The potent bioactive sphingolipid mediator, <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P), is produced by 2 <em>sphingosine</em> kinase isoenzymes, SphK<em>1</em> and SphK2. Expression of SphK<em>1</em> is up-regulated in cancers, including leukemia, and associated with cancer progression. A screen of <em>sphingosine</em> analogs identified (2R,3S,4E)-N-methyl-5-(4'-pentylphenyl)-2-aminopent-4-ene-<em>1</em>,3-diol, designated SK<em>1</em>-I (BML-258), as a potent, water-soluble, isoenzyme-specific inhibitor of SphK<em>1</em>. In contrast to pan-SphK inhibitors, SK<em>1</em>-I did not inhibit SphK2, PKC, or numerous other protein kinases. SK<em>1</em>-I decreased growth and survival of human leukemia U937 and Jurkat cells, and enhanced apoptosis and cleavage of Bcl-2. Lethality of SK<em>1</em>-I was reversed by caspase inhibitors and by expression of Bcl-2. SK<em>1</em>-I not only decreased S<em>1</em>P levels but concomitantly increased levels of its proapoptotic precursor ceramide. Conversely, S<em>1</em>P protected against SK<em>1</em>-I-induced apoptosis. SK<em>1</em>-I also induced multiple perturbations in activation of signaling and survival-related proteins, including diminished phosphorylation of ERK<em>1</em>/2 and Akt. Expression of constitutively active Akt protected against SK<em>1</em>-I-induced apoptosis. Notably, SK<em>1</em>-I potently induced apoptosis in leukemic blasts isolated from patients with acute myelogenous leukemia but was relatively sparing of normal peripheral blood mononuclear leukocytes. Moreover, SK<em>1</em>-I markedly reduced growth of AML xenograft tumors. Our results suggest that specific inhibitors of SphK<em>1</em> warrant attention as potential additions to the therapeutic armamentarium in leukemia.

Sphingolipids have emerged as molecules whose metabolism is regulated leading to generation of bioactive products including ceramide, <em>sphingosine</em>, and <em>sphingosine</em>-<em>1</em>-<em>phosphate</em>. The balance between cellular levels of these bioactive products is increasingly recognized to be critical to cell regulation; whereby, ceramide and <em>sphingosine</em> cause apoptosis and growth arrest phenotypes, and <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> mediates proliferative and angiogenic responses. <em>Sphingosine</em> kinase is a key enzyme in modulating the levels of these lipids and is emerging as an important and regulated enzyme. This review is geared at mechanisms of regulation of <em>sphingosine</em> kinase and the coming to light of its role in disease.

We used a HPLC-MS/MS methodology for determination of a basic metabolomic profile (<em>1</em>8:<em>1</em>,<em>1</em>8:0 sphingoid backbone, C(<em>1</em>4)-C(26) N-acyl part) of "normal" sphingolipid levels in human serum and plasma. Blood was collected from healthy males and nonpregnant females under fasting and nonfasting conditions with and without anticoagulants. Sphingolipids analyzed included sphingoid bases, <em>sphingosine</em> and dihydro<em>sphingosine</em>, their <em>1</em>-<em>phosphates</em> (S<em>1</em>P and dhS<em>1</em>P), molecular species (C(n)-) of ceramide (Cer), sphingomyelin (SM), hexosylceramide (HexCer), lactosylceramide (LacCer), and Cer <em>1</em>-<em>phosphate</em> (Cer<em>1</em>P). SM, LacCer, HexCer, Cer, and Cer<em>1</em>P constituted 87.7, 5.8, 3.4, 2.8, and 0.<em>1</em>5% of total sphingolipids, respectively. The abundant circulating SM was C(<em>1</em>6)-SM (64.0 µM), and it increased with fasting (<em>1</em>00 µM). The abundant LacCer was C(<em>1</em>6)-LacCer (<em>1</em>0.0 µM) and the abundant HexCer was C(24)-HexCer (2.5 µM). The abundant Cer, C(24)-Cer (4.0 µM), was not influenced by fasting; however, levels of C(<em>1</em>6)-C(20) Cers were decreased in response to fasting. S<em>1</em>P levels were higher in serum than plasma (0.68 µM vs. 0.32 µM). We also determined levels of sphingoid bases and SM species in isolated lipoprotein classes. HDL(3) was the major carrier of S<em>1</em>P, dhS<em>1</em>P, and Sph, and LDL was the major carrier of Cer and dhSph. Per particle, VLDL contained the highest levels of SM, Cer, and S<em>1</em>P. HPLC-MS/MS should provide a tool for clinical testing of circulating bioactive sphingolipids in human blood.

