S1PR2 deficiency enhances neuropathic pain induced by partial sciatic nerve ligation

Background/aim: Sphingosine 1-phosphate receptor 2 (S1PR2), a member of the seven-transmembrane receptor family, can be activated by its natural ligand sphingosine 1-phosphate (S1P) to initiate signal transduction and is involved in a wide range of biological effects such as immune cell migration and vascular permeability. Its relationship with neuropathic pain (NP) has not been reported. In this study, the effects of S1PR2 on the development of NP were studied. Materials and methods: We generated a model of NP by partial sciatic nerve ligation (pSNL). The 50% paw withdrawal threshold of the wild-type (WT) group and the S1PR2 deficiency group were measured at several time points after surgery. The inflammatory factor levels of the two groups were measured by real-time quantitative polymerase chain reaction (RT-PCR). Neutrophil infiltration and glial cell activation were detected by immunofluorescence. Matrix metalloproteinase 9 (MMP9) and its substrate myelin basic protein (MBP) were measured by RT-PCR, western blotting, and immunofluorescence. Result: The S1PR2 deficiency group showed a reduction in 50% paw withdrawal threshold compared with WT mice (P < 0.05) at 3 days after the operation. In the ligated sciatic nerve of the S1PR2 deficiency group, the mRNA expression of IL-1ß was increased; the numbers of infiltrating neutrophils and activated astrocytes were also increased. The expression of MMP9 was elevated while MBP was decreased. Conclusion: S1PR2 deficiency could increase the pain sensitivity of a NP mouse model and promote the development of NP

Sphingosine 1-phosphate (S1P) reduces hepatocyte growth factor-induced migration of hepatocellular carcinoma cells via S1P receptor 2.

A bioactive lipid, sphingosine 1-phosphate (S1P), acts extracellularly as a potent mediator, and is implicated in the progression of various cancers including hepatocellular carcinoma (HCC). S1P exerts its functions by binding to five types of specific receptors, S1P receptor 1 (S1PR1), S1PR2, S1PR3, S1PR4 and S1PR5 on the plasma membrane. However, the exact roles of S1P and each S1PR in HCC cells remain to be clarified. In the present study, we investigated the effect of S1P on the hepatocyte growth factor (HGF)-induced migration of human HCC-derived HuH7 cells, and the involvement of each S1PR. S1P dose-dependently reduced the HGF-induced migration of HuH7 cells. We found that all S1PRs exist in the HuH7 cells. Among each selective agonist for five S1PRs, CYM5520, a selective S1PR2 agonist, significantly suppressed the HGF-induced HuH7 cell migration whereas selective agonists for S1PR1, S1PR3, S1PR4 or S1PR5 failed to affect the migration. The reduction of the HGF-induced migration by S1P was markedly reversed by treatment of JTE013, a selective antagonist for S1PR2, and S1PR2- siRNA. These results strongly suggest that S1P reduces the HGF-induced HCC cell migration via S1PR2. Our findings may provide a novel potential of S1PR2 to therapeutic strategy for metastasis of HCC.

Sphingosine-1-phosphate receptor-2 facilitates pulmonary fibrosis through potentiating IL-13 pathway in macrophages.

