• Bioactive sphingolipids in health and disease: lipidomic analysis, metabolism and roles in membrane signaling and autophagy.


    A. H. Merrill, Jr.

    School of Biology, Chemistry and Biochemistry and the Petit Institute of Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, Georgia 30332-0230, USA.

    Organisms usually contain large numbers of sphingolipid subspecies (for a pathway based compilation, see and knowledge about the types and amounts is imperative because they influence membrane structure, interactions with the extracellular matrix and neighboring cells, vesicular traffic and the formation of specialized structures such as phagosomes and autophagosomes, as well as participate in intracellular and extracellular signaling. Fortunately, “sphingolipidomic” analysis is becoming feasible for important subsets such as all of the backbone “signaling” subspecies (ceramides, ceramide 1-phosphates, sphingoid bases, sphingoid base 1-phosphates, inter alia) using mass spectrometry,and these profiles are revealing many surprises,such as that under certain conditions cells contain significant amounts of “unusual” species such as N-mono-, di-, and tri- methyl-sphingoid bases (including N,N-dimethylsphingosine) and dihydroceramides (DHCer), in the latter case, in very high proportions when cells are treated with the anticancer drug fenretinide (4- hydroxyphenylretinamide). The elevation of DHCer by fenretinide is befuddling because the 4,5-trans-double bond of Cer has been thought to be required for biological activity; however, DHCer induces autophagy and may be important in the regulation of this important cellular process. The complexity of the sphingolipidome is hard to imagine, but one hopes that, when partnered with other systems biology approaches, the causes and consequences of the complexity will explain how these intriguing compounds are involved in almost every aspect of cell behavior and the malfunctions of many diseases.

    1Merrill et al., Methods 2005 36:207. 2Zheng et al., Biochim. Biophys. Acta 2006 in press.

  • Influence of Sphingolipids on Expression of the Multidrug Resistance Phenotype1,2.


    V. Gouaze-Andersson, A. J. Kreitenberg, A. E. Giuliano, M. C. Cabot. Department of Experimental Therapeutics, John Wayne Cancer Institute, 2200 Santa Monica Blvd., Santa Monica, CA 90404, USA.

    Natural product anticancer agents enhance intracellular levels of ceramide, a sphingolipid that promotes cell apoptosis. However, multidrug-resistant (MDR) cancer cells may avoid the cytotoxic effects of chemotherapy by glycosylating ceramide. In the present study we examined the relationship between ceramide, ceramide metabolites and expression of the MDR1 gene in human breast cancer cell lines. MDR1 codes for P-glycoprotein (P-gp), a member of the ABC transporter superfamily of proteins that promote cellular efflux of chemotherapeutic agents.

    Four wild-type (drug-sensitive) human breast cancer cell lines (MCF-7, T47D,MDA-MB-231, MDA-MB-435) were used to evaluate the influence of acute and chronic exposure to ceramide and ceramide metabolites on MDR1 mRNA,P-gp, and resistance to chemotherapeutic agents. Acute exposure (<72 hours) to C8- ceramide (5 μg/ml culture medium), a cell-permeable analog of ceramide, enhanced MDR1 mRNA levels by 3- and 5-fold in T47D and inMDA-MB-435 cells, respectively, but did not affect MCF-7 cells.D-erythro-sphingosine exposure (5 μg/ml, 72 hr) increased MDR1 mRNA levels in MDA-MB-435 cells by 3.5-fold. Chronic exposure (> 72 hours) of MDA-MB-231cells to C8-ceramide enhanced MDR1 mRNA levels by 45- and 370-fold(real-time RT-PCR) at passages 12 and 22, respectively, and elicited expression of P-gp (Western blot). These cells not only demonstrated a 3-foldincrease in resistance to doxorubicin and a 9-fold increase in resistance to paclitaxel, but also showed increased efflux of rhodamine. Acute exposure ofMCF-7 and MDA-MB-231 cells to C8-glucosylceramide (10 μg/ml culture medium), a cell-permeable analog of glucosylceramide, increased MDR1 mRNA levels by 2- and 4-fold, respectively. Exposure of cells to either octanoic acid (C8:0), a C8-ceramide hydrolysis product, or oleic acid (C18:1) did not affect MDR1 expression.

    These data are the first evidence that chronic exposure to ceramide and its metabolites enhances expression of the MDR phenotype in cancer cells. The impact of sphingolipids on MDR1 gene expression in cancer appears to reflect the myriad intracellular signaling pathways of ceramide and its metabolites. Although the mechanisms of gene activation are still unclear, ceramide’s role as a messenger of cytotoxic response to chemotherapy might be linked to the MDR pathway.

    Supported by Susan B. Komen Breast Cancer Foundation; Department of Defense Breast Cancer Research Program; Associates for Breast and Prostate Cancer Studies, Los Angeles; and Fashion Footwear Association of New York Charitable Foundation (Shoes on Sale/QVC).

    1Gouaze et al., Mol. Cancer Ther. 2004 3:633. 2Gouaze et al., Cancer Res. 2005 65:3861.

  • Balancing (grants) between peroxisomal and sphingolipid metabolism.


    Paul P. Van Veldhoven.

    Farmakologie, Dept. Mol. Cell Biol., K.U.Leuven, Campus Gasthuisberg, Herestraat, B-3000 Leuven, Belgium.

    Over the last three decades, the role of peroxisomes in mammalian lipid metabolism has been actively studied by our group. Although not obvious at first sight, a few links between peroxisomes and sphingolipid metabolism have been explored or discovered in recent years. A first one concerns the degradation of alpha-hydroxylated fatty acids (2-OH-FA), best known as constituents of certain sphingolipids (cerebrosides, ceramides). Our analysis of ceramide-phosphate in mouse tissues revealed also the presence of a2-OH- fatty N-acylated species in cerebellum. These 2-OH-FAs appear to be degraded by a modified alpha-oxidation pathway, involving an activation step and a cleavage reaction, generating formyl-CoA and a fatty aldehyde. The latter reaction is catalyzed by a peroxisomal enzyme that is also required for the removal of 3-methylbranched fatty acids (classical alpha-oxidation). In case of thiamine depletion or in peroxisome disorders, degradation of 2-OH-FAs is affected. Another example is related to C2-ceramide, the formation of which has been proposed to be dependent on platelet activating factor (PAF). Based on studies in a mouse model lacking peroxisomes, and unable to form (vinyl)etherlipids, such link seems highly questionable. Finally, given the fact that fatty aldehydes, produced during the last step of sphingolipid catabolism, are incorporated into (vinyl)etherlipids (plasmalogens), the responsible enzyme, sphingosine-phosphate lyase, was studied. This finally leads to the generation of a knock-out mouse, providing new insights insphingosine-1-phosphate mediated biology.

  • Cannabinoids and ceramide: two lipids acting hand by hand.


    M. Guzmán.

    Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Químicas, Universidad Complutense de Madrid, Spain.

    Cannabinoids, the active components of Cannabis sativa (marijuana) and their endogenous counterparts, exert their effects by binding to specific Gi/o-protein-coupled receptors that modulate adenylyl cyclase, ion channels andmitogen-activated protein kinases. Research carried out by our group during the last years has shown that cannabinoid receptors are also coupled to the generation of ceramide via two different pathways: sphingomyelin hydrolysis and ceramide synthesis de novo. Sustained ceramide accumulation in tumor cells mediates cannabinoid-induced apoptosis, as evidenced by in vitro andin vivo studies. Former experiments supported that this effect was due to the impact of ceramide on key cell signaling pathways such as the extracellularsignal-regulated kinase cascade and the Akt pathway. More recently we have identified a new route that mediates cannabinoid-induced apoptosis of tumor cells via de novo-synthesized ceramide. This route involves the stress- regulated protein p8 (also designated as candidate of metastasis 1) and its downstream targets ATF-4, CHOP and TRB3, which had been previously implicated in the endoplasmic reticulum stress response. These findings provide a new conceptual view on how cannabinoids act, and raise interesting pathophysiological questions.

  • Biological evaluation of sphingolipid analogues as potential antifungal agents.


    D. Mormeneo1,2, J. Casas1, A. Manresa3, A. Delgado1,2.

    1RUBAM, Department of Biological Organic Chemistry, IIQAB-CSIC, C/ Jordi Girona 18-26, 08034 Barcelona and Units of 2Pharmaceutical Chemistry and3Microbiology, School of Pharmacy, University of Barcelona, Avda. Joan XXIII s/n; 08029 Barcelona, Spain.

    Over the last 20 years, the number of patients with serious fungal opportunistic infections has been rising because of increased number of immune suppressed patients, either by infection with HIV, intense chemotherapy for treatment of cancer or organ transplantation. Unfortunately, the structural and metabolic similarities between fungal and mammalian cells have made the identification of novel antifungal targets extremely difficult. In this sense, differences between sphingolipid metabolism in fungi and mammals might lead to new pharmacological targets. The present work presents the design, synthesis and biological evaluation of phytosphingosine (PHS) and phytoceramide (PHC) analogues as fungal growth inhibitors.From a chemical standpoint, the new entities differ from the natural sphingoid bases in the nature of Cposition, side chain at Cposition and stereochemistry of Cand Cstereogenic centers (figure). All these analogues were screened preliminarily over Saccharomyces cerevisiaecultures and the most promising structures were evaluated over different yeasts and

    The present work has been funded by an Education and Science Ministry fellowship (ref.AP2002-0567)moulds. In addition to these in vivo approaches, some of PHC analogues were also screened as in vitro inhibitors over the essential fungal enzyme inositolposphorylceramide synthase (IPCS).2

    1Chung et al., J. Biol. Chem. 2001 276:35614. 2Georgopapadakou, Expert Opin. Investig. Drugs 2000 9:1787.

  • Synthesis of novel scyphostatin analogues and evaluation as neutral sphingomyelinase inhibitors.


    E. N. Pitsinos1A. Cruz1, A. Giannis2, V. Wascholowski2.

    1Lab. of Natural Products Synthesis & Bioorganic Chemistry, Inst. of Physical Chemistry, NCSR “DEMOKRITOS”, P.O. Box 60228, 153 10 Ag. Paraskevi, Greece. 2University of Leipzig, Institute of Organic Chemistry, Johannisallee 29, 04103 Leipzig, Germany.

    Sphingomyelinases are enzymes that, in response to specific stimuli, catalyse the hydrolysis of sphingomyelin to ceramide. Of the various isotypes described to date, the plasma membrane-located neutral sphingomyelinase (N-SMase) is of particular interest as a target for the development of new drugs for the treatment of neurodegenerative diseases, such as Alzheimer’s disease.1


    We have recently developed a short, versatile and efficient strategy towards the pharmacophoric polar core of Scyphostatin.
    Modification of this strategy has allowed us to prepare several analogues of the natural product (2-3).Details of the synthetic route employed as well as their activity as N-SMaseinhibitors will be presented.4The natural product Scyphostatin (1) is a potent and selective inhibitor (IC50 = 1 μΜ) of N-SMase. However, its chemical stability is limited due to the lipophilic polyunsaturated side chain.2

    1Wascholowski and Giannis, Drug News & Perspectives 2001 14:581. 2Yang et al., Neurobiology of Disease 2004 17, 99; Nara et al., J. Antibiot. 1999 52:525. 3Pitsinos and Cruz, Org. Lett. 2005 7:2245. 4Wascholowski et al., ChemMedChem 2006 1:718.

  • Phospholipid metabolism modulators in pathogenic organisms.


    P. González-Bulnes1, J. Casas1, C. Ben Mamounand A. Llebaria1.1Department of Organic Biological Chemistry (IIQAB-CSIC), Jordi Girona 18- 26, 08034 Barcelona, Spain. 2Department of Genetics and Developmental Biology (UCHC), 263 Farmington Avenue, Farmington CT 06030, USA.

    Aminohydroxamic acids represent a new class of compounds which can affect phospholipid metabolism. The compounds presented here were originally designed as inhibitors of PC-PLCBc1, a phospholipase that catalyzes the hydrolysis of phosphatidylcholine to form DAG and phosphorylcholine. Study of their biological activity showed that they inhibited the targeted enzyme with IC50 values ranging between 4 and 100 µM. The similarity between the phospholipase of Bacillus cereus and that of Clostridium perfringens2, the bacterium which causes gas gangrene, suggested that the aforementioned compounds could also inhibit this enzyme. However, in vitro studies demonstrated that these compounds had no effect against the phospholipase C of Clostridium perfringens for most of the compounds. The fact that sphingomyelin synthase uses the same substrate than phospholipase C and catalyzes a similar reaction made us think that these compounds might inhibit sphingomyelin synthase. Interestingly all compounds were found to inhibit this enzyme albeit at a higher inhibitory concentration. Finally, the report of a phospholipase C as a good target for synthesis of antimalarialdrugs made us think that these aminohydroxamic acids may inhibit Plasmodium falciparum, the parasite that causes malaria. A preeliminary screening made by the WHO showed that they inhibited the growth of Plasmodium falciparum as well as that of Leishmania donovani, Trypanosoma cruzi and Trypanosoma brucei rhodesiense with little or no cytotoxicity in mammalian cells. Because of their strong antimalarial activity, a more thorough study of the mode of action of these compounds in P. falciparum was performed. The structural similarity between them and miltefosinemade us think that the effect against the parasite could be the same. Being miltefoxine a lipid inhibitor and affecting our compounds enzymes implicated in lipid metabolism the first thing to do was to assess the effect over lipid biosynthesis. The fact that the stage of P. falciparum life cycle inhibited by these compounds was the trophozoite stage, when the production of lipids increases to form the membranes of the merozoites, reinforced this idea. Labelling studies showed a dose-dependent inhibition of synthesis of phosphatidylcholine, the predominant phospholipid in the parasite’s membrane, and no effect on the synthesis of phosphatidylethanolamine. The IC50 values of these compounds were not affected by the availability of choline and were similar between wild type and transgenic parasites overexpressing or lacking an enzyme involved in the early steps of synthesis of phosphatidylcholine from serine. Together these studies suggest that aminohydroxamic acids exert their antimalarial activity via inhibition of the last essential steps of synthesis of phosphatidylcholine.

    1González-Roura et al., Angew. Chem. Int. Ed. 2004 43:852. 2Naylor et al., Nat. Struc. Biol. 1998 5:738. 3Hanada et al., J. Exp. Med. 2002 195:23. 4Pessi et al., Proc. Natl. Acad. Sci. 2004, 101:6206.


  • Suppression of the gene expression of GD3 synthase in human melanoma cells blocks the ability of the tumor cells to induce neoangiogenesis upon xenografting in nude mice.


    I. Popa, S. Trompezinski, L. Thomas, J. Portoukalian.

    University of Lyon-1, Dermatology, Edouard Herriot Hospital, 69437 Lyon Cx 03, France.

    We established several years ago a model of human metastatic melanoma with immunosuppressed newborn rats by subcutaneously grafting a single human melanoma cell line. The lung metastases were repeatedly excised, grown in culture and grafted again to produce increasingly metastatic variants. After many cycles, a set of ten variants was obtained, each one showing a stable and reproducible metastatic potential that ranged from 1 to more than 300 lung metastases in the newborn rats. Along with the lung metastases, all variants gave a tumor at the s.c. injection site in three weeks following grafting of one million cells. The analysis of gangliosides of the variants showed a highly significant reverse correlation between the GD3 ganglioside content and the metastatic potential.The highly metastatic variants had a strongly reduced ganglioside content with a nearly complete absence of GD3, a much lower amount of GM3 and a strong increase in lactosylceramide as compared to the poorly metastatic ones. However, one striking observation was that keeping in culture the highly metastatic variant TW12 over a ten passage period progressively brought back its ganglioside pattern to that of the non-metastatic one IC8 (containing high amounts of both GM3 and GD3), along with a loss of its metastatic potential. These changes of the ganglioside pattern were also seen in vivo since all the tumors grown at the s.c. injection site in rats had the ganglioside pattern of the original non-metastatic variant, regardless of their lung metastatic potential, even when cloned cells were injected.The possible role of GD3 synthase gene expression in the formation of melanoma metastases was studied after transfection of the non-metastatic variant IC8 with plasmids containing antisense sequences for GD3 synthase. Highly metastatic IP-1 cells were selected from GD3 antisense-transfected IC8 cells using Matrigel-coatedBoyden chambers. The cells that crossed the chambers were expanded and the sphingolipid pattern was found to match exactly the one of the highly metastatic one TW12. Neo-angiogenesis is critical for the growth of metastases and the role of GD3 was investigated in vitro with human vein endothelial cells (HUVEC) and in vivo with Matrigel-embedded melanoma cells injected in nude mice. The results showed that exogenously added GD3 strongly increases in vitro angiogenesis. Moreover, blocking the GD3 synthase gene expression in IP-1 cells nearly abolished angiogenesis in nude mice, whereas IC8 induced a strong angiogenesis in a two-week period. Interestingly, TW12 embedded in Matrigel and injected to the nude mice expressed GD3 and induced angiogenesis, whereas GD3 antisense-transfectedTW12 did not, supporting the hypothesis that GD3 may be produced again by proliferating highly metastatic cells to ensure a proper neo-angiogenesis.

    1Thomas et al., C. R. Acad. Sci. III. 1995, 318:1233. 2Zebda et al., FEBS Lett. 1995, 362:161.

  • Developmental expression of the sialidase Neu2 ortholog gene in zebrafish and characterization of the knock-down model.


    A. Fanzani1, F. Colombo1, S. Nicoli2, R. Giuliani1, D. Zizioli1, F. Cotelli3, S. Rossi1, D. Maiolo1, M. Presta2, A. Preti1, S. Marchesini1.

    Department of Biomedical Sciences and Biotechnology, Unit of 1Biochemistry and 2Pathology, University of Brescia, Italy and 3Department of Biology, University of Milan, Italy.

    Neu2 is a soluble sialidase that has been suggested to play an important role in skeletal muscle differentiation. However, the role of Neu2 gene during embryogenesis has not been ever investigated. In the present study, in silico analysis identified in zebrafish genome six genes encoding sialidase-likeproteins. Here we reported the cloning of cytosolic sialidase Neu2 ortholog in zebrafish. The Neu2 gene is located on chromosome 21 with 3 ORF exons encoding a 376 amino acid protein which has 57-59% homology to mammalian Neu2 forms. This protein contains all the conserved sialidase amino acid motifs, and the enzyme heterologously expressed in mammalian cells is present in the cytosol and it is functional toward the artificial substrate 4-MU-NeuAc in a range of neutral pH typical of soluble sialidases. During zebrafish embryogenesis, Neu2 resulted strongly expressed in the early stages along the somitogenesis, while the expression after 24-48 hours post fertilization resulted in the anterior and caudal regions of the embryos. The knock-downmodel of Neu2 gene obtained by morpholinos microinjection revealed a severe phenotype, underlying an emerging role of this enzyme during vertebrate development.

  • Ceramide kinase in inflammation: What do we learn from KO mice?


    S. Niwa, B. Zemann, C. Graf, P. Rovina, R. Reuschel, D. Mechtcheriakova, N. Urtz, B. Kinzel, M. Müller, T. Baumruker, A. Billich, F. Bornancin.

    Novartis Institutes for BioMedical Research, Vienna, Austria, and Basel, Switzerland.

    Ceramide kinase (CerK) uses ATP to phosphorylate ceramide, leading toceramide-1-phosphate (C1P). CerK/C1P have been recognized as new players in inflammation, controlling mechanisms such as phagocytosis, mast cell degranulation and cPLA2 activation. We generated CerK-/- mice by homologous recombination in Balb/C background. The resulting animals are viable, breed normally, and do not display any overt phenotype. By using in vitro as well as in vivo models, we have started investigating the dependency of critical inflammatory responses on the presence of CerK. The results of these ongoing studies will be presented.

  • Construction of a conditional knockout mouse model of the ceramide- kinase like gene (CERKL).


    A. Garanto, M. Tuson, R. González-Duarte, G. Marfany.

    Departament de Genètica, Facultat de Biologia, Universitat de Barcelona, Avda. Diagonal 645, 08028-Barcelona, Spain.

    Retinitis pigmentosa (RP) is the main cause of blindness in human, affecting1:3000-1:5000 adults. RP defines a clinically and genetically group of retinal disorders caused by progressive photoreceptor neurodegeneration and for which there is not any effective therapy yet. Mutations in at least 32 genes have been associated to this disorder and more than 18 genes are responsible for the autosomal recessive forms (arRP). Recently, our group identified a novel causative gene, CERKL (ceramide kinase-like gene), in three consanguineous pedigrees from Spain. Remarkably, the mutation introduced a STOP codon within the lipid kinase domain, generating a prematurely truncated protein. Although sequence homology clearly involves the CERKL protein in the sphingolipid metabolism, its substrate(s) and precise physiological function remain as yet unknown. Ceramide has already been shown to be a key regulator of retinal cell apoptosis. We surmise that CERKL activity may protect the cell against ceramide production in stressed retinal cells. Therefore, this mutation of CERKL would preclude the phosphorylation of ceramide, triggering photoreceptor cell death and eventually causing blindness.

    Although natural mouse mutants that serve as models for retinal diseases have been collected since the beginning of the XXth century, very few of them are good models for retinitis pigmentosa. Besides, given the high number of genes involved in RP, the available knockout models are also very scarce. In order to shed light on the biological function of CERKL as well as on the pathogenic mechanisms by which the disfunction of the sphingolipid metabolism causes retinitis, we designed a conditional knockout mouse model (cre-loxP) to disrupt the CERKL gene. Last but not least, this knockout mouse will provide a model in which to validate the effectiveness of new therapeutic approaches.

  • Lipid rafts rich in sphingomyelin and cholesterol content in the nucleus: a platform for transcription process.


    G. Cascianelli, M. Villani, M. Tosti, F. Marini, M. P. Viola Magni, E. Albi Department of Clinical and Experimental Medicine, Physiopathology, University of Perugia, Italy.

    The role of cholesterol (CHO) and sphingolipid in the generation of lipid raft domains in cell membrane has been widely described1. A relation between sphingomyelin (SM) and cholesterol (CHO) regulated by neutral sphingomyelinase (N-SMase) activity has been shown inside the nucleus2. The CHO SM-linked fraction changed during cell proliferation. The aim of the work was to ascertain first the existence of lipid microdomains in the nucleus. The microdomains were extracted from purified nuclei using TritonX-100 detergent at low temperature and were isolated by sucrose density gradient. The results showed the existence of nuclear lipid rafts which had a morphology similar to that previously reported for microvillar membrane. The biochemical analysia showed that nuclear microdomains were characterized by the presence of CHO, SM and phosphatidylcholine (PC) in the 1:1:1 ratio. The SM metabolism enzyme assay demonstrated the N-SMase,SM-synthase and reverse-SM-synthase activity suggesting that the SM was metabolized directly in lipid rafts probably in relation to cell function. To study the possible involvement in nuclear processes, we have analyzed the incorporation of radioactive uridine at 24 hours after partial hepatectomy which resulted very high in the nuclear lipid rafts. The presence of lamin B as a specific protein of nuclear membrane and STAT3 transcription factor suggested that the nuclear lipid rafts could represent a platform for transcription process in nuclear membrane.

    1Edidin M. Ann. Rev. Biophys. Biomol. Struct. 2003 32: 257¸ 2Albi E and Viola Magni MP. J. Hepatol. 2002 36: 395

  • Modulation of sphingolipid metabolism alters vascular responses to angiotensin II and muscarinic receptor agonists.


    A. C. M. Mulders, M. C. Hendriks-Balk, M.-J. Mathy, M. C. Michel, A. E. Alewijnse, S. L. M. Peters.

    Dept. Pharmacology and Pharmacotherapy, Academic Medical Center, Amsterdam, The Netherlands.

    Sphingolipids are major constituents of the cellular membrane and play important roles in maintaining cellular homeostasis. The sphingolipid metabolites sphingomyelin, ceramide, sphingosine and sphingosine-1-phosphate can be metabolized into eachother and can have opposite effects on cell growth and survival. It has been suggested for programmed cell death, cell survival and cell growth that it is not the absolute amount, but the relative balance between these sphingolipid metabolites which is decisive. Next to this, sphingolipids also have vasoactive properties, and can induce vascular relaxation or constriction depending, amongst others, on vessel type. Besides their occurrence in plasma, they can also be formed locally in the vascular wall itself in response to external stimuli, for example TNFα. We investigated whether the known vasoactive compounds angiotensin II and methacholine modulate the sphingolipid metabolism in the vascular wall and how this might contribute to the vascular responses.

    In isolated rat carotid arteries, the contractile responses to angiotensin II were enhanced by the sphingosine kinase inhibitor N,N dimethylsphingosine (DMS, 10 µM). Endothelium removal or NO synthase inhibition withnω-nitro-L- arginine (L-NNA, 100 µM) resulted in a similar enhancement. Applied together, DMS had no additional effect over the L-NNA-induced shift of the concentration response curve. Angiotensin IIconcentration-dependently induced NO production in an endothelial cell line, which could be diminished by DMS. Using immunoblotting and intracellular calcium measurements, we demonstrated that this sphingosinekinase-dependent endothelial NO synthase activation was mediated via both phosphatidylinositol 3-kinase/Akt and calcium-dependent pathways.

    In translocation experiments in endothelial cells we showed that activation of the muscarinic M3 receptor lead to translocation of YFP-labelled sphingosine kinase from the cytoplasm to the cellular membrane. In relaxation experiments, inhibition of sphingosine kinase reduced the potency of relaxation response to the muscarinic receptor agonist methacholine in isolated rat carotid arteries and aorta. The endogenously formed S1P had a relaxant effect, in analogy to the angiotensin II pathway we have shown. Interestingly, in contrast to conduit vessels, DMS induced a significant enhancement of methacholine-induced relaxation in mesenteric artery preparations. This suggests that the endogenously formed S1P in this resistance vessel is a contractile factor in muscarinic M3-induced vascular relaxation.

    We conclude that modulation of sphingolipid metabolism by vasoactive compounds such as angiotensin II and muscarinic receptor agonists, contributes to their vasoactive properties. For the muscarinic M3 receptor, this modulation of sphingolipid metabolism leads to differential effects in conduit and resistance vessels. The involvement of endogenous sphingolipid formation may be of importance under pathological circumstances with reduced NO bioavailability. Moreover, a disturbed sphingolipid metabolism in the vascular wall may lead to reduced NO bioavailability and endothelial dysfunction.

  • Involvement of the endoplasmic-reticulum during serum-withdrawal dependent apoptosis in hippocampal neuroblasts.


    V. Voccoli,F. Mazzoni,L. Colombaioni,M. García-Gil,2

    1Istituto di Neuroscienze CNR, Via G.Moruzzi 1, 56100 Pisa, Italy and 2Dipartimento di Biologia, Unità di Fisiologia, Università di Pisa, Via S. Zeno 31, 56127 Pisa, Italy.

    Apoptotic death caused by diseases or toxic insults is preceded and determined by endoplasmic reticulum dysfunction and altered intraluminar calcium homeostasis in many different cell types. With the present study we have explored the possibility that the ER stress could be involved also in apoptotic death induced by serum deprivation in neuronal cells. We have previously shown that in the embryonic hippocampal cell line HN9, serum deprivation induces nuclear sphingomyelinase activation together withsphingomyelin-synthase inhibition and a consequent increase of nuclear ceramide pool; this is followed by relocation of cytochrome c to cytosol and of Bax to mitochondria, deregulation of calcium metabolism without loss of mitochondrial functionality, and activation of caspase-3. The Ca++concentration in the lumen of the ER has been evaluated by using the low affinity Ca++ probe Mag-fluo-4. We show that serum deprivation lowers the ER Ca++ concentration with a time course closely related to the increase of apoptosis incidence. Serum deprivation also enhances the expression of a well known marker of ER stress, the glucose regulated protein-78 (GRP-78), a member of the heat shock/stress response protein family. Moreover, in serum deprived neuroblasts, following GRP-78 up-regulation, the ER-associatedprocaspase-12 is cleaved with a time course which parallels the ER calcium loss. These findings indicate that, in hippocampal neuroblasts, Ca++mobilization from ER and caspase-12 activation are components of the molecular pathway that leads to apoptosis triggered by serum deprivation and may constitute an amplifying loop of the mitochondrial pathway.

  • Sphingolipid modulation of immune cell function.


    E. A. Martinova.

    State Institute of Nutrition Russian Academy of Medical Sciences, Moscow, Russian Federation.

    Simple sphingolipids such as sphingosine, sphinganine, C2-ceramide as well as inhibitor of ceramide synthase – fumonisin B1 were found to modulate many signaling pathways into cell. This allows them to regulate the immune cell function. Previously we have shown that exogenously added sphingolipids changed the receptor expression in immunological synapse including CD3, CD4, CD8, CD45, CD54 and some others.This causes the disruption in T- helper cell differentiation and prevalence Th2 cells on Th1 cells.As a result the primary immune response to T-dependent antigens can be modulated by exogenous sphingolipids. The most effective lipid from mentioned above is sphingosine. D-sphinganine is less effective compared with D,L-sphinganinewhich is more toxic for immune cells and different cell lines. Sphingolipids activate the macrophage function via elevation of receptor F4/80 expression and chemokine production. The excellent tolls for immune cell regulation are three fumonisins – B1, B2, and B3 which may elevate or impair immune response dependent on its percentages in the mixture. Fumonisin B1 regulates the memory cell formation and secondary immune response toT-dependent antigens.Fumonisin B1 inhibits the DNA synthesis in normal lymphocytes in response to mitogenes, more effective in T cells compared with B cells.Under condition of fumonisin exposure it causes the prolonged immune deficiency and activation of growth of transformed cells. Effects of sphingolipids on immune cells are strong connected with cell cycle.It is not possible to initiate an apoptosis in some conditions in the synchronized cultured cells. In the absence of specific antigen sphingolipids lead to the activation induced apoptosis of lymphocytes. The practical implication of this one is an induction of apoptosis in the tumor-associated lymphocytes which lost the! normal anti-tumor defense. Endogenous sphingolipids participate as si gnaling molecules in all types of apoptosis such as receptor-dependent,mitochondrial-connected or stress-initiated. We also found that cell death after sphingolipid exposure may be rather by necrosis but not apoptosis. It may be under several conditions including additional activation of TNF signaling pathwaysor ATP depletion. We found the cross-talk betweensphingolipid-derived signaling and mitochondrial respiration. Inhibitors of respiration may modulate the sphingolipid initiated apoptosis in different cell types. One of mechanisms of fumonisin effect on lymphocytes is an activation of the neutral sphingomyelinase of plasma membrane and generation of sphingomyeline cycle.Our last data discovered the new mechanism of sphingolipid effect on immune cells. We found that exogenous added sphingosine, sphinganine or fumonisin B1 activated the stress-dependentsignaling pathways in endoplasmic reticulum followed by the expression of some mitochondrial proteins including Lon protease. This causes an activation of ATP-dependent proteolysis and perturbation of regulatory proteins data in press). Under stimulation of normal immune response, Lon protease expression is activated simultaneously with proteolysis of regulatory proteins. Simultaneous exposure of sphingolipids and inhibitors of Lon protease expression amplifies the apoptotic signals into immune cells. Conclusion: exogenous sphingosine, sphinganine, C2-ceramide and fumonisin affect some stages of immune cell differentiation, proliferation, and apoptosis followed by modulation of immune response.

    1Martinova, Adv. Exp. Med. Biol. 1996 391:331. 2Martinova, Toxicon. 1997 35:497. 3Martinova and Merrill, Mycopathologia 1995 130:163. 4Martinova et al., Biomed. Khim. 2003 49:35.5Martinova et al., Biochemistry (Moscow) 1995 60:461.

  • The tricyclodecan-9-yl-xanthogenate D609 triggers ceramide increase and enhances FasL-induced caspase-dependent and -independent cell death in T lymphocytes.


    D. Milhas, N. Therville, T. Levade, H. Benoist*, B. Segui*

    INSERM U466, IFR31 BP 84225 31432 Toulouse cedex 4 FRANCE.

    D609 is known to modulate death receptor-induced ceramide generation and cell death. We show that in Jurkat cells, non-toxic D609 concentrations inhibit sphingomyelin synthase and, to a lesser extent, glucosylceramide synthase, and transiently increase intracellular ceramide level. D609 significantly enhanced FasL-induced caspase activation and apoptosis. D609 stimulated FasL-induced cell death in caspase-8-deficient Jurkat cells indicating that D609 acts downstream of caspase-8. At high FasL concentration (500 ng/mL), cell death was significantly, but not completely, inhibited byzVAD-fmk, a broad-spectrum caspase inhibitor, indicating that FasL can activate both caspase-dependent and -independent cell death signaling pathways. FasL- induced caspase activation was abolished by zVAD-fmkwhereas ceramide production was only partially impaired. D609 enhancedcaspase-independent ceramide increase and cell death in response to FasL. Also, D609 overcame zVAD-fmk-conferred resistance to a FasL concentration as low as 50 ng/mL and bypassed Rip deficiency. Mitochondrial events were likely involved since Bcl-xL over-expression impaired D609 effects. InPHA-activated human T lymphocytes, D609 enhanced FasL-induced cell death in the presence or absence of zVAD-fmk. Altogether, our data strongly indicate that the inhibition of ceramide conversion to complex sphingolipids by D609 is accompanied by an enhancement of FasL-inducedcaspase-dependent and - independent cell death in T lymphocytes.

  • Ceramide Mediates Amyloid Beta- and Tumor Necrosis Factor Alpha- Induced NADPH Oxidase Activation and Subsequent Oxidation of the Neuronal Actin Cytoskeleton.


    B. M. Barth1,2, D. L. LaVictorie1, S. J. Brown1, C. M. McGill1, T. P. Clausen1, T. B. Kuhn1,2.

    1Department of Chemistry and Biochemistry and 2Alaska Basic Neuroscience Program, University of Alaska-Fairbanks, Fairbanks, AK 99775 USA.

    Inflammation and its mediators are implicated in the orchestration and progression of acute and chronic degenerative pathologies of the central nervous system (CNS). In particular, oxidative stress plays a major role in the progressive nature of CNS inflammation. Additionally, dysfunction of the neuronal actin cytoskeleton represents a crucial step in many degenerative CNS pathologies. The integrity and dynamics of the neuronal actin cytoskeleton is pivotal for growth cone motility, axon outgrowth and regeneration, and dendritic spine dynamics. In this study, we explored neurodegenerative mechanisms in SH-SY5Y human neuroblastoma cells exposed to either the proinflammatory cytokine tumor necrosis factor alpha (TNFα), or the amyloid beta fragment (Aβ) associated with Alzheimer’s pathology. We elucidated a molecular pathway linking TNFα or Aβ-exposureto the destruction of the neuronal actin cytoskeleton and dysfunction of neuronal motility processes. TNFα and Aβ both stimulated a rapid increase in ceramide by activating a neutral sphingomyelinase. The role of the neutral sphingomyelinase was evaluated pharmacologically, and by assay of enzymatic activity. Increases in ceramide further resulted in an activation of a neuronal NADPH oxidase (NOX) activity as revealed by phosphorylation and membrane translocation of cytosolic NOX components, and the production of oxygen radicals as revealed by fluorescent imaging. Lastly, 2,4- dinitrophenylhydrazine derivatization of protein carbonyls revealed irreversible oxidative damage to immuno-precipitated actin.

    Together, our results implicate ceramide as a key intermediate of Aβ- andTNFα-induced NOX activation in neuronal cells. Moreover, this investigation links neuronal NOX activation with an irreversible oxidative damage to the neuronal actin cytoskeleton. Thus, inflammatory processes associated with acute and chronic CNS degeneration could directly contribute to the failure of axon regeneration. A more detailed understanding of this pathway in primary neurons could allow for the development of more specific therapeutic approaches to improve axonal regeneration and neuronal connectivity.

    Supported by USDA grant number 2005-34495-16519 and NIH grant number U54 NS41069.

  • Ceramide regulates free calcium levels in endoplasmic reticulum of hippocampal neuroblasts: possible role in initiation of neuronal differentiation.


    V. Voccoli, L. Colombaioni.

    Istituto di Neuroscienze, CNR. Via G. Moruzzi, 1 56100 Pisa, Italy.

    Ceramide is largely known as a lipid second messenger capable of regulating different cellular functions. Increases in intracellular ceramide levels have been mainly related to the apoptosis onset, but also the processes of growth suppression or terminal differentiation are potentially controlled by this lipid or by its metabolites, such as ceramide-1-phosphate (C1P) or glucosylceramide (GlcCer). With this study we have analyzed the cellular signaling correlated to the early phases of neuronal differentiation in the hippocampal cell line HN9.10e. We have found that, in response to pro- differentiating stimuli, endoplasmic reticulum (ER) undergoes functional modifications that are closely correlated to the progression of the neuritic growth. In particular, the enhancement of ER free calcium, appears to be a key factor in promoting differentiation. We have observed that also the application of C2Cer, a membrane-permeant analogous of ceramide, at low doses (100-500nM) rapidly stimulates neuritic growth. C2Cer induces a free calcium increase in ER comparable to that observed during differentiation induced by agents such as retinoic acid (RA). Also the treatment with the ceramidase inhibitor N-oleoylethanolamine (NOE) at low doses (5 uM) and for short periods (2-5 hours) mimics the pro-differentiating effects of C2Cer, and produces a free calcium increase in ER. This indicates that also endogenous ceramide is capable of controlling the early phases of differentiation. We have previously demonstrated that 25-75 uM C2Cer has a pro-apoptotic action in HN9.10e neuroblasts. These data suggest that while relatively high levels of intracellular ceramide can trigger the cellular apoptotic death, lower levels are inductors of neuritic growth and differentiation. A direct action on calcium homeostasis in ER appears to be the major event in the intracellular signalling triggered by ceramide. It is possible that the observed effects on ER calcium homeostasis are not directly due to ceramide, but rather to some ceramide metabolite.

  • Metabolic and functional role of the ceramide binding protein CERT in glioma cells.


    P. Giussani1, T. Colleoni1, R. Bassi1, L. Brioschi1, G. Tettamanti1, K. Hanada2, L. Riboni1, P. Viani1.

    1Department of Medical Chemistry, Biochemistry and Biotechnology, Faculty of Medicine, University of Milan, Milan, Italy. 2Department of Biochemistry and Cell Biology, National Institute of Infection Diseases, Tokyo, Japan.

    Different studies demonstrate that in glial cells ceramide (Cer) exerts antiproliferative and proapoptotic effects, and strongly support that Cer- signalling is altered in glial tumors. The control of ceramide levels in glial cells involves specific enzymes which are known to be localized in different subcellular compartments as well as Cer movements along the sphingomyelin biosynthetic pathway. A key element in defining the role of Cer in sphingolipid metabolism and signalling is its hydrophobic nature, and its consequent inability to spontaneously move among different subcellular sites where the enzymes of its metabolism and its molecular targets are located. Based on this evidence, the biological effect exerted by Cer may depend on the presence of specific signalling pools of this bioactive sphingoid in the cell, as well as the regulation of its intracellular traffic. Recently, the identification in CHO cells of the Cer specific carrier protein CERT have revealed a novel pathway for the delivery of Cer to the Golgi apparatus for sphingomyelin biosynthesis. In this study we investigated the metabolic and functional role of CERT in glioma cells. All glioma cells analyzed constitutively express CERT, the protein being mainly associated to the cytosolic fraction. Metabolic experiments performed with different radioactive metabolic precursors of sphigolipids indicate that, in all analyzed glioma cells, downregulation of CERT by RNA interference technology promoted a significant but not complete reduction of the amount of Cer converted to SM. This suggests that in glioma cells CERT mediated Cer transport contributes in addressing ceramide toward sphingomyelin biosynthesis. Since the regulation of sphingomyelin biosynthesis represents a crucial step in the role of ceramide on glial cell proliferation we evaluated the possible role of CERT in the control of glioma cell proliferation. We found that in all glioma cells down regulation of CERT resulted in an increased cell proliferation associated to higher ERK1/2 phosphorylation. In vitro and in vivo experiments indicated that CERT is necessary for the inhibition exerted by Cer on ERK activation. In conclusion these results suggest that CERT, besides the involvement in sphingolipid metabolism, participates to Cer signalling and could play a role in the control of glioma cell proliferation.

  • Ceramide 1- phosphate stimulates cell proliferation in bone marrow- derived macrophages through activation of the PI3-K/PKB (Akt) and MAPK pathways.


    P. Gangoiti1, M. Granado1, J. Y. Kong2, U. P. Steinbrecher2, A. Gómez- Muñoz1.

    1Department of Biochemistry and Molecular Biology, School of Science and Technology, University of the Basque Country, 48080 Bilbao, Spain.2Department of Medicine, University of British Columbia, Vancouver, Canada.

    We previously reported that ceramide 1-phosphate (Cer 1-P) was bioactive as it stimulated DNA synthesis and cell division in rat or mouse fibroblasts, and prevented cells from entering apoptosis. A major mechanism whereby Cer 1-Pblocks apoptosis involves the inhibition of acid sphingomyelinase. However, the mechanisms or signaling pathways that are implicated in the mitogenic effect of Cer 1-P are incompletely understood.

    In the present work, we show that Cer 1-P stimulates DNA synthesis and cell proliferation in bone marrow-derived macrophages. This effect was independent of phospholipase C or phospholipase D activation, it did not require intracellular calcium mobilization and did not alter the intracellular levels of cAMP. However, Cer 1-P induced strong and rapid phosphorylation of ERK1-2 in the macrophages, and inhibition of this pathway by PD098059 or UO126, which are selective inhibitors of mitogen-activated protein kinase kinase (MEK), blocked macrophage proliferation. In addition, Cer 1-P caused potent activation of PKB, as determined by its phosphorylation on Ser 473. PKB phosphorylation was blocked by the PI3-K inhibitor LY290042, suggesting that PKB is downstream of PI3-K in this system. Of importance, this PI3-Kinhibitor also blocked the mitogenic effect of Cer 1-P in the macrophages. We also found that treatment of the cells with Cer 1-P resulted in phosphorylation of glycogen synthase kinase-3 beta (GSK-3b), which is a kinase downstream of ERK1-2 or PKB. GSK-3b has been shown to stabilize the levels of proteins that are involved in the control of cell cycle, such as for instance cyclin D1 (CD1). Interestingly, Cer 1-P was also able to increase the expression of CD1 in the macropahges.

