OBJECTIVE

We have previously shown that the <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P)/S<em>1</em>P receptor-<em>1</em> (S<em>1</em>P(<em>1</em>)R) axis contributes to the migration of transplanted neural progenitor cells (NPCs) toward areas of spinal cord injury. In the current study, we examined a strategy to increase endogenous NPC migration toward the injured central nervous system to modify S<em>1</em>PR.

METHODS

S<em>1</em>P concentration in the ischemic brain was measured in a mouse thrombosis model of the middle cerebral artery. NPC migration in vitro was assessed by a Boyden chamber assay. Endogenous NPC migration toward the insult was evaluated after ventricular administration of the S<em>1</em>P(2)R antagonist JTE-0<em>1</em>3.

RESULTS

The concentration of S<em>1</em>P in the brain was increased after ischemia and was maximal <em>1</em>4 days after the insult. The increase in S<em>1</em>P in the infarcted brain was primarily caused by accumulation of microglia at the insult. Mouse NPCs mainly expressed S<em>1</em>P(<em>1</em>)R and S<em>1</em>P(2)R as S<em>1</em>PRs, and S<em>1</em>P significantly induced the migration of NPCs in vitro through activation of S<em>1</em>P(<em>1</em>)R. However, an S<em>1</em>P(<em>1</em>)R agonist failed to have any synergistic effect on S<em>1</em>P-mediated NPC migration, whereas pharmacologic or genetic inhibition of S<em>1</em>P(2)R by JTE-0<em>1</em>3 or short hairpin RNA expression enhanced S<em>1</em>P-mediated NPC migration but did not affect proliferation and differentiation. Interestingly, administration of JTE-0<em>1</em>3 into a brain ventricle significantly enhanced endogenous NPC migration toward the area of ischemia.

CONCLUSIONS

Our findings suggest that S<em>1</em>P is a chemoattractant for NPCs released from an infarcted area and regulation of S<em>1</em>P(2)R function further enhances the migration of NPCs toward a brain infarction.

Sphingolipids are a fascinating class of signaling molecules derived from the membrane lipid sphingomyelin. They show abundant expression in the brain. Complex sphingolipids such as glycosphingolipids (gangliosides and cerebrosides) regulate vesicular transport and lysosomal degradation and their dysregulation can lead to storage diseases with a neurological phenotype. More recently, simple sphingolipids such ceramide, <em>sphingosine</em> and <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) were discovered to signal in response to many extracellular stimuli. Forming an intricate signaling network, the balance of these readily interchangeable mediators is decisive for cell fate under stressful conditions. The immunomodulator fingolimod is the prodrug of an S<em>1</em>P receptor agonist. Following receptor activation, the drug leads to downregulation of the S<em>1</em>P<em>1</em> receptor inducing functional antagonism. As the first drug to modulate the sphingolipid signaling pathway, it was marketed in 20<em>1</em>0 for the treatment of multiple sclerosis (MS). At that time, immunomodulation was widely accepted as the key mechanism of fingolimod's efficacy in MS. But given the excellent passage of this lipophilic compound into the brain and its massive brain accumulation as well as the abundant expression of S<em>1</em>P receptors on brain cells, it is conceivable that fingolimod also affects brain cells directly. Indeed, a seminal study showed that the protective effect of fingolimod in experimental autoimmune encephalitis (EAE), a murine MS model, is lost in mice lacking the S<em>1</em>P<em>1</em> receptor on astrocytes, arguing for a specific role of astrocytic S<em>1</em>P signaling in MS. In this review, we discuss the role of sphingolipid mediators and their metabolizing enzymes in neurologic diseases and putative therapeutic strategies arising thereof.

Graft-versus-host disease (GvHD) mediated by donor T cells recognizing host alloantigens is associated with beneficial graft-versus-tumor effects in recipients of allogeneic hematopoietic cell transplants. Since leukemias and lymphomas reside largely within the lymphohematopoietic system, we have proposed that the desired graft-versus-leukemia or graft-versus-lymphoma effect can be separated from the complication of GvHD by confinement of the graft-versus-host alloresponse to the lymphohematopoietic tissues. Since the new <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> receptor agonist immunosuppressive drug FTY720 leads to trapping of T cells in secondary lymphoid tissues, we evaluated the possibility that this drug could diminish GvHD, a disease involving epithelial target tissues, while permitting a beneficial alloresponse to take place within the lymphohematopoietic system, leading to graft-versus-lymphoma effects. We demonstrate here that FTY720 markedly reduces GvHD in a clinically relevant, haploidentical strain combination, while permitting antitumor effects against a T cell lymphoma of unshared host MHC haplotype to proceed unhindered. These results establish a potential new immunotherapeutic approach to separating graft-versus-leukemia effects from GvHD.

