• Sphingolipids as regulators of cell death: facts and uncertainties.

    T. Levade , N. Andrieu-Abadie, E. Bonhoure, S. Carpentier, O. Cuvillier, V. Garcia, V. Gouazé, S. Malagarie, I. Popa # , J. Portoukalian # , B. Ségui, C. Tardy, R.J. Veldman *

    INSERM U.466, Lab. Biochimie, CHU Rangueil, Toulouse, France ; # INSERM U.346, Lyon, France ; * (present address) The Netherlands Cancer Institute, Amsterdam

    Programmed cell death is a regulated process that is required for normal development and tissue homeostasis. The major biochemical pathways involved in apoptosis include the activation of caspases and mitochondrial events. Since the pioneering work of Obeid and Hannun on ceramide and TNF-induced apoptosis one decade ago, a wealth of reports have been published supporting a role for sphingolipids in the initiation and/or regulation of the apoptotic program [1]. Different sphingolipids have been implicated, including ceramide, sphingosine, sphingosine-1-phosphate as well as some glycolipids, which could trigger or modulate either programmed cell death or cell survival [2]. Despite intensive search, the molecular mechanisms that underlie the production of action of (bioactive) sphingolipids, and more generally the functions of sphingolipids in cell death/growth regulation remain to be clarified. Several instances of still unsolved issues will be presented. These include i) the contribution of acid and neutral sphingomyelinases to the generation of ceramide as a potential proapoptotic second messenger, both in cultured cell systems and in animals, ii) the subcellular location of this bioactive ceramide, and especially its localization to membrane microdomains, iii) the nature of ceramide targets (for example, the role of cathepsin D as a ceramide-controlled protease in apoptosis will be discussed), iv) the role of enzymatic conversion of ceramide to glycolipids in cell survival/resistance, and v) the role of ganglioside production in apoptosis signalling. These various issues will be critically discussed in the light of recent observations .


    1 - Hannun YA, Luberto C. (2000) Trends Cell Biol . 10 : 73-80

    2 - Levade T, Hannun YA, Spiegel S. (2002) Biochim. Biophys. Acta 1585 : 51-226

  • Design and synthesis of ceramide analogues as a new strategy for anticancer drug development.

    Marco Macchia
    Dipartimento di Scienze Farmaceutiche, Università di Pisa

    Sphingomyelins play a primary role in the regulation of cellular responses to exogenous stimuli as well as cell growth, differentiation, transformation, and cell-cell contact. In particular, membrane sphingomyelin is hydrolyzed in response to extracellular stimuli, such as tumour necrosis factor-alpha (TNF- a ) and interleukin 1, generating the putative second messenger ceramide. Ceramide, in turn, is thought to propagate the signal into the cell interior by the activation of phosphatases and mitogen-activated protein kinases. The mode of action of ceramide and the regulation of its production have recently attracted great attention due to the emerging role of this lipid messenger as an intracellular effector molecule in apoptosis. On the other hand, growth factors, such as platelet-derived growth factor, trigger further hydrolysis of ceramide to sphingosine and activate sphingosine kinase to form sphingosine 1-phosphate with promotion of cell growth. For these reasons, the dynamic balance between levels of ceramide and sphingosine 1-phosphate, is an important factor that determines whether a cell survives or dies .

    In view of the emerging role of ceramide as an intracellular effector molecule, possible therapeutic targets of new pharmacological interventions directed at regulating abnormal tumour growth include the development of analogues that mimic the effects of endogenous ceramide, are metabolically stable to prevent further metabolism to sphingosine, and possess an inhibitory activity on ceramidase to prevent generation of sphingosine from endogenous sources of ceramide: on this basis, a series of conformationally restrained and metabolically stable ceramide analogues have been developed in our laboratory; these compounds proved to induce cytotoxicity and apoptosis in human cancer cells.

  • Sphingomyelin metabolism in response to the effect of the UV cosmic (stratospheric) radiation.

    Perrella G. °, Cataldi S.*, Albi E.*, Toller M., Meli A., Del Terra E., Casani S.,

    °Department of Experimental and Clinical Pathology and Medicine University of Udine

    *Department of Biochemical Sciences and Molecular Biotechnology, Physiopathology, Perugia

    Classic radiation dosimetry, performed with traditional physical methods, does not evaluate the kinetics of radiation absorbance or the biological response(s) to radiation. Both are key aspects in biological systems and cells, which possess elaborate radiation response mechanisms (DNA repair, cellular components turnover, apoptosis, etc.).

    Biological dosimetry on eukaryotic cell cultures provides nowadays elaborate experimental targets very close to animal and human organs or organisms, which can be used as radiation target systems.

    In this study, the available technologies allow us to use FRTL5 cells to evaluatey the biological radiation effects under the following different physiological conditions: 1) in proliferative state, in Thyroid Stimulating Hormone (TSH) additioned growth medium; 2) in non proliferative state, in TSH-deprived culture medium. For both conditions the FRTL5 cells have been exposed to ionizing radiation (11,23 m Sv/h) by mean of stratospheric balloons or UVC (20 J/m 2 ). Shielded cells have been used as non irradiated control in the stratospheric balloons.

    The FRTL5 cells are a strain of normal and differentiated follicular thyroid cells, obtained, characterized and also patented by us some years ago, presently an international standard for the clinical or autoimmune thyroid diseases. The FRTL5 cell line permanently express in vitro most of the in vivo tissue-specific thyroid characteristics, such as Thyroglobulin synthesis and secretion, Iodide active transport, Peroxidase production and Thyrotropin (TSH) sensitivity, etc. Other cell types (in particular of human origins) now under test in our laboratory may possibly be used in addition.

    We studied sphingomyelin metabolism modifications in response to the effect of UV and stratospheric radiation related to the apoptosis process.

    The results: exposure of FRTL5 cell lines to U.V. radiation appears to inhibit neutral sphingomyelinase activity and to increase sphingomyelin synthase activity in the nuclear cellular compartment. This effect is comparable to the enzymatic activities registered during the G2/M cell cycle phase and apoptosis of hepatocytes.

    Both inhibition of neutral sphingomyelinase and enhancing of sphingomyelin synthase activities by high altitude (stratospheric) radiations occur in all FRTL5 samples, including controls.

    Previous studies have already demonstrated that neutral sphingomyelinase stimulates cell proliferation; thus, it might be assumed that the reduced activity of this enzyme by radiations could determine a decreased cell growth rate. Moreover, the radiation-induced increase of sphingomyelin synthase activity could indicate that FRTL5 cells stop their cell cycle at G2/M, thus avoiding to proceed to mitosis. In other words, exposure of cells to high altitude radiations could determine alteration of metabolic pathways, so that cells are with the time be induced to enter the apoptosis process.

    The increased sphingomyelin intracellular concentration due to the radiation-induced enhanced enzymatic activity could modify membrane fluidity, which will be reduced, and thus interfering with nucleus-cytoplasm exchange processes. Moreover, as G2/M nuclear transition step should slow down, mitotic fuse formation could be affected.

  • Serum deprivation activates the sphingomyelin metabolism in normal neuronal nuclei.

    Elisabetta Albi 1 , Samuela Cataldi 1 , Laura Colombaioni 2 , Francesca Mazzoni 3 , Mariapia Viola Magni 1 , Vladimir Voccoli 3 , and Mercedes Garcia-Gil 3 .

    • Department of Biochemical Sciences and Molecular Biotechnology, Physiopathology, Policlinico Monteluce, 06100, Perugia; 2) Institute of Neuroscience CNR, Pisa; 3) Dpt. of Physiology and Biochemistry, University of Pisa.

    Sphingolipids are known as molecules involved in cell proliferation, differentiation and apoptosis processes. Recent evidences suggest that the subcellular localization of ceramide may have a critical role in determining the cellular response. Since enzymes involved in ceramide metabolism such as sphingomyelinase, sphingomyelin synthase, sphingosine kinase and ceramidase have been found in the nucleus (Albi and Viola Magni, 1997, 1999; Tsugane et al., 1999, Jaffrezou et al., 2001; Kleuser et al., 2001), we have studied whether nuclear sphingomyelin metabolism varies during apoptosis induced by serum deprivation in the embryonic hippocampal cell line (HN9.10e), by measuring the activites of sphingomyelinase and sphingomyelin-synthase. We have previously shown, during the apoptosis induced by serum deprivation, an increase of cell ceramide levels, a translocation of Bax, a release of cytochrome c, and a maintenance of mitochondrial functionality followed by calcium deregulation and caspase-3 activation. We report now that nuclear sphingomyelinase increases biphasically, with an earlier peak one hour after deprivation, and a second at 15 h until 48h after deprivation. In contrast, nuclear sphingomyelin-synthase decreases after serum deprivation with a minimun after 1 hour. These changes in nuclear metabolism of sphingomyelin occur before the nuclear translocation of the NFkB transcription factor, and the nuclear raise of Ca ++ . Our results suggest that the formation of ceramide through nuclear sphingomyelinase activation may have a role in serum deprivation-induced apoptosis.


    Albi E. and Viola-Magni M. (1997) Biochem. Biophys. Res. Commun. 236 , 29-33.

    Albi E. and Viola-Magni M. (1999) FEBS Lett. 460 , 369-372.

    Jaffrezou J.P., Bruno A.P., Moisand A., Levade T. and Laurent G. (2001) FASEB J. 15 , 123-133.

    Kleuser B., Maceyka M., Milstien S., and Spiegel S. (2001) FEBS Lett. 503 , 85-90.

    Tsugane K., Tamiya-Koizumi K., Nagino M., Nimura Y. and Yoshida S. (1999) J. Hepatol. 31 , 8-17.

  • Lysosomes, cell death and drug resistance in cancer: when sphingolipids and cathepsins get together.

    Roberta Castino, Marzia Mirabelli, Giuseppina Nicotra, Anna Aspesi, Marina Démoz, and Ciro Isidoro

    Università del Piemonte Orientale “A. Avogadro”, Dipartimento di Scienze Mediche di Novara
    Altered regulation of cell survival and cell death is considered an important factor contributing to tumour development and progression, as well as to the resistance to anti-cancer therapy. Recent data emphasise the role of lysosomes in the induction of cell death, not only in necrosis and autophagic type cell death but also in apoptosis. At least two classes of lysosomal hydrolases contribute to the regulation of the cellular response toward cytotoxic cytokines and antiblastic drugs: (1) those involved in sphingolipid metabolism, such as acid sphingomyelinase and acid ceramidase, and (2) acid endopeptidases such as Cathepsins B and D.

    A central molecule that mediates the death signal triggered by cytotoxic cytokines and chemotherapeutic drugs is ceramide. Hyper-expression of acid ceramidase has been shown to confer resistance to cytotoxic treatments with TNF a , whereas pharmacological inhibition of acid ceramidase renders the cells highly susceptible to the cytokine. In accord with the above observation, the induction of apoptosis in cancer cells by ionising radiation, cytotoxic cytokines and various chemotherapeutic drugs has been shown to require the activation of Acid Sphingomyelinase, the enzyme that leads to ceramide production within lysosomes. Finally, sphingosine, that is formed by hydrolysis of ceramide by acid ceramidase, exerts a cytotoxic effect via destabilisation of the lysosomal membrane and consequent leakage of lysosomal hydrolases. Another class of lysosomal hydrolases that has been shown to play a role in drug resistance comprises the proteases Cathepsins B and D. Both this acid endopeptidases have been shown to mediate the death signal triggered by cytotoxic cytokines and various chemotherapeutic drugs. Of note, the expression of Cathepsin D is regulated also by the levels of p53. In addition, the maturation and activation of pro-cathepsin D is catalysed by lysosomal ceramide.

    These data suggest that defective functioning of the lysosomal metabolism (of certain lipids and proteins) in cancer cells may constitute a novel mechanism of chemo-resistance to antineoplastic drugs as well as of immune-resistance against cytotoxic cytokines.

    We will review the literature on this subject and will present some original data recently obtained in our laboratory.

    Funded by MIUR-Cofin2001, AIRC, Regione Piemonte and CNR-target project on Biotechnology

  • The impact of ceramide on lysosomal targeting: a study with wild-type and glycosylation knock-out mutated cathepsin d-green fluorescent protein chimeras.

    Camilo Gianinazzi , Nicol Trincheri, Carlo Follo, Anna Aspesi, Marina Démoz, Roberta Castino and Ciro Isidoro

    Università del Piemonte Orientale “A. Avogadro”, Dipartimento di Scienze Mediche di Novara

    Ceramide is a sphingosine-based lipid naturally occurring within the cell that acts as a pleiotropic second messenger in a number of cellular functions ranging from proliferation and differentiation to senescence and apoptosis. Onset of cell death is associated with morphological changes that in an ordered, as yet not known, sequence involve the various organelles and the cytoskeleton. This implies that location of organelles has to change in order to provide a topological organisation of membranes that favours the biochemical cross-talk among the organelles involved in the cell death process. Also, molecules must exit from one compartment and enter to another in order to propagate the death signal. Leakage of molecules from an organelle imply (a transient) loss of stability of membranes. The aim of the present work was to assess the impact of ceramide on protein transport and membrane traffic, with a particular focus on lysosomes and lysosomal targeting of cathepsin D, a lysosomal protease involved in the cell death process. To this end we have followed by confocal immunofluorescence the localisation of the fusion protein Cathepsin D-Green Fluorescent Protein (CD-GFP) in cells treated with N-acetyl sphingosine (NAS), a synthetic analogue of ceramide. Cathepsin D (CD) is synthesised in the rough endoplasmic reticulum as a di-glycosylated precursor that is transported to the Golgi apparatus and eventually to the endosomal-lysosomal compartments via Mannose-6-Phosphate (M6P)-dependent and M6P-independent pathways. The latter pathway can be studied by expressing mutagenized cDNA coding for CD in which either one or both N-glycosylation sites have been knocked-out. We and other have already shown that ceramide influences the transport and proteolytic maturation of Cathepsins D. In the present work we describe the effects of NAS on the M6P-dependent and M6P-independent targeting and subcellular localisation of CD in cells expressing the wild-type or the N-Glycosylation mutated CD-GFP constructs.

    Funded by MIUR-Cofin2001 and CNR-target project on Biotechnology

  • Role of ceramide in the control of autophagy in colon cancer cells.

    Francesca Scarlatti , Chantal Bauvy*, Giusy Sala, Daniel Meley*, Patrice Codogno*, Riccardo Ghidoni

    Lab. Biochemistry and Molecular Biology, San Paolo Hospital medical School, University of Milan; * INSERM U504, Villejuif, France

    Autophagy is a degradative pathway that culminates in the lysosomal compartment after the formation of a cytoplasmic vacuole engulfing macromolecules and organelles. The autophagic mechanism triggers a non-apoptotic programmed cell death which inhibits the tumor development. Consequently, the autophagic pathway is an important player in the elimination of cancer cells

    A regulatory signaling mechanism of autophagy involves the class I PI3K/PKB pathway, that is known to inhibit autophagy in human cancer cells.

    Our study aims to investigate the relationship between the autophagic signaling pathway and the increase of endogenous ceramide levels.

    The literature suggests ceramide as an important mediator of apoptosis. However, very little is known on its ability to interfere with the autophagic cellular death. We tested this possibility in HT-29 colorectal cancer cells, that are known to undergo autophagy, either spontaneously and after serum deprival stimulus. In this cell line, C2-ceramide induces apoptosis and the morphological signs, along with DNA fragmentation, occur after 24-48 h of incubation, while PARP occurs not earlier than 8 h from incubation.

    We found that a 3-hours treatment of HT-29 cells with C2-ceramide induces autophagy in a dose-dependend manner, as suggested by increase in protein degradation. Moreover, we found that HT-29 cells treated with C2-ceramide reduce expression levels of protein kinase B/Akt by activating Serine 473 dephosphorilylation. C2-ceramide-induced dephosphorylation promotes autophagy by reverting PI3kinase/PKB inhibitory pathway.

    In addition, we found that in HT-29 cells the treatment with C2-ceramide induces a dramatic increase (up to 8-fold) of the endogenous long-chain ceramide, which is inhibited by FB 1 , a ceramide synthase inhibitor, but not by myriocin, an SPT inhibitor. Consequently our results show that in HT-29 cancer cells, after treatment with C2-ceramide, endogenous ceramide controls autophagy by interfering with the class I PI3K signaling pathway.

  • The sphingomyelinase activity regulates the cholesterol level in the nucleus.

    Albi E ., Cataldi S., Lazzarini R, Rossi G., Tringali A.R., Tringali S., Viola Magni,MP.

    Department of Biochemical Sciences and Molecular Biotechnology, Physiopathology, Perugia, Italy.

    Cholesterol (CHO) exchange between membranes containing sphingomyelin (SM) occurs at a much slower rate than between membranes lacking SM (1). Numerous studies have shown that the strong interaction between unesterified CHO and SM arises from the van der Waals interactions between CHO and the saturated lipid acyl chains, whereas the hydroxy groups of SM are less important (2, 3). The cellular concentrations of SM and CHO are positively correlated in several pathological and experimental conditions (4). In atherosclerosis, abnormal deposition of CHO, cholesteryl esters, and SM has been demonstrated (5 ); SM and CHO accumulate in the intima as a function of ageing (6). Co-localization of SM and CHO has been demonstrated using bacterial sphingomyelinase; hydrolysis of SM results in a movement of CHO to the interior of the cell where it is esterified and reduces sterol synthesis, repressing 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase activity (7). On the other hand, the 25-hydroxycholesterol (25 OH-CHO), an inhibitor of HMG-CoA reductase, reduces the CHO synthesis and stimulates SM synthesis (4). The CHO-SM relation suggests a mechanism for sterol distribution in which sterol levels are determined by the intrinsic characteristics of each membrane (8). The aim of this work is to establish the relationship between CHO and SM in a intranuclear complex formed by a small amount of DNA, proteins, double-strand RNA, SM and phosphatidylcholine (PC). The results show that in the in intranuclear complex, the amount of CHO is similar to that of SM, and it increases after digestion with exogenous sphingomyelinase. After this treatment the double strand RNA which is RNase-resistent becames RNAse-sensitive. Moreover the cholesterol inhibits the chromatin sphingomyelin-synthase activity. It may be concluded that in intranuclear complex the CHO play a role on SM metabolism modifying the bridge between two RNA strands and consequently regulating transcription.

    • Kan CC, Ruan Z, and Bittman R. Biochemistry 1991; 30: 7759-7766; 2) Lund-Katz S, Laboda HM, McLean LR, and Phillipis MC. Biochemistry 1988; 27: 3416-3423; 3) McIntosh TJ, Simon SA, Needham D, and Huang C. Biochemistry 1992; 31: 2020-2024; 4) Ridgway ND. J Lipid Res 1995; 36: 1345-1358; 5) Smith EB, and Cantab BA. Lancet 1960; 1: 799-803; 6) Eisenberg S, Stein Y, and Stein O. Biochim Biophys Acta 1969; 176: 557-569; 7) Slotte JP, and Bierman EL. Biochem J 1988; 250: 653-658; 8) Wattenberg BW, and Silbert DF. J Biol Chem 1983; 258: 2284-2289.


  • Possible mechanism of inhibition of human PBL proliferation by DMS an inhibitor of sphingosine kinase.

    Lucia Cavallini, Nada Sonda, Francesca Pistollato and Adolfo Alexandre

    Dipartimento di Chimica Biologica-Università di Padova
    The activation of T lymphocytes is the central event in the development of immunity. IL-2 is produced early upon stimulation, together with the high affinity IL-2 receptor, (CD25). They mediate proliferation and differentiation responses, as well as apoptosis at the end of the process of clonal expansion. We performed experiments on the role of the sphingolipid intermediates on lymphocyte proliferation induced by phytohemoagglutinin (PHA) by using some inhibitors of the sphingolipid pathway.

    Previous studies in our lab showed the inhibition of proliferation of activated T cell by by fumonisin B1, an inhibitor of ceramide synthase and perhaps of a neutral sphingomyelinase, and by N,N-dimethylsphingosine (DMS) and threo-dihydrosphingosine (DHS), both inhibitors of Sphingosine kinase (SPHK). Alltogether these experiments suggest the involvement of some sphingolipid compound as obligatory intermediate of lymphocyte proliferation. Among the inhibitors tested DMS is the most efficient, inducing an almost complete inhibition at 2-3 uM, followed by DHS at 5-10 mM. IL2 expression is slightly decreased by DMS and more affected by DHS. The expression of CD25 expression is strongly inhibited by DMS, but not by DHS. The DMS inhibition (75-100%) is also observed in a model of IL-2-dependent proliferation where PBL are pre-stimulated with PHA for 74 hours to induce a high expression of CD25 , and after washing proliferation is induced by IL2. In this system DHS is again a poor inhibitor. In all cases the effects of DMS are evidenced by inhibition of the increase of cyclin D2 and D3, pRb phosphorylation and an almost complete absence of expression of cyclin A and cyclin E; the data are consistent with a block in the G1-S transition of the cell cycle. SPHK activity measured in extracts of PHA and IL2 restimulated PBL showed an increase. The increment of the activity was completely abrogated by PBL traetment with DMS but not with DHS at similar concentration, suggesting a positive correlation between the enzyme activity and proliferation.

    The effect of inhibition of SPHK could be due to an increase of ceramide. Ceramide has been reported as involved in the CD28 costimulation of lymphocytes, but also in numerous different effects (e.g. activation of PP-1 and -2 phosphatases, activation of caspases). Direct measurements of ceramide in PHA stimulated and IL-2 restimulated PBL revealed a slight decrease in the ceramide content which was marginally and not significant prevented by DMS. Sphingosine and SPP levels in human PBL are so low to be nearly undetectable. Besides they did not show extensive variation in the activated and DMS inhibited cells. So a significant mass variation of the different metabolites seems not correlated with the massive effect of DMS. A direct effect of DMS seems a more probable explanation for its effect, and the target could be a discrete pattern of enzymes. Parallel measurements of SPHK and SPP-Lyase and Diacyl-glycerol kinase activities show inhibition by DMS, but also the increase of retinoblastoma protein is impaired and that of cyclins A and E whose synthesis depend on Rb transcription/ activation. Lactate dehydrogenase variations (used as marker of protein content) induced by DMS are however far less evident than those on SPHK. Preliminary data comparing the effects of DMS and sphingosine on the inhibition of PBL proliferation show that DMS has the same efficacy as > 10 mM sphingosine, reported to be a lysosomotropic agent. An increase of Sphingosine or DMS could be the physiological cause of apoptosis at the end of the process of clonal expansion. This aspect is under investigation.


  • Composition in polyunsatured fatty acids of membrane phospholipid is involved in the regulation of cell proliferation in tumor human cell lines.

    Marina Maggiora, Antonella Trombetta, Germana Martinasso, Rosa Angela Canuto and Giuliana Muzio

    Dipartimento di Medicina ed Oncologia Sperimentale, Università di Torino

    The length and the number of unsaturation of phospholipid fatty acids determine not only modification in the physic behaviour of membranes, but also influence its function, such as interaction ligand-receptor, hormone and growth factor receptors, signalling transduction, enzyme metabolising xenobiotics. These interactions can affect cell proliferation and death for apoptosis. Our previous results demonstrated that enriching rat hepatoma cells with arachidonic acid inhibits cell proliferation and reverts tumor-associated phenotype of class 2 and 3 aldehyde dehydrogenases (1). These effects due to arachidonic acid enrichment are consequent to the restoration of normal content of this fatty acid in membrane phospholipids, being lower in rat hepatoma cells in comparison with normal hepatocytes. In the light of these observations we extended our study on human tumor cell lines by using different polyunsaturated fatty acids (linoleic, conjugated linoleic and arachidonic acids).

    Different types of human tumor cells are used: two hepatoma cell lines with different tumorigenicity, non-tumorigenic HepG2 cells and tumorigenic SK-HEP-1 cells, and a lung cell line, A549 cells.

    Preliminary results show that polyunsaturated fatty acids inhibit cell proliferation and induce apoptosis in a dose-dependent manner. Moreover, the entity of the cytostatic and cytotoxic effect differs in relation with the cell type and, in the hepatoma cell lines it is directly correlated with the degree of malignancy. Flow cytometry analysis of DNA distribution evidences that in cells undergoing growth inhibition an increased G0/G1 population occurs; whereas in cells undergoing apoptosis an increased percentage of cells in S phase is present. About the mechanisms underlying the inhibition of cell proliferation induction MAPK pathway is under investigation; about apoptosis induction ceramide involvement and mitochondria pathway are investigated.

    This research is supported by a grant from Compagnia di San Paolo, Turin .

  • Involvement of the de novo ceramide synthesis in igfbp-3-mediated endothelial cell apoptosis.

    Riccarda Granata a , Marzia De Petrini a , Marina Taliano a , Ada Castelli a , G. Garbarino a , Giulia Somenzi b , Riccardo Ghidoni b , Ezio Ghigo a .

    a Division Endocrinology and Metabolism, Department Internal Medicine; University of Turin, Italy; b Laboratory of Genetics and Biochemistry, San Paolo University Hospital; University of Milan, Italy.

    IGFBP-3 modulates IGFs action and exerts direct IGF-independent actions inducing apoptosis or promoting cell growth in different cell types. IGFBP-3 expression has been shown in both cardiomyocytes and endothelial cells. Doxorubicin exerts its apoptotic effect through the ceramide death pathway and we have found that IGFBP-3 mediates apoptosis in cardiomyocytes. In this study we aimed to investigate the role of IGFBP-3 in endothelial cell apoptosis induced by doxorubicin and its relation with ceramide in the induction of the apoptotic response. We evaluated 1) the expression of IGFBP-3 and IGF-I in human endothelial cells (HUVEC) exposed to doxorubicin (0.5 m M) by Western blot and radio-immunoassay; 2) the effect of IGFBP-3 (1000 ng/ml) on cell survival and apoptosis of HUVEC exposed to doxorubicin and to the ceramide synthase inhibitor fumonisin B1 (FB1) by MTT, and FACS analysis; 3) IGF-I secretion in cells exposed to IGFBP-3 with or without doxorubicin and/or FB1; 4) the effect of IGFBP-3 antisense oligonucleotides on cell survival and apoptosis (by HOECHST staining) in all experimental conditions; 5) the variations in ceramide levels; 6) cell motility of cells treated with IGFBP-3, doxorubicin and/or FB1. An inhibitor of the first step in the de novo synthesis of ceramide, myriocin (50 nM), was also tested for the apoptotic effect of IGFBP-3 in association with doxorubicin. We found that IGFBP-3 was up-regulated upon doxorubicin-induced cell death, with increased ceramide production and no change in IGF-I secretion. Exogenous IGFBP-3 reduced apoptosis in serum deprived cells also decreasing ceramide levels. When associated to doxorubicin, IGFBP-3 enhanced apoptosis and increased ceramide levels. The protective effect of FB1 over doxorubicin-induced cell death and apoptosis was unexpectedly enhanced by IGFBP-3 with reduced ceramide levels and dramatically increased IGF-I secretion. IGFBP-3 antisense oligonucleotides counteracted apoptosis induced by doxorubicin but also reduced the protective effect of both FB1 and FB1+IGFBP-3 over doxorubicin-induced cell death. Finally, IGFBP-3 increased cell motility of HUVEC exposed to doxorubicin and FB1. Preliminary results indicate that IGFBP-3 even attenuates doxorubicin-induced apoptosis in the presence of myriocin. In conclusion, IGFBP-3 modulates endothelial cell fate, protecting or enhancing serum starvation- and doxorubicin-induced apoptosis and potentiating the protective effect of fumonisin B1 over doxorubicin. These actions appear to be paralleled by ceramide variations and increase in IGF-I secretion. Thus, IGFBP-3 likely exerts complex modulation of the ceramide signaling pathway directly or indirectly through the de novo ceramide synthesis, regulating apoptosis in endothelial cells and even IGF-I release that, in turn, would per se exert antiapoptotic action.

    Study supported by MURST, University of Turin and SMEM Foundation


  • Modifying the cellular response to ceramide-mediated stress by gene manipulation of lysosomal cathepsin d expression.

    Anna Aspesi , Roberta Castino, Camilo Gianinazzi, Marzia Mirabelli, Carlo Follo, Marina Démoz, and Ciro Isidoro

    Università del Piemonte Orientale “A. Avogadro”, Dipartimento di Scienze Mediche di Novara

    Ceramide is generated as a second messenger in response to various environmental stimuli such as cytokines, serum deprivation and cytotoxic chemotherapeutic drugs, and it is believed to play a major role in mediating their cellular effects. Ectopic administration of ceramide analogues (for instance, C2-ceramide) can mimic these cellular effects. We have assessed culture conditions to induce apoptotic cell death in L929 fibrosarcoma cells by treatments with C2-ceramide or chemotherapeutic drugs known to rise the intracellular levels of ceramide. Cathepsin D is a lysosomal protease that has been shown to play an active role in the onset of cytokine- and antiblastic drug-triggered apoptosis in tumour cells. Cathepsin D is also a target of endosomal-lysosomal ceramide generated by activation of acid sphingomyelinase under cytotoxic conditions. In the present work we describe the consequences of the transgenic hyperexpression of Cathepsin D in L929 cells subjected to increased levels of ceramide. We show that sensitivity of transfected cells to ceramide is proportional to the rate of Cathepsin D expression. The molecular mechanism underlying ceramide-induced cytotoxicity include the destabilisation of lysosomal membranes, likely associated with leakage of lysosomal cathepsins.

    Funded by MIUR-Cofin2001, AIRC, Regione Piemonte and CNR-target project on Biotechnology

  • Differentiation and cell signaling: ceramide-dependent modulation of neuroblastoma cell viability.

    Deborah Pace , Raffaele Picco , F abrizio Condorelli , Pier Luigi Canonico

    DISCAFF Dept. Università del Piemonte Orientale, Novara, Italy

    Ceramide, either produced by sphingomyelin hydrolysis or by neosynthesis, has been proposed as a possible second messenger for different extracellular signals. The increase in the intracellular ceramide levels may lead to induction of apoptotic death or to cell differentiation depending on experimental models including neuronal-derived cell lines. Since retinoic acid (RA) is known as the most effective inducer of differentiation (mainly in hematopoyetic and neuronal models) we decided to study the possible interactions of the ceramide-dependent pathways with RA-induced neuronal differentiation. Two different human neuroblastoma cell lines (SK-N-BE, SH-SY5Y) were exposed for increasing lengths of time (4 h minimal, 1 week maximal) to different concentrations of RA (10 nM-1 mM) and/or cell-permeable C2 ceramide (C2 10 nM-10 mM) to evaluate their response in terms of neuronal phenotype acquisition and viability. In similar conditions high C2 concentrations (1-10 mM) showed an addictive effect on RA (10 mM) induced differentiation evaluated both morphologically and by western-blot analysis of neuronal protein marker expression. Paradoxically, lower concentrations of C2 were cytotoxic according to trypan-blue exclusion assay (10 nM = 45 ± 8% and 100 nM = 35 ± 6% blue positive), an effect antagonized by RA (10 mM) co-treatment. This effect was probably due to induction of apoptosis as shown by DAPI-stained cells nuclei. Moreover we evaluated by western-blot analysis the subcellular localization of some member of Bcl-2 family and cytochrome c in SK-N-BE cells. These results set the basis to investigate further ceramide involvement in RA-induced differentiation.

  • Preliminary data on glyco(sphingo)lipid amount and distribution in food.

    Bruno Berra , Gigliola Montorfano, Attilio Giacosa *

    Institute of General Physiology and Biochemistry “G. Esposito” University of Milano, Italy; Gastroenterology and Nutrition Department, National Cancer Institute, Genova, Italy

    Sphingolipids are commonly assumed to be present mainly in neuronal tissue; however, they also appear in lipoproteins, milk fat, globule membranes, the lamellar barrier of skin, the Golgi apparatus and the lisosomes. The amounts vary considerably, with the highest proportions being in membrane-rich tissues. They are also present in relatively large amounts in some foods, such as dairy products and soybeans, which contain more gangliosides and glucosylcerebrosides, respectively than brain.

    There is no known nutritional requirement for sphingolipids. Nonetheless studies with experimental animals have shown that consumption of shingolipids inhibits colon carcinogenesis, reduces serum low-density lipoprotein cholesterol and elevates high-density lipoproteins, which suggest that they are “functional” components of food.

    No human clinical trials have yet evaluated sphingolipids for the prevention or treatment of colon cancer, nor any epidemiological studies of possible associations between sphingolipid consumption and cancer risk was made. Nonetheless, there is indirect evidence to suggest that sphingolipids can inhibit colon cancer in humans: (1) shingosine and ceramide induce apoptosis in a human adenocarcinoma cell line (HT29); and (2) feeding sphingolipids to Min mice (Min mice have a genetic defect similar to that found in human familial adenomatous polyposis and which is also found in a large percentage of sporadic human colon cancers) reduced the number of intestinal tumours by 55%.

    An epidemiologic programme is ongoing in Italy to evaluate the association between sphingolipids content of diet and colorectal cancer risk. This is part of case-control study that has been performed in six Italian areas.

    In this view detailed programme for the analysis of the sphingolipid content in different foods has been developed in our Institute.






    Fresh Cheese

    Soy Bean

    Neutral glycolipids

    mg/g of fresh weight




    358.94 – 551.49


    mg/g of fresh weight




    67.14 – 247.95


    mg/g of fresh weight




    291.35 – 496.57


    mg/g of fresh weight





    Other minor glycolipids

    mg/g of fresh weight




    25.73 – 91.75


    Table show the preliminary data we have found when we analyzed foods normally consumed in our population (like milk and fresh cheese) or less popular foods as different kinds of soy beans (were large variations were observed). In this study we included also a variety of bacterial strains used in fermented milks, due to the therapeutical potential of these food in the treatment of colon cancer.


  • Overexpression of cytosolic sialidase neu2 induces myoblast differentiation in c2c12 cells.


    A. Fanzani , R. Giuliani, F. Colombo, D. Zizioli, *M. Presta, A. Preti, and S. Marchesini

    Unit of Biochemistry and *Unit of General Pathology and Immunology, Department of Biomedical Sciences and Biotechnology, University of Brescia

    Cytosolic sialidase Neu2 has been implicated in myoblast differentiation. Here we observed a significant upregulation of Neu2 expression during differentiation of murine C2C12 myoblasts. This was evidenced both as an increase in Neu2 mRNA steady-state levels and in the cytosolic sialidase enzymatic activity.

