Sphingosine 1-Phosphate Produced by Sphingosine Kinase 2 Intrinsically Controls Platelet Aggregation In Vitro and In Vivo.

RATIONALE: Platelets are known to play a crucial role in hemostasis. Sphingosine kinases (Sphk) 1 and 2 catalyze the conversion of sphingosine to the bioactive metabolite sphingosine 1-phosphate (S1P). Although platelets are able to secrete S1P on activation, little is known about a potential intrinsic effect of S1P on platelet function. OBJECTIVE: To investigate the role of Sphk1- and Sphk2-derived S1P in the regulation of platelet function. METHODS AND RESULTS: We found a 100-fold reduction in intracellular S1P levels in platelets derived from Sphk2(-/-) mutants compared with Sphk1(-/-) or wild-type mice, as analyzed by mass spectrometry. Sphk2(-/-) platelets also failed to secrete S1P on stimulation. Blood from Sphk2-deficient mice showed decreased aggregation after protease-activated receptor 4-peptide and adenosine diphosphate stimulation in vitro, as assessed by whole blood impedance aggregometry. We revealed that S1P controls platelet aggregation via the sphingosine 1-phosphate receptor 1 through modulation of protease-activated receptor 4-peptide and adenosine diphosphate-induced platelet activation. Finally, we show by intravital microscopy that defective platelet aggregation in Sphk2-deficient mice translates into reduced arterial thrombus stability in vivo. CONCLUSIONS: We demonstrate that Sphk2 is the major Sphk isoform responsible for the generation of S1P in platelets and plays a pivotal intrinsic role in the control of platelet activation. Correspondingly, Sphk2-deficient mice are protected from arterial thrombosis after vascular injury, but have normal bleeding times. Targeting this pathway could therefore present a new therapeutic strategy to prevent thrombosis.

Ovalbumin-induced plasma interleukin-4 levels are reduced in ceramide kinase-deficient DO11.10 RAG1-/- mice.

Ceramide kinase (CERK) produces the bioactive lipid ceramide-1-phosphate (C1P) and is a key regulator of ceramide and dihydroceramide levels. It is likely that CERK and C1P play a role in inflammatory processes but the cells involved and the mechanisms used remain to be clarified. In particular, the impact of CERK on T-cell biology has not been studied so far. Here, we used Cerk-/- mice backcrossed with DO11.10/RAG1-/- mice to probe the effect of CERK ablation on T-cell activation. Levels of interleukin (IL)-2, IL-4, IL-5, IL-13, of tumor necrosis factor (TNF)-alpha, and of interferon (INF)-gamma were recorded following ovalbumin challenge in vivo and using ovalbumin-treated splenocytes ex- vivo. Absence of CERK led to a significant decrease in the production of IL-4, thus suggesting that CERK may polarize T cells towards the TH2 cell subtype. However, the importance of CERK to TH2 cell biology will have to be investigated further because in a model of asthma, which is TH2-cell driven, Cerk-/- mice responded like wild-type animals.

Sphingolipid metabolizing enzymes as novel therapeutic targets.

Pharmacological interference with sphingolipid metabolizing enzymes promises to provide novel ways to modulate cellular pathways relevant in multiple diseases. In this review, we focus on two sphingolipid signaling molecules, sphingosine-1-phosphate (S1P) and ceramide, as they are involved in cell fate decisions (survival vs. apoptosis) and in a wide range of pathophysiological processes. For S1P, we will discuss sphingosine kinases and S1P lyase as the enzymes which are crucial for its production and degradation, respectively, emphasizing the potential therapeutic usefulness of inhibitors of these enzymes. For ceramide, we will concentrate on acid sphingomyelinase, and critically review the substantial literature which implicates this enzyme as a worthwhile target for pharmacological inhibitors. It will become clear that the task to validate these enzymes as drug targets is not finished and many questions regarding the therapeutic usefulness of their inhibitors remain unanswered. Still this approach holds promise for a number of totally new therapies, and, on the way, detailed insight into sphingolipid signaling pathways can be gained.

Targeting ceramide metabolism with a potent and specific ceramide kinase inhibitor.

