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Sphingosine 1-phosphate (S1P) is a membrane-derived lysophospholipid that acts primarily as an extracellular signaling molecule. Signals initiated by S1P are transduced by five G protein-coupled receptors, named S1P1–5. Cellular and temporal expression of the S1P receptors (S1PRs) determine their specific roles in various organ systems, but they are particularly critical for regulation of the cardiovascular, immune, and nervous systems, with the most well-known contributions of S1PR signaling being modulation of vascular barrier function, vascular tone, and regulation of lymphocyte trafficking. However, our knowledge of S1PR biology is rapidly increasing as they become attractive therapeutic targets in several diseases, such as chronic inflammatory pathologies, autoimmunity, and cancer. Understanding how the S1PRs regulate interactions between biological systems will allow for greater efficacy in this novel therapeutic strategy as well as characterization of complex physiological networks. Because of the rapidly expanding body of research, this review will focus on the most recent advances in S1PRs.
Sphingosine 1-phosphate [2S-amino-1-(dihydrogen phosphate)-4E-octadecene-1,3R-diol] (S1P) is a simple membrane-derived lysophospholipid with regulatory roles in almost all facets of mammalian biology (
). Expression patterns of the five S1P receptors (S1PRs) vary in tissues and also during development and ageing. S1P1, S1P2, and S1P3 are essentially ubiquitously expressed, whereas expression of S1P4 and S1P5 are highly restricted to distinct cell types (
). How albumin or ApoM deliver S1P to specific S1PRs has yet to be characterized.
AGONISTS AND ANTAGONISTS
There are several well-characterized agonists and antagonists of S1PRs; however, most compounds have been directed toward modulating the activity of S1P1. FTY720 is the prototypical S1PR agonist and was approved by the US Food and Drug Administration as a first line oral therapy for relapsing-remitting multiple sclerosis (MS) (
). Although FTY720 acts as an agonist at picomolar to nanomolar concentrations on S1P1 and S1P3–5, it also acts as a functional antagonist for S1P1 by inducing receptor endocytosis and degradation of this receptor (
). Initial results from rodent studies indicated that FTY720 phosphate activation of S1P3 was responsible for both bradycardia and hypertension; however, treatment of humans with more selective agonists indicated that S1P1 agonism was responsible for reduced heart rate, whereas S1P3 signaling contributed to the development of hypertension (
). The divergent utilization of S1P1 and S1P3 in rodents versus primates for the regulation of these coordinated physiological functions highlights the difficulties encountered upon extrapolation from rodent model-based characterization of S1PR function to human disease therapies.
SEW2871 is an S1P1-specific agonist that activates ERK1/2, AKT, and Rac signals at nanomolar concentrations and induces receptor internalization and recycling; however, it has a relatively short half-life in vivo (
). The only known compound with activity at S1P2 is JTE-013, an antagonist with an IC50 of approximately 20 nM, which blocks S1P2 signaling through Rho-associated protein kinase (ROCK) and phosphatase and tensin homologue (
Many effects of S1P on the vasculature are due to expression of S1P1 by the endothelium. S1P1, originally named EDG1 (endothelial differentiation gene), was discovered during a search for immediate early genes regulating endothelial cell differentiation (
). The generation of inducible cell-specific S1P1 knockout mice has clarified the roles of endothelial cells (ECs) or VSMC S1P1 in the regulation of postnatal vascular development, maturation, and function. In the developing retinal vasculature, S1P1 expression is restricted to the ECs and increases with vessel maturity, as the lowest levels of expression are found at the vascular leading front (
). Postnatal deletion of EC S1pr1 did not affect mural cell recruitment or vessel coverage in the retina; however, angiogenic hypersprouting occurred, characterized by dilated vessels and increases in the number of branch points and tip cells. Induced over-expression of EC S1P1 suppressed vascular sprouting (
). Changes in the vascular architecture of EC S1pr1−/− mice were accompanied by increased vascular permeability, resulting from altered vascular endothelial cadherin localization at endothelial cell-cell junctions (
