Sphingosine-1-phosphate Lyase Activity Research Articles

Nuclear Sphingosine-1-phosphate Lyase Generated ∆2-hexadecenal is A Regulator of HDAC Activity and Chromatin Remodeling in Lung Epithelial Cells.

Sphingosine-1-phosphate (S1P), a bioactive lipid mediator, is generated from sphingosine by sphingosine kinases (SPHKs) 1 and 2 and is metabolized to ∆2-hexadecenal (∆2-HDE) and ethanolamine phosphate by S1P lyase (S1PL) in mammalian cells. We have recently demonstrated the activation of nuclear SPHK2 and the generation of S1P in the nucleus of lung epithelial cells exposed to Pseudomonas aeruginosa. Here, we have investigated the nuclear localization of S1PL and the role of ∆2-HDE generated from S1P in the nucleus as a modulator of histone deacetylase (HDAC) activity and histone acetylation. Electron micrographs of the nuclear fractions isolated from MLE-12 cells showed nuclei free of ER contamination, and S1PL activity was detected in nuclear fractions isolated from primary lung bronchial epithelial cells and alveolar epithelial MLE-12 cells. Pseudomonas aeruginosa-mediated nuclear ∆2-HDE generation, and H3/H4 histone acetylation was attenuated by S1PL inhibitors in MLE-12 cells and human bronchial epithelial cells. In vitro, the addition of exogenous ∆2-HDE (100-10,000 nM) to lung epithelial cell nuclear preparations inhibited HDAC1/2 activity, and increased acetylation of Histone H3 and H4, whereas similar concentrations of S1P did not show a significant change. In addition, incubation of ∆2-HDE with rHDAC1 generated five different amino acid adducts as detected by LC-MS/MS; the predominant adduct being ∆2-HDE with lysine residues of HDAC1. Together, these data show an important role for the nuclear S1PL-derived ∆2-HDE in the modification of HDAC activity, histone acetylation, and chromatin remodeling in lung epithelial cells.

A Bioassay Using a Pentadecanal Derivative to Measure S1P Lyase Activity.

Sphingosine-1-phosphate (S1P) is a unique lipid ligand binding to S1P receptors to transduce various cell survival or proliferation signals via small G proteins. S1P lyase (S1PL) is the specific enzyme that degrades S1P to phosphoethanolamine and (2E)-hexadecenal and therefore regulates S1P levels. S1PL also degrades dihydrosphingosine-1-phosphate (Sa1P), with a higher affinity to produce hexadecanal. Here, we developed a newly designed assay using a C17-Sa1P substrate that degrades into pentadecanal and phosphoethanolamine. For higher sensitivity in pentadecanal analysis, we developed a quantitative protocol as well as a 5,5-dimethyl cyclohexanedione (5,5-dimethyl CHD) derivatization method. The derivatization conditions were optimized for the reaction time, temperature, and concentrations of the 5,5-dimethyl CHD reagent, acetic acid, and ammonium acetate. The S1PL reaction in the cell lysate after spiking 20 µM of C17-Sa1P for 20 min was linear to the total protein concentrations of 50 µg. The S1PL levels (4 pmol/mg/min) were readily detected in this HPLC with fluorescence detection (λex = 366 nm, λem = 455 nm). The S1PL-catalyzed reaction was linear over 30 min and yielded a Km value of 2.68 μM for C17-Sa1P. This new method was validated to measure the S1PL activity of mouse embryonal carcinoma cell lines of the standard cell (F9-0), S1PL knockdown cells (F9-2), and S1PL-overexpressed cells (F9-4). Furthermore, we treated F9-4 cells with different S1PL inhibitors such as FTY720, 4-deoxypyridoxine (DOP), and the deletion of pyridoxal-5-phosphate (P5P), an essential cofactor for S1PL activity, and observed a significant decrease in pentadecanal relative to the untreated cells. In conclusion, we developed a highly sensitive S1PL assay using a C17-Sa1P substrate for pentadecanal quantification for application in the characterization of S1PL activity in vitro.

