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  • Regulation of pulmonary inflammation in cystic fibrosis by ceramide.

    I1

    Erich Gulbins, Yang Zhang, Katrin Anne Becker

    Dept. of Molecular Biology, University of Duisburg-Essen, Hufelandstrasse 55, 45122 Essen, Germany

    Cystic fibrosis (CF), caused by mutations of the CF transmembrane conductance regulator (CFTR) molecule, is characterized by chronic pulmonary inflammation, reduced mucociliary clearance, and increased susceptibility to infection. We have recently demonstrated an important role of sphingolipids in CF. Cftr-knock-out or B6.129P2(CF/3)-CftrTgH(neoim)Hgu mice that produce low levels of Cftr accumulate ceramide in their lungs. This ceramide triggers enhanced cell death of respiratory epithelial cells, release of pro-inflammatory cytokines in the lung, release of DNA into the mucus, chronic inflammation and high sensitivity to P. aeruginosa infections. Human post-mortem specimens also have high levels of ceramide in lung tissue of CF patients. Using mass spectrometry, fluorescence microscopy and biochemical measurements, we show an accumulation of ceramide also in alveolar macrophages, the trachea and intestinum of CF mice. Ceramide induces a constitutive activation of caspase 1 and an increased rate of cell death in CF mice, which might result in inflammation and increased susceptibility of CF mice. Heterozygosity of the acid sphingomyelinase or systemic application or inhalation of inhibitors of the acid sphingomyelinase reduced ceramide levels in bronchial epithelial cells of CF mice. Normalization of ceramide levels prevented inflammation in the lung of CF mice and infection with P. aeruginosa. These studies indicate a central role of ceramide in the pathophysiology of CF and suggest the acid sphingomyelinase as a novel target to treat cystic fibrosis.

  • Glycosylation and signaling: glycosphingolipids get their share.

    I2

    Sandrine Pizette1 

    1Institute of Developmental Biology and Cancer, Universite de Nice Sophia Antipolis, France 

    Studies of the Notch receptor have shown that glycosylation of the receptor or its ligand is a means to regulate ligand-receptor interaction. To address whether glycosylation is a general mechanism controling signaling, we undertook the characterisation of two Drosophila genes, egghead (egh) and brainiac (brn), which harbor sequence similarities to genes encoding glycosyltransferases. In addition, mutations in these genes result in phenotypes reminiscent of alterations in Notch and EGFR signaling. 

    Using in vitro enzymatic assays, we first demonstrated that Egh and Brn are glycosyltransferases that catalyze the synthesis of disaccharides specific to the glycosphingolipid (GSL) pathway. We then showed by biochemical analysis of GSL species and by immunofluorescence with an antibody specific to a short form of GSL, that the egh and brn mutant animals accumulate truncated GSLs. This indicates that Egh and Brn act on GSL biosynthesis in vivo. Finally, we made use of the egh and brn mutants to investigate the role of GSLs in signaling in vivo. We found that during oogenesis, GSLs shape the extracellular gradient of the Drosophila TGFa-like EGFR ligand by controling its diffusion. These results thus reveal an unexpected role for GSLs in signaling. 

    I will discuss the molecular mechanism underlying this novel function, and will present preliminary data indicating new in vivo roles for GSLs at both the organismal and subcellular level. 

  • Ceramide kinase, ceramide-1-phosphate: roles and regulation.

    I3

    Frederic Bornancin 

    Novartis Institutes for BioMedical Research, Basel, Switzerland 

    It has been almost a decade since ceramide kinase (CERK) was cloned. Phosphorylation of ceramide by CERK remains the only known mechanism for production of ceramide 1-phosphate (C1P). There is, however, mounting evidence for an as yet unidentified alternative C1P producing pathway. Therefore, the knowledge acquired using C1P as active principle may only partially reflect modulation of CERK. Nevertheless, CERK appears to be key for controlling ceramide levels, and C1P produced by CERK has emerged as a genuine signaling entity. 

    The current understanding of CERK at protein level as well as some insights into the regulation of ceramide metabolism by CERK will be presented, including new data on the modulation of CERK in innate immunity. 

  • Tumor gangliosides condition the microenvironment and favor tumor progression.

    I4

    Stephan Ladisch 

    Children’s Rresearch Institute, Children’s National Medical Center, 111 Michigan Ave. NW, Washington, DC, 20010, USA 

    To better understand the pathogenesis of human cancer, increasing attention has been directed to identifying potential in vivo interactions between the tumor cell and the surrounding tumor microenvironment–-the tumor-host interaction. Our findings on the role of key membrane molecules, gangliosides, in influencing these tumor-host interactions will be presented. Briefly, by their synthesis and subsequent shedding from tumor cells, tumor cell gangliosides enhance, and interference with their synthesis impedes, tumor development and progression in vivo. Specifically, the characteristically rapid tumor cell ganglioside metabolism, i.e., substantial synthesis and shedding into the tumor microenvironment, results in transfer to surrounding normal cells. Subsequently, these ganglioside-enriched normal cells have altered functions. Basic cellular mechanisms of ganglioside effects in the tumor microenvironment–-inhibition of the immune response, enhancement of stromal cell proliferation, and enhancement of the angiogenic response–-will be highlighted. The critical question raised is: Is tumor formation and progression itself affected? To directly address this, we created a genetically stable, complete, and specific model of tumor cell ganglioside depletion. Early findings on the consequences of selective and complete depletion of tumor cell gangliosides in this murine sarcoma cell model will be summarized, and their implications for cancer treatment considered. [NIH grants R01CA42361-17 and R01 CA61010-14.] 

  • Targeting the sphingosine 1-phosphate signalling pathway in cancer.

    I5

    Nigel J. Pyne, JS Long, C. Loveridge, F. Tonelli, KG Lim, S. Pyne 

    Cell Biology Group, Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, 27 Taylor St, Glasgow, G4 0NR, Scotland, UK. 

    Sphingosine 1-phosphate is a bioactive lipid that is formed by the sphingosine kinase-catalysed phosphorylation of sphingosine, and which binds to a family of G-protein coupled receptors termed S1P1-5 to elicit biological responses. We will demonstrate in this presentation that high expression of S1P1, S1P3 receptors and sphingosine kinase 1 (SK1) in ER+ breast cancer tumours are associated with reduced patient survival and recurrence on tamoxifen when compared with patients with low expression of these proteins in their tumours. We will also provide evidence for how S1P1 receptors participate in tumour neovascularisation and how SK1/S1P3 function together to induce a metastatic phenotype in ER+ breast cancer cells. A novel promiscuous interaction between S1P4 and the HER2 oncogene in ER- breast cancer cells will also be described. The highlighting of the role of SK1 and S1P receptors in breast cancer provide impetus for targeting these proteins with compounds that inhibit their function. In this regard, we will also demonstrate novel actions of SK1 inhibitors, which reveal a new mechanism by which SK1 induces chemotherapeutic resistance in androgen-independent prostate cancer cells. 

    Supported by CRUK [A7536]. 

  • Dual and opposite effects of fumonisin B1 on ceramide and sphingomyelin contents in piglet’s lung and liver.

    S1 

    N. Loiseau1, N. Therville2, T. Levade2, J. Bertrand-Michel3 

    1UR66, Laboratoire de Pharmacologie-Toxicologie, INRA, 2U858, Institut de Médecine Moléculaire de Rangueil, INSERM, and 3U563, Plateau technique de Lipidomique, IFR 30, INSERM, Toulouse, France 

    Fumonisin B1 (FB1) is one of the most known inhibitor of Ceramide Synthases (CerS). In this study, we hypothesized that FB1 may interact with a specific CerS subgroup to explain the interspecies differences in the clinical symptoms. 

    Since each CerS uses a preferential subset of fatty acyl-CoAs, we compared the sphingolipid contents of lungs and liver from normal and FB1-contaminated piglets (fed with 1.5 mg FB1/kg body weight daily for 9 days). This study focused on the analysis of type 2 ceramide (Cer2) and sphingomyelin (SM) contents. Total Cer2 and total SM contents decreased 2-fold and 1.6-fold, respectively in lungs of FB1-contaminated piglets. In contrast, surprisingly, in liver of FB1-contaminated animals, total Cer2 and total SM contents increased 5.8-fold and 1.5-fold, respectively. By analyzing the FB1 effects on individual Cer2 and SM species, we deduced the effect of this toxin on each CerS family. Our data indicate that FB1 inhibits CerS5/6 whatever the tissues. Moreover, this toxin interacts with piglet’s CerS2/4 to deplete lung and to enrich liver with ceramides containing long fatty acids. These changes could explain the species-specific toxicity of FB1. 

    [Supported by DAER-Recherche/06001316/07006301] 

  • Increased expression of sphingosine kinase 1 mediates prosaposin-induced cytokine production.

    S2

    Lysann Sauer1, Dmitry Pshezhetskiy1 and Jonathan Waxman

    1Tumour Microenvironment and Chemotherapy Group, Hammersmith Campus, Faculty of Medicine, Imperial College London, UK 

    Sphingosine kinase 1 (Sphk1) upregulation has been implicated in chemo-resistance in prostate cancer and its inhibition can sensitize to chemo-induced apoptosis. Prosaposin, a neurotrophic protein has been recently shown to induce prostate cancer cell proliferation, migration and chemoresistance. Furthermore an increased expression of prosaposin was linked with progression of human prostate cancer. 

    Prosaposin knockout mice exhibited an involution of the prostate gland which correlated with a significant reduction of prostate SphK1 activity. In prostate cancer cell lines SphK1 activity correlated with the amount of secreted, but not intracellular prosaposin, indicating a role for the prosaposin/GPCR signalling. The specific knockdown of prosaposin in prostate cancer cells induced a significant decrease in SphK1 activity and expression on mRNA and protein level. Additionally, prosaptide TX14A, derived from the trophic sequence of saposin C, enhanced SphK1 activity and up-regulated SphK1 mRNA expression via ERK1/2-mediated mechanism. Furthermore, TX14A induced an up-regulation of the proliferative factor interleukin 6, which was abrogated by pre-treatment with SphK1-specific siRNA. 

    Overall, our data indicate that SphK1 upregulation is required for prosaposin-induced cytokine expression in prostate cancer cells.

  • Role of sphingolipid enzymes in oxidized phospholipid (oxPL) induced RAW 267.4 macrophage cell death.

    S3

    L Marlingapla Halasiddappa,D Koller, U Stemmer, Z Dunai, E Zenzmaier, A Hermetter 

    Institute of Biochemistry, Graz University of Technology, Petersgasse 12/2, 8010 Graz, Austria. 

