sphingosine-kinase and Multiple-Sclerosis

Sphingosine-1-phosphate signaling is emerging as a critical regulator of cellular processes that is initiated by the intracellular production of bioactive lipid molecule, sphingosine-1-phosphate. Binding of sphingosine-1-phosphate to its extracellular receptors activates diverse downstream signaling that play a critical role in governing physiological processes. Increasing evidence suggests that this signaling pathway often gets impaired during pathophysiological and diseased conditions and hence manipulation of this signaling pathway may be beneficial in providing treatment. In this review, we summarized the recent findings of S1P signaling pathway and the versatile role of the participating candidates in context with several disease conditions. Finally, we discussed its possible role as a novel drug target in different diseases.

Topics: Arthritis, Rheumatoid; Ceramidases; Diabetes Mellitus; Humans; Lysophospholipids; Molecular Targeted Therapy; Multiple Sclerosis; Neoplasms; Phosphotransferases (Alcohol Group Acceptor); Signal Transduction; Sphingosine

FTY720 is a recently approved first line therapy for relapsing forms of multiple sclerosis. In this context, FTY720 is a pro-drug, with its anti-multiple sclerosis, immunosuppressive effects largely elicited following its phosphorylation by sphingosine kinase 2 and subsequent modulation of G protein-coupled sphingosine 1-phosphate (S1P) receptor 1 that induces lymphopenia by altering lymphocyte trafficking. A number of other biological effects of FTY720 have, however, been described, including considerable evidence that this drug also has anti-cancer properties. These other effects of FTY720 are independent of S1P receptors, and appear facilitated by modulation of a range of other recently described protein targets by nonphosphorylated FTY720. Here, we review the direct targets of FTY720 that contribute to its anti-cancer properties. We also discuss other recently described protein effectors that, in combination with S1P receptors, appear to contribute to its immunosuppressive effects.

Topics: Antineoplastic Agents; Apoptosis; Fingolimod Hydrochloride; Humans; Immunosuppressive Agents; Lymphopenia; Lysophospholipids; Multiple Sclerosis; Neoplasms; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Propylene Glycols; Receptors, Lysosphingolipid; Sphingosine

FTY720 (fingolimod) is a first-in-class sphingosine 1-phosphate (S1P) receptor modulator that was highly effective in Phase II clinical trials for Multiple Sclerosis (MS). FTY720 is phosphorylated in vivo by sphingosine kinase-2 to form the active moiety FTY720-phosphate that binds to four of the five G protein-coupled S1P receptor subtypes. Studies using conditional S1P1 receptor-deficient and sphingosine kinase-deficient mice showed that the egress of lymphocytes from lymph nodes requires signalling of lymphocytic S1P1 receptors by the endogenous ligand S1P. The S1P mimetic FTY720-phosphate causes internalization and degradation of cell membrane-expressed S1P1, thereby antagonizing S1P action at the receptor. In models of human MS and demyelinating polyneuropathies, functional antagonism of lymphocytic S1P1 slows S1P-driven egress of lymphocytes from lymph nodes, thereby reducing the numbers of autoaggressive TH17 cells that recirculate via lymph and blood to the central nervous system and the sciatic/ischiatic nerves. Based on its lipophilic nature, FTY720 crosses the blood-brain barrier, and ongoing experiments suggest that the drug also down-modulates S1P1 in neural cells/astrocytes to reduce astrogliosis, a phenomenon associated with neurodegeneration in MS. This may help restore gap-junctional communication of astrocytes with neurons and cells of the blood-brain barrier. Additional effects may result from (down-) modulation of S1P3 in astrocytes and of S1P1 and S1P5 in oligodendrocytes. In conclusion, FTY720 may act through immune-based and central mechanisms to reduce inflammation and support structural restoration of the central nervous system parenchyma. Beyond the autoimmune indications, very recent studies suggest that short-term, low-dose administration of FTY720 could help treat chronic (viral) infections. Differential effects of the drug on the trafficking of naïve, central memory and effector memory T cell subsets are discussed.