Tumor necrosis factor-alpha (TNF) receptor-associated factor 2 (TRAF2) is one of the major mediators of TNF receptor superfamily transducing TNF signaling to various functional targets, including activation of NF-kappa B, JNK, and antiapoptosis. We investigated how TRAF2 mediates differentially the distinct downstream signals. We now report a novel mechanism of TRAF2-mediated signal transduction revealed by an association of TRAF2 with <em>sphingosine</em> kinase (SphK), a lipid kinase that is responsible for the production of <em>sphingosine</em> <em>1</em>-<em>phosphate</em>. We identified a TRAF2-binding motif of SphK that mediated the interaction between TRAF2 and SphK resulting in the activation of the enzyme, which in turn is required for TRAF2-mediated activation of NF-kappa B but not JNK. In addition, by using a kinase inactive dominant-negative SphK and a mutant SphK that lacks TRAF2-binding motif we show that the interaction of TRAF2 with SphK and subsequent activation of SphK are critical for prevention of apoptosis during TNF stimulation. These findings show a role for SphK in the signal transduction by TRAF2 specifically leading to activation of NF-kappa B and antiapoptosis.

Sphingolipids have emerged as bioeffector molecules, controlling various aspects of cell growth and proliferation in cancer, which is becoming the deadliest disease in the world. These lipid molecules have also been implicated in the mechanism of action of cancer chemotherapeutics. Ceramide, the central molecule of sphingolipid metabolism, generally mediates antiproliferative responses, such as cell growth inhibition, apoptosis induction, senescence modulation, endoplasmic reticulum stress responses and/or autophagy. Interestingly, recent studies suggest de novo-generated ceramides may have distinct and opposing roles in the promotion/suppression of tumors, and that these activities are based on their fatty acid chain lengths, subcellular localization and/or direct downstream targets. For example, in head and neck cancer cells, ceramide synthase 6/C(<em>1</em>6)-ceramide addiction was revealed, and this was associated with increased tumor growth, whereas downregulation of its synthesis resulted in ER stress-induced apoptosis. By contrast, ceramide synthase <em>1</em>-generated C(<em>1</em>8)-ceramide has been shown to suppress tumor growth in various cancer models, both in situ and in vivo. In addition, ceramide metabolism to generate <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) by <em>sphingosine</em> kinases <em>1</em> and 2 mediates, with or without the involvement of G-protein-coupled S<em>1</em>P receptor signaling, prosurvival, angiogenesis, metastasis and/or resistance to drug-induced apoptosis. Importantly, recent findings regarding the mechanisms by which sphingolipid metabolism and signaling regulate tumor growth and progression, such as identifying direct intracellular protein targets of sphingolipids, have been key for the development of new chemotherapeutic strategies. Thus, in this article, we will present conclusions of recent studies that describe opposing roles of de novo-generated ceramides by ceramide synthases and/or S<em>1</em>P in the regulation of cancer pathogenesis, as well as the development of sphingolipid-based cancer therapeutics and drug resistance.

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) exerts diverse physiological actions by activating its cognate G protein-coupled receptors. Five S<em>1</em>P receptors have been identified in mammals: LP(B<em>1</em>)/EDG-<em>1</em>, LP(B2)/H2<em>1</em>8/AGR<em>1</em>6/EDG-5, LP(B3)/EDG-3, LP(B4)/NRG-<em>1</em>/EDG-8, and LP(C<em>1</em>)/EDG-6. One of these receptors, LP(B<em>1</em>), has recently been shown to be essential for mouse embryonic development. Here we disrupted the lp(B3) gene in mice, resulting in the complete absence of lp(B3) gene, transcript, and LP(B3) protein. LP(B3)-null mice were viable and fertile and developed normally with no obvious phenotypic abnormality. We prepared mouse embryonic fibroblast (MEF) cells to examine effects of LP(B3) deletion on S<em>1</em>P-induced signal transduction pathways. Wild-type MEF cells expressed lp(B<em>1</em>), lp(B2), and lp(B3) but neither lp(B4) nor lp(C<em>1</em>), and they were highly responsive to S<em>1</em>P in phospholipase C (PLC) activation, adenylyl cyclase inhibition, and Rho activation. Identically prepared LP(B3)-null MEF cells showed significant decreases in PLC activation, slight decreases in adenylyl cyclase inhibition, and no change in Rho activation. Retrovirus-mediated rescue of the LP(B3) receptor in LP(B3)-null MEF cells restored S<em>1</em>P-dependent PLC activation and adenylyl cyclase inhibition. These results indicate a nonessential role for LP(B3) in normal development of mouse but show nonredundant cellular signaling mediated by a single type of S<em>1</em>P receptor.