<em>Sphingosine</em> kinase <em>1</em> (SK<em>1</em>), a key enzyme in <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) synthesis, regulates various aspects of cell behavior, including cell survival and proliferation. DNA damaging anti-neoplastic agents have been shown to induce p53, ceramide levels, and apoptosis; however, the effects of anti-neoplastic agents on SK have not been assessed. In this study, we investigated the effects of a DNA damaging agent, actinomycin D (Act D), on the function of <em>sphingosine</em> kinase (SK<em>1</em>). Act D caused a reduction in the protein levels of SK<em>1</em>, as indicated by Western blot analysis, with a concomitant decrease in SK activity. The down-regulation was post-transcriptional, because the mRNA levels of SK<em>1</em> remained unchanged. Similar decreases in SK<em>1</em> protein were observed with other DNA damaging agents such as doxorubicin, etoposide, and gamma-irradiation. ZVAD, the pancaspase inhibitor, and Bcl-2 annulled the effect of Act D on SK<em>1</em>, demonstrating a role for cysteine proteases downstream of Bcl-2 in the down-regulation of SK<em>1</em>. Inhibition of caspases 3, 6, 7, and 9 only partially reversed Act D-induced SK<em>1</em> loss. Inhibition of cathepsin B, a lysosomal protease, produced a significant reversal of SK<em>1</em> decline by Act D, suggesting that a multitude of ZVAD-sensitive cysteine proteases downstream of Bcl-2 mediated the SK<em>1</em> decrease. When p53 up-regulation after Act D treatment was inhibited, SK<em>1</em> down-regulation was rescued, demonstrating p53 dependence of SK<em>1</em> modulation. Treatment of cells with S<em>1</em>P, the product of SK<em>1</em>, partially inhibited Act D-induced cell death, raising the possibility that a decrease in SK<em>1</em> may be in part necessary for cell death to occur. Furthermore, the knockdown of SK<em>1</em> by small interfering RNA in MCF-7 cells resulted in a significant reduction in cell viability. These studies demonstrate that SK<em>1</em> is down-regulated by genotoxic stress, and that basal SK<em>1</em> function may be necessary for the maintenance of tumor cell growth.

Potential <em>sphingosine</em> (Sph) metabolites include phosphorylated, N-acylated, and N-methylated derivatives. Phosphorylated Sph, i.e., <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (Sph-<em>1</em>-P), may act as an autocrine stimulator of blood platelets, as it is abundantly stored in platelets and released extracellularly and its exogenous addition induces platelet activation. In this study, we evaluated Sph-<em>1</em>-P formation and its effects in human platelets in the presence of other Sph metabolites. On addition of [3H]Sph to intact platelets, the label was rapidly converted to Sph-<em>1</em>-P. This conversion into [3H]Sph-<em>1</em>-P was inhibited by N,N-dimethyl<em>sphingosine</em> (DMS) in a dose-dependent manner, but not by other structurally related Sph derivatives, including ceramide. The inhibition of Sph-<em>1</em>-P formation by DMS was reproduced using a cell-free system (Sph kinase obtained from platelet cytosolic fractions) and much stronger than that by DL-threo-dihydro<em>sphingosine</em>, which had been considered to be the strongest inhibitor of Sph kinase. Administration of DMS to intact platelets resulted in a decrease in Sph-<em>1</em>-P mass and an increase in Sph mass. Furthermore, DMS inhibited the release of Sph-<em>1</em>-P from platelets stimulated with <em>1</em>2-O-tetradecanoylphorbol <em>1</em>3-acetate and inhibited platelet aggregation induced by exogenous addition of Sph-<em>1</em>-P. Collectively, our results indicate that DMS is useful as a Sph kinase inhibitor and that Sph-<em>1</em>-P actions as an autocrine stimulator of platelets are inhibited by DMS.

<em>Sphingosine</em> kinase <em>1</em> (SK<em>1</em>) and its product <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) have been implicated in the regulation of many cellular processes including growth regulation, protection from apoptosis, stimulation of angiogenesis, and most recently as mediators of the TNF-alpha inflammatory response. In this study we set out to examine the role of SK<em>1</em>/S<em>1</em>P in the RAW macrophage response to the potent inflammatory stimulus lipopolysaccharide (LPS). We show that LPS increases cellular levels of SK<em>1</em> message and protein. This increase is at the transcriptional level and is accompanied by increased SK activity and generation of S<em>1</em>P. S<em>1</em>P is able to cause increases in COX-2 and PGE2 levels in RAW cells. Knockdown of SK<em>1</em> using siRNA is able to inhibit the TNF but not the LPS inflammatory response. Moreover, knockdown of SK<em>1</em> enhances both TNF- and LPS-induced apoptosis. These data indicate that there is a dual and distinct role for SK<em>1</em> and S<em>1</em>P in the TNF and the LPS inflammatory pathways.

