Characterization of the human and mouse sphingosine 1-phosphate receptor, S1P5 (Edg-8): structure-activity relationship of sphingosine1-phosphate receptors.

Five G protein-coupled receptors (S1P(1)/Edg-1, S1P(3)/Edg-3, S1P(2)/Edg-5, S1P(4)/Edg-6, and S1P(5)/Edg-8) for the intercellular lipid mediator sphingosine 1-phosphate have been cloned and characterized. We found human and mouse sequences closely related to rat S1P(5) (97% identical amino acids) and report now the characterization of the human and mouse S1P(5) gene products as encoding sphingosine 1-phosphate receptors. When HEK293T cells were cotransfected with S1P(5) and G protein DNAs, prepared membranes showed sphingosine 1-phosphate concentration-dependent increases in [gamma-(35)S]GTP binding (EC(50) = 12.7 nM). The lipid mediator inhibited forskolin-driven rises in cAMP by greater than 80% after introduction of the mouse or human S1P(5) DNAs into rat hepatoma RH7777 cells (IC(50) = 0.22 nM). This response is blocked fully by prior treatment of cultures with pertussis toxin, thus implicating signaling through G(i/o)alpha proteins. Northern blot analysis showed high expression of human S1P(5) mRNA in spleen, corpus collosum, peripheral blood leukocytes, placenta, lung, aorta, and fetal tissues. Mouse S1P(5) mRNA is also expressed in spleen and brain. Finally, we found that one enantiomer of a sphingosine 1-phosphate analogue wherein the 3-hydroxyl and 4,5-olefin are replaced by an amide functionality shows some selectivity as an agonist S1P(1) and S1P(3) vs S1P(2) and S1P(5).     

The immune modulator FTY720 targets sphingosine 1-phosphate receptors

Immunosuppressant drugs such as cyclosporin have allowed widespread organ transplantation, but their utility remains limited by toxicities, and they are ineffective in chronic management of autoimmune diseases such as multiple sclerosis. In contrast, the immune modulating drug FTY720 is efficacious in a variety of transplant and autoimmune models without inducing a generalized immunosuppressed state and is effective in human kidney transplantation. FTY720 elicits a lymphopenia resulting from a reversible redistribution of lymphocytes from circulation to secondary lymphoid tissues by unknown mechanisms. Using FTY720 and several analogs, we show now that FTY720 is phosphorylated by sphingosine kinase; the phosphorylated compound is a potent agonist at four sphingosine 1-phosphate receptors and represents the therapeutic principle in a rodent model of multiple sclerosis. Our results suggest that FTY720, after phosphorylation, acts through sphingosine 1-phosphate signaling pathways to modulate chemotactic responses and lymphocyte trafficking.     

Immunological effects of transgenic constitutive expression of the type 1 sphingosine 1-phosphate receptor by mouse lymphocytes

The type 1 sphingosine 1-phosphate (S1P) G protein-coupled receptor (S1P1) normally transduces S1P effects on lymph node (LN) egress and tissue migration of naive lymphocytes. We now show that persistent expression of S1P1 by lymphocytes of S1P1-transgenic (Tg) mice suppresses delayed-type hypersensitivity and results in production of significantly more IgE Ab and less IgG2 Ab than in wild-type (wt) mice. wt host LN homing of 51Cr-labeled T cells from S1P1-Tg mice was only 30-40% of that for wt T cells. Adoptive-transfer of dye-labeled activated T cells from S1P1-Tg mice into wt mice resulted in 2.2-fold more in blood and 60% less in LNs than for activated wt T cells after 1 day. Proliferative responses of stimulated T cells from S1P1-Tg mice were only 10-34% of those for wt T cells. Disordered cellular and humoral immunity of S1P1-Tg mice thus may be attributable to both altered T cell traffic and depressed T cell functions, suggesting that S1P1-specific agonists may represent a novel therapeutic approach to autoimmunity and transplant rejection.     