    To understand the biological significance of Neu2 upregulation in myoblast differentiation, C2C12 cells were stably transfected with the rat cytosolic sialidase Neu2 cDNA. Neu2 overexpressing clones were characterized by a marked decrement of cell proliferation and by the capacity to undergo spontaneous myoblast differentiation also when maintained under standard growth conditions. This was evidenced by the formation of myogenin-positive myotubes and by a significant decrease in the nuclear levels of cyclin D1 protein. No differentiation was instead observed in parental and mock transfected cells under the same experimental conditions.

    The results indicate that Neu2 upregulation is per sè sufficient to trigger myoblast differentiation in C2C12 cells.

  • Calnexin suppresses GD3 synthase - induced apoptosis.

    Barbara Tomassini *, Florence Malisan*, Luigi Franchi*, Chiara Nicolo'*, Gloria Brea Calvo* and Roberto Testi*

    *Laboratory of Immunology and Signal Transduction, Department of Experimental Medicine and Biochemical Sciences, University of Rome Tor Vergata, Roma

    The accelerated activity of the GD3 synthase, with consequent GD3 accumulation, is part of the response to environmental stressors in different cell types. Depending on specific, yet largely undefined, cellular settings, this can be followed by cellular adaptation or apoptosis, in large part due to GD3-induced mitochondrial damage. Subcellular localization of the GD3 synthase could significantly affect the biological outcome of GD3 accumulation. We found that binding to the molecular chaperone calnexin causes the retention of the GD3 synthase within the endoplasmic reticulum (ER) and prevents its relocalization to the Golgi. Calnexin-dependent ER retention does not affect the activity of the GD3 synthase, yet the de novo synthesized GD3 largely fails to reach the mitochondria. Accordingly, overexpression of calnexin suppresses the pro-apoptotic activity of the GD3 synthase, and the loss of calnexin sensitizes cells to stress-induced apoptosis. Consequently, we conclude that calnexin has the ability to affect the subcellular distribution of the GD3 synthase and the biological outcome of GD3 accumulation during the stress response process.

  • Isolation and characterization of the GM3 synthase cDNA from human placenta.

    E. Sottocornola , P. Berselli, S. Zava, , B. Berra and I. Colombo

    Inst. of General Physiology and Biochemistry, University of Milan

    It is known that gangliosides have various important biological functions, and their functions as well as their biosynthesis are currently clarified (1, 2). In vertebrates, almost all the ganglio-series gangliosides are synthesized from a common precursor, ganglioside GM 3 , which has the simplest structure among the major gangliosides. GM 3 itself is known to participate in induction of differentiation, modulation of proliferation, signal transduction and integrin-mediated cell adhesion. GM 3 synthase (EC, ST3Gal V) is the enzyme involved in the last step of GM 3 biosynthesis: it catalyses the transfer of a sialic acid moiety from CMP-sialic acid onto lactosylceramide, forming an a2-3 linkage. Whereas GM 3 is ubiquitously distributed in the plasma membranes of all eukaryotic cells, GM 3 synthase results expressed in a tissue specific manner, especially in brain, placenta, muscle and testis (3). Many important issues, such as human cDNA identification and characterization, genomic structure and regulation of gene expression, are still open.

    To isolate the coding sequence of the gene of GM 3 synthase from human placenta we used the 5'- and 3'-Rapid Amplification of cDNA Ends technology (SMART RACE cDNA Amplification Kit, Clontech) using, as specific primers, oligonucleotides derived from the human GM 3 synthase cDNA sequence from differentiated HL60 cells (3). The different PCR products were cloned into the pCR2.1 vector (TA Cloning Kit, InVitrogen) and the nucleotide sequence was determined.

    A cDNA, showing high sequence homology with that encoding the human GM 3 synthase from TPA-differentiated HL60 cells (3), has been successfully isolated and cloned from human placenta. The major difference between these two cDNAs is in the 5'-end, according to the existence of different promoter regions, responsible for tissue-specific expression of the gene. Furthermore, the cDNA from the human placenta contains, upstream and in frame with the ATG indicated as translation initiation site for the GM 3 synthase of HL60 cells, another ATG codon inserted in a sequence compatible with Kozak's rule, suggesting that the protein of the human placenta has an additional portion in NH 2 -terminus. The complete coding region of the human placenta cDNA is going to be cloned in an expression vector, under the control of the CMV promoter, in order to evaluate its activity. On the other hand, in vitro translation experiments are going to be carried out to define the first start codon.

    1) Hakomori S.I. (2000): Glycoconj. J. 17, 627-647

    2) Kolter T. et al. (2002): J.Biol.Chem . 277, 25859-25862

    3) Ishii A. et al. (1998): J.B.C . 273, 31652-31655


  • Cancer-associated suppression of β 3gal-T5 reduces sialy-lewis a and enhances sialyl-lewis X and poly-N-acetyllactosamines.

    Lidia Mare and Marco Trinchera

    Dipartimento di Scienze Biomediche Sperimentali e Cliniche, Università dell'Insubria, Varese
    Glycosylation of glycoproteins and glycolipids is one of many molecular changes that accompany malignant transformation. Poly-N-acetyllactosamine (Gal β 1-4GlcNAc β 1-3) n sequences frequently terminated by the sialyl-Lewis x antigenic determinant (NeuAc α 2-3Gal β 1-4[Fuc a 1-3]GlcNAc, have been observed to increase in some cancer, and to correlate with poor prognosis. We reported that the expression of a specific β 1,3 galactosyltransferase (β 3Gal-T5) prevents poly-N-acetyllactosamine and sialyl-Lewis x expression on N-glycans in CHO cells. We also found that β 3Gal-T5 transcript is down-regulated in colon adenocarcinomas and presumably responsible for the differential glycosylation of CEA in cancer, where it acquires N-linked poly-N-acetyllactosamine chains, absent in the normal counterpart. The very low levels of β 3Gal-T5 transcript detectable in colon cancer specimens also open the question as to whether the CA19.9 antigen, that is sialyl-Lewis a epitope carried by a mucin backbone and circulating in cancer patient serum, is synthesized by β 3Gal-T5. Our working hypothesis is that β 3Gal-T5 is an enzyme associated with the normal phenotype and potentially able to opposite the malignant phenotype by preventing poly-N-acetyllactosamine extension, and thus synthesis of sialyl-Lewis x and related antigens.

    To assess this hypothesis, we first try to prove that β 3Gal-T5 is actually the enzyme responsible for β 1,3Gal-T activity in gastrointestinal tissues. To this aim, we measured β 1,3Gal-T activity and transcript levels in cancer, normal colon mucosa, and various cancer cell lines and recombinant clones, and compared the kinetic properties of the activity detected. We also determined the amount of sialyl-Lewis a synthesized and secreted by such sources. We found that one β 1,3Gal-T activity, corresponding to β 3Gal-T5, is expressed in tissues and cells and responsible for Lewis antigen synthesis and secretion on O-glycans. Moreover, almost no sialyl-Lewis a was detectable in colon cancer specimens by dot-blot. We then studied the effect of β 3Gal-T5 suppression in a cancer cell line. To this purpose we transfected BxPc3 cells with a β 3Gal-T5 fragment placed in the antisense orientation under the control of a very strong promoter, and isolated a recombinant clone that stably expresses high levels of the antisense transcript. We found that the clone expresses much less sialyl-Lewis a on the cell surface than BxPc3 cells, and β 1,3Gal-T activity is faintly detectable. These data strongly confirm that β 3Gal-T5 is the gene responsible for β 1,3Gal-T activity and sialyl-Lewis a antigen in these cells.

    Moreover the clone became positive to sialyl-Lewis x, that is undetectable in BxPc3 cells. A relevant amount of sialyl-Lewis x is also detected in the culture medium of the clone, while sialyl-Lewis a, that is secreted by BxPc3 cells, is not detectable. Sialyl-Lewis x expression in the antisense clone is reduced by treatment with b -benzyl-GalNAc and not affected by swainsonine. Similar results are obtained with sialyl-Lewis a in BxPc3 cells. Analysis of radioactive glycoconiugates in the antisense clone upon metabolic labeling with tritiated Gal indicated increased synthesis and secretion of poly-N-actetyllactosamine chains with respect to parental BxPc3 cells. Altogether the results suggest that sialyl-Lewis a synthesis is controlled by β 3Gal-T5 and reduced in cancer.

  • Role of lipid molecules in tumor cell biology: old facts and new perspectives.

    Ruggieri S.

    Department of Experimental Pathology and Oncology, University of Florence,, tel. 055 4282323, fax 055 4282333.
    Studies of tumor lipids have developed in step with those regarding the role of lipid molecules in cell biology. The parallel development of these studies has represented an interesting example of a fruitful interaction in the investigation of cell physiology and tumor biology. In the sixties, the explosive interest in cholesterol regulation in the body in order to understand the pathogenesis of atherosclerosis led to the discovery that hepatomas lack the feedback inhibition of cholesterol synthesis. Much attention has been devoted to phospholipids in tumor cells, due to the recognition that phospholipids are essential membrane components involved in enzymatic activities, receptorial properties, cellular permeability -- all of which are implicated in tumor cell biology. Structural analyses of phospholipids in different types of tumors revealed a profound alteration in their diacyl choline-glycerophospholipid molecular species. These analyses also showed an accumulation of ether-linked lipids in tumor cells, particularly evident in those with a high metastatic potential. This finding stimulated a great deal of research on the biosynthesis, distribution, and biological functions of ether-linked lipids.

    Moreover, awareness that glycolipids and gangliosides are involved in cellular recognition, particularly in the altered social behavior of tumor cells, has opened the way to understanding the complexity of the family of glycolipid molecules, for which Hakomori’s great contribution should be aknowledged. Unlike phospholipids, a specific glycolipid pattern was not found in different tumors examined. In fact, the glycolipid biosynthesis exhibits a lineage-dependent specificity which leads to peculiarities in the glycolipid patterns in tumor cells of different origin. For instance, a reduction of the more complex gangliosides in virally-transformed cell lines has been reported by different laboratories, while an accumulation of fucose-containing glycolipids has been noticed in epithelial tumors of a different origin. Moreover, in a fibrosarcoma line, the metastatic properties appeared to be correlated with the content and cell surface expression of Gb3ose, a glycolipid characteristic of this line.

    The tremendous expansion of research on inflammatory lipid mediators (platelet-activating factor, prostaglandins, thromboxanes, leukotrienes, hydroxyeicosatetraenoic, lipoxins) showed the importance of these mediators in several homeostatic reactions, including some which are implicated in various aspects of tumor progression, such as inflammatory processes, immunological responses and activation of platelets and endothelial cells. Several laboratories documented the involvement of arachidonic acid metabolites in growth rate and metastatic diffusion of tumor cells. Concentratingon the role played by platelet-activating factor (PAF) in certain steps of metastatic diffusion, we observed that: a) PAF is synthesized by different transformed cell lines under basal conditions and after stimulation with cytokines; b) PAF influences the interaction of melanoma and colon carcinoma cells with adhesive proteins of endothelial cells; and c) PAF promotes mobility of tumor cells by influencing the cytoskeleton.

    The long-lasting consensus on the pro-tumoral activity of high fat diets focused attention on the different families (n-6, n-3) of polyunsaturated fatty acids (PUFA) as possible modulators of tumor progression. Indeed, contrasting effects on carcinogenesis and metastatic diffusion were observed in animals fed diets enriched with different families of PUFA : stimulatory with PUFA n-6, inhibitory with PUFA n-3.

    A further insight on the role played by lipid molecules on carcinogenesis and tumor progression will be offered by the incoming investigation on the influence of lipid molecules on gene expression. This consideration is sustained by the recent report that the antitumoral effects of certain PUFA are mediated by peroxisome proliferator-activated receptors, a ligand-activated nuclear receptor superfamily which plays a regulatory role in several biological responses.

  • Biophysical properties of short- and long-chain ceramides.

    Goñi, FM, Contreras, FX, Montes, LR, Sot, J and Alonso, A.

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

    In the past decade the long-neglected ceramides (N-acylsphingosines) have become one of the most attractive lipid molecules in molecular cell biology, because of their involvement in essential structures (stratum corneum) and processes (cell signalling). Most natural ceramides have a long (16-24 C atoms) N-acyl chain, but short N-acyl chain ceramides (2-6 C atoms also exist in nature, apart from being extensively used in experimentation, because they can be easily dispersed in water. Long-chain ceramides are among the most hydrophobic molecules in nature, they are totally insoluble in water and they hardly mix with phospholipids in membranes, giving rise to ceramide-enriched domains. In situ enzymic generation, or external addition, of ceramides in membranes has at least three important effects: (i) lipid monolayer tendency to adopt a negative curvature, e.g. through a transition to an inverted hexagonal structure, is increased, (ii) bilayer permeability to aqueous solutes is notoriously enhanced, and (iii) transbilayer (flip-flop) lipid motion is promoted. Short-chain ceramides mix much better with phospholipids, promote a positive curvature in lipid monolayers, and their capacities to increase bilayer permeability or transbilayer motion are very low or inexistent.

  • Subcellular distribution of neutral ceramidase: localization to the outer mitochondrial membrane of rat liver.

    *&°Porcelli AM, §Novgorodov SA, *Luberto C, &Rugolo M, §Obeid LM,and *Hannun YA.

    *Department of Biochemistry and Molecular Biology and §Department of Medicine, MUSC, Charleston, SC (USA); &Dipartimento di Biologia E.S., Università di Bologna, Bologna, Italy.

    °Via Irnerio 42, 40126, Bologna, Italy. Tel. 051/2091286; Fax 051/242576; Email:

    Ceramide, sphingosine and sphingosine-1P play important roles in the regulatory functions of cellular processes such as apoptosis, proliferation and differentiation. Ceramidases (CDases) are the enzymes that hydrolyze ceramide to free fatty acid and sphingosine, thus contributing to change the intracellular levels of these bioactive molecules. These enzymes have been classified in three groups: acid, neutral and alkaline. Although the acid and alkaline CDases appear to be clearly localized to the lysosomes and ER/Golgi, respectively (1-3), the intracellular localization of the neutral enzyme is not clear. In this regard we had noted from our own studies (4) and from published studies a significant variation in the subcellular localization of neutral CDase when overe-xpressed in various cell lines.

    The aim of this work was to define the intracellular localization of endogenous CDase and toreconcile the results from tissue culture studies with subfractionation results. Rat liver subfractions were prepared using differential centrifugation and subsequent separation of pure mitochondria on Percoll gradient. CDase activity assay using D-e-C12-NBD-ceramide as substrate revealed that the activity was enriched in the pure mitochondrial fraction. Further, submitochondrial fractionation using digitonin showed that the enzyme resided preferentially in the outer mitochondrial membrane. Western blot analysis also supported these results and indicated that the molecular mass of neutral CDase in rat liver is around 85kDa. Unlike liver, most cell line are not very rich in mitochondria, and we hypothesized that whereas mitochondria may be enriched in the enzyme, over-expression may overwhelm the transfected cells. Therefore we investigatedthe subcellular localization of human neutral CDase in HEK 293 cells after transfectionwith GFP empty vector or vector in which GFP was at the C-terminus of human CDase. Subcellular fractionation of HEK cells showed that the specific activity of neutral ceramidase was highest in mitochondria. Nevertheless, mitochondria accounted for only a small fraction of the total activity. Confocal microscopy revealed that the over-expressed CDase exhibited a tubular pattern in the cytoplasm in close proximity to mitochondria and partially colocalized with an ER marker protein.

    All together these results indicate that the endogenous neutral CDase is enriched in mitochondria from rat liver, and that the over-expression of the enzyme likely causes a mislocalization. This finding warrants caution in interpreting results from over-expression studies, in particular when applied to membrane-bound proteins.


    Sugita M, Dulaney JT, Moser HW. Ceramidase deficiency in Farber’s disease. Science. 1972; 178:1100-1102.

    Mao C, Xu R, Szulc ZM, Bielawska A, Galadari SH, Obeid LM. Cloning and characterization of a novel human alkaline ceramidase. J. Biol. Chem. 2001; 276:26577-26588.

    Mao C, Xu R, Szulc ZM, Bielawska J, Becker KP, Bielawska A, Galadari SH, Hu W, Obeid LM. Cloning and characterization of a mouse endoplasmic reticulum alkaline ceramidase. J. Biol. Chem. 2003; 278:31184-31191.

    El Bawab S, Roddy P, Quin T, Bielawska A, Lemasters JJ, Hannun YA. Molecular cloning and characterization of a human mitochondrial ceramidase. J. Biol. Chem. 2000; 275: 21508-21513.



  • Gangliosides are present in all subcellular fractions of rat liver.

    1Popa I, 2Ardail D, 1Portoukalian J

    1Department of Dermatology, Edouard Herriot Hospital, 69437 Lyon Cx 03, France, 2Laboratory of Radiobiology, Lyon-Sud Medical School (Popa I, phone +33-4-72110607; fax +33-4-72110290; e-mail :

    The site of biosynthesis of gangliosides has long been thought to be exclusively the Golgi apparatus and their localization to be mostly at the outer surface of plasma membranes. However, recent evidence suggest that the mitochondria-associated membrane (MAM) can also synthesize gangliosides. Moreover, the presence of gangliosides has been documented in mitochondria and nuclei. In order to gain insight on the presence of gangliosides in subcellular fractions, we have carried out analyses of the gangliosides isolated from highly purified subcellular fractionsof rat liver. The major gangliosides species of the total rat liver were GD1a>GM1>GM3>GD1b>GT1b>GD3>GM2. Gangliosides were identified by co-migration with standards and by immunostaining on thin-layer plates with specific antibodies. The subcellular fractions purified from rat liver were the following : whole mitochondria, mitochondrial outer and inner membranes, mitochondria-associated membranes (MAM), microsomes, Golgi apparatus, nuclei and plasma membranes. All subcellular fractions analyzed were found to contain gangliosides. The ganglioside contents and the quantitative patterns were different in each organite, but no qualitative differences could be observed. Our results show that gangliosides are present in all cellular membranes, but the respective patterns do not reflect the global ganglioside composition of a tissue.


  • Sphingomyelin metabolism changes after serum deprivation in neuronal cell nuclei.

    Albi E1, Cataldi S 1, Bartoccini E1,Mazzoni F 2, Voccoli V3, Viola Magni M1,Lazzarini R1, and Garcia-GilM2.

    Department of Biochemical Sciences and Molecular Biotechnology, Physiopathology, Policlinico Monteluce, 06100, Perugia;2) Dpt. of Physiology and Biochemistry, University of Pisa, 3)Institute ofNeuroscience, CNR, Pisa.

    We have previously shown that in murine embryonic hippocampal HN9 cells, serum deprivation induces variations in the nuclear enzyme activities involved in the sphingomyelin (SM) metabolism. Nuclear sphingomyelinase (SMase) increases biphasically, with an earlier peak one hour after serum deprivation, and a secondraise starting at 15 hrs. In contrast, nuclear sphingomyelin-synthase (SM-synthase) decreases after serum deprivation with a minimun after 1 hr. No variations of SM metabolism were observed in the homogenate. The aim of this work is to clarify if the enzymes present in the nuclei differ from those present in the homogenate on the basis of the physical chemical parameters of the SMase and the SM-synthase in homogenate and nuclei isolated from HN9 cells and so to ascertain the specificity of the above modificationsandevaluatethe SM and ceramide content changes in relation to the serum deprivation-induced apoptosis. 3H-PC and 14C-SM were used as precursors to evaluate the SM-synthase and SMase activityrespectively. The neutral SMase and SM-synthase have a pH optimum 7.2 in homogenate and 7.6 in nuclei, whereas the acid SMase, measured in the homogenate, has a pH optimum 5.00.Their activity is linear during the first 60 min and in the range from 50 to 200 mg protein. The enzyme activities obey a regular Michaelis-Menten kinetics in both preparations. The Km values of the neutral SMase are 1.48 x 10-3M for the homogenate and 1.26 x 10-4 M for nuclei whereas that of acid SMase is 4.55 x 10-5 M. The Vmax were 344, 113 and 183 pmol/mg protein/min respectively. The Km values of SM-synthase are 4.6 x 10-3M for homogenate and 1.59 x 10-4 for nuclei. The Vmax are 140 and 109 pmol/mg protein/min respectively. The physical chemical parameters of the nuclear enzymes in HN9 cells are similar to those reported previously for hepatocyte nuclei. The lower Kms of the nuclear SMase and SM-synthase indicate a higher affinity for their substrate compared to those of the homogenate. Our results suggest that the enzymes present in the homogenate and nuclei are different. The modifications of nuclear levels of SM and ceramide during the first hours of serum deprivation were studied by labeling the cells with 3H palmitic acid and measuring 3H-SM and 3H-ceramide in the purified nuclei. We have found that aftertwo hours of serum deprivation, the nuclear 3H-SM decreases while 3H-ceramide increases significantly, in agreement with the increase of SMase activity and inhibition of SM-synthase activity inhibited as previously reported.


  • Development of a high throughput screening assay for ceramidases and application to the screening of combinatorial libraries of sphingolipid analogs.

    Bedia C, Triola G, Badalassi F, Grijalvo S, Casas J, Delgado A, Llebaria A and Fabriàs G.

    Research Unit on BioActive Molecules (RUBAM), Department of Biological Organic Chemistry, IIQAB, CSIC. Jordi Girona 18. 08034-Barcelona. Spain. - FAX: 34-93-2045904 Phone: 34-93-4006115

    The first step in the catabolic route of ceramide is its amide hydrolysis by ceramidases. Several types of ceramidases have been reported that differ in their optimum pH, intracellular location and substrate specificity. Mutations in the acidic form gives rise to Farber disease (1), a rare lysosomal storage disorder for which there is no current therapy. A promising novel strategy that has afforded promising results in Gaucher and Fabry diseases is the active site directed chaperone approach. This strategy relies on the use of competitive enzyme inhibitors at sub-inhibitory concentrations to aid the correct folding of the mutated enzyme, thus favouring its transport to the lysosome and increasing its residual activity. Although several inhibitors of ceramidases have been reported (2-4), potent inhibitors of the acidic enzyme are not available. The only acid ceramidase inhibitor so far described is N-oleoylethanolamine, and its potency is rather low (4). In contrast, potent and selective inhibitors of mechanistically similar hydrolases are known (5). In this context, we have recently undertaken a project aimed at the synthesis of combinatorial libraries of sphingolipid analogs with a general structure combining the diverse structural motifs present in reported inhibitors of ceramidases and mechanistically similar amide hydrolases. To identify and select competitive inhibitors within the combinatorial lipid libraries, the development of a high throughput screening (HTS) procedure was necessary.

    In this work we describe the development of a HTS procedure to determine ceramidase activity and its application to the screening of combinatorial libraries of lipid analogs. As detailed in the figure, the method is based on the Reymond’s concept (6), which consists on the use of a fluorogenic substrate, which, upon amide hydrolysis, affords an aminoalcohol. Its oxidation with NaIO4 gives rise to an aldehyde, which is broken down to fluorescent coumarine in BSA at pH = 8.0.

     Missing figure


    The fluorescent substrate can be easily prepared as outlined in the scheme.


    As depicted in the figure, experiments with the neutral ceramidase have shown that compound CHA is a substrate of this enzyme with a Km of 120 µM and a Vmax = 212 nmole/min. Using this fluorimetric assay, combinatorial libraries of sphingolipid analogs can be screened and ceramidase inhibitors can be identified



    1. He X, Okino N, Dhami R, Dagan A, Gatt S, Schulze H, Sandhoff K, Schuchman EH.J. Biol. Chem. 2003;278:32978-32986.

    2. Bielawska A, Greenberg MS, Perry D, Jayadev S, Shayman JA, McKay C, Hannun YA. J. Biol. Chem. 1996;271:12646-12654.

    3. Selzner M, Bielawska A, Morse MA, Rudiger H. A, Sindram D, Hannun YA, Clavien PA Cancer Res. 2001;61:1233-1240.\

    5. Boger DL, Sato H, Lerner AE, Hedrick MP, Fecik RA, Miyauchi H, Wilkie GD, Austin BJ, Patricelli MP, Cravatt BF. Proc. Natl. Acad. Sci. USA. 2000;97:5044-5049.

    6. Wahler D, Badalassi F, Crotti P, Reymond, JL. Chemistry--A European Journal. 2002;8:3211-3228.





  • Double strand RNA and sphingomyelin.

    Rossi G., Viola Magni MP, Albi E.

    Department of Biochemical Sciences and Molecular Biotechnology, Physiopathology, Policlinico Monteluce, 06100 Perugia, Italy. e-mail:

    A complex formed by 97% protein, 1.7% RNA, 0.4% DNA and 0.18% phospholipids (PLs), constituted by sphingomyelin (SM) and phosphatidylcholine (PC), was isolated from hepatocyte nuclei (1). The RNA appeared to be double-strand since was resistant to RNase treatment and became sensitive to enzymatic hydrolysis when submitted to thermal denaturation. The digestion of SM with sphingomyelinase (SMase) trasformed Rnase-undigested RNA in RNase-sensitive RNA, thus suggesting that the SM may form a bridge between two RNA strands (1). The EM analysis, using double labeling with anti-RNA polymerase II antibody and N-SMase-gold complex showed that SM colocalizes with RNA-polymerase II in the transcription sites (unpublished data). The aim of this work was to highlight the relationship between SM and RNA transcription in the intranuclear complex. Hepatectomised animals were killed 24 hrs after operation, 100mCi 3H-uridine was injected 1h prior to killing. The 3H-uridine incorporation value was 1893 cpm/mg RNA in the homogenate and increased by 2.9 and 3.7 times in the nuclei and in the intranuclear complex respectively. The data suggest that the RNA present in the complex is neosynthesised. To investigate if the RNA was synthesised directly in the intranuclear complex, the possible presence of a transcription factor STAT3 was detected by immunoblotting with specific antibodies. The results showed that the STAT3 is present in the intranuclear complex, its molecular weight is 180 KDa which is a dimeric active structure formed in the cytoplasm and transferred in the nucleus. After SMase treatment the dimeric structure decreased strongly and a spot corresponding to 80 KDa, a monomeric form, appeared. It is possible that SM and STAT3 interact in the transcription process and facilitate the formation of the RNA loops. The SMase degrades STAT3 and SM favouring the splicing process.

    1) Micheli,M., Albi,E, Leray,C., and Viola Magni,M.P. "Nuclear sphingomyelin protects RNA from RNase action" FEBS Letters 431, 443-447, 1998.

  • Sphingosine kinase as a ‘sensor’ during chemotherapy-induced apoptosis in prostate cancer.

    Cuvillier O1, Pchejestki D1,Bonhoure E1, Levade T1, Calvet C2, Malavaud B2

    1 Inserm U466 and 2 Urology Department, Rangueil Hospital, 1 avenue Poulhes, 31059 Toulouse Cedex 9, France

    Tel : (+33) 561.32.20.60 ; Fax : (+33) 561.32.20.84

    Prostate cancer is the most common malignancy and the second leading cause of cancer death in men, killing more than 200,000 men annually worldwide. Many forms of prostate cancer initially are androgen dependent, but the response to androgen-ablation therapy is transient, and after a few years, the majority of prostate cancers relapse to the status of androgen independence resulting in death. Despite the availability of various therapeutic approaches (ionizing radiation, chemotherapy) that kill tumor cells by apoptosis, many cancer cells develop resistance to apoptosis.

    Sphingolipid metabolites - including ceramide, sphingosine and sphingosine 1-phosphate (S1P)- are active mediators that play essential roles in cell growth, survival and death. Abundant evidence suggests that ceramide is a critical messenger of ionizing radiation- and chemotherapeutic drug-induced apoptosis. In contrast to the growth-inhibitory and pro-apoptotic effects of ceramide, S1P, which is produced by phosphorylation of sphingosine by the oncogenic sphingosine kinase, has been implicated in cell growth and inhibition of ceramide-mediated apoptosis. It has been hence suggested that the dynamic balance between levels of ceramide versus S1P, and consequent regulation of opposing signaling pathways, is an important factor that could determine whether a cell survives or dies.

    Herein, we report that sphingosine kinase could act as a ‘sensor’ during chemotherapy-induced apoptosis in prostate cancer. In other words, we have found that the chemosensitivity/chemoresistance status is well correlated with sphingosine kinase activity. In prostate cancer cells in which chemotherapeutics induced a strong apoptosis, sphingosine kinase activity was rapidly down regulated. Accordingly, levels of endogenous ceramide and S1P were respectively increased and diminished. Conversely, there were no changes in sphingosine kinase activity (nor in ceramide and S1P levels) in tumor cells in which chemotherapeutics have no or little effect on cell viability. To demonstrate that sphingosine kinase inhibition was instrumental for chemotherapeutic-induced apoptosis, we over expressed its gene in two prostate cancer cell lines.As anticipated, sphingosine kinase overexpression markedly protected cells from chemotherapeutic-induced apoptosis.

    Ability of sphingosine kinase to determine the resistance of cancer cells to chemotherapy might propose inhibition of this enzyme for potential application in cancer treatment. Strategies that elevate cellular ceramide and diminish S1P by switching off sphingosine kinase could be used for therapies aimed to arrest growth or promote apoptosis. To this end, we have utilized newly developed sphingosine kinase inhibitors to alter this signaling pathway. These inhibitors could successfully overcome chemoresistance by triggering apoptosis in the prostate cancer cell lines used in this study.

    By its very nature, the sphingolipid biostat is poised for pharmacologic manipulation, and we can anticipate therapeutic success from the thoughtful use of molecules that can interfere with it by inhibiting the key enzyme, sphingosine kinase.


  • Sphingosine 1 Phosphate (S1P) protects mice from the radiation–induced GI syndrome and LPS-induced death.

    1Bonnaud S, 1Niaudet C, 2Fuks Z, 2Kolesnick R, 1Paris F.

    1INSERM UMR601, Nantes, France and 2Memorial Sloan-Kettering Cancer Center, New York, NY. Paris F. INSERM UMR601, IFR26, 9 quai Moncousu, 44000 Nantes, France. Tel : +33.(0) Fax : +33.(0)

    While ceramide is considered a pro-apoptotic factor, its metabolite, S1P, acts as a proliferative, anti-apoptotic factor. Recent results showed that acid sphingomyelinase gene disrupted (asmase-/-) oocytes and S1P treated oocytes resist radiation-induced apoptosis ex vivo (1). Moreover, in adult mice, oocyte loss, ovarian aplasia and sterility, observed in 0.1 Gy irradiated wild-type mice, were prevented by injection of S1P into the ovary prior to radiation (2). This study showed that S1P might be a promising agent for the protection of critical tissues against toxicity of anticancer drugs. The gastrointestinal (GI) syndrome is a common side effect of anticancer treatments and limits their efficacy. Our previous data showed that microvascular endothelial apoptosis after whole body irradiation at 15 Gy leads to stem cell dysfunction, crypt damage, organ failure, and death from the GI syndrome (3). These events were prevented when endothelial apoptosis was inhibited pharmacologically, by i.v. basic fibroblast growth factor (bFGF) injection, or genetically, by deletion of asmase. bFGF-treated mice or asmase-/- mice died later from bone marrow aplasia. We now report that retro-orbital injection of S1P in wild-type C57Bl/6 mice, prior to irradiation, mimics the effect of asmase disruption. S1P protected mice from death caused by the GI syndrome. Survival of 15 Gy-irradiated mice after S1P treatment was increased (median survival 9 days vs. 6 days for 15 Gy control mice; p<0.05). Autopsies showed that 80% of 15 Gy irradiated animals treated with S1P died from marrow failure rather than the GI syndrome. Furthermore, published data already showed that LPS-induced mouse death by a disseminated form of endothelial apoptosis was mediated through ceramide generation, and inhibitable by bFGF injection or asmase deletion (4). Similarly, S1P-pretreated mice were protected from lethal LPS injection (76% of mice survive 175 mg LPS/25g body weight after S1P pretreatment vs. 19% of control mice; p<0.05). In vivo investigations showed that S1P radioprotection against tissue damage and death in these models is due to direct inhibition of endothelial cell apoptosis.

    In order to better describe the molecular pathway used by S1P to inhibit endothelial cell apoptosis, we decided to study the effect of S1P in irradiated microvascular endothelial cells (HMEC-1). We first showed that a 15 Gy irradiation kills 40% of cells by apoptosis after 24 hours. Pretreatment with 1mM S1P 2 hours before irradiation was able to inhibit apoptosis by 50% in HMEC-1 cells. In order to know whether the S1P was acting directly against ceramide generation or through S1P receptors and G protein activation, we pretreated endothelial cells with an inhibitor of Gi/G0 proteins activation, the Pertussis Toxin (PTX), before S1P treatment and irradiation. This PTX treatment reverted radioprotection of S1P in HMEC-1 cells. In vivo investigation are underway to determine whether PTX pretreament can block the radioprotection of the microvascular endothelium, tissue damage and death caused by S1P.


    1. Morita Y, Perez GI, Paris F, Miranda SR, Ehleiter D, Haimovitz-Friedman A, Fuks Z, Xie Z, Reed JC, Schuchman EH, Kolesnick RN, Tilly JL. Oocyte apoptosis is suppressed by disruption of the acid sphingomyelinase gene or by sphingosine-1-phosphate therapy. Nature Medicine. 2000;6(10):1109-14.

    2. Paris F, Perez GI, Fuks Z, Haimovitz-Friedman A, Nguyen H, Bose M, Ilagan A, Hunt PA, Morgan WF, Tilly JL, Kolesnick R. Sphingosine 1-phosphate preserves fertility in irradiated female mice without propagating genomic damage in offspring. Nat Med. 2002 Sep;8(9):901-2. Nature Medicine. 2002;8(11):1329

    3. Paris F, Fuks Z, Kang A, Capodieci P, Juan G, Ehleiter D, Haimovitz-Friedman A, Cordon-Cardo C, Kolesnick R. Endothelial apoptosis as the primary lesion initiating intestinal radiation damage in mice. Science. 2001;293(5528):293-7.

    4. Haimovitz-Friedman A, Cordon-Cardo C, Bayoumy S, Garzotto M, McLoughlin M, Gallily R, Edwards CK 3rd, Schuchman EH, Fuks Z, Kolesnick R. Lipopolysaccharide induces disseminated endothelial apoptosis requiring ceramide generation.J Exp Med. 1997;186(11):1831-41.