Δ‐2 Hexadecenal Generated from S1P by Nuclear S1P Lyase Is a Regulator of HDAC1/2 Activity and Histone Acetylation in Lung Epithelial Cells

Sphingosine‐1‐phosphate (S1P), a bioactive lipid mediator, is generated from sphingosine by sphingosine kinases (SPHKs) 1 and 2 and is metabolized to Δ‐2 hexadecenal (Δ2HD) and ethanolamine‐phosphate by S1P lyase (S1PL) in mammalian cells. While the role of S1P as a key regulator of cellular functions in normal and pathological conditions is well established, the physiological role of fatty aldehyde released from S1P by S1PL is still unclear. We have recently demonstrated activation of nuclear SPHK2 and generation of S1P and Δ‐2 Hexadecenal in the nucleus of lung epithelial cells exposed to Pseudomonas aeruginosa (PA). Here, we have investigated the nuclear localization of S1PL and the role of Δ2HD as a modulator of nuclear HDAC activity and histone acetylation.Nuclear extracts, free from cytosolic and ER contamination, prepared by detergent fractionation method, were probed for purity by Western blotting using organelle‐specific markers Lamin B, ERp72, and GAPDH. Nuclear fractions derived from primary lung bronchial epithelial cells and alveolar epithelial MLE‐12 cell line exhibited S1PL activity, as measured by a fluorogenic substrate assay and mass spectrometric analysis and immunostained for S1PL by Western blotting. Activation of lung epithelial cells with heat‐inactivated Pseudomonas aeruginosa (PA) stimulated Δ2HD generation in the nucleus, which was S1PL dependent. Blocking S1PL activity with an inhibitor, compound C1 (5μM), in MLE‐12 cells inhibited PA‐induced nuclear Δ2HD production and H3 and H4 histone acetylation. In vitro, addition of exogenous Δ2HD (100–10000 nM) to lung epithelial cell nuclear preparation inhibited HDAC 1/2 activity, and increased acetylation of Histone H3 and H4. Overexpressing wild‐type Sgpl1 gene into mouse embryonic fibroblasts (MEFs) deficient in Sgpl1 decreased basal and PA‐induced nuclear HDAC activity, underlining the role of S1P degradative products in modulating HDAC activity. Furthermore, in Sgpl1 −/−MEFs, PA‐induced HDAC activity was higher compared to Sgpl1+/+MEFs treated with PA. Additionally, incubation of Δ2HD with HDAC1 generated 5 different amino acid adducts as detected by LC‐MS/MS; the predominant adduct being Δ2HD with lysine residues of HDAC1.These data show nuclear localization of S1PL in lung epithelial cells and indicate a crucial epigenetic role for Δ2HD, released from S1P by S1PL in the nucleus, in regulating HDAC1/2 activity and histone acetylation. Thus, nuclear S1PL and generation Δ2HD may have a physiological and pathophysiological relevance to lung inflammatory injury.Support or Funding InformationThis work was supported by NIH grant HL 098050 to VN.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Acquired modification of sphingosine-1-phosphate lyase activity is not related to adrenal insufficiency

