Tissue-resident memory T cells provide local immune protection in barrier tissues, such as skin and mucosa. However, the molecular mechanisms controlling effector T cell retention and subsequent memory formation in those locations are not fully understood. In this study, we analyzed the role of CD69, an early leukocyte activation marker, in regulating effector T cell egress from peripheral tissues. We provide evidence that CD69 surface expression by skin-infiltrating CD8 T cells can be regulated at multiple levels, including local Ag stimulation and signaling through type I IFNRs, and it coincides with the transcriptional downregulation of the <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> receptor S<em>1</em>P<em>1</em>. Importantly, we demonstrate that expression of CD69, by interfering with <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> receptor function, is a critical determinant of prolonged T cell retention and local memory formation. Our results define an important step in the generation of long-lived adaptive immune memory at body surfaces.

FTY720, a potent immunomodulatory drug in phase 2/3 clinical trials, induces rapid and reversible sequestration of lymphocytes into secondary lymphoid organs, thereby preventing their migration to sites of inflammation. As prerequisite for its function, phosphorylation of FTY720 to yield a potent agonist of the <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> receptor S<em>1</em>P(<em>1</em>) is required in vivo, catalyzed by an as-yet-unknown kinase. Here, we report on the generation of <em>sphingosine</em> kinase 2 (SPHK2) knockout mice and demonstrate that this enzyme is essential for FTY720 <em>phosphate</em> formation in vivo. Consequently, administration of FTY720 does not induce lymphopenia in SPHK2-deficient mice. After direct dosage of FTY720 <em>phosphate</em>, lymphopenia is only transient in this strain, indicating that SPHK2 is constantly required to maintain FTY720 <em>phosphate</em> levels in vivo.

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) is a lysophospholipid mediator that evokes a variety of cell and tissue responses via a set of cell surface receptors. The recent development of S<em>1</em>P receptor agonists, led by the immunomodulatory pro-drug FTY720, has revealed that S<em>1</em>P signaling is an important regulator of lymphocyte trafficking. With the twin goals of understanding structure-activity relationships of S<em>1</em>P ligands and developing tool compounds to explore S<em>1</em>P biology, we synthesized and tested numerous S<em>1</em>P analogs. We report herein that a subset of our aryl amide-containing compounds are antagonists at the S<em>1</em>P(<em>1</em>) and S<em>1</em>P(3) receptors. The lead compound in series, VPC230<em>1</em>9, was found in broken cell and whole cell assays to behave as a competitive antagonist at the S<em>1</em>P(<em>1</em>) and S<em>1</em>P(3) receptors. The structure-activity relationship of this series is steep; for example, a slight modification of the lead compound resulted in VPC25239, which was one log order more potent at the S<em>1</em>P(3) receptor. These new chemical entities will enable further understanding of S<em>1</em>P signaling and provide leads for further S<em>1</em>P receptor antagonist development.

Although <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (Sph-<em>1</em>-P) is reportedly involved in diverse cellular processes and the physiological roles of this bioactive sphingolipid have been strongly suggested, few studies have revealed the presence of Sph-<em>1</em>-P in human samples, including body fluids and cells, under physiological conditions. In this study, we identified Sph-<em>1</em>-P as a normal constituent of human plasma and serum. The Sph-<em>1</em>-P levels in plasma and serum were <em>1</em>9<em>1</em>+/-79 and 484+/-82 pmol/ml (mean+/-SD, n=8), respectively. Furthermore, when Sph-<em>1</em>-P was measured in paired plasma and serum samples obtained from 6 healthy adults, the serum Sph-<em>1</em>-P/plasma Sph-<em>1</em>-P ratio was found to be 2.65+/-<em>1</em>.26 (mean+/-SD). It is most likely that the source of discharged Sph-<em>1</em>-P during blood clotting is platelets, because platelets abundantly store Sph-<em>1</em>-P compared with other blood cells, and release part of their stored Sph-<em>1</em>-P extracellularly upon stimulation. We also studied Sph-<em>1</em>-P-related metabolism in plasma. [3H]Sph was stable and not metabolized at all in plasma, but was rapidly incorporated into platelets and metabolized mainly to Sph-<em>1</em>-P in platelet-rich plasma. [3H]Sph-<em>1</em>-P was found to be unchanged in plasma, revealing that plasma does not contain the enzymes needed for Sph-<em>1</em>-P degradation. In summary, platelets can convert Sph into Sph-<em>1</em>-P, and are storage sites for the latter in the blood. In view of the diverse biological effects of Sph-<em>1</em>-P, the release of Sph-<em>1</em>-P from activated platelets may be involved in a variety of physiological and pathophysiological processes, including thrombosis, hemostasis, atherosclerosis and wound healing.