Pathways of transduction engaged by sphingosine 1-phosphate through G protein-coupled receptors

Pathways of transduction employed by receptors for sphingosine 1-phosphate (S1P) are identified by the nature of second messengers and/or downstream targets regulated and, more formally, by direct assays of heterotrimeric G protein activation. The different methods generally agree. S1P1 couples to members of the Gi family, apparently selectively, although reported pertussis toxin (PTX)-insensitive actions make categorical statements regarding exclusivity difficult. S1P2 and S1P3 couple to members of the Gi, Gq, and G12/13 families. S1P4 couples to Gi and possibly G12/13, while S1P5 couples to Gi and G12/13 but not to Gq. In virtually all circumstances, coupling of S1P receptors to Gi is reflected in PTX-sensitive inhibition of adenylyl cyclase, activation of extracellular-regulated kinases (ERKs), and, depending on the cell, activation of phospholipase C (PLC). Coupling to Gq is reflected in PTX-insensitive activation of phospholipase C. Coupling to G12/13 is reflected in activation of Rho and subsequent activation of serum response factor (SRF). Specific linkages have been verified in almost all instances by receptor-promoted [35S]GTPgammaS/GDP exchange on identified G proteins.     

Ethanol triggers sphingosine 1-phosphate elevation along with neuroapoptosis in the developing mouse brain

Recent studies suggest that l-3,4 dihydroxyphenylalanine (L-DOPA)-induced dyskinesia (LID), a severe complication of conventional L-DOPA therapy of Parkinson's disease, may be caused by dopamine (DA) release originating in serotonergic neurons. To evaluate the in vivo effect of a 5-HT(1A) agonist [(±)-8-hydroxy-2-(dipropylamino) tetralin hydrobromide, 8-OHDPAT] on the L-DOPA-induced increase in extracellular DA and decrease in [(11) C]raclopride binding in an animal model of advanced Parkinson's disease and LID, we measured extracellular DA in response to L-DOPA or a combination of L-DOPA and the 5-HT(1A) agonist, 8-OHDPAT, with microdialysis, and determined [(11) C]raclopride binding to DA receptors, with micro-positron emission tomography, as the surrogate marker of DA release. Rats with unilateral 6-hydroxydopamine lesions had micro-positron emission tomography scans with [(11) C]raclopride at baseline and after two pharmacological challenges with L-DOPA?+?benserazide with or without 8-OHDPAT co-treatment. Identical challenge regimens were used with the subsequent microdialysis concomitant with ratings of LID severity. The baseline increase of [(11) C]raclopride-binding potential (BP(ND) ) in lesioned striatum was eliminated by the L-DOPA challenge, while the concurrent administration of 8-OHDPAT prevented this L-DOPA-induced displacement of [(11) C]raclopride significantly in lesioned ventral striatum and near significantly in the dorsal striatum. With microdialysis, the L-DOPA challenge raised the extracellular DA in parallel with the emergence of strong LID. Co-treatment with 8-OHDPAT significantly attenuated the release of extracellular DA and LID. The 8-OHDPAT co-treatment reversed the L-DOPA-induced decrease of [(11) C]raclopride binding and increase of extracellular DA and reduced the severity of LID. The reversal of the effect of L-DOPA on [(11) C]raclopride binding, extracellular DA and LID by 5-HT agonist administration is consistent with the notion that part of the DA increase associated with LID originates in serotonergic neurons.     

