair the chemotactic activity of α-chemokine stromal-derived factor-<em>1</em> (SDF-<em>1</em>). As SDF-<em>1</em> is considered a crucial BM chemoattractant for transplanted hematopoietic stem/progenitor cells (HSPCs), we sought to determine whether other factors that are resistant to proteolytic enzymes have a role in this process, focusing on proteolysis-resistant bioactive lipids. We found that the concentrations of <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) increase in the BM after conditioning for transplantation and that both S<em>1</em>P and, as we show here for the first time, C<em>1</em>P are potent chemoattractants for HSPCs. Next, we observed that C5-deficient mice that do not generate MAC show impaired engraftment of HSPCs. In support of a role for MAC in homing and engraftment, we found that soluble MAC enhances in a CR3 (CD<em>1</em><em>1</em>b/CD<em>1</em>8)-dependent manner the adhesion of HSPCs to BM stromal cells and increases the secretion of SDF-<em>1</em> by BM stroma. We conclude that an increase in BM levels of proteolytic enzyme-resistant S<em>1</em>P and C<em>1</em>P and activation of CC, which leads to the generation of MAC, has an important and previously underappreciated role in the homing of transplanted HSPCs.

T cell death-associated gene 8 (TDAG8) has been reported to be a receptor for psychosine. Ovarian cancer G-protein-coupled receptor <em>1</em> (OGR<em>1</em>) and GPR4, G-protein-coupled receptors (GPCRs) closely related to TDAG8, however, have recently been identified as proton-sensing or extracellular pH-responsive GPCRs that stimulate inositol <em>phosphate</em> and cAMP production, respectively. In the present study, we examined whether TDAG8 senses extracellular pH change. In the several cell types that were transfected with TDAG8 cDNA, cAMP was markedly accumulated in response to neutral to acidic extracellular pH, with a peak response at approximately pH 7.0-6.5. The pH effect was inhibited by copper ions and was reduced or lost in cells expressing mutated TDAG8 in which histidine residues were changed to phenylalanine. In the membrane fractions prepared from TDAG8-transfected cells, guanosine 5'-O-(3-thiotri<em>phosphate</em>) binding activity and adenylyl cyclase activity were remarkably stimulated in response to neutral and acidic pH. The concentration-dependent effect of extracellular protons on cAMP accumulation was shifted to the right in the presence of psychosine. The inhibitory psychosine effect was also observed for pH-dependent actions in OGR<em>1</em>- and GPR4-expressing cells but not for prostaglandin E(2)- and <em>sphingosine</em> <em>1</em>-<em>phosphate</em>-induced actions in any pH in native and <em>sphingosine</em> <em>1</em>-<em>phosphate</em> receptor-expressing cells. Glucosyl<em>sphingosine</em> and sphingosylphosphorylcholine similarly inhibited the pH-dependent action, although to a lesser extent. Psychosine-sensitive and pH-dependent cAMP accumulation was also observed in mouse thymocytes. We concluded that TDAG8 is one of the proton-sensing GPCRs coupling to adenylyl cyclase and psychosine, and its related lysosphingolipids behave as if they were antagonists against protein-sensing receptors, including TDAG8, GPR4, and OGR<em>1</em>.

Ample evidence indicates that <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (SPP) can serve as an intracellular second messenger regulating calcium mobilization, and cell growth and survival. Moreover, the dynamic balance between levels of the sphingolipid metabolites, ceramide and SPP, and consequent regulation of opposing signaling pathways, is an important factor that determines whether a cell survives or dies. SPP has recently also been shown to be the ligand for the EDG-<em>1</em> family of G-protein-coupled receptors, which now includes EDG-<em>1</em>, -3, -5, -6 and -8. SPP is thus a lipid mediator that has novel dual actions signaling inside and outside of the cell.