BackgroundCongenital sphingosine-1-phosphate (S1P) lyase deficiency due to biallelic mutations in SGPL1 gene has recently been described in association with primary adrenal insufficiency and steroid-resistant nephrotic syndrome. S1P lyase, on the other hand, is therapeutically inhibited by fingolimod which is an oral drug for relapsing multiple sclerosis (MS). Effects of this treatment on adrenal function has not yet been evaluated. We aimed to test adrenal function of MS patients receiving long-term fingolimod treatment.MethodsNineteen patients (14 women) with MS receiving oral fingolimod (Gilenya®, Novartis) therapy were included. Median age was 34.2 years (range; 21.3–44.6 years). Median duration of fingolimod treatment was 32 months (range; 6–52 months) at a dose of 0.5 mg/day. Basal and ACTH-stimulated adrenal steroid measurements were evaluated simultaneously employing LC-MS/MS based steroid panel. Basal steroid concentrations were also compared to that of sex- and age-matched healthy subjects. Cortisol and 11-deoxycortisol, 11-deoxycorticosterone and dehydroepiandrosterone were used to assess glucocorticoid, mineralocorticoid and sex steroid producing pathways, respectively.ResultsBasal ACTH concentrations of the patients were 20.8 pg/mL (6.8–37.8 pg/mL) (normal range; 5–65 pg/mL). There was no significant difference in the basal concentrations of cortisol, 11-deoxycortisol, 11-deoxycorticosterone and dehydroepiandrosterone between patients and controls (p = 0.11, 0.058, 0.74, 0.15; respectively). All patients showed adequate cortisol response to 250 mcg IV ACTH stimulation (243 ng/mL, range; 197–362 ng/mL). There was no significant correlation between duration of fingolimod treatment and basal or ACTH-stimulated cortisol or change in cortisol concentrations during ACTH stimulation test (p = 0.57, 0.66 and 0.21, respectively).ConclusionModification and inhibition of S1P lyase activity by the long-term therapeutic use of fingolimod is not associated with adrenal insufficiency in adult patients with MS. This suggests that S1P lyase has potentially a critical role on adrenal development rather than the function of a fully mature adrenal gland.

Aryl hydrocarbon receptor signaling promotes ORMDL3-dependent generation of sphingosine-1-phosphate by inhibiting sphingosine-1-phosphate lyase.

Aryl hydrocarbon receptor (AhR), a cellular chemical sensor, controls cellular homeostasis, and sphingosine-1-phosphate (S1P), a bioactive intermediate of sphingolipid metabolism, is believed to have a role in immunity and inflammation, but their potential crosstalk is currently unknown. We aimed to determine whether there is a functional linkage between AhR signaling and sphingolipid metabolism. We showed that AhR ligands, including an environmental polycyclic aromatic hydrocarbon (PAH), induced S1P generation, and inhibited S1P lyase (S1PL) activity in resting cells, antigen/IgE-activated mast cells, and mouse lungs exposed to the AhR ligand alone or in combination with antigen challenge. The reduction of S1PL activity was due to AhR-mediated oxidation of S1PL at residue 317, which was reversible by the addition of an antioxidant or in cells with knockdown of the ORMDL3 gene encoding an ER transmembrane protein, whereas C317A S1PL mutant-transfected cells were resistant to the AhR-mediated effect. Furthermore, analysis of AhR ligand-treated cells showed a time-dependent increase of the ORMDL3-S1PL complex, which was confirmed by FRET analysis. This change increased the S1P levels, which in turn, induced mast cell degranulation via S1PR2 signaling. In addition, elevated levels of plasma S1P were found in children with asthma compared to non-asthmatic subjects. These results suggest a new regulatory pathway whereby the AhR-ligand axis induces ORMDL3-dependent S1P generation by inhibiting S1PL, which may contribute to the expression of allergic diseases.

Drosophila DNA/RNA methyltransferase contributes to robust host defense in aging animals by regulating sphingolipid metabolism.

Drosophila methyltransferase (Mt2) has been implicated in the methylation of both DNA and tRNA. In this study, we demonstrate that loss of Mt2 activity leads to an age-dependent decline of immune function in the adult fly. A newly eclosed adult has mild immune defects that are exacerbated in a 15day old Mt2-/- fly. The age-dependent effects appear to be systemic, including disturbances in lipid metabolism, changes in cell shape of hemocytes and significant fold-changes in levels of transcripts related to host defense. Lipid imbalance, as measured by quantitative lipidomics, correlates with immune dysfunction, with high levels of immunomodulatory lipids, sphingosine-1-phosphate (S1P) and ceramides, along with low levels of storage lipids. Activity assays on fly lysates confirm the age-dependent increase in S1P and concomitant reduction of S1P lyase activity. We hypothesize that Mt2 functions to regulate genetic loci such as S1P lyase and this regulation is essential for robust host defense as the animal ages. Our study uncovers novel links between age--dependent Mt2 function, innate immune response and lipid homeostasis.

Sphingosine kinase 1 activation enhances epidermal innate immunity through sphingosine-1-phosphate stimulation of cathelicidin production

BackgroundThe ceramide metabolite, sphingosine-1-phosphate (S1P), regulates multiple cellular functions in keratinocytes (KC). We recently discovered that production of a key innate immune element, cathelicidin antimicrobial peptide (CAMP), is stimulated via a NF-κB-dependent mechanism that is activated by S1P when S1P is generated by sphingosine kinase (SPHK) 1. ObjectiveWe investigated whether pharmacological modulation of SPHK1 activity, using a novel synthetic SPHK1 activator, (S)-methyl 2-(hexanamide)-3-(4-hydroxyphenyl) propanoate (MHP), stimulates CAMP expression. MethodsMHP-mediated changes in both S1P and CAMP downstream mediators were analyzed in normal cultured human KC by qRT-PCR, Western immunoblot, ELISA, confocal microscopy for immunohistochemistry, HPLC and ESI–LC/MS/MS, and microbial pathogen invasion/colonization in a human epidermal organotypic model. ResultsTreatment with MHP directly activated SPHK1 and increased cellular S1P content in normal cultured human KC. Because MHP did not inhibit S1P lyase activity, which hydrolyses S1P, augumented S1P levels could be attributed to increased synthesis rather than blockade of S1P degradation. Next, we found that exogenous MHP significantly stimulated CAMP mRNA and protein production in KC, increases that were significantly suppressed by siRNA directed against SPHK1, but not by a scrambled control siRNA. NF-κB activation, assessed by nuclear translocation of NF-κB, occurred in cells following incubation with MHP. Conversely, pretreatment with a specific inhibitor of SPHK1 decreased MHP-induced nuclear translocation of NF-κB, and significantly attenuated the MHP-mediated increase in CAMP production. Finally, topical MHP significantly suppressed invasion of the virulent Staphylococcus aureus into murine skin explants. ConclusionMHP activation of SPHK1, a target enzyme of CAMP production, can stimulate innate immunity.

Nuclear S1P Lyase Regulates Histone Acetylation In Pseudomonas aeruginosa ‐Induced Lung Inflammation

Sphingosine -1-Phosphate (S1P), a bioactive sphingolipid, regulates various cellular processes, including vascular integrity and inflammation. Sphingosine-1-Phosphate Lyase (S1PL), the enzyme that irreversibly degrades S1P to hexadecenal and phosphoethanolamine, regulates the intracellular levels of S1P and is also recognized to modulate inflammatory responses. Pseudomonas aeruginosa, a gram-negative opportunistic pathogen, is the common cause of pneumonia in ventilator-induced and immuno-compromised patients that is associated with increased morbidity and mortality. Understanding the role of sphingolipid metabolism in P. aeruginosa-induced lung inflammation is critical to improve clinical outcome of sepsis patients. Our study aims to define the epigenetic role of S1PL in modulating inflammatory responses in P. aeruginosa induced lung inflammation. Nuclei isolated from mouse lung epithelial cells (MLE-12) exhibited S1PL activity as determined by LC-MS/MS.Over-expression of S1PL resulted in elevated S1PL levels in both the cytosol and nucleus, and inhibition of S1PL by 4-deoxy pyridoxine elevated nuclear S1P levels. Also, blocking S1PL expression attenuated P. aeruginosa-induced Histone H3 and H4 acetylation. Interestingly, exogenous addition of Δ2-hexadecenal (1-100 nM) to isolated intact nuclei of MLE-12 cells induced Histone H3 and H4 acetylation, suggesting an epigenetic role of fatty aldehyde released from S1P by S1PL. Thus, targeting S1PL enzyme could be a novel mechanism for the treatment of bacterial infection of the lungs.

Reduced Activity of Sphingosine-1-Phosphate Lyase Induces Podocyte-related Glomerular Proteinuria, Skin Irritation, and Platelet Activation.

