6th Meeting

Bilbao, Spain, Nov 22-25, 2007

Inside and out the “sphingolipid rheostat”

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

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

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

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

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

Bioactive sphingolipids in inflammation and cancer.

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

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

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

Shimon Gatt and Arie Dagan
Department of Biochemistry, Hebrew University-Hadassah school of Medicine, Jerusalem, Israel 91120.

The purpose of our studies was to synthesize non-natural analogs of sphingolipids that would affect their metabolism in two directions: a. Elevate ceramide, for killing cancers. b. Decrease the levels of sphingolipids for therapy of sphingolipidoses.
1.We have synthesized sphingolipids to each of which a fluorescent molecule is linked, enabling us to omit the use of radioactivity and analyze effects on sphingolipid metabolism by quantification of the fluorescence of the respective products.
2.We have synthesized novel, non-natural synthetic analogs of sphingolipids that inhibited the hydrolysis of ceramide or its conversion to more complex sphingolipids.
This led to an elevation of the ceramide, leading to death by apoptosis of a variety of cancer cells.
3.Administration of the synthetic sphingolipid-analogs to tumor-bearing mice by intradermal or intraperitoneal injections, or by giving them per-os, ie, in their drinking water, resulted in a reduction and even disappearance of the tumors, while in mice that have not received our synthetic analogs the tumors continued growing.
4.Other, synthetic analogs of sphingolipids were prepared for therapy of two sphingolipid storage diseases, ie, Niemann-Pick and Gaucher diseases, in which sphingomyelin and glucosylceramide accumulate respectively. The analogs were tested for two therapeutic approaches. In the first (Substrate Reduction Therapy) the analogs inhibited the biosyntheses of sphingomyelin (for Niemann-Pick disease) and/or glucosylceramide (for Gaucher disease), thereby decreasing their respective levels.
In the second therapeutic approach (Chaperone Therapy) the analogs linked to the mutated enzyme (eg. acid sphingomyelinase) and protected it from being inactivated in the endoplasmic reticulum. The enzyme-analog complex was then transported to the lysosomes of Niemann-Pick disease cells, where the accumulated sphingomyelin displaced the analog and was hydrolyzed by the residual activity of the mutated sphingomyelinase.

The Extended Family of Neutral Sphingomyelinases: roles and regulation.

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

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

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

Luz Camacho-Castillo1, Carmen Bedia1, Josefina Casas1, Paul P. Van Veldhoven2, Gemma Fabriàs1.
1 Research Unit on BioActive Molecules (RUBAM), IIQAB-CSIC, Barcelona, Spain 2 Katholieke Universiteit Leuven, Campus Gasthuisberg, LIPIT, Leuven, Belgium.

Sphingosine-1-phosphate lyase (SPL) is an endoplasmic reticulum resident enzyme responsible for the degradation of the bioactive lipid compound sphingosine-1- phosphate (S1P). The disruption of SPL function by small molecules has been shown to induce lymphopenia in mice.1 This suggests that this enzyme represents a novel target for immunomodulatory therapy. Screening for new SPL inhibitors requires however the availability of a reliable and fast enzyme assay. The measurement of the SPL activity has been carried out for years using radiolabeled dihydrosphingosine-1- phosphate as substrate in a rather tedious assay.2 A new assay using a fluorescent substrate has been recently reported, but requires still extraction and RP-HPLC.3 A simple and reproducible assay based on the release of umbeliferone from an appropriate substrate (RU13) was developed in our laboratories.4
In this communication we describe the optimization of this assay and its validation by using several cell lines with different degree of SPL activity. We also report on a simplified format for library screening using semi-purified human SPL expressed in E. coli5 and its application to the discovery of SPL inhibitors. Finally, the effect of RU13 on the sphingolipidome, as determined by LC-MS, is also reported.

1. S.R. Schwab, J.P. Pereira, M. Matloubian, Y. Xu, Y. Huang, J.G. Cyster, Lymphocyte sequestration through S1P lyase inhibition and disruption of S1P gradients. Science, 2005.309, 1735-9
2.P. P. Van Veldhoven, G. P. Mannaerts, Sphingosine-phosphate lyase. Adv. Lipid Res. 1993, 26, 69-98.
3.P. Bandhuvula, H. Fyrst, J. D. Saba, A rapid fluorescent assay for sphingosine-1-phosphate lyase enzyme activity J. Lipid Res. 2007, in press.
4.C. Bedia, J. Casas, G. Fabrias. Fluorimetric determination of sphingosine-1- phosphate lyase activity. 5th International Meeting of the Sphingolipid Club. Calella,Spain, 2006.
5. P. P. Van Veldhoven, S. Gijsbers, G .P. Mannaerts, J. R. Vermeesch, V. Brys, Human sphingosine 1-phosphate lyase: cDNA cloning, functional expression studies and mapping to chromosome 10q22. Biochim. Biophys. Acta 2000, 1487, 128-134.

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

L-Ruth Montes1, Alicia Alonso1, Felix Goñi1 and Luis A. Bagatolli2*
1 Unidad de Biofisica (Centro Mixto CSIC-UPV/EHU), Depto. de Bioquimica, Universidad del Pais Vasco. 2 Dpt. Biochemistry and Molecular Biology/MEMPHYS, University of Southern Denmark.

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

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

F.Betul Kavun, Kutlu Ulgen
Department of Chemical Engineering, Bogazici University, Bebek 34342, Istanbul,Turkey

Sphingolipids are a class of lipids regulating cellular processes that are critically important in cell’s fate. These cellular processes function in cancer development and progression, thus this fact underlies the basics of the novel cancer therapy approach. The pharmacological manipulation of the sphingolipid metabolism in cancer therapeutics necessitates the detailed understanding of the pathway. Mathematical modeling is employed to unravel the complexity of biological systems, where the information we have gained from the use of two the computational systems biology tools in this paper is expected to shed light on the sphingolipid metabolism.
In this study, Saccharomyces cerevisiae is employed as the model organism since detailed and complete data for this organism are available in the literature. Moreover, the sphingolipid metabolic and regulatory pathways are conserved across species and they resemble each other in all eukaryotes. Metabolic control analysis and metabolic pathway analysis are here performed in a comparative manner to identify potential drug target enzymes among sphingolipid pathway that can be further utilized in drug design studies for cancer therapy. Our aim was to rank the enzymes in SL pathway according to their roles in controlling the metabolic network by MCA and to identify the physiologically connected reactions, i.e. biologically significant and functional modules of network by MPA. The ultimate goal of this work is to achieve a set of candidate drug target enzymes and to compare the resulting critical control enzymes identified from both metabolic control analysis and metabolic pathway analysis. In fact this will be the comparison of metabolic control analysis and metabolic pathway analysis in terms of their efficiency in drug target identification. The final set of candidate drug target enzymes are selected by handling the results from both metabolic control and pathway analysis. Palmitoyl-CoA Synthase (rxn 1),Glycerol-3- Phosphate Acyltransferase (rxn 2), CDP-DAG Synthase (rxn 4), Phosphatidylinositol synthase (rxn 5), Inositol-1-phosphate synthase (rxn 6),Phosphoserine-Phosphatase (rxn 9), Serine Palmitoyl Transferase (rxn 11), Acyl-CoAbinding protein (rxn 12), Sphingolipid long chain base kinase (rxn 15 and 20),4-Hydroxylase (rxn 21), Ceramide Sythase (rxn 23), GPI Remodelase (rxn 27) andAcetyl-CoA Synthethase (rxn 34) are proposed as potential drug targets by both MCA and MPA. The mathematical tools’ efficiency for drug target identification performed in this study is also validated by the clinically available drugs.

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

David J. Lopez,1 Meritxell Egido-Gabas, 2 Josefina Casas,2 Marie Garnier,3 Banafshe Larijani,3 Felix M. Goni1 and Alicia Alonso1*

For years, sphingolipids have been thought to work as mere structural lipids in cells, however in the past decade their role in cell signalling has been underlined. A particularly well studied sphingolipid is ceramide, which acts as an important second messenger in cell death processes, although its putative role as an inducer of cell proliferation has also been described. Ceramide may be synthesized following two pathways: de novo synthesis and sphingomyelin (SM) hydrolysis. The first one occurs in the endoplasmic reticulum (ER) via condensation of L-serine with palmitoyl-CoA to form 3-ketosphinganine. The second pathway appears to occur mainly in the plasma membrane, as a result of sphingomyelinase (SMase) activation by an appropriate effector of the so-calledsphingolipid signaling pathway.
Lang et al., studying the processes occurring during erythrocyte death, described an increase in ceramide content after 15 hour-incubation of human red blood cells (RBC) in a hypertonic medium. Gatt, Loyter and co-workers had reported some years back that incubation of chicken erythrocytes in a hypotonic medium resulted in the hydrolysis of SM. Because of the newly acknowledged importance of ceramide signaling, in RBC as well as in other cells, it was important to explore and define any latent sphingomyelinase activity in human erythrocytes. This work deals with the characterization of a sphingomyelinase activity in human RBC that is elicited upon incubation in either hypotonic or hypertonic media. The decrease in SM content has been monitored by three different techniques, namely thin layer chromatography (TLC), reverse phase high performance liquid chromatography (RP-HPLC) and electrospray mass spectrometry (ESI-MS). This intrinsic activity is calcium and magnesium-dependent and causes an increase in ceramide content whose maximum is reached 3-6 minutes after establishing the osmotic shock at neutral pH. The activity is blunted after addition of neutral sphingomyelinase (nSMase) inhibitors such as GW4869 or spiroepoxide. Some studies have used detergents as activators of sphingomyelinases; in this case, Triton X-100 (TX-100) slightly increases the SM hydrolysis.
Up to seven different groups of sphingomyelinases have been described. Among the ones present in mammals, some are active at neutral pH only in the presence of magnesium ions. One type of neutral sphingomyelinases, nSMase 2, has been localized in the plasma membrane (PM), is magnesium-dependent and is inhibited by nSMase inhibitors such as GW4869. The activity described in the present work might belong to this group of neutral sphingomyelinases.
*Corresponding author: alicia.alonso@ehu.es

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

Delphine Milhas1, Hervé Benoist1, Virginie Garcia1, Zhe-Xiong Jin2, Hisanori Umehara2, Toshiro Okazaki3, Thierry Levade1, and Bruno Ségui1
1 Inserm U858, Institut de Médecine Moléculaire de Rangueil, BP 84225, 31432 Toulouse Cédex 4, France. 2 Division of Hematology and Immunology, Department of Internal Medicine, Kanazawa Medical University, Ishikawa 920-0293, Japan. 3 Department of Clinical Laboratory, Medicine/Hematology, Faculty of Medicine, Tottori University, Yonago, Tottori683-8504, Japan.

