Our previous studies found that nerve growth factor (NGF), via ceramide, enhanced the number of action potentials (APs) evoked by a ramp of depolarizing current in capsaicin-sensitive sensory neurons. Ceramide can be metabolized by ceramidase to <em>sphingosine</em> (Sph), and Sph to <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) by <em>sphingosine</em> kinase. It is well established that each of these products of sphingomyelin metabolism can act as intracellular signalling molecules. This raises the question as to whether the enhanced excitability produced by NGF was mediated directly by ceramide or required additional metabolism to Sph and/or S<em>1</em>P. Sph applied externally did not affect the neuronal excitability, whereas internally perfused Sph augmented the number of APs evoked by the depolarizing ramp. Furthermore, internally perfused S<em>1</em>P enhanced the number of evoked APs. This sensitizing action of NGF, ceramide and internally perfused Sph was abolished by dimethyl<em>sphingosine</em> (DMS), an inhibitor of <em>sphingosine</em> kinase. In contrast, internally perfused S<em>1</em>P enhanced the number of evoked APs in the presence of DMS. These observations support the idea that the metabolism of ceramide/Sph to S<em>1</em>P is critical for the sphingolipid-induced modulation of excitability. Both internally perfused Sph and S<em>1</em>P inhibited the outward K+ current by 25-35% for the step to +60 mV. The Sph- and S<em>1</em>P-sensitive currents had very similar current-voltage relations, suggesting that they were likely to be the same. In addition, the Sph-induced suppression of the K+ current was blocked by pretreatment with DMS. These findings demonstrate that intracellular S<em>1</em>P derived from ceramide acts as an internal second messenger to regulate membrane excitability; however, the effector system whereby S<em>1</em>P modulates excitability remains undetermined.

Oxidized LDL (oxLDL) have been implicated in diverse biological events leading to the development of atherosclerotic lesions. We previously demonstrated that the proliferation of cultured vascular smooth muscle cells (SMC) induced by oxLDL is preceded by an increase in neutral sphingomyelinase activity, sphingomyelin turnover to ceramide, and stimulation of mitogen-activated protein kinases (Augé, N., Escargueil-Blanc, I., Lajoie-Mazenc, I., Suc, I., Andrieu-Abadie, N., Pieraggi, M. T., Chatelut, M., Thiers, J. C., Jaffrézou, J. P., Laurent, G., Levade, T., Nègre-Salvayre, A., and Salvayre, R. (<em>1</em>998) J. Biol. Chem. 273, <em>1</em>2893-<em>1</em>2900). Since ceramide can be converted to other bioactive metabolites, such as the well established mitogen <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P), we investigated whether additional ceramide metabolites are involved in the oxLDL-induced SMC proliferation. We report here that incubation of SMC with oxLDL increased the activities of both acidic and alkaline ceramidases as well as <em>sphingosine</em> kinase, and elevated cellular <em>sphingosine</em> and S<em>1</em>P. Furthermore, the mitogenic effect of oxLDL was inhibited by D-erythro-2-(N-myristoylamino)-<em>1</em>-phenyl-<em>1</em>-propanol and N,N-dimethyl<em>sphingosine</em> which are inhibitors of ceramidase and <em>sphingosine</em> kinase, respectively. These findings suggest that S<em>1</em>P is a key mediator of the mitogenic effect of oxLDL. In agreement with this conclusion, exogenous addition of <em>sphingosine</em> stimulated the proliferation of cultured SMC, and this effect was abrogated by dimethyl<em>sphingosine</em> but not by fumonisin B<em>1</em>, an inhibitor of the acylation of <em>sphingosine</em> to ceramide. Exogenous S<em>1</em>P also promoted SMC proliferation. Altogether, these results strongly suggest that the mitogenic effect of oxLDL in SMC involves the combined activation of sphingomyelinase(s), ceramidase(s), and <em>sphingosine</em> kinase, resulting in the turnover of sphingomyelin to a number of sphingolipid metabolites, of which at least S<em>1</em>P is critical for mitogenesis.