    Oxidized lipoproteins and their oxidized phospholipids (oxPL) induce apoptosis in vascular cells. Ceramide (Cer) mediates and propagates apoptosis by activation of JNK and p38 MAPkinases. Cer is generated instantly by aSMase via sphingomyelin hydrolysis and by de novo synthesis in the hours. Ceramide synthase (CerS) isoforms regulate de novo generation of ceramides as they display substrate specificity for the chain lengths of fatty acyl-CoAs. Depending on the stimulus and cell type, specific ceramide species synthesized by these isoforms (CerS1-6) are likely to contribute differently to the apoptosis pathway. Here we report that only a subset of CerS is activated in RAW 264.7 cells by oxPL treatment in a time-dependent manner. Levels of apoptotic ceramide are also influenced by modification and/or degradation. So far, we have studied the activities of ceramidase in oxPL-treated macrophages. This enzyme is almost unaffected and seems not to play any particular role in ceramide-induced cell death. Currently we study the role of other enzymes regulating the apoptotic ceramide pool in cultured RAW 267.4 macrophage cells under the influence of oxPL (POVPC and PGPC). Analyzing the sphingolipidome, as well as expression, localization and activity at various stages of programmed cell death should help understanding the toxicity of oxPL and role of sphingolipid species in mediating apoptosis. 

  • Involvement of SphK1 in LPS-induced TLR 4-mediated accumulation of HIF-1α protein, activation of ASK1 and production of IL-6.

    S4 

    Pshezhetskiy D1*, Nunes J1, Coughlan K2, Lall H2, Waxman J1, Sumbayev V2* 

    1Dept Oncology, ICL, 2 Medway School of Pharmacy, Univ. Kent 

    Toll-like receptors (TLRs) lie at the core of resistance to infectious diseases detecting various pathogens. While both plasma membrane associated TLR4 (recognises bacterial LPS) and endosomal TLR7/8 (recognise viral single-stranded RNA) induce expression of proinflammatory cytokines through redox-dependent upregulation of HIF-1α, the intracellular mechanisms mediating this expression vary. Recently sphingosine kinase 1 (SphK1) was reported to act downstream of TLR4. 

    In this present study we have identified the implication of the SphK1 signalling in TLR-mediated inflammatory response. In THP-1 and RAW264.7 macrophages ligand-induced activation of the TLR4 but not TLR7/8 induced activation and transcriptional upregulation of SphK1. Activation of SphK1 was dependent on both ERK and PLC-1γ/PI3 kinase pathways and in turn mediated production of ROS and an increase in HIF-1α expression and ASK1 activity. Importantly, TLR4-mediated SphK1 activation was critical for the prevention of LPS-induced activation of caspase 3 and the expression of pro-inflammatory cytokine IL-6. 

    In conclusion, our findings suggest a novel SphK1-mediated mechanism of TLR4-induced activation of ASK1/HIF-1α axis and cytokine production. Our data strongly suggest that SphK1 inhibition may prove effective to fight bacterial sepsis. 

  • Involvement of the S1P3 receptor in neuroinflammation.

    S5 

    I Fischer1, C Alliod1, N Martinier1, M Frossard1, C Brana1, S Pouly1

    1TA Neurodegenerative Diseases, Merckserono International S.A., Switzerland 

    Reactive astrocytes are implicated in the development and maintenance of neuro-inflammation in multiple sclerosis (MS) lesions. The sphingosine signalling pathway, including the S1P3 receptor was shown to be involved in the mediation of the inflammatory response in different cell types. So far, the potential role of S1P3 receptor signalling in reactive astrocytes has, however not been defined. 

    Our data show for the first time the specific expression of the receptor S1P3 on astrocytes in human MS-brain tissue. In cultures of primary rat astrocytes, the treatments with the pro-inflammatory stimuli LPS or with a combination of TNFα and IFNγ increased the mRNA expression of SphK1 and S1P3 receptor. In addition, the protein expression of the SphK1 and S1P3 were increased on the plasma membrane, suggesting increased signalling. Furthermore, LPS treatment increased specific ERK1/2-phosphorylation, which could be mediated via S1P3 as demonstrated by using a S1P3 specific agonist. Moreover LPS, cytokines or sphingosine-1-phosphate (S1P) induced the migration of astrocytes and the release of CXCL1, a known inducer of oligodendrocyte proliferation and chemo-attractant for immune cells. 

    Together these findings provide new evidence for the implication of SphK1 and S1P3 signalling in reactive astrocytes under inflammatory conditions. 

  • Sphingosine 1-phosphate induces differentiation of mesoangioblasts towards smooth muscle cells.

    S6 

    Donati C1,2., Marseglia G.3, Magi A.3, Cencetti F.1,2, Bernacchioni C.1,2, Benelli M.3, Torricelli F.3, Bruni P.1,2 

    1Dip. Scienze Biochimiche, 2Istituto Interuniversitario Miologia (IIM), Università di Firenze and 3UO Citogenetica e Genetica AOUC Firenze, Italy 

    Smooth muscle cells (SMCs) control fundamental functions such as arterial tone and airway resistance. Recent studies proved that circulating, SMC progenitor cells can contribute to tissue repair following vascular injury. Mesoangioblasts (Mbs) are a new type of stem cells, capable of differentiating into mesoderm cell types, such as muscle and bone. Sphingosine 1-phosphate (S1P) is a lipid mediator that regulates many biological processes as vascular development and SMC growth and migration. We previously demonstrated that S1P acts as potent mitogen and antiapoptotic agent in Mbs. We also showed that TGFβ exerts a marked antiapoptotic action in Mbs, involving the regulation of SK1. In order to exploit the therapeutic potential of these cells, we performed a microarray study to establish transcriptional profiles of human Mbs treated with S1P for 6 h and 24 h. Obtained result, validated by Real Time PCR, Western blotting and immunofluorescence analysis, demonstrate that S1P promotes differentiation of human Mbs towards SMC. Moreover, we provide here evidence that TGFβ-induced differentiation of Mbs into SMC relies on SK regulation. This study highlights a new role for S1P in Mbs which can be used to favour vascular regeneration. 

  • Pharmacological regulation of sphingolipid metabolites in blood.

    S7 

    YM Lee1,T Hla2, K Venkataramann2, S Oh3, SM Kim1, KO Shin 1, HS Yoo1.

    1College of Pharmacy and CBITRC, Chungbuk National Univ., 2Center for Vascular Biology, Cornell Univ., 3Dept. Neurosci. and Medical Research Institute, Ewha Womans Univ., South Korea 

    Intracellular and extracellular S1P is tightly regulated by the relative enzyme activities of Sphk1 and Sphk2, S1P lyase and lipid phosphate phosphohydrolases. Extracellular S1P exported by the aid of ABC transporters binds to S1P receptors to transfer its signaling. Recent studies have concluded that S1P gradient and S1P receptor signaling enable immune cell egress from lymphoid tissues. However, the maintenance and regulating factors on S1P level in blood is still under investigation. We found that anti-Fas antibody (Jo2) or combination of LPS and D-GalN treatment into C57/BL6 mice significantly induced liver damage and showed reduced plasma S1P level, but not plasma ceramides. The forced exercise also reduced sphingolipid metabolites including S1P in plasma. The combined treatment of LPS and D-GalN greatly reduced Sphk1 activity in whole blood, while no changes were observed in plasma Sphk1 activity. Notably, the half-life of injected C17-S1P in this condition was not changed. The Sphk1 activities in RBCs and cultured HUVEC cells were not significantly changed by Jo2 or LPS+D-GalN treatment. The Sphk1 activity in liver tissues was slightly increased after liver injury. However, partial hepatectomy did not reduce plasma S1P concentration. Thus, Sphk1 activity in whole blood mainly participated to maintain plasma S1P level. In future the pharmacological effects of ABC transporters inhibitors and Sphk2 activity will be investigated. 

  • Sphingosine 1-phosphate inhibits angiogenesis via interaction with vascular smooth muscle cells.

    S8 

    KS Mascall, GR Small, GF Nixon 

    Institute of Medical Sciences, University of Aberdeen, Aberdeen, UK 

    Following myocardial infarction, new blood vessels sprout from coronary arteries. Sphingolipids released from the blood clot could have an effect on this response. Evidence shows that sphingosine 1-phosphate (S1P) may be an important pro-angiogenic factor. This is predominantly through the actions of S1P1 receptors although the S1P2 receptor subtype may inhibit angiogenesis. The aim of this study was to examine the effects of S1P on angiogenesis using ex vivo human arteries and a co-culture in vitro model. 

    In human mammary artery placed in Matrigel, S1P inhibited endothelial tubule formation. Co-culture of primary cultured human fibroblasts, human coronary artery smooth muscle (HCASM) cells and human coronary artery endothelial cells also revealed a significant inhibition of endothelial tubule formation following treatment with S1P. When HCASM cells were omitted from the co-culture, tubule formation was not affected by S1P incubation. Incubation with specific antagonists determined that this inhibition occurred through activation of S1P2 receptors linked to the rho-kinase pathway. Conditioned medium from S1P-treated HCASM cells added to the co-culture also inhibited endothelial tubule growth suggesting the inhibitory factor(s) are released from smooth muscle cells. In conclusion, S1P inhibits angiogenesis through activation of S1P2 receptor and this involves interaction with smooth muscle cells. Such sphingolipid-mediated inhibition of angiogenesis may occur following myocardial infarction. 

  • Sphingosylphosphorylcholine, but not sphingosine 1-phosphate, is a pro- inflammatory mediator in rat cerebral artery vascular smooth muscle.

    S9 

    C Wirrig, FA Mathieson, I Hunter, GF Nixon 

    Institute of Medical Sciences, University of Aberdeen, Aberdeen, UK. 

    Following subarachnoid haemorrhage (SAH) of the cerebral arteries a marked inflammation occurs. This is caused by an adventitial blood clot and contributes to the subsequent cerebral artery vasospasm and ischaemia. Pro-inflammatory mediators in SAH have not yet been identified. As sphingosylphosphorylcholine (SPC) and sphingosine 1-phosphate (S1P) are elevated in serum, we investigated the pro-inflammatory potential of these sphingolipids in rat cerebral arteries using an ex vivo model. 

    SPC, but not S1P, induced activation of p38 mitogen-activated protein kinase (MAPK), a kinase involved in inflammatory signalling. A transcription factor array identified SPC-induced DNA binding for the transcription factors nuclear factor-κB and CCAAT-enhancer-binding proteins in cerebral arteries, confirmed by electromobility shift assays. S1P did not increase the activity of these transcription factors. In the rat vascular smooth muscle cell line, A7r5, an inflammatory protein array revealed that SPC elicited release of only one pro-inflammatory chemokine, monocyte chemotactic protein (MCP)-1. Further investigation also showed SPC-induced MCP-1 production in rat cerebral arteries. 

    In summary we demonstrate that SPC can act as a pro-inflammatory mediator in cerebral arteries via increased activity of inflammatory transcription factors and subsequent release of MCP-1. This may contribute to vasospasm following SAH. 

