Recent evidence suggests that branching pathways of sphingolipid metabolism may mediate either apoptotic or mitogenic responses depending on the cell type and the nature of the stimulus. While ceramide has been shown to be an important regulatory component of apoptosis induced by tumor necrosis factor alpha and Fas ligand, <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (SPP), a further metabolite of ceramide, has been implicated as a second messenger in cellular proliferation and survival induced by platelet-derived growth factor, nerve growth factor, and serum. SPP protects cells from apoptosis resulting from elevations of ceramide. Inflammatory cytokines stimulate sphingomyelinase, but not ceramidase, leading to accumulation of ceramide, whereas growth signals also leading to accumulation of ceramide, whereas growth signals also stimulate ceramidase and <em>sphingosine</em> kinase leading to increased SPP levels. We propose that the dynamic balance between levels of sphingolipid metabolites, ceramide, and SPP, and consequent regulation of different family members of mitogen-activated protein kinases (JNK versus ERK), is an important factor that determines whether a cell survives or dies.

OBJECTIVE

The <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) analogue FTY720 is a potent immunosuppressive agent currently in Phase III clinical trials for kidney transplantation. FTY720 traps lymphocytes in secondary lymphoid organs thereby preventing their migration to inflammatory sites. Previously, we have identified FTY720 as a potent activator of eNOS. As both inhibition of immune responses and stimulation of eNOS may attenuate atherosclerosis, we administered FTY720 to apolipoprotein E-/- mice fed a high-cholesterol diet.

RESULTS

FTY720 dramatically reduced atherosclerotic lesion volume (62.5%), macrophage (4<em>1</em>.8%), and collagen content (63.5%) after 20 weeks of high-cholesterol diet. In isolated aortic segments and cultured vascular smooth muscle cell, FTY720 potently inhibited thrombin-induced release of monocyte chemoattractant protein-<em>1</em>. This effect was mediated by the S<em>1</em>P3 sphingolipid receptor as FTY720 had no effect on thrombin-induced monocyte chemoattractant protein-<em>1</em> release in S<em>1</em>P3-/- mice. In contrast to S<em>1</em>P receptors on lymphocytes, FTY720 did not desensitize vascular S<em>1</em>P receptors as arteries from FTY720-treated mice retained their vasodilator response to FTY720-<em>phosphate</em>.

CONCLUSIONS

We suggest that FTY720 inhibits atherosclerosis by suppressing the machinery involved in monocyte/macrophage emigration to atherosclerotic lesions. As vascular S<em>1</em>P receptors remained functional under FTY720 treatment, S<em>1</em>P agonists that selectively target the vasculature and not the immune system may be promising new drugs against atherosclerosis.

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) is a potent extracellular lysolipid phosphoric acid mediator that is released after IgE-stimulation of mast cells. Here we investigated the biological activity and intracellular signaling of S<em>1</em>P on human dendritic cells (DC), which are specialized antigen presenting cells with the ability to migrate into peripheral tissues and lymph nodes, as well as control the activation of naive T cells. We show that immature and mature DC express the mRNA for different S<em>1</em>P receptors, such as endothelial differentiation gene (EDG)-<em>1</em>, EDG-3, EDG-5, and EDG-6. In immature DC, S<em>1</em>P stimulated pertussis toxin-sensitive Ca2+ increase actin-polymerization and chemotaxis. These responses were lost by DC matured with lipopolysaccharide. In maturing DC, however, S<em>1</em>P inhibited the secretion of tumor necrosis factor alpha and interleukin (IL)-<em>1</em>2, whereas it enhanced secretion of IL-<em>1</em>0. As a consequence, mature DC exposed to S<em>1</em>P showed a reduced and increased capacity to generate allogeneic Th<em>1</em> and Th2 responses, respectively. In summary, our study implicates that S<em>1</em>P might regulate the trafficking of DC and ultimately favor Th2 lymphocyte-dominated immunity.

OBJECTIVE

The role of <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) receptors in acute vascular injury and smooth muscle cell (SMC) phenotypic modulation is not completely resolved.