    Taken together these results suggest that the PI3-K/PKB and MEK/ERK1-2pathways are implicated in the stimulation of macrophage proliferation by Cer 1-P.

    This study was supported by grants BFU2006-13689/BFI from “Ministerio de Educación y Ciencia” (Madrid, Spain) and 9/UPV 00042.310-15852/2004 from “Universidad del País Vasco” (UPV/EHU) (Bilbao, Basque Country).

  • Sphinganine and sphinganine to sphingosine ratio as a biomarker of dietary fumonisins during chronic exposure in ducks.


    J. D. Bailly1, D. Tardieu1, A. Auvergne2, R. Babilé2, P. Guerre1.

    1Department of mycotoxicology, National veterinary school of Toulouse, 23 chemin des capelles, 31076 Toulouse, France and 2National agronomy school of Toulouse, BP 107, 31326 Castanet Tolosan cedex, France.

    Fumonisin B1 is structurally similar to sphinganine (Sa) and sphingosine (So) and inhibits ceramide synthase. It causes an elevation in Sa concentration, leading to an increase in Sa/So ratio in exposed animals. This increase often precedes fumonisins associated toxicity and that is why Sa/So ratio and free Sa have been proposed as biomarkers of fumonisin exposure in many species. The purpose of this study was to investigate the kinetics of Sa and So concentrations and Sa/So ratio in serum,liver and kidney over a 77 days exposure of ducks to different doses of FB1 to determine their use as biomarker of dietary fumonisins. The kinetics of 1P derivatives was also investigated in liver. One hundred and thirty mule ducks, 7 days of age, were randomly distributed into 5 groups receiving daily via oral route a dose of FB1 equivalent to ingestion of 0, 2, 8, 32 and 128 mg FB1/kg feed. On treatment days 7, 14, 21 and 28, 5 animals per group were bled and killed using carbon dioxide. The five remaining animals in each group were bled once a week during 7 further weeks. Neither mortality nor sign of mycotoxicosis were observed during the 77 days of treatment, no macroscopic nor microscopic lesions were observed on organs at the end of the treatment period. Determination of the kinetics of Sa/So ratio in serum revealed that Sa/So has increased dramatically from day 0 to day 7 (more than 3000% by using 128 mg FB1/kg feed). Then from day 7 to 28, a strong decrease occurred. The ratio continued to decrease slowly from day 28 to day 49 then stabilized throughout day 77. These variations were mainly due to modifications of Sa concentration in treated animals whereas only moderate effects were observed on So. Sa/So ratio in liver showed a very good correlation with that observed in serum. The kinetics was similar with a maximum reached at day 7 followed by a decrease until day 77. In the kidney, Sa/So analysis revealed that the ratio increased in all treated ducks to reach a maximum at day 7. Then after a slow decrease from day 7 to 14, the ratio stabilized until day 77. Liver sphinganine 1-phosphate was strongly increased in ducks fed 32 and 128 mg FB1/kg feed. Interestingly this increase is proportional to the amount of Sa within cells since Sa1P/Sa ratio remained nearly constant throughout the study, whatever the FB1 concentration in feed. In conclusion, although they can be considered as resistant to fumonisin toxicity, mule ducks are sensitive to the disruption of sphingolipid metabolism generated by these mycotoxins. However, this modification is also depending on the duration of exposure. Therefore, it seems that Sa/So ratio and Sa can be good biomarkers of exposure when ducks are exposed over short periods (1-2 weeks). If birds are exposed over 7 weeks, the determination of these parameters will probably fail to detect exposure. Although the mechanism of resistance of ducks to FB1 toxicity is still uncertain, it might be linked to the conversion of sphingoid bases into their 1P or other metabolite, and their elimination from the target tissues.


  • Purification and characterization of human intestinal neutral ceramidase.


    L. Ohlsson1, C. Palmberg2, R.-D. Duan1, M. Olsson1, T. Bergman2Å. Nilsson1.

    1Gastroenterology and Nutrition Laboratory, Biomedical Centre B11, Lund University, S-22184 Lund, Sweden. 2Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S-171 77 Stockholm, Sweden.

    Sphingolipids are degraded by sphingomyelinase and ceramidase in the gut to ceramide and sphingosine, which may inhibit cell proliferation and induce apoptosis, and thus may have antitumour and anti-inflammatory effects in the gut. Although previous rodent studiesincluding studies on neutral ceramidase (-/-) (ash2 -/-) miceindicate a role of neutral ceramidase in ceramide digestion, the human intestinal ceramidase has never been purified and characterized. We here report the purification and characterization of neutral ceramidase from human ileostomy content, using14C-octanoyl-sphingosine as substrate. After four chromatographic steps, HiTrapQ, PD10, CHT hydroxyappatite and Superdex 200 HR chromatography, a homogenous protein band with molecular mass 116 kDa was obtained. MALDI mass spectrometry identified 16 peptide masses matching human mitochondrial ceramidase3. By RT-PCR and 5'RACE method, a predicted partial nucleotide sequence of neutral ceramidase was obtained from a human duodenum biopsy sample, which was homologous to that of known neutral/alkaline ceramidases. The enzyme has neutral pH optimum and catalyzes both hydrolysis and formation of ceramide in presence of bile salts. Distinct stimulation by any specific bile salt, was, however, not observed. The ceramidase activity is inhibited by Cu2+ and Zn2+ ions and by low concentrations of cholesterol. The enzyme is a glycoprotein but deglycosylation does not affect its activity. Our study indicates that neutral ceramidase is expressed in human intestine, released in the intestinal lumen and plays a major role in ceramide metabolism in the human gut.

    1Olsson et al., Am. J. Physiol. 2004 287:G929. 2Kono et al., J. Biol. Chem. 2006 281:7324. 3El Bawab et al., J. Biol. Chem. 2000 275:21508.

  • Effects of bile diversion in rats on intestinal sphingomyelinase and ceramidase.


    R. D. Duan1, H.J. Verkade2, Y. Cheng1, R. Havinga2, A. Nilsson1.

    1Gastroenterology Laboratory, Biomedical Center, B11, Lund University, Lund, Sweden. 2Pediatric Gastroenterology, University Medical Center Groningen, University of Groningen, Groningen, Netherlands.

    Alkaline sphingomyelinase (Alk-SMase) and neutral ceramidase (N-Case) are localized in the intestinal microvillar membrane as ecto-enzymes and released into the gut lumen in an active form by bile salts and trypsin. The activities of these enzymes are also increased by bile salts at micellar concentrations. These enzymes have been shown to play important roles in sphingomyelin digestion in the small intestine and have implications in tumorigenesis in the colon. It is unclear to what extent that the intestinal presence of bile salts is critical for the intraluminal activities of these enzymes. In the present study, we compared the activities of Alk-SMase,N-CDase, and other types of SMases in control and permanently bile diverted rats. Bile deficiency rats were equipped with permanent catheters in bile duct and continuously bile drained. Animals were allowed to recover from surgery for 1 week and were subjected to overnight fasting before experiment. We found that in the intestinal tract of control rats, the activity of Alk-SMase was profoundly higher than those of acid and neutral SMases; the activity of neutral SMase in the lumen is very low, being less than 1% of that of Alk-SMase. Bile diversion reduced Alk-SMase activity by 85% in the small intestinal content, and by 68% in the faeces. Western blot showed that the reduced activity was caused by decreased level of enzyme protein in the intestinal lumen. In the mucosa of small intestine, the activities of acid and neutral SMase were only about 1-3 % of that of Alk-SMase. Bile diversion did not significantly affect Alk-SMase in the mucosa of small intestine. N-CDasedistributed in parallel with Alk-SMase in the intestinal tract. Bile diversion significantly reduced N-CDase activity both in the intestinal mucosa and content. The activity of alkaline phosphatase, another brush border enzyme, was not significantly changed in the intestinal mucosa by bile diversion. In conclusion, the intestinal presence of bile salts is important for maintaining high intraluminal levels of Alk-SMase and N-CDase, two key enzymes forstep-wise hydrolysis of sphingomyelin in the gut, and the digestion and absorption of sphingomyelin in cholestatic conditions could be impaired by the reduced hydrolytic capacities of the enzymes.

  • Influence of β-amyloid peptide, tumor necrosis factor-α and mexidole on the activity of neutral sphingomyelinase in rat brain.


    A.V. Alessenko, A.E. Bugrova.

    Institute of Biochemical Physics of the Russian Academy of Sciences, 4 Kosygin str. Moscow 119991, Russia.

    Alzheimer’s disease (AD) is characterized by progressive decline in cognition, memory and intellect. It has been hypothesized that amyloid-beta peptide (Aβ) and TNF-α may have prominent role in neurodegeneration. It is wellknown that neuronal death is developed according to apoptotic program. Most signaling pathway that trigger apoptosis remain unknown, but the sphingomyelin pathway has been recognized as a ubiquitous signaling system that links specific cell-surface receptors and environmental stresses to the nucleus. This pathway is initiated by the hydrolysis of sphingomyelin via the action of sphingomyelinases to generate ceramide. Ceramide then serves as a second messenger in this system, leading to apoptosis. Recently it was shown that cross-talk between oxidation system and sphingomyelin cycle exists in cells and could have important implication for the induction phase and evolution of AD. TNF-α activates receptors linked to multiple effector systems, including a sphingomyelin pathway and peroxide oxidation. Taking together the role of free radical reactions in pathogenesis of AD and participation of sphingomyelin pathway in apoptosis of neuronal cells, we have monitored induction of sphingomyelin cycle and activation of lipid peroxidation in rat brain sections after a single intracerebral injection ofAβ(25-35), TNF-α and their combination. For protection of brain against Aβ toxicity we injected mexidole- wellknown membraneprotector used for neurology for treatment ischemia and insult. We have determined the changes of neutral sphingomyelinase activity, sphingomyelin (SPM) and ceramide contents and level of lipid peroxide products (conjugated dienes and ketodienes) in the cerebral cortex, hippocampus and cerebellum of rats within 3 hrs and 7 days after Aβ (3 nmol per rat) and TNF- α (2 mkg per rat) intracerebral injection and mexidole after interperitonal injection in doses 100 and 200 mg per kg of rats. Maximal changes in the activity of sphingomyelinase, SPM, ceramide contents after single TNF-α and Aβadministration were found in the hippocampus, and were less expressed in the cerebral cortex and cerebellum. We found intensification of lipid peroxidation after 3 hrs of peptides injection to the rat brain, but activity of sphingomyelinase was not increased. Remarkable increases in sphingomyelinase activity and content of ceramide were found after 7 days of injection of Aβ. Increase in the level of peroxide products in hippocampus after 7 days of Aβ and TNF-α injection was still remarkable, but combinative injection of TNF-α and Aβ in the rat brain decreased activity of sphingomyelinase and level of ketodienes to normal level after 7 days. It was proposed that TNF-α may protect brain cells from injury induced by Aβ. The role of TNF-α in the pathogenesis of AD is unclear, because it has been shown to be involved in both neuroprotection and neurodegeneration depending on doses and age of animals. Small doses of this cytokine induced protective effect against Aβ neurotoxicity. In our experiments we used low dose of TNF-α. We suggest that using low doses of TNF-α in clinic for prevention of development of AD might be perspective. Mexidole decreased the level of lipid peroxides and activity of sphingomyelinase and content of ceramide in hippocampus. We suppose also that new drugs, protected brain cells from sphingomyelin digestion could be used for preventing or slowing the onset of AD.

  • Critical role for Sphingosine Kinase-1 in regulating survival of neuroblastoma cells exposed to amyloid-beta peptide.


    A. Gómez-Brouchet1,2,3, D. Pchejetski1,2,4, M.-L. Maddelein2,4, V. García1, M.- F. Altié1, M.-B. Delisle1,2,3O. Cuvillier1,2,4.

    1INSERM, U466, Toulouse, F-31000 France. 2Université Toulouse III Paul Sabatier, Toulouse, F-31000 France. 3CHU Toulouse, Service d’Anatomie et de Cytologie Pathologiques, Toulouse, F-31000 France. 4CNRS, Institut de Pharmacologie et de Biologie Structurale, UMR 5089, Toulouse, F-31000France.

    The amyloid-beta (Aβ) peptide, the main constituent of amyloid plaques, is believed to play a causative role in the neurodegenerative process occuring in Alzheimer’s disease. Although Aβ-mediated neuronal cell death demonstrates biochemical characteristics of apoptosis, the molecular mechanism underlying Aβ toxicity remains largely undefined. Noteworthy, increased levels of ceramide have been found in the brain of Alzheimer’s disease patients, thereby implying that ceramide accumulation could contribute to Alzheimer’s disease pathogenesis. In addition Aβ toxicity was recently shown to be linked with ceramide generation both in cell culture models and in animal model. Compelling data have revealed the potential involvement of insulin-like growth factor I (IGF-I) in the Alzheimer’s disease pathophysiology. Low serum levels of IGF-I is correlated with premature brain amyloidosis and IGF-I has been found to regulate Aβ clearance from the brain. Furthermore, IGF-I prevents Aβ-induced neuronal cell death including inSH-SY5Y cells. The TGF- b1 peptide growth factor also protects neurons from a variety of insults including Aβ.

    We examined the role of sphingosine kinase-1 (SphK1), a critical regulator of the ceramide/sphingosine 1-phosphate (S1P) biostat, in the regulation of death and survival of SH-SY5Y neuroblastoma cells in response to Aβ peptide(25-35). Upon incubation with Aβ, SH-SY5Y cells displayed a marked down- regulation of SphK1 activity coupled with an increase in the ceramide/S1P ratio followed by cell death. This mechanism was redox-sensitive as N- acetylcysteine totally abrogated the down-regulation of SphK1 activity and strongly inhibited Aβ-induced cell death. SphK1 overexpression impaired the cytoxicity of Ab while SphK1 silencing by RNA interference mimicked Aβ- induced cell death hence establishing a critical role for SphK. We further demonstrated that SphK1 could mediate the well-established cytoprotective action of IGF-I against Aβ toxicity through a PI3 kinase-dependent mechanism as wortmannin and LY294002 blocked SphK1 activation. A dominant-negativeform of SphK1 or its pharmacological inhibition not only abrogated IGF-I-triggered stimulation of SphK1 but also hampered IGF-I protective effect. Similarly to IGF-I, the neuroprotective action of TGF-b1 was also dependent on SphK1 activity as activation of SphK1 as well as cell survival were impeded by a dominant-negative form of SphK1. Collectively, these results provide the first illustration of SphK1 role as a critical regulator of death and survival ofAβ-treated cells.

  • Inhibition of sphingosine-1-phosphate-induced endothelial cell chemotaxis and PAF-synthesis by red grape and bilberry polyphenols.


    C. Barthomeuf1,2, S. Lamy3, P. Chollet1,4, R. Beliveau3.

    1INSERM-484, Université d’Auvergne, Centre hospitalier Jean Perrin, rue Montalembert, 63005 Clermont-Fd Cedex, France, 2Laboratoire de Pharmacognosie et Biotechnologies, Université d’Auvergne, Faculté de Pharmacie, Place H. Dunant, 63001 Clermont-Fd Cedex, France, 3Laboratoire de Médecine Moléculaire de l’Hôpital Sainte-Justine, Centre de Cancérologie Charles-Bruneau, Montréal, Québec, Canada and 4Unité de recherche clinique, Centre hospitalier Jean Perrin, Centre Anticancéreux, 63011 Clermont-Fd, France.

    Sphingosine-1-phosphate (S1P) is a biologically active sphingolipid and a pro- inflammatory mediator, secreted mainly by platelets at clotting sites and, by several cell-types (including cancer cells) in response to various external stimuli. Five members of the endothelial differentiation gene (EDG) family have been identified as S1P receptors in a wide range of cell-lines. EDG- 1/S1P, a member of this receptor family, has recently been identified as an inducible transcript expressed during endothelial cell (EC) differentiation. By promoting the synthesis of endothelial platelet-activating factor (PAF) which stimulates EC migration, S1P enhances inflammation and pathological angiogenesis and, thereby, aggressive tumour proliferation and metastasis. We have recently reported that a red grape skin polyphenolic extract (SGE) preventsVEGF-induced endothelium neutrophil adherenceand S1P-induced EC chemotaxisby decreasing the acute synthesis of PAF in S1P-stimulated ECs. These data provided the first demonstration that grape polyphenols may inhibit the S1P/endothelial differentiation gene-1 cascade. Inhibiting this cascade appears of great interest in cancer patients and in patients with cardiological disfunction. New experiments have been done to understand how SGE prevented S1P/EDG1 activation and,to compare the preventive effect of SGE and a commercially bioavailable highly purified bilberry extract (Antho50®) on S1P-induced HUVEC chemotaxis.Antho50® at 25 µM efficiently inhibited theserum-induced migration of androgen-resistant PC3 prostate cancer cells but, conversely to SGE, was unable to reverse the S1P/endothelial differentiation gene-1cascade activation in S1P-stimulated endothelial cells. Western blot, biochemical andMS-MS analysis revealed that SGE at 25 µM inhibited the immediate release of lysophophosphatidylcholine and of C16- and C18-alkyl PAF in HUVECs elicited by S1P. They correlated these effects with a lower activity and expression of group V secreted PLA2 (VsPLA2) in HUVECs' membrane and a down-regulation of p38 MAPK and Akt in cells. In contrast to SGE, Antho50# at 25 µM was unable to inhibit the phosphorylation of p38 MAPK. These new data show that inhibitingV-sPLA2 activity and expression and subsequent p38 activation is critical for the prevention of S1P-induced signaling .

    Barthomeuf et al., Bull. Cancer 2006 93:S123. 2Barthomeuf et al., Free Rad. Biol. Med. 2006 40:581.


  • Sphingosine 1-phosphate: a novel stimulator of aldosterone secretion.


    L. Brizuela, M. Rábano, J. M. Macarulla, M. Trueba, A. Gómez-Muñoz.

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

    Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid capable of regulating critical physiological and pathological functions. We have reported for the first time that S1P stimulates aldosterone secretion in cells of the zona glomerulosa of the adrenal gland. Regulation of aldosterone secretion is important because this hormone controls electrolyte and fluid balance and is implicated in cardiovascular homeostasis. S1P-stimulated aldosterone secretion was dependent upon the protein kinase C (PKC) isoforms α and δ and extracellular Ca2+ and it was inhibited by pertussis toxin (PTX). S1P activated phospholipase D (PLD) through a PTX-sensitive mechanism, also involving PKC α and δ and extracellular Ca2+. Primary alcohols, which attenuate the formation of phosphatidic acid (the product of PLD), and cell- permeable ceramides, which inhibit PLD activity, blocked S1P-stimulatedaldosterone secretion. Furthermore, propranolol and chlorpromazine, which are potent inhibitors of phosphatidate phosphohydrolase (PAP) (the enzyme that produces diacylglycerol from phosphatidate), also blocked aldosterone secretion. These data suggest that the PLD/PAP pathway plays a crucial role in the regulation of aldosterone secretion by S1P and that Gi protein-coupledreceptors, extracellular Ca2+, and the PKC isoforms α and δ are all important components in the cascade of events controlling this process.

    This study was supported by grants 9/UPV 00042.310-15852/2004 from “Universidad del País Vasco” (UPV/EHU) (Bilbao, Basque Country), and BFU2006-13689/BFI from “Ministerio de Educación y Ciencia” (Madrid, Spain).

  • Internalisation of sphingosine-1-phosphate receptors.


    M. Jongsma, U. M. Florczyk, M. C. Hendriks-Balk, P. B. van Loenen, M. C. Michel, S. L. M. Peters, A. E. Alewijnse.

    Department of Pharmacology and Pharmacotherapy, Academic Medical Center, Amsterdam, The Netherlands.

    Internalisation is an important regulatory process that controls the signalling via G-protein coupled receptors. Upon ligand binding, receptors can be phosphorylated and translocated from the cell membrane to internal vessicles. From there, they are either recycled or broken down. Forsphingosine-1-phosphate (S1P) receptors, regulatory processes are of particular interest as there now is a drug in clinical trials which mechanism of action is suggested to involve downregulation of S1Preceptors on T-cells. In this study we aimed to compare internalisation of different S1P receptor subtypes, which requires a sensitive and quantitative assay. While several methods have been used to study receptor internalisation, none of them allows a precise quantitative description. We have therefore set up a new method to quantitatively measure internalisation with N-terminal HisG- tagged receptors stably transfected into CHO-FlpIn cells. Agonist exposure caused internalisation of S1P1, S1Pand S1Preceptors, but that of the S1P3receptor differed from that of the other subtypes in several ways: It required much lower S1P- concentrations despite S1P having comparable affinity and potency at all subtypes. While the S1P concentrations required to increase intracellular calcium and/or affect cAMP accumulation were similar to those required for internalisation at the S1Preceptor, the other subtypes required a much higher S1P concentration to induce internalisation than to activate signalling. Moreover, S1Preceptor internalisation was much faster than that of S1Pand S1Preceptors, requiring 5 min vs. up to 30 min. This may be explained by a recent finding that internalisation of the S1Preceptor is independent of phosphorylation in contrast to that of the S1Preceptor. Preliminary results suggest that also resensitisation differs among these three S1P receptor subtypes. In conclusion, our study shows that S1P1, S1Pand S1P3receptors differ markedly with regard to agonist-induced internalisation. We hope to elucidate the mechanisms responsible for these differences in future studies.

  • Sphingosine-1-phosphate induces apoptosis in lyase deficient primary cultured murine cerebellar neurons.


    N. Hagen1, R. Broere1, P. P. Van Veldhoven2, G. van Echten-Deckert1.

    1Kekulé-Institut für Organische Chemie und Biochemie, Rheinische Friedrich-Wilhelms-Universitaet Bonn, Gerhard-Domagk-Strasse 1, 53121 Bonn, Germany. 2Katholieke Universiteit Leuven, Campus Gasthuisberg, Afdeling Farmakologie, Herestraat Box 601, B-3000 Leuven, Belgium.

    Sphingosine-1-phosphate (S1P) was shown to regulate a wide range of physiological processes including proliferation, differentiation, motility, cytoskeleton rearrangements and calcium homeostasis1. Using cis-4-methylsphingosine, a synthetic sphingosine analogue pro-drug that is phosphorylated intracellulary to a metabolically stable S1P analogue, we have, however, demonstrated that in terminally differentiated post-mitoticneurons S1P has an apoptotic rather than an anti-apoptotic effect2. Accordingly, primary cultured neurons from 6-days-old lyase deficient mice, which are unable to cleave phosphorylated sphingosine, underwent apoptosis in response to exogenously added S1P. As in case of the lyase resistant phosphorylated cis-4-methylsphingosine, an abortive reactivation of the cell cycle on the one hand and activation of caspases on the other hand appear to be essential for S1P-induced apoptosis in lyase deficient neurons. Note that lyase deficient mice exhibit a complex phenotype including pronounced growth impairment and shortened life span that generally does not exceed the nursing period3.

    The (patho)physiological significance of our observations is not yet clear but neuronal death could be indicative for an involvement of S1P in neurodegenerative disorders.

    1Spiegel and Milstien, Nat. Rev. Mol. Cell. Biol. 2003 4:397. 2Naetzker et al., Genes Cells 2006 11:269. 3Van Veldhoven, Chem. Phys. Lipids 2005 136:164.

  • Sphingosine kinase-1 is a downstream regulator of imatinib-induced apotosis in LAMA84 chronic myeloid leukemia cells.


    E. Bonhoure1,2, A. Lauret1, B. Malavaud2,3, T. Kohama4, J. Melo5, O. Cuvillier1,2.

    1INSERM, U466, Toulouse, F-31000 France, 2CNRS, Institut de Pharmacologie et de Biologie Structurale, UMR5089, Toulouse, F-31000 France, 3Université Toulouse III Paul Sabatier, Toulouse, F-31000 France, 4Sankyo Co. Ltf., Core Technology Research Laboratories, Tokyo, 140-8710, Japan and 5Department of Haematology, Falculty of Medicine, Imperial College London, Hammersmith Hospital, London, United Kingdom.

    Chronic myeloid leukaemia (CML) is a myeloproliferative disease characterized by the expression of the oncogenic fusion protein tyrosine kinase BCR-ABL that is essential for leukemogenesis and which occurs in 95% of CML. Inhibition of this tyrosine kinase activity appears to be the most effective way to abolish the activity of this oncoprotein. Imatinib mesylate (or STI571), a specific inhibitor of BCR-ABL, exhibits potent antileukemic effects in vitro and in vivo, and is now the first-line treatment for CML. Despite imatinib is highly effective in the treatment of CML, the emergence of resistance has now become a significant issue. A better understanding of the signaling pathways initiated by imatinib in CML cells is a prerequisite step in order to develop new therapeutic strategies to treat patients who do not respond anymore to imatinib.

    In our study, we examined the involvement of the oncogenic sphingosinekinase-1, a critical regulator of the ceramide/sphingosine 1-phosphatesphingolipid balance, in the imatinib resistance of LAMA-84 cells. Contrary to sensitive LAMA-84s cells, imatinib failed to induce apoptosis in chemoresistant LAMA-84r cells overexpressing Bcr-Abl. The chemosensitivity of LAMA-84s to imatinib was associated with a decrease in sphingosinekinase-1 activity and S1P level and an increase in ceramide level. In contrast, chemoresistant LAMA-84r had sustained sphingosine kinase-1 activity and did not produce ceramide during treatment with imatinib. Overexpression of sphingosine kinase-1 in LAMA-84s cells resulted in marked inhibition ofimatinib-induced apoptosis.

    It is well established that the Bcr-Abl signaling involves the Ras/Raf/MEK/ERK pathway. Inhibition of Ras and MEK has been shown to circumvent imatinib- induced resistance. Herein, we show that Ras farnesyl-transferase inhibitor (FTI) and MEK inhibitor (UO126) triggered sphingosine kinase-1 inhibition during apoptosis of LAMA-84r cells. Enforced expression of sphingosine kinase- 1 prevented cell death induced by FTI and UO126. To further establish the critical role for sphingosine kinase-1 in regulating CML apoptosis, we next studied the effect of cytosine β-D-arabinofuranoside (Ara-C), a known CML chemotherapy that has recently been shown to overcome imatinib-resistancein the LAMA84-r cell model. Interestingly, Ara-C induced marked inhibition of sphingosine kinase-1 activity during apoptosis, and sphingosine kinase-1overexpression counteracted its effect.

    We next established the proof-of-concept that sphingosine kinase-1 inhibition is an essential event to mediate apoptosis of CML cells by showing that sphingosine kinase-1 inhibition (pharmacological inhibitor, RNAi strategy) could kill CML cells regardless of their imatinib-resistance status. Overall, this work demonstrates that sphingosine kinase-1 could be a good target for efficiently overcoming the imatinib-resistance problem in CML.

  • The pleiotropic effects of S1P are mitigated by an anti-S1P mAb in multiple murine models of cancer, age-related macular degeneration and heart failure.


    R. A. Sabbadini.

    San Diego State University and Lpath, Inc., San Diego, CA, USA.

    It has been recognized that alterations in lipid metabolism can lead to cancer, cardiovascular disease, diabetes, neurodegenerative disorders, immune function, pain, mental disorders and inflammation. However, only as a consequence of our recent appreciation that bioactive lipids are bona fide signaling molecules have key members of the functional lipidome been viewed as targets for rational drug design in mitigating lipid-associateddisorders. Recent research has demonstrated that the bioactive signaling molecule, sphingosine-1-phosphate (S1P) plays a pivotal role in cancer progression. We hypothesize that S1P may also be associated with maladaptive wound healing and angiogenesis in various ocular and cardiovascular disorders as well. We have developed a bio-specificmonoclonal anti-S1P antibody (anti-S1P mAb) that could be used as a therapeutic molecular sponge to selectively neutralize S1P. The therapeutic potential of the anti-S1P mAb was analyzed in several murine models of cancer as well as a model of Age-related Macular Degeneration (AMD) and in awell-established murine model of heart failure. For cancer, theanti-tumorigenic effects of the anti-S1P mAb were evaluated in multiple cell lines representing a spectrum of histological cancer subtypes. The ability of S1P to stimulate proliferation, promotes cell invasion and protects tumor cell from apoptosis induced by chemotherapeutic drugs was neutralized by theanti-S1P mAb. We also demonstrate the ability of our antibody to inhibit angiogenesis. The anti-S1P mAb blocked both the migration of endothelial cells and their ability to form de novo capillary-like structures that resemble blood vessels using in vitro Matrigel models. In vivo, we investigated the ability of the anti-S1P mAb to reduce tumor volume and inhibit angiogenesis in multiple murine models. The anti-angiogenic effects of the antibody were also validated in a murine model of the ocular disorder, AMD, induced by laser burn or Bruch's membrane. The resulting choroid neovascular lesions were almost completely prevented by intravitreal injection of the antibody. Moreover, the profibrotic effects of S1P were mitigated by this treatment such that substantial reductions in scarring, inflammation and other aspects of maladaptive wound healing were observed. The anti-scarring effect of theanti-S1P mAb was also studied in a murine model of post-MI heart failure, a condition associated with maladaptive fibrosis and scarring that results in cardiac dysfunction and death. The antibody dramatically reduced scarring and improved cardiac function and animal survivability. Taken together, the findings from multiple murine disease models suggests that the anti-S1PmAbis not only a useful research tool to investigate S1P's role in tumorigenesis, angiogenesis and maladaptive wound healing, but it may represent a novel and innovative approach to the treatment of disease. The anti-S1P mAb may be the first example of targeting an extracellular bioactive lipid for use as a molecular sponge to prevent key ligand-receptor interactions. This leads one to argue that the antibody may be a first-in-class drug of an emerging discipline that may be termed lipidomic- based therapeutics.

  • Critical role of acidic sphingomyelinase in murine hepatic ischemia reperfusion injury.


    L. Llacuna, M. Marí, R. Paris, C. García-Ruiz, J. C. Fernandez-Checa, A. Morales.

    Liver Unit, Hospital Clinic, IDIBAPS, IIBB-CSIC, Barcelona, Spain.

    Hepatic ischemia reperfusion (I/R) damage can occur in diverse settings including liver transplantation, trauma, hemorrhagic shock or liver surgery, and represents a serious clinical complication that may compromise liver function because of extensive hepatocellular loss. However, the molecular mechanisms of hepatic ischemia reperfusion (I/R) damage are not completely known. Thus, we investigated the role of ceramide in a murine model of warm hepatic I/R injury, as this sphingolipid induces cell death and participates in TNF signaling. Determination of hepatic ceramide homeostasis and its metabolic regulation during I/R. Inhibition or suppression of ceramide- regulating enzymes and consequences in hepatic I/R damage. Hepatic ceramide levels transiently increased after the reperfusion phase of the ischemic liver in mice, due to an early activation of acidic sphingomyelinase (ASMase) followed by acid ceramidase stimulation. In vivo administration of an ASMase inhibitor, imipramine, or ASMase knockdown by siRNA, which decreased ceramide generation during I/R, significantly attenuated serum ALT levels, hepatocellular necrosis, cytochrome c release and caspase-3 activation, by preventing JNK activation and Bim translocation to mitochondria. In contrast, blockade of ceramide catabolism by N- oleyolethanolamine (NOE), a ceramidase inhibitor, enhanced ceramide levels and potentiated I/R injury compared to vehicle-treated mice. Pentoxifylline treatment prevented both TNF up-regulation and ASMase activation and protected the liver against I/R injury. Moreover, 80-90% of rats treated with imipramine survived 7 days after total liver ischemia compared to 30-40%survival of vehicle-treated animals, while 100% NOE-treated rats died within 2 days of total liver ischemia. Ceramide generated from ASMase plays a key role in I/R-induced liver damage and its modulation may be of therapeutic relevance.

  • Searching for biomarkers of Gaucher disease using proteomic tools.


    L. Quintana1, A. Monasterio2, L. Simón2, A. Martínez2, P. Giraldo1, M. Pocoví1

    1Instituto Aragonés de Ciencias de la Salud, Edificio CEA, Avda. Gómez Laguna 25, 50009 Zaragoza. 2Proteomika SL, Parque Tecnológico de Vizcaya, Edificio 801B, 48160 Derio.

    Introduction. Gaucher disease (GD) is a lysosomal glycolipid storage disease caused by mutations in the acid β-glucosidase gene that shows autosomal recessive inheritance. This defect causes a deficiency in the glucocerebrosidase enzyme that leads to the accumulation of glucocerebroside in the lysosomes of macrophages. Many biomarkers show altered accumulation in the plasma of GD patients and can be used for disease diagnosis and follow-up. Chitotriosidase protein (ChT) is the most important biomarker described in GD. However, its clinical application is restricted because a common genetic defect that results in the absence of detectable ChT in the plasma of 6% of Caucasian population. Thus, new surrogate biomarkers are necessary for effective diagnosis and to decide therapeutic intervention, such as the recently described chemokine CCL18/PARC. Proteomics has recently emerged as a new technology for global analysis of protein profiles and biomarker identification in biological fluids. This technology can play a critical role in the elucidation of GD pathophysiology and for monitoring patient response to therapies.

    Materials and Methods. In this work, a population of 39 individuals was studied for differentially expressed plasma proteins grouped as follows: 16 GD patients and 23 healthy controls. For patient sample collection, plasma was retrieved immediately following diagnosis and prior to treatment. Plasma samples were analysed using proteomic tools: proteins were resolved by two dimensional gel electrophoresis (2-DE) and silver stained. Image analysis was performed using Progenesis PG220 software (Nonlinear Dynamics). This analysis includes detection, quantization and normalization of each protein spot in every 2-DE image. Significant differences in protein expression levels between controls and GD patients were determined by t Student test with a set significance value of p < 0.05.

    Results. Following this methodology a set of 30 differentially-expressedplasma proteins has been identified by mass spectrometry. This set includes proteins that are involved in regulation of immune system and inflammation, metabolism of lipoproteins and coagulation cascade. Potential plasma biomarkers of GD are being still validated using specific antibodies bywestern-blot.

  • RNAi-mediated inhibition of the glucosylceramide synthase (GCS) gene: a preliminary study towards a therapeutic strategy for Gaucher disease and other glycosphingolipid storage diseases


    A. Diaz-Font1, A. Chabás2, D. Grinberg1, L. Vilageliu1.

    1Departament de Genètica, Facultat de Biologia, Universitat de Barcelona and 2Institut de Bioquímica Clínica, Corporació Sanitària Clínic. Barcelona, Spain.

    In the last few years, small interference RNAs (siRNAs) have been used in a number of different experimental settings to silence gene expression. In some of them, chemically synthesized or in vitro transcribed siRNAs have been transfected into cells. In others, siRNAs have been expressed endogenously from siRNA expression vectors. Enzyme replacement and substrate deprivation therapies are the current approaches to treat Gaucher disease. Although good results have been reported there are several limitations and side effects that make interesting to search for new alternatives. We present here a new approach based on the inhibition of theGCS gene using siRNAs as a possible future therapeutic strategy for Gaucher disease. We have designed four siRNAs for the human GCS gene and transfected them into HeLa cells. With two of them, a clear reduction of GCSRNA levels and enzyme activity was obtained. Consistently, a reduction in glucosylceramide synthesis was observed. Similar results were obtained when plasmids expressing shRNAs (targeting the same sequences) were transfected into the cells. The inhibition of the mouse homolog Ugcg gene was also achieved, using a siRNA that targeted both human and mouse sequences. In summary, we have shown that siRNAs are a good tool to silence GCS gene expression and, thus, to reduce glucosylceramide formation. This success at the cellular level, including human and mouse cells, should be followed by experiments in animal models to develop a new therapeutic strategy for Gaucher disease.

  • The Molecular Characterization of Gaucher Disease in Spain.


    P. Alfonso1,3 M. Pocovi1,3, P. Giraldo2,3.

    1Departamento de Bioquímica y Biología Molecular y Celular, Universidad de Zaragoza, Zaragoza, Spain. 2Miguel Servet University Hospital, Zaragoza, Spain. 3Institute Aragones of Health Sciences (I+CS), Zaragoza, Spain.

    Mutations in the β-glucocerebrosidase (GBA) gene cause Gaucher disease (GD), an autosomal recesive inherited disorder associated to organomegaly, thrombocytopenia, anaemia, bone disease and in the most severe cases neurological involvement. The aim of this study was to characterize the GBA mutations in a group of 193 apparently non-related from the Spanish GD Registry. We have screened all patients for the presence of N370S and L444P previously described mutations. To identify other unknown mutations the promoter region and the 11 exons with their flanking intron sequences of the GBA gene were screened by DNA sequencing. The allelic frequencies of Spanish are reported and the mutation profile is analyzed. This approach led to the identification of 98.7% of mutant GBA alleles. We have found 55 different GBA mutations and 65 genotypes causing GD in Spain: 49 mutations previously described, and 9 novel mutations: 4 missense R395C, R463H, W312R, V398I; one nonsense R359X, 3 frame-shift c.708delC, c.1214- 1215delGC, c.1439-1445del7, and one in-frame c.42-65 del 24. The most prevalent mutations were N370S and L444 P accounting for the 68.7 % of the mutant alleles. Among the type 1 GD patients, 93.8 % carried the mutation N370S. The wide phenotypic differences observed within the genotypic groups as well as between siblings implicate a significant contribution of other modifying genetic and/or non-genetic factors. All of these findings indicate that there is significant genotypic heterogeneity among Spanish GD patients.

    *on behalf of the Spanish Registry of Gaucher’s disease.

  • Effective Gene Therapy in a Mouse Model of Sandhoff Disease.


    B. Cachón-Gonzaléz1, S. Z. Wang1, R. Ziegler2, S. H. Cheng2, T. M. Cox.1

    1Department of Medicine, University of Cambridge, Cambridge, CB2 2QQ, UK and 2Genzyme Corporation, Framingham, MA, 01701-9322, USA.

    The inborn neurodegenerative Tay-Sachs and Sandhoff diseases are caused by mutations in the α- and β-subunits of β-hexosaminidase A (αβ) [HexA] and B (ββ) [HexB] isozymes, respectively. In the absence of active enzyme, GM2 ganglioside and related glycosphingolipids accumulate prominently in the lysosomes of neurones. This storage is associated with inflammation, cell loss and neurological dysfunction.

    The Sandhoff mouse, homozygous for a disrupted β-hexosaminidase β subunit allele (hex β -/-), serves as a model of human GM2 gangliosidosis: although normal at birth, it develops a neurodegenerative disease requiring euthanasia by the age of 16 weeks.

    We have used AAV-derived vectors to direct expression of human hex β (hhexβ) and hex α (hhexα) in the brain of Sandhoff mice in an attempt to improve outcome by gene therapy and thereby further to investigate the pathogenesis of this disease.

    One-month old Sandhoff mice were injected with a mixture of AAV2/2 expressing hhexβ (AAVhhexβ) and hhexα (AAVhhexα) or AAVhhexβ and AAVhhexα singly, in the presence of 6% mannitol. Single striatal injections, as well as four-site injections (two in the striatum and two in the cerebellum), were performed. All animals treated with AAVhhexα and β or AAVhhexβ alone, and culled at different post-injection times, showed widespread therapeutic gene expression ipsilaterally and contralaterally. Pathological glycolipid storage was cleared with reduced microglial expansion/activation and fewer apoptotic cells. The onset of symptoms in six untreated Sandhoff mice was 9613 (sd) days; they reached their humane end-point at 1204 days. Mice receiving control injections of AAVhhexα alone had a similar evolution with symptom onset and end points at 101 8 and 120 5 days, respectively. In contrast, six mice injected with a mixture of AAVhhexα and AAVhhexβ at a single site had delayed symptoms and reached the humane end point at 19917 days. More than 40% of animals receiving four-site injections of AAVhhexβ alone survived beyond one year, (p<0.0001).

    In this acute model of Sandhoff disease, our gene therapy strategy greatly increased survival and improved neuronal function with concomitant maintenance of motor coordination, balance and body weight – onset of ataxia and tremor was delayed. The intervention also decreased neuro- inflammatory and apoptotic responses, as well as glycosphingolipid storage.Co-injection of AAVhhexα with AAVhhexβ was not required for this salutary outcome.

  • Cyclodextrin-Mediated Chiral Separation of Sphingolipids and High Resolution Detection by Capillary Electrophoresis.


    B. M. Barth1,2,3, D. L. Kirschner2,3, T. B. Kuhn1,2,3, T. K. Green2,3.

    1Alaska Basic Neuroscience Program, 2Institute of Arctic Biology and3Department of Chemistry and Biochemistry, University of Alaska-Fairbanks,Fairbanks, Alaska 99775 USA.

    Sphingolipids are a unique and major class of lipids important not only to membrane structure and dynamics, but also to cellular function and signaling. The bioactive properties of sphingolipids are as diverse as the lipids themselves and range from mediation of survival and development to apoptosis. Numerous analytical techniques have been developed to study sphingolipids, yet the ability to analyze chirality of sphingolipids in biological samples remains rather impractical due to the large amount of sample often needed. In this study, we sought to develop a new method to detect and analyze sphingolipids that would be applicable to small biological samples.

    Capillary Electrophoresis (CE) is a powerful analytical tool for high resolution analysis of biological species including amino acids, peptides, and viruses. Micellular Electrokinetic Chromatography (MEKC) is a commonly employed CE technique often used for separation of hydrophobic molecules with charged or neutral functional groups. Cyclodextrins (CDs) are water soluble macrocyclic sugars commonly used as buffer additives in MEKC to improve separation efficiency and allow for chiral resolution. The CDs act by forming chiral host- guest inclusion complexes with the hydrophobic guest molecules. To date, we are unaware of any CE methods developed for analysis of sphingolipids.

    In this study we derivatized a mixture of D and L threo-dihydrosphingosinewith naphthalene-2,3-dicarboxaldehyde (NDA) and sodium cyanide to yield the highly fluorescent cyanobenz[f]isoindole derivatives. The analytes were separated and detected using CE with laser induced fluorescence (LIF) detection. Various buffer parameters affecting separation were investigated including a combination of 30 mM SDS and 20 mM α-CD, which achieved baseline resolution of dihydrosphingosine. The method was applied to the analysis of other sphingolipids and amino acids as well.