Lymphocyte egress from lymph nodes requires the G-protein-coupled <em>sphingosine</em> <em>1</em>-<em>phosphate</em> receptor-<em>1</em> (S<em>1</em>P(<em>1</em>)). The activation antigen CD69 associates with and inhibits the function of S<em>1</em>P(<em>1</em>), inhibiting egress. Here we undertook biochemical characterization of the requirements for S<em>1</em>P(<em>1</em>)-CD69 complex formation. Domain swapping experiments between CD69 and the related type II transmembrane protein, NKRp<em>1</em>A, identified a requirement for the transmembrane and membrane proximal domains for specific interaction. Mutagenesis of S<em>1</em>P(<em>1</em>) showed a lack of requirement for N-linked glycosylation, tyrosine sulfation, or desensitization motifs but identified a requirement for transmembrane helix 4. Expression of CD69 led to a reduction of S<em>1</em>P(<em>1</em>) in cell lysates, likely reflecting degradation. Unexpectedly, the S<em>1</em>P(<em>1</em>)-CD69 complex exhibited a much longer half-life for binding of S<em>1</em>P than S<em>1</em>P(<em>1</em>) alone. In contrast to wild-type CD69, a non-S<em>1</em>P(<em>1</em>) binding mutant of CD69 failed to inhibit T cell egress from lymph nodes. These findings identify an integral membrane interaction between CD69 and S<em>1</em>P(<em>1</em>) and suggest that CD69 induces an S<em>1</em>P(<em>1</em>) conformation that shares some properties of the ligand-bound state, thereby facilitating S<em>1</em>P(<em>1</em>) internalization and degradation.

Excessive mechanical stress is a key component of ventilator-associated lung injury, resulting in profound vascular leak and an intense inflammatory response. To extend our in vitro observations concerning the barrier-protective effects of the lipid growth factor <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (Sph <em>1</em>-P), we assessed the ability of Sph <em>1</em>-P to prevent regional pulmonary edema accumulation in clinically relevant rodent and canine models of acute lung injury induced by combined intrabronchial endotoxin administration and high tidal volume mechanical ventilation. Intravenously delivered Sph <em>1</em>-P significantly attenuated both alveolar and vascular barrier dysfunction while significantly reducing shunt formation associated with lung injury. Whole lung computed tomographic image analysis demonstrated the capability of Sph <em>1</em>-P to abrogate significantly the accumulation of extravascular lung water evoked by 6-hour exposure to endotoxin. Axial density profiles and vertical density gradients localized the Sph <em>1</em>-P response to transitional zones between aerated and consolidated lung regions. Together, these results indicate that Sph <em>1</em>-P represents a novel therapeutic intervention for the prevention of pulmonary edema related to inflammatory injury and increased vascular permeability.

BACKGROUND

The oral immunomodulator FTY720 has shown efficacy in patients with relapsing multiple sclerosis (MS). FTY720 functionally antagonizes <em>sphingosine</em> <em>1</em>-<em>phosphate</em> receptor-<em>1</em> (S<em>1</em>P<em>1</em>) on T cells and consequently inhibits S<em>1</em>P/S<em>1</em>P<em>1</em>-dependent lymphocyte egress from secondary lymphoid organs. Little is known about the phenotype and function of T cells remaining in peripheral blood during long-term FTY720 treatment.

METHODS

T cells from FTY720-treated, interferon-beta (IFNbeta)-treated and untreated patients with MS, and healthy donors (HD) were analyzed with respect to T cell subpopulation composition, proliferation, and cytokine production.