). These data confirmed numerous earlier in vitro studies describing the necessity of EC S1P1 for the maintenance of vascular barrier function through adherens junction formation induced by activation of Rac after Gαi coupling to S1P1 (Fig. 2) (
Maintenance and formation of adherens junctions was dependent on S1P1 signaling initiated not only by ligand, but also by fluid shear stress (Fig. 2). Examination of murine aortae found that areas of turbulent flow (the lesser curvature) had poor endothelial cell alignment and S1P1 relocalized from the EC surface to endocytic vesicles, whereas in the descending aorta, an area of laminar flow, S1P1 and vascular endothelial cadherin colocalized to the cell surface (
). Additionally, maintenance of vascular homeostasis by the endothelial glycocalyx, which is also susceptible to changes in flow dynamics, was dependent upon S1P1-induced inhibition of matrix metalloproteinase (
Mice with endothelium-specific deletion of S1P1 developed severe pathology in a model of renal ischemia/reperfusion injury, both in the kidneys and the liver, characterized by elevated plasma creatinine, alanine transferase, and tissue necrosis (
). Conversely, of the five S1PRs, S1P2 mRNA in the kidney was most increased upon renal ischemia/reperfusion, and mice deficient in S1P2 developed significantly less pathology compared with WT controls (
). When S1pr2−/− mice were treated with the S1P1 antagonist, W146, before ischemia/reperfusion, they were no longer protected from renal injury, suggesting that S1P1 and S1P2 in the renal vasculare endothelium play protective and injurious roles, respectively, in kidney injury and disease (
The pro-inflammatory tendency of S1P2 is supported by in vitro studies suggesting a paracrine feedback loop involving EC TNFα induction of S1P2 expression leading to activation of nuclear factor (NF)-κB and increases in intracellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 (
). In vivo studies utilizing S1pr2−/− mice and a model of acute inflammation, endotoxemia, further support the conclusion that S1P2 is an important regulator of vascular activation and therefore, permeability (
). Induction of endotoxemia in mice lacking S1pr2 in the stroma and not in the bone marrow (BM) compartment resulted in decreased vascular permeability, VCAM-1 and ICAM-1 expression, and more rapid resolution (
). Examination of murine iliac collecting lymph vessels demonstrated that while S1P does not induce nitric oxide or prostaglandin release, signaling via S1P2 regulates tonic contractility of lymph vessels, as shown using S1P2 inhibition by JTE013 (
). However, this is not the only role for S1P1 in lymphocytes, and roles for the other four S1PRs in the immune system have recently been revealed.
The contribution of S1PRs to regulation of the immune response has been studied extensively in the context of experimental autoimmune encephalomyelitis (EAE), the most commonly used animal model of MS (
). Although EAE and MS are considered to be primarily diseases of the immune system, the role of S1PRs on neural cells is also gaining an appreciation and will be discussed later. FTY720 is a Sph analog that is phosphorylated, acts on S1P1,3–5, and was the first US Food and Drug Administration approved oral therapy for MS (
). However, T cell S1P1 may also regulate the activation and differentiation status of these immune cells. Deletion of T cell S1P1 significantly suppresses the ability of these cells to be polarized to T-helper (Th)17 in vitro (
). Conversely, when EAE was induced in mice expressing an internalization-defective S1P1 (S5A), this significantly increased polarization of T cells to the Th17 phenotype resulting in increased disease pathology and immune cell infiltration into the CNS (
). In collagen-induced models of rheumatoid arthritis, a S1P1-specific antagonist prevented or ameliorated disease by upregulating lymphocyte CD69 expression, which downregulates S1P1 surface expression, blocking thymic egress (
). S1P1 suppresses Treg development via the AKT/mammalian target of rapamyacin pathway and affects their migration from the thymus and out of the periphery by counteracting CCR7 retention signals, similar to the mechanism regulating the egress of effector T cells from lymph nodes (
). In a specific subset of T memory cells, nonlymphoid resident memory cells (TRM), cytokines that induce the TRM phenotype also downregulate the transcription factor Krüppel-like factor2 and its target gene, S1pr1 (
). Subsequently, TRM are unable to sense S1P in circulation and are maintained in the periphery.
