5th Meeting

Calella de la Costa, Spain, Nov 2-4, 2006

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 www.sphingomap.org) 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,1 and these profiles are revealing many surprises,2 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.1 From a chemical standpoint, the new entities differ from the natural sphingoid bases in the nature of C1 position, side chain at C2 position and stereochemistry of C3 and C4 stereogenic 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. Pitsinos1, A. 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.3 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 Mamoun2 and 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 antimalarial3 drugs 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 miltefosine4 made 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.1 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.2 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,1 F. Mazzoni,1 L. Colombaioni,1 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.1 This causes the disruption in T- helper cell differentiation and prevalence Th2 cells on Th1 cells.2 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.3 Fumonisin B1 inhibits the DNA synthesis in normal lymphocytes in response to mitogenes, more effective in T cells compared with B cells.1 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.4 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 pathways4 or 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.5 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. Bergman2, Å. 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 studies1 including studies on neutral ceramidase (-/-) (ash2 -/-) mice2 indicate 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. Delisle1,2,3, O. 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 adherence1 and S1P-induced EC chemotaxis2 by 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 .
1 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 S1P1 receptors 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, S1P2 and S1P3 receptors, 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 S1P3 receptor, the other subtypes required a much higher S1P concentration to induce internalisation than to activate signalling. Moreover, S1P3 receptor internalisation was much faster than that of S1P1 and S1P2 receptors, requiring 5 min vs. up to 30 min. This may be explained by a recent finding that internalisation of the S1P3 receptor is independent of phosphorylation in contrast to that of the S1P1 receptor. Preliminary results suggest that also resensitisation differs among these three S1P receptor subtypes. In conclusion, our study shows that S1P1, S1P2 and 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 and 3Department 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 function1 supports 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. Chollet1, C. 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.1 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.2 In combination with ionizing radiation, PS also enhances apoptotic cell death inradiation-resistant cancer cells through ROS-dependent and –independent AIF release.3 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,1 G. Villorbina,1 S. Grijalvo1,2, A. Delgado1,2, A. Llebaria,1 G. Fabriàs,1 J. Casas1.
1Research Unit on BioActive Molecules, Department of Biological Organic Chemistry, IIQAB, CSIC. Jordi Girona 18, 08034-Barcelona, Spain and 2University 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.1 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.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 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. 3 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)6 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 and 2Department 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 and 2Memphys 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.
1 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. Ribar1, I. 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. Bruni1,4, E. 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. Strigini1 and 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’.