Topics: Animals; Astrocytes; Brain; Encephalomyelitis, Autoimmune, Experimental; Fingolimod Hydrochloride; Gap Junctions; Humans; Immune System; Inflammation; Lymph Nodes; Lysophospholipids; Multiple Sclerosis; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Propylene Glycols; Receptors, Lysosphingolipid; Sphingosine; T-Lymphocytes

Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid metabolite involved in many critical cellular processes including proliferation, survival, and migration, as well as angiogenesis and allergic responses. S1P levels inside cells are tightly regulated by the balance between its synthesis by sphingosine kinases and degradation. S1P is interconvertible with ceramide, which is a critical mediator of apoptosis. It has been postulated that the ratio between S1P and ceramide determines cell fate. Activation of sphingosine kinase by a variety of agonists increases intracellular S1P, which in turn can function intracellularly as a second messenger or be secreted out of the cell and act extracellularly by binding to and signaling through S1P receptors in autocrine and/or paracrine manners. Recent studies suggest that this "inside-out" signaling by S1P may play a role in many human diseases, including cancer, atherosclerosis, inflammation, and autoimmune disorders such as multiple sclerosis. In this review we summarize metabolism of S1P, mechanisms of sphingosine kinase activation, and S1P receptors and their downstream signaling pathways and examine relationships to multiple disease processes. In particular, we describe recent preclinical and clinical trials of therapies targeting S1P signaling, including 2-amino-2-propane-1,3-diol hydrochloride (FTY720, fingolimod), S1P receptor agonists, sphingosine kinase inhibitors, and anti-S1P monoclonal antibody.

Topics: Animals; Antibodies, Monoclonal; Antineoplastic Agents; Apoptosis; Enzyme Activation; Fingolimod Hydrochloride; Humans; Hypersensitivity; Immunosuppressive Agents; Lysophospholipids; Multiple Sclerosis; Neoplasms; Phosphotransferases (Alcohol Group Acceptor); Propylene Glycols; Receptors, Lysosphingolipid; Signal Transduction; Sphingosine; Sulfhydryl Compounds

Topics: Animals; Autoimmunity; CD4-Positive T-Lymphocytes; Central Nervous System; Encephalomyelitis, Autoimmune, Experimental; Female; Humans; Killer Cells, Natural; Mesenchymal Stem Cells; Mice; Mice, Inbred C57BL; Multiple Sclerosis; Phosphotransferases (Alcohol Group Acceptor); T-Lymphocytes, Regulatory; Th17 Cells; Transfection; Umbilical Cord

Berberine (BBR) has shown neuroprotective properties. The present study aims to investigate the effects of BBR on experimental autoimmune encephalomyelitis (EAE), a murine model of multiple sclerosis (MS), and SphK1/S1P signaling, which plays a key role in MS. EAE was induced in mice, followed by treatment with BBR at 50, 100, or 300 mg/kg/d. Neurophysiological function was evaluated daily; inflammation, cell infiltration, and the severity of demyelination were also examined. The SphK1, SphK2, and S1P levels in the animals and primary astrocyte culture were measured. We found that treatment with BBR reduced the loss of neurophysiological function and the degree of demyelination. Moreover, BBR was associated with a decrease in SphK1 and S1P levels both in the animals and in culture. These results indicated that BBR suppresses demyelination and loss of neurophysiological function by inhibiting the SphK1/S1P signaling pathway. The use of BBR as a treatment of MS warrant further exploration.

Topics: Animals; Berberine; Encephalomyelitis, Autoimmune, Experimental; Female; Lysophospholipids; Mice; Mice, Inbred C57BL; Multiple Sclerosis; Neuroprotective Agents; Phosphotransferases (Alcohol Group Acceptor); Signal Transduction; Sphingosine; Spinal Cord

Multiple sclerosis patients are treated with fingolimod (FTY720), a prodrug that acts as an immune modulator. FTY720 is first phosphorylated to FTY720-P and then internalizes sphingosine-1-phosphate receptors, preventing lymphocyte sequestration. IL-33 is released from necrotic endothelial cells and contributes to MS severity by coactivating T cells. Herein we analyzed the influence of FTY720, FTY720-P, and S1P on IL-33 induced formation of IL-2 and IFN-γ, by using IL-33 receptor overexpressing EL4 cells, primary CD8(+) T cells, and splenocytes. EL4-ST2 cells released IL-2 after IL-33 stimulation that was inhibited dose-dependently by FTY720-P but not FTY720. In this system, S1P increased IL-2, and accordingly, inhibition of S1P producing sphingosine kinases diminished IL-2 release. In primary CD8(+) T cells and splenocytes IL-33/IL-12 stimulation induced IFN-γ, which was prevented by FTY720 but not FTY720-P, independently from intracellular phosphorylation. The inhibition of IFN-γ by nonphosphorylated FTY720 was mediated via the SET/protein phosphatase 2A (PP2A) pathway, since a SET peptide antagonist also prevented IFN-γ formation and the inhibition of IFN-γ by FTY720 was reversible by a PP2A inhibitor. While our findings directly improve the understanding of FTY720 therapy in MS, they could also contribute to side effects of FTY720 treatment, like progressive multifocal leukoencephalopathy, caused by an insufficient immune response to a viral infection.