Critical role of ABCA1 transporter in sphingosine 1-phosphate release from astrocytes

Sphingosine 1-phosphate (S1P) is accumulated in lipoproteins, especially high-density lipoprotein (HDL), in plasma. However, it remains uncharacterized how extracellular S1P is produced in the CNS. The treatment of rat astrocytes with retinoic acid and dibutyryl cAMP, which induce apolipoprotein E (apoE) synthesis and HDL-like lipoprotein formation, stimulated extracellular S1P accumulation in the presence of its precursor sphingosine. The released S1P was present together with apoE particles in the HDL fraction. S1P release from astrocytes was inhibited by the treatment of the cells with glybenclamide or small interfering RNAs specific to ATP-binding cassette transporter A1 (ABCA1). Astrocytes from Abca1−/− mice also showed impairment of retinoic acid/dibutyryl cAMP-induced S1P release in association with the blockage of HDL-like lipoprotein formation. However, the formation of either apoE or lipoprotein itself was not sufficient, and additional up-regulation of ABCA1 was requisite to stimulate S1P release. We conclude that the S1P release from astrocytes is coupled with lipoprotein formation through ABCA1.     

Delivery of sphingosine 1-phosphate from poly(ethylene glycol) hydrogels

While protein growth factors promote therapeutic angiogenesis, delivery of lipid factors such as sphingosine 1-phosphate (S1P) may provide better stabilization of newly formed vessels. We developed a biomaterial for the controlled delivery of S1P, a bioactive lipid released from activated platelets. Multiarm poly(ethylene glycol)-vinyl sulfone was cross-linked with albumin, a lipid-transporting protein, to form hydrogels. The rate of S1P release from the materials followed Fickian kinetics and was dependent upon the presence of lipid carriers in the release solution. Delivery of S1P from RGD-modified hydrogels increased the cell migration speed of endothelial cells growing on the materials. The materials also induced angiogenesis in the chorioallantoic membrane assay. Our data demonstrate that the storage and release of lipid factors provides a new route for the induction of angiogenesis by artificial materials.     

An efficient, one-pot synthesis of various ceramide 1-phosphates from sphingosine 1-phosphate

An efficient, one-pot procedure for the synthesis of ceramide 1-phosphates with varying N-acyl substituents, to serve as tool compounds for analytical and biological investigations, was developed. Sphingosine 1-phosphate was silylated in situ to increase its solubility and to protect the 3-hydroxy functionality and then allowed to react with activated acid derivatives in the presence of diisopropylethylamine. Simultaneous cleavage of the silyl protecting groups and separation from reagents and by-products was achieved by medium pressure chromatography on reversed phase material. Thus, ceramide 1-phosphates with various fatty acid chains and with fluorescent and affinity labels attached to the sphingoid backbone were prepared in good yields.     

Insulin-like growth factors mediate heterotrimeric G protein-dependent ERK1/2 activation by transactivating sphingosine 1-phosphate receptors

Although several studies have shown that a subset of insulin-like growth factor (IGF) signals require the activation of heterotrimeric G proteins, the molecular mechanisms underlying IGF-stimulated G protein signaling remain poorly understood. Here, we have studied the mechanism by which endogenous IGF receptors activate the ERK1/2 mitogen-activated protein kinase cascade in HEK293 cells. In these cells, treatment with pertussis toxin and expression of a Galpha(q/11)-(305-359) peptide that inhibits G(q/11) signaling additively inhibited IGF-stimulated ERK1/2 activation, indicating that the signal was almost completely G protein-dependent. Treatment with IGF-1 or IGF-2 promoted translocation of green fluorescent protein (GFP)-tagged sphingosine kinase (SK) 1 from the cytosol to the plasma membrane, increased endogenous SK activity within 30 s of stimulation, and caused a statistically significant increase in intracellular and extracellular sphingosine 1-phosphate (S1P) concentration. Using a GFP-tagged S1P1 receptor as a biological sensor for the generation of physiologically relevant S1P levels, we found that IGF-1 and IGF-2 induced GFP-S1P receptor internalization and that the effect was blocked by pretreatment with the SK inhibitor, dimethylsphingosine. Treating cells with dimethylsphingosine, silencing SK1 expression by RNA interference, and blocking endogenous S1P receptors with the competitive antagonist VPC23019 all significantly inhibited IGF-stimulated ERK1/2 activation, suggesting that IGFs elicit G protein-dependent ERK1/2 activation by stimulating SK1-dependent transactivation of S1P receptors. Given the ubiquity of SK and S1P receptor expression, S1P receptor transactivation may represent a general mechanism for G protein-dependent signaling by non-G protein-coupled receptors.     