    Lastly, biological samples obtained from developing chicken embryos were investigated utilizing this unique CE method. Altogether, our research is an initial step towards the development of a unique method that allows for accurate and quantifiable low level detection of various chiral sphingolipids in biological samples.

  • Changes in glycosphingolipid content and overexpression of glucosyl- ceramide synthase are associated with acquired resistance to unrelated drugs in T98G human glioma cells.


    R. Bassi, P. Giussani, V. Anelli, P. Viani, L. Riboni.

    Department of Medical Chemistry, Biochemistry and Biotechnology, LITA- Segrate, Milan, Italy .

    Different studies support that glycosphingolipids (GSLs) act as important players in tumor biology, and emerging evidence suggests their involvement in drug resistance too. Glucosylceramide synthase (GCS) is a pivotal enzyme in sphingolipid metabolism which converts ceramide to glucosylceramide (GlcCer). In drug-resistant cancer cells and in tumor specimens from patients with scarce response to chemotherapy, the activity of GCS and the levels of GlcCer were found elevated. Moreover, inhibitors of GCS have been shown to increase the cellular levels of ceramide, a key player in the regulation of apoptosis, and to reverse drug resistance. Notwithstanding, little is known on the involvement of GCS in malignant gliomas. These are the most frequent and deadly human primary brain tumors, their intrinsic or acquired resistance limiting therapy effectiveness. In this study, we investigated the role of GCS in the resistance of T98G human glioma cells to paclitaxel and temozolomide, two unrelated drugs with clinical activity against gliomas. By selection with gradually increasing drug concentrations, we generated a paclitaxel (Tax-R)and a temozolomide (Tmz-R) resistant cell line. The analysis of resistance markers by immunoblotting showed that MDR1 (P-gp) expression was exclusive of Tax-R cells, whereas alkylguanine methyltransferase was present in all cell lines, and overexpressed exclusively in Tmz-R cells. After labeling at equilibrium with 3H-sphingosine or 3H-serine, Tax-R and Tmz-R were both characterized, with respect to sensitive T98G, by increased levels of both GlcCer and gangliosides. On the contrary, the levels of lactosyl-ceramide, the major neutral glycosphingolipid of T98G cells, were significantly lower inTmz-R, but not in Tax-R cells. In addition, the ceramide levels were found lower in resistant vs. sensitive cells. In both Tax-R and Tmz-R cells the in vitro activity of GCS was significantly higher than in sensitive cells. Moreover, a semi-quantitative RT-PCR analysis showed that a higher expression of the mRNA for the GCS gene was evident in both resistant cell lines. Finally, cytotoxicity assays revealed differences between the cell lines with respect to their sensitivity toward GCS inhibitors, cell survival in resistant cells being significantly lower than in sensitive ones. Noteworthy, exposure of Tmz-R cells to the GCS inhibitor PDMP restored cell sensitivity to temozolomide. Altogether our data demonstrate that overexpression of GCS and alterations of glycosphingolipid level, with increased GlcCer and ganglioside content, are associated to T98G resistance to unrelated cytotoxic drugs. These variations occur independently of MDR1 expression, and are associated to the attenuation of ceramide levels. This suggests that the increase of specific glycosphingolipids might offer glioma cells a survival advantage, resulting in resistance to chemotherapeutic drugs.

    Grant support: MURST PRIN 2004 to LR.

  • Fluorimetric determination of sphingosine-1-phosphate lyase activity.


    C. Bedia, J. Casas, G. Fabrias.

    Research Unit on BioActive Molecules, Department of Biological Organic Chemistry, IIQAB, CSIC. Jordi Girona 18, 08034-Barcelona, Spain.

    Intracellular sphingosine 1-phosphate (S1P) levels are regulated by its synthesis from sphingosine by sphingosine kinases and its catabolism by lipid phosphatases and by sphingosine 1-phosphate lyase (SPL). The latter is a pyridoxal 5-phosphate-dependent enzyme residing in the endoplasmic reticulum and is responsible for the irreversible retroaldolic cleavage of S1P to ethanolamine phosphate and hexadecenal. The notion that inhibition of sphingosine 1-phosphate lyase may account for some effects of FTY720 on immune functionsupports that SPL may be a potential target for immunomodulatory therapy and that the search for potent and selective inhibitors of SPL may offer novel immunosupressant drugs. The discovery of SPL inhibitors requires the availability of a reliable enzyme assay. The measurement of the SPL activity has been carried out for years using radiolabeled dihydrosphingosine as substrate in an poorly reproducible assay. In this work we describe the development of a feasible and consistent procedure to determine SPL activity. This method uses a fluorogenicsphinganine-1-phosphate analog as substrate, which, after the carbon-carbonbond cleavage produced by SPL, releases an aldehyde, which in turn suffers a spontaneous β-elimination to liberate the fluorescent molecule umbelliferone. The kinetic parameters of the SPL-mediated cleavage of this substrate and the implementation of the method for the high throughput screening of libraries of compounds both in vitro and living mammalian cells are reported.

    1Bandhuvula et al., J. Biol. Chem. 2005 280:33697.

  • Cholesterol and Sphingomyelin relationship in the cancer.


    I. Bernardini, L. Pugliese, L. Cecchetti, E. Bartoccini, M. P. Viola Magni, E. Albi.

    Department of Clinical and Experimental Medicine, Physiopathology, University of Perugia. Italy.

    In the last thirty years it has been described a strong hypocholesterolemy in patients affected with tumour not due to synthesis decrease1. Numerous studies have shown that exists a strong interaction between unesterified cholesterol (CHO) and sphingomyelin (SM) which arises from the Van der Waals interaction. Since SM was important in the physiological and pathological proliferation, the aim of the present work was to study the possible hyposphingomyelinemia associated to hypocholesterolemia in the patients with cancer. The blood of not drug treated 25 patients with monoclonal gammopathy were analyzed for their proteins and lipids content. The results demonstrated that the group with high level of gamma proteins presented a strong decrease of CHO and SM in blood. To investigate about the possible mechanism, we have evaluated the content of CHO, CHO esters and SM in the culture medium culture of SUP-T1 lymphoma cells at 0 and 48 hours of culture. The results showed that there was a strong reduction of CHO and SM without a variation of CHO esters accompanied by an increase of these lipids inside the cells. Enriching the culture medium by 800nM CHO, the cell content of CHO-SM increased together with an increase of 60% 3H-tymidineincorporation. The data suggest that the tumour cells need to incorporate these lipids to growth and that it could to explain the hypocholesterolemia and hyposphingomyelinemia in patients with cancer.

    1Pugliese L. et al Albi E. Int.J. Immunopath. Pharm. 2006, in press.

  • Sphingosine 1-phosphate stimulates aldosterone secretion through activation of the PI3-K/PKB and MEK/ERK 1/2 pathways.


    L. Brizuela, M. Rábano, J. M. Macarulla, M. Trueba, A. Gómez-Muñoz.Department of Biochemistry and Molecular Biology. Faculty of Science and Technology. University of the Basque Country.

    The Phosphoinositide 3-kinase/protein kinase B (PI3-kinase/PKB) pathway and extracellular signal-regulated kinases 1/2 (ERK1/2) play essential roles in the regulation of cell growth and survival, chemotaxis, cell motility, and secretion. These pathways can be activated by a variety of agonists, including bioactive sphingolipids. In particular, sphingosine 1-phosphate (Sph 1-P) has been implicated in the regulation of cell proliferation and survival, cell differentiation, tumour cell invasion, smooth muscle contraction and angiogenesis. Of interest, our group has preliminary evidence suggesting that Sph 1-P might be involved in steroidogenesis. Although Sph 1-P can act intracellularly as a second messenger, many of its effects are elicited through interaction with specific Gi protein- coupled receptors (S1P1-5) that are ubiquitously expressed in cells. In the present work, we show that Sph 1-Pstimulates aldosterone secretion in glomerulosa cells of bovine adrenal glands, and that the PI3-kinase/PKB and ERK 1/2 are essential pathways implicated in the regulation of this process.

    This study was supported by grants 9/UPV 00042.310-15852/2004 from “Universidad del País Vasco” (UPV/EHU) (Bilbao, Basque Country), and BFU2006-13689/BFI from “Ministerio de Educación y Ciencia” (Madrid, Spain).

  • In vitro, phytosphingosine enhances the cytotoxicity of drugs used forhormone-refractory prostate cancer treatment.


    A. Cabrespine1, E. Debiton2, J.-O. Bay1, P. Chollet1C. Barthomeuf2.

    1Centre Jean Perrin, Unité de recherche clinique Clermont-Ferrand, France; UMR484 INSERM, Clermont-Ferrand (France) and 2Université d’Auvergne, Laboratoire de Pharmacognosie et Biotechnologies, UMR484 INSERM,Clermont-Ferrand (France).

    Prostate cancer is the most frequently diagnosed malignancy and the second leading cause of cancer-related death of men in industrialized countries. Chemotherapy is the treatment of choice for patients with hormone- refractory prostate cancer (HRPC). Mitoxanthrone, carboplatin and paclitaxel have demonstrated some clinical value. However, in monotherapy, they have only limited efficacy. Combined chemotherapy appears a valuable approach to enhance their clinical value. In vitro drug combination studies conducted in conditions that mimic what happened in vivo could be helpful in the design of protocols that would have greater clinical efficacy. We previously reported that combination of paclitaxel and 13-cis-retinoic acid and of chelerythrine, mitoxanthrone and paclitaxel, may have interest for the treatment of HRPC.Phytosphingosine (PS) induces apoptotic cell death via caspase 8 activation and Bax translocation in human T-cell lymphoma (Jurkat) and in NCI-H460 non-small cell lung cancer cells.In combination with ionizing radiation, PS also enhances apoptotic cell death inradiation-resistant cancer cells through ROS-dependent and –independent AIF release.As these mechanisms may be helpful for cancer treatment, we have developed a mutant fungic strain overexpressing PS and a purification process leading to obtain high amounts of pure PS (Barthomeuf, personal communication). The aim of this study was to evaluate the value of PS in combination with mitoxanthrone, carboplatin, 13- cis-retinoic acid or paclitaxel vs PS alone and vs each drug alone or in combination. Tests were conducted on PC3 androgen-independent prostate cancer cells cultured insteroid-free conditions. Synergism, additivity and antagonism were determined by the combination index and calculated for each combination by the median-effect method. Data confirm that in combination, this sphingoid base may have clinical value for the treatment of HRPC.

    1Cabrespine et al., Anticancer drugs 2005 16:417. 2Park et al., Clin. Cancer Res. 2003 9:878.3Park et al., Blood 2005 105:1724.

  • Sphingolipid analogs: Searching new drugs against COPD.


    D. Canals,G. Villorbina,S. Grijalvo1,2, A. Delgado1,2, A. Llebaria,G. Fabriàs,J. Casas1.

    1Research Unit on BioActive Molecules, Department of Biological Organic Chemistry, IIQAB, CSIC. Jordi Girona 18, 08034-Barcelona, Spain and2University of Barcelona, School of Pharmacy, Unit of Pharmaceutical Chemistry (CSIC Associated Unit), Juan XXIII, s/n, 08028, Barcelona, Spain.

    Chronic obstructive pulmonary disease (COPD) is characterised by chronic inflammation of the airways and progressive destruction of lung parenchyma, a process that in most cases is initiated by cigarette smoking. Among the several mechanisms postulated to be involved in the pathogenesis of COPD, disruption of the balance between apoptosis and replenishment of structural cells in the lung has been recently reviewed.There is an increase in apoptotic alveolar epithelial and endothelial cells in the lungs of COPD patients. Moreover, Petrache et al reported increased lung ceramide levels in emphysema patients, suggesting that ceramide upregulation might be an important pathogenetic element in the development of emphysema.2

    The putative apoptotic activity of D-erythro-dihydroceramides (DHC’s) is a matter of controversy. Thus, it has been published that exogenously added short chain DHC’s do not exhibit apoptotic activity in different cell lines,3,4while L-threo-N-acetyl-sphinganine causes cell death in HL-60 cells.3Additionally, accumulation of natural DHC’s, but not ceramides, along with cell death occurs in human leukaemia cells treated with gamma-tocopherol,as well as in prostate cancer cells.Finally, a recent report describes that both long and short chain erythro-DHC’s do not simply lack apoptogenic activity, but counteract the apoptotic effect of ceramide by inhibiting Cer channel formation in mitochondria in early apoptosis.6

    In this context, the synthesis of a small combinatorial DHC library obtained by systematic variation of both the sphingoid and aliphatic and acyl chains is reported in this work. In addition, preliminary data on the activity of the synthesized compounds in A549 cells, a human alveolar epithelial cell line used in COPD studies, are also presented.

    1Demedts et al., Respir. Res. 2006 30:7. 2Petrache et al., Nat. Med. 2005 11:471. 3Bielawska et al., J. Biol. Chem. 1993 268:26226. 4Ogretmen et al., J. Biol. Chem. 2001 276:32506; 5Jiang et al., Proc. Natl. Acad. Sci. USA 2004 101:17825. 6Stiban et al., Apoptosis 2006 11:773.

  • Neutral sphingomyelinase-induced ceramide triggers germinal vesicle breakdown and apoptosis in Xenopus laevis oocytes


    O. Coll, C. García-Ruiz, J. C. Fernández-Checa,.

    Liver Unit, Hospital Clinic, IDIBAPS, IIBB-CSIC, Barcelona, Spain.

    Sphingolipids, in particular ceramide, are dynamic regulators of many cellular processes, including cell growth, differentiation, apoptosis, and inflammatory responses. These diverse modes of action likely reflect that ceramide acts at different targets determined upon the site and/or mechanism mediating ceramide generation. For instance intracellular ceramide generation in cells transfected with neutral sphingomyelinase (NSMase) has been shown to result in apoptosis as opposed to when cells were exposed to exogenous NSMase. In addition, previous studies have indicated that NSMase triggered meiotic cell cycle progression in Xenopus laevis oocytes. Thus, our aim was to compare the effect of incubation vs microinjection of SMase from Bacillus cereus(bNSMase) on the maturation or apoptosis in Xenopus laevis oocytes. We show that while incubation of mature stage VI oocytes with bNSMase induces maturation measured as the germinal vesicle breakdown, its microinjection resulted in apoptosis, characterized by reactive oxygen species generation, GSH depletion, release of cytochrome c and Smac/Diablo from mitochondria, and caspase-3 activation. Preincubation of oocytes with with reduced GSH- ethyl ester prior to bNSMase microinjection, prevented these events and protected oocytes from bSMase-induced death. Taken together, these findings show a divergent action of bNSMase-induced ceramide on oocyte maturation or apoptosis depending on the site where ceramide is generated.

  • Effect of protein kinase C inhibitors on tumour cells via cellular and nuclear sphingomyelinase.


    E. Damaskopoulou, G. Cascianelli, I. Bernardini, M. P. Viola Magni, E. Albi.

    Department of Clinical and Experimental Medicine, Physiopathology, University of Perugia, Italy.

    Diacylglycerol (DAG), 3-phosphoinositides and ceramide acted on protein kinase C (PKC) which partecipated to signal transduction in many cells and mediated a number of intracellular functions1. Twelve different isoforms of PKC(α, βI, βII, γ, δ, ε, ζ, η, θ, ι, λ and μ) have been characterised. The atypical PKC ζ, ι and λ activity depended on phospholipids, such as ceramide and phosphatidylinositol 3,4,5 triphosphate, and not on DAG, phorbol ester and Ca++. PKC ζ activation ceramide- dependent stimulated cell proliferation. In order to study the effect of PKC ζ inhibition on DNA/RNA synthesis and on sphingomyelinase (SMase) activity, hepatoma cells were cultured in the presence of 9 different specific inhibitors for 48 hours. 3H-tymidine or 3H- uridine were added to the culture medium 3 hours before. The results showed that all inhibitors reduced the 3H-tymidine incorporation about40%-50%. 3H- uridine incorporation was reduced in the cells of 5-20% by all inhibitors except inhibitor I-1 and I-4 which induced a reduction of about 35%and in the nuclei of 35%-40% by all inhibitors except I-3 which had very little effect and I-2 which determined a reduction of 56%. The activity of cellular SMase was reduced 19-29% whereas the nuclear SMase activity was reduced about 45%- 70% by all inhibitors except I-9 which reduced the activity of 84%. It can be concluded that the inhibitors of PKC ζ induce a reduction of DNA and nuclear RNA synthesis accompanied by a strong inhibition of nuclear SMase.

    1Musashi M et al., Int J Hematol 2000 72:12

  • Effect of aminocyclitols on mutant acid β-glucosidase activity in Gaucher disease fibroblasts. Their potential role as chemical chaperones.


    J. Duque1, M. Lluch1, G. Sánchez-Ollé2, M. Egido-Gabás3, J. Casas3, A. Delgado3,4, A. Llebaria3, D. Grinberg2, L. Vilageliu2, A. Chabás1.

    1Institut de Bioquímica Clínica, Hospital Clínic. Barcelona, 2Departament de Genètica, Facultat de Biología, Universitat de Barcelona, 3Research Unit on BioActive Molecules, Department of Biological Organic Chemistry, IIQAB, CSIC. Barcelona. 4University of Barcelona, Faculty of Pharmacy, Unit of Pharmaceutical Chemistry (Associated Unit to CSIC), Barcelona.

    Chemical chaperone therapy has been developed recently for several sphingolipidoses, including Gaucher disease. In this disorder, mutation N370S is the most prevalent one associated with important residual acid β- Glucosidase (β-Glu) activity, and different iminosugars has been reported to increase the mutant enzyme activity when added to patient fibroblast culture medium. We have studied the effect of several 2.5-60 μM aminocyclitols (C10, C9, C8, C4-PhBu derivatives of 1-deoxy-1-alkyl-amino inositol) on β-Gluactivity in fibroblast cultures derived from homozygous N370S patients. These compounds were potent inhibitors of fibroblast enzyme activity in vitro. For comparison, the effect of addition of the iminosugar N-Nonyl-DNJ (N-NDNJ)to WT and mutant fibroblast cultures was also carried out. N-NDNJ 5-20μMmarkedly increased the N370S mutant enzyme activity in both intact and lysed cells (1.5-2.2-fold), confirming previous reports with this iminosugar. WT and N370S patient fibroblast cultures were incubated for 4-6 days with aminocyclitols and β-Glu activity measured in intact and lysed cells. C10 andC4-PhBu (2.5-40 μM) inhibit enzyme activity in WT cells (27% with C10 30 μM; 50% with C4-PhBu 40 μM). In contrast, the mutant N370S enzyme activity is not affected or slightly stimulated with both compounds. The effect of C9 is similar in both WT and mutant cells. In the concentration range 2.5-60 μM, enzyme activity is moderately inhibited (33-55% with 40μM). The addition of C8 (2.5-60 μM) does not affect the WT enzyme, despite their inhibitory effect observed previously in vitro. The mutant enzyme activity is not affected or slightly increased (1.2–fold) at high C8 concentration.

    Studies under different experimental conditions are needed for C10 and C4- PhBu to confirm the effect of these aminocyclitols on the N370S mutant activity fibroblasts.

  • New aminocyclitol libraries as candidates to chemical chaperone therapy for Gaucher Disease.


    M. Egido-Gabás1, P. Serrano1,2, J. Casas1, M. Zucco3, G. Emeric3, A. Llebaria1, A. Delgado1,2.

    1Research Unit on Bioactive Molecules (RUBAM); Department of Organic and Biological Chemistry, Chemical and Environmental Research Institute of Barcelona (IIQAB-CSIC); Jordi Girona 18-26, 08034 Barcelona, Spain. 2University of Barcelona, Faculty of Pharmacy, Unit of Pharmaceutical Chemistry (Associated Unit to CSIC), Juan XXIII, s/n, 08028, Barcelona, Spain.BayerCropScience GmbH Chemistry Frankfurt, Industriepark Höchst, G836. Frankfurt am Main, Germany.

    Lysosomal storage diseases are a group of rare human disorders. One of them is Gaucher disease, a sphingolipidosis caused by a marked decreased in glucocerebrosidase (EC activity. This deficiency cause to a progressive accumulation of glucosylceramide in macrophages, leading to hepatosplenomegaly, anemia, skeletal lesions, sometimes involving the central nervous system. The current therapeutic strategies to treat Gaucher disease consist of enzyme replacement therapy (ERT) or substrate reduction therapy (SRT). However, they are expensive and relatively ineffective for therapies involving the CNS. The molecular therapeutic strategy or chemical chaperone therapy for genetic metabolic diseases seems a promising alternative to restore the activity of mutant glucocerebrosidase.1-4

    We present libraries of scyllo and racemic chiro N-substituted aminocyclitol derivatives. All compounds were synthesized by parallel solution-phasemethodology with the assistance of robotic technology. Chemical diversity were introduced by reaction of selected scaffolds with a set of aldehydes, acyl chlorides, sulfonyl chlorides, chloroformates, and amines to afford the corresponding amines, amides, sulfonamides, carbamates and ureas, respectively.5

    Selected compounds were evaluated as inhibitors for recombinant glucocerebrosidase (Imiglucerase, Cerezyme®, Genzyme)with Ki values in the low micromolar range for the most active members. In conclusion, these compounds could become candidates for chemical chaperone therapy of Gaucher disease.

    1Ishii et al. Bioch. Biophys. Acta 2004 1690:250. 2Matsuda et al. Proc. Natl. Acad. Sci. U S A 2003 100:15912. 3Sawkar et al. Proc. Natl. Acad. Sci. U S A. 2002 99:15428. 4Lin et al. Biochim. Biophys. Acta 2004 1689:19. 5J. Comb. Chem. In press. 6A generous gift of Cerezyme® from Genzyme Corporation is gratefully acknowledged.

  • Effects of ceramide 1-phosphate and its synthetic analogues on inflammatory pathways like stimulation of acid sphingomyelinase and phospholipase A2 in bone marrow derived macrophages.


    P. Gangoiti1, S. Grijalvo2,3, M. González1, M. Granado1, A. Delgado2,3, J. Casas2, A. Gómez-Muñoz1.

    1Department of Biochemistry and Molecular Biology, School of Science and Technology, University of the Basque Country 48080 Bilbao, Spain,2Department of Biological-Organic Chemistry (IIQAB-CSIC), Barcelona, Spain and 3Unit of Pharmaceutical Chemistry, School of Pharmacy, University of Barcelona, Avda. Joan XXIII s/n; 08029 Barcelona, Spain.

    The stimulation of acid sphingomyelinase (A-SMase) and the subsequent formation of ceramides are events that are associated to the pro- inflammatory actions of a variety of agonists including platellet activating factor (PAF). Therefore, there is increasing interest in biomedicine to develop drugs or compounds that are able to inhibit A-SMase activity in the absence of unwanted side effects. In this concern, we recently discovered that ceramide 1-phosphate (C1P) is a potent inhibitor of A-SMase both in intact macrophages as well as in cell free-systems. However, this putativeanti-inflammatory effect of C1P may be overcome by its ability to also activate the generation of arachidonic acid (through activation of PLA2) and the subsequent synthesis of eicosanoids, an action that is pro-inflammatory.In the present work, we have developed specific C1P analogs that are able to inhibit A-SMase activity in primary cultures of bone marrow-derivedmacrophages in the absence of PLA2 activation.

    This study was supported by grants 040732 from Fundació la Marató de TV3, Barcelona (Spain), and 9/UPV 00042.310-15852/2004 from “Universidad del País Vasco” (UPV/EHU) (Bilbao, Basque Country).

  • Sphingosine-1-phosphate effects on the gel-fluid and lamellar-hexagonaltransitions of aqueous phospholipid dispersions.


    M. García-Pacios, M. I. Collado, M. A. Requero, A. Alonso, J. L. R. Arrondo, F. M. Goñi.

    Unidad de Biofísica (CSIC-UPV/EHU) and Departamento de Bioquímica, Universidad del País Vasco, Aptdo. 644, 48080 Bilbao, Spain.

    Sphingosine-1-phosphate (SPP) has been recognized as a signalling molecule in cell metabolism. We have studied the effect of increasing proportions of SPP in aqueous phospholipid dispersions, using differential scanning calorimetry and infrared spectroscopy. The glycerophospholipids were either dielaidoylphosphatidyletanolamine (DEPE) or deuterated dipalmitoylphosphatidylcholine (d62-DPPC). SPP modified the gel-fluidtransition of both glycerophospholipids, shifting it to lower temperatures, and decreasing the reansition enthalpy. IR spectroscopy of natural SPP mixed with deuterated DPPC allows the independent observation of transitions in each mole. The DPPC CO group stretching vibration was shifted to lower frequencies, by about 2 cm-1, in the presence equimolar SPP. Pure SPP displayed a broad landothemic transition at ca. 65º C, that was accompanied by an increased lipid order. This transition was not detected in equimolar SPP: d62-DPPC mixtures. Finally, SPP was found to have a notorious effect on the lamellar-to-inverted hexagonal transition of DEPE, increasing the transition temperature and decreasing the transition enthalpy, i.e. stabilizing the lamellar vs. the inverted hexagonal phase.

  • Serum removal causes apoptosis in cultured alveolar macrophage through de novo syntesis of ceramide. Inhibition by ceramide 1-phosphate.


    M. Granado, P. Gangoiti, A. Gómez-Muñoz.

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

    Macrophages are involved in developing of pulmonary inflammation and parenchymal damage that occur in chronic obstructive pulmonary disease (COPD). The fundamental observation of this report is that incubation of alveolar macrophages NR8383 in the absence of serum leads to cell death. Serum removal also produced a concomitant increase in ceramide levels, which are detrimental for cells, and are usually implicated in the induction of apoptosis. In fact, incubation of the macrophages in the absence of serum caused phosphatidylserine exposure, which is a marker of apoptosis. Ceramide generation was mainly caused by de novo synthesis, as determined by inhibition of the incorporation of [3H]-palmitate into ceramides in the presence of myriocin, a selective inhibitor of serinepalmitoyltransferase. Another key observation of this work was that ceramide 1-phosphateinhibited the generation of the de novo synthesized ceramides, thereby preventing the macrophages from entering apoptosis.

  • Dependence of sphingoid bases concentration on growth phase and addition of zeolite in brewer’s yeast.


    I. Karmelic1, F. Ivušic2, S. Ribar1, V. Maric2, M. Mesaric1.

    1Department of Chemistry and Biochemistry, School of Medicine and2Department of Biochemical Engineering, Faculty of Food Technology and Biotechnology, University of Zagreb, Croatia.

    Sphingolipids having a long-chain sphingoid base backbone are primarily located in the yeast's plasma membrane. The two types of sphingoid bases in yeasts are sphinganine and its 4-hydroxy derivative, phytosphingosine, containing 16, 18 or 20 carbons. Sphinganine, phytosphingosine and ceramide, which are precursors of sphingolipids are involved in membrane signalling, regulation of cell wall biosynthesis, phospholipid biosynthesis and binding of cell surface glycoproteins. In addition, they are proven to play important roles in signal transduction during heat stress response, regulation of calcium homeostasis or components in calcium-mediate signalling pathways and in regulation of the cell cycle. Since little is known about the regulation of de novo synthesis of complex sphingolipids in any organism, an investigation into the regulation of the sphingoid bases’ biosynthesis in yeast may shed some light on this issue. The aim of our research was to investigate the effect of growth phase on the concentration and composition of sphingoid bases from brewer's yeast. The second aspect of our research was to investigate the effect of natural zeolite clinoptilolite on sphingolipid metabolism in brewer's yeast. The yeast cells were cultured in semi-industrialbioreactors in growth medium unhopped worth and in zeolite-supplementgrowth medium (1% zeolite into basic medium). Total sphingoid bases were extracted according to Riley at al. O-Phthaladehyde derivatives were analysed using reversed-phase high performance liquid chromatography (HPLC). The results point to the following conclusions: brewer's yeast is a good source of sphingolipids and the predominant sphingoid base is phytosphingosine; the level of total sphingoid bases in brewer’s yeast changes depending on culture growth phase; the most obvious effect of the growth phase on the sphingoid base production has been observed in the case of phytosphingosine, the concentration of which is highest in the exponential phase; natural zeolite clinoptilolite increased the concentration of pytosphingosine which may indicate that zeolite acted at the level of phytosphingosine formation.

  • Ceramide formation by sphingomyelinases in red blood cells.


    D. J. López1, L.-R. Montes1, L. A. Bagatolli2, F. M. Goñi1, A. Alonso1.

    1Unidad de Biofísica (CSIC-UPV/EHU), P.O. Box 644, E-48080 Bilbao, Spain and2Memphys Center for Biomembrane Physics, Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark.

    Red blood cell membranes are particularly rich in sphingomyelin, thus they are potentially good substrates for sphingomyelinases. In this communication we summarize some recent results obtained with either an intrinsic sphingomyelinase activity or with an externally added (bacterial) enzyme. A latent intrinsic sphingomyelinase activity can be elicited by subjecting the erythrocytes to a hypertonic shock. This activity is magnesium–dependent,and its maximum value is reached 3-6 min after establishing the hypertonic conditions. The specific activity is 0.1 nmol/min x mg protein. When erythrocytes are stored at 4ºC, the activity remains intact for the first 48 h, then decreases. The intrinsic sphingomyelinase activity isdetergent–sensitive, increasing by ≈ 5-fold in the presence of sodium dodecylsulphate at subsolubilizing concentrations. Concominant measurements of intrinsic sphingomyelinase activity and ceramide concentrations reveal that the latter remain constant with time, indicating rapid conversion of the newly formed ceramide into other sphingolipids. Moreover, erythrocyte treatment with a bacterial sphingomyelinase induces rapid shape changes and generation of ceramide-rich domains in the red blood cell membranes.

  • Acidic sphingomyelinase is required for TNF-induced mitochondrial permeabilization and hepatocellular death via cardiolipin peroxidation.


    M. Marí, A. Colell, C. García-Ruiz, J. C. Fernández-Checa. 

    Liver Unit, Hospital Clinic, IDIBAPS, IIBB-CSIC; Barcelona, Spain.

    TNF-α apoptotic signaling is a complex process that involves protein-proteininteractions and the participation of several intermediates. Sphingolipid generation through sphingomyelinase activation has been involved in apoptotic pathways induced by death ligands. In previous studies we have observed that the intrinsic resistance of hepatocytes to TNF is overcome by selectively depleting mitochondrial glutathione (mGSH) with apoptotic/necrotic hepatocyte death, and that acidic sphingomyelinase (ASMase) is necessary for efficient TNF-α–mediated hepatocellular damage.1Thus our aim was to examine the signaling in wild type and ASMase knockout hepatocytes responsible for the different outcome observed after TNF challenge following mGSH depletion. Cultured hepatocytes from 8-10 week- old wild type and ASMase KO mice (C57BL/6) were obtained by collagenase perfusion. Mouse hepatocytes were depleted of mGSH by (R/S)-3-hydroxy-4-pentenoate (HP, 0.5mM). Survival and chromatin morphology were assessed by GST release and Hoechst/PI staining. Bax, tBid and pBim translocation to mitochondria, JNK phosphorylation, release of cathepsin B, and caspase-3activation were determined by western blot. Mitochondrial membrane permeabilization (MMP) was assessed by the distribution of calcein/TMRM. Cardiolipin levels were analyzed by HPLC. ROS generation was analyzed fluorimetrically. The signaling events upstream of mitochondria such as Bax translocation to mitochondria, Bid truncation, NF-κB activation and transient JNK phosphorylation, were preserved in ASMase-deficient hepatocytes regardless of mGSH depletion. Only wild type mGSH-depleted cells underwent MMP with lower intact cardiolipin levels (40-50%) due to enhanced cardiolipin hydroperoxides content, cyt c release into the cytosol and caspase 3 activation followed by cell death. None of these features were observed inmGSH-depleted ASMase knockout cell. Interestingly, an early burst of ROS (beginning 15-30 min after TNF addition) was also only observed in mGSH- depleted wild type hepatocytes, and not in TNF-alone treated hepatocytes, nor in ASMase knockout hepatocytes despite mGSH depletion. Furthermore, in previous studies we have monitored the cellular distribution of ganglioside GD3 during TNF-α signaling indicating its mitochondrial targeting (as soon as 30 min after TNF addition) in wild type but not in ASMase knockout hepatocytes, suggesting that ASMase is necessary for this event. These suggest that mGSH modulates hepatocellular sensitivity to TNF through control of mitochondrial ROS generation targeting mitochondrial cardiolipin status.

    1García- Ruiz et al., J. Clin. Invest. 2003 111:197; Marí et al., J. Clin. Invest. 2004 113:895.

  • Involvement of nuclear sphingomyelinase in embryonic hippocampal cell differentiation vitamin D3-induced.


    F. Marini1, E. Bartoccini1, G. Cascianelli1, M. García-Gil2, M. P. Viola Magni1, E. Albi1.

    1Department of Clinical and Experimental Medicine, Physiopathology, Policlinico Monteluce, 06100 Perugia, Italy and 2Department of Physiology and Biochemistry, University of Pisa, Italy.

    It is known that vitamin D3 induces cell differentiation by activating neutral sphingomyelinase (N-SMase). Recently it has been demonstrated that nuclearN-SMase rather that cellular N-SMase is involved in the early signal transduction after cell stimulation to proliferation and/or differentiation 1. The aim of the present work was to study the N-SMase activity modifications in the differentiation of embryonic hippocampal cells induced by vitamin D3 treatment. At this end HN9.10e, which exhibited morphologic and trophic activity typical of primary hippocampal neurons, were used. Vitamin D3 was added to culture medium at the final concentration from 50 to 400 nM and the morphological differentiation and nuclear N-SMase activity were evaluated. The results showed that 50-100 nM was the optimal concentration to obtain cell differentiation whereas 400nM concentration induced the cells to apoptosis. In fact after 50-100nM vitamin D3 treatment, the cells showed the lengthening of the soma while neurite and dendrites appeared after 5 days of culture. Bcl2 expression increased 16 times higher respect to the control and also NGF resulted strongly increased. Only 2 hours of treatment, the nuclear N-SMase activity increased of two time and 14C palmitic acid incorporation in lipids showed an increase of ceramide production. The results suggested the involvement of nuclear SM pathway in HN9.10 differentiation.

    Albi E and Viola Magni MP Biology of the Cell 2004 96:657.

  • A new family of dihydroceramide desaturase inhibitors.


    X. Matabosch1*J. M. Muñoz-Olaya1*, J. Casas1, A. Delgado1,2, A. Llebaria1, G. Fabriàs1.

    1Research Unit on BioActive Molecules, Department of Biological Organic Chemistry, IIQAB, CSIC. Jordi Girona 18, 08034-Barcelona, Spain and 2Unit of Pharmaceutical Chemistry, School of Pharmacy, University of Barcelona, Avda. Joan XXIII s/n; 08029 Barcelona, Spain.

    The last step in the de novo biosynthetic pathway of ceramide involves the introduction of the (E)-4 double bond in dihydroceramide by dihydroceramide desaturase (DHCD). The only inhibitors of this enzyme so far described include a family of cyclopropene analogs of ceramide (GT11 and analogs). By means of a novel procedure to determine DHCD activity, a new family of DHCD inhibitors has been identified among a library of dihydroceramide analogs. The biochemical assay utilizesN-[6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl]-D-erythro-sphinganine as substrate and aHPLC-fluorimetric method, and can be used both in vitro and cultured cells. From a structural standpoint, the inhibitors are analogs of dihydroceramide in which a specific methylene group of the long chain base has been replaced by an heteroatom. The several compounds differ in both the heteroatom substituents and the N-acyl group. Their preliminary activities in both rat liver microsomes and mammalian cells are reported.

    *Both authors contributed equally to this work and are listed in alphabetical order.

  • Analysis of epidermal lipids in dogs. A preparative study for validation of an animal model of atopic dermatitis.


    I. Popa1, A. Piekutowska1, J. Chapman1, H. Gatto2, D. Pin3, M. Haftek1, J. Portoukalian1.

    1University Lyon-I, Dermatology, Edouard Herriot Hospital 69437 Lyon Cx 3, France. 2Virbac, Carros, France. 3Ecole Nationale Vétérinaire de Lyon, Marcy l'Etoile, 69280, France.

    Atopic dermatitis (AD) is a well characterized clinical entity in dogs. We have observed ultrastructural changes in stratum corneum (SC) morphology and lamellar lipid organization in the non-lesional skin of atopic dogs using osmium and ruthenium tetroxide postfixation. Because of the observed clinical and morphological similarities between the human and dog atopic skin, we speculated that biochemical modifications concerning SC lipid expression described in man could also underlie AD in dogs. Dog SC was taken using strips. Ten consecutive strips were needed to reach the viable epidermis. We purified and analyzed individually for each strip the different classes of free lipids (free fatty acids, ceramides, phospholipids, glycolipids, gangliosides) and protein-bound lipids (ceramides and fatty acids) and compare to the lipid profile of human epidermis. This comparison showed us that dog epidermis contain similar ceramides species as in humans with the same three sphingoid bases coupled to long-chain fatty acids, especially toomega-hydroxylated fatty acids. In the case of human AD, it has been reported that the deficiency in acylceramides was correlated to the defects in skin barrier. We have compared the lipid profiles of normal dog epidermis with those of atopic dermatitis dogs, and found that the epidermis of atopic dogs have a much lower lipid content with a marked deficit in ceramides. A detailed knowledge about the lipid composition of normal SC in dogs is an indispensable step towards the biochemical characterization of canine AD and should help to define an animal model of this disease.

  • Sphingoid bases and gangliosides in infant formulas.


    S. Ribar1I. Karmelic1, D. Ivankovic2, M. Mesaric1.

    1Department of Chemistry and Biochemistry, School of Medicine, University of Zagreb, Šalata 3, 10000 Zagreb, Croatia. 2School of Medicine, University of Zagreb, Šalata 3, 10000 Zagreb, Croatia.

    Sphingolipids are a group of lipids present in all eukaryotic cells. They consist of a long-chain sphingoid base as the backbone. The prevalent long-chainbases of most mammals are D-erythro-sphinganine and sphingosine. Gangliosides comprise a group of glycosphingolipids that contain one or more sialic acid moieties. They are one of the components of human milk that have been demonstrated to play important roles in neuronal development, neuropathologic processes, and receptor functions with respect to protein hormones, interferon, fibronection and bacterial toxins. The aim of our research was to establish if there are the diferences in sphingoid bases concentrations between human milk and infant formulas. The second aspect of the research was to ascertain whether there was any difference in the concentrations of gangliosides between human milk and infant formulas. Sphingolipids were extracted from human milk and infant formulas. Free and total sphingoid bases were obtained by base and acid hydrolysis respectively. Sphingosine and sphinganine were derivatized with the OPA (O- Phtaldialdehyde) reagent and analysed by high performance liquid chromatography. After sphingolipid extraction, gangliosides were quantitatively determined in supernatant with a colorimetric resorcinol- hydrochloric acid method. The results of this research indicate the significant differences between the concentrations of sphingoid bases and gangliosides in infant formulas and human milk. Based on the obtained results, it can be concluded that despite all efforts made to produce infant formulas as similar to human milk as possible, in terms of their structure and the amount of their constituents, there are differences that could be biologically significant and thus need to be further researched.

  • Preliminary study of the chaperone effect on mutated glucocerebrosidases as a treatment for Gaucher disease.


    G. Sánchez-Ollé1, M. Egido-Gabás2, J. Duque3, J. Casas2, A. Chabás3, L. Vilageliu1, D. Grinberg1.

    1Departament de Genètica, Facultat de Biologia, Universitat de Barcelona, Barcelona, 2RUBAM (Research Unit on BioActive Molecules), Departament de Química Orgànica Biològica, IIQAB-CSIC, Barcelona and 3Institut de Bioquímica Clínica, Corporació Sanitària Clínic, Barcelona.

    Gaucher disease (GD) is a glycosphingolipid storage disorder caused by deficiencies in lysosomal acid β-glucosidase (GBA), resulting in progressive accumulation of glucosylceramide in macrophages of liver, spleen, bone marrow and, sometimes, central nervous system. It has been reported that some iminosugar compounds are able to work as chemical chaperones, at subinhibitor concentrations, and increase the activity of wild-type and mutated GBAs.

    The aim of this work is to express and characterize mutant GBA alleles and assay the chaperone effect of the iminosugars N-(n-nonyl)deoxynojirimycin(NN-DNJ) and N-(n-butyl)deoxyno-jirimycin (NB-DNJ) on the expressed proteins. Eleven different mutated alleles have been studied. COS-7 cells have been transfected with the pcDNA3 expression vector carrying the wild- type or the mutated cDNAs. The residual enzymatic activities, given as percentage of the wild-type enzyme activity are the following: N188S: 53.84%, G202R: 24.84%, H255Q: 77.09%, E326K: 30.51%, N370S: 8.11%, G377S: 11.06%, I402T: 19.37%, D409H: 16.46%, L444P: 2.42%, [N188S;E326K]: 12.78% and [H255Q;D409H]: 16.05%.

    Stable transfected cells, selected with geneticin, were treated for 6 days with NN-DNJ (2.5 and 5 μM) or NB-DNJ (5 and 10 μM). Preliminary results show an increase of the enzymatic activity for the wild-type enzyme (at 2.5 and 5 μM) and for the mutated N188S (at 2.5 μM) and [N188S;E326K] (at 5 μM) GBAs. Surprisingly, a decrease in protein activity was also observed for the N188S (at 5 μM) and [N188S;E326K] (at 2.5 μM) mutated enzymes. After treating the cells with NB-DNJ, the increase in activity was observed for the wild type enzyme (at 10 μM), but also for the N188S (at 5 and 10 μM), N370S (at 10 μM) and [N188S;E326K] (at 10 μM) mutated proteins.

    After these promising preliminary results, new compounds (aminociclitol derivatives), will be assayed.

  • Localization of sphingosine kinase-1 in detergent-resistant membranes of C2C12 myoblasts.


    E. Sarchielli1, C. Sassoli2, L. Becciolini1, L. Formigli2,4, C.Donati1,4, G. Nemoz3, P. Bruni1,4E. Meacci1,4.

    Departments of 1Biochemical Sciences, 2Anatomy, Histology and Forensic Medicine, University of Florence, 3Laboratoire de Physiopathologie des Lipides et Membranes, Institut National des Sciences Appliquees de Lyon, Villeurbanne, France. 4Interuniversity Institute of Myology (IIM), Italy.