Idiopathic pulmonary fibrosis is a devastating disease with poor prognosis. The pathogenic role of the lysophospholipid mediator sphingosine-1-phosphate and its receptor S1PR2 in lung fibrosis is unknown. We show here that genetic deletion of S1pr2 strikingly attenuated lung fibrosis induced by repeated injections of bleomycin in mice. We observed by using S1pr2LacZ/+ mice that S1PR2 was expressed in alveolar macrophages, vascular endothelial cells and alveolar epithelial cells in the lung and that S1PR2-expressing cells accumulated in the fibrotic legions. Bone marrow chimera experiments suggested that S1PR2 in bone marrow-derived cells contributes to the development of lung fibrosis. Depletion of macrophages greatly attenuated lung fibrosis. Bleomycin administration stimulated the mRNA expression of the profibrotic cytokines IL-13 and IL-4 and the M2 markers including arginase 1, Fizz1/Retnla, Ccl17 and Ccl24 in cells collected from broncho-alveolar lavage fluids (BALF), and S1pr2 deletion markedly diminished the stimulated expression of these genes. BALF cells from bleomycin-administered wild-type mice showed a marked increase in phosphorylation of STAT6, a transcription factor which is activated downstream of IL-13, compared with saline-administered wild-type mice. Interestingly, in bleomycin-administered S1pr2-/- mice, STAT6 phosphorylation in BALF cells was substantially diminished compared with wild-type mice. Finally, pharmacological S1PR2 blockade in S1pr2+/+ mice alleviated bleomycin-induced lung fibrosis. Thus, S1PR2 facilitates lung fibrosis through the mechanisms involving augmentation of IL-13 expression and its signaling in BALF cells, and represents a novel target for treating lung fibrosis.

Sphingosine-1-phosphate Receptor 2 Signaling Promotes Caspase-11-dependent Macrophage Pyroptosis and Worsens Escherichia coli Sepsis Outcome.

WHAT WE ALREADY KNOW ABOUT THIS TOPIC: WHAT THIS ARTICLE TELLS US THAT IS NEW: BACKGROUND:: Pyroptosis, a type of proinflammatory programmed cell death, drives cytokine storm. Caspase-11-dependent macrophage pyroptosis contributes to mortality during sepsis. Sphingosine-1-phosphate receptor 2 (S1PR2) signaling can amplify interleukin-1ß secretion in endotoxin-induced inflammation. Here, we hypothesized that S1PR2 signaling increases caspase-11-dependent macrophage pyroptosis and worsens Gram-negative sepsis outcome. METHODS: A Gram-negative sepsis model was induced through intraperitoneal injection of Escherichia coli. Primary peritoneal macrophages isolated from wild-type, S1pr2-deficient (S1pr2), or nucleotide-binding oligomerization domain-like receptor protein-3-deficient mice were treated with E. coli. Caspase-11 activation, macrophage pyroptosis, and Ras homolog gene family, member A-guanosine triphosphate levels were assessed in those cells. Additionally, monocyte caspase-4 (an analog of caspase-11) expression and its correlation with S1PR2 expression were determined in patients with Gram-negative sepsis (n = 11). RESULTS: Genetic deficiency of S1PR2 significantly improved survival rate (2/10 [20%] in wild-type vs. 7/10 [70%] in S1pr2, P = 0.004) and decreased peritoneal macrophage pyroptosis (pyroptosis rate: 35 ± 3% in wild-type vs. 10 ± 3% in S1pr2, P < 0.001). Decreased caspase-11 activation in S1PR2 deficiency cells contributed to the reduced macrophage pyroptosis. In addition, RhoA inhibitor abrogated the amplified caspase-11 activation in wild-type or S1PR2-overexpressing cells. In patients with Gram-negative sepsis, caspase-4 increased significantly in monocytes compared to nonseptic controls and was positively correlated with S1PR2 (r = 0.636, P = 0.035). CONCLUSIONS: S1PR2 deficiency decreased macrophage pyroptosis and improved survival in E. coli sepsis. These beneficial effects were attributed to the decreased caspase-11 activation of S1PR2-deficient macrophages. S1PR2 and caspase-11 may be promising new targets for treatment of sepsis.

Sexual dimorphism of metabolic and vascular dysfunction in aged mice and those lacking the sphingosine 1-phosphate receptor 3.

