Generation and metabolism of bioactive sphingosine-1-phosphate

Sphingosine-1-phosphate (S1P) is a bioactive lysosphingophospholipid that has been implicated in the regulation of vital biological processes. Abundant evidence indicates that S1P acts as both an intracellular messenger and an extracellular ligand for a family of five specific G protein-coupled S1P receptors (S1PRs). Cellular levels of S1P are tightly regulated in a spatio-temporal manner through its synthesis catalyzed by sphingosine kinases (SphKs) and degradation by S1P lyase (SPL) and specific S1P phosphohydrolases. Over the past decade, the identification and cloning of genes encoding S1P metabolizing enzymes has increased rapidly. Overexpression and deletion of these enzymes has provided important insights into the intracellular and the "inside-out" functions of S1P. The purpose of this review is to summarize the current knowledge of S1P metabolizing enzymes, their enzymatic properties, and their roles in the control of cellular functions by S1P.     

bFGF induces S1P1 receptor expression and functionality in human pulmonary artery smooth muscle cells

Sphingosine-1-phosphate (S1P), acting through five closely related G-protein coupled receptors termed S1P1-5, has recently emerged as a possible regulator of smooth muscle cell (SMC) physiology with the potential to induce contraction, proliferation and stress fiber formation. In the present study, real-time quantitative PCR was used to determine the expression patterns of S1P receptor subtypes in human primary pulmonary artery smooth muscle cells (PASMC). We report here that subconfluent PASMC express predominantly S1P2 and S1P3 receptors and we show that S1P1 receptor mRNA levels are significantly up-regulated following basic fibroblast growth factor (bFGF) treatment. As a consequence, increased responsiveness, as measured by impedance and ERK1/2 phosphorylation, was observed upon stimulation with a specific S1P1 receptor agonist SEW2871. We therefore demonstrate, for the first time, that a growth factor that was previously shown to be involved in physiological and pathological changes of SMC function induced S1P1 receptor expression and we propose that S1P1 receptor up-regulation could contribute to vascular remodeling.     

Effects of S1P on skeletal muscle repair/regeneration during eccentric contraction

Skeletal muscle regeneration is severely compromised in the case of extended damage. The current challenge is to find factors capable of limiting muscle degeneration and/or potentiating the inherent regenerative program mediated by a specific type of myoblastic cells, the satellite cells. Recent studies from our groups and others have shown that the bioactive lipid, sphingosine 1-phosphate (S1P), promotes myoblast differentiation and exerts a trophic action on denervated skeletal muscle fibres. In the present study, we examined the effects of S1P on eccentric contraction (EC)-injured extensor digitorum longus muscle fibres and resident satellite cells. After EC, skeletal muscle showed evidence of structural and biochemical damage along with significant electrophysiological changes, i.e. reduced plasma membrane resistance and resting membrane potential and altered Na(+) and Ca(2+) current amplitude and kinetics. Treatment with exogenous S1P attenuated the EC-induced tissue damage, protecting skeletal muscle fibre from apoptosis, preserving satellite cell viability and affecting extracellular matrix remodelling, through the up-regulation of matrix metalloproteinase 9 (MMP-9) expression. S1P also promoted satellite cell renewal and differentiation in the damaged muscle. Notably, EC was associated with the activation of sphingosine kinase 1 (SphK1) and with increased endogenous S1P synthesis, further stressing the relevance of S1P in skeletal muscle protection and repair/regeneration. In line with this, the treatment with a selective SphK1 inhibitor during EC, caused an exacerbation of the muscle damage and attenuated MMP-9 expression. Together, these findings are in favour for a role of S1P in skeletal muscle healing and offer new clues for the identification of novel therapeutic approaches to counteract skeletal muscle damage and disease.     

