Targeting the sphingosine kinase/sphingosine 1-phosphate pathway in disease: Review of sphingosine kinase inhibitors

Sphingosine 1-phosphate (S1P) is an important bioactive sphingolipid metabolite that has been implicated in numerous physiological and cellular processes. Not only does S1P play a structural role in cells by defining the components of the plasma membrane, but in the last 20 years it has been implicated in various significant cell signaling pathways and physiological processes: for example, cell migration, survival and proliferation, cellular architecture, cell–cell contacts and adhesions, vascular development, atherosclerosis, acute pulmonary injury and respiratory distress, inflammation and immunity, and tumorogenesis and metastasis [ and ]. Given the wide variety of cellular and physiological processes in which S1P is involved, it is immediately obvious why the mechanisms governing S1P synthesis and degradation, and the manner in which these processes are regulated, are necessary to understand. In gaining more knowledge about regulation of the sphingosine kinase (SK)/S1P pathway, many potential therapeutic targets may be revealed. This review explores the roles of the SK/S1P pathway in disease, summarizes available SK enzyme inhibitors and examines their potential as therapeutic agents. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.     

An assay system for measuring the acute production of sphingosine 1-phosphate in intact monolayers

Sphingosine kinase (SK) is a signaling enzyme that phosphorylates sphingosine to produce sphingosine 1-phosphate. Sphingosine and sphingosine 1-phosphate (S1P) belong to a class of bioactive sphingolipid metabolites that are critical in a number of cellular processes, yet often have opposing biological functions. The intracellular localization of sphingosine kinase has been demonstrated in multiple studies to be a critical aspect of its signaling function. To date, assays of sphingosine kinase activity have been developed for measuring activity in lysates, where the effects of localization are lost. Here we outline a system in which the rate of production of S1P can be measured in intact cells using exogenously added radiolabeled ATP instead of tritiated sphingosine. The surprising ability of ATP to enter unpermeabilized monolayers is one aspect that makes this assay simple, efficient, and inexpensive, yet sensitive enough to measure endogenous enzyme activity. The assay is well behaved in terms of kinetics and substrate dependence. Overall, this assay is ideal for future studies to identify changes in S1P production in intact cells such as those that result from the differential intracellular targeting of sphingosine kinase.     

Altered expression of sphingosine kinase 1 and sphingosine-1-phosphate receptor 1 in mouse hippocampus after kainic acid treatment

Kainic acid (KA) induces hippocampal cell death and astrocyte proliferation. There are reports that sphingosine kinase (SPHK)1 and sphingosine-1- phosphate (S1P) receptor 1 (S1P₁) signaling axis controls astrocyte proliferation. Here we examined the temporal changes of SPHK1/S1P₁ in mouse hippocampus during KA-induced hippocampal cell death. Mice were killed at 2, 6, 24, or 48 h after KA (30 mg/kg) injection. There was an increase in Fluoro-Jade B-positive cells in the hippocampus of KA-treated mice with temporal changes of glial fibrillary acidic protein (GFAP) expression. The lowest level of SPHK1 protein expression was found 2 h after KA treatment. Six hours after KA treatment, the expression of SPHK1 and S1P₁ proteins steadily increased in the hippocampus. In immunohistochemical analysis, SPHK1 and S1P₁ are more immunoreactive in astrocytes within the hippocampus of KA-treated mice than in hippocampus of control mice. These results indicate that SPHK1/S1P₁ signaling axis may play an important role in astrocytes proliferation during KA-induced excitotoxicity.     

