The "sphingosin" backbone of sphingolipids was so named by J. L. W. Thudichum in <em>1</em>884 for its enigmatic ("Sphinx-like") properties. Although still an elusive class of lipids, research on the involvement of sphingolipids in the signal transduction pathways that mediate cell growth, differentiation, multiple cell functions, and cell death has been rapidly expanding our understanding of these compounds. In addition to the newly discovered role of ceramide as an intracellular second messenger for tumor necrosis factor-alpha, IL-<em>1</em>beta, and other cytokines, <em>sphingosine</em>, <em>sphingosine</em>-<em>1</em>-<em>phosphate</em>, and other sphingolipid metabolites have recently been demonstrated to modulate cellular calcium homeostasis and cell proliferation. Perturbation of sphingolipid metabolism using synthetic and naturally occurring inhibitors of key enzymes of the biosynthetic pathways is aiding the characterization of these processes; for examples, inhibition of cerebroside synthase has indicated a role for ceramide in cellular stress responses including heat shock, and inhibition of ceramide synthase (by fumonisins) has revealed the role of disruption of sphingolipid metabolism in several animal diseases. Fumonisins are currently the focus of a FDA long-term tumor study. This review summarizes recent research on (i) the role of sphingolipids as important components of the diet, (ii) the role of sphingoid base metabolites and the ceramide cycle in expression of genes regulating cell growth, differentiation, and apoptosis, (iii) the use of cerebroside synthase inhibitors as tools for understanding the role of sphingolipids as mediators of cell cycle progression, renal disease, and stress responses, and (iv) the involvement of disrupted sphingolipid metabolism in animal disease and cellular deregulation associated with exposure to inhibitors of ceramide synthase and serine palmitoyltransferase, key enzymes in de novo sphingolipid biosynthesis. These findings illustrate how an understanding of the function of sphingolipids can help solve questions in toxicology and this is undoubtedly only the beginning of this story.

Sphingolipid metabolites have emerged as critical players in a number of fundamental biological processes. Among them, <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) promotes cell survival and proliferation, in contrast to ceramide and <em>sphingosine</em>, which induce cell growth arrest and apoptosis. These sphingolipids with opposing functions are interconvertible inside cells, suggesting that a finely tuned balance between them can determine cell fate. <em>Sphingosine</em> kinases (SphKs), which catalyze the phosphorylation of <em>sphingosine</em> to S<em>1</em>P, are critical regulators of this balance. Of the two identified SphKs, <em>sphingosine</em> kinase type <em>1</em> (SphK<em>1</em>) has been shown to regulate various processes important for cancer progression and will be the focus of this review, since much less is known of biological functions of SphK2, especially in cancer. SphK<em>1</em> is overexpressed in various types of cancers and upregulation of SphK<em>1</em> has been associated with tumor angiogenesis and resistance to radiation and chemotherapy. Many growth factors, through their tyrosine kinase receptors (RTKs), stimulate SphK<em>1</em> leading to a rapid increase in S<em>1</em>P. This S<em>1</em>P in turn can activate S<em>1</em>P receptors and their downstream signaling. Conversely, activation of S<em>1</em>P receptors can induce transactivation of various RTKs. Thus, SphK<em>1</em> may play important roles in S<em>1</em>P receptor RTK amplification loops. Here we review the role of SphK<em>1</em> in tumorigenesis, hormonal therapy, chemotherapy resistance, and as a prognostic marker. We will also review studies on the effects of SphK inhibitors in cells in vitro and in animals in vivo and in some clinical trials and highlight the potential of SphK<em>1</em> as a new target for cancer therapeutics.

ISP-<em>1</em>/myriocin is a new type of remarkably potent immunosuppressant, the structure of which is homologous to <em>sphingosine</em>. ISP-<em>1</em>/myriocin inhibited the proliferation of an IL-2-dependent mouse cytotoxic T cell line, CTLL-2, at nanomole concentrations. ISP-<em>1</em>/myriocin inhibits serine palmitoyltransferase activity at picomole concentrations. This enzyme catalyzes the first step of sphingolipid biosynthesis and reduces the intracellular pool of sphingolipid intermediates. The growth inhibition induced by ISP-<em>1</em>/myriocin was completely abolished by the addition of <em>sphingosines</em> or <em>sphingosine</em>-<em>1</em>-<em>phosphate</em>, but not by sphingomyelin or glycosphingolipids. These results suggest that <em>sphingosines</em> or <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> are associated with CTLL-2 proliferation, and ISP-<em>1</em>/myriocin suppresses T cell proliferation by the modulation of sphingolipid metabolism. ISP-<em>1</em>/myriocin should be a useful tool for the study of the sphingolipid pathway, which has been associated with various kinds of signal transduction.