RESULTS

In FTY720-treated patients (n = <em>1</em>6), peripheral blood CD4+ and CD8+ T cell counts were reduced by approximately 80% and 60% when compared to the other groups (IFN beta: n = 7; untreated: n = 5; HD: n = <em>1</em>0). This related to selective reduction of naive (CCR7+CD45RA+) and central memory (CCR7+CD45RA-) T cells (TCM), and resulted in a relative increase of peripheral effector memory (CCR7-CD45RA- [TEM] and CCR7-CD45RA+ [TEMRA]) T cells. The remaining blood T cell populations displayed a reduced potential to secrete IL-2 and to proliferate in vitro, but rapidly produced interferon-gamma upon reactivation, confirming a functional TEM/TEMRA phenotype. Neither FTY720 nor FTY720-P directly suppressed proliferation or cytokine production by T cells.

CONCLUSIONS

Therapeutic dosing of FTY720 reduces naïve T cells and TCM, but not TEM, in blood, without affecting T cell function. This is presumably because naive T cells and TCM express the homing receptor CCR7, allowing recirculation to secondary lymphoid tissues on a regular basis and, thus, trapping of the cells by FTY720 in lymph nodes.

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P), produced by Sphks (<em>sphingosine</em> kinases), is a multifunctional lipid mediator that regulates immune cell trafficking and vascular development. Mammals maintain a large concentration gradient of S<em>1</em>P between vascular and extravascular compartments. Mechanisms by which S<em>1</em>P is released from cells and concentrated in the plasma are poorly understood. We recently demonstrated [Ancellin, Colmont, Su, Li, Mittereder, Chae, Stefansson, Liau and Hla (2002) J. Biol. Chem. 277, 6667-6675] that Sphk<em>1</em> activity is constitutively secreted by vascular endothelial cells. In the present study, we show that among the five Sphk isoforms expressed in endothelial cells, the Sphk-<em>1</em>a isoform is selectively secreted in HEK-293 cells (human embryonic kidney cells) and human umbilical-vein endothelial cells. In sharp contrast, Sphk2 is not secreted. The exported Sphk-<em>1</em>a isoform is enzymatically active and produced sufficient S<em>1</em>P to induce S<em>1</em>P receptor internalization. Wild-type mouse plasma contains significant Sphk activity (<em>1</em>79 pmol x min(-<em>1</em>) x g(-<em>1</em>)). In contrast, Sphk<em>1</em>-/- mouse plasma has undetectable Sphk activity and approx. 65% reduction in S<em>1</em>P levels. Moreover, human plasma contains enzymatically active Sphk<em>1</em> (46 pmol x min(-<em>1</em>) x g(-<em>1</em>)). These results suggest that export of Sphk-<em>1</em>a occurs under physiological conditions and may contribute to the establishment of the vascular S<em>1</em>P gradient.

The role of the protein kinase Akt in cell migration is incompletely understood. Here we show that <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P)-induced endothelial cell migration requires the Akt-mediated phosphorylation of the G protein-coupled receptor (GPCR) EDG-<em>1</em>. Activated Akt binds to EDG-<em>1</em> and phosphorylates the third intracellular loop at the T(236) residue. Transactivation of EDG-<em>1</em> by Akt is not required for G(i)-dependent signaling but is indispensable for Rac activation, cortical actin assembly, and chemotaxis. Indeed, T236AEDG-<em>1</em> mutant sequestered Akt and acted as a dominant-negative GPCR to inhibit S<em>1</em>P-induced Rac activation, chemotaxis, and angiogenesis. Transactivation of GPCRs by Akt may constitute a specificity switch to integrate rapid G protein-dependent signals into long-term cellular phenomena such as cell migration.