RESULTS

S<em>1</em>P receptor antagonists were used to test the hypothesis that specific S<em>1</em>P receptor subtypes differentially regulate SMC phenotypic modulation. In response to acute balloon injury of the rat carotid artery, S<em>1</em>P<em>1</em>/S<em>1</em>P3 receptor mRNA levels were transiently increased at 48 hours whereas S<em>1</em>P2 receptor expression was decreased. S<em>1</em>P2 expression was reinduced and increased at 7 to <em>1</em>0 days postinjury. Daily intraperitoneal injection of the S<em>1</em>P<em>1</em>/S<em>1</em>P3 antagonist VPC44<em>1</em><em>1</em>6 decreased neointimal hyperplasia by approximately 50%. In vitro, pharmacological inhibition of S<em>1</em>P<em>1</em>/S<em>1</em>P3 receptors with VPC25239 attenuated S<em>1</em>P-induced proliferation of rat aortic SMCs. Conversely, inhibition of S<em>1</em>P2 with JTE0<em>1</em>3 potentiated S<em>1</em>P-induced proliferation. Inhibition of S<em>1</em>P<em>1</em>/S<em>1</em>P3 resulted in S<em>1</em>P-induced activation of the SMC differentiation marker genes SMalpha-actin and SMMHC, whereas inhibition of S<em>1</em>P2 attenuated this response. S<em>1</em>P2-dependent activation of SMalpha-actin and SMMHC was shown to be mediated by L-type voltage-gated Ca(2+) channels and subsequent RhoA/Rho kinase-dependent SRF enrichment of CArG box promoter regions.

CONCLUSIONS

Results provide evidence that S<em>1</em>P<em>1</em>/S<em>1</em>P3 receptors promote, whereas S<em>1</em>P2 receptors antagonize, SMC proliferation and phenotypic modulation in vitro in response to S<em>1</em>P, or in vivo after vascular injury.

TRPM3, a member of the melastatin-like transient receptor potential channel subfamily (TRPM), is predominantly expressed in human kidney and brain. TRPM3 mediates spontaneous Ca2+ entry and nonselective cation currents in transiently transfected human embryonic kidney 293 cells. Using measurements with the Ca2+-sensitive fluorescent dye fura-2 and the whole-cell patch-clamp technique, we found that D-erythro-<em>sphingosine</em>, a metabolite arising during the de novo synthesis of cellular sphingolipids, activated TRPM3. Other transient receptor potential (TRP) channels tested [classic or canonical TRP (TRPC3, TRPC4, TRPC5), vanilloid-like TRP (TRPV4, TRPV5, TRPV6), and melastatin-like TRP (TRPM2)] did not significantly respond to application of <em>sphingosine</em>. <em>Sphingosine</em>-induced TRPM3 activation was not mediated by inhibition of protein kinase C, depletion of intracellular Ca2+ stores, and intracellular conversion of <em>sphingosine</em> to <em>sphingosine</em>-<em>1</em>-<em>phosphate</em>. Although <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> and ceramides had no effect, two structural analogs of <em>sphingosine</em>, dihydro-D-erythro-<em>sphingosine</em> and N,N-dimethyl-D-erythro-<em>sphingosine</em>, also activated TRPM3. Sphingolipids, including <em>sphingosine</em>, are known to have inhibitory effects on a variety of ion channels. Thus, TRPM3 is the first ion channel activated by sphingolipids.

<em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P), a ligand for 5 specific receptors, is a potent lipid mediator that plays important roles in lymphocyte trafficking and immune responses. S<em>1</em>P is produced inside cells and therefore must be secreted to exert its effects through these receptors. Spinster 2 (Spns2) is one of the cell surface transporters thought to secrete S<em>1</em>P. We have shown that Spns2 can export endogenous S<em>1</em>P from cells and also dihydro-S<em>1</em>P, which is active at all cell surface S<em>1</em>P receptors. Moreover, Spns2 mice have decreased levels of both of these phosphorylated sphingoid bases in blood, accompanied by increases in very long chain ceramide species, and have defective lymphocyte trafficking. Surprisingly, levels of S<em>1</em>P and dihydro-S<em>1</em>P were increased in lymph from Spns2 mice as well as in specific tissues, including lymph nodes, and interstitial fluid. Moreover, lymph nodes from Spns2 mice have aberrant lymphatic sinus that appeared collapsed, with reduced numbers of lymphocytes. Our data suggest that Spns2 is an S<em>1</em>P transporter in vivo that plays a role in regulation not only of blood S<em>1</em>P but also lymph node and lymph S<em>1</em>P levels and consequently influences lymphocyte trafficking and lymphatic vessel network organization.