Although S1P1 has been the focus of much research, not much is known of the roles of the other S1PRs. In CD8 effector T cells, S1P4 may influence their trafficking to lymph nodes (LNs), although it appears not to be a primary regulator (
S1PR expression choreographs many aspects of B cell subset localization within lymphoid organs, thereby affecting their functionality; however, there are some direct effects of S1P signaling on B cell survival (Fig. 3) (
). While S1P1 has some regulatory functions in B cells, it appears that S1P2 has a greater impact on these cells. Aged S1pr2−/− mice develop diffuse large B cell lymphoma (DLBCL), characterized by increased germinal center (GC) B cells and spontaneous GC formation, which correlates with an approximate 26% mutation incidence for S1PR2 in human DLBCL (
). Additionally, trafficking of marginal zone (MZ) B cells between the MZ and the follicle is regulated by S1P1, which maintains these cells in the MZ in order for them to capture blood-borne antigens (
). However, it may be important for positioning of immature B cells and their progenitors within the BM, whereas S1P1 participates in directing their migration from the BM parenchyma into sinusoids and subsequently into circulation (
). S1P5 normally antagonizes NK CXCR4 BM retention signals, and S1pr5−/− mice have decreased numbers of NK cells in the periphery and increased numbers in lymph nodes and BM due to defective migration (
). This phenotype is also observed in the mouse model of Niemann-Pick disease type C, a lysosomal storage disorder presenting as an accumulation of cholesterol and sphingolipids in the lysosome and decreased concentrations of circulating S1P in human patients (
). Studies utilizing FTY720 indicated that S1P1 also contributes to NK cell migration from LN to lymph, but the contribution is relatively minor compared with that of S1P5, which is not subject to CD69 regulation (
). Decidual NK (dNK) cells are a specialized NK cell subset that regulates trophoblast invasion during early pregnancy by secreting pro-angiogenic and growth factors, including vascular endothelial growth factor (VEGF) (
). S1PRs expressed by monocytes and macrophages regulate their migration and activation, and the receptors responsible are cell subtype- and situation-specific (Fig. 4). In general, S1P1 and S1P3 appear to induce migration toward S1P, whereas S1P2 expression repulses macrophages from S1P (
). S1pr2−/− mice on a pro-atherogenic genetic background (Apoe−/−) developed significantly less atherosclerosis, accompanied by decreased macrophage and monocyte retention in atherosclerotic plaques, indicating effects on migration, tissue retention, and activation (
). In WT mice, treatment with FTY720 results in decreased circulating monocytes; however, use of the S1P1/4/5 agonist, BAF312, yielded similar results, both at homeostasis and during EAE, indicating that S1P3 is not the sole regulator of monocyte circulation (
A report utilizing the zymosan peritonitis model proposed that the resulting apoptotic neutrophils induced S1P1 expression on recruited macrophages and that S1P1 is necessary for emigration from the inflamed peritoneum, but has no role in efferocytosis or activation (
). S1P2 on alveolar macrophages may regulate their phagocytic capacity, as S1pr2−/− alveolar macrophages displayed decreased phagocytosis of the fungus Cryptococcus neoformans due to decreased expression of Fc receptors necessary for phagocytosis of antibody-opsonized fungus (
). Although S1P4 deficiency in Sgpl knockouts resulted in circulating neutrophil numbers that were close to WT, S1P4 was not specifically deleted in neutrophils, raising the possibility that multiple cell types were responsible for the effect. Specific deletion of neutrophil S1P1 did not normalize neutrophil numbers in Sgpl−/− mice. However, in rat models of hyperalgesia dependent upon neutrophil infiltration, S1P1 was necessary for neutrophil recruitment (
Migration of CD4 T cells and dendritic cells toward sphingosine 1-phosphate (S1P) is mediated by different receptor subtypes: S1P regulates the functions of murine mature dendritic cells via S1P receptor type 3.
). In EAE, although S1P1 agonism decreased disease pathology, it did not affect entry into the CNS of a subset of DCs (plasmacytoid DCs). However, plasmacytoid DCs in the CNS were necessary for the efficacy of S1P1 agonist treatment (
). In models of autoimmune disease, Th2-type immune responses such as allergic airway inflammation and cutaneous hypersensitivity, S1pr4−/− mice had increased pathology and up to 50% increase in DCs in draining LN after topical antigen application (
). S1P2 may also play a role in regulating neural progenitors, as postischemic administration of the S1P2 antagonist JTE-013 or short hairpin RNA against S1P2 significantly increased progenitor migration to the ischemic region (
Although analyses of entire mouse dorsal root ganglion found that S1P3 was the most highly expressed S1PR, single cell mRNA analysis of individual neurons found that S1P1 was most highly expressed, regardless of neuronal subtype, indicating that high expression of S1P3 occurs in ganglion cell types other than neurons (Fig. 5) (
). One group found that pain responses induced by intradermal S1P injection or models of postoperative pain were significantly decreased in S1pr3−/− mice, whereas minimal differences were seen in S1pr1−/− mice (
). Finally, in the murine model of the neurodevelopmental disease, Rett syndrome, FTY720 or S1P1-specific agonist SEW2871 in vivo treatment increased neuron production of brain-derived neurotrophic factor and decreased neurological symptoms (
). Ex vivo studies using cerebellar slice cultures indicated that S1PR agonism, particularly S1P1, could prevent or reverse demyelination, explaining the ability of FTY720 to induce remyelination and process extension in the same system (