    Sphingosine kinase (SphK), which catalyzes the formation of sphingosine 1- phosphate starting from sphingosine and ATP, has been recognized to be critical for the control of cell proliferation, differentiation and motility in a large number of cells as well as in C2C12 myoblasts1,2. Recently, we demonstrated that SphK activity as well as SphK1 protein content are increased upon the attainment of the cellular confluence and the terminal differentiation in myotubes, consistently with an important role of the enzyme in cell growth arrest and myogenesis3. Although in many circumstances the major enzymatic isoform, SphK1, is largely localized in the cytoplasm, it is generally accepted that SphK functionality requires membrane localization.

    The study of membrane localization of SphK1 in C2C12 myoblasts revealed that the enzymatic activity increase observed in confluent myoblasts, was paralleled by a significant rise of membrane-associated enzyme and the its enrichment in detergent-resistant membranes, as indicated by the enhancedco-localization with specific lipid raft markers such as cholera toxin B subunit and co-immunoprecipitation with caveolin-1, the caveolae scaffold protein.

    Given that, phosphatidic acid (PA), generated by phospholipase D, has been proposed to direct SphK1 membrane translocation4, we have investigated the possible role of PLD/PA system in the recruitment of SphK1 in these membrane compartments. Interestingly, [3H]PA locally increased when the cells reached the confluence and co-localized with SphK1 in fully confluent myoblasts as judged by confocal microscopy analysis. Interestingly, cell treatment with PLD1-siRNA or 1-butanol drastically reduced PA formation, SphK1 detergent-resistant membrane activity and association, while the overexpression of PLD1 augmented SphK activity as well as SphK1 protein content in that membrane compartment. Both experimental conditions also greatly affected cell growth and myogenic differentiation.

    In conclusion, these data support that PLD/PA system play an important role in SphK1 translocation to detergent-resistant membranes of confluent myoblasts and represent a crucial event in C2C12 myoblast growth and myogenesis

    This work was supported in part by funds from Ministero dell’Istruzione, dell’Università e della Ricerca (MIUR-PRIN 2003 to P.B.; MIUR-PRIN 2004 to E.M.), University of Florence (ex- 60%) to P.B. and E.M., Ente Cassa di Risparmio di Pistoia e Pescia to E.M.

    1Liu et al., Prog Nucleic Acid Res Mol Biol. 2002 71:493. 2Donati et al., FASEB J. 2005 19, 449.3Meacci et al. (2006) submitted. 4Delon et al., J. Biol. Chem. 2004 279:44763.


  • Resveratrol induces apoptosis in human melanoma A375 cells by ceramide signalling.


    P. Signorelli1, C. A. La Porta2, F. Scarlatti1, R. Maffei1, G. Sala1, R. Ghidoni1.

    1Laboratory of Biochemistry and Molecular Biology, San Paolo University Hospital and 2Department of Biomolecular Science and Biotechnology, University of Milan. Milan, Italy.

    Resveratrol is a plant-derived antioxidant and a phytoestrogen polyphenol that has been extensively studied in vivo and in cultured cells lines for its antitumoral activity. The aim of this study was to investigate the effects of resveratrol on the growth and survival of human melanoma A375 cells and to identify the mechanism of resveratrol action in this model. Resveratrol reduced proliferation and viability and increased the intracellular concentration of the pro-apoptotic sphingolipid mediator ceramide. The increase in ceramide was due to both its de novo synthesis and to sphingomyelin hydrolysis since it was reduced by myriocin, an inhibitor ofserine-palmitoyl-transferase, and completely impaired by glutathione, an inhibitor of neutral sphingomyelinase. The abrogation of ceramide increase by glutathione but not its reduction by myriocin rescued cells from the loss of viability induced by resveratrol. Moreover resveratrol induced cell death was related to the cleavage of poly-(ADP-ribose)-polymerase and this event was prevented by glutathione and not by myriocin.

    We can conclude that resveratrol triggered ceramide signalling in human melanoma A375 cells and the pool of ceramide derived from neutral sphingomyelinase activity was required for PARP cleavage and for apoptosis induction.

  • The role of sphingolipids, glycolipids and phospholipids in the Drosophila epithelial and glial permeability barriers.


    M. Striginiand D. Karagogeos1,2.

    1IMBB/FORTH, Iraklio, Crete, Greece and 2Medical School, University of Crete, Iraklio, Crete, Greece.

    Cell adhesion depends on specialised structures, such as occluding and adherens junctions. These junctions localize to specific membrane regions, for example to distinct domains along the apical-basal axis in polarised cells. Indeed, junction formation and cell polarization are interdependent. Polarised cells compartmentalize proteins as well as lipids in their membranes. While much progress has been made in learning how several junction proteins contribute to cell polarity and how they get localised, little is known about the contribution of lipids to cell polarization and cell adhesion.

    Drosophila is an attractive model system to study in vivo the genetic, molecular and cellular basis of developmental and cell biological processes. We try to exploit such system to study the role of lipids (and in particular sphingolipids, glycosphingolipids, and phospholipids) in the establishment of cell polarity and cell adhesion. We concentrate on the formation of septate junctions (SJ), a type of occluding junctions found among epithelial cells and among glial cells that enwrap the nervous system. SJ sustain the epithelial and glial permeability diffusion barriers. Several proteins are known to localise to SJ, and their role in the establishment and maintenance of SJ has been investigated by many groups, including ours. We have now turned our attention to lipids.

    We have just embarked in the characterization of the subcellular distribution of different lipid subtypes in the polarised cells of Drosophila epithelia. In addition, as a handle to the genetic study of lipids in this context, we investigate the contribution of lipid metabolism genes to the process of SJ formation. We are screening mutants in sphingolipid, glycosphingolipid, and phospholipid metabolism genes using established dye penetration assays, to probe the permeability of the epithelial and glial barriers. We will then proceed to assess the molecular, cellular and structural defects in the mutants with disrupted barriers, by looking at the organization of SJ and the morphogenesis of the glia forming the so called blood-brain barrier. We will report on our preliminary results.

  • Quantitation and composition of ceramide-1-phosphate in mouse tissues.


    H. Van Overloop, P.P. Van Veldhoven.

    Pharmacology, Katholieke Universiteit Leuven, Belgium.

    Ceramide-1-phosphate (Cer-1-P) appears to be powerful bioactive sphingolipid, involved in different physiological processes, like mitosis, inflammation, phagocytosis and apoptosis. Until now, not much is known about the basal levels and composition of Cer-1-P in mammalian tissues and cells. To determine this sphingolipid in tissues/cells, we developed a method, based on the strong retention of phosphate esters on aminopropyl SPE columns, followed by elution and dephosphorylation with alkaline phosphatase, and rephosphorylation with recombinant ceramide kinase (CERK) in the presence of [γ-32P]-ATP. This protocol allows the quantification of Cer- 1-P in the low nmole range. When applied to mouse tissues, TLC separation of the 32P-labeled ceramides revealed at least three major species, differing in the N-acyl chain: long chain, very long chain and2-hydroxy (very) long chain fatty acids. Both non-hydroxy- and 2-hydroxyfatty acid containing Cer-1-P are most enriched in cerebellum, estimated at ~ 20 nmol and 8 nmol/g tissue, respectively. Lowest Cer-1-P levels are found in liver and kidney (~ 5 nmol/g).

    This work was supported by the Flemish government (Geconcerteerde Onderzoeksacties GOA/2004/08), the Belgian Ministery of 'Federaal Wetenschapsbeleid' (Interuniversitaire Attractiepolen IAP-P5/05) and the Flemish ‘Fonds voor Wetenschappelijk Onderzoek’ (G.0405.02). H.V.O. is an aspirant from the Flemish 'Fonds voor Wetenschappelijk Onderzoek'.

  • Inside and out the "sphingolipid rheostat"


    Riccardo Ghidoni

    Lab. Biochemistry & Molecular Biology, San Paolo University Hospital, Medical School, University of Milan - Via A. di Rudinì, 8, 20142 Milan - Italy

    A "sphingolipid rheostat" has been proposed to explain the opposite roles in cell growth arrest and survival of ceramide and sphingosine-1-phosphate [Cuvillier O. etal, Nature, 381, 800, 1996]. Based on the fact that these two metabolites are interconvertible, it has been proposed that it is not the absolute amounts of these metabolites but rather their relative levels that determines the cell fate [Maceyka M. et al, BBA, 1585, 193, 2002]. After a decade, the complexity of the signaling pathways by the integrated sphingolipid network requires a rivisitation of this paradigm. The body of such revisitation focuses on: i) the consistency and size of subcellular pools of individual sphingolipids involved in the signaling pathways, ii) the evidence that other species (as ceramide-1-phosphate) may participate in the cell fate, iii) the still obscure role of the "intermediate" free sphingosine, iv) the "time" factor, including the half-life of each mediator, v) the occurrence of potential "dominant" and "recessive" signals.

  • Ceramide-1-phosphate: a lipid second messenger in eicosanoid synthesis.


    Charles E. Chalfant

    Department of Biochemistry and Molecular Biology, Virginia CommonwealthUniversity-School of Medicine, Richmond, VA 23298-0614; Research Service, Hunter Holmes McGuire Veterans Administration Medical Center, Richmond, VA 23249; and The Massey Cancer Center, Richmond, VA 23298.

    Ceramide-1-phosphate (C1P) is a new addition to the growing group of bioactive sphingolipids, which includes ceramide and sphingosine-1-phosphate. Previous findings from our laboratory have demonstrated the interaction between C1P and group IVA cytosolic phospholipase A2 (cPLA2a) at the mechanistic level. Specifically, C1P was shown to enhance the activity of cPLA2a in a specific manner thereby acting as a positive activator of cPLA2a (Subramanian, P., et al. (2005) J.Biol.Chem. 280, 17601-7). In this study, we have identified several key amino acids adjacent to the CBR II region of the CaLB domain of cPLA2a? required for interaction with C1P. Therefore, the interaction site for C1P lies within the CaLB domain of cPLA2a, and is thus, distinctly different from the binding site for the anionic lipid, PIP2. Mutagenesis of the interaction site for C1P also inhibited the ability of cPLA2a to translocate in response to several inflammatory agonists. The requirement of the C1P/cPLA2a interaction for activation of enzyme will be discussed in detail. The regulation of the C1P-generating enzyme, ceramide kinase, by inflammatory agonists and in vivo will also be discussed.

  • Bioactive sphingolipids in inflammation and cancer.


    Lina M. Obeid, M.D.

    Departments of Medicine and Biochemistry and Molecular Biology, Medical University of South Carolina, and The Ralph H. Johnson Veterans Affairs Medical Center, Charleston, SC.

    Our laboratory focuses on the role of the bioactive sphingolipids ceramide andsphingosine-1-phosphate (S1P) in regulation of cell growth. To this end, we study the regulation of enzymes involved in ceramide and S1P synthesis and breakdown. Here we describe new data implicating a golgi alkaline ceramidase enzyme in generation of S1P. Moreover we show that sphingosine kinase 1 (SK1), the predominant enzyme that generates S1P in cells, is regulated in inflammation and in cancer. Studies on regulation of SK1 by hypoxia will be presented. In addition, using a cancer cell/endothelial co-culturemodel and a mouse matrigel plug model, we demonstrate that SK1 expression in the tumor causes S1P generation leading to de novo blood vessel formation. Finally in very recent studies we demonstrate a critical role for SK1 in animal models of inflammatory bowel disease and of colon cancer. Taken together these studies implicate the bioactive sphingolipid SK1-S1P pathway in inflammatory diseases and in carcinogenesis.

  • Therapies of cancers and sphingolipid storage diseases by novel synthetic analogs of sphingolipids.


    Shimon Gatt and Arie Dagan

    Department of Biochemistry, Hebrew University-Hadassah school of Medicine, Jerusalem, Israel 91120.

    The purpose of our studies was to synthesize non-natural analogs of sphingolipids that would affect their metabolism in two directions: a. Elevate ceramide, for killing cancers. b. Decrease the levels of sphingolipids for therapy of sphingolipidoses.

    1.We have synthesized sphingolipids to each of which a fluorescent molecule is linked, enabling us to omit the use of radioactivity and analyze effects on sphingolipid metabolism by quantification of the fluorescence of the respective products.

    2.We have synthesized novel, non-natural synthetic analogs of sphingolipids that inhibited the hydrolysis of ceramide or its conversion to more complex sphingolipids.

    This led to an elevation of the ceramide, leading to death by apoptosis of a variety of cancer cells.

    3.Administration of the synthetic sphingolipid-analogs to tumor-bearing mice by intradermal or intraperitoneal injections, or by giving them per-os, ie, in their drinking water, resulted in a reduction and even disappearance of the tumors, while in mice that have not received our synthetic analogs the tumors continued growing.

    4.Other, synthetic analogs of sphingolipids were prepared for therapy of two sphingolipid storage diseases, ie, Niemann-Pick and Gaucher diseases, in which sphingomyelin and glucosylceramide accumulate respectively. The analogs were tested for two therapeutic approaches. In the first (Substrate Reduction Therapy) the analogs inhibited the biosyntheses of sphingomyelin (for Niemann-Pick disease) and/or glucosylceramide (for Gaucher disease), thereby decreasing their respective levels.

    In the second therapeutic approach (Chaperone Therapy) the analogs linked to the mutated enzyme (eg. acid sphingomyelinase) and protected it from being inactivated in the endoplasmic reticulum. The enzyme-analog complex was then transported to the lysosomes of Niemann-Pick disease cells, where the accumulated sphingomyelin displaced the analog and was hydrolyzed by the residual activity of the mutated sphingomyelinase.

  • The Extended Family of Neutral Sphingomyelinases: roles and regulation.


    Yusuf A Hannun

    Department of Biochemistry and Molecular Biology, Medical University of South Carolina,175 Ashley Ave, Charleston, SC 29425,USA.

    Sphingomyelinases (SMases) are key enzymes that regulate the formation of ceramide through the hydrolysis of sphingomyelin substrates. Recent results have led to the identification of a novel family of neutral SMases including the yeast Isc1 and mammalian nSMase2. These enzymes belong to an extended family of enzymes that appear to share similar in vitro catalytic mechanisms and similar mechanisms of regulation by anionic phospholipids. The yeast Isc1 hydrolyzes yeast inositol phosphoceramides, localizes to mitochondria, is activated by mitochondrial anionic phospholipids, and participates in thepost-diauxic response of yeast cells. In human cells, TNF and other cytokines cause acute activation of nSMase2 over 5-30 minutes. This is accompanied by translocation of the enzyme to the plasma membrane. Evidence will be presented demonstrating palmitoylation of the enzyme and its localization to the inner leaflet of the plasma membrane. Activation of nSMase2 in response to TNF plays an important role in mediating regulation of expression of adhesion molecules and prostaglandin formation. These and other studies are beginning to define specific functional pathways of ceramide and sphingolipid metabolism, but they also introduce two additional layers of complexity to the understanding of sphingolipid action: a) the interconnection of lipid metabolism, and 2) the compartmentalized metabolism and action of these lipids.

  • Optimization and validation of the fluorogenic sphingosine-1-phosphate lyase assay and application to library screening.


    Luz Camacho-Castillo1, Carmen Bedia1, Josefina Casas1, Paul P. Van Veldhoven2, Gemma Fabriàs1.

    1Research Unit on BioActive Molecules (RUBAM), IIQAB-CSIC, Barcelona, Spain2Katholieke Universiteit Leuven, Campus Gasthuisberg, LIPIT, Leuven, Belgium.

    Sphingosine-1-phosphate lyase (SPL) is an endoplasmic reticulum resident enzyme responsible for the degradation of the bioactive lipid compound sphingosine-1- phosphate (S1P). The disruption of SPL function by small molecules has been shown to induce lymphopenia in mice.This suggests that this enzyme represents a novel target for immunomodulatory therapy. Screening for new SPL inhibitors requires however the availability of a reliable and fast enzyme assay. The measurement of the SPL activity has been carried out for years using radiolabeled dihydrosphingosine-1- phosphate as substrate in a rather tedious assay.A new assay using a fluorescent substrate has been recently reported, but requires still extraction and RP-HPLC.3 A simple and reproducible assay based on the release of umbeliferone from an appropriate substrate (RU13) was developed in our laboratories.4

    In this communication we describe the optimization of this assay and its validation by using several cell lines with different degree of SPL activity. We also report on a simplified format for library screening using semi-purified human SPL expressed in E. coliand its application to the discovery of SPL inhibitors. Finally, the effect of RU13 on the sphingolipidome, as determined by LC-MS, is also reported.

    1. S.R. Schwab, J.P. Pereira, M. Matloubian, Y. Xu, Y. Huang, J.G. Cyster, Lymphocyte sequestration through S1P lyase inhibition and disruption of S1P gradients. Science, 2005.309, 1735-9

    2.P. P. Van Veldhoven, G. P. Mannaerts, Sphingosine-phosphate lyase. Adv. Lipid Res. 1993, 26, 69-98.

    3.P. Bandhuvula, H. Fyrst, J. D. Saba, A rapid fluorescent assay for sphingosine-1-phosphate lyase enzyme activity J. Lipid Res. 2007, in press.

    4.C. Bedia, J. Casas, G. Fabrias. Fluorimetric determination of sphingosine-1- phosphate lyase activity. 5th International Meeting of the Sphingolipid Club. Calella,Spain, 2006.

    5. P. P. Van Veldhoven, S. Gijsbers, G .P. Mannaerts, J. R. Vermeesch, V. Brys, Human sphingosine 1-phosphate lyase: cDNA cloning, functional expression studies and mapping to chromosome 10q22. Biochim. Biophys. Acta 2000, 1487, 128-134.

  • Sphingolipid-based giant unilamellar vesicles electroformed under physiological ionic strength conditions.


    L-Ruth Montes1, Alicia Alonso1, Felix Goñiand Luis A. Bagatolli 2*

    1Unidad de Biofisica (Centro Mixto CSIC-UPV/EHU), Depto. de Bioquimica, Universidad del Pais Vasco. 2Dpt. Biochemistry and Molecular Biology/MEMPHYS, University of Southern Denmark.

    In the last years giant unilamellar vesicles (GUVs) have become objects of intense scrutiny by chemists, biologists, and physicists who are interested in the many aspects of biological membranes. In particular, this "cell size" model system allows direct visualization of particular membrane-related phenomena at the level of single vesicles using fluorescencemicroscopy-related techniques. However, the conventional preparation of GUVs currently requires very low salt concentration, thus precluding experimentation under physiological conditions. Here we show that GUVs can be prepared using a new protocol based on the electroformation method either from native membranes or organic lipid mixtures at physiological ionic strength. Additionally, for the GUVs composed of native membranes we show that membrane proteins and glycosphingolipids preserve their natural orientation after electroformation. We anticipate our result to be important in order to revisit a vast variety of findings performed with GUVs under low or no salt conditions. As an example, we show images of GUV composed of sphingolipids, and the effect of a sphingomyelinase on sphingomyelin-containing vesicles.


  • Drug target identification in sphingolipid metabolism by computational systems biology tools : MCA and MPA.


    F.Betul Kavun, Kutlu Ulgen

    Department of Chemical Engineering, Bogazici University, Bebek 34342, Istanbul,Turkey

    Sphingolipids are a class of lipids regulating cellular processes that are critically important in cell’s fate. These cellular processes function in cancer development and progression, thus this fact underlies the basics of the novel cancer therapy approach. The pharmacological manipulation of the sphingolipid metabolism in cancer therapeutics necessitates the detailed understanding of the pathway. Mathematical modeling is employed to unravel the complexity of biological systems, where the information we have gained from the use of two the computational systems biology tools in this paper is expected to shed light on the sphingolipid metabolism.

    In this study, Saccharomyces cerevisiae is employed as the model organism since detailed and complete data for this organism are available in the literature. Moreover, the sphingolipid metabolic and regulatory pathways are conserved across species and they resemble each other in all eukaryotes. Metabolic control analysis and metabolic pathway analysis are here performed in a comparative manner to identify potential drug target enzymes among sphingolipid pathway that can be further utilized in drug design studies for cancer therapy. Our aim was to rank the enzymes in SL pathway according to their roles in controlling the metabolic network by MCA and to identify the physiologically connected reactions, i.e. biologically significant and functional modules of network by MPA. The ultimate goal of this work is to achieve a set of candidate drug target enzymes and to compare the resulting critical control enzymes identified from both metabolic control analysis and metabolic pathway analysis. In fact this will be the comparison of metabolic control analysis and metabolic pathway analysis in terms of their efficiency in drug target identification. The final set of candidate drug target enzymes are selected by handling the results from both metabolic control and pathway analysis. Palmitoyl-CoA Synthase (rxn 1),Glycerol-3- Phosphate Acyltransferase (rxn 2), CDP-DAG Synthase (rxn 4), Phosphatidylinositol synthase (rxn 5), Inositol-1-phosphate synthase (rxn 6),Phosphoserine-Phosphatase (rxn 9), Serine Palmitoyl Transferase (rxn 11), Acyl-CoAbinding protein (rxn 12), Sphingolipid long chain base kinase (rxn 15 and 20),4-Hydroxylase (rxn 21), Ceramide Sythase (rxn 23), GPI Remodelase (rxn 27) andAcetyl-CoA Synthethase (rxn 34) are proposed as potential drug targets by both MCA and MPA. The mathematical tools’ efficiency for drug target identification performed in this study is also validated by the clinically available drugs.

  • Osmotic shock-elicited neutral sphingomyelinase activity in human red blood cells.


    David J. Lopez,Meritxell Egido-Gabas, Josefina Casas,Marie Garnier,Banafshe Larijani,Felix M. Goniand Alicia Alonso1*


    For years, sphingolipids have been thought to work as mere structural lipids in cells, however in the past decade their role in cell signalling has been underlined. A particularly well studied sphingolipid is ceramide, which acts as an important second messenger in cell death processes, although its putative role as an inducer of cell proliferation has also been described. Ceramide may be synthesized following two pathways: de novo synthesis and sphingomyelin (SM) hydrolysis. The first one occurs in the endoplasmic reticulum (ER) via condensation of L-serine with palmitoyl-CoA to form 3-ketosphinganine. The second pathway appears to occur mainly in the plasma membrane, as a result of sphingomyelinase (SMase) activation by an appropriate effector of the so-calledsphingolipid signaling pathway.

    Lang et al., studying the processes occurring during erythrocyte death, described an increase in ceramide content after 15 hour-incubation of human red blood cells (RBC) in a hypertonic medium. Gatt, Loyter and co-workers had reported some years back that incubation of chicken erythrocytes in a hypotonic medium resulted in the hydrolysis of SM. Because of the newly acknowledged importance of ceramide signaling, in RBC as well as in other cells, it was important to explore and define any latent sphingomyelinase activity in human erythrocytes. This work deals with the characterization of a sphingomyelinase activity in human RBC that is elicited upon incubation in either hypotonic or hypertonic media. The decrease in SM content has been monitored by three different techniques, namely thin layer chromatography (TLC), reverse phase high performance liquid chromatography (RP-HPLC) and electrospray mass spectrometry (ESI-MS). This intrinsic activity is calcium and magnesium-dependent and causes an increase in ceramide content whose maximum is reached 3-6 minutes after establishing the osmotic shock at neutral pH. The activity is blunted after addition of neutral sphingomyelinase (nSMase) inhibitors such as GW4869 or spiroepoxide. Some studies have used detergents as activators of sphingomyelinases; in this case, Triton X-100 (TX-100) slightly increases the SM hydrolysis.

    Up to seven different groups of sphingomyelinases have been described. Among the ones present in mammals, some are active at neutral pH only in the presence of magnesium ions. One type of neutral sphingomyelinases, nSMase 2, has been localized in the plasma membrane (PM), is magnesium-dependent and is inhibited by nSMase inhibitors such as GW4869. The activity described in the present work might belong to this group of neutral sphingomyelinases.

    *Corresponding author:

  • FasL-triggered inhibition of sphingomyelin synthesis is involved in ceramide increase and cell death in Jurkat leukemia T cells.


    Delphine Milhas1, Hervé Benoist1, Virginie Garcia1, Zhe-Xiong Jin2, Hisanori Umehara2, Toshiro Okazaki3, Thierry Levade1, and Bruno Ségui1

    1Inserm U858, Institut de Médecine Moléculaire de Rangueil, BP 84225, 31432 Toulouse Cédex 4, France. 2Division of Hematology and Immunology, Department of Internal Medicine, Kanazawa Medical University, Ishikawa 920-0293, Japan. 3Department of Clinical Laboratory, Medicine/Hematology, Faculty of Medicine, Tottori University, Yonago, Tottori683-8504, Japan.

    Ceramide is converted to sphingomyelin (SM) by SM synthase (SMS). Herein, we show that in human leukemia Jurkat cells, FasL treatment inhibited SMS activity in a dose- andtime-dependent manner. SMS inhibition elicited by FasL was abrogated by zVAD-fmk, abroad-spectrum caspase inhibitor, and did not occur in caspase-8- deficient cells. Thus,FasL-mediated SMS inhibition likely depends on caspase activation. Mechanisms involved in SMS inhibition upon FasL treatment are currently under investigation. RT-PCR analysis indicated that Jurkat cells expressed SMS1 but not SMS2. Jurkat cells stably expressing SMS1 siRNA displayed approximately 40-50% reduction of SM synthase activity and 15% decrease of SM content. Whereas PC level was increased by 20%, basal ceramide and diacylglycerol contents were not modified by SMS1 silencing. FasL-induced apoptosis and ceramide production were enhanced in SMS1 knockdown Jurkat cells. Our data indicate that the inhibition of ceramide conversion to SM is accompanied by an enhancement ofFasL-induced cell death in Jurkat cells.

  • Expression of alkaline sphingomyelinase (NPP7) in human liver and liver tumorigenesis.


    Yajun Cheng1, Jun Wu2, Erik Hertervig3, Stefan Lindgren4, Deming Duan2, Åke Nilsson3,Rui-Dong Duan1

    1.Biomedical Center, B11, Institute of Clinical Sciences, Lund University, Lund Sweden.

    2.Institute of Biotechnology, Beijing, China.

    3.Gastroenterology Division, Lund University Hospital, Lund, Sweden.

    4.Gastroenterology Division, Lund University Hospital, Malmö, Sweden.

    Alkaline sphingomyelinase (alk-SMase) is a novel member of nucleotide pyrophosphatase / phosphodiesterase (NPP) family and is therefore also called NPP7. It is an ecto-enzymeexpressed in the intestinal mucosa of many species. High activity was previously also found in the bile of human but not that of other species including rat, mouse, guinea pig, rabbit, pig, and baboon. In the intestine, the enzyme may prevent colonic tumorigenesis by generating ceramide, reducing the formation of lysophosphatidic acid and inactivatingplatelet-activating factor. Loss of function mutation of alk-SMase has been identified in colon cancer HT29 cells. The present study investigates alk-SMase expression in normal and diseased human liver. Northern blot identified positive mRNA band in human liver but not in rat liver. Western blot detected 3 protein bands in normal human liver and the largest one is released into human bile. Deglycosylation studies indicated that the three bands represent the enzyme with different degrees of glycosylation. No band was identified in rat liver by Western blot. Searching GenBank of expressed sequence tag (EST) found that the enzyme might be expressed in human fetal liver as early as 20-weekgestation. Analysis of SMase activities in 30 human liver biopsy samples showed relatively high activity of alk-SMase in cholestatic diseases and low activity in steatosis and primary sclerosing cholangitis (PSC). In Hep G2 liver cancer cells, RT-PCR identified 3 transcripts with 1.4 kb, 1.2 kb and 0.4 kb, respectively. DNA sequence showed that the 1.4 kb form is the wild type cDNA of alk-SMases with 5 translated exons, the 1.2 kb product lacks exon 4, and the 0.4 kb form is only a combination of exon 1 and 5. Genomic sequence indicated that these aberrant transcripts are resulted from the alternative splicing. Transient expression of the 1.2 kb did not show any alk-SMase activity. When HepG2 cells were cultured, the alk-SMase activity was low in monolayer cells but increased in polarized HepG2 cells. In addition, searching GenBank with full insert sequence (FIS) identified a "big"alk-SMase from a human liver tumor, which contains the full sequence of the enzyme plus a 73 amino acid tag at the N-terminal. The N-terminal tag changed the signal peptide ofalk-SMase that originally presents at the N-terminal, and significantly reduced the enzyme activity. In conclusion, liver expression of alk-SMase occurs specifically in human, and the expression is changed in association with hepatic tumorigenesis and diseases.

  • Nitric oxide boosts chemoimmunotherapy via inhibition of acid sphingomyelinase in a mouse model of melanoma.


    Cristiana Perrotta1, Laura Bizzozero2, Sestina Falcone2, Patricia Rovere-Querini1, Alessandro Prinetti3, Edward H. Schuchman4, Sandro Sonnino3, Angelo A. Manfredi1,5, and Emilio Clementi2,6.

    1H San Raffaele Scientific Institute, 20132 Milano, Italy; 2E. Medea Scientific Institute, 23842 Bosisio Parini, Italy; 3Department of Medical Chemistry, Biochemistry and Biotechnology, University of Milano, 20090 Segrate, Italy; 4Department of Human Genetics, Mount Sinai School of Medicine, New York, NY 10029, USA; 5Università Vita- Salute San Raffaele, 20132 Milano, Italy; 6Department of Preclinical Sciences, University of Milano, 20157 Milano, Italy.

    Cisplatin is one of the most effective anticancer drugs, but its severe toxic effects, including depletion of immune-competent cells, limit its efficacy. One promising strategy to reduce the toxicity of chemotherapy maintaining its efficacy is the combination of chemotherapy with immunotherapy. Among immunotherapies, approaches based on dendritic cells (DCs) vaccines are particularly promising. A major limitation for combination chemoimmunotherapy is that the chemotherapeutic drugs induce apoptotic death of immune competent cells used for immunotherapies. A strategy that protects selectively DCs but not tumour cells from the cytotoxic action of chemotherapeutics would therefore significantly enhance the efficacy of combined chemoimmunotherapy allowing reduced dosage of the chemotherapeutics to limit their toxic effects against normal tissues. To address the issue we took advantage of the properties of nitric oxide (NO). NO enhances resistance of DCs to the toxic effect of the tumour environment in vivo and increases DC cytotoxic, endocytic and antigen presenting functions. Here we report the results of a chemoimmunotherapy in which systemic treatment with cisplatin was combined with intratumour delivery of DCs, previously treated ex vivo with a pulse of NO released by the NO donor DETA-NO. The therapy, tested in the B16 mouse model of melanoma, was significantly more efficacious than cisplatin alone, leading to tumour regression and animal survival at low doses of cisplatin that alone had no therapeutic effect. Tumour cure was not observed combining cisplatin with DCs not exposed toDETA-NO, indicating the key role of the NO pretreatment. We investigated the mechanisms responsible for the synergic effect of NO-treated DCs and cisplatin and found that NO-treated DCs were protected both in vitro and in vivo from cisplatin-inducedcytotoxicity. Cisplatin triggered DC apoptosis through increased expression and activation of acid sphingomyelinase (A-SMase); pre-treatment of DCs with DETA-NO prevented such activation and inhibited activation of the downstream proapoptotic events, including generation of ceramide, activation of caspases 3 and 9 and mitochondrial depolarization. The effects of NO were mediated through generation of its physiological messenger cyclic GMP. We conclude that NO and NO generating drugs represent promising tools to increase the efficacy of chemoimmunotherapies in vivo, promoting the survival and increasing the function of injected cells by targeting a key pathway in cisplatin-induced cytotoxicity.

  • Ceramide kinase mediates toll-like receptor 4-induced inflammation in vascular smooth muscle cells.


    Kristy L Houck, Todd E Fox, Mark Kester

    Penn State University, 500 University Drive, Mail Code R130,Hershey PA 17033, USA

    Sphingolipid metabolites have recently been implicated in toll-like receptor 4 (TLR4) signaling. Activation of lipopolysaccharide (LPS)-induced TLR4 signaling is dependent upon production of ceramide by acid sphingomyelinase (Cuschieri et al, Surg Infect, 2007). Yet, the role of ceramide metabolites to contribute to TLR4-induced inflammatory responses has not been investigated. Specifically, we now hypothesize that TLR4-inducedproliferation and inflammation in vascular smooth muscle cells (VSMC) is dependent upon activated ceramide kinase (CERK) and resulting production of ceramide-1-phosphate(C1P). We demonstrate that LPS treatment of A7r5 VSMC leads to elevated CERK mRNA and protein expression. This increased expression of CERK correlates with increasedLPS-induced C1P mass, quantified by LC/MS/MS analysis. We show that LPS- andceramide-1-phosphate-, but not ceramide-treatment, leads to hallmarks of vascular inflammatory disease, including increased VSMC proliferation, positive remodeling, and production of inflammatory mediators. In addition, LPS-treated mouse blood samples had increased levels of ceramide-1- phosphate at the expense of ceramide. Taken together, our studies implicate ceramide-1-phosphate as a potential biomarker and /or mediator of vascular inflammation in diseases such as atherosclerosis or restenosis after angioplasty. These studies were funded, in part, by NIH RO1 HL076789 to MK.

  • Grape polyphenols regulate a critical cross-link between sphingosine-1-phosphate (S1P) and vascular endothelial growth factor- signalingpathwaysS1P-mediated transactivation of VEGF-receptor-2 (Flk-1/KDR).


    Chantal Barthomeuf 1,2 and Richard Beliveau 3

    1 Univ Auvergne, Lab de Pharmacognosie et Biotechnologies, Clermont-Fd (France)2INSERM-484, 3 Hôpital Ste Justine-UQAM, Service d'Hémato-Oncologie, Hôpital Ste- Justine/UQAM, Montréal, (Québec, Canada).

    Sphingosine-1-phosphate (S1P), a bioactive lipid released by activated platelets has emerged as an essential mediator of angiogenesis, a stimulus of the metastatic cascade and, a crucial link between inflammation, coagulation and angiogenesis. S1P is the ligand of G-protein coupled receptors (GPCR) of the endothelial differentiation gene (Edg) family, namely edg-1, edg-3, edg-5, edg-6 and edg-8. Ligation to Edg-1, the preferential Edg receptor in ECs, promotes Edg-1 translocation into caveolae and, pertuissis toxin (PTx)- sensitive dependent activation of phosphoinositide 3-kinase (PI3-K)/Akt, MEK/ERK and p38 mitogen activated protein kinase (MAPK) pathways. Edg-1 plays a major role in EC and smooth muscle cell function. It is most notably involved in migration, proliferation and differentiation of endothelial cells (ECs). Recent studies have shown that S1P promotes Src family kinase-dependent transactivation of vascular the endothelial growth factor Flk- 1/KDR also called VEGFR-2. Flk-1/KDR transactivation enhances eNOS and Akt activation in elicited cells (1). We have recently reported that grape polyphenols regulates a PI3K- dependent process: serum-mediated invasiveness of estrogen-receptor (ER)-negativebreast cancer cells (2) and, inhibit S1P and VEGF-induced EC chemotaxis (3). We have established a link between inhibition of S1P-mediated chemotaxis, reduction of platelet- activating factor (PAF) synthesis and down-regulation of p38 MAPK activation in elicited cells (2, 3). The aim of this study was to verify if the regulation of these S1P-dependenteffects could be due to inhibition of Flk-1/KDR transactivation. Western blots unambiguously demonstrate that in S1P-stimulated cells, Flk-1/KDR phosphorylation occurs as early as 3 min after S1P stimulation and, is decreased by pre-treatment with SGE or PTx. It has been found that although SGE decreases PAF synthesis and EC chemotaxis in S1P-stimulated cells, it did not modify the time-course of these events. They reach a maximum 7.5 min after S1P stimulation. Given the stimulatory effect of Flk-1-KDR on EC migration and PAF synthesis and, the kinetic of these events in SGE- pretreated cells, the suppression of Flk-1-KDR transactivation in S1P-elicited cells plays certainly a crucial role in the regulation of S1P-mediated chemotaxis and PAF synthesis by grape polyphenols. Since GPCR-dependent processes are involved in the promotion of inflammation, angiogenesis, coagulation and tumor invasiveness by S1P (5), this study has evidenced a mechanism playing a crucial role in the prevention of angiogenesis- dependent diseases by wine and grape polyphenols and, their tumor chemopreventive andanti-metastasis effects. Note that these data are the first to establish that certain dietary polyphenols regulate a critical cross-link between VEGF- and S1P-dependent signaling.

    1 Tanimoto T et al. J Biol Chem 2002, 277: 42997-3001

    2 Barthomeuf C et al. 2007 AACR Proceed, April 14-18, LA, Abstract 2656 3 Barthomeuf C. et al. Free Rad Biol Med 2006, 40:581-90

    4 Barthomeuf C et al. NS-Arch Pharmacol 2007 374: 331-3325 Barthomeuf C Free Rad Biol Med 2007 42:312-313

  • Role for sphingosine kinase-1 in survival and progression of LNCaP prostate cancer cells to androgen independence.


    Audrey Dayon 1,2, Nelly Pirot 1,2, Nicolas Doumerc 2,3, Catherine Mazerolles 4, Leonor Nogueira 5, Muriel Golzio 1,2, Justin Teissié 1,2, Guy Serre 5, Pascal Rischmann 1,2,3, Bernard Malavaud 1,2,3, Olivier Cuvillier 1,2,3

    1 CNRS, Sphingolipids and Cancer Research Laboratory, Institut de Pharmacologie et de Biologie Structurale, UMR5089, Toulouse, F-31000 France

    2 Université Toulouse III Paul Sabatier, Toulouse, F-31000 France

    3 CHU Toulouse, Hôpital Rangueil, Service d'Urologie et de Transplantation Rénale, Toulouse, F-31000 France

    4 CHU Toulouse, Hôpital Rangueil, Laboratoire Anatomie Pathologique et Histologie- Cytologie, Toulouse, F-31000 France

    5 CHU Toulouse, Hôpital Purpan, Laboratoire de Biologie Cellulaire et Cytologie, Toulouse,F-31000 France

    Androgen depletion, which is known to be associated with decreased cell proliferation and lower prostate specific antigen (PSA) secretion in LNCaP prostate cancer cells, triggers a rapid and significant but transient (< 48 h) inhibition of the oncogenic lipid kinase Sphingosine Kinase-1 (SphK1). The inhibition of SphK1 is instrumental as its overexpression can counteract the loss of proliferation induced by androgen privation. In orthotopically xenotransplanted animal models (SCID mice), the loss of tumor volume after castration is also correlated with a marked SphK1 inhibition in the prostatic tissues.

    The addition of dihydrotestosterone (DHT) to androgen deprived LNCaP cells restablishes cell proliferation as well as PSA secretion. SphK1 activity is implicated in this action since the overexpression of a dominant negative form of SphK1 or treatment with specific pharmacological SphK1 inhibitors inhibit the proliferative effects of DHT.

    Noteworthy, longer exposure of LNCaP cells to androgen-free conditions (beyond 48h and up to 42 days) is linked to a progressive increase in SphK1 activity (up to 4-fold increase at 42 days), Akt activation and ERK 1/2 stimulation. These events are associated with a neuroendocrine differentiation of these cells (as shown by the morphological changes in cell phenotype and increased levels of the Neuron Specific Enolase (NSE) marker).

    Collectively, these results implicate SphK1 in the progression of prostate cancer cells to androgen independence. The mechanisms by which SphK1 activity is increased during this phenomenon are still under investigation.

  • Genetic deletion of the sphingosine-1-phosphate lyase confers resistance to anticancer drugs.


    Colié S. 1, Kedjouar B. 1, Garcia V. 1, Carpentier S. 1, Van Veldhoven P.P. 2, Levade T. 1and Andrieu-Abadie N. 1

    1INSERM U858, Institut de Mèdecine Moléculaire de Rangueil, BP84225, 31432 Toulouse Cedex 4, France. 2LIPIT, Katholieke Universiteit Leuven, Begium.

    Sphingosine 1-phosphate (S1P) is a bioactive metabolite of sphingolipid metabolism, which modulates cell survival, proliferation and migration, as well as angiogenesis and lymphocyte trafficking. The intracellular concentration of this oncogenic lipid is governed by the activities of three different enzymes that catalyse its synthesis or removal. One of them, sphingosine-1-phosphate lyase (SPL), encoded by the Sgpl1 gene, is responsible for the irreversible degradation of S1P.

    In order to investigate the role of SPL in cell survival, we used embryonic fibroblasts derived from SPL-deficient mice (KO), and compared their susceptibility to various apoptotic inducers to that of cells from heterozygous (HET) or wild type (WT) animals. Here we demonstrated that SPL-deficient cells are resistant to the toxic effect of etoposide, doxorubicin or other cytotoxic agents such as staurosporine. In mutant cells,stress-induced caspase activation and phosphatidylserine externalization were strongly inhibited. Moreover, the ability of SPL to protect cells from apoptosis was correlated with an accumulation of S1P in SPL-deficient cells. Interestingly, heterozygous disruption of the gene encoding for SPL was associated with a partial resistance to apoptosis and moderately elevated S1P levels. Finally, SPL deficiency led to increased cell migration in an in vitro scratch-wound assay resulting in accelerated wound closure, indicating an important role for SPL in this process.

    These results suggest that, by affecting S1P metabolism, SPL may contribute to cell resistance upon antitumor treatment and could favor tumor progression.

    (Supported by INSERM, Université Paul Sabatier, and Ligue contre le Cancer)

  • Role of sphingosine kinase in the antiapoptotic action of TGF-beta in murine mesoangioblasts D16.


    Chiara Donati1, Francesca Cencetti1, Paola Nincheri1, Silvia Brunelli2,3, Emilio Clementi4, Giulio Cossu2,5, Marta Farnararoe Paola Bruni1

    1Department of Biochemical Sciences, University of Florence, Florence 2Stem Cell Research Institute, Ospedale San Raffaele, Milan, 3Dipartimento di Medicina Sperimentale ed Ambientale e Biotecnologie Mediche, Università di Milano-Bicocca, Monza 4Dipartimento di Scienze Precliniche LITA-Vialba, Università di Milano, 5Dipartimento di Biologia, Università di Milano

    Mesoangioblasts are a novel class of stem cells able to differentiate into diverse mesodermal cell types such as adipocytes, osteoblasts, smooth and skeletal muscle cells. These cells are presently regarded as suitable candidates for cell therapy of muscle degenerative diseases since they have been demonstrated capable of regenerating, in animal models, the dystrophic muscle [1]. The characterization of the physiological agonists that regulate proliferation and survival of these stem cells becomes fundamental to enhance their therapeutic efficacy.