Sphingosine-1-phosphate (S1P) lyase is considered as a drug target in autoimmune diseases based on the protective effect of reducing activity of the enzyme in animal models of inflammation. Since S1P lyase deficiency in mice causes a severe, lethal phenotype, it was of interest to investigate any pathological alterations associated with only partially reduced activity of S1P lyase as may be encountered upon pharmacological inhibition. Both genetic reduction of S1P lyase activity in mice and inhibition of S1P lyase with a low-molecular-weight compound in rats consistently resulted in podocyte-based kidney toxicity, which is the most severe finding. In addition, skin irritation and platelet activation were observed in both instances. The similarity of the findings in both the genetic model and the pharmacological study supports the value of analyzing inducible partially target-deficient mice for safety assessment. If the findings described in rodents translate to humans, target-related toxicity, particularly podocyte dysfunction, may limit chronic systemic treatment of autoimmune diseases with S1P lyase inhibitors. Furthermore, partial deficiency or inhibition of S1P lyase appears to provide an in vivo rodent model to enable studies on the mechanism of podocyte dysfunction.

Sphingosine-1-phosphate (S1P) displays sustained S1P1 receptor agonism and signaling through S1P lyase-dependent receptor recycling

The sphingosine-1-phosphate (S1P) type 1 receptor (S1P1R) is a novel therapeutic target in lymphocyte-mediated autoimmune diseases. S1P1 receptor desensitization caused by synthetic S1P1 receptor agonists prevents T-lymphocyte egress from secondary lymphoid organs into the circulation. The selective S1P1 receptor agonist ponesimod, which is in development for the treatment of autoimmune diseases, efficiently reduces peripheral lymphocyte counts and displays efficacy in animal models of autoimmune disease. Using ponesimod and the natural ligand S1P, we investigated the molecular mechanisms leading to different signaling, desensitization and trafficking behavior of S1P1 receptors. In recombinant S1P1 receptor-expressing cells, ponesimod and S1P triggered Gαi protein-mediated signaling and β-arrestin recruitment with comparable potency and efficiency, but only ponesimod efficiently induced intracellular receptor accumulation. In human umbilical vein endothelial cells (HUVEC), ponesimod and S1P triggered translocation of the endogenous S1P1 receptor to the Golgi compartment. However, only ponesimod treatment caused efficient surface receptor depletion, receptor accumulation in the Golgi and degradation. Impedance measurements in HUVEC showed that ponesimod induced only short-lived Gαi protein-mediated signaling followed by resistance to further stimulation, whereas S1P induced sustained Gαi protein-mediated signaling without desensitization. Inhibition of S1P lyase activity in HUVEC rendered S1P an efficient S1P1 receptor internalizing compound and abrogated S1P-mediated sustained signaling. This suggests that S1P lyase – by facilitating S1P1 receptor recycling – is essential for S1P-mediated sustained signaling, and that synthetic agonists are functional antagonists because they are not S1P lyase substrates.

S1P Is Associated with Protection in Human and Experimental Cerebral Malaria

Cerebral malaria (CM) is associated with excessive inflammatory responses and endothelial activation. Sphingosine 1-phosphate (S1P) is a signaling sphingolipid implicated in regulating vascular integrity, inflammation and T-cell migration. We hypothesized that altered S1P signaling during malaria contributes to endothelial activation and inflammation, and show that plasma S1P levels were decreased in Ugandan children with CM compared with children with uncomplicated malaria. Using the Plasmodium berghei ANKA (PbA) model of experimental CM (ECM), we demonstrate that humanized S1P lyase (hS1PL)(-/-) mice with reduced S1P lyase activity (resulting in increased bio-available S1P) had improved survival compared with wild-type littermates. Prophylactic and therapeutic treatment of infected mice with compounds that modulate the S1P pathway and are in human trials for other conditions (FTY720 or LX2931) significantly improved survival in ECM. FTY720 treatment improved vascular integrity as indicated by reduced levels of soluble intercellular adhesion molecule (sICAM), increased angiopoietin 1 (Ang1) (regulator of endothelial quiescence) levels, and decreased Evans blue dye leakage into brain parenchyma. Furthermore, treatment with FTY720 decreased IFNγ levels in plasma as well as CD4(+) and CD8(+) T-cell infiltration into the brain. Finally, when administered during infection in combination with artesunate, FTY720 treatment resulted in increased survival to ECM. These findings implicate dysregulation of the S1P pathway in the pathogenesis of human and murine CM and suggest a novel therapeutic strategy to improve clinical outcome in severe malaria.

Sphingosine-1-phosphate Lyase Deficiency Produces a Pro-inflammatory Response While Impairing Neutrophil Trafficking

Sphingosine-1-phosphate (S1P) lyase catalyzes the degradation of S1P, a potent signaling lysosphingolipid. Mice with an inactive S1P lyase gene are impaired in the capacity to degrade S1P, resulting in highly elevated S1P levels. These S1P lyase-deficient mice have low numbers of lymphocytes and high numbers of neutrophils in their blood. We found that the S1P lyase-deficient mice exhibited features of an inflammatory response including elevated levels of pro-inflammatory cytokines and an increased expression of genes in liver associated with an acute-phase response. However, the recruitment of their neutrophils into inflamed tissues was impaired and their neutrophils were defective in migration to chemotactic stimulus. The IL-23/IL-17/granulocyte-colony stimulating factor (G-CSF) cytokine-controlled loop regulating neutrophil homeostasis, which is dependent on neutrophil trafficking to tissues, was disturbed in S1P lyase-deficient mice. Deletion of the S1P4 receptor partially decreased the neutrophilia and inflammation in S1P lyase-deficient mice, implicating S1P receptor signaling in the phenotype. Thus, a genetic block in S1P degradation elicits a pro-inflammatory response but impairs neutrophil migration from blood into tissues.

Characterization of sphingosine-1-phosphate lyase activity by electrospray ionization–liquid chromatography/tandem mass spectrometry quantitation of (2 E)-hexadecenal

Sphingosine-1-phosphate (S1P) is a sphingolipid signaling molecule crucial for cell survival and proliferation. S1P-mediated signaling is largely controlled through its biosynthesis and degradation, and S1P lyase (S1PL) is the only known enzyme that irreversibly degrades sphingoid base-1-phosphates to phosphoethanolamine and the corresponding fatty aldehydes. S1PL-mediated degradation of S1P results in the formation of (2 E)-hexadecenal, whereas hexadecanal is the product of dihydrosphingosine-1-phosphate (DHS1P) degradation. Fatty aldehydes can undergo biotransformation to fatty acids and/or alcohols, making them elusive and rendering the task of fatty aldehyde quantitation challenging. We have developed a simple, highly sensitive, and high-throughput protocol for (2 E)-hexadecenal quantitation as a semicarbazone derivative by liquid chromatography–electrospray ionization–tandem mass spectrometry. The approach was applied to determining S1PL activity in vitro with the ability to use as low as 0.25 μg of microsomal protein per assay. The method is also applicable to the use of total tissue homogenate as the source of S1PL. A correction for (2 E)-hexadecenal disappearance due to its biotransformation during enzymatic reaction is required, especially at higher protein concentrations. The method was applied to confirm FTY720 as the inhibitor of S1PL with an IC 50 value of 52.4 μM.

Inhibition of Sphingosine-1-Phosphate Lyase for the Treatment of Autoimmune Disorders

During nearly a decade of research dedicated to the study of sphingosine signaling pathways, we identified sphingosine-1-phosphate lyase (S1PL) as a drug target for the treatment of autoimmune disorders. S1PL catalyzes the irreversible decomposition of sphingosine-1-phosphate (S1P) by a retro-aldol fragmentation that yields hexadecanaldehyde and phosphoethanolamine. Genetic models demonstrated that mice expressing reduced S1PL activity had decreased numbers of circulating lymphocytes due to altered lymphocyte trafficking, which prevented disease development in multiple models of autoimmune disease. Mechanistic studies of lymphoid tissue following oral administration of 2-acetyl-4(5)-(1(R),2(S),3(R),4-tetrahydroxybutyl)-imidazole (THI) 3 showed a clear relationship between reduced lyase activity, elevated S1P levels, and lower levels of circulating lymphocytes. Our internal medicinal chemistry efforts discovered potent analogues of 3 bearing heterocycles as chemical equivalents of the pendant carbonyl present in the parent structure. Reduction of S1PL activity by oral administration of these analogues recapitulated the phenotype of mice with genetically reduced S1PL expression.

Involvement of sphingosine kinase in plant cell signalling

In mammalian cells sphingosine-1-phosphate (S1P) is a well-established messenger molecule that participates in a wide range of signalling pathways. The objective of the work reported here was to investigate the extent to which phosphorylated long-chain sphingoid bases, such as sphingosine-1-phosphate and phytosphingosine-1-phosphate (phytoS1P) are used in plant cell signalling. To do this, we manipulated Arabidopsis genes capable of metabolizing these messenger molecules. We show that Sphingosine kinase1 (SPHK1) encodes an enzyme that phosphorylates sphingosine, phytosphingosine and other sphingoid long-chain bases. The stomata of SPHK1-KD Arabidopsis plants were less sensitive, whereas the stomata of SPHK1-OE plants were more sensitive, than wild type to ABA. The rate of germination of SPHK1-KD was enhanced, whereas the converse was true for SPHK1-OE seed. Reducing expression of either the putative Arabidopsis S1P phosphatase (SPPASE) or the DPL1 gene, which encodes an enzyme with S1P lyase activity, individually, had no effect on guard-cell ABA signalling; however, stomatal responses to ABA in SPPASEDPL1 RNAi plants were compromised. Reducing the expression of DPL1 had no effect on germination; however, germination of SPPASE RNAi seeds was more sensitive to applied ABA. We also found evidence that expression of SPHK1 and SPPASE were coordinately regulated, and discuss how this might contribute to robustness in guard-cell signalling. In summary, our data establish SPHK1 as a component in two separate plant signalling systems, opening the possibility that phosphorylated long-chain sphingoid bases such as S1P and phytoS1P are ubiquitous messengers in plants.

Genetic and Chemical Inhibition of Sphingosine 1-Phosphate Lyase Results in Peripheral Lymphopenia and Alleviates Disease Development in Animal Models of Inflammation and Autoimmunity.

The ceramide metabolite sphingosine 1-phosphate (S1P) is an important modulator of a wide variety of physiologic functions via its action as a second messenger in intracellular signaling pathways and its extracellular interaction with specific G protein-coupled receptors on the cell surface. In the immune system, interaction of S1P with one of its receptors, S1P1, leads to inhibition of lymphocyte egress from primary and secondary lymphoid tissues resulting in peripheral lymphopenia, a mechanism with demonstrated effects in recent human clinical studies using the S1P1 receptor agonist, FTY720. S1P lyase (S1PL), which catalyzes the only irreversible step in sphingosine metabolism, the degradation of S1P to hexadecenal and ethanolamine phosphate, has recently been suggested to be a target of 2-acetyl-4-tetrahydroxybutylimidazole (THI), a compound known to increase tissue S1P levels and alter lymphocyte trafficking. In the current study, genetic inactivation of S1PL in knockout (KO) mice resulted in severe depletion of lymphocytes in the peripheral blood accompanied by accumulation of mature T cells in the thymus. This abnormal pattern of lymphocyte distribution suggests that results previously seen in THI treated mice were indeed due to THI's inhibition of S1PL rather than another unknown activity. S1PL KO mice also demonstrated significantly reduced viability, perhaps due to increased numbers of myeloid cells which infiltrated the lungs. In a S1PL humanized allele, in which the human S1PL has been introduced into S1PL KO mice and restores ∼17% of wild type S1PL activity, the reduced viability trait is rescued while peripheral lymphopenia is maintained. Since the phenotypes are genetically separable by an allele that reduces S1PL activity, the lack of observed toxicity at doses of THI resulting in lymphopenia is likely due to its partial inhibition of S1PL activity at those doses, perhaps due to differences in S1P or S1P receptor expression or metabolism in different cell types. The potential for separation of lymphopenia and myeloid cell lung pathology effects by chemical inhibition was confirmed by chronic treatment of mice with the oral, small molecule S1PL inhibitor, LX2931, for 47 days during which no effects on viability were observed while the altered profile of lymphocyte migration seen in the partially inhibited humanized S1PL allele was maintained. Rats and monkeys treated with LX2931 also exhibited peripheral lymphopenia suggesting that the S1PL signaling pathway is conserved across mammalian species. Both S1PL KO and LX2931 treated mice were protected in the oxazolone challenge assay of delayed type hypersensitivity and the collagen induced model of rheumatoid arthritis, demonstrating that the reduction of peripheral lymphocytes previously observed with S1PL inhibition can beneficially affect the development of autoimmune disease.

Mechanisms of sphingosine and sphingosine 1-phosphate generation in human platelets

The bioactive molecule sphingosine 1-phosphate (S1P) is abundantly stored in platelets and can be released extracellularly. However, although they have high sphingosine (Sph) kinase activity, platelets lack the de novo sphingolipid biosynthesis necessary to provide the substrates. Here, we reveal a generation pathway for Sph, the precursor of S1P, in human platelets. Platelets incorporated extracellular 3H-labeled Sph much faster than human megakaryoblastic cells and rapidly converted it to S1P. Furthermore, Sph formed from plasma sphingomyelin (SM) by bacterial sphingomyelinase (SMase) and neutral ceramidase (CDase) was rapidly incorporated into platelets and converted to S1P, suggesting that platelets use extracellular Sph as a source of S1P. Platelets abundantly express SM, possibly supplied from plasma lipoproteins, at the cell surface. Treating platelets with bacterial SMase resulted in Sph generation at the cell surface, conceivably by the action of membrane-bound neutral CDase. Simultaneously, a time-dependent increase in S1P levels was observed. Finally, we demonstrated that secretory acid SMase also induces S1P increases in platelets. In conclusion, our results suggest that in platelets, Sph is supplied from at least two sources: generation in the plasma followed by incorporation, and generation at the outer leaflet of the plasma membrane, initiated by cell surface SM degradation.

Lymphocyte Sequestration Through S1P Lyase Inhibition and Disruption of S1P Gradients

Lymphocyte egress from the thymus and from peripheral lymphoid organs depends on sphingosine 1-phosphate (S1P) receptor-1 and is thought to occur in response to circulatory S1P. However, the existence of an S1P gradient between lymphoid organs and blood or lymph has not been established. To further define egress requirements, we addressed why treatment with the food colorant 2-acetyl-4-tetrahydroxybutylimidazole (THI) induces lymphopenia. We found that S1P abundance in lymphoid tissues of mice is normally low but increases more than 100-fold after THI treatment and that this treatment inhibits the S1P-degrading enzyme S1P lyase. We conclude that lymphocyte egress is mediated by S1P gradients that are established by S1P lyase activity and that the lyase may represent a novel immunosuppressant drug target.

Sphingosine-1-phosphate Lyase Is Involved in the Differentiation of F9 Embryonal Carcinoma Cells to Primitive Endoderm

Sphingosine 1-phosphate (S1P) is a bioactive lipid molecule that acts both extracellularly and intracellularly. The SPL gene encodes a mammalian S1P lyase that degrades S1P. Here, we have disrupted the SPL gene in mouse F9 embryonal carcinoma cells by gene targeting. This is the first report of gene disruption of mammalian S1P lyase. The SPL-null cells exhibited no S1P lyase activity, and intracellular S1P was increased approximately 2-fold, compared with wild-type cells. Treatment of F9 embryonal carcinoma cells with retinoic acid induces differentiation to primitive endoderm (PrE). An acceleration in this PrE differentiation was observed in the SPL-null cells. This effect was apparently caused by the accumulated S1P, since N,N-dimethylsphingosine, a S1P synthesis inhibitor, had an inhibitory effect on the PrE differentiation. Moreover, F9 cells stably expressing sphingosine kinase also exhibited an acceleration in the differentiation. Exogenous S1P had no effect on differentiation, indicating that intracellular but not extracellular S1P is involved. Moreover, we determined that expression of the SPL protein is up-regulated during the progression to PrE. We also showed that sphingosine kinase activity is increased in PrE-differentiated cells. These results suggest that intracellular S1P has a role in the PrE differentiation and that SPL may be involved in the regulation of intracellular S1P levels during this differentiation.