). Data from a different in vitro system, myelinated neurospheres, indicated that FTY720 decreased microglial activation and oligodenrocyte apoptosis, and induced remyelination primarily by S1P5 agonism (
). An in vivo study provides conflicting evidence to these in vitro studies, reporting no effects on myelin repair with FTY720 treatment; however, the models of demyelination utilized in both the in vitro and in vivo studies were induced chemically and were meant to exclude possible effects of immune or vascular cells (
). In vitro studies indicated that FTY720 downregulated production of pro-inflammatory molecules by microglia while increasing neurotrophic factor production, resulting in an overall neuroprotective phenotype (
). In vivo, specific deletion of astrocyte S1P1 resulted in decreased EAE pathology and a loss of FTY720 efficacy, indicating that the primary target of FTY720 during EAE was S1P1 specifically on astrocytes (
). Another target of FTY720, S1P3, was also found on reactive astrocytes in human MS lesions and upregulated by lipopolysaccharide stimulation of astrocytes in vitro, although it is unknown if expression of S1P3 is protective or pathogenic in the context of MS/EAE (
Brain volume changes, on-study correlations and the link to disability in three fingolimod phase 3 studies (Abstract in 29th Congress of the European Committee for Treatment and Research in Multiple Sclerosis. Copenhagen,
Relapse rates and disability remain consistently low with long-term fingolimod therapy: five year interim results of the LONGTERMS extension study (Abstract in 29th Congress of the European Committee for Treatment and Research in Multiple Sclerosis. Copenhagen,
The blood brain barrier (BBB) forms through unique interactions between brain endothelial cells, astrocyte foot-processes, and pericytes, and regulates interactions between the immune and nervous systems (
). Studies of FTY720 treatment in the context of transient cerebral ischemia and reperfusion have demonstrated neuroprotection in mouse and rat models; however, these effects may be due to effects on interactions between the neurovasculature and immune cells (
). Additionally, when transient cerebral ischemia was induced in lymphocyte-deficient Rag1−/− mice, the protective effect of FTY720 was lost, further implying that FTY720-mediated protection is due to effects on the neurovasculature and its interactions with immune cells (
). Conversely, a study utilizing a model of permanent cerebral ischemia demonstrated no effect on pathology with FTY720 treatment, whereas another group demonstrated efficacy after delaying FTY720 treatment for 3 days after photothrombosis induction, with increased functional capacity and decreased astrogliosis (
). WT hamster lung fibroblasts were protected from nutrient deprivation-induced apoptosis by expression of S1P1, which induced the anti-apoptotic protein Mcl1 via the phosphoinositide 3-kinase and PKC pathways (
High expression of sphingosine 1-phosphate receptors, S1P1 and S1P3, sphingosine kinase 1, and extracellular signal-regulated kinase-1/2 is associated with development of tamoxifen resistance in estrogen receptor-positive breast cancer patients.
Identification of novel functional and spatial associations between sphingosine kinase 1, sphingosine 1-phosphate receptors and other signaling proteins that affect prognostic outcome in estrogen receptor-positive breast cancer.
). In a model of colitis-associated cancer, S1P1 signaling was necessary for persistent activation of nuclear factor-kB and signal transducer and activator of transcription3 transcription factors needed for maintaining the chronic inflammatory state and could be blocked by FTY720 treatment (
). Interestingly, FTY720 treatment impaired the mucosal immune response to the extracellular bacterium, Citrobacter rodentium, including decreased DC numbers, as well as macrophages and T cells in the colon, while increasing bacterial burden (
). AKT and Bcl-associated death promoter pro-survival pathways were also reduced by FTY720 administration to neuroblastoma cells in an in vitro and an in vivo xenograft model, resulting in decreased cancer cell viability (
S1PR expression in several hematological malignancies has also been described, including S1P1 expression by classical Hodgkin's lymphoma (CHL) cells, B cell chronic lymphocytic leukemia (B-CLL) cells, and activated B cell-like DLBCL cells (
). Expression of S1PRs by blood cancer cells may directly regulate their survival or by controlling the localization of cells within permissive environments such as the lymph nodes.
S1PRs are gaining appreciation as powerful modulators of homeostasis and pathogenesis. In all biological systems, S1PRs play some role in regulating cell survival, migration, phenotype, activation status, and proliferation. In the current review, we have attempted to summarize the most recent advances in the field of S1PR biology and to provide novel insights into the biological responses regulated. As more cell-specific animal models of gene deletion or over-expression are created, and agonists and antagonists with greater S1PR subtype specificity are developed, further studies with such tools will clarify the contributions of specific S1PRs in each physiological or pathological context. This is especially true of the less explored members of the S1PR family, S1P4 and S1P5. Additionally, we anticipate that the development of more compounds for clinical use will expand our understanding of the complex signaling networks regulated by S1PRs and their role in human homeostasis and disease.
Regulation of mammalian physiology, development, and disease by the sphingosine 1-phosphate and lysophosphatidic acid receptors.
Migration of CD4 T cells and dendritic cells toward sphingosine 1-phosphate (S1P) is mediated by different receptor subtypes: S1P regulates the functions of murine mature dendritic cells via S1P receptor type 3.