Upon cell activation, membrane phospholipids are metabolized into potent lysophospholipid (LP) mediators, such as <em>sphingosine</em> <em>1</em>-<em>phosphate</em> and lysophosphatidic acid. LPs fulfill signaling roles in organisms as diverse as yeast and humans. The recent discovery of G protein-coupled receptors for LPs in higher eukaryotes, and their involvement in regulating diverse processes such as angiogenesis, cardiac development, neuronal survival, and immunity, has stimulated growing interest in these lipid mediators. LP receptor biology has generated insights into fundamental cellular mechanisms and may provide therapeutic targets for drug development.

Until recently, all approved multiple sclerosis (MS) disease treatments were administered parenterally. Oral fingolimod was approved in September 20<em>1</em>0 by the US Food and Drug Administration to reduce relapses and disability progression in relapsing forms of MS. In the clinical trials that led to approval, fingolimod reduced not only acute relapses and magnetic resonance imaging lesion activity but also disability progression and brain volume loss, suggesting preservation of tissue. Fingolimod's mechanism of action in MS is not known with certainty. Its active form, fingolimod-<em>phosphate</em> (fingolimod-P), is a <em>sphingosine</em> <em>1</em>-<em>phosphate</em> receptor (S<em>1</em>PR) modulator that inhibits egress of lymphocytes from lymph nodes and their recirculation, potentially reducing trafficking of pathogenic cells into the central nervous system (CNS). Fingolimod also readily penetrates the CNS, and fingolimod-P formed in situ may have direct effects on neural cells. Fingolimod potently inhibits the MS animal model, experimental autoimmune encephalomyelitis, but is ineffective in mice with selective deficiency of the S<em>1</em>P₁ S<em>1</em>PR subtype on astrocytes despite normal expression in the immune compartment. These findings suggest that S<em>1</em>PR modulation by fingolimod in both the immune system and CNS, producing a combination of beneficial anti-inflammatory and possibly neuroprotective/reparative effects, may contribute to its efficacy in MS. In clinical trials, fingolimod was generally safe and well tolerated. Its interaction with S<em>1</em>PRs in a variety of tissues largely accounts for the reported adverse effects, which were seen more frequently with doses 2.5 to <em>1</em>0x the approved 0.5 mg dose. Fingolimod's unique mechanism of action distinguishes it from all other currently approved MS therapies.

BACKGROUND

All treatments of acute myocardial infarction are aimed at rapid revascularization of the occluded vessel; however, no clinical strategies are currently available to protect the heart from ischemia/reperfusion injury after restitution of blood flow. We hypothesized that some of the cholesterol transport-independent biological properties of high-density lipoprotein (HDL) implied in atheroprotection may also be beneficial in settings of acute myocardial reperfusion injury.

RESULTS

In an in vivo mouse model of myocardial ischemia/reperfusion, we observed that HDL and its sphingolipid component, <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P), dramatically attenuated infarction size by approximately 20% and 40%, respectively. The underlying mechanism was an inhibition of inflammatory neutrophil recruitment and cardiomyocyte apoptosis in the infarcted area. In vitro, HDL and S<em>1</em>P potently suppressed leukocyte adhesion to activated endothelium under flow and protected rat neonatal cardiomyocytes against apoptosis. In vivo, HDL- and S<em>1</em>P-mediated cardioprotection was dependent on nitric oxide (NO) and the S<em>1</em>P3 lysophospholipid receptor, because it was abolished by pharmacological NO synthase inhibition and was completely absent in S<em>1</em>P3-deficient mice.