Topics: Animals; CD8-Positive T-Lymphocytes; Cell Line, Tumor; DNA-Binding Proteins; Female; Fingolimod Hydrochloride; Histone Chaperones; Interferon-gamma; Interleukin-2; Interleukin-33; Lysophospholipids; Mice; Mice, Inbred C57BL; Multiple Sclerosis; Oncogene Proteins; Organophosphates; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Protein Phosphatase 2; Sphingosine; Spleen

The prevalence of allergies, including rhinitis, eczema, and anaphylaxis, is rising dramatically worldwide. This increase is especially problematic in children who bear the greatest burden of this rising trend. Increasing evidence identifies neutrophils as primary perpetrators of the more severe and difficult to manage forms of inflammation. A newly recognized mechanism by which neutrophils are recruited during the early phase of histamine-induced inflammation involves the sphingosine kinase (SK)/sphingosine-1-phosphate axis. This study examines whether topical application of fingolimod, an established SK/sphingosine-1-phosphate antagonist already in clinical use to treat multiple sclerosis, may be repurposed to treat cutaneous inflammation. Using two mouse models of ear skin inflammation (histamine- and IgE-mediated passive cutaneous anaphylaxis) we topically applied fingolimod prophylactically, as well as after establishment of the inflammatory response, and examined ear swelling, SK activity, vascular permeability, leukocyte recruitment, and production of proinflammatory mediators. The present study reveals that when applied topically, fingolimod attenuates both immediate and late-phase responses to histamine with reduced extravasation of fluid, SK-1 activity, proinflammatory cytokine and chemokine production, and neutrophil influx and prevents ear swelling. Intravital microscopy demonstrates that histamine-induced neutrophil rolling and adhesion to the postcapillary venules in the mouse ears is significantly attenuated even after 24 h. More importantly, these effects are achievable even once inflammation is established. Translation into humans was also accomplished with epicutaneous application of fingolimod resolving histamine-induced and allergen-induced inflammatory reactions in forearm skin. Overall, this study demonstrates, to our knowledge for the first time, that fingolimod may be repurposed to treat cutaneous inflammation.

Topics: Administration, Topical; Animals; Capillary Permeability; Cell Movement; Cells, Cultured; Cytokines; Dermatitis; Disease Models, Animal; Fingolimod Hydrochloride; Histamine; Humans; Immunoglobulin E; Inflammation Mediators; Mice; Mice, Inbred C57BL; Mice, Knockout; Multiple Sclerosis; Neutrophils; Phosphotransferases (Alcohol Group Acceptor); Skin

FTY720 (Fingolimod; Gilenya®) is an immune-modulatory prodrug which, after intracellular phosphorylation by sphingosine kinase 2 (SphK2) and export, mimics effects of the endogenous lipid mediator sphingosine-1-phosphate. Fingolimod has been introduced to treat relapsing-remitting multiple sclerosis. However, little has been published about the immune cell membrane penetration and subcellular distribution of FTY720 and FTY720-P. Thus, we applied a newly established LC-MS/MS method to analyze the subcellular distribution of FTY720 and FTY720-P in subcellular compartments of spleen cells of wild type, SphK1- and SphK2-deficient mice. These studies demonstrated that, when normalized to the original cell volume and calculated on molar basis, FTY720 and FTY720-P dramatically accumulated several hundredfold within immune cells reaching micromolar concentrations. The amount and distribution of FTY720 was differentially affected by SphK1- and SphK2-deficiency. On the background of recently described relevant intracellular FTY720 effects in the nanomolar range and the prolonged application in multiple sclerosis, this data showing a substantial intracellular accumulation of FTY720, has to be considered for benefit/risk ratio estimates.