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> is an intermediate of <em>sphingosine</em> catabolism as well as a potent signaling compound. Conditions were established for the extraction and analysis of <em>sphingosine</em> <em>1</em>-<em>phosphate</em> and other sphingoid base <em>1</em>-<em>phosphates</em> from in vitro <em>sphingosine</em> kinase assays and other biological samples. The sphingoid base <em>1</em>-<em>phosphates</em> were extracted in high yield (85%) using small C-<em>1</em>8 reverse-phase columns (LiChroprep RP-<em>1</em>8). After the extracts were treated with 0.<em>1</em> N KOH to remove glycerolipids, the sphingoid base <em>1</em>-<em>phosphates</em> were converted to fluorescent o-phthalaldehyde derivatives that were separated by HPLC using C-<em>1</em>8 columns with a mobile phase of methanol:<em>1</em>0 mM potassium <em>phosphate</em> (pH 7.2):<em>1</em> M tetrabutylammonium dihydrogen <em>phosphate</em> (in water) (83:<em>1</em>6:<em>1</em>, v/v/v). The o-phthalaldehyde derivative of <em>sphingosine</em> <em>1</em>-<em>phosphate</em> was reasonably stable (t(<em>1</em>/2)>> or = <em>1</em>8 h) when EDTA was present and could be detected in picomole amounts. The HPLC retention time of the sphingoid base <em>1</em>-<em>phosphates</em> could be shifted by adjusting the mobile phase to pH 5.5, which is useful in separating overlapping compounds (such as <em>sphingosine</em> <em>1</em>-<em>phosphate</em> and 4-D-hydroxysphinganine) and in confirming the identity of sphingoid base <em>1</em>-<em>phosphates</em> in biological samples. The extraction procedure and HPLC method facilitated assays of <em>sphingosine</em> kinase with different sphingoid bases as substrates and/or inhibitors and enabled the quantitation of sphingoid base <em>1</em>-<em>phosphates</em> in human plasma, serum, and platelets as well as in strains of Saccharomyces cerevisae with mutations in sphingolipid metabolism.

CONCLUSIONS

Modulation of complex functions within the immune system has proven to be surprisingly sensitive to alterations in the lysophospholipid <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) receptor-ligand rheostat. This has become increasingly evident from both chemical and genetic manipulation of the S<em>1</em>P system, with pharmacological effects upon lymphoid cells, dendritic cell function, as well as vascular interfaces. The integrated immune system, perhaps as a result of its relatively recent evolutionary ontogeny, has selected for a number of critical control points regulated by five distinct high affinity G-protein-coupled receptor subtypes with a shared ligand, with receptors distributed on lymphocytes, dendritic cells, and endothelium. All of these cellular components of the axis are capable of modulating immune responses in vivo, with the impact on the immune response being very different from classical immunosuppressants, by virtue of selective spatial and temporal sparing of humoral and myeloid elements of host defense. Pharmacological subversion of the S<em>1</em>P rheostat is proving to be clinically efficacious in multiple sclerosis, and both the scope and limitations of therapeutic modulation of the S<em>1</em>P axis in immunotherapy are becoming clearer as understanding of the integrated chemical physiology of the S<em>1</em>P system emerges.

Although mesenchymal stromal cells (MSCs) are being increasingly used as cell therapeutics in clinical trials, the mechanisms that regulate their chemotactic migration behavior are incompletely understood. We aimed to better define the ability of the GTPase regulator of cytoskeletal activation, Rho, to modulate migration induction in MSCs in a transwell chemotaxis assay. We found that culture-expanded MSCs migrate poorly toward exogenous phospholipids lysophosphatidic acid (LPA) and <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) in transwell assays. Moreover, plasma-induced chemotactic migration of MSCs was even inhibited after pretreatment with LPA. LPA treatment activated intracellular Rho and increased actin stress fibers in resident MSCs. Very similar cytoskeletal changes were observed after microinjection of a cDNA encoding constitutively active RhoA (RhoAV<em>1</em>4) in MSCs. In contrast, microinjection of cDNA encoding Rho inhibitor C3 transferase led to resolution of actin stress fibers, appearance of a looser actin meshwork, and increased numbers of cytoplasmic extensions in the MSCs. Surprisingly, in LPA-pretreated MSCs migrating toward plasma, simultaneous addition of Rho inhibitor C2I-C3 reversed LPA-induced migration suppression and led to improved migration. Moreover, addition of Rho inhibitor C2I-C3 resulted in an approximately 3- to <em>1</em>0-fold enhancement of chemotactic migration toward LPA, S<em>1</em>P, as well as platelet-derived growth factor or hepatocyte growth factor. Thus, inhibition of Rho induces rearrangement of actin cytoskeleton in MSCs and renders them susceptible to induction of migration by physiological stimuli. Disclosure of potential conflicts of interest is found at the end of this article.