<em>Sphingosine</em> kinase <em>1</em> (SphK<em>1</em>) phosphorylates <em>sphingosine</em> to form <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) and is a critical regulator of sphingolipid-mediated functions. Cell-based studies suggest a tumor-promoting function for the SphK<em>1</em>/S<em>1</em>P pathway. Also, our previous studies implicated the SphK<em>1</em>/S<em>1</em>P pathway in the induction of the arachidonic acid cascade, a major inflammatory pathway involved in colon carcinogenesis. Therefore, we investigated whether the SphK<em>1</em>/S<em>1</em>P pathway is necessary for mediating carcinogenesis in vivo. Here, we report that 89% (42/47) of human colon cancer samples stained positively for SphK<em>1</em>, whereas normal colon mucosa had negative or weak staining. Adenomas had higher expression of SphK<em>1</em> vs. normal mucosa, and colon cancers with metastasis had higher expression of SphK<em>1</em> than those without metastasis. In the azoxymethane (AOM) murine model of colon cancer, SphK<em>1</em> and S<em>1</em>P were significantly elevated in colon cancer tissues compared to normal mucosa. Moreover, blood levels of S<em>1</em>P were higher in mice with colon cancers than in those without cancers. Notably, SphK<em>1</em>(-/-) mice subjected to AOM had significantly less aberrant crypt foci (ACF) formation and significantly reduced colon cancer development. These results are the first in vivo evidence that the SphK<em>1</em>/S<em>1</em>P pathway contributes to colon carcinogenesis and that inhibition of this pathway is a potential target for chemoprevention.

<em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) is a bioactive sphingolipid metabolite that has been implicated in many biological processes, including cell migration, survival, proliferation, angiogenesis and immune and allergic responses. S<em>1</em>P levels inside cells are regulated tightly by the balance between its synthesis by <em>sphingosine</em> kinases and degradation by S<em>1</em>P lyases and S<em>1</em>P phosphatases. Activation of <em>sphingosine</em> kinase by any of a variety of agonists increases S<em>1</em>P levels, which in turn can function intracellularly as a second messenger or in an autocrine and/or paracrine fashion to activate and signal through S<em>1</em>P receptors present on the surface of the cell. This review summarizes recent findings on the roles of S<em>1</em>P as a mediator of the actions of cytokines, growth factors and hormones.

FTY720 [2-amino-2-[2-(4-octylphenyl) ethyl]propane-<em>1</em>,3-diol hydrochloride] is an oral <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> receptor modulator under development for the treatment of multiple sclerosis (MS). The drug is phosphorylated in vivo by <em>sphingosine</em> kinase 2 to its bioactive form, FTY720-P. Although treatment with FTY720 is accompanied by a reduction of the peripheral lymphocyte count, its efficacy in MS and experimental autoimmune encephalomyelitis (EAE) may be due to additional, direct effects in the central nervous system (CNS). We now show that FTY720 localizes to the CNS white matter, preferentially along myelin sheaths. Brain trough levels of FTY720 and FTY720-P in rat EAE are of the same magnitude and dose dependently increase; they are in the range of 40 to 540 ng/g in the brain tissue at efficacious doses and exceed blood concentrations severalfold. In a rat model of chronic EAE, prolonged treatment with 0.03 mg/kg was efficacious, but limiting the dosing period failed to prevent EAE despite a significant decrease in blood lymphocytes. FTY720 effectiveness is likely due to a culmination of mechanisms involving reduction of autoreactive T cells, neuroprotective influence of FTY720-P in the CNS, and inhibition of inflammatory mediators in the brain.