    TGF-beta plays a critical role in the maintenance of the homeostasis between cell growth and apoptosis, however, depending on the cell type, a dual role of the citokine able of inducing proapoptotic or survival responses has been reported [2]. As regards the molecular mechanism of action, in the last few years a cross-talk between TGF-beta and sphingolipid signalling has been demonstrated to take place. It has been recently demonstrated that sphingosine 1-phosphate (S1P), a sphingolipid derived from sphingomyelin catabolism, involved in the regulation of fundamental biological effects [3], exerts a potent antiapoptotic action in murine mesoangioblasts [4].

    In this study it is reported that TGf-beta exerts a marked antiapoptotic action in mesoangioblasts with a mechanism that involves activation of sphingosine kinase (SphK), which is the enzyme responsible for S1P formation. Treatment with the cytokine indeed efficaciously protected mesoangioblasts from apoptosis induced by serum starvation or staurosporine or C2-ceramide treatment assessed by various means such as activation ofcaspase-3 and determination of cytoplasmic histone-associated-DNA-fragments. The protective action of TGF-beta from staurosporine-induced apoptosis resulted strongly reduced when SphK activity was inhibited either by pharmacological inhibitors or by overexpression of the dominant negative mutant of the enzyme and when its expression levels were downregulated by specific siRNA transfection. Finally, staurosporine-induceddownregulation of SphK was counteracted by TGF-beta treatment. Interestingly, TGF–betaantiapoptic action was impaired when ERK1/2 activation was inhibited by the specific inhibitor U0126.


    [1] Sampaolesi M., Blot S., D'Antona G., Granger N., Tonlorenzi R., Innocenzi A., Mognol P., Thibaud J.L., Galvez B.G., Barthelemy I., Perani L., Mantero S., Guttinger M., Pansarasa O., Rinaldi C., Cusella De Angelis M.G., Torrente Y., Bordignon C., Bottinelli R., Cossu G. (2006) Nature 444(7119), 574-579

    [2]Sanchez-Capelo A., (2005) Cytokine Growth Factor Rev. 16, 15-34

    [3]Spiegel S. and Milstein S. (2002) J. Biol. Chem, 277, 25851-25854

    [4]Donati C., Cencetti F., Nincheri P., Bernacchioni C., Brunelli S., Clementi E., Cossu G. and Bruni P. (2007) Stem Cells, 25(7), 1713-1719

  • Sphingosine 1-phosphate stimulates aldosterone secretion through a mechanism involving the PI3-kinase/PKB and MEK/ERK 1/2 pathways.


    Leyre Brizuela, Miriam Rábano, Patricia Gangoiti, Natalia Narbona, José María Macarulla, Miguel Trueba, and Antonio Gómez-Muñoz

    Department of Biochemistry and Molecular Biology. Faculty of Science and Technology. University of the Basque Country. P.O. Box 644. 48080 - Bilbao (Spain)

    We reported recently that sphingosine 1-phosphate (S1P) is a novel regulator of aldosterone secretion in zona glomerulosa cells of adrenal glands, and that phospholipase D (PLD) is implicated in this process. We now show that S1P causes phosphorylation of protein kinase B (PKB) and extracellularly regulated kinases 1/2 (ERK1/2), which is an indication of their activation, in these cells. These effects are probably mediated through interaction of S1P with the Gi protein-coupled receptors S1P1/3, as pre-treatment with pertussis toxin or with the S1P1/3 antagonist VPC 23019 completely abolished phosphorylation of these kinases. Inhibitors of phosphatidylinositol 3-kinase (PI3K) ormitogen-activated kinase kinase (MEK) blocked S1P-stimulated aldosterone secretion. This inhibition was only partial when the cells were incubated independently with inhibitors of each pathway. However, aldosterone output was completely blocked when the cells werepre-treated withLY294002 and PD98059, simultaneously. These inhibitors also blocked PLD activation, which indicates that this enzyme is downstream of PI3K and MEK in this system. We propose a working model for S1P in which stimulation of the PI3K/PKB and MEK/ERK pathways leads to stimulation of PLD and aldosterone secretion.

    Key words: adrenal gland; ceramide; mitogen-activated protein kinase; glomerulosa cells; mineralocorticoids; phospholipase D; protein kinase B; sphingolipids; steroids.

    J. Lipid Res. 2007. 48:2264-2274.

  • Enhancement of NO-mediated and attenuation of EDHF-mediated vasorelaxation via activation of sphingosine kinase by muscarinic receptor stimulation.


    Stephan L.M. Peters, Arthur C.M. Mulders, Marie-Jeanne Mathy, Dagmar Meyer zu Heringdorf1, Michael ter Braak1, Najat Hajji, Dominique C. Olthof, Martin C. Michel and Astrid E. Alewijnse.

    Department of Pharmacology and Pharmacotherapy, Academic Medical Center, Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands.

    Institute of Pharmacology, University Hospital Essen, Hufelandstrasse 55, 45122 Essen, Germany.

    Local formation of the sphingomyelin metabolite sphingosine-1-phosphate (S1P) within the vascular wall has been shown to modulate vascular contraction and relaxation. In the present study we investigated whether sphingosine kinase plays a role in muscarinicreceptor-mediated NO production and relaxation in different vascular beds. In a vascular endothelial cell line, expressing muscarinic Mreceptors and YFP-tagged sphingosine kinase, activation of the M3-receptor leads to a rapid translocation of sphingosine kinase from the cytosol to the plasma membrane. Moreover, the sphingosine kinase inhibitor dimethylsphingosine (DMS, 10 mM) attenuated NO production in this cell line after muscarinic receptor stimulation with the agonist methacholine. Accordingly, in isolated rat aorta the relaxant responses to methacholine were attenuated in the presence of DMS. Interestingly, DMS enhanced the relaxant responses to methacholine in mesenteric artery preparations. In this preparation, methacholine-induced endothelium-derivedhyperpolarizing factor- mediated (i.e. non NO and non-eicosanoid-dependent) relaxation was enhanced by DMS. In addition, DMS enhanced the dilatory actions of the putative endothelium- derived hyperpolarizing factor C-type natriuretic peptide.

    We conclude that stimulation of endothelial muscarinic receptors induces sphingosine kinase signalling subsequently resulting in differential effects in different vessel types. Whereas locally produced S1P enhances NO-mediated vasorelaxation, it attenuatesEDHF-mediated vasodilation. A disturbed regulation of sphingolipid metabolism in the vascular wall may therefore play a role in the aetiology/pathology of disease states characterized by endothelial dysfunction.

  • Sulfogalactosylceramides in motor and psycho-cognitive adult metachromatic leukodystrophy: relations between clinical, biochemical analysis and molecular aspects.


    Benoit Colsch(1,2), Carlos Afonso (1), Jean-Claude Turpin (3), Jacques Portoukalian (4),Jean-Claude Tabet (1) and Nicole Baumann(1,2).

    (1)University Pierre and Marie Curie UMR CNRS 7613, 4 Place Jussieu, 75252 Paris cedex 05, France; (2)UMR INSERM 711 Salpetriere Hospital, 47 Boulevard de l’Hopital, 75651 Paris cedex 13, France; (3) Association pour la Recherche en Neurochimie Salpetriere Hospital, 47 Boulevard de l’Hôpital, 75651 Paris cedex 13, France; (4) Laboratory for dermatologic research EA 4169 University Lyon 1, Edouard Herriot hospital, 5 Place d’Arsonval, 69437 Lyon cedex 03, France.

    Metachromatic leukodystrophy (MLD) is a human autosomal recessive lysosomal neurodegenerative disorder that results from the accumulation of sulfatides in the central and peripheral nervous system. It is due to the enzyme deficiency of the sulfatide sulfatase i.e. arylsulfatase A (ASA). During adolescence and/or adulthood , there are 2 clinical presentations. It may be that of a degenerative disease of the central nervous system with mainly spastic manifestations or a spino-cerebellar ataxia, or that of a psychosis.As several lines of evidence indicate that the psychotic form of MLD could be a model of psychosis, we decided to do a pluridisciplinary study on 11 psycho-cognitivecases involving mental and psychiatric testing in comparison with 5 motor cases, a biochemical study with enzyme assays and quantitative mass spectrometry of urinary sulfatides, so as to determine whether there were biochemical particularities related to the psychotic forms. For quantitative mass spectrometry, a non physiological sulfatide with C17:0 fatty acid was synthesized. Urinary sulfatides consisted of C22:1/d18:1, and /or C22:0/d18:2 (m/z 862.7),C22:0 (OH)/d18:1 (m/z 878,8), C24:0/d18:1 and / or C24:0/C23:1(OH)/d18:2 (m/z 890,7), C24:0(OH)/d18:1(m/z 906.5). We had shown previously that there were different ASA mutations in the psychiatric adult form (heterozygous I179S) versus the adult motor form (homozygous P426L) and also characteristic polymorphisms in the psychiatric forms. We show here that there were no relations with the level of ASA and with the mass spectrometric study of the sulfatide isoforms which were identical in the 2 clinical forms.

  • Cellular Antioxidant Mechanisms Regulate TNF-α and Doxorubicin-InducedNADPH Oxidase Activity Through Glucosylceramide Synthase.


    B.M. Barth, S.J. Gustafson, and T.B. Kuhn

    Alaskan Basic Neuroscience Program, Department of Chemistry and Biochemistry, University of Alaska-Fairbanks, 900 Yukon Drive, Room 194, Fairbanks, Alaska 99775 USA

    The treatment of advanced malignancies involves various therapeutic agents as well as radiation therapy. The most advanced cancers have emerged as being highly invasive and resistant to chemotherapy and radiation. Cancers of the nervous system are especially detrimental because of the dramatic impact on the everyday lives of patients. The sensitive location can make these cancers inoperable or make surgical intervention highly impractical. Furthermore, certain cancers of the brain respond minimally at best to chemotherapy and radiation, altogether giving poor prognosis to those effected. Chemotherapy results in increases in ceramide that can in turn induce cell stress, and ultimately apoptosis. Clearance of ceramide, in addition to regulation of drug transporters has linked the up-regulation of glucosylceramide synthase to cancer multi-drug resistance. Additionally, the up-regulation of sphingosine kinase has been linked to increased cancer cell survival and invasiveness.

    Previously we had demonstrated that TNF-αinduced the NADPH oxidase, and subsequent reactive oxygen species, in human SH-SY5Y neuroblastoma cells in a neutral sphingomyelinase-dependentmechanism. In this study, we demonstrated that TNF-α and doxorubicin-induced NADPH oxidase activity resulted in oxidation and down-regulation of glucosylceramide synthase and sphingosine kinase in SH-SY5Y neuroblastoma cells. We further demonstrated that glucosylceramide inhibited the NADPH oxidase, demonstrating an intriguing cellular antioxidant mechanism. Despite the presence of an NADPH oxidase, two human glioblastoma cell lines, U-87 MG and LN-18, responded to TNF-αand doxorubicin with no significant increase in reactive oxygen species. We determined through enzyme assays that the glioblastomas had increased superoxide dismutase, catalase, and glucosylceramide synthase activities compared to the neuroblastoma cell line. We propose that the increased cellular antioxidant capacity is another mechanism that promotes the resistance of these advanced cancers to chemotherapy, by protecting enzymes with important function in invasiveness and multi-drug resistance. Therefore selective targeting of the cellular antioxidant enzymes, including glucosylceramide synthase, or the up- regulation of the NADPH oxidase may be a useful strategy for the treatment of advanced drug resistant and invasive cancers.

  • Inhibition of glucosylceramide synthesis induces a cytokinesis arrest and blocks stage differentiation in Giardia lamblia.


    Sonda S,Š tefanićS. and Hehl AB.

    Institute of Parasitology, University of Zurich, Winterthurerstrasse 266a, CH-8057 Zurich, Switzerland.

    Sphingolipid biosynthesis pathways have recently emerged as a promising target for therapeutic intervention against pathogens, including parasites. A key step in the synthesis of complex sphingolipids is the glucosylation of ceramide, mediated by glucosylceramide (GlcCer) synthase, whose activity can be inhibited by PPMP (1-Phenyl-2-palmitoylamino-3-morpholino-1-propanol). Here we investigated whether PPMP affects the pathogen parasite Giardia lamblia, a major cause of intestinal disease worldwide and morbidity in young children. PPMP treatment was found to block in vitro G. lamblia replication at concentrations not affecting host cell metabolism. Importantly, PPMP inhibited the completion of cell division at a specific stage in late cytokinesis. In addition, PPMP inhibited the formation of cysts, the parasite stage responsible for the disease transmission. Moreover, an active glucosylceramide synthase inhibited by PPMP was for the first time identified and characterized in the parasite.These results show that PPMP is a powerful inhibitor of G. lamblia in vitro and that as yet uncharacterized sphingolipid biosynthetic pathways are potential targets for the development of anti-G. lamblia agents.

  • The cytosolic sialidase Neu2 is substrate of degradation by autophagy during myoblast atrophy.


    Alessandro Fanzani a, Stefani a Rossia, Elena Stoppani a, Roberta Giuliani a, Francesca Colombo a, Wim Martinet b, Augusto Preti and Sergio Marchesini a

    Department of Biomedical Sciences and Biotechnology, Unit of Biochemistry,University of Brescia, Italy. Division of Pharmacology, University of Antwerp, Wilrijk, Belgium.

    The sialidase Neu2 is an exoglycosidase able to remove sialic acid residues from glycoproteins and glycolipids. Here we investigated the behavior of Neu2 during muscle atrophy, being this enzyme expressed in the cytosol of muscle myofibers. To this purpose, we employed an in vitro model consisting of terminally differentiated C2C12 myotubes which underwent atrophy when exposed to either starvation medium or dexamethasone treatment. Neu2 expression resulted significantly down-regulated in atrophic myofibers, as both the transcript level and enzymatic activity were significantly delayed. Interestingly, the loss of Neu2 activity in atrophic myotubes occurred by protein degradation mediated by an autophagic-dependent proteolytic system. In particular, Neu2 was degraded in atrophic myofibers by lysosomal cathepsin L and B forms, as demonstrated by in vivo and in vitro degradation assays. Therefore, the impairment of sialidase Neu2 during muscle atrophy further suggests the expression of this enzyme as causally linked to proper differentiation and maturation of muscle myofibers.

  • A role for furin in TNFalpha-induced activation of the MMP/sphingolipid mitogenic pathway.


    Edwige Tellier 1, Anne Nègre-Salvayre 1, Beatrice Bocquet 1, Shigeyoshi Itohara Yusuf A. Hannun Robert Salvayre 1Nathalie Augé 1

    1.SERM UMR-466, Dept of Biochemistry, IFR-31, CHU Rangueil, Toulouse, France

    2.RIKEN Brain Research Institute, Wako-Shi, Saitama 351-0198, Japan.

    3.Medical University South Carolina, Dept of Biochemistry and Molecular Biology, Charleston, SC, 29425, USA.

    Neutral sphingomyelinase (nSMase), the initial enzyme of the sphingolipid signaling pathway, is thought to play a key role in cellular responses to TNFa, such as inflammation, proliferation and apoptosis. The mechanism of TNFa-induced nSMase activation is only partly understood. Using biochemical, molecular and pharmacologicpharmacological approaches, we find that nSMase activation triggered by TNFa is required forTNFa-induced proliferation and in turn requires a proteolytic cascade involving furin,MT1-MMP and MMP2, and leading finally to ERK1/2 phosphorylation and DNA synthesis, in smooth muscle cells (SMC) and fibroblasts. PharmacologicPharmacological and molecular inhibitors of MMPs (Batimastat), furin (a1-PDX inhibitor-transfected SMC), MT1-MMP (SMC overexpressing a catalytically inactive iMT1-MMP), MMP2 (fibroblasts from MMP2-/- mice), and various siRNA strategies (siRNAs targeting furin, MT1-MMP, MMP2 and nSMase) resulted in near complete inhibition of the activation of nSMase, SK-1, and ERK1/2 and of subsequent DNA synthesis. Exogenous MT1-MMP activated nSMase and SMC proliferation in normal but not in MMP2-/- fibroblasts, whereas exogenous MMP2 was active on both normal and MMP2-/- fibroblasts.

    Altogether these findings highlight a pivotal role for furin, MT1-MMP and MMP2 inTNFa-induced sphingolipid signaling, and they identify this system as a possible target to inhibit SMC proliferation in vascular diseases.

  • C2-Ceramide effect on hippocampal pyramidal neurons: an electrophysiological approach by using patch clamp tight- seal whole cell recording technique.





    H.Saybasili, Ö. Genç, T. Köker, Y.P. Tan

    Boğaziçi University, Biomedical Engineering Institute, 34342, Be bek, Istanbul, Turkey

    Ceramide, besides its structural role in cell membrane as a sphingolipid, has essential roles in apoptosis, cell growth and differentiation. In this study, the effect of C2- ceramide (10 µM) application on whole cell currents recorded by patch-clamp technique from soma of hippocampal CA1 pyramidal neurons was investigated. Evoked post-synaptic currents were recorded with low frequency (0.1 Hz) stimulation of Schaffer Collateral. It was observed that ceramide depressed the amplitude of NMDA currents (23 %±8 pA, n=5); the NMDA current was blocked with AP5(60 µM) application to artificial cerebrospinal fluid (ACSF). Non-NMDA currents were isolated by applying AP5 at -50mV, C2- ceramide application did not cause significant change in the non-NMDA current amplitude (n = 4). Thus, C2-ceramide leads to a selective depression on the NMDA current; this indicates that sphingolipids have a modulatory effect on the cell membrane besides their roles as secondary messengers inside the cell.

    Keywords: C2-ceramide, hippocampal synaptic currents, NMDA and AMPA receptors,patch-clamp tight-seal whole cell technique.

    * This work wasğaziçi supported University by grants Research 02S104 Fund and. 07HX101 from Bo

  • FAN (Factor Associated with Neutral sphingomyelinase activation) modulatesTNF-induced ceramide production, cytokine gene expression and neutrophil recruitment.


    Anne Montfort1, Victorine Douin-Echinard1, Pascal Martin2, Virginie Garcia1, Jason Iacovoni1, Caroline Nevoit1, Thierry Levade1, Hervé Benoistand Bruno Ségui1

    1Inserm U858, Institut de Médecine Moléculaire de Rangueil, BP 84225, 31432 Toulouse Cédex 4, France. 2Laboratoire de Pharmacologie-Toxicologie, INRA BP3, 180 chemin de Tournefeuille, 31931 Toulouse Cédex 9, France.

    Tumor Necrosis Factor alpha (TNF) is a pleiotropic inflammatory cytokine that signals the majority of its biological effects through its receptor TNFR1. FAN (Factor associated with neutral sphingomyelinase activation) is an adaptor protein that constitutively binds to a membrane proximal domain of TNFR1, referred to as the Neutral Sphingomyelinase Domain (NSD). In response to TNF, both the NSD and FAN are essential for the activation of neutral sphingomyelinase, which hydrolyses plasma membrane sphingomyelin (SM) into ceramide, a putative bioactive lipid involved in cell death and inflammation.Over-expression of a dominant-negative form of FAN (DFAN) inhibited TNF-induced SM breakdown and ceramide generation in fibroblasts. In addition, TNF-mediated IL-6secretion was impaired in cells over-expressing DFAN. Microarray analysis was performed in fibroblasts derived from wild-type or FAN knock out mouse embryos to evaluate FAN's role in TNF-induced gene expression. Approximately 50% of TNF-induced genes exhibited lower expression levels in FAN- deficient fibroblasts. We noticed that the main biological function associated with FAN- dependent gene expression is chemotaxis. Of particular interest, TNF-induced expression of cytokines/chemokines, such as IL-6, CXCL-2 and GM1960 was impaired in FAN-deficient cells. This observation was confirmed by real time PCR as well as by ELISA. To evaluate the role of FAN in TNF signalling in vivo, intraperitoneal TNF injection was performed in wild-type and in FAN-deficient mice. Neutrophil recruitment into the peritoneal cavity was reduced by 70% in FAN-deficient mice. Altogether, our results indicate the involvement of FAN in the activation of the TNF- inducedsphingomyelin-ceramide signalling pathway, which may participate in both gene expression and inflammatory responses.

    (Supported by INSERM, Université Paul Sabatier, Association pour la recherche sur le cancer and Ligue contre le Cancer)

  • Involvement of ceramide in the tissue protective effect of ectoin during ischemia/reperfusion of small bowel.


    Lai Wei1, René H. Tolba1, Reinhard Büttner2, Annika Wedeking, Gerhild vanEchten-Deckert

    Kekulé-Institute of Organic Chemistry and Biochemistry and 1House of Experimental Therapy, 2Institute of Pathology, University of Bonn, Germany.

    Using a standardized surgical rodent model of intestinal manipulation we have reported recently that sphingosine 1-phosphate (S1P) and ceramide 1-phosphate (C1P) were elevated in intestinal muscularis post-surgery. In addition, our data suggested that these two ceramide-derived molecules might be involved in the local inflammatory reaction occurring after surgery [1]. Based on these results, we hypothesized that a reduction of ceramide, the common precursor of both, S1P and C1P might improve morbidity known to occur after abdominal surgery. Since ectoin, a compatible solute, that enables microorganisms to survive under extreme conditions, was reported to prevent UVA induced ceramide increase thus rescuing keratinocytes from apoptotic cell death [2], we extended our working hypothesis. We assumed that reduction of ceramide, a well-knownmediator of cellular stress by ectoin might improve tissue quality following different kinds of stress. To simplify matters we analyzed in a first approach whether ectoin is able to improve tissue quality in rat bowel subjected to ischemia/reperfusion and whether this effect is correlated with tissue ceramide content. It is generally accepted that ischemia/reperfusion is accompanied by a complex damage mechanism that primarily affects intestinal mucosa but also involves intestinal muscularis, the latter having been neglected for quite a while [3]. Although intestinal mucosa has high regenerative ability, morphological recovery of the injured ileal mucosa after 24-hours cold storage requires at least 1 month [4]. This prolonged damage from ischemia/reperfusion injury makes it a major obstacle for successful small bowel transplantation. It is therefore of primary interest to reduce ischemia/reperfusion injury after organ preservation thus improving the clinical outcome of small bowel transplantation. Our results demonstrate that ectoin indeed convincingly prevents tissue damage following ischemia and this effect is accompanied by the reduction of ceramide in intestinal tissue. Of interest, a synthetic derivative of ectoin, incapable to overcome tissue injury, did not affect the content of ceramide, either.

    [1]M. Dragusin, S. Wehner, S. Kelly, E. Wang, A.H. Merrill, Jr., J.C. Kalff and G. vanEchten-Deckert, FASEB J 20, 1930-1932 (2006).

    [2]j. Buenger and H. Driller, Skin Pharmacol.Physiol 17, 232-237 (2004).

    [3]A. Turler, J.C. Kalff, P. Heeckt, K.M. Abu-Elmagd, W.H. Schraut, G.J. Bond, B.A. Moore, G. Brunagel and A.J. Bauer, Gastroenterology 122,1886-1897 (2002).

    [4] I. Takeyoshi, S. Zhang, M. Nomoto, Y. Zhu, Y. Kokudo, T. Suzuki, N. Hamada, A. Nemoto, T.E. Starzl and S. Todo, Transplantation 71, 1-7 (2001).

  • Inhibition of angiogesis in Krabbe’s disease: endothelial cytoskeleton disassembly by the glycosphingolipid psychosine.


    1Belleri M., 1Coltrini D., 2Nico B., 2Ribatti D., 1Alessi P., 3Poliani P.L., 4Marchesini S.,5Bongarzone E., and 1Presta M.

    1Unit of General Pathology and Immunology and 4Unit of Biochemistry, Department of Biomedical Sciences and Biotechnology, School of Medicine, University of Brescia;2Department of Human Anatomy and Histology, University of Bari; 3Department of Pathology, University of Brescia, Italy; 5Department of Anatomy and Cell Biology, University of Illinois, Chicago, IL, USA.

    Globoid cell leukodystrophy (GLD), or Krabbe’s disease, is a neurological disorder of infants caused by the reduction of the lysosomal enzyme b-galactosylceramidase activity. This results in the accumulation of the intermediate metabolite psychosine (b- galactosylsphingosine) at micromolar levels in the CNS. The accumulation of psychosine induces apoptosis of oligodendrocytes, progressive demyelination, and the formation of multinuclear (globoid) cells derived from perivascular microglia, accompanied with block of cytokinesis.

    Neovascularization plays an important role in CNS development. Here, we assessed the effect of psychosine on angiogenesis in vitro and in vivo. Also, the neovascularization process was investigated in Twitcher mice, a genetically and enzymatically authentic mouse model of human GLD.

    When tested on endothelial cells of bovine and murine origin, micromolar levels of psychosine inhibited cell proliferation and cell attachment/spreading on different substrata. Moreover, psychosine reduced cell migration after mechanical wounding of the endothelial cell monolayer by impairing the repositioning of the microtubule organization center and the formation of membrane ruffles. Also, psychosine impaired the capacity of Fibroblast Growth Factor-2 (FGF2)-transfected murine aortic endothelial cell aggregates to invade a 3D-fibrin gel and to form solid sprouts. Similarly, psychosine prevented bovine aortic endothelial cells to enter a 3D-collagen gel and to organize capillary-like structures when stimulated by FGF2 plus Vascular Endothelial Growth Factor (VEGF). Finally, psychosine caused cytoskeleton disorganization inducing the disruption of actin stress fibers in bovine aortic endothelial cells. In keeping with the in vitro observations, psychosine inhibited FGF2-induced in vivo neovascularization when delivered on the top of the chick embryo chorioallantoic membrane.

    On this basis, we performed histological and ultrastructural analysis of the brain of Twitcher mice at 2-5 weeks after birth. The cerebral cortex of Twitcher mice was characterized by reduced FVIII-vWF+ vascularity with dilated and structurally altered blood vessels when compared to control heterozygous and wild type littermates. Also, Twitcher mice showed a reduced capacity to mount an angiogenic response when challenged by a subcutaneous Matrigel implant containing angiogenic FGF2 plus VEGF. These results point to a primary defect in neovascularization of the nervous system of mutant animals.

    Our study demonstrates that psychosine has antiangiogenic properties causing the disassembling of endothelial cell actin structures. This may be responsible for the reduced angiogenic potential of Twitcher mice, leading to defects in brain vascularization. Alterations in blood vessel development may contribute to the CNS damage that occurs in Krabbe’s disease.


  • THC induces ER stress, autophagy and tumor cell death via accumulation of de novo synthesized ceramide.


    María Salazar1*, Arkaitz Carrracedo1*, §, Íñigo J. Salanueva1, Sonia Hernández1, Cristina Blázquez1, Mar Lorente1, Ainara Egia1, §, Patricia Vázquez2, Sofía Torres1, Stephane García3, Juan Iovanna3, Manuel Guzmán1, Patricia Boyaand Guillermo Velasco1#

    THC exerts a wide variety of biological effects by mimicking endogenous substances - the endocannabinoids anandamide and 2-arachidonoylglycerol - that bind to and activate specific cannabinoid receptors: CB_1 and CB_2. One of the most exciting areas of research in the cannabinoid field is the study of the potential application of cannabinoids as antitumoral agents. Recent findings have shown that cannabinoid treatment of tumor cells leads to accumulation of /de novo/-synthesized ceramide,up-regulation of the transcriptional co-activator p8 and of several endoplasmic reticulum (ER) stress-related downstream targets, including the pseudo-kinase Tribbles homologue 3 (TRB3). Activation of this pathway plays a major role in mediating the pro-apoptotic and antitumoral activity of cannabinoids. In this study, we investigated the role of this ERstress-related pathway in the antitumoral action of cannabinoids. Our data show that THC, via ceramide accumulation and eIF2α phosphorylation, up-regulates the expression of p8 and TRB3, which in induces autophagy. We show as well that autophagy is upstream of apoptosis in cannabinoid- induced tumor cell death. These findings define a new route for promoting the autophagic death of tumor cells and suggest that its activation constitutes a potential therapeutic strategy for inhibiting tumor growth.

  • Combination therapy for Tay-Sachs and related neurodegenerative diseases.


    M.B. Cachón-González, S.Z. Wang and T.M Cox

    Department of Medicine, University of Cambridge, Cambridge, UK

    The GM2 gangliosidosis are classical lysosomal storage diseases, in which neurodegeneration is caused by storage of GM2 ganglioside and related glycosphingolipids, primarily in neurones. β-hexosaminidase A - a heterodimer of the alpha and beta subunits - and the GM2 Activator Protein are absolutely required for GM2 degradation. The GM2 gangliosidoses, Tay-Sachs (TSD) and Sandhoff disease, are caused by defects in the alpha and beta subunits of β-hexosaminidase, respectively, in which failure to breakdown GM2 leads to motor and visual impairment, ataxia and dementia due to neuronal dysfunction and neuroinflammation. We have demonstrated that gene transfer (GT) can correct these diseases in mice. Substrate reduction therapy (SRT) has also been shown to improve outcome in a variety of lysosomal storage diseases, provided residual enzymatic activity remains. By addressing distinct but complementing pathways of disease, the combination of GT and SRT would be predicted to improve outcomes over and above those obtained with each individual therapy. We thus tested for therapeutic cooperation between these two treatment modalities. Murine models of Sandhoff disease, lacking the beta hexosaminidase subunit, develop typical signs of TSD and die by 20 weeks of age. We treated Sandhoff mice by stereotactic inoculation of recombinant adeno-associated viral vectors (rAAV) encoding the complementing human β-hexosaminidase genes at a single site in the striatum, and/or by SRT at a dose of 600mg/Kg/daily of the iminosugar,N-butyldeoxynojirimycin (NB-DNJ; miglustat), which was added to their diet. Each treatment was provided either alone or in combination. GT was given at 4 weeks of age, and miglustat started one week later. A single intracranial injection of rAAV extended the mean survival of Sandhoff mice from the humane end point of 135±7 (sd., n=19) to 198±24 days (n=8) (p<0.001), and miglustat alone to 171±8 (n=8) (p<0.001). The combined regimen of GT and SRT extended life only to 211±26 days (n=8), which is not significantly different from that obtained by gene transfer alone (p=0.33). As predicted, GT and SRT are therapeutic in murine GM2 gangliosidosis since each improved survival and delayed the onset of disease, but they proved neither synergistic nor additive in this animal model of TSD. Furthermore, when brain gangliosides from untreated andmiglustat-treated animals was analysed by HPTLC at the age of 112 days the quantitities of GM2 ganglioside and the related GA2 were similar in both groups. Our findings suggest that the observed therapeutic effect of the iminosugar, NB-DNJ, miglustat in GM2 gangliosidoses is brought about by a mechanism independent of substrate inhibition -eventhough the agent is licensed for the related sphingolipidosis, Gaucher disease, as asubstrate-reducing agent.

  • Involvement of neutral-sphingomyelinase on differentiation and/or apoptosis of embryonic hippocampal cells.


    Marini F1, Bartoccini E1, Bernardini I1, Cascianelli G1, Garcia-Gil MViola-Magni M1, Albi E1.

    1Dept. Clinical and Experimental Medicine, University of Perugia; 2Dept. of Biology, University of Pisa.

    During nervous system development, the precursors of neurons reside within the epithelium of the neural plate and then in the neural tube. Embryonic cells can be used for their therapeutic potential to treat neurological disorders. In fact they can be incorporated into the adult central nervous system following transplantation thanks to their ability of migration and differentiation. A critical problem of the neural transplantation is how to reduce their apoptosis and improve cell survival. Since nuclear sphingomyelin (SM) metabolism is involved in differentiation and/or apoptosis process (1), the aim of the present study was to analyse the neutral- sphingomyelinase (N-SMase) activity in nuclei free lysate (NFL) and purified nuclei of embryonic hippocampal cells. At this end HN9.10e cell line is used as experimental model. These cells are a somatic fusion product of hippocampal cells from embryonic day 18 C57BL/6 mice and N18TG2 neuroblastoma cells and have morphological and cytoskeletal features similar to their neuronal precursors (2). The differentiation is induced by vitamin D3 (3) whereas apoptosis is induced by serum deprivation (4).The results show that the nuclear N-SMase activity increases during the S-phase of the cell cycle in correspondence of the SM reduction and ceramide production. No changes are observed in NFL N-SMase. Vitamin D3 reduces DNA synthesis together to PCNA and cyclin D expression whereas bcl2 increases indicating a delay of cell cycle and an induction to differentiation supported by microscopy observations that highlight the dendrite and neurite growth. The NFL N-SMase and nuclear N-SMase activities increase after 2 and 12 hours respectively from Vitamin D3 treatment, before the S-phase of the cell cycle. The time for nuclear N-SMase activation is justified by the time necessary to vitamin D3 to reach the nucleus equal to 10 hours. Serum deprivation is accompanied by strong DNA synthesis inhibition, an early activation of nuclear N-SMase (after 1 hour) and a later activation of NFL N-SMase (after 12 hours). It can be concluded that cellular and nuclear SMases behave in different way in relation to the cell fate.

    1)Ledeen RW, and Wu G. Biochim Biophys Acta 2006; 1761: 588-598.

    2)Lee HJ, Hammond DN, Large TH, Roback JD, Sim JA, Brown DA, Otten UH,Wainer

    BH.1990. J Neurosci 10: 1779-1787

    3)Brown J, Bianco JI, McGrath JJ, Eyles DW. Neurosci Lett. 2003 Jun 5;343(2):139-


    4)Albi E, Cataldi S, Bartoccini E, Viola Magni M, Marini F, Mazzoni F, Rainaldi G, Evangelisti M, Garcia-Gil M. J Cell Physiol, 2006, 206(1):189-195

  • Involvement of dihydroceramides in cell fate determination.


    José M. Munoz-Olaya1, Xavier Matabosch1, Carmen Bedia1, Meritxell Egido-Gabás1, Josefina Casas1, Amadeu Llebaria1, Antonio Delgado1,2, Gemma Fabrias1.

    1Research Unit on BioActive Molecules (RUBAM), IIQAB-CSIC, Barcelona, Spain.2Pharmaceutical Chemistry Unit (CSIC Associated Unit), Faculty of Pharmacy, University of Barcelona, Spain.

    Ceramide is a well-known mediator of apoptosis that can be induced by a variety of signalling molecules or stress events, causing a transient intracellular increase in this bioactive lipid. Ceramide rise results from sphingomyelinase activity or de novo biosynthesis. In the latter, ceramide is biosynthesized from L-serine in four distinct steps, the last one being the 4-desaturation of dihydroceramide to ceramide by dihydroceramide desaturase.

    For years, dihydroceramides have been regarded as innocuous lipids and mere precursors of their ceramide counterparts. This assumption was sustained by the failure of exogenous dihydroceramides with short N-acyl chains to mimic the antimitogenic effects caused by the respective ceramides in cultured cells. However, recent articles point towards a possible role of dihydroceramide in programmed cell death1.

    XM462 is a dihydroceramide analogue containing a sulphur atom at the C5 position of the long chain base. This compound inhibits dihydroceramide desaturase both in vitro and in intact cells, it features high chemical and metabolic stability and it is easily available by synthesis. Flow cytometry analysis of Jurkat A3 human leukemia cells revealed that XM462 produces a concentration-dependent increase in the number of annexin V-positive cells. Measurements by LC-MS confirmed accumulation of dihydroceramides at these concentrations. The design, synthesis and biological activity of this compound are presented and discussed.

    1W. Zheng, J. Kollmeyer, H. Symolon, A. Momin, E. Munter, E. Wang, S. Kelly, J. C. Allegood, Y. Liu, Q. Peng, H. Ramaraju, M. C. Sullards, M. Cabot, A. H. Merrill, Biochim. Biophys. Acta 2006, 1758, 1864-1884; J. Stiban, D. Fistere, M. Colombini, Apoptosis 2006, 11, 773-780; J. M. Kraveka, L. Li, Z. M. Szulc, J. Bielawski, B. Ogretmen, Y. A. Hannun, L. M. Obied, A. Bielawska, J. Biol. Chem. 2007, 282, 16718- 16728



  • Expression of intestianl and lung alkaline sphingomyelinase and neutral ceramidase in cystic fibrosis F508 transgenic mice.


    Lena Ohlsson*, Lena Hjelte**, Michael Hϋhn †, Bob J. Scholte ††, Martina Wilke ††, MalinFlodström-Tullberg ††, Ake Nilsson*.

    *Department of Clinical Sciences, Medicine (Gastroenterology and Nutrition), Lund University Hospital, S221 85 Lund, Sweden

    **Stockholm CF Center, Department of Pediatrics, Karolinska University Hospital, Huddinge, S-141 86 Stockholm, Sweden

    †Centerfor Infectious Medicine (CIM), Deparment of Medicine, Karolinska Institutet, Karoliska University Hospital Huddinge, S-141 86 Stockholm, Sweden

    ††Departments of Cell Biology and Biochemistry, Erasmus Medical Center P.O. Box2040, 3000 CARotterdam, The Netherlands.

    Objetives: The instestinal brush border enzymes alkaline sphingomylinase (alk- SMase) and neutral ceramidasa (CDase) digest milk sphingomyelin (SM) in suckling neonates. Futhermore they have been implicated in sphingolipid signalling, which exhibits abnormalities in cystic fibrosis (CF). We asked wheter these enzyme are normally expressed in mice with DF508 (Cftrtm1Eur) mutation, a CF mouse model with well- characterized intestinal pathology. Methods: Alk-SMase and CDase were measured in tissues and intestinal contents of homozygous and heterozygous DF508 mice and wild type mice. Results: No difference was found in the level (activity per mg protein) of enzymes in small intestinal mucosa or content or in their longitudinal distribution. Despite a lower body weightF508of micethe the length of the small intestine and

    the weight per centimetre colon were larger than in wild type mice. Alk-SMase and CDase levels in the lung was manifold lower than in the gut and did not differ between the groups. The level of acid SMase in the distal half of the small intestine was lower in CF than in wild type mice. Conclusion: Alk-SMase and neutral CDase are normally expressed in DF508 CF mice and may generate ceramide and sphingosine from milk and endogenous sphingolipid and normal rate.

  • Lateral segregation in a palmitoyl-sphingomyelin/ palmitoyl-ceramide lipid mixture.


    Jon V. Busto*1, María Laura Fanani 2, Luisina De Tullio 2, Jesús Sot 1, Bruno Maggio 2, Félix M. Goñi 1, Alicia Alonso 1

    1Unidad de Biofísica (Centro Mixto CSIC-UPV/EHU), and Departamento de Bioquímica, Universidad del País Vasco (UPV/EHU) 2Departamento de Química Biológica-CIQUIBIC,Facultad de Ciencias Químicas-CONICET, Universidad Nacional de Córdoba, Argentina

    (*) presenting author email:

    Monomolecular films (monolayers) have been used to study the intermolecular organization of a lipid mixture containing palmitoyl-sphingomyelin (pSM) andpalmitoyl-ceramide (pCer). Lipid monolayers were formed after spreading a mixture of pSM including increasing proportions of pCer onto a clean air/water interface at 26ºC. The monolayers were then compressed while measuring surface pressure, area and potential. Multilamellar vesicles of the same lipid mixture have been analysed under differential scanning calorimetry (DSC). Lipid monolayer compression isotherms show a clear pSMliquid-expanded to liquid-condensed phase transition which disappears with increasing proportions of pCer. A critical point is observed at 0.4 XpCer in the molecular area vs. composition study, with an ideal behaviour of the mixture below and above that composition. The mixture exhibits a clear hyperpolarization in the 0- 0.4 XpCer range with a marked decrease in the excess free energy of mixing, showing a favourable interaction of both molecules. Moreover, the phase diagram reflects an isobaric pSM phase transition in the same XpCer range, while an increase in monolayer stability is observed as an increase of the collapse pressure. Taken together, these data indicate a clear lateral segregation ortwo-phase separation, with a pSM-enriched less ordered phase and a pCer-enriched more ordered phase; this is corroborated by the calorimetric data on vesicles. This study may be relevant in terms of sphingolipid signalling and domain platform formation by ceramides.

  • Role of nuclear sphingolipids on vitamin D3 -induced differentiation in embryonic hippocampal HN9 cells.


    Albi E1, Marini F1, Bartoccini E1, Meacci E2, Chico L3, Viola-Magni M1,Garcia-Gil M

    1Dept. Clinical and Experimental Medicine, University of Perugia; 2Dept.Biochemical Sciences, University of Florence; 3Dept. of Biology, University of Pisa.

    Although 1a,25-dihydroxyvitamin D3 (vitamin D3) modulates a number of functions in the nervous system, its mechanism of action is unclear.Sphingolipids are known to regulate cell fate but their effects depend on subcellular localization and the ratio betweenceramide/sphingosine-1-phosphate concentration. In the present work, we aimed to investigate whether vitamin D3 induces differentiation in the embryonic hippocampal HN9 cells, and whether nuclear sphingolipid metabolism is involved. Cells were treated with different concentrations of vitamin D3 for different periods of time; morphological differentiation was evaluated as process extension; NGF expression was measured byRT-PCR; nuclear neutral sphingomyelinase, sphingosine kinase-1 actities and ceramide content were measured as previously described (1-3). We have found that treatment with 50 nM vitamin D3 for two hours induces increase of nuclear neutral sphingomielinase activity; prolonged treatment (3 days) induces neurite growth and increases of NGF expression without altering nuclear sphingosine kinase-1 activity. Higher concentrations of vitamin D3 induce cell death. Therefore, vitamin D3- induced differentiation in murine embryonic hippocampal cells is accompanied by an early increase of nuclear neutral sphingomyelinase activity and ceramide content, followed by a raise in NGF expression.