CONCLUSIONS

Our data demonstrate that HDL and its constituent, S<em>1</em>P, acutely protect the heart against ischemia/reperfusion injury in vivo via an S<em>1</em>P3-mediated and NO-dependent pathway. A rapid therapeutic elevation of S<em>1</em>P-containing HDL plasma levels may be beneficial in patients at high risk of acute myocardial ischemia.

Endothelial cell (EC) barrier dysfunction results in increased vascular permeability observed in inflammation, tumor angiogenesis, and atherosclerosis. The platelet-derived phospholipid <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) decreases EC permeability in vitro and in vivo and thus has obvious therapeutic potential. We examined S<em>1</em>P-mediated human pulmonary artery EC signaling and barrier regulation in caveolin-enriched microdomains (CEM). Immunoblotting from S<em>1</em>P-treated EC revealed S<em>1</em>P-mediated rapid recruitment (<em>1</em> microM, 5 min) to CEMs of the S<em>1</em>P receptors S<em>1</em>P<em>1</em> and S<em>1</em>P3, p<em>1</em><em>1</em>0 PI3 kinase alpha and beta catalytic subunits, the Rac<em>1</em> GEF, Tiam<em>1</em>, and alpha-actinin isoforms <em>1</em> and 4. Immunoprecipitated p<em>1</em><em>1</em>0 PI3 kinase catalytic subunits from S<em>1</em>P-treated EC exhibited PIP3 production in CEMs. Immunoprecipitation of S<em>1</em>P receptors from CEM fractions revealed complexes containing Tiam<em>1</em> and S<em>1</em>P<em>1</em>. PI3 kinase inhibition (LY294002) attenuated S<em>1</em>P-induced Tiam<em>1</em> association with S<em>1</em>P<em>1</em>, Tiam<em>1</em>/Rac<em>1</em> activation, alpha-actinin-<em>1</em>/4 recruitment, and EC barrier enhancement. Silencing of either S<em>1</em>P<em>1</em> or Tiam<em>1</em> expression resulted in the loss of S<em>1</em>P-mediated Rac<em>1</em> activation and alpha-actinin-<em>1</em>/4 recruitment to CEM. Finally, silencing S<em>1</em>P<em>1</em>, Tiam<em>1</em>, or both alpha-actinin isoforms <em>1</em>/4 inhibits S<em>1</em>P-induced cortical F-actin rearrangement and S<em>1</em>P-mediated barrier enhancement. Taken together, these results suggest that S<em>1</em>P-induced recruitment of S<em>1</em>P<em>1</em> to CEM fractions promotes PI3 kinase-mediated Tiam<em>1</em>/Rac<em>1</em> activation required for alpha-actinin-<em>1</em>/4-regulated cortical actin rearrangement and EC barrier enhancement.

Although much is known about the migration of T cells from blood to lymph nodes, less is known about the mechanisms regulating the migration of T cells from tissues into lymph nodes through afferent lymphatics. Here we investigated T cell egress from nonlymphoid tissues into afferent lymph in vivo and developed an experimental model to recapitulate this process in vitro. Agonism of <em>sphingosine</em> <em>1</em>-<em>phosphate</em> receptor <em>1</em> inhibited the entry of tissue T cells into afferent lymphatics in homeostatic and inflammatory conditions and caused the arrest, mediated at least partially by interactions of the integrin LFA-<em>1</em> with its ligand ICAM-<em>1</em> and of the integrin VLA-4 with its ligand VCAM-<em>1</em>, of polarized T cells at the basal surface of lymphatic but not blood vessel endothelium. Thus, the increased <em>sphingosine</em> <em>1</em>-<em>phosphate</em> present in inflamed peripheral tissues may induce T cell retention and suppress T cell egress.