Selective activation of G alpha i mediated signalling of S1P3 by FTY720-phosphate

The immune modulator FTY720 is phosphorylated in vivo to FTY720 phosphate (FTY-P), which activates four sphingosine 1-phosphate (S1P) receptors including S1P₃. Upon activation with S1P, S1P₃ couples to G_i- and G_q-protein-dependent signalling pathways. Here we show that FTY-P selectively activates the S1P₃-mediated and G_i-coupled inhibition of adenylyl cyclase. Contemporaneously, it antagonizes the S1P-induced activation of G_q via S1P₃ in intracellular calcium flux measurements, GTP-binding experiments, and flow cytometric analyses of activation-induced receptor down-regulation. In contrast to S1P, pre-treatment with FTY-P did not desensitize S1P-induced calcium flux or chemotaxis via S1P₃. The lack of receptor desensitization prevented S1P₃-mediated migration to FTY-P. Human umbilical vein endothelial cells express S1P₁ and S1P₃, and respond to S1P and FTY-P by ERK1/2 phosphorylation and by intracellular calcium release in a pertussis toxin-sensitive manner. But whereas a mixture of S1P and FTY-P was not affecting ERK1/2 phosphorylation, the intracellular calcium flux was hampered with increasing amounts of FTY-P, which points to a cross-talk between S1P₁ and S1P₃. FTY-P is therefore one of the rare ligands which bind to a receptor that couples multiple G-proteins but selectively activates only one signalling pathway.     

Role of sphingosine kinase and sphingosine-1-phosphate in inflammatory arthritis

The importance of sphingosine kinase (SphK) and sphingosine-1-phosphate (S1P) in inflammation has been extensively demonstrated. As an intracellular second messenger, S1P plays an important role in calcium signaling and mobilization, and cell proliferation and survival. Activation of various plasma membrane receptors, such as the formyl methionyl leucyl phenylalanine receptor, C5a receptor, and tumor necrosis factor α receptor, leads to a rapid increase in intracellular S1P level via SphK stimulation. SphK and S1P are implicated in various chronic autoimmune conditions such as rheumatoid arthritis, primary Sjögren’s syndrome, and inflammatory bowel disease. Recent studies have demonstrated the important role of SphK and S1P in the development of arthritis by regulating the pro-inflammatory responses. These novel pathways represent exciting potential therapeutic targets.     

Inhibition of chemotactic motility and trans-endothelial migration of human neutrophils by sphingosine 1-phosphate

In previous studies, we reported that sphingosine 1-phosphate (Sph-1-P) inhibits the chemotactic motility of some cancer cell lines such as mouse melanoma cells, as well as human smooth muscle cells, at a very low concentration, as demonstrated by a transwell migration assay method (Proc. Natl. Acad. Sci. USA 89, 9698, 1992; J. Cell Biol. 130, 193, 1995). In this study, we investigated the effect of Sph-1-P on the chemotactic motility and invasiveness of human neutrophils, utilizing three different assay systems: (a) a transwell migration assay where IL-8 or fLMP was added as a chemotactic factor, (b) a phagokinetic assay with gold colloids, and (c) a trans-endothelial migration assay with human umbilical vein endothelial cells (HUVECs) plated on collagen layers. We found that among various sphingosine derivatives, Sph-1-P specifically inhibited the IL-8- or fLMP-induced chemotactic migration of neutrophils at concentrations below 1 μM. Phagokinetic activity of neutrophils was also suppressed by Sph-1-P, but more moderately than by the PKC inhibitory sphingosine analog, trimethylsphingosine. Finally, Sph-1-P inhibited trans-endothelial migration and invasiveness of neutrophils into HUVEC-covered collagen layers, whereas no effect on their adhesion to HUVECs was observed. These observations strongly suggest that Sph-1-P can act as a specific and effective motility regulator of human neutrophils, raising the possibility of future applications of Sph-1-P, or its analogs, as anti-inflammatory agents regulating invasive migration of neutrophils through endothelial layers at injured vascular sites.     

Dimethylsphingosine and FTY720 inhibit the SK1 form but activate the SK2 form of sphingosine kinase from rat heart

Fractionation of cytosolic sphingosine kinase (SKase) activity by gel filtration chromatography gave rise to a 96-kDa peak that contained only the SK2 form of SKase (by Western analysis) and a broad ca. 46 kDa peak that contained only SK1 forms. SK2 appeared to have a bound accessory protein. When tested with the classic SKase inhibitor dimethylsphingosine (DMS), SK1 was extensively inhibited; however, SK2 was not inhibited but unexpectedly was activated. Activation of SK2 was the result of DMS enhancing the affinity of the enzyme for sphingosine, and, at low concentrations of ATP and sphingosine, activated by more than 100%. Activation of SK2 could be demonstrated in the cytosolic fraction indicating it was unrelated to the purification step. The immunomodulator FTY720 also activated SK2 (although to a lesser extent), but was a potent inhibitor of SK1. SK2 from rat liver and spleen was also not inhibited by DMS. L-Sphingosine and to a lesser extent dihydrosphingosine and phytosphingosine were effective inhibitors of both forms.     

Involvement of released sphingosine 1-phosphate/sphingosine 1-phosphate receptor axis in skeletal muscle atrophy

Skeletal muscle (SkM) atrophy is caused by several and heterogeneous conditions, such as cancer, neuromuscular disorders and aging. In most types of SkM atrophy overall rates of protein synthesis are suppressed, protein degradation is consistently elevated and atrogenes, such as the ubiquitin ligase Atrogin-1/MAFbx, are up-regulated. The molecular regulators of SkM waste are multiple and only in part known.Sphingolipids represent a class of bioactive molecules capable of modulating the destiny of many cell types, including SkM cells. In particular, we and others have shown that sphingosine 1phosphate (S1P), formed by sphingosine kinase (SphK), is able to act as trophic and morphogenic factor in myoblasts.Here, we report the first evidence that the atrophic phenotype observed in both muscle obtained from mice bearing the C26 adenocarcinoma and C2C12 myotubes treated with dexamethasone was characterized by reduced levels of active phospho-SphK1. The importance of SphK1 activity is also confirmed by the specific pharmacological inhibition of SphK1 able to increase Atrogin-1/MAFbx expression and reduce myotube size and myonuclei number. Furthermore, we found that SkM atrophy was accomplished by significant increase of S1P transporter Spns2 and in changes in the pattern of S1P receptor (S1PRs) subtype expression paralleled by increased Atrogin-1/MAFbx expression, suggesting a role for the released S1P and of specific S1PR-mediated signaling pathways in the control of the ubiquitin ligase. Altogether, these findings provide the first evidence that SphK1/released S1P/S1PR axis acts as a molecular regulator of SkM atrophy, thereby representing a new possible target for therapy in many patho-physiological conditions.     

Sphingosine kinase and sphingosine 1-phosphate in the heart: A decade of progress

Activation of sphingosine kinase/sphingosine 1-phosphate (SK/S1P)‐mediated signaling has emerged as a critical cardioprotective pathway in response to acute ischemia/reperfusion injury. S1P is released in both ischemic pre- and post-conditioning. Application of exogenous S1P to cultured cardiac myocytes subjected to hypoxia or treatment of isolated hearts either before ischemia or at the onset of reperfusion exerts prosurvival effects. Synthetic congeners of S1P such as FTY720 mimic these responses. Gene targeted mice null for the SK1 isoform whose hearts are subjected to ischemia/reperfusion injury exhibit increased infarct size and respond poorly either to ischemic pre- or postconditioning. Measurements of cardiac SK activity and S1P parallel these observations. Experiments in SK2 knockout mice have revealed that this isoform is necessary for survival in the heart. High density lipoprotein (HDL) is a major carrier of S1P, and studies of hearts in which selected S1P receptors have been inhibited implicate the S1P cargo of HDL in cardioprotection. Inhibition of S1P lyase, an endogenous enzyme that degrades S1P, also leads to cardioprotection. These observations have considerable relevance for future therapeutic approaches to acute and chronic myocardial injury. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.     

Design and synthesis of pyrazolopyridine derivatives as sphingosine 1-phosphate receptor 2 ligands

Eleven new sphingosine 1-phosphate receptor 2 (S1PR2) ligands were synthesized by modifying lead compound N-(2,6-dichloropyridin-4-yl)-2-(4-isopropyl-1,3-dimethyl-1H-pyrazolo[3,4-b]pyridin-6-yl)hydrazine-1-carboxamide (JTE-013) and their binding affinities toward S1PRs were determined in vitro using [³²P]S1P and cell membranes expressing recombinant human S1PRs. Among these ligands, 35a (IC₅₀?=?29.1?±?2.6?nM) and 35b (IC₅₀?=?56.5?±?4.0?nM) exhibit binding potency toward S1PR2 comparable to JTE-013 (IC₅₀?=?58.4?±?7.4?nM) with good selectivity for S1PR2 over the other S1PRs (IC₅₀?>?1000?nM). Further optimization of these analogues may identify additional and more potent and selective compounds targeting S1PR2.     

Role of sphingosine 1-phosphate in anti-atherogenic actions of high-density lipoprotein

The reverse cholesterol transport mediated by high-density lipoprotein (HDL) is an important mechanism for maintaining body cholesterol, and hence, the crucial anti-atherogenic action of the lipoprotein. Recent studies, however, have shown that HDL exerts a variety of anti-inflammatory and anti-atherogenic actions independently of cholesterol metabolism. The present review provides an overview of the roles of sphingosine 1-phosphate (S1P)/S1P receptor and apolipoprotein A-I/scavenger receptor class B type I systems in the anti-atherogenic HDL actions. In addition, the physiological significance of the existence of S1P in the HDL particles is discussed.     

A splicing isoform of LPP1, LPP1a, exhibits high phosphatase activity toward FTY720 phosphate

The sphingolipid metabolite sphingosine 1-phosphate (S1P) plays an essential function in the egress of T cells from the thymus and secondary lymphoid organs. The novel immunomodulating agent FTY720 is phosphorylated in vivo to the functional form FTY720 phosphate (FTY720-P), which is structurally similar to S1P. FTY720-P inhibits the S1P-mediated T cell egress as an agonist of S1P receptors. FTY720-P is not stable in plasma and is dephosphorylated to FTY720. In the present study, we investigated activities toward FTY720-P of LPP family members (LPP1, LPP1a, LPP2, and LPP3), which exhibit broad substrate specificity. Of the four, LPP1a, the splicing isoform of LPP1, had the highest activity toward FTY720-P, and the highest affinity. Among blood-facing cells tested, only endothelial cells displayed high phosphatase activity for FTY720-P. Significant levels of LPP1a expression were found in endothelial cells, suggesting that LPP1a is important for the dephosphorylation of FTY720-P in plasma.     

Fluid shear stress enhances the sphingosine 1-phosphate responses in cell-cell interactions between platelets and endothelial cells

Fluid shear stress modulates the functional responses of platelets and vascular cells, and plays an important role in the pathogenesis of vascular disorders, including atherosclerosis and restenosis. Since shear stress induces activation of platelets, which abundantly store sphingosine 1-phosphate (Sph-1-P), and upregulates the mRNA expression of S1P(1), the most important Sph-1-P receptor expressed on the endothelial cells, we examined the effects of shear stress on the Sph-1-P-related responses involving these cells. Shear stress was found to induce Sph-1-P release from the platelets in a shear intensity- and time-dependent manner. Inhibitors of protein kinase C suppressed this mechanical force-induced Sph-1-P release, suggesting involvement of this kinase. On the other hand, in vascular endothelial cells, shear stress increased S1P(1) protein expression, as revealed by flow-cytometric analysis, and the responsiveness to Sph-1-P, which was assessed by monitoring the intracellular Ca(2+) concentration. These results indicate that shear stress enhances the Sph-1-P responses in cell-cell interactions between platelets and endothelial cells.     

A house divided: Ceramide, sphingosine, and sphingosine-1-phosphate in programmed cell death

Programmed cell death is an important physiological response to many forms of cellular stress. The signaling cascades that result in programmed cell death are as elaborate as those that promote cell survival, and it is clear that coordination of both protein- and lipid-mediated signals is crucial for proper cell execution. Sphingolipids are a large class of lipids whose diverse members share the common feature of a long-chain sphingoid base, e.g., sphingosine. Many sphingolipids have been shown to play essential roles in both death signaling and survival. Ceramide, an N-acylsphingosine, has been implicated in cell death following a myriad of cellular stresses. Sphingosine itself can induce cell death but via pathways both similar and dissimilar to those of ceramide. Sphingosine-1-phosphate, on the other hand, is an anti-apoptotic molecule that mediates a host of cellular effects antagonistic to those of its pro-apoptotic sphingolipid siblings. Extraordinarily, these lipid mediators are metabolically juxtaposed, suggesting that the regulation of their metabolism is of the utmost importance in determining cell fate. In this review, we briefly examine the role of ceramide, sphingosine, and sphingosine-1-phosphate in programmed cell death and highlight the potential roles that these lipids play in the pathway to apoptosis.     

FTY720 (Gilenya) phosphate selectivity of sphingosine 1-phosphate receptor subtype 1 (S1P1) G protein-coupled receptor requires motifs in intracellular loop 1 and transmembrane domain 2

FTY720 phosphate (FTY720P) is a high potency agonist for all the endothelial differentiation gene family sphingosine 1-phosphate (S1P) receptors except S1P receptor subtype 2 (S1P(2)). To map the distinguishing features of S1P(2) ligand recognition, we applied a computational modeling-guided mutagenesis strategy that was based on the high degree of sequence homology between S1P(1) and S1P(2). S1P(2) point mutants of the ligand-binding pocket were characterized. The head group-interacting residues Arg3.28, Glu3.29, and Lys7.34 were essential for activation. Mutation of residues Ala3.32, Leu3.36, Val5.41, Phe6.44, Trp6.48, Ser7.42, and Ser7.46, predicted to interact with the S1P hydrophobic tail, impaired activation by S1P. Replacing individual or multiple residues in the ligand-binding pocket of S1P(2) with S1P(1) sequence did not impart activation by FTY720P. Chimeric S1P(1)/S1P(2) receptors were generated and characterized for activation by S1P or FTY720P. The S1P(2) chimera with S1P(1) sequence from the N terminus to transmembrane domain 2 (TM2) was activated by FTY720P, and the S1P(2)(IC1-TM2)(S1P1) domain insertion chimera showed S1P(1)-like activation. Twelve residues in this domain, distributed in four motifs a-d, differ between S1P(1) and S1P(2). Insertion of (78)RPMYY in motif b alone or simultaneous swapping of five other residues in motifs c and d from S1P(1) into S1P(2) introduced FTY720P responsiveness. Molecular dynamics calculations indicate that FTY720P binding selectivity is a function of the entropic contribution to the binding free energy rather than enthalpic contributions and that preferred agonists retain substantial flexibility when bound. After exposure to FTY720P, the S1P(2)(IC1-TM2)(S1P1) receptor recycled to the plasma membrane, indicating that additional structural elements are required for the selective degradative trafficking of S1P(1).     

Regulation by sphingosine 1-phosphate of Bax and Bad activities during apoptosis in a MEK-dependent manner

Herein we report that the prosurvival sphingolipid sphingosine 1-phosphate regulates the activities of both Bad and Bax during apoptosis of Jurkat cells. First, sphingosine 1-phosphate treatment results in Bad inactivation via the ERK/Rsk-1 pathway. Second, sphingosine 1-phosphate blocks the translocation of Bax to the mitochondria induced by Fas ligation. MEK inhibition by PD98059 or U0126 not only abrogates sphingosine 1-phosphate-induced Bad phosphorylation, but also its cytoprotective effect. Furthermore, inhibition of both mitochondrial cytochrome c efflux and Bax translocation to the mitochondria by sphingosine 1-phosphate could be overcome by PD98059 or U0126. Hence, the MEK/ERK pathway seems to be crucial for the survival effects initiated by sphingosine 1-phosphate.     

Sphingosine and FTY720 modulate pacemaking activity in interstitial cells of Cajal from mouse small intestine

Interstitial cells of Cajal (ICCs) are the pacemakers of the gastrointestinal tract, and transient receptor potential melastatin type 7 (TRPM7) and Ca²⁺ activated Cl⁻ channels (ANO1) are candidate the generators of pacemaker potentials in ICCs. The effects of D-erythro-sphingosine (SPH) and structural analogues of SPH, that is, N,N-dimethyl-Derythro-sphingosine (N,N-DMS), FTY720, and FTY720-P on the pacemaking activities of ICCs were examined using the whole cell patch clamp technique. SPH, N,N-DMS, and FTY720 decreased the amplitudes of pacemaker potentials in ICC clusters, but resting membrane potentials displayed little change. Also, perfusing SPH, N,N-DMS, or FTY720 in the bath reduced both inward and outward TRPM7-like currents in single ICCs, and inhibited ANO1 currents. The another structural analogue of SPH, FTY720-P was ineffective at the pacemaker potentials in ICC clusters and the TRPM7-like currents in single ICCs. Furthermore, FTY720- P had no effect on ANO1. These results suggest that SPH, N,N-DMS, and FTY720 modulate the pacemaker activities of ICCs, and that TRPM7 and ANO1 channels affect intestinal motility.     

Ginsenoside Rg1 provides neuroprotection against blood brain barrier disruption and neurological injury in a rat model of cerebral ischemia/reperfusion through downregulation of aquaporin 4 expression

Ginsenoside Rg1 is regarded as one of main bioactive compounds responsible for pharmaceutical actions of ginseng with little toxicity and has been shown to have possibly neuroprotective effects. However, the mechanism of its neuroprotection for acute ischemic stroke is still elusive. The purpose of present study is thus to assess the neuroprotective effects of the ginsenoside Rg1 against blood brain barrier disruption and neurological injury in a rat model of cerebral ischemia/reperfusion, and then to explore the mechanisms for these neuroprotective effects by targeting aquaporin 4. Focal cerebral ischemia was induced by middle cerebral artery occlusion. Neurological examinations were performed by using Longa's 5-point scale. Evans blue dye was used to investigate the effects of ginsenoside Rg1 on blood brain barrier permeability. Immunohistochemical analysis and real-time fluorescence quantitative polymerase chain reaction were used to assess aquaporin 4 expression. As a result, general linear model with repeated measures analysis of variance for neurological scores at 5 repeated measures showed that ginsenoside Rg1-treated group could significantly reduce the changing trend of neurological deficit scores when compared with the middle cerebral artery occlusion model group (p < 0.05). Compared with the middle cerebral artery occlusion model group, ginsenoside Rg1 group has significantly decreased Evans blue content and reduced aquaporin 4 expression at each time point (p < 0.05). In conclusion, ginsenoside Rg1 as a ginsenoside neuroprotective agent could improve neurological injury, attenuate blood brain barrier disruption and downregulate aquaporin 4 expression induced by cerebral ischemia/reperfusion insults in rats.     

Regulatory role of sphingosine kinase and sphingosine-1-phosphate receptor signaling in progenitor/stem cells

Balanced sphingolipid signaling is important for the maintenance of homeostasis. Sphingolipids were demonstrated to function as structural components, second messengers, and regulators of cell growth and survival in normal and disease-affected tissues. Particularly, sphingosine kinase 1 (SphK1) and its product sphingosine-1-phosphate (S1P) operate as mediators and facilitators of proliferation-linked signaling. Unlimited proliferation (selfrenewal) within the regulated environment is a hallmark of progenitor/stem cells that was recently associated with the S1P signaling network in vasculature, nervous,muscular, and immune systems. S1P was shown to regulate progenitor-related characteristics in normal and cancerstemcells(CSCs) viaG-protein coupled receptorsS1Pn(n=1 to 5). The SphK/S1P axis is crucially involved in the regulation of embryonic development of vasculature and the nervous system, hematopoietic stem cell migration, regeneration of skeletal muscle, and development of multiple sclerosis. The ratio of the S1P receptor expression, localization, and specific S1P receptoractivated downstream effectors influenced the rate of selfrenewal and should be further explored as regeneration related targets. Considering malignant transformation,it is essential to control the level of self-renewal capacity.Proliferation of the progenitor cell should be synchronized with differentiation to provide healthy lifelong function of blood, immune systems, and replacement of damaged ordead cells. The differentiation-related role of SphK/S1P remains poorly assessed. A few pioneering investigations exploredpharmacologicaltoolsthattargetsphingolipid signaling and can potentially confine and direct self-renewal towards normal differentiation. Further investigation is required to test the role of the SphK/S1P axis in regulation of self-renewal and differentiation.