Piperazinyl-oxadiazoles as selective sphingosine-1-phosphate receptor agonists

The discovery of a new series of selective S1P₁ agonists is described. This series of piperazinyl-oxadiazole derivatives was rapidly optimized starting from high-throughput screening hit 1 to afford potent and selective lead compound 10d. Further SAR studies showed that 10d was converted to the active phosphate metabolite 29 in vivo. Oral administration of compound 10d to rats was shown to induce lymphopenia at 3 mg/kg.     

Shaping the landscape: Metabolic regulation of S1P gradients

Sphingosine-1-phosphate (S1P) is a lipid that functions as a metabolic intermediate and a cellular signaling molecule. These roles are integrated when compartments with differing extracellular S1P concentrations are formed that serve to regulate functions within the immune and vascular systems, as well as during pathologic conditions. Gradients of S1P concentration are achieved by the organization of cells with specialized expression of S1P metabolic pathways within tissues. S1P concentration gradients underpin the ability of S1P signaling to regulate in vivo physiology. This review will discuss the mechanisms that are necessary for the formation and maintenance of S1P gradients, with the aim of understanding how a simple lipid controls complex physiology. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.     

S1P lyase regulates DNA damage responses through a novel sphingolipid feedback mechanism

The injurious consequences of ionizing radiation (IR) to normal human cells and the acquired radioresistance of cancer cells represent limitations to cancer radiotherapy. IR induces DNA damage response pathways that orchestrate cell cycle arrest, DNA repair or apoptosis such that irradiated cells are either repaired or eliminated. Concomitantly and independent of DNA damage, IR activates acid sphingomyelinase (ASMase), which generates ceramide, thereby promoting radiation-induced apoptosis. However, ceramide can also be metabolized to sphingosine-1-phosphate (S1P), which acts paradoxically as a radioprotectant. Thus, sphingolipid metabolism represents a radiosensitivity pivot point, a notion supported by genetic evidence in IR-resistant cancer cells. S1P lyase (SPL) catalyzes the irreversible degradation of S1P in the final step of sphingolipid metabolism. We show that SPL modulates the kinetics of DNA repair, speed of recovery from G2 cell cycle arrest and the extent of apoptosis after IR. SPL acts through a novel feedback mechanism that amplifies stress-induced ceramide accumulation, and downregulation/inhibition of either SPL or ASMase prevents premature cell cycle progression and mitotic death. Further, oral administration of an SPL inhibitor to mice prolonged their survival after exposure to a lethal dose of total body IR. Our findings reveal SPL to be a regulator of ASMase, the G2 checkpoint and DNA repair and a novel target for radioprotection.     

A benzo[b]thiophene-based selective type 4 S1P receptor agonist

S1P receptors (S1PR1-5) are a group of GPCRs activated by a high affinity binding with S1P that have important roles in the regulation of the immune system. A potent S1PR agonist FTY720 is an immunomodulator used to treat multiple sclerosis and several ‘second generation’ drugs are under clinical development. Subtype-selective agonists have been reported for each S1PR isotype, some of which are used as pharmacological tools for functional studies. Here we report the discovery and initial characterization of compound 5c, a benzo[b]thiophene amino carboxylate which exhibits potent and selective agonist activity for S1PR4. Compound 5c has an EC₅₀ = 200 nM as an agonist in GTPγ³⁵S binding assay for S1PR4 and exhibits no activity against S1PR1,2,3,5. We confirmed its potent activity and decent S1PR subtype selectivity using biochemical and cellular assays.     

Individual variation of human S1P1 coding sequence leads to heterogeneity in receptor function and drug interactions