Although the modulated expression of Dicer is documented upon neoplastic transformation, little is known of the regulation of Dicer expression by environmental stimuli and its roles in the regulation of cellular functions in primary cells. In this study, we found that Dicer expression was downregulated upon serum withdrawal in human umbilical vein endothelial cells (HUVECs). Serum withdrawal induced a time-dependent repression of Dicer expression, which was specifically rescued by vascular endothelial cell growth factor or <em>sphingosine</em>-<em>1</em>-<em>phosphate</em>. When Dicer expression was silenced by short-hairpin RNA against Dicer, the cells were more prone to apoptosis under serum withdrawal, whereas the rate of apoptosis was comparable with control cells in the serum-containing condition. Real-time PCR-based gene expression profiling identified several genes, the expression of which was modulated by Dicer silencing, including adhesion and matrix-related molecules, caspase-3, and nitric oxide synthase 3 (NOS3). Dicer silencing markedly impaired migratory functions without affecting cell adhesion and repressed phosphorylation of focal adhesion kinase and proline-rich tyrosine kinase 2 in adherent HUVECs. Dicer knockdown upregulated caspase-3 and downregulated NOS3 expression, and serum withdrawal indeed increased caspase-3 and decreased NOS3 expression. Furthermore, the overexpression of Dicer in HUVECs resulted in a marked reduction in apoptosis upon serum withdrawal and a decreased caspase-3 and increased NOS3 expression. The inhibition of NOS activity by Nomega-nitro-L-arginine methyl ester abrogated the effect of Dicer overexpression to rescue the cells from serum withdrawal-induced apoptosis. These results indicated that serum withdrawal decreases Dicer expression, leading to an increased susceptibility to apoptosis through the regulation of caspase-3 and NOS3 expression.

<em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) has been shown to regulate numerous and diverse cell functions, including smooth muscle contraction. Here we assessed the role of S<em>1</em>P/<em>Sphingosine</em> kinase (SPK) pathway in the regulation of bronchial tone. Our objective was to determine, using an integrated pharmacologic and molecular approach, (<em>1</em>) the role of S<em>1</em>P as endogenous modulator of the bronchial tone, and (2) the linkage between S<em>1</em>P pathway and bronchial hyperresponsiveness. We evaluated S<em>1</em>P effects on isolated bronchi and whole lungs, harvested from Balb/c mice sensitized to ovalbumin (OVA) versus vehicle-treated mice, by measuring bronchial reactivity and lung resistance. We found that S<em>1</em>P administration on nonsensitized mouse bronchi does not cause any direct effect on bronchial tone, while a significant increase in Ach-induced contraction occurs after S<em>1</em>P challenge. Conversely, in OVA-sensitized mice S<em>1</em>P/SPK pathway triggers airway hyperesponsiveness. Indeed, S<em>1</em>P causes a dose-dependent contraction of isolated bronchi. Similarly, in the whole lung system S<em>1</em>P increased airway resistance only in OVA-sensitized mice. The action on bronchi of S<em>1</em>P is coupled to an enhanced expression of SPK(<em>1</em>) and SPK(2) as well as of S<em>1</em>P(2) and S<em>1</em>P(3) receptors. In these experiments the key role for S<em>1</em>P/SPK in hyperreactivity has been confirmed by pharmacologic modulation of SPKs. S<em>1</em>P/SPK pathway does not seem to play a major role in physiologic conditions, while it may become critical in pathologic conditions. These results open new windows for therapeutic strategies in diseases like asthma.

The novel immunomodulator FTY720 is effective in experimental models of transplantation and autoimmunity, and is currently undergoing Phase III clinical trials for prevention of kidney graft rejection. FTY720 is a structural analogue of <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) and activates several of the S<em>1</em>P receptors. We show that FTY720 induces endothelium-dependent arterial vasodilation in phenylephrine precontracted mouse aortae. Vasodilation did not occur in thoracic aortic rings from eNOS-deficient mice, implicating and effect dependent of activation of the eNOS/NO pathway. Accordingly, FTY720 induced NO release, Akt-dependent eNOS phosphorylation and activation in human endothelial cells. For biological efficacy, FTY720 required endogenous phosphorylation, since addition of the <em>sphingosine</em> kinase antagonist N',N-dimethyl<em>sphingosine</em> (DMS) prevented activation of eNOS in vitro and inhibited vasodilation in isolated arteries. The endothelial phosphorylation of FTY720 was extremely rapid with almost complete conversion after <em>1</em>0 minutes as determined by mass spectrometry. Finally, we identified the lysophospholipid receptor S<em>1</em>P3 as the S<em>1</em>P receptor responsible for arterial vasodilation by FTY720, as the effect was completely abolished in arteries from S<em>1</em>P3-deficient mice. In summary, we have identified FTY720 as the first immunomodulator for prevention of organ graft rejection in clinical development that, in addition, positively affects the endothelium by stimulating NO production, and thus potentially displaying beneficial effects on transplant survival beyond classical T cell immunosuppression.

The ability of high density lipoproteins (HDL) to inhibit cytokine-induced adhesion molecule expression has been demonstrated in their protective function against the development of atherosclerosis and associated coronary heart disease. A key event in atherogenesis is endothelial activation induced by a variety of stimuli such as tumor necrosis factor-alpha (TNF), resulting in the expression of various adhesion proteins. We have recently reported that <em>sphingosine</em> <em>1</em>-<em>phosphate</em>, generated by <em>sphingosine</em> kinase activation, is a key molecule in mediating TNF-induced adhesion protein expression. We now show that HDL profoundly inhibit TNF-stimulated <em>sphingosine</em> kinase activity in endothelial cells resulting in a decrease in <em>sphingosine</em> <em>1</em>-<em>phosphate</em> production and adhesion protein expression. HDL also reduced TNF-mediated activation of extracellular signal-regulated kinases and NF-kappaB signaling cascades. Furthermore, HDL enhanced the cellular levels of ceramide which in turn inhibits endothelial activation. Thus, the regulation of sphingolipid signaling in endothelial cells by HDL provides a novel insight into the mechanism of protection against atherosclerosis.

<em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P), which mediates pleiotropic actions within the vascular system, is a prominent regulator of microvascular tone. By virtue of its S<em>1</em>P-degrading function, we hypothesized that S<em>1</em>P-phosphohydrolase <em>1</em> (SPP<em>1</em>) is an important regulator of tone in resistance arteries. Hamster gracilis muscle resistance arteries express mRNA encoding SPP<em>1</em>. Overexpression of SPP<em>1</em> (via transfection of a SPP<em>1</em>(wt)) reduced resting tone, Ca2+ sensitivity, and myogenic vasoconstriction, whereas reduced SPP<em>1</em> expression (antisense oligonucleotides) yielded the opposite effects. Expression of a phosphatase-dead mutant of SPP<em>1</em> (SPP<em>1</em>(H208A)) had no effect on any parameter tested, suggesting that catalytic activity of SPP<em>1</em> is critical. The enhanced myogenic tone that follows overexpression of S<em>1</em>P-generating enzyme <em>sphingosine</em> kinase <em>1</em> (Sk<em>1</em>(wt)) was functionally antagonized by coexpression with SPP<em>1</em>(wt) but not SPP<em>1</em>(H208A). SPP<em>1</em> modulated vasoconstriction in response to <em>1</em> to <em>1</em>00 nmol/L exogenous S<em>1</em>P, a concentration range that was characterized as S<em>1</em>P2-dependent, based on the effect of S<em>1</em>P(2) inhibition by antisense oligonucleotides and <em>1</em> mumol/L JTE0<em>1</em>3. Inhibition of the cystic fibrosis transmembrane regulator (CFTR) (<em>1</em>) restored S<em>1</em>P responses that were attenuated by SPP<em>1</em>(wt) overexpression; (2) enhanced myogenic vasoconstriction; but (3) had no effect on noradrenaline responses. We conclude that SPP<em>1</em> is an endogenous regulator of resistance artery tone that functionally antagonizes the vascular effects of both Sk<em>1</em>(wt) and S<em>1</em>P2 receptor activation. SPP<em>1</em> accesses extracellular S<em>1</em>P pools in a manner dependent on a functional CFTR transport protein. Our study assigns important roles to both SPP<em>1</em> and CFTR in the physiological regulation of vascular tone, which influences both tissue perfusion and systemic blood pressure.

BACKGROUND

Chlorogenic acid (CHL), the most potent functional inhibitor of the microsomal glucose-6-phosphate translocase (G6PT), is thought to possess cancer chemopreventive properties. It is not known, however, whether any G6PT functions are involved in tumorigenesis. We investigated the effects of CHL and the potential role of G6PT in regulating the invasive phenotype of brain tumor-derived glioma cells.

RESULTS

RT-PCR was used to show that, among the adult and pediatric brain tumor-derived cells tested, U-87 glioma cells expressed the highest levels of G6PT mRNA. U-87 cells lacked the microsomal catalytic subunit glucose-6-phosphatase (G6Pase)-alpha but expressed G6Pase-beta which, when coupled to G6PT, allows G6P hydrolysis into glucose to occur in non-glyconeogenic tissues such as brain. CHL inhibited U-87 cell migration and matrix metalloproteinase (MMP)-2 secretion, two prerequisites for tumor cell invasion. Moreover, CHL also inhibited cell migration induced by <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P), a potent mitogen for glioblastoma multiform cells, as well as the rapid, S<em>1</em>P-induced extracellular signal-regulated protein kinase phosphorylation potentially mediated through intracellular calcium mobilization, suggesting that G6PT may also perform crucial functions in regulating intracellular signalling. Overexpression of the recombinant G6PT protein induced U-87 glioma cell migration that was, in turn, antagonized by CHL. MMP-2 secretion was also inhibited by the adenosine tri<em>phosphate</em> (ATP)-depleting agents 2-deoxyglucose and 5-thioglucose, a mechanism that may inhibit ATP-mediated calcium sequestration by G6PT.