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

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

Yajun Cheng1, Jun Wu2, Erik Hertervig3, Stefan Lindgren4, Deming Duan2, Åke Nilsson3,Rui-Dong Duan1
1.Biomedical Center, B11, Institute of Clinical Sciences, Lund University, Lund Sweden.
2.Institute of Biotechnology, Beijing, China.
3.Gastroenterology Division, Lund University Hospital, Lund, Sweden.
4.Gastroenterology Division, Lund University Hospital, Malmö, Sweden.

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

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

Cristiana Perrotta1, Laura Bizzozero2, Sestina Falcone2, Patricia Rovere-Querini1, Alessandro Prinetti3, Edward H. Schuchman4, Sandro Sonnino3, Angelo A. Manfredi1,5 and Emilio Clementi2,6
1H San Raffaele Scientific Institute, 20132 Milano, Italy; 2E. Medea Scientific Institute, 23842 Bosisio Parini, Italy; 3Department of Medical Chemistry, Biochemistry and Biotechnology, University of Milano, 20090 Segrate, Italy; 4Department of Human Genetics, Mount Sinai School of Medicine, New York, NY 10029, USA; 5Università Vita- Salute San Raffaele, 20132 Milano, Italy; 6Department of Preclinical Sciences, University of Milano, 20157 Milano, Italy.

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

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

Kristy L Houck, Todd E Fox, Mark Kester
Penn State University, 500 University Drive, Mail Code R130,Hershey PA 17033, USA

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

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

Chantal Barthomeuf1,2 and Richard Beliveau3
1 Univ Auvergne, Lab de Pharmacognosie et Biotechnologies, Clermont-Fd (France)2INSERM-484, 3 Hôpital Ste Justine-UQAM, Service d’Hémato-Oncologie, Hôpital Ste- Justine/UQAM, Montréal, (Québec, Canada).

Sphingosine-1-phosphate (S1P), a bioactive lipid released by activated platelets has emerged as an essential mediator of angiogenesis, a stimulus of the metastatic cascade and, a crucial link between inflammation, coagulation and angiogenesis. S1P is the ligand of G-protein coupled receptors (GPCR) of the endothelial differentiation gene (Edg) family, namely edg-1, edg-3, edg-5, edg-6 and edg-8. Ligation to Edg-1, the preferential Edg receptor in ECs, promotes Edg-1 translocation into caveolae and, pertuissis toxin (PTx)- sensitive dependent activation of phosphoinositide 3-kinase (PI3-K)/Akt, MEK/ERK and p38 mitogen activated protein kinase (MAPK) pathways. Edg-1 plays a major role in EC and smooth muscle cell function. It is most notably involved in migration, proliferation and differentiation of endothelial cells (ECs). Recent studies have shown that S1P promotes Src family kinase-dependent transactivation of vascular the endothelial growth factor Flk- 1/KDR also called VEGFR-2. Flk-1/KDR transactivation enhances eNOS and Akt activation in elicited cells (1). We have recently reported that grape polyphenols regulates a PI3K- dependent process: serum-mediated invasiveness of estrogen-receptor (ER)-negativebreast cancer cells (2) and, inhibit S1P and VEGF-induced EC chemotaxis (3). We have established a link between inhibition of S1P-mediated chemotaxis, reduction of platelet- activating factor (PAF) synthesis and down-regulation of p38 MAPK activation in elicited cells (2, 3). The aim of this study was to verify if the regulation of these S1P-dependenteffects could be due to inhibition of Flk-1/KDR transactivation. Western blots unambiguously demonstrate that in S1P-stimulated cells, Flk-1/KDR phosphorylation occurs as early as 3 min after S1P stimulation and, is decreased by pre-treatment with SGE or PTx. It has been found that although SGE decreases PAF synthesis and EC chemotaxis in S1P-stimulated cells, it did not modify the time-course of these events. They reach a maximum 7.5 min after S1P stimulation. Given the stimulatory effect of Flk-1-KDR on EC migration and PAF synthesis and, the kinetic of these events in SGE- pretreated cells, the suppression of Flk-1-KDR transactivation in S1P-elicited cells plays certainly a crucial role in the regulation of S1P-mediated chemotaxis and PAF synthesis by grape polyphenols. Since GPCR-dependent processes are involved in the promotion of inflammation, angiogenesis, coagulation and tumor invasiveness by S1P (5), this study has evidenced a mechanism playing a crucial role in the prevention of angiogenesis- dependent diseases by wine and grape polyphenols and, their tumor chemopreventive andanti-metastasis effects. Note that these data are the first to establish that certain dietary polyphenols regulate a critical cross-link between VEGF- and S1P-dependent signaling.
1 Tanimoto T et al. J Biol Chem 2002, 277: 42997-3001
2 Barthomeuf C et al. 2007 AACR Proceed, April 14-18, LA, Abstract 2656 3 Barthomeuf C. et al. Free Rad Biol Med 2006, 40:581-90
4 Barthomeuf C et al. NS-Arch Pharmacol 2007 374: 331-3325 Barthomeuf C Free Rad Biol Med 2007 42:312-313

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

Audrey Dayon 1,2, Nelly Pirot 1,2, Nicolas Doumerc 2,3, Catherine Mazerolles 4, Leonor Nogueira 5, Muriel Golzio 1,2, Justin Teissié 1,2, Guy Serre 5, Pascal Rischmann 1,2,3, Bernard Malavaud 1,2,3, Olivier Cuvillier 1,2,3
1 CNRS, Sphingolipids and Cancer Research Laboratory, Institut de Pharmacologie et de Biologie Structurale, UMR5089, Toulouse, F-31000 France
2 Université Toulouse III Paul Sabatier, Toulouse, F-31000 France
3 CHU Toulouse, Hôpital Rangueil, Service d’Urologie et de Transplantation Rénale, Toulouse, F-31000 France
4 CHU Toulouse, Hôpital Rangueil, Laboratoire Anatomie Pathologique et Histologie- Cytologie, Toulouse, F-31000 France
5 CHU Toulouse, Hôpital Purpan, Laboratoire de Biologie Cellulaire et Cytologie, Toulouse,F-31000 France

Androgen depletion, which is known to be associated with decreased cell proliferation and lower prostate specific antigen (PSA) secretion in LNCaP prostate cancer cells, triggers a rapid and significant but transient (< 48 h) inhibition of the oncogenic lipid kinase Sphingosine Kinase-1 (SphK1). The inhibition of SphK1 is instrumental as its overexpression can counteract the loss of proliferation induced by androgen privation. In orthotopically xenotransplanted animal models (SCID mice), the loss of tumor volume after castration is also correlated with a marked SphK1 inhibition in the prostatic tissues.
The addition of dihydrotestosterone (DHT) to androgen deprived LNCaP cells restablishes cell proliferation as well as PSA secretion. SphK1 activity is implicated in this action since the overexpression of a dominant negative form of SphK1 or treatment with specific pharmacological SphK1 inhibitors inhibit the proliferative effects of DHT.
Noteworthy, longer exposure of LNCaP cells to androgen-free conditions (beyond 48h and up to 42 days) is linked to a progressive increase in SphK1 activity (up to 4-fold increase at 42 days), Akt activation and ERK 1/2 stimulation. These events are associated with a neuroendocrine differentiation of these cells (as shown by the morphological changes in cell phenotype and increased levels of the Neuron Specific Enolase (NSE) marker).
Collectively, these results implicate SphK1 in the progression of prostate cancer cells to androgen independence. The mechanisms by which SphK1 activity is increased during this phenomenon are still under investigation.

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

Colié S. 1, Kedjouar B. 1, Garcia V. 1, Carpentier S. 1, Van Veldhoven P.P. 2, Levade T. 1 and Andrieu-Abadie N. 1
1 INSERM U858, Institut de Mèdecine Moléculaire de Rangueil, BP84225, 31432 Toulouse Cedex 4, France.
2 LIPIT, Katholieke Universiteit Leuven, Begium.

Sphingosine 1-phosphate (S1P) is a bioactive metabolite of sphingolipid metabolism, which modulates cell survival, proliferation and migration, as well as angiogenesis and lymphocyte trafficking. The intracellular concentration of this oncogenic lipid is governed by the activities of three different enzymes that catalyse its synthesis or removal. One of them, sphingosine-1-phosphate lyase (SPL), encoded by the Sgpl1 gene, is responsible for the irreversible degradation of S1P.
In order to investigate the role of SPL in cell survival, we used embryonic fibroblasts derived from SPL-deficient mice (KO), and compared their susceptibility to various apoptotic inducers to that of cells from heterozygous (HET) or wild type (WT) animals. Here we demonstrated that SPL-deficient cells are resistant to the toxic effect of etoposide, doxorubicin or other cytotoxic agents such as staurosporine. In mutant cells,stress-induced caspase activation and phosphatidylserine externalization were strongly inhibited. Moreover, the ability of SPL to protect cells from apoptosis was correlated with an accumulation of S1P in SPL-deficient cells. Interestingly, heterozygous disruption of the gene encoding for SPL was associated with a partial resistance to apoptosis and moderately elevated S1P levels. Finally, SPL deficiency led to increased cell migration in an in vitro scratch-wound assay resulting in accelerated wound closure, indicating an important role for SPL in this process.
These results suggest that, by affecting S1P metabolism, SPL may contribute to cell resistance upon antitumor treatment and could favor tumor progression.
(Supported by INSERM, Université Paul Sabatier, and Ligue contre le Cancer)

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

Chiara Donati1, Francesca Cencetti1, Paola Nincheri1, Silvia Brunelli2,3, Emilio Clementi4, Giulio Cossu2,5, Marta Farnararo1 e Paola Bruni1
1 Department of Biochemical Sciences, University of Florence, Florence
2 Stem Cell Research Institute, Ospedale San Raffaele, Milan
3 Dipartimento di Medicina Sperimentale ed Ambientale e Biotecnologie Mediche, Università di Milano-Bicocca, Monza
4 Dipartimento di Scienze Precliniche LITA-Vialba, Università di Milano
5 Dipartimento di Biologia, Università di Milano