In this study a novel biological activity of <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) in C2C<em>1</em>2 myoblasts was identified. In these cells the bioactive lipid profoundly regulated myogenesis exerting an antimitogenic activity, by reducing serum-induced cell proliferation, and acting as powerful prodifferentiating agent by enhancing the expression of myogenic differentiation markers such as myogenin, myosin heavy chain, and caveolin-3. The S<em>1</em>P-dependent diminution of serum-induced labeled thymidine incorporation was abrogated by antisense oligodeoxyribonucleotides (ODN) to S<em>1</em>P2, but not to S<em>1</em>P<em>1</em> or S<em>1</em>P3 receptor, also expressed in C2C<em>1</em>2 cells, implicating S<em>1</em>P2 in the biological response. Using antisense ODN and short interfering RNA treatment, we highlighted the key role played by S<em>1</em>P2 in the S<em>1</em>P-dependent induction of muscle-specific gene products. Notably, S<em>1</em>P2 overexpression increased the content of myogenic markers and hastened the onset of differentiated muscle phenotype in comparison with control cells. Cell treatment with pertussis toxin did not affect the biological responses to S<em>1</em>P, ruling out the involvement of Gi-mediated events in the signaling promoted by the sphingolipid. Among the various signaling pathways activated by S<em>1</em>P, the activation of ERK<em>1</em>/ERK2 and p38 MAPK, both identified as downstream effectors of S<em>1</em>P2, was required for the inhibition of cell proliferation and the stimulation of myogenic differentiation, respectively.

Bile salts play crucial roles in allowing the gastrointestinal system to digest, transport and metabolize nutrients. They function as nutrient signaling hormones by activating specific nuclear receptors (FXR, PXR, Vitamin D) and G-protein coupled receptors [TGR5, <em>sphingosine</em>-<em>1</em> <em>phosphate</em> receptor 2 (S<em>1</em>PR2), muscarinic receptors]. Bile acids and insulin appear to collaborate in regulating the metabolism of nutrients in the liver. They both activate the AKT and ERK<em>1</em>/2 signaling pathways. Bile acid induction of the FXR-α target gene, small heterodimer partner (SHP), is highly dependent on the activation PKCζ, a branch of the insulin signaling pathway. SHP is an important regulator of glucose and lipid metabolism in the liver. One might hypothesize that chronic low grade inflammation which is associated with insulin resistance, may inhibit bile acid signaling and disrupt lipid metabolism. The disruption of these signaling pathways may increase the risk of fatty liver and non-alcoholic fatty liver disease (NAFLD). Finally, conjugated bile acids appear to promote cholangiocarcinoma growth via the activation of S<em>1</em>PR2.

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> subtype <em>1</em> (S<em>1</em>P(<em>1</em>)) receptor agonists alter lymphocyte trafficking and endothelial barrier integrity in vivo. Among these is the potent, non-selective agonist, FTY720-P, whose mechanism of action has been suggested to correlate with S<em>1</em>P(<em>1</em>) down-regulation. Discovery of the in vivo active S<em>1</em>P(<em>1</em>)-selective agonist, SEW287<em>1</em>, has broadened our understanding of minimal requirements for S<em>1</em>P(<em>1</em>) function while highlighting differences regarding agonist effect on S<em>1</em>P(<em>1</em>) fate, because SEW287<em>1</em> does not degrade S<em>1</em>P(<em>1</em>). To further understand the mechanism of agonist-induced S<em>1</em>P(<em>1</em>) down-regulation, we compared signaling and fate of human S<em>1</em>P(<em>1</em>)-green fluorescent protein (GFP) in stable 293 cells, using AFD-R, a chiral analog of FTY720-P, SEW287<em>1</em>, and S<em>1</em>P. Although all agonists acutely internalized S<em>1</em>P(<em>1</em>) to late endosomal vesicles and activated GTPgammaS(35) binding and pERK to similar maxima, only AFD-R led to significant S<em>1</em>P(<em>1</em>) down-regulation, as shown by GFP immunoprecipitation studies. Down-regulation was time- and concentration-dependent, was partially blocked by proteasomal inhibition and reversed by chloroquine and an antagonist to S<em>1</em>P(<em>1</em>). All agonists induced a receptor-associated increase in ubiquitination, with AFD-R inducing 3-fold more accumulation than S<em>1</em>P and being 3-4 logs more potent than SEW287<em>1</em>. The formation of AFD-R-receptor ubiquitin complex was inhibited by antagonist and chloroquine and was enhanced by proteasomal inhibition. Identification of proteins by PAGE liquid chromatography-tandem mass spectrometry in cells treated with AFD-R confirmed the co-migration of ubiquitin peptides with those of S<em>1</em>P(<em>1</em>) and GFP, relative to vehicle alone. These data suggest that the hierarchy of ubiquitin recruitment to S<em>1</em>P(<em>1</em>) (AFD-R>> S<em>1</em>P>> SEW287<em>1</em>) correlates with the efficiency of lysosomal receptor degradation and reflects intrinsic differences between agonists.