Elderly people often suffer adverse health because of inflammation associated with poor metabolism and cardiovascular dysfunction, but these conditions present differently in men and women. We performed experiments in aged male and female mice to understand this sexual dimorphism. We focused on sphingosine 1-phosphate (S1P) signaling, which has both protective and detrimental effects on vascular and metabolic function. We examined vascular function of mesenteric (resistance) arteries from aged male and female wild-type (WT) mice compared to littermate S1P receptor 3 (S1PR3) knockouts (KO). We also measured plasma glucose, insulin, triglycerides, adiponectin, corticosterone and inflammatory cytokines. The novel results of this study are: 1) methacholine-induced vasodilation relied completely on S1PR3 in both sexes, but was dependent on nitric oxide synthase (NOS) only in arteries from aged female mice; 2) S1P-induced vasoconstriction depended solely on S1PR3 in arteries from males, but only partly in females; 3) vasoconstriction to a thromboxane mimetic was decreased by endogenous NOS activity only in arteries from females, regardless of genotype; 4) myogenic responses were lower in arteries from aged WT males compared to females and responses in arteries from KO females were lower than WT females, while the opposite was true of arteries from male mice; 5) aged male mice showed higher fasting glucose and triglycerides with lower plasma adiponectin compared to females and 6) lack of signaling through S1PR3 in females was associated with decreased plasma adiponectin and increased inflammatory mediators. This study showed that there is considerable sexual dimorphism in the vascular and metabolic responses of aged mice and that reduced signaling through S1PR3 could be one mechanism to explain these effects. These results also emphasize that different treatments for mitigating the deleterious effects on vascular health in aged males versus females should be considered.

Ablation of S1P3 receptor protects mouse soleus from age-related drop in muscle mass, force, and regenerative capacity.

We investigated the effects of S1P3 deficiency on the age-related atrophy, decline in force, and regenerative capacity of soleus muscle from 23-mo-old male (old) mice. Compared with muscle from 5-mo-old (adult) mice, soleus mass and muscle fiber cross-sectional area (CSA) in old wild-type mice were reduced by ~26% and 24%, respectively. By contrast, the mass and fiber CSA of soleus muscle in old S1P3-null mice were comparable to those of adult muscle. Moreover, in soleus muscle of wild-type mice, twitch and tetanic tensions diminished from adulthood to old age. A slowing of contractile properties was also observed in soleus from old wild-type mice. In S1P3-null mice, neither force nor the contractile properties of soleus changed during aging. We also evaluated the regenerative capacity of soleus in old S1P3-null mice by stimulating muscle regeneration through myotoxic injury. After 10 days of regeneration, the mean fiber CSA of soleus in old wild-type mice was significantly smaller (-28%) compared with that of regenerated muscle in adult mice. On the contrary, the mean fiber CSA of regenerated soleus in old S1P3-null mice was similar to that of muscle in adult mice. We conclude that in the absence of S1P3, soleus muscle is protected from the decrease in muscle mass and force, and the attenuation of regenerative capacity, all of which are typical characteristics of aging.

T cell-intrinsic S1PR1 regulates endogenous effector T-cell egress dynamics from lymph nodes during infection.

Viral clearance requires effector T-cell egress from the draining lymph node (dLN). The mechanisms that regulate the complex process of effector T-cell egress from the dLN after infection are poorly understood. Here, we visualized endogenous pathogen-specific effector T-cell migration within, and from, the dLN. We used an inducible mouse model with a temporally disrupted sphingosine-1-phosphate receptor-1 (S1PR1) gene specifically in endogenous effector T cells. Early after infection, WT and S1PR1(-/-) effector T cells localized exclusively within the paracortex. This localization in the paracortex by CD8 T cells was followed by intranodal migration by both WT and S1PR1(-/-) T cells to positions adjacent to both cortical and medullary lymphatic sinuses where the T cells exhibited intense probing behavior. However, in contrast to WT, S1PR1(-/-) effector T cells failed to enter the sinuses. We demonstrate that, even when LN retention signals such as CC chemokine receptor 7 (CCR7) are down-regulated, T cell intrinsic S1PR1 is the master regulator of effector T-cell emigration from the dLN.

The hematopoietic oncoprotein FOXP1 promotes tumor cell survival in diffuse large B-cell lymphoma by repressing S1PR2 signaling.

Aberrant expression of the oncogenic transcription factor forkhead box protein 1 (FOXP1) is a common feature of diffuse large B-cell lymphoma (DLBCL). We have combined chromatin immunoprecipitation and gene expression profiling after FOXP1 depletion with functional screening to identify targets of FOXP1 contributing to tumor cell survival. We find that the sphingosine-1-phosphate receptor 2 (S1PR2) is repressed by FOXP1 in activated B-cell (ABC) and germinal center B-cell (GCB) DLBCL cell lines with aberrantly high FOXP1 levels; S1PR2 expression is further inversely correlated with FOXP1 expression in 3 patient cohorts. Ectopic expression of wild-type S1PR2, but not a point mutant incapable of activating downstream signaling pathways, induces apoptosis in DLBCL cells and restricts tumor growth in subcutaneous and orthotopic models of the disease. The proapoptotic effects of S1PR2 are phenocopied by ectopic expression of the small G protein Gα13 but are independent of AKT signaling. We further show that low S1PR2 expression is a strong negative prognosticator of patient survival, alone and especially in combination with high FOXP1 expression. The S1PR2 locus has previously been demonstrated to be recurrently mutated in GCB DLBCL; the transcriptional silencing of S1PR2 by FOXP1 represents an alternative mechanism leading to inactivation of this important hematopoietic tumor suppressor.

S1P2/G12/13 Signaling Negatively Regulates Macrophage Activation and Indirectly Shapes the Atheroprotective B1-Cell Population.

OBJECTIVES: Monocyte/macrophage recruitment and activation at vascular predilection sites plays a central role in the pathogenesis of atherosclerosis. Heterotrimeric G proteins of the G12/13 family have been implicated in the control of migration and inflammatory gene expression, but their function in myeloid cells, especially during atherogenesis, is unknown. APPROACH AND RESULTS: Mice with myeloid-specific deficiency for G12/13 show reduced atherosclerosis with a clear shift to anti-inflammatory gene expression in aortal macrophages. These changes are because of neither altered monocyte/macrophage migration nor reduced activation of inflammatory gene expression; on the contrary, G12/13-deficient macrophages show an increased nuclear factor-κB-dependent gene expression in the resting state. Chronically increased inflammatory gene expression in resident peritoneal macrophages results in myeloid-specific G12/13-deficient mice in an altered peritoneal micromilieu with secondary expansion of peritoneal B1 cells. Titers of B1-derived atheroprotective antibodies are increased, and adoptive transfer of peritoneal cells from mutant mice conveys atheroprotection to wild-type mice. With respect to the mechanism of G12/13-mediated transcriptional control, we identify an autocrine feedback loop that suppresses nuclear factor-κB-dependent gene expression through a signaling cascade involving sphingosine 1-phosphate receptor subtype 2, G12/13, and RhoA. CONCLUSIONS: Together, these data show that selective inhibition of G12/13 signaling in macrophages can augment atheroprotective B-cell populations and ameliorate atherosclerosis.

Sphingosine 1-Phosphate Receptor 3-Deficient Dendritic Cells Modulate Splenic Responses to Ischemia-Reperfusion Injury.

The plasticity of dendritic cells (DCs) permits phenotypic modulation ex vivo by gene expression or pharmacologic agents, and these modified DCs can exert therapeutic immunosuppressive effects in vivo through direct interactions with T cells, either inducing T regulatory cells (T(REG)s) or causing anergy. Sphingosine 1-phosphate (S1P) is a sphingolipid and the natural ligand for five G protein-coupled receptors (S1P1, S1P2, S1P3, S1P4, and S1P5), and S1PR agonists reduce kidney ischemia-reperfusion injury (IRI) in mice. S1pr3(-/-)mice are protected from kidney IRI, because DCs do not mature. We tested the therapeutic advantage of S1pr3(-/-) bone marrow-derived dendritic cell (BMDC) transfers in kidney IRI. IRI produced a rise in plasma creatinine (PCr) levels in mice receiving no cells (NCs) and mice pretreated with wild-type (WT) BMDCs. However, S1pr3(-/-) BMDC-pretreated mice were protected from kidney IRI. S1pr3(-/-) BMDC-pretreated mice had significantly higher numbers of splenic T(REG)s compared with NC and WT BMDC-pretreated mice. S1pr3(-/-) BMDCs did not attenuate IRI in splenectomized, Rag-1(-/-), or CD11c(+) DC-depleted mice. Additionally, S1pr3(-/-) BMDC-dependent protection required CD169(+)marginal zone macrophages and the macrophage-derived chemokine CCL22 to increase splenic CD4(+)Foxp3(+) T(REG)s. Pretreatment with S1pr3(-/-) BMDCs also induced T(REG)-dependent protection against IRI in an allogeneic mouse model. In summary, adoptively transferred S1pr3(-/-) BMDCs prevent kidney IRI through interactions within the spleen and expansion of splenic CD4(+)Foxp3(+) T(REG)s. We conclude that genetically induced deficiency of S1pr3 in allogenic BMDCs could serve as a therapeutic approach to prevent IRI-induced AKI.

S1P1 deletion in oligodendroglial lineage cells: Effect on differentiation and myelination.

Sphingosine 1-phosphate (S1P) receptors are G protein-coupled receptors expressed by many cell types, including cells of oligodendrocyte (OLG) lineage. We had previously shown that targeted deletion of S1P1 in OLG lineage cells did not result in obvious clinical phenotype or altered number of OLGs at 3 months, but there were subtle abnormalities in myelin. In this study, we examined the role of S1P1 in developmental myelination and cell survival, focusing on age 3 weeks. We found that S1P1 deficiency led to delayed differentiation of OLG progenitors (OPCs) into OLGs that is independent of p38 phosphorylation. This was accompanied by decreased levels of myelin basic protein (MBP) but not of myelin-OLG glycoprotein (MOG), and slight decrease in myelin thickness in the corpus callosum of S1P1 conditional knockout (CKO) mice. S1P1 -deficient OLGs exhibited slower process extension, which was associated with attenuated phosphorylation of extracellular signal regulated kinases (ERKs) and p21-activated kinases (PAKs), and with upregulation of tropomodulin1. Basal levels of pAkt were not affected, though expectedly, no response to a selective S1P1 agonist SEW2871 was observed. S1P1 -deficient OLGs did not exhibit increased cell death in response to cuprizone, tumor necrosis factor-α, or deprivation of nutrients and growth factors. We conclude that S1P1 signaling regulates OLG development, morphological maturation and early myelination.

Enhanced Chemokine Receptor Recycling and Impaired S1P1 Expression Promote Leukemic Cell Infiltration of Lymph Nodes in Chronic Lymphocytic Leukemia.

Lymphocyte trafficking is orchestrated by chemokine and sphingosine 1-phosphate (S1P) receptors that enable homing and egress from secondary lymphoid organs (SLO). These receptors undergo rapid internalization and plasma membrane recycling to calibrate cellular responses to local chemoattractants. Circulating chronic lymphocytic leukemia (CLL) cells display an abnormal increase in the surface levels of the homing receptors CCR7 and CXCR4 concomitant with low S1P receptor 1 (S1P1) expression. In this study, we investigated the role of receptor recycling on CXCR4/CCR7 surface levels in CLL cells and addressed the impact of quantitative alterations of these receptors and S1P1 on the ability of leukemic cells to accumulate in SLOs. We show that recycling accounts, to a major extent, for the high levels of surface CXCR4/CCR7 on CLL cells. In addition, increased expression of these receptors, together with S1P1 deficiency, is detectable not only in circulating leukemic cells, but also in SLOs of CLL patients with lymphoadenopathy. We further provide evidence that ibrutinib, a Btk inhibitor that promotes mobilization of leukemic cells from SLOs, normalizes the imbalance between CXCR4/CCR7 and S1P1. Taken together, our results highlight the relevance of chemokine and S1P receptor recycling in CLL pathogenesis and clinical outcome.

Sphingosine 1-phosphate Receptor 2 Signaling Suppresses Macrophage Phagocytosis and Impairs Host Defense against Sepsis.

BACKGROUND: Sepsis is characterized by an inappropriate systemic inflammatory response and bacteremia that promote multiorgan failure and mortality. Sphingosine 1-phosphate receptor 2 (S1PR2) modulates endotoxin-induced inflammation in endothelium. However, as a highly expressed S1P receptor in macrophages, its role in regulating macrophage response to bacterial infection remains unclear. METHODS: Cecal ligation and puncture or intratracheal instillation of Escherichia coli was induced in wild-type or S1pr2-deficient mice. The antibacterial ability of cell-specific S1PR2 was tested in bone marrow reconstitution mice or mice with macrophage-specific deletion. Signaling molecules responsible for S1PR2-mediated phagocytosis were also measured in the bone marrow-derived macrophages. In addition, S1PR2 expression levels and its correlation with severity of sepsis were determined in critically ill patients (n = 25). RESULTS: Both genetic deletion and pharmaceutical inhibition of S1PR2 significantly limited bacterial burden, reduced lung damage, and improved survival (genetic deletion, 0% in S1pr2 vs. 78.6% in S1pr2, P < 0.001; pharmaceutical inhibition, 9.1% in vehicle vs. 22.2% in S1PR2 antagonist, P < 0.05). This protection was attributed to the enhanced phagocytic function of S1PR2-deficient macrophages (mean fluorescent intensity, 2035.2 ± 202.1 vs. 407.8 ± 71.6, P < 0.001). Absence of S1PR2 in macrophage inhibits RhoA-dependent cell contraction and promotes IQGAP1-Rac1-dependent lamellipodial protrusion, whose signaling pathways depend on extracellular stimulators. In septic patients, increased S1PR2 levels in peripheral blood mononuclear cells were positively correlated with the severity of sepsis (r = 0.845, P < 0.001). CONCLUSIONS: This study implies that S1PR2, as a critical receptor in macrophage, impairs phagocytosis and antimicrobial defense in the pathogenesis of sepsis. Interventions targeting S1PR2 signaling may serve as promising therapeutic approaches for sepsis.

Loss of signalling via Gα13 in germinal centre B-cell-derived lymphoma.

Germinal centre B-cell-like diffuse large B-cell lymphoma (GCB-DLBCL) is a common malignancy, yet the signalling pathways that are deregulated and the factors leading to its systemic dissemination are poorly defined. Work in mice showed that sphingosine-1-phosphate receptor-2 (S1PR2), a Gα12 and Gα13 coupled receptor, promotes growth regulation and local confinement of germinal centre B cells. Recent deep sequencing studies of GCB-DLBCL have revealed mutations in many genes in this cancer, including in GNA13 (encoding Gα13) and S1PR2 (refs 5,6, 7). Here we show, using in vitro and in vivo assays, that GCB-DLBCL-associated mutations occurring in S1PR2 frequently disrupt the receptor's Akt and migration inhibitory functions. Gα13-deficient mouse germinal centre B cells and human GCB-DLBCL cells were unable to suppress pAkt and migration in response to S1P, and Gα13-deficient mice developed germinal centre B-cell-derived lymphoma. Germinal centre B cells, unlike most lymphocytes, are tightly confined in lymphoid organs and do not recirculate. Remarkably, deficiency in Gα13, but not S1PR2, led to germinal centre B-cell dissemination into lymph and blood. GCB-DLBCL cell lines frequently carried mutations in the Gα13 effector ARHGEF1, and Arhgef1 deficiency also led to germinal centre B-cell dissemination. The incomplete phenocopy of Gα13- and S1PR2 deficiency led us to discover that P2RY8, an orphan receptor that is mutated in GCB-DLBCL and another germinal centre B-cell-derived malignancy, Burkitt's lymphoma, also represses germinal centre B-cell growth and promotes confinement via Gα13. These findings identify a Gα13-dependent pathway that exerts dual actions in suppressing growth and blocking dissemination of germinal centre B cells that is frequently disrupted in germinal centre B-cell-derived lymphoma.

Enhanced sphingosine-1-phosphate receptor 2 expression underlies female CNS autoimmunity susceptibility.

Multiple sclerosis (MS) is an inflammatory disease of the CNS that is characterized by BBB dysfunction and has a much higher incidence in females. Compared with other strains of mice, EAE in the SJL mouse strain models multiple features of MS, including an enhanced sensitivity of female mice to disease; however, the molecular mechanisms that underlie the sex- and strain-dependent differences in disease susceptibility have not been described. We identified sphingosine-1-phosphate receptor 2 (S1PR2) as a sex- and strain-specific, disease-modifying molecule that regulates BBB permeability by destabilizing adherens junctions. S1PR2 expression was increased in disease-susceptible regions of the CNS of both female SJL EAE mice and female patients with MS compared with their male counterparts. Pharmacological blockade or lack of S1PR2 signaling decreased EAE disease severity as the result of enhanced endothelial barrier function. Enhanced S1PR2 signaling in an in vitro BBB model altered adherens junction formation via activation of Rho/ROCK, CDC42, and caveolin endocytosis-dependent pathways, resulting in loss of apicobasal polarity and relocation of abluminal CXCL12 to vessel lumina. Furthermore, S1PR2-dependent BBB disruption and CXCL12 relocation were observed in vivo. These results identify a link between S1PR2 signaling and BBB polarity and implicate S1PR2 in sex-specific patterns of disease during CNS autoimmunity.

Sphingosine 1-phosphate induces filopodia formation through S1PR2 activation of ERM proteins.

Previously we demonstrated that the sphingolipids ceramide and S1P (sphingosine 1-phosphate) regulate phosphorylation of the ERM (ezrin/radixin/moesin) family of cytoskeletal proteins [Canals, Jenkins, Roddy, Hernande-Corbacho, Obeid and Hannun (2010) J. Biol. Chem. 285, 32476-3285]. In the present article, we show that exogenously applied or endogenously generated S1P (in a sphingosine kinase-dependent manner) results in significant increases in phosphorylation of ERM proteins as well as filopodia formation. Using phosphomimetic and non-phosphorylatable ezrin mutants, we show that the S1P-induced cytoskeletal protrusions are dependent on ERM phosphorylation. Employing various pharmacological S1PR (S1P receptor) agonists and antagonists, along with siRNA (small interfering RNA) techniques and genetic knockout approaches, we identify the S1PR2 as the specific and necessary receptor to induce phosphorylation of ERM proteins and subsequent filopodia formation. Taken together, the results demonstrate a novel mechanism by which S1P regulates cellular architecture that requires S1PR2 and subsequent phosphorylation of ERM proteins.

[Sphingosine 1-phosphate receptors: from biology to physiopathology]. / Les récepteurs de la sphingosine 1-phosphate - De la biologie à la physiopathologie.

Sphingosine 1-phosphate (S1P) mediates critical physiological responses by its binding to G protein-coupled receptor (GPCR) subtypes, known as S1P receptors. Five distinct mammalian S1P receptors, designated S1P1-5 have been identified, each with a different cellular pattern of expression which influences the responses to S1P. In this review, we briefly outline our understanding of the modes of action and the roles of S1P receptors in the regulation of physiological and pathological functions in the cardiovascular, immune and central nervous system.

A novel role of sphingosine 1-phosphate receptor S1pr1 in mouse thrombopoiesis.

Millions of platelets are produced each hour by bone marrow (BM) megakaryocytes (MKs). MKs extend transendothelial proplatelet (PP) extensions into BM sinusoids and shed new platelets into the blood. The mechanisms that control platelet generation remain incompletely understood. Using conditional mutants and intravital multiphoton microscopy, we show here that the lipid mediator sphingosine 1-phosphate (S1P) serves as a critical directional cue guiding the elongation of megakaryocytic PP extensions from the interstitium into BM sinusoids and triggering the subsequent shedding of PPs into the blood. Correspondingly, mice lacking the S1P receptor S1pr1 develop severe thrombocytopenia caused by both formation of aberrant extravascular PPs and defective intravascular PP shedding. In contrast, activation of S1pr1 signaling leads to the prompt release of new platelets into the circulating blood. Collectively, our findings uncover a novel function of the S1P-S1pr1 axis as master regulator of efficient thrombopoiesis and might raise new therapeutic options for patients with thrombocytopenia.

The sphingosine-1-phosphate receptor S1PR1 restricts sprouting angiogenesis by regulating the interplay between VE-cadherin and VEGFR2.

Angiogenesis, the process by which new blood vessels arise from preexisting ones, is critical for embryonic development and is an integral part of many disease processes. Recent studies have provided detailed information on how angiogenic sprouts initiate, elongate, and branch, but less is known about how these processes cease. Here, we show that S1PR1, a receptor for the blood-borne bioactive lipid sphingosine-1-phosphate (S1P), is critical for inhibition of angiogenesis and acquisition of vascular stability. Loss of S1PR1 leads to increased endothelial cell sprouting and the formation of ectopic vessel branches. Conversely, S1PR1 signaling inhibits angiogenic sprouting and enhances cell-to-cell adhesion. This correlates with inhibition of vascular endothelial growth factor-A (VEGF-A)-induced signaling and stabilization of vascular endothelial (VE)-cadherin localization at endothelial junctions. Our data suggest that S1PR1 signaling acts as a vascular-intrinsic stabilization mechanism, protecting developing blood vessels against aberrant angiogenic responses.

Dendritic cell sphingosine 1-phosphate receptor-3 regulates Th1-Th2 polarity in kidney ischemia-reperfusion injury.

Dendritic cells (DCs) are central to innate and adaptive immunity of early kidney ischemia-reperfusion injury (IRI), and strategies to alter DC function may provide new therapeutic opportunities. Sphingosine 1-phosphate (S1P) modulates immunity through binding to its receptors (S1P1-5), and protection from kidney IRI occurs in S1P3-deficient mice. Through a series of experiments we determined that this protective effect was owing in part to differences between S1P3-sufficient and -deficient DCs. Mice lacking S1P3 on bone marrow cells were protected from IRI, and S1P3-deficient DCs displayed an immature phenotype. Wild-type (WT) but not S1P3-deficient DCs injected into mice depleted of DCs prior to kidney IR reconstituted injury. Adoptive transfer (i.e., i.v. injection) of glycolipid (Ag)-loaded WT but not S1P3-deficient DCs into WT mice exacerbated IRI, suggesting that WT but not S1P3-deficient DCs activated NKT cells. Whereas WT DC transfers activated the Th1/IFN-γ pathway, S1P3-deficient DCs activated the Th2/IL-4 pathway, and an IL-4-blocking Ab reversed protection from IRI, supporting the concept that IL-4 mediates the protective effect of S1P3-deficient DCs. Administration of S1P3-deficient DCs 7 d prior to or 3 h after IRI protected mice from IRI and suggests their potential use in cell-based therapy. We conclude that absence of DC S1P3 prevents DC maturation and promotes a Th2/IL-4 response. These findings highlight the importance of DC S1P3 in modulating NKT cell function and IRI and support development of selective S1P3 antagonists for tolerizing DCs for cell-based therapy or for systemic administration for the prevention and treatment of IRI and autoimmune diseases.