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

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

Ca2+ signaling induced by sphingosine 1-phosphate and lysophosphatidic acid in mouse B cells

Lysophospholipids (LPLs) such as lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) are chemotactic for lymphocytes, and increases of in cytosolic [Ca²⁺] signal the regulation of lymphocyte activation and migration. Here, the authors investigated the effects of LPA and S1P on [Ca²⁺]_c in mouse B cell lines (WEHI-231 and Bal-17) and primary B cells isolated from mouse spleen and bone marrow, and focused on the modulation of store-operated Ca²⁺ entry (SOCE) by LPLs. In Bal-17 (a mature B cell line) both LPA and S1P induced a transient [Ca²⁺]_c increase via a phospholipase C pathway. In addition, pretreatment with LPLs was found to augment thapsigargin-induced SOCE in Bal-17 cells. However, in WEHI-231 (an immature B cell line) LPLs had no significant effect on [Ca²⁺]_c or SOCE. Furthermore, in freshly isolated splenic B cells (SBCs) and bone marrow B cells (BMBCs), LPLs induced only a small increase in [Ca²⁺]_c. Interestingly, however, pretreatment with LPLs markedly increased SOCE in primary B cells, and this augmentation was more prominent in BMBCs than SBCs. The unidirectional influx of Ca²⁺ was measured using Ba²⁺ as a surrogate ion. Similarly, Ba²⁺ influx was also found to be markedly increased by LPLs in SBCs and BMBCs. Summarizing, LPLs were found to strongly augment SOCE-mediated Ca²⁺-signaling in mouse B cells. However, unlike the mature Bal-17 cell line, PLC-dependent Ca²⁺ release was insignificant in primary B cells and inWEHI-231.     

Distinctive G protein-dependent signaling in smooth muscle by sphingosine 1-phosphate receptors S1P1 and S1P2

We examined expression of sphingosine 1-phosphate (S1P) receptors and sphingosine kinase (SPK) in gastric smooth muscle cells and characterized signaling pathways mediating S1P-induced 20-kDa myosin light chain (MLC₂₀) phosphorylation and contraction. RT-PCR demonstrated expression of SPK1 and SPK2 and S1P₁ and S1P₂ receptors. S1P activated G_q, G₁₃, and all G_i isoforms and stimulated PLC-1, PLC-3, and Rho kinase activities. PLC- activity was partially inhibited by pertussis toxin (PTX), G or G_q antibody, PLC-1 or PLC-3 antibody, and by expression of G_q or G_i minigene, and was abolished by a combination of antibodies or minigenes. S1P-stimulated Rho kinase activity was partially inhibited by expression of G₁₃ or G_q minigene and abolished by expression of both. S1P stimulated Ca²⁺ release that was inhibited by U-73122 and heparin and induced concentration-dependent contraction of smooth muscle cells (EC₅₀ 1 nM). Initial contraction and MLC₂₀ phosphorylation were abolished by U-73122 and MLC kinase (MLCK) inhibitor ML-9. Initial contraction was also partially inhibited by PTX and G_q or G antibody and abolished by a combination of both antibodies. In contrast, sustained contraction and MLC₂₀ phosphorylation were partially inhibited by a PKC or Rho kinase inhibitor (bisindolylmaleimide and Y-27632) and abolished by a combination of both inhibitors but not affected by U-73122 or ML-9. These results indicate that S1P induces 1) initial contraction mediated by S1P₂ and S1P₁ involving concurrent activation of PLC-1 and PLC-3 via G_q and G_i, respectively, resulting in inositol 1,4,5-trisphosphate-dependent Ca²⁺ release and MLCK-mediated MLC₂₀ phosphorylation, and 2) sustained contraction exclusively mediated by S1P₂ involving activation of RhoA via G_q and G₁₃, resulting in Rho kinase- and PKC-dependent MLC₂₀ phosphorylation. muscle contraction; signal transduction     

Constrained azacyclic analogues of the immunomodulatory agent FTY720 as molecular probes for sphingosine 1-phosphate receptors

Constrained azacyclic analogues of FTY720 were prepared starting with d- and l-pyroglutamic acids. One enantiomer was shown to be a substrate for sphingosine kinase 2, being phosphorylated 4-fold more efficiently than FTY720. Among the corresponding phosphates, two were found to have unusual specificity in binding to S1P receptors: while being inactive on S1P1 and S1P3, they acted as potent agonists on S1P4 and S1P5. The phosphates may be useful to explore the biology and binding site of these receptors.     

Th17 augmentation in OTII TCR plus T cell-selective type 1 sphingosine 1-phosphate receptor double transgenic mice

Sphingosine 1-phosphate (S1P) in blood and lymph controls lymphoid traffic and tissue migration of T cells through signals from the type 1 S1PR (S1P(1)), but less is known of effects of the S1P-S1P(1) axis on nonmigration functions of T cells. CD4 T cells from a double transgenic (DTG) mouse express OTII TCRs specific for OVA peptide 323-339 (OVA) and a high level of transgenic S1P(1), resistant to suppression by T cell activation. OVA-activated DTG CD4 T cells respond as expected to S1P by chemotactic migration and reduction in secretion of IFN-gamma. In addition, DTG CD4 T cells stimulated by OVA secrete a mean of 2.5-fold more IL-17 than those from OTII single transgenic mice with concomitantly higher levels of mRNA encoding IL-17 by real-time PCR and of CD4 T cells with intracellular IL-17 detected by ELISPOT assays. OVA challenge of s.c. air pockets elicited influx of more OTII TCR-positive T cells producing a higher level of IL-17 in DTG mice than OTII control mice. Augmentation of the number and activity of Th17 cells by the S1P-S1P(1) axis may thus enhance host defense against microbes and in other settings increase host susceptibility to autoimmune diseases.     

Regulation of limb development by the sphingosine 1-phosphate receptor S1p1/EDG-1 occurs via the hypoxia/VEGF axis

Angiogenesis, also known as new blood vessel formation, is regulated coordinately with other tissue differentiation events during limb development. Although vascular endothelial cell growth factor (VEGF) is important in the regulation of angiogenesis, chondrogenesis and osteogenesis during limb development, the role of other angiogenic factors is not well understood. Sphingosine 1-phosphate, a platelet-derived lipid mediator, regulates angiogenesis and vascular maturation via its action on the G-protein-coupled receptor S1P(1) (also known as EDG-1). In addition to vascular defects, abnormal limb development was also observed in S1p(1)(-/-) mice. Here we show that strong induction of S1P(1) expression is observed in the blood vessels and the interdigital mesenchymal cells during limb development. Deletion of S1P(1) results in aberrant chondrocyte condensation and defective digit morphogenesis. Interestingly, the vasculature in the S1p(1)(-/-) limbs was hyperplastic and morphologically altered. In addition, the hypoxia inducible factor (HIF)-1 alpha and its response gene VEGF were induced in S1p(1)(-/-) limbs. However, aberrant regulation of HIF-1 alpha and VEGF were not observed in embryonic fibroblasts derived from S1p(1)(-/-) mice, suggesting a non-cell autonomous effect of S1P(1) on VEGF expression. Indeed, similar limb defects were observed in endothelium-specific S1P(1) null mice in vivo. These data suggest that the function of S1P(1) in the developing vasculature is essential for proper limb development.     