Mesoangioblasts are a novel class of stem cells able to differentiate into diverse mesodermal cell types such as adipocytes, osteoblasts, smooth and skeletal muscle cells. These cells are presently regarded as suitable candidates for cell therapy of muscle degenerative diseases since they have been demonstrated capable of regenerating, in animal models, the dystrophic muscle [1]. The characterization of the physiological agonists that regulate proliferation and survival of these stem cells becomes fundamental to enhance their therapeutic efficacy.
TGF-beta plays a critical role in the maintenance of the homeostasis between cell growth and apoptosis, however, depending on the cell type, a dual role of the citokine able of inducing proapoptotic or survival responses has been reported [2]. As regards the molecular mechanism of action, in the last few years a cross-talk between TGF-beta and sphingolipid signalling has been demonstrated to take place. It has been recently demonstrated that sphingosine 1-phosphate (S1P), a sphingolipid derived from sphingomyelin catabolism, involved in the regulation of fundamental biological effects [3], exerts a potent antiapoptotic action in murine mesoangioblasts [4].
In this study it is reported that TGf-beta exerts a marked antiapoptotic action in mesoangioblasts with a mechanism that involves activation of sphingosine kinase (SphK), which is the enzyme responsible for S1P formation. Treatment with the cytokine indeed efficaciously protected mesoangioblasts from apoptosis induced by serum starvation or staurosporine or C2-ceramide treatment assessed by various means such as activation ofcaspase-3 and determination of cytoplasmic histone-associated-DNA-fragments. The protective action of TGF-beta from staurosporine-induced apoptosis resulted strongly reduced when SphK activity was inhibited either by pharmacological inhibitors or by overexpression of the dominant negative mutant of the enzyme and when its expression levels were downregulated by specific siRNA transfection. Finally, staurosporine-induceddownregulation of SphK was counteracted by TGF-beta treatment. Interestingly, TGF–betaantiapoptic action was impaired when ERK1/2 activation was inhibited by the specific inhibitor U0126.
References[1] Sampaolesi M., Blot S., D’Antona G., Granger N., Tonlorenzi R., Innocenzi A., Mognol P., Thibaud J.L., Galvez B.G., Barthelemy I., Perani L., Mantero S., Guttinger M., Pansarasa O., Rinaldi C., Cusella De Angelis M.G., Torrente Y., Bordignon C., Bottinelli R., Cossu G. (2006) Nature 444(7119), 574-579[2]Sanchez-Capelo A., (2005) Cytokine Growth Factor Rev. 16, 15-34[3]Spiegel S. and Milstein S. (2002) J. Biol. Chem, 277, 25851-25854[4]Donati C., Cencetti F., Nincheri P., Bernacchioni C., Brunelli S., Clementi E., Cossu G. and Bruni P. (2007) Stem Cells, 25(7), 1713-1719

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

Leyre Brizuela, Miriam Rábano, Patricia Gangoiti, Natalia Narbona, José María Macarulla, Miguel Trueba, and Antonio Gómez-Muñoz
Department of Biochemistry and Molecular Biology. Faculty of Science and Technology. University of the Basque Country. P.O. Box 644. 48080 – Bilbao (Spain)

We reported recently that sphingosine 1-phosphate (S1P) is a novel regulator of aldosterone secretion in zona glomerulosa cells of adrenal glands, and that phospholipase D (PLD) is implicated in this process. We now show that S1P causes phosphorylation of protein kinase B (PKB) and extracellularly regulated kinases 1/2 (ERK1/2), which is an indication of their activation, in these cells. These effects are probably mediated through interaction of S1P with the Gi protein-coupled receptors S1P1/3, as pre-treatment with pertussis toxin or with the S1P1/3 antagonist VPC 23019 completely abolished phosphorylation of these kinases. Inhibitors of phosphatidylinositol 3-kinase (PI3K) ormitogen-activated kinase kinase (MEK) blocked S1P-stimulated aldosterone secretion. This inhibition was only partial when the cells were incubated independently with inhibitors of each pathway. However, aldosterone output was completely blocked when the cells werepre-treated withLY294002 and PD98059, simultaneously. These inhibitors also blocked PLD activation, which indicates that this enzyme is downstream of PI3K and MEK in this system. We propose a working model for S1P in which stimulation of the PI3K/PKB and MEK/ERK pathways leads to stimulation of PLD and aldosterone secretion.
Key words: adrenal gland; ceramide; mitogen-activated protein kinase; glomerulosa cells; mineralocorticoids; phospholipase D; protein kinase B; sphingolipids; steroids.
J. Lipid Res. 2007. 48:2264-2274.

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

Stephan L.M. Peters, Arthur C.M. Mulders, Marie-Jeanne Mathy, Dagmar Meyer zu Heringdorf1, Michael ter Braak1, Najat Hajji, Dominique C. Olthof, Martin C. Michel and Astrid E. Alewijnse.
Department of Pharmacology and Pharmacotherapy, Academic Medical Center, Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands.
1 Institute of Pharmacology, University Hospital Essen, Hufelandstrasse 55, 45122 Essen, Germany.

Local formation of the sphingomyelin metabolite sphingosine-1-phosphate (S1P) within the vascular wall has been shown to modulate vascular contraction and relaxation. In the present study we investigated whether sphingosine kinase plays a role in muscarinicreceptor-mediated NO production and relaxation in different vascular beds. In a vascular endothelial cell line, expressing muscarinic M3 receptors and YFP-tagged sphingosine kinase, activation of the M3-receptor leads to a rapid translocation of sphingosine kinase from the cytosol to the plasma membrane. Moreover, the sphingosine kinase inhibitor dimethylsphingosine (DMS, 10 mM) attenuated NO production in this cell line after muscarinic receptor stimulation with the agonist methacholine. Accordingly, in isolated rat aorta the relaxant responses to methacholine were attenuated in the presence of DMS. Interestingly, DMS enhanced the relaxant responses to methacholine in mesenteric artery preparations. In this preparation, methacholine-induced endothelium-derivedhyperpolarizing factor- mediated (i.e. non NO and non-eicosanoid-dependent) relaxation was enhanced by DMS. In addition, DMS enhanced the dilatory actions of the putative endothelium- derived hyperpolarizing factor C-type natriuretic peptide.
We conclude that stimulation of endothelial muscarinic receptors induces sphingosine kinase signalling subsequently resulting in differential effects in different vessel types. Whereas locally produced S1P enhances NO-mediated vasorelaxation, it attenuatesEDHF-mediated vasodilation. A disturbed regulation of sphingolipid metabolism in the vascular wall may therefore play a role in the aetiology/pathology of disease states characterized by endothelial dysfunction.

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

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

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

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

B.M. Barth, S.J. Gustafson, and T.B. Kuhn
Alaskan Basic Neuroscience Program, Department of Chemistry and Biochemistry, University of Alaska-Fairbanks, 900 Yukon Drive, Room 194, Fairbanks, Alaska 99775 USA

The treatment of advanced malignancies involves various therapeutic agents as well as radiation therapy. The most advanced cancers have emerged as being highly invasive and resistant to chemotherapy and radiation. Cancers of the nervous system are especially detrimental because of the dramatic impact on the everyday lives of patients. The sensitive location can make these cancers inoperable or make surgical intervention highly impractical. Furthermore, certain cancers of the brain respond minimally at best to chemotherapy and radiation, altogether giving poor prognosis to those effected. Chemotherapy results in increases in ceramide that can in turn induce cell stress, and ultimately apoptosis. Clearance of ceramide, in addition to regulation of drug transporters has linked the up-regulation of glucosylceramide synthase to cancer multi-drug resistance. Additionally, the up-regulation of sphingosine kinase has been linked to increased cancer cell survival and invasiveness.
Previously we had demonstrated that TNF-αinduced the NADPH oxidase, and subsequent reactive oxygen species, in human SH-SY5Y neuroblastoma cells in a neutral sphingomyelinase-dependentmechanism. In this study, we demonstrated that TNF-α and doxorubicin-induced NADPH oxidase activity resulted in oxidation and down-regulation of glucosylceramide synthase and sphingosine kinase in SH-SY5Y neuroblastoma cells. We further demonstrated that glucosylceramide inhibited the NADPH oxidase, demonstrating an intriguing cellular antioxidant mechanism. Despite the presence of an NADPH oxidase, two human glioblastoma cell lines, U-87 MG and LN-18, responded to TNF-αand doxorubicin with no significant increase in reactive oxygen species. We determined through enzyme assays that the glioblastomas had increased superoxide dismutase, catalase, and glucosylceramide synthase activities compared to the neuroblastoma cell line. We propose that the increased cellular antioxidant capacity is another mechanism that promotes the resistance of these advanced cancers to chemotherapy, by protecting enzymes with important function in invasiveness and multi-drug resistance. Therefore selective targeting of the cellular antioxidant enzymes, including glucosylceramide synthase, or the up- regulation of the NADPH oxidase may be a useful strategy for the treatment of advanced drug resistant and invasive cancers.

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

Sonda S, Štefanić S. and Hehl AB.
Institute of Parasitology, University of Zurich, Winterthurerstrasse 266a, CH-8057 Zurich, Switzerland.

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

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

Alessandro Fanzani a, Stefani a Rossia, Elena Stoppani a, Roberta Giuliani a, Francesca Colombo a, Wim Martinet b, Augusto Preti a and Sergio Marchesini a
a Department of Biomedical Sciences and Biotechnology, Unit of Biochemistry,University of Brescia, Italy. b Division of Pharmacology, University of Antwerp, Wilrijk, Belgium.

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

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

Edwige Tellier 1, Anne Nègre-Salvayre 1, Beatrice Bocquet 1, Shigeyoshi Itohara 2 Yusuf A. Hannun 3 Robert Salvayre 1, Nathalie Augé 1
1. SERM UMR-466, Dept of Biochemistry, IFR-31, CHU Rangueil, Toulouse, France
2. RIKEN Brain Research Institute, Wako-Shi, Saitama 351-0198, Japan.
3. Medical University South Carolina, Dept of Biochemistry and Molecular Biology, Charleston, SC, 29425, USA.

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

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

H.Saybasili, Ö. Genç, T. Köker, Y.P. Tan
Boğaziçi University, Biomedical Engineering Institute, 34342, Be bek, Istanbul, Turkey

Ceramide, besides its structural role in cell membrane as a sphingolipid, has essential roles in apoptosis, cell growth and differentiation. In this study, the effect of C2- ceramide (10 µM) application on whole cell currents recorded by patch-clamp technique from soma of hippocampal CA1 pyramidal neurons was investigated. Evoked post-synaptic currents were recorded with low frequency (0.1 Hz) stimulation of Schaffer Collateral. It was observed that ceramide depressed the amplitude of NMDA currents (23 %±8 pA, n=5); the NMDA current was blocked with AP5(60 µM) application to artificial cerebrospinal fluid (ACSF). Non-NMDA currents were isolated by applying AP5 at -50mV, C2- ceramide application did not cause significant change in the non-NMDA current amplitude (n = 4). Thus, C2-ceramide leads to a selective depression on the NMDA current; this indicates that sphingolipids have a modulatory effect on the cell membrane besides their roles as secondary messengers inside the cell.
Keywords: C2-ceramide, hippocampal synaptic currents, NMDA and AMPA receptors,patch-clamp tight-seal whole cell technique.
* This work wasğaziçi supported University by grants Research 02S104 Fund and. 07HX101 from Bo

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

Anne Montfort1, Victorine Douin-Echinard1, Pascal Martin2, Virginie Garcia1, Jason Iacovoni1, Caroline Nevoit1, Thierry Levade1, Hervé Benoist1 and Bruno Ségui1
1 Inserm U858, Institut de Médecine Moléculaire de Rangueil, BP 84225, 31432 Toulouse Cédex 4, France.
2 Laboratoire de Pharmacologie-Toxicologie, INRA BP3, 180 chemin de Tournefeuille, 31931 Toulouse Cédex 9, France.

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

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

Lai Wei1, René H. Tolba1, Reinhard Büttner2, Annika Wedeking, Gerhild vanEchten-Deckert
Kekulé-Institute of Organic Chemistry and Biochemistry and 1House of Experimental Therapy, 2Institute of Pathology, University of Bonn, Germany.

Using a standardized surgical rodent model of intestinal manipulation we have reported recently that sphingosine 1-phosphate (S1P) and ceramide 1-phosphate (C1P) were elevated in intestinal muscularis post-surgery. In addition, our data suggested that these two ceramide-derived molecules might be involved in the local inflammatory reaction occurring after surgery [1]. Based on these results, we hypothesized that a reduction of ceramide, the common precursor of both, S1P and C1P might improve morbidity known to occur after abdominal surgery. Since ectoin, a compatible solute, that enables microorganisms to survive under extreme conditions, was reported to prevent UVA induced ceramide increase thus rescuing keratinocytes from apoptotic cell death [2], we extended our working hypothesis. We assumed that reduction of ceramide, a well-knownmediator of cellular stress by ectoin might improve tissue quality following different kinds of stress. To simplify matters we analyzed in a first approach whether ectoin is able to improve tissue quality in rat bowel subjected to ischemia/reperfusion and whether this effect is correlated with tissue ceramide content. It is generally accepted that ischemia/reperfusion is accompanied by a complex damage mechanism that primarily affects intestinal mucosa but also involves intestinal muscularis, the latter having been neglected for quite a while [3]. Although intestinal mucosa has high regenerative ability, morphological recovery of the injured ileal mucosa after 24-hours cold storage requires at least 1 month [4]. This prolonged damage from ischemia/reperfusion injury makes it a major obstacle for successful small bowel transplantation. It is therefore of primary interest to reduce ischemia/reperfusion injury after organ preservation thus improving the clinical outcome of small bowel transplantation. Our results demonstrate that ectoin indeed convincingly prevents tissue damage following ischemia and this effect is accompanied by the reduction of ceramide in intestinal tissue. Of interest, a synthetic derivative of ectoin, incapable to overcome tissue injury, did not affect the content of ceramide, either.[1]M. Dragusin, S. Wehner, S. Kelly, E. Wang, A.H. Merrill, Jr., J.C. Kalff and G. vanEchten-Deckert, FASEB J 20, 1930-1932 (2006).[2]j. Buenger and H. Driller, Skin Pharmacol.Physiol 17, 232-237 (2004).[3]A. Turler, J.C. Kalff, P. Heeckt, K.M. Abu-Elmagd, W.H. Schraut, G.J. Bond, B.A. Moore, G. Brunagel and A.J. Bauer, Gastroenterology 122,1886-1897 (2002).[4] I. Takeyoshi, S. Zhang, M. Nomoto, Y. Zhu, Y. Kokudo, T. Suzuki, N. Hamada, A. Nemoto, T.E. Starzl and S. Todo, Transplantation 71, 1-7 (2001).

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

1Belleri M., 1Coltrini D., 2Nico B., 2Ribatti D., 1Alessi P., 3Poliani P.L., 4Marchesini S.,5Bongarzone E., and 1Presta M.
1 Unit of General Pathology and Immunology and 4 Unit of Biochemistry, Department of Biomedical Sciences and Biotechnology, School of Medicine, University of Brescia;
2 Department of Human Anatomy and Histology, University of Bari;
3 Department of Pathology, University of Brescia, Italy;
5 Department of Anatomy and Cell Biology, University of Illinois, Chicago, IL, USA.

Globoid cell leukodystrophy (GLD), or Krabbe’s disease, is a neurological disorder of infants caused by the reduction of the lysosomal enzyme b-galactosylceramidase activity. This results in the accumulation of the intermediate metabolite psychosine (b- galactosylsphingosine) at micromolar levels in the CNS. The accumulation of psychosine induces apoptosis of oligodendrocytes, progressive demyelination, and the formation of multinuclear (globoid) cells derived from perivascular microglia, accompanied with block of cytokinesis.
Neovascularization plays an important role in CNS development. Here, we assessed the effect of psychosine on angiogenesis in vitro and in vivo. Also, the neovascularization process was investigated in Twitcher mice, a genetically and enzymatically authentic mouse model of human GLD.
When tested on endothelial cells of bovine and murine origin, micromolar levels of psychosine inhibited cell proliferation and cell attachment/spreading on different substrata. Moreover, psychosine reduced cell migration after mechanical wounding of the endothelial cell monolayer by impairing the repositioning of the microtubule organization center and the formation of membrane ruffles. Also, psychosine impaired the capacity of Fibroblast Growth Factor-2 (FGF2)-transfected murine aortic endothelial cell aggregates to invade a 3D-fibrin gel and to form solid sprouts. Similarly, psychosine prevented bovine aortic endothelial cells to enter a 3D-collagen gel and to organize capillary-like structures when stimulated by FGF2 plus Vascular Endothelial Growth Factor (VEGF). Finally, psychosine caused cytoskeleton disorganization inducing the disruption of actin stress fibers in bovine aortic endothelial cells. In keeping with the in vitro observations, psychosine inhibited FGF2-induced in vivo neovascularization when delivered on the top of the chick embryo chorioallantoic membrane.
On this basis, we performed histological and ultrastructural analysis of the brain of Twitcher mice at 2-5 weeks after birth. The cerebral cortex of Twitcher mice was characterized by reduced FVIII-vWF+ vascularity with dilated and structurally altered blood vessels when compared to control heterozygous and wild type littermates. Also, Twitcher mice showed a reduced capacity to mount an angiogenic response when challenged by a subcutaneous Matrigel implant containing angiogenic FGF2 plus VEGF. These results point to a primary defect in neovascularization of the nervous system of mutant animals.
Our study demonstrates that psychosine has antiangiogenic properties causing the disassembling of endothelial cell actin structures. This may be responsible for the reduced angiogenic potential of Twitcher mice, leading to defects in brain vascularization. Alterations in blood vessel development may contribute to the CNS damage that occurs in Krabbe’s disease.

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

María Salazar1*, Arkaitz Carrracedo1*, §, Íñigo J. Salanueva1, Sonia Hernández1, Cristina Blázquez1, Mar Lorente1, Ainara Egia1, §, Patricia Vázquez2, Sofía Torres1, Stephane García3, Juan Iovanna3, Manuel Guzmán1, Patricia Boya2 and Guillermo Velasco1#

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

Combination therapy for Tay-Sachs and related neurodegenerative diseases.

M.B. Cachón-González, S.Z. Wang and T.M Cox
Department of Medicine, University of Cambridge, Cambridge, UK

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

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

Marini F1, Bartoccini E1, Bernardini I1, Cascianelli G1, Garcia-Gil M2 Viola-Magni M1, Albi E1.
1 Dept. Clinical and Experimental Medicine, University of Perugia;
2Dept. of Biology, University of Pisa.

During nervous system development, the precursors of neurons reside within the epithelium of the neural plate and then in the neural tube. Embryonic cells can be used for their therapeutic potential to treat neurological disorders. In fact they can be incorporated into the adult central nervous system following transplantation thanks to their ability of migration and differentiation. A critical problem of the neural transplantation is how to reduce their apoptosis and improve cell survival. Since nuclear sphingomyelin (SM) metabolism is involved in differentiation and/or apoptosis process (1), the aim of the present study was to analyse the neutral- sphingomyelinase (N-SMase) activity in nuclei free lysate (NFL) and purified nuclei of embryonic hippocampal cells. At this end HN9.10e cell line is used as experimental model. These cells are a somatic fusion product of hippocampal cells from embryonic day 18 C57BL/6 mice and N18TG2 neuroblastoma cells and have morphological and cytoskeletal features similar to their neuronal precursors (2). The differentiation is induced by vitamin D3 (3) whereas apoptosis is induced by serum deprivation (4).The results show that the nuclear N-SMase activity increases during the S-phase of the cell cycle in correspondence of the SM reduction and ceramide production. No changes are observed in NFL N-SMase. Vitamin D3 reduces DNA synthesis together to PCNA and cyclin D expression whereas bcl2 increases indicating a delay of cell cycle and an induction to differentiation supported by microscopy observations that highlight the dendrite and neurite growth. The NFL N-SMase and nuclear N-SMase activities increase after 2 and 12 hours respectively from Vitamin D3 treatment, before the S-phase of the cell cycle. The time for nuclear N-SMase activation is justified by the time necessary to vitamin D3 to reach the nucleus equal to 10 hours. Serum deprivation is accompanied by strong DNA synthesis inhibition, an early activation of nuclear N-SMase (after 1 hour) and a later activation of NFL N-SMase (after 12 hours). It can be concluded that cellular and nuclear SMases behave in different way in relation to the cell fate.
1)Ledeen RW, and Wu G. Biochim Biophys Acta 2006; 1761: 588-598.
2)Lee HJ, Hammond DN, Large TH, Roback JD, Sim JA, Brown DA, Otten UH,Wainer
BH.1990. J Neurosci 10: 1779-1787
3)Brown J, Bianco JI, McGrath JJ, Eyles DW. Neurosci Lett. 2003 Jun 5;343(2):139-
4)Albi E, Cataldi S, Bartoccini E, Viola Magni M, Marini F, Mazzoni F, Rainaldi G, Evangelisti M, Garcia-Gil M. J Cell Physiol, 2006, 206(1):189-195

Involvement of dihydroceramides in cell fate determination.

José M. Munoz-Olaya1, Xavier Matabosch1, Carmen Bedia1, Meritxell Egido-Gabás1, Josefina Casas1, Amadeu Llebaria1, Antonio Delgado1,2, Gemma Fabrias1.
1 Research Unit on BioActive Molecules (RUBAM), IIQAB-CSIC, Barcelona, Spain.
2 Pharmaceutical Chemistry Unit (CSIC Associated Unit), Faculty of Pharmacy, University of Barcelona, Spain.

