Non-selective Sphingosine 1-phosphate Research Articles

Abstract 14871: APD588, a Novel, Selective S1P Receptor Modulator, Regulates Inflammatory Responses and Attenuates Cardiac Dysfunction Following Experimental Myocardial Infarction in Mice

Recent studies have determined that chronic inflammation contributes to left ventricular (LV) remodeling following myocardial infarction (MI) leading to heart failure (HF). Evidence points to a critical role of T lymphocytes in driving the maladaptive inflammatory process associated with adverse tissue remodeling and cardiac dysfunction. FTY720, a non-selective sphingosine 1-phosphate (S1P) receptor modulator, elicits anti-inflammatory effects via inhibition of lymphocyte egress from secondary lymphoid organs and has been reported to improve cardiac remodeling and function post-MI. We hypothesized that APD588, a next-generation S1P receptor modulator with optimized S1P 1,5 receptor selectivity, would prevent cardiac remodeling and dysfunction following MI through modulation of T lymphocyte-mediated inflammatory responses. Efficacy of APD588 on cardiac function was assessed using C57BL/6N mice (n=7-10 mice/group) with experimental MI induced by coronary artery occlusion. Vehicle (VEH) or APD588 (0.3 mg/kg) was administered orally once daily starting at Day 3 post-MI and continued until Day 28. Blood samples were collected at Days 7 and 28 for lymphocyte count. Echocardiographic measurements, including LV ejection fraction (LVEF), were performed on Days 7, 14, and 28 post-MI. Administration of APD588 following MI resulted in a reduction of lymphocyte count at Days 7 and 28 post-MI, but only reached significance in the latter (VEH: 3.3 ± 0.5 x10 3 /μL; APD588: 1.3 ± 0.2 x10 3 /μL). Decreased cardiac function was observed at Day 7 post-MI (LVEF: 27.7 ± 3.6%, MI vs 45.7 ± 0.6%, Sham). No difference in LVEF was observed 7 days post-MI, however APD588 significantly improved LVEF at Day 28 post-MI, compared to vehicle (APD588: 35.7 ± 3.9%; VEH: 25.3 ± 3.9%). Therapies targeting inflammation in the pathophysiology of HF have shown limited success in the clinic. Novel strategies based on advancements in understanding the underlying immunobiology are warranted. Our results demonstrate that APD588 was effective at regulating lymphocyte activity and improved cardiac functional recovery following MI in mice. This supports the potential of S1P receptor modulation and other T lymphocyte-directed strategies as a targeted anti-inflammatory approach in HF.

Sphingosine 1-Phosphate Receptors in Cerebral Ischemia.

Sphingosine 1-phosphate (S1P) is an important lipid biomolecule that exerts pleiotropic cellular actions as it binds to and activates its five G-protein-coupled receptors, S1P1-5. Through these receptors, S1P can mediate diverse biological activities in both healthy and diseased conditions. S1P is produced by S1P-producing enzymes, sphingosine kinases (SphK1 and SphK2), and is abundantly present in different organs, including the brain. The medically important roles of receptor-mediated S1P signaling are well characterized in multiple sclerosis because FTY720 (Gilenya™, Novartis), a non-selective S1P receptor modulator, is currently used as a treatment for this disease. In cerebral ischemia, its role is also notable because of FTY720's efficacy in both rodent models and human patients with cerebral ischemia. In particular, some of the S1P receptors, including S1P1, S1P2, and S1P3, have been identified as pathogenic players in cerebral ischemia. Other than these receptors, S1P itself and S1P-producing enzymes have been shown to play certain roles in cerebral ischemia. This review aims to compile the current updates and overviews about the roles of S1P signaling, along with a focus on S1P receptors in cerebral ischemia, based on recent studies that used in vivo rodent models of cerebral ischemia.

Identification of Sphingosine 1-Phosphate Receptor Subtype 1 (S1P1) as a Pathogenic Factor in Transient Focal Cerebral Ischemia.