Topics: Animals; Cells, Cultured; Female; Fingolimod Hydrochloride; Lysophospholipids; Mice; Mice, Inbred C57BL; Multiple Sclerosis; Organophosphates; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Sphingosine; Spleen; Tandem Mass Spectrometry

Neuropathic pain is a chronic, refractory condition that arises after damage to the nervous system. We previously showed that an increased level of the endogenous metabolite N,N-dimethylsphingosine (DMS) in the central nervous system (CNS) is sufficient to induce neuropathic pain-like behavior in rats. However, several important questions remain. First, it has not yet been demonstrated that DMS is produced in humans and its value as a therapeutic target is therefore unknown. Second, the cell types within the CNS that produce DMS are currently unidentified. Here we provide evidence that DMS is present in human CNS tissue. We show that DMS levels increase in demyelinating lesions isolated from patients with multiple sclerosis, an autoimmune disease in which the majority of patients experience chronic pain. On the basis of these results, we hypothesized that oligodendrocytes may be a cellular source of DMS. We show that human oligodendrocytes produce DMS in culture and that the levels of DMS increase when oligodendrocytes are challenged with agents that damage white matter. These results suggest that damage to oligodendrocytes leads to increased DMS production which in turn drives inflammatory astrocyte responses involved in sensory neuron sensitization. Interruption of this pathway in patients may provide analgesia without the debilitating side effects that are commonly observed with other chronic pain therapies.

Topics: Animals; Astrocytes; Cell Line; Humans; Multiple Sclerosis; Oligodendroglia; Phosphotransferases (Alcohol Group Acceptor); Rats; Sphingosine

Reactive astrocytes are implicated in the development and maintenance of neuroinflammation in the demyelinating disease multiple sclerosis (MS). The sphingosine kinase 1 (SphK1)/sphingosine1-phosphate (S1P) receptor signaling pathway is involved in modulation of the inflammatory response in many cell types, but the role of S1P receptor subtype 3 (S1P(3)) signaling and SphK1 in activated rat astrocytes has not been defined.. Using immunohistochemistry we observed the upregulation of S1P(3) and SphK1 expression on reactive astrocytes and SphK1 on macrophages in MS lesions. Increased mRNA and protein expression of S1P(3) and SphK1, as measured by qPCR and Western blotting respectively, was observed after treatment of rat primary astrocyte cultures with the pro-inflammatory stimulus lipopolysaccharide (LPS). Activation of SphK by LPS stimulation was confirmed by SphK activity assay and was blocked by the use of the SphK inhibitor SKI (2-(p-hydroxyanilino)-4-(p-chlorphenyl) thiazole. Treatment of astrocytes with a selective S1P(3) agonist led to increased phosphorylation of extracellular signal-regulated kinase (ERK)-1/2), which was further elevated with a LPS pre-challenge, suggesting that S1P(3) upregulation can lead to increased functionality. Moreover, astrocyte migration in a scratch assay was induced by S1P and LPS and this LPS-induced migration was sensitive to inhibition of SphK1, and independent of cell proliferation. In addition, S1P induced secretion of the potentially neuroprotective chemokine CXCL1, which was increased when astrocytes were pre-challenged with LPS. A more prominent role of S1P(3) signaling compared to S1P(1) signaling was demonstrated by the use of selective S1P(3) or S1P(1) agonists.. In summary, our data demonstrate that the SphK1/S1P(3) signaling axis is upregulated when astrocytes are activated by LPS. This signaling pathway appears to play a role in the establishment and maintenance of astrocyte activation. Upregulation of the pathway in MS may be detrimental, e.g. through enhancing astrogliosis, or beneficial through increased remyelination via CXCL1.

Topics: Animals; Astrocytes; Chemokine CXCL1; Inflammation; Lipopolysaccharides; MAP Kinase Signaling System; Multiple Sclerosis; Phosphotransferases (Alcohol Group Acceptor); Rats; Receptors, Lysosphingolipid; Signal Transduction; Up-Regulation

Modifying FTY720, an immunosuppressant modulator, led to a new series of well phosphorylated tetralin analogs as potent S1P1 receptor agonists. The stereochemistry effect of tetralin ring was probed, and (-)-(R)-2-amino-2-((S)-6-octyl-1,2,3,4-tetrahydronaphthalen-2-yl)propan-1-ol was identified as a good SphK2 substrate and potent S1P1 agonist with good oral bioavailability.

Topics: Administration, Oral; Animals; Crystallography, X-Ray; Immunosuppressive Agents; Lymphopenia; Mice; Models, Molecular; Multiple Sclerosis; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Prodrugs; Receptors, Lysosphingolipid; Structure-Activity Relationship; Tetrahydronaphthalenes