  • Sphingosine-1-phosphate signalling in neuronal cells – Studies with a synthetic analogue.

    van Echten-Deckert G and Nätzker S

    Kekulé-Institut für Organische Chemie und Biochemie der Universität Bonn, Gerhard-Domagk-Strasse 1, 53121 Bonn, Germany.

    phone 0049-228-732703; fax 0049-228-737778; e-mail:

    cis-4-Methylsphingosine is a synthetic cell permeable pro-drug, that is taken up by cells and phosphorylated to a metabolically stable cis-4-methylsphingosine-1-phosphate (1). This compound was shown to mimic the mitogenic effect of its short living physiological counterpart sphingosine-1-phosphate (S1P) in Swiss 3T3 fibroblasts (2). In neuroblastoma cells, however, cis-4-methylsphingosine-phosphate was found to induce apoptosis and this effect was mediated by an opposite regulation of p38 and of ERK MAPKs (3).

    We have now investigated the effect of both, S1P and its synthetic analogue in highly differentiated primary cultured neurons. In contrast to S1P, which had no effect on growth of these post-mitotic cells, cis-4-methylsphingosine-1-phosphate induced apoptosis. Like in neuroblastoma cells MAPK pathways were involved in this process. In addition, however, an aberrant activation of the cell cycle machinery was found to be required.

    Interestingly, our results document that the different physiological effects, apoptosis in case of the accumulating metabolically stable synthetic compound versus no apoptosis in case of the short-living S1P, rely only on nuances of impact. In other words - similar pathways are affected by both compounds albeit in a sustained and more pronounced manner in case of the metabolically stable synthetic compound.


    1 - van Echten-Deckert G, Zschoche A, Bär T, Schmidt RR, Raths A, Heinemann T, Sandhoff K. cis-4-Methylsphingosine decreases sphingolipid biosynthesis by specifically interfering with serine palmitoyltransferase activity in primary cultured neurons. J. Biol. Chem. 1997;272:15825-33.

    2 - van Echten-Deckert G, Schick A, Heinemann T, Schnieders B. Phosphorylated cis-4-methylsphingosine mimics the mitogenic effect of sphingosine-1-phosphate in Swiss 3T3 fibroblasts. J. Biol. Chem. 1998;273:23585-89.

    3 - Nätzker S, Heinemann T, Figueroa-Perez S, Schnieders B, Schmidt RR, Sandhoff K, van Echten-Deckert G. cis-4-Methylsphingosine-phosphate induces apoptosis in neuroblastoma cells by opposite effects on p38 and ERK MAPKs. Biol. Chem. 2002;383:1885-94.

  • Sphingosine kinase activity is required for sphingosine-mediated phospholipase d activationin C2C12 myoblasts.

    Meacci E, Donati C, Cencetti F, Nuti F, Becciolini L and BruniP

    Dipartimento di Scienze Biochimiche, Università degli Studi di Firenze,Viale G.B. Morgagni 50, 50134 Firenze; Istituto Interuniversitario di Miologia (IIM); tel ++39055413765; fax ++390554222725

    Sphingosine (Sph) has been implicated as modulator of membrane signal transduction systems and as regulatory element of cardiac and skeletal muscle physiology but scarce information is presently available on its precise mechanism of action. Recent studies have shown that sphingosine 1-phosphate (S1P), generated by sphingosine kinase (SphK) action on Sph, possesses also biological activity, acting as intracellular messenger as well as extracellular ligand for specific membrane receptors. At present however, it is not clear whether biological effects elicited by Sph are attributable to its conversion into S1P. In the present study this issue has been investigated.

    C2C12 cells were routinely grown in DMEM supplemented with 10% fetal calf serum in an atmosphere of 95% air 5% CO2. For their differentiation into myotubes confluent cells were shifted to low serum medium (DMEM added with 2% horse serum). Mature phenotype was obtained after 5-6 days. Initially, sphingosine (Sph) was examined for its ability to activate phospholipase D (PLD), as observed in other cell types (1). Indeed, 1mM Sph appreciably stimulated PLD activity. Intriguingly, stimulation of PLD by Sph was found to be inhibited by pertussis toxin (PTx), as also observed for S1P-induced PLD activation (2) and could not be detected in differentiated C2C12 cells, in which S1P is uncoupled from PLD stimulation (3), supporting the hypothesis that a common pathway accounts for the action of both sphingoid bases. Furthermore, experiments performed in C2C12 myoblasts overexpressing S1P2 or in which endogenous receptor was down-regulated by specific antisense oligonucleotides, were in favour of a major role of S1P2 in the activation of PLD by Sph. Moreover, Sph-induced activation of PLD was inhibited by N,N-dimethyl-D-erythro-sphingosine (DMS), at concentrations capable to block specifically SphK. The crucial role of SphK-derived S1P in the activation of PLD by Sph was confirmed by the observed potentiated effect of Sph in myoblasts where SphK1 was overexpressed and the attenuated response in cells transfected with the dominant negative form of SphK1. Notably, the in vivo measurement of S1P formation by employing labelled ATP revealed that cell-associated SphK active in the extracellular compartment largely contributed to the transformation of Sph into S1P, being negligible the amount of SphK released into the medium. It will be important to establish whether the here identified mechanism of action is implicated in the multiple biological effects elicited by Sph in muscle cells.

    Spiegel S and Milstien S. Sphingoid bases and phospholipase D activation. Chem. Phys. Lipids 1996;80: 27-36.

    Meacci E, Vasta V, Donati C, Farnararo M, Bruni P Receptor-mediated activation of phospholipase D by sphingosine 1-phosphate in skeletal muscle C2C12 cells. A role for protein kinase C. FEBS Lett. 1999; 457: 184-188.

    Meacci E, Cencetti F, Donati C, Nuti F, Farnararo M, Kohno T, Igarashi Y, Bruni P. Down-regulation of EDG5/S1P2 during myogenic differentiation results in the specific uncoupling of sphingosine 1-phosphate signalling to phospholipase D. Biochim Biophys Acta 2003; 1633:133-142.



  • Involvement of sphingosine in glucocorticoid-induced apoptosis of thymocytes.

    Lépine S,* Lakatos B,*† Courageot M-P,* Le Stunff H,§ Sulpice J-C,* Giraud* F.

    *Biomembranes et Messagers Cellulaires and §Activation Cellulaire et Transduction des Signaux,CNRS UMR8619,Université Paris XI-Orsay, France; †present address: Department of Biochemistry and Microbiology, Slovak University of Technology, Bratislava, Slovak Republic.

    Corresponding author: Dr. Françoise Giraud, Biomembranes et Messagers Cellulaires, Bat 440, UniversitéParis XI-Orsay, 91405 Orsay Cedex, France. Phone: 33-1-69 15 76 44, Fax: 33-1 69 15 49 61, E-mail:

    During the selection process in the thymus, most thymocytes are eliminated by apoptosis through signaling via TCR or glucocorticoids. The involvement of ceramide (Cer) and sphingosine (SP), important apoptotic mediators, remains poorly defined in glucocorticoid-induced apoptosis. We report that, in mouse thymocytes, apoptosis triggered by 10-6 M dexamethasone was preceded by a caspase-dependent Cer and SP generation, together with activation of acidic and neutral ceramidases. Apoptosis was drastically reduced by blocking either sphingolipid production (by acid sphingomyelinase inhibitor) or SP production (by ceramidase inhibitors), but not by inhibition of de novo Cer synthesis. Thus, SP generated through acid sphingomyelinase and ceramidase activity would contribute to the apoptotic effect of dexamethasone. Consistent with this hypothesis, SP addition or inhibition of SP kinase induced thymocyte apoptosis. Dexamethasone induced a proteasome-dependent loss of mitochondrial membrane potential DYm and caspase-8, -3 and -9 processing. Apoptosis was abolished by inhibition of DYm loss or caspases-8 or -3, but not caspase-9. DYm loss was independent of SP production and caspase-8, -3 and -9 activation. However, inhibition of SP production reduced caspase-8 and -3, but not caspase-9 processing. Proteasome inhibition impaired activation of the three caspases, whereas inhibition of DYm loss solely blocked caspase-9 activation. These data indicate that dexamethasone-induced apoptosis is mediated in part by SP which contributes, together with proteasome activity, to caspase-8/-3 processing independently of mitochondria, and in part by the proteasome/mitochondria pathway, although independently of caspase-9 activation.


  • Acid ceramidase controls daunorubicin resistance of human hepatoma cells via the ceramide/sphingosine-1-phosphate switch.

    Morales A1, París R1, Marí M1, Villanueva A2, García-Ruiz C1, 3 and Fernández-Checa JC 1, 3

    1 Liver Unit, Institut de Malalties Digestives, Hospital Clínic i Provincial, Institut d’Investigacions Biomèdiques August Pi i Sunyer, 2 Institut Català d’Oncologia, Hospital Duran i Reynals, Granvia Km 2.7, 08907 Hospitalet, and 3Departmento de Patología Experimental, Instituto Investigaciones Biomédicas Barcelona, Consejo Superior de Investigaciones Científicas, Barcelona, 08036, Spain. Correspondence JC F-Ch (Liver Unit, Hospital Clinic, C/Villarroel, 170, 08036-Barcelona,

    Sphingolipids comprise a family of bioactive lipids that have been described to regulate and mediate diverse cellular functions with opposing outcomes such as in cell proliferation and growth arrest (1-3). In this regard, ceramide and sphingosine-1-phosphate (S1P), play an antagonizing role in cell death. Since ceramidases contribute to the conversion of ceramide to S1P, we analyzed the role of acid ceramidase in the response of tumor cells to chemotherapy. We report that daunorubicin (DNR) selectively stimulated acid ceramidase, decreasing the cellular ceramide to S1P balance. Acid ceramidase silencing by small interfering RNA or inhibition by N-oleoylethanolamine (NOE) enhanced the ceramide/S1P ratio, sensitizing human hepatoblastoma cell lines HepG2, SK-Hep, Hep-3B and human colon HT-29 cells to DNR-induced apoptosis. The sensitisation to DNR by NOE was reversed by extracellular S1P via a pertussis toxin-independent mechanism. The combination of DNR plus NOE resulted in ultrastructural changes in mitochondria, triggering reactive oxygen species generation, cytoplasmic release of Smac/DIABLO and cytochrome c, and caspase-3 activation. Importantly, the apoptotic potential of DNR plus NOE was selective for liver tumor cells, as primary hepatocytes were insensitive to DNR with or without NOE. Thus, acid ceramidase mediates the resistance of tumor cells to DNR through the sphingolipid ceramide/S1P rheostat and hence its antagonism may be of therapeutic value.


    1. Obeid, LM, Linardic CM, Karolak LA, Hannun YA.Programmed cell death induced by ceramide. Science.1993. 1769-1771.

    2. Hannun YA, and Obeid LM The Ceramide-centric universe of lipid-mediated cell regulation: stress encounters of the lipid kind. J. Biol. Chem. 2002. 277:25847-25850.

    3. Spiegel S, andMilstein S. Sphingosine-1-phosphate: an enigmatic signaling lipid. Nat. Rev. Mol. Cell Biol. 2003. 4:397-407.



  • Biological activity of resveratrol via ceramide signaling.

    P. Signorelli, R. Ghidoni

    Laboratory of Biochemistry and Molecular Biology San Paolo Medical School, Milan, Italy – via A. di Rudinì, 8 – 20142 Milan –

    Resveratrol (3,4',5-trans-trihydroxystilbene) is a polyphenol small molecule, naturally occurring in a variety of dietary vegetables (roots, peanuts, grape skin) and produced by plants in response to external stresses such as UV and pathogens attack. In human cells, resveratrol treatment affects a variety of cellular targets and the compound acts as anti-oxidant, radical scavenger, anti-inflammatory and antitumoral agent. Plants produce resveratrol to eliminate injured cells via an apoptotic mechanisms; similarly resveratrol induces apoptosis in human cancer cells, in a dose dependant fashion. We demonstrated that in human tumors of different origins (mammary adenocarcinoma, prostate carcinoma and melanoma) resveratrol treatment induces accumulation of endogenous ceramide. Ceramide formation is associated with an increased rate of sphingomyelin hydrolysis at plasmamembrane (nSMase) and of de novo synthesis of sphingolipids. Impairing ceramide accumulation, via metabolic inhibitors, rescued from resveratrol induced cell death. Thus ceramide is a necessary mediator of resveratrol-induced apoptotic mechanism. Since many chemotherapic agents that are effective in cell culture do not show equal efficacy in vivo, due to intrinsic resistance and reduced exposure to drugs of a solid tumoral mass, we tested resveratrol effect on a three dimensional culture of mammary adenocarcinoma cells. Resveratrol determined impairment of three-dimensional growth, disruption of a grown mass of tumoral cells and death of dispersed cells. Moreover the remaining aggregated cells were not able to proliferate if isolated and re-seeded (colony formation), showing a reduced metastasising activity. Even in three-dimensional culture resveratrol induced ceramide intracellular accumulation that is dose dependent and parallels the cytostatic and cytotoxic effect of the compound. In order to investigate on structure-function relation, resveratrol analogues were administered to prostate cancer cells. Whereas trans-stilbene (no hydroxylated) had no effect on cell proliferation, survival and induction of ceramide intracellular increase, the substitution of methoxyl groups in lieu of hydroxyl-groups resulted in an extremely toxic compound inducing cell death by a non apoptotic mechanism and unable to increase ceramide intracellular content. Piceatannol (3,4,3',5'-trans-tetrahydroxystilbene), another analogue with an additional hydroxyl group, showed biological effects comparable to resveratrol. These results assess that resveratrol antitumoral activity is related to its phenolic moiety that targets, either directly or indirectly, ceramide metabolism.

  • De novo-synthesized ceramide is involved in cannabinoid-induced apoptosis of LeukAemia cell lines.

    Herrera B, Carracedo A, Gómez del Pulgar T, Guzmán M and Velasco G

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

    Fax: +34 913944672; Phone: +34 913944668; e-mail:

    Cannabinoids, the active components of Cannabis sativa and their derivatives, exert a wide spectrum of effects both in the central nervous system and in peripheral locations due to their ability to bind to the specific CB1 and CB2 cannabinoid receptors.

    During the last few years, research from several laboratories has ravelled the therapeutic potential of cannabinoids as antitumoral agents. However, the mechanisms involved in these antiproliferative actions have been only partially elucidated. Interestingly, cannabinoid-triggered ceramide accumulation has been identified as one of the mechanisms involved in the antitumoral actions of these compounds in gliomas.

    Here, we employed the human leukaemic cell lines Jurkat and SupT1 -that had been previously shown to undergo apoptosis in response to cannabinoids- to gain insight into the intracellular pathways leading to cannabinoid-induced apoptosis of tumoral cells. For this purpose, cells were incubated with cannabinoids in the presence or absence of several inhibitors and antagonists, and cell viability, apoptosis, ceramide levels and serine palmitoyltransferase activity were determined. Western blot analyses were performed to measure expression, distribution and phosphorylation of several proteins.

    Challenge of Jurkat and SupT1 cells to different cannabinoid receptor agonists and antagonists show that apoptosis occurs via CB2 receptor. Regarding intracellular signaling, data show that: (i) the pro-apoptotic effect of cannabinoids was prevented by pharmacological abrogation of the ceramide synthesis de novo pathway; (ii) cannabinoid treatment triggered a rapid accumulation of ceramide and a parallel stimulation of serine palmitoyltransferase activity; (iii) activation of c-Jun N-terminal kinase and p38 mitogen-activated protein kinase was observed after cannabinoid challenge; (iv) cannabinoid treatment led to cytochrome c release from mitochondria and to activation of caspase 3.

    Taken together, these data provide clues about the mechanism of cannabinoid pro-apoptotic action, and point to the involvement of de novo-synthesized ceramide in the process of CB2-induced apoptosis of human leukaemia cells.

  • Frataxin prevents ceramide-induced apoptosis.

    Ventura N*, Condò I*, Malisan F and Testi R $

    equally contributed to this work
    Laboratory of Immunology and Signal Transduction, Department of Experimental Medicine,
    University of Rome "Tor Vergata". Via Montpellier, 1. 00133 ROMA.

    Tel: 0672596502- Fax: 0672596505

    Defective frataxin expression in humans causes Friedreich's Ataxia (FA), an autosomal recessive neurodegenerative syndrome characterized by progressive ataxia, dysarthria, skeletal abnormalities, glucose intolerance and cardiomyopathy. Frataxin is a highly conserved mitochondrial protein involved in the biogenesis of iron/sulfur clusters (ISC) and iron homeostasis. Low frataxin levels affect mitochondrial energy metabolism as a consequence of altered biogenesys of ISC enzymes, which include several components of the mitochondrial electron transport chain.

    FA cells are more sensitive to oxidative stress, yet the involvement of frataxin in cell survival is still obscure. Herein, we show that the transient overexpression of frataxin prevents ceramide-induced ROS accumulation and apoptosis in human tumor cells, with the same efficiency of the anti-apoptotic protein Bcl-2. GD3-mediated apoptosis, driven by GD3 synthase overexpression, is also inhibited by co-transfection of the cells with frataxin. Moreover, differently from Bcl-2, frataxin protects against anti-Fas induced apoptosis. Accordingly, frataxin-deficient FA cells are more sensitive to Fas, ceramide- and GD3-induced cell death. Taken together, these results suggest a protective role of frataxin in apoptosis.

  • Regulation of macroautophagy by ceramide in mammalian cells.

    Scarlatti F.1,2, Bauvy C.1, Ghidoni R.2 and Codogno P.1

    1INSERM U504, 16 avenue Paul Vaillant Couturier, 94807 Villejuif Cedex, France. Patrice Codogno Tel: 33145595042, Fax: 33146770233, e-mail:

    2Laboratory of Biochemistry and Molecular Biology San Paolo Medical School, via Rudini 8, 20142 Milan Italy.

    Macroautophagy is an evolutionary conserved lysosomal pathway involved in the turnover of long-lived proteins and organelles (1). Macroautophagy starts with the formation of a multilayer membrane bound autophagosome that sequesters fractions of the cytoplasm. In mammalian cells, most of autophagosomes receive inputs from endocytic compartments before fusing with lysosomes where the degradation of the sequestered material is completed. The discovery of a family of genes (ATG: AuTophaGy-related genes. Reviewed in Refs. 2,3) conserved from yeast to humans, and involved in the formation of autophagosomes has shed light on the importance of macroautophagy in physiology (adaptation to starvation, development and longevity) (4) and pathophysiology (cancer, myopathies, neurodegeneratives diseases) (5). The elucidation of the molecular control of autophagy will also lead to a better understanding of the role of macroautophagy during cell death (6). As a great number of extracellular stimuli (starvation, hormonal or therapeutic treatment) and intracellular stimuli (accumulation of misfolded proteins, invasion of microorganisms) is able to modulate the autophagic response, it is not surprising that several signaling pathways are involved in the control of macroautophagy. The class I PI3K/PKB/mammalian Target of Rapamycin (mTOR) signaling pathway plays a major role in transmitting autophagic stimuli because of its ability to sense nutrient, metabolic and hormonal signals (7).

    Macroautophagy is under the control of different classes of phosphatidylinositol 3-kinases (PI3Ks). 3-methyladenine inhibits the formation of autophagosomes by interfering with the activity of the class III PI3K suggesting that the production of PtdIns3P is required during early steps of macroautophagy (8). By contrast the stimulation of the activity of class I PI3K inhibits the autophagic pathway. The production of PtdIns(3,4)P2 and PtdIns(3,4,5)P3recruits the serine/threonine kinase Akt/protein kinase B (PKB) and its activator the kinase PDK1. This signaling pathway which is involved in many cellular processes including proliferation and cell survival is negatively regulated by the tumor suppressor PTEN which dephosphorylates the D-3 position of PtdIns(3,4)P2 and PtdIns(3,4,5)P3. The overexpression of PTEN counteracts the down-regulation of macroautophagy by the stimulation of class I PI3K signaling pathway whereas the expression of a constitutively active form of PKB has an inhibitory effect on macroautophagy (9). More recently we have shown that the sphingolipid ceramide, which is involved many stress responses, upregulates macroautophagy (10). Exogenous C2-ceramide (C2-Cer) stimulates macroautophagy in the human colon cancer HT-29 cells by increasing the endogenous pool of long chain ceramides as demonstrated by the use of the ceramide synthase inhibitor fumonisin B1. Ceramide reverted the class I PI3K-dependent inhibition of macroautophagy, by interfering with the activation of PKB, and stimulated the expression of the autophagy gene beclin 1 (the ortholog of yeast ATG6) (11). Ceramide is also the mediator of the tamoxifen (TAM)-dependent accumulation of autophagic vacuoles in the human breast cancer MCF-7 cells (12). Myriocin, an inhibitor of de novo ceramide synthesis abrogates the accumulation of autophagic vacuoles in TAM-treated MCF-7 cells. Interestingly, the glucosylceramide synthase inhibitor PDMP mimicked the TAM-dependent accumulation of vacuoles. PDMP, TAM and C2-Cer stimulated the expression of beclin 1 whereas myriocin antagonized the TAM-dependent upregulation of beclin 1 and the PKB inhibition by ceramide.

    In conclusion, macroautophagy is controlled by the interplay between protein and lipid signaling. The control of macroautophagy by ceramide provides a novel function for this lipid mediator in a cell process with major biological outcomes.


    1. Klionsky, D. J., and Emr, S. D. (2000) Science 290, 1717-1721.

    2. Ohsumi, Y. (2001) Nat. Rev. Mol. Cell Biol. 2, 211-216.

    3. Klionsky et al. (2003) Dev. Cell 5, 539-545.

    4. Levine, B., and Klionsky, D. J. (2004) Dev. Cell 6, 463-477.

    5. Cuervo, A. M. (2004) Trends Cell Biol. 14, 70-77.

    6. Gozuacik, D., and Kimchi, A. (2004) Oncogene 23, 2891-2906.

    7. Codogno, P., and Meijer, A. J. (2004) in Autophagy (Klionsky, D. J., ed), pp. 26-48., Landes Bioscience, Georgetown, TX.

    8. Petiot et al.(2000) J. Biol. Chem. 275, 992-998.

    9. Arico et al. (2001) J. Biol. Chem. 276, 35243-35246.

    10. Scarlatti et al. (2004) J. Biol. Chem. 279, 18334-18391.

    11. Liang et al. (1999) Nature 402, 672-676.

    12 Bursch et al. (2000) J. Cell Sci. 113, 1189-1198.


  • Mutation of CERKL, a novel human ceramide kinase gene, causes autosomal recessive retinitis pigmentosa.

    Tuson M, Marfany G, Gonzàlez-Duarte R.

    Departament de Genètica. Facultat de Biologia. Universitat de Barcelona, Av. Diagonal 645, E-08028-Barcelona, Catalonia, Spain - Phone: +34934021500. Fax: +34934110969. E-mail:

    Retinitis pigmentosa (RP) is an inherited retinal degenerative disease characterized by the progressive attrition of mature photoreceptor cells through apoptosis, and which eventually results in complete blindness in early adulthood. At present, 38 genes and loci have been implicated in this monogenic, albeit complex, disorder, emphasizing the high genetic heterogeneity of RP. These include genes encoding proteins of the phototransduction cascade, proteins of the visual cycle, proteins responsible for the structure and polarity of the photoreceptors, and regulatory proteins, such as transcription and splicing factors (1). To date, none of the described RP genes have been directly involved in the apoptotic pathways that lead to photoreceptor loss. In the present study, we report a novel RP gene encoding a ceramide kinase enzyme (CERKL), which is responsible for the disease in a Spanish consanguineous family with autosomal recessive retinitis pigmentosa (arRP). Previous linkage analysis in this family allowed us to define a new arRP locus (RP26) within an 11-centiMorgan (cM) interval (17.4 Megabases, Mb) on 2q31.2-q32.3 (2). We further refined the RP26 locus down to 2.5 Mb by microsatellite and single-nucleotide polymorphism (SNP) homozygosity mapping. This 2.5-Mb candidate region harboured nine genes, which were analyzed for mutations, but none showed any pathogenic variant in the patients of this family. Subsequently, an exhaustive gene search based on expressed-sequence-tag (EST) data unveiled two partial sequences corresponding to a novel gene. This new gene spanned 13 exons and encoded a predicted protein of 532 amino acids that shared the highest similarity (29% identity; 50% similarity) with the human ceramide kinase (CERK [3]). Hence, it was named human ceramide kinase–like (CERKL [4]). Database searches also revealed two partial murine sequences, which, upon assembly and conceptual translation, produced a protein that was highly homologous to human CERKL (75% identity; 85.6% similarity). The human CERKL coding exons were sequenced in all members of the RP26 family. Remarkably, all patients were homozygous for a nonsense mutation (R257X; CGAÕTGA) in exon 5, which prematurely truncated the protein within the predicted kinase catalytic domain. RT-PCR analysis revealed that human CERKL is expressed in the retina, among other adult and fetal tissues. In situ hybridization on mouse eye sections showed expression of the mouse CERKL ortholog in the retina ganglion cell layer, and a faint signal was also detected in the inner nuclear and photoreceptor cell layers.

    Nowadays, it is widely accepted that sphingolipid (SL) metabolism plays a fundamental role in the regulation of cell proliferation, differentiation, and apoptosis. Ceramide is at the core of sphingolipid metabolism and is the precursor of complex and bioactive sphingolipids (5). Both the central position of ceramide in SL metabolism and the increasing evidence of its involvement apoptosis highlight the significance of a finely tuned regulation of ceramide levels. Therefore, the enzymes involved in the production and clearance of ceramide must be precisely regulated. Our results suggest that CERKL may play a role in the survival of photoreceptors and other retinal cells. Moreover, recent data support ceramide-1-phosphate as a novel regulator of cell functions (6–8). Within this context, the observation that ceramide-1-phospate prevents the accumulation of ceramide by inhibiting acid sphingomyelinase in macrophages, thus avoiding apoptosis (8), is highly suggestive.

    Our findings are the first genetic report linking retinal neurodegeneration in RP and ceramide-induced apoptosis, highlight SL metabolism enzymes as functional candidates for retinal disorders and establish novel targets for therapeutic intervention of these diseases.


    1. Hims MM, Daiger SP, Inglehearn, CF. Retinitis pigmentosa: genes, proteins and prospects. Dev Ophthalmol. 2003;109-125.

    2. Bayés M, Goldaracena B, Martínez-Mir A, Iragui-Madoz MI, Solans T, Chivelet P, Bussaglia E, Ramos-Arroyo MA, Baiget M, Vilageliu L, Balcells S, Gonzàlez-Duarte R, Grinberg D. A new autosomal recessive retinitis pigmentosa locus maps on chromosome 2q31-q33. J Med Genet. 1998;2:141-145.

    3. Sugiura M, Kono K, Liu H, Shimizugawa T, Minekura H, Spiegel S, Kohama, T. Ceramide kinase, a novel lipid kinase. Molecular cloning and functional characterization. J Biol Chem. 2002;26:23294-23300.

    4. Tuson M, Marfany G, Gonzàlez-Duarte R. Mutation of CERKL, a novel human ceramide kinase gene, causes autosomal recessive retinitis pigmentosa (RP26). Am J Hum Genet. 2004;1:128-138.

    5. Hannun YA, Obeid LM. The Ceramide-centric universe of lipid-mediated cell regulation: stress encounters of the lipid kind. J Biol Chem. 2002;29:25847-25850.

    6. Pettus BJ, Bielawska A, Spiegel S, Roddy P, Hannun YA, Chalfant, CE. Ceramide kinase mediates cytokine- and calcium ionophore-induced arachidonic acid release. J Biol Chem. 2003;40:38206-38213.

    7. Pettus BJ, Bielawska A, Subramanian P, Wijesinghe DS, Maceyka M, Leslie CC, Evans JH, Freiberg J, Roddy P, Hannun YA, Chalfant CE. Ceramide 1-phosphate is a direct activator of cytosolic phospholipase A2. J Biol Chem. 2004;12:11320-11326.

    8. Gómez-Muñoz A, Kong JY, Salh B, Steinbrecher UP. Ceramide-1-phosphate blocks apoptosis through inhibition of acid sphingomyelinase in macrophages. J Lipid Res. 2004;1:99-105.

  • Sphingolipid synthesis as a target for anti-T. Gondii drugs.

    Sonda S. 1, 2 and Pieters J. 2

    1Institut für Parasitologie, Winterthurerstrasse 266a, 8057 Zürich, Switzerland Phone: +41-1-635-8514 Fax: +41-1-635-8907 e-mail:

    2Department of Biochemistry, Biozentrum der Universitaet Basel, 4056 Basel, Switzerland

    Sphingolipids are ubiquitous components of plasma membranes, where they contribute to the functional heterogeneity of the lipid bilayer. In addition, sphingolipids act as second messengers in important signaling systems that regulate a wide range of cellular functions, including growth and cell cycle, apoptosis and differentiation.

    Inositol phosphorylceramide (IPC) is a sphingolipid present in plants, yeast, fungi and some protozoa, but is absent in mammalian cells. The synthesis of IPC is an essential process in fungi, as inhibition of IPC synthase causes arrest of cell cycle progression followed by loss of viability.

    Based on the fact that T. gondii contains an apicoplast, a plant-derived organelle that is involved in lipid synthesis, we tested the effect of Aureobasidin A, a pharmacological inhibitor of IPC synthase, on the parasite. We discovered that Aureobasidin A treatment exerts a significant dose-dependent anti-proliferative effect on T. gondii, without affecting host cell viability. Moreover the presence of the compound inhibits host cell invasion by the parasite.

    We hypothesize that characterization of the IPC biosynthetic pathways present in T. gondii will (1) provide the first indication of a co-existence of plant-type and animal-type sphingolipid pathways in T. gondii, and (2) provide a potential new target for developing drugs specifically affecting T. gondii with low toxicity for humans.


  • Fabry disease: molecular studies in italian patients and x-inactivation analysis in female carriers.

    A. Morrone1, C. Cavicchi1, L. Carraresi1, T. Bardelli1, M.A. Donati1, S. Funghini1, D. Antuzzi2, A. Frustaci2, R. Parini3, M. Di Rocco4, S. Feriozzi5, O. Gabrielli6, R. Barone7, G. Pistone8, C. Spisni9, R. Ricci2, E. Zammarchi1

    1Dip. di Pediatria, Firenze;, fax 055570380, tel. 0555662543; 2Univ. Cattolica, Roma; 3Osp. ICP Milano; 4Ist. Gaslini, Genova; 5Osp. Belcolle, Viterbo; 6Clin. Ped., Ancona; 7Dip. Ped., Catania, 8Dip. di Dermatologia, Palermo; 9Un. Nefrologia, Chieti

    Anderson-Fabry disease is an X-linked lysosomal storage disorder of glycosphingolipid metabolism. This multisystemic disease is caused by a primary deficiency of a-Galactosidase A enzyme (GAL A). The enzymatic GAL A defect leads to accumulation of neutral glycosphingolipids, with terminal linked galactosyl moieties, primarily in vascular endothelium and in tissue lysosomes (kidney cells, neurons, cardiomyocytes).

    Fabry disease can be classified in two clinical phenotypes: the classic and the cardiac form. The former is mainly characterized, in affected hemizygous males, by angiokeratoma, acroparesthesias, hypohidrosis, pains, fever crises involvement of kidneys, brain and heart and sometimes, as the disease evolves, psychological manifestations with personality disturbances. The latter is characterized by late onset cardiomyopathy and conduction defects. Fabry patients with classic phenotype show a marked reduction in GAL A enzymatic activity, while patients with cardiac variant show residual enzymatic activity. A prevalence of Fabry disease in a referential population of male patients with a clinical diagnosis of late onset Hypertrophic Cardiomyopaty (HCM) has also been reported. Female carriers can be asymptomatic or clinically affected, usually with the mild and late onset form of the disease. They show corneal abnormalities as the most frequent clinical manifestations. Up to now more than 300 mutations have been identified in the GLA gene, mapped on Xq22. Recently, a model of the GAL A enzyme was constructed and 245 genetic lesions were plotted. This structural model could be helpful to predict, from the genetic mutation, the phenotype of the disease.

    With the aim of a genotype/phenotype correlation and for better patient care and therapeutic management molecular studies were carried out on 24 Italian Fabry patients. Six new (c126-127insCATG, L167P, c617-618delTT, Q279K, c946delG, de novo A352D,) and eleven known (P40L, c124-125delAT, S78X, C174Y, IVS3+1G>A, N215S, R220X, R227Q, W236C, de novo Y365X, C378Y) mutations were identified in the genomic DNA and/or in total RNA of patients’ GLA gene. An aberrant GLA transcript c486-547del62bp, that leads to an early stop codon, was detected in a male patient carrying the IVS3+1G>A splicing defect. These results confirm molecular heterogeneity also in Italian Fabry patients.

    We also detected GLA gene mutations in 26 females from 19 unrelated families with family history of Fabry disease. Mutation analysis is the only certain method of detecting female carrier status. The diagnosis in male patients can easily be made by enzimatic GAL A assay, while heterozygous females can show normal activity because of X-chromosome inactivation.

    Random X-inactivation has been reported for the GLA gene. X-inactivation studies in two carrier female monozygotic twins showing a discordant expression of the same mutation of GLA gene were also described. Up to now no further studies on X-inactivation have been carried out on manifesting Fabry carriers. In order to determine an eventual correlation with clinical manifestations in Fabry carriers, X-inactivation studies were carried out on peripheral blood DNA of 26 Fabry carriers. Eleven females showed a random X-inactivation pattern and fifteen showed a moderately skewed and/or skewed X-inactivation pattern. Nine manifesting carriers showed a skewed pattern in favour of the mutant allele, while five asymptomatic carriers showed a moderate and/or skewed pattern in favour of the wild type allele. In these carriers the X-inactivation studies performed suggest a correlation between clinical manifestations and the skewing of X-inactivation. Such studies could be helpful to predict female phenotypes and give useful indications for therapeutic management.

    Supported by TKT Europe 5S and Genzyme Corporation


  • Sphingosine 1-phosphate inhibits cell motility of C2C12 mouse myoblasts. A role for S1P2 receptor.

    Becciolini L, Donati C, Nuti F, Farnararo M, Meacci E and Bruni P.