    1) Albi et al. J Cell Physiol. 2006 206(1):189-95. 2) De Palma et al. Arterioscler Thromb Vasc Biol. 2006 26(1):99-105. 3) Albi et al. Arch. Biochem Biophys. 2005 438:156-61.

  • Patterns of free and protein-bound epidermal sphingolipids in dogs.


    Popa I, Thuy L., Pietukowska A, *Pin D, Haftek M, Portoukalian J.

    Laboratory of Dermatological Research, EA3732 University Lyon-1, Edouard Herriot Hospital, Lyon; * Veterinary School of Lyon

    The major role of the skin is to protect the body against harmful agents and water loss. There are several skin diseases in which the lipid composition in the intercellular matrix of the stratum corneum is different from that of healthy human skin, especially atopic dermatitis (AD). A comparable clinical picture of this illness can be found in atopic dogs of various breeds. Canine AD also presents evidence of lamellar lipids disorganization in the stratum corneum, similar to that described in humans. 12 tape strips were taken from the thorax of one normal dog and one AD dog. The epidermal free lipid were then extracted from these strips by a mixture of chloroform/methanol and the protein-bound lipids were released by alkaline treatment. The lipids were separated in different classes, analyzed by thin layer chromatography. Differences in the level of lipid components were found in each layer. Regarding the normal dog, the lipid components increase from the first to the 10th layer, then decrease in the lowest strips. In AD skin, we observed a high quantity of lipid in the medium strips and a marked reduction in the level of ceramide, -hydroxyl ceramide and the augmentation of free fatty and -hydroxy fatty acids. Furthermore, in the involved and non-involved skin of AD dog, different from those GM3, GD3, GT1, GQ1 gangliosides were present along with lactosylceramide.




  • Sphingolipid analogues: searching new bioactive drugs


    Daniel Canals, Gemma Fabrias, Josefina Casas.

    Research Unit on Bioactive Molecules (RUBAM); Department of Organic and Biological Chemistry,Chemical and Environmental Research Institute of Barcelona (IIQAB-CSIC); Jordi Girona 18-26, 08034 Barcelona,Spain.

    The apoptotic activity of D-erythro-dihydroceramides (DHC’s) is a matter of controversy. Thus, it has been published that exogenously added short chain DHC’s do not exhibit apoptotic activity in different cell lines1,2, while L-threo-N-acetyl- sphinganine causes cell death in HL-60 cells1,3. Additionally, accumulation of natural DHC’s, but not ceramides, along with cell death occurs in human leukaemia cells treated with gamma-tocopherol,human neuroblastoma cells treated with fenretinide, as well as in prostate cancer cells4,5. Finally, a recent report describes that both long and short chain erythro-DHC’s do not simply lack apoptogenic activity, but counteract the apoptotic effect of ceramide by inhibiting ceramide channel formation in mitochondria in early apoptosis6. In this context, the synthesis of a small combinatorial dihydroceramide analogue library obtained by systematic variation of both the sphingoid and aliphatic and acyl chains has been carried out in our group7. Among them compound GVC8PH exhibited a well defined profile of time and dose- dependent apoptosis induction, and it has been selected to perform deeper studies. Results regarding its molecular mechanism, and its effect on the sphingolipid profile in A549 cells, a human alveolar epithelial cell line used in COPD studies, are reported.

    1Bielawska A, et al., J Biol Chem., 1993268, 26226. 2Ogretmen B, et al., J Biol Chem.,2001276, 32506. 3Dragusin M, et al.,. J Lip Res2003. 44,1772. 4Jiang Q, et al., Proc. Natl Acad. Sci USA2004101, 17825. 5Kraveka K et al, J. Biol. Chem.2007, in press6Stiban J, et al. Apoptosis200611, 773.  Villorbina G, et al. Bioorg. Med Chem2007, 15, 50.





  • A new family of sphingomyelin synthase inhibitors.


    Meritxell Egido-Gabása, Xavier Mataboscha, Gemma Fabriàsa, Antonio Delgadoa,b, Josefina Casasand Amadeu Llebariaa

    a) Research Unit on Bioactive Molecules (RUBAM); Department of Organic and Biological Chemistry, Chemical and Environmental Research Institute of Barcelona (IIQAB-CSIC);Jordi Girona 18-26, 08034 Barcelona, Spain. b)Pharmaceutical Chemistry Unit (CSIC Associated Unit), Faculty of Pharmacy, University of Barcelona, Spain.

    Sphingomyelin (SM) plays an important role both in cell signaling and membrane structure and dynamics. Alteration of its concentration may have important implications in the development of diseases, such as atherosclerosis and diabetes. Sphingomyelin synthase (SMS), the last enzyme involved in SM biosynthesis, transfers the phosphorylcholine moiety from phosphatidylcholine to the primary hydroxyl group of ceramide (Cer) to produce SM and diacylglycerol.

    In this presentation we report on the synthesis and activity of a series of ceramide and sphingomyelin analogues. Initially, the SMS activity was determined in rat liver microsomes by using the fluorescent ceramide substrate NBD-C6-Cer, and monitoring its transformation to the corresponding NBD-C6-sphingomyelin by HPLC. Among all compounds tested, the most potent one inhibited SMS with an IC50 valueµ of 5 M. Kinetic experiments revealed that this compound was a non competitive inhibitor with a KiµM.ofPreliminary3 results in intact cells using Jurkat A3 and A549 cell lines indicated that the most active compounds caused a significant decrease in SMS activity without affecting glucosylceramide synthase activity. Measurements of sphingolipid concentrations by LC-MS confirmed these observations.

  • A new family of sphingomyelinase inhibitors.


    Fabio Simbaria, Xavier Mataboscha, Gemma Fabriasa, Antonio Delgadoa,b, Amadeu Llebariaa, Josefina Casasa.

    aResearch Unit on BioActive Molecules (RUBAM), IIQAB-CSIC, Barcelona, Spain.bPharmaceutical Chemistry Unit (CSIC Associated Unit), Faculty of Pharmacy, University of Barcelona, Spain

    Sphingomyelin (SM) is the primary sphingolipid in mammalian cells and plays important structural and functional roles. SM hydrolysis can be carried out by different isoforms of sphingomyelinase (SMase), producing phosphorylcholine and the intracellular effector ceramide. Ceramide diffuses within membranes acting as a second messenger. Generation of ceramide in various cellular systems is currently recognized as critical to the initiation of vital cellular processes such as differentiation, cell proliferation and apoptosis.

    Inhibitory activity of acid SMase by a small library of SM analogues synthesized in our group is reported. SMase activity was determined using A549 cell homogenates and6-hexadecanoylamino-4-methylumbelliferylphosphorylcholine (HMU-PC) as substrate. The same compounds were also tested in intact A549 cells. Flow cytometry analysis suggested that these molecules caused a time and dose dependent apoptotic cell death. Their effect on the cellular sphingolipid profile was also studied for the most active inhibitors.




  • Role of COPI vesicles in lipid sorting.


    Contreras F-Xabier*; Haberkant Per*, Thiele Christoph† Brügger Britta* and Wieland F*.

    *Heidelberg University Biochemistry Center (BZH), Im Neuenheimer Feld 328, 69120 Heidelberg, Germany.

    †Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstr. 108, 01307 Dresden, Germany.

    Transport of proteins within the early secretory pathway is mediated by COPI and COPII vesicles, with p24 family membrane proteins as receptors for their coats. These types I transmembrane proteins are enriched in COPI vesicles and belong to the minimal machinery required for the formation of these vesicles. p24 proteins cycle within the secretory pathway, and exist as monomers or dimers depending on their subcellular localization. Recent results have shown that sphingomyelin (SM) and cholesterol are segregated from in vitro generated COPI vesicles. However, against this segregation, one defined sphingomyelin species, N-stearoyl-SM (SM 18:0), is enriched in COPI vesicles. In order to investigate the mechanism of this lipid sorting, we analyzed the affinities of p24 proteins with membrane lipids under in vivo and in vitro condition. To this end, we made use of photolabile phosphatidylcholine, cholesterol and sphingolipids. Here we show thatp24-proteins do not partition into detergent resistant membranes (DRMs), indicating that COPI vesicle budding occurs from embrane platforms with a low concentration of SM and cholesterol. Furthermore, we investigated affinities of the p24-proteins to phosphatidylcholine, cholesterol and sphingolipids, and observed a specific interaction of p24 with SM. Finally we set up a new in vitro system in which polyenic fatty acid containing membrane lipids can undergo FRET with suitable donor molecules. Using this new biophycal approach we observed a specific p24 transmembrane domain-SMinteraction. From these in vivo and in vitro data we suggest that p24 proteins interact with SM and might play an important role in sorting of this lipid within the early secretory pathway.

  • Synthesis and evaluation of new aminocyclitols as analogues of the immunostimulant -galactosyl ceramide (KRN7000).


    Youssef Harrak,a Carmen Bedia,a Carolina Barra,c Raúl Castaño,c Antonio Delgadoa,b, Amadeu Llebariaa

    a) Research Unit on BioActive Molecules, Departamento de Química Orgánica Biológica,IIQAB-CSIC, Jordi Girona 18-26, 08034 Barcelona, España; b) Universidad de Barcelona, Facultad de Farmacia, Unidad de Química Farmacéutica (Unidad Asociada al CSIC), Avda. Joan XXIII s/n; 08029 Barcelona, España; c) Institut de Biotecnología i de Biomedicina (IBB), Universitat Autònoma de Barcelona. 





    Interest in glycosphingolipids has increased in recent years, due, in part due to their ability to modulate the immuneresponses.1-3 For example, the β-galactosylceramide plakoside (A), isolated from the marine sponge Plakoris simplex, was found to be a non- cytotoxic immunosuppressant agent. On the other hand, KRN7000, an - galactosyl ceramide identified from SAR studies at Kirin Brewery, has been shown to be a potent activador of the immune system.




    Over the last years, KRN7000 has become the most representative glycolipid used in the study on NKT cell stimulation. As described above, KRN7000 stimulates the production of both Th1 and Th2-related cytokines by NKT cells. In this work, we have prepared a series of KRN7000 analogues based on the aminocyclitol core structure with N-axial orN-equatorial sphingoid side chains. Preliminary studies on the immune responses mediated by NKT cells are reported.

    Acknowledgements: Ministerio de Educación y Ciencia.

    (1)Merrill, A. H.; Sweeley, C. C. In Biochemistry of Lipids, Lipoproteins and Membranes 1996,31, 309-339.

    (2)Hannum, Y. A. Sphingolipid-Mediated Signal Transduction; R. G.Landes Company: Austin,1997.

    (3) Porcelli, S. A.; Modlin, R. L. Annu. ReV. Immunol. 199917, 297-329.

  • Chaperone effect of several iminosugars and aminocyclitols derivatives on defective glucocerebrosidases causing Gaucher disease.


    G. Sánchez-Ollé1,2, M. Egido-Gabás3, J. Casas3, A. Chabás2,4, D. Grinberg1,2 and L. Vilageliu1,2

    1Departament de Genètica, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain.

    2CIBERER, Barcelona, Spain.

    3RUBAM (Research Unit on BioActive Molècules), Departament de Química Orgànica Biològica, Institut d'Investigacions Químiques i Ambientals de Barcelona (IIQAB-CSIC),Barcelona, Spain.

    4Institut de Bioquímica Clínica, Hospital Clínic, Barcelona, Spain.

    Gaucher disease (GD) is caused by the defective activity of lysosomal glucocerebrosidase (GBA) due mainly to mutations in the GBA gene. It results in the accumulation of glucosylceramide in the lysosomes of phagocytic cells, leading to several symptoms including hepatosplenomegaly, skeletal lesions and, sometimes, central nervous system involvement. Some of the GBA mutated enzymes retain the catalytic activity but impair proper folding and trafficking in the cell. It has been demonstrated that several compounds are able to act as pharmacological chaperones by increasing endoplasmic reticulum (ER) folding efficiency and lysosomal trafficking. However, this process is not general and different GBA mutations showed distinct chaperoning profiles.

    Here we report the chaperone effect of two iminosugars N-(n-nonyl)deoxynojirimycin (NN- DNJ) and N-(n-butyl)deoxynojirimycin (NB-DNJ), and four aminocyclitols derivatives (C4- Ph, C8, C9 and C10) on different expressed mutant glucocerebrosidases. In particular, mutations N188S, N188S;E326K, N370S, I402T, D409H and L444P were analysed. COS-7cells were transfected with an expression vector carrying the wild-type or the mutated cDNAs. Stable transfectants were obtained with geneticin (G418) selection (500 µg/ml) and cultured either with or without NB-DNJ (5 and 10 µM), NN-DNJ (2.5 and 50µM), C4-Ph(10, 15 and 20 µM), C8 (10, 15 and 20 µM), C9 (10, 15 and 20 µM) and C10 (2.5, 5, 10, 15 and 20 µM). The ratio of enzyme activity with/without the compound was calculated. The values shown are the results of at least two experiments with 3 replicates.

    NN-DNJ (5 µM) led to an increase (1.2 fold) in the activity of wild-type enzyme, as reported. Moreover, a 1.2 (5 µM) to 1.4 (2.5 µM) fold increase was shown for the N188S GBA and a 1.2 fold for the G377S enzyme. No significant changes in activity for the rest of the mutated enzymes were shown. NB-DNJ induced a 1.2-fold increase in the activity of the N188S and N188S;E326K GBAs, at 5 µM. No other significant effect was observed. C4- Ph seemed to produce a dose-dependant pattern on the wild-type and N188S GBAs. At 15µM, 1.2-fold increase for the N188S and the N188S; E326K enzyme and a 1.3-foldincreased for the wild-type GBA were detected. At 20µM, 1.3 and 1.4-fold increase was shown for the N188S and wild-type GBA respectively. No effect was showed at 10 µM. The other compounds, C8, C9 and C10, did not seem to have any chaperoning effect on the mutated enzymes at the concentrations used.

    We can conclude that, in some conditions, the NN-DNJ, NB-DNJ and C4-Ph seem to have an effect as pharmacological chaperones on particular mutant enzymes. However, new conditions and new compounds should be assayed.




  • Characterization of the vasoactive properties of S1P receptors in rat aorta.


    AE Alewijnse, M Jongsma, M-J Mathy, PB van Loenen, N Hajji, MC Michel and SLM Peters

    Dept. Pharmacology & Pharmacotherapy, Academic Medical Center, Amsterdam, the Netherlands

    In the cardiovascular system, the bioactive sphingolipid sphingosine-1- phosphate (S1P) exerts its vasoactive effects predominantly via three specific G-protein coupled receptors, named S1P1, S1Pand S1P3. These receptors are differentially expressed in vascular smooth muscle cells (VSMCs) and endothelial cells (ECs).

    We have explored the contribution of these three receptors to vascular reactivity in the rat aorta and to related signaling mechanisms in VSMCs and ECs isolated from rat aorta using some new S1P receptor ligands. We show that in intact isolated rat aortic rings, all agonists at the S1Pand/or S1Preceptor induced endothelium-dependent vasorelaxation in pre-contracted preparations. In rat aortic ECs, which showed a high mRNA expression of the Gi-coupled S1Preceptor, all ligands, including the S1Pspecific agonist SEW2871, decreased the forskolin-induced cAMP accumulation. In addition, VPC23019 competitively antagonized the FTY720-P- induced inhibition of the cAMP accumulation. The tested ligands did not induce major changes in intracellular calcium in ECs.

    In VSMCs, which express mRNA for all three S1P receptors, predominantly Gq-coupled S1P receptor signaling was observed via the S1Preceptor, resulting in increases in intracellular calcium.

    In conclusion, we have shown that the S1P receptor agonists tested in this study can act on both ECs and VSMCs from the rat aorta. S1Preceptor stimulation in ECs leads to Giactivation (measured as inhibition of cAMP accumulation). Endothelial Gactivation can cause relaxation via activation of the PI3K/Akt pathway subsequently leading to the production of NO. Stimulation of mainly the S1Preceptor in VSMCs results in calcium elevation, a response typically linked to contraction. At least for agonists devoid of S1P2activity, the net effect observed on aortic tone is vasorelaxation.




  • Role of Sphingosine Kinase-1 in Calcium Signalling and Growth of Mouse Embryonic Fibroblasts.


    B. Hegen1, M. ter Braak1, V. Hardel1, T. Hla2, K.H. Jakobs1, D. Meyer zu Heringdorf1

    1Institut für Pharmakologie, Universitätsklinikum Essen, 45122 Essen, Germany; 2Center for Vascular Biology, University of Connecticut Health Center, Farmington, Connecticut, USA

    Sphingosine kinases (SphKs) catalyse the phosphorylation of sphingosine tosphingosine-1-phosphate (S1P), which acts as a ubiquitous mediator regulating cell growth and survival, migration, cytoskeleton organization and Ca2+ homoeostasis. Many plasma membrane receptors stimulate SphK activity, thereby producing S1P that can either be secreted and activate specific G protein-coupled receptors or act on so far unknown intracellular target sites. We have analysed the role of SphK1 in Ca2+ signalling and cell growth using mouse embryonic fibroblasts (MEFs) deficient in SphK1. [Ca2+]i increases induced by lysophosphatidic acid (LPA) were identical in SphK1 expressing (SPHK1+/+) and SphK1 deficient (SPHK1-/-) MEFs with respect to velocity of [Ca2+]i increase, peak [Ca2+]i values, time course of decay and concentration dependence. Extracellular S1P increased [Ca2+]i in SPHK1-/- cells maximally by only 441±50 nM (n=10) compared to 747±49 nM (n=9) in SPHK1+/+ cells, while the EC50 was not different. Similarly, [Ca2+]i increases by ATP and thrombin in SPHK1-/- cells amounted to only about 25 % and 50 % of those in control cells, respectively. Bradykinin induced [Ca2+]i increases only in SPHK1+/+ cells, while angiotensin increased [Ca2+]i only inSPHK1-/- cells. These differences were in part paralleled by differences ininositol-1,4,5-trisphosphate (IP3) production, suggesting that the receptor expression profile was different in the two cell populations. However, LPA, which induced maximum [Ca2+]i increases of a similar magnitude as bradykinin, did not significantly stimulate IP3 production in either cell type, while bradykinin elevated IP3 levels by about 9-fold in SPHK1+/+ cells. Therefore, LPA-induced [Ca2+]i increases were independent of IP3 and SphK1. [Ca2+]i increases by LPA in both SPHK1+/+ and SPHK1-/- cells were strongly inhibited by the sphingosine kinase inhibitor, 2-(p-hydroxyanilino)-4-(p-chlorophenyl)thµM, 60 min),azolesuggesting(30 that (an)other SphK(s) might be

    involved. A major difference in [Ca2+]i signalling was observed with sphingosine, which mobilized Ca2+ in SPHK1+/+ cells but did not cause measurable [Ca2+]i increases inSPHK1-/- cells. These data support the hypothesis that sphingosine has to be phosphorylated for inducing Ca2+ mobilization and implicate SphK1 in this process. Finally, growth of SPHK1-/- cells was strongly delayed compared to SPHK1+/+ cells. This difference was not abrogated by treatment with extracellular S1P (10-1000 nM), supporting prior data showing that SphK1-dependent cell growth is mediated by intracellular, not extracellular S1P.




  • Involvement of PI3K/Akt in the ER to Golgi traffic of ceramide in glioma cells: a link between lipid signaling pathways involved in the control of cell survival.


    P. Giussani, L. Brioschi, T. Colleoni, R. Bassi, G. Tettamanti, L. Riboni and P. Viani

    Dept. Medical Chemistry, Biochemistry and Biotechnology, Faculty of Medicine, University of Milan, Italy

    The role of ceramide (Cer) as lipid mediator governing the fate of glioma cells is supported by different studies which demonstrate that the modulation of Cer levels in these cells plays a fundamental role in the control of cell proliferation, differentiation and apoptosis. The metabolic pathways involved in the control of Cer levels in glial cell proliferation and death include both specific enzymes of Cer metabolism, such as serine palmitoyl- transferase and sphingomyelin-synthase, and the transport of Cer from endoplasmic reticulum (ER) to the Golgi apparatus, for the biosynthesis of complex sphingolipids, thus suggesting that the modulation of Cer levels in the ER/Golgi compartment can be crucial for glial cell survival. Recent results obtained in our laboratories indicate that in C6 glioma cells the ER-to-Golgi Cer transport involves both the Cer binding protein CERT and avesicle-mediated mechanism which can separately contribute to the control of sphingolipid metabolism and Cer levels in these cells. In the present study we investigated the mechanisms involved in the regulation of Cer transport in glioma cells. The presence on CERT of a N-terminal pleckstrin homology (PH) domain with selectivity towardphosphatidylinositol-4-phosphate (PI4P), suggests that phosphatidylinositol-4-kinases(PI4K) are involved in the control of CERT-mediated Cer transport. Very recently, PI4KIIIbeta was identified as the enzyme specifically involved in the control of Cer flow from the ER to the Golgi apparatus. In this study we found that phenylarsine oxide (PAO), an inhibitor of all PI4K enzymes, and wortmannin (WT), a widely used PI3K inhibitor that also specifically inhibits type III PI4K, strongly inhibited the ER-to-Golgi transport ofBODIPY-Cer and significantly limit the metabolic utilization of Cer for the biosynthesis of sphingomyelin (SM) and, in a lesser extent, of glucosylceramide (GlcCer). However, down regulation of PI4KIIIbeta did not cause any modification on Cer transport and metabolism in C6 glioma cells, thus indicating that multiple PI4Ks are involved in the control of PI4P required for CERT functioning. Interestingly the use of 100 nM WT, that selectively inhibit PI3K, significantly reduced the transport and metabolic use of Cer. In addition, in cells down regulated for CERT, 100 nM WT was still able to inhibit Cer transport and the biosynthesis of SM and GlcCer. The results obtained with LY294002 a potent and specific inhibitor of PI3K confirmed the role of PI3K in regulating Cer traffic. Moreover, preliminary results indicate that Akt, the downstream target of PI3K in the PI3K/Akt survival pathway, is involved in the effect of PI3K on Cer traffic and sphingolipid metabolism. Taken together our results indicate that PI3K/Akt pathway could regulate the ER-to-Golgi transport of Cer independently from CERT, and contribute to the control of Cer levels in glioma cells. This could represent a crucial link between Cer signaling and PI3K/Akt pathway functional to maintain Cer levels low and/or to stimulate the biosynthesis of membrane complex sphingolipids as a requirement for cell proliferation and growth.




  • THC-induced ceramide accumulation induces autophagy via TRB3 inhibition of the mTORC1 complex.


    María Salazar1, Arkaitz Carrracedo1Íñigo J. Salanueva1, Sonia Hernández1, Cristina Blázquez1, Mar Lorente1, Ainara Egia1, Patricia Vázquez2, Sofía Torres1, Stephane García3, Juan Iovanna3, Manuel Guzmán1, Patricia Boya2 and Guillermo Velasco1

    1.Department of Biochemistry and Molecular Biology I, School of Biology, Complutense University, 28040 Madrid, Spain

    2.Department of Cell and Developmental Biology, Centro de Investigaciones Biológicas, CSIC, 28040 Madrid, Spain.

    3.U624 INSERM, Campus de Luminy, 13288 Marseille, France.

    THC, the main active component of the plant Cannabis sativa, exerts a wide variety of biological effects by mimicking endogenous substances (the endocannabinoids) that bind to and activate specific cannabinoid cell receptors: CB1 and CB2. One of the most exciting areas of research in the cannabinoid field is the study of the application of cannabinoids as potential antitumoral agents. Recent findings have shown that cannabinoid treatment of tumor cells leads to accumulation of de novo-synthesized ceramide, up-regulation of the transcriptional co-activator p8 and of several endoplasmic reticulum (ER) stress-relateddownstream targets, including the pseudo- kinase Tribbles homologue 3 (TRB3). Activation of this pathway plays a major role in mediating the pro-apoptotic and antitumoral activity of cannabinoids. In the accompanying abstract we show that activation of this ER-stress related pathway leads to autophagy and cell death. In this study, we investigated the mechanisms through which ceramide-induced p8 and TRB3up-regulation leads to activation of autophagy, showing that THC promotes inhibition of the mTORC1 complex via TRB3 inhibition of Akt. We also show that activation of this signaling route takes place in vivo. These findings further support that activation of this THC and ceramide- regulated autophagic cell death pathway constitutes a potential therapeutic strategy for inhibiting tumor growth.




  • Altered calcium signalling in mouse embryonic fibroblasts deficient insphingosine-1-phosphate lyase.


    M. ter Braak1, V. Hardel1, K.H. Jakobs1, P.P. Van Veldhoven2D. Meyer zu Heringdorf1

    1.Institut für Pharmakologie, Universitätsklinikum Essen, 45122 Essen, Germany 2.K.U.Leuven, Department Molecular Cell Biology, LIPIT, Leuven, B-3000, Belgium

    Sphingosine-1-phosphate (S1P) regulates cell growth and survival, migration, cytoskeleton organization and Ca2+ homoeostasis in many cell types via activation of specific Gprotein-coupled receptors, termed S1P1-5. Intracellular S1P has furthermore been shown to mobilize stored Ca2+ independently of G-protein-coupled receptors. To further elucidate the mechanism of S1P-induced Ca2+ mobilization, we have analysed Ca2+ signalling in mouse embryonic fibroblasts (MEFs) deficient in S1P lyase (Sgpl1-/-),displaying elevated S1P levels [1]. Overall [Ca2+]i was measured in suspended cells loaded with fura-2. In Sgpl1-/- MEFs, basal [Ca2+]i was strongly elevated (~300 nM versus ~120 nM in control MEFs). Basal [Ca2+]i levels were also elevated in Ca2+-freemedium (252±38 nM compared to 123±16 nM, n=6 each) indicating that the effect was not caused by Ca2+ influx. [Ca2+]i increases induced by agonists were enhanced inlyase-deficient MEFs. [Ca2+]i increases by LPA, bradykinin, andµMS1Peach)(1 amounted to 156±17 % (n=5), 400±52 % (n=3), and 198±28

    % (n=3) of those in control cells, respectively. Furthermore, [Ca2+]i increases induced by the SERCA inhibitor, thapsigargin, were augmented (302±25 nM versus 203±18 nM, n=5), and the velocity of [Ca2+]i increases induced by thapsigargin was elevated (13.1±1.7 versus 7.4±0.9 nM/s, n=5), suggesting enhanced Ca2+ storage in Sgpl1-/-cells. Interestingly, treatment with µMa sphingosine2 kinase inhibitor (30 -(p-hydroxyanilino)-4-(p-chlorophenyl)thiazole overnight) largely decreased elevated basal [Ca2+]i levels in lyase-deficient MEFs, suggesting that the observed alterations in Ca2+ signalling are caused by or linked to S1P accumulation. Analysis of fluo-4- loaded MEFs by confocal microscopy revealed enhanced fluorescence of subcellular compartments inSgpl1-/- MEFs, while the fluorescence was evenly distributed throughout the cytosol of control MEFs. Two major staining patterns were observed, a reticular staining that colocalized with ER markers, and a punctuate staining that colocalized with a lysosomal marker. Treatment with thapsigargin decreased the fluorescence within subcellular compartments, indicating that these were thapsigargin-sensitive Ca2+ stores. It is concluded that MEFs lacking a functional S1P lyase display an enhanced Ca2+ storage in endoplasmic reticulum and lysosomes that leads to augmented [Ca2+]i increases by agonists.

    [1] Van Veldhoven P.P. Sphingosine 1-phosphate lyase deficient mice. Chem. Phys. Lip. (2005) 136, 164-165.




  • Regulation of acid sphingomyelinase by Gi proteins. Role in macrophage apoptosis.


    Shih Wei WangaPatricia Gangoitib, Urs P. Steinbrechera, and Antonio Gómez-Muñozb.

    aDepartment of Medicine, University of British Columbia, Vancouver (Canada), and 

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

    Apoptosis is an important mechanism involved in regulating the number of macrophages present at sites of inflammation. Several lines of evidence indicate that blocking macrophage apoptosis can promote the progression of atherosclerotic plaques. In previous studies we showed that apoptosis of bone marrow-derived macrophages (BMDM) induced by M-CSF withdrawal involves stimulation of acid sphingomyelinase (A-SMase)and ceramide accumulation. Treatment of macrophages with oxidized LDL, sphingosine1-phosphate (S1P) or ceramide 1-phosphate (C1P) prevented ceramide accumulation partially and blocked macrophage death. This action involved inhibition of A-SMaseactivity.

    In the present study we used pertussis toxin (PTX) to test whether G protein coupled receptors are involved in the regulation of A-SMase activity. PTX is a bacterial toxin that inhibits Gactivation by ADP-ribosylating the α subunit of Gi, thereby preventing the subunit from interacting with receptors. Unexpectedly, we found that PTX blocked macrophage apoptosis by itself. This effect was dose-dependent and involved inhibition ofA-SMase and the subsequent generation of ceramide. In addition the Gactivator peptide mastoparan, but not its inactive analogue mastoparan 17, stimulated A-SMase and the increase of ceramide levels in macrophage which induced apoptosis. Interestingly,pre-treatment with PTX partially overrode mastoparan- induced apoptosis.

    Taken together these results suggest that Gprotein coupled receptors may be involved in the control of cell survival through regulation of A-SMase activity.

    This study was supported by grants from the Heart and Stroke Foundation of British Columbia and Yukon (Canada), and BFU2006-13689/BFI from Ministerio de Educacion y Ciencia (Madrid, Spain).




  • Involvement of JNK but not p38 in the mitogenic effect of ceramide 1- phosphate.


    Patricia Gangoiti , María Granado , and Antonio Gómez-Muñoz .

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

    Our previous work on fibroblasts and macrophages showed that ceramide 1- phosphate (C1P) is mitogenic and antipoptotic. Recently we demonstrated that a major mechanism whereby C1P blocks apoptosis involves inhibition of acid sphingomyelinase and the subsequent generation of ceramides. However, the mechanisms or signaling pathways that are implicated in the mitogenic effect of C1P have not been thoroughly studied.

    In a recent study we demonstrated that C1P stimulates DNA synthesis and cell division in bone marrow-derived macrophages. This effect was dependent upon activation of PI3-K/PKB (Akt) and ERK 1-2 pathways, but did not require the activity of phospholipase D, or adenylyl cyclase. We now observed that C1P stimulated a rapid phosphorylation of c-Jun terminal kinase (JNK) in the macrophages and that inhibition of this pathway by SP600125 blocked macrophage proliferation. In addition, treatment with SB202190, which is a selective inhibitor of p38, did not affect C1P-induced DNA synthesis in these cells. A classical transcriptional factor involved in the regulation of cell proliferation is NF-κB. In a previous report, we demonstrated its implication in macrophage survival. In this work, we show that C1P stimulates the DNA binding activity of NF-κB in macrophages, and that selective inhibitors of this transcriptional factor, such as CAPE or SC-514 blocked the stimulatory effect of C1P on cell proliferation. In addition, we found that the inhibitors of PI3-K, MEK and JNK, (LY290042, PD098059 and SP600125 respectively), completely blocked the DNA binding activity of NF-κB. Lastly, another key finding of the present work is that monocyte-colony stimulating factor (MCSF), which promotes macrophage survival and proliferation, caused an increase in the intracellular levels of C1P.

    It can be concluded that the mechanism whereby C1P stimulates cell proliferation in bone marrow-derived macrophages involves the activation of JNK but not p38.

    This study was supported by grants BFU2006-13689/BFI from “Ministerio de Educación y Ciencia” (Madrid, Spain) and 9/UPV 00042.310-15852/2004 from “Universidad del País Vasco” (UPV/EHU) (Bilbao, Basque Country).




  • Implication of the PI3-Kinase/PKB pathway in Ceramide 1-Phosphate-stimulated arachidonic acid release.


    María Granado, Patricia Gangoiti, and Antonio Gómez-Muñoz

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

    Ceramide 1-phosphate (C1P) is a bioactive sphingolipid capable of regulating important pathophysiological functions including cell proliferation, apoptosis, phagocytosis, and inflammation. Chalfant and co-workers first showed that C1P, which was delivered to cells in a solution of ethanol:dodecane, stimulated the calcium dependent cytosolic form of phospholipase A(cPLA2α) in lung cancer A549 cells. Using rat alveolar NR8383 macrophages and C1P dispersed in water, we demonstrate in this work that the stimulation of arachidonic acid release by C1P is independent of the solvent system used. Also, this effect is specific as other related phospholipids such as ceramide, or phosphatidic acid (PA) failed to stimulate secretion of this fatty acid. UsingN-{(2S,4R)-4-(Biphenyl-2-ylmethyl-isobutyl-amino)-1-[2-(2,4-difluorobenzoyl)-benzoyl]-pyrrolidin-2-ylmethyl}-3-[4-(2,4-dioxothiazolidin-5-ylidenemethyl)-phenyl]acrylamide, HCl , a selective inhibitor of cPLA2-α, we show that this enzyme is directly implicated in the stimulation of arachidonic acid release by C1P in alveolar macrophages, which is in agreement with work mentioned above.

    Further investigation into the mechanism whereby C1P increases the release of arachidonic acid revealed that selective inhibitors of phosphatidylinositol 3-kinase (PI3-K) blockedC1P-stimulated arachidonic acid release in the macrophages, suggesting an involvement of this enzyme in the pro-inflammatory effect of C1P. The latter also induced phosphorylation of protein kinase B (Akt), which is downstream of PI3-K. However, although C1P caused phosphorylation of the mitogen activated protein kinases (MAPK) ERK1/2 (extracellularly regulated kinases 1 and 2), specific inhibitors of this pathway did not affect the stimulation of arachidonic acid by C1P.

    It can be concluded that stimulation of arachidonic acid release by C1P in alveolar macrophages can be achieved by direct stimulation of cPLA2 or through stimulation of thePI3-K/PKB (Akt) pathway, but not ERK1/2 or p38.

    This study was supported by grants numbers 040732 from “Fundació la Marató de TV3”, Barcelona, Spain, 9/UPV 00042.310-15852/2004, from “Universidad del Pais Vasco (UPV/EHU)” Bilbao, Spain, and S-PE06UN 13 from “Departamento de Industria, Comercio y Turismo del Gobierno Vasco, Gasteiz-Vitoria, Spain.




  • Regulation of acid sphingomyelinase trafficking and activity: nitric oxide role and new players.


    1Laura Bizzozero2Cristiana Perrotta, 3Patrizia Rosa and 1,4Emilio Clementi.

    1E. Medea Scientific Institute, 23842 Bosisio Parini, Italy; 2Stem Cell Research Institute, H. San Raffaele Scientific Institute, 20132, Milan, Italy; 3CNR Institute of Neuroscience, Department of Medical Pharmacology, University of Milan, 20133 Milan, Italy; 4Department of Preclinical Sciences, LITA Vialba, L. Sacco Hospital, University of Milan, 20157 Milan, Italy.

    The gaseous messenger nitric oxide plays an important role in a variety of biological functions such as cell differentiation, proliferation and death. Physiological concentrations of NO inhibit apoptosis triggered by a variety of apoptogens, including those activating death receptors of the TNF- (TNFRI)/ CD95 superfamily. Recent evidences indicate that NO is able to regulate ceramide levels and in U937 monocyte-derived cells it has been shown that NO inhibits the apoptotic responses induced by CD95 and TNF-RI by reducing the ability of these receptors to generate ceramide. One of the enzymes responsible for ceramide generation is the lysosomal phosphodiesterase Acid Sphingomyelinase. In recent papers we demonstrated that NO is able to abolish activation of A-SMase triggered by the apoptogenic receptors and lipopopolysaccharide (LPS) and that the anti-apoptotic function of NO is mediated through activation of soluble guanylyl cyclase and cGMP-dependentprotein kinase.

    We therefore decided to investigate the mechanisms of ASMase inhibition by NO in U373 glioma cell line. Recent experiments have demonstrated that activation of this enzyme involves its trafficking from intracellular compartments to the plasma membrane by processes of exo-endocytosis. We observed that CD95 triggers A-SMase activation in atime-dependent and transient way and induces A-SMase translocation to the plasma membrane with the same trend of the activation. The same sets of experiments carried out in the presence of a NO donor demonstrated that NO inhibits A-SMase activity through the blockade of enzyme translocation. Immunofluorescence studies carried out under resting condition, i.e. prior to CD95 stimulation, in order to understand the A-SMasetranslocation further, showed that A-SMase co-localised with the lysosomal marker cathepsin D and with the SNARE protein syntaxin 4 suggesting this protein as responsible of A-SMase trafficking and as a target of the inhibitory action of NO. To this end we performed immunofluorescence experiments in the presence/absence of DETA-NO/8Br-cGMP in which we found that the two proteins no longer colocalized.

    In addition we observed a very strong reduction of the levels of syntaxin 4 as measured by western blotting experiments carried out in the same conditions. Experiments intended to investigate the mechanism by which NO regulates the level of syntaxin 4 are now in progress; preliminary evidences indicate that NO promotes degradation of this protein. Evidence accumulated over the last few years indicates that the cross talk of A- SMase with NO plays prominent roles during key patho- physiological processes such as inflammation, proliferation, death and differentiation. Therefore, the identification of the mechanisms of this tightly regulated event and of its molecular intermediates might be relevant for understanding these important, as yet not fully defined, processes.





  • Tumor necrosis factor receptor 1 and 2 deficiency or acidic sphingomyelinase knockout ameliorates ethanol-induced liver damage.


    Ana Fernández, Anna Colell, Fran Caballero, Carmen García-Ruiz, and José Carlos,Fernández-Checa.

    Liver Unit, Hospital Clínic i Provincial. CIBEREHD- IDIBAPS, IIBB-CSIC, Barcelona, Spain.

    Tumor necrosis factor (TNF) is a proinflammatory cytokine that induces different cellular responses including inflammation, proliferation and cell death. TNF binds to two different plasma membrane receptors, TNF receptor 1 (TNFR1) and receptor 2 (TNFR2). Evidence from studies using anti-TNF therapy and TNFR1 knockout mice indicated an important role for TNF in the pathogenesis of alcoholic liver disease (ALD). Recent studies in experimental models of alcoholic (ASH) and non-alcoholic steatohepatitis (NASH) questioned the role of TNFR1 in disease progression and hepatic steatosis. Moreover, acidic sphingomyelinase (ASMase) has been shown to play a key role in the hepatocellular death induced by TNF. However, since in the absence of TNFR1 TNF can signal through TNFR2, and because the role of the ablation of both TNFR1 and TNFR2 or ASMase in ALD have not been previously examined, the aim of this study was to examine the contribution of TNFR1/TNFR2 and ASMase in ALD. Methods: 8 weeks-old male C57BL/6 mice deficient in both TNFR1 and TNFR2 (DKO) and ASMase-/- mice were used. Animals were fed the Lieber-DeCarli diet for up to 4-6 weeks. Animals received a single dose of LPS (5mg/kg) 24 hours before sacrifice. Liver damage was assessed by histochemical hematoxilin/eosin staining and serum transaminase levels. Hepatic steatosis and lipid infiltration were examined by oil red staining and biochemical analyses. Hepatic inflammation was monitored by myeloperoxidase (MPO) staining.

    Results: the hepatic inflammation and necrosis observed in wild type ethanol-fed mice were attenuated significantly in DKO knockout mice. In vivo LPS injection in ethanol wild animals increased AST serum levels 8 times over pair-fed control mice (1440±210 U/dL vs. 135±14 U/dL) respectively. This increase was significantly lower in mice lacking both TNF receptors (ethanol DKO +LPS vs. ethanol wild type + LPS: 235±45 U/dL vs.1320±157U/dL). The increase in hepatic triglycerides, cholesterol and free fatty acids induced by ethanol feeding was also reduced (72±9, 68±11, 59±8%, respectively) in the DKO mice fed ethanol. Moreover, inflammatory cell foci reflected by MPO staining showed a marked reduction in DKO-fed ethanol mice vs wild-type ethanol fed mice following in vivo LPS challenge. Interestingly, ASMase knockout mice exhibited hepatic steatosis, although alcohol feeding did not further increase the levels of TG, FFA and cholesterol. However, unlike DKO, ASMase-/- mice are sensitive to concanavalin-induced liver injury, indicating a functional role for TNFR2 in and a link between TNFR1 and ASMase.

    Conclusion: These results support the hypothesis that TNF plays an important role in inflammation and necrosis in alcohol-induced liver injury and that the targeting of TNF or its downstream mediator ASMase may be therapeutically useful in this disease.




  • Ganglioside GD3 synthase overexpression sensitizes human hepatoma cells to hypoxia by suppressing the nuclear factor-kB-dependent survival pathway.


    Albert Morales1, Josep M. Lluis1, Scott Welford2, Amato Giaccia2 and José C. Fernandez- Checa1

    1Liver Unit, Instituto Malalties Digestives, Hospital Clinic I Provincial, IDIBAPS, CSIC, UB, Barcelona, Spain. 2Division of Radiation and Cancer Biology, Stanford University, Stanford, California, USA.