Medically relevant roles of receptor-mediated sphingosine 1-phosphate (S1P) signaling have become a successful or promising target for multiple sclerosis or cerebral ischemia. Animal-based proof-of-concept validation for the latter is particularly through the neuroprotective efficacy of FTY720, a non-selective S1P receptor modulator, presumably via activation of S1P1. In spite of a clear link between S1P signaling and cerebral ischemia, it remains unknown whether the role of S1P1 is pathogenic or neuroprotective. Here, we investigated the involvement of S1P1 along with its role in cerebral ischemia using a transient middle cerebral artery occlusion ("tMCAO") model. Brain damage following tMCAO, as assessed by brain infarction, neurological deficit score, and neural cell death, was reduced by oral administration of AUY954, a selective S1P1 modulator as a functional antagonist, in a therapeutic paradigm, indicating that S1P1 is a pathogenic mediator rather than a neuroprotective mediator. This pathogenic role of S1P1 in cerebral ischemia was reaffirmed because tMCAO-induced brain damage was reduced by genetic knockdown with an intracerebroventricular microinjection of S1P1 shRNA lentivirus into the brain. Genetic knockdown of S1P1 or AUY954 exposure reduced microglial activation, as assessed by reduction in the number of activated microglia and reversed morphology from amoeboid to ramified, and microglial proliferation in ischemic brain. Its role in microglial activation was recapitulated in lipopolysaccharide-stimulated primary mouse microglia, in which the mRNA expression level of TNF-α and IL-1β, well-known markers for microglial activation, was reduced in microglia transfected with S1P1 siRNA. These data suggest that the pathogenic role of S1P1 is associated with microglial activation in ischemic brain. Additionally, the pathogenic role of S1P1 in cerebral ischemia appears to be associated with the blood-brain barrier disruption and brain-derived neurotrophic factor (BDNF) downregulation. Overall, findings from the current study clearly identify S1P1 signaling as a pathogenic factor in transient focal cerebral ischemia, further implicating S1P1 antagonists including functional antagonists as plausible therapeutic agents for human stroke.

Modulation of sphingosine-1-phosphate in inflammatory bowel disease.

Inflammatory bowel diseases (IBD), including ulcerative colitis and Crohn's disease, involve an inappropriate immune reaction in the digestive tract, causing a variety of disabling symptoms. The advent of monoclonal antibodies (anti-tumor necrosis factor, anti-integrin, anti-interleukin -23) has revolutionized IBD management. Nevertheless, these agents, with potential for immunogenicity, are associated with high rates of response loss and disease relapse over time. They are also associated with high production costs. Sphingosine-1-phosphate (S1P), a membrane-derived lysophospholipid signaling molecule, is implicated in a vast array of physiological and pathophysiological processes, primarily via extracellular activation of S1P1-S1P5 receptors. S1P1, S1P4 and S1P5 are involved in regulation of the immune system, while S1P2 and S1P3 may be associated with cardiovascular, pulmonary, and theoretical cancer-related risks. Targeting S1P receptors for inflammatory conditions has been successful in clinical trials leading to approval of the non-selective S1P modulator, fingolimod, for relapsing forms of multiple sclerosis. However, the association of this non-selective S1P modulator with serious adverse events provides the rationale for developing more selective S1P receptor modulators. Until recently, three S1P modulators with differing selectivity for S1P receptors were in clinical development for IBD: ozanimod (RPC1063), etrasimod (APD334) and amiselimod (MT-1303). The development of amiselimod has been stopped as Biogen are currently focusing on other drugs in its portfolio. Following encouraging results from the Phase 2 TOUCHSTONE trial, a Phase 3 trial of the S1P modulator ozanimod in patients with moderate-to-severe ulcerative colitis is ongoing. Etrasimod is also being tested in a phase 2 trial in ulcerative colitis. These pipeline medications can be administered orally and may avoid the formation of anti-drug antibodies that can lead to treatment failure with injectable biologic therapies for IBD. Data from ongoing clinical trials will establish the relationship between the selectivity of S1P modulators and their safety and efficacy in IBD, as well as their potential place in the clinical armamentarium for IBD.

Ozanimod (RPC1063) is a potent sphingosine-1-phosphate receptor-1 (S1P1 ) and receptor-5 (S1P5 ) agonist with autoimmune disease-modifying activity.