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) and vascular endothelial growth factor (VEGF) elicit numerous biological responses including cell survival, growth, migration, and differentiation in endothelial cells mediated by the endothelial differentiation gene, a family of G-protein-coupled receptors, and fetal liver kinase-<em>1</em>/kinase-insert domain-containing receptor (Flk-<em>1</em>/KDR), one of VEGF receptors, respectively. Recently, it was reported that S<em>1</em>P or VEGF treatment of endothelial cells leads to phosphorylation at Ser-<em>1</em><em>1</em>79 in bovine endothelial nitric oxide synthase (eNOS), and this phosphorylation is critical for eNOS activation. S<em>1</em>P stimulation of eNOS phosphorylation was shown to involve G(i) protein, phosphoinositide 3-kinase, and Akt. VEGF also activates eNOS through Flk-<em>1</em>/KDR, phosphoinositide 3-kinase, and Akt, which suggested that S<em>1</em>P and VEGF may share upstream signaling mediators. We now report that S<em>1</em>P treatment of bovine aortic endothelial cells acutely increases the tyrosine phosphorylation of Flk-<em>1</em>/KDR, similar to VEGF treatment. S<em>1</em>P-mediated phosphorylation of Flk-<em>1</em>/KDR, Akt, and eNOS were all inhibited by VEGF receptor tyrosine kinase inhibitors and by antisense Flk-<em>1</em>/KDR oligonucleotides. Our study suggests that S<em>1</em>P activation of eNOS involves G(i), calcium, and Src family kinase-dependent transactivation of Flk-<em>1</em>/KDR. These data are the first to establish a critical role of Flk-<em>1</em>/KDR in S<em>1</em>P-stimulated eNOS phosphorylation and activation.

Membrane type <em>1</em> matrix metalloproteinase (MT<em>1</em>-MMP) is a transmembrane MMP that plays important roles in migratory processes underlying tumor invasion and angiogenesis. In addition to its matrix degrading activity, MT<em>1</em>-MMP also contains a short cytoplasmic domain whose involvement in cell locomotion seems important but remains poorly understood. In this study, we show that MT<em>1</em>-MMP is phosphorylated on the unique tyrosine residue located within this cytoplasmic sequence (Tyr(573)) and that this phosphorylation requires the kinase Src. Using phosphospecific antibodies recognizing MT<em>1</em>-MMP phosphorylated on Tyr(573), we observed that tyrosine phosphorylation of the enzyme is rapidly induced upon stimulation of tumor and endothelial cells with the platelet-derived chemoattractant <em>sphingosine</em>-<em>1</em>-<em>phosphate</em>, suggesting a role in migration triggered by this lysophospholipid. Accordingly, overexpression of a nonphosphorylable MT<em>1</em>-MMP mutant (Y573F) blocked <em>sphingosine</em>-<em>1</em>-<em>phosphate</em>-induced migration of Human umbilical vein endothelial cells and HT-<em>1</em>080 (human fibrosarcoma) cells and failed to stimulate migration of cells lacking the enzyme (bovine aortic endothelial cells). Altogether, these findings strongly suggest that the Src-dependent tyrosine phosphorylation of MT<em>1</em>-MMP plays a key role in cell migration and further emphasize the importance of the cytoplasmic domain of the enzyme in this process.

Sphingolipids are major lipid constituents of the eukaryotic plasma membrane. Without certain sphingolipids, cells and/or embryos cannot survive, indicating that sphingolipids possess important physiological functions that are not substituted for by other lipids. One such role may be signaling. Recent studies have revealed that some sphingolipid metabolites, such as long-chain bases (LCBs; <em>sphingosine</em> (Sph) in mammals), long-chain base <em>1</em>-<em>phosphates</em> (LCBPs; <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) in mammals), ceramide (Cer), and ceramide <em>1</em>-<em>phosphate</em> (C<em>1</em>P), act as signaling molecules. The addition of <em>phosphate</em> groups to LCB/Sph and Cer generates LCBP/S<em>1</em>P and C<em>1</em>P, respectively. These phospholipids exhibit completely different functions than those of their precursors. In this review, we describe recent advances in understanding the functions of LCBP/S<em>1</em>P and C<em>1</em>P in mammals and in the yeast Saccharomyces cerevisiae. Since LCB/Sph, LCBP/S<em>1</em>P, Cer, and C<em>1</em>P are mutually convertible, regulation of not only the total amount of the each lipid but also of the overall balance in cellular levels is important. Therefore, we describe in detail their metabolic pathways, as well as the genes involved in each reaction.