    Dipartimento di Scienze Biochimiche, Università degli Studi di Firenze,Viale G.B. Morgagni 50, 50134 Firenze; Istituto Interuniversitario di Miologia (IIM); tel ++39055413765; fax ++390554222725

    Migration of skeletal muscle precursor cells is a crucial step in skeletal muscle development and post-lesional muscle regeneration. A number of growth factors including insulin-like growth factor-1 (IGF-1), hepatocyte growth factor and platelet derived growth factor have been shown to regulate positively migration of C2C12 mouse myoblasts (1). Recent studies have demonstrated that sphingosine 1-phosphate (S1P), a lipid mediator capable to exert pleiotropic actions in a variety of cell systems, is a quite unique regulator of cell motility, in that it acts as chemoattractant or chemorepellant, depending on the cell type (2). C2C12 myoblasts bear at least three different S1P receptor subtypes, namely S1P1, S1P2, S1P3 (3) and a number of different signalling pathways are initiated by the bioactive lipid in these cells (4). Here we have investigated the effect of S1P on the cell motility of these cells.

    For the experiments C2C12 mouse myoblasts were routinely maintained in DMEM supplemented with 10% fetal calf serum in an atmosphere of 95% air 5% CO2. Cell migration was measured in modified Boyden chamber using polycarbonate filters, 8mm pores coated with Matrigel (4). Cells were harvested by trypsinization, washed with serum-free DMEM and were added to the upper wells at 2x104 cells per well. In some experiments cells were transfected with antisense oligodeoxynucleotides (ODN) to individual S1P receptors (5) or with pcDNA plasmid encoding for S1P2 or short interfering RNA to silence S1P2. Agonists were added to the bottom wells of the Boyden chambers. After 2 h at 37°C the filters were fixed with methanol and successively stained with Diff-Quik. The non-migratory cells on the upper membrane surface were removed and the cells which traversed and spread on the lower surface were enumerated using a Nikon microscope.

    The bioactive lipid (10 nM-1 mM) dose-dependently inhibited random cell motility. Moreover, S1P (1 mM ) exerted a powerful inhibitory effect on the chemotactic response evoked by IGF-1 (10 ng/ml). The inhibition of random motility and of the IGF-1-chemotactic response by S1P resulted to be significantly attenuated in cells loaded with antisense ODN designed to inhibit S1P2 receptor. Interestingly, S1P-induced inhibition of cell motility was appreciably enhanced in S1P2-overexpressing myoblasts. Pertussis toxin treatment (200 ng/ml, 16 h) diminished random cell motility in unstimulated myoblasts but did not affect the S1P-induced biological response, suggesting that the inhibitory effect of S1P does not involve Gi-coupled receptor. These findings are in favour of a major role of signalling downstream to S1P2 receptor in the inhibition of skeletal myoblast motility induced by S1P.



    Chargé SBP and Rudnicki MA. Cellular and molecular regulation of muscle regeneration. Physiol. Rev. 2004; 84: 209-238.

    Takuwa Y. Subtype-specific differential regulation of Rho family G proteins and cell migration by the Edg family sphingosine-1-phosphate receptors. Biochim Biophys Acta. 2002; 1582:112-120.

    Meacci E, Vasta V, Donati C, Farnararo M, Bruni P. Receptor-mediated activation of phospholipase D by sphingosine 1-phosphate in skeletal muscle C2C12 cells. A role for protein kinase C. FEBS Lett. 1999; 457: 184-188.

    Okamoto H, Takuwa N, Yokomizo T, Sugimoto N, Sakurada S, Shigematsu H, Takuwa Y. Inhibitory regulation of Rac activation, membrane ruffling, and cell migration by the G protein-coupled sphingosine-1-phosphate receptor EDG5 but not EDG1 or EDG3.
    Mol Cell Biol. 2000; 20: 9247-61.

    Meacci E, Cencetti F, Formigli L, Squecco R, Donati C, Tiribilli B, Quercioli F, Zecchi Orlandini S, Francini F, Bruni P. Sphingosine 1-phosphate evokes calcium signals in C2C12 myoblasts via Edg3 and Edg5 receptors. Biochem J. 2002; 362: 349-357

  • Sphingosine 1-phosphate metabolism and fate in cells from the nervous system.

    Riboni L, Anelli V, Bassi R, Giussani P, Viani P and Tettamanti G

    Department of Medical Chemistry, Biochemistry and Biotechnology, University of Milan, via Fratelli Cervi, 93, LITA-Segrate, 20090 Segrate, Milan, Italy. E-mail,; fax, +39-02-50330365; phone: +39-02-50330357.

    Sphingosine-1-phosphate (S1P) has long been known as an intermediate of sphingolipid catabolism. In cells, it originates by phosphorylation of sphingosine catalyzed by sphingosine kinase and can be degraded by S1P lyase. It can also be dephosphorylated back to sphingosine by S1P phosphatase. In different cells, including cells from the nervous system, S1P has emerged as important regulator of different cellular processes including proliferation, shape and apoptosis. This sphingoid molecule is able to act both as intracellular and intercellular messenger, and different membrane receptors for it are expressed in neurons and astrocytes. Notwithstanding this, little is known on S1P metabolism and turnover in cells of the nervous system and in particular on its origin and fate in the extracellular compartment.

    In this study we investigated S1P metabolism in different cells from the nervous system, including glial, neurons and brain-derived endothelial cells. To this purpose we first performed experiments by feeding cells with radiolabeled sphingosine at different doses (nM-µM). After short time pulse, 3H-sphingosine was rapidly internalized into cells in a similar fashion in all cell types; radioactive S1P was rapidly produced. The amount of 3H-sphingosine that was submitted to phosphorylation varied among cell types and increased with sphingosine doses. In all cells, 3H-S1P was submitted to degradation, detected as tritiated water in the medium. This process represented the major fate of the newly synthesized S1P in both glial and endothelial cells. In these conditions, cell medium analysis revealed the presence of extracellular S1P in all cell types. No sphingosine kinase activity was detectable in the medium, supporting that the different cells can release S1P in the extracellular compartment. In different cells, growth factors and phorbol esters stimulated sphingosine phosphorylation, without affecting its degradation. In parallel, an increase of extracellular S1P was also observed. The administration of the S1P lyase inhibitor 4-deoxypyridoxine caused a marked reduction of S1P degradation, paralleled by an increase of cellular S1P. However, this inhibitor did not affect the levels of extracellular S1P. Within the different cell types, a brain-derived endothelial cell line was found the most efficient in S1P secretion, suggesting a possible role for these cells in contributing to the extracellular S1P levels in the nervous system.

    In further experiments, aiming to evaluate the turnover of extracellular S1P, 3H-S1P (50nM-5µM) was administered to cells for different times up to 1h. At all concentrations, 3H-S1P was efficiently taken up in a time-dependent fashion and about 40% of the administered radioactivity was found incorporated into cells at 1h pulse. However, intracellular 3H-S1P represented only a minor portion of the cell-associated radioactivity, the majority being associated to N-acylated sphingolipids (prevailing at lower doses), and tritiated water (prevailing at higher doses). In the presence of deoxypyridoxine, no difference in the uptake of S1P was detectable even if a marked decrease in radioactive water was observed. In this condition cellular S1P accumulated, accounting for about 50% of total uptake.

    Collectively, our results demonstrate that cells from the nervous system are able to: a) regulate their S1P levels in a rapid and efficient manner; different enzymes characterized by different capacity and localization appear to be involved; to b) release in (and take up from) the extracellular compartment S1P; their metabolic equipment is also implicated in the rapid turnover of extracellular S1P.

    Grant support: Italian Ministry of University, Scientific and Technological Research PRIN 2002 to L.R.

  • Sphingolipid metabolism in mitochondria and MAM purified from rat liver.

    1Ardail D, 2Popa I, 1Bionda C, 2Portoukalian J

    1Laboratory of Radiobiology, Lyon-Sud Medical School, 2Department of Dermatology, Edouard Herriot Hospital, 69437 Lyon Cx 03, France (Portoukalian J, phone +33-4-72110607; fax +33-4-72110290; e-mail :

    GSLs biosynthesis starts with the formation of ceramide in the endoplasmic reticulum (ER) which is transported by controversial mechanisms to the Golgi apparatus where stepwise addition of monosaccharides onto ceramides takes place. We have reported the presence of GSLs biosynthetic enzymes in a subcompartment of the ER previously characterized and termed as “mitochondria-associated-membrane” (MAM). MAM is a membrane bridge between the ER and mitochondria that is involved in the biosynthesis and trafficking of phospholipids between the two organelles. Using exogenous acceptors coated on silica gel, we demonstrate the presence of ceramide-glucosyl transferase (Cer-Glc-T), glucosyl-ceramide-galactosyl transferase (Gal-T1) and sialyl transferase activities in the MAM. These enzymatic activities were found to represent at least 65% of the activities recovered in the purified Golgi apparatus, although the cross-contamination of MAM by Golgi membranes did not exceed 7.5%. Sialyl transferases activities in MAM led to the synthesis of GM3 ganglioside and small amounts of GD3.. We show that purified mitochondria as well as MAM are able to generate ceramide in vitro through both ceramide synthase or reverse ceramidase, whereas the latter enzyme activity is barely detectable in microsomes. Moreover, ceramide synthase activities were recovered in outer mitochondrial membranes as well as in inner mitochondrial membranes. Using radiolabeled sphingosine as a substrate, mitochondria could generate ceramide and phytoceramide. However, the in vitro sensitivity of ceramide synthase toward fumonisin B1 (FB1) in mitochondria as well as in MAM was found to depend upon the sphingoid base : whereas dihydrosphingosine-N-acyltransferase was inhibited by FB1 in a concentration-dependent manner, FB1 actually activated the ceramide synthase when using sphingosine as a substrate. Acylation of sphingosine-1-phosphate and dihydrosphingosine-1-phosphate generating ceramide-1-phosphate was also shown with both subcellular fractions. Moreover, the same difference in sensivity toward FB1 for the ceramide synthase activities was seen between the two phosphorylated sphingoid bases, raising the possibility that distinct base-specific enzymes may be involved as ceramide synthases.

  • Sphingosine-1-phosphate and liver fibrosis: friend or foe?

    Sophie Lotersztajn

    INSERM U581, Hôpital Henri Mondor, 94010 Créteil, France.

    Liver fibrosis is the common response to chronic liver injury, and is characterized by increased deposition and altered composition of extracellular matrix. Its progression leads tocirrhosis and its complications (portal hypertension, hepatic failure and hepatocarcinoma), responsible for a high morbidity and mortality. The fibrogenic process is consecutive to migration of the cells to the lesions, intense proliferation and hepatic accumulation of myofibroblasts that synthetize fibrosis components and inhibitors of matrix degradation. These cells also contribute to the development of portal hypertension, due to their contractile properties. We have recently focussed on the role of S1P on the liver fibrogenesis, and used a model of cultured human hepatic myofibroblasts, that we obtain by outgrowth of explants prepared from surgical specimens of normal human liver.

    We have identified S1P1, S1P2 and S1P3 mRNAs in human hepatic myofbroblasts. These receptors are functional and coupled to pertussis toxin (PTX)-sensitive and insensitive G proteins, as demonstrated in GTPgS binding assays. S1P exhibited pro and antifibrogenic properties of human hepatic myofibroblasts.

    Antifibrogenic properties of S1P included inhibition of proliferation, migration and of procollagen I mRNA expression, as well as induction of apoptosis. These effects were reproduced by dihydro S1P, indicating a receptor-dependent mechanism. Analysis of the pathways involved revealed that inhibitory effects of S1P on proliferation and migration by S1P were mediated by PGE2 derived from cyclooxygenase-2 (COX-2). Indeed, S1P potently induced COX-2 protein expression. Blocking COX-2 by NS-398, a selective COX-2 inhibitor, blunted both the inhibition of proliferation and of migration by S1P, but did not affect procollagen I mRNA expression. In addition, S1P induced apoptosis of human hepatic myofibroblasts, but this occurred via a receptor-independent mechanism, since dihydro-S1P did not affect hepatic myofibroblast viability.

    In contrast, S1P elicited profibrogenic properties, since the sphingolipid stimulated a receptor-dependent survival pathway. This antiapoptotic effect of S1P was PTX-sensitive and was mediated by simultaneous, but independent activations of ERK and of PI3Kinase/Akt. In addition, activation of S1P receptors triggered PTX-dependent contraction of human hepatic fibroblasts, that followed increase in intracellular calcium.

    In conclusion, our results identify S1P as a novel regulator of liver fibrogenesis. The sphingolipid shows both anti and profibrogenic properties. On the one hand, S1P is antifibrogenic since it blunts proliferation, migration and procollagen I mRNA expression via a receptor-dependent pathway, and is proapoptotic via a receptor-independent mechanism. However, S1P is also profibrogenic, by promoting cell survival via a receptor-dependent mechanism. Finally, the contractile properties of S1P suggest that the sphingolipid, by regulating sinusoidal tone, may contribute to the pathogenesis portal hypertension during chronic liver diseases. Elucidation of the S1P receptors that mediate the pro and the antifibrogenic properties of S1P is underway.

  • Role of sphingosine 1-phosphate on NADPH oxidase activation and redox regulation of c-Src in NIH3T3 fibroblasts.

    Catarzi S., Biagioni C, Favilli F., Giannoni E., Iantomasi T. and Vincenzini M.T.

    Department of Biochemical Sciences, University of Florence, Viale Morgagni 50, 50134 Florence, Italy. M.T. Vincenzini: Fax 055/4222725; Tel.055/416686; e-mail:

    Sphingosine 1-phosphate (S1P) belongs to groups of platelet-derived lipid mediators that regulate various biological effects. Extracellular S1P can act through specific G-protein coupled receptors (S1PRs) of the endothelial differentiation gene (EDG) family, in various types of cells including NIH3T3 fibroblasts. In these cells, intracellular S1P is also related to PDGF mitogenic signalling as second messenger and PDGF-induced cell motility by autocrin mechanism of S1PRs stimulation (1). Our previous data demonstrated that S1P and H2O2 production induced by PDGF stimulation are related to tyrosine autophosphorylation of PDGFr (2). Indeed, reactive oxygen species (ROS) such as O2-and H2O2 are signal mediators of various factors including cytokines and growth factors. A multicomponent NADPH oxidase is the enzyme mainly involved in production of ROS (3) and it is widely studied in phagocyte cells, whereas few data on activation mechanisms of this enzyme in non-phagocyte cells are reported.

    The results of this study demonstrate a time dependent increase of H2O2 content in NIH3T3 fibroblasts after S1P stimulation measured by a fluorescence-based assay with DCF-DA as compared with non-stimulated cells. S1P-induced H2O2 production decreases remarkable in cells pretreated by DPI or AEBSF, specific inhibitors of flavine oxidase and NADPH oxidase respectively, indicating the involvement of this enzyme. Subsequently, we demonstrated using specific inhibitors that also phosphatidylinositol-3-kinase (PI3K) and protein kinase C (PKC) activities, enzymes involved in S1P signalling, are related to the activation mechanisms of NADPH oxidase due to S1P stimulation. Indeed, both PI3K and PKC can contribute to the translocation and assembly of various components of NADPH oxidase in phagocyte cells stimulated by cytokines or other factors, but to date there are not data in non-phagocyte cells after S1P stimulation. Experiments with pertussis toxin, which inactivates Gi-coupled S1PRs, showed that S1P induces H2O2 production through the stimulation of these receptors.

    Considering that c-Src, non-receptor tyrosine kinase, is related to both PDGFr and S1PRs signalling (4, 5) and is involved in signal events stimulated by ROS (6), we studied the effect of H2O2 production due to S1P stimulation on c-Src activation, in order to clarify the relationship previously found among S1P, H2O2 production and PDGF-induced tyrosine phosphorylation.

    First results obtained by catalase treatment demonstrate in NIH 3T3 overexpressing c-Src that S1P-induced H2O2 generation in the presence of vanadate (inhibitor of tyrosine phosphatase) contributes to full c-Src phosphorylation and activation as compared with vanadate treated cells alone. c-Src activation is detected by immunoblotting with specific antibody of c-Src activated form that is tyr-416- phosphorylated. The treatment with inhibitors of NADPH oxidase (DPI and AEBSF) decreases c-Src autophosphorylation, suggesting the involvement of this enzyme in S1P-induced c-Src activation. To confirm a possible redox regulation of c-Src kinase activity, we exposed NIH3T3 overexpressing c-Src to 0.5 mM H2O2 for 30 min in the presence of vanadate. The results obtained show under these conditions the phosphorylation and activation of c-Src. Furthermore, no inhibition of tyr 416-phosphorylated c-Src was observed after treatment with the inhibitors of NADPH oxidase, PI3K and PKC activity indicating that S1P activation of these enzymes is an upstream event of c-Src activation. These data demonstrate that S1P and its receptors are involved in NADPH oxidase activation and redox regulation of c-Src kinase activation and these events can be important factors for the cross communication between receptor tyrosine kinase and G-protein coupled receptors.


    1. Hobson JP, Rosenfeldt HM, Barak LS, Olivera A, Poulton S, Caron MG, Milstien S, Spiegel S. Role of the sphingosine-1-phosphate receptor EDG-1 in PDGF-induced cell motility. Science. 2001; 291:1800-1803.

    2. Catarzi S, Iantomasi T, Biagioni C, Favilli F, Vincenzini MT Sphingosine-1-phosphate receptor involvement on the tyrosine phosphorylation of PDGF receptor in NIH3T3 fibroblasts. Fifty European Symposium of the protein society2003, Florence 29 Marzo -2 Aprile Abstract

    3. Finkel T Oxidant signals and oxidative stress Curr. Opin. Cell Biol. 2003; 15, 247-254

    4. Rakhit S, Conway AM, Tate R, Bower T, Pyne NJ, Pyne S. Sphingosine 1-phosphate stimulation of the p42/p44 mitogen-activated protein kinase pathway in airway smooth muscle. Role of endothelial differentiation gene 1, c-Src tyrosine kinase and phosphoinositide 3-kinase. Biochem J. 1999; 338:643-649

    5. Conway AM, Rakhit S, Pyne S, Pyne Platelet-derived-growth-factor stimulation of the p42/p44 mitogen-activated protein kinase pathway in airway smooth muscle: role of pertussis-toxin-sensitive G-proteins, c-Src tyrosine kinases and phosphoinositide 3-kinase. Biochem J. 1999; 337:171-177.

    6. Yoshizumi M, Abe J, Haendeler J, Huang Src and Cas mediate JNK activation but not ERK1/2 and p38 kinases by reactive oxygen species. J Biol Chem. 2000; 275:11706-11712.

    This work was supported by grants from MIUR and by a donation from the Cassa di Risparmio di Firenze


  • Characterization of the ceramide moieties of sphingoglycolipids containing sphingadienine fromhuman brain with Alzheimer's disease by ESI-MS/MS.

    Colsch B 1,2, Afonso C 2,Fournier F 2, Portoukalian J 3, Tabet JC 2, Baumann N 1.

    1INSERM U495, Paris, France; 2CNRS UMR 7613, Paris, France ;3INSERM U346, Lyon, France.

    Mailing address: Nicole Baumann INSERM U 495, Salpetriere Hospital, 75651 Paris cedex 13; Fax: 33 1 4584 8008; Tel: 33 1 4216 2155 ; e-mail:

    We have shown previously by ESI tandem mass spectrometry, that adult mouse brain contains ceramides from galactocerebrosides and sulfatides with both sphingosine and sphingadienine [1]. In this study we were interested in using a similar methodology for a comparative analysis of sphingoglycolipids present in humain brain with Alzheimer’s disease.

    Lipids extracts were obtained by chloroform-methanol extraction, and Folch partition from human brain. The upper phase containing the gangliosides and a sulfatide fraction were further purified on C18-silicic acid cartridges followed by RP-HPLC separation. The lower phase containing galactocerebrosides and free ceramides were purified using LC-NH2 cartridges. Control of the fractions purity was carried out by HP-TLC. Mass spectrometry characterization was performed by electrospray ionization using both negative and positive mode. An ESI-ion trap mass spectrometer (Bruker Esquire 3000) was used in MSn in order to characterize ceramide structures. A triple quadrupole instrument (Micromass Quattro) was employed in parent and neutral loss scan mode to obtain additional information.

    By using the same methology that was used in our previous study on mouse brain [1], we have found that sphingadienine is present in human brain ceramide moieties of the sphingoglycolipids. The relative fatty acid composition is different than what is observed in the mouse with a lower proportion of short chain fatty acids [C16:0 to C22:0 (OH)] and a greater proportion of very long chain fatty acids [C24:1 to C26:0 (OH)]. In Alzheimer's disease, a reduction of the most abundant sulfatide species and an increase of ceramides in the total lipid extract has been observed [2]. After purification of the isoforms of sulfatides, galactocerebrosides and ceramides, we have confirmed that the sulfatide abundance was decreased. It concerns both the sulfatides with sphingosine and those with sphingadienine. We have also observed an increase of various ceramide isoforms.


    [1] Colsch B, Afonso C, Popa I, Portoukalian J, Fournier F, Tabet JC, Baumann N. Characterization of ceramide moieties of sphingoglycolipids from mouse brain by electrospray-tandem mass spectrometry : identification of ceramides containing sphingadienine. J Lipid Res. 2004, 45: 281-286

    [2] Han X, Holtzman D, McKeel D, Kelley J, Morris J. Substantial sulfatide deficiency andceramide elevation in very early Alzheimer’s disease : potential role in disease pathogenesis. J Neurochem 2002, 82 : 809-818.


  • Cytosolic sialidase Neu2 upregulation during PC12 cells differentiation.

    F. Colombo, A. Fanzani, R. Giuliani, M. Bresciani, A. Preti, and S. Marchesini

    Unit of Biochemistry, Department of Biomedical Sciences and Biotechnology, University of Brescia, Italy.

    The cytosolic sialidase Neu2 is known to be involved in myoblast differentiation (1,2). Despite its relative abundance in skeletal muscle, cytosolic sialidase was found also in other tissues, such as liver (3), thymus (4) and, although at low levels, brain (5).

    RT-PCR experiments and "gene reporter" analysis of a 1.4 Kb fragment of Neu2 rat promoter were performed to obtain the expression levels of Neu2 sialidase in PC12 cells, a favored model to study neuronal differentiation. As a consequence, a Neu2 transcriptional induction during nerve growth factor (NGF), fibroblast growth factor 2 (FGF-2) and epidermal growth factor (EGF) treatments was found in this cell line, whereas the Neu2 transcript was absent in untreated cells. In addition, the expression analysis of four Neu2 deleted promoters revealed a remarkable increase of luciferase activity in NGF treated PC12 cells, suggesting that the Neu2 transcriptional level is highly regulated. Interestingly, the major increase of luciferase activity was found after deletion of a fragment containing a rat dispersed repetitive element, a genomic element able in some cases to modulate transcription and translation efficiency (6).

    Enzymatic assays revealed a cytosolic sialidase activity only during the early steps of NGF differentiation at days 1-2 of treatment, and the low enzymatic activity strictly correlated with the transcript expression levels observed. As a consequence, PC12 cells stably transfected with an expression vector harboring the rat Neu2 cDNA showed a sialidase activity up to 25-times higher than the NGF induced endogenous activity. These data suggest a possible involvement of cytosolic sialidase Neu2 in differentiation of PC12 cells (7).


    [1] Sato, K. and Miyagi, T.(1996) Bioch. Bioph. Res. Comm. 221, 826-830.

    [2] Fanzani, A., Giuliani, R., Colombo, F., Zizioli, D., Presta, M., Preti, A., Marchesini S. (2003) FEBS Lett. 547, 183-8.

    [3] Miyagi, T. and Tsuiki, S.(1985) J. Biol. Chem. 260, 6710-6.

    [4] Kotani, K., Kuroiwa, A., Saito, T., Matsuda, Y., Koda, T., Kijimoto-Ochiai, S.(2001) Biochem. Biophys. Res. Commun. 286, 250-8.

    [5] Hasegawa, T., Feijoo Carnero, C., Wada, T., Itoyama, Y., Miyagi, T.(2001) Biochem. Biophys. Res. Commun. 280, 726-32.

    [6] Landry, J.R., Medstrand, P., Mager, D.L. (2001) Genomics 76, 110-6.

    [7] Fanzani, A., Colombo, F., Giuliani, R., Preti, A., Marchesini S. (2004) FEBS Lett. 566, 178-82.

  • IGFBP-3 is a survival factor for human endothelial cells and promotes in vitro angiogenesis through activation of sphingosine kinase.

    Riccarda Granata1, Letizia Trovato1, Giusy Sala2, Giovanni Garbarino1, Riccardo Ghidoni2, Ezio Ghigo1

    1Dept Internal Medicine, Unversity of Turin; 2Laboratory of Biochemistry and Molecular Biology San Paolo Medical School, Milan, Italy

    Insulin-like growth factor binding protein-3 (IGFBP-3), the major carrier of IGF, exerts either growth promoting or growth inhibiting effects in different cell types. We have previously found that IGFBP-3 inhibits human umbilical vein endothelial cell (HUVEC) apoptosis induced by serum starvation by decreasing the proapoptotic sphingolipid ceramide. Moreover IGFBP-3 was found able to enhance cell motility and to increase IGF-I release, suggesting a new role in endothelial cell survival and angiogenesis. Herein we show that IGFBP-3 (1000 ng/ml) promoted HUVEC survival and inhibited apoptosis in serum-free conditions by activating the enzyme sphingosine kinase (SK). SK catalyses the formation of sphingosine-1-phosphate (S1P), a lipid second messenger implicated in the stimulation of cellular proliferation and inhibition of apoptosis. N,N-dimethylsphingosine, an inhibitor of SK potently blocked IGFBP-3 survival effect, suggesting that IGFBP-3 acts through the SK signaling pathway. Furthermore, IGFBP-3 stimulated the IGF-I receptor (IGF-IR) tyrosine phosphorylation and IGFBP-3 antiapoptotic effect was blocked by the IGF-IR tyrosine kinase inhibitor tyrphostin AG1024. IGFBP-3 even activated the survival pathways PI3K/Akt and ERK which are known to be involved in both IGF-I and S1P signaling. Finally we show that IGFBP-3 dose-dependently induced in vitro formation of vessel-like tubular structures by HUVEC in growth factor-reduced Matrigel. In all, our findings demonstrate that IGFBP-3 promotes endothelial cell survival and inhibits apoptosis via the sphingolipid pathway down-regulating ceramide and activating SK to increase S1P. These effects are likely mediated by the IGF-IR signaling pathway.Enhancement of HUVEC motility and induction of vessel-like structure formation suggest a role for IGFBP-3 to promote angiogenesis.

    Study supported by MURST, University of Turin and SMEM Foundation


  • Resveratrol impairs the 3D aggregation of MDA-MB-231 through a ceramide-mediated mechanism.

    Ersilia Dolfini1, Leda Roncoroni1, Giusy Sala2, Paola Signorelli2, Elena Dogliotti1 , Nicoletta Sacchi3, Riccardo Ghidoni2

    1Dept Biology and Genetics, Univ Milan, Milan, Italy, 22Laboratory of Biochemistry and Molecular Biology San Paolo Medical School, Milan, Italy, 3Dept Cancer Genetics, Roswell Park Cancer Institute, Buffalo, NY-

    Resveratrol, a polyphenol present in grapes and wine, exerts a drastic growth-inhibitory effect on the human breast cancer MDA-MB-231 cell line grown in the 2D-culture. We recently described the mechanistic relationship between growth inhibitory/pro-apoptotic effect of resveratrol and de novo synthesis of ceramide [Scarlatti et al FASEB J (2003) 17,2339-41].

    Here we report that ceramide mediates the growth inhibitory effects of resveratrol in MDA-MB-231 cells grown in three-dimensional (3D) cultures. MDA-MB-231 when grown in 3D-cultures can form multicellular spheroids (MCSs). First we treated fully grown MCSs with resveratrol (32 mM, 48 hours) and observed an accumulation of ceramide in the MCSs, in concomitance with the disruption of the most external spheroid cell layer(s) and loss of the clonogenic potential.

    Second, we observed that treatment of MDA-MB-231 cells with 32 mM resveratrol significantly impaired the formation (number and size) of 3D-MCSs. This effect correlated with a time-dependent accumulation of endogenous ceramide. In addition, cells disaggregated from the few MCSs that were able to form MCSs after resveratrol treatment lost their clonogenic potential.

    Thus, resveratrol can both reduce full-grown MCSs and prevent the formation of MCSs by triggering ceramide-dependent pathways. These results indicate that resveratrol-ceramide signaling is indipendent from the sovracellular culture level.

  • Sphingomyelin metabolism modifications in response to the effect of high neutron energy.

    Perrella G.°, Cataldi S.*, Toller M°., Meli A°., Ranaldi F•, Messina C#., Viola Magni M.P.* and Albi E.*.

    °Department of Experimental and Clinical Pathology and Medicine University of Udine.

    *Department of Biochemical Science and Molecular Biotechnology, Physiopathology, Perugia.

    •Department of Biochemical Science University of Firenze

    #Dipartimento di Biologia Marina Universita di Trapani.

    Classic radiation dosimetry, performed with traditional physical methods, does not evaluate the kinetics of radiation absorbance or the biological response(s) to radiation. Those are key aspects in biological systems and cells, which possess elaborate radiation response mechanisms (DNA repair, cellular components turnover, apoptosis, etc.). Biological dosimetry on eukaryotic cell cultures provides nowadays elaborate experimental targets very close to animal and human organs or organisms, which can be used as radiation target systems. In this study, we used FRTL5 cells, strain of normal and differentiated follicular thyroid cells, well characterized, presently an international standard for bioassay of autoimmune thyroid diseases. The FRTL5 cell strain permanently expresses in vitro most of the in vivo tissue-specific thyroid functions, such as for Thyrotropin (TSH) sensitivity for Thyroglobulin synthesis and secretion, Iodide active transport, Peroxidase production, etc. The aim of this study is to investigate the sphingomyelin metabolism modifications in response to neutron irradiation in under the following different physiological conditions: 1) in proliferative state, under Thyrotropin effect. 2) in non proliferative state, in TSH-deprived culture medium. In both conditions the FRTL5 cells have been exposed to neutron irradiation (558 mSv/h) in the laboratory of CERN of Gineva. Moreover for both conditions non irradiated FRTL5 cells have been used as control. The obtained results show that: 1) In the cellular homogenate, exposure of FRTL5 cells to neutron irradiation appears to strongly inhibit the neutral sphingomyelinase activity and to slowlyinhibit sphingomyelin synthase and phosphatidylcholine-dependent phospholipase C activity only in proliferative state. 2) At nuclear level, exposure of FRTL5 cells to neutron irradiation appears to increase significantly the neutral sphingomyelinase activity and to inhibit the phosphatidylcholine-dependent phospholipase C activity in non proliferative cells. No significant effects are reported for sphingomyelin-synthase. We can speculate that the different responses of the high neutron energy on the cellular and nuclear sphingomyelinase and phosphatidylcholine-dependent phospholipase C activities are due to the presence of different isoforms with different functions. In the active proliferating cells the sphingomyelinase produced in the homogenate decreases whereas in the non proliferating cells there is an increment in synthesis of sphingomyelinase in the nucleus and also an inhibition of phosphatidylcholine-dependent phospholipase C activity. In non proliferative cells the data show an incrementof nuclear ceramide and a decrement of diacylglycerol pool, probably involved in cell deth. The correct balance ofceramide and diacylglycerol in the nucleus is essential for cell viability. Alteration in this balance could be related to apoptosis.

  • Dexametasone reduces cell growth via nuclear sphingomyelin metabolism.

    Tringali Sabina, Tringali Anna Rita, Viola Magni MP,Albi E

    Department of Biochemical Sciences and Molecular Biotechnology, Physiopathology, Policlinico Monteluce, 06100, Perugia.

    Corticosteroids have effects on the function of most cells. In the whole animal deficiency or chronic excess is incompatible with survival. Glucocorticoid hormones have been implicated as regulators of T-lymphocyte growth and differentiation e/o apoptosis. Recently studies show that dexamethasone acts via sphingomyelin (SM) metabolism. In fact, it increases SM content, regulates the SM-synthase and sphingomyelinase (SMase) activity. The biological effects of dexamethasone are generally thought to be mediated by an intracellular receptor protein which transduces the hormone signal to the nucleus and participates directly in gene regulation. Since inside the nucleus the metabolism of the SM in relation to cell function has been described, the aim of this work is to establish the possible effects of dexamethasone on cellular and nuclear SM metabolism in tumour cells. At this end, the lymphoma cells (SUP-T1) were seeded in RPMI 1640 in the presence of 10% fetal bovine serum, at a concentration of 100,000/ml and cultivated until 6 days in a 5% CO2 humid atmosphere.Two lots of 5 ml were prepared: one control and one cultivatedin the presence of dexamethasone (500mg/ml). The cell growth was monitored and theSMase and SM-synthase assayed in the homogenate and nuclei.

    The results show a growth inhibition of 66% after dexamethasone treatment respect to the control cells. In the same cells an increase of homogenate SMase activity reachs a peakafter 2 hours when the SM-synthase activity is inhibited. The nuclear SMase activity show aprogressiveinhibition between 2 and 24 hours. It is possible that the dexametasone inhibits the cell growth acting not only on cellular sphingomyelin metabolism enriching early the ceramide pool but also on nuclear SM. The inhibition of nuclear SMase induces a reduction of nuclear ceramide pool and an increase of SM. Since it may be postulated that SM acts by stabilising chromatin structure, it is possible that changes in nuclear SMase activity may be involved in the regulation of cell cycle not through the action of ceramide production but through the influence of SM at the level of chromatin structure. After dexamethasone treatment, SM could contribute to favour chromatin clamps formation in apoptotic process.

  • Cholesterol/sphingomyelin relationships in oncohaematologic patients.

    Pugliese L., Bernardini I., Viola Magni MP,Albi E.

    Department of Biochemical Sciences and Molecular Biotechnology, Physiopathology, Policlinico Monteluce, 06100, Perugia.