Sphingosine 1-phosphate receptor 1 (S1P₁), an abundantly-expressed G protein-coupled receptor which regulates key vascular and immune responses, is a therapeutic target in autoimmune diseases. Fingolimod/Gilenya (FTY720), an oral medication for relapsing-remitting multiple sclerosis, targets S1P₁ receptors on immune and neural cells to suppress neuroinflammation. However, suppression of endothelial S1P₁ receptors is associated with cardiac and vascular adverse effects. Here we report the genetic variations of the S1P₁ coding region from exon sequencing of >12,000 individuals and their functional consequences. We conducted functional analyses of 14 nonsynonymous single nucleotide polymorphisms (SNPs) of the S1PR1 gene. One SNP mutant (Arg¹²⁰ to Pro) failed to transmit sphingosine 1-phosphate (S1P)-induced intracellular signals such as calcium increase and activation of p44/42 MAPK and Akt. Two other mutants (Ile⁴⁵ to Thr and Gly³⁰⁵ to Cys) showed normal intracellular signals but impaired S1P-induced endocytosis, which made the receptor resistant to FTY720-induced degradation. Another SNP mutant (Arg¹³ to Gly) demonstrated protection from coronary artery disease in a high cardiovascular risk population. Individuals with this mutation showed a significantly lower percentage of multi-vessel coronary obstruction in a risk factor-matched case-control study. This study suggests that individual genetic variations of S1P₁ can influence receptor function and, therefore, infer differential disease risks and interaction with S1P₁-targeted therapeutics.     

The sphingosine 1-phosphate receptor S1P4 regulates cell shape and motility via coupling to Gi and G12/13

Sphingosine 1-phosphate (S1P) receptors represent a novel subfamily of G-protein-coupled receptors binding S1P specifically and with high affinity. Although their in vivo functions remain largely unknown, in vitro extracellular application of S1P induces distinct S1P receptor-dependent cellular responses including proliferation, differentiation, and migration. We have analyzed signaling pathways engaged by S1P(4), which is highly expressed in the lymphoid system. Here we show that S1P(4) couples directly to Galpha(i) and even more effectively to Galpha(12/13)-subunits of trimeric G-proteins, but not to Galpha(q) unlike other S1P receptors. Consequently, CHO-K1 cells ectopically expressing S1P(4) potently activate the small GTPase Rho and undergo cytoskeletal rearrangements, inducing peripheral stress fiber formation and cell rounding, upon S1P stimulation. Overexpression of S1P(4) in Jurkat T cells induces pertussis toxin-sensitive cell motility even in the absence of exogenously added S1P. In addition, S1P(4) is internalized upon binding of S1P. The capacity of S1P(4) to mediate cellular responses, such as motility and shape change through Galpha(i)- and Galpha(12/13)-coupled signaling pathways may be important for its in vivo function which is currently under investigation.     

S1PR3 is essential for phosphorylated fingolimod to protect astrocytes against oxygen‐glucose deprivation‐induced neuroinflammation via inhibiting TLR2/4‐NFκB signalling

Fingolimod (FTY720) is used as an immunosuppressant for multiple sclerosis. Numerous studies indicated its neuroprotective effects in stroke. However, the mechanism remains to be elucidated. This study was intended to investigate the mechanisms of phosphorylated FTY720 (pFTY720), which was the principle active molecule in regulating astrocyte‐mediated inflammatory responses induced by oxygen‐glucose deprivation (OGD). Results demonstrated that pFTY720 could protect astrocytes against OGD‐induced injury and inflammatory responses. It significantly decreased pro‐inflammatory cytokines, including high mobility group box 1 (HMGB1) and tumour necrosis factor‐α (TNF‐α). Further, studies displayed that pFTY720 could prevent up‐regulation of Toll‐like receptor 2 (TLR2), phosphorylation of phosphoinositide 3‐kinase (PI3K) and nuclear translocation of nuclear factor kappa B (NFκB) p65 subunit caused by OGD. Sphingosine‐1‐phosphate receptor 3 (S1PR3) knockdown could reverse the above change. Moreover, administration of TLR2/4 blocker abolished the protective effects of pFTY720. Taken together, this study reveals that pFTY720 depends on S1PR3 to protect astrocytes against OGD‐induced neuroinflammation, due to inhibiting TLR2/4‐PI3K‐NFκB signalling pathway.     