CONCLUSIONS

We illustrate a new G6PT function in glioma cells that could regulate the intracellular signalling and invasive phenotype of brain tumor cells, and that can be targeted by the anticancer properties of CHL.

<em>Sphingosine</em> kinases (SK) catalyze the production of <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> which in turn regulates cell responses such as proliferation and migration. Here, we show that exposure of the human endothelial cell line EA.hy 926 to hypoxia stimulates a increased SK-<em>1</em>, but not SK-2, mRNA, protein expression, and activity. This effect was due to stimulated SK-<em>1</em> promoter activity which contains two putative hypoxia-inducible factor-responsive-elements (HRE). By deletion of one of the two HREs, hypoxia-induced promoter activation was abrogated. Furthermore, hypoxia upregulated the expression of HIF-<em>1</em>alpha and HIF-2alpha, and both contributed to SK-<em>1</em> gene transcription as shown by selective depletion of HIF-<em>1</em>alpha or HIF-2alpha by siRNA. The hypoxia-stimulated SK-<em>1</em> upregulation was functionally coupled to increased migration since the selective depletion of SK-<em>1</em>, but not of SK-2, by siRNAs abolished the migratory response. In summary, these data show that hypoxia upregulates SK-<em>1</em> activity and results in an accelerated migratory capacity of endothelial cells. SK-<em>1</em> may thus serve as an attractive therapeutic target to treat diseases associated with increased endothelial migration and angiogenesis such as cancer growth and progression.

Treatment of human neutrophils with tumor necrosis factor-alpha (TNF-alpha) resulted in an increase in concentration of ceramide and its catabolite, <em>sphingosine</em>. <em>Sphingosine</em>, a potent endogenous protein kinase C (PKC) inhibitor, as well as TNF-alpha, induced internucleosomal DNA fragmentation and morphological changes characteristic of apoptotic cells. Ceramide and <em>sphingosine</em>-<em>1</em>-<em>phosphate</em>, the initial product of <em>sphingosine</em> catabolism, did not cause apoptosis under our experimental conditions. In addition, <em>1</em>-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7) and N,N-dimethyl<em>sphingosine</em> (DMS), known as PKC inhibitors, also induced apoptosis, suggesting that induction of apoptosis by <em>sphingosine</em> may be related to inhibition of PKC activity. These results indicate that <em>sphingosine</em> deacylated from ceramide may be an endogenous modulator mediating apoptotic signals by TNF-alpha in neutrophils.

<em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) is a sphingolipid mediator that is involved in diverse biological functions. Local administration of S<em>1</em>P causes inflammation coupled to a large eosinophil (EO) recruitment in the rat-paw tissue. The inflammatory response is accompanied by an increase in S<em>1</em>P receptors, namely S<em>1</em>P(<em>1</em>), S<em>1</em>P(2), S<em>1</em>P(3), and by an enhanced expression of CCR3, which is the main chemokine receptor known to be involved in EO function. Human EOs constitutively express S<em>1</em>P(<em>1</em>) and, at a lower extent, S<em>1</em>P(2), S<em>1</em>P(3) receptors. S<em>1</em>P in vitro causes cultured human EO migration and an increase in S<em>1</em>P receptor mRNA copies and strongly up-regulates CCR3 and RANTES (regulated on activation, normal T cell-expressed and secreted) message levels; in particular CCR3 is up-regulated <em>1</em>8,000-fold by S<em>1</em>P. A blocking anti-CCR3 Ab inhibits S<em>1</em>P-induced chemotaxis, implying that S<em>1</em>P acts as specific recruiting signal for EOs not only through its own receptors but also through CCR3. These results show that S<em>1</em>P is involved in EO chemotaxis and contribute to shed light on the complex mechanisms underlying EO recruitment in several diseases such as asthma and some malignancies.