<em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> receptor <em>1</em> (S<em>1</em>P(<em>1</em>)) was recently shown to be required for lymphocyte egress from lymphoid organs. Here we have examined the relationship between S<em>1</em>P(<em>1</em>) abundance on the cell and egress efficiency. Using an integrin neutralization approach to separate the processes of entry and exit, we show that pertussis toxin treatment reduces lymphocyte egress from lymph nodes. Retrovirally mediated S<em>1</em>P(<em>1</em>) overexpression is sufficient to reduce B cell accumulation in the splenic white pulp and to promote egress of activated T cells from lymph nodes, whereas S<em>1</em>P(<em>1</em>)(+/-) cells have reduced lymph node exit efficiency. Furthermore, lymphocyte S<em>1</em>P(<em>1</em>) is down-regulated in the blood, up-regulated in lymphoid organs, and down-regulated again in the lymph. We propose that cyclical ligand-induced modulation of S<em>1</em>P(<em>1</em>) on circulating lymphocytes contributes to establishing their lymphoid organ transit time.

A growing number of orphan G-protein-coupled receptors (GPCRs) have been reported to be activated by lipid ligands, such as lysophosphatidic acid, <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P), and cannabinoids, for which there are already well established receptors. These new ligand claims are controversial due to either lack of independent confirmations or conflicting reports. We used the beta-arrestin PathHunter assay system, a newly developed, generic GPCR assay format that measures beta-arrestin binding to GPCRs, to evaluate lipid receptor and ligand pairing. This assay eliminates interference from endogenous receptors on the parental cells because it measures a signal that is specifically generated by the tagged receptor and is immediately downstream of receptor activation. We screened a large number of newly "deorphaned" receptors (GPR23, GPR92, GPR55, G2A, GPR<em>1</em>8, GPR3, GPR6, GPR<em>1</em>2, and GPR63) and control receptors against a collection of approximately 400 lipid molecules to try to identify the receptor ligand in an unbiased fashion. GPR92 was confirmed to be a lysophosphatidic acid receptor with weaker responses to farnesyl pyro<em>phosphate</em> and geranylgeranyl di<em>phosphate</em>. The putative cannabinoid receptor GPR55 responded strongly to AM25<em>1</em>, rimonabant, and lysophosphatidylinositol but only very weakly to endocannabinoids. G2A receptor was confirmed to be an oxidized free fatty acid receptor. In addition, we discovered that 3,3'-diindolylmethane, a dietary molecule from cruciferous vegetables, which has known anti-cancer properties, to be a CB(2) receptor partial agonist, with binding affinity around <em>1</em> microm. The anti-inflammatory effect of 3,3'-diindolylmethane in RAW264.7 cells was shown to be partially mediated by CB(2).

The signaling pathways that couple tumor necrosis factor-alpha (TNFalpha) receptors to functional, especially inflammatory, responses have remained elusive. We report here that TNFalpha induces endothelial cell activation, as measured by the expression of adhesion protein E-selectin and vascular adhesion molecule-<em>1</em>, through the <em>sphingosine</em> kinase (SKase) signaling pathway. Treatment of human umbilical vein endothelial cells with TNFalpha resulted in a rapid SKase activation and <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) generation. S<em>1</em>P, but not ceramide or <em>sphingosine</em>, was a potent dose-dependent stimulator of adhesion protein expression. S<em>1</em>P was able to mimic the effect of TNFalpha on endothelial cells leading to extracellular signal-regulated kinases and NF-kappaB activation, whereas ceramide or <em>sphingosine</em> was not. Furthermore, N, N-dimethyl<em>sphingosine</em>, an inhibitor of SKase, profoundly inhibited TNFalpha-induced extracellular signal-regulated kinases and NF-kappaB activation and adhesion protein expression. Thus we demonstrate that the SKase pathway through the generation of S<em>1</em>P is critically involved in mediating TNFalpha-induced endothelial cell activation.

