<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P), produced by two <em>sphingosine</em> kinase isoenzymes, denoted SphK<em>1</em> and SphK2, is the ligand for a family of five specific G protein-coupled receptors that regulate cytoskeletal rearrangements and cell motility. Whereas many growth factors stimulate SphK<em>1</em>, much less is known of the regulation of SphK2. Here we report that epidermal growth factor (EGF) stimulated SphK2 in HEK 293 cells. This is the first example of an agonist-dependent regulation of SphK2. Chemotaxis of HEK 293 cells toward EGF was inhibited by N,N-dimethyl<em>sphingosine</em>, a competitive inhibitor of both SphKs, implicating S<em>1</em>P generation in this process. Down-regulating expression of SphK<em>1</em> in HEK 293 cells with a specific siRNA abrogated migration toward EGF, whereas decreasing SphK2 expression had no effect. EGF contributes to the invasiveness of human breast cancer cells, and EGF receptor expression is associated with poor prognosis. EGF also stimulated SphK2 in MDA-MB-453 breast cancer cells. Surprisingly, however, down-regulation of SphK2 in these cells completely eliminated migration toward EGF without affecting fibronectin-induced haptotaxis. Our results suggest that SphK2 plays an important role in migration of MDA-MB-453 cells toward EGF.

<em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> is a potent lipid mediator formed by phosphorylation of <em>sphingosine</em>, a metabolite of sphingolipids, catalyzed by two <em>sphingosine</em> kinase (SphK) isoenzymes, SphK<em>1</em> and SphK2. Expression of SphK2, which is enriched in the nucleus of MCF7 human breast cancer cells, increased expression of the cyclin-dependent kinase inhibitor p2<em>1</em> but had no effect on p53 or its phosphorylation. The anticancer drug doxorubicin is known to increase p2<em>1</em> via p53-dependent and p53-independent mechanisms. Down-regulation of endogenous SphK2 with small interfering RNA targeted to unique mRNA sequences decreased basal and doxorubicin-induced expression of p2<em>1</em> without affecting increased expression of p53. Down-regulation of SphK2 also decreased G(2)-M arrest and markedly enhanced apoptosis induced by doxorubicin. Moreover, siSphK2 reduced doxorubicin-induced p2<em>1</em> expression in p53-inactivated MCF7 cells. Likewise, in human wild-type p53- and p2<em>1</em>-expressing HCT<em>1</em><em>1</em>6 colon carcinoma cells, as well as in p53-null counterparts, down-regulation of SphK2 markedly reduced p2<em>1</em> induction by doxorubicin. Knockdown of SphK2 sensitized HCT<em>1</em><em>1</em>6 cells to apoptosis induced by doxorubicin with concomitant cleavage of poly(ADP-ribose) polymerase. Collectively, our results show that endogenous SphK2 is important for p53-independent induction of p2<em>1</em> expression by doxorubicin and suggest that SphK2 may influence the balance between cytostasis and apoptosis of human cancer cells.

<em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P), a lipid mediator enriched in blood, controls the dynamic migration of osteoclast (OC) precursors (OPs) between the blood and bone, in part via the S<em>1</em>P receptor <em>1</em> (S<em>1</em>PR<em>1</em>) which directs positive chemotaxis toward S<em>1</em>P. We show that OPs also express S<em>1</em>PR2, an S<em>1</em>P receptor which mediates negative chemotaxis (or chemorepulsion). OP-positive chemotaxis is prominent in gradients with low maximal concentrations of S<em>1</em>P, whereas such behavior is minimal in fields with high maximal S<em>1</em>P concentrations. This reverse-directional behavior is caused by S<em>1</em>PR2-mediated chemorepulsion acting to override S<em>1</em>PR<em>1</em> upgradient motion. S<em>1</em>PR2-deficient mice exhibit moderate osteopetrosis as a result of a decrease in osteoclastic bone resorption, suggesting that S<em>1</em>PR2 contributes to OP localization on the bones mediated by chemorepulsion away from the blood where S<em>1</em>P levels are high. Inhibition of S<em>1</em>PR2 function by the antagonist JTE0<em>1</em>3 changed the migratory behavior of monocytoid cells, including OPs, and relieved osteoporosis in a mouse model by limiting OP localization and reducing the number of mature OCs attached to the bone surface. Thus, reciprocal regulation of S<em>1</em>P-dependent chemotaxis controls bone remodeling by finely regulating OP localization. This regulatory axis may be promising as a therapeutic target in diseases affecting OC-dependent bone remodeling.