    Hypoxia is a common characteristic of locally advanced tumors and emerging evidence indicates that it has a profound effect in malignant progression and responsiveness to therapy. The cellular adaptation to hypoxia is mediated by mitochondrial reactive oxygen species (ROS) generation. Hypoxia-induced ROS play a key role in HIF-1a stabilization andNF-kB activation, which are responsible for promoting survival of cancer cells, angiogenesis, vascularization, glycolytic ATP and tumor invasion. Ganglioside GD3 has been identified as a lipid death effector due to its dual

    function of interacting with and recruiting mitochondria to apoptotic pathways and counteracting survival signals by suppressing NF-kB activation. We have recently shown that NF-kB plays a critical role in the survival of hepatoma cells during hypoxia. Thus, the aim of our work was to examine the ROS generation, NF-kB and HIF-1a activation, and survival of hepatoma cells that overexpress GD3 during hypoxia. Methods. GD3 synthasefull-lenght cDNA (1.2 kb) was cloned into pcDNATM 3.1D/v5-His-TOPO®. Hep3B were transfected with pcDNATM-GD3 (Hep3B-GD3) or pcDNATM (no insert, Hep3Bwt) and selected during 3-4 weeks based on expression of GD3 synthase gene, GD3 synthase activity and ganglioside GD3 levels. Hypoxia (2%) was induced in Hep3B cells for 24-72hours. Peroxides and superoxide anion were monitored with fluorescent probes 2´-7´-dichlorofluorescein diacetate (DCFH-DA) and hydroethidine (HE), respectively. NF-kB andHIF-1a transactivation were measured by luciferase reporters. Results. Immunostaining analyses indicated that GD3-expressing Hep3B clones accumulated GD3 both at the cell surface and in internal membranes, including mitochondria. These cells did not exhibit changes in morphology, growth and apoptosis compared to Hep3Bwt during normoxia. However, the exposure of Hep3B-GD3 cells to 2%O2 elicited a time-dependent decrease in cell survival reaching 55% at 72 hours while in Hep3Bwt was 90%. This was accompanied by greater ROS generation (1.5-2 fold) in Hep3B-GD3 versus Hep3Bwt. Hypoxic NF-kBactivation observed in Hep3Bwt (2-3 fold) was abrogated in Hep3B-GD3 cells (1.2-1.5fold) whereas HIF-1a levels (6 fold versus normoxia) remained similar in both clones. We next, focused on the regulation of Mn-SOD, a known kB-controlled antioxidant enzyme. Interestingly, hypoxia induced Mn-SOD protein and mRNA levels (2-3 fold, respectively) in Hep3Bwt that were reduced upon NF-kB downregulation in Hep3B- GD3 (1.2 fold). Moreover, MnTBAP, a SOD mimetic, reduced ROS generation and protected Hep3B-GD3cells against 2%O2 at 72 hours (viability of 75%). Conclusions. Endogenous expression of ganglioside GD3 sensitizes Hep3B cells to hypoxia through suppression of inducible kB- dependent antioxidant Mn-SOD resulting in cytotoxic ROS overgeneration. These findings suggest that ganglioside GD3 may be useful in cancer gene therapy as a sensitizing agent in hypoxic-resistant cancer cells.




  • Sphingosine Kinase 1 as an effective target to inhibit proliferation of myeloid leukemia cells: insights into the mechanism of action of "SK Inhibitor”.


    Clara Ricci, MS(1,2), Francesco Onida, MD(2,3,4), Federica Servida, MS (2), Kati Todoerti, MS(3), Antonino Neri, MD(3,4), Giorgio Lambertenghi Deliliers, MD(2,3,4) and Riccardo Ghidoni, PhD(1,4)

    1.Lab. of Biochemistry and Molecular Biology, San Paolo University Hosp., University of Milan, Milan, Italy, 20142;

    2.Fondazione Matarelli, Milan, Italy, 20122;

    3.Hematology Dept., Fondazione IRCCS Osp. Maggiore Policlinico, Mangiagalli e Regina Elena, Milan, Italy, 20122

    4.University of Milan, Milan, Italy, 20122.

    In the last decades, several studies have highlighted the role of SPL metabolism in the process of carcinogenesis. Particularly, the impairment of intracellular levels of enzymes controlling the generation of key mediators, such as SphK1 that produces S1P, contributes to cell growth, survival and transformation and to the acquisition of drug resistance in models of solid tumors as well as of leukemias. However, few data are available on specific SphK1 inhibitors for in vitro and in vivo studies. The hypothesis of this experimental work is that targeting SphK1 in myeloid leukaemia cells, particularly in CML, constitutes the basis for an effective anti-cancer therapy. Therefore, the aim of this study is to gain insights into the mechanism of action of a non competitive inhibitor of the ATP binding site of SphK1, “SK Inhibitor” (SKI), whose anti-tumor activity has been demonstrated only in in vitro and in vivo models of solid tumors. First, we show that in the panel of 5 different cell lines chosen to represent different subtypes of myeloid leukemias, SphK1 is expressed and active, and then we demonstrate that effective targeting of SphK1 by SKI causes an anti-proliferative, cytotoxic effect in all cell lines tested, hence supporting the hypothesis on which this work is based. Next, we focused on K562 as a model ofp210Bcr/Abl+-CML cells, to elucidate the mechanism of action of SKI. We show that the compound induces an early, significant inhibition of SphK1 activity without affecting kinase expression, confirming its selective, inhibitory mechanism of action. It also induces apoptosis, as evidenced by PARP cleavage and cell cycle analysis, and affects cell cycle progression, leading to accumulation of cells in the G0/G1 phase. At the molecular level, SKI does not inhibit ERK1/2, most likely acting upstream of SphK1, and induces changes of the expression of genes involved in many different cellular processes. Finally, we provide evidence for the usefulness of designing combination therapies based on the inhibition of both Bcr/Abl and SphK1 and present preliminary results on the effect of SKI on the clonogenic potential of hematopoietic progenitor cells. Our study supports the hypothesis that SphK1 is an effective target for killing leukemia cells, and may thus provide insights into the mechanism of action of the new SphK1 inhibitor SKI as an anti-leukemic agent, to be used alone or in combination with IM. Indeed, due to the fact that the phenomenon of resistance to IM remains a major issue in the treatment of patients with CML, the identification of alternative targets and new drugs is of clinical relevance.


  • Calmodulin is a potential intracellular receptor for the lipid mediator sphingosylphosphorylcholine.


    Erika Kovacs and Karoly Liliom

    Affiliation and address: Institute of Enzymology, Hungarian Academy of Sciences, P.O.Box 7, Budapest, H-1518 Hungary


    Sphingosine-1-phosphate (S1P) and sphingosylphosphorylcholine (SPC) are physiologically relevant mediators of signaling, regulating basic cellular processes like growth, apoptosis, motility, and Ca2+ homeostasis. They can act as first as well as second messengers. While their activation of cell surface G protein-coupled receptors is intensively studied, not much is known about their intracellular mechanisms of action, their target proteins are yet to be identified. Here we examined whether these sphingolipids interact with calmodulin (CaM), the ubiquitous intracellular Ca2+ sensor. Binding assays utilizing intrinsic tyrosine fluorescence of the protein, dansyl-labeled CaM, and surface plasmon resonance showed that SPC selectively binds to both apo and Ca2+-saturated CaM, characterized by a submicromolar dissociation constant. Experiments carried out with the model CaM-binding domain melittin revealed that SPC dissociates the CaM-peptide complex, suggesting an inhibitory role. The functional effect of the interaction was examined on two target proteins, phosphodiesterase and calcineurin. We found that SPC inhibited the Ca2+-CaM- dependent activation of both enzymes. Thus, we propose that CaM is an intracellular receptor for SPC, and raise the possibility for a novel endogenous regulation of CaM.




  • Sphingosine 1-phosphate modulates Transient Receptor Potential Channel 1 (TRCP1) in skeletal muscle cells. Relevance for Connexin43-up- Regulation and myogenesis.


    Sassoli C. Martinesi M. 1, Squecco R. 3, Bini F.1, Zecchi-Orlandini S. 2, Francini F. 3, Formigli L2., and Meacci.E1.

    Departments of 1Biochemical Sciences, 2Anatomy, Histology and Forensic Medicine,3Physiological Sciences, University of Florence and -Interuniversity Institute of Myology (IIM), Italy.

    Sphingosine 1-phosphate (S1P), a lipid mediator originated by sphingomyelin degradation, is able to regulate a wide variety of important cellular functions (1). Although, S1P can act as pro-differentiating agent in C2C12 myoblasts, its downstream effectors are poorly known.        Recently, we have demonstrated that the induction of myogenesis by S1P required the activation of several signaling pathways, including the up-regulation of Cx43 expression and its interaction to cytoskeleton (2).Mechanosensitive channels (stretch-activatedchannel, SACs) are present in many cell types, and partecipate to the mechano- transduction, converting membrane stretch into electrical cationic current and biochemical signalling (3). Increasing evidence support the important role of SACs in cell homeostasis, cell differentiation and in the pathogenesis of several muscle diseases, such as muscular dystrophy, muscle hypertrophy and cardiac arrhythmias. Despite these observations, the involvement of canonical transient receptor potential 1 (TRPC1), identified as a critical componenet of SACs (3), in S1P-induced signalling remains to be elucidated. To this purpose, in the present study, we examined the expression and localization of TRCP1 in C2C12 myoblasts and its role in S1P-induced Cx43 expression, gap junction functionality and myogenesis.                                                                           

    Interestingly, we found that TRCP1 appears to be responsible for the majority of stretch- activated channel (SAC) activity induced by S1P, as demonstrated by the reduced SAC activity in cells in which TRCP1 protein is silenced by siRNA. TRCP1 preferentially co- localizes with Cx43 in lipid rafts and directly interact with cytoskeleton actin. The treatment with dihydrocytochalasin B, a selective actin disrupter agent, with gadolinium chloride, a specific pharmacological inhibitor of SACs, and the transfection of C2C12 myoblasts with siRNA-TRCP1 significantly reduces the up-regulation of Cx43 as well as myogenic marker expression promoted by S1P.

    Overall, these findings provide novel information regarding the mechanisms by which S1P affects muscle cell biology, including in the list of the downstream effectors of the bioactive lipid, TRCP1. Importantly, these results suggest that, in myoblasts stimulated with S1P, cytoskeleton remodeling and the following activation of cation currents through TRPC1/SACs are essential steps in the up-regulation of Cx43 expression, event that is physiologically required for myogenesis in vivo (4). In addition, the discovery of the functional interaction between sphingolipid signalling and TRP channels will inevitably reveals a new possible target for therapy in diseases, in which alterations of cation concentration appears to be crucially involved in the activation of proteolytic enzymes and cell degeneration, such as muscle distrophy.

    Grants from university of Florence, Ente CRPP and Fondazione Banche di Pistoia e Vignole




  • Sphingomyelin metabolism of thyroid cells changes in the space.


    Albi E1, Bartoccini E1, Marini F1, Cascianelli G1, Bernardini I1 , Viola-Magni M1, Perrella G2.

    1Dept. Clinical and Experimental Medicine, University of Perugia; 2Department of Experimental and Clinical Pathology and Medicine, University of Udine.

    The radiation absorbed by astronauts during interplanetary flights is mainly due to cosmic rays that can cause thyroid disorders through the activation of intracellular signals. Since the ionizing radiation exposure induces the release of ceramide and diacylglycerol as second messengers for proapoptotic and antiapoptotic signals respectively (1), the aim of the present work was to evaluate the effect of the cosmic enviroment on lipid fraction of the FRTL-5 cells. This cell line permanently expresses in vitro most of the in vivotissue-specific thyroid characteristics, such as thyroglobulin synthesis and secretion, iodide active transport, peroxidase production and thyrotropin sensitivity. FRTL-5 cells are induced to proliferate only when they are cultured in the presence of TSH (TSH+) whereas in the THS-free medium the cells remain in a quiescent state for long time (TSH-, 2). The study has been performed on TSHand TSHFRTL5 cell during the 10-days Italian Soyuz Mission(ISM) “Eneide” (April 15 to April 25) on international Space Station. The activity of neutral– sphingomyelinas (N-SMase) and sphingomyelin-synthase (SM-synthase) were evaluated in nuclei-free lysates (NFL) and purified nuclei. Moreover the same experiments have been made in the samples maintained in colture at Udine and Baikonur. The results show that in the space the N-SMase activity increases and SM- synthase activity decreases and it occurs more in the purified nuclei that in the NFL. The variations obtained in the TSHFRTL5 cells are similar whereas those obtained in the TSHFRTL5 cells are lower of those previously reported for the same cells treated with UV rays (3). These data can be correlated with the reduced proliferative activity of TSHFRTL5 cells.




  • Unexpected role of glycosphingo-lipids in cell physiology.


    G van Meer, J Wolthoorn, S Groux-Degroote, D van den Heuvel1, H Gerritsen1, D Halter, S Neumann, S van der Poel, H Sprong

    Bijvoet Center and Institute of Biomembranes, 1Molecular Biophysics, Utrecht University, The Netherlands

    Until a decade ago, the biosynthesis of the sphingolipids seemed simple: sphingosine, newly synthesized or recycled from the lysosomes, is converted to ceramide in the ER, where after vesicular transport it receives a phosphocholine or glucose headgroup in the cis-Golgi lumen. Now we know that there are at least 6 ceramide synthases, that sphingomyelin is synthesized in thetrans-Golgi, that the ceramide is supplied via the ceramide transfer protein CERT, and that CERT is regulated via PI4P on the Golgi and phosphorylation. Unexpectedly, glucosylceramide is synthesized on the cytosolic surface of the Golgi, and must cross the membrane to reach the site of complex glycolipid synthesis in the Golgi lumen.

    We found that natural glucosylceramide does not cross the Golgi membrane and is not a substrate for the multidrug transporters ABCB1 and C1. Instead, glucosylceramide was transported back to the ER by a mechanism involving the glucosylceramide binding protein FAPP2 bound to the trans- Golgi via PI4P. It then crosses towards the ER lumen and reaches the Golgi lumen by vesicular transport, possibly generating ER sphingolipid domains along the way. Mean- while, glucosylceramide is required for protein sorting in the secretory pathway by affecting conditions in the lumen. As a possible explanation, we found that glucosylceramide affects the lumenal pH.

  • The (patho)physiological role of sphingolipids in vascular biology.


    SLM Peters, LJA Spijkers, RFP van den Akker, PB van Loenen, N Hajji and AE Alewijnse

    Dept. Pharmacology & Pharmacotherapy, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands

    Besides their well-documented effects on cellular growth, sphingolipids have vasoactive properties. Although sphingolipids are present in blood in high concentrations, both endothelial as well a vascular smooth muscle cells can synthesize the different sphingomyelin metabolites. Since these celle also express the different target molecules, sphingomyelin metabolites can be regarded as auto- or paracrine factors in the vascular system. Indeed, several vasoactive factors exert their vasoactive effects partly by modulation of sphingolipid metabolism in the vessel wall. For instance, both angiotensin II as wel as muscarinic receptor agonist can activate endothelial NO synthase by activation of sphingosine kinase. Interestingly, the effect of locally formed S1P proved to be vessel type dependent. Whereas in larger conduit vessels endothelial S1P production leads to NO synthase activation, in resistance vessels locally formed S1P has an inhibitory effect on the release or action of endothelium- derived hyperpolarizing factor. Hypertension is associated with marked alterations in sphingolipid homeostasis; Inhibition of sphingosine kinase induces major constrictions in isolated vessels of hypertensive rats. Interestingly, these contractions are mediat- ed by an endothelium-derived cyclooxygenase 1 product. Endothelial sphingolipid metabolism plays an important role in controlling the release or action of dilatory or contracting factors.

  • P38-mediated endothelial cell death after ionizing radiation is under the control of ceramide generation and membrane remodeling.


    C Niaudet1, S Bonnaud1, S Gouard1, MH Gaugler1,2, I Corre1, F Paris1

    1Centre de Recherche en Cancérologie Nantes-Angers, Inserm U892, Nantes, France; 2IRSN, Fontenay aux Roses, France

    Despite the involvement of ceramide and acid sphingomyelinase (aSM) in irradiated endothelial cells (EC) apoptosis, molecular mechanisms involved are poorly understood. MAPK p38 has been considered as a crucial actor in EC radiosensitivity. If p38 activation has been shown to be dependent of DNA damage induction in tumor models, no relation has been described between ceramide and p38 under genotoxic stress. In the present study, we highlighted a direct link as p38 phosphostain- ing detected in intestines EC from 15 Gy-irradiated mouse was not observed in intestines EC from irradiated aSM KO mouse. In HMEC-1 EC, adding exogenous C16-ceramide or bacterial SM induced raft microdomain reorganisation from a discrete pattern in the cell surface to large and polarised areas, followed by p38 activation and cell death. Similar membrane remodeling following ceramide generation was also observed in 15 Gy- irradiated ECs and this membrane remodeling is a pre-requisite to p38 phosphorylation. The two concomitant phenomena, i.e. ceramide-induced raft coalescence and p38 death-pathway, have been validated by use of drugs, such as desipramine (aSM inhibitor), and nystatin (cholesterol scavenger, disorganizing lipid raft formation), which inactivated p38 and the subsequent EC death.

  • Ceramide: a novel modulator of toxoplasma gondii survival?


    S Sonda, I Bottova, AB Hehl

    Institute of Parasitology, University of Zurich, Winterthurerstrasse 266a, CH-8057 Zurich, Switzerland.

    Toxoplasma gondii, the most versatile and widespread member of Apicomplexan parasites, is responsible for a disease of major medical and veterinary importance worldwide. Despite the impact of toxoplasmosis, there are few currently available treatments, often resulting in general side-effects. Interest in invertebrates, that sustain the diffusion permeability barriers of epithelia and the glial blood-brain barrier. Trafficking of junction components to the correct membrane domain is interlocked to the process of cell polarization and relies on the correct compartmental- ization of proteins as well as lipids in membranes. Little is known about the contribution of lipids to cell junction formation. We use Drosophila to study the role of lipids (in particular sphingolipids, glycosphingo- lipids, and phospholipids) in these cell biological processes. As a handle to the genetic study of lipids in this context, we investigate the contribution of lipid metabolism genes to the process of SJ formation. We screened mutants in sphingolipid,glyco-sphingolipid, and phospholipid metabolism genes using an established dye penetration assay, which probes the tightness’ of the epithelial permeability barrier. We now proceed to assess the molecular, cellular and ultrastructural defects in the mutants with disrupted barriers. In addition, we try to identify/develop reagents to visualize lipid subtypes in our system, in order to characterize the subcellular distribution of different lipids in the polarised cells of Drosophila epithelia.

  • Role of dihydroceramides in resveratrol induced autophagy.


    P Signorelli1, JM Munoz-Olaya2, V Gagliostro1, J Casas2, R Ghidoni1, G Fabrias.2

    1Dept. Medicine, Surgery and Dentistry, San Paolo Hospital Medical School, University of Milan, Italy, 2Dept. of Biomedi- cinal Chemistry, IQAC CSIC, Barcelona, Spain

    Dihydroceramides, the metabolic precursors of ceramides along the de novo synthesis pathway, have always been considered as non signaling lipids. We report that resveratrol induced autophagy in gastric cancer cells is mediated by dihydroceramides. Treatment ofHGC-27 cells with resveratrol produced a marked autophago- some localization of GFP-LC3 as found by fluorescence micros- copy, and an accumulation of its PE-conjugated protein as measured by western blotting. UPLC-MS analysis of resveratrol treated cells showed unchanged levels of ceramides and a dose- dependent increase in dihydroceramides. These results suggested that the buildup of dihydroceramides was responsible for the observed autophagic flux and that resveratrol might inhibit dihydroceramide desaturase. In support of this, dihydroceramide desaturase activity in treated cells showed a robust inhibition by both resveratrol and a synthetic dihydroceramide desaturase inhibitor. Furthermore, the latter mimicked the effects of resver- atrol in terms of autophagy induction and accumulation of dihydroceramides. Collectively, these results demonstrate that resveratrol induced autophagy occurs by a rise in the intracellular dihydroceramide pool as a result of inhibition of dihydroceramide desaturase.

  • Dihydroceramide accumulation and oxidative stress are distinct events in 4-HPR induced cell death.


    A Apraiz1, J Idkowiak-Baldys2, N Nieto-Rementeria1, MD Boyano1, YA Hannun2, A Asumendi1

    1Dept. Cell Biology and Histology, University of the Basque Country, Spain, and 2Dept. Biochemistry and Molecular Biology, Medical University of South Carolina, USA

    N-(4-hydroxyphenyl) retinamide (4-HPR or fenretinide) is a synthetic derivate of retinoic acid used as chemopreventive agent in both, in vitro and clinical trials. In vitro studies show a dose and/or cell type dependent growth-inhibition/apoptosis-induction. In addition, 4- HPR mediated cytotoxicity has been widely linked to oxidative stress and ceramide increase. Previous studies in our group described strong apoptotic effect of 4-HPR on Acute Lymphoblastic Leukemia cells (CCRF-CEM and Jurkat) which was linked to oxidative stress. It was also shown that oxidative stress and dihydroceramide (but not ceramide) accumulation are early events following 4-HPR treatment. The aim of this work was to stablish the relationship between 4-HPR-induced oxidative stress, changes in sphingolipids profile and cell death. Interestingly, we observed that 4-HPR-induced dihydroceramide accumulation even at suble- thal (<1microM) 4-HPR concentrations. Inhibition of sphingolipid de novo synthesis by myriocin prevented dihydroceramide accu- mulation but did not protect from 4-HPR mediated cytotoxicity. On the other hand, pretreatment with antioxidants protected cells from4-HPR induced viability loss suggesting that oxidative stress (not dihydroceramide accumulation) is the cell death inducing event.

  • Caspase-mediated inhibition of sphingomyelin synthesis is involved in fasl-triggered cell death.


    D Milhas, E Lafont, T Levade, H Benoist, B Ségui

    U858 INSERM, Equipe 14, BP84225, 31432 Toulouse cedex 4, France

    Ceramide can be converted to sphingomyelin (SM) by SM synthases (SMS) 1 and 2. Here, we show that in human leukemia Jurkat cells, which express mainly SMS1, FasL treatment inhibited SMS activity in a dose- and time-dependent manner. SMS inhibition elicited by FasL (i) was abrogated by zVAD-fmk, a broad-spectrum caspase inhibitor, (ii) did not occur in caspase-8- and -10 doubly deficient cells, (iii) was only partially affected in caspase-9 deficient cells and (iv) was not impaired in Bcl-xL over-expressing cells. Western blot experiments showed early SMS1 cleavage in a caspase-dependentmanner upon FasL treatment. In a cell-free system, caspase-2, -8 and-9, but not caspase-3 and -10, cleaved SMS1. Thus, FasL-mediatedSMS inhibition depends on caspase activation and is an early event in Fas signaling, occurring upstream of mitochondria. FasL-inducedceramide production and cell death were enhanced in cells stably expressing a siRNA against SMS1. Conversely, in cells stably over- expressing SMS1, FasL did neither increase ceramide generation nor efficiently induced cell death. Altogether, our data demonstrate that SMS1 is a caspase target that is functionally involved in the regulation of FasL-induced apoptosis.

  • Transcriptional regulation of sphingosine kinase 1 by hypoxia in human endothelial cells.


    A Huwiler

    Institute of Pharmacology, University of Bern, Friedbühlstrasse 49, CH-3010 Bern, Switzerland.

    Sphingosine kinases (SK) catalyse the formation of sphingosine 1- phosphate which plays an important role in cell growth and survival.

    So far, two subtypes of SK have been cloned and partially characterized. Previously, it was shown that SK-1 is rapidly activated by various growth factors through ERK-mediated direct phosphor- ylation. Here, we show that SK-1 is also regulated on the transcriptional level. Low oxygen levels (hypoxia) stimulates a delayed increase of SK-1 activity which is preceded by increased mRNA expression and de novo protein synthesis of SK-1. This effect is due to stimulated SK-1-promoter activity which contains two putative hypoxia-inducible factor-responsive-elements (HRE). By deletion of one of the two HREs, hypoxia-induced promoter activation is abrogated. Furthermore, hypoxia upregulates the expression of HIF-1α and HIF-2α, and both contribute to SK-1 gene transcription as shown by selective depletion of HIF-1α or HIF-2αby siRNA. The hypoxia-stimulated SK-1 upregulation was function- ally coupled to increased migration since the selective depletion ofSK-1 by siRNAs abolishes the migratory response. In summary, these data show that hypoxia upregulates SK-1activity and results in an accelerated migratory capacity of endothelial cells. SK-1 may thus serve as an attractive therapeutic target to treat diseases associated with increased endothelial migration and angiogenesis such as cancer growth and progression.

  • Sphingosine kinase-1: a new modulator of hif-1α during hypoxia in human cancer cells.


    I Ader1,2, L Brizuela1,2, P Bouquerel1,2, B Malavaud1,2O Cuvillier1,2 

    1CNRS, Sphingolipids and Cancer Research Laboratory, Institut de Pharmacologie et de Biologie Structurale, UMR5089, Toulouse,F-31000 France

    2Université Toulouse III Paul Sabatier, Toulouse, F-31000 France

    We provide the first evidence that sphingosine kinase-1 (SphK1) - an oncogenic lipid kinase balancing the intracellular level of key signaling sphingolipids - modulates the transcription factor HIF-1α,master regulator of hypoxia. SphK1 activity is stimulated under low oxygen conditions and regulated by reactive oxygen species. The SphK1- dependent stabilization of HIF-1α levels is mediated by the Akt/ glycogen synthase kinase-3β signaling pathway that prevents its von Hippel-Lindau protein-mediated degradation by the proteasome. The pharmacological and RNA silencing inhibition of SphK1 activity prevents the accumulation of HIF-1α and its transcriptional activity in several human cancer cell lineages (prostate, brain, breast, kidney, and lung) suggesting a canonical pathway. Therefore, we propose that SphK1 can act as a master regulator for hypoxia giving support to its inhibition as a valid strategy to control tumor hypoxia and its molecular consequences.

  • Sphingosine kinase is implicated in the transforming growth factor-β-dependent myofibroblastic differentiation of myoblasts.


    C Donati, F Cencetti, C Bernacchioni, E Rapizzi, P Nincheri, P Bruni Dipartimento di Scienze Biochimiche, Università di Firenze, Firenze Italy

    The pleiotropic cytokine transforming growth factor-β (TGFβ) has been reported to impair myoblast differentiation during myogenesis and favour muscle fibrogenesis. Recently, the occur- rence of a direct functional interaction between the TGFβ- and sphingosine1-phosphate- (S1P) regulated signalling pathways has been established. In this context, here to gain insights into the mechanism of action of TGFβ in myoblasts, the effect of the cytokine on SphK has been examined together with its possible implication in the evoked biological response.                  TGFβ was found to exert a biphasic effect on SK: the enzymatic activity was indeed inhibited within the first 4 h of incubation whereas it was enhanced at more prolonged times of incubation (18-72 h). The enhancement of SK activity was accompanied by the up-regulation of SK1, indicating that TGFβexerts a transcriptional control of SK1. The pro-fibrotic effect of TGFβ, was attenuated when SphK1 was inhibited or down- regulated by specific silencing. Moreover, by employing selective agonists and antagonists for S1PR, as well as siRNA, it was shown that the TGFβ-regulated expression of fibrosis marker was downstream of S1P3. These data demonstrate that SphK/S1P axis is exploited by TGFβ to transform skeletal myoblasts into myofibroblasts.

  • Characterizing and targeting survival factors in a hepatocellular carcinoma stem cell population.


    BM Barth1, W Ding2, SL Lehman3, ND Molyneaux4, JM Kaiser1, CB Rountree1,2 and M Kester1

    1Dept. of Pharmacology, and 2Dept. of Pediatrics, Penn State University College of Medicine, USA, and 3Dept. of Biology, Elizabethtown College, USA, and 4Dept. of Health and Human Development, Penn State University, USA

    Aggressive forms of cancer have recently been hypothesized to arise from small sub-populations of cells with stem cell-likecharacteristics. These cancer stem cells are suggested to arise from either normal stem cell populations or a reversion of cancerous cells to a stem cell phenotype. Cancer stem cells are suggested to be the underlying cause of chemotherapy failure in many cancers. In our study we evaluated Mat1a-/- and liver-specific PTEN-/- models of hepatocellular carcinoma for CD133cellular sub- populations, as well as expression of anti-apoptotic proteins and developmental regulators. We determined that the inhibitor of apoptosis protein survivin, and the transcription factor Evi-1, were significantly up regulated in the PTEN-/- model. Specific siRNA targeting survivin or Evi-1, delivered using our novel cationic nanoliposomes, sensitized and synergistically augmented doxorubicin-inducedPTEN-/- cancer stem cell death. We further found thatnanoliposomal-delivered ceramide dramatically re- duced the cellular levels of survivin and Evi-1. This study demonstrates, for the first time, increased expression of survivin and Evi-1 in a model of aggressive hepatocellular carcinoma with distinct cancer stem cell characteristics.

  • Critical role of acid sphingomyelinase (asm) in intestinal dysfunction after radiation exposure.


    MH Gaugler1,2, N Ripoche2, S Gouard2, M Benderitter1, F Paris1

    1IRSN, DRPH, SRBE, LRPAT, Fontenay-aux-Roses, F-92260 France; 2Inserm, U892, Nantes, F-44000 France

    Endothelial cell (EC) response to high dose of irradiation is implicated in the induction of intestinal damage. The crucial role of ASM release is shown in the pathogenesis of tissue dysfunction other than radiation-induced intestinal injuries. We hypothesize that ASM release after endothelial apoptosis and activation contributes to intestinal dysfunction after radiation exposure.

    First we validate that irradiation of EC results in the induction of epithelial cell damage via paracrine pathway. In fact, we show in an in vitro non contact endothelial-epithelial cell coculture model that a 15 Gy irradiation of EC induces a decreased epithelial cell number (29%), and percentage in mitosis (66%) as well as increased epithelial apoptosis (1.5-fold) and cell-surface area (1.5-fold).Endothelial ASM may be a potential mediator of the effects observed in epithelial cells. Indeed radiation-induced apoptotic and activated EC secrete 1.4-fold more ASM than non irradiated EC, and a 100 mU/ml of exogenous ASM recapitulates the effects triggered by irradiated EC in epithelial cells. Second we corroborate these in vitro results in 15 Gy total body irradiated mice. We show for the first time an upregulation of serum and intestinal ASM activity (up to 2-fold). Furthermore, intestinal ceramide levels are increased up to 4-fold after irradiation.

  • Pkcζ inhibition promotes apoptosis in tumor cells via the sphingomyelin pathway.


    E Damaskopoulou1, G Keri2, I Bernardini1, MP Viola Magni1,E Albi1

    1Dept. Clinical and Experimental Medicine, University of Perugia, Italy, and 2Rational Drug Design Laboratory CRC, Semmelweis University, Hungary

    It has been demonstrated that different inhibitors of PKCζ delay hepatoma cell growth.The aim of the present study was to evaluate the effect of 9 inhibitors on sphingomyelin cycle enzymatic activity (SMase,SM-synthase and PLC). The relation with Bax, Cyclin D, STAT3 and RNA polymerase II was analyzed by Western blot. Our results showed a reduction of the SMase and of the SM-synthase activity either in cells or in nuclei. The inhibitors 4,5,7,8 and 9 had the most potent effect on the Smase and in particular the inhibitor 9 caused a decrease in its activity of 84%. The SM-synthase was more affected by the inhibitors 4,5,8 and 9. The PLC activity was not significantly different from the control cells. Inhibitors 4,5,7,8 and 9 increased Bax level and decreased STAT3 level. Cyclin D was particularly inhibited by inhibitors 2,6 and 7. All the inhibitors caused reduction of RNA polymerase II levels, except of the inhibitor 3 that had no influence. The PKCζ have recently been proposed to play critical roles in signalling pathways that control cell growth, differentiation and survival. Our findings provide an evidence that Bax, Cyclin D, STAT3 and RNA polymerase II levels were regulated by PKCζvia the SMase pathway. Thus, its inhibition can promote apoptosis and be a novel therapeutic approach in cancer.

  • Acid ceramidase promotes cell survival and can enhance the outcome of in vitrofertilization.


    EH Schuchman, N Shtraizent, X He, E Eliyahu

    Dept. Genetics and Genomic Sciences, Mount Sinai School of Medicine, New York, NY

    It has been suggested that acid ceramidase (AC) is an importantreostat” regulating the levels of ceramide and sphingosine- phosphate (S1P) in cells. Previously, we showed that AC is an essential enzyme that is needed for mouse embryos to survive beyond the two-cell stage (Eliyahu et al., FASEB J., 2007). We have also produced Chinese hamster ovary (CHO) cells that overexpress and secrete the recombinant human enzyme, and found that thisconditioned” media or pure protein can prevent apoptosis and enhance the survival of several cell types in culture. In particular, AC can prevent apoptosis of unfertilized oocytes and preimplantation embryos, and enhance the outcome of in vitro fertilization (IVF). Moreover, we found that AC is expressed at high levels in human embryos, as well as in follicular fluid containing healthy oocytes. To explore these findings further, we have also produced floxed” mice that can be used to inactive AC at later stages of development and/or in a cell-specific manner. Based on these findings, we suggest that AC is needed for oocyte and embryo survival in vivo, and is an important component that is missing from IVF/embryo cell culture media. We also hypothesize that AC can be used to enhance the survival of other primary cells in culture, as well as embryonic and other types of stem cells.

  • Spatial aspects of signalling by sphingosine1-phosphate.


    S Pyne, SC Lee, J Long, L Gillies and NJ Pyne

    Cell Biology Group, Strathclyde Institute for Pharmacy and Biomedical Science, University of Strathclyde, Glasgow, G4 0NR, Scotland, U.K.

    Sphingosine kinase (SK) catalyses the phosphorylation of sphin- gosine to produce sphingosine 1-phosphate (S1P). S1P is a bioactive lipid that regulates cell proliferation, survival, migration  etc. Extracellular S1P mediates its effects by binding to a family of five closely related G protein coupled receptors, S1P1-5. In addition, intracellular S1P may bind to undefined intracellular targets to elicit cellular responses. The focus of our recent work has been on defining the spatial aspects of S1P signalling. In this regard, SK1 interacts with phospholipase D-derived phosphatidic acid in the Golgi apparatus, where S1P may be linked to cell survival. However, we have also detected SK1 and SK2, together with S1P5, in the centrosome, raising the possibility of a role for S1P in mitosis. Additionally, the signalling outcome of extracellular S1P binding to its G protein coupled receptors can be influenced by growth factor receptors in cancer. These various aspects will be discussed.

  • Sphingosine kinase-1 as therapy target.


    D Pchejetski1, L Sauer, O Cuvillier1, J Waxman1

    1Dept. Oncology, Imperial College London, and 2Dept. of therapeutic targets, IPBS, Toulouse

    Recently we have identified sphingosine kinase-1 (SK1) as a therapy target in prostate cancer (Pchejetski et al, Cancer Res 2005). Our studies indicate that targeting SK1 with specific inhibitors (Pchejetski et al, Mol Cancer Ther, 2008) or FTY720 (Pchejetski et al, submitted) may chemo- or radio-sensitize prostate cancer cells. In the current study we have investigated chemotherapy-induced modulation of the SK1 activity and expression in several prostate cancer cell lines. In PC-3 prostate cancer cells 20 nM docetaxel was inducing a loss of SK1 expression and activity, which correlated with the loss of cell viability. On the contrary, 5 nM docetaxel was inefficient in modulating SK1 expression and activity and had much lower effect on cell viability. Pharmacological SK1 inhibition or specific  siRNA have sensitized PC-3 cells to docetaxel. On the contrary, in LNCaP and DU145 prostate cancer cells, docetaxel has induced an increase in SK1 expression and activity. Yet, in LNCaP cells this has resulted in a resistance to docetaxel-induced apoptosis, whereas DU145 cells were still sensitive to this drug. Both cell types were still sensitive to pharmacological SK1 inhibition, which could also sensitize them to docetaxel. Overall, our data indicate that SK1 inhibition is not required forchemotherapy-induced cancer cell apoptosis. We have shown that depending on cell line, same therapy can drive SK1 expression in different directions, which doesnt predict the therapeutic out- come. Yet, direct SK1 inhibition proves to be a universal tool to induce prostate cancer cell apoptosis and sensitize them to chemotherapy.

  • Expression of GM3 and GD3 synthases in human melanoma cell lines with different metastatic potential.


    I Popa, Y Wang, L Thomas, J Portoukalian

    Laboratory of Dermatological Research, University of Lyon-1 and Edouard Herriot Hospital, Lyon, France

    We have previously established a set of human melanoma variants with increasing metastatic potential from a low metastatic human melanoma cell line IC8 injected in immunosuppressed newborn 

    rats. The increase in metastatic potential was found to be correlated with a decrease in GM3 and mostly GD3 gangliosides in the variants. The purpose of the present work was to compare the gene expression of GD3 and GM3 synthases in the human melanoma variants with low (IC8) and high metastatic potential (TW12, IP1). Western blot analysis to detect the proteins of GD3 synthase and GM3 synthase showed the presence of both enzymes in the three melanoma variants. RT-PCR technique applied to the intact RNA of GD3 synthase and GM3 synthase from human cell lines IP1, IC8 and TW12 showed also a high expression of the genes in the three variants. By immunohistochemical staining, GD3 was proven to be highly expressed in primary melanoma, but its reactivity was low in small metastases, whereas CDH was weakly stained in primary melanomas and strongly stained in small metastases. The results suggest that GM3 and GD3 synthases are expressed in both primary tumors and small metastases, but they are in an inactive form in small metastases.

  • Non-natural sphingolipid analogues in anticancer therapy: synthesis and biological properites.


    AA Ali1, CM Barzilay1, A Dagan2

    1Dept. Medicinal Chemistry and Natural Products, School of Pharmacy, 2Dept. Experimental Medicine and Cancer Research, School of Medicine, The Hebrew University of Jerusalem, Israel

    Sphingolipids participate in signal transduction and regulation of the cell growth. Ceramide is generally associated with growth arrest and cell death while sphingosine-1-phosphate (S1P) enhances growth and survival. Attenuation of ceramide levels and/or increased levels of S1P are increasingly implicated in various stages of cancer pathogenesis, including an anti-apoptotic phenotype, metastasis and escape from senescence. Thus, inhibition of the metabolic pathways of those metabolites might lead to ceramide accumulation and/or S1P reduction that served as a target in the anticancer therapy. Therefore, many inhibitors and analogues have been developed, but so far, none of those have been approved for clinical use. Accordingly, we have synthesized novel non-natural sphingolipid analogues by utilizing epoxide chemistry. These compounds have shown better anticancer activity in comparison with cis-Pt in colon, lung and ovarian cancer cell-lines. Moreover, a SAR study was performed in order to understand the importance of the lipophilic groups of the compounds. A systematic change of lipophilicity in the two different sites of the molecule were studied and presented. A fluorescent procedure was utilized in order to study their enzymatic inhibition of the relevant enzymes, which may demonstrate the possible mechanism of ceramide accumula- tion resulting in apoptosis.

  • Hypertension associated alterations in the vasoactive effects of sphingomyelin metabolites.


    LJA Spijkers, RFP van den Akker, MC Michel, AE Alewijnse, SLM Peters

    Dept. Pharmacology and Pharmacotherapy, Academic Medical Center, Amsterdam, The Netherlands

    Hypertension is associated with pronounced alterations in vessel growth and contractile properties, including the contractile responses to endothelin-1 (ET-1). Since the sphingomyelin metab- olites ceramide, sphingosine and sphingosine-1-phosphate have vasoactive properties, we investigated whether modulation of sphingolipid metabolism induces altered contractile responses in isolated arteries of six months old male, spontaneously hyperten- sive rats (SHR) compared to normotensive Wistar Kyoto rats (WKY). In SHR aorta, inhibition of sphingosine kinase by dimethylsphingosine (DMS, 10μM) significantly augmented ET-1 induced constrictions, but not in WKY. In addition, inhibition of cyclooxygenase (COX) by indomethacin (10μM) decreased con- striction by ET-1 more strongly in SHR aorta than in WKY. In SHR carotid artery, DMS itself induced a major transient contraction but not in WKY. Furthermore, exogenous application of sphingomye- linase (SMase, 0.1 U/ml) induced a pronounced constriction in carotid arteries from SHR, whereas in WKYonly minor constriction to SMase was observed. Interestingly, the DMS and SMase-induced contractions in SHR could be prevented by indomethacine or prior removal of the endothelium. We conclude that hypertension is associated with pronounced alterations in sphingolipid homeosta- sis, which accounts for drastically altered contractile responses.

  • Crosstalk between pi3k-akt-pten and ceramide signaling in the control of glioma cell fate.


    P Giussani, L Brioschi, R Bassi, L Riboni, P Viani

    Dept. of Medical Chemistry, Biochemistry and Biotechnology, University of Milano, LITA Segrate, Via Fratelli Cervi, 93, 20090 Segrate (MI) Italy

    Alterations in specific cell signaling mechanisms is a common feature of glioblastoma multiforme (GBM), the most malignant brain tumor in humans. PI3K-Akt-PTEN signaling pathway has been identified as an important oncogenic one in GBMs. In addition a disregulation of ceramide (Cer)-dependent signaling in GBMs is supported by the evidence that Cer levels are inversely associated to malignant progression of these tumors. Moreover, in glioma cells a Cer increase is functional to the activity of many cytotoxic treatments. Both apoptotic and non apoptotic Cer- induced cell death can be inhibited by the activation of the Akt pathway. The control of Cer levels in glioma cells involves specific enzymes as well as the transport of Cer from ER to the Golgi apparatus in the sphingolipid biosynthetic pathway. Moreover, the accumulation of the de-novo synhesized Cer is crucial in cannabinoid triggered ER stress and apoptosis in glioma, thus suggesting that Cer levels in the ER/Golgi compartment can be crucial for glioma cell fate. In this study we demonstrated that in C6 glioma cells, in which PI3K/Akt pathway is constitutively activated, inhibition of PI3K and transfection with a dominant negative Akt strongly inhibited Cer utilization for the biosynthesis of complex sphingolipids by controlling the ER-to Golgi vesicular transport of Cer. This suggests that maintaining Cer levels low in the ER can contribute to the prosurvival activity of PI3K/Akt.

  • Acid sphingomyelinase, nitric oxide and syntaxin 4: an important link between apoptosis and exocytosis.


    C Perrotta1, L Bizzozero1, S Morlacchi1, P Rosa2, E Gulbins3, E Clementi1

    1Dept. Preclinical Science and 2Inst. of Neuroscience, University of Milan, Italy, 3Inst. für Molekularbiologie Universitätsklinikum Essen

    Acid sphingomyelinase (A-SMase) plays an important role inCD95-induced apoptosis. Activation of this enzyme involves a process of exocytosis through which it translocates from intracel- lular compartments to the plasma membrane. The molecular mechanisms underlying this translocation are not known.

    Here we report that syntaxin 4 (synt 4), is an important player inA-SMase trafficking. We found that synt 4 colocalises with A- SMase and, when its expression is silenced by a specific siRNA, exposure of A-SMase induced by CD95 is reduced and CD95 internalization and apoptosis inhibited. We investigated whether synt 4 is regulated by messengers known to regulate A-SMaseactivity, in particular Nitric Oxide (NO) which inhibits apoptosis through inhibition of A-SMase. We found that NO blocks ASMase translocation in the same way of synt 4 siRNA. Molecular analyses showed that the mechanism through which NO inhibits A-SMase activity is the degradation of synt 4 through the activation of the proteasome, indicating synt 4 as the target of NO pathway.