Ceramide is a well-known mediator of apoptosis that can be induced by a variety of signalling molecules or stress events, causing a transient intracellular increase in this bioactive lipid. Ceramide rise results from sphingomyelinase activity or de novo biosynthesis. In the latter, ceramide is biosynthesized from L-serine in four distinct steps, the last one being the 4-desaturation of dihydroceramide to ceramide by dihydroceramide desaturase.
For years, dihydroceramides have been regarded as innocuous lipids and mere precursors of their ceramide counterparts. This assumption was sustained by the failure of exogenous dihydroceramides with short N-acyl chains to mimic the antimitogenic effects caused by the respective ceramides in cultured cells. However, recent articles point towards a possible role of dihydroceramide in programmed cell death1.

XM462 is a dihydroceramide analogue containing a sulphur atom at the C5 position of the long chain base. This compound inhibits dihydroceramide desaturase both in vitro and in intact cells, it features high chemical and metabolic stability and it is easily available by synthesis. Flow cytometry analysis of Jurkat A3 human leukemia cells revealed that XM462 produces a concentration-dependent increase in the number of annexin V-positive cells. Measurements by LC-MS confirmed accumulation of dihydroceramides at these concentrations. The design, synthesis and biological activity of this compound are presented and discussed.
1W. Zheng, J. Kollmeyer, H. Symolon, A. Momin, E. Munter, E. Wang, S. Kelly, J. C. Allegood, Y. Liu, Q. Peng, H. Ramaraju, M. C. Sullards, M. Cabot, A. H. Merrill, Biochim. Biophys. Acta 2006, 1758, 1864-1884; J. Stiban, D. Fistere, M. Colombini, Apoptosis 2006, 11, 773-780; J. M. Kraveka, L. Li, Z. M. Szulc, J. Bielawski, B. Ogretmen, Y. A. Hannun, L. M. Obied, A. Bielawska, J. Biol. Chem. 2007, 282, 16718- 16728

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

Lena Ohlsson*, Lena Hjelte**, Michael Hϋhn †, Bob J. Scholte ††, Martina Wilke ††, MalinFlodström-Tullberg ††, Ake Nilsson*.
*Department of Clinical Sciences, Medicine (Gastroenterology and Nutrition), Lund University Hospital, S221 85 Lund, Sweden
**Stockholm CF Center, Department of Pediatrics, Karolinska University Hospital, Huddinge, S-141 86 Stockholm, Sweden
†Centerfor Infectious Medicine (CIM), Deparment of Medicine, Karolinska Institutet, Karoliska University Hospital Huddinge, S-141 86 Stockholm, Sweden
††Departments of Cell Biology and Biochemistry, Erasmus Medical Center P.O. Box2040, 3000 CARotterdam, The Netherlands.

Objetives: The instestinal brush border enzymes alkaline sphingomylinase (alk- SMase) and neutral ceramidasa (CDase) digest milk sphingomyelin (SM) in suckling neonates. Futhermore they have been implicated in sphingolipid signalling, which exhibits abnormalities in cystic fibrosis (CF). We asked wheter these enzyme are normally expressed in mice with DF508 (Cftrtm1Eur) mutation, a CF mouse model with well- characterized intestinal pathology. Methods: Alk-SMase and CDase were measured in tissues and intestinal contents of homozygous and heterozygous DF508 mice and wild type mice. Results: No difference was found in the level (activity per mg protein) of enzymes in small intestinal mucosa or content or in their longitudinal distribution. Despite a lower body weightF508of micethe the length of the small intestine and
the weight per centimetre colon were larger than in wild type mice. Alk-SMase and CDase levels in the lung was manifold lower than in the gut and did not differ between the groups. The level of acid SMase in the distal half of the small intestine was lower in CF than in wild type mice. Conclusion: Alk-SMase and neutral CDase are normally expressed in DF508 CF mice and may generate ceramide and sphingosine from milk and endogenous sphingolipid and normal rate.

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

Jon V. Busto*1, María Laura Fanani 2, Luisina De Tullio 2, Jesús Sot 1, Bruno Maggio 2, Félix M. Goñi 1, Alicia Alonso 1
1 Unidad de Biofísica (Centro Mixto CSIC-UPV/EHU), and Departamento de Bioquímica, Universidad del País Vasco (UPV/EHU)
2 Departamento de Química Biológica-CIQUIBIC,Facultad de Ciencias Químicas-CONICET, Universidad Nacional de Córdoba, Argentina (*) presenting author email: gbbbuvej@lg.ehu.es

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

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

Albi E1, Marini F1, Bartoccini E1, Meacci E2, Chico L3, Viola-Magni M1,Garcia-Gil M3
1 Dept. Clinical and Experimental Medicine, University of Perugia;
2 Dept.Biochemical Sciences, University of Florence;
3 Dept. of Biology, University of Pisa.

Although 1a,25-dihydroxyvitamin D3 (vitamin D3) modulates a number of functions in the nervous system, its mechanism of action is unclear.Sphingolipids are known to regulate cell fate but their effects depend on subcellular localization and the ratio betweenceramide/sphingosine-1-phosphate concentration. In the present work, we aimed to investigate whether vitamin D3 induces differentiation in the embryonic hippocampal HN9 cells, and whether nuclear sphingolipid metabolism is involved. Cells were treated with different concentrations of vitamin D3 for different periods of time; morphological differentiation was evaluated as process extension; NGF expression was measured byRT-PCR; nuclear neutral sphingomyelinase, sphingosine kinase-1 actities and ceramide content were measured as previously described (1-3). We have found that treatment with 50 nM vitamin D3 for two hours induces increase of nuclear neutral sphingomielinase activity; prolonged treatment (3 days) induces neurite growth and increases of NGF expression without altering nuclear sphingosine kinase-1 activity. Higher concentrations of vitamin D3 induce cell death. Therefore, vitamin D3- induced differentiation in murine embryonic hippocampal cells is accompanied by an early increase of nuclear neutral sphingomyelinase activity and ceramide content, followed by a raise in NGF expression.
1) Albi et al. J Cell Physiol. 2006 206(1):189-95. 2) De Palma et al. Arterioscler Thromb Vasc Biol. 2006 26(1):99-105. 3) Albi et al. Arch. Biochem Biophys. 2005 438:156-61.

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

Popa I, Thuy L., Pietukowska A, *Pin D, Haftek M, Portoukalian J.
Laboratory of Dermatological Research, EA3732 University Lyon-1, Edouard Herriot Hospital, Lyon; * Veterinary School of Lyon

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

Sphingolipid analogues: searching new bioactive drugs

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

The apoptotic activity of D-erythro-dihydroceramides (DHC’s) is a matter of controversy. Thus, it has been published that exogenously added short chain DHC’s do not exhibit apoptotic activity in different cell lines1,2, while L-threo-N-acetyl- sphinganine causes cell death in HL-60 cells1,3. Additionally, accumulation of natural DHC’s, but not ceramides, along with cell death occurs in human leukaemia cells treated with gamma-tocopherol,human neuroblastoma cells treated with fenretinide, as well as in prostate cancer cells4,5. Finally, a recent report describes that both long and short chain erythro-DHC’s do not simply lack apoptogenic activity, but counteract the apoptotic effect of ceramide by inhibiting ceramide channel formation in mitochondria in early apoptosis6. In this context, the synthesis of a small combinatorial dihydroceramide analogue library obtained by systematic variation of both the sphingoid and aliphatic and acyl chains has been carried out in our group7. Among them compound GVC8PH exhibited a well defined profile of time and dose- dependent apoptosis induction, and it has been selected to perform deeper studies. Results regarding its molecular mechanism, and its effect on the sphingolipid profile in A549 cells, a human alveolar epithelial cell line used in COPD studies, are reported.
1Bielawska A, et al., J Biol Chem., 1993, 268, 26226. 2Ogretmen B, et al., J Biol Chem.,2001, 276, 32506. 3Dragusin M, et al.,. J Lip Res, 2003. 44,1772. 4Jiang Q, et al., Proc. Natl Acad. Sci USA, 2004, 101, 17825. 5Kraveka K et al, J. Biol. Chem.2007, in press. 6Stiban J, et al. Apoptosis, 2006, 11, 773. 7 Villorbina G, et al. Bioorg. Med Chem, 2007, 15, 50.

A new family of sphingomyelin synthase inhibitors.

Meritxell Egido-Gabása, Xavier Mataboscha, Gemma Fabriàsa, Antonio Delgadoa,b, Josefina Casasa and Amadeu Llebariaa
a) Research Unit on Bioactive Molecules (RUBAM); Department of Organic and Biological Chemistry, Chemical and Environmental Research Institute of Barcelona (IIQAB-CSIC);Jordi Girona 18-26, 08034 Barcelona, Spain. b)Pharmaceutical Chemistry Unit (CSIC Associated Unit), Faculty of Pharmacy, University of Barcelona, Spain.

Sphingomyelin (SM) plays an important role both in cell signaling and membrane structure and dynamics. Alteration of its concentration may have important implications in the development of diseases, such as atherosclerosis and diabetes. Sphingomyelin synthase (SMS), the last enzyme involved in SM biosynthesis, transfers the phosphorylcholine moiety from phosphatidylcholine to the primary hydroxyl group of ceramide (Cer) to produce SM and diacylglycerol.
In this presentation we report on the synthesis and activity of a series of ceramide and sphingomyelin analogues. Initially, the SMS activity was determined in rat liver microsomes by using the fluorescent ceramide substrate NBD-C6-Cer, and monitoring its transformation to the corresponding NBD-C6-sphingomyelin by HPLC. Among all compounds tested, the most potent one inhibited SMS with an IC50 valueµ of 5 M. Kinetic experiments revealed that this compound was a non competitive inhibitor with a KiµM.ofPreliminary3 results in intact cells using Jurkat A3 and A549 cell lines indicated that the most active compounds caused a significant decrease in SMS activity without affecting glucosylceramide synthase activity. Measurements of sphingolipid concentrations by LC-MS confirmed these observations.

A new family of sphingomyelinase inhibitors.

Fabio Simbaria, Xavier Mataboscha, Gemma Fabriasa, Antonio Delgadoa,b, Amadeu Llebariaa, Josefina Casasa.
aResearch Unit on BioActive Molecules (RUBAM), IIQAB-CSIC, Barcelona, Spain.bPharmaceutical Chemistry Unit (CSIC Associated Unit), Faculty of Pharmacy, University of Barcelona, Spain

Sphingomyelin (SM) is the primary sphingolipid in mammalian cells and plays important structural and functional roles. SM hydrolysis can be carried out by different isoforms of sphingomyelinase (SMase), producing phosphorylcholine and the intracellular effector ceramide. Ceramide diffuses within membranes acting as a second messenger. Generation of ceramide in various cellular systems is currently recognized as critical to the initiation of vital cellular processes such as differentiation, cell proliferation and apoptosis.
Inhibitory activity of acid SMase by a small library of SM analogues synthesized in our group is reported. SMase activity was determined using A549 cell homogenates and6-hexadecanoylamino-4-methylumbelliferylphosphorylcholine (HMU-PC) as substrate. The same compounds were also tested in intact A549 cells. Flow cytometry analysis suggested that these molecules caused a time and dose dependent apoptotic cell death. Their effect on the cellular sphingolipid profile was also studied for the most active inhibitors.

Role of COPI vesicles in lipid sorting.

Contreras F-Xabier*; Haberkant Per*, Thiele Christoph† Brügger Britta* and Wieland F*.
*Heidelberg University Biochemistry Center (BZH), Im Neuenheimer Feld 328, 69120 Heidelberg, Germany.
†Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstr. 108, 01307 Dresden, Germany.

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

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

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

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

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

Acknowledgements: Ministerio de Educación y Ciencia.
(1)Merrill, A. H.; Sweeley, C. C. In Biochemistry of Lipids, Lipoproteins and Membranes 1996,31, 309-339.
(2)Hannum, Y. A. Sphingolipid-Mediated Signal Transduction; R. G.Landes Company: Austin,1997.
(3) Porcelli, S. A.; Modlin, R. L. Annu. ReV. Immunol. 1999, 17, 297-329.

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

G. Sánchez-Ollé1,2, M. Egido-Gabás3, J. Casas3, A. Chabás2,4, D. Grinberg1,2 and L. Vilageliu1,2
1 Departament de Genètica, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain.
2 CIBERER, Barcelona, Spain.
3 RUBAM (Research Unit on BioActive Molècules), Departament de Química Orgànica Biològica, Institut d’Investigacions Químiques i Ambientals de Barcelona (IIQAB-CSIC),Barcelona, Spain.
4 Institut de Bioquímica Clínica, Hospital Clínic, Barcelona, Spain.

Gaucher disease (GD) is caused by the defective activity of lysosomal glucocerebrosidase (GBA) due mainly to mutations in the GBA gene. It results in the accumulation of glucosylceramide in the lysosomes of phagocytic cells, leading to several symptoms including hepatosplenomegaly, skeletal lesions and, sometimes, central nervous system involvement. Some of the GBA mutated enzymes retain the catalytic activity but impair proper folding and trafficking in the cell. It has been demonstrated that several compounds are able to act as pharmacological chaperones by increasing endoplasmic reticulum (ER) folding efficiency and lysosomal trafficking. However, this process is not general and different GBA mutations showed distinct chaperoning profiles.
Here we report the chaperone effect of two iminosugars N-(n-nonyl)deoxynojirimycin (NN- DNJ) and N-(n-butyl)deoxynojirimycin (NB-DNJ), and four aminocyclitols derivatives (C4- Ph, C8, C9 and C10) on different expressed mutant glucocerebrosidases. In particular, mutations N188S, N188S;E326K, N370S, I402T, D409H and L444P were analysed. COS-7cells were transfected with an expression vector carrying the wild-type or the mutated cDNAs. Stable transfectants were obtained with geneticin (G418) selection (500 µg/ml) and cultured either with or without NB-DNJ (5 and 10 µM), NN-DNJ (2.5 and 50µM), C4-Ph(10, 15 and 20 µM), C8 (10, 15 and 20 µM), C9 (10, 15 and 20 µM) and C10 (2.5, 5, 10, 15 and 20 µM). The ratio of enzyme activity with/without the compound was calculated. The values shown are the results of at least two experiments with 3 replicates.
NN-DNJ (5 µM) led to an increase (1.2 fold) in the activity of wild-type enzyme, as reported. Moreover, a 1.2 (5 µM) to 1.4 (2.5 µM) fold increase was shown for the N188S GBA and a 1.2 fold for the G377S enzyme. No significant changes in activity for the rest of the mutated enzymes were shown. NB-DNJ induced a 1.2-fold increase in the activity of the N188S and N188S;E326K GBAs, at 5 µM. No other significant effect was observed. C4- Ph seemed to produce a dose-dependant pattern on the wild-type and N188S GBAs. At 15µM, 1.2-fold increase for the N188S and the N188S; E326K enzyme and a 1.3-foldincreased for the wild-type GBA were detected. At 20µM, 1.3 and 1.4-fold increase was shown for the N188S and wild-type GBA respectively. No effect was showed at 10 µM. The other compounds, C8, C9 and C10, did not seem to have any chaperoning effect on the mutated enzymes at the concentrations used.
We can conclude that, in some conditions, the NN-DNJ, NB-DNJ and C4-Ph seem to have an effect as pharmacological chaperones on particular mutant enzymes. However, new conditions and new compounds should be assayed.

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

AE Alewijnse, M Jongsma, M-J Mathy, PB van Loenen, N Hajji, MC Michel and SLM Peters
Dept. Pharmacology & Pharmacotherapy, Academic Medical Center, Amsterdam, the Netherlands

In the cardiovascular system, the bioactive sphingolipid sphingosine-1- phosphate (S1P) exerts its vasoactive effects predominantly via three specific G-protein coupled receptors, named S1P1, S1P2 and S1P3. These receptors are differentially expressed in vascular smooth muscle cells (VSMCs) and endothelial cells (ECs).
We have explored the contribution of these three receptors to vascular reactivity in the rat aorta and to related signaling mechanisms in VSMCs and ECs isolated from rat aorta using some new S1P receptor ligands. We show that in intact isolated rat aortic rings, all agonists at the S1P1 and/or S1P3 receptor induced endothelium-dependent vasorelaxation in pre-contracted preparations. In rat aortic ECs, which showed a high mRNA expression of the Gi-coupled S1P1 receptor, all ligands, including the S1P1 specific agonist SEW2871, decreased the forskolin-induced cAMP accumulation. In addition, VPC23019 competitively antagonized the FTY720-P- induced inhibition of the cAMP accumulation. The tested ligands did not induce major changes in intracellular calcium in ECs.
In VSMCs, which express mRNA for all three S1P receptors, predominantly Gq-coupled S1P receptor signaling was observed via the S1P2 receptor, resulting in increases in intracellular calcium.
In conclusion, we have shown that the S1P receptor agonists tested in this study can act on both ECs and VSMCs from the rat aorta. S1P1 receptor stimulation in ECs leads to Giactivation (measured as inhibition of cAMP accumulation). Endothelial Gi activation can cause relaxation via activation of the PI3K/Akt pathway subsequently leading to the production of NO. Stimulation of mainly the S1P2 receptor in VSMCs results in calcium elevation, a response typically linked to contraction. At least for agonists devoid of S1P2activity, the net effect observed on aortic tone is vasorelaxation.

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

B. Hegen1, M. ter Braak1, V. Hardel1, T. Hla2, K.H. Jakobs1, D. Meyer zu Heringdorf1
1 Institut für Pharmakologie, Universitätsklinikum Essen, 45122 Essen, Germany;
2 Center for Vascular Biology, University of Connecticut Health Center, Farmington, Connecticut, USA

Sphingosine kinases (SphKs) catalyse the phosphorylation of sphingosine tosphingosine-1-phosphate (S1P), which acts as a ubiquitous mediator regulating cell growth and survival, migration, cytoskeleton organization and Ca2+ homoeostasis. Many plasma membrane receptors stimulate SphK activity, thereby producing S1P that can either be secreted and activate specific G protein-coupled receptors or act on so far unknown intracellular target sites. We have analysed the role of SphK1 in Ca2+ signalling and cell growth using mouse embryonic fibroblasts (MEFs) deficient in SphK1. [Ca2+]i increases induced by lysophosphatidic acid (LPA) were identical in SphK1 expressing (SPHK1+/+) and SphK1 deficient (SPHK1-/-) MEFs with respect to velocity of [Ca2+]i increase, peak [Ca2+]i values, time course of decay and concentration dependence. Extracellular S1P increased [Ca2+]i in SPHK1-/- cells maximally by only 441±50 nM (n=10) compared to 747±49 nM (n=9) in SPHK1+/+ cells, while the EC50 was not different. Similarly, [Ca2+]i increases by ATP and thrombin in SPHK1-/- cells amounted to only about 25 % and 50 % of those in control cells, respectively. Bradykinin induced [Ca2+]i increases only in SPHK1+/+ cells, while angiotensin increased [Ca2+]i only inSPHK1-/- cells. These differences were in part paralleled by differences ininositol-1,4,5-trisphosphate (IP3) production, suggesting that the receptor expression profile was different in the two cell populations. However, LPA, which induced maximum [Ca2+]i increases of a similar magnitude as bradykinin, did not significantly stimulate IP3 production in either cell type, while bradykinin elevated IP3 levels by about 9-fold in SPHK1+/+ cells. Therefore, LPA-induced [Ca2+]i increases were independent of IP3 and SphK1. [Ca2+]i increases by LPA in both SPHK1+/+ and SPHK1-/- cells were strongly inhibited by the sphingosine kinase inhibitor, 2-(p-hydroxyanilino)-4-(p-chlorophenyl)thµM, 60 min),azolesuggesting(30 that (an)other SphK(s) might be
involved. A major difference in [Ca2+]i signalling was observed with sphingosine, which mobilized Ca2+ in SPHK1+/+ cells but did not cause measurable [Ca2+]i increases inSPHK1-/- cells. These data support the hypothesis that sphingosine has to be phosphorylated for inducing Ca2+ mobilization and implicate SphK1 in this process. Finally, growth of SPHK1-/- cells was strongly delayed compared to SPHK1+/+ cells. This difference was not abrogated by treatment with extracellular S1P (10-1000 nM), supporting prior data showing that SphK1-dependent cell growth is mediated by intracellular, not extracellular S1P.

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

P. Giussani, L. Brioschi, T. Colleoni, R. Bassi, G. Tettamanti, L. Riboni and P. Viani
Dept. Medical Chemistry, Biochemistry and Biotechnology, Faculty of Medicine, University of Milan, Italy

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

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

María Salazar1, Arkaitz Carrracedo1, Íñigo J. Salanueva1, Sonia Hernández1, Cristina Blázquez1, Mar Lorente1, Ainara Egia1, Patricia Vázquez2, Sofía Torres1, Stephane García3, Juan Iovanna3, Manuel Guzmán1, Patricia Boya2 and Guillermo Velasco1
1.Department of Biochemistry and Molecular Biology I, School of Biology, Complutense University, 28040 Madrid, Spain
2.Department of Cell and Developmental Biology, Centro de Investigaciones Biológicas, CSIC, 28040 Madrid, Spain.
3.U624 INSERM, Campus de Luminy, 13288 Marseille, France.

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

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

M. ter Braak1, V. Hardel1, K.H. Jakobs1, P.P. Van Veldhoven2, D. Meyer zu Heringdorf1
1.Institut für Pharmakologie, Universitätsklinikum Essen, 45122 Essen, Germany 2.K.U.Leuven, Department Molecular Cell Biology, LIPIT, Leuven, B-3000, Belgium

Sphingosine-1-phosphate (S1P) regulates cell growth and survival, migration, cytoskeleton organization and Ca2+ homoeostasis in many cell types via activation of specific Gprotein-coupled receptors, termed S1P1-5. Intracellular S1P has furthermore been shown to mobilize stored Ca2+ independently of G-protein-coupled receptors. To further elucidate the mechanism of S1P-induced Ca2+ mobilization, we have analysed Ca2+ signalling in mouse embryonic fibroblasts (MEFs) deficient in S1P lyase (Sgpl1-/-),displaying elevated S1P levels [1]. Overall [Ca2+]i was measured in suspended cells loaded with fura-2. In Sgpl1-/- MEFs, basal [Ca2+]i was strongly elevated (~300 nM versus ~120 nM in control MEFs). Basal [Ca2+]i levels were also elevated in Ca2+-freemedium (252±38 nM compared to 123±16 nM, n=6 each) indicating that the effect was not caused by Ca2+ influx. [Ca2+]i increases induced by agonists were enhanced inlyase-deficient MEFs. [Ca2+]i increases by LPA, bradykinin, andµMS1Peach)(1 amounted to 156±17 % (n=5), 400±52 % (n=3), and 198±28
% (n=3) of those in control cells, respectively. Furthermore, [Ca2+]i increases induced by the SERCA inhibitor, thapsigargin, were augmented (302±25 nM versus 203±18 nM, n=5), and the velocity of [Ca2+]i increases induced by thapsigargin was elevated (13.1±1.7 versus 7.4±0.9 nM/s, n=5), suggesting enhanced Ca2+ storage in Sgpl1-/-cells. Interestingly, treatment with µMa sphingosine2 kinase inhibitor (30 -(p-hydroxyanilino)-4-(p-chlorophenyl)thiazole overnight) largely decreased elevated basal [Ca2+]i levels in lyase-deficient MEFs, suggesting that the observed alterations in Ca2+ signalling are caused by or linked to S1P accumulation. Analysis of fluo-4- loaded MEFs by confocal microscopy revealed enhanced fluorescence of subcellular compartments inSgpl1-/- MEFs, while the fluorescence was evenly distributed throughout the cytosol of control MEFs. Two major staining patterns were observed, a reticular staining that colocalized with ER markers, and a punctuate staining that colocalized with a lysosomal marker. Treatment with thapsigargin decreased the fluorescence within subcellular compartments, indicating that these were thapsigargin-sensitive Ca2+ stores. It is concluded that MEFs lacking a functional S1P lyase display an enhanced Ca2+ storage in endoplasmic reticulum and lysosomes that leads to augmented [Ca2+]i increases by agonists.[1] Van Veldhoven P.P. Sphingosine 1-phosphate lyase deficient mice. Chem. Phys. Lip. (2005) 136, 164-165.

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

Shih Wei Wanga, Patricia Gangoitib, Urs P. Steinbrechera, and Antonio Gómez-Muñozb.
aDepartment of Medicine, University of British Columbia, Vancouver (Canada), and
bDepartment of Biochemistry and Molecular Biology, Faculty of Science and Technology, University of the Basque Country, 48080 Bilbao (Spain).

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

In the present study we used pertussis toxin (PTX) to test whether G protein coupled receptors are involved in the regulation of A-SMase activity. PTX is a bacterial toxin that inhibits Gi activation by ADP-ribosylating the α subunit of Gi, thereby preventing the subunit from interacting with receptors. Unexpectedly, we found that PTX blocked macrophage apoptosis by itself. This effect was dose-dependent and involved inhibition ofA-SMase and the subsequent generation of ceramide. In addition the Gi activator peptide mastoparan, but not its inactive analogue mastoparan 17, stimulated A-SMase and the increase of ceramide levels in macrophage which induced apoptosis. Interestingly,pre-treatment with PTX partially overrode mastoparan- induced apoptosis.
Taken together these results suggest that Gi protein coupled receptors may be involved in the control of cell survival through regulation of A-SMase activity.
This study was supported by grants from the Heart and Stroke Foundation of British Columbia and Yukon (Canada), and BFU2006-13689/BFI from Ministerio de Educacion y Ciencia (Madrid, Spain).

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

Patricia Gangoiti , María Granado , and Antonio Gómez-Muñoz .
Department of Biochemistry and Molecular Biology, Faculty of Science and Technology, University of the Basque Country, 48080 Bilbao, Spain.

Our previous work on fibroblasts and macrophages showed that ceramide 1- phosphate (C1P) is mitogenic and antipoptotic. Recently we demonstrated that a major mechanism whereby C1P blocks apoptosis involves inhibition of acid sphingomyelinase and the subsequent generation of ceramides. However, the mechanisms or signaling pathways that are implicated in the mitogenic effect of C1P have not been thoroughly studied.
In a recent study we demonstrated that C1P stimulates DNA synthesis and cell division in bone marrow-derived macrophages. This effect was dependent upon activation of PI3-K/PKB (Akt) and ERK 1-2 pathways, but did not require the activity of phospholipase D, or adenylyl cyclase. We now observed that C1P stimulated a rapid phosphorylation of c-Jun terminal kinase (JNK) in the macrophages and that inhibition of this pathway by SP600125 blocked macrophage proliferation. In addition, treatment with SB202190, which is a selective inhibitor of p38, did not affect C1P-induced DNA synthesis in these cells. A classical transcriptional factor involved in the regulation of cell proliferation is NF-κB. In a previous report, we demonstrated its implication in macrophage survival. In this work, we show that C1P stimulates the DNA binding activity of NF-κB in macrophages, and that selective inhibitors of this transcriptional factor, such as CAPE or SC-514 blocked the stimulatory effect of C1P on cell proliferation. In addition, we found that the inhibitors of PI3-K, MEK and JNK, (LY290042, PD098059 and SP600125 respectively), completely blocked the DNA binding activity of NF-κB. Lastly, another key finding of the present work is that monocyte-colony stimulating factor (MCSF), which promotes macrophage survival and proliferation, caused an increase in the intracellular levels of C1P.
It can be concluded that the mechanism whereby C1P stimulates cell proliferation in bone marrow-derived macrophages involves the activation of JNK but not p38.
This study was supported by grants BFU2006-13689/BFI from “Ministerio de Educación y Ciencia” (Madrid, Spain) and 9/UPV 00042.310-15852/2004 from “Universidad del País Vasco” (UPV/EHU) (Bilbao, Basque Country).

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

María Granado, Patricia Gangoiti, and Antonio Gómez-Muñoz
Department of Biochemistry and Molecular Biology, Faculty of Science and Technology, University of the Basque Country, 48080 Bilbao, Spain.

Ceramide 1-phosphate (C1P) is a bioactive sphingolipid capable of regulating important pathophysiological functions including cell proliferation, apoptosis, phagocytosis, and inflammation. Chalfant and co-workers first showed that C1P, which was delivered to cells in a solution of ethanol:dodecane, stimulated the calcium dependent cytosolic form of phospholipase A2 (cPLA2α) in lung cancer A549 cells. Using rat alveolar NR8383 macrophages and C1P dispersed in water, we demonstrate in this work that the stimulation of arachidonic acid release by C1P is independent of the solvent system used. Also, this effect is specific as other related phospholipids such as ceramide, or phosphatidic acid (PA) failed to stimulate secretion of this fatty acid. UsingN-{(2S,4R)-4-(Biphenyl-2-ylmethyl-isobutyl-amino)-1-[2-(2,4-difluorobenzoyl)-benzoyl]-pyrrolidin-2-ylmethyl}-3-[4-(2,4-dioxothiazolidin-5-ylidenemethyl)-phenyl]acrylamide, HCl , a selective inhibitor of cPLA2-α, we show that this enzyme is directly implicated in the stimulation of arachidonic acid release by C1P in alveolar macrophages, which is in agreement with work mentioned above.
Further investigation into the mechanism whereby C1P increases the release of arachidonic acid revealed that selective inhibitors of phosphatidylinositol 3-kinase (PI3-K) blockedC1P-stimulated arachidonic acid release in the macrophages, suggesting an involvement of this enzyme in the pro-inflammatory effect of C1P. The latter also induced phosphorylation of protein kinase B (Akt), which is downstream of PI3-K. However, although C1P caused phosphorylation of the mitogen activated protein kinases (MAPK) ERK1/2 (extracellularly regulated kinases 1 and 2), specific inhibitors of this pathway did not affect the stimulation of arachidonic acid by C1P.
It can be concluded that stimulation of arachidonic acid release by C1P in alveolar macrophages can be achieved by direct stimulation of cPLA2 or through stimulation of thePI3-K/PKB (Akt) pathway, but not ERK1/2 or p38.
This study was supported by grants numbers 040732 from “Fundació la Marató de TV3”, Barcelona, Spain, 9/UPV 00042.310-15852/2004, from “Universidad del Pais Vasco (UPV/EHU)” Bilbao, Spain, and S-PE06UN 13 from “Departamento de Industria, Comercio y Turismo del Gobierno Vasco, Gasteiz-Vitoria, Spain.

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

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

The gaseous messenger nitric oxide plays an important role in a variety of biological functions such as cell differentiation, proliferation and death. Physiological concentrations of NO inhibit apoptosis triggered by a variety of apoptogens, including those activating death receptors of the TNF- (TNFRI)/ CD95 superfamily. Recent evidences indicate that NO is able to regulate ceramide levels and in U937 monocyte-derived cells it has been shown that NO inhibits the apoptotic responses induced by CD95 and TNF-RI by reducing the ability of these receptors to generate ceramide. One of the enzymes responsible for ceramide generation is the lysosomal phosphodiesterase Acid Sphingomyelinase. In recent papers we demonstrated that NO is able to abolish activation of A-SMase triggered by the apoptogenic receptors and lipopopolysaccharide (LPS) and that the anti-apoptotic function of NO is mediated through activation of soluble guanylyl cyclase and cGMP-dependentprotein kinase.
We therefore decided to investigate the mechanisms of ASMase inhibition by NO in U373 glioma cell line. Recent experiments have demonstrated that activation of this enzyme involves its trafficking from intracellular compartments to the plasma membrane by processes of exo-endocytosis. We observed that CD95 triggers A-SMase activation in atime-dependent and transient way and induces A-SMase translocation to the plasma membrane with the same trend of the activation. The same sets of experiments carried out in the presence of a NO donor demonstrated that NO inhibits A-SMase activity through the blockade of enzyme translocation. Immunofluorescence studies carried out under resting condition, i.e. prior to CD95 stimulation, in order to understand the A-SMasetranslocation further, showed that A-SMase co-localised with the lysosomal marker cathepsin D and with the SNARE protein syntaxin 4 suggesting this protein as responsible of A-SMase trafficking and as a target of the inhibitory action of NO. To this end we performed immunofluorescence experiments in the presence/absence of DETA-NO/8Br-cGMP in which we found that the two proteins no longer colocalized.
In addition we observed a very strong reduction of the levels of syntaxin 4 as measured by western blotting experiments carried out in the same conditions. Experiments intended to investigate the mechanism by which NO regulates the level of syntaxin 4 are now in progress; preliminary evidences indicate that NO promotes degradation of this protein. Evidence accumulated over the last few years indicates that the cross talk of A- SMase with NO plays prominent roles during key patho- physiological processes such as inflammation, proliferation, death and differentiation. Therefore, the identification of the mechanisms of this tightly regulated event and of its molecular intermediates might be relevant for understanding these important, as yet not fully defined, processes.

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

Ana Fernández, Anna Colell, Fran Caballero, Carmen García-Ruiz, and José Carlos,Fernández-Checa.
Liver Unit, Hospital Clínic i Provincial. CIBEREHD- IDIBAPS, IIBB-CSIC, Barcelona, Spain.

Tumor necrosis factor (TNF) is a proinflammatory cytokine that induces different cellular responses including inflammation, proliferation and cell death. TNF binds to two different plasma membrane receptors, TNF receptor 1 (TNFR1) and receptor 2 (TNFR2). Evidence from studies using anti-TNF therapy and TNFR1 knockout mice indicated an important role for TNF in the pathogenesis of alcoholic liver disease (ALD). Recent studies in experimental models of alcoholic (ASH) and non-alcoholic steatohepatitis (NASH) questioned the role of TNFR1 in disease progression and hepatic steatosis. Moreover, acidic sphingomyelinase (ASMase) has been shown to play a key role in the hepatocellular death induced by TNF. However, since in the absence of TNFR1 TNF can signal through TNFR2, and because the role of the ablation of both TNFR1 and TNFR2 or ASMase in ALD have not been previously examined, the aim of this study was to examine the contribution of TNFR1/TNFR2 and ASMase in ALD. Methods: 8 weeks-old male C57BL/6 mice deficient in both TNFR1 and TNFR2 (DKO) and ASMase-/- mice were used. Animals were fed the Lieber-DeCarli diet for up to 4-6 weeks. Animals received a single dose of LPS (5mg/kg) 24 hours before sacrifice. Liver damage was assessed by histochemical hematoxilin/eosin staining and serum transaminase levels. Hepatic steatosis and lipid infiltration were examined by oil red staining and biochemical analyses. Hepatic inflammation was monitored by myeloperoxidase (MPO) staining.
Results: the hepatic inflammation and necrosis observed in wild type ethanol-fed mice were attenuated significantly in DKO knockout mice. In vivo LPS injection in ethanol wild animals increased AST serum levels 8 times over pair-fed control mice (1440±210 U/dL vs. 135±14 U/dL) respectively. This increase was significantly lower in mice lacking both TNF receptors (ethanol DKO +LPS vs. ethanol wild type + LPS: 235±45 U/dL vs.1320±157U/dL). The increase in hepatic triglycerides, cholesterol and free fatty acids induced by ethanol feeding was also reduced (72±9, 68±11, 59±8%, respectively) in the DKO mice fed ethanol. Moreover, inflammatory cell foci reflected by MPO staining showed a marked reduction in DKO-fed ethanol mice vs wild-type ethanol fed mice following in vivo LPS challenge. Interestingly, ASMase knockout mice exhibited hepatic steatosis, although alcohol feeding did not further increase the levels of TG, FFA and cholesterol. However, unlike DKO, ASMase-/- mice are sensitive to concanavalin-induced liver injury, indicating a functional role for TNFR2 in and a link between TNFR1 and ASMase.
Conclusion: These results support the hypothesis that TNF plays an important role in inflammation and necrosis in alcohol-induced liver injury and that the targeting of TNF or its downstream mediator ASMase may be therapeutically useful in this disease.

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

Albert Morales1, Josep M. Lluis1, Scott Welford2, Amato Giaccia2 and José C. Fernandez- Checa1
1Liver Unit, Instituto Malalties Digestives, Hospital Clinic I Provincial, IDIBAPS, CSIC, UB, Barcelona, Spain.
2Division of Radiation and Cancer Biology, Stanford University, Stanford, California, USA.

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

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

Clara Ricci, MS(1,2), Francesco Onida, MD(2,3,4), Federica Servida, MS (2), Kati Todoerti, MS(3), Antonino Neri, MD(3,4), Giorgio Lambertenghi Deliliers, MD(2,3,4) and Riccardo Ghidoni, PhD(1,4)
1.Lab. of Biochemistry and Molecular Biology, San Paolo University Hosp., University of Milan, Milan, Italy, 20142;
2.Fondazione Matarelli, Milan, Italy, 20122;
3.Hematology Dept., Fondazione IRCCS Osp. Maggiore Policlinico, Mangiagalli e Regina Elena, Milan, Italy, 20122
4.University of Milan, Milan, Italy, 20122.

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

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

Erika Kovacs and Karoly Liliom
Affiliation and address: Institute of Enzymology, Hungarian Academy of Sciences, P.O.Box 7, Budapest, H-1518 Hungary
e-mail: liliom@enzim.hu

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

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

Sassoli C. 2 Martinesi M. 1, Squecco R. 3, Bini F.1, Zecchi-Orlandini S. 2, Francini F. 3, Formigli L. 2, and Meacci.E1.
Departments of 1Biochemical Sciences, 2Anatomy, Histology and Forensic Medicine, 3Physiological Sciences, University of Florence and -Interuniversity Institute of Myology (IIM), Italy.

Sphingosine 1-phosphate (S1P), a lipid mediator originated by sphingomyelin degradation, is able to regulate a wide variety of important cellular functions (1). Although, S1P can act as pro-differentiating agent in C2C12 myoblasts, its downstream effectors are poorly known. Recently, we have demonstrated that the induction of myogenesis by S1P required the activation of several signaling pathways, including the up-regulation of Cx43 expression and its interaction to cytoskeleton (2).Mechanosensitive channels (stretch-activatedchannel, SACs) are present in many cell types, and partecipate to the mechano- transduction, converting membrane stretch into electrical cationic current and biochemical signalling (3). Increasing evidence support the important role of SACs in cell homeostasis, cell differentiation and in the pathogenesis of several muscle diseases, such as muscular dystrophy, muscle hypertrophy and cardiac arrhythmias. Despite these observations, the involvement of canonical transient receptor potential 1 (TRPC1), identified as a critical componenet of SACs (3), in S1P-induced signalling remains to be elucidated. To this purpose, in the present study, we examined the expression and localization of TRCP1 in C2C12 myoblasts and its role in S1P-induced Cx43 expression, gap junction functionality and myogenesis.
Interestingly, we found that TRCP1 appears to be responsible for the majority of stretch- activated channel (SAC) activity induced by S1P, as demonstrated by the reduced SAC activity in cells in which TRCP1 protein is silenced by siRNA. TRCP1 preferentially co- localizes with Cx43 in lipid rafts and directly interact with cytoskeleton actin. The treatment with dihydrocytochalasin B, a selective actin disrupter agent, with gadolinium chloride, a specific pharmacological inhibitor of SACs, and the transfection of C2C12 myoblasts with siRNA-TRCP1 significantly reduces the up-regulation of Cx43 as well as myogenic marker expression promoted by S1P.
Overall, these findings provide novel information regarding the mechanisms by which S1P affects muscle cell biology, including in the list of the downstream effectors of the bioactive lipid, TRCP1. Importantly, these results suggest that, in myoblasts stimulated with S1P, cytoskeleton remodeling and the following activation of cation currents through TRPC1/SACs are essential steps in the up-regulation of Cx43 expression, event that is physiologically required for myogenesis in vivo (4). In addition, the discovery of the functional interaction between sphingolipid signalling and TRP channels will inevitably reveals a new possible target for therapy in diseases, in which alterations of cation concentration appears to be crucially involved in the activation of proteolytic enzymes and cell degeneration, such as muscle distrophy.
Grants from university of Florence, Ente CRPP and Fondazione Banche di Pistoia e Vignole

Sphingomyelin metabolism of thyroid cells changes in the space.

Albi E1, Bartoccini E1, Marini F1, Cascianelli G1, Bernardini I1 , Viola-Magni M1, Perrella G2.
1Dept. Clinical and Experimental Medicine, University of Perugia;
2Department of Experimental and Clinical Pathology and Medicine, University of Udine.

The radiation absorbed by astronauts during interplanetary flights is mainly due to cosmic rays that can cause thyroid disorders through the activation of intracellular signals. Since the ionizing radiation exposure induces the release of ceramide and diacylglycerol as second messengers for proapoptotic and antiapoptotic signals respectively (1), the aim of the present work was to evaluate the effect of the cosmic enviroment on lipid fraction of the FRTL-5 cells. This cell line permanently expresses in vitro most of the in vivotissue-specific thyroid characteristics, such as thyroglobulin synthesis and secretion, iodide active transport, peroxidase production and thyrotropin sensitivity. FRTL-5 cells are induced to proliferate only when they are cultured in the presence of TSH (TSH+) whereas in the THS-free medium the cells remain in a quiescent state for long time (TSH-, 2). The study has been performed on TSH+ and TSH- FRTL5 cell during the 10-days Italian Soyuz Mission(ISM) “Eneide” (April 15 to April 25) on international Space Station. The activity of neutral– sphingomyelinas (N-SMase) and sphingomyelin-synthase (SM-synthase) were evaluated in nuclei-free lysates (NFL) and purified nuclei. Moreover the same experiments have been made in the samples maintained in colture at Udine and Baikonur. The results show that in the space the N-SMase activity increases and SM- synthase activity decreases and it occurs more in the purified nuclei that in the NFL. The variations obtained in the TSH- FRTL5 cells are similar whereas those obtained in the TSH+ FRTL5 cells are lower of those previously reported for the same cells treated with UV rays (3). These data can be correlated with the reduced proliferative activity of TSH+ FRTL5 cells.