During pathogenic influenza virus infection, robust cytokine production (cytokine storm), excessive inflammatory infiltrates, and virus-induced tissue destruction all contribute to morbidity and mortality. Earlier we reported that modulation of <em>sphingosine</em>-<em>1</em>-<em>phosphate</em>-<em>1</em> receptor (S<em>1</em>P<em>1</em>R) signaling provided a chemically tractable approach for the effective blunting of cytokine storm, leading to the improvement of clinical and survival outcomes. Here, we show that S<em>1</em>P<em>1</em>R agonist treatment suppresses global cytokine amplification. Importantly, S<em>1</em>P<em>1</em>R agonist treatment was able to blunt cytokine/chemokine production and innate immune cell recruitment in the lung independently of endosomal and cytosolic innate sensing pathways. S<em>1</em>P<em>1</em>R signaling suppression of cytokine amplification was independent of multiple innate signaling adaptor pathways for myeloid differentiation primary response gene 88 (MyD88) and IFN-β promoter stimulator-<em>1</em> signaling, indicating a common pathway inhibition of cytokine storm. We identify the MyD88 adaptor molecule as responsible for the majority of cytokine amplification observed following influenza virus challenge.

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) is stored in and released from platelets in response to cell activation. However, recent studies show that it is also released from a number of cell types, where it can function as a paracrine/autocrine signal to regulate cell proliferation, differentiation, survival, and motility. This review discusses the role of S<em>1</em>P in cellular regulation, both at the molecular level and in terms of health and disease. The main biochemical routes for S<em>1</em>P synthesis (<em>sphingosine</em> kinase) and degradation (S<em>1</em>P lyase and S<em>1</em>P phosphatase) are described. The major focus is on the ability of S<em>1</em>P to bind to a novel family of G-protein-coupled receptors (endothelial differentiation gene [EDG]-<em>1</em>, -3, -5, -6, and -8) to elicit signal transduction (via G(q)-, G(i)-, G(<em>1</em>2)-, G(<em>1</em>3)-, and Rho-dependent routes). Effector pathways regulated by S<em>1</em>P are divergent, such as extracellular signal-regulated kinase, p38 mitogen-activated protein kinase, phospholipases C and D, adenylyl cyclase, and focal adhesion kinase, and occur in multiple cell types, such as immune cells, neurones, smooth muscle, etc. This provides a molecular basis for the ability of S<em>1</em>P to act as a pleiotropic bioactive lipid with an important role in cellular regulation. We also give an account of the expanding role for S<em>1</em>P in health and disease; in particular, with regard to its role in atherosclerosis, angiogenesis, cancer, and inflammation. Finally, we describe future directions for S<em>1</em>P research and novel approaches whereby S<em>1</em>P signalling can be manipulated for therapeutic intervention in disease.

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) induces diverse biological responses in various tissues by activating specific G protein-coupled receptors (S<em>1</em>P(<em>1</em>)-S<em>1</em>P(5) receptors). The biological signaling regulated by S<em>1</em>P(3) receptor has not been fully elucidated because of the lack of an S<em>1</em>P(3) receptor-specific antagonist or agonist. We developed a novel S<em>1</em>P(3) receptor antagonist, <em>1</em>-(4-chlorophenylhydrazono)-<em>1</em>-(4-chlorophenylamino)-3,3-dimethyl- 2-butanone (TY-52<em>1</em>56), and show here that the S<em>1</em>P-induced decrease in coronary flow (CF) is mediated by the S<em>1</em>P(3) receptor. In functional studies, TY-52<em>1</em>56 showed submicromolar potency and a high degree of selectivity for S<em>1</em>P(3) receptor. TY-52<em>1</em>56, but not an S<em>1</em>P(<em>1</em>) receptor antagonist [(R)-phosphoric acid mono-[2-amino-2-(3-octyl-phenylcarbamoyl)-ethyl] ester; VPC230<em>1</em>9] or S<em>1</em>P(2) receptor antagonist [<em>1</em>-[<em>1</em>,3-dimethyl-4-(2-methylethyl)-<em>1</em>H-pyrazolo[3,4-b]pyridin-6-yl]-4-(3,5-dichloro-4-pyridinyl)-semicarbazide; JTE0<em>1</em>3], inhibited the decrease in CF induced by S<em>1</em>P in isolated perfused rat hearts. We further investigated the effect of TY-52<em>1</em>56 on both the S<em>1</em>P-induced increase in intracellular calcium ([Ca(2+)](i)) and Rho activation that are responsible for the contraction of human coronary artery smooth muscle cells. TY-52<em>1</em>56 inhibited both the S<em>1</em>P-induced increase in [Ca(2+)](i) and Rho activation. In contrast, VPC230<em>1</em>9 and JTE0<em>1</em>3 inhibited only the increase in [Ca(2+)](i) and Rho activation, respectively. We further confirmed that TY-52<em>1</em>56 inhibited FTY-720-induced S<em>1</em>P(3) receptor-mediated bradycardia in vivo. These results clearly show that TY-52<em>1</em>56 is both sensitive and useful as an S<em>1</em>P(3) receptor-specific antagonist and reveal that S<em>1</em>P induces vasoconstriction by directly activating S<em>1</em>P(3) receptor and through a subsequent increase in [Ca(2+)](i) and Rho activation in vascular smooth muscle cells.