    The identification of the interaction between ASMase, synt 4 and NO establishes a strong link between three key players in intracellular trafficking and apoptosis and might open new vistas in understanding the complex events underlying the process of cell death.

  • Regulation of ryanodine receptors by signaling sphingolipids: calmodulin is the missing link?


    E Kovács, K Liliom

    1Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, Budapest, Hungary

    Sphingosine-1-phosphate (S1P) and sphingosylphosphoryl-choline(SPC) are novel mediators of cell signaling. Interestingly, they can act as first and second messengers as well. While their activation of cell surface receptors has been characterized, not much information is 

    available about their intracellular target proteins. They can mobilize calcium from the endoplasmic reticulum in a yet unknown manner. SPC has been shown to activate ryanodine receptors through an undefined mechanism. Calmodulin binds to ryanodine receptors and regulates their activity. We have demonstrated that SPC binds to calmodulin and inhibits its action on target enzymes (Kovacs and Liliom, Biochem J. 2008, 410(2): 427-37). Here we hypothesised that SPC liberates calcium from intracellular stores through dissociating the complex between calmodulin and ryanodine receptors, hence regulating the channels activity. To prove our assumption, we studied the interaction between the calmodulin-binding domain of the ryanodine receptor and calmodulin in the presence of SPC and S1P. Monitoring the fluorescence of both dansyl-labeled calmodulin and the tryptophan of the peptide, we have found that SPC dissociates the calmodulin - ryanodine receptor peptide complex, while S1P does not. We also studied SPC-elicited calcium currents from microsomal preparations in the presence of calmodulin inhibitors or additional calmodulin. Based on our results, we suggest that calmodulin might be the missing link between SPC and ryanodine receptors.

  • The role of sphingolipids, glycolipids and phospholipids in the drosophilaepithelial permeability barriers.


    G Tsikala1,2, D Karagogeos1,3, M Strigini1

    1Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology Hellas, Iraklio, Greece, 2Department of Biology, and 3Medical School, University of Crete, Iraklio, Greece

    Cell adhesion depends on specialised structures that localize to specific membrane domains. An example are the septate junctions (SJ) of invertebrates, that sustain the diffusion permeability barriers of epithelia and the glial blood-brain barrier. Trafficking of junction components to the correct membrane domain is interlocked to the process of cell polarization and relies on the correct compartmental- ization of proteins as well as lipids in membranes. Little is known about the contribution of lipids to cell junction formation. We use Drosophila to study the role of lipids (in particular sphingolipids, glycosphingo- lipids, and phospholipids) in these cell biological processes. As a handle to the genetic study of lipids in this context, we investigate the contribution of lipid metabolism genes to the process of SJ formation. We screened mutants in sphingolipid,glyco-sphingolipid, and phospholipid metabolism genes using an established dye penetration assay, which probes the tightness’ of the epithelial permeability barrier. We now proceed to assess the molecular, cellular and ultrastructural defects in the mutants with disrupted barriers. In addition, we try to identify/develop reagents to visualize lipid subtypes in our system, in order to characterize the subcellular distribution of different lipids in the polarised cells of Drosophila epithelia.

  • Involvement of nitric oxide in the promotion of cell survival by ceramide 1-phosphate.


    P Gangoiti, MH Granado, L Arana, A Ouro, A Gómez-Muñoz

    Dept. Department of Biochemistry and Molecular Biology. Faculty of Science and Technology. University of the Basque Country. P.O. Box 644, 48080 - Bilbao (Spain)

    Macrophages play critical roles in the inflammatory response. In addition to their ability to migrate to the sites of lesions or infections, macrophage number is strictly regulated by cell death and division. In primary monocytes, obtained from the bone marrow, cell homeostasis is regulated by bioactive sphingolipids. In particular, ceramides cause cell cycle arrest and are potent inducers of apoptosis. By contrast, the phosphorylated form of ceramide, ceramide 1-phosphate (C1P), has mitogenic properties, and promote cell survival. We now demonstrate that C1P upregulates the expression of the inducible form of nitric oxide synthase (iNOS) and that production of nitric oxide (NO) is a major mechanism whereby C1P inhibits apoptosis in the macro- phages. Paradoxically, NO failed to stimulate DNA synthesis and macrophage proliferation. We have now found that the prosurvival effect of C1P is blocked by inhibitors of iNOS, and that both iNOS expression and the antiapoptotic effect of C1P are blocked by selective inhibitors of phosphatidylinositol 3-kinase, or nuclearfactor-kappa B. Another mechanism whereby C1P blocks apoptosis is through inhibition of acidic sphingomyelinase (A-SMase),which produces ceramides from sphingomyelin. However, al- though NO reversed the inhibitory effect of C1P on A-SMase, this pahtways was not involved in the prosurvival effect of C1P.

  • Ceramide 1-phosphate inhibits serine palmitoyltransferase and blocks apoptosis in alveolar macrophages.


    MH Granado, P Gangoiti, A Ouro, L Arana and A Gómez-Muñoz

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

    Ceramide 1-phosphate (C1P) is a bioactive sphingolipid capable of regulating vital cellular functions including cell proliferation, apoptosis, phagocytosis, or inflammatory responses. As expected, incubation of alveolar NR8383 macrophages in the absence of growth factors caused accumulation of ceramides, and cell death by apoptosis. However, there were very little activation of A-SMaseand neutral SMase when these cells were incubated in the absence of growth factors. An important observation in this work was that synthesis de novo is the major pathway involved in the production of ceramides in apoptotic macrophages. Stimulation of this pathway requires SPT activation, as demonstrated by measuring this enzyme activity and protein expression of the two SPT subunits (SPT1 and SPT2). Another key finding is that C1P inhibits both SPT activation (probably through direct interaction with the enzyme) and ceramide accumulation, leading to inhibition of apoptosis and promotion of macrophage survival. These observations add a new dimension to the understanding of the prosurvival actions of C1P in mammalian cells.

  • A ceramide-1-phosphate analog, PCERA-1,suppresses TNFα and induces IL-10 production in activated macro-phages.


    M Goldsmith1, D Avni1, M Levi1, O Ernst1, MM Meijler2, NS Gray3, H Rosen4 and T Zor1

    1Dept of Biochemistry, Tel-Aviv University, Tel-Aviv, Israel, 2Dept of Chemistry, Ben-Gurion University, Be'er-Sheva, Israel, 3Dept of Biological Chemistry, Genomics Institute of the Novartis Research Foundation, La Jolla, CA, USA, and 4Dept of Immunology, The Scripps Research Institute, La Jolla, CA, USA

    Tight regulation of the production of the key pro-inflammatorycytokine TNFa is essential for the prevention of chronic inflamma- tory diseases. In vivo administration of a synthetic phospholipid, named PCERA-1 (Phospho-CERamide Analog-1), was previously found to suppress LPS-induced TNFa blood levels. Here we show that extra-cellular PCERA-1 potently suppresses production of thepro-inflammatory cytokine TNFa in stimulated RAW264.7 macro- phages, and in addition, it independently and reciprocally regulates the production of the anti-inflammatory cytokine IL-10. Specificity is demonstrated by the inability of the phospholipids C1P, S1P and LPA to perform these activities. Regulation of cytokine production is demonstrated at the mRNA and protein levels. Finally, we show that while PCERA-1 does not block activation of NFkB and MAP kinases by LPS, it elevates intra- cellular cAMP level. In conclusion, the anti-inflammatory activity of PCERA-1 seems to be mediated by a cell membrane receptor, upstream to cAMP production and eventually TNFa suppression and IL-10 induction. Thus, identification of the PCERA-1 receptor may provide new pharmacological means to block inflammation.

  • Effects of dimebon on the TNF-alpha-induced disorders of sphingolipids in mice brain sections.


    AV Alessenko1, YO Karatasso1, AA Korotayeva1, EF Shevtsova1, LN Shingarova1, SO Bachurin

    1Institute of Biochemical Physics , Moscow, Russia

    Institute of Physiologically Active Substances, Chernogolovka, Russia

    There is substantial evidence that inflammatory mechanisms, induced by TNF-alpha are involved in Alzheimers Disease (AD). Metabolism of sphingolipids is of particular interest due to their high concentration in the brain. In our experiments we used dimebon for prevention of sphingolipids disorders in mice brain sections (hippocampus, cerebellum and cerebral cortex) induced byTNF-alpha. Recently Dimebon has attracted renewed interest after being shown to have positive effects on persons suffering from AD.TNF-alpha (10mkg per mouse), dimebon (0,2 mg/kg ) and their combination were injected to mice interperitonealy. Changes in level of sphingomyelin and galactosylceramides molecular species were detected by chromato-mass-spectrometry in hippocampus, cerebellum and cerebral cortex within 30 min, 2, 4 and 24 hours after injection. Maximal changes in sphingomyelin and galactosyl- ceramides contents of different sphingolipids molecular species after single TNF-alpha administration were found in the hippocam- pus, and were less expressed in the cerebral cortex and cerebellum after 2, 4 and 24 hours. Dimebon itself did not induce changes in sphingolipids spectrum in brain sections, but protected lipids against disorders induced by TNF-alpha in mice brain.

  • Expression of NPC1i1061t mutant in the neuronal cell line HN9.10e: a cellular model of niemann-pick C disease.


    V Voccoli, G Della Sala, L Colombaioni

    Istituto di Neuroscienze, Area della Ricerca CNR, Pisa, Italy

    Niemann-Pick type C (NPC) disease is a fatal lipid storage disorder characterized, at neuronal level, by accumulation of cholesterol, gangliosides and glycosphingolipids as well as by the presence of dendritic and axonal abnormalities. NPC is caused by mutations on two genes: NPC1 and NPC2. Mutations in the NPC1 gene are responsible for approximately 95% of human NPC disease and the most prevalent mutation, NPC1 I1061T, is associated with the classic juvenile-onset phenotype of NPC disorder. To acquire more information on the molecular pathways involved in NPC neurodegeneration, we have transfected the hippocampal neuronal cell line HN9.10e with the mutant protein NPC1I1061T as well as with the wild type, functional, NPC1. Then, we have compared NPC1 mutant with wild type neurons. In particular, we have assessed the effects of the expression of the mutant form NPC1I1061T on the physiological properties of endoplasmic reticulum, mitochondria, Golgi apparatus and endo- cytic and lysosomal compartments. Aberrant organelle structures and localization as well as alterations of the physiological properties of the mitochondrial network were evidenced in neurons lacking functional NPC1.

  • Pharmacological manipulation of glycosphingolipids


    JM Aerts

    Dept. Medical Biochemistry, Academic Medical Center, University of Amsterdam, The Netherlands

    Enormous progress has been made regarding therapy of type I Gaucher disease, a disorder characterized by intralysosomal storage of glucosylceramide in tissue macrophages. Effective enzyme replacement therapy, based on chronic intravenous administration of recombinant human glucocerebrosidase, has now been very successfully applied in several thousand patients.

    An alternative therapeutic approach, so-called substrate reduction therapy, is based on partial reduction of the synthesis of glucosylcer- amide and hence of subsequent metabolites. Oral administration of an inhibitor of glucosylceramide synthesis (N-butyldeoxynojirimycin,registered as miglustat (Zavesca)), is effective in reversing clinical symptoms in type I Gaucher patients with mild to moderate disease manifestations. More optimal small compounds are being developed for substrate reduction therapy. Such therapy, in conjunction with enzyme replacement therapy, may play an important role in the future clinical management of patients suffering from type I Gaucher disease and potentially also for patients suffering from storage disorders with neurological disease manifestations.Very recently the therapeutic potential of two classes of inhibitors of glycosphingolipid synthesis has been demonstrated for obesity-induced type 2 diabetes. The future for pharmacolog- ical manipulation of glycosphingolipids is discussed.

  • DRMs exist in sphingolipid- and cholesterol- depleted cells. impact on localization and function of MRP1.


    K Klappe1, A-J Dijkhuis1, I Hummel1, A de Boer1, A-M Huls1, A van Dam2, H. Permentier2, B-J Kroesen3, H Sietsma3 and JW Kok1

    1Department of Cell Biology, University Medical Center Groningen, 2Center for Pharmacy, University of Groningen, 3Department of Pathology and Medical Biology, University Medical Center Groningen, The Netherlands

    Extensive depletion of sphingolipids or cholesterol in two different cell lines does not abrogate the ability to isolate lubrol-baseddetergent-resistant membranes (DRMs) from these cells. Compared to control, DRM fractions contain equal amounts of protein, while the classical DRM markers Src and caveolin-1(Cav-1) display a similar gradient distribution. DRMs themselves are severely depleted of sphingolipids and cholesterol, respectively. Also detergent-free lipid rafts can be isolated from sphingolipid- orcholesterol-depleted cells. Concerning functional consequences of sphingolipid depletion, neither DRM/detergent-free lipid rafts localization nor efflux function of multidrug resistance-relatedprotein 1 (MRP1) was affected in the two cell lines. On the contrary, cholesterol depletion caused a shift of MRP1 out ofdetergent-free lipid raft fractions to higher density gradient fractions and concurrently resulted in inhibition of MRP1 efflux activity, especially in Neuro-2a cells. We conclude that in contrast to sphingolipids, cholesterol depletion affects MRP1 function in neuroblastoma cells and this may be related to its localization indetergent-free lipid rafts.

  • Neuroprotective effect of vitamin D on the 1-42 beta-amyloid peptide-induced cytotoxicity in differentiated SH-SY5 ycells. A role for sphingolipids.


    F Bini1, M Martinesi1, M Garcia-Gil2, B Meacci1

    1Department of Biochemical Sciences, University of Florence, Italy 

    2Department of Biology, University of Pisa, Italy

    Sphingolipids, mainly ceramide and sphingosine-1-phosphate(S1P) are bioactive molecules involved in the regulation of cell proliferation, differentiation and apoptosis in many cell types, acting in opposite manner: ceramide induces cell death, whereas S1P is mainly a pro-survival factor. Although the role of 1!,25- dihydroxyvitamin D3 (VitD) in the homeostasis of Ca2+ and phosphate is well known in classic target tissues, its role in neuronal cells is less clear. Here, we have investigated whether VitD modulate the death and survival of differentiated SH-SH5Hcells in response to beta amyloid peptide and the involvement of ceramide and S1P in hormone action. We found that both VitD and beta amyloid peptide induced cell death and markedly reduced S1P synthesis by sphingosine kinase in BDNF-differentiated SH- SY5Y cells. However, when VitD was added to the cell medium before beta amyloid peptide treatment, the apoptotic effect was markedly reduced, suggesting a neuroprotective action of VitD on the cytotoxicity induced by the peptide. Notably, although both agonists reduced the synthesis of the pro-survival factor S1P, VitD could prevent amyloid peptide-induced toxicity affecting ceramide content. Taken together, these results provide the first evidence of the ability of VitD to prevent the neurodegeneration upon beta amyloid treatment through the regulation of ceramide/S1P ratio.

  • Lateral distribution of very long chain sphingolipids in mixed membranes.


    YJE Björkqvist, JP Slotte and B Westerlund

    Department of Biochemistry, Åbo Akademi University, Turku, Finland

    Sphingolipids containing very long acyl chains (VLC-SLs) are abundant in certain specialized tissues and are minor components of plasma membranes in most mammalian cells. There is evidence of cellular processes in which VLC-SLs are required, and their function is thought to be mediated through sphingolipid-richmembrane domains. This study was conducted to explore how very long (C24) saturated or monounsaturated acyl chains influence the lateral distribution of sphingolipids in model membranes. Differential scanning calorimetry was applied to compare the thermotropic behavior of 24:0- and 24:1-sphingoli- pids and to analyze their miscibility with palmitoylsphingomyelin. The results from fluorescence quenching measurements showed that the24:0-sphingolipids formed ordered domains in multicom- ponent membranes. The 24:0-sphingolipid enriched domains had a high chain packing density which appeared to hinder the partitioning of sterols into them. The effect of a 24:1 acyl chain was found to be dependent on the head group structure of the sphingolipid and could either stabilize or disrupt ordered sphingo- lipid/cholesterol domains. We conclude that VLC-SLs may be components of functional sphingolipid-rich domains in biological membranes and their presence may affect the distribution of sterols between ordered and disordered membrane regions.

  • Free sphingenine-1-phosphate plasma levels in sgpl1-/- mice.


    PP Van Veldhoven

    LIPIT, Dept. Molecular Cell Biology, K.U.Leuven, Leuven,Belgium

    Sphingenine-1-phosphate (S1P) is an important lipid mediator in the body circulation. In Sgpl1-/- mice, lacking S1P-lyase, S1P is accumulating in most tissues, with concomitant immunosuppression, inflammation, and apoptosis [1]. Given the elevated plasma S1P levels, it is postulated that Edg/S1P-receptors.S1P-receptor are continously activated/downregulated in these mice. Indeed, S1P levels in sick pups (2035µM) are well above the reported Kvalues,210 nM for S1P5 (Edg8) to 2027 nM for S1P2 (Edg5) [2]. On the other hand, the same holds true for the wild type values(24µM). According to Murata et al. [3], more than 60% of human plasma S1P is however bound to lipoproteins and active (unbound) S1P was estimated at 7.3 nM (based on a radioreceptor assay). Hence, attempts were made to estimate the unbound S1P in Sgpl1-/-plasma. Given the small amount of plasma available and the peculiar properties of S1P, not all approaches appeared useful. The most reliable assay turned out to be gel filtration using low volume spin columns. Recoveries, based on samples spiked with [4,5-3H]-sphinganine-1-phosphate, reached 95 %. Mass measurements indicated that ~ 90% of mouse plasma S1P was protein bound, both in +/+ and -/- pups. Hence in -/- pups, free S1P plasma levels are estimated at 1µM (versus 20 nM in +/+ pups), and indeed able to attenuate S1P-receptors.

    1.Van Veldhoven PP (2005) Chem Phys Lipids 136: 164-165.

    2.Anliker B, Chun J (2004) J Biol Chem 279: 20555-20558.

    3.Murata N et al (2000) Biochem J 352: 809-815.

  • Sphingenine-1-phosphate  induced permeability changes in sgpl1-/red blood cells?


    PP Van Veldhoven

    LIPIT, Dept. Molecular Cell Biology, K.U.Leuven, Leuven, Belgium

    Red blood cells (RBC) are apparently intimately involved insphingenine-1-phosphate (S1P) mediated signalling. Although the enzymes required for generation and removal of S1P are absent or low, RBC do store and release S1P and in human blood, the majority of blood S1P is associated with RBC [1,2]. Given the accumulation of S1P in tissues of mice lacking S1P-lyase [3], an enzyme required for the degradation of catabolic and bioactive S1P and encoded by Sgpl1, it was of interest to analyze RBC in more depth in this model.

    Hereto, blood was collected on heparin and subjected to Histopaque step gradient centrifugation. Due to the limiting amount of -/- blood, this procedure was optimized with 1020µl blood. In wild type samples, about 30 % of blood SeP was recovered in plasma, 60 % in the RBC and 12 % was associated with the other blood cells. When applied to -/- samples, RBC failed however to sediment through the Histopaque gradient. Analysis of total blood cells of -/- pups indicated that their SeP content was increased ~25-fold compared to wild type cells (332 versus 12.4.pmol/µl cells; n=4); in C57Bl/6 background). Appar- ently, the accumulating zwitterionic S1P is responsible for a change in membrane permeability of RBC and this could explain a small degree of hemolysis seen in -/- samples. If so, increase in plasma S1P in Sgpl1-/- mice might be partly due to leakage from RBC during blood collection.

    1.Ito K et al (2007) Biochem Biophys Res Commun 357: 212217.

    2.Hänel P et al (2007) 21: 12021209.

    3.Van Veldhoven PP (2005) Chem Phys Lipids 136: 164-165.


  • Intracellular membrane bound pools of sphingenine-1-phosphate?


    PP Van Veldhoven

    LIPIT, Dept. Molecular Cell Biology, K.U.Leuven, Leuven, Belgium

    Cellular levels of bioactive phosphorylated sphingoid bases (PSB) such as sphingenine-1-phosphate (S1P) are controlled by sphingosine kinases, S1P-phosphatases and S1P-lyase in mam- mals. Based on the use of inhibitors, siRNA technology andknock-out models, the lyase appears most important. In tissues of mice lacking S1P-lyase (Sgpl1-/-), S1P and other PSBs indeed accumulate in an age-dependent manner to mM concentrations, especially in thymus, lung and Harderian gland [1]. In order to understand how cells can cope with such amounts of a zwitterionicdetergent-like compound, major organelles were studied in liver ofSgpl1-/- mice, and the subcellular localization of S1P was analyzed. Based on their sedimentation in density gradients, membrane properties of lysosomes and mitochondria appear to be slightly altered in Sgpl1-/- liver. In control liver, S1P was only measurable in the nuclear fraction, whereas in -/- samples 60 % was recovered in the post-nuclear fraction. Surprisingly, the majority of S1P present in this fraction was not cytoplasmic, but appeared to be associated with a (vesicular) compartment. Based on density gradient centrifugation, this compartment appears to be different from the major organelles, including endoplasmic reticulum- derived vesicles.

    1. Van Veldhoven PP (2005) Chem Phys Lipids 136: 164-165.

  • Ceramide-enriched membrane domains in red blood cells and the mechanism of sphingomyelinase-induced hot-cold haemolysis.


    DJ López1, L-R Montes1, J Sot1, LA Bagatolli2, MJ Stonehouse3, ML Vasil3, BX Wu4, YA Hannun4, FM Goñi1 and A Alonso1

    1Unidad de Biofísica (CSIC-UPV/EHU), Bilbao, Spain. 2MEM- PHYS Center for Biomembrane Physics, University of Southern Denmark, Odense, Denmark. 3Department of Microbiology, University of Colorado, Aurora, USA. 4Department of Biochem- istry and Molecular Biology, Medical University of South Carolina, Charleston, USA.

    Hot-cold haemolysis is the phenomenon whereby red blood cells, preincubated at 37 C in the presence of certain agents, undergo rapid haemolysis when transferred to 4 C. PlcHR2, a phospholipase C/ sphingomyelinase from Pseudomonas aeruginosa, induces hot-coldhaemolysis. We found that the sphingomyelinase, but not the phospholipase C activity, is essential for hot-cold haemolysis. Fluorescence microscopy observations confirm the formation ofceramide-enriched domains as a result of PlcHRactivity. We suggest that formation of these rigid domains in the originally flexible cell make it fragile thus highly susceptible to haemolysis. Ceramidase, that is known to facilitate slow haemolysis at 37 C, actually hindershot-cold haemolysis. Differential scanning calorimetry of erytrocyte membranes treated with PlcHRdemonstrates the presence ofceramide-rich domains that are rigid at 4 C, but fluid at 37 C. Finally, in liposomes composed of SM, PC and cholesterol, which exhibit slow release of aqueous contents at 37 C, addition of 10 mol % ceramide and transfer to 4 C cause a large increase in the rate of solute efflux.

  • Cholesterol displacement by ceramide in sphingomyelin-containing liquid-ordered domains, and generation of gel regions in giant lipidic vesicles.


    M Ibarguren, J Sot, JV Busto, L-R Montes, FM Goñi, A Alonso

    Unidad de Biofisíca (Centro Mixto CSIC-UPV/EHU) and Depar- tamento de Bioquímica, Universidad del País Vasco, P. O. Box 644, 48080 Bilbao, Spain

    Fluorescence confocal microscopy and differential scanning calorimetry are used in combination to study the phase behaviour of bilayers composed of PC:PE:SM:Chol equimolecular mixtures, in the presence or absence of 10 mol% egg ceramide. In the absence of ceramide, separate liquid-ordered and liquid-disordereddomains are observed in giant unilamellar vesicles. In the presence of ceramide, gel-like domains appear within the liquid-orderedregions. The melting properties of these gel-like domains resemble those of SM:ceramide binary mixtures, suggesting Chol displace- ment by ceramide from SM:Chol-rich liquid-ordered regions. Thus three kinds of domains coexist within a single vesicle in the presence of ceramide: gel, liquid-ordered, and liquid-disordered. In contrast, when 10 mol% egg diacylglycerol is added instead of ceramide, homogeneous vesicles, consisting only of liquid- disordered bilayers, are observed.

  • Ceramide 1-phosphate stimulates proliferation and differentiation of mouse myoblasts.


    P Gangoiti1, C Bernacchioni2, MH Granado1, C Donati2, AGómez-Muñoz1, P Bruni2

    1Department of Biochemistry and Molecular Biology, University of the Basque Country, Bilbao, Spain and 2Department of Biochemical Sciences, University of Firenze, Firenze, Italy

    Bioactive sphingolipids including sphingosine, sphingosine 1- phosphate, ceramide, GM3 ganglioside have been recently emerged as important regulators of skeletal muscle cell biology being capable of regulating key cellular parameters including myoblast proliferation and myogenesis. However, no information is presently available on the possible biological effects of ceramide1-phosphate (C1P) in these cells. Here we provide the first experimental evidence that C1P acts both as mitogen and pro- myogenic agent in C2C12 myoblasts. C1P (15µM) was found to stimulate DNA synthesis and accelerate cell cycle. Specific inhibitors of PI3K or ERK1/2 signaling pathways strongly attenuated the C1P action, whereas pertussis toxin treatment, or inhibition of p38 MAPK or JNK were uneffective. In agreement, C1P was responsible for a rapid phosphorylation of Akt and ERK1/2. Notably, C1P was disclosed to exert also a robust promyogenic effect. It enhanced the expression of myogenic markers such as myosin heavy chain and caveolin-3 and hastened the fusion of mononucleated myoblasts. Thus, in this study a novel important biological action of C1P in myoblasts has been highlighted, which possibly will be exploited in the future to enhance skeletal muscle regeneration.

  • The first irreversible inhibitor of acid ceramidase. Preliminary characterization and cytotoxicity studies.


    L Camacho, C Bedia, J Casas, G Fabrias

    Dept. of Biomedicinal Chemistry, Instituto de Química Avanzada de Cataluña, Consejo Superior de Investigaciones Científicas (CSIC)

    Acid ceramidase (AC) catalyzes the degradation of ceramide into sphingosine and free fatty acids within the lysosomes. Mutations in the AC gene (ASAH1) result in Farber disease, a fatal human genetic disorder. The involvement of AC in cell death in both physiological and pathological conditions has been extensively documentedand different studies support AC as a drug target in cancer.In agreement, AC inhibitors have been successfully used to slow the growth of cancer cells, alone or in combination with other anticancer treatments. Continuing with ongoing projects aimed at the discovery of novel AC inhibitors, in this communication we report on the activity of compound RBM15 as the first irreversible inhibitor of AC. Using Farber cells transduced to overexpress AC, this com- pound inhibited the hydrolysis of CerC12NBD dose- dependently both in intact cells and in cell lysates with IC50 values in the low µM range in both cases.Time-course and dilution experiments proved that inhibition was irreversible. AC inhibition was also evident in the ceramidome, as concluded form HPLC-MS analyses of sphingolipid extracts from cells treated with RBM15. A preliminary account of the cytotoxicity of this molecule over the human adenocarcinoma cell line A459 is presented.

  • Reduced hiv-1 infection by inhibition of dihydroceramide desaturase.


    JM Munoz-Olaya1, C Vieira2, J Sot3, J Casas1, SJimenez-Baranda2, JL Abad1, S Grijalvo1, A Delgado1,4, A Llebaria1, A Alonso3, FM Goñi3, S Mañes2, G Fabrias1

    1Dept. of Biomedicinal Chemistry, IQAC-CSIC, Barcelona, Spain

    2Dept. of Immunology and Oncology, CNB-CSIC, Madrid, Spain 

    3Dept. of Biochemistry, and Unidad de Biofísica CSIC-UPV/EHU, University of the Basque Country, Bilbao, Spain4Pharmaceutical chemistry Unit, School of Pharmacy, University of Barcelona, Barcelona, Spain

    HIV-1 infects permissive cells by the gp41-mediated fusion of viral and targets cell membranes. This fusion event requires the sequential interactions of the gp120 viral envelope with CD4, the primary viral receptor, and CXCR4 or CCR5, the coreceptors required for HIV-1 cell entry and infection. Accumulated evidence indicates that lipid rafts have an essential role in the HIV-1-induced lateral associations required for viral infection. Disruption of cell membrane rafts by cholesterol depletion before viral exposure inhibits virus entry. Similar effects are observed with either inhibitors of de novo sphingolipid biosynthesis or inhibitors of glucosylceramide synthase. Here we describe the synthesis of compound GT11pyr and its use as pharmacological tool against HIV cell entry. This compound inhibited the HIV-1 envelope-mediated fusion with target cell membranes in a dose-dependent manner. Biophysical studies with model membranes agree with the biological data. The overall findings reinforce the therapeutical interest of dihydrocer- amide desaturase in anti HIV-1 therapy and other raft-associated diseases.

  • Acidic and neutral sphingomyelinase inhibition by dihydroceramide and dihydrosphingomyelin analogues.


    F Simbari1, X Matabosch1, G Fabriàs1, A Llebaria1, A Delgado1,2, T Levade3, J Casas1

    1Dept. of Biomedicinal Chemistry, Instituto de Química Avanzada de Cataluña (CSIC), Barcelona, Spain 

    2Unidad de Química Farmacéutica (Unidad Asociada al CSIC), Facultad de Farmacia, Universidad de Barcelona, Barcelona, Spain

     3INSERM U858, Institut Louis Bugnard, Centre Hospitalier Universitaire de Rangueil, Toulouse, France

    Sphingomyelin (SM) is the primary sphingolipid in mamma- lian cells and plays important structural and functional roles. SM hydrolysis can be carried out by different isoforms of sphingomye- linase (SMase), producing phosphorylcholine and the intracellular effector ceramide. Ceramide diffuses within membranes acting as a second messenger. Generation of ceramide in various cellular systems is currently recognized as critical to the initiation of vital cellular processes such as differentiation, cell proliferation and apoptosis. In this communication we describe the inhibitory activity of acidic and neutral SMases by dihydroceramide and dihydrosphin- gomyelin analogues, using C6-NBD-sphingomyelin as substrate. Acidic SMase activity was determined with A549 cell homoge- nates whereas homogenates from acidic SMase-deficientNiemann-Pick cells were used as neutral SMase enzymatic source. The most active compounds were tested in intact cells and their effect on the cellular sphingolipid profile was also studied.

  • Regulation of DHS1P release in F9 mutant cells by fumonisin- B1.


    SM Pak1, NY Park1, SM Kim1, KO Shin1, JY Kwak1, HS Yoo1, S Oh2, WJ Kim3, YM Lee1

    1College of Pharmacy and CBITRC, Chungbuk National University 

    2Department of Neuroscience, College of Medicine, Ehwa Womans University 3Department of Life Sciences, Soonchun- hyang University, Korea

    The main goal of our study was to determine the regulation mechanism of dihydrosphingosine 1-phosphate (DHS1P) release from F9 mutant cells. We used a specific mutant (F9-12) of F9 cells over-expressing Sphk1 and showing null of S1P lyase. FB1 treatment (50 uM) exhibited concurrent increases of DHS and DHS1P in F9-12 cells. In this condition, the increased DHS and DHS1P also efficiently released outside. ISP-1 and DMS treatment largely attenuated the accumulation of DHS and DHS1P in both inside and outside of cells. FB1 treatment induced a small percentage of apoptotic cells, indicating the releases of DHS and DHS1P were not mediated by apoptosis. DHS1P release may be regulated by the rate of sphingolipid biosynthesis and Sphk activity. We further investigated the effects of ABC transporters on DHS1P release. By using ABC transporters inhibitors, verap- amil, MK-571 and glyburide, the involvement of ABC trans- porters on DHS1P release was verified.

    This work was supported by the Korea Research Foundation Grant funded by the Korean Government (MOEHRD)" (The Regional Research Universities Program/Chungbuk BITResearch-Oriented University Consortium)

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


    L Bizzozero1, G Milani2, E Clementi1,3, C Perrotta1

    1Department of Preclinical Sciences, University of Milano, Milano, Italy; 2H. San Raffaele Scientific Institute, Milano, Italy; 3E. Medea Scientific Institute, Bosisio Parini, Italy

    Recent evidence indicates that Acid Sphingomyelinase (A-SMase),plays important role in tumour biology, including tumour response to chemotherapeutic drugs. The effects of A-SMase were shown in vivo to results from a combination of effects of the enzyme expressed by the tumour cells themselves, and the surrounding microvascular endothelial and inflammatory cells. We decided to investigate the specific role of A-SMase expressed by melanoma B16 cells which show different tumorigenic and metastatic properties dependent on their melanin content. In order to investigate a possible correlation between A-SMase expression and B16 cells phenotype we isolated several B16 clones on the basis of 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 preliminary experiments showed that the white and the black clones differ in terms of growth rate, invasiveness and histological characteristics. These data suggest a strong relationship betweenA-SMase expression and tumour behav- iour. These studies might help refine therapeutic strategies against tumour based on regulation of A-SMase activity/ expression.

  • Ceramide induces autophagic death and HMGB1 release in human glioma cells.


    P Giussani, R Bassi, V Anelli, F De Zen, L Brioschi, P Viani, L Riboni

    Dept Medical Chemistry, Biochemistry and Biotechnology,LITA-Segrate, University of Milan, Milan, Italy

    Gliomas are among the deadliest of human cancers, and exhibit striking resistance to therapy. Glioma cells are frequently resistant to apoptosis, the induction of autophagy recently emerging as a potential effective therapeutic approach. The molecular mechanisms underlying autophagic death in glioma cells remain largerly unknown. The aim of this research was to investigate whether the sphingoid mediator ceramide (Cer) may play a role in glioma cell autophagic death. Treatment of human glioma cells with the alkylating agent temozolomide (TMZ) results in the increase of Cer levels, followed by cell death. The administration of short-chainCer analogues mimics TMZ in inducing glioma cell toxicity. After TMZ and Cer treatment, no formation of apoptotic small bodies occurs. Acridine orange staining and western blot of the autophago- some marker LC3B-II indicate TMZ- and Cer- induce autoph- agy. In agreement, co-treatment of cells with the autophagic inhibitor 3-methyladenine reverses toxicity. Moreover, glioma cells undergoing Cer-induced autophagy release large amounts of HMGB1 (high mobility group box 1), after its translocation from the nucleus to cytosol. On its turn, HMGB1 acts as an autocrine stimulus via RAGE receptors, by inducing cell growth and migration. In conclusion, our data implicate Cer as mediator of TMZ-induced autophagic death in glioma cells and suggest glioma cells dying by autophagy provide HMGB1 as extracellular signal to enhance the growth of viable cells.

  • Desensitization and internalization of sphingosine-1-phosphate receptors induced by cis-4-methylsphingosine.


    M ter Braak1, B Hegen1, V Hardel1, KH Jakobs1, G van Echten- Deckert2, D Meyer zu Heringdorf 1,3

    1Institut für Pharmakologie, Universitätsklinikum Essen, 2Kekulé- Institut, LIMES Membrane Biology and Lipid Biochemistry, Universität Bonn and 3Institut für Pharmakologie, Universitätsklinikum Frankfurt, Germany

    Cis-4-methylsphingosine (cis-4 M-Sph) is a synthetic sphingosine analogue that is readily taken up by cells and phosphorylated to cis-4-methylsphingosine-1-phosphate (cis-4 M-S1P), which accumu- lates intracellularly. Previous reports demonstrated that cis-4 M-Sphmimicked the mitogenic effect of sphingosine-1-phosphate (S1P) in Swiss 3 T3 fibroblasts, but induced apoptosis in neuronal cells. Here, we analysed the influence of cis-4 M-Sph on S1P receptor signalling in HEK-293 cells and mouse embryonic fibroblasts. Addition of cis-4M-Sph to the cells induced increases in intracellular Ca2+concentrations ([Ca2+]i) that were slower than those by S1P but faster than those by sphingosine. Pre-incubation with cis-4 M-Sph, but not with sphingosine, reduced ([Ca2+]i increases by S1P in a time- andconcentration-dependent manner, while it had no influence on ([Ca2+]i increases by LPA. Both the stimulation of acute ([Ca2+]i increases and the inhibition of S1P-induced ([Ca2+]i increases were dependent on the presence of sphingosine kinase-1, indicating that they were mediated by cis-4 M-S1P. Confocal microscopy revealed that cis-4 M-Sphinduced an internalization of S1P receptors. It is concluded that cellular production of cis-4 M-S1P can lead to S1P receptor desensitization and internalization.

  • Space environment and lipid domains rich in sphingomyelin-cholesterolcontent.


    E Albi1, M Peverini1, R Lazzarini1, M Viola Magni1, G Perrella2

    1Department of Clinical and Experimental Medicine, Physiopathology Section, University of Perugia. 

    2Department of Experimental and Clinical Pathology and Medicine, University of Udine

    It has been shown that in FRTL-5 cells UV treatment induced an increase ofN-SMase activity higher in proliferating cells (TSH+) than in quiescent cells(TSH-). Differently, in the space the enzyme activity increase of TSHcells was similar to that of TSHcells. The aim of the present work was to evaluate the effect of the cosmic environment on lipid domains in relation to FRTL-5 TSH+cell activity during the 15-days on the Space Shuttle (Experia Mission).The activity of N-SMase and the content of RNA polymerase II were evaluated in the cells whereas the lipid composition was analyzed in the culture medium at the re-entry of the shuttle or after 48 hours of cell culture at 37 C. The results showed low value of N-SMase activity and RNA polymerase II content. After 48 hours at 37 C the SMase activity and the content of RNA polymerase II increased in the cell. In the culture medium the content of PE, PI, and PS was similar in the two experimental conditions whereas the PC, SM and CHO content decreased in the space with respect to the medium of the cells after 48 hours at 37 C. It is possible that in space the cells are hungry of lipid domains that are able to make them more stable, slowing down the proliferative activity and protecting them from the damage.

  • Role of the third amino acid of the e/dry motif in g protein selectivity of s1p receptors.


    M Jongsma, FS den Boon, PB van Loenen, MC Michel, SLM Peters, AE Alewijnse

    Dept. Pharmacology & Pharmacotherapy, Academic Medical Center Amsterdam, The Netherlands

    Sphingosine-1-phosphate (S1P) receptors show variance in the third amino acid of the conserved E/DRY motif. This study investigates whether this amino acid is important in G-proteinselectivity of these receptors. The most remarkable differences were observed for the S1Pmutant receptors. The S1P1ERH and S1P1ERA mutant receptor showed a decrease in the potency of S1P and SEW2871 to inhibitforskolin-induced cAMP accumulation whereas the potency for FTY720-P was unchanged. The potency of S1P and FTY720-P to induce increases in intracellular calcium concentrations were shifted to the left at the S1P1ERH mutant receptor. The ERH to ERY or ERA mutations of the S1P2 and S1P3 receptor did not induce major changes in signaling. In conclusion, this study is a first indication that the tyrosine residue of the E/DRY motif may restrict the S1P1 receptor from activating multiple G-proteins. However, introducing a tyrosine residue in the S1P2 or S1P3 receptor did affect but did not limit the signaling of these receptors. Interestingly, besides these findings we show that some mutations differentially affected the signaling of the tested ligands.

  • Importance of connexin-43 in the regulation of sphingolipid metabolism in primary skeletal muscle cells.


    F Bini1, J von Maltzahn2, V Wulf2, K Willecke2, Meacci E1

    1Department of Biochemical Sciences, University of Florence, Italy; 2Institut für Genetik, Abteilung Molekulargenetik, Universität Bonn, Germany;

    Diverse intercellular signalling pathways influence the regulation of myoblast differentiation of skeletal muscle during development and regeneration. One of them is mediated by gap junctions, i.e. intercellular conduits formed by docking of two hemichannels composed of connexin (Cx) proteins. The expression of Cx43 is upregulated during the regeneration of adult skeletal muscle. In vitro studies revealed that Cx43 expression is required for normal myo- genesis. In agreement with this evidence, we have recently shown that the regulation and assembly of connexins into gap junctions and Cx43 protein in particular represent critical events in myoblast differentiation elicited by sphingosine 1-phosphate (S1P), a bioactive lipid involved in this biological process and in the activation of muscle stem cells. Here we investigated the possible interaction between Cx43 expression and function on sphingolipid metabolism in primary cultures of myoblasts obtained from wild type andCx43-knockout (Cx43-/-) newborn mice. Interestingly, we found that sphingolipid metabolism is significantly altered in Cx43-/- cells compared to wild type and heterozygous Cx43-/+ cells. These findings suggest that the ability of myoblasts to share information and/or signalling pathways mediated by Cx43 is required for the correct balance between sphingolipids, the ceramide and S1P. Ceramide and S1P act in opposite manner regulating the coordinated differentiation of myoblasts to their final fate.

    (Grants from Fondazione Cassa di Risparmio di Pistoia e Pescia, Programma Vigoni)

  • S1P and its analogs are killing neurons.


    N Hagen, AH Merrill Jr1, G van Echten-Deckert 

    Kekulé-Institute,LIMES Membrane Biology & Lipid Biochemistry, University Bonn, Germany

    1School of Biology and Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, USA

    Sphingosine-1-phosphate (S1P) is known to induce proliferation in most cell types described so far. However, in the present study we demonstrate that terminally differentiated neurons, which store S1P as a result of S1P-lyase deficiency, are directed into apoptosis. Using liquid chromatography (LC) electrospray ionization (ESI) mass spectrometry we determined the amounts of different bioactive sphingolipids in neurons prepared from lyase deficient versus wildtype and heterozygous mice. Our results confirm the hypothesis based on results obtained with the metabolically stable sphingoid phosphate cis-4-methylsphingosine-phosphate that S1P induces neuronal apoptosis when it reaches a certain threshold value. In addition the molecular mechanism responsible for this effect involves aberrant cell cycle activation as described before in wild type neurons accumulating the semi-synthetic cis-4-methylsphingo-sine-phosphate. Of interest, FTY720 showed a similar yet less pronounced apoptotic effect in wild type neurons. We thus propose cis-4-methylsphingosine as a new synthetic prodrug, which might help understand the role of S1P in neuronal cell death.