Sphingolipid metabolites such as <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) and ceramide modulate apoptosis during development and in response to stress. In general, ceramide promotes apoptosis, whereas S<em>1</em>P stimulates cell proliferation and protects against apoptosis. S<em>1</em>P is irreversibly degraded by the enzyme S<em>1</em>P lyase (SPL). In this study, we show a crucial role for SPL in mediating cellular responses to stress. SPL expression in HEK293 cells potentiated apoptosis in response to stressful stimuli including DNA damage. This effect seemed to be independent of ceramide generation but required SPL enzymatic activity and the actions of p38 MAP kinase, p53, p53-inducible death domain protein (PIDD), and caspase-2 as shown by molecular and chemical inhibition of each of these targets. Further, SPL expression led to constitutive activation of p38. Endogenous SPL expression was induced by DNA damage in WT cells, whereas SPL knockdown diminished apoptotic responses. Importantly, SPL expression was significantly down-regulated in human colon cancer tissues in comparison with normal adjacent tissues, as determined by quantitative real-time PCR (Q-PCR) and immunohistochemical analysis. Down-regulation of S<em>1</em>P phosphatases was also observed, suggesting that colon cancer cells manifest a block in S<em>1</em>P catabolism. In addition, SPL expression and activity were down-regulated in adenomatous lesions of the Min mouse model of intestinal tumorigenesis. Taken together, these results indicate that endogenous SPL may play a physiological role in stress-induced apoptosis and provide an example of altered SPL expression in a human tumor. Our findings suggest that genetic or epigenetic changes affecting intestinal S<em>1</em>P metabolism may correlate with and potentially contribute to carcinogenesis.

The sphingolipid ceramide induces macroautophagy (here called autophagy) and cell death with autophagic features in cancer cells. Here we show that overexpression of <em>sphingosine</em> kinase <em>1</em> (SK<em>1</em>), an enzyme responsible for the production of <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P), in MCF-7 cells stimulates autophagy by increasing the formation of LC3-positive autophagosomes and the rate of proteolysis sensitive to the autophagy inhibitor 3-methyladenine. Autophagy was blocked in the presence of dimethyl<em>sphingosine</em>, an inhibitor of SK activity, and in cells expressing a catalytically inactive form of SK<em>1</em>. In SK<em>1</em>(wt)-overexpressing cells, however, autophagy was not sensitive to fumonisin B<em>1</em>, an inhibitor of ceramide synthase. In contrast to ceramide-induced autophagy, SK<em>1</em>(S<em>1</em>P)-induced autophagy is characterized by (i) the inhibition of mammalian target of rapamycin signaling independently of the Akt/protein kinase B signaling arm and (ii) the lack of robust accumulation of the autophagy protein Beclin <em>1</em>. In addition, nutrient starvation induced both the stimulation of autophagy and SK activity. Knocking down the expression of the autophagy protein Atg7 or that of SK<em>1</em> by siRNA abolished starvation-induced autophagy and increased cell death with apoptotic hallmarks. In conclusion, these results show that SK<em>1</em>(S<em>1</em>P)-induced autophagy protects cells from death with apoptotic features during nutrient starvation.

The enzyme <em>sphingosine</em> kinase (SK) catalyzes the formation of <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P), a bioactive lipid that acts extracellularly on G protein-coupled receptors of the S<em>1</em>P(<em>1</em>)/EDG-<em>1</em> subfamily. Although S<em>1</em>P is formed in the cytosol of various cells, S<em>1</em>P release is not understood and is controversial because this lipid mediator is also regarded as a second messenger. In this report, we describe the existence of an extracellular S<em>1</em>P-generating system in vascular endothelial cells. Endothelial cells release SK constitutively and form S<em>1</em>P in the range of receptor stimulation. Levels of <em>sphingosine</em> but not ATP in the extracellular environment are rate-limiting. Treatment of endothelial cells with small interfering RNA for SK-<em>1</em> transcript specifically inhibited SK export, and SK-<em>1</em>-transfected human embryonic kidney 293 cells exhibited enhanced release of SK-<em>1</em>. The export of SK-<em>1</em> is constitutive and is inhibited by cytochalasin D and treatment at 4 degrees C but not by brefeldin A or nocodazole, suggesting that a nonclassical secretory pathway that requires the actin cytoskeleton dynamics is involved. Because S<em>1</em>P regulates angiogenesis and vascular maturation, we overexpressed SK-<em>1</em> using an adenoviral vector in vivo in the Matrigel system of angiogenesis. Overexpression of SK-<em>1</em> resulted in enhanced release of SK activity and induced angiogenesis and vascular maturation. These findings suggest that S<em>1</em>P is made in the extracellular milieu and that extracellular export of SK contributes to the action of S<em>1</em>P in the vascular system.