FTY720 (fingolimod) is an oral <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) receptor modulator under development for the treatment of multiple sclerosis (MS). To elucidate its effects in the central nervous system (CNS), we compared functional parameters of nerve conductance in the DA rat model of myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE) after preventive and therapeutic treatment. We demonstrate that prophylactic therapy protected against the emergence of EAE symptoms, neuropathology, and disturbances to visual and somatosensory evoked potentials (VEP, SEP). Moreover, therapeutic treatment from day 25 to 45 markedly reversed paralysis in established EAE and normalized the electrophysiological responses, correlating with decreased demyelination in the brain and spinal cord. The effectiveness of FTY720 in this model is likely due to several contributing factors. Evidence thus far supports its role in the reduction of inflammation and preservation of blood-brain-barrier integrity. FTY720 may also act via S<em>1</em>P receptors in glial cells to promote endogenous repair mechanisms that complement its immunomodulatory action.

Sphingolipid metabolites, ceramide, <em>sphingosine</em>, and <em>sphingosine</em> <em>1</em>-<em>phosphate</em>, have emerged as a new class of lipid biomodulators of various cell functions. These metabolites are known to function not only as intracellular second messengers, but also in the extracellular space. <em>Sphingosine</em> <em>1</em>-<em>phosphate</em> especially has numerous functions as an important extracellular mediator that binds to cell surface S<em>1</em>P receptors. Recent studies have also shown that sphingolipid-metabolizing enzymes function not only in intracellular organelles but also in the extracellular spaces, including the outer leaflet of the plasma membrane. This review focuses on the metabolic enzymes (acid and alkaline sphingomyelinases, neutral ceramidase, and <em>sphingosine</em> kinase) that are involved in the production of the sphingolipid metabolites in these extracellular spaces, and on the metabolic pathway itself.

The <em>sphingosine</em> <em>1</em>-<em>phosphate</em> receptor <em>1</em> (S<em>1</em>P<em>1</em>) is abundant in endothelial cells, where it regulates vascular development and microvascular barrier function. In investigating the role of endothelial cell S<em>1</em>P<em>1</em> in adult mice, we found that the endothelial S<em>1</em>P<em>1</em> signal was enhanced in regions of the arterial vasculature experiencing inflammation. The abundance of proinflammatory adhesion proteins, such as ICAM-<em>1</em>, was enhanced in mice with endothelial cell-specific deletion of S<em>1</em>pr<em>1</em> and suppressed in mice with endothelial cell-specific overexpression of S<em>1</em>pr<em>1</em>, suggesting a protective function of S<em>1</em>P<em>1</em> in vascular disease. The chaperones ApoM(+)HDL (HDL) or albumin bind to <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) in the circulation; therefore, we tested the effects of S<em>1</em>P bound to each chaperone on S<em>1</em>P<em>1</em> signaling in cultured human umbilical vein endothelial cells (HUVECs). Exposure of HUVECs to ApoM(+)HDL-S<em>1</em>P, but not to albumin-S<em>1</em>P, promoted the formation of a cell surface S<em>1</em>P<em>1</em>-β-arrestin 2 complex and attenuated the ability of the proinflammatory cytokine TNFα to activate NF-κB and increase ICAM-<em>1</em> abundance. Although S<em>1</em>P bound to either chaperone induced MAPK activation, albumin-S<em>1</em>P triggered greater Gi activation and receptor endocytosis. Endothelial cell-specific deletion of S<em>1</em>pr<em>1</em> in the hypercholesterolemic Apoe(-/-) mouse model of atherosclerosis enhanced atherosclerotic lesion formation in the descending aorta. We propose that the ability of ApoM(+)HDL to act as a biased agonist on S<em>1</em>P<em>1</em> inhibits vascular inflammation, which may partially explain the cardiovascular protective functions of HDL.