Ceramide kinase (CerK) produces the bioactive lipid ceramide-1-phosphate (C1P) and appears as a key enzyme for controlling ceramide levels. In this study, we discovered and characterized adamantane-1-carboxylic acid (2-benzoylamino-benzothiazol-6-yl)amide (NVP-231), a potent, specific, and reversible CerK inhibitor that competitively inhibits binding of ceramide to CerK. NVP-231 is active in the low nanomolar range on purified as well as cellular CerK and abrogates phosphorylation of ceramide, resulting in decreased endogenous C1P levels. When combined with another ceramide metabolizing inhibitor, such as tamoxifen, NVP-231 synergistically increased ceramide levels and reduced cell growth. Therefore, NVP-231 represents a novel and promising compound for controlling ceramide metabolism that may provide insight into CerK physiological function.

Sphingosine 1-phosphate phosphatase 2 is induced during inflammatory responses.

Sphingosine 1-phosphate (S1P) levels in cells and, consequently, its bioactivity as a signalling molecule are controlled by the action of enzymes responsible for its synthesis and degradation. In the present report, we examined alterations in expression patterns of enzymes involved in S1P-metabolism (sphingosine kinases including their splice variants, sphingosine 1-phosphate phosphatases, and sphingosine 1-phosphate lyase) under certain inflammatory conditions. We found that sphingosine kinase type 1 (SPHK1) mRNA could be triggered in a cell type-specific manner; individual SPHK1 splice variants were induced with similar kinetics. Remarkably, expression and activity of S1P phosphatase 2 (SPP2) was found to be highly upregulated by inflammatory stimuli in a variety of cells (e.g., neutrophils, endothelial cells). Bandshift analysis using oligonucleotides spanning predicted NFkappaB sites within the SPP2 promoter and silencing of NFkappaB/RelA via RelA-directed siRNA demonstrated that SPP2 is an NFkappaB-dependent gene. Silencing of SPP2 expression in endothelial cells, in turn, led to a marked reduction of TNF-alpha-induced IL-1beta mRNA and protein and to a partial reduction of induced IL-8, suggesting a pro-inflammatory role of SPP2. Notably, up-regulation of SPP2 was detected in samples of lesional skin of patients with psoriasis, an inflammatory skin disease. This study provides detailed insights into the regulation of SPP2 gene expression and suggests that SPP2 might be a novel player in pro-inflammatory signalling.

FTY720-phosphate is dephosphorylated by lipid phosphate phosphatase 3.

FTY720 is a novel immunomodulatory drug efficacious in the treatment of multiple sclerosis. The drug is converted in vivo to the monophosphate, FTY720-P, by sphingosine kinase 2. This conversion is incomplete, suggesting opposing actions of kinase and phosphatase activities. To address which of the known lipid phosphatases might dephosphorylate FTY720-P, we overexpressed the broad specificity lipid phosphatases LPP1-3, and the specific S1P phosphatases (SPP1 and 2) in HEK293 cells, and performed in vitro assays using lysates of transfected cells. Among LPPs, only LPP3 was able to dephosphorylate FTY720-P; among SPPs, only SPP1 showed activity against FTY720-P. On intact cells, LPP3 acted as an ecto-phosphatase or FTY720-P, thus representing the major phosphatase involved in the equilibrium between FTY720 and FTY720-P observed in vivo.

Normal neutrophil functions in sphingosine kinase type 1 and 2 knockout mice.

Sphingosine kinase (SPHK) has been implicated as an important element in neutrophil responses to diverse stimulatory agents. To get more insight into the role of the type 1 and 2 isoforms of SPHK in neutrophil functions, we made use of the respective SPHK knockout mice. Neutrophils isolated from the bone marrow of these mice showed normal increase of intracellular Ca(2+) when stimulated in vitro by fMLP, platelet-activating factor, the anaphylatoxin C5a, or ATP, and normal migration towards fMLP and C5a. Also, recruitment of neutrophils into the peritoneum towards the chemokines KC and MIP-2 or to LPS, and into the peripheral blood after fMLP injection was similar in SPHK knockout strains and wild-type animals. An in vivo model of bacterial lung infection revealed an accelerated progression of disease in SPHK2 (but not SPHK1) knockout mice as compared to wild-type controls. However, effector functions of SPHK-deficient neutrophils, such as superoxide production, beta-glucuronidase release and their capacity to kill bacteria were unchanged as compared to wild-type cells. To conclude, the data derived from SPHK knockout mice do not support the hypothesis that any of the two lipid kinases plays a crucial role in signalling downstream of various neutrophil stimuli; SPHKs appear not to be essential for neutrophil recruitment and effector functions.