    Cholesterol (CHO) is a molecule widely distributed in living organisms. It is located mainly at the level of cell membranes in the unesterified form and in the blood as a component of lipoproteins mainly in the esterified form and it has a role in many physiological and pathological processes. Numerous studies have shown that a strong interaction exists between unesterified CHO and sphingomyelin (SM) which arises from the van der Waals interaction between CHO and the saturated lipid acyl chains. The cellular concentrations of SM and CHO are positively correlated in several pathological and experimental conditions. The CHO-SM relation suggests a mechanism for sterol distribution in which sterol levels are determined by the intrinsic characteristics of each membrane. Epidemiologic studies have indicated a relation between lipids and cancer and abnormal blood lipid profiles have been reported in human malignancies. Statistically significant values of CHO were reported in oncohaematologic patients. A significant increase in CHO levels was observed in all patients responding to therapy. The aim of this work was to highlight if the low CHO levels in patients with Monoclonal Gammopathy are accompanied by modification in the SM level. Blood samples of hospitalized patients with Monoclonal Gammopathy with low CHO level (Experimental) were collected. Patients with Monoclonal Gammopathy with a normal CHO level were used as control (Control) . Serum samples were stored at -20°C until used. The level of CHO and SM was measured in untreated and sphingomyelinase treated samples. The results show that the serum CHO and SM levels in experimental patients are 55% and 61% compared to control. After digestion of SM with SMase in both group of patients a decrease in CHO and an increase in CHO esters, was observed. The effect of SMase on SM hydrolysis was low. After three washings of the inorganic phase with chloroform/methanol and solvent containing NaCl, an increase of CHO and CHO was reported; the values were higher in experimental samples.It is possible that in the patients with Monoclonal Gammopathy there is a metabolic and structural modification of the sphingomyelin which forms a pathological complex with CHO influencing the effect of the therapy.


  • Lipid rafts in hepatocyte nuclei.

    Villani M.*, Cascianelli G.*, Tosti M.#, Viola Magni MP.*, Albi E.*

    *Department of Biochemical Sciences and Molecular Biotechnology, Physiopathology, Policlinico Monteluce, 06100 Perugia, Italy. e-mail:

    #Dipartimento scienze biopatologiche veterinarie, Università di Perugia.

    Lipid composition has been described as being responsible for the presence of domains in the membrane. The main role in the process of domain formation is played by cholesterol (CHO) and sphingolipids.These cholesterol-enriched domains may be stabilised by the presence of saturated alkyl chains of sphingomyelin. Recent biophysical data have provided clues to show that changes in membrane structure are a critical function. Lipid raft microdomains were conceived as part of a mechanism for intracellular trafficking of lipids and lipid-anchored proteins.The composition and function of membrane rafts can be modulated in response to a variety of factors and stress conditions. In fact the membrane rafts are the specific sites for ceramide generation in response to various agonists. Aida et al. (2002, 1) suggest that sphingomyelin-cholesterol microdomains or rafts form a stable lipid matrix, which act as an ordered support for receptor-mediated signaling events. Recently we have demonstrated, inside the nucleus, that the amount of cholesterol is similar to that of sphingomyelin, and it increases in chromatin digested with exogenous sphingomyelinase or proteinaseK, suggesting the existence of a chromatin CHO-SM linked fraction (2).

    Moreover, during liver regeneration, an increase in chromatin cholesterol was observed between 6 and 18 hours after hepatectomy, when the neutral-sphingomyelinase activity increases and the sphingomyelin-synthase is inhibited. In the present work the first aim was to isolate the lipid rafts from hepatocyte nuclei. After nuclei Triton X-100 treatment, isolation of lipid rafts by sucrose density gradient centrifugation was performedaccording to Danielsen (1995, 3). On isolated rafts, the lipid, sphingomyelinase activity and the electron microscope activitywere studied. The results show that the morphology of lipid rafts is similar to that previously reported for microvillar membrane and they are enriched with spherical vesicles, suggesting that these are the mechanism for intranuclear molecule transport in relation to cell function. The nuclear lipid rafts contain CHO and SM and a high activity of sphingomyelinase is detected. Since the nuclear SM, its metabolites and CHO play a role in inducing cell proliferation or apoptosis, it is possible that their function is due to microdomain organization.

    1) Aida et al., (2002) FEBS Letters 531, 47-53

    2) Albi and Viola Magni (2002) Journal of Hepatology 36, 395-400

    3) Danielsen (1995)Biochemistry 34, 1596-1605

  • Effect of protein kinase C delta on nuclear sphingomyelin metabolism.

    La Porta C.A.M.*, Cataldi S., Lazzarini, R, Viola Magni,MP., Albi E.

    *Department of Biomolecular Science and Biotechnology, University of Milan; Department of Biochemical Sciences and Molecular Biotechnology Physiopathology, Policlinico Monteluce, 06100 Perugia, Italy. e-mail:

    Nuclear sphingomyelin metabolism changes during cell proliferation and apoptosis due to modifications in sphingomyelinase (SMase) and sphingomyelin-synthase (SM-synthase) activity (1). The first enzyme hydrolyses sphingomyelin (SM) to produce ceramide, and SM-synthase forms SM by using the phosphorylcholine of phosphatidylcholine (PC) so producing diacylglycerol (DAG). Until now the mechanism of SM metabolism modification in relation to cell function has not been described. Since the protein kinase C delta (PKC delta) has an inhibitory effect on SMase activity (2), in fact specific antibodies anti PKC delta increase the SMase activity significantly, and SM-synthase activation attenuates SMase-induced signalling (3), the present research was aimed at investigating the effect of PKC delta overexpression on nuclear SM metabolism in relation to the induced apoptotic process. At this end we chose B murine melanoma cells (BL6), that do not express the PKC delta and in which overexpression of this enzyme was induced, as the experimental model (4). The overexpression of such an isoform induces a slight increase of cells into apoptosis and an increase of arrested cells into G0/G1 (4). Radioactive PC and SM were used as precursors for evaluating either PC-PLC or SM-synthase and SMase respectively. Incubation of cell homogenate showed no significant modifications of PC-PLC, SMase and SM-synthase activities whereas in the nucleus inhibition of SMase and activation of SM-synthase were demonstrated in transfected cells. No modifications were found for nuclear PC-PLC activity. In chromatin, isolated from the tumoral mass, the overexpression of PKC delta determined the PC-PLC and SMase inhibition whereas the SM-synthase reached a higher level of the activity than that found in the isolated nuclei. Therefore the PC content decreased and the SM content increased. The decrease in PC was due mainly to the SM-synthase activity whereas the pathway through PC-PLC is inhibited. In conclusion, the overexpression of PKC delta, which in murine melanoma cells shifts from the cytoplasm to the nucleus in relation to cell cycle (5) determines an increase in SM content in whole nuclei, so probably modifying the nuclear membrane structure and a greater increase in chromatin SM which appears to be related to protein synthesis present in the apoptotic process.

    1) Albi,E. et al.. J. 2003 Cell Physiol. 196:354-61; 2) Visnjic D et 1999 Biochem. J. 344, 921-928; 3) Hiutema, K. et al. 2004 EMBO J. 23, 33-44; 4) C.A.M. La Porta et al.2000 Melanoma Research 10:93-102; 5)Perego C., et al., 2002. Biochem Biophys Res Commun 294, 127-131.

  • Clinical and genetic heterogeneity in GM1 gangliosidosis patients as a result of alteration in β-galactosidase enzyme and elastin binding protein.

    A Caciotti,MA Donati, A Boneh1, A d’Azzo2, T Bardelli, S. Malvagia, V Kimonis3, R Parini4, D Antuzzi5, A Federico6, E Zammarchi, A Morrone.

    Dept. of Pediatrics, Meyer Hospital, Florence, Italy,, fax 055570380, tel. 0555662543; Metab Serv, Victoria Australia1, Genetics St. Jude H, Memphis, USA2, Children's Hospital, Boston, USA3, Neurology, Siena, Italy4, Catholic Univ, Rome, Italy5, Pediatrics, Monza, Italy6

    A deficiency of the human acid b-galactosidase (GLB1) results in two lysosomal storage disorders: the sfingolipidosis GM1-gangliosidosis and the mucopolisaccharidosis Morquio B disease. The clinical heterogeneity of GM1-gangliosidosis is expressed by three major phenotypes: infantile (type I), late infantile or juvenile (type II), and adult or chronic (type III). To date several therapeutic approaches, including substrate deprivation and treatment with “chemical chaperone” molecules, have been evaluated for some lysosomal storage disorders, but most of these diseases, such as GM1-gangliosidosis and Morquio B, remain untreated.

    GLB1 gene gives rise to the GLB1 lysosomal enzyme and to the elastin binding protein (EBP), whose primary deficiency is linked to impaired elastogenesis. In addition, galactosugar-bearing moieties have been demonstrated to alter EBP function and in turn to cause impaired elastogenesis. GLB1 forms an high molecular weight multienzyme complex with the glycosidases [beta galactosidase (GLB1), alfa Neuraminidase (NEU1) and N-acetylaminogalacto-6-sulfate sulfatase (GALNS)] and the protease [(lysosomal carboxypeptidase A (PPCA)] inside lysosomes, while EBP is bound to PPCA and NEU1 on cell surface.

    Here we report the clinical, biochemical and genetic characterisation of 11 GM1 patients covering the entire spectrum of GM1- gangliosidosis phenotypes. Molecular analysis was carried out on the patients' genomic DNA and total RNA isolated from cultured fibroblasts, by direct sequencing of GLB1 cDNA, genomic DNA and enzymatic restriction analysis. With exception of a juvenile patient, carrying the new C230Y and the known R201Hlocalized in a region of GLB1 pre-mRNA encoding only for the lysosomalenzyme, all other genetic lesions identified affect both GLB1 and EBP cDNA and these proteins, altered in function and distribution, contribute differently to the specific clinical manifestations. With the aim of assigning a putative role of these mutated amino acids in the GLB1 stability or catalysis, expression studies and enzyme assays were performed. These studies enabled a genotype/phenotype correlation. Sequence analyses of GLB1 related proteins between species, suggest or confirm correlation between pathological molecular and clinical findings. Expression studies on COS1 cells and Western Blot analyses showed that the identified mutations affect GLB1 enzyme activity and/or stability. In addition, some GLB1 gene mutations do not appear to be stabilized by PPCA probably because these mutations hamper the interaction between GLB1/EBP and PPCA inside multienzyme complex. Impaired elastogenesis in fibroblasts of some patients with infantile form, as detected by elastic fibers assembly by immunofluorescence studies, might be associated with a primary defect of EBP function, given that its normal amount remained unchanged. In a patient with the juvenile form, who shows large urinary keratan sulfate excretion and mutations affecting only GLB1 enzyme, a mild EBP reduction and a decreased elastin deposition were present. These data suggest that the keratan sulfate accumulation leads to secondary EBP deficiency with impaired elastogenesis.

    In order to study the effect of the GSL biosynthesis inhibitors (NB-DNJ) and of the chaperone molecules (DJG and galactose), the skin fibroblasts of three patients with three different phenotypes were pharmacological supplemented with galactose and galactose inhibitors. Interestingly the GLB1 enzymatic activity assayed in the adult patient’s fibroblasts, cultured in the presence of galactose, showed a considerable increase of GLB1 activity. These results could be useful for the development of therapeutic strategies.

    Supported by Azienda O-U Meyer, AMMEC and MPS Italy

  • The extended family of neutral sphingomyelinases.

    Hannun YA

    Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC 29425

    The levels of ceramide are under complex regulation by the action of a combination of enzymes whose own regulation serves to transduce the actions of stimuli into specific cell responses. Neutral(N-SMases) regulate the formation of ceramide through the hydrolysis of sphingomyelin substrates. A combination of genetic, informatic and biochemical approaches has led to the identification of a novel family of N-SMases including the yeast Isc1 and mammalian nSMase2. These enzymes share similarin vitro catalytic mechanisms and a similar requirement for anionic phospholipids. Yeast Isc1 is activated by mitochondrial anionic phospholipids, localizes to mitochondria, and participates in thepost-diauxic response of yeast cells. In human cells, nSMase2 is activated by cell-cell contact in a mechanism involving translocation to the plasma membrane. This enzyme appears to regulate the formation of specific very long chain ceramide species with suggested roles in mediating dephosphorylation of the retinoblastoma protein and the induction of cell cycle arrest in response tocell-cell contact and confluence. These results underscore the specific functions of individual pathways of ceramide metabolism in eukaryotic cell regulation

  • Design and synthesis of acidic ceramidase inhibitors and their characterization using a novel fluorogenic substrate.

    C Bedia1, G Triola1, S Grijalvo1,2, J Casas1, A Delgado1,2, A Llebaria1, T Levadeand

    G Fabriàs1.

    1Research Unit on BioActive Molecules (RUBAM), Department of Biological Organic Chemistry, IIQAB, CSIC. Jordi Girona 18. 08034-Barcelona. Spain. Departamento de Farmacología y Química Terapéutica, Facultad de Farmacia, Universidad de Barcelona, Unidad de Química Farmacéutica (Unidad Asociada al CSIC), Avda. Juan XXIII s/n. 08029- Barcelona. Spain. 3Laboratoire de Biochimie et Maladies Metaboliques, INSERM U466, CHU Rangueil. 1, avenue Jean Poulhes. 31059 Toulouse cedex. France.

    The first step in the catabolic route of ceramide is its amide hydrolysis by ceramidases. Several types of ceramidases have been reported that differ in their optimum pH, intracellular location and substrate specificity. Mutations in the acidic form gives rise to Farber disease (1), a rare lysosomal storage disorder for which there is no current therapy. A novel strategy that has afforded promising results in Gaucher and Fabry diseases is the active site directed chaperone approach. This strategy relies on the use of competitive enzyme inhibitors at sub-inhibitory concentrations to aid the correct folding of the mutated enzyme, thus favouring its transport to the lysosome and increasing its residual activity. Although several inhibitors of ceramidases have been reported (2-4), potent inhibitors of the acidic enzyme are not available. The only acid ceramidase inhibitor so far described is N-oleoylethanolamine, and its potency is rather low (4). In contrast, potent and selective inhibitors of mechanistically similar hydrolases are known (5). Some of these inhibitors include anelectron-deficient carbonyl group that reacts with the active-site nucleophylic amino acid to form an hemiacetal intermediate, which mimics the reaction transition state thus inhibiting the hydrolysis

    (6). Taking into account these studies and considering that N-acyl sphingosines with acyl chain lengths below C10 are poor substrates of the neutral ceramidase (7), two families of compounds were designed, synthesized and their activity as selective inhibitors of the acidic ceramidase was determined both in vitro and in cultured cells using a fluorogenic substrate in a 96-well plate format.

    Table. Inhibition of the acidic ceramidase.



    IC50 (µM)

    In vitro


    In cell culture
































    Among the compounds tested, a family of ketoamides exhibited inhibitory activity of the acidic ceramidase, both in enzyme preparations in vitro and in cultured Farber cells that overexpress the acidic ceramidase (Table). Kinetic studies conducted with GT85 revealed that this compound is a competitive inhibitor with a Kof 16 µM (Figure). These ketoamides, which probably behave as reversible transition state mimics, are the first inhibitors of the acid ceramidase with potencies in the low µM range. Remarkably, these compounds have a good selectivity as compared to the neutral ceramidase in vitro, which requires around 20 fold higher concentrations for inhibition. Thus, these ketoamides are attractive candidates as chemical chaperones of potential utility in the therapy of Farber disease.

    Some of the ketoamides bear a cyclopropene ring in the long chain base and have dual inhibitory activity of both dihydroceramide desaturase and the acidic ceramidase. Besides their putative usefulness as chemical chaperones, these compounds also produce a decrease of intracellular ceramide and are thus interesting as substrate reduction agents for Farber disease treatment.


    1. He X, Okino N, Dhami R, et al., J. Biol. Chem. 2003, 278, 32978-32986. 2. Bielawska A, Greenberg MS, Perry D, et al., J. Biol. Chem. 1996, 271, 12646-12654. 3. Selzner M, Bielawska A, Morse MA, et al., Cancer Res. 2001, 61, 1233- 1240. 4. Strelow A, Bernardo K, Adam-Klages S, et al., J. Exp. Med. 2000, 192, 601-612. 5. Boger DL, Sato H, Lerner AE, et al., Proc. Natl. Acad. Sci. USA. 2000, 97, 5044-5049. 6. Koutek, B, Prestwich, GD, Howlett, et al., J. Biol. Chem. 1994, 269, 22937-22940. 7. El Bawab, S, Usta, J, Roddy, et al., J. Lipid Res. 2002, 43, 141-148.

  • CD1 glycosphingolipid antigen synthesis for the study of protein-carbohydrate interactions.

    Panza L,D. Colombo,F. Compostella, L. Franchiniand F. Ronchetti2

    1DISCAFF, Università degli Studi del Piemonte Orientale, Novara, Italy

    2Dipartimento di Chimica, Biochimica e Biotecnologie per la Medicina , Università di Milano, Via Saldini 50, 20133-Milano, Italy

    Different classes of foreign and self lipid antigens are known to be presented by CD1 antigen presenting molecules to T cells.[1] Several studies suggest that CD1-restricted T cells perform a variety of important roles in cell-mediated immunity, although different responses have been observed after stimulation by foreign or self antigens. While the role of these proteins in bacterial infections can in general be considered clearly evaluated, their functions in the balance between the maintenance of tolerance and the development of autoimmune disease require further investigations.

    Sulfatide (a mixture of 3-sulfated b-D-galactosylceramides with different fatty acids at the ceramide moiety) is one of the glycolipid antigens so far characterized that bind group I CD1 molecules.[2] Much interest is now addressed to the elucidation of the mechanism of antigen presentation. In this context a series of derivatives structurally related to sulfatide have been synthesized as pure compounds for biological assays to shed light on how differences in antigen structures can influence the activity or as probes for binding assays.

    For the elucidation of the role of the lipidic moiety structure in antigen presentation, a family of sulfatides, constituents of the natural mixture and bearing palmitic, stearic, behenic or nervonic fatty acid chains, has been synthesized; these compounds have been shown to be capable of stimulating sulfatide-specific andCD1-restricted T-cell clones.[3]

    In order to have a deeper knowledge of the various parameters affecting lipid binding to the different CD1 isoforms, a fluorescent sulfatide has been prepared, with a fluorophor attached on positon 6 of galactose, thus not interfering with the binding; the fluorescent probe binds to soluble recombinant human CD1a and is a valuable tool for the study of CD1 antigen binding properties.[4] Further biological tests will show if the activity of this derivative is maintained also with the other CD1 isoforms.

    Finally, a b-D-galactosylceramide fully protected with selectively removable protecting groups has been synthesized; this derivative is a valuable scaffold which allows the access to a series of sulfatide related compounds, e.g. sulfated galactosylceramides differing in the position of the sulfate group on galactose for the study of the influence of the sulfate group position on T cells activation.

    [1]Brigl, M.; Brenner, M. B. (2004) Annu. Rev. Immunol. 22, 817-890.

    [2]Shamshiev, A.; Gober, H.-J.; Donda, A.; Mazorra, Z.; Mori, L.; De Libero, G. (2002) J. Exp. Med. 195, 1013-1021.

    [3]Compostella F.; Franchini, L.; De Libero, G.; Palmisano, G.; Ronchetti, F.; Panza, L. (2002) Tetrahedron 58, 8703- 8708.

    [4]Franchini L.; Compostella F.; Donda A.; Mori L.; Colombo D.; De Libero G.; Matto P.; Ronchetti F.; Panza L. (2004)

    Eur. J. Org. Chem. 4755-4761.

  • Synthetic hydroxylated aryl-naphthalene derivatives as resveratrol rigid analogues showing ceramide-mediatedantiproliferative and pro-apoptotic properties.

    Filippo Minutolo1, Giusy Sala2, Annalisa Bagnacani2, Simone Bertini1, Isabella

    Carboni1, Giovanni Prota1, Simona Rapposelli1, Nicoletta Sacchi3, Marco Macchia1,

    Riccardo Ghidoni2

    1Dept. Pharmaceutical Sciences, University of Pisa

    2Lab. Biochemistry & Molecular Biology, San Paolo University Hospital, Medical School, University of Milan

    3Dept. Cancer Genetics, Roswell Park Cancer Institute, Buffalo, NY, USA

    The cancer chemoprevention and the antiproliferative effect associated with dietary polyphenol resveratrol has prompted many research groups to devote their efforts in the search for new and more efficient analogues. The antiproliferative and proapoptotic properties of resveratrol have been shown to be due to an accumulation of endogenous ceramide, a pro-apoptotic natural sphingolipid, in cancer cell lines. In particular, the apoptotic cell death has been attributed more likely to the de novo synthesized ceramide, rather than to sphingomyelin hydrolysis. Moreover, the presence of phenolic hydroxy-groups have been demonstrated to be crucial in preserving this mechanism of action, since tri-O-methylated resveratrol, although showing potent growth inhibition properties in cancer cells, did not induce any accumulation of ceramide. The trans-configuration of the stilbene double bond of resveratrol also seems to be important in preserving the ceramide accumulation mediated pro-apoptotic properties.

    Nevertheless, the trihydroxylated trans-stilbene chemical structure of resveratrol may undergo, as well as most stilbene derivatives, to several chemical and metabolic degradations. One well-known transformation is represented by the photochemical (E)/(Z)-isomerization of the double bond, to produce compounds of (Z)- configuration that are generally less active to inhibit cellular growth and promote apoptosis. The main metabolic reactions on stilbene derivatives involve oxidation reactions on the double bond and on theelectron-rich phenolic rings, producing highly reactive oxidized metabolites (e.g. epoxides, arene oxides, benzylic alcohols, etc.). These modifications may constitute a significant problem because in vitro biological data may be misinterpreted and, if these compounds are submitted to in vivo assays, there might be a concurrence of several metabolites to define the activity and toxicity profiles. For these reasons we decided to investigate the possibility of preparing resveratrol analogues possessing a more rigid and stable scaffold, by replacing the 4-hydroxy-styrene portion with a beta-naphthol portion. We then designed and synthesized a series of naphthalene-based resveratrol analogues, which possess either "all-free" hydroxyls, or various combinations of O-methylated OH substituents, to assess the importance of the phenolic groups also in this new class of resveratrol analogues.

    The biological assays proved that the presence of a naphalene ring generally increases functional proapoptotic properties of the new molecules when compared to similarly substituted stilbene analogues. Furthermore, the concurrent presence of three hydroxyls on the phenyl-naphthalene core is still mandatory, as for resveratrol itself, to induce ceramide-mediated apoptotic cell death, since methoxy-substituted naphthalene derivatives, in spite of some good antiproliferative activity found, did not cause significant ceramide accumulation levels.

  • New aminocyclitols as candidates to chemical chaperone therapy for gaucher disease.

    Egido-Gabás M1, Duque J2, Murillo A,Canals D1, Delgado A1,3, Llebaria A1, Chabás

    A2, Casas, J 1

    1Research Unit on BioActive Molecules (RUBAM), Department of Biological Organic Chemistry, IIQAB, CSIC. Jordi Girona 18.08034-Barcelona. Spain. 2Institut de Bioquímica Clínica, Corporació Sanitària Clínic, Mejia Lequerica, s/n. 08028-Barcelona. Spain.3Departamento de Farmacología y Química Terapéutica, Unidad de Química Farmacéutica (Unidad Asociada al CSIC), Facultad de Farmacia, Universidad de Barcelona, Avda. Juan XXIII s/n. 08034-Barcelona. Spain.

    Glycosphingolipids are amphipathic molecules that contain a hydrophobic moiety (ceramide) and a hydrophilic residue (sugar). They play an important role in cell recognition, proliferation and differentiation, immunorecognition and signal transduction1.

    Glycosphingolipidoses are lysosomal storage diseases, a group of rare human disorders. One of them is Gaucher disease, a sphingolipidosis caused by a marked decreased in glucocerebrosidase (EC GBA) activity. This deficiency leads to a progressive accumulation of glucosylceramide in macrophages, resulting in hepatosplenomegaly, anaemia, skeletal lesions and, sometimes, central nervous system (CNS) involvement.

    The current therapeutic strategies to treat Gaucher disease involve enzyme replacement therapy (Imiglucerase, Cerezyme®, Genzyme) or substrate reduction therapy (Miglustat, Zavesca®, Actelion). However, they are expensive and relatively ineffective for CNS involvement cases2. The molecular therapeutic strategy for genetic metabolic diseases based on the use of chemical chaperones can be a promising alternative for restoration of mutant glucocerebrosidase activity3-4.

    We present a new series of aminocyclitol derivatives that have been synthesized and tested as glucocerebrosidase inhibitors5. Some of them are potent and competitive inhibitors at low concentrations (Ki<3microM) of lysosomal GBA from rat liver and Imiglucerase (Cerezyme®, Genzyme6), and they are also active on the residual GBA activity present in fibroblasts of Gaucher disease patients with different genotypes (N370S/N370S, L444P/L444P, and N370S/L444P). These compounds are specific inhibitors of glucocerebrosidase since they are not inhibitors of other glucose-metabolizing enzymes (alpha-glucosidasefrom Baker's Yeast, beta-glucosidase from Crude Almonds and microsomal glucosylceramide synthase from rat liver). They are also inactive against other lysosomal enzymes involved in ganglioside and glycosphingolipid metabolism, (beta-galactosidase, alpha-galactosidase A and N-acetyl-alpha-galactosaminidase from human fibroblasts).

    Among the active compounds, three of them protect GBA from thermal denaturation. Consequently, these compounds might be considered as good candidates for a chemical chaperone therapy of Gaucher disease.


    1. Kolter, T., Sandhoff, K. Angew. Chem. Int. Ed. 1999, 38, 1535-1568. 2. Germain, D.P., Clin. Genet., 2004, 65, 77- 86 3. Sawkar, A.R., Cheng, W., Beutler, E., Wong, C., Balch, W.E. and Kelly, J.W., PNAS, 2002, 99, 15428-15433. 4. Lin, H., Sugimoto, Y., Ohsaki, Y., Ninomiya, H., Oka, A., Taniguchi et al. Biochim. Biophys. Acta 2004, 1689, 19-228. 5.Egido-Gabás, M., Serrano, P., Casas, J., Llebaria, A., Delgado, A. Org. Biomol. Chem. 2005, 3, 1195-201. 6. A generous gift of Cerezyme® from Genzyme Corporation is gratefully acknowledged.

  • Membrane fusion induced by PlcHR2, a novel sphingomyelinase / phospholipase C from pseudomonas aeruginosa.

    L.-Ruth Montes1, Maitane Ibarguren1, Félix M. Goñi1, Michael L.Vasiland

    Alicia Alonso1

    1Unidad de Biofísica (Centro Mixto CSIC-UPV/EHU), Universidad del País Vasco, 48080 Bilbao, Spain. 2Department of Microbiology, University of Colorado, Denver, CO 80262, USA.

    PlcHR2 from P. aeruginosa is a heterodimeric complex, formed by PlcH, the subunit containing the sphingomyelinase/phospholipase C active center, and PlcR2, a chaperone that modulates the catalytic activity.

    PlcHR2 is the paradigm enzyme of a novel phospholipase C/phosphatase superfamily, with members in a variety of bacterial genera. In the present work we study the structural effects of PlcHR2 when acting on liposomal and cell membranes containing sphingomyelin together with other lipids. Both PlcHR2 and its separate components bind Ca2+ and, while this cation has no detectable effect on the hydrolytic activity, it inhibits PlcHR2 -induced haemolysis. In liposomes (large unilamellar vesicles) the enzyme complex inducesvesicle-vesicle fusion at similar rates, and to similar extents, in the presence and absence of Ca2+. However, this cation increases dramatically the rate of vesicle aggregation, and the rate and extent of enzyme-inducedrelease of vesicular aqueous contents ("leakage").

  • Plasma membranes purified from rat liver are made mostly of detergent-resistant  microdomains (lipid rafts).

    Popa I1, Bionda C1, Ardail D.2, Portoukalian J.1

    1Department. of Dermatology, Edouard Herriot Hospital,69437 Lyon Cx 03, France; 2Laboratory of Radiobiology, Lyon-Sud Medical School 69921 Oullins Cx, France

    Plasma membranes are thought to be made of assembled detergent-resistant (rafts) and detergent-soluble(non-rafts) microdomains. However, most studies isolate lipid rafts from whole cells and very few investigations aim to determine the respective proportions of rafts and non-rafts domains in plasma membranes.

    In the present study, the plasma membrane fraction of rat liver has been purified, then the raft and non-raftdomains have been isolated from the plasma membrane. Following solubilization of the membranes in TritonX-100 1%, fractions were separated by ultracentrifugation on Optiprep gradient. The specific marker proteins flottilin and glyceraldehyde-3 phosphate dehydrogenase (Gapdh) were monitored by western blot to characterize respectively the raft and non-raft domains in the fractions recovered in our experimental conditions.

    Nearly 80% of the membrane proteins were in the flotillin-positive fractions. Whereas the Gadph-positivefractions contained a low amount of cholesterol, free fatty acids and phosphatidylcholine, about 90% of cholesterol and phospholipids were found in the rafts that contained also all plasma membrane-derivedsphingolipids, i.e.ceramides, neutral glycolipids, sphingomyelin and gangliosides. Our results suggest that the plasma membranes of rat hepatocytes are made mostly of domains highly resistant to the action of detergents.

  • Sphingomyelin and STAT3 in nuclear lipid rafts.

    G. Cascianelli, M. Villani, M. Tosti, MP Viola Magni, E. Albi

    Department of Clinical and Experimental Medicine, Physiopathology Section, Policlinico Monteluce, 06100, Perugia.

    Lipid rafts are microdomains enriched in cholesterol and glycosphingolipids and are detected mainly in cellular membranes. Because of the high amount of cholesterol and sphingolipids, the fluidity of these regions is lower than the other regions of the membrane. The fatty-acid chains of lipids within the rafts tend to be extended, highly saturated and so more tightly packed, creating domains with higher order, and a new phase calledliquid-ordered phase (lphase, Zuckermann, 1993).

    Many proteins with a high affinity for the lphase have been found in this well organised phase: GPI- anchored proteins, Src-family kinases and heteromeric G proteins.

    The presence of different proteins endows to the rafts with many functions including cholesterol transport, endocytosis, protein trafficking processes and signal transduction (Moffet et al., 2000).

    The demonstration of the existence of a nuclear lipid fraction highly enriched in sphingomyelin and cholesterol allowed us to hypothesise the existence of lipid rafts also in the nuclear membrane.

    Therefore the aim of the present work was to study the possible presence of lipid rafts in hepatocyte nuclei. The lipid rafts were purified, after Triton X-100 treatment at 4°C, according to Danielsen (1995).

    The electron microscopy analysis revealed microdomains with a typical and defined bilayer and a morphology similar to those previously studied in the cellular membrane.

    The biochemical analysis showed a higher amount of sphingomyelin than that detected in other nuclear compartments, particularly the nuclear membrane. Likewise, the cholesterol content in nuclear rafts was higher than the cholesterol detected in other nuclear structures, and the ratio cholesterol-sphingomyelin is quite similar to cellular membrane lipid rafts, confirming the typical composition of these structures. Sphingomyelinase activity was also present in nuclear lipid raft.

    Protein analysis evidenced many proteins of different molecular weight, with a very evident spot corresponding to a molecular weight of 90 KDa. Since sphingomyelinase is involved in STAT 3 activation, we evaluated the possible presence of this protein by immunoblotting analysis using specific antibodies. The analysis showed positivity for the signal corresponding to 90 KDa.

    Thus, this protein is STAT 3, a polypeptide involved in signal transduction, able to modulate the expression of different genes involved in the cell cycle, proliferation and apoptosis. This confirms the important role that rafts can play as a platform for mediators of cell processes, also at a nuclear level. The next step will be the characterisation of the other proteins found to determine all the roles of these microdomains.

    Danielsen E.M., Biochemistry 34 (1995), 1596-1605

    Moffet S. Brown D.A., and Linder M.E., J. Biol. Chem. 275 (2000), 2191 – 2198

    Jørgensen K., J.H. Ipsen, O.G. Mouritsen, and M.J. Zuckermann, Chem Phys. Lipids 65 (1993), 205 – 216

  • Regulation of cell migration by lipid phosphates and their phosphatases.

    D. Brindley

    Signal Transduction Research Group, Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2S2


  • Regulation of cell migration by lipid phosphates and their phosphatases.

    D. Brindley

    Signal Transduction Research Group, Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2S2


  • Modeling and mutagenesis study of the pleckstrin homology domain of ceramide kinase.

    Frédéric Bornancin, Markus Jaritz, Piroska Dévay, Siegfried Hoefinger, Philipp

    Rovina, Andreas Billich, Thomas Baumruker

    Novartis Institutes for BioMedical Research, Brunner strasse 59, A-1235 Vienna, Austria

    The N-terminus of ceramide kinase (CERK) bears a Pleckstrin Homology (PH) domain. We recently showed that this domain is essential for kinase activity and localization as well as vesicular trafficking of the enzyme1. We now used computational modeling and site-directed mutagenesis to investigate the properties of the CERKPH-domain. In silico protein folding analysis unambiguously confirmed that the N-terminal part of CERK (a.a.8-121) is best fitted to a PH-domain fold. The top hit obtained after threading is mouse Tapp2 N- terminal PH domain, and we therefore modeled CERK PH-domain on the basis of the known 3D-structure of Tapp2. Analysis of the CERK PH-domain model using Poisson-Boltzmann calculations, indicates a strong positive electrostatic potential, in line with the ability of CERK to bind membranes and phosphoinositides. InPH-domains, three variable loops, VL1, VL2 and VL3, interconnecting strands β1-β2, β3-β4 and β6-β7,respectively, provide key charged residues interacting with phosphate groups of phosphoinositides. The VL3 loop of CERK PH-domain appears to be unusually long and to play an important role, as seen from the behavior of recombinant proteins mutated within this region. Furthermore, the profile of phosphoinositide binding to the CERK PH-domain is consistent with the ability of the enzyme to readily associate with various intracellular components, e.g. Golgi complex, vesicles and plasma membrane.

    1Carré et al. Biochem. Biophys. Res. Comm. (2004) 324:1215-19.

  • Sphingomyelin synthase 2 regulates NF-KB activation in hela cells.