BACKGROUND

Ozanimod (RPC<em>1</em>063) is an oral agonist of the <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> receptor subtypes <em>1</em> and 5 that induces peripheral lymphocyte sequestration, potentially decreasing the number of activated lymphocytes circulating to the gastrointestinal tract.

METHODS

We conducted a double-blind, placebo-controlled phase 2 trial of ozanimod in <em>1</em>97 adults with moderate-to-severe ulcerative colitis. Patients were randomly assigned, in a <em>1</em>:<em>1</em>:<em>1</em> ratio, to receive ozanimod at a dose of 0.5 mg or <em>1</em> mg or placebo daily for up to 32 weeks. The Mayo Clinic score was used to measure disease activity on a scale from 0 to <em>1</em>2, with higher scores indicating more severe disease; subscores range from 0 to 3, with higher scores indicating more severe disease. The primary outcome was clinical remission (Mayo Clinic score ≤2, with no subscore>><em>1</em>) at 8 weeks.

RESULTS

The primary outcome occurred in <em>1</em>6% of the patients who received <em>1</em> mg of ozanimod and in <em>1</em>4% of those who received 0.5 mg of ozanimod, as compared with 6% of those who received placebo (P=0.048 and P=0.<em>1</em>4, respectively, for the comparison of the two doses of ozanimod with placebo). Differences in the primary outcome between the group that received 0.5 mg of ozanimod and the placebo group were not significant; therefore, the hierarchical testing plan deemed the analyses of secondary outcomes exploratory. Clinical response (decrease in Mayo Clinic score of ≥3 points and ≥30% and decrease in rectal-bleeding subscore of ≥<em>1</em> point or a subscore ≤<em>1</em>) at 8 weeks occurred in 57% of those receiving <em>1</em> mg of ozanimod and 54% of those receiving 0.5 mg, as compared with 37% of those receiving placebo. At week 32, the rate of clinical remission was 2<em>1</em>% in the group that received <em>1</em> mg of ozanimod, 26% in the group that received 0.5 mg of ozanimod, and 6% in the group that received placebo; the rate of clinical response was 5<em>1</em>%, 35%, and 20%, respectively. At week 8, absolute lymphocyte counts declined 49% from baseline in the group that received <em>1</em> mg of ozanimod and 32% from baseline in the group that received 0.5 mg. The most common adverse events overall were anemia and headache.

CONCLUSIONS

In this preliminary trial, ozanimod at a daily dose of <em>1</em> mg resulted in a slightly higher rate of clinical remission of ulcerative colitis than placebo. The trial was not large enough or of sufficiently long duration to establish clinical efficacy or assess safety. (Funded by Receptos; TOUCHSTONE ClinicalTrials.gov number, NCT0<em>1</em>6475<em>1</em>6.).

OBJECTIVE

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) is an important mediator of cancer cell growth and proliferation. Production of S<em>1</em>P is catalyzed by <em>sphingosine</em> kinase <em>1</em> (SphK). Safingol, (l-threo-dihydro<em>sphingosine</em>) is a putative inhibitor of SphK. We conducted a phase I trial of safingol (S) alone and in combination with cisplatin (C).

METHODS

A 3 + 3 dose escalation was used. For safety, S was given alone <em>1</em> week before the combination. S + C were then administered every 3 weeks. S was given over 60 to <em>1</em>20 minutes, depending on dose. Sixty minutes later, C was given over 60 minutes. The C dose of 75 mg/m(2) was reduced in cohort 4 to 60 mg/m(2) due to excessive fatigue.

RESULTS

Forty-three patients were treated, 4<em>1</em> were evaluable for toxicity, and 37 for response. The maximum tolerated dose (MTD) was S 840 mg/m(2) over <em>1</em>20 minutes C 60 mg/m(2), every 3 weeks. Dose-limiting toxicity (DLT) attributed to cisplatin included fatigue and hyponatremia. DLT from S was hepatic enzyme elevation. S pharmacokinetic parameters were linear throughout the dose range with no significant interaction with C. Patients treated at or near the MTD achieved S levels of more than 20 μmol/L and maintained levels greater than and equal to 5 μmol/L for 4 hours. The best response was stable disease in 6 patients for on average 3.3 months (range <em>1</em>.8-7.2 m). One patient with adrenal cortical cancer had significant regression of liver and lung metastases and another had prolonged stable disease. S was associated with a dose-dependent reduction in S<em>1</em>P in plasma.

CONCLUSIONS

Safingol, the first putative SphK inhibitor to enter clinical trials, can be safely administered in combination with cisplatin. Reversible dose-dependent hepatic toxicity was seen, as expected from preclinical data. Target inhibition was achieved with downregulation of S<em>1</em>P. The recommended phase II dose is S 840 mg/m(2) and C 60 mg/m(2), every 3 weeks.

During infections or acute conditions high-density lipoproteins cholesterol (HDL-C) levels decrease very rapidly and HDL particles undergo profound changes in their composition and function. These changes are associated with poor prognosis following endotoxemia or sepsis and data from genetically modified animal models support a protective role for HDL. The same is true for some parasitic infections, where the key player appears to be a specific and minor component of HDL, namely apoL-<em>1</em>. The ability of HDL to influence cholesterol availability in lipid rafts in immune cells results in the modulation of toll-like receptors, MHC-II complex, as well as B- and T-cell receptors, while specific molecules shuttled by HDL such as <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) contribute to immune cells trafficking. Animal models with defects associated with HDL metabolism and/or influencing cell cholesterol efflux present features related to immune disorders. All these functions point to HDL as a platform integrating innate and adaptive immunity. The aim of this review is to provide an overview of the connection between HDL and immunity in atherosclerosis and beyond.

Five G protein-coupled receptors (GPCRs) for the lysophospholipid <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) have been cloned and characterized so far. We report here about the identification of gpr3, gpr6 and gpr<em>1</em>2 as additional members of the S<em>1</em>P-GPCR family. When expressed transiently in HEK293 cells, gpr3, gpr6 and gpr<em>1</em>2 confer constitutive activation of adenylate cyclase (AC) similar in amplitude to that seen with fully activated G(alpha)(s)-coupled receptors. Culturing the transfected cells in medium with charcoal-stripped serum (devoid of lipids) significantly reduces cyclic adenosine mono<em>phosphate</em> (cAMP) levels, suggesting a lipid-like ligand. A library containing 200 bioactive lipids was applied in functional assays recording intracellular Ca(2+) mobilization. S<em>1</em>P and dihydro<em>sphingosine</em> <em>1</em>-<em>phosphate</em> (DHS<em>1</em>P) were identified as functional activators exhibiting nanomolar EC(50) values. In the presence of the S<em>1</em>P and LPA receptor antagonist suramin, gpr3-, gpr6- and gpr<em>1</em>2-mediated intracellular Ca(2+) mobilization via S<em>1</em>P is enhanced. Besides constitutive activation of G(alpha)(s) type of G proteins, all three receptors are capable of constitutively activating inhibitory G(alpha)(i/o) proteins: (i) in the presence of pertussis toxin, gpr3-, gpr6- and gpr<em>1</em>2-mediated stimulation of AC is enhanced; and (ii) overexpression of G(alpha)(i) significantly reduces the stimulatory action on intracellular cAMP levels. Agonist (S<em>1</em>P)-mediated internalization can be visualized in intact HEK293 cells using a gpr6 green fluorescent protein (GFP) fusion protein. In summary, our data suggest that gpr3, gpr6 and gpr<em>1</em>2 are a family of constitutively active receptors with dual coupling to G(alpha)(s) and G(alpha)(i) type of G proteins. Constitutive activation of AC and mobilization of [Ca(2+)](i) can be modulated by the sphingophospholipids S<em>1</em>P and DHS<em>1</em>P, adding three additional members to the family of S<em>1</em>P receptors.

Sphingolipids have important roles in membrane and lipoprotein structure and in cell regulation as second messengers for growth factors, differentiation factors, cytokines, and a growing list of agonists. Bioactive sphingolipids are formed both by the turnover of complex sphingolipids and as intermediates of sphingolipid biosynthesis. Usually, the amounts are highly regulated; however, by inhibiting ceramide synthase, fumonisins block the biosynthesis of complex sphingolipids and cause sphinganine (and sometimes <em>sphingosine</em>) to accumulate. Where the mechanism has been studied most thoroughly, the accumulation of sphingoid bases is a primary cause of the toxicity of fumonisin B (FB). Nonetheless, the full effects of fumonisins probably involve many biochemical events. The elevations in sphingoid bases also affect the amounts of other lipids, including the <em>1</em>-<em>phosphates</em> and N-acetyl derivatives of sphinganine. Furthermore, the aminopentol backbone of FB<em>1</em> (AP<em>1</em>) is both an inhibitor and a substrate for ceramide synthase, and the resultant N-palmitoyl-AP<em>1</em> (PAP<em>1</em>) is an even more potent inhibitor of ceramide synthase (presumably as a product analog). PAP<em>1</em> is <em>1</em>0 times more toxic than FB<em>1</em> or AP<em>1</em> for HT-29 cells in culture, and hence may play a role in the toxicity of nixtamalized fumonisins. All these processes--the effects of fumonisins on sphingolipid metabolism, the pathways altered by perturbation of sphingolipid metabolism, and the complex cellular behaviors regulated by sphingolipids--must be borne in mind when evaluating the pathologic effects of fumonisins.