  • C1P and its synthetic analog, PCERA-1, have distinct receptors in macrophages.

    S10 

    T Zor1, A Gómez-Muñoz2, M. Meijler3, H. Rosen4, D Avni1, A Philosoph1, M Levi1, L Arana2, A Ouro2 

    1Dept. Biochemistry, Tel-Aviv University, Tel-Aviv, Israel, 2Dept. Biochemistry & molecular biology, University of the Basque country, Bilbao, Spain, 3Dept. Chemistry, Ben-Gurion University, Be'er-Sheva, Israel, and 4 Dept. of Immunology, The Scripps Research Institute, La Jolla, CA, USA 

    Tight regulation of the production of pro- and anti-inflammatory cytokines is essential for the prevention of chronic inflammatory diseases. A synthetic C1P analog, named PCERA-1 (Phospho-CERamide Analog-1), suppressed production of the pro-inflammatory cytokine TNFα, and elevated production of the anti-inflammatory cytokine IL-10, in LPS-stimulated macrophages. PCERA-1 and bovine brain-derived C1P activated distinct signaling pathways in RAW264.7 macrophages. PCERA-1 modulated cytokine expression via the Gs protein and the cAMP pathway, whereas C1P stimulated macrophages migration via the Gi protein and the NFκB pathway. Neither PCERA-1 nor C1P mimicked or antagonized the activities of each other, and PCERA-1 failed to interfere with a C1P binding assay. These results thus indicate that PCERA-1 and C1P bind and activate distinct GPCRs expressed in RAW264.7 macrophages. The orphan receptor GPR3 is predicted to be a phospholipid-binding receptor, due to sequence homology with the S1P and LPA receptor families. Our preliminary results indicate that GPR3 is a receptor for PCERA-1, and thus suggest that GPR3 regulates inflammation. 

  • Ceramide 1-phosphate stimulates reactive oxygen species (ROS) formation. implication in macrophage growth.

    S11 

    L Arana, A Ouro, P Gangoiti , A Gomez-Muñoz 

    1Dept. Biochemistry and Molecular Biology, Faculty of science and technology, University of the Basque Country, 48080 Bilbao, Spain 

    The pro-mitogenic and anti-apoptotic actions of Ceramide 1-phosphate (C1P) are now well established. Major pathways involved in the mitogenic effect of C1P include mitogen-activated protein kinase kinase (MEK)/extracellularly regulated kinases (ERK1-2), phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB, also known as Akt), c-Jun N terminal kinase (JNK), and protein kinase C-alpha. We report here that C1P induces ROS formation through activation of NADPH oxidase. C1P-stimulated ROS production was inhibited by the NADPH oxidase inhibitor apocynin, the cell-permeable ROS scavenger N-acetyl cystein (NAC), the protein kinase C (PKC) inhibitor Go6976, the PKC-delta inhibitor rottlerin, and by long-term treatment with the phorbol ester PMA, a condition known to downregulate PKC activity. Moreover, a specific cytosolic phospholipase A2-alpha inhibitor potently blocked C1P-stimulated ROS production, and all of the ROS inhibitors blocked C1P-stimulated macrophage proliferation. These findings suggest that ROS are implicated in the mitogenic effect of C1P in macrophages. 

    This work was supported by grants BFU2009-13314 from MCINN and S-PE09UN42 from the Basque Government (Spain) 

  • Role of the sphingolipid biostat in apoptosis of pancreatic β cells induced by palmitate.

    S12 

    Hervé Le Stunff1, Julien Veret1, Evgeny V. Berdyshev2, Anastasia Skobeleva2, Viswanathan Natarajan2 and Bernard Portha1 

    1Laboratoire de Biologie et Pathologie du Pancréas Endocrine, Unité BFA, CNRS EAC 4413, Université Paris Diderot-7, Paris, France. 2Department of Medicine, Section of Pulmonary and Critical Care Medicine, University of Chicago, Chicago, IL, USA. 

    Prolonged exposure to fatty acids with high glucose induced pancreatic β cell apoptosis. This study aimed to ascertain the role of sphingolipid in β cell apoptosis induced by palmitate + high glucose. Lipidomic analyses revealed that palmitate but also 30 mM glucose increased de novo ceramide synthesis. Importantly, thirty mM glucose potentiated palmitate-induced accumulation of dihydro-sphingosine and ceramide levels, especially C18:0, C22:0 and C24:1. Apoptosis induced by palmitate with high glucose was partially blocked by fumonisin-B1, an inhibitor of ceramide synthase. Interestingly, palmitate + high glucose stimulated accumulation of dihydrosphingosine-1-phosphate (DHS1P). N,N-dimethyl-sphingosine, a potent inhibitor of sphingosine kinase, increased by 2-fold palmitate + high glucose-induced caspase activity. Furthermore, SphK1 over-expression in β cells drastically increased DHS1P levels and partially inhibited palmitate-induced caspase activation. Together, these results indicate that regulation of the dynamic balance between ceramide and DHS1P levels, the sphingolipid biostat, by palmitate with high glucose concentrations will play a critical role in the survival of pancreatic β cells during the development of type 2 diabetes. 

  • Sphingosine kinase-1 contributes to the anti-fibrotic effect of PPARγ agonists.

    S13  

    A Völzke1, A Koch1, A Huwiler2, J Pfeilschifter1 

    1pharmazentrum frankfurt/ZAFES, Goethe University Frankfurt, Frankfurt am Main, Germany

    2Institute of Pharmacology, University of Bern, Bern, Switzerland 

    Peroxisome proliferator-activated receptor (PPAR)γ agonists (thiazolidinediones; TZDs) are known to act in an anti-fibrotic manner. Previously, it was shown that sphingosine kinase-1 (SK-1) depletion or its pharmacological inhibition led to accelerated expression of the profibrogenic molecule connective tissue growth factor (CTGF) suggesting a protective role of SK-1 activity in the fibrotic process. 

    Here, we investigated the effect of TZDs on the transcriptional regulation of SK-1 in vitro (rat mesangial cells) and in vivo (mice glomerula) and evaluated the putative role of SK-1 in the anti-fibrotic effect of TZDs. Stimulation with troglitazone and rosiglitazone led to increased SK-1 activity which was preceded by elevated mRNA and protein expression. This effect is due to enhanced SK-1-promotor activity which contains seven putative PPAR response elements. Furthermore, pre-incubation with the PPARγ antagonist GW-9662 inhibited the stimulating effect of TZDs on SK-1 mRNA expression and activity. Finally, the up-regulation of SK-1 by TZDs was paralleled by decreased CTGF expression, an effect which was abolished using the SK-1-inhibitor SKI II. In summary, TZDs dependent SK-1 activation results in lower CTGF demonstrating an essential role of SK-1 in the anti-fibrotic effect of PPARγ agonists. 

  • Synthesis and biological activity of novel inhibitors of acid sphingomyelinase.

    S14 

    Christoph Arenz

    1Institut für Chemie, Humboldt Universität zu Berlin, Germany 

    The acid sphingomyelinase (aSMase) is a key enzyme involved in lipid signalling and an emerging drug target. 

    Many inhibitors of this enzyme are either unspecific, not active in cell culture or have an indirect way of action. We used the potent inhibitor of aSMase, phosphatidyl-3,5-bisphosphate (PtdIns3,5P2) as a lead for the development of novel aSMase inhibitors with improved features. Our inositol- or carbohydrate-based inhibitors are at least of the same potency as PtdIns3,5P2 and inhibit dexamethasone (Dex)-induced apoptosis in HEK-293 cells. In addition, we have synthesized a class of novel bisphosphonate inhibitors that are the most potent inhibitors of aSMase so far. The IC50 for aSMase is 20 nM and >2000fold lower than for nSMAse. The selectivity versus the remote homologue phsophatase PP1 is at least 200fold. Moreover the most potent bisphosphonate completely suppresses Dex-induced apoptosis in HEK-293 cells and inhibits the formation of PAF-induced pulmonary edema in an ex-vivo model. However, many aspects of the biological activity of these substances need to be clarified. So far, the usability of theses inhibitors for medical and in vivo applications is unclear.

  • Role of sphingolipid trafficking in cannabinoid-induced autophagy.

    S15 

    S Hernández-Tiedra1 , IJ Salanueva1, M Salazar1, G Fabriàs2, J Casas2, K Hanada3, M Guzmán1, G Velasco1 

    1Dept of Biochem and Mol Biol I, Complutense University, Madrid, Spain 2Research Unit on BioActive Molecules, Dept de Química Biomèdica, (IQAC), Barcelona, Spain 3Dept of Biochemistry and Cell Biology, National Institute of Infectious Diseases, 1-23-1, Toyama, Shinjuku-ku, 

    Macroautophagy is a conserved degradative process implicated in the turnover of damage organelles and long-live macromolecules. Under starvation conditions, autophagy promotes cell survival providing cells with nutrients derived from the lysosomal degradation of the autophagosome content. However, under specific situations autophagy can lead to cell death. 

    Work by our laboratory has unraveled that the mechanisms of cannabinoid antitumoral action relies on a ceramide-dependent stimulation of the stress regulated protein p8 and its downstream target TRB3 which leads to stimulation of autophagy and apoptosis in cancer cells. In this work, we investigated whether modulation of sphingolipid trafficking could play a role in the stimulation of autophagy by cannabinoids. 

    Our results show that THC but not other autophagy inducers alters the sub-cellular distribution of fluorescent analogous of ceramide and promotes the accumulation of long chain sphingolipids species in the microsomal fraction of U87MG glioma cells. Moreover, we also found that THC administration promotes the translocation to the autophagosomes of the ceramide transporter protein (CERT). Our data suggest that changes in sphingolipids distribution play a pivotal role in the stimulation of autophagy-mediated cell death by cannabinoids. 

  • Nuclear import of sphingomyelinase in space environment.

    S16  

    E. Albi1, M. Peverini1, E. Damaskopoulou1, F. Cingolani1, R. Lazzarini1, F. Curcio2, F.S. Ambesi-Impiombato2, G. Perrella

    1Dept. Clinical and Experimental Medicine, Faculty of Medicine and Surgery, Perugia University, Italy and 2Department of Experimental and Clinical Pathology and Medicine, University of Udine, p.le S.M. della Misericordia, 33100 Udine, Italy 

    It has been reported that during space missions the thyroid cells cultured in the presence of thyrotropin (TSH) decreased their proliferation rate and both their cell growth and lipid metabolism were similar to those of quiescent cells. A rearrangement of cell membrane lipid rafts with an alteration of TSH-TSH receptor interaction has been suggested (1). To study whether the sphingomyelinase (SMase) could be involved in the modification of the cell function in space environment, the SMase localization was analysed either in vitro or in vivo experiments during space TEXUS-44 and STS-129 mission respectively. FRTL5 thyroid cells in culture and thyroid of mice were analysed after the return to earth. The SMase was localized with indirect immunofluorescence by using anti-SMase antibodies alone or in combination with propidium iodine. The results showed that in the ground SMase is localized either in cell membrane or in the nucleus. After space mission the nuclear concentration of this enzyme increases thus indicating a cytoplasmic-nucleus translocation by influencing signal transduction. 

    1) E. Albi, F.S. Ambesi-Impiombato, E. Damaskopuolou, M. Peverini, A. Lazzarini, R. Lazzarini and G. Perrella. ESA Publications, 2009 

  • Metabolic reconstruction of in silico yeast sphingolipid metabolism incorporating hydroxylation levels of ceramide.

    S17 

    F. Betul Kavun Ozbayraktar1, Kutlu O. Ulgen1 

    1Dept. of Chem. Eng., Bogazici University, 34342 Bebek, Istanbul, Turkey 

    The hydroxylation level diversifies the identity, structure and location of complex sphingolipids in higher eukaryotes. In order to clarify the significance of hydroxylation on sphingolipids, the first metabolic model of Sacchoramyces cerevisiae sphingolipid metabolism was reconstructed in silico, taking into consideration five levels of hydroxylation derived from dihydroceramide, alpha-dihydroceramide, phytoceramide, alpha-phytoceramide and alpha-alpha-phytoceramide. The reconstructed model, which is validated with experimental findings from literature resources, equipped us with the analysis of complex sphingolipid content of the plasma membrane coupled with DAG and phosphatidic acid biosynthesis and ATP consumption rates of in silico cell. These critical metabolites with utmost importance to cell’s viability are utilized as criteria in studying all single deletion mutants of in silico model to propose novel potential drug targets for inducing apoptosis to be further used in cancer therapy. The computational systems biology tools used for interpreting simulation results in terms of their metabolic consequences, flexibility and robustness are flux balance and variability analysis, minimal cut set calculations and principal component analysis. First and second order hydroxylated phytoceramide derived mature sphingolipid synthesis happen to be the most robust part among the others.

  • Sphingolipids in retinal degenerations.

    S18 

    I. Piano1, E. Novelli2, G. Sala3, P. Gasco4, C. Gargini1, E. Strettoi5, R. Ghidoni3 

    Dept. Psychiatry & Neurobiology, Univ. Pisa1; G.B. Bietti F’dn for Ophthalmology, Rome2; DMCO, San Paolo Med. School, Univ. Milan3; Nanovector srl, Turin4; Neuroscience Inst. CNR, Pisa5, Italy 

    Ceramide is a known pro-apoptotic messenger whose de-novo biosynthesis is associated with cell death initiation. In Retinitis Pigmentosa (RP) photoreceptor death occurs by apoptosis. We investigated the role of ceramide in RP using the rd10 mutant mouse, a model of this disease. Myriocin, an inhibitor of serine palmitoyltransferase (SPT, the rate-limiting enzyme of ceramide biosynthesis) was either injected intraocular or administered daily to rd10 mice as eye drops preparations of Solid Lipid Nanoparticles (SLNs). Control mice were administered with vehicle alone or unloaded lipid particles, respectively. We found that retinal ceramide levels double during the time interval of maximum photoreceptor death in rd10 mice (from P14 to P30). Intraocular Myriocin decreases the number of rd10 pycnotic photoreceptors by approximately 50%. Electroretinogram (ERG) recordings were obtained from animals chronically treated with Myriocin-SLNs. ERG a-waves persist after P30 in treated mice while these responses are virtually extinct in control littermates. Confocal microscopy of retinal sections from ERG recorded animals aged P24 (peak of rod apoptosis) up to P30 showed prolonged survival of photoreceptors in treated animals. 

    This study is the first in vivo demonstration of the possibility to decrease photoreceptor apoptosis by lowering retinal ceramide levels through non-invasive administrations of SPT inhibitors.

  • Acid ceramidase expression can modulate the sensitivity of A375 melanoma cells to dacarbazine.

    S19 

    C Bedia1, G Fabriàs2, J Casas2, T Levade.

    1Institut de Médecine Moleculaire de Rangueil, INSERM U858, CHU Rangueil, Toulouse, France, 2Department of Biomedicinal Chemistry, IQAC-CSIC, Barcelona, Spain. 

    Melanoma is still a dreadful cancer with high mortality and morbidity. In most cases (around 90%), melanoma appears as a skin tumor, but this cancer exhibits a strong metastatic potential. Its incidence has steadily risen during the last decades. Dacarbazine is the most commonly used therapy for metastatic melanoma. Response to dacarbazine remains very low. Its mechanism of action is not yet completely known but it has been shown to induce autophagy-associated cell death. 

    Here we show that dacarbazine causes a post-transcriptional degradation of acid ceramidase (aCDase) in A375 human melanoma cells in a dose and time-dependent manner. As a consequence, intracellular levels of ceramide increase considerably while those of sphingosine decrease. Overexpression of aCDase in A375 cells protects them to death induced by low concentrations of dacarbazine. Also, this overexpression prevents the increase of ceramide levels and the autophagic features. Reciprocally, down-regulation of aCDase by siRNA sensitizes A375 cells to dacarbazine-induced cell death in a synergistic manner. The melanoma cell line UACC 257, more resistant to dacarbazine, also shows an increased sensitivity through aCDase silencing. This suggests that combination of dacarbazine and inhibition of aCDase activity may improve current therapies for melanoma

  • Measles virus (MV) induced smase activation in T and dendritic cells: implications for viral uptake and immunosuppression.

    S20 

    E Avota1, E Gassert1, E Gulbins2, S Schneider-Schaulies

    1Dept.for Virology and Immunobiology, University of Wuerzburg, and 2Dept. Molecular Medicine, University of Essen, Germany 

    Smase activation in response to receptor ligation has been described to affect T cell signaling, yet the role of Smases in regulating immune cell function and APC-dependent T cell activation is poorly understood. 

    We have shown the measles virus (MV), which causes profound T cell paralysis in vivo, efficiently induces SMAse activation and membrane ceramide accumulation in both T cells and dendritic cells (DCs) in vitro. For the latter, this occurs DC-SIGN dependently and essentially mediates enhancement of viral uptake into these cells. In T cells, MV interaction also activates NSM and ASM and thereby membrane ceramides within minutes. This accounts for the breakdown of actin based protrusions on these cells and their inability to organize actin cytoskeletal dependent processes such as morphological polarization, receptor redistribution, adherence and chemotactic responses. These findings indicate that activation of membrane ceramides may be central to the understanding of viral interference with T cell activation and thereby, immunosuppression.

  • Lysosomal sphingosine storage induced by mycolic acid mediated inhibition of the npc1 protein is central to Mycobacterium tuberculosis induced pathogenesis.

    S21 

    Emyr Lloyd-Evans, Paul D. Fineran and Frances M. Platt 

    Dept. of Pharmacology, University of Oxford, Oxford, OX1 3QT 

    Niemann-Pick type C1 (NPC) disease is a lysosomal storage disorder that causes defective endocytosis, defective phago-lysosome fusion and intralysosomal multi-lipid storage. We recently reported that, in NPC cells, primary lysosomal accumulation of sphingosine induces a lysosomal calcium defect resulting in the secondary phenotypes listed above. 

    In this study we demonstrate that mycobacterial infection induces a phenotype very similar to NPC. We discovered striking similarities between these two disorders, including intralysosomal (not phagosomal) accumulation of cholesterol, sphingomyelin and gangliosides, defective late endosome-lysosome lipid efflux, and defective lysosomal calcium signalling which inhibits phago-lysosome fusion. The lysosomal calcium defect was caused by primary accumulation of sphingosine. 

    Secretion of lipids (mycolic acids) from the coat of M. tuberculosis or BCG but not M. smegmatis is essential for inducing these phenotypes. As mycolic acids mimic sterols we tested their effect on cells expressing differential levels of the sterol regulated lysosomal NPC1 protein. We discovered that NPC1 heterozygous cells are more sensitive to mycolic acids whilst NPC1 overexpressing cells are less sensitive, indicating that the NPC1 protein is a target for successful mycobacterial infection and that sphingosine storage underlies the pathology of tuberculosis. 

  • Sphingosine 1-phosphate protects primary human keratinocytes from apoptosis via nitric oxide formation through the receptor subtype S1P3.

    S22 

    M Schüppel1, EI Schmitz1, H Potteck2, B Kleuser2 

    1Institute of Pharmacy, Dept. Pharmacology, Free University of Berlin, and 2Institute of Nutritional Science, Dept. Nutritional Toxicology, University of Potsdam, Germany 

    Although sphingosine 1-phosphate (S1P) has been identified to induce cell growth arrest of human keratinocytes, the sphingolipid effectively protects epidermal cells from apoptosis. The molecular mechanism of the antiapoptotic action induced by S1P is less characterized. Apart from S1P, endogenously produced nitric oxide (NO) has been recognized as a potent modulator of apoptosis in keratinocytes. Therefore, it was of great interest to elucidate whether S1P protects human keratinocytes via an NO-dependent signaling pathway. 

    In this study we proofed the ability of S1P to modulate NO-synthases and subsequent NO-formation in primary human keratinocytes. Moreover, the involvement of G-protein coupled S1P receptor subtypes was examined concerning the antiapoptotic action as well as NO-generation. 

    The constitutive NO-synthases, eNOS and neuronal NO-synthase (nNOS) are both present in primary human keratinocytes. Our data suggests, that S1P induces activation of eNOS leading to the formation of NO and an NO-dependent protection against apoptosis. Moreover, this study clearly shows that S1P3 is the exclusive receptor subtype mediating eNOS activation, NO-formation and the resulting cytoprotection by S1P.

  • Specific activation of sphingomyelin synthase by 2-hydroxyoleic acid (Minerval), a potent antitumor drug.

    S23 

    M. Laura Martin1, Gwendolyn Barceló-Coblijn1, Rodrigo de Almeida2 and Pablo V Escribá1 

    1Department of Biology, University of the Balearic Islands, Spain and 2Centro de Química e Bioquímica, Universidade de Lisboa, Portugal 

    The mechanism of action of 2-hydroxyoleic acid (2-OHOA, Minerval), a potent antitumor drug, involves membrane lipid structure modifications and changes in the MAPK signaling pathway. In U118 human glioma cells, 2-OHOA dramatically alters cell morphology and lipid metabolism. Thus, cell lipid analysis showed that while SM mass was significantly increased, PC and PE mass was decreased after treatment with 2-OHOA (200 μM, 24-72h). Taking into account these changes, we investigated whether 2-OHOA affected sphingomyelin synthase activity (SMS). So, incubation of cells with NBD-C6-ceramide demonstrated that SMS activity was already increased after 5 min. of treatment. Interestingly, treating cells with different fatty acids differing in the saturation degree and chain length, we could establish a clear relationship between the fatty acid structure and the SM mass increase. In addition, we studied biophysical properties of liposomes mimicking the lipid composition of control and treated cells. Results showed that 2-OHOA induces an increased packing of the ordered domains with a consequent enhancement of global membrane order, due to the substantial increase in SM content. All together, our research indicates that the rapid activation of SMS by 2-OHOA may play a key role in the induction of the signaling pathways responsible for the antitumor effects of 2-OHOA. 

  • Syntaxin 4 is required for acid sphingomyelinase activity and apoptotic function in a pathway regulated by NO.

    S24  

    C Perrotta1, L Bizzozero2, D Cazzato2, S Morlacchi2, P Rosa3, E Gulbins4, E Clementi

    1Unit of Clinical Pharmacology, Dept Preclinical Science, L.Sacco University Hosp., 2Dept. Preclinical Science and 3Inst. of Neuroscience, University of Milan, Italy, 4Inst. für Molekularbiologie Universitätsklinikum Essen 

    Acid sphingomyelinase (A-SMase) plays key roles in apoptosis, immunity, development and cancer and mediates cytotoxicity of chemotherapeutic drugs. 

    We now demonstrate that A-SMase is activated throught translocation in an exocytic pathway requiring the t-SNARE protein syntaxin 4 (synt 4) and regulated by NO. 

    NO induces proteasome-dependent degradation of synt 4, leading to inhibition of A-SMase translocation, activation and its biological effects. Indeed, synt 4 down-regulation induces inhibition of caspases activity, activation of the survival pathway involving Akt and modification of the cell cycle profile, inducing cell proliferation even in the presence of death stimulus. 

    The molecular interaction among A-SMase, synt 4 and NO were not known and clarify the mechanism of A-SMase activation. In addition the novel actions of syntaxin 4 and NO in sphingolipid metabolism and exocytosis we describe here define signaling mechanisms of broad relevance in cell phatophysiology.

  • Sphingoid base phosphates, a novel link in the process of neurodegeneration?

    S25 

    N Hagen1, M Hans2, D Swandulla2, G van Echten-Deckert

    1LIMES Institute Membrane Biology and Lipid Biochemistry, Rhein. Friedr.-Wilh. Universität, 53121 Bonn, Germany, and 2Institute of Physiology, Rhein. Friedr.-Wilh. Universität, 53111 Bonn, Germany 

    The bioactive lipid sphingosine-1-phosphate (S1P) usually signaling proliferation and anti-apoptosis, was recently reported to induce apoptosis in S1P-lyase deficient terminally-differentiated postmitotic neurons, when generated by sphingosine-kinase2 (Hagen et al., 2009). This is similar to what we observed in wildtype neurons with the metabolically stable S1P analog cimes-P (cis-4-methylsphingosine phosphate) (Naetzker et al., 2006). The aim of the present study was to identify the signaling cascade involved in the neurotoxic effect of sphingoid base phosphates using cimes-P in neurons derived from wildtype and sphingosine kinase deficient mice. We demonstrate that the calcium-dependent cysteine protease calpain mediates neurotoxicity by induction of the ER-stress specific caspase cascade and in addition activation of cyclin dependent kinase5 (CDK5). The latter is not only involved in an abortive reactivation of the cell cycle but also enhances Tau phosphorylation. Since deregulation of calpain activity has been proposed as a key cytotoxic event in neurodegenerative disorders, our results suggest that calpain-mediated S1P-induced apoptosis may constitute a key mechanism in the process of neurodegeneration 

    - Hagen N, Van Veldhoven PP, Proia RL, Park H, Merrill AH, Jr., van Echten-Deckert G (2009). J Biol Chem 284:11346-11353. 

    - Naetzker S, Hagen N, Echten-Deckert G (2006). Genes Cells 11:269-279. 

  • The neutral-sphingomyelinase leaves the nucleus during apoptosis of hepatoma cells induced by daunorubicin.

    P1 

    M. Peverini, F. Cingolani, E. Damaskopoulou, R. Lazzarini, E. Albi 

    Dept. Clinical and Experimental Medicine, Faculty of Medicine and Surgery, Perugia University, Italy. 

    It is known that neutral-sphingomyelinase (NSMase) plays a role in daunorubicin (DA)-induced cell death. DA remarkably increased the NSMase2 message and protein, whereas little change in NSMase1 and NSMase3 mRNAs and only a mild increase in acid SMase mRNA were observed(1). A large part of nuclear NSMase was NSMase1 but so far there is no indication about his behaviour after DA treatment. In the present study, the optimal dose of drug to induce hepatoma cell death was analysed with morphological and immunoblotting analysis. This dose was used to localize nuclear NSMase with indirect immunofluorescence by using anti SMase antibodies combined with propidium iodine nuclei count-staining technology 

    The results showed that after 5μM DA treatment, the H35 cells presented morphological signs of apoptoic death with cytoplasmic vesciculation and apoptotic bodies, increase of Bax and PKCzeta level and reduction of RNApolimerase II. The nucleus changed its morphology with chromatin condensation and release of NSMase which moved away from the nucleus and probably was relocated to apoptotic bodies on the cell surface of early apoptotic cells. 

    1. Ito et al., Biochim Biophys Acta. 2009 Nov-Dec;1789(11-12):681-90 

  • A simple, novel fluorogenic method for determination of acid ceramidase activity and diagnosis of Farber disease.

    P2 

    C. Bedia1, L. Camacho2, JL Abad2, G. Fabriàs2, T. Levade1 

    1Institut de Médecine Moleculaire de Rangueil, INSERM U858, CHU Rangueil, Toulouse, 2Department of Biomedicinal Chemistry, IQAC-CSIC, Barcelona, Spain

    Farber disease (FD) is a rare lipid storage disorder characterized by accumulation of ceramide in different tissues due to deficient activity of acid ceramidase. Up to now, methods for diagnosis of FD include an extremely complex in vitro assay of acid ceramidase, and the use of radiolabeled sphingolipids like sphingomyelin in loading tests on intact cultured cells. Another alternative approach is the quantification of intracellular stored ceramide by the diacylglycerol kinase assay. All of these methods use radiolabeled compounds, most of them no longer commercially available. Therefore, the specific determination of acid ceramidase activity and diagnosis of FD are restricted to a very limited number of specialized laboratories. 

    We present a new and easy method for Farber disease diagnosis by measuring acid ceramidase activity. The high specificity of the substrate for acid ceramidase and its fluorogenic properties make the assay quicker and simpler. Very little amounts of protein are needed and the entire assay can be performed in a 96-well plate, with no post-incubation separation of products. 

    This assay was validated for routine FD diagnosis, by using different fibroblast and lymphoid cell lines derived from FD and control patients. 

  • Differential effects of SKi-II on SK1 isoforms SK1a and SK1b in androgen-dependent and –independent prostate cancer cells.

    P3 

    F. Tonelli, C. Loveridge, K.G. Lim, NJ Pyne, S Pyne 

    Cell Biology Group, SIPBS, University of Strathclyde, Glasgow, UK 

    Evidence supports a key role for sphingosine kinase 1 (SK1), which synthesises sphingosine 1-phosphate, in prostate cancer biology. SK1 promotes cell survival, chemotherapeutic resistance and is increased upon androgen depletion. Here we investigated the effects of a SK inhibitor on SK1 expression in androgen-dependent (LNCaP) and androgen-independent (LNCaP-AI) prostate cancer cells. 

    SK1 was expressed as two isoforms, SK1a and SK1b, in both cell types and to a greater extent in LNCaP-AI cells versus LNCaP cells. SKi-II (2-(p-hydroxyanilino)-4-(p-chlorophenyl) thiazole) induced apoptosis of LNCaP but not LNCaP-AI cells. This was accompanied by the SKi induced the proteasomal degradation of both isoforms of SK1 in LNCaP cells but of only SK1a in androgen-independent LNCaP-AI cells. This suggests that SK1b is modified in some way in LNCaP-AI cells and is ‘protected’. This may account for the increased resistance of androgen-independent prostate cancer cells to apoptotic stimuli that is associated with the transition of prostate cancer to androgen independence 

    This study identifies an entirely new mechanism of action of SKi-II and suggests that targeting the SK1b isoform might be an appropriate strategy for treating androgen-independent prostate cancer, which is currently unable to be successfully managed. 

    [Funded by University of Strathclyde scholarships to FT and KGL] 

  • Dual action of sphingosine 1-phosphate in eliciting proinflammatory responses in primary cultured ratintestinal smooth muscle cells.

    P4 

    M Gurgui, R Broere and G van Echten-Deckert 

    Kekulé-Institute for Organic Chemistry and Biochemistry, University Bonn, Germany 

    Sphingosine 1-phosphate (S1P) is involved in the local inflammatory response within the intestinal muscularis, which has been suggested to play a major role in the pathogenesis of postoperative ileus. The aim of the present study was to elucidate the role of S1P and the molecular mechanisms underlying regulation of inflammatory mediators in primary cultured rat intestinal smooth muscle (RISM) cells. Although our experimental data clearly show the mediatory role of sphingosine kinase (SK)-derived S1P in the TNF-α and the LPS induced activation of NF-kB, exogenously added S1P failed to trigger this transcription factor. Instead, exogenous S1P induced early growth response-1 (Egr-1), which was reported to play a proinflammatory role in postoperative ileus. Using RNA interference we found that Egr-1 is required primarily for S1P induced expression of IL-1 and COX2. Conversely, IL-6 expression following S1P treatment was mediated by STAT3 (signal transducer and activator of transcription-3). In addition our data indicate that the proinflammatory effect of S1P is mediated by its receptors S1P1-3 and requires activation of MAP-kinases. In conclusion, Egr-1 and STAT3 cooperatively mediate S1P-induced inflammatory responses in RISM cells, providing novel targets for attenuation of postoperative ileus. 

  • Sphingosine 1-phosphate receptor 1 (S1P1) and VEGFR-2 form a signalling complex with ERK1/2 and PKC-α regulating ML-1 tyroid carcinoma cell migration .

    P5 

    N Bergelin1,2,3, C Löf1,2, S Balthasar1, V Kalhori1, K Törnquist13,

    1Dept of Biology, Åbo Akademi University, Finland. 2Turku Graduate School of Biomedical Sciences, Finland, 3The Minerva Foundation Institute for Medical Research, Biomedicum Helsinki, Finland. 

    S1P- and VEGFR-2-signalling has been shown to integrate in many biological processes. The follicular thyroid carcinoma cell line ML-1 expresses VEGFR-2, and secretes substantial amounts of both VEGF-A and VEGF-C. ML-1 cells also express S1P-receptors (S1P1-3,5). S1P is able to phosphorylate VEGFR-2, and inhibiting VEGFR-2 attenuates S1P-induced migration and down-regulates S1P1-expression in ML-1 cells. In the present study we focus on the interactions between S1P1 and VEGFR-2. We show that S1P-receptors form complexes with VEGFR-2, and that the S1P1/VEGFR-2 complex associates with PKC-α and ERK1/2. Furthermore, the complex evokes bidirectional signalling, as the S1P-induced ERK1/2 phosphorylation is sensitive to VEGFR-2 kinase inhibition and VEGF-A-induced ERK1/2 phosphorylation is sensitive to Ptx-treatment as well as S1P1 siRNA treatment. Both S1P- and VEGF-A-induced haptotaxis is sensitive to Ptx-treatment and S1P1 siRNA treatment. Phosphorylation of ERK1/2 evoked by both VEGF-A and the S1P1 agonist SEW-2871 is inhibited by PKC-α and PKC-βI siRNA. We hypothesise that VEGFR-2 forms a signalling complex with S1P1, evoking bidirectional signalling regulating ERK1/2 phosphorylation and haptotaxis of ML-1 cells.

  • Tumour biology of melanoma: a novel role of acid sphingomyelinase.

    P6 

    L. Bizzozero1, D.Cazzato1, C. Verdelli1, E. Clementi1,3, C. Perrotta1 

    1Department of Preclinical Sciences, University of Milano, Milano, Italy; 3E. Medea Scientific Institute, Bosisio Parini, Italy 

    Recent studies show that sphingolipids and the enzymes responsable for their intracellular metabolism are important in cellular processes such as proliferation, apoptosis and differentiation. Recent evidence suggested a role for this enzymes in tumorigenesis. In particular, Acid sphingomyelinase (A-SMase) plays pivotal role in tumor biology and tumor response to chemotherapeutic drugs. We investigate the specific role of A-SMase expressed by melanoma B16 cells which show different tumorigenic and metastatic properties dependent on their melanin content. We isolated several B16 clones on the basis of their pigmentation, indicating the pigmented clones as “black”, and the not-pigmented as “white” and we observed a higher expression and activity of A-SMase in the white B16 clones. in vivo and in vitro experiments showed that the white and the black clones differ in terms of growth rate, metastatic capacity and histological characteristics. These data suggest a strong relationship between A-SMase expression and tumour behaviour. These studies suggest A-SMase as possible prognostic factor in tumor and might help refine therapeutic strategies against tumour based on regulation of A-SMase activity/expression.

  • Gαq mediates sphingosine kinase-1 translocation by GPCRs.

    P7 

    RF Claas1, K Ihlefeld1, S Offermanns2, T Wieland3, D Meyer zu Heringdorf1 

    1Institut für Allgemeine Phamakologie und Toxikologie, Klinikum der Goethe-Universität, Frankfurt am Main, 2Max-Planck-Institut für Herz- und Lungenforschung, Bad Nauheim, 3Experimentelle Pharmakologie, Medizinische Fakultät Mannheim der Universität Heidelberg, Germany 

    The activation of Gq-coupled receptors leads to a rapid and long-lasting translocation of sphingosine kinase-1 (SK1) to the plasma membrane. We have shown previously that overexpression of constitutively active Gαq or Gα11, but not Gαi, Gα12 or Gα13, was accompanied by a permanent localisation of SK1 at the plasma membrane. Here, the role of Gq in SK1 translocation by G-protein-coupled receptors (GPCRs) was further characterised. First, SK1 translocation induced by the M3 receptor in HEK-293 cells was inhibited by co-expression of a catalytically inactive mutant of GRK2, which is able to sequester activated Gαq/11 subunits. Second, bradykinin failed to induce SK1 translocation in Gαq/11-double deficient mouse embryonic fibroblasts over-expressing the B2 receptor. Expression of wild type Gαq restored B2-mediated SK1 translocation in these cells. Expression of Gαq-Y261N, Gαq-D321A and Gαq-Y356K likewise restored SK1 translocation without an influence on the translocation half-time, whereas Gαq-W263D mediated a significantly retarded SK1 translocation, and Gαq-T257E was nearly ineffective. Our data confirm an essential role of Gαq in GPCR-mediated translocation of SK1 and reveal an involvement of Gαq residues that play a role in activation of effectors such as PLCβ and p63RhoGEF. 

  • Differential hepatic lipid accumulation in ASMase-/- mice on HFD is associated with hyperglycaemia and liver damage.

    P8 

    R Fucho, C García-Ruiz, JC Fernández-Checa

    Dept. Cell Death and Proliferation, IIBB-CSIC, and Liver Unit, Hospital Clínic i Provincial, IDIBAPS-CIBEK, CIBEREHD, Barcelona, Spain

    It is known that ceramides can blunt the insulin-signaling pathway thereby affecting the metabolic homeostasis. Acid sphingomyelinase (ASMase) is one of the key regulators of ceramide synthesis. In this study, we tested the metabolic effects of a diet-induced obesity (DIO) on ASMase deficient mice. A 60% KCal diet derived from fat was administered for 12 weeks to ASMase , ASMase mice. Blood glucose was monitored every 4 weeks and glucose and insulin tolerance tests were performed at week 10 and 14. Mice were sacrificed at 16 weeks of age, analyzing blood and tissue lipid profiles. ASMase yet ASMase mice showed a higher body weight and higher blood glucose levels. They also showed less glucose tolerance. ASMase mice increased their fat stores but ASMase mice were blind to this dietary effect, yet they only increased their liver weight. Lipid analysis showed increased total liver cholesterol but decreased in liver triglyceride content in ASMase mice compared to ASMase mice. Serum analysis showed a slightly increase in total cholesterol and an increase in AST levels. In conclusion, ASMase deficiency decreases fat mass but exacerbates the deleterious effects of DIO leading to hyperglycaemia and liver damage.

  • Critical role for acid sphingomyelinase in hepatic stellate cell activation and liver fibrosis.

    P9

    A Moles3, N Tarrats3, A Morales2,4, M Domínguez1, R Bataller1,2, J Caballería1,2, C García-Ruiz,2,4, JC Fernández-Checa2,4, M Marí2,4

    1Liver Unit, Hospital Clínic i Provincial, 2CIBEREHD, 3IDIBAPS, 4IIBB-CSIC, Barcelona. 

    Hepatic stellate cell (HSC) activation and proliferation are key events in liver fibrosis. Although acidic sphingomyelinase (ASMase) has been previously shown to play a key role in hepatocyte apoptosis, its contribution to fibrogenesis is largely unknown. Thus, our aim was to analyze the contribution of ASMase to HSC activation and liver fibrosis. We observed a selective ASMase stimulation, preceding the in vitro activation of primary mouse HSC, coinciding with cathepsin B (CtsB) and D (CtsD) processing. Pharmacological ASMase inhibition or genetic silencing by siRNA blunted CtsB/D processing and subsequent HSC activation and proliferation. The role of ASMase in wild type and ASMase+/- mice, which exhibited 60% less of ASMase activity, was analyzed in two experimental fibrogenesis animal models. Bile duct ligation (BDL) produces hepatocyte damage mediated in part by ASMase. In contrast, CCl4-induced liver damage is independent of ASMase. Consistent with these findings, BDL in ASMase+/- mice results in less hepatic damage and fibrosis, because of the dual role of ASMase in the hepatocellular injury and the HSC activation, while CCl4 leads to a less hepatic fibrosis by directly regulating HSC activation. These findings illustrate a novel role of ASMase in HSC biology and liver fibrosis, emerging as a potential therapeutic target for liver fibrosis. 

  • Implication of mTOR in the mitogenic effect of ceramide 1-phosphate.

    P10 

    P Gangoiti, L Arana, A Ouro, A Gomez Muñoz 

    Department of Biochemistry and Molecular Biology, Faculty of Science and Technology. University of the Basque Country, 48080 Bilbao, Spain 

    Ceramide 1-phosphate (C1P) regulates important biological functions including cell proliferation, apoptosis, cell migration and inflammation. Major mechanisms by which C1P stimulates cell division in macrophages include activation of the phosphoinositide 3-kinase (PI3-K)/ protein kinase B (PKB, or Akt) pathway, mitogen-activated protein kinase kinases (MEK), ERK1-2 and JNK. Another key enzyme involved in the regulation of cell growth is the mammalian target of rapamycin (mTOR), which is usually downstream of the PI3-K/PKB pathway or the MAP kinases ERK1/2. mTOR controls protein synthesis and cell proliferation via activation of p70 ribosomal S6 kinase (p70S6K) and inhibition of eIF-4E binding protein (4E-BP1). Here we demonstrate that C1P causes phosphorylation of mTOR and p70S6K in primary (bone marrow-derived) macrophages through activation of the PI3-K/PKB pathway. In addition, C1P caused phosphorylation of PRAS40, a component of the mTOR complex 1 (mTORC1). Furthermore, inhibition of the small G protein Ras homolog enriched in brain (Rheb), which is also a specific component of mTORC1, completely blocked C1P-stimulated macrophage growth. It can be concluded that mTORC1 and its direct target p70S6K are essential components of the mechanism whereby C1P stimulates macrophage proliferation. 

    This work was supported by grants BFU2009-13314 from MCINN and S-PE09UN42 from the Basque Government (Spain) 

  • HDAC inhibition contributes to the phenotype of S1P lyase-deficient fibroblasts.

    P11  

    1Katja Ihlefeld, 1Ralf Frederik Claas, 1Alexander Koch, 2Paul P. Van Veldhoven, 1Dagmar Meyer zu Heringdorf 

    1Institut für Allgemeine Pharmakologie und Toxikologie, Klinikum der Goethe-Universität, Frankfurt am Main, Germany; 2K.U. Leuven, Department Molecular Cell Biology, LIPIT, Leuven, B-3000, Belgium 

    Sphingosine-1-phosphate (S1P) lyase catalyses the irreversible degradation of the bioactive lipid, S1P. In S1P lyase-deficient mouse embryonic fibroblasts (MEFs), S1P levels are elevated, cytosolic [Ca2+]i is high and Ca2+ storage is enhanced. It was shown recently that nuclear S1P, produced by sphingosine kinase-2, inhibits histone deacetylases (HDACs). Since the accumulation of S1P in S1P lyase-deficient cells probably also includes the nuclei, we hypothesised that HDAC inhibition might occur in S1P lyase-deficient MEFs. Indeed, HDAC activity was reduced and acetylation of lysine-9 of histone-3 was enhanced in S1P lyase-deficient compared to wild type MEFs. The HDAC-regulated gene, p21, was strongly upregulated in S1P lyase-deficient MEFs. Pretreatment of wild type MEFs with the HDAC inhibitor, trichostatin A (TsA), increased basal [Ca2+]i and enhanced Ca2+ storage, thus mimicking the dysregulation of Ca2+ homoeostasis in S1P lyase-deficient MEFs. In agreement, mRNA levels of SERCA-2 and -3 were upregulated by about 2-3fold in S1P lyase-deficient MEFs, and particularly SERCA-3 was strongly upregulated by TsA. From these data we conclude that the elevated S1P levels in S1P lyase-deficient MEFs lead to HDAC inhibition which in turn contributes to the altered Ca2+ homoeostasis in these cells. 

  • Sphingosine-induced changes in the bilayer structure.

    P12 

    Noemí Jiménez- Rojo, Félix M. Goñi, Alicia Alonso 

    Unidad de Biofísica (CSIC-UPV/EHU) and Departamento de Bioquímica, Universidad del País Vasco, Barrio Sarriena s/n, 48940 Leioa, Spain. 

    Sphingosine [(2S, 3R, 4E)-2-amino-4-octadecen-1,3-diol] is the basic building block of sphingolipids. In its free state it is known to exist in the cells only in minute amounts. In the last decade it has been shown to act as a potent metabolic signaling molecule, by activating a number of protein kinases. The present contribution intends to describe some physical properties of sphingosine in lipid bilayers. Sphingosine increases the permeability of phospholipid bilayers, giving rise to vesicle leakage. Since at the physiological pH sphingosine has a net positive charge, its interaction with negatively charged phospholipids (e.g. in bilayers containing phosphatidic acid together with phosphatidylcholine and cholesterol) gives rise to a complex pattern of pH-dependent effects. Moreover, sphingosine appears to be sensitive to lipid oxidation: only when bilayer lipids are partially oxidized does sphingosine elicit vesicle aggregation. 

  • Shingosine 1-phosphate as a regulator of hypoxia-induced factor-1α in thyroid follicular ML-1 carcinoma cells.

    P13 

    Veronica Kalhori1, Kati Kemppainen1 and Kid Törnquist1,2 

    1Department of Biology, Åbo Akademi University, Turku, Finland and 2Minerva Foundation Institute, Biomedicum, Helsinki, Finland 

    Sphingosine-1-phosphate (S1P) is a bioactive lipid that regulates the proliferation and migration of cells by binding to S1P receptors and activating G-proteins. Hypoxia induced factor 1α (HIF-1α) is a transcription factor that is degraded under normoxic conditions, while hypoxic conditions stabilize its expression and activity. In the present study, we investigated the effect of S1P on HIF-1α in thyroid follicular carcinoma ML-1 cells. We showed that S1P increased HIF-1α in a time-dependent fashion. The effect was abolished by inhibition of S1P receptors 1, 2 and 3 as well as inhibition of Gi proteins with Ptx. S1P did not enhance the level of HIF-1α mRNA. Inhibiting VEGF receptor 2 decreased HIF-1α levels and if the VEGF receptor 2 was blocked, S1P evoked a rapid, transient increase in HIF-1α. In addition to increasing HIF-1α, SIP also transiently increased the amount of P70S6 kinase, The proliferation and migration of ML-1 cells was also investigated. Our results suggest that HIF-1α and P70S6K, a kinase upstream of HIF-1α, regulate the proliferation of these cells. HIF-1α and P70S6K did not regulate the basal migration of ML-1-cells, but the S1P-induced migration was blunted when either P70S6K or HIF-1α was blocked. Taken together, these results suggest that S1P and VEGFR2 together regulate the expression of HIF-1α. These results can be of importance and should be considered with regard to finding new anti-angiogenetic drugs.

  • Is acid sphingomyelinase required for the anti-proliferative response to Rituximab?

    P14  

    F Sabourdy1,2, J Selves3, E Baeza1, L Astudillo2, P Brousset3, MB Delisle4, C Recher5, T Levade1,2 

    1Service de Biochimie-IFB, 2INSERM U858, 3Service d’Anatomo-Pathologie Purpan, 4Service d’Anatomo-Pathologie Rangueil, and 5Service d’Hématologie, CHU Toulouse, Toulouse, France 

    Acid sphingomyelinase and its product ceramide have been proposed to mediate the growth inhibitory effect of anti-CD20 antibodies (such as Rituximab) on malignant lymphoid B cells (1). 

    Here, we describe the case of an adult patient who developed at age 65 a marginal zone lymphoma. After 5 cycles of a combination of Rituximab, cyclophosphamide, vincristine and prednisone, splenomegaly was still present, leading to treatment interruption and splenectomy. Microscopic examination of the spleen revealed no lymphoma cells but the presence of foamy cells suggestive of a lipid storage disorder. Enzyme studies on peripheral blood cells demonstrated deficient activity of acid sphingomyelinase. Diagnosis of Niemann-Pick disease (type B) was further confirmed by documenting sphingomyelin storage in spleen and identification of point mutations in the SMPD1 gene. EBV-transformed lymphoid cells from the patient, as well as from other patients with Niemann-Pick type A or B, and from control individuals proved to be equally sensitive to the anti-proliferative effects of Rituximab. 

    Thus, not only this case report indicates the occurrence of lymphoma in Niemann-Pick patients, but also suggests that acid sphingomyelinase is dispensable for Rituximab efficacy. 

    1. Bezombes et al. (2004) Blood 104:1166-73. 

  • Sphingosylphosphorylcholine binds to calmodulin and inhibits its function.

    P15 

    E Kovacs1, V Harmat2, J Toth1, BG Vertessy1, K Modos3, J Kardos2, K Liliom

    1Institute of Enzymology, Hungarian Academy of Sciences, 2Institute of Biology, Eötvös Loránd University, and 3Institute of Biophysics, Semmelweis University, Budapest, Hungary 

    Sphingosylphosphorylcholine (SPC) is a dual messenger as it acts on specific heptahelical receptors and mobilize calcium from the endoplasmic reticulum directly as well. SPC has been shown to activate ryanodine receptors through an undefined mechanism. Calmodulin (CaM), the ubiquitous calcium sensor, binds to ryanodine receptors and regulates their activity. Previously we have identified SPC as an endogenous inhibitor of CaM. Here we give mechanistic insight into CaM interaction with SPC and present a crystal structure of their complex. Based on studies with the model CaM-binding domain melittin we demonstrate that both the peptide and SPC micelles bind to CaM in a rapid and reversible manner with comparable affinities. We present evidence that SPC is a competitive inhibitor of CaM-target peptide interaction. SPC also disrupts the complex of CaM and the CaM-binding domain of ryanodine receptor type 1, IP3 receptor type 1, and the plama membrane Ca2+ pump. By interfering with these interactions, thus inhibiting the negative feedback that CaM has on Ca2+ signaling, we hypothesize that SPC could lead to Ca2+ mobilization in vivo. Moreover, we demonstrate that SPC disrupts the complex between both the apo and the Ca2+-saturated form of the protein and the target peptide, suggesting a completely novel regulation for target proteins that constitutively bind CaM, such as ryanodine receptors. 

  • S1P receptors regulate individual cell behaviours in directed migration during zebrafish gastrulation.

    P16  

    Masatake Kai1, Carl-Philipp Heisenberg2 and Masazumi Tada1  

    1Department of Cell & Developmental Biology, University College London, UK. 2Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany 

    Acid sphingomyelinase (A-SMase) plays key roles in apoptosis, During gastrulation, cells forming the prechordal plate, the most anterior mesendodermal structure, undergo directed migration as a cohesive cluster. Recent studies showed that E-cadherin plays an important role in effective anterior migration as a cohesive cluster, and that PDGF may act as a guidance cue in anterior migration of prechordal plate progenitor cells. In a morpholino (MO)-based screen in zebrafish, we identified miles apart (mil)/edg5 as a suppressor of defective anterior migration of the prechordal plate in silberblick(slb)/wnt11 mutant embryos, in which E-cadherin mediated coherence of cell movement is reduced. Here, we examined the roles of Edg5 in anterior migration of prechordal plate cells and found that in wnt11 embryos injected with edg5-MO cells migrate with increased motility but decreased directionality without restoring the reduced coherence of cell migration. This indicates that prechordal plate cells can migrate effectively as individuals as well as in a coherent cluster of cells. Moreover, we demonstrate that Edg5 regulates cell motility and polarization through PDGF and its intracellular effecter PI3K but modulates cell coherence independently of the PDGF/PI3K pathway. Thus, S1P signalling co-ordinates cell motility and coherence underlying directed migration of prechordal plate progenitors. 

  • Involvement of the ABC-transporter ABCC1 and the sphingosine 1-phosphate receptor subtype S1P3 in the cytoprotection of human fibroblasts by the glucocorticoid dexamethasone.

    P17

    1B Nieuwenhuis, 1A Lüth, 2M Jakobi, 1B Kleuser 

    1Institute of Nutritional Science, Dept. Nutritional Toxicology, University of Potsdam, Germany and 2Institute of Pharmacy, Dept. Pharmacology, Free University of Berlin, Germany 

    Glucocorticoids (GC) represent the most commonly used drugs for the treatment of acute and chronic inflammatory skin diseases. However, the topical long term therapy of GC is limited by the occurrence of skin atrophy. Most interestingly although GC inhibit proliferation of human fibroblasts, they exert a pronounced anti-apoptotic action. In the present study we further elucidated the molecular mechanim of the GC dexamethasone (Dex) to protect human fibroblasts from programmed cell death. Dex not only significantly alters the expression of the cytosolic isoenzyme sphingosine kinase 1 but also initiated an enhanced intracellular formation of the sphingolipid sphingosine 1-phosphate (S1P). Investigations using S1P3(-/-)-fibroblasts revealed that this S1P-receptor subtype is essential for the Dex-induced cytoprotection. Moreover, we demonstrate that the ATP-binding cassette (ABC)-transporter ABCC1 is upregulated by Dex and may represent a crucial carrier to transport S1P from the cytosol to the S1P3-receptor subtype. 

  • Influence of NO-donors on the sphingo-myelinase activity and accumulation of lipid peroxide products in mice liver.

    P18  

    M.A. Shupik, A.V. Alessenko 

    Institute of Biochemical Physics of the RAS, Moscow, Russia 

    Proposing a link between NO generation, activation of sphingomyelin cycle and accumulation of lipid peroxide products we investigated interfering of biochemical signaling pathways. Therefore, we tested the ability of NO-releasing compounds S-nitrosoglutathione (GSNO), dinitrosyl iron complexe (DNIC) and NaNO2 to change the activity of n sphingomyelinase (n-SMase) and level of conjugated dienes in liver after their injection to mice. GSNO was injected in doses 0,3; 1,5 and 3 mg, DNIC in doses- 0,3; 0,6; 0,9; and 1,2 mg , NaNO2 – in doses 1 mg per mouse. All NO donors caused changes in the activity of n-SMase and level of lipid conjugated dienes in a time- and concentration-dependent manner. We found that the activity of n-SMase is changed during 4 hours after administration of NO donors by a dual mechanism. On the one hand NO inhibits n-SMase during 15-30 minutes, and on the other hand it stimulates n-SMase after 4 hours action of GSNO and normalizes activity of enzyme after 4 hours DNIC’s action. n-SMase activity coincided in time with level of lipid peroxidation products (decreased level of conjugated dienes within 15-30 min ) after injection of GSNO, DNIC and NaNO2. This data proposed that both sphingomyelinase and messengers of oxidative systems could be targets of NO-derived oxidants. n-SMase could be inhibited by NO or peroxynitrite, which is the product of reaction of NO with superoxide. In vivo peroxynitrite formation has been described during sepsis, autoimmune and inflammatory conditions. 

  • Sterol affinity for bilayer membranes is affected by their ceramide content and the ceramide chain length.

    P19 

    TKM Nyholm, P-.M Grandell, B Westerlund, JP Slotte 

    Biochemistry, Dept.Biosciences, Åbo Akademi University, 20520 Turku, Finland 

    Ceramides have been shown to alter the composition of cholesterol and sphingolipid enriched nanoscopic domains, by displacing cholesterol, and forming gel phase domains with sphingomyelin. We have investigated how the bilayer content of ceramides and their chain length influence sterol partitioning into the membranes. The effect of ceramides with saturated chains ranging from 4 to 24 carbons in length was investigated. In addition, unsaturated 18:1- and 24:1-ceramides were also examined. The sterol partitioning into bilayer membranes was studied by measuring the distribution of cholestatrienol, a fluorescent cholesterol analogue, between methyl-β-cyclodextrin and large unilamellar vesicle with defined lipid composition. Up to 15 mol% ceramide was added to bilayers composed of DOPC:PSM:cholesterol (3:1:1), and the effect on sterol partitioning was measured. Both at 23 and 37 oC addition of ceramide affected the sterol partitioning in a chain length dependent manner, so that the ceramides with intermediate chain lengths were the most effective in reducing sterol partitioning into the membranes. In conclusion, this work shows how the distribution of sterols within sphingomyelin-containing membranes is affected by the acyl chain composition in ceramides. The overall membrane partitioning measured in this study reflects the differential partitioning of sterol into ordered domains where ceramides compete with the sterol for association with.

  • S1P affects skeletal muscle repair/regeneration after eccentric contraction-induced damage.

    P20 

    E.Meacci1, C.Sassoli2, F.Bini1, R.Squecco3, M.Martinesi1, L.Formigli2, F.Francini3 

    Departments of 1Biochemical Sciences, 2Anatomy, Histology and Forensic Medicine 3Physiological Sciences, University of Florence, Florence, Italy 

    Skeletal muscle regeneration is severely compromised in case of extended damage. The current challenge is to find factors capable of limiting muscle degeneration and/or activating the inherent regenerative program. Recent studies have shown that the bioactive lipid, sphingosine 1-phosphate (S1P), promotes myoblast differentiation and exerts a trophic action on denervated skeletal muscle fibres. In this study, we examined the effects of the sphingolipid on eccentric contraction (EC)-injured mouse EDL muscle fibers and the resident satellite cells. Treatment with exogenous S1P attenuated the EC-induced tissue damage, protected skeletal muscle cells from apoptosis and affected extracellullar matrix remodelling. Moreover, S1P greatly potentiated satellite cell activation and enhanced their attitude to fuse into multinucleated myotubes once isolated from the single fibers. Notably, the activity of sphingosine kinase 1 (SphK1) and the levels of endogenous S1P were significantly higher in the injured fibres and satellite cells, stressing the involvement of SphK1/S1P axis in skeletal muscle protection and repair, which may represents a novel therapeutic approaches to manage skeletal muscle damage and disease.(Grant from Fondazione Banche di Pistoia e Vignole and A.R.S.I.A to EM)

  • Elevation of sphingosine-1-phosphate caused neurodegenerative changes in brain of rats after immobilized stress.

    P21 

    Yeonju Lee, Sohyeon Moon and Seikwan Oh 

    Department of Neuroscience and Medical Research Institute, School of Medicine, Ewha Womans University, Seoul 158-710, Korea 

    This study investigated the changes of sphingolipid metabolites as a biomarker of physiological change in serum after three weeks of repeated immobilization. The level of sphingolipids was measured in order to assess the changes in sphingolipid metabolites in the serum of rats exposed to stress. 

    Interestingly, the level of So-1-P was increased in the plasma of rats subjected to 6-hr immobilization stress than repeated immobilization. To further investigate the modulating effect of increased So-1-P in various brain regions, So-1-P was infused into the lateral cerebroventricle at a rate of 100 pmol/10μl/hr for 7 days. 

    Immunoreactivity for GFAP and vimentin, as a marker of reactive gliosis, was also elevated in the cortex and hippocampus. The expression of iNOS and NR1 was elevated in the cortex, hippocampus, striatum, and cerebellum after So-1-P infusion into the cerebroventricle, while the level of GFAP was elevated in the hippocampus and striatum. Interestingly, the expression levels of iNOS, GFAP, and NR1 were increased by the direct application of So-1-P to cultured cortical cells. These results suggest that NO production via iNOS expression, the NR1 expression, the activation of astrocytes, and the elevation of So-1-P may cause neurodegenerative changes in rats subjected to chronic immobilization and that the elevation of So-1-P by stress exposure would be one of the stress signal molecules. 

  • The scopolamine- and a β-induced memory impairment was ameliorated by the treatment with phytoceramide.

    P22  

    Yeonju Lee1, Sohyeon Moon1, Jae-Chul Jung1, Hwan-Soo Yoo2, Yong-Moon Lee2 and Seikwan Oh1 

    1Department of Neuroscience and Medical Research Institute, School of Medicine, Ewha Womans University, Seoul 158-710, Korea 

    2College of Pharmacy, Chungbuk National University, Cheongju 361-763, Korea 

    The ameliorative effect of phytoceramide and phytosphingosine was evaluated in scopolamine- and beta-amyloid (Aβ)-induced amnesia by passive avoidance test. Phytoceramide improved the scopolamine- and beta-amyloid (Aβ)-induced learning and memory impairment while phytosphingosine did not. 

    The level of sphingosine was increased after chronic administration of phytoceramide. 

    The expression of S1P-R1 was significantly reduced in Aβ-induced hippocampus of mice, while phytoceramide inhibited the suppression of S1P-R1 expression) in hippocampus of amnesic mice with Aβ (i.c.v injection) after administration of phytoceramide. In addition, the mRNA level of pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6) was measured in Aβ-injected mice, because it was known that sphingolipids were involved in inflammation. The expression of IL-1β was robustly increased by Aβ, while phytoceramide decreased it. Although further study is needed to find precise mechanisms of ceramide and/or sphingolipids on neuronal functions, it could be suggested that phytoceramide could be used to ameliorate the memory impairment. 

  • Activation of the RhoA/ROCK pathway is essential for stimulation of cell proliferation by ceramide 1-phosphate.

    P23

    A Ouro, P Gangoiti, L Arana , A Gomez-Muñoz 

    1Dept. Biochemistry and Molecular Biology, Faculty of science and technology, Basque Country University Leioa, Spain 

    Ceramide 1-phosphate (C1P) was previously described as pro-mitogenic, antiapoptotic, and pro-inflammatory. In addition, C1P was shown to bind to a specific plasma membrane receptor to stimulate macrophage migration, and this action was independent of the intracellular generation of C1P. Using a “caged” ceramide 1-phosphate analogue: 4-bromo-5-hydroxy-2-nitrobenzhydryl-ceramide-1-phosphate (BHNB-C1P), we have demonstrated that C1P-stimulated cell proliferation is independent of receptor interaction, but dependent upon the intracellular accumulation of C1P. A key enzyme that is involved in the regulation of macrophage proliferation by C1P is the mammalian target of rapamycin (mTOR) (P. Gangoiti, and A. Gómez-Muñoz, unpublished work). Here, we demonstrate that activation of mTOR requires prior stimulation of the RhoA/ROCK pathway, and that this action is essential for the mitogenic effect of C1P through this pathway. 

    This work was supported by grants BFU2009-13314 from MCINN and S-PE09UN42 from the Basque Government (Spain)

  • Towards an understanding of the role of an acid sphingomyelinase in the maturation of the fly respiratory system.

    P24  

    M Strigini1 

    1Institute of Molecular Biology and Biotechnology, FORTH, Iraklio, Crete, Greece 

    The Drosophila respiratory system consists of an epithelial tubular network that develops during embryogenesis. The lumen of developing tracheal tubes is filled with a fluid that is cleared and replaced by air shortly before hatching. I study an acid sphingomyelinase specifically expressed in the trachea. The mutants I generated for this enzyme fail to fill their trachea with air. Trachea maturation proceeds through three steps in a strict developmental order: first, a burst of secretion deposits proteins into the lumen and expands it; second, solid luminal material is removed by endocytosis; third, liquid is cleared (Tsarouchas et al., 2007). In light of recent findings that sphingosine and sphingosine metabolism enzymes may modulate vesicular trafficking in neuronal terminals and secretory T-cells (Darios et al., 2009; Herz et al., 2009), I am assessing the role of aSMase in the control of epithelial secretion and endocytosis during maturation of the fly airways. This work may establish a suitable system for further genetic analysis of sphingolipid function in membrane dynamics.

  • Sphingosine kinase-1 is critically involved in nitric oxide-mediated human endothelial cell migration and tube formation.

    P25 

    Stephanie Schwalm1,2, Josef Pfeilschifter1 and Andrea Huwiler

    1Pharmazentrum Frankfurt/ZAFES, Goethe University Frankfurt, Frankfurt am Main, Germany. 

    2Institute of Pharmacology, University of Bern, Bern, Switzerland. 

    Sphingosine kinases (SK) convert sphingosine to sphingosine-1-phosphate (S1P) which is a bioactive lipid that regulates a variety of cellular processes including migration. In this study, we have used the human endothelial cell line EA.hy 926 and investigated the effect of nitric oxide (NO)-donors on SK-1 expression and on cell migration and tube formation. We show that exposure of EA.hy 926 cells to Deta-NO resulted in a time- and concentration-dependent upregulation of SK-1 mRNA and protein expression, and activity. In mechanistic terms, the NO-triggered upregulation of SK-1 occurred independently of cGMP, but involved the classical mitogen-activated protein kinase cascade and a redox-dependent mechanism. Functionally, Deta-NO triggered an increased endothelial cell migration in an adapted Boyden chamber assay and also increased endothelial tube formation in a Matrigel assay. These responses were both abolished in cells depleted of SK-1. In summary, these data show that NO-donors upregulate SK-1 expression and activity in human endothelial cells and SK-1 in turn critically contributes to the migratory capability and tube formation of endothelial cells. Thus, SK-1 may be considered an attractive novel target to interfere with pathological processes involving angiogenesis.


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