Sphingosine-1-phosphate promotes mouse melanocyte survival via ERK and Akt activation

In this study, we investigated the signalling pathways induced by ultraviolet B (UVB) and the effects of sphingosine-1-phosphate on UVB-induced apoptosis of mouse melanocytes, Mel-Ab, and observed the cytoprotective effects of sphingosine-1-phosphate on UVB-induced apoptosis. Since sphingosine-1-phosphate is a well-known mitogenic agent, we thought it possible that the mitogenic effect of sphingosine-1-phosphate might contribute to cell survival. However, we found that sphingosine-1-phosphate significantly inhibits DNA synthesis. We next examined the regulation of the three major subfamilies of mitogen-activated protein (MAP) kinases and of the Akt pathway by sphingosine-1-phosphate against UVB-induced apoptosis. UVB irradiation resulted in the remarkable and sustained activation of c-Jun N-terminal kinase (JNK), while p38 MAP kinase was only transiently activated. The basal level of extracellular signal-regulated protein kinase (ERK) phosphorylation decreased 30 min after UVB irradiation, whereas the basal level of Akt phosphorylation was unaffected by UVB. We also found that sphingosine-1-phosphate potently stimulates the phosphorylation of both ERK and Akt, which are involved in the cell survival-signalling cascade. Furthermore, the specific inhibition of the ERK and Akt pathways by PD98059 and LY294002, respectively, restored the cytoprotective effect induced by sphingosine-1-phosphate. On the other hand, the p38 inhibitor SB203580 additively enhanced the cytoprotective effect on sphingosine-1-phosphate. Based on these results, we conclude that the activation of p38 MAP kinase plays an important role in UVB-induced apoptosis, and that sphingosine-1-phosphate probably exert its cytoprotective effect in Mel-Ab cells through ERK and Akt activation.     

Expression and characterization of Edg-1 receptors in rat cardiomyocytes: calcium deregulation in response to sphingosine 1-phosphate.

Recent evidence indicates that sphingolipids are produced by the heart during hypoxic stress and by blood platelets during thrombus formation. It is therefore possible that sphingolipids may influence heart cell function by interacting with G-protein-coupled receptors of the Edg family. In the present study, it was found that sphingosine 1-phosphate (Sph1P), the prototypical ligand for Edg receptors, produced calcium overload in rat cardiomyocytes. The cDNA for Edg-1 was cloned from rat cardiomyocytes and, when transfected in an antisense orientation, effectively blocked Edg-1 protein expression and reduced the Sph1P-mediated calcium deregulation. Taken together, these results demonstrate that cardiomyocytes express an extracellular lipid-sensitive receptorsystem that can respond to sphingolipid mediators. Because the major source of Sph1P is from blood platelets, we speculate that Edg-mediated Sph1P negative inotropic and cardiotoxic effects may play important roles in acute myocardial ischemia where Sph1P levels are probably elevated in response to thrombus.     

Development of electrical activity in cardiac myocyte aggregates derived from mouse embryonic stem cells

Embryonic stem cells differentiate into cardiac myocytes, repeating in vitro the structural and molecular changes associated with cardiac development. Currently, it is not clear whether the electrophysiological properties of the multicellular cardiac structure follow cardiac maturation as well. In long-term recordings of extracellular field potentials with microelectrode arrays consisting of 60 substrate-integrated electrodes, we examined the electrophysiological properties during the ongoing differentiation process. The beating frequency of the growing preparations increased from 1 to 5 Hz concomitant to a decrease of the action potential duration and action potential rise time. A developmental increase of the conduction velocity could be attributed to an increased expression of connexin43 gap junction channels. Whereas isoprenalin elicited a positive chronotropic response from the first day of spontaneous beating onward, a concentration-dependent negative chronotropic effect of carbachol only developed after approximately 4 days. The in vitro development of the three-dimensional cardiac preparation thus closely follows the development described for the mouse embryonic heart, making it an ideal model to monitor the differentiation of electrical activity in embryonic cardiomyocytes.