Sphingosine1-phosphate (S1P) receptors mediate multiple events including lymphocyte trafficking, cardiac function, and endothelial barrier integrity. Stimulation of S1P1 receptors sequesters lymphocyte subsets in peripheral lymphoid organs, preventing their trafficking to inflamed tissue sites, modulating immunity. Targeting S1P receptors for treating autoimmune disease has been established in clinical studies with the non-selective S1P modulator, FTY720 (fingolimod, Gilenya™). The purpose of this study was to assess RPC1063 for its therapeutic utility in autoimmune diseases. The specificity and potency of RPC1063 (ozanimod) was evaluated for all five S1P receptors, and its effect on cell surface S1P1 receptor expression, was characterized in vitro. The oral pharmacokinetic (PK) parameters and pharmacodynamic effects were established in rodents, and its activity in three models of autoimmune disease (experimental autoimmune encephalitis, 2,4,6-trinitrobenzenesulfonic acid colitis and CD4(+) CD45RB(hi) T cell adoptive transfer colitis) was assessed. RPC1063 was specific for S1P1 and S1P5 receptors, induced S1P1 receptor internalization and induced a reversible reduction in circulating B and CCR7(+) T lymphocytes in vivo. RPC1063 showed high oral bioavailability and volume of distribution, and a circulatory half-life that supports once daily dosing. Oral RPC1063 reduced inflammation and disease parameters in all three autoimmune disease models. S1P receptor selectivity, favourable PK properties and efficacy in three distinct disease models supports the clinical development of RPC1063 for the treatment of relapsing multiple sclerosis and inflammatory bowel disease, differentiates RPC1063 from other S1P receptor agonists, and could result in improved safety outcomes in the clinic.

Therapeutic Potential of the Modulation of Sphingosine-1-Phosphate Receptors.

It is accepted that sphingolipids (SL) are not only structural lipids in cellular membranes, but also key regulators of different cell process. Sphingosine-1-phosphate (S1P) is a member of this family involved, inter alia, in cell migration, angiogenesis and cell proliferation processes, being able to play different intracellular and extracellular roles. When S1P is transported out of the cell, it binds S1P specific G protein-coupled receptors, which are mainly involved in the regulation of the immune, vascular and nervous systems. These effects account for the vast diversity of functions that arise from the activation of S1P receptors. Deregulation of S1P levels is correlated with several pathologies, such as autoimmune disorders and cancer. Consequently, the correct modulation of these receptors represents a valuable approach for the development of new therapeutic strategies. Along this line, the non-selective S1P receptor agonist fingolimod (FTY720) has been commercialized recently for the treatment of multiple sclerosis and several related S1P receptor modulators are ongoing clinical trials. However, despite the progress in this field, the biological functions of S1P receptors are not still well elucidated. For this reason, several studies are being developed in order to better understand the functions of these receptors, making use of new selective S1P receptor agonists and antagonists as pharmacological tools.

Sphingosine-1-phosphate receptor 1 agonism attenuates lung ischemia-reperfusion injury.

Outcomes for lung transplantation are the worst of any solid organ, and ischemia-reperfusion injury (IRI) limits both short- and long-term outcomes. Presently no therapeutic agents are available to prevent IRI. Sphingosine 1-phosphate (S1P) modulates immune function through binding to a set of G protein-coupled receptors (S1PR1-5). Although S1P has been shown to attenuate lung IRI, the S1P receptors responsible for protection have not been defined. The present study tests the hypothesis that protection from lung IRI is primarily mediated through S1PR1 activation. Mice were treated with either vehicle, FTY720 (a nonselective S1P receptor agonist), or VPC01091 (a selective S1PR1 agonist and S1PR3 antagonist) before left lung IR. Function, vascular permeability, cytokine expression, neutrophil infiltration, and myeloperoxidase levels were measured in lungs. After IR, both FTY720 and VPC01091 significantly improved lung function (reduced pulmonary artery pressure and increased pulmonary compliance) vs. vehicle control. In addition, FTY720 and VPC01091 significantly reduced vascular permeability, expression of proinflammatory cytokines (IL-6, IL-17, IL-12/IL-23 p40, CC chemokine ligand-2, and TNF-α), myeloperoxidase levels, and neutrophil infiltration compared with control. No significant differences were observed between VPC01091 and FTY720 treatment groups. VPC01091 did not significantly affect elevated invariant natural killer T cell infiltration after IR, and administration of an S1PR1 antagonist reversed VPC01091-mediated protection after IR. In conclusion, VPC01091 and FTY720 provide comparable protection from lung injury and dysfunction after IR. These findings suggest that S1P-mediated protection from IRI is mediated by S1PR1 activation, independent of S1PR3, and that selective S1PR1 agonists may provide a novel therapeutic strategy to prevent lung IRI.

ASP4058, a novel agonist for sphingosine 1-phosphate receptors 1 and 5, ameliorates rodent experimental autoimmune encephalomyelitis with a favorable safety profile.

Sphingosine-1-phosphate (S1P) is a biologically active sphingolipid that acts through the members of a family of five G protein-coupled receptors (S1P1–S1P5). S1P1 is a major regulator of lymphocyte trafficking, and fingolimod, whose active metabolite fingolimod phosphate acts as a nonselective S1P receptor agonist, exerts its immunomodulatory effect, at least in part, by regulating the lymphocyte trafficking by inducing down regulation of lymphocyte S1P1. Here, we detail the pharmacological profile of 5-{5-[3-(trifluoromethyl)-4-{[(2S)-1,1,1-trifluoropropan-2-yl]oxy}phenyl]-1,2,4-oxadiazol-3-yl}-1H-benzimidazole (ASP4058), a novel next-generation S1P receptor agonist selective for S1P1 and S1P5. ASP4058 preferentially activates S1P1 and S1P5 compared with S1P2, 3, 4 in GTPγS binding assays in vitro. Oral administration of ASP4058 reduced the number of peripheral lymphocytes and inhibited the development of experimental autoimmune encephalomyelitis (EAE) in Lewis rats. Further, ASP4058 prevented relapse of disease in a mouse model of relapsing-remitting EAE. Although these immunomodulatory effects were comparable to those of fingolimod, ASP4058 showed a wider safety margin than fingolimod for bradycardia and bronchoconstriction in rodents. These observations suggest that ASP4058 represents a new therapeutic option for treating multiple sclerosis that is safer than nonselective S1P receptor agonists such as fingolimod.

Calcitonin controls bone formation by inhibiting the release of sphingosine 1-phosphate from osteoclasts.

The hormone calcitonin (CT) is primarily known for its pharmacologic action as an inhibitor of bone resorption, yet CT-deficient mice display increased bone formation. These findings raised the question about the underlying cellular and molecular mechanism of CT action. Here we show that either ubiquitous or osteoclast-specific inactivation of the murine CT receptor (CTR) causes increased bone formation. CT negatively regulates the osteoclast expression of Spns2 gene, which encodes a transporter for the signalling lipid sphingosine 1-phosphate (S1P). CTR-deficient mice show increased S1P levels, and their skeletal phenotype is normalized by deletion of the S1P receptor S1P3. Finally, pharmacologic treatment with the nonselective S1P receptor agonist FTY720 causes increased bone formation in wild-type, but not in S1P3-deficient mice. This study redefines the role of CT in skeletal biology, confirms that S1P acts as an osteoanabolic molecule in vivo and provides evidence for a pharmacologically exploitable crosstalk between osteoclasts and osteoblasts.

Prolonging survival of corneal transplantation by selective sphingosine-1-phosphate receptor 1 agonist.

Corneal transplantation is the most used therapy for eye disorders. Although the cornea is somewhat an immune privileged organ, immune rejection is still the major problem that reduces the success rate. Therefore, effective chemical drugs that regulate immunoreactions are needed to improve the outcome of corneal transplantations. Here, a sphingosine-1-phosphate receptor 1 (S1P1) selective agonist was systematically evaluated in mouse allogeneic corneal transplantation and compared with the commonly used immunosuppressive agents. Compared with CsA and the non-selective sphingosine 1-phosphate (S1P) receptor agonist FTY720, the S1P1 selective agonist can prolong the survival corneal transplantation for more than 30 days with a low immune response. More importantly, the optimal dose of the S1P1 selective agonist was much less than non-selective S1P receptor agonist FTY720, which would reduce the dose-dependent toxicity in drug application. Then we analyzed the mechanisms of the selected S1P1 selective agonist on the immunosuppression. The results shown that the S1P1 selective agonist could regulate the distribution of the immune cells with less CD4+ T cells and enhanced Treg cells in the allograft, moreover the expression of anti-inflammatory cytokines TGF-β1 and IL-10 unregulated which can reduce the immunoreactions. These findings suggest that S1P1 selective agonist may be a more appropriate immunosuppressive compound to effectively prolong mouse allogeneic corneal grafts survival.

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

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

Effect of sphingosine 1-phosphate (S1P) receptor agonists FTY720 and CYM5442 on atherosclerosis development in LDL receptor deficient (LDL-R−/−) mice

ObjectivesSphingosine 1-phosphate (S1P) – a lysosphingolipid present in HDL – exerts atheroprotective effects in vitro, while FTY720, a non-selective S1P mimetic inhibits atherosclerosis in LDL receptor-deficient (LDL-R−/−) mice under conditions of severe hypercholesterolemia. We here examined the effect of FTY720 and a selective S1P receptor type 1 agonist CYM5442 on atherosclerosis in moderately hypercholesterolemic LDL-R−/− mice. Methods and resultsLDL-R−/− mice fed Western diet (0.25% cholesterol) were given FTY720 (0.4mg/kg/day) or CYM5442 (2.0mg/kg/day) for 18weeks. FTY720 but not CYM5422 persistently lowered blood lymphocytes, depleted CD4+ and CD8+ T cells in spleen and lymph nodes, and reduced splenocyte IL-2 secretion. However, both compounds reduced the activity of splenic and peritoneal macrophages as inferred from the down-regulated CD68 and MHC-II expression in CD11b+ cells and the reduced IL-6 secretion in response to LPS, respectively. CYM5442 and FTY720 reduced weight gain, white adipose tissue depots and fasting glucose suggesting improvement of metabolic control, but failed to influence atherosclerosis in LDL-R−/− mice. ConclusionDespite down-regulating macrophage function and – in case of FTY720 – altering lymphocyte distribution CYM5442 and FTY720 fail to affect atherosclerosis in moderately hypercholesterolemic LDL-R−/− mice. We hypothesize that S1P mimetics exert atheroprotective effects only under conditions of increased cholesterol burden exacerbating vascular inflammation.

Cardiomyocyte S1P1 Receptor-mediated Extracellular Signal-related Kinase Signaling and Desensitization

We examined the ability of sphingosine-1-phosphate (S1P) to desensitize extracellular signal-related kinase (ERK), a mitogen-activated protein kinase linked to antiapoptotic responses in the heart. In isolated adult mouse cardiomyocytes, S1P (10 nM-5 microM) induced ERK phosphorylation in a time- and dose-dependent manner. S1P stimulation of ERK was completely inhibited by an S1P1/3 subtype receptor antagonist (VPC23019), by a Gi protein inhibitor (pertussis toxin) and by a mitogen-activated protein kinase/ERK kinase inhibitor (PD98059). A selective S1P3 receptor antagonist (CAY10444) had no effect on S1P-induced ERK activation. The selective S1P1 agonist SEW2871 also induced ERK phosphorylation. Activation of ERK by restimulation with 100 nM S1P was suppressed after 1 hour of preincubation with 100 nM S1P but recovered fully the next day, suggesting receptor recycling. Similar results were obtained in protein kinase C epsilon-null cardiomyocytes. Treatment with the nonselective S1P receptor agonist FTY720 for 1 hour also reduced phospho-ERK expression in response to subsequent S1P stimulation. In contrast to S1P, some desensitization to FTY720 persisted after overnight exposure. Cell death induced by hypoxia/reoxygenation was reduced by pretreatment with exogenous S1P. This enhanced survival was abrogated by pretreatment with PD98059, VPC23019, or pertussis toxin. Thus, exogenous S1P induces rapid and reversible S1P1-mediated ERK phosphorylation. S1P-induced adult mouse cardiomyocyte survival requires ERK activation mediated via an S1P1-Gi pathway.

Lysophospholipid Receptors as Potential Drug Targets in Tissue Transplantation and Autoimmune Diseases

New therapies directed at ameliorating or altering autoimmune diseases represent an area of significant medical need. Included amongst autoimmune diseases are problems related to transplantation rejection, as well as a number of neurological diseases such as Multiple Sclerosis (MS). A new group of molecular targets that may lead to novel therapies are lysophospholipid (LP) receptors. A large range of biological activities has been attributed to the actions of these simple phospholipids that include well-studied members lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P). Documented cellular effects of these lipid molecules encompass growth-factor-like influences on cells, including but not limited to survival, migration, adhesion differentiation, as well as pathophysiological actions associated with cancer. In turn, these cellular effects have roles in developing and adult organ systems such as the nervous system, cardiovascular system, reproductive system and, of relevance here, the immune system. The mechanisms for these actions can be attributed to a growing family of cognate, 7-transmembrane G protein-coupled receptors (GPCRs), with documented validation through studies utilizing pharmacology, molecular genetics and an enlarging repertoire of chemical tools having agonist or antagonist properties. The growing literature on immunological effects of LP receptors, particularly those mediating the effects of S1P, has suggested possible therapeutic roles for this class of receptors. In particular, entry into humans of a non-selective S1P receptor agonist, FTY720, for kidney transplantation and possibly other indications (e.g., Multiple Sclerosis), has raised prospects for efficacious treatment of human diseases based on LP receptor targets. Here we provide a brief introduction to receptor-mediated lysophospholipid signaling and discuss its basic and potential therapeutic roles in autoimmune-related diseases.

CD69 down‐modulation and inhibition of thymic egress by short‐ and long‐term selective chemical agonism of sphingosine 1‐phosphate receptors

Thymic development requires proliferation, selection, maturation and release of mature single-positive CD4 and CD8 T cells into the periphery. In mice, non-selective sphingosine-1 phosphate (S1P) receptor agonists, active on four of the five known S1P receptors, alter thymocyte phenotype and egress. Here, we show that down-modulation of CD69 occurs acutely and transiently at a discrete and late stage of medullary development after a single-dose administration of S1P(1) receptor-selective agonist, which induces long-term tonic receptor activation in the absence of receptor degradation. In addition, agonist acutely inhibited egress of mature thymocytes into peripheral lymphoid organs, suggesting that both the phenotype and migration of medullary thymocytes are regulated simultaneously and coordinately by agonism of S1P(1) alone. Long-term dosing shifted the early/late medullary thymocyte ratio with an expansion of the late medullary compartment, as mature CD69(-) thymocytes were retained within the thymus. Therefore, chemical agonism of S1P(1) accelerates medullary phenotypic maturation and inhibits egress, leading to the expansion and accumulation of the recent thymocyte emigrant population in the medulla. However, chemical agonism fails to replicate the S1P(1)-null CD69(hi) late medullary phenotype, suggesting that agonism and gene deletion operate by distinct mechanisms, and that functional receptor antagonism may not be required for lymphocyte sequestration.

Sphingosine 1-Phosphate (S1P) Receptor Subtypes S1P1 and S1P3, Respectively, Regulate Lymphocyte Recirculation and Heart Rate

Sphingosine 1-phosphate (S1P) influences heart rate, coronary artery caliber, endothelial integrity, and lymphocyte recirculation through five related high affinity G-protein-coupled receptors. Inhibition of lymphocyte recirculation by non-selective S1P receptor agonists produces clinical immunosuppression preventing transplant rejection but is associated with transient bradycardia. Understanding the contribution of individual receptors has been limited by the embryonic lethality of the S1P(1) knock-out and the unavailability of selective agonists or antagonists. A potent, S1P(1)-receptor selective agonist structurally unrelated to S1P was found to activate multiple signals triggered by S1P, including guanosine 5'-3-O-(thio)triphosphate binding, calcium flux, Akt and ERK1/2 phosphorylation, and stimulation of migration of S1P(1)- but not S1P(3)-expressing cells in vitro. The agonist also alters lymphocyte trafficking in vivo. Use of selective agonism together with deletant mice lacking S1P(3) receptor reveals that agonism of S1P(1) receptor alone is sufficient to control lymphocyte recirculation. Moreover, S1P(1) receptor agonist plasma levels are causally associated with induction and maintenance of lymphopenia. S1P(3), and not S1P(1), is directly implicated in sinus bradycardia. The sustained bradycardia induced by S1P receptor non-selective immunosuppressive agonists in wild-type mice is abolished in S1P(3)-/- mice, whereas S1P(1)-selective agonist does not produce bradycardia. Separation of receptor subtype usage for control of lymphocyte recirculation and heart rate may allow the identification of selective immunosuppressive S1P(1) receptor agonists with an enhanced therapeutic window. S1P(1)-selective agonists will be of broad utility in understanding cell functions in vitro, and vascular physiology in vivo, and the success of the chemical approach for S1P(1) suggests that selective tools for the resolution of function across this broad lipid receptor family are now possible.