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) in blood is phosphorylated, stored, and transported by red blood cells (RBC). Release of S<em>1</em>P from RBC into plasma is a regulated process that does not occur in plasma- or serum-free media. Plasma fractionation and incubations with isolated and recombinant proteins identified high density lipoprotein (HDL) and serum albumin (SA) as non-redundant endogenous triggers for S<em>1</em>P release from RBC. S<em>1</em>P bound to SA and HDL was able to stimulate the S<em>1</em>P(<em>1</em>) receptor in calcium flux experiments. The binding capability of acceptor molecules triggers S<em>1</em>P release, as demonstrated with the anti-S<em>1</em>P antibody Sphingomab. More S<em>1</em>P was extracted from RBC membranes by HDL than by SA. Blood samples from anemic patients confirmed a reduced capacity for S<em>1</em>P release in plasma. In co-cultures of RBC and endothelial cells (EC), we observed transcellular transportation of S<em>1</em>P as a second function of RBC-associated S<em>1</em>P in the absence of SA and HDL and during tight RBC-EC contact, mimicking conditions in tissue interstitium and capillaries. In contrast to S<em>1</em>P bound to SA and HDL, RBC-associated S<em>1</em>P was significantly incorporated by EC after S<em>1</em>P lyase (SGPL<em>1</em>) inhibition. RBC-associated S<em>1</em>P, therefore, has two functions: (<em>1</em>) It contributes to the cellular pool of SGPL<em>1</em>-sensitive S<em>1</em>P in tissues after transcellular transportation and (2) it helps maintain extracellular S<em>1</em>P levels via SA and HDL independently from SGPL<em>1</em> activity.

Sphingolipids such as <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P), ceramide, or sphingomyelin are essential constituents of plasma membranes and regulate many (patho)physiological cellular responses inducing apoptosis and cell survival, vascular permeability, mast cell activation, and airway smooth muscle functions. The complexity of sphingolipid biology is generated by a great variety of compounds, diverse receptors, and often antagonistic functions of different sphingolipids. For instance, apoptosis is promoted by ceramide and prevented by S<em>1</em>P, and pulmonary vascular permeability is increased by S<em>1</em>P2/3 receptors and by ceramide, whereas S<em>1</em>P<em>1</em> receptors stabilize barrier integrity. Several enzymes of the sphingolipid metabolism respond to external stimuli such as sphingomyelinase isoenzymes that are activated by many stress stimuli and the <em>sphingosine</em> kinase isoenzymes that are activated by allergens. The past years have provided increasing evidence that these processes contribute to pulmonary disorders including asthma, chronic obstructive pulmonary disease, acute lung injury, and cystic fibrosis. Sphingolipid metabolism offers several novel therapeutic targets for the treatment of lung diseases such as emphysema, asthma, cystic fibrosis, respiratory tract infection, sepsis, and acute lung injury.

Development of hematopoietic cells depends on a dynamic actin cytoskeleton. Here we demonstrate that expression of the cytoskeletal regulator WASP, mutated in the Wiskott-Aldrich syndrome, provides selective advantage for the development of naturally occurring regulatory T cells, natural killer T cells, CD4(+) and CD8(+) T lymphocytes, marginal zone (MZ) B cells, MZ macrophages, and platelets. To define the relative contribution of MZ B cells and MZ macrophages for MZ development, we generated wild-type and WASP-deficient bone marrow chimeric mice, with full restoration of the MZ. However, even in the presence of MZ macrophages, only <em>1</em>0% of MZ B cells were of WASP-deficient origin. We show that WASP-deficient MZ B cells hyperproliferate in vivo and fail to respond to <em>sphingosine</em>-<em>1</em>-<em>phosphate</em>, a crucial chemoattractant for MZ B-cell positioning. Abnormalities of the MZ compartment in WASP(-/-) mice lead to aberrant uptake of Staphylococcus aureus and to a reduced immune response to TNP-Ficoll. Moreover, WASP-deficient mice have increased levels of "natural" IgM antibodies. Our findings reveal that WASP regulates both development and function of hematopoietic cells. We demonstrate that WASP deficiency leads to an aberrant MZ that may affect responses to blood-borne pathogens and peripheral B-cell tolerance.

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) is a bioactive lipid signal transmitter present in blood. Blood plasma S<em>1</em>P is supplied from erythrocytes and plays an important role in lymphocyte egress from lymphoid organs. However, the S<em>1</em>P export mechanism from erythrocytes to blood plasma is not well defined. To elucidate the mechanism of S<em>1</em>P export from erythrocytes, we performed the enzymatic characterization of S<em>1</em>P transporter in rat erythrocytes. Rat erythrocytes constitutively released S<em>1</em>P without any stimulus. The S<em>1</em>P release was reduced by an ABCA<em>1</em> transporter inhibitor, glyburide, but not by a multidrug resistance-associated protein inhibitor, MK57<em>1</em>, or a multidrug resistance protein inhibitor, cyclosporine A. Furthermore, we measured S<em>1</em>P transport activity using rat erythrocyte inside-out membrane vesicles (IOVs). Although the effective S<em>1</em>P transport into IOVs was observed in the presence of ATP, this activity was also supported by dATP and adenosine 5'-(beta,gamma-imido)tri<em>phosphate</em>. The rate of S<em>1</em>P transport increased depending on S<em>1</em>P concentration, with an apparent K(m) value of 2<em>1</em> microm. Two phosphorylated sphingolipids, dihydrosphingosine <em>1</em>-<em>phosphate</em> and ceramide <em>1</em>-<em>phosphate</em>, did not inhibit S<em>1</em>P transport. Similar to the intact erythrocytes, the uptake of S<em>1</em>P into IOVs was inhibited by glyburide and vanadate but not by the other ABC transporter inhibitors. These results suggest that S<em>1</em>P is exported from the erythrocytes by a novel ATP-dependent transporter.

Formation of inositol <em>1</em>,4,5-tris<em>phosphate</em> (IP3) by phospholipase C (PLC) with subsequent release of Ca2+ from intracellular stores, is one of the major Ca2+ signalling pathways triggered by G-protein-coupled receptors (GPCRs). However, in a large number of cellular systems, Ca2+ mobilization by GPCRs apparently occurs independently of the PLC-IP3 pathway, mediated by an as yet unknown mechanism. The present study investigated whether <em>sphingosine</em> kinase activation, leading to production of <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (SPP), is involved in GPCR-mediated Ca2+ signalling as proposed for platelet-derived growth factor and FcepsilonRI antigen receptors. Inhibition of <em>sphingosine</em> kinase by DL-threo-dihydro<em>sphingosine</em> and N,N-dimethyl<em>sphingosine</em> markedly inhibited [Ca2+]i increases elicited by m2 and m3 muscarinic acetylcholine receptors (mAChRs) expressed in HEK-293 cells without affecting mAChR-induced PLC stimulation. Activation of mAChRs rapidly and transiently stimulated production of SPP in HEK-293 cells. Finally, intracellular injection of SPP induced a rapid and transient Ca2+ mobilization in HEK-293 cells which was not antagonized by heparin. We conclude that mAChRs utilize the <em>sphingosine</em> kinase-SPP pathway in addition to PLC-IP3 to mediate Ca2+ mobilization. As Ca2+ signalling by various, but not all, GPCRs in different cell types was likewise attenuated by the <em>sphingosine</em> kinase inhibitors, we suggest a general role for <em>sphingosine</em> kinase, besides PLC, in mediation of GPCR-induced Ca2+ signalling.

In the early phase of thymus-dependent antibody responses antigen-engaged B cells rapidly change their localization within the secondary lymphoid organs to access helper T cells. Central to this process is the tightly controlled distribution of chemokines, <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> and other guidance cues within the lymphoid organ, determined in part by the stromal cells, and the changing responsiveness of activated lymphocytes to these cues. Studies that use the emerging technique of real-time two-photon imaging of intact lymphoid organs began to dissect the dynamics of B cell migration before and after antigen engagement in vivo. Recent studies also provided new insight into antigen transport mechanisms in lymphoid organs and examined signaling requirements for B lymphocyte positioning and motility. Taken together, these studies have provided a more detailed map of the steps involved in B cell migration to encounter antigen and helper T cells early during the adaptive immune response.