Sphingosine 1-phosphate receptors

Sphingosine-1-phosphate (SPP) is a bioactive lipid produced from the metabolism of sphingomyelin. It is an important constituent of serum and regulates cell growth, survival, migration, differentiation and gene expression. Its mode of action has been enigmatic; however, recent findings have shown that a family of G-protein-coupled receptors (GPCR) of the endothelial differentiation gene (EDG) family serve as plasma membrane-localized receptors for SPP. Furthermore, the EDG receptors appear to be SPP receptor subtypes with distinct signaling characteristics. In vascular endothelial cells, SPP acts on EDG-1 and EDG-3 subtypes of receptors to induce cell survival and morphogenesis. Such pathways appear to be critical for SPP-induced angiogenic response in vivo. In addition, the EDG-1 gene is essential for vascular maturation in development. Moreover, developmental studies in Zebrafish have indicated that SPP signaling via the EDG-5 like receptor Miles Apart (Mil) is essential for heart development. These data strongly suggest that a physiological role of SPP is in the formation of the cardiovascular system. Despite these recent findings, much needs to be clarified with respect to the physiological role of SPP synthesis and action. This review will focus on the recent findings on SPP receptors and the effects on the cardiovascular system.     

Sphingosine 1-phosphate signalling and termination at lipid phosphate receptors

Sphingosine 1-phosphate (S1P) is a polar lysophospholipid metabolite that is stored in platelets and released upon their activation. However, diverse stimuli such as growth factors, cytokines, G-protein coupled receptor (GPCR) agonists and antigens have been shown to increase sphingosine kinase activity and S1P formation in other cell types, such as smooth muscle. Indeed, S1P has been implicated in the regulation of several important cellular processes, such as proliferation, differentiation, apoptosis and migration in these cells. Over the past few years, there has been a major advance in our understanding of how S1P can act as an intercellular mediator by binding to a new class of G-protein coupled receptors to regulate cell function. This review focuses on the enzymatic regulation of S1P formation and degradation and its interaction with a novel tethered receptor complex containing the S1P receptor (S1P(1)) and the platelet-derived growth factor (PDGF) beta receptor. This tethered receptor complex enables coincident integrative signalling to p42/p44 MAPK. This is compared with a sequential model in which PDGF promotes S1P release, which in turn acts on S1P(1) to promote Rac signalling.     

Sphingosine 1-phosphate receptors modulate intracellular Ca2+ homeostasis

Ligation of sphingosine 1-phosphate (S1P) to a set of specific receptors named S1P receptors (S1PRs) regulates important biological processes. Although the ability of S1P to increase cytosolic Ca2+ in various cell types is well known, the role of the individual S1PRs has not been fully characterized. Here, we provide a complete analysis of S1P-dependent intracellular Ca2+ homeostasis in HeLa cells. Overexpression of S1P2, or S1P3, but not S1P1, leads to a significant increase in cytosolic and mitochondrial [Ca2+] in response to S1P challenge. Moreover, cells ectopically expressing S1P2, or S1P3 exhibited an appreciable decrease of the free Ca2+ concentration in the endoplasmic reticulum, dependent on stimulation of receptors by S1P endogenously present in the culture medium which was accompanied by a reduced susceptibility to C2-ceramide-induced cell death. These results demonstrate a differential contribution of individual S1PRs to Ca2+ homeostasis and its possible implication in the regulation of cell survival.     

Sphingosine 1-phosphate chemical biology

A dozen years ago, the term 'S1P' (sphingosine 1-phosphate) was not in the lexicons of scientific literature databases. By early 2008, this query term retrieved well over 1000 citations from PubMed - about 225 of these appeared in 2007. Indeed, S1P is arguably the most heavily studied lipid molecule at present. What happened to distinguish S1P among many other signaling lipids? We believe that the seminal event was the linking of the investigational drug, FTY720 (fingolimod), to S1P signaling. This realization profoundly altered understanding of S1P biology, revealing both that S1P is prominent in lymphocyte trafficking and that mimicking S1P signaling with an agonist drug can modulate the immune system to considerable therapeutic benefit. Neither fact was known prior to FTY720; indeed, this molecule is testament to the power of chemical biology. In this communication, we attempt to summarize progress to date in S1P chemical biology.     