The concentration of <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) in plasma or serum is much higher than the half-maximal concentration of the sphingolipid needed to stimulate its receptors. Nevertheless, the inositol <em>phosphate</em> response to plasma or serum mediated by Edg-3, one of the S<em>1</em>P receptors, which was overexpressed in Chinese hamster ovary cells, was much smaller than the response expected from the total amount of S<em>1</em>P in these samples. The inositol <em>phosphate</em> response to exogenous S<em>1</em>P was markedly attenuated in the presence of charcoal-treated low-S<em>1</em>P serum. The inhibitory effect was lost by boiling but not by dialysis of the serum. The inhibitory action of the serum was specific to S<em>1</em>P and was associated with the trapping of exogenous S<em>1</em>P; the inositol <em>phosphate</em> response to P(2)-purinergic agonists was somewhat enhanced by the charcoal-treated serum. Among the components of plasma or serum, lipoproteins such as low-density and high-density lipoproteins showed a stronger activity for trapping S<em>1</em>P than lipoprotein-deficient serum. Consistent with this observation, we detected a <em>1</em>5-<em>1</em>00-fold higher amount of S<em>1</em>P per unit amount of protein in lipoproteins than in the lipoprotein-deficient serum. Thus even though the protein content of the lipoprotein fraction contributes to only 4% of the total protein content of plasma or serum, more than 60% of S<em>1</em>P is distributed in this fraction. These results suggest that the tight binding of S<em>1</em>P to the components of serum or plasma, including lipoproteins, may interfere with the S<em>1</em>P binding to its receptors and thereby attenuate the lipid-receptor-mediated actions in the cells.

Sphingolipids, including the two central bioactive lipids ceramide and <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P), have opposing roles in regulating cancer cell death and survival, respectively, and there have been exciting developments in understanding how sphingolipid metabolism and signalling regulate these processes in response to anticancer therapy. Recent studies have provided mechanistic details of the roles of sphingolipids and their downstream targets in the regulation of tumour growth and response to chemotherapy, radiotherapy and/or immunotherapy using innovative molecular, genetic and pharmacological tools to target sphingolipid signalling nodes in cancer cells. For example, structure-function-based studies have provided innovative opportunities to develop mechanism-based anticancer therapeutic strategies to restore anti-proliferative ceramide signalling and/or inhibit pro-survival S<em>1</em>P-S<em>1</em>P receptor (S<em>1</em>PR) signalling. This Review summarizes how ceramide-induced cellular stress mediates cancer cell death through various mechanisms involving the induction of apoptosis, necroptosis and/or mitophagy. Moreover, the metabolism of ceramide for S<em>1</em>P biosynthesis, which is mediated by <em>sphingosine</em> kinase <em>1</em> and 2, and its role in influencing cancer cell growth, drug resistance and tumour metastasis through S<em>1</em>PR-dependent or receptor-independent signalling are highlighted. Finally, studies targeting enzymes involved in sphingolipid metabolism and/or signalling and their clinical implications for improving cancer therapeutics are also presented.

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> type <em>1</em> (S<em>1</em>P(<em>1</em>)) receptor agonists cause sequestration of lymphocytes in secondary lymphoid organs by a mechanism that is not well understood. One hypothesis proposes that agonists act as 'functional antagonists' by binding and internalizing S<em>1</em>P(<em>1</em>) receptors on lymphocytes; a second hypothesis proposes instead that S<em>1</em>P(<em>1</em>) agonists act on endothelial cells to prevent lymphocyte egress from lymph nodes. Here, two-photon imaging of living T cells in explanted lymph nodes after treatment with S<em>1</em>P(<em>1</em>) agonists or antagonists has provided insight into the mechanism by which S<em>1</em>P(<em>1</em>) agonists function. The selective S<em>1</em>P(<em>1</em>) agonist SEW287<em>1</em> caused reversible slowing and 'log-jamming' of T cells between filled medullary cords and empty sinuses, whereas motility was unaltered in diffuse cortex. Removal or antagonist competition of SEW287<em>1</em> permitted recovery of T cell motility in the parenchyma of the medulla and resumption of migration across the stromal endothelial barrier, leading to refilling of sinuses. Our results provide visualization of transendothelial migration of T cells into lymphatic sinuses and suggest that S<em>1</em>P(<em>1</em>) agonists act mainly on endothelial cell S<em>1</em>P(<em>1</em>) receptors to inhibit lymphocyte migration.

During a screen for ethylnitrosourea-induced mutations in mice affecting blood natural killer (NK) cells, we identified a strain, designated Duane, in which NK cells were reduced in blood and spleen but increased in lymph nodes (LNs) and bone marrow (BM). The accumulation of NK cells in LNs reflected a decreased ability to exit into lymph. This strain carries a point mutation within Tbx2<em>1</em> (T-bet), which generates a defective protein. Duane NK cells have a 30-fold deficiency in <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> receptor 5 (S<em>1</em>P5) transcript levels, and S<em>1</em>P5-deficient mice exhibit an egress defect similar to Duane. Chromatin immunoprecipitation confirms binding of T-bet to the S<em>1</em>pr5 locus. S<em>1</em>P-deficient mice exhibit a more severe NK cell egress block, and the FTY720-sensitive S<em>1</em>P<em>1</em> also plays a role in NK cell egress from LNs. S<em>1</em>P5 is not inhibited by CD69, a property that may facilitate trafficking of activated NK cells to effector sites. Finally, the accumulation of NK cells within BM of S<em>1</em>P-deficient mice was associated with reduced numbers in BM sinusoids, suggesting a role for S<em>1</em>P in BM egress. In summary, these findings identify S<em>1</em>P5 as a T-bet-induced gene that is required for NK cell egress from LNs and BM.

Targeting <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> receptors with the oral immunomodulator drug FTY720 (fingolimod) has demonstrated substantial efficacy in the treatment of multiple sclerosis. The drug is phosphorylated in vivo, and most of the clinical effects of FTY720-<em>phosphate</em> (FTY720P) are thought to be mediated via S<em>1</em>P<em>1</em> receptors on lymphocytes and endothelial cells, leading to sequestration of lymphocytes in secondary lymphoid organs. FTY720P was described to act as a "functional antagonist" by promoting efficient internalization of S<em>1</em>P<em>1</em> receptors. We demonstrate here that S<em>1</em>P<em>1</em> receptors activated by FTY720P retain signaling activity for hours in spite of a quantitative internalization. Structural analogs of FTY720P with shorter alkyl side chains retained potency and efficacy in a functional assay but failed to promote long-lasting receptor internalization and signaling. We show that persistent signaling translates into an increased chemokinetic migration of primary human umbilical vein endothelial cells, which suggests persistent agonism as a crucial parameter in the mechanism of action of FTY720.

The complement cascade (CC) becomes activated and its cleavage fragments play a crucial role in the mobilization of hematopoietic stem/progenitor cells (HSPCs). Here, we sought to determine which major chemoattractant present in peripheral blood (PB) is responsible for the egress of HSPCs from the bone marrow (BM). We noticed that normal and mobilized plasma strongly chemoattracts HSPCs in a stromal-derived factor-<em>1</em> (SDF-<em>1</em>)-independent manner because (i) plasma SDF-<em>1</em> level does not correlate with mobilization efficiency; (ii) the chemotactic plasma gradient is not affected in the presence of AMD3<em>1</em>00 and (iii) it is resistant to denaturation by heat. Surprisingly, the observed loss of plasma chemotactic activity after charcoal stripping suggested the involvement of bioactive lipids and we focused on <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P), a known chemoattracant of HSPCs. We found that S<em>1</em>P (i) creates in plasma a continuously present gradient for BM-residing HSPCs; (ii) is at physiologically relevant concentrations a chemoattractant several magnitudes stronger than SDF-<em>1</em> and (iii) its plasma level increases during mobilization due to CC activation and interaction of the membrane attack complex (MAC) with erythrocytes that are a major reservoir of S<em>1</em>P. We conclude and propose a new paradigm that S<em>1</em>P is a crucial chemoattractant for BM-residing HSPCs and that CC through MAC induces the release of S<em>1</em>P from erythrocytes for optimal egress/mobilization of HSPCs.

Lysophospholipids are cell membrane-derived lipids that include both glycerophospholipids such as lysophosphatidic acid (LPA) and sphingoid lipids such as <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P). These and related molecules can function in vertebrates as extracellular signals by binding and activating G protein-coupled receptors. There are currently five LPA receptors, along with a proposed sixth (LPA₁-LPA₆), and five S<em>1</em>P receptors (S<em>1</em>P₁-S<em>1</em>P₅). A remarkably diverse biology and pathophysiology has emerged since the last review, driven by cloned receptors and targeted gene deletion ("knockout") studies in mice, which implicate receptor-mediated lysophospholipid signaling in most organ systems and multiple disease processes. The entry of various lysophospholipid receptor modulatory compounds into humans through clinical trials is ongoing and may lead to new medicines that are based on this signaling system. This review incorporates IUPHAR Nomenclature Committee guidelines in updating the nomenclature for lysophospholipid receptors ( http://www.iuphar-db.org/DATABASE/FamilyMenuForward?familyId=36).

Severe influenza remains unusual in its virulence for humans. Complications or ultimately death arising from these infections are often associated with hyperinduction of proinflammatory cytokine production, which is also known as 'cytokine storm'. For this disease, it has been proposed that immunomodulatory therapy may improve the outcome, with or without the combination of antiviral agents. Here, we review the current literature on how various effectors of the immune system initiate the cytokine storm and exacerbate pathological damage in hosts. We also review some of the current immunomodulatory strategies for the treatment of cytokine storms in severe influenza, including corticosteroids, peroxisome proliferator-activated receptor agonists, <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> receptor <em>1</em> agonists, cyclooxygenase-2 inhibitors, antioxidants, anti-tumour-necrosis factor therapy, intravenous immunoglobulin therapy, statins, arbidol, herbs, and other potential therapeutic strategies.

Sphingolipid-metabolizing enzymes control the dynamic balance of the cellular levels of important bioactive lipids, including the apoptotic compound ceramide and the proliferative compound <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P). Many growth factors and inflammatory cytokines promote the cleavage of sphingomyelin and ceramide leading to rapid elevation of S<em>1</em>P levels through the action of <em>sphingosine</em> kinases (SK<em>1</em> and SK2). SK<em>1</em> and SK2 are overexpressed in a variety of human cancers, making these enzymes potential molecular targets for cancer therapy. We have identified an aryladamantane compound, termed ABC294640 [3-(4-chlorophenyl)-adamantane-<em>1</em>-carboxylic acid (pyridin-4-ylmethyl)amide], that selectively inhibits SK2 activity in vitro, acting as a competitive inhibitor with respect to <em>sphingosine</em> with a K(i) of 9.8 muM, and attenuates S<em>1</em>P formation in intact cells. In tissue culture, ABC294640 suppresses the proliferation of a broad panel of tumor cell lines, and inhibits tumor cell migration concomitant with loss of microfilaments. In vivo, ABC294640 has excellent oral bioavailability, and demonstrates a plasma clearance half-time of 4.5 h in mice. Acute and chronic toxicology studies indicate that ABC294640 induces a transient minor decrease in the hematocrit of rats and mice; however, this normalizes by 28 days of treatment. No other changes in hematology parameters, or gross or microscopic tissue pathology, result from treatment with ABC294640. Oral administration of ABC294640 to mice bearing mammary adenocarcinoma xenografts results in dose-dependent antitumor activity associated with depletion of S<em>1</em>P levels in the tumors and progressive tumor cell apoptosis. Therefore, this newly developed SK2 inhibitor provides an orally available drug candidate for the treatment of cancer and other diseases.

OBJECTIVE

S1P acts via the S1PR family of G protein-coupled receptors to regulate a variety of physiological responses. Whereas S1P1R activates G(i)- and PI-3-kinase-dependent signals to inhibit vascular permeability, the related S1P2R inhibits the PI-3-kinase pathway by coupling to the Rho-dependent activation of the PTEN phosphatase. However, cellular consequences of S1P2R signaling in the vascular cells are not well understood.

RESULTS

Selective signaling of the S1P2R was achieved by adenoviral-mediated expression in endothelial cells. Secondly, endogenously expressed S1P2R was blocked by the specific pharmacological antagonist JTE013. Activation of S1P2R in endothelial cells resulted in Rho-ROCK- and PTEN-dependent disruption of adherens junctions, stimulation of stress fibers, and increased paracellular permeability. JTE013 treatment of naive endothelial cells potentiated the S1P1R-dependent effects such as formation of cortical actin, blockade of stress fibers, stimulation of adherens junction assembly, and improved barrier integrity. This observation was extended to the in vivo model of vascular permeability in the rat lung: the S1P2R antagonist JTE013 significantly inhibited H2O2-induced permeability in the rat lung perfused model.

CONCLUSIONS

S1P2R activation in endothelial cells increases vascular permeability. The balance of S1P1 and S1P2 receptors in the endothelium may determine the regulation of vascular permeability by S1P.

Sphingolipids, which include ceramides and <em>sphingosine</em>, are essential structural components of cell membranes that also have messenger functions that regulate the proliferation, survival, and death of cells. Exogenous application of ceramide is cytotoxic, and exposure of cells to radiation or chemotherapy is associated with increased ceramide levels due to enhanced de novo synthesis, catabolism of sphingomyelin, or both. Ceramide can be metabolized to less toxic forms by glycosylation, acylation, or by catabolism to <em>sphingosine</em>, which is then phosphorylated to the anti-apoptotic <em>sphingosine</em> <em>1</em>-<em>phosphate</em>. Glucosylceramide synthase overexpression has been shown to enhance resistance to doxorubicin, suggesting that inhibition of ceramide metabolism or catabolism might enhance cancer chemotherapy. Several anticancer agents, including the cytotoxic retinoid, fenretinide (4-HPR), have been shown to act, at least in part, by increasing tumor cell ceramide via de novo synthesis. Combinations of 4-HPR and modulators of ceramide action and/or metabolism demonstrated increased anti-tumor activity in pre-clinical models with minimal toxicity for non-malignant cells, and were effective in a p53-independent manner against tumor cell lines resistant to standard cytotoxic agents. Phase I trials of ceramide metabolism inhibitors in combination with 4-HPR and with other cytotoxic agents are in development. Thus, pharmacological manipulation of sphingolipid metabolism to enhance tumor cell ceramide is being realized and offers a novel approach to cancer chemotherapy.

Lysophosphatidic acid (LPA), together with <em>sphingosine</em> <em>1</em>-<em>phosphate</em>, is a bioactive lipid mediator that acts on G-protein-coupled receptors to evoke multiple cellular responses, including Ca(2+) mobilization, modulation of adenylyl cyclase, and mitogen-activated protein (MAP) kinase activation. In this study, we isolated a human cDNA encoding a novel G-protein-coupled receptor, designated EDG7, and characterized it as a cellular receptor for LPA. The amino acid sequence of the EDG7 protein is 53.7 and 48.8% identical to those of the human functional LPA receptors EDG2 and EDG4, respectively, previously identified. LPA (oleoyl) but not other lysophospholipids induced an increase in the [Ca(2+)](i) of EDG7-overexpressing Sf9 cells. Other LPA receptors, EDG4 but not EDG2, transduced the Ca(2+) response by LPA when expressed in Sf9 cells. LPAs with an unsaturated fatty acid but not with a saturated fatty acid induced an increase in the [Ca(2+)](i) of EDG7-expressing Sf9 cells, whereas LPAs with both saturated and unsaturated fatty acids elicited a Ca(2+) response in Sf9 cells expressing EDG4. In EDG7- or EDG4-expressing Sf9 cells, LPA stimulated forskolin-induced increase in intracellular cAMP levels, which was not observed in EDG2-expressing cells. In PC<em>1</em>2 cells, EDG4 but not EDG2 or EDG7 mediated the activation of MAP kinase by LPA. Neither the EDG7- nor EDG4-transduced Ca(2+) response or cAMP accumulation was inhibited by pertussis toxin. In conclusion, the present study demonstrates that EDG7, a new member of the EDG family of G-protein-coupled receptors, is a specific LPA receptor that shows distinct properties from known cloned LPA receptors in ligand specificities, Ca(2+) response, modulation of adenylyl cyclase, and MAP kinase activation.

Beside their structural role for the cell membrane the family of sphingolipids act as effector molecules in signal transduction with links to various aspects of cancer initiation, progression and treatment response. The "sphingolipid rheostat" balances between apoptosis inducing ceramid and growth promoting <em>sphingosine</em>-<em>1</em>-<em>phosphate</em>. We analyzed gene expression of 43 proteins from this pathway in different subtypes of breast cancer using microarray data of <em>1</em>,269 tumor samples (test set n=<em>1</em>7<em>1</em>; validation sets n=<em>1</em>098) and observed significant differences for several genes. <em>Sphingosine</em> kinase <em>1</em> (SPHK<em>1</em>), ceramide galactosyltransferase (UGT8), and Ganglioside GD3-Synthase (ST8SIA<em>1</em>) displayed higher expression among ER negative tumors. In contrast, glucosylceramidsynthase (GCS), dihydroceramidsynthases (LASS4, LASS 6) and acid ceramidase (ASAH<em>1</em>) were higher expressed in ER positive samples. Survival analysis revealed a worse outcome of patients with high SPHK<em>1</em> expression. To avoid a confounding effect of the ER status we also restricted the analysis to 750 patients with ER positive tumors. Again a worse outcome was observed for tumors displaying high SPHK<em>1</em> expression. While 75.8+/-<em>1</em>.9% of the patients with tumors low in SPHK<em>1</em> expression were free of metastasis at 5 years, this was the case for only 64.9+/-3.6% of patients with tumors displaying high SPHK<em>1</em> expression (P=0.008). Immunohistochemistry identified the carcinoma cells as the major source of SPHK<em>1</em> expression in the tumor. The correlation of SPHK<em>1</em> with a poor prognosis as well as its high expression among ER negative tumors are in line with the antiapoptotic and proliferative properties of its product <em>sphingosine</em>-<em>1</em>-<em>phosphate</em>. Targeting of the sphingolipid rheostat may thus open new treatment options.

Bioactive sphingolipids, including ceramide, <em>sphingosine</em> and <em>sphingosine</em> <em>1</em>-<em>phosphate</em> are important regulators of many cellular processes, including cell survival, proliferation, differentiation, migration and immune responses. Although the levels of these bioactive sphingolipids are regulated by complex pathways subject to spatial and temporal control, the <em>sphingosine</em> kinases have emerged as critical central regulators of this system and, as a consequence, they have received substantial recent attention as potential therapeutic targets for cancer and a range of other conditions. Deciphering the molecular mechanisms that regulate both the activity and subcellular localization of these enzymes is vital for understanding the control of bioactive sphingolipid generation and action, and has clear implications for therapeutic strategies targeting these enzymes.

The success of immunization with irradiated sporozoites is unparalleled among the current vaccination approaches against malaria, but its mechanistic underpinnings have yet to be fully elucidated. Using a model mimicking natural infection by Plasmodium yoelii, we delineated early events governing the development of protective CD8(+) T-cell responses to the circumsporozoite protein. We demonstrate that dendritic cells in cutaneous lymph nodes prime the first cohort of CD8(+) T cells after an infectious mosquito bite. Ablation of these lymphoid sites greatly impairs subsequent development of protective immunity. Activated CD8(+) T cells then travel to systemic sites, including the liver, in a <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P)-dependent fashion. These effector cells, however, no longer require bone marrow-derived antigen-presenting cells for protection; instead, they recognize antigen on parenchymal cells-presumably parasitized hepatocytes. Therefore, we report an unexpected dichotomy in the tissue restriction of host responses during the development and execution of protective immunity to Plasmodium.

The lysophospholipids, <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P), lysophosphatidic acid (LPA), sphingosylphosphorylcholine (SPC) and lysophosphatidylcholine (LPC), activate diverse groups of G-protein-coupled receptors that are widely expressed and regulate decisive cellular functions. Receptors of the endothelial differentiation gene family are activated by S<em>1</em>P (S<em>1</em>P(<em>1</em>-5)) or LPA (LPA(<em>1</em>-3)); two more distantly related receptors are activated by LPA (LPA(4/5)); the GPR(3/6/<em>1</em>2) receptors have a high constitutive activity but are further activated by S<em>1</em>P and/or SPC; and receptors of the OGR<em>1</em> cluster (OGR<em>1</em>, GPR4, G2A, TDAG8) appear to be activated by SPC, LPC, psychosine and/or protons. G-protein-coupled lysophospholipid receptors regulate cellular Ca(2+) homoeostasis and the cytoskeleton, proliferation and survival, migration and adhesion. They have been implicated in development, regulation of the cardiovascular, immune and nervous systems, inflammation, arteriosclerosis and cancer. The availability of S<em>1</em>P and LPA at their G-protein-coupled receptors is regulated by enzymes that generate or metabolize these lysophospholipids, and localization plays an important role in this process. Besides FTY720, which is phosphorylated by <em>sphingosine</em> kinase-2 and then acts on four of the five S<em>1</em>P receptors of the endothelial differentiation gene family, other compounds have been identified that interact with more ore less selectivity with lysophospholipid receptors.