The many biological responses documented for lysophospholipids that include lysophosphatidic acid and <em>sphingosine</em> <em>1</em>-<em>phosphate</em> can be mechanistically attributed to signaling through specific G protein-coupled receptors. At least nine receptors have now been identified, and the total number is likely to be larger. In this brief review, we note cogent features of lysophospholipid receptors, including the current nomenclature, signaling properties, development of agonists and antagonists, and physiological functions.

Calcium mobilization through antigen receptors, including high-affinity IgE receptors (Fc epsilon RI), is thought to be mediated by inositol-<em>1</em>,4,5-tris<em>phosphate</em> production (InsP3). Here we show that antigen clustering of Fc epsilon RI on the rat mast-cell line (RBL-2H3) activates a <em>sphingosine</em> kinase (SK) and produces <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P), and alternative second messenger for intracellular calcium mobilization. The <em>sphingosine</em> analogue, D-L-threo-dihydro<em>sphingosine</em> (DHS), inhibits the SK enzyme competitively with a dissociation constant, K<em>1</em>, of 5 to <em>1</em>8 microM. This inhibition substantially suppresses the Fc epsilon RI-mediated calcium signal, but leaves intact the syk tyrosine kinase activation and the small InsP3 production. The entire InsP3-dependent pathway activated by a transfected G-protein coupled receptor, used here as a positive control, also remained intact. Thus Fc epsilon RI principally utilizes a SK pathway to mobilize calcium.

<em>Sphingosine</em> kinase <em>1</em> (SK<em>1</em>) phosphorylates <em>sphingosine</em> to form <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P), which has the ability to promote cell proliferation and survival and stimulate angiogenesis. The SK<em>1</em>/S<em>1</em>P pathway also plays a critical role in regulation of cyclooxygenase-2 (COX-2), a well-established pathogenic factor in colon carcinogenesis. Therefore, we examined the expression of SK<em>1</em> and COX-2 in rat colon tumors induced by azoxymethane (AOM) and the relationship of these two proteins in normal and malignant intestinal epithelial cells. Strongly positive SK<em>1</em> staining was found in 2<em>1</em>/28 (75%) of rat colon adenocarcinomas induced by AOM, whereas no positive SK<em>1</em> staining was observed in normal mucosa. The increase in SK<em>1</em> and COX-2 expression in AOM-induced rat colon adenocarcinoma was confirmed at the level of mRNA by real-time RT-PCR. In addition, it was found that <em>1</em>) down-regulation of SK<em>1</em> in HT-29 human colon cancer cells by small interfering RNA (siRNA) decreases COX-2 expression and PGE2 production; 2) overexpression of SK<em>1</em> in RIE-<em>1</em> rat intestinal epithelial cells induces COX-2 expression; and 3) S<em>1</em>P stimulates COX-2 expression and PGE2 production in HT-29 cells. These results suggest that the SK<em>1</em>/S<em>1</em>P pathway may play an important role in colon carcinogenesis, in part, by regulating COX-2 expression and PGE2 production.

Airway DCs play a crucial role in the pathogenesis of allergic asthma, and interfering with their function could constitute a novel form of therapy. The <em>sphingosine</em> <em>1</em>-<em>phosphate</em> receptor agonist FTY720 is an oral immunosuppressant that retains lymphocytes in lymph nodes and spleen, thus preventing lymphocyte migration to inflammatory sites. The accompanying lymphopenia could be a serious side effect that would preclude the use of FTY720 as an antiasthmatic drug. Here we show in a murine asthma model that local application of FTY720 via inhalation prior to or during ongoing allergen challenge suppresses Th2-dependent eosinophilic airway inflammation and bronchial hyperresponsiveness without causing lymphopenia and T cell retention in the lymph nodes. Effectiveness of local treatment was achieved by inhibition of the migration of lung DCs to the mediastinal lymph nodes, which in turn inhibited the formation of allergen-specific Th2 cells in lymph nodes. Also, FTY720-treated DCs were intrinsically less potent in activating naive and effector Th2 cells due to a reduced capacity to form stable interactions with T cells and thus to form an immunological synapse. These data support the concept that targeting the function of airway DCs with locally acting drugs is a powerful new strategy in the treatment of asthma.

<em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (Sph-<em>1</em>-P) is the initial product of catabolism of <em>sphingosine</em> by <em>sphingosine</em> kinase and is cleaved by Sph-<em>1</em>-P lyase to a fatty aldehyde and ethanolamine <em>phosphate</em>. This phosphorylated sphingoid base is not only an intermediary catabolite, but also a bioactive lipid with important functions, including stimulation of cell proliferation in Swiss 3T3 fibroblasts and inhibition of tumor cell motility. In the present study, we examined functional roles of Sph-<em>1</em>-P in human platelets. Sph-<em>1</em>-P induced platelet shape change and aggregation reactions, although it failed to elicit secretion. <em>Sphingosine</em>, ceramide, sphingomyelin, and N,N-dimethyl<em>sphingosine</em> did not mimic the positive effects of Sph-<em>1</em>-P on platelets. Subthreshold concentrations of Sph-<em>1</em>-P and weak platelet agonists such as adenosine di<em>phosphate</em> (ADP) and epinephrine synergistically elicited aggregation, which may be important for efficient amplification of platelet activation. Sph-<em>1</em>-P induced intracellular Ca2+ mobilization and the dose-response for Ca2+ release correlated closely with the concentration required for induction of shape change. On addition of [3H]<em>sphingosine</em> to intact platelets, the label was rapidly converted to Sph-<em>1</em>-P, and subsequently to ceramide and sphingomyelin. Interestingly, the Sph-<em>1</em>-P formed was specifically released into medium on stimulation of platelets with physiologic agonists. The amount of Sph-<em>1</em>-P in platelets, as measured by its conversion into radiolabeled N-acetyl-Sph-<em>1</em>-P, was <em>1</em>.4 nmol/<em>1</em>0(9) cells and was about four times higher than the mass of Sph present. When compared by mole percent Sph-<em>1</em>-P/phospholipid, the value for platelets is over <em>1</em>0 times higher than that for neutrophils. Our results suggest that Sph-<em>1</em>-P, rapidly converted from <em>sphingosine</em>, abundantly stored in platelets, and released on the cell activation, may play a physiologic role in thrombosis, hemostasis, and the natural wound-healing processes.

The functional relevance of brain-derived neurotrophic factor (BDNF) is beginning to be well appreciated not only in mice, but also in humans. Because reduced levels typically correlate with impaired neuronal function, increasing BDNF levels with well-tolerated drugs diffusing into the central nervous system may help in ameliorating functional deficits. With this objective in mind, we used the <em>sphingosine</em>-<em>1</em> <em>phosphate</em> receptor agonist fingolimod, a drug that crosses the blood-brain barrier. In addition, fingolimod has recently been introduced as the first oral treatment for multiple sclerosis. In cultured neurons, fingolimod increases BDNF levels and counteracts NMDA-induced neuronal death in a BDNF-dependent manner. Ongoing synaptic activity and MAPK signaling is required for fingolimod-induced BDNF increase, a pathway that can also be activated in vivo by systemic fingolimod administration. Mice lacking Mecp2, a gene frequently mutated in Rett syndrome, show decreased levels of BDNF, and fingolimod administration was found to partially rescue these levels as well as the size of the striatum, a volumetric sensor of BDNF signaling in rodents. These changes correlate with increased locomotor activity of the Mecp2-deficient animals, suggesting that fingolimod may improve the functional output of the nervous system, in addition to its well-documented effects on lymphocyte egress from lymph nodes.

<em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) is a highly bioactive sphingolipid involved in diverse biological processes leading to changes in cell growth, differentiation, motility, and survival. S<em>1</em>P generation is regulated via <em>sphingosine</em> kinase (SK), and many of its effects are mediated through extracelluar action on G-protein-coupled receptors. In this study, we have investigated the mechanisms regulating SK, where this occurs in the cell, and whether this leads to release of S<em>1</em>P extracellularly. The protein kinase C (PKC) activator, phorbol <em>1</em>2-myristate <em>1</em>3-acetate (PMA), induced early activation of SK in HEK 293 cells, and this activation was more specific to the membrane-associated SK. Therefore, we next investigated whether PMA induced translocation of SK to the plasma membrane. PMA induced translocation of both endogenous and green fluorescent protein (GFP)-tagged human SK<em>1</em> (hSK<em>1</em>) to the plasma membrane. PMA also induced phosphorylation of GFP-hSK<em>1</em>. The PMA-induced translocation was abrogated by preincubation with known PKC inhibitors (bisindoylmaleimide and calphostin-c) as well as by the indirect inhibitor of PKC, C(6)-ceramide, supporting a role for PKC in mediating translocation of SK to the plasma membrane. SK activity was not necessary for translocation, because a dominant negative G82D mutation also translocated in response to PMA. Importantly, PKC regulation of SK was accompanied by a 4-fold increase in S<em>1</em>P in the media. These results demonstrate a novel mechanism by which PKC regulates SK and increases secretion of S<em>1</em>P, allowing for autocrine/paracrine signaling.