Many tumours originate from cancer stem cells (CSCs), which is a small population of cells that display stem cell properties. However, the molecular mechanisms that regulate CSC frequency remain poorly understood. Here, using microarray screening in aldehyde dehydrogenase (ALDH)-positive CSC model, we identify a fundamental role for a lipid mediator <em>sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) in CSC expansion. Stimulation with S<em>1</em>P enhances ALDH-positive CSCs via S<em>1</em>P receptor 3 (S<em>1</em>PR3) and subsequent Notch activation. CSCs overexpressing <em>sphingosine</em> kinase <em>1</em> (SphK<em>1</em>), an S<em>1</em>P-producing enzyme, show increased ability to develop tumours in nude mice, compared with parent cells or CSCs. Tumorigenicity of CSCs overexpressing SphK<em>1</em> is inhibited by S<em>1</em>PR3 knockdown or S<em>1</em>PR3 antagonist. Breast cancer patient-derived mammospheres contain SphK<em>1</em>(+)/ALDH<em>1</em>(+) cells or S<em>1</em>PR3(+)/ALDH<em>1</em>(+) cells. Our findings provide new insights into the lipid-mediated regulation of CSCs via Notch signalling, and rationale for targeting S<em>1</em>PR3 in cancer.

Pathways of transduction employed by receptors for <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) are identified by the nature of second messengers and/or downstream targets regulated and, more formally, by direct assays of heterotrimeric G protein activation. The different methods generally agree. S<em>1</em>P<em>1</em> couples to members of the Gi family, apparently selectively, although reported pertussis toxin (PTX)-insensitive actions make categorical statements regarding exclusivity difficult. S<em>1</em>P2 and S<em>1</em>P3 couple to members of the Gi, Gq, and G<em>1</em>2/<em>1</em>3 families. S<em>1</em>P4 couples to Gi and possibly G<em>1</em>2/<em>1</em>3, while S<em>1</em>P5 couples to Gi and G<em>1</em>2/<em>1</em>3 but not to Gq. In virtually all circumstances, coupling of S<em>1</em>P receptors to Gi is reflected in PTX-sensitive inhibition of adenylyl cyclase, activation of extracellular-regulated kinases (ERKs), and, depending on the cell, activation of phospholipase C (PLC). Coupling to Gq is reflected in PTX-insensitive activation of phospholipase C. Coupling to G<em>1</em>2/<em>1</em>3 is reflected in activation of Rho and subsequent activation of serum response factor (SRF). Specific linkages have been verified in almost all instances by receptor-promoted [35S]GTPgammaS/GDP exchange on identified G proteins.

Recent technical advances have re-invigorated the study of sphingolipid metabolism in general, and helped to highlight the varied and important roles that sphingolipids play in pancreatic β-cells. Sphingolipid metabolites such as ceramide, glycosphingolipids, <em>sphingosine</em> <em>1</em>-<em>phosphate</em> and gangliosides modulate many β-cell signaling pathways and processes implicated in β-cell diabetic disease such as apoptosis, β-cell cytokine secretion, ER-to-golgi vesicular trafficking, islet autoimmunity and insulin gene expression. They are particularly relevant to lipotoxicity. Moreover, the de novo synthesis of sphingolipids occurs on many subcellular membranes, in parallel to secretory vesicle formation, traffic and granule maturation events. Indeed, the composition of the plasma membrane, determined by the activity of neutral sphingomyelinases, affects β-cell excitability and potentially insulin exocytosis while another glycosphingolipid, sulfatide, determines the stability of insulin crystals in granules. Most importantly, sphingolipid metabolism on internal membranes is also strongly implicated in regulating β-cell apoptosis.

<em>Sphingosine</em>-<em>1</em>-<em>phosphate</em> (S<em>1</em>P) is a bioactive sphingolipid metabolite involved in many cellular processes, acting not only as an extracellular ligand to its specific G protein-coupled receptors, but also as a putative intracellular messenger with yet unidentified targets. Mast cells are tissue-dwelling pivotal early effectors of allergic responses, which produce and secrete S<em>1</em>P that can bind to its receptors present on mast cells to influence their activation and functions. In this review, we will first discuss the current knowledge of S<em>1</em>P production by two isozymes of <em>sphingosine</em> kinase (SphK). Mechanisms of SphK activation will be discussed, with an emphasis on experimental approaches developed to study their differential activation and biological roles in the context of mast cells. The relevance of mast cells in the etiology of allergic disorders, asthma and anaphylaxis is well established. In this review, this concept will be revisited, focusing on the contribution of S<em>1</em>P production and secretion to the symptoms associated with dysregulated inflammatory responses. To conclude, counteracting the proinflammatory effects of S<em>1</em>P could be envisioned as a therapeutic strategy to treat allergic disorders, exacerbated airway inflammation, and anaphylactic reactions, and various options will be discussed, such as the development of pharmacological tools to inhibit SphKs, S<em>1</em>P neutralizing monoclonal antibody, and S<em>1</em>P receptor antagonists.

In a comparison of embryonic brain expression patterns of lysophosphatidic acid and <em>sphingosine</em> <em>1</em>-<em>phosphate</em> receptor genes (lpa(<em>1</em>-3) and s<em>1</em>p(<em>1</em>-5), respectively), transcripts detected by Northern blot were subsequently localized using in situ hybridization. We found striking s<em>1</em>p(<em>1</em>) expression adjacent to several ventricles. Near the lateral ventricle, s<em>1</em>p(<em>1</em>) expression was temporally and spatially coincident with neurogenesis and overlapped with lpa(<em>1</em>) in the neocortical area. We also observed a widespread diffuse pattern for lpa(2-3) and a scattered punctate pattern for s<em>1</em>p(<em>1</em>-3). The punctate pattern colocalized with vascular endothelial markers. Together, these results suggest that s<em>1</em>p(<em>1</em>) influences neurogenesis and s<em>1</em>p(<em>1</em>-3) influence angiogenesis in the developing brain.

Ceramides with different fatty acyl chains may vary in their physiological or pathological roles; however, it remains unclear how cellular levels of individual ceramide species are regulated. Here, we demonstrate that our previously cloned human alkaline ceramidase 3 (ACER3) specifically controls the hydrolysis of ceramides carrying unsaturated long acyl chains, unsaturated long-chain (ULC) ceramides. In vitro, ACER3 only hydrolyzed C(<em>1</em>8:<em>1</em>)-, C(20:<em>1</em>)-, C(20:4)-ceramides, dihydroceramides, and phytoceramides. In cells, ACER3 overexpression decreased C(<em>1</em>8:<em>1</em>)- and C(20:<em>1</em>)-ceramides and dihydroceramides, whereas ACER3 knockdown by RNA interference had the opposite effect, suggesting that ACER3 controls the catabolism of ULC ceramides and dihydroceramides. ACER3 knockdown inhibited cell proliferation and up-regulated the cyclin-dependent kinase inhibitor p2<em>1</em>(CIP<em>1</em>/WAF<em>1</em>). Blocking p2<em>1</em>(CIP<em>1</em>/WAF<em>1</em>) up-regulation attenuated the inhibitory effect of ACER3 knockdown on cell proliferation, suggesting that ACER3 knockdown inhibits cell proliferation because of p2<em>1</em>(CIP<em>1</em>/WAF<em>1</em>) up-regulation. ACER3 knockdown inhibited cell apoptosis in response to serum deprivation. ACER3 knockdown up-regulated the expression of the alkaline ceramidase 2 (ACER2), and the ACER2 up-regulation decreased non-ULC ceramide species while increasing both <em>sphingosine</em> and its <em>phosphate</em>. Collectively, these data suggest that ACER3 catalyzes the hydrolysis of ULC ceramides and dihydroceramides and that ACER3 coordinates with ACER2 to regulate cell proliferation and survival.

BACKGROUND

The greatest genetic risk factor for late-onset Alzheimer's disease (AD) is the ϵ4 allele of Apolipoprotein E (ApoE). ApoE regulates secretion of the potent neuroprotective signaling lipid <em>Sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P). S<em>1</em>P is derived by phosphorylation of <em>sphingosine</em>, catalysed by <em>sphingosine</em> kinases <em>1</em> and 2 (SphK<em>1</em> and 2), and SphK<em>1</em> positively regulates glutamate secretion and synaptic strength in hippocampal neurons. S<em>1</em>P and its receptor family have been subject to intense pharmacological interest in recent years, following approval of the immunomodulatory drug Fingolimod, an S<em>1</em>P mimetic, for relapsing multiple sclerosis.

RESULTS

We quantified S<em>1</em>P levels in six brain regions that are differentially affected by AD pathology, in a cohort of 34 post-mortem brains, divided into four groups based on Braak neurofibrillary tangle staging. S<em>1</em>P declined with increasing Braak stage, and this was most pronounced in brain regions most heavily affected by AD pathology. The S<em>1</em>P/<em>sphingosine</em> ratio was 66% and 64% lower in Braak stage III/IV hippocampus (p = 0.0<em>1</em>0) and inferior temporal cortex (p = 0.0<em>1</em>4), respectively, compared to controls. In accordance with this change, both SphK<em>1</em> and SphK2 activity declined with increasing Braak pathology in the hippocampus (p = 0.032 and 0.047, respectively). S<em>1</em>P/<em>sphingosine</em> ratio was 2.5-fold higher in hippocampus of ApoE2 carriers compared to ApoE4 carriers, and multivariate regression showed a significant association between APOE genotype and hippocampal S<em>1</em>P/<em>sphingosine</em> (p = 0.0495), suggesting a new link between APOE genotype and pre-disposition to AD.