<em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) is a pleiotropic bioactive lipid mediator that promotes breast cancer progression by diverse mechanisms that remain somewhat unclear. Here we report pharmacologic evidence of a critical role for <em>sphingosine</em> kinase <em>1</em> (SphK<em>1</em>) in producing S<em>1</em>P and mediating tumor-induced hemangiogenesis and lymphangiogenesis in a murine model of breast cancer metastasis. S<em>1</em>P levels increased both in the tumor and the circulation. In agreement, serum S<em>1</em>P levels were significantly elevated in stage IIIA human breast cancer patients, compared with age/ethnicity-matched healthy volunteers. However, treatment with the specific SphK<em>1</em> inhibitor SK<em>1</em>-I suppressed S<em>1</em>P levels, reduced metastases to lymph nodes and lungs, and decreased overall tumor burden of our murine model. Both S<em>1</em>P and angiopoietin 2 (Ang2) stimulated hemangiogenesis and lymphangiogenesis in vitro, whereas SK<em>1</em>-I inhibited each process. We quantified both processes in vivo from the same specimen by combining directed in vivo angiogenesis assays with fluorescence-activated cell sorting, thereby confirming the results obtained in vitro. Notably, SK<em>1</em>-I decreased both processes not only at the primary tumor but also in lymph nodes, with peritumoral lymphatic vessel density reduced in SK<em>1</em>-I-treated animals. Taken together, our findings show that SphK<em>1</em>-produced S<em>1</em>P is a crucial mediator of breast cancer-induced hemangiogenesis and lymphangiogenesis. Our results implicate SphK<em>1</em> along with S<em>1</em>P as therapeutic targets in breast cancer.

Coordinated cell migration is essential in many fundamental biological processes including embryonic development, organogenesis, wound healing and the immune response. During organogenesis, groups of cells are directed to specific locations within the embryo. Here we show that the zebrafish miles apart (mil) mutation specifically affects the migration of the heart precursors to the midline. We found that mutant cells transplanted into a wild-type embryo migrate normally and that wild-type cells in a mutant embryo fail to migrate, suggesting that mil may be involved in generating an environment permissive for migration. We isolated mil by positional cloning and show that it encodes a member of the lysosphingolipid G-protein-coupled receptor family. We also show that <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> is a ligand for Mil, and that it activates several downstream signalling events that are not activated by the mutant alleles. These data reveal a new role for lysosphingolipids in regulating cell migration during vertebrate development and provide the first molecular clues into the fusion of the bilateral heart primordia during organogenesis of the heart.

<em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) is a bioactive lipid that regulates myriad important cellular processes, including growth, survival, cytoskeleton rearrangements, motility, and immunity. Here we report that treatment of Jurkat and U937 leukemia cells with the pan-<em>sphingosine</em> kinase (SphK) inhibitor N,N-dimethyl<em>sphingosine</em> to block S<em>1</em>P formation surprisingly caused a large increase in expression of SphK<em>1</em> concomitant with induction of apoptosis. Another SphK inhibitor, D,L-threo-dihydro<em>sphingosine</em>, also induced apoptosis and produced dramatic increases in SphK<em>1</em> expression. However, up-regulation of SphK<em>1</em> was not a specific effect of its inhibition but rather was a consequence of apoptotic stress. The chemotherapeutic drug doxorubicin, a potent inducer of apoptosis in these cells, also stimulated SphK<em>1</em> expression and activity and promoted S<em>1</em>P secretion. The caspase inhibitor ZVAD reduced not only doxorubicin-induced lethality but also the increased expression of SphK<em>1</em> and secretion of S<em>1</em>P. Apoptotic cells secrete chemotactic factors to attract phagocytic cells, and we found that S<em>1</em>P potently stimulated chemotaxis of monocytic THP-<em>1</em> and U937 cells and primary monocytes and macrophages. Collectively, our data suggest that apoptotic cells may up-regulate SphK<em>1</em> to produce and secrete S<em>1</em>P that serves as a "come-and-get-me" signal for scavenger cells to engulf them in order to prevent necrosis.

Membrane sphingolipids are metabolized to <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P), a bioactive lipid mediator that regulates many processes in vertebrate development, physiology, and pathology. Once exported out of cells by cell-specific transporters, chaperone-bound S<em>1</em>P is spatially compartmentalized in the circulatory system. Extracellular S<em>1</em>P interacts with five GPCRs that are widely expressed and transduce intracellular signals to regulate cellular behavior, such as migration, adhesion, survival, and proliferation. While many organ systems are affected, S<em>1</em>P signaling is essential for vascular development, neurogenesis, and lymphocyte trafficking. Recently, a pharmacological S<em>1</em>P receptor antagonist has won approval to control autoimmune neuroinflammation in multiple sclerosis. The availability of pharmacological tools as well as mouse genetic models has revealed several physiological actions of S<em>1</em>P and begun to shed light on its pathological roles. The unique mode of signaling of this lysophospholipid mediator is providing novel opportunities for therapeutic intervention, with possibilities to target not only GPCRs but also transporters, metabolic enzymes, and chaperones.

<em>Sphingosine</em> kinase (Sphk) enzymes are important in intracellular sphingolipid metabolism as well as in the biosynthesis of <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P), an extracellular lipid mediator. Here, we show that Sphk<em>1</em> is expressed and is required for small intestinal tumor cell proliferation in Apc Min/+ mice. Adenoma size but not incidence was dramatically reduced in Apc Min/+ Sphk(-/-) mice. Concomitantly, epithelial cell proliferation in the polyps was significantly attenuated, suggesting that Sphk<em>1</em> regulates adenoma progression. Although the S<em>1</em>P receptors (S<em>1</em>P<em>1</em>R, S<em>1</em>P2R, and S<em>1</em>P3R) are expressed, polyp incidence or size was unaltered in Apc Min/+ S<em>1</em>p2r(-/-), Apc Min/+ S<em>1</em>p3r(-/-), and Apc Min/+ S<em>1</em>p<em>1</em>r(+/-) bigenic mice. These data suggest that extracellular S<em>1</em>P signaling via its receptors is not involved in adenoma cell proliferation. Interestingly, tissue <em>sphingosine</em> content was elevated in the adenomas of Apc Min/+ Sphk<em>1</em>(-/-) mice, whereas S<em>1</em>P levels were not significantly altered. Concomitantly, epithelial cell proliferation and the expression of the G<em>1</em>/S cell cycle regulator CDK4 and c-myc were diminished in the polyps of Apc Min/+ Sphk<em>1</em>(-/-) mice. In rat intestinal epithelial (RIE) cells in vitro, Sphk<em>1</em> overexpression enhanced cell cycle traverse at the G<em>1</em>/S boundary. In addition, RIE cells treated with <em>sphingosine</em> but not C6-ceramide exhibited reduced cell proliferation, reduced retinoblastoma protein phosphorylation, and cyclin-dependent kinase 4 (Cdk4) expression. Our findings suggest that Sphk<em>1</em> plays a critical role in intestinal tumor cell proliferation and that inhibitors of Sphk<em>1</em> may be useful in the control of intestinal cancer.

<em>Sphingosine</em>-<em>1</em>-<em>phosphate</em>, a key mediator in immune cell trafficking, is elevated in the lungs of asthmatic patients and regulates pulmonary epithelium permeability. Stimulation of mast cells by allergens induces two mammalian <em>sphingosine</em> kinases (Sphk<em>1</em> and Sphk2) to produce <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P). Little is known about the individual role of these kinases in regulating immune cell function. Here we show that in mast cells, Sphk2 is required for production of S<em>1</em>P, for calcium influx, for activation of protein kinase C, and for cytokine production and degranulation. However, susceptibility to in vivo anaphylaxis is determined both by S<em>1</em>P within the mast cell compartment and by circulating S<em>1</em>P generated by Sphk<em>1</em> predominantly from a non-mast cell source(s). Thus, <em>sphingosine</em> kinases are determinants of mast cell responsiveness, demonstrating a previously unrecognized relationship with anaphylaxis.

Immature CD4+CD8+ thymocytes, which are generated in the thymic cortex, are induced upon positive selection to differentiate into mature T lymphocytes and relocate to the thymic medulla. It was recently shown that a chemokine signal via CCR7 is essential for the cortex-to-medulla migration of positively selected thymocytes in the thymus. However, the role of the cortex-to-medulla migration in T cell development and selection has remained unclear. The present study shows that the developmental kinetics and the thymic export of mature thymocytes were undisturbed in adult mice lacking CCR7 or its ligands (CCR7L). The inhibition of <em>sphingosine</em>-<em>1</em>-<em>phosphate</em>-mediated lymphocyte egress from the thymus led to the accumulation of mature thymocytes in the cortex of CCR7- or CCR7L-deficient mice, unlike the accumulation in the medulla of normal mice, thereby suggesting that mature thymocytes may be exported directly from the cortex in the absence of CCR7 signals. However, the thymocytes that were generated in the absence of CCR7 or CCR7L were potent in causing autoimmune dacryoadenitis and sialadenitis in mice and were thus incapable of establishing central tolerance to organ-specific antigens. These results indicate that CCR7-mediated cortex-to-medulla migration of thymocytes is essential for establishing central tolerance rather than for supporting the maturation or export of thymocytes.

<em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) is a bioactive lipid mediator with crucial roles in a wide variety of cellular functions across a broad range of organisms. Though a simple molecule in structure, S<em>1</em>P functions are complex. The formation of S<em>1</em>P is catalyzed by one of two <em>sphingosine</em> kinases that have differential cellular distributions as well as both overlapping and opposing functions and which are activated by many different stimuli. S<em>1</em>P can act on a family of G protein-coupled receptors (S<em>1</em>PRs) that are also differentially expressed in different cell types, which influences the cellular responses to S<em>1</em>P. In addition to acting on receptors located on the plasma membrane, S<em>1</em>P can also function inside the cell, independently of S<em>1</em>PRs. It also appears that both the intracellular location and the isotype of <em>sphingosine</em> kinase involved are major determinants of inside-out signaling of S<em>1</em>P in response to many extracellular stimuli. This chapter is focused on the current literature on extracellular and intracellular actions of S<em>1</em>P.

<em>Sphingosine</em> kinase (SK) <em>1</em> catalyzes the formation of the bioactive lipid <em>sphingosine</em> <em>1</em>-<em>phosphate</em>, and has been implicated in several biological processes in mammalian cells, including enhanced proliferation, inhibition of apoptosis, and oncogenesis. Human SK (hSK) <em>1</em> possesses high instrinsic catalytic activity which can be further increased by a diverse array of cellular agonists. We have shown previously that this activation occurs as a direct consequence of extracellular signal-regulated kinase <em>1</em>/2-mediated phosphorylation at Ser225, which not only increases catalytic activity, but is also necessary for agonist-induced translocation of hSK<em>1</em> to the plasma membrane. In this study, we report that the oncogenic effects of overexpressed hSK<em>1</em> are blocked by mutation of the phosphorylation site despite the phosphorylation-deficient form of the enzyme retaining full instrinsic catalytic activity. This indicates that oncogenic signaling by hSK<em>1</em> relies on a phosphorylation-dependent function beyond increasing enzyme activity. We demonstrate, through constitutive localization of the phosphorylation-deficient form of hSK<em>1</em> to the plasma membrane, that hSK<em>1</em> translocation is the key effect of phosphorylation in oncogenic signaling by this enzyme. Thus, phosphorylation of hSK<em>1</em> is essential for oncogenic signaling, and is brought about through phosphorylation-induced translocation of hSK<em>1</em> to the plasma membrane, rather than from enhanced catalytic activity of this enzyme.

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) is a bioactive lysolipid with pleiotropic functions mediated through a family of G protein-coupled receptors, S<em>1</em>P(<em>1</em>,2,3,4,5). Physiological effects of S<em>1</em>P receptor agonists include regulation of cardiovascular function and immunosuppression via redistribution of lymphocytes from blood to secondary lymphoid organs. The phosphorylated metabolite of the immunosuppressant agent FTY720 (2-amino-2-(2-[4-octylphenyl]ethyl)-<em>1</em>,3-propanediol) and other phosphonate analogs with differential receptor selectivity were investigated. No significant species differences in compound potency or rank order of activity on receptors cloned from human, murine, and rat sources were observed. All synthetic analogs were high-affinity agonists on S<em>1</em>P(<em>1</em>), with IC(50) values for ligand binding between 0.3 and <em>1</em>4 nM. The correlation between S<em>1</em>P(<em>1</em>) receptor activation and the ED(50) for lymphocyte reduction was highly significant (p < 0.00<em>1</em>) and lower for the other receptors. In contrast to S<em>1</em>P(<em>1</em>)-mediated effects on lymphocyte recirculation, three lines of evidence link S<em>1</em>P(3) receptor activity with acute toxicity and cardiovascular regulation: compound potency on S<em>1</em>P(3) correlated with toxicity and bradycardia; the shift in potency of phosphorylated-FTY720 for inducing lymphopenia versus bradycardia and hypertension was consistent with affinity for S<em>1</em>P(<em>1</em>) relative to S<em>1</em>P(3); and toxicity, bradycardia, and hypertension were absent in S<em>1</em>P(3)(-/-) mice. Blood pressure effects of agonists in anesthetized rats were complex, whereas hypertension was the predominant effect in conscious rats and mice. Immunolocalization of S<em>1</em>P(3) in rodent heart revealed abundant expression on myocytes and perivascular smooth muscle cells consistent with regulation of bradycardia and hypertension, whereas S<em>1</em>P(<em>1</em>) expression was restricted to the vascular endothelium.

In animals, the sphingolipid metabolite <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) functions as both an intracellular messenger and an extracellular ligand for G-protein-coupled receptors of the S<em>1</em>P receptor family, regulating diverse biological processes ranging from cell proliferation to apoptosis. Recently, it was discovered in plants that S<em>1</em>P is a signalling molecule involved in abscisic acid (ABA) regulation of guard cell turgor. Here we report that the enzyme responsible for S<em>1</em>P production, <em>sphingosine</em> kinase (SphK), is activated by ABA in Arabidopsis thaliana, and is involved in both ABA inhibition of stomatal opening and promotion of stomatal closure. Consistent with this observation, inhibition of SphK attenuates ABA regulation of guard cell inward K(+) channels and slow anion channels, which are involved in the regulation of stomatal pore size. Surprisingly, S<em>1</em>P regulates stomatal apertures and guard cell ion channel activities in wild-type plants, but not in knockout lines of the sole prototypical heterotrimeric G-protein alpha-subunit gene, GPA<em>1</em> (refs 5, 6, 7-8). Our results implicate heterotrimeric G proteins as downstream elements in the S<em>1</em>P signalling pathway that mediates ABA regulation of stomatal function, and suggest that the interplay between S<em>1</em>P and heterotrimeric G proteins represents an evolutionarily conserved signalling mechanism.

OBJECTIVE

The <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) receptor agonist fingolimod (FTY720), that has shown efficacy in advanced multiple sclerosis clinical trials, decreases reperfusion injury in heart, liver, and kidney. We therefore tested the therapeutic effects of fingolimod in several rodent models of focal cerebral ischemia. To assess the translational significance of these findings, we asked whether fingolimod improved long-term behavioral outcomes, whether delayed treatment was still effective, and whether neuroprotection can be obtained in a second species.

METHODS

We used rodent models of middle cerebral artery occlusion and cell-culture models of neurotoxicity and inflammation to examine the therapeutic potential and mechanisms of neuroprotection by fingolimod.

RESULTS

In a transient mouse model, fingolimod reduced infarct size, neurological deficit, edema, and the number of dying cells in the core and periinfarct area. Neuroprotection was accompanied by decreased inflammation, as fingolimod-treated mice had fewer activated neutrophils, microglia/macrophages, and intercellular adhesion molecule-<em>1</em> (ICAM-<em>1</em>)-positive blood vessels. Fingolimod-treated mice showed a smaller infarct and performed better in behavioral tests up to <em>1</em>5 days after ischemia. Reduced infarct was observed in a permanent model even when mice were treated 4 hours after ischemic onset. Fingolimod also decreased infarct size in a rat model of focal ischemia. Fingolimod did not protect primary neurons against glutamate excitotoxicity or hydrogen peroxide, but decreased ICAM-<em>1</em> expression in brain endothelial cells stimulated by tumor necrosis factor alpha.

CONCLUSIONS

These findings suggest that anti-inflammatory mechanisms, and possibly vasculoprotection, rather than direct effects on neurons, underlie the beneficial effects of fingolimod after stroke. S<em>1</em>P receptors are a highly promising target in stroke treatment.

The potent lipid mediator <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) regulates diverse physiological processes by binding to 5 specific GPCRs, although it also has intracellular targets. Here, we demonstrate that S<em>1</em>P, produced in the mitochondria mainly by <em>sphingosine</em> kinase 2 (SphK2), binds with high affinity and specificity to prohibitin 2 (PHB2), a highly conserved protein that regulates mitochondrial assembly and function. In contrast, S<em>1</em>P did not bind to the closely related protein PHB<em>1</em>, which forms large, multimeric complexes with PHB2. In mitochondria from SphK2-null mice, a new aberrant band of cytochrome-c oxidase was detected by blue native PAGE, and interaction between subunit IV of cytochrome-c oxidase and PHB2 was greatly reduced. Moreover, depletion of SphK2 or PHB2 led to a dysfunction in mitochondrial respiration through cytochrome-c oxidase. Our data point to a new action of S<em>1</em>P in mitochondria and suggest that interaction of S<em>1</em>P with homomeric PHB2 is important for cytochrome-c oxidase assembly and mitochondrial respiration.

Heterotrimeric G proteins composed of alpha, beta, and gamma subunits link ligand perception by G protein-coupled receptors (GPCRs) with downstream effectors, providing a ubiquitous signaling mechanism in eukaryotes. The Arabidopsis thaliana genome encodes single prototypical Galpha (GPA<em>1</em>) and Gbeta (AGB<em>1</em>) subunits, and two probable Ggamma subunits (AGG<em>1</em> and AGG2). One Arabidopsis gene, GCR<em>1</em>, encodes a protein with significant sequence similarity to nonplant GPCRs and a predicted 7-transmembrane domain structure characteristic of GPCRs. However, whether GCR<em>1</em> actually interacts with GPA<em>1</em> was unknown. We demonstrate by in vitro pull-down assays, by yeast split-ubiquitin assays, and by coimmunoprecipitation from plant tissue that GCR<em>1</em> and GPA<em>1</em> are indeed physically coupled. GCR<em>1</em>-GPA<em>1</em> interaction depends on intracellular domains of GCR<em>1</em>. gcr<em>1</em> T-DNA insertional mutants exhibit hypersensitivity to abscisic acid (ABA) in assays of root growth, gene regulation, and stomatal response. gcr<em>1</em> guard cells are also hypersensitive to the lipid metabolite, <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P), which is a transducer of the ABA signal upstream of GPA<em>1</em>. Because gpa<em>1</em> mutants exhibit insensitivity in aspects of guard cell ABA and S<em>1</em>P responses, whereas gcr<em>1</em> mutants exhibit hypersensitivity, GCR<em>1</em> may act as a negative regulator of GPA<em>1</em>-mediated ABA responses in guard cells.