    Maristella Villani1,2, Young Choi1, Maurizio Del Poeta1, Y. A. Hannun1, and

    Chiara Luberto1

    1Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC 29425 and 2Department of Biochemical Sciences and Molecular Biotechnology, Physiopathology, Policlinico Monteluce, 06100 Perugia, Italy

    Sphingomyelin synthase (SMS) represents an important class of enzymes regulating sphingolipid metabolism. In particular, SMS transfers the phosphorylcholine moiety from phosphatidylcoline onto ceramide forming sphingomyelin and diacylglycerol (DAG). Because of the ability to modulate in opposing directions the levels of ceramide, a negative regulator of cell growth, and DAG, a well-established mitogenic factor, SMS activity has been proposed to play a significant role in the regulation of those processes associated with aberrant cell proliferation. Recently, two sphingomyelin synthases, SMS1 and SMS2 have been identified. Both enzymes showed bidirectional activity when tested in vitro after expression in yeast cells, and when expressed in mammalian cells, SMS1 localized to the Golgi apparatus whereas SMS2 localized to the Golgi and plasma membrane. Our laboratory has previously shown that activation of a plasma membrane associated SMS would lead to nuclear translocation of NF-kB, a transcription factor which promotes proliferation and inflammation. Based on these observations, we investigated the function of SMS2 in mammalian cells and its role in the regulation of NF-kB activation. Treatment of Hela cells with silencing RNA (siRNA) to specifically down regulate SMS2 led to a 40-45% decrease of total SMS activity in vitro. Although no change in the mass level of DAG was found upon SMS2 down-regulation, an increase of ceramide and decrease of sphingomyelin levels were observed, suggesting a basal SMS function for SMS2 in cells. Down-regulation of SMS2 caused inhibition of basal as well as serum- and TNF-induced NF-kB activation as determined by decreased translocation to the nucleus (western blot and immunofluorescence) and DNA-binding activity (Electrophoretic Mobility Shift Assay). Finally, down-regulation of SMS2 significantly inhibited basal and induced expression of a NF-kB target gene, such as cox-2 as determined by real time PCR and luciferase reporter gene assay. All together these results suggest that SMS2 functions as a SMS in Hela cells, and participates in maintaining the molecular machinery that controls proper NF-kB activity.

    This work was in part supported by COBRE Grant #5NIHP20RR17677 to LM Obeid (project #6 to C. Luberto; mentor, Dr James S. Norris).

  • Ceramide-1-phosphate: a key metabolite for regulation of cell survival.

    Antonio Gómez-Muñoz1, Jennifer Kong2, Kuljit Parhar2, Sherry Wang2, Patricia Gangoiti1, Mónica González, Vince Duronio2, and Urs P. Steinbrecher2

    1Department of Biochemistry and Molecular Biology, University of the BasqueCountry, Bilbao (Spain), and 2Department of Medicine, University of British Columbia, Vancouver (Canada).

    Ceramide-1-phosphate (C1P) is a bioactive sphingolipid that regulates vital cellularprocesses. We first showed that C1P is mitogenic for fibroblasts, and more recently wehave demonstrated that it prevents apoptosis in bone marrow-derived macrophages.

    This action of C1P involves direct inhibition of acid sphingomyelinase (A-SMase) andblockade of the caspase 9/caspase 3 pathway. Current work in our laboratory indicatesthat C1P is a potent stimulator of the phosphatidylinositol 3-kinase (PI3-K)/proteinkinase B (PKB, Akt) pathway, which is a major mechanism whereby growth factorspromote cell survival. Pretreatment of the macrophages with the selective PI3-Kinhibitors LY294002 and wortmannin blocked the inhibitory effect of C1P on cellapoptosis. However, C1P did not stimulate mitogen-activated protein kinases (MAPK), and the MAPK kinase inhibitors PD98059 and UO126 did not alter the effect of C1P onmacrophage survival. By looking into downstream effectors of PKB that might beaffected by C1P we were able to show that C1P enhanced the DNA binding activity ofthe transcription factorNF-kB. Interestingly, the selective NF-kB inhibitors, caffeic acidphenylethyl ester and SC- 514, blocked the prosurvival effect of C1P. In addition, apoptotic macrophages showed a marked reduction of Bcl-XL levels, and this decreasewas prevented by C1P. Taken together, these findings suggest that C1P blocksapoptosis, at least in part, by stimulating the PI3-K/PKB/NF-kB pathway andmaintaining the production of antiapoptoticBcl-XL.

    We propose a working model for C1P in which inhibition of A-SMase and thesubsequent decrease in ceramide levels would allow cell signaling of C1P through stimulation of the PI3-K/PKB pathway to promote cell survival.

    This work was supported by grants MT8630 from CIHR (Canada) and SAF-2002-03184 from Ministerio de Ciencia y Tecnología (Spain).

  • Relative ceramide and ceramide-1-phosphate levels: a cellular gauge for cell death during development.

    Y. León1,2, T. Homan1, E.J. de la Rosa3, I. Varela-Nieto1.

    1Instituto de Investigaciones Biomédicas Alberto Sols, CSIC-UAM. Arturo Duperier 4, 28029 Madrid, Spain. 2Dpto. Biología (Fisiología Animal), UAM. Darwin 2, 28049 Madrid, Spain.

    3Centro de Investigaciones Biológicas, CSIC. 28040 Madrid, Spain.

    Development involves the dynamic balance of cell division, differentiation and programmed cell death. The ex vivo culture of intact inner ear primordium, the otic vesicle, provides an excellent model system to study the physiological interactions among extrinsic factors during early development.

    We have described the opposite actions of nerve growth factor (NGF) and insulin-like growth factor-I (IGF-I)during inner ear ontogenesis. In the otic vesicle, IGF-I is the strongest promoter of inner ear proliferation and morphogenesis, whereas NGF induces programmed cell death by binding to its low affinity p75 neurotrophin receptor (p75NTR). Activation of the p75NTR death domain triggers the generation of ceramide, the activation of caspase-3, the degradation of PARP and finally DNA fragmentation and cell death. Ceramide is generated during cellular stress and apoptosis and has been demonstrated to be a potent inductor of apoptosis within the otic vesicle neuroepithelium (Frago et al., J. Cell Sci. 116, 475. 2003). The exogenous administration of a synthetic short-chain ceramide (C2-cer) induces apoptosis in the otic vesicle. On the contrary,ceramide-1-phosphate (Cer-1-P) is a cytoprotector for otic vesicle explants, acting as a suppressor of cell death upon withdrawal of serum. Addition of Cer-1-P to otic vesicles cultures induces the phosphorylation of Akt and increases PCNA levels. Other lipidic mediators such as phosphatidic acid, sphingosine,sphingosine-1-phosphate or sphingosyl-phosphorylcholine have no appreciable survival actions on this system. NGF induces ceramide generation by activating both de novo synthesis and by increasing the activity of the acid sphingomyelinase. Both NGF- and short-chain ceramide-induced apoptosis are blocked by caspase inhibitors and by IGF-I to different extents. The protective role of IGF-I on NGF- induced cell death was accompanied by a decrease in the intracellular levels of ceramide and by the activation of the Akt kinase pathway. These results suggested that ceramide clearance by means of ceramide kinase activation could be a target of IGF-I in our system. Accordingly, IGF-I actions can be blocked by R59949, an inhibitor of lipid kinases containing a DAGK domain and RT-PCR studies show that ceramide kinase is expressed in the developing chicken otic vesicle. Being its expression up regulated at the stages crucial for cell survival and proliferation. Therefore, the conversion of the pro-apoptotic ceramide to its phosphorylated cytoprotective metabolite ceramide-1-phosphate is one of the pathways underlying the protective action of IGF-I against apoptosis induced by NGF or C2-cer.

    Our data suggests that the relative levels of ceramide and ceramide-1-phosphate form part of the panel of the intracellular indicators that regulate cell death decisions during normal development.

  • Sphingosine-1-phosphate in human glioma cells.

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

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

    Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid metabolite, that plays important roles in the regulation of vital cellular processes, acting as both an intracellular messenger and an extracellular ligand for specific transmembrane receptors. As it occurs in many cells, a hallmark of S1P action in glioma cells is the induction of proliferation and protection against apoptosis, implicating S1P in glial transformation. Although information on receptor-mediated functions of S1P is available, little is known on the cell type(s) that may produce extracellular S1P in gliomas. In this study we searched for the possible origin of extracellular S1P in human glioma cells, using T98G, a human glioblastoma cell line, as model. After feeding T98G cells with tritiated sphingosine, S1P was rapidly synthesized, the majority of it undergoing degradation. In these conditions, cell medium analysis revealed the presence of extracellular radioactive S1P, whereas sphingosine kinase activity was undetectable. The extracellular export of S1P was induced by different mitogenic stimuli. In growth-stimulated astrocytes, the administration of N,N-dimethylsphingosine (a sphingosine kinase inhibitor) resulted in a significant reduction of S1P release and cell proliferation. Moreover, the administration of S1P to quiescent T98G cells was followed by the stimulation of cell growth. a rapid activation of ERK1/2, (mediated through a PTX-dependent pathway) and. In addition, we found that bEnd5, a brain-derived endothelial cell line, can release in the extracellular milieu both S1P and sphingosine kinase. The amount of S1P released from bEnd5 was markedly increased after co-culture with T98G cells. Altogether this study supports that S1P acts as an autocrine/paracrine growth factor in T98G glioma cells, and may be implicated in glial tumors.

  • Sphingosine kinase-1 mediates IGFBP-3 proangiogenic effects in human endothelial cells.

    1,2Riccarda Granata, 3Enrico Lupia, 1,2Letizia Trovato, 1,2Fabio Settanni, 1,2Silvia Destefanis, 1,2Cristina Barbirato, 1,2Davide Gallo, 2Giovanni Garbarino, 4Riccardo Ghidoni, 2,5Giovanni Camussi, 2Ezio Ghigo

    1Laboratory of Molecular and Cellular Endocrinology, 2Department of Internal Medicine and 3Clinical Physiopathology, University of Torino, Torino, Italy; 4Laboratory of Biochemistry and Molecular Biology, San Paolo Medical School; University of Milano, Milano, Italy; 5Research Center for Experimental Medicine (CeRMS), Ospedale S. Giovanni Battista, Torino, Italy.

    Angiogenesis, the formation of new vessels from pre-existing vessels is a key step in developmental and pathological events, including wound healing and tumorigenesis. We have recently shown that insulin-likegrowth factor binding protein-(IGFBP)-3, the major carrier of IGF, inhibits human endothelial cell apoptosis induced by serum starvation by decreasing the proapoptotic sphingolipid ceramide. Moreover, IGFBP-3activated sphingosine kinase (SphK), that catalyses the formation of sphingosine-1-phosphate (S1P), a lipid second messenger known to inhibit endothelial cell apoptosis and to induce angiogenesis. IGFBP-3 even increased IGF-I expression, induced IGF-I receptor, Akt and ERK1/2 phosphorylation and enhanced cell motility. On the basis of the above result, we investigated the role of IGFBP-3 in human endothelial cell angiogenic signaling. We show that IGFBP-3 dose-dependently promoted capillary-like structure formation by endothelial cells on growth factor-reduced Matrigel. cDNA array analysis showed that IGFBP-3 enhanced the expression of 8 angiogenic-related genes. The use of other methods (RT-PCR, ELISA and gelatin zymogram analysis) provided further evidence of increased expression of vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMP)-2 and -9. Importantly, IGFBP-3 also increased the production ofmembrane-type 1 MMP (MT1-MMP), that plays a key role in MMP-2 activation. Decreased SphK1 expression by small interfering RNA (siRNA), blocked the proangiogenic effects of IGFBP-3 on capillary-like formation and inhibited VEGF and MMPs but not the previously observed IGF-I up-regulation. Finally, IGFBP-3dose-dependently stimulated the growth of neovessels into subcutaneous (s.c.) implants of Matrigel in vivo. We propose IGFBP-3 to be a positive regulator of angiogenesis. Moreover, we suggest that SphK1 mediatesIGFBP-3 proangiogenic effects in human endothelial cells.

  • Regulation of autophagy by sphingosine -1-phosphate.

    Lavieu G1, Scarlatti F2, Sala G2, , Levade T3, Ghidoni R2, Botti J1, Codogno P1

    1INSERM U504 Villejuif France; 2University of Milan Italy;3INSERM U466 Toulouse France

    Autophagy is a process by which cytoplasm is delivered to the lysosome and degraded. Autophagy is involved in the control of cell fate by promoting cell survival (under starvation or during cell invasion by bacterial and viral pathogens). Moreover autophagy is associated with non classical cell death under anti- tumoral treatment (as tamoxifen treatment in MCF-7 cell line).

    Sphingolipid are equally involved in regulation of cell viability by an equilibrium between ceramide induced cell death and sphingosine-1-phosphate induced cell survival.

    We have recently shown that ceramide is a mediator of tamoxifen induced autophagy and cell death in MCF- 7 cells (Scarlatti et al 2004 J. Biol. Chem.).

    Here we show that sphigosine-1-phosphate stimulates autophagy by increasing the formation of LC3 positive autophagosomes and the rate of 3-methyladenine sensitive proteolysis without modification of the level of ceramide. Sphingosine kinase 1 (SK1) overexpression recapitulated the stimulation of S1P supplementation on autophagy. This effect was counteracted by the SK inhibitor dimethysphingosine and by expression of a dominant negative form of SK1. Dihydrosphingosine 1-P, an agonist of S1P cell surface receptors, did not stimulate autophagy supporting the notion that autophagy is mediated by the intracellular second messenger activity of S1P. In contrast to ceramide-mediated autophagy, S1P-mediated autophagy is characterized by inhibition of mTOR signaling independently of the Akt/PKB signaling arm. In contrast to ceramide, S1P did not trigger autophagic cell death. Moreover S1P-mediated autophagy has a protective effect toward the induction of apoptosis by nutrient and serum free medium. In conclusion both ceramide and SP1 are able to stimulate autophagy by different pathways with opposite outcomes on cell survival and death.

  • Sphingosine kinase-1 as a chemotherapy sensor in prostate adenocarcinoma cell and mouse models.

    Dimitri Pchejetski1, Muriel Golzio2, Cyril Calvet1,3, Elisabeth Bonhoure1, Nicolas

    Doumerc1,3, Virginie Garcia1, Catherine Mazerolles4, Pascal Rischmann3, Justin

    Teissié2, Bernard Malavaud3, Olivier Cuvillier1

    1Inserm U466, Institut Louis Bugnard, BP 84225, 31432 Toulouse Cedex 4, France ; 2Institut de Pharmacologie et de Biologie Structurale, CNRS UMR5089, Route de Narbonne, 31077 Toulouse; 3Service d’Urologie et de Transplantation Rénale, Hôpital Rangueil, TSA 50032, 31059 Toulouse Cedex 9; 4Service d’Anatomie et Cytologie Pathologiques, Hôpital Rangueil, TSA 50032, 31059 Toulouse Cedex 9, France.

    Systemic chemotherapy was considered of modest efficacy in prostate cancer until the recent introduction of taxanes. We took advantage of the known differential impact of camptothecin and docetaxel on human PC-3and LNCaP prostate cancer cells to determine their effect on sphingosine kinase-1 activity and subsequent ceramide/sphingosine 1-phosphate balance, in relation to cell survival. In vitro, docetaxel and camptothecin induced strong inhibition of SphK1 and elevation of ceramide/S1P ratio only in cell lines sensitive to the drugs. Sphingosine kinase-1 overexpression in both cell lines impaired the efficacy of chemotherapy by decreasing ceramide/S1P ratio. Alternatively, silencing sphingosine kinase-1 by RNAi or pharmacological inhibition induced apoptosis coupled with elevation of ceramide and loss of S1P. Differential effect of both chemotherapeutics was confirmed in an orthotopic PC-3/GFP model established in nude mice. Docetaxel induced stronger sphingosine kinase-1 inhibition and ceramide/S1P ratio elevation than camptothecin. This was accompanied by smaller tumor volume and reduced occurrence and number of metastases. Sphingosine kinase-1overexpressing PC-3 cells implanted in animals developed - not only remarkably larger tumors - but also resistance to docetaxel treatment. These results represent the first in vivo demonstration of sphingosinekinase-1 as a sensor to chemoth.

  • Resveratrol affects the formation of multicellular spheroids of breast cancer cells: involvement of ceramide and adhesion molecules.

    Leda Roncoroni1, Giusy Sala2, Floriana Facchetti3, Elena Dogliotti1, Nicoletta Sacchi4, Caterina La Porta3, Ersilia DolfiniRiccardo Ghidoni2

    1Dept Biology and Genetics and 2Lab Biochemistry and Molecular Biology, San Paolo Medical School, 3Dept of Biomolecular Science and Biotechnology, University of Milan, Italy; 4Dept Cancer Genetics, Roswell Park Cancer Institute, Buffalo, NY, USA

    Resveratrol, a polyphenol present in grapes and wine, exerts a drastic growth-inhibitory effect on the human breast cancer MDA-MB-231 cell line grown in the 2D-culture. We recently described the mechanistic relationship between growth inhibitory/pro-apoptotic effect of resveratrol and de novo synthesis of ceramide [Scarlatti et al FASEB J (2003) 17,2339-41].

    Here we report that ceramide mediates the growth inhibitory effects of resveratrol in MDA-MB-231 cells grown in three-dimensional (3D) cultures. MDA-MB-231 when grown in 3D-cultures can form multicellular tumour spheroids (MCTSs), which revealed an ovoid or spherical shape with well-defined and compact cells, smooth boundaries and regular surface.

    First, we observed that treatment of MDA-MB-231 cells with 32 µM resveratrol significantly impaired the formation (number and size) of 3D-MCTSs. This effect correlated with a time-dependent accumulation of endogenous ceramide. In addition, cells disaggregated from the few MCTSs that were able to form MCTSs after resveratrol treatment lost their clonogenic potential.

    The levels of expression of E-cadherin, N-cadherin, P-cadherin, PECAM (CD31), P-selectin and VCAM were also analysed by RT-PCR in MDA-MB231 3D-MCTSs. For E-cadherin and VCAM factors no significant expression were detected. In contrast, N-cadherin, P-selectin and P cadherin factors were expressed byMDA-MB231 cells, but 3D conditions (7 and 10 days spheroids) did not modify significantly their level of expression.

    The treatment with resveratrol dramatically decreased the level of expression of N-and P-cadherin, while the level of CD31 appeared unmodified in all the growth condition (2D or 3D). Interestingly, in cells grown as spheroids resveratrol was more efficacious than in cells grown on plastic plate. In particular, 32 µM resveratrol was efficacious up to 10 days.

    Thus, resveratrol can affect the formation of MCTSs by triggering ceramide-dependent pathways and modifying expression of adhesion molecules.

  • Cannabinoids induce apoptosis of glioma cells via ceramide- dependent up-regulation of the pro-apoptoticgene p8.

    Arkaitz Carracedo, Cedric Malicet§, Ainara Egia, Cristina Blázquez, Mar Llorente, Raquel Villuendas #, M. A. Piris #, Juan L. Iovanna§, Manuel Guzmán and Guillermo Velasco

    Departamento de Bioquímica y Biología Molecular I, Facultad de Biología, Universidad Complutense, 28040 Madrid Centro Nacional de Investigaciones Oncológicas, 28029 Madrid#

    U624 INSERM, Campus de Luminy, Marsella, Francia§

    Cannabinoids - and in particular D9-tetrahydrocannabinol (THC) - have been shown to exert antiproliferative actions both in vitro and in vivo on a wide spectrum of tumour cells. In the case of gliomas and leukaemias, this effect is - at least partially - due to the ability of these compounds to induce apoptosis of these cells via stimulation of ceramide synthesis de novo. In order to understand the molecular mechanisms underlying thispro-apoptotic effect, we have studied the gene expression profile of two sub-clones of the rat glioma cell line C6 that exhibit different sensitivity to the treatment with cannabinoids.

    Our results show that one of the genes that are differentially expressed in the cannabinoid-treated sensitive cells is the stress-regulated gene p8. By using p8 siRNA and a retroviral vector to over-express this protein, we further confirmed that expression of this gene was necessary for the pro-apoptotic effect of cannabinoids to be observed in glioma cells. Interestingly, pharmacological inhibition of ceramide synthesis de novo prevented not only cannabinoid-induced apoptosis but also p8 up-regulation. Moreover, abrogation of this pathway also prevented up-regulation of several downstream targets of p8 that seem to participate in cannabinoid-inducedapoptosis.

    In summary, our results suggest that treatment with cannabinoids leads to apoptosis of glioma cells via aceramide-dependent up-regulation of the pro-apoptotic gene p8.

  • Effect of vitamin D3 on embryonic hippocampal cells via neutral sphingomyelinase.

    F Marini1, Bartoccini E1, Viola Magni M1, Garcia-Gil Mand Albi E1,

    1Department of Clinical and Experimental Medicine, Physiopathology Section, Policlinico Monteluce, 06100, Perugia.

    2Dept. of Physiology and Biochemistry, University of Pisa

    Over the last ten years abundant evidence has emerged for new roles of vitamin D3. This molecule is known for its activity in calcium preservation and phosphorous homeostasis but it is also involved as a defensive agent against multiple sclerosis and in the prevention of encephalomyelitis progression, suggesting an immunosuppressive action (1). Recently, it has been demonstrated that Vitamin D3 induces erythroleucemic cell differentiation via sphingomyelinase (SMase) and prevents serum starvation apoptosis (2). Since in HN9.10e embryonic hippocampal cell serum deprivation induces apoptosis first increasing the nuclear SMase activity and after cellular SMase activity (3), the aim of the present work was to establish if vitamin D3 could have a role in slowing down the cell cycle inducing differentiation and preventing serum deprivation induced apoptosis via sphingomyelin (SM) metabolism.

    HN9.10e cells were synchronized by serum free DMEM for 24h and then were grown in DMEM supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 IU/ml penicillin, 100 µg/ml streptomycin and 2.5 µg/ml amphotericin B (fungizone). In experimental cells 400 nM vitamin D3 was added. Cells were maintained at 37 °C in a 5% COhumid atmosphere. Cell cycle was studied using 3H- thymidine whereas the SM metabolism was studied incubating the cells with 3H palmitic acid.

    The results show that, after synchronization, the cells present two peaks of 3H-thymidine incorporation at 36 and 60 hrs respectively, corresponding to the first and second mitotic wave. Vitamin D3 does not influence behavior in time but reduces the first peak by 32% and the second by 47%, suggesting a reduction of the cell number entering in the S phase. At 48h hrs of 3H palmitic acid incorporation, the saturation conditions were reached in SM and ceramide. After this time, in the control sample the radioactivity was 45 nmol/mg protein and 35 nmol/mg protein for SM and ceramide respectively and the values did not change up to 120 min. The vitamin D3 determined a 6.6 fold increase in SM at 120 min and may influence the reduction of the S phase of the cell cycle and induce differentiation. Serum deprivation did not change the SM levels, as previously reported(3) but the vitamin D3 in these cells increased the SM levels after 30 min of treatment. The early increase of SM could play a role in preventing serum deprivation induced apoptosis.

    1- A.J. Brown, A. Dusso and . Slatopolsky. Am J Physiol Renal Physiol, (1999) 277: F157-175

    2- Witasp E, Gustafsson AC, Cotgreave I, Lind M, Fadeel B. Biochem Biophys Res Commun. (2005) 330:891-7.

    3- E.Albi, S. Cataldi, E. Bartoccini, M. Viola Magni, F. Marini, F. Mazzoni, G. Rainaldi, M. Evangelista, M. Garcia-Gil2005 J. Cell Physiol, in press.

  • Sphingomyelin derivatives affect the progress of denervation of rat soleus muscle.

    M.Zanin1, E. Germinario1, R.A. Sabbadini2, R, Bettoand D. Danieli1

    1Department of Human Anatomy and Physiology, University of Padova, Italy; 2Department of Biology, San Diego State University, USA, 3C.N.R. Neuroscience Institute, Muscle Biology and Physiopathology Unit, Padova, Italy.Via Marzolo 3,

    35131 Padova. Tel. 049 8275305, Fax. 049 8275301

    Present work is aimed at studying the effects of bioactive sphingomyelin derivatives on the progress of denervation of slow-twitch skeletal muscle. The study is based on the fact that sphingomyelin derivatives are elevated in serum so that we hypothesize they may exert trophic functions on muscles. Denervation was bilaterally performed cutting the sciatic nerve at the level of trochanter in adult rats. Sphingosine (SPH), sphingosine 1-phosphate (S1P) or sphingosylphosphorylcholine (SPC) were continuously released over soleus muscle by a mini-osmotic pump implanted subcutaneously in the scapular region and connected to the muscle through a catheter. Effects of individual sphingomyelin derivatives were evaluated on the fibre cross sectional area, myosin heavy chains composition (MHC), TUNEL-positive nuclei, and expression of MRFs. In general, either of the three lipids decrease the development of atrophy, with SPH and SPC being the most effective. However, the action of the lipids appears to be to some extent distinctive. The protective action of SPC has an early onset and is transient, while that of SPH is delayed. SPH slows down the slow- to-fast transformation of MHC isoforms normally occurring in denervation, while S1P and SPC do not seem to influence this process. SPH, S1P and SPC modulate the expression of MyoD and myogenin, suggesting that the three lipids influence satellite cells.

    In general, SPH and S1P/SPC exert distinctive and opposite actions. SPH inhibits cell proliferation and promote apoptosis, while the extracellular action of S1P/SPC stimulates cell growth and protect from apoptosis. In contrast, present results show that the three lipids produce common effects, even though distinctive, on the progress of soleus muscle denervation. We speculate that the expected negative effect of SPH, that is the worsening of atrophy, is abolished by the presence at muscle fibre surface of SPH kinase, which convert SPH in S1P. Preliminary data show that in the presence of SPH kinase inhibitor, DMS, the SPH positive effect is lost. Future work will further investigate this hypothesis and the molecular mechanisms at the base of the distinctive actions of sphingolipids on skeletal muscle.

    Funded by PRIN 2003, NIH, AHA and CNR.

  • Control of bioactive sphingolipid levels by retinoic acid receptors in breast cancer cells.

    Giulia Somenzi1, Giusy Sala2, MingQiang Ren1, Manuela Amelotti1, Riccardo

    Ghidoni2Nicoletta Sacchi 1

    1Dept. Cancer Genetics, Roswell Park Cancer Institute, Buffalo, NY, 14263, USA

    2Lab. Biochemistry & Mol Biology, San Paolo University Hospital, Medical School, University of Milan, Milan, Italy

    All-trans-retinoic acid (RA) is a bioactive natural derivative of Vitamin A. RA action is mediated by nuclear RA receptors, RARs. More than one RA-receptor is involved in mediating RA-signal. RA binds RARα, which in turn regulates another RA receptor, RAR β2, with potent tumor suppressor activity. RA-resistant cancer cells often lack both RARα and RARβ2 expression. Lack of both RARs has been traced to epigenetic silencing, due to aberrant DNA methylation and histone modifications of the RARs’ chromatin. Breast cancer cells lacking both RARα and RARβ2 are unable to respond to the growth inhibitory, differentiating and apoptotic effects of RA. Biologically active sphingolipids like ceramide and sphingosine-1-phosphate (S1P) are pivotal regulators of these fundamental cellular processes. We found that sphingolipid metabolism is different in RA-resistantbreast cancer cells lacking both RARα and RARβ2 and RA-sensitive cells with intact RARα and RARβ2. We hypothesized that RARs control sphingolipid metabolism. Here we show that as a consequence of functional RARα disruption, breast cancer cells become unable of a) re-inducing RARβ2 in response to RA, b) accumulating ceramide via neutral sphingomyelinase and c) reducing the level of sphingosine kinase transcription and activity. Moreover, cells instead of being growth-inhibited are growth- promoted by RA. However, cells retain the ability of accumulating endogenous ceramide and undergoing apoptosis in response to the synthetic retinoid fenretinide, which action is RAR-independent.

    The overall data lead to the conclusion that RARs control distinct sphingolipid metabolic pathways.

  • Activation of APP1 transcription by ipc1-dag pathway is regulated by ATF2 in Cryptococcus neoformans.

    Roberta Iatta1,2, Lydia Mare2, Chiara Luberto2, Maria Teresa Montagnae

    Maurizio Del Poeta2

    1Dipartimento di Medicina Interna e Medicina Pubblica Sezione Igiene, Università degli Studi di Bari, Bari, Italia2Department of Biochemistry and Molecular Biology Medical University of South Carolina, Charleston, South Carolina, USA

    Inositol-phosphoryl ceramide synthase 1 (Ipc1) is a fungal specific enzyme that regulates the level of two bioactive molecules, phytoceramide and diacylglycerol (DAG) (1). In previous studies, we demonstrated that Ipc1 regulates the expression of antiphagocytic protein 1 (App1), a new factor involved in the pathogenicity of Cryptococcus neoformans (2). Here, we investigated the molecular mechanism by which Ipc1 regulates App1. To this end, the APP1 promoter was fused to the firefly luciferase gene in C. neoformans wild-type and in GAL7::IPC1 mutant, in which Ipc1 expression can be modulated. We found that luciferase activity is indeed regulated by modulation of Ipc1, suggesting that the lipid(s) regulated by Ipc1 may trigger APP1 transcription. Next, we investigated the role of DAGs and other sphingolipids in the activation of APP1 promoter and we found that treatment with 1,2-dioctanoyl-glycerol increases luciferase activity whereas treatment with phytosphingosine or ceramides did not affect luciferase activity. Importantly, deletion of ATF2 consensus sequence in the APP1 promoter or deletion of ATF2 gene from the genome abolishes luciferase activation by Ipc1 or DAG, suggesting that Atf2 transcription factor is sufficient and necessary for regulation of APP1 gene transcription. Since App1 is found in sera of AIDS patients affected by cryptococcosis, these studies may have important implications in understanding mechanisms of pathogenicity of C. neoformans.


    Luberto C, Toffaletti D, Wills E, Tucker S, Casadevall A, Perfect J, Hannun Y, Del Poeta M. Roles for inositol-phosphorylceramide synthase 1 (IPC) in pathogenesis of C. neoformans . Gene & Development 2001; 15: 201-212.

    Luberto C, Martinez-Marino B, Taraskiewicz D, Bolanos B, Chitano P, Toffaletti D, Cox G, Perfect J, Hannun Y, Balish E, Del Poeta M. Identification of App1 as a regulator of phagocytosis and virulence of Cryptococcus neoformans. J. Clin. Invest. 2003; 112: 1080-1094.

  • The presence of the inositolphosphoceramide synthase AUR1p is critical for the survival of lac1 lag1 yeast mutants lacking the acyl-coa-dependent ceramide synthase.

    Isabelle Guillas1, Vanessa Cerontola, S. Michal Jazwinskiand Andreas Conzelmann

    Department of Medicine, University of Fribourg, Switzerland and 2Department of Biochemistry and Molecular Biology,

    Louisiana State University Health Sciences Center, New Orleans, USA.

    The lag1∆ lac1∆ double mutants lack acyl-CoA-dependent ceramide synthase and are either very sick or nonviable. Growth of lag1∆lac1∆ cells can be greatly improved by overexpression of Ypc1p or Ydc1p, two homologous alkaline ceramide hydrolases that also catalyze the reverse reaction, i.e. the condensation of free fatty acids with phyto- or dihydrosphingosine (Schorling et al., 2001; Mao et al., 2000a; Mao et al., 2000b). Overexpression of Ypc1p restores viability and growth YPK9 lag1∆ lac1∆ and allows for the synthesis of large amounts of structurally normal inositolphosphorylceramides containing fatty acids with no less than 26 C atoms. Overexpression of Ydc1p also restores viability and slow growth to lag1∆ lac1∆ cells but allows for synthesis of only small amounts of a single C26-containg non-hydroxylated inositolphosphorylceramide. The lag1∆ lac1∆ cells making only limited amounts of ceramides can during a few cell devisions in the presence of low concentrations of Aureobasidine A, a specific inhibitor of inositolphosphorylceramide sybnthase. The drug is taken up as it efficiently blocks residual inositolphosphorylceramide biosynthesis in those cells. Ultimately however, cells stop growing. Also, the AUR1 gene cannot be deleted in these lag1∆ lac1∆ cells overexpressing Ydc1p. The results indicate that the inositolphosphorylceramide synthase AUR1 remains essential in the absence of acyl-CoAdependent ceramides synthase. AUR1 may allow cells to make very small amounts of essential inositolphosphorylceramides or may serve another, unrelated, but essential function.

  • Involvement of bioactive sphingolipids in the postoperative intestinal trauma.

    Mihaela Dragusin, Roland Broere, Sven Nätzker, Sven Wehner§, Jörg C.Kalff§, Nicolas Schwarz§, Elaine Wang*, Cameron Sullards*, Alfred H. Merrill Jr.*, Gerhild van Echten-Deckert

    Kekulé-Institut für Organische Chemie und Biochemie der Universität Bonn, Gerhard-Domagk-Strasse 1, 53121 Bonn, Germany § Klinik und Poliklinik für Allgemein-, Viszeral-, Thorax-, und Gefäßchirurgie, Sigmund-Freud-Strasse 25, 53105 Bonn, Germany *School of Biology, Georgia Institute of Technology, Atlanta, GA 30332-0230,USA

    There is growing evidence that inflammatory processes are accompanied by increasing levels of bioactive sphingolipids including ceramide and sphingosin-1-phosphate (S1P). Recently, a novel bioactive sphingolipid,ceramide-1-phosphate (Cer1P) has emerged as an inducer of inflammatory responses by activation of PLA2. However, no information concerning the role of these sphingolipids in inflammatory events during the postoperative intestinal trauma is yet available.

    In the present study we analysed the levels of bioactive sphingolipids of mucosa and muscularis from small intestine of i) a standardized surgical rat model of intestinal manipulation, as well as of ii) lipopolysaccharide (LPS) treated rats. In addition, we have used cultured muscularis smooth muscle cells in order to investigate the bioactive sphingolipid downstream targets involved in the inflammatory cascade.

    Using thin layer chromatography we detected a slight increase (up to 2 fold) of ceramide levels at 6 h and 3 h after mechanical manipulation in intestinal mucosa and muscularis, respectively, compared to sham controls. Concerning LPS treatment the increase of ceramide was less pronounced and occurred only after 24 h in both intestinal mucosa and muscularis, when compared with sham controls.

    Results obtained from ESI tandem mass spectrometry (MS) measurements indicate that not ceramide but rather its metabolic products, Cer1P and S1P are increased in the intestinal muscularis, the mainly affected tissue during postoperative ileus.

    Experiments performed in primary cultured muscularis smooth muscle cells indicate that both, ceramide and S1P induce an activation of cox2, a key player in inflammatory processes. Accordingly, increased levels of prostaglandins could be detected. Furthermore, using DNA chip technology, up-regulation of pro- inflammatory cytokines including IL-1 was observed.