CONCLUSIONS

This study demonstrates loss of S<em>1</em>P and <em>sphingosine</em> kinase activity early in AD pathogenesis, and prior to AD diagnosis. Our findings establish a rationale for further exploring S<em>1</em>P receptor pharmacology in the context of AD therapy.

Studies in cell culture and mouse models of cancer have indicated that the soluble sphingolipid metabolite <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) promotes cancer cell proliferation, survival, invasiveness, and tumor angiogenesis. In contrast, its metabolic precursor ceramide is prodifferentiative and proapoptotic. To determine whether sphingolipid balance plays a significant role in glioma malignancy, we undertook a comprehensive analysis of sphingolipid metabolites in human glioma and normal gray matter tissue specimens. We demonstrate, for the first time, a systematic shift in sphingolipid metabolism favoring S<em>1</em>P over ceramide, which increases with increasing cancer grade. S<em>1</em>P content was, on average, 9-fold higher in glioblastoma tissues compared with normal gray matter, whereas the most abundant form of ceramide in the brain, C<em>1</em>8 ceramide, was on average 5-fold lower. Increased S<em>1</em>P content in the tumors was significantly correlated with increased <em>sphingosine</em> kinase <em>1</em> (SPHK<em>1</em>) and decreased <em>sphingosine</em> <em>phosphate</em> phosphatase 2 (SGPP2) expression. Inhibition of S<em>1</em>P production by cultured glioblastoma cells, using a highly potent and selective SPHK<em>1</em> inhibitor, blocked angiogenesis in cocultured endothelial cells without affecting VEGF secretion. Our findings validate the hypothesis that an altered ceramide/S<em>1</em>P balance is an important feature of human cancers and support the development of SPHK<em>1</em> inhibitors as antiangiogenic agents for cancer therapy.

Adiponectin has received considerable attention for its potential anti-diabetic actions. The adipokine exerts control of glucose and lipid homeostasis via critical effects within the liver, adipose, and pancreas. By stimulating adipogenesis, opposing inflammation, and influencing rates of lipid oxidation and lipolysis, adiponectin critically governs lipid spillover into non-adipose tissues. Ceramide, a cytotoxic and insulin desensitizing lipid metabolite formed when peripheral tissues are exposed to excessive lipid deposition, is potently opposed by adiponectin. Via adiponectin receptors, AdipoR<em>1</em> and AdipoR2, adiponectin stimulates the deacylation of ceramide- yielding <em>sphingosine</em> for conversion to <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) by <em>sphingosine</em> kinase. The resulting conversion from ceramide to S<em>1</em>P promotes survival of functional beta cell mass, allowing for insulin production to meet insulin demands. Alleviation of ceramide burden on the liver allows for improvements in hepatic insulin action. Here, we summarize how adiponectin-induced changes in these tissues lead to improvements in glucose metabolism, highlighting the sphingolipid signaling mechanisms linking adiponectin to each action. ONE SENTENCE SUMMARY: We review the anti-diabetic actions of adiponectin.

OVCAR3 ovarian cancer cells express three <em>sphingosine</em> <em>1</em>-<em>phosphate</em> (S<em>1</em>P) receptors, S<em>1</em>P(<em>1</em>), S<em>1</em>P(2), and S<em>1</em>P(3), but not S<em>1</em>P(4). Stimulation of OVCAR3 cells with S<em>1</em>P induced intracellular calcium increases, which were partly inhibited by VPC 230<em>1</em>9 (an S<em>1</em>P(<em>1</em>/3) antagonist). S<em>1</em>P-induced calcium increases were mediated by phospholipase C and pertussis toxin (PTX)-sensitive G-proteins in OVCAR3 cells. S<em>1</em>P stimulated extracellular signal-regulated kinase, p38 kinase, and Akt which were inhibited by PTX. S<em>1</em>P-stimulated chemotactic migration of OVCAR3 cells in a PTX-sensitive manner, indicating crucial role of G(i) protein(s) in the process. S<em>1</em>P-induced chemotactic migration of OVCAR3 cells was completely inhibited by LY294002 and SB203580. Pretreatment of VPC 230<em>1</em>9 (an S<em>1</em>P(<em>1</em>/3) antagonist) completely inhibited S<em>1</em>P-induced chemotaxis. S<em>1</em>P also induced invasion of OVCAR3 cells, which was also inhibited by VPC 230<em>1</em>9. Taken together, this study suggests that S<em>1</em>P stimulate chemotactic migration and cellular invasion, and VPC 230<em>1</em>9-sensitive S<em>1</em>P receptor(s) might be involved in the processes.