The lipid mediator <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P), the product of <em>sphingosine</em> kinase (SPHK)-induced phosphorylation of <em>sphingosine</em>, is known to stabilize interendothelial junctions and prevent microvessel leakiness. Here, we investigated the role of SPHK<em>1</em> activation in regulating the increase in pulmonary microvessel permeability induced by challenge of mice with lipopolysaccharide or thrombin ligation of protease-activating receptor (PAR)-<em>1</em>. Both lipopolysaccharide and thrombin increased mouse lung microvascular permeability and resulted in a delayed activation of SPHK<em>1</em> that was coupled to the onset of restoration of permeability. In contrast to wild-type mice, Sphk<em>1</em>(-/-) mice showed markedly enhanced pulmonary edema formation in response to lipopolysaccharide and PAR-<em>1</em> activation. Using endothelial cells challenged with thrombin concentration (50 nmol/L) that elicited a transient but reversible increase in endothelial permeability, we observed that increased SPHK<em>1</em> activity and decreased intracellular S<em>1</em>P concentration preceded the onset of barrier recovery. Thus, we tested the hypothesis that released S<em>1</em>P in a paracrine manner activates its receptor S<em>1</em>P<em>1</em> to restore the endothelial barrier. Knockdown of SPHK<em>1</em> decreased basal S<em>1</em>P production and Rac<em>1</em> activity but increased basal endothelial permeability. In SPHK<em>1</em>-depleted cells, PAR-<em>1</em> activation failed to induce Rac<em>1</em> activation but augmented RhoA activation and endothelial hyperpermeability response. Knockdown of S<em>1</em>P<em>1</em> receptor in endothelial cells also enhanced the increase in endothelial permeability following PAR-<em>1</em> activation. S<em>1</em>P treatment of Sphk<em>1</em>(-/-) lungs or SPHK<em>1</em>-deficient endothelial cells restored endothelial barrier function. Our results suggest the crucial role of activation of the SPHK<em>1</em>->>S<em>1</em>P->>S<em>1</em>P<em>1</em> signaling pathway in response to inflammatory mediators in endothelial cells in regulating endothelial barrier homeostasis.

Sphingolipids (SLs) have been considered for many years as predominant building blocks of biological membranes with key structural functions and little relevance in cellular signaling. However, this view has changed dramatically in recent years with the recognition that certain SLs such as ceramide, <em>sphingosine</em> <em>1</em>-<em>phosphate</em> and gangliosides, participate actively in signal transduction pathways, regulating many different cell functions such as proliferation, differentiation, adhesion and cell death. In particular, ceramide has attracted considerable attention in cell biology and biophysics due to its key role in the modulation of membrane physical properties, signaling and cell death regulation. This latter function is largely exerted by the ability of ceramide to activate the major pathways governing cell death such as the endoplasmic reticulum and mitochondria. Overall, the evidence so far indicates a key function of SLs in disease pathogenesis and hence their regulation may be of potential therapeutic relevance in different pathologies including liver diseases, neurodegeneration and cancer biology and therapy.

The potent immunosuppressive drug FTY720, a <em>sphingosine</em> analog, induces redistribution of lymphocytes from circulation to secondary lymphoid tissues. FTY720 is phosphorylated in vivo and functions as an agonist for four G-protein-coupled <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> receptors. The identity of the kinase that phosphorylates FTY720 is still not known. Here we report that although both <em>sphingosine</em> kinase type <em>1</em> (SphK<em>1</em>) and type 2 (SphK2) can phosphorylate FTY720 with low efficiency, SphK2 is much more effective than SphK<em>1</em>. FTY720 inhibited phosphorylation of <em>sphingosine</em> catalyzed by SphK2 to a greater extent than it inhibits SphK<em>1</em>. Thus, SphK2 may be the relevant enzyme that is responsible for in vivo phosphorylation of FTY720.