    Our latest results obtained in primary cultured intestinal smooth muscle cells concerning sphingolipid mediatedpro-inflammatory signalling cascades will be presented.

  • Psychotic forms of late manifesting adult metachromatic leukodystrophies : phenotype / genotype relationships.

    Nicole Baumann, Benoit Colsch, Mireille Lefevre and Jean-Claude Turpin

    Laboratory of Neurochimie Unité Mixte de Recherche INSERM U711-UPMC , Hopital de Salpetriere, Paris, France

    Metachromatic leukodystrophy (MLD) is due to an abnormal catabolism of sulfogalactosylceramides (sulfatides)and other sulfogalactolipids (seminolipid) because of a deficient cerebroside sulfate sulfatase (arylsulfatase A i.e. ASA).Diagnosis is based on sulfatiduria and ASA determination.

    In childhood this disease is multisystemic and its symptomatology and natural history is well known. Surprisingly, this genetic disease can manifest for the first time during adolescence and/or adulthood. In this case, the clinical presentation and evolutivity are very different.

    Its presentation 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.

    To try to understand this psychotic phenomena, we decided to do a complete study reviewing cases already published for which we could have more information, and also unpublished cases from clinics and laboratories including our own. Interestingly several patients were institutionalized in psychiatry departments and considered as schizophrenia. From the survey of the literature and our own experience there seems to be in opposite to infantile forms, a phenotype-genotype relationship in the adult . 10 psychotic adult cases and 3 psychotic juvenile cases responded to the classical criteria of Metachromatic Leukodystrophy, with 5- 10 % normal value, and sulfatiduria. There were common psychiatric pattern according to criteria of the DSM4 for Schizophrenia: delusions, hallucinations,disorganized behavior, affective flattening, social occupational dysfunction. This was associated to cognitive deficiency. Many years could occur till neurological manifestations became evident. Molecular studies were performed both by PCR restriction and sequencing. Motor forms involve the major adult mutation P426L in a homozygote form in contrast to psychocognitive forms which involved as a compound heterozygote a specific I179S mutation of ASA, the second mutation being mainly of the infantile type The I179S mutation was present as a compound heterozygote in the 13 psychiatric cases we studied. Possibly other mutations are also involved in psychiatric cases but I179S is the major one and there is definitely a phenotype/genotype correlation.

  • Human sialidases: molecular biology, biochemistry and physiopathological implications.


    Guido Tettamanti

    Department of Medical Chemistry, Biochemistry and Biotechnology, The Medical School, University of Milan, Via F.lli Cervi 93, 20090 Segrate, Milan, Italy

    Sialidases are expressed in viruses, bacteria, protozoa and vertebrates. Viral and bacterial sialidases display about 35% homology in their primary structure, and share some specific motifs: six four-stranded antiparallelβ-sheets arranged as the blades of a propeller; a F(Y)RIP motif located toward the aminoterminal region, a repetitive motif SXDXGXXT/W in the active site region; and some Asp-boxes. In mammalian (including human) organs and tissues, sialidases display different subcellular locations (lysosomes, plasma membrane, cytosol and, possibly, other locations) and substrate specificities. The lysosomal, cytosolic and plasma membrane sialidases, also from the same animal, have different protein structure, antigenic properties and cDNAs.

    The following human sialidases have been cloned so far: the lysosomal sialidase NEU1 coded by NEU1 gene mapped in 6p21, the cytosoluble sialidase NEU2 coded by NEU2 gene mapped in 2q37, the plasma membrane bound sialidase NEU3 coded by NEU3 gene mapped in 11q13, and the newly discovered sialidase NEU4 coded by NEU4 gene mapped in 2q37. The corresponding recombinant sialidases constitute powerful tools for studying the structure-function relationships of these enzymes. As an example, recombinant human cytosolic sialidase (Hs NEU2), expressed in E. Coli, was purified to homogeneity and its crystal structure defined. The presence of the canonical six blades β-propeller structure, F (Y)RIP motifs and three Asp- boxes, typical of all studied sialidases, was confirmed. In addition, with the use of the inhibitor DANA (2,3–dehydro–3–deoxy–N–acetylneuraminic acid) the dynamic nature of substrate recognition was assessed. Recombinant Hs NEU2 was proven to differentially recognise: (a) the type of sialosyl linkage (the α 2-3sialosyl linkage to galactose is preferred); (b) the aglycone part of the substrate (gangliosides act as the best substrates); and (c) the supra-molecular organization (monomer, micelle, vesicle) of the ganglioside substrate. The latter ability might be relevant in sialidase interactions with gangliosides under physiological conditions.

    Some plasma membrane bound sialidases appear to be attached to the membrane via a glycan- phosphoinositide (GPI) anchor, possibly indicating their location in specialized plasma membrane "domains" (lipid rafts). Surface sialidase NEU3 is able to affect sialoglycolipids exposed on the surface of vicinal cells and was shown to be directly involved in neural and myoblast differentiation. In the latter case the block of sialidase expression led to inhibition of the differentiation toward myocytes. Sialidases appear also to be involved in human erythrocytes maturation and ageing under physiological and pathological conditions. Very recent results point to the implication of sialidase NEU3, and its hydrolysis products gangliosides GM1 and GM2, in the insulin post-receptor signaling events, and of sialidases NEU2 and NEU3 in tumor growth. The genetic lack of sialidase NEU1 is responsible of a very severe lysosomal disease.

  • Resveratrol sensitization of DU145 prostate cancer cells to ionizing radiation is associated to endogenous ceramide increase.

    Scarlatti F., Maioli C.$, Sala G., Maffei R., Milani F.$, and Ghidoni R.

    Lab Biochem & Molecular Biology – San Paolo University Hospital, Medical School,

    Institute of Radiological Sciences, University of Milan, Milan

    Radiotherapy is an established therapeutic modality for prostate cancer. Since it is well known that the restriction of radiotherapy is its severe toxicity on normal cells at high dose and minimal effect at low dose. The search for biological compounds able to increase the sensitivity of tumors cells to radiation may improve the efficacy of therapy. Resveratrol, a natural antioxidant, was shown to inhibit carcinogenesis in animal models [1], and to block the process of tumor initiation and progression [2]. The purpose of this study was to examine whether resveratrol can sensitize DU145, an androgen-independent human prostate cancer cell line, to ionizing radiation (IR).

    We observed that, although DU145 cells are relatively sensitive to IR-induced cell death, pretreatment with resveratrol significantly enhances tumor cell death induced by IR alone. Resveratrol acts synergistically with IR to inhibit colony formation of DU145 cells in vitro. Moreover resveratrol potentiates IR-induced ceramide accumulation in DU145 cells. We supposed that the clonogenic survival of DU145 cells after combined treatment of resveratrol with IR is associated to the generation of ceramide. The synergistic effect at biologically relevant doses of radiation (0.5-2 Gy) suggests that the combination of resveratrol with IR allows reduction of radiation doses and potentially reduce treatment-related morbidity.

    1.Yang CS. et al. Inhibition of carcinogenesis by dietary polyphenolic compounds. (2001), Annu. Rev. Nutr. 21:381-406.

    2.Jang M. et al. Cancer chemopreventive activity of resveratrol, a natural product derived from grapes. (1997)Science 275: 218-220.

  • Effect of dexamethasone on cellular and nuclear sphingomyelin- synthase.

    Tringali A. R., Tringali S. , Viola Magni M.P., Albi E.

    Department of Clinical and Experimental Medicine, Physiopathology Section, Policlinico Monteluce, 06100, Perugia.

    It has been determined that glucocorticoids are essential for cell growth in culture acting upon receptors, enzyme activity and membrane transport (1). On the other hand, these molecules induced growth inhibition and lysis in certain sensitive leukemic cells (2) and apoptosis in mature, activated, peripheral T lymphocytes

    (3). The induction of apoptosis could be one of the key mechanisms mediating the therapeutic effect of glucocorticoids in the treatment of leukemias, lymphomas and various autoimmune disorders.

    The biological effects of dexamethasone were generally thought to be mediated by an intracellular protein receptor which transduces the hormone signal to the nucleus and participates directly in gene regulation (4). Recently, we have demonstrated that dexamethasone inhibited the growth of lymphoblastic lymphoma cells(SUP-T1) by stimulating the cellular sphingomyelinase (SMase) activity and inhibiting the nuclear SMase activity after two hours of treatment (data not shown). This effect induced an increase in cellular ceramide and a reduction in the nuclear ceramide pool enriching sphingomyelin (SM) in the nucleus. Since the SM- synthase activity was present in the nuclei, the aim of this work was to establish the possible effects of dexamethasone on cellular and nuclear SM-synthase activity in tumor cells. To this end, the lymphoma cells (SUP-T1) were seeded in RPMI 1640 in the presence of 10% fetal bovine serum, at the concentration of 100.000/ml and cultivated up to 6 days in a 5% CO2 humid atmosphere. Two 5% lots were prepared: one control and one cultivated in the presence of dexamethasone (500 µg/ml). The SM-synthase activity was assayed in the homogenate and nuclei by using 3H-phosphatidylcholine as substrate. The results show that in the homogenate the SM-synthase activity is 10 pmol/mg protein/min at 0 min, increases of 80 times after 30 min, then decreases slowly until 2 hours and after remains constant for all considered times. In the nuclei,SM-synthase activity is inhibited by 50% compared to the control after 30 min and then increases progressively up to 2 hours. In conclusion the nuclear SM metabolism is independent from that of in the whole cell and the SM-synthase activity contributes to the SM enrichment pool after two hours of dexamethasone treatment. Since it has been postulated that SM acts on chromatin structure, it is possible that dexamethasone induces apoptosis by enriching the nuclear SM pool and favouring chromatin destabilization.

    (1)Nelson DH, Murray DK., Proc. Natl. Acad. Sci. USA. 1982 Nov. 79: 6690-6692.

    (2)Yuh YS, Thompson EB., J Biol Chem. 1989 Jun 25; 264(18): 10904-10.

    (3)Galili U, J. Steroid Biochem. 1983 ; 19: 483-490.

    (4)McEwan IJ, Wright APH, Gustafsson JÅ, BioEssays 1997 ; 19: 153-160

  • Role of sphingosine kinase 1 in the regulation of proliferation and autophagy in myeloid leukemia cells.

    Ricci C.1,2, Sala G.1, Onida F.3, Soligo D.and Ghidoni R1.

    1Lab Biochem & Molecular Biology – San Paolo University Hospital, Medical School, University of Milan, 2Fondazione

    Matarelli, Milan, 3IRCCS Ospedale Maggiore and University of Milan

    Sphingosine Kinase 1 (SK1) is a key enzyme of the sphingolipid metabolism. By phosphorylating sphingosine (Sph), it induces intracellular accumulation of sphingosine 1 phosphate (S1P). As a second messenger, S1P stimulates proliferation and survival and protects cells against ceramide-induced apoptosis [1]. Although oncogenic activity of SK1 has been demonstrated in solid tumors [2], its role in the proliferation of leukemia cells remains unclear. In particular, we aimed to investigate SK1 involvement in cell growth regulation of chronic myeloid leukemia (CML), an hematopoietic disorder characterized by the malignant expansion of bone marrow pluripotent stem cells. The fusion protein Bcr/Abl, through its constitutive tyrosine kinase activity, is the pathogenetic event that causes CML and thus specific inhibitors have been developed. Among those, Imatinib Mesylate (IM) represents a successful strategy in CML treatment. However, most patients who are treated in advanced stages of the disease relapse after an initial response to IM. Therefore, the existence of a functional link between Bcr/Abl and SK1 would provide alternative targets to develop new strategies to overcome acquired resistance to IM in CML.

    Recently, the ERK1/2 and PI3K/AKT pathways were shown to be activated by SK1 overexpression [3]. Those molecules are involved also in the negative control of macroautophagy [4], a constitutive cellular mechanism for the turnover of organelles and long-lived proteins. According to the activating signal, increased autophagy can lead to either cell survival or death. First evidences showed a decrease of autophagic activity in tumor cells, but its exact role in oncogenic transformation needs to be clarified. Autophagosome formation requires the recruitment of Beclin-1, which is the product of a tumor-suppressor gene frequently deleted in human cancer, and LC3, marker of autophagic vacuoles. We aim to investigate the contribution of autophagy to CML proliferation and to define whether there is any relationship between this process and SK1.

    As an in vitro model, we used a panel of CML cell lines (KU-812, K562, RWLeu4 and AR230) and evaluated the basal expression level of SK1, Beclin 1 and LC3 by semi-quantitative RT-PCR. Afterwards, the effect of increasing doses (0, 1, 5 and 10 µM) of a novel SK1 inhibitor was determined at different time points (0, 24, 48 and 72 hrs) on cell proliferation and viability. Preliminary results showed that pharmacological inhibition of SK1 caused a decrease of the percentage of viable cells, suggesting that this kinase may play a role in the growth of leukemia cells.

    After establishing the IC50 of SK inhibitor for each cell line, the correlations between SK1 and Bcr/Abl will be investigated by treating cells with both SK1 inhibitor and IM. In particular, possible changes in the expression of mRNA and protein together with the kinase activity will be evaluated. Under the same conditions, we will also explore whether autophagy is consequently modulated.


    1.Cuvillier O et al, Nature, 381: 800-3, 1996

    2.French KJ et al, Cancer Res, 63: 5962-9, 2003

    3.Lescolan E et al, Blood (published online), 2005

    4.Arico S et al, J Biol Chem, 276 (38): 35243-6, 2001

  • Different ceramide-mediated cell responses to drugs in prostate cancer cells.

    Roberta Maffei, Giusy Sala & Riccardo Ghidoni

    Lab Biochem & Molecular Biology – San Paolo University Hospital, Medical School, University of Milan

    The sphingolipid ceramide is an important bioactive lipid that regulates diverse signaling pathways involving apoptosis, cell senescence, cell cycle and differentiation. It can be generated through different pathways. The various biosynthetic pathways seem to reflect the distinct cellular compartments where ceramide is localized, and to lead to different cellular responses.

    Our purpose is to correlate ceramide increase with i) its way of generation, and ii) the biological cell outcome. In this perspective we assayed a panel of different drugs to induce endogenous ceramide increase inandrogen-sensitive prostate cancer cell line (LNCaP).

    Celecoxib, CoCl2, doxorubicin, etoposide, fenretinide (4-HPR), resveratrol and tamoxifen have been used. Treated LNCaP have been evaluated for cell growth, induction of apoptosis and ceramide accumulation.

    All drugs, with the exception of tamoxifen, decreased viability. Growth curves showed a dose-dependentinhibition of proliferation for each treatment.

    With celecoxib, CoCl2, etoposide, 4-HPR and resveratrol we observed a small increase in intracellular ceramide, which is dose-dependent only with 4-HPR.

    Ceramide increase is not followed by PARP cleavage, with the exception of 4-HPR, and DNA laddering formation, in a wide concentration range.

    These preliminary results suggest that i) the LNCaP biological response to different drugs is not significantlyceramide-dependent, ii) only 4-HPR unequivocally induces apoptosis and this is associated with ceramide increase. The LNCaP genotype (i.e. this cell line exhibits acid ceramidase overexpression [1]) may explain the modest correlation between ceramide accumulation and cell response.


    [1] Seelan RS, Qian C, Yokomizo A, Bostwick DG, Smith DI, Liu W. “Human acid ceramidase is overexpressed but not mutated in prostate cancer.” Genes Chromosomes Cancer. 2000;29:137-46.

  • Anti-sphingomyelin antibodies in oncologic patients.

    Pugliese L., Bernardini I., Viola Magni MP., Albi E.

    Department of Clinical and Experimental Medicine, Physiopathology Section, Policlinico Monteluce, 06100, Perugia.

    Antiphospholipid (aPL) antibodies are a heterogeneous group of immunoglobulinsthat exhibit a broad range of target specificities and affinities, all recognizing various combinations of phospholipids, phospholipid-bindingproteins or both and phospholipid- protein complexes. It is known that the high values of Antiphospholipid (aPL) antibodies are indices of Antiphospholipid Syndrome characterised by vascular thrombosis, recurrent pregnancy loss and thrombocytopenia. Recently, the aPL antibody positivity was observed in patients with leukaemia, lymphoma and monoclonal gammopathy3-5.

    The aim of this work was to study the possibility of the aPL antibody positivity in patients with solid cancers in which other classic tumour markers were positive.

    First, according to the immunoassay currently performed, we used the ELISA Test (ALFA WASSERMANN,Cardiolipina-aK Combi test). Since anticardiolipin antibodies are believed to be polyspecific antibodies thatcross-react with all the anionic phospholipids6, we also proposed the use of phatidylinositol and sphingomyelin as antigens.

    Fifty six patients entered the study (35 F; 21 M) average age 62 yrs (range 30-92) with high tumour markers levels (CEA; CA 19.9; Ca 125; CA 15.3). None of the patients had SLE or other conditions known to be associated with aPL antibodies such as chronic infections, recent acute bacterial or viral infections. Patients with inherited causes of venous hypercoagulability (protein C/S, antithrobin III deficiencies) were excluded. As control, 48 healthy donor blood was used. Serum samples were stored at –20 ° C until used.

    The results showed that 28 of 56 patients (50.0%) were aPL positive and 25/28 (89.0%), 45% of all patients, had moderate or high antiphospholipid antibody levels. In particular we noticed a positivity for aPL antibodies in patients with high levels of CA 19.09 (6/7 corresponding to 85,7%) or CEA plus CA19.09 (8/10 corresponding to 80%). Nevertheless we also detected a strong association between aSM antibodies and these markers compared to aCL and aPI antibodies. In fact, the results showed that in patients with high level of CEA plus Ca19.09, the IgG positivity was 3/10 (30%) for the CL and PI and 6/10 (60%) for the SM and the IgM positivity were 1/10 (10%) for the CL and PI and 3/10 (30%) for the SM. In patients with Ca19.9 we noticed an IgG positivity for the CL 3/7 (42,8%), for the PI 4/7 (57,1%) and for the SM 3/7 (42,8%), the IgM positivity resulted 0/7 for the CL and PI and 3/7 (42,8%) for the SM.

    1.Hughes GRV BMJ 1983; 287: 1088; 2. Jerrold S. Levine M.D., Ware Branch M.D and Joyce Rauch PH.D NEJM, 2002, Vol.346, No.10; 3.Soltez P.,Szekanec Z., Vegh J., Lakos G., Toth L., Szakall S., Veres K., Szegedi G. Haematologia 2000; 30 (4):303-11; 4. Asherson RA, Khamashta MA, Ordi-Ros J, et al. Medicine (Baltimore)1989;68:366-74. 5. Vianna JL, Khamashta MA, Ordi-Ros J, et al. Am J Med 1994;96:3-9.Galli M, Comfurius P, Maassen C, et al. Lancet 1990;335:1544-7.

  • Effect of energetic stress and the ceramide analogue C6-ceramide on thyroid oncocytoma cell lines.

    A.M.Porcelli, A.Ghelli, L.Iommarini, M. Hoque, C. Zanna, V.Carelli*, G. Romeo§ and M.Rugolo

    Dip. di Biologia Evol. Sperim.; *Dip. di Scienze Neurologiche and

    §Dip. di Medicina Interna, Cardioangiologia, Epatologia, Università di Bologna

    Thyroid oncocytomas or Hürthle cell tumours (HCT) are characterized by a specific cytoplasmic eosinophilia. This reflects the abundance of mitochondria, which leads to the typical oxyphilic phenotype. Previous biochemical studies have suggested that a mitochondrial dysfunction might underlie HCT (1, 2). Recently, a high frequency of mtDNA mutations in complex I and IV genes has been shown to be involved in thyroid tumours (3, 4), suggesting that a coupling defect in oxidative phosphorylation might be a cause of compensatory mitochondrial hyperplasia in these tumours (5, 6). In order to study the bioenergetic efficiency of mitochondria in HCT we have determined cell growth in a medium where glucose is substituted by galactose. Under these conditions, the rate of glycolysis is greatly reduced and cells are forced to rely only on mitochondria for ATP production. Experiments have been carried out with the XTC-1 cell line (thyroid follicular oxyphilic cells) and the TPC-1 cell line (thyroid papillary carcinoma with no oxyphilic feature). In galactose medium, the TPC-1 cells were able to grow, albeit at a slower rate than in glucose medium. XTC-1 cells on the other hand exhibited a significant decrease in both cell viability and ATP levels. These results suggest that a severe energetic impairment exists in XTC-1 cells, which is likely to be responsible for the compensatory mitochondrial proliferation.

    In contrast to the cell death observed in galactose medium, XTC-1 cells were resistant to the toxic effect of the ceramide analogue C6-ceramide (20µM). The ATP content of these cells was maintained after treatment with the ceramide analogue, whereas the ATP level of TPC-1 cells was remarkably reduced. However, both cell lines appeared to be stressed, in particular the mitochondrial network, evaluated after loading with the specific dye Mitotracker Red, was found to be dramatically fragmented and swollen. We observed that Lysotracker Red, which specifically accumulates in the acidic compartments and lysosomes, was significantly increased in XTC-1 in comparison to TPC-1 cells. Our hypothesis is therefore that an autophagic process might be constitutively activated in the oxyphilic cell line, perhaps as a result of the mitochondrial energetic impairment. To investigate this specific issue, we will determine the effect of specific inhibitors of autophagy, such as 3-methyladenine (10mM) and wortmannin (100nM) on both cell viability and ATP levels ofC6-ceramide-treated XTC-1 cells. Furthermore, the incorporation of the fluorescent dye monodansylcadaverine, a specific marker for autophagic vacuoles (7), will be utilized to quantify the changes occurring in these organelles during incubation with the ceramide analogue. These preliminary results suggest that XTC-1 cells incubated with C6-ceramide might represent a useful model with which to investigate the autophagic process.

    1.Tallini G. (1998) Virchows Arch. 433:5-12.

    2.Maximo V. et al. (2000) Virchows Arch. 437:107-115.

    3.Maximo V. et al. (2002) Am J Pathol. 160:1857-1865.

    4.Rogounovitch T. et al. (2004) Endocr J. 51: 265-277.

    5.Savagner F. et al. (2001) Thyroid 11: 327-333.

    6.Savagner F. et al. (2001) J Clin Endocrinol Metab. 86:4920-4925.

    7.Munafò D.B. and Colombo M.I. (2001) J. Cell Sci. 114, 3619-3629

    This work is supported by a grant from AIRC.

  • Regulation of ceramide metabolism in glioma cells: evidence on the role of phosphatidylinositol- 4-kinases.

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

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

    A great number of evidence supports the role of ceramide (Cer), a key intermediate of sphingolipid metabolism as signalling molecule in mechanisms governing growth, differentiation and death in many cell types including glial cells. Different studies demonstrate that in glial cells Cer exerts antiproliferative and proapoptotic effects, and strongly support that Cer-signaling is altered in glial tumors. The control of Cer levels is a very complex process which involves both specific enzymes localized in different subcellular compartments and the regulation of Cer intracellular movements. Recent evidence suggests that the biological effects exerted by Cer depends on the variation of Cer levels in specific subcellular compartments, therefore correlated to the regulation of its intracellular traffic. Notwithstanding the mechanisms involved in Cer transport play a crucial role in Cer metabolism and signalling, the information on this process is very limited. Recently, a protein (CERT) that acts as ceramide carrier protein between endoplasmic reticulum (ER) and Golgi apparatus and is involved in sphingomyelin (SM) biosynthesis has been identified. The amino-terminal region of CERT contains a pleckstrin homology (PH) domain with selectivity towards phosphatidylinositol-4-phosphate (PI4P), to allow its targeting to the Golgi apparatus. In addition, recent evidence demonstrates that the interaction ofoxysterol-binding protein (OSBP) with ER and Golgi apparatus is regulated by its PH domain and is involved in the vesicle -mediated Cer transport from ER to the the Golgi for SM biosynthesis. This suggests the possibility that phosphoinositides, in particular PI4P a lipid known to be enriched in Golgi membranes, contributes to the regulation of ceramide metabolism and signalling. A number of different isoforms ofPI4-kinase (PI4-K) have been characterized. The isoenzymes differ from subcellular distribution and sensivity to inhibitors. Most of the isoenzymes are inhibited by phenylarsine oxide (PAO), whereas only the class IIIPI4-Ks, mainly associated to Golgi membranes, are sensitive to wortmannin (WT).

    In the present study we investigate the effect of PI4-Ks on ceramide metabolism and traffic in C6 glioma cells. Treatment of C6 glioma cells with both PAO and WT results in a dose-dependent decrease of phosphatidylinositol phosphorylation. In pulse/chase experiments with 3H-sphingosine both inhibitors promote a dose-dependent increase of 3H-Cer with a concomitant decrease of 3H-SM and in a lesser extent of 3H-glucosylceramide. Metabolic experiments performed with N-hexanoyl-[3H]sphingosine and 3H-SM allow to exclude an effect of PI4-K inhibitors and/or polyphosphoinositides directly on SM-synthase, SMase orglucosylceramide-synthase activity. Moreover, PI4-K inhibitors strongly affect the intracellular distribution ofBODIPY-Cer. In fact in cells treated with WT and PAO the accumulation of the fluorescence in the perinuclear region representative of the Golgi apparatus was strongly reduced. Staining with NBD Cer a specific marker of the Golgi demonstrate the integrity of Golgi apparatus in WT and PAO treated cells.Taken together these results strongly suggest that the inhibition of a PI4-K localized in the Golgi apparatus impairs the biosynthesis of sphingomyelin and in a lesser extent of glucosylceramide, possibly reducing the dockng sites, namely PI4P, for ceramide targeting to the Golgi apparatus. This could represent a further element in the cross-talkbetween phosphoinositides and sphingolipid signaling involved in the control of glial cell fate.

  • Regulation of pulmonary inflammation in cystic fibrosis by ceramide.


    Erich Gulbins, Yang Zhang, Katrin Anne Becker

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

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

  • Glycosylation and signaling: glycosphingolipids get their share.


    Sandrine Pizette1 

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

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

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

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

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


    Frederic Bornancin 

    Novartis Institutes for BioMedical Research, Basel, Switzerland 

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

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

  • Tumor gangliosides condition the microenvironment and favor tumor progression.


    Stephan Ladisch 

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

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

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


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

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

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

    Supported by CRUK [A7536]. 

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


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

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

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

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

    [Supported by DAER-Recherche/06001316/07006301] 

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


    Lysann Sauer1, Dmitry Pshezhetskiy1 and Jonathan Waxman

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

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

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

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

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


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

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

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

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


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

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

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

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

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

  • Involvement of the S1P3 receptor in neuroinflammation.


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

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

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

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

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

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


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

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

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

  • Pharmacological regulation of sphingolipid metabolites in blood.


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

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

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

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


    KS Mascall, GR Small, GF Nixon 

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

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

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

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


    C Wirrig, FA Mathieson, I Hunter, GF Nixon 

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

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

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

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

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


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

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

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

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


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

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

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

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

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


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

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

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

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


    A Völzke1, A Koch1, A Huwiler2, J Pfeilschifter1 

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

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

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

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

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


    Christoph Arenz

    1Institut für Chemie, Humboldt Universität zu Berlin, Germany 

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

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

  • Role of sphingolipid trafficking in cannabinoid-induced autophagy.


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

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

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

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

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

  • Nuclear import of sphingomyelinase in space environment.


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

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

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

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

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


    F. Betul Kavun Ozbayraktar1, Kutlu O. Ulgen1 

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

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

  • Sphingolipids in retinal degenerations.


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

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

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

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

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


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

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

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

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

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


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

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

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

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

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


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

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

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

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

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

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


    M Schüppel1, EI Schmitz1, H Potteck2, B Kleuser2 

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

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

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

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

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


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

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

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

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


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

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

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

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

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

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

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


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

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

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

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

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

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


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

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

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

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

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

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


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

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

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

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

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

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


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

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

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

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

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

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

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


    M Gurgui, R Broere and G van Echten-Deckert 

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

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

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


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

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

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

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


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

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

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

  • Gαq mediates sphingosine kinase-1 translocation by GPCRs.


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

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

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

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


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

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

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

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


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

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

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

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


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

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

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

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

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


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

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

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

  • Sphingosine-induced changes in the bilayer structure.


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

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

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

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


    Veronica Kalhori1, Kati Kemppainen1 and Kid Törnquist1,2 

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

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

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


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

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

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

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

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

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

  • Sphingosylphosphorylcholine binds to calmodulin and inhibits its function.


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

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

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

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


    Masatake Kai1, Carl-Philipp Heisenberg2 and Masazumi Tada1  

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

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

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


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

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

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

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


    M.A. Shupik, A.V. Alessenko 

    Institute of Biochemical Physics of the RAS, Moscow, Russia 

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

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


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

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

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

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


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

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

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

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


    Yeonju Lee, Sohyeon Moon and Seikwan Oh 

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

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

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

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

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


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

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

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

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

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

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

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


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

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

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

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

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


    M Strigini1 

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

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

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


    Stephanie Schwalm1,2, Josef Pfeilschifter1 and Andrea Huwiler

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

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

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

  • In situ synthesis of fluorescent membrane lipids (ceramides) using click chemistry.


    María Garrido1, José Luis Abad1, Alicia Alonso2, Antonio Delgado1,3, L. Ruth Montes2, Félix M. Goñi2

    1Consejo Superior de Investigaciones Científicas (CSIC), Institut de Química Avançada de Catalunya (IQAC), Research Unit on Bioactive Molecules (RUBAM). Jordi Girona 18‑26, 08034 Barcelona, Spain.2Unidad de Biofísica (CSIC-UPV/EHU), and Departamento de Bioquímica, Universidad del País Vasco, Aptdo. 688, 48080 Bilbao, Spain.L. Ruth Montes, Phone: +34 946 018 050, Email: lidiaruth.montes@ehu.es3Universidad de Barcelona, Facultad de Farmacia, Unidad de Química Farmacéutica, Avda. Joan XXIII s/n, 08028 Barcelona, Spain.

    Ceramide analogues containing azide groups either in the polar head or in the hydrocarbon chains are non-fluorescent. When incorporated into phospholipid bilayers they can react in situ with a non-fluorescent 1,8‑naphtalimide using click chemistry giving rise to fluorescent ceramide derivatives emitting at ≈440 nm. When incorporated into giant unilamellar vesicles, two-photon excitation at 760 nm allows visualization of the ceramide-containing bilayers. This kind of method may be of general applicability in the study of membrane and cell membranes.

  • On line hptlc-maldi for the qualitative and quantitative analysis of neutral and acidic glycosphingolipids.


    Torretta E. 1, Vasso M. 1,2 , Fania C. 1, Capitanio D. 1, Bergante S. 1,3, Piccoli M. 1,3, Anastasia L. 1,3, Gelfi C. 1,2

    1 Department of Biomedical Sciences for Health, University of Milan, Segrate (Milan), Italy; 2 Institute of Molecular Bioimaging and Physiology (IBFM), CNR, Cefalù (Palermo) Segrate (Milan), Italy; 3 Laboratory of Stem Cells for Tissue Engineering, IRCCS Policlinico San Donato (Milan), Italy

    Glycosphingolipids are a wide class of ubiquitous lipids, characterized by a great structural and functional variety. Altered levels have been correlated with different diseases, suggesting their crucial role in health. To date, characterization is mainly based on TLC, followed by antigen antibody reactions or by radioactive labelling. Recently, new mass spectrometry strategies have been developed. Herein we set up an online HPTLC-MALDI method combining high resolving power and mass accuracy.

    Different matrices solutions were tested to set up the best conditions for matrix delivery to improve GSLs detection, resulting in DHB matrix in 50% acetonitrile as the best matrix, 50mg/mL for neutral GSLs and 100 mg/mL for acidic GSLs. Calibration curve of a neutral GSL (Gb3) and of a ganglioside (GM3) were constructed to demonstrate the applicability of HPTLC-MALDI method to quantitative analyses. The aqueous and organic phases extracted from wild-type and overexpressing NEU3 sialidase C2C12 murine myoblasts, were analysed by HPTLC, followed by MALDI-TOF and results compared to [3H]sphingolipids radiolabeled HPTLC. MS resolution allowed to identify several species with similar retention factor on the HTPLC plate, and that could not be resolved with the radiolabelling. Neutral GSLs (SM, Gb3, LacCer, GlcCer) and gangliosides (GM1, GM2, GM3, GD1a) were identified as C16:0, C22:0, C24:1 and C24:0 chains. GM3, the physiological substrate of NEU3, is decreased in NEU3 over-expressing cells, as assessed by radioactive quantitative measurements. Interestingly, by HPTLC-MALDI, spectra indicated that two GM3 isoforms decreased (d18:1 C16:0 and d18:1 C22:0) while d18:1 C24:1 increased.

    HPTLC-MALDI is an easy and high-throughput analysis for the qualitative and quantitative characterization of GSLs suggesting its use for their profiling with high specificity and sensitivity.

  • Polyene sphingolipids with latent fluorescence: new tools to study the biophysical properties of cellular membranes.


    Ingrid Nieves1, Ruth Montes2, Jesús Sot2, Alicia Alonso2, José Luís Abad1, Antonio Delgado1,3

    1Research Unit on Bioactive Molecules (RUBAM), Dept. of Biomedicinal Chemistry, IQAC-CSIC, Barcelona, Spain;  2University of País Vasco, Dept. of Biochemistry, Unit of Biophysics, CSIC‑UPV/EHU, Bilbao, Spain

    3University of Barcelona, Faculty of Pharmacy, Unit of Pharmaceutical Chemistry (Associated Unit to CSIC), Barcelona, Spain.

    Experiments with bioactive sphingolipids (SLs) in model membranes are currently hampered by a lack of adequate experimental conditions able to mimic the physiological environment. To analyze membrane microdomains that are temporarily enriched in certain lipids, it is necessary to employ high molar concentrations of SLs analogues, or fluorescence tags that strongly influence the properties of lipid bilayers, such as BODIPY or NBD. Herein, we describe the synthesis of a series of GABA-polyene-n‑Doxyl fluorescent probes, specifically designed as biochemical tools to allow the study of model membranes at low lipid concentrations, closer to physiological conditions. These compounds contain a fluorescent polyene system, acylated with an internal n‑Doxyl radical able to act as a fluorescence quencher. This probe has enabled the development of a simple biophysical method to experimentally identify the formation of lipid domains. Thus, fluorescence of the GABA-polyene-n‑Doxyl system depends on the rigidity or fluidity of the lipid environment. The absorption and fluorescence properties of the probes, when incorporated into artificial vesicles, were monitored by espectrofluorimetry (for LUVs) or by fluorescence confocal microscopy (for GUVs). Preparation of rigid‑ordered liposomes with DSPC or DPPC showed a decrease of the fluorescence intensity versus fluid-disordered membranes of DOPC or eggPC. Moreover, it was observed that the conformational mobility of the n-Doxyl group was crucial for intramolecular fluorescence quenching, a property that was used as diagnostic to determine the characteristics of the membrane phosphoglycerolipids.