Early activation of sphingosine kinase in mast cells and recruitment to FcepsilonRI are mediated by its interaction with Lyn kinase.

Sphingosine kinase has been recognized as an essential signaling molecule that mediates the intracellular conversion of sphingosine to sphingosine-1-phosphate. In mast cells, induction of sphingosine kinase and generation of sphingosine-1-phosphate have been linked to the initial rise in Ca(2+), released from internal stores, and to degranulation. These events either precede or are concomitant with the activation of phospholipase C-gamma and the generation of inositol trisphosphate. Here we show that sphingosine kinase type 1 (SPHK1) interacts directly with the tyrosine kinase Lyn and that this interaction leads to the recruitment of this lipid kinase to the high-affinity receptor for immunoglobulin E (FcepsilonRI). The interaction of SPHK1 with Lyn caused enhanced lipid and tyrosine kinase activity. After FcepsilonRI triggering, enhanced sphingosine kinase activity was associated with FcepsilonRI in sphingolipid-enriched rafts of mast cells. Bone marrow-derived mast cells from Lyn(-/)(-) mice, compared to syngeneic wild-type cells, were defective in the initial induction of SPHK1 activity, and the defect was overcome by retroviral Lyn expression. These findings position the activation of SPHK1 as an FcepsilonRI proximal event.

Pulmonary and vascular pharmacology of sphingosine 1-phosphate.

Dysregulation of vasomotor tone, endothelial barrier function and immune cell trafficking are central to the pathology of many lung diseases, including acute lung injury, adult respiratory distress syndrome, chronic obstructive pulmonary disease and asthma. There is increasing evidence that the serum sphingolipid sphingosine 1-phosphate and its G-protein-coupled receptors are pivotal not only in the regulation of lymphocyte migration, but also in the maintenance of vascular homeostasis and the preservation of permeability barriers that separate discrete compartments in the lung.

A critical beta6-beta7 loop in the pleckstrin homology domain of ceramide kinase.

CerK (ceramide kinase) produces ceramide 1-phosphate, a sphingophospholipid with recognized signalling properties. It localizes to the Golgi complex and fractionates essentially between detergent-soluble and -insoluble fractions; however, the determinants are unknown. Here, we made a detailed mutagenesis study of the N-terminal PH domain (pleckstrin homology domain) of CerK, based on modelling, and identified key positively charged amino acid residues within an unusual motif in the loop interconnecting beta-strands 6 and 7. These residues are critical for CerK membrane association and polyphosphoinositide binding and activity. Their mutagenesis results in increased thermolability, sensitivity to proteolysis, reduced apparent molecular mass as well as propensity of the recombinant mutant protein to aggregate, indicating that this loop impacts the overall conformation of the CerK protein. This is in contrast with most PH domains whose function strongly relies on charges located in the beta1-beta2 loop.

Characterization of a ceramide kinase-like protein.

Ceramide is a key player governing cell fate, and its conversion to ceramide-1-phosphate by ceramide kinase (CERK) is emerging as an important mean to regulate apoptosis and inflammatory processes. We identified a new ceramide kinase homolog, designated CERK-like protein (CERKL) and we compared it to the known CERK. Real time-PCR analysis of human tissues revealed a restricted pattern of expression for CERKL mRNA. Surprisingly, various ceramides, known substrates for CERK, were not phosphorylated by CERKL in vitro. Upon 32P(i)-pulse labeling of COS-1 cells transiently expressing CERKL, or incubation with NBD-C6-ceramide, ceramide-1-phosphate was not detected. After recombinant expression in COS-1 cells, CERKL was partially recovered in the soluble fraction, as a phosphorylated protein. Live cell imaging indicated localization of GFP-tagged CERKL to many cell compartments, including specific association with nucleoli. Two splice variants of CERKL did not localize to nucleoli nor did a CERKL variant with a point mutation in the putative ATP binding site. We also studied a naturally occurring CERKL mutant (R257X), recently linked to the pathology of retinitis pigmentosa. It accumulated in the nucleus but was not associated with nucleoli. Treatment with the calcium ionophore A23187 led to clearing of CERKL from nucleoli, but had no effect on the R257X CERKL mutant. Collectively, although kinase activity of CERKL remains to be proven, these findings suggest a functional link between CERKL and its nucleolar localization. Furthermore, we propose that the cause for retinitis pigmentosa in patients bearing the CERKL R257X mutation might be the accumulation of a truncated CERKL protein in the nucleus.