Sphingosine 1-phosphate and its receptors: an autocrine and paracrine network

Sphingosine 1-phosphate (S1P) is a biologically active lysophospholipid that transmits signals through a family of G-protein-coupled receptors to control cellular differentiation and survival, as well as the vital functions of several types of immune cell. In this Review article, we discuss recent results that indicate that S1P and its receptors are required for the emigration of thymocytes from the thymus, the trafficking of lymphocytes in secondary lymphoid organs and the migration of B cells into splenic follicles. In an autocrine manner, through interactions with different G-protein-coupled receptors, S1P also enhances optimal mast-cell migration and release of pro-inflammatory mediators in allergic reactions. S1P-S1P-receptor regulatory systems might therefore be novel targets for the therapy of diverse immunological diseases.     

Sphingosine 1-phosphate signalling in cancer

There is an increasing body of evidence demonstrating a critical role for the bioactive lipid S1P (sphingosine 1-phosphate) in cancer. S1P is synthesized and metabolized by a number of enzymes, including sphingosine kinase, S1P lyase and S1P phosphatases. S1P binds to cell-surface G-protein-coupled receptors (S1P1-S1P5) to elicit cell responses and can also regulate, by direct binding, a number of intracellular targets such as HDAC (histone deacetylase) 1/2 to induce epigenetic regulation. S1P is involved in cancer progression including cell transformation/oncogenesis, cell survival/apoptosis, cell migration/metastasis and tumour microenvironment neovascularization. In the present paper, we describe our research findings regarding the correlation of sphingosine kinase 1 and S1P receptor expression in tumours with clinical outcome and we define some of the molecular mechanisms underlying the involvement of sphingosine kinase 1 and S1P receptors in the formation of a cancer cell migratory phenotype. The role of sphingosine kinase 1 in the acquisition of chemotherapeutic resistance and the interaction of S1P receptors with oncogenes such as HER2 is also reviewed. We also discuss novel aspects of the use of small-molecule inhibitors of sphingosine kinase 1 in terms of allosterism, ubiquitin-proteasomal degradation of sphingosine kinase 1 and anticancer activity. Finally, we describe how S1P receptor-modulating agents abrogate S1P receptor-receptor tyrosine kinase interactions, with potential to inhibit growth-factor-dependent cancer progression.     

Sphingosine 1-phosphate breakdown in platelets

We examined the formation of sphingolipid mediators in platelets, which abundantly store, and release extracellularly, sphingosine 1-phosphate (Sph-1-P). Challenging [(3)H]Sph-labeled platelet suspensions with thrombin or 12-O-tetradecanoylphorbol 13-acetate (TPA) resulted in a decrease in Sph-1-P formation and an increase in sphingosine (Sph), ceramide (Cer), and sphingomyelin formation. Sph conversion into Cer, and Cer conversion into sphingomyelin were not affected upon activation, suggesting that Sph-1-P dephosphorylation may initiate the formation of sphingolipid signaling molecules. In fact, Sph-1-P phosphatase (but not lyase) activity was detected in platelets, but this activity was not enhanced by thrombin or TPA. When quantified with [(3)H]acetic anhydride acetylation, followed by HPLC separation, the amounts of Sph-1-P and Sph decreased and increased, respectively, upon stimulation with thrombin or TPA, and these changes were attenuated by staurosporine. Under these TPA treatment conditions, over half of the [(3)H]Sph-1-P (formed in platelets incubated with [(3)H]Sph) was detected extracellularly, possibly due to its release from platelets, which was completely inhibited by staurosporine pretreatment. Furthermore, when TPA-induced Sph-1-P release was blocked by staurosporine after the stimulation, the extracellular [(3)H]Sph-1-P radioactivity decreased, suggesting that the Sph-1-P released may undergo dephosphorylation extracellularly. To support this, [(32)P]Sph-1-P, when added extracellularly to platelet suspensions, was rapidly degraded, possibly due to the ecto-phosphatase activity. Our results suggest the presence in anucleate platelets of a transmembrane cycling pathway starting with Sph-1-P dephosphorylation and leading to the formation of other sphingolipid mediators.     

Sphingosine 1-phosphate and its G protein-coupled receptors constitute a multifunctional immunoregulatory system

The lysophospholipid growth factors sphingosine 1-phosphate (S1P) and lysophosphatidic acid (LPA) are generated by many cells involved in immunity, including macrophages, dendritic cells, mast cells, and platelets, with resultant lymph and plasma concentrations of 0.1-1 microM. All immune cells express distinctive profiles of G protein-coupled receptors (GPCRs) for S1P and LPA, which are regulated developmentally and by cellular activation. For T-cells, constitutive S1P signaling through their principal S1P(1) GPCR inhibits chemotactic responses to chemokines, with lesser suppression of proliferation and cytokine production. These S1P-S1P(1) GPCR signals tonically reduce T-cell chemotactic sensitivity to chemokines and thereby limit homing of blood and spleen T-cells to secondary lymphoid tissues. S1P(1) GPCR antagonists evoke lymphopenia by permitting blood T-cells to enter lymph nodes and blocking S1P(1) GPCR-dependent T-cell efflux from lymph nodes. Inversely, there is a longer than normal persistance in blood and a decrease in lymphoid transit time for T-cells overexpressing transgenic S1P(1) GPCRs. The immunotherapeutic potential of S1P(1) GPCR antagonists derives from their capacity to limit T-cell access to organ grafts and autoimmune antigens without reducing their other intrinsic functional capabilities. Lysophospholipids and their GPCRs thus constitute an immunoregulatory system of sufficient prominence for pharmacological targeting in transplantation, autoimmunity and immunodeficiency.     

Modulators of the Sphingosine 1-phosphate receptor 1

The Sphingosine 1-phosphate receptor (S1P-R) signaling system has proven to be of biological and medical importance in autoimmune settings. S1P₁-R is a validated drug target for multiple sclerosis (MS) for which FTY720 (Fingolimod), a S1P_1,3–5-R pan-agonist, was recently approved as the first orally active drug for the treatment of relapsing-remitting MS. Transient bradycardia and long half-life are the FTY720 critical pitfalls. This review provides the latest advances on next-generation S1P₁-R modulators from 2012 up to date, with an overview of the chemical structures, structure–activity relationships, and relevant biological and clinical properties.     

Sphingosine 1-phosphate: synthesis and release

Sphingosine 1-phosphate (Sph-1-P) is a bioactive sphingolipid, acting both as an intracellular second messenger and extracellular mediator, in mammalian cells. In cell types where Sph-1-P acts as an intracellular messenger, stimulation-dependent synthesis of Sph-1-P, possibly resulting from sphingosine (Sph) kinase activation, is essential. Since this important kinase has recently been cloned, precise regulation of intracellular Sph-1-P synthesis will be clarified in the near future. As an intercellular mediator, elucidation of sources for extracellular Sph-1-P is important, in addition to identification of the cell surface receptors for this phospholipid. Blood platelets are very unique in that they store Sph-1-P abundantly (possibly due to the existence of highly active Sph kinase and a lack of Sph-1-P lyase) and release this bioactive lipid extracellularly upon stimulation. It is likely that platelets are an important source for extracellular Sph-1-P, especially for plasma and serum Sph-1-P. Platelet-derived Sph-1-P seems to play an important role in vascular biology.     

Sphingosine 1-phosphate metabolism and insulin signaling

Insulin is the main anabolic hormone secreted by β-cells of the pancreas stimulating the assimilation and storage of glucose in muscle and fat cells. It modulates the postprandial balance of carbohydrates, lipids and proteins via enhancing lipogenesis, glycogen and protein synthesis and suppressing glucose generation and its release from the liver. Resistance to insulin is a severe metabolic disorder related to a diminished response of peripheral tissues to the insulin action and signaling. This leads to a disturbed glucose homeostasis that precedes the onset of type 2 diabetes (T2D), a disease reaching epidemic proportions. A large number of studies reported an association between elevated circulating fatty acids and the development of insulin resistance. The increased fatty acid lipid flux results in the accumulation of lipid droplets in a variety of tissues. However, lipid intermediates such as diacylglycerols and ceramides are also formed in response to elevated fatty acid levels. These bioactive lipids have been associated with the pathogenesis of insulin resistance. More recently, sphingosine 1-phosphate (S1P), another bioactive sphingolipid derivative, has also been shown to increase in T2D and obesity. Although many studies propose a protective role of S1P metabolism on insulin signaling in peripheral tissues, other studies suggest a causal role of S1P on insulin resistance. In this review, we critically summarize the current state of knowledge of S1P metabolism and its modulating role on insulin resistance. A particular emphasis is placed on S1P and insulin signaling in hepatocytes, skeletal muscle cells, adipocytes and pancreatic β-cells. In particular, modulation of receptors and enzymes that regulate S1P metabolism can be considered as a new therapeutic option for the treatment of insulin resistance and T2D.     

Sphingosine 1-phosphate modulates spinal nociceptive processing

Sphingosine 1-Phosphate (S1P) modulates various cellular functions such as apoptosis, cell differentiation, and migration. Although S1P is an abundant signaling molecule in the central nervous system, very little is known about its influence on neuronal functions. We found that S1P concentrations were selectively decreased in the cerebrospinal fluid of adult rats in an acute and an inflammatory pain model. Pharmacological inhibition of sphingosine kinases (SPHK) decreased basal pain thresholds and SphK2 knock-out mice, but not SphK1 knock-out mice, had a significant decrease in withdrawal latency. Intrathecal application of S1P or sphinganine 1-phosphate (dihydro-S1P) reduced the pain-related (nociceptive) behavior in the formalin assay. S1P and dihydro-S1P inhibited cyclic AMP (cAMP) synthesis, a key second messenger of spinal nociceptive processing, in spinal cord neurons. By combining fluorescence resonance energy transfer (FRET)-based cAMP measurements with Multi Epitope Ligand Cartography (MELC), we showed that S1P decreased cAMP synthesis in excitatory dorsal horn neurons. Accordingly, intrathecal application of dihydro-S1P abolished the cAMP-dependent phosphorylation of NMDA receptors in the outer laminae of the spinal cord. Taken together, the data show that S1P modulates spinal nociceptive processing through inhibition of neuronal cAMP synthesis.     

Sphingosine 1-phosphate analogs as receptor antagonists

Sphingosine 1-phosphate (S1P) is a lysophospholipid mediator that evokes a variety of cell and tissue responses via a set of cell surface receptors. The recent development of S1P receptor agonists, led by the immunomodulatory pro-drug FTY720, has revealed that S1P signaling is an important regulator of lymphocyte trafficking. With the twin goals of understanding structure-activity relationships of S1P ligands and developing tool compounds to explore S1P biology, we synthesized and tested numerous S1P analogs. We report herein that a subset of our aryl amide-containing compounds are antagonists at the S1P(1) and S1P(3) receptors. The lead compound in series, VPC23019, was found in broken cell and whole cell assays to behave as a competitive antagonist at the S1P(1) and S1P(3) receptors. The structure-activity relationship of this series is steep; for example, a slight modification of the lead compound resulted in VPC25239, which was one log order more potent at the S1P(3) receptor. These new chemical entities will enable further understanding of S1P signaling and provide leads for further S1P receptor antagonist development.     

Sphingosine 1-phosphate: synthesis and release

Sphingosine 1-phosphate (Sph-1-P) is a bioactive sphingolipid, acting both as an intracellular second messenger and extracellular mediator, in mammalian cells. In cell types where Sph-1-P acts as an intracellular messenger, stimulation-dependent synthesis of Sph-1-P, possibly resulting from sphingosine (Sph) kinase activation, is essential. Since this important kinase has recently been cloned, precise regulation of intracellular Sph-1-P synthesis will be clarified in the near future. As an intercellular mediator, elucidation of sources for extracellular Sph-1-P is important, in addition to identification of the cell surface receptors for this phospholipid. Blood platelets are very unique in that they store Sph-1-P abundantly (possibly due to the existence of highly active Sph kinase and a lack of Sph-1-P lyase) and release this bioactive lipid extracellularly upon stimulation. It is likely that platelets are an important source for extracellular Sph-1-P, especially for plasma and serum Sph-1-P. Platelet-derived Sph-1-P seems to play an important role in vascular biology.     

Sphingosine 1-phosphate stimulates proliferation and migration of satellite cells: role of S1P receptors

Satellite cells are resident stem cells of skeletal muscle; they are normally quiescent but upon post-trauma activation start to proliferate and fuse with damaged fibers contributing to muscle regeneration. In this study the effect of the bioactive sphingolipid sphingosine 1-phosphate (S1P) on the proliferative and migratory response of murine satellite cells has been examined. S1P was found to stimulate labeled thymidine incorporation in a phosphatidylinositol 3-kinase-dependent manner. Moreover, by employing selective S1P receptor agonists and antagonists and silencing individual S1P receptors, the mitogenic action of S1P in satellite cells was shown to depend on S1P2 and S1P3. Notably, by using different experimental approaches S1P was found to positively influence satellite cell migration, necessary for their recruitment at the site of muscle damage. Interestingly, the specific silencing of individual S1P receptor subtypes demonstrated the pivotal role of S1P1 and S1P4 in mediating the S1P migratory effect. This latter result demonstrates for the first time that S1P4 receptor has a role in skeletal muscle cells, supporting the notion that this receptor subtype plays a biological action broader than that so far identified in lymphoid tissue. On the contrary, S1P2 was found to negatively regulate cell migration. Collectively, these results are in favour of an important function of S1P in satellite cell biology that could in principle be exploited as novel pharmacological target for improving skeletal muscle regeneration.     

Sphingosine kinase and sphingosine 1-phosphate in asthma

Sphingolipids are amphiphatic molecules ubiquitously expressed in all eukaryotic cell membranes. Initially characterized as structural components of cell membranes, sphingolipids have emerged as sources of important signalling molecules over the past decade. Sphingolipid metabolites, such as ceramide and S1P (sphingosine 1-phosphate), have been demonstrated to have roles as potent bioactive messengers involved in cell differentiation, proliferation, apoptosis, migration and angiogenesis. The importance of SphK (sphingosine kinase) and S1P in inflammation has been demonstrated extensively. The prevalence of asthma is increasing in many developed nations. Consequently, there is an urgent need for the development of new agents for the treatment of asthma, especially for patients who respond poorly to conventional therapy. Recent studies have demonstrated the important role of SphK and S1P in the development of asthma by regulating pro-inflammatory responses. These novel pathways represent exciting potential therapeutic targets in the treatment of asthma and are described in the present review.     

Sphingosine 1-phosphate and control of vascular tone

Sphingosine-1-phosphate (S1P) is a platelet-derived lipid mediator that activates the endothelial isoform of nitric oxide synthase (eNOS) in endothelial cells. However, the role of S1P in endothelium-dependent vasodilation and the signaling pathways elicited by S1P in intact vessels are largely unknown. We found that S1P induces dose-dependent transient relaxation of isolated pressurized mesenteric arterioles (EC(50) 10 +/- 3 nM); maximal vasodilation (55 +/- 8%) is seen approximately 2 min after S1P addition and returns to baseline by 5 min. S1P promotes comparable responses in arterioles from wild-type but not eNOS(null) mice. S1P-induced vasodilation is abrogated by removal of endothelium or by the addition of the NOS inhibitor N(omega)-monomethyl-l-arginine but is not affected by the cyclooxygenase inhibitor indomethacin, nor by the blockade of K(+) channels by using 4-aminopyridine. S1P-induced vasodilation is attenuated by pertussis toxin, by the phosphoinositide 3-kinase (PI3-kinase) inhibitor wortmannin, and by the calcium chelator BAPTA. With the use of high-sensitivity protein immunoblots in extracts from single pressurized vessels, we found that S1P, but not bradykinin, promotes the phosphorylation of eNOS at Ser(1179). Maximum S1P-induced eNOS Ser(1179) phosphorylation was reached at the time of maximum vasorelaxation, but enzyme phosphorylation persisted for several minutes after vasodilation had resolved. Thus regulatory pathways distinct from eNOS Ser(1179) dephosphorylation serve to terminate agonist-promoted vasorelaxation. Taken together, our findings demonstrate that S1P, an important intercellular mediator of platelet-vessel wall interactions, is a effective arteriolar vasodilator that acts via G protein-dependent, calcium-sensitive, and PI3-kinase-modulated signaling pathways.     

Sphingosine kinases, sphingosine-1-phosphate and sphingolipidomics

It has become abundantly clear over the past decade that sphingolipids and their metabolites are key signaling molecules. Ceramide, the backbone of all sphingolipids, predominantly inhibits cell growth and induces apoptosis, while its metabolite, sphingosine-1-phosphate promotes growth and survival. Given the interconvertibility of these two opposing signaling molecules, it is essential that any study that examines the effects of one also look at the other. The newly available technology of liquid chromatography-tandem mass spectroscopy (LC-MS/MS) is increasingly being applied for this purpose, as it can quickly identify and measure many different sphingolipids simultaneously. An added benefit of LC-MS/MS is that it is several orders of magnitude more sensitive than enzymatic methods or more traditional chromatographic techniques, allowing smaller sample sizes and increased throughput. Here, we briefly discuss the importance of LC-MS/MS for measuring sphingolipid metabolites and some future directions researchers may take given the increasingly accessibility to this technology.     

Sphingosine 1-phosphate signal transduction in muscle cells

Sphingosine 1-phosphate (S1P) is a powerful bioactive sphingolipid recently recognized to act as extracellular ligand for various subtypes of G protein-coupled receptors belonging to the S1P family. In our study, focused on mouse skeletal muscle cells, we showed that S1P activated enzymes crucial for membrane signal transduction, such as phospholipase D (PLD) and protein kinase C; it promoted also a significant increase of cytosolic Ca2+ via ligation to S1P2 and S1P3 receptor subtypes. Interestingly, myogenic differentiation was found to be accompanied by a profound variation of S1P receptor expression levels and the progressive uncoupling of S1P from PLD activation, suggesting that this signaling pathway is exclusively required for S1P action on proliferating cells.     

Sphingosine 1-phosphate receptors regulate chemokine-driven transendothelial migration of lymph node but not splenic T cells

Chemokines and chemokine receptors are required for T cell trafficking and migration. Recent evidence shows that sphingosine 1-phosphate (S1P) and S1PRs are also important for some aspects of T cell migration, but how these two important receptor-ligand systems are integrated and coregulated is not known. In this study, we have investigated CCL19-CCR7 and CXCL12-CXCR4-driven migration of both splenic and peripheral lymph node (PLN) nonactivated and naive T cells, and used both S1P and the S1PR ligand, FTY720, to probe these interactions. The results demonstrate that splenic T cell migration to CCL19 or CXCL12 is enhanced by, but does not require, S1PR stimulation. In contrast, PLN T cell migration to CXCL12, but not CCL19, requires both chemokine and S1PR stimulation, and the requirement for dual receptor stimulation is particularly important for steps involving transendothelial migration. The results also demonstrate that: 1) splenic and PLN nonactivated and naive T cells use different molecular migration mechanisms; 2) CCR7 and CXCR4 stimulation engage different migration mechanisms; and 3) S1P and FTY720 have distinct S1PR agonist and antagonist properties. The results have important implications for understanding naive T cell entry into and egress from peripheral lymphoid organs, and we present a model for how S1P and chemokine receptor signaling may be integrated within a T cell.