Platelets are known to store a large amount of the bioactive lipid molecule <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) and to release it into the plasma in a stimuli-dependent manner. Erythrocytes can also release S<em>1</em>P, independently from any stimuli. We measured the S<em>1</em>P and <em>sphingosine</em> (Sph) levels in erythrocytes by HPLC and found that the contribution of erythrocyte S<em>1</em>P to whole blood S<em>1</em>P levels is actually higher than that of platelets. In vitro assays demonstrated that erythrocytes possess much weaker Sph kinase activity compared to platelets but lack the S<em>1</em>P-degrading activities of either S<em>1</em>P lyase or S<em>1</em>P phosphohydrolase. This combination may enable erythrocytes to maintain a high S<em>1</em>P content relative to Sph. The absence of both S<em>1</em>P-degrading enzymes has not been reported for other cell types. Thus, erythrocytes may be specialized cells for storing and supplying plasma S<em>1</em>P.

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P), a product of sphingomyelin metabolism, is enriched in the circulatory system whereas it is estimated to be much lower in interstitial fluids of tissues. This concentration gradient, termed the vascular S<em>1</em>P gradient appears to form as a result of substrate availability and the action of metabolic enzymes. S<em>1</em>P levels in blood and lymph are estimated to be in the muM range. In the immune system, the S<em>1</em>P gradient is needed as a spatial cue for lymphocyte and hematopoietic cell trafficking. During inflammatory reactions in which enhanced vascular permeability occurs, a burst of S<em>1</em>P becomes available to its receptors in the extravascular compartment, which likely contributes to the tissue reactions. Thus, the presence of the vascular S<em>1</em>P gradient is thought to contribute to physiological and pathological conditions. From an evolutionary perspective, S<em>1</em>P receptors may have co-evolved with the advent of a closed vascular system and the trafficking paradigms for hematopoietic cells to navigate in and out of the vascular system.

BACKGROUND

The role of <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) and its receptors in the pathogenesis of atherosclerosis has not been investigated.

OBJECTIVE

We hypothesized that the S<em>1</em>P receptor 3 (S<em>1</em>P(3)) plays a causal role in the pathogenesis of atherosclerosis.

RESULTS

We examined atherosclerotic lesion development in mice deficient for S<em>1</em>P(3) and apolipoprotein (Apo)E. Although S<em>1</em>P(3) deficiency did not affect lesion size after 25 or 45 weeks of normal chow diet, it resulted in a dramatic reduction of the monocyte/macrophage content in lesions of S<em>1</em>P(3)(-/-)/ApoE(-/-) double knockout mice. To search for putative defects in monocyte/macrophage recruitment, we examined macrophage-driven inflammation during thioglycollate-induced peritonitis. Elicited peritoneal macrophages were reduced in S<em>1</em>P(3)-deficient mice and expressed lower levels of tumor necrosis factor-α and monocyte chemoattractant protein-<em>1</em>. Bone marrow-derived S<em>1</em>P(3)-deficient macrophages produced less MCP-<em>1</em> in response to lipopolysaccharide stimulation. In vitro, S<em>1</em>P was chemotactic for wild-type but not S<em>1</em>P(3)-deficient peritoneal macrophages. In vivo, S<em>1</em>P concentration increased rapidly in the peritoneal cavity after initiation of peritonitis. Treatment with the S<em>1</em>P analog FTY720 attenuated macrophage recruitment to the peritoneum. Studies in bone marrow chimeras showed that S<em>1</em>P(3) in both hematopoietic and nonhematopoietic cells contributed to monocyte/macrophage accumulation in atherosclerotic lesions. Finally, S<em>1</em>P(3) deficiency increased the smooth muscle cell content of atherosclerotic lesions and enhanced neointima formation after carotid ligation arguing for an antiproliferative/antimigratory role of S<em>1</em>P(3) in the arterial injury response.

CONCLUSIONS

Our data suggest that S<em>1</em>P(3) mediates the chemotactic effect of S<em>1</em>P in macrophages in vitro and in vivo and plays a causal role in atherosclerosis by promoting inflammatory monocyte/macrophage recruitment and altering smooth muscle cell behavior.

<em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) represents a potent modulator of diverse cellular activities, including lymphocyte trafficking and maintenance of lymphocyte homeostasis. The five known receptors for S<em>1</em>P (S<em>1</em>P(<em>1</em>-5)) belong to the family of G protein-coupled receptors. Upon binding S<em>1</em>P, they act downstream via heterotrimeric G proteins on members of the small GTPase family (Cdc42/Rac/Rho), evoking a S<em>1</em>P receptor-dependent activation pattern of Cdc42, Rac, and Rho, respectively. This, in turn, triggers cytoskeletal rearrangements determining cellular morphology and movement. In this study we investigated the effects of S<em>1</em>P on murine dendritic cells (DC). Mature DC, but not immature in vitro differentiated DC, were found to migrate to S<em>1</em>P, a phenomenon that correlated to the up-regulation of S<em>1</em>P<em>1</em> and S<em>1</em>P3 in maturing DC. The same pattern of S<em>1</em>P receptor regulation could be observed in vivo on skin DC after their activation and migration into the lymph node. The migration-inducing effect of S<em>1</em>P could be severely hampered by application of the S<em>1</em>P analogon FTY720 in vitro and in vivo. A similar, yet more pronounced, block was observed upon preventing Cdc42/Rac and/or Rho activation by specific inhibitors. These results suggest that S<em>1</em>P-mediated signaling plays a pivotal role in the life cycle of DC.

Autotaxin (ATX) is an exoenzyme that potently induces tumor cell motility, and enhances experimental metastasis and angiogenesis. ATX was shown recently to be identical to serum lysophospholipase D activity, producing lysophosphatidic acid (LPA) from lyso-glycerophospholipids. LPA, itself a strong chemoattractant for tumor cells, may mediate the actions of ATX. We now extend the substrate specificity to sphingosylphosphorylcholine (SPC), which ATX hydrolyzes to <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P). Under migration assay conditions, this novel reaction for the production of S<em>1</em>P has a substrate (SPC) K(m) = 0.23 +/- 0.07 mM. In our responder cell lines (NIH3T3 clone7 and A2058), S<em>1</em>P exerts maximal biological effects at concentrations of <em>1</em>0-<em>1</em>00 nM and is mimicked in its biological effects by ATX plus SPC. These effects include inhibition of ATX- and LPA-stimulated motility, and elevation of activated Rho. In NIH3T3 clone7 cells stimulated with platelet-derived growth factor and treated with <em>1</em>0-25 nM S<em>1</em>P, motility is not inhibited and activation of Rho is unaffected, indicating that S<em>1</em>P possesses specificity in its effects. The exoenzyme ATX can potentially regulate diverse processes such as motility and angiogenesis via the S<em>1</em>P family of receptors. Because ATX hydrolyzes nucleotides, lyso-glycerophospholipids, and phosphosphingolipids into bioactive products, it possesses the ability, depending on the availability of substrates, to act as positive or negative regulator of receptor-mediated activity in the cellular microenvironment.

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (SPP), a platelet-derived bioactive lysophospholipid, is a regulator of angiogenesis. However, molecular mechanisms involved in SPP-induced angiogenic responses are not fully defined. Here we report the molecular mechanisms involved in SPP-induced human umbilical vein endothelial cell (HUVEC) adhesion and migration. SPP-induced HUVEC migration is potently inhibited by antisense phosphothioate oligonucleotides against EDG-<em>1</em> as well as EDG-3 receptors. In addition, C3 exotoxin blocked SPP-induced cell attachment, spreading and migration on fibronectin-, vitronectin- and Matrigel-coated surfaces, suggesting that endothelial differentiation gene receptor signaling via the Rho pathway is critical for SPP-induced cell migration. Indeed, SPP induced Rho activation in an adherence-independent manner, whereas Rac activation was dispensible for cell attachment and focal contact formation. Interestingly, both EDG-<em>1</em> and -3 receptors were required for Rho activation. Since integrins are critical for cell adhesion, migration, and angiogenesis, we examined the effects of blocking antibodies against alpha(v)beta(3), beta(<em>1</em>), or beta(3) integrins. SPP induced Rho-dependent integrin clustering into focal contact sites, which was essential for cell adhesion, spreading and migration. Blockage of alpha(v)beta(3)- or beta(<em>1</em>)-containing integrins inhibited SPP-induced HUVEC migration. Together our results suggest that endothelial differentiation gene receptor-mediated Rho signaling is required for the activation of integrin alpha(v)beta(3) as well as beta(<em>1</em>)-containing integrins, leading to the formation of initial focal contacts and endothelial cell migration.

Some of the simplest sphingolipids, namely <em>sphingosine</em>, ceramide, some closely related molecules (eicosa<em>sphingosine</em>, phyto<em>sphingosine</em>), and their phosphorylated compounds (<em>sphingosine</em>-<em>1</em>-<em>phosphate</em>, ceramide-<em>1</em>-<em>phosphate</em>), are potent metabolic regulators. Each of these lipids modifies in marked and specific ways the physical properties of the cell membranes, in what can be the basis for some of their physiological actions. This paper reviews the mechanisms by which these sphingolipid signals, <em>sphingosine</em> and ceramide in particular, are able to modify the properties of cell membranes.

<em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) lyase catalyzes the degradation of S<em>1</em>P, a potent signaling lysosphingolipid. Mice with an inactive S<em>1</em>P lyase gene are impaired in the capacity to degrade S<em>1</em>P, resulting in highly elevated S<em>1</em>P levels. These S<em>1</em>P lyase-deficient mice have low numbers of lymphocytes and high numbers of neutrophils in their blood. We found that the S<em>1</em>P lyase-deficient mice exhibited features of an inflammatory response including elevated levels of pro-inflammatory cytokines and an increased expression of genes in liver associated with an acute-phase response. However, the recruitment of their neutrophils into inflamed tissues was impaired and their neutrophils were defective in migration to chemotactic stimulus. The IL-23/IL-<em>1</em>7/granulocyte-colony stimulating factor (G-CSF) cytokine-controlled loop regulating neutrophil homeostasis, which is dependent on neutrophil trafficking to tissues, was disturbed in S<em>1</em>P lyase-deficient mice. Deletion of the S<em>1</em>P4 receptor partially decreased the neutrophilia and inflammation in S<em>1</em>P lyase-deficient mice, implicating S<em>1</em>P receptor signaling in the phenotype. Thus, a genetic block in S<em>1</em>P degradation elicits a pro-inflammatory response but impairs neutrophil migration from blood into tissues.

Agonist activation of a subset of G protein coupled receptors (GPCRs) stimulates cell proliferation, mimicking the better known effects of tyrosine kinase growth factors. Cell survival or apoptosis is also regulated via pathways initiated by stimulation of these same GPCRs. This review focuses on aspects of signaling by the lysophospholipid mediators, lysophosphatidic acid (LPA), and <em>sphingosine</em> <em>1</em> <em>phosphate</em> (S<em>1</em>P), which make these agonists uniquely capable of modulating cell growth and survival. The general features of GPCR coupling to specific G proteins, downstream effectors and signaling cascades are first reviewed. GPCR coupling to G(i) and Ras/MAPK or to G(q) and phospholipase generated second messengers are insufficient to regulate cell proliferation while G(<em>1</em>2/<em>1</em>3)/Rho engagement provides additional complementary signals required for cell proliferation. Survival is best predicted by coupling to G(i) pathways that regulate PI3K and Akt, but other signals generated through different G protein pathways are also implicated. The unique ability of LPA and S<em>1</em>P to concomitantly stimulate G(i), G(q), and G(<em>1</em>2/<em>1</em>3) pathways, given the proper complement of expressed LPA or S<em>1</em>P receptors, allows these receptors to support cell survival and proliferation. In pathophysiological situations, e.g., vascular disease, cancer, brain injury, and inflammation, components of the signaling cascade downstream of lysophospholipid receptors, in particular those involving Ras or Rho, may be altered. In addition, up or downregulation of LPA or S<em>1</em>P receptor subtypes, altering their ratio, and increased availability of the lysophospholipid ligands at sites of injury or inflammation, likely contribute to disease and may be important targets for therapeutic intervention.

Tumor necrosis factor (TNF)-alpha signals cell death and simultaneously induces the generation of ceramide, which is metabolized to <em>sphingosine</em> and <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) by ceramidase (CDase) and <em>sphingosine</em> kinase. Because the dynamic balance between the intracellular levels of ceramide and S<em>1</em>P (the "ceramide/S<em>1</em>P rheostat") may determine cell survival, we investigated these sphingolipid signaling pathways in TNF-alpha-induced apoptosis of primary hepatocytes. Endogenous C<em>1</em>6-ceramide was elevated during TNF-alpha-induced apoptosis in both rat and mouse primary hepatocytes. The putative acid sphingomyelinase (ASMase) inhibitor imipramine inhibited TNF-alpha-induced apoptosis and C<em>1</em>6-ceramide increase as did the knock out of ASMase. Overexpression of neutral CDase (NCDase) inhibited the TNF-alpha-induced increase of C<em>1</em>6-ceramide and apoptosis in rat primary hepatocytes. Moreover, NCDase inhibited liver injury and hepatocyte apoptosis in mice treated with D-galactosamine plus TNF-alpha. This protective effect was abrogated by the <em>sphingosine</em> kinase inhibitor N,N-demethyl<em>sphingosine</em>, suggesting that the survival effect of NCDase is due to not only C<em>1</em>6-ceramide reduction but also S<em>1</em>P formation. Administration of S<em>1</em>P or overexpression of NCDase activated the pro-survival kinase AKT, and overexpression of dominant negative AKT blocked the survival effect of NCDase. In conclusion, activation of ASMase and generation of C<em>1</em>6-ceramide contributed to TNF-alpha-induced hepatocyte apoptosis. NCDase prevented apoptosis both by reducing C<em>1</em>6-ceramide and by activation of AKT through S<em>1</em>P formation. Therefore, the cross-talk between sphingolipids and AKT pathway may determine hepatocyte apoptosis by TNF-alpha.

Sphingolipids have been suggested to act as second messengers for an array of cellular signaling activities in plant cells, including stress responses and programmed cell death (PCD). However, the mechanisms underpinning these processes are not well understood. Here, we report that an Arabidopsis mutant, fumonisin B<em>1</em> resistant <em>1</em><em>1</em>-<em>1</em> (fbr <em>1</em><em>1</em>-<em>1</em>), which fails to generate reactive oxygen intermediates (ROIs), is incapable of initiating PCD when the mutant is challenged by fumonisin B(<em>1</em>) (FB(<em>1</em>)), a specific inhibitor of ceramide synthase. Molecular analysis indicated that FBR<em>1</em><em>1</em> encodes a long-chain base <em>1</em> (LCB<em>1</em>) subunit of serine palmitoyltransferase (SPT), which catalyzes the first rate-limiting step of de novo sphingolipid synthesis. Mass spectrometric analysis of the sphingolipid concentrations revealed that whereas the fbr <em>1</em><em>1</em>-<em>1</em> mutation did not affect basal levels of sphingoid bases, the mutant showed attenuated formation of sphingoid bases in response to FB(<em>1</em>). By a direct feeding experiment, we show that the free sphingoid bases dihydro<em>sphingosine</em>, phyto<em>sphingosine</em> and <em>sphingosine</em> efficiently induce ROI generation followed by cell death. Conversely, ROI generation and cell death induced by dihydro<em>sphingosine</em> were specifically blocked by its phosphorylated form dihydro<em>sphingosine</em>-<em>1</em>-<em>phosphate</em> in a dose-dependent manner, suggesting that the maintenance of homeostasis between a free sphingoid base and its phosphorylated derivative is critical to determining the cell fate. Because alterations of the sphingolipid level occur prior to the ROI production, we propose that the free sphingoid bases are involved in the control of PCD in Arabidopsis, presumably through the regulation of the ROI level upon receiving different developmental or environmental cues.

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) is a platelet-derived sphingolipid that binds to S<em>1</em>P<em>1</em> (EDG-<em>1</em>) receptors and activates the endothelial isoform of NO synthase (eNOS). S<em>1</em>P and the polypeptide growth factor vascular endothelial growth factor (VEGF) act independently to modulate angiogenesis and activate eNOS. In these studies, we explored the cross-talk between S<em>1</em>P and VEGF signaling pathways. When cultured bovine aortic endothelial cells were treated with VEGF (<em>1</em>0 ng/ml), the expression of S<em>1</em>P<em>1</em> protein and mRNA increased by approximately 4-fold. S<em>1</em>P<em>1</em> up-regulation by VEGF was seen within 30 min of VEGF addition and reached a maximum after <em>1</em>.5 h. By contrast, expression of neither bradykinin B2 receptors nor the scaffolding protein caveolin-<em>1</em> was altered by VEGF treatment. The EC50 for VEGF-promoted induction of S<em>1</em>P<em>1</em> expression was approximately 2 ng/ml, within its physiological concentration range. S<em>1</em>P<em>1</em> induction by VEGF was attenuated by the tyrosine kinase inhibitor genistein and by the PKC inhibitor calphostin C. Preincubation of bovine aortic endothelial cells with VEGF (<em>1</em>0 ng/ml for 90 min) markedly enhanced subsequent S<em>1</em>P-dependent eNOS activation. VEGF pretreatment of cultured endothelial cells also markedly potentiated S<em>1</em>P-promoted eNOS phosphorylation at Ser-<em>1</em><em>1</em>79, as well as S<em>1</em>P-mediated activation of kinase Akt. In isolated rat arteries, VEGF pretreatment markedly potentiated S<em>1</em>P-mediated vasorelaxation and eNOS Ser-<em>1</em><em>1</em>79 phosphorylation. Taken together, these data indicate that VEGF specifically induces expression of S<em>1</em>P<em>1</em> receptors, associated with enhanced intracellular signaling responses to S<em>1</em>P and the potentiation of S<em>1</em>P-mediated vasorelaxation. We suggest that VEGF acts to sensitize the vascular endothelium to the effects of lipid mediators by promoting the induction of S<em>1</em>P<em>1</em> receptors, representing a potentially important point of cross-talk between receptor-regulated eNOS signaling pathways in the vasculature.

Engagement of the high affinity receptor for IgE (FcepsilonRI) on mast cells results in the production and secretion of <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P), a lipid metabolite present in the lungs of allergen-challenged asthmatics. Herein we report that two isoforms of <em>sphingosine</em> kinase (SphK<em>1</em> and SphK2) are expressed and activated upon FcepsilonRI engagement of bone marrow-derived mast cells (BMMC). Fyn kinase is required for FcepsilonRI coupling to SphK<em>1</em> and -2 and for subsequent S<em>1</em>P production. Normal activation of SphK<em>1</em> and -2 was restored by expression of wild type Fyn but only partly with a kinase-defective Fyn, indicating that induction of SphK<em>1</em> and SphK2 depended on both catalytic and noncatalytic properties of Fyn. Downstream of Fyn, the requirements for SphK<em>1</em> activation differed from that of SphK2. Whereas SphK<em>1</em> was considerably dependent on the adapter Grb2-associated binder 2 and phosphatidylinositol 3-OH kinase, SphK2 showed minimal dependence on these molecules. Fyn-deficient BMMC were defective in chemotaxis and, as previously reported, in degranulation. These functional responses were partly reconstituted by the addition of exogenous S<em>1</em>P to FcepsilonRI-stimulated cells. Taken together with our previous study, which demonstrated delayed SphK activation in Lyn-deficient BMMC, we propose a cooperative role between Fyn and Lyn kinases in the activation of SphKs, which contributes to mast cell responses.

A critical step in the mechanism of action of inflammatory cytokines is the stimulation of sphingolipid metabolism, including activation of <em>sphingosine</em> kinase (SK), which produces the mitogenic and proinflammatory lipid <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P). We have developed orally bioavailable compounds that effectively inhibit SK activity in vitro in intact cells and in cancer models in vivo. In this study, we assessed the effects of these SK inhibitors on cellular responses to tumor necrosis factor alpha (TNFalpha) and evaluated their efficacy in the dextran sulfate sodium (DSS) model of ulcerative colitis in mice. Using several cell systems, it was shown that the SK inhibitors block the ability of TNFalpha to activate nuclear factor kappa B (NFkappaB), induce expression of adhesion proteins, and promote production of prostaglandin E(2) (PGE(2)). In an acute model of DSS-induced ulcerative colitis, SK inhibitors were equivalent to or more effective than Dipentum in reducing disease progression, colon shortening, and neutrophil infiltration into the colon. The effects of SK inhibitors were associated with decreased colonic levels of inflammatory cytokines TNFalpha, interleukin (IL)-<em>1</em>beta, interferon gamma (IFN)-gamma, IL-6, and reduction of S<em>1</em>P levels. A similar reduction in disease progression was provided by SK inhibitors in a chronic model of ulcerative colitis in which the mice received 3-week-long cycles of DSS interspaced with week-long recovery periods. In the chronic model, immunohistochemistry for SK showed increased expression in DSS-treated mice (compared with water-treated controls) that was reduced by drug treatment. S<em>1</em>P levels were also elevated in the DSS group and significantly reduced by drug treatment. Together, these data indicate that SK is a critical component in inflammation and that inhibitors of this enzyme may be useful in treating inflammatory bowel diseases.

Bioactive phospholipids, which include <em>sphingosine</em>-<em>1</em>-<em>phosphate</em>, lysophosphatidic acid, ceramide and their derivatives regulate a wide variety of cellular functions in culture such as proliferation, apoptosis and differentiation. The availability of these lipids and their products is regulated by the lipid <em>phosphate</em> phosphatases (LPPs). Here we show that mouse embryos deficient for LPP3 fail to form a chorio-allantoic placenta and yolk sac vasculature. A subset of embryos also show a shortening of the anterior-posterior axis and frequent duplication of axial structures that are strikingly similar to the phenotypes associated with axin deficiency, a critical regulator of Wnt signaling. Loss of LPP3 results in a marked increase in beta-catenin-mediated TCF transcription, whereas elevated levels of LPP3 inhibit beta-catenin-mediated TCF transcription. LPP3 also inhibits axis duplication and leads to mild ventralization in Xenopus embryo development. Although LPP3 null fibroblasts show altered levels of bioactive phospholipids, consistent with loss of LPP3 phosphatase activity, mutant forms of LPP3, specifically lacking phosphatase activity, were able to inhibit beta-catenin-mediated TCF transcription and also suppress axis duplication, although not as effectively as intact LPP3. These results reveal that LPP3 is essential to formation of the chorio-allantoic placenta and extra-embryonic vasculature. LPP3 also mediates gastrulation and axis formation, probably by influencing the canonical Wnt signaling pathway. The exact biochemical roles of LPP3 phosphatase activity and its undefined effect on beta-catenin-mediated TCF transcription remain to be determined.

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) from platelets and macrophages stimulates migration and enhances survival of T cells. Mouse spleen CD4 and CD8 T cells are shown to express predominantly S<em>1</em>P<em>1</em> (Edg-<em>1</em>) and S<em>1</em>P4 (Edg-6) G-protein-coupled receptors with only minimal representation of S<em>1</em>P2, S<em>1</em>P3, and S<em>1</em>P5. At and below plasma concentrations of healthy mammals (<em>1</em> nM-<em>1</em> microM), S<em>1</em>P evokes trans-Matrigel chemotaxis of mouse CD4 and CD8 T cells and recruits T cells into subcutaneous air pouches. T cell receptor-mediated activation of CD4 T cells suppresses expression of S<em>1</em>P<em>1</em> and S<em>1</em>P4 receptors and eliminates their chemotactic responses to S<em>1</em>P. The immunoregulator FTY720, a structural homologue of S<em>1</em>P, lacks T cell chemotactic activity and competitively inhibits T cell chemotactic responses to S<em>1</em>P in vitro and in vivo. S<em>1</em>P may be a distinctive contributor to compartmental immunity by attracting naïve and memory T cells preferentially over activated effector T cells.