Roles for N-glycosylation in the dynamics of Edg-1/S1P1 in sphingosine 1-phosphate-stimulated cells

Sphingosine 1-phosphate (Sph-1-P) is a bioactive lipid mediator released from activated platelets. To date, 5 seven-transmembrane-spanning receptors, Edg-1/S1P1, Edg-3/S1P3, Edg-5/S1P2, Edg-6/S1P4 and Edg-8/S1P5, have been identified as specific Sph-1-P receptors. Our recent novel studies established that Edg-1/S1P1 is glycosylated in its N-terminal extracellular portion and further identified the specific glycosylation site as asparagine 30. We also demonstrated that the structure of the N-terminal ectodomain of Edg-1/S1P1 affects both its transport to the cell surface and the N-glycosylation process. These studies revealed a possible regulatory role for the N-glycan on Edg-1/S1P1 in the dynamics of the receptor, such as its lateral and internal movements within the membrane, in ligand-stimulated mammalian cells. Published in 2004.     

The role of sphingosine 1‐phosphate and its receptors in cardiovascular diseases

There are many different types of cardiovascular diseases, which impose a huge economic burden due to their extremely high mortality rates, so it is necessary to explore the underlying mechanisms to achieve better supportive and curative care outcomes. Sphingosine 1‐phosphate (S1P) is a bioactive lipid mediator with paracrine and autocrine activities that acts through its cell surface S1P receptors (S1PRs) and intracellular signals. In the circulatory system, S1P is indispensable for both normal and disease conditions; however, there are very different views on its diverse roles, and its specific relevance to cardiovascular pathogenesis remains elusive. Here, we review the synthesis, release and functions of S1P, specifically detail the roles of S1P and S1PRs in some common cardiovascular diseases, and then address several controversial points, finally, we focus on the development of S1P‐based therapeutic approaches in cardiovascular diseases, such as the selective S1PR1 modulator amiselimod (MT‐1303) and the non‐selective S1PR1 and S1PR3 agonist fingolimod, which may provide valuable insights into potential therapeutic strategies for cardiovascular diseases.     

Sphingosine 1-Phosphate (S1P) Receptor Agonists Mediate Pro-fibrotic Responses in Normal Human Lung Fibroblasts via S1P2 and S1P3 Receptors and Smad-independent Signaling

Synthetic sphingosine 1-phosphate receptor 1 modulators constitute a new class of drugs for the treatment of autoimmune diseases. Sphingosine 1-phosphate (S1P) signaling, however, is also involved in the development of fibrosis. Using normal human lung fibroblasts, we investigated the induction of fibrotic responses by the S1P receptor (S1PR) agonists S1P, FTY720-P, ponesimod, and SEW2871 and compared them with the responses induced by the known fibrotic mediator TGF-β1. In contrast to TGF-β1, S1PR agonists did not induce expression of the myofibroblast marker α-smooth muscle actin. However, TGF-β1, S1P, and FTY720-P caused robust stimulation of extracellular matrix (ECM) synthesis and increased pro-fibrotic marker gene expression including connective tissue growth factor. Ponesimod showed limited and SEW2871 showed no pro-fibrotic potential in these readouts. Analysis of pro-fibrotic signaling pathways showed that in contrast to TGF-β1, S1PR agonists did not activate Smad2/3 signaling but rather activated PI3K/Akt and ERK1/2 signaling to induce ECM synthesis. The strong induction of ECM synthesis by the nonselective agonists S1P and FTY720-P was due to the stimulation of S1P₂ and S1P₃ receptors, whereas the weaker induction of ECM synthesis at high concentrations of ponesimod was due to a low potency activation of S1P₃ receptors. Finally, in normal human lung fibroblast-derived myofibroblasts that were generated by TGF-β1 pretreatment, S1P and FTY720-P were effective stimulators of ECM synthesis, whereas ponesimod was inactive, because of the down-regulation of S1P₃R expression in myofibroblasts. These data demonstrate that S1PR agonists are pro-fibrotic via S1P₂R and S1P₃R stimulation using Smad-independent pathways.     

Structural requirements of nociceptin antagonist Ac-RYYRIK-NH2 for receptor binding.

Ac-RYYRIK-NH2 is a peptide isolated from the peptide library as an antagonist that inhibits the biological activities of nociceptin, a hyperalgesic neuropeptide. In order to clarify the structural requirements of this peptide for binding to the nociceptin receptor ORL1, systematic structure-activity studies were carried out. The result of Ala-scanning indicated that the N-terminal tripeptide RYY(= Arg-Tyr-Tyr) is crucially important for binding to the ORL1 receptor. Residual truncations from the N- or C-terminus revealed the special importance of the N-terminal Arg residue. The removal of protecting groups indicated that the N-terminal acetyl group is essential, but the C-terminal amide group is insignificant. These results indicated the conspicuous importance of acetyl-Arg at position 1 of Ac-RYYRIK-NH2 as a key structure allowing binding to the receptor. To investigate the binding site of this peptide in the ORL1 receptor, we synthesized and assayed a series of analogues of the nociceptin dibasic repeat region, residues 8-13 of RKSARK. None of the derivatives were active. Ac-RYYRIK-NH2 was inactive for the mu opioid receptor to which nociceptin binds with considerable strength. All the results suggested that the mode of binding between Ac-RYYRIK-NH2 and the ORL1 receptor is different to that between the ORL1 receptor and nociceptin, and that it may consist of interaction with the receptor site to which nociceptin(1-7) or -(14-17) binds.     

Discovery of S1P agonists with a dihydronaphthalene scaffold

Structure-activity relationship of sphingosine-1-phosphate receptor agonists was examined. Cinnamyl derivative 1 was modified to improve S1P₁ agonistic activity as well as selectivity over S1P₃ agonistic activity. Dihydronaphthalene derivative 10d was identified as a potent S1P₁ receptor agonist with high selectivity against S1P₃ and enhanced efficacy in lowering peripheral lymphocyte counts in mice.     

S1P-lyase independent clearance of extracellular sphingosine 1-phosphate after dephosphorylation and cellular uptake

Sphingosine 1-phosphate (S1P) is the natural ligand for a specific family of G protein-coupled receptors (-Rs). The type 1 S1P-R (S1P(1)) is important for lymphocyte egress, and blood-borne S1P as the natural ligand for S1P(1) is involved in the maintenance of lymphocyte circulation. This report reveals that extracellular S1P was cleared by all tested primary cells and cell lines with exponential progression. Clearance of S1P, but not sphingosine (Sph) was inhibited with the protein phosphatase inhibitor sodium orthovanadate. Fluorescence microscopy and flow cytometry using fluorescently labeled S1P and Sph showed a major cellular uptake of Sph, but not S1P. HPLC-analyses with C17-Sph demonstrated that cellular Sph accumulation was transient in tested cell lines, but enduring in mouse splenocytes. Sub cellular fractionation resulted in dephosphorylation of S1P to Sph by nuclear, membrane, and cytosolic fractions. Degradation of Sph however only occurred in combined membrane and cytosolic fractions. Inhibitors for Sph kinases 1/2, ceramide synthase, and S1P-lyase, as well as S1P-lyase deficiency did not block clearance of extracellular S1P. In vivo experiments revealed a transient increase in plasma S1P levels after single intravenous injection into C57BL/6 mice. This exogenously added S1P was cleared within 15-30 min in contrast to ex vivo incubation of whole blood which required more than 8 h for comparable clearance from plasma. Our data thus show that extracellular S1P is dephosphorylated and subsequently converted by cells, which appears to be important for clearance of the signaling molecule S1P in the local tissue environment after infections or injuries.     

鞘氨醇激酶-磷酸鞘氨醇轴在血管生成相关性疾病中的作用

血管生成是指在原有血管的基础上形成新血管的过程.病理性血管生成是癌症、心血管类疾病和视网膜病变等一系列疾病的标志.1-磷酸鞘氨醇(sphingosine-1-phosphate,S1P)是一种信号脂质,由鞘氨醇激酶(sphingosine kinases,SPHK)合成,通过5种G蛋白偶联受体(sphingosine-...     