Basal and induced sphingosine kinase 1 activity in A549 carcinoma cells: function in cell survival and IL-1beta and TNF-alpha induced production of inflammatory mediators.

Sphingosine-1-phosphate, a lipid mediator produced by sphingosine kinases, regulates diverse cellular processes, ranging from cell growth and survival to effector functions, such as proinflammatory mediator synthesis. Using human A549 epithelial lung carcinoma cells as a model system, we observed transient upregulation of sphingosine kinase type 1 (SPHK1) enzyme activity upon stimulation with both TNF-alpha or IL-1beta. This transient activation of SPHK1 was found to be required for cytokine-induced COX-2 transcription and PGE2 production, since not only specific siRNA (abolishing both basal and induced SPHK1 enzyme activity), but also a dominant-negative SPHK1 mutant (suppressing induced SPHK1 activity only) both reduced COX-2 and PGE2. Furthermore, TNF-alpha- or IL-1beta-induced transcription of selected cytokines, chemokines, and adhesion molecules (IL-6, RANTES, MCP-1, and VCAM-1) was found to require SPHK1 activation. Suppression of SPHK1 activation led to reduction of cytokine-induced IkappaBalpha phosphorylation and consequently diminished NFkappaB activity due to reduced nuclear translocation of RelA (p65), explaining the dependence of inflammatory mediator production on SPHK1 activation. Inhibition of basal SPHK1 activity by N,N-dimethylsphingosine or by downregulation of its expression using siRNA induced spontaneous apoptosis in A549 cells, an effect that can be explained through interference with constitutive NFkappaB activity in this cell type. In contrast, expression of the dominant-negative mutant did not induce apoptosis. Taken together, these findings demonstrate a role of SPHK1 activation in proinflammatory signalling and of SPHK1 basal activity in survival of A549 lung carcinoma cells.

The role of sphingosine and ceramide kinases in inflammatory responses.

The 1-phosphates of sphingosine and ceramide (S1P and C1P) have emerged as key representatives of a new group of lipid signalling molecules. S1P is known to act both as an extracellular mediator and as an intracellular 'second messenger,' while C1P currently is only known for its intracellular actions. Therefore, sphingosine and ceramide kinases, the enzymes involved in the generation of these lipid mediators, are now in the spotlight. This review summarizes current information on structure, localization, substrate specificity, activation, and binding partners of these kinases, and then focuses on discoveries in relation to immune cell regulation and inflammation, addressing in particular mast cell activation and degranulation, IL-12 signalling, prostaglandin biosynthesis, monocyte activation, and neutrophil priming.

Suppression of early T-cell-receptor-triggered cellular activation by the Janus kinase 3 inhibitor WHI-P-154.

BACKGROUND: Therapeutic targeting of Janus kinase 3 (JAK3) has received particular attention, because it is associated with the common gamma signaling of cytokine receptors and thus vitally influences T-cell growth and survival. Recent evidence, however, indicates a critical role for JAK3 in signaling linked to the T-cell antigen receptor. METHODS: In this study we investigated whether targeting JAK3 with a rationally designed inhibitor affects early T-cell activation events. T cells were stimulated by CD3 and CD28 cross-linking, and interleukin (IL)-2 production, activation marker expression, increase of free intracellular Ca2+ concentration, activation of the extracellular-related kinase, and nuclear translocation of transcription factors were evaluated. RESULTS: We found that JAK3 inhibitor treatment dramatically impaired T-cell-receptor (TCR)-induced IL-2 production, surface activation marker expression (CD69, CD154), and homotypic T-cell aggregation. Accordingly, mRNA production of IL-2, interferon-gamma, and IL-10 was profoundly inhibited. Molecular analysis revealed that TCR-triggered phosphorylation of phospholipase C-gamma1, increase in cytoplasmic Ca2+ concentration, and activation of extracellular-related kinase were markedly reduced by the JAK3 inhibitor, resulting in substantially decreased DNA binding of nuclear factor of activated T cells and alkaline phosphatase-1 and subsequent IL-2 promoter activation. Remarkably, on TCR-independent stimulation, IL-2 production, CD69 expression, and blast formation were completely insensitive to JAK3 inhibitor treatment. CONCLUSION: These data indicate that pharmacologic targeting of JAK3 uncouples early TCR-triggered signaling from essential downstream events, which may have important implications for the use of such compounds in T-cell-mediated disorders such as allograft rejection or graft-versus-host disease.

Partial deficiency of sphingosine-1-phosphate lyase confers protection in experimental autoimmune encephalomyelitis.

BACKGROUND: Sphingosine-1-phosphate (S1P) regulates the egress of T cells from lymphoid organs; levels of S1P in the tissues are controlled by S1P lyase (Sgpl1). Hence, Sgpl1 offers a target to block T cell-dependent inflammatory processes. However, the involvement of Sgpl1 in models of disease has not been fully elucidated yet, since Sgpl1 KO mice have a short life-span. METHODOLOGY: We generated inducible Sgpl1 KO mice featuring partial reduction of Sgpl1 activity and analyzed them with respect to sphingolipid levels, T-cell distribution, and response in models of inflammation. PRINCIPAL FINDINGS: The partially Sgpl1 deficient mice are viable but feature profound reduction of peripheral T cells, similar to the constitutive KO mice. While thymic T cell development in these mice appears normal, mature T cells are retained in thymus and lymph nodes, leading to reduced T cell numbers in spleen and blood, with a skewing towards increased proportions of memory T cells and T regulatory cells. The therapeutic relevance of Sgpl1 is demonstrated by the fact that the inducible KO mice are protected in experimental autoimmune encephalomyelitis (EAE). T cell immigration into the CNS was found to be profoundly reduced. Since S1P levels in the brain of the animals are unchanged, we conclude that protection in EAE is due to the peripheral effect on T cells, leading to reduced CNS immigration, rather than on local effects in the CNS. SIGNIFICANCE: The data suggest Sgpl1 as a novel therapeutic target for the treatment of multiple sclerosis.

Fingolimod (FTY720): discovery and development of an oral drug to treat multiple sclerosis.

The discovery of fingolimod (FTY720/Gilenya; Novartis), an orally active immunomodulatory drug, has opened up new approaches to the treatment of multiple sclerosis, the most common inflammatory disorder of the central nervous system. Elucidation of the effects of fingolimod--mediated by the modulation of sphingosine 1-phosphate (S1P) receptors--has indicated that its therapeutic activity could be due to regulation of the migration of selected lymphocyte subsets into the central nervous system and direct effects on neural cells, particularly astrocytes. An improved understanding of the biology of S1P receptors has also been gained. This article describes the discovery and development of fingolimod, which was approved by the US Food and Drug Administration in September 2010 as a first-line treatment for relapsing forms of multiple sclerosis, thereby becoming the first oral disease-modifying therapy to be approved for multiple sclerosis in the United States.

Sphingosine kinase 1 is essential for proteinase-activated receptor-1 signalling in epithelial and endothelial cells.

There is accumulating evidence that activation of sphingosine kinase 1 (SPHK1) is an important element in intracellular signalling cascades initiated by stimulation of multiple receptors, including certain growth factor, cytokine, and also G-protein coupled receptors. We here report that stimulation of the lung epithelial cell line A549 by thrombin leads to transient increase of SPHK1 activity and elevation of intracellular sphingosine-1-phosphate (S1P); abrogation of this stimulation by SPHK1-specific siRNA, pharmacological inhibition, or expression of a dominant-negative SPHK1 mutant blocks the response to thrombin, as measured by secretion of MCP-1, IL-6, IL-8, and PGE(2). Using selective stimulation of proteinase-activated receptors (PARs) a specific involvement of SPHK1 in the PAR-1 induced responses in A549 cell, including activation of NFkappaB, was evident, while PAR-2 and PAR-4 responses were independent of SPHK1. Moreover, PAR-1 or thrombin-induced cytokine production and adhesion factor expression of human umbilical vein endothelial cells was also seen to depend on SPHK1. Using dermal microvascular endothelial cells from SPHK1-deficient mice, we showed that absence of the enzyme abrogates MCP-1 production induced in these cells upon treatment with thrombin or PAR-1 activating peptide. We propose SPHK1 inhibition as a novel way to block PAR-1 mediated signalling, which could be useful in treatment of a number of diseases, in particular in atherosclerosis.

FTY720 rescue therapy in the dark agouti rat model of experimental autoimmune encephalomyelitis: expression of central nervous system genes and reversal of blood-brain-barrier damage.

FTY720 (fingolimod) is an oral sphingosine-1 phosphate (S1P) receptor modulator in phase III development for the treatment of multiple sclerosis. To further investigate its mode of action, we analyzed gene expression in the central nervous system (CNS) during experimental autoimmune encephalomyelitis (EAE). FTY720 downregulated inflammatory genes in addition to vascular adhesion molecules. It decreased the matrix metalloproteinase gene MMP-9 and increased its counterregulator--tissue inhibitor of metalloproteinase, TIMP-1--resulting in a proteolytic balance that favors preservation of blood-brain-barrier (BBB) integrity. Furthermore, FTY720 reduced S1P lyase that increases the S1P concentration in the brain, in line with a marked reversal of neurological deficits and raising the possibility for enhanced triggering of S1P receptors on resident brain cells. This is accompanied by an increase in S1P(1) and S1P(5) in contrast with the attenuation of S1P(3) and S1P(4). Late-stage rescue therapy with FTY720, even up to 1 month after EAE onset, reversed BBB leakiness and reduced demyelination, along with normalization of neurologic function. Our results indicate rapid blockade of ongoing disease processes by FTY720, and structural restoration of the CNS parenchyma, which is likely caused by the inhibition of autoimmune T cell infiltration and direct modulation of microvascular and/or glial cells.

Neutropenia with impaired immune response to Streptococcus pneumoniae in ceramide kinase-deficient mice.

In mammals, ceramide kinase (CerK)-mediated phosphorylation of ceramide is the only known pathway to ceramide-1-phosphate (C1P), a recently identified signaling sphingolipid metabolite. To help delineate the roles of CerK and C1P, we knocked out the gene of CerK in BALB/c mice by homologous recombination. All in vitro as well as cell-based assays indicated that CerK activity is completely abolished in Cerk-/- mice. Labeling with radioactive orthophosphate showed a profound reduction in the levels of de novo C1P formed in Cerk-/- macrophages. Consistently, mass spectrometry analysis revealed a major contribution of CerK to the formation of C16-C1P. However, the significant residual C1P levels in Cerk-/- animals indicate that alternative routes to C1P exist. Furthermore, serum levels of proapoptotic ceramide in these animals were significantly increased while levels of dihydroceramide as the biosynthetic precursor were reduced. Previous literature pointed to a role of CerK or C1P in innate immune cell function. Using a variety of mechanistic and disease models, as well as primary cells, we found that macrophage- and mast cell-dependent readouts are barely affected in the absence of CerK. However, the number of neutrophils was strikingly reduced in blood and spleen of Cerk-/- animals. When tested in a model of fulminant pneumonia, Cerk-/- animals developed a more severe disease, lending support to a defect in neutrophil homeostasis following CerK ablation. These results identify ceramide kinase as a key regulator of C1P, dihydroceramide and ceramide levels, with important implications for neutrophil homeostasis and innate immunity regulation.

FTY720, an immunomodulatory sphingolipid mimetic: translation of a novel mechanism into clinical benefit in multiple sclerosis.

FTY720 (fingolimod; 2-amino-2[2-(4-octylphenyl)ethyl]-1,3-propanediol, Novartis) is the prototype of a new generation of immunomodulators. The drug is the result of extensive chemical derivatisation based on the natural product myriocin, isolated from the ascomycete Isaria sinclairii. FTY720 bears structural similarity to sphingosine, a naturally occurring sphingolipid. As with sphingosine, FTY720 is effectively phosphorylated by sphingosine kinases in vivo and the phosphorylated drug targets G-protein-coupled receptors for sphingosine-1-phosphate (S1P). Gene deletion and reverse pharmacology studies have shown that FTY720 acts at S1P1 receptors on lymphocytes and the endothelium, thereby inhibiting the egress of T- and B cells from secondary lymphoid organs into the blood and their recirculation to inflamed tissues. Animal studies suggest that this novel mechanism translates into effective treatments for several autoimmune diseases and a recently completed Phase II clinical trial highlighted FTY720 as a potential therapy for relapsing-remitting multiple sclerosis.