Phospholipids and sphingolipids play critical roles in signal transduction, intracellular membrane trafficking, and control of cell growth and survival. We discuss recent progress in the identification and characterization of a family of integral membrane proteins with central roles in bioactive lipid metabolism and signalling. These five groups of homologous proteins, which we collectively term LPTs (lipid phosphatases/phosphotransferases), are characterized by a core domain containing six transmembrane-spanning alpha-helices connected by extramembrane loops, two of which interact to form the catalytic site. LPT family members are localized to all major membrane compartments of the cell. The transmembrane topology of these proteins places their active site facing the lumen of endomembrane compartments or the extracellular face of the plasma membrane. Sequence conservation between the active site of the LPPs (lipid <em>phosphate</em> phosphatases), SPPs (<em>sphingosine</em> <em>phosphate</em> phosphatases) and the recently identified SMSs (sphingomyelin synthases) with vanadium-dependent fungal oxidases provides a framework for understanding their common catalytic mechanism. LPPs hydrolyse LPA (lysophosphatidic acid), S<em>1</em>P (<em>sphingosine</em> <em>1</em>-<em>phosphate</em>) and structurally-related substrates. Although LPPs can dephosphorylate intracellularly generated substrates to control intracellular lipid metabolism and signalling, their best understood function is to regulate cell surface receptor-mediated signalling by LPA and S<em>1</em>P by inactivating these lipids at the plasma membrane or in the extracellular space. SPPs are intracellularly localized S<em>1</em>P-selective phosphatases, with key roles in the pathways of sphingolipid metabolism linked to control of cell growth and survival. The SMS enzymes catalyse the interconversion of phosphatidylcholine and ceramide with sphingomyelin and diacylglycerol, suggesting a pivotal role in both housekeeping lipid synthesis and regulation of bioactive lipid mediators. The remaining members of the LPT family, the LPR/PRGs (lipid phosphatase-related proteins/plasticity-related genes) and CSS2s (type 2 candidate sphingomyelin synthases), are presently much less well studied. These two groups include proteins that lack critical amino acids within the catalytic site, and could therefore not use the conserved LPT reaction mechanism to catalyse lipid phosphatase or phosphotransferase reactions. In this review, we discuss recent ideas about their possible biological activities and functions, which appear to involve regulation of cellular morphology and, possibly, lipid metabolism and signalling in the nuclear envelope.

No treatment has consistently shown efficacy in slowing disability progression in patients with secondary progressive multiple sclerosis (SPMS). We assessed the effect of siponimod, a selective <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) receptor<em>1</em>,5 modulator, on disability progression in patients with SPMS.

This event-driven and exposure-driven, double-blind, phase 3 trial was done at 292 hospital clinics and specialised multiple sclerosis centres in 3<em>1</em> countries. Using interactive response technology to assign numbers linked to treatment arms, patients (age <em>1</em>8-60 years) with SPMS and an Expanded Disability Status Scale score of 3·0-6·5 were randomly assigned (2:<em>1</em>) to once daily oral siponimod 2 mg or placebo for up to 3 years or until the occurrence of a prespecified number of confirmed disability progression (CDP) events. The primary endpoint was time to 3-month CDP. Efficacy was assessed for the full analysis set (ie, all randomly assigned and treated patients); safety was assessed for the safety set. This trial is registered with ClinicalTrials.gov, number NCT0<em>1</em>665<em>1</em>44.

<em>1</em>65<em>1</em> patients were randomly assigned between Feb 5, 20<em>1</em>3, and June 2, 20<em>1</em>5 (<em>1</em><em>1</em>05 to the siponimod group, and 546 to the placebo group). One patient did not sign the consent form, and five patients did not receive study drug, all of whom were in the siponimod group. <em>1</em>645 patients were included in the analyses (<em>1</em>099 in the siponimod group and 546 in the placebo). At baseline, the mean time since first multiple sclerosis symptoms was <em>1</em>6·8 years (SD 8·3), and the mean time since conversion to SPMS was 3·8 years (SD 3·5); <em>1</em>055 (64%) patients had not relapsed in the previous 2 years, and 9<em>1</em>8 (56%) of <em>1</em>65<em>1</em> needed walking assistance. 903 (82%) patients receiving siponimod and 424 (78%) patients receiving placebo completed the study. 288 (26%) of <em>1</em>096 patients receiving siponimod and <em>1</em>73 (32%) of 545 patients receiving placebo had 3-month CDP (hazard ratio 0·79, 95% CI 0·65-0·95; relative risk reduction 2<em>1</em>%; p=0·0<em>1</em>3). Adverse events occurred in 975 (89%) of <em>1</em>099 patients receiving siponimod versus 445 (82%) of 546 patients receiving placebo; serious adverse events were reported for <em>1</em>97 (<em>1</em>8%) patients in the siponimod group versus 83 (<em>1</em>5%) patients in the placebo group. Lymphopenia, increased liver transaminase concentration, bradycardia and bradyarrhythmia at treatment initiation, macular oedema, hypertension, varicella zoster reactivation, and convulsions occurred more frequently with siponimod than with placebo. Initial dose titration mitigated cardiac first-dose effects. Frequencies of infections, malignancies, and fatalities did not differ between groups.

Siponimod reduced the risk of disability progression with a safety profile similar to that of other S<em>1</em>P modulators and is likely to be a useful treatment for SPMS.

Novartis Pharma AG.

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) is accumulated in lipoproteins, especially high-density lipoprotein (HDL), in plasma. However, it remains uncharacterized how extracellular S<em>1</em>P is produced in the CNS. The treatment of rat astrocytes with retinoic acid and dibutyryl cAMP, which induce apolipoprotein E (apoE) synthesis and HDL-like lipoprotein formation, stimulated extracellular S<em>1</em>P accumulation in the presence of its precursor <em>sphingosine</em>. The released S<em>1</em>P was present together with apoE particles in the HDL fraction. S<em>1</em>P release from astrocytes was inhibited by the treatment of the cells with glybenclamide or small interfering RNAs specific to ATP-binding cassette transporter A<em>1</em> (ABCA<em>1</em>). Astrocytes from Abca<em>1</em>-/- mice also showed impairment of retinoic acid/dibutyryl cAMP-induced S<em>1</em>P release in association with the blockage of HDL-like lipoprotein formation. However, the formation of either apoE or lipoprotein itself was not sufficient, and additional up-regulation of ABCA<em>1</em> was requisite to stimulate S<em>1</em>P release. We conclude that the S<em>1</em>P release from astrocytes is coupled with lipoprotein formation through ABCA<em>1</em>.

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (Sph-<em>1</em>-P), a bioactive lysophospholipid capable of inducing a wide spectrum of biological responses, acts as an intercellular mediator, through interaction with the endothelial differentiation gene (EDG)/S<em>1</em>P family of G protein-coupled receptors. In this study, the effects of JTE-0<em>1</em>3, a specific antagonist of the migration-inhibitory receptor EDG-5, on Sph-<em>1</em>-P-elicited responses were examined in human umbilical vein endothelial cells (HUVECs) and vascular smooth muscle cells (SMCs), which expressed EDG-5 protein weakly and abundantly, respectively. This pyrazolopyridine compound reversed the inhibitory effect of Sph-<em>1</em>-P on SMC migration and further enhanced Sph-<em>1</em>-P-stimulated HUVEC migration. In contrast, its effect on Sph-<em>1</em>-P-induced intracellular Ca(2+) mobilization was marginal. Our results indicate that specific regulation of Sph-<em>1</em>-P-modulated migration responses in vascular cells can be achieved by EDG-5 antagonists and that manipulation of Sph-<em>1</em>-P biological activities by each EDG antagonist may lead to a therapeutical application to control vascular diseases.

OBJECTIVE

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) is involved in various pathologic conditions and has been implicated as an important mediator of angiogenesis, inflammation, cancer, and autoimmunity. This study was undertaken to examine the role of S<em>1</em>P/S<em>1</em>P<em>1</em> signaling in the pathogenesis of rheumatoid arthritis (RA).

METHODS

We examined S<em>1</em>P<em>1</em> messenger RNA (mRNA) and protein levels in RA synoviocytes and MH7A cells by reverse transcriptase-polymerase chain reaction and Western blotting. We also performed S<em>1</em>P<em>1</em> immunohistochemistry analysis in synovial tissue from 28 RA patients and <em>1</em>8 osteoarthritis (OA) patients. We investigated the effects of S<em>1</em>P on proliferation by WST-<em>1</em> assay, and its effects on tumor necrosis factor alpha (TNFalpha)- or interleukin-<em>1</em>beta (IL-<em>1</em>beta)-induced cyclooxygenase 2 (COX-2) expression and prostaglandin E2 (PGE2) production in RA synoviocytes and MH7A cells by Western blotting and enzyme-linked immunosorbent assay, respectively. Finally, we examined whether these effects of S<em>1</em>P were sensitive to pertussis toxin (PTX), an inhibitor of the Gi/Go proteins.

RESULTS

S<em>1</em>P<em>1</em> mRNA and protein were detected in RA synoviocytes and MH7A cells. S<em>1</em>P<em>1</em> was more strongly expressed in synovial lining cells, vascular endothelial cells, and inflammatory mononuclear cells of RA synovium compared with OA synovium. S<em>1</em>P increased the proliferation of RA synoviocytes and MH7A cells. S<em>1</em>P alone significantly enhanced COX-2 expression and PGE2 production. Moreover, S<em>1</em>P enhanced expression of COX-2 and production of PGE2 induced by stimulation with TNFalpha or IL-<em>1</em>beta in RA synoviocytes and MH7A cells. These effects of S<em>1</em>P were inhibited by pretreatment with PTX.

CONCLUSIONS

These findings suggest that S<em>1</em>P signaling via S<em>1</em>P receptors plays an important role in cell proliferation and inflammatory cytokine-induced COX-2 expression and PGE2 production by RA synoviocytes. Thus, regulation of S<em>1</em>P/S<em>1</em>P<em>1</em> signaling may represent a novel therapeutic target in RA.