Sphingosine‐1‐phosphate (S1P) enhances glomerular endothelial cells activation mediated by anti‐myeloperoxidase antibody‐positive IgG

Cumulating evidences suggested an important role of sphingosine‐1‐phosphate (S1P) and its receptors in regulating endothelial barrier integrity. Our previous study revealed that the circulating S1P levels and renal expression of S1PRs correlated with disease activity and renal damage in patients with antineutrophil cytoplasmic antibody (ANCA)‐associated vasculitis (AAV). This study investigated the role of S1P and its receptors in myeloperoxidase (MPO)‐ANCA‐positive IgG‐mediated glomerular endothelial cell (GEnC) activation. The effect of S1P on morphological alteration of GEnCs in the presence of MPO‐ANCA‐positive IgG was observed. Permeability assay was performed to determine endothelial monolayer activation in quantity. Both membrane‐bound and soluble ICAM‐1 and VCAM‐1 levels were measured. Furthermore, antagonists and/or agonists of various S1PRs were employed to determine the role of different S1PRs. S1P enhanced MPO‐ANCA‐positive IgG‐induced disruption of tight junction and disorganization of cytoskeleton in GEnCs. S1P induced further increase in monolayer permeability of GEnC monolayers in the presence of MPO‐ANCA‐positive IgG. S1P enhanced MPO‐ANCA‐positive IgG‐induced membrane‐bound and soluble ICAM‐1/VCAM‐1 up‐regulation of GEnCs. Soluble ICAM‐1 levels in the supernatants of GEnCs stimulated by S1P and MPO‐ANCA‐positive IgG increased upon pre‐incubation of S1PR1 antagonist, while pre‐incubation of GEnCs with the S1PR1 agonist down‐regulated sICAM‐1 level. Blocking S1PR2‐4 reduced sICAM‐1 levels in the supernatants of GEnCs stimulated by S1P and MPO‐ANCA‐positive IgG. Pre‐incubation with S1PR5 agonist could increase sICAM‐1 level in the supernatants of GEnC stimulated by S1P and MPO‐ANCA‐positive IgG. S1P can enhance MPO‐ANCA‐positive IgG‐mediated GEnC activation through S1PR2‐5.     

Second generation S1P pathway modulators: Research strategies and clinical developments

Multiple Sclerosis (MS) is a chronic autoimmune disorder affecting the central nervous system (CNS) through demyelination and neurodegeneration. Until recently, major therapeutic treatments have relied on agents requiring injection delivery. In September 2010, fingolimod/FTY720 (Gilenya, Novartis) was approved as the first oral treatment for relapsing forms of MS. Fingolimod causes down-modulation of S1P1 receptors on lymphocytes which prevents the invasion of autoaggressive T cells into the CNS. In astrocytes, down-modulation of S1P1 by the drug reduces astrogliosis, a hallmark of MS, thereby allowing restoration of productive astrocyte communication with other neural cells and the blood brain barrier. Animal data further suggest that the drug directly supports the recovery of nerve conduction and remyelination. In human MS, such mechanisms may explain the significant decrease in the number of inflammatory markers on brain magnetic resonance imaging in recent clinical trials, and the reduction of brain atrophy by the drug. Fingolimod binds to 4 of the 5 known S1P receptor subtypes, and significant efforts were made over the past 5 years to develop next generation S1P receptor modulators and determine the minimal receptor selectivity needed for maximal therapeutic efficacy in MS patients. Other approaches considered were competitive antagonists of the S1P1 receptor, inhibitors of the S1P lyase to prevent S1P degradation, and anti-S1P antibodies. Below we discuss the current status of the field, and the functional properties of the most advanced compounds. This article is part of a Special Issue entitled New Frontiers in Sphingolipid Biology.