Thymic T cell progenitor (TCP) importation is a periodic, gated event that is dependent on the expression of functional P-selectin ligands on TCPs. Occupancy of intrathymic TCP niches is believed to negatively regulate TCP importation, but the nature of this feedback mechanism is not yet resolved. We show that P-selectin and CCL25 are periodically expressed in the thymus and are essential parts of the thymic gate-keeping mechanism. Periodicity of thymic TCP receptivity and the size of the earliest intrathymic TCP pool were dependent on the presence of functional P-selectin ligand on TCPs. Furthermore, we show that the numbers of peripheral blood lymphocytes directly affected thymic P-selectin expression and TCP receptivity. We identified <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) as one feedback signal that could mediate influence of the peripheral lymphocyte pool on thymic TCP receptivity. Our findings suggest a model whereby thymic TCP importation is controlled by both early thymic niche occupancy and the peripheral lymphocyte pool via S<em>1</em>P.

The migration and positioning of osteoclast precursor monocytes are controlled by the blood-enriched lipid mediator <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) and have recently been shown to be critical points of control in osteoclastogenesis and bone homeostasis. Here, we show that calcitriol, which is the hormonally active form of vitamin D, and its therapeutically used analog, eldecalcitol, inhibit bone resorption by modulating this mechanism. Vitamin D analogs have been used clinically for treating osteoporosis, although the mode of its pharmacologic action remains to be fully elucidated. In this study, we found that active vitamin D reduced the expression of S<em>1</em>PR2, a chemorepulsive receptor for blood S<em>1</em>P, on circulating osteoclast precursor monocytes both in vitro and in vivo. Calcitriol- or eldecalcitol-treated monocytoid RAW264.7 cells, which display osteoclast precursor-like properties, migrated readily to S<em>1</em>P. Concordantly, the mobility of circulating CX3CR<em>1</em>(+) osteoclast precursor monocytes was significantly increased on systemic administration of active vitamin D. These results show a mechanism for active vitamin D in controlling the migratory behavior of circulating osteoclast precursors, and this action should be conducive to limiting osteoclastic bone resorption in vivo.

<em>Sphingosine</em> kinase <em>1</em> (SphK<em>1</em>) is an enzyme that converts <em>sphingosine</em> to bioactive <em>sphingosine</em>-<em>1</em>-<em>phosphate</em>. Recent in vitro data suggest a potential role of SphK<em>1</em> in TNF-alpha-mediated inflammation. Our aims in this study were to determine the in vivo significance of SphK<em>1</em> in TNF-alpha-mediated chronic inflammation and to define which pathogenic mechanisms induced by TNF-alpha are SphK<em>1</em> dependent. To pursue these aims, we studied the effect of SphK<em>1</em> deficiency in an in vivo model of TNF-alpha-induced chronic inflammatory arthritis. Transgenic hTNF-alpha mice, which develop spontaneous inflammatory erosive arthritis beginning at <em>1</em>4-<em>1</em>6 wk, were crossed with SphK<em>1</em> null mice (SphK<em>1</em>(-/-)), on the C57BL6 genetic background. Beginning at 4 mo of age, hTNF/SphK<em>1</em>(-/-) mice had significantly less severe clinically evident paw swelling and deformity, less synovial and periarticular inflammation, and markedly decreased bone erosions as measured quantitatively through micro-CT images. Mechanistically, the mice lacking SphK<em>1</em> had less articular cyclooxygenase 2 protein and fewer synovial Th<em>1</em>7 cells than did hTNF/SphK<em>1</em>(+/+) littermates. Microarray analysis and real-time RT-PCR of the ankle synovial tissue demonstrated that hTNF/SphK<em>1</em>(-/-) mice had increased transcript levels of suppressor of cytokine signaling 3 compared with hTNF/SphK<em>1</em>(+/+) mice, likely also contributing to the decreased inflammation in the SphK<em>1</em>-deficient mice. Finally, significantly fewer mature osteoclasts were detected in the ankle joints of hTNF/SphK<em>1</em>(-/-) mice compared with hTNF/SphK<em>1</em>(+/+) mice. These data indicate that SphK<em>1</em> plays a key role in hTNF-alpha-induced inflammatory arthritis via impacting synovial inflammation and osteoclast number.

Extracellular calcium (Ca2+(o)) potently induces the growth arrest and differentiation of human epidermal keratinocytes (HEKs). We report that Ca2+(o) markedly upregulates the human alkaline ceramidase <em>1</em> (haCER<em>1</em>) in HEKs; and its upregulation mediates the Ca2+(o)-induced growth arrest and differentiation of HEKs. haCER<em>1</em> is the human ortholog of mouse alkaline ceramidase <em>1</em> that we previously identified. haCER<em>1</em> catalyzed the hydrolysis of very long-chain ceramides to generate <em>sphingosine</em> (SPH). This in vitro activity required Ca2+. Ectopic expression of haCER<em>1</em> in HEKs decreased the levels of D-e-C(24:<em>1</em>)-ceramide and D-e-C(24:0)-ceramide but elevated the levels of both SPH and its <em>phosphate</em> (S<em>1</em>P), whereas RNA interference-mediated knockdown of haCER<em>1</em> caused the opposite effects on the levels of these sphingolipids in HEKs. Similar to haCER<em>1</em> overexpression, Ca2+(o) increased the levels of SPH and S<em>1</em>P, and this was attenuated by haCER<em>1</em> knockdown. haCER<em>1</em> knockdown also inhibited the Ca2+(o)-induced growth arrest of HEKs and the Ca2+(o)-induced expression of keratin <em>1</em> and involucrin in HEKs. In addition, the acid ceramidase (AC) was also upregulated by Ca2+(o); and its knockdown attenuated the Ca2+(o)-induced expression of keratin <em>1</em> and involucrin in HEKs. These results strongly suggest that upregulation of haCER<em>1</em> and AC mediates the Ca2+(o)-induced growth arrest and differentiation of HEKs by generating SPH and S<em>1</em>P.

<em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) is lipid messenger involved in the regulation of embryonic development, immune system functions, and many other physiological processes. However, the mechanisms of S<em>1</em>P transport across cellular membranes remain poorly understood, with several ATP-binding cassette family members and the spinster 2 (Spns2) member of the major facilitator superfamily known to mediate S<em>1</em>P transport in cell culture. Spns2 was also shown to control S<em>1</em>P activities in zebrafish in vivo and to play a critical role in zebrafish cardiovascular development. However, the in vivo roles of Spns2 in mammals and its involvement in the different S<em>1</em>P-dependent physiological processes have not been investigated. In this study, we characterized Spns2-null mouse line carrying the Spns2(tm<em>1</em>a(KOMP)Wtsi) allele (Spns2(tm<em>1</em>a)). The Spns2(tm<em>1</em>a/tm<em>1</em>a) animals were viable, indicating a divergence in Spns2 function from its zebrafish ortholog. However, the immunological phenotype of the Spns2(tm<em>1</em>a/tm<em>1</em>a) mice closely mimicked the phenotypes of partial S<em>1</em>P deficiency and impaired S<em>1</em>P-dependent lymphocyte trafficking, with a depletion of lymphocytes in circulation, an increase in mature single-positive T cells in the thymus, and a selective reduction in mature B cells in the spleen and bone marrow. Spns2 activity in the nonhematopoietic cells was critical for normal lymphocyte development and localization. Overall, Spns2(tm<em>1</em>a/tm<em>1</em>a) resulted in impaired humoral immune responses to immunization. This study thus demonstrated a physiological role for Spns2 in mammalian immune system functions but not in cardiovascular development. Other components of the S<em>1</em>P signaling network are investigated as drug targets for immunosuppressive therapy, but the selective action of Spns2 may present an advantage in this regard.

T and B lymphocytes, as well as endothelial cells, express distinctive profiles of G protein-coupled receptors for <em>sphingosine</em> <em>1</em>-<em>phosphate</em>, which is a major regulator of T cell development, B and T cell recirculation, tissue homing patterns, and chemotactic responses to chemokines. The capacity of drugs that act on type <em>1</em> <em>sphingosine</em> <em>1</em>-<em>phosphate</em> receptors to suppress organ graft rejection in humans and autoimmunity in animal models without apparent impairment of host defenses against infections suggests that this system is a promising target for new forms of immunotherapy.

Protease activated receptor <em>1</em> (PAR<em>1</em>) signaling can play opposing roles in sepsis, either promoting dendritic cell (DC)-dependent coagulation and inflammation or reducing sepsis lethality due to activated protein C (aPC) therapy. To further define this PAR<em>1</em> paradox, we focused on the vascular effects of PAR<em>1</em> signaling. Pharmacological perturbations of the intravascular coagulant balance were combined with genetic mouse models to dissect the roles of endogenously generated thrombin and aPC during escalating systemic inflammation. Acute blockade of the aPC pathway with a potent inhibitory antibody revealed that thrombin-PAR<em>1</em> signaling increases inflammation-induced vascular hyperpermeability. Conversely, aPC-PAR<em>1</em> signaling and the endothelial cell PC receptor (EPCR) prevented vascular leakage, and pharmacologic or genetic blockade of this pathway sensitized mice to LPS-induced lethality. Signaling-selective aPC variants rescued mice with defective PC activation from vascular leakage and lethality. Defects in the aPC pathway were fully compensated by <em>sphingosine</em> <em>1</em> <em>phosphate</em> receptor 3 (S<em>1</em>P3) deficiency or by selective agonists of the S<em>1</em>P receptor <em>1</em> (S<em>1</em>P<em>1</em>), indicating that PAR<em>1</em> signaling contributes to setting the tone for the vascular S<em>1</em>P<em>1</em>/S<em>1</em>P3 balance. Thus, the activating proteases and selectivity in coupling to S<em>1</em>P receptor subtypes determine vascular PAR<em>1</em> signaling specificity in systemic inflammatory response syndromes in vivo.

Blood-borne lymphocytes home to lymph nodes by interacting with and crossing high endothelial venules (HEVs). The transendothelial migration (TEM) step is poorly understood. Autotaxin (ATX) is an ectoenzyme that catalyzes the conversion of lysophosphatidylcholine (LPC) to lysophosphatidic acid (LPA), a bioactive lipid and a close relative of <em>sphingosine</em> <em>1</em>-<em>phosphate</em>. HEVs produce and secrete ATX into the blood. A prior study implicated ATX in the overall homing process, but the step in which it functions and its mechanism of action have not been defined. In this article, we show that HA<em>1</em>30, an inhibitor of the enzymatic activity of ATX, slows T cell migration across lymph node HEVs in vivo. Ex vivo, ATX plus LPC or LPA itself induces the polarization of mouse naive T cells and stimulates their motility on an ICAM-<em>1</em> substratum. Under physiologic shear conditions in a flow chamber, LPA or ATX/LPC strongly enhances TEM of integrin-arrested T cells across an endothelial monolayer. HA<em>1</em>30 blunts the TEM-promoting activity of ATX, paralleling its in vivo effects. T cells possess Mn(+2)-activatable receptors for ATX, which are localized at the leading edge of polarized cells. ATX must bind to these receptors to elicit a maximal TEM response, providing a mechanism to focus the action of LPA onto arrested lymphocytes in flowing blood. Our results indicate that LPA produced via ATX facilitates T cell entry into lymph nodes by stimulating TEM, substantiating an additional step in the homing cascade. This entry role for LPA complements the efflux function of <em>sphingosine</em> <em>1</em>-<em>phosphate</em>.

BACKGROUND

Fingolimod, a <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> receptor agonist, is used for the treatment of multiple sclerosis and exerts antiapoptotic properties. We hypothesized that <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> receptor activation with fingolimod during acute myocardial infarction (MI) inhibits apoptosis, leading to increased myocardial salvage, reduced infarct size, and mitigated left ventricular (LV) remodeling in a porcine model of ischemia/reperfusion.

RESULTS

Ischemia/reperfusion was induced in pigs by balloon occlusion of the left anterior descending artery, followed by reperfusion. Animals randomly received fingolimod or saline (control). In short-term experiments, fingolimod treatment activated the cardioprotective reperfusion injury salvage kinase and survivor activating factor enhancement pathways in the infarct border zone 24 hours after MI, leading to decreased cardiomyocyte apoptosis and reduced myocardial oxidative stress. These effects were abolished by specific inhibitors of both pathways, demonstrating that fingolimod-induced cardioprotection was mediated by reperfusion injury salvage kinase and survivor activating factor enhancement pathways. In long-term experiments, fingolimod significantly improved myocardial salvage, reduced infarct size, and improved systolic LV function measured by cardiac magnetic resonance <em>1</em> week and <em>1</em> month after MI. Importantly, fingolimod mitigated the development of adverse post-MI LV remodeling <em>1</em> month after MI. Specifically, fingolimod treatment led to a significant reduction in LV mass, LV dilatation, and neurohormonal activation, and it preserved LV geometry. Furthermore, fingolimod decreased interstitial fibrosis, cardiomyocyte hypertrophy, and chronic activation of Akt and extracellular receptor kinase <em>1</em>/2 in the remote noninfarcted myocardium.

CONCLUSIONS

Sphingosine-<em>1</em>-<em>phosphate</em> receptor activation with fingolimod during acute MI reduced infarct size via the reperfusion injury salvage kinase and survivor activating factor enhancement pathways, improved systolic LV function, and mitigated post-MI LV remodeling. Our data strongly support a cardioprotective role for <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> receptor activation during MI.

Exogenous <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) is an effective cardioprotectant against ischemic injury. We have investigated the hypothesis that S<em>1</em>P is also an important endogenous cardioprotectant released during both ischemic preconditioning (IPC) and ischemic postconditioning (IPOST). IPC of ex vivo rat hearts was instituted by two cycles of 3 min ischemia-5 min reperfusion prior to 40 min of index ischemia and then 40 min of reperfusion. IPC resulted in 70% recovery of left ventricular developed pressure (LVDP) upon reperfusion and a small infarct size (<em>1</em>0%). VPC230<em>1</em>9 (VPC), a specific antagonist of S<em>1</em>P(<em>1</em> and 3) G protein-coupled receptors (GPCRs), when present during preconditioning blocked protection afforded by two cycles of IPC. VPC also blocked preconditioning of isolated rat cardiac myocytes subjected to hypoxia-reoxygenation injury. Increased release of S<em>1</em>P from myocytes in response to IPC was also demonstrated. These data indicate that S<em>1</em>P is released from myocytes in response to IPC and protects by binding to S<em>1</em>P GPCRs. In the ex vivo heart, if a third cycle of IPC was added to increase release of endogenous mediators, then the need for any individual mediator (e.g., S<em>1</em>P) was diminished and VPC had little effect. The adenosine antagonist 8-(p-sulfophenyl)-theophylline (8-SPT) likewise inhibited protection by two cycles but not three cycles of IPC, but VPC plus 8-SPT inhibited protection by three cycles of IPC. Similar to IPC, IPOST induced by four postindex ischemia cycles of <em>1</em>5 s reperfusion-<em>1</em>5 s ischemia resulted in 66% recovery of LVDP and a 7% infarct size. When VPC was present during postconditioning and reperfusion, LVDP only recovered by 26% and the infarct size increased to 27%. Adding an additional cycle of IPOST reduced the inhibitory effect of VPC and 8-SPT individually, but not their combined effect. These studies reveal that S<em>1</em>P is an important mediator of both IPC and IPOST that is released along with adenosine during each cycle of IPC or IPOST.

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (Sph-<em>1</em>-P) has been implicated as an intracellular second messenger in many studies. We investigated the metabolism of Sph-<em>1</em>-P and the mechanism by which Sph-<em>1</em>-P induces activation in enucleated and highly differentiated platelets. Platelets lack Sph-<em>1</em>-P lyase activity, possess persistently active <em>sphingosine</em> (Sph) kinase, and abundantly store Sph-<em>1</em>-P. Although exogenous Sph-<em>1</em>-P activated platelets, intracellular Sph-<em>1</em>-P, formed from exogenously added Sph by cytosolic Sph kinase, failed to do so. To support the notion that exogenous Sph-<em>1</em>-P stimulates platelets from outside, contact of platelet surfaces with immobilized Sph-<em>1</em>-P covalently linked to glass particles resulted in platelet activation. Furthermore, we detected the specific binding sites for radiolabeled Sph-<em>1</em>-P on the platelet surface, suggesting extracellular effects of Sph-<em>1</em>-P on plasma membrane receptors. This specific Sph-<em>1</em>-P binding was inhibited not by other sphingolipids but by lysophosphatidic acid (LPA), and platelet aggregation response to LPA was specifically desensitized by prior addition of Sph-<em>1</em>-P. Finally, internally stored Sph-<em>1</em>-P is released extracellularly upon stimulation, and the release correlated well with protein kinase C activation in intact platelets. These results suggest that Sph-<em>1</em>-P acts not intracellularly but intercellularly, following discharge from activated platelets, and shares a platelet surface receptor with LPA.

Lipid biology continues to emerge as an area of significant therapeutic interest, particularly as the result of an enhanced understanding of the wealth of signaling molecules with diverse physiological properties. This growth in knowledge is epitomized by lysophosphatidic acid (LPA), which functions through interactions with at least six cognate G protein-coupled receptors. Herein, we present three crystal structures of LPA<em>1</em> in complex with antagonist tool compounds selected and designed through structural and stability analyses. Structural analysis combined with molecular dynamics identified a basis for ligand access to the LPA<em>1</em> binding pocket from the extracellular space contrasting with the proposed access for the <em>sphingosine</em> <em>1</em>-<em>phosphate</em> receptor. Characteristics of the LPA<em>1</em> binding pocket raise the possibility of promiscuous ligand recognition of phosphorylated endocannabinoids. Cell-based assays confirmed this hypothesis, linking the distinct receptor systems through metabolically related ligands with potential functional and therapeutic implications for treatment of disease.

Ceramide, a sphingolipid generated by the hydrolysis of membrane-associated sphingomyelin, appears to play a role as a gauge of apoptosis. A further metabolite of ceramide, <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (SPP), prevents ceramide-mediated apoptosis, and it has been suggested that the balance between intracellular ceramide and SPP levels may determine the cell fate (Cuvillier, O., Pirianov, G, Kleuser, B., Vanek, P. G., Coso, O. A., Gutkind, J. S., and Spiegel, S. (<em>1</em>996) Nature 38<em>1</em>, 800-803). Here, we investigated the role of SPP and the protein kinase C activator, phorbol ester <em>1</em>2-O-tetradecanoylphorbol-<em>1</em>3-acetate (TPA), in the caspase cascade leading to the proteolysis of poly(ADP-ribose) polymerase (PARP) and lamins. In Jurkat T cells, Fas ligation or addition of exogenous C2-ceramide induced activations of caspase-3/CPP32 and caspase-7/Mch3 followed by PARP cleavage, effects that can be blocked either by SPP or TPA. Furthermore, both SPP and TPA inhibit the activation of caspase-6/Mch2 and subsequent lamin B cleavage. Ceramide, in contrast to Fas ligation, did not induce activation of caspase-8/FLICE and neither SPP nor TPA were able to prevent this activation. Thus, SPP, likely generated via protein kinase C-mediated activation of <em>sphingosine</em> kinase, suppresses the apoptotic pathway downstream of FLICE but upstream of the executioner caspases, caspase-3, -6, and -7.

The <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) receptor agonist, phosphorylated FTY720 (FTY-P), causes lymphopenia, lymphocyte sequestration in mesenteric lymph nodes (MLNs), and immunosuppression. Using multiple techniques to analyze MLN cells harvested from mice treated with S<em>1</em>P receptor agonists, we saw a redistribution of lymphocytes out of nodal sinuses and an expansion of follicles. Although changes in circulating monocytes were not observed with overnight exposure to FTY720, we saw a significant increase in S<em>1</em>P receptor <em>1</em> (S<em>1</em>P<em>1</em>)-expressing CD68+ macrophages in subcapsular sinuses of FTY-P-treated MLNs. This was confirmed by quantitative analysis of F4/80+ cells in MLN suspensions. The sinus volume and number of S<em>1</em>P<em>1</em>-positive cells within sinuses were also increased by FTY-P. High endothelial venules and lymphatic endothelium expressed high levels of S<em>1</em>P<em>1</em>, and treatment with FTY-P resulted in intense staining and colocalization of CD3<em>1</em>, beta-catenin, and zona occludens <em>1</em> in junctions between sinus cells. Transmission electron microscopy showed that FTY-P greatly reduced lymphocyte microvilli and increased cell-cell contacts in the parenchyma. Immunoelectron microscopy revealed that intranodal lymphocytes lacked surface expression of S<em>1</em>P<em>1</em>, whereas S<em>1</em>P<em>1</em> was evident on the surface and within the cytoplasm of macrophages, endothelial cells, and stromal cells. This subcellular pattern of intranodal receptor distribution was unchanged by treatment with FTY-P. We conclude that S<em>1</em>P<em>1</em> agonists have profound effects on macrophages and endothelial cells, in addition to inducing lymphopenia.

Monocyte recruitment and adhesion to vascular endothelium are key early events in atherosclerosis. We examined the role of <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) on modulating monocyte/endothelial interactions in the NOD/LtJ (NOD) mouse model of type <em>1</em> diabetes. Aortas from nondiabetic and diabetic NOD mice were incubated in the absence or presence of <em>1</em>00 nmol/L S<em>1</em>P. Fluorescently labeled monocytes were incubated with the aortas. Aortas from NOD diabetic mice bound 7-fold more monocytes than nondiabetic littermates (<em>1</em>0+/-<em>1</em> monocytes bound/field for nondiabetic mice vs 74+/-<em>1</em>2 monocytes bound/field for diabetic mice, P<0.000<em>1</em>). Incubation of diabetic aortas with <em>1</em>00 nmol/L S<em>1</em>P reduced monocyte adhesion to endothelium by 90%. We found expression of S<em>1</em>P<em>1</em>, S<em>1</em>P2, and S<em>1</em>P3 receptors on NOD aortic endothelial cells. The S<em>1</em>P<em>1</em> receptor-specific agonist SEW287<em>1</em> inhibited monocyte adhesion to diabetic aortas. Studies in diabetic S<em>1</em>P3-deficient mice revealed that the S<em>1</em>P3 receptor did not play a pivotal role in this process. S<em>1</em>P reduced endothelial VCAM-<em>1</em> induction in type <em>1</em> diabetic NOD mice, most likely through inhibition of nuclear factor kappaB translocation to the nucleus. Thus, S<em>1</em>P activation of the S<em>1</em>P<em>1</em> receptor functions in an antiinflammatory manner in type <em>1</em> diabetic vascular endothelium to prevent monocyte/endothelial interactions. S<em>1</em>P may play an important role in the prevention of vascular complications of type <em>1</em> diabetes.