    As a result, GABA-polyene-nDoxyl compounds may be of utility as novel fluorescent lipid probes to identify rigid-fluid domains in model membranes. 

  • UFLC MS/MS to study the response of sphingomyelin to proton radiations in FRTL-5 cells.


    Andrea Lazzarini1,2 Elisabetta Albi1*†, Giuseppina Perrella2† , Remo Lazzarini1, Samuela Cataldi1, Alessandro Floridi1, Emanuela Floridi1, Francesco Saverio Ambesi-Impiombato2, Francesco Curcio2

    1 Laboratory of Nuclear Lipid BioPathology, Research Center of Biochemical-Specialized Analyses CRABioN, 06100 Perugia (Italy); 2Department of Clinical and Biological Sciences, University of Udine, 33100 Udine (Italy)

    In cell nucleus the sphingomyelin (SM) cycle is regulated by an exact internal mechanism. In no-stimulated cells exist a balance between  neutral-sphingomyelinase (N-SMase) and sphingomyelin-synthase (SM-synthase) activity that maintains constant the SM level, stabilizing the double helix of DNA thanks to  the  trimethylammonio group that binds to the phosphate group of DNA and apolar fatty acids that bind to the hydrophobic centres of the helical DNA. The increase of the nuclear SM concentration during cell proliferation stimulates the SMase activity  during the S phase of the cell cycle, thus favouring the destabilization and the opening of the double helix of DNA and the beginning of its synthesis. The winding of a double helix being restored as soon as DNA synthesis finishes and SM-synthase activity is stimulated. During apoptosis specific the gene expression and DNA fragmentation are accompanied by the change of nuclear SM content. Recently the nuclear N-SMase/SM-synthase ratio was considered a marker of cell function. In fact it was very high in apoptotic cells, medium in pro-apoptotic cells, low in proliferating cells and very low in quiescent cells. Here we studied for the first time whether in thyroid FRTL-5 cells the proton beams could change nuclear SM metabolism by comparing the results with that occurred in nuclei-free lysates (NFL)  at the end to identify their role on functional state of the cells. In the nucleus proton beams stimulated  1.09 times N-SMase activity and  inhibited 1.9 times that of SM-synthase in nuclei purified from quiescent cells whereas stimulated 12.44 times N-SMase activity and unchanged that of SM-synthase. The effect was very low in NFL lipid enzymes. As a consequence, the nuclear N-SMase/SM-synthase ratio was 1.06 and 1.77 in nuclei of quiescent and proliferating cells respectively, indicating that protons induced quiescent cells in a propaoptotic state whereas its value in proliferating cells was between the proapoptotic state and apoptotic state, as supported by bax expression that was higher in proliferating than in quiescent cells. The analysis of nuclear SM and ceramides by UFLC-MS/MS showed the variation of these lipids after proton treatment. In conclusion, the damage of proton beams on epithelial thyroid cells depends on their physiological state, whether they are stimulated or not with thyrotropin to proliferate. The nuclear SM metabolism is involved in  the mechanism of action of protons.

  • Recent contributions of chemistry to sphingolipid research.


    Gemma Fabrias,1 José Luis Abad,1 Josefina Casas,1 Maria Garrido,1 Ingrid Nieves,1 Pedro Rayo,1 and Antonio Delgado1,2

    1Consejo Superior de Investigaciones Científicas (CSIC), Institut de Química Avançada de Catalunya (IQAC-CSIC), Research Unit on Bioactive Molecules (RUBAM), Barcelona, Spain. 2Universidad de Barcelona (UB), Facultad de Farmacia, Unidad de Química Farmacéutica (Unidad Asociada al CSIC), Barcelona, Spain.

    Progress in the understanding of sphingolipid metabolism and functions has benefited from the contribution of chemistry. Structural analogs, enzyme inhibitors and receptor agonists and antagonists have been employed as pharmacological tools and some of them have reached relevance in a therapeutic context.

    An overview of recently reported compounds of use in sphingolipid research is given in this talk. Furthermore, unpublished ongoing studies carried out with novel 1-deoxysphingolipid analogs are presented. Biological screening of these analogs has revealed the appropriateness of some of the compounds as probes to profile ceramide synthase activities in intact cells, while potent and selective neutral ceramidase inhibitory activity has been disclosed by other molecules of the library. Future directions include the development of suitably functionalized derivatives amenable to orthogonal reactions with fluorescence tags for subcellular trafficking studies.

  • Inhibition of S1P degradation rescues 661w cells from oxidative stress.


    Carlotta Fabiani 1, Anna Caretti 1, Annalisa Guala 1, Josefina Casas 2, Gemma Fabriàs 2, Riccardo Ghidoni 1, Paola Signorelli 1.

    1 , Department of Health Sciences, University of Milan, San Paolo Hospital, Italy;2, Research Unit on BioActive Molecules, Department of Biomedicinal Chemistry, Catalan Institute of Advanced Chemistry (IQAC/CSIC), Barcelona, Spain.

    Sphingolipids have crucial roles in retinal pathologies (Rostein et al 2010). Recently, increase in Ceramides has been correlated with rat retinal light-induced oxidative stress and degeneration  (Chen et al. 2013). In a murine model of human Retinitis Pigmentosa (rd10 mutant mice), we demonstrated that inhibition of Ceramide synthesis and accumulation rescued photoreceptors from age-related apoptosis (Strettoi et al. 2010).Our aim is targeting sphingolipid metabolites to reduce oxidative damage in a retinal photoreceptor cell colture, 661W cone-like cells. In this study we focalized our attention on sphingosine-1-phosphate (S1P) and its pro-survival and proliferative activity. We inhibited S1P degradation, by THI treatment, reducing stress response signaling, pro-apoptosis Ceramide accumulation and promoting survival to oxidative stress. Murine 661W cone-like cell line, was treated with 75µM 2-acetyl-4-(1(R),2(S),3(R),4-tetrahydroxybutyl)-imidazole (THI), an inhibitor of S1PLyase, for 2 hours; next,  cells were treated with 1mM H2O2 for different times. We showed that enhanced stability of S1P, obtained through THI administration, reduces H2O2 inhibitory effect on cell viability.

    The evaluation of Ceramides content by UPLC/TOF analysis, showed an augment in short-chain Ceramides (16:0 and 18:0) in stressed cells, that is contrasted by THI. At the same time, oxidative stress produced a decrease in long-chain Ceramide (24:0), which was not altered by THI. MAP kinase phosphorylation is involved in photoreceptors protection from oxidative stress (German et al. 2006). We demonstrated that THI reverses H2O2 -induced ERK1/2 de-phosphorylation. Activation of AKT plays an important role in cell survival. Specifically phosphorylation of Ser473 was shown to be enhanced under stress in different cell models (Yung et al. 2011), including oxidative stress in retinal pigment epithelial cells (Lee et al. 2011). Pre-treatment with THI reduced H2O2 -induced Akt phosphorylation on Ser473.

  • Inhibition of dihydroceramide desaturase activity by the sphingosine kinase inhibitor SKI II.


    Francesca Cingolani, Cindy Nájera, Josefina Casas and Gemma Fabrias

    Consejo Superior de Investigaciones Científicas (CSIC), Institut de Química Avançada de Catalunya (IQAC-CSIC), Research Unit on Bioactive Molecules (RUBAM), Barcelona, Spain.

    Compound SKI II (4-((4-(4-chlorophenyl)thiazol-2-yl)amino)phenol) is a dual inhibitor of sphingosine kinases 1 and 21 exhibiting anti-proliferative activity on several human cancer cell lines with IC50 values in the low µM range (0.9-4.6 µM).2 In their study on the role of sphingosine kinases in the resistance of A2780 ovarian cancer cell line to N-(4-hydroxyphenyl)retinamide (HPR), Illuzi et al.3 showed that cells treated with SKI II produced significantly more dihydroceramides than controls, although remarkably less than cells treated with HPR alone or in combination with SKI II. On the other hand, Tonelli et al.4 have recently reported that SKI II induces oxidative stress, which is known to inhibit Des1 activity.5 These overall data suggested that SKI II might inhibit Des1, thereby not being specific for sphingosine kinases.

    In this communication we report that, at the concentrations used to inhibit sphingosine kinases, SKI II inhibits the desaturation of dihydroceramide-C6NBD to ceramide-C6NBD both in intact cells and in cell lysates. In the first case, SKI II inhibits proliferation of gastric adenocarcinoma HGC27 cells with a concomitant increase in dihydroceramide levels. Similar activities were elicited by the non-phenolic polyaromatic drug celecoxib, also reported to inhibit Des1.6 The effects of both SKI II and celecoxib on autophagy and cell cycIe in correlation with changes in the sphingolipidome are presented and discussed.

    In conclusion, SKI II inhibits Des1 and therefore, caution must be taken when interpreting data based on the use of this compound as a pharmacological tool.

  • Modulation of S1P receptors by the fingolimod derivatives, ST968 and ST1071.

    OP8 poster

    Kira Blankenbach1, Holger Stark2, Josef Pfeilschifter1, Andrea Huwiler3, Dagmar Meyer zu Heringdorf1

    1Institut für Allgemeine Pharmakologie und Toxikologie, Klinikum der Goethe-Universität Frankfurt am Main, Germany; 2Institut für Pharmazeutische Chemie, Goethe-Universität Frankfurt am Main, Germany; 3Institut für Pharmakologie, Universität Bern, Switzerland

    Fingolimod is a novel immunosuppressive drug which has been approved for the treatment of multiple sclerosis. It is a prodrug of fingolimod-phosphate which acts as superagonist at the S1P receptors, S1P1,3,4,5, and induces lymphopenia by internalizing the S1P1 receptor.

    Here, we analysed whether the novel compounds, ST968 and ST1071, which are structurally related to fingolimod, interacted with S1P1-3. Internalization of S1P1, S1P2 and S1P3 was studied with GFP-tagged receptors transiently expressed in CHO cells. After 2 h of exposure, both ST968 and ST1071 (1 µM each) induced internalization of S1P1 and S1P3, but not S1P2, similar to fingolimod. When the cells were incubated for 2 h with the respective agonists, washed, and analysed ~22 h later, S1P1 was still internalized in cells treated with ST968, ST1071, and fingolimod-phosphate, but not in S1P-treated cells. S1P receptor-induced [Ca2+]i increases were analysed in CHO cells stably transfected with GFP-tagged S1P1, S1P2 or S1P3 plus the Gαqi5 protein which links Gi-coupled receptors to Gq signalling pathways. S1P (1 µM) induced [Ca2+]i increases in all three cell lines, while fingolimod-phosphate (1 µM) was active in S1P1- and S1P3-, but not S1P2-expressing cells. ST968 and ST1071 (1 µM each) induced [Ca2+]i increases in S1P3-expressing cells but were inactive in S1P1- or S1P2-expressing cells. In conclusion, ST968 and ST1071 represent interesting novel structures which interact with S1P receptors.

  • Design and synthesis of new sphingosine-1-phosphate lyase (S1PL) inhibitors.

    OP9 poster

    Pol Sanllehí1, Jordi Bujons2, Josefina Casas1 and Antonio Delgado1,3

    1Research Unit on Bioactive Molecules (RUBAM), Departament de Química Biomèdica, Institut de Química Avançada de Catalunya (IQAC-CSIC), 2Department de Química Biològica i Modelització Molecular, Institut de Química Avançada de Catalunya (IQAC-CSIC), 3Facultat de Farmàcia, Unitat de Química Farmacèutica (Unitat Associada al CSIC), Universitat de Barcelona

    Accumulating evidence indicate that sphingosine-1-phosphate (S1P) modulates many noxious processes that follow CNS injury. Thus, the discovery of novel neuropretective therapies may arise from interventions on S1P metabolism. S1PL is a PLP-dependent enzyme that irreversibly cleaves S1P into 2‑trans‑hexadecenal and phosphoethanolamine. Despite the capital role of S1PL in controlling the intracellular levels of S1P, the number of available pharmacological tools to modulate its activity is scarce.

    A homology model of human S1PL was built using templates derived from the structures of prokaryotic and yeast S1PLs (PDB 3MAD and 3MC6, respectively), which show around 40 % sequence identity with the human protein. The obtained modelled protein allowed a first massive in silico screening of potential S1PL inhibitors. Therefore, MOE’s Lead-Like database of 653.200 compounds was screened against the 3MAD-derived model using standard docking protocols. Among the best scored compounds, a subset of 28 molecules were chosen (mainly based on availability criteria), purchased and tested using our previously reported assays. It is worthy of mention the inhibitory activities exerted by, at least, 4 compounds of the selected set, which were comparable to those of the reference compound FTY-720. Finally, a general pharmacophore for the S1PL inhibitory activity was elucidated regarding the common structural features of the more active compounds. This general pharmacophore led us to the design of a series of new putative S1PL inhibitors.

    Herein we present the homology modelling and structure-base drug design (SBDD) studies of human S1PL, which allowed us to find a small series of compounds with promising S1PL inhibitory activity. As a result of the resulting pharmacophore, a family of new potential inhibitors was designed and synthesized.

  • Highly potent acid ceramidase inhibitors with marked cancer chemosensitization activity.

    OP10 poster

    1Realini Natalia, 1Pizzirani Daniela, 1Pagliuca Chiara, 1Armirotti Andrea, 1Bandiera Tiziano, 1Marco De Vivo, 1,2Piomelli Daniele

    1Unit of Drug Discovery and Development, Italian Institute of Technology, 16163 Genoa, Italy

    2Departments of Pharmacology and Biological Chemistry, University of California, Irvine, Irvine, California 92697-4625

    New therapeutic strategies for overcoming chemo-resistance include the stimulation of ceramide accumulation through increased ceramide synthesis or inhibition of ceramide degradation. Acid ceramidase (AC)is an ubiquitous cysteine hydrolase that catalizes the hydrolysis of ceramide into sphingosine and fatty acid within the lysosome. The expression of AC is up-regulated in several types of human cancer, making this enzyme an interesting pharmacological target for cancer therapy. We here report that the anticancer drug, 1-hexylcarbamoyl-5-fluorouracil (carmofur), is a potent AC inhibitor and that this mechanism may contribute to its anti-cancer properties. Carmofur inhibits native and recombinant rat AC activity in broken cell preparations with a median inhibitory concentration (IC50) of 29±5 nM (n=4). The effect is partially reversible and occurs through a non-competitive mechanism. Studies in various cancer cell lines (including SW403 and LNCaP) showed that carmofur inhibits AC activity and increases ceramide levels in intact cells. Furthermore, in vivo administration of carmofur in mice resulted in a dose- and time-dependent inhibition of AC activity, which was accompanied by an elevation of ceramide levels in various organs and tissues (Realini et al., Sci. Rep., 2013). A systematic SAR study around the uracil scaffold allowed a first elucidation of critical structural features associated with AC inhibition and the identification of the first single-digit nanomolar inhibitors (Pizzirani et al., J.Med.Chem., 2013). Selected compounds were found to inhibit AC activity and elevate ceramide levels with high potency. Importantly, such compounds displayed a marked synergistic effect with 5-FU and other chemotherapeutic drugs in decreasing colon cancer cell viability.

    Our studies identify the first class of potent AC inhibitors. These compounds will be useful to investigate the functions of ceramide in vivo, and may represent a starting point  for the development  of novel chemosensitizing agents for cancer therapy. 

  • Specific sphingolipid content decrease in cerkl knockdown mouse retinas.

    OP11 poster

    Alejandro Garanto a,b,c, Nawajes M. Mandal d,e , Meritxell Egido-Gabás f, Gemma Marfany a,b,c, Gemma Fabriàs f, Robert E. Anderson d,e,g, Josefina Casas f, Roser Gonzàlez-Duarte a,b,c

    a Departament de Genètica, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain. b Institut de Biomedicina (IBUB), Universitat de Barcelona, Barcelona, Spain. c CIBERER, Instituto de Salud Carlos III, Barcelona, Spain. d Dean McGee Eye Institute, Oklahoma City, USA. e Department of Ophtalmology, University Health Sciences Center, Oklahoma City, USA. f Research Unit on BioActive Molecules (RUBAM), Departamento de Química BioMédica, Instituto de Química Avanzada de Catalunya, CSIC, Barcelona, Spain. g Department of Cell Biology, Oklahoma University Health Sciences Center, Oklahoma City, USA

    Sphingolipids (SPLs) are finely tuned structural compounds and bioactive molecules involved in membrane fluidity and cellular homeostasis. The core sphingolipid, ceramide (CER), and its derivatives, regulate several crucial processes in neuronal cells, among them cell differentiation, cell-cell interactions, membrane conductance, synaptic transmission, and apoptosis. Ceramide Kinase-Like (CERKL) is a gene causing autosomal recessive Retinitis Pigmentosa and Cone Rod Dystrophy. The presence of a conserved lipid kinase domain and the overall similarity with CERK suggested that CERKL might play a role in the SPL metabolism as a CER kinase. Unfortunately, CERKL function and substrate(s), as well as its contribution to the retinal ethiopathology, remain as yet unknown. In this work we aimed to characterize the mouse retinal sphingolipidome by UPLC-TOF to first, thoroughly investigate the SPL composition of the murine retina, compare it to our Cerkl -/- model, and finally assess new possible CERKL substrates by phosphorus quantification and protein-lipid overlay. Our results showed a consistent and notable decrease of the retinal SPL content (mainly ranging from 30% to 65%), particularly evident in the glucosyl/galactosylceramide species (Glc/GalCer), while the fatty acids, phospholipids and neutral lipids remained unaltered. Moreover, evidence in favor of CERKL binding to GlcCer, GalCer and sphingomyelin has been gathered. Altogether, these results highlight the involvement of CERKL in the SPL metabolism, question its role as a kinase, and open new scenarios.

  • The complex biology of acid sphingomyelinase in Human Diseases and membrane microdomains.


    Edward H. Schuchman

    Icahn School of Medicine at Mount Sinai, New York, USA

    The enzyme acid sphingomyelinase (ASM) was first described in rat brain homogenates by Professor Shimon Gatt at the Hadassah Medical Center in Israel in the early 1960s, and was later found to be the enzyme deficient in the lysosomal storage disorder, Niemann-Pick disease (NPD) Types A & B, by Dr. Roscoe Brady at the National Institutes of Health in the United States.  Due to its acidic pH optimum and the predominant lysosomal storage of sphingomyelin in NPD patients, ASM was presumed to be an exclusively lysosomal housekeeping protein involved in membrane turnover within this cellular compartment.  In the mid-1990s Richard Kolesnick at the Sloan Kettering Cancer Center in New York postulated that ASM had an extra-lysosomal function in radiation-induced apoptosis, and also participated in the re-organization of lipid signaling domains in the plasma membrane through the generation of ceramide.  This hypothesis was confirmed using mice deficient in ASM activity (ASMKO mice) constructed at the Mount Sinai School of Medicine in New York, which were protected from radiation-induced apoptosis.  Subsequent studies by numerous investigators have confirmed the broad role of ASM in stress-induced cell signaling, and the importance of the enzyme in diverse diseases such as fibrosis, diabetes, cystic fibrosis and others.  Numerous outstanding questions remain, however, including the mechanism of transport of intracellular ASM to the cell surface upon stress induction, and the precise compartment(s) at the cell surface in which it functions.  In addition, since recombinant ASM is currently in clinical development as a therapy for NPD patients, the potential of this enzyme for cancer therapy also has been suggested.  This overview will discuss the complex biology of ASM and its role in cell signaling and human disease.

  • Role of nuclear lipid microdomains on cell function.


    Alessandro Floridi1, Giacomo Cascianelli1, Andrea Lazzarini1,2, Remo Lazzarini1, Emanuela Floridi1, Francesco Saverio Ambesi-Impiombato2, Elisabetta Albi1*

    1 Laboratory of Nuclear Lipid BioPathology, Research Center of Biochemical-Specialized Analyses CRABioN, 06100 Perugia (Italy); 2Department of Clinical and Biological Sciences, University of Udine, 33100 Udine (Italy)

    The inner nuclear membrane is internally covered with the nuclear lamina, which comprises a meshwork of intermediate filaments located on the endonuclear surface associated with chromatin transcriptionally active.  We have recently demonstrated in inner nuclear membrane the presence of the lipid microdomains that act as platform for active chromatin. Like cell membrane lipid rafts, the nuclear lipid microdomains (NLM) are rich in sphingomyelin (SM) and cholesterol (CHO) content and appear as a homogenous population of closed, spherical or ovoid vesicle-like structures with an average diameter in the range of 300-600 nm. The marker of nuclear lipid microdomains is the Signal Transducer and Activator of Transcription-3. Their plasticity, due to neutral-sphingomyelinase (N-SMase), sphingomyelin-synthase (SM-synthase) and reverse sphingomyelin-synthase (RSM-synthase) activity,  influences nuclear function. In cell proliferation the INM play a role as platform for on DNA synthesis and transcription process. It has been supposed that the decrease of SM, due to the activation of N-SMase at the beginning of the S phase of the cell cycle, could be responsible for the destabilization of the double strand DNA, favouring its despiralization and new synthesis. Differently the increase of SM, due to the activation of SM-synthase at the end of the S phase of the cell cycle, could be responsible  for the transcription process. Here we have performed for the first time a lipidomic study of the NLM purified from liver nuclei by UFLC-MS/MS mehod.  

    Our results show that the species of SM more representative of nuclear membrane such as SM 18:1 12:0, SM 18:1 16:0, SM 18:1 18:1, SM 24:0 are present also in NLM but the analysis of the difference of the two structures highlights that NLM is very rich of SM 24:0. Among all ceramide species considered, such as ceramide  18:1 16:0, ceramide 18:1 18:0, ceramide 18:1 20:0, ceramide 18:1 24:0, the level of ceramide 18:1 24:0 is strongly reduced in comparison to that of the nuclear membrane. Also the value of phosphatidylcholine (PC) 16:0 18:1, PC 16:0 20:4 and PC 18:1 18:0 are lower than those present in nuclear membrane. 2-arachidonoyl glycerol  is absent in NLM.

  • Improving doxorubicin efficacy through nanoliposomes equipped with selective tumor cell membrane.


    Lília R. Cordeiro Pedrosaa, Albert van Hellb, Regine Süssc, Wim van Blitterswijk e, Ann L.B Seynhaeve a, Wiggert A. van Cappellend, Alexander M.M Eggermonta,e, Timo ten Hagena, Marcel Verheijb,e,f and Gerben A. Koninga

    aLaboratory Experimental Surgical Oncology, Section Surgical Oncology, Department of Surgery, Erasmus MC - Daniel den Hoed Cancer Center, Rotterdam,3000 CA, The Netherlands bDivision of Biological Stress Response, eDivision of Cellular Biochemistry, and fDepartment of Radiotherapy, The Netherlands Cancer Institute - Antoni van Leeuwenhoek Hospital, Amsterdam,1066 CX, The Netherlands cDepartment of Pharmaceutical Technology and Biopharmacy, Albert-Ludwigs University, Freiburg,D79104, Germany, dOptical Imaging Centre, Erasmus MC Center, Rotterdam, 3000 CA,The Netherlands, eInstitut de Cancerologie Gustave Roussy, Villejuif, Paris, 94800,France

    To improve nanoliposomal-doxorubicin (DoxNL) delivery in tumor cells using liposome membrane-incorporated short-chain sphingolipids (SCS) with selective membrane-permeabilizing properties. DoxNL bilayers contained synthetic short-chain derivatives of known membrane microdomain-forming sphingolipids; C8-glucosylceramide (C8-GluCer) C8-galactosylceramide (C8-GalCer) or C8-lactosylceramide (C8-LacCer). Methods: DoxNL enriched with C8-GluCer or C8-GalCer were developed, optimized and characterized with regard to size, stability and drug retention. In vitro cytotoxic activity was studied in a panel of human tumor cell lines and normal cells. Intracellular Dox delivery was measured by flow cytometry and visualized by fluorescence microscopy. For a further understanding of the involved drug delivery mechanism confocal microscopy studies addressed the cellular fate of the nanoliposomes, the SCS and Dox in living cells. In vivo efficacy studies were performed in nude mice bearing human A431 xenografts. Standard and SCS-enriched Dox-NL were administered intravenously via the tail vein and tumor growth and body weight measurements were conducted for a period of 14–20 d. Results: C8-LacCer-DoxNL aggregated upon Dox loading. In tumor cell lines SCS-DoxNL with C8-GluCer or C8-GalCer demonstrated strongly increased Dox delivery and cytotoxicity compared to standard DoxNL. Surprisingly, this effect was much less pronounced in normal cells. Nanoliposomes were not internalized and SCS transfer from the nanoliposomal bilayer to the cell membrane preceded cellular uptake and subsequent nuclear localization of Dox. In vivo efficacy showed treatment of nude mice bearing human A431 xenografts with 6 mg/kg GluCer-Dox-NL almost doubled the tumor growth delay compared with standard DoxNL. A second administration of 5 mg/kg after 3 d induced even 3-fold delay in tumor growth, while no systemic toxicity was found. Conclusion: SCS incorporated in DoxNL selectively improved intracellular drug delivery through transfer to tumor cell membranes by local enhancement.

  • Sphingomyelin synthases modulate death-receptor ligand-induced apoptosis.


    Guillaume Poiroux1, Elodie Lafont1, Hervé Benoist1, Toshiro Okazaki2, Thierry Levade1 and Bruno Ségui1

    1 INSERM UMR1037-Cancer Research Center of Toulouse, BP84225, 31432 Toulouse cedex 4, France. 2 Department of Medicine, Kanazawa Medical University, Uchinada, Ishikawa 920-0293, Japan.

    Ceramide, a biologically active sphingolipid, is considered as an anti-oncometabolite, promoting apoptosis and non-apoptotic forms of cell death. The intracellular levels of ceramide are highly regulated via complex metabolic pathways. Sphingomyelin synthases (SMS) 1 and 2 convert ceramide to sphingomyelin (SM), a ubiquitous phospholipid in mammals. Controversy exists in the literature concerning the role of SMSs in stress-induced apoptosis.

    We previously reported that SMS1 and, albeit to a lesser extent, SMS2 inhibit CD95-triggered ceramide increase and apoptosis (Lafont et al, 2010 Cell Death Diff.; Lafont et al, 2012 Biochim Biophys Acta). Our unpublished data indicated that SMS1 is also able to partially inhibit TRAIL-induced apoptosis in HeLa cells. Whereas SMS1 overexpression impaired TRAIL-triggered ceramide increase, caspase activation and apoptosis, SMS1 knockdown had opposite effects. One should note however that, under our experimental conditions, sphingomyelin level was not or minimally affected by the modulation of SMS1 expression, most likely as a consequence of sphingolipid metabolism adaptation. We are currently investigating the role of SMS1 and 2 in death receptor signalling, using SV40-transformed mouse embryonic fibroblasts (MEFs) derived from SMS1 knockout (KO), SMS2 KO or SMS1 and 2 double KO embryos. Our very recent observations indicate that SMS KO MEFs partially resist CD95L and TRAIL-induced apoptosis. The resistance was associated with profound alteration of sphingolipid composition, including sphingomyelin decrease, as evaluated by mass spectrometry.

    On the one hand, SMSs inhibit death receptor signalling, most likely through their ability to convert ceramide to sphingomyelin. On the other hand, complete and sustained inhibition.

  • Regulation of protein phosphatase 1 alpha by plasma membrane ceramide.

    OP16 poster

    Daniel Canals and Yusuf A. Hannun

    Cancer Center, Stony Brook University, Stony Brook, NY 11794, US. 

    Different reports have identified activation of a few proteins by ceramide (e.g. PKC, protein phosphatases, cathepsin D, etc). However, the mechanism of action of ceramide is not known. We have recently published that ceramide and sphingosine 1-phosphate (S1P) differentially regulate the cytoskeletal protein ezrin. Thus, S1P activates ezrin through receptor S1P2, while plasma membrane ceramide drives to ezrin dephosphorylation. We have identified protein phosphatase 1 isoform alpha (PP1a) necessary for the ceramide effect. Resolving how ceramide generation results in PP1a activation will shed some light on the molecular mechanism of ceramide not only on PP1a but also on other proteins known to be directly regulated by ceramide.

    After describing PP1a as necessary for ceramide-dependent ezrin dephosphorylation, we have found that PP1a is also activated in vitro by ceramide towards general phosphatase substrates and towards recombinant phosphorylated ezrin. After proving the direct in vitro interaction, we also showed that a pool of PP1a translocate to ceramide-enriched plasma membrane fractions in vivo, and this interaction is dependent of ceramide as we showed by the use of recombinant sphingomyelinase and ceramidase enzymes treatment.

    For the last half century, ceramide have been described as a bioactive lipid. However, no molecular mechanism has been described. We found a protein that gets activated in vitro and in vivo by ceramide. This activation seems to occur by direct interaction between the lipid and the phosphatase. If we are able to demonstrate the existence of a ceramide binding motif, it will be the first time to understand the molecular mechanism of how this lipid works. 

  • Role of ceramide synthases/ceramide in tumor necrosis factor alpha (TNFα)-induced plasma membrane permeabilization.

    OP17 poster

    María Jose Hernández-Corbacho1, Daniel Canals1, Yusuf A. Hannun1, Lina M. Obeid1, 2

    1 Department of Medicine, Stony Brook University, Stony Brook, NY 11794, U.S.A.; 2 The Northport VA Medical Center, Northport, NY 11768, U.S.A.

    In the last decades, the important role of the sphingolipd ceramide as mediator and regulator of cell death has emerged. The enzymes ceramide synthases (CerS) catalyze the N-acetylation of the (dihydro)sphingosine backbone to produce (dihydro)ceramide. In mammals, six different ceramide synthases have been described (CerS1-6), each of which synthesizes ceramides with different acyl chain lengths. We hypothesized that CerSs/ceramide mediate plasma membrane permeabilization during programmed cell death induced by TNFα.

    Our group has recently described the role of CerS5 and CerS6 on plasma membrane permeabilization in UV-induced programmed-cell death (Mullen TD et al, J Biol Chem. 2011), and more recently, preliminary data supports the role of Cers on TNFα-induced programmed cell death. Pre-treatment of breast cancer cells MCF-7 with the ceramide synthase inhibitor fumonisin B1 (FB1) protected TNFα-induced cell death (measured by MTT reductase activity) and the lost of plasma membrane integrity (measured by LDH release into the medium as well as by trypan blue dye exclusion). However, the inhibitor of serine-palmitoyl transferase myriocin (Myr), the first step of the de novo pathway, failed to do so. Furthermore, FB1 partially blocked the activation of caspase-7 after TNFα, as well some downstream events such as DNA fragmentation, cell rounding and phosphatidylserine externalization.

    These data suggest a key role for CerSs/ceramide regulating TNFα-induced plasma membrane permeabilization, a late event of programmed cell death. Furthermore, CerSs most likely are involved in the salvage pathway and not the de novo pathway synthesis of ceramide as FB1, but not  Myr, prevented TNFα-induced cell death.

  • Sphingosine 1-phosphate signaling axis: a flexible pathway essential for skeletal muscle cell biology.


    Paola Bruni, Francesca Cencetti, Chiara Donati                                                                

    Dipartimento di Scienze Biomediche, Sperimentali e Cliniche,Università di Firenze,Viale GB Morgagni 50 50134 Firenze, Italy

    Sphingosine 1 phosphate (S1P) is a potent lipid signaling molecule that plays a pivotal role in the control of key biological processes including proliferation, differentiation, motility and survival in numerous cell systems. Studies performed in the last 20 years have clarified that S1P metabolism within cells is highly regulated and S1P evokes specific biological responses in a cell-specific manner acting either as intracellular messenger or as ligand of five distinct G protein coupled receptors named S1P1-5.

    Past investigation of the biological role exerted by S1P signaling axis in cultured mouse skeletal myoblasts accomplished in our laboratory have clearly demonstrated that exogenous S1P influences key biological parameters. Moreover, S1P metabolism appears to be critical for the control of myoblast proliferation as well as their differentiation. More recently, inside-out S1P signaling has been shown to be required for the signaling of growth factors that physiologically control skeletal muscle regeneration. For example, PDGF and IGF1, are capable of regulating S1P metabolism and, via the engagement of distinct S1P receptors, address their complex biological action towards a reduced proliferation rate and enhanced motility or differentiation. A complex cross-talk between TGFbeta and S1P signaling axis has also been highlighted, that accounts in part for the detrimental pro-fibrotic and pro-apoptotic effects of this cytokine.

    Overall these results are in favor of a pleiotropic role of S1P signaling axis in the complex process of skeletal muscle regeneration. Moreover, they suggest that a focused pharmacological intervention could represent an innovative strategy to improve skeletal repair that is impaired in many myopathies.

  • Effect of exercise intensity and duration on tissue content and arterio-venous difference in sphingolipid concentration in human skeletal muscle.


    Marcin Baranowski1, Agnieszka U. Błachnio-Zabielska1, Fleming Dela2, Jørn W. Helge2

    1Department of Physiology, Medical University of Białystok, Białystok, Poland; 2Department of Biomedical Sciences, University of Copenhagen, Copenhagen, Denmark

    We have previously reported that exercise acutely increased plasma levels of sphingoid base-1-phosphates. This finding prompted us to examine the possible role of skeletal muscle in this effect.

    Ten healthy males were subjected to three consecutive periods of one-leg knee extension exercise separated by 30min of rest. First subjects exercised with one leg for 30min at 25% of maximal workload (Wmax), then with the other leg for 120min at 55% of Wmax, and finally again with the first leg for 30min at 85% of Wmax. The biopsies of vastus lateralis muscle from the working leg, and blood samples from the radial artery as well as from both femoral veins were taken before and after 30, and 120 minutes of exercise. The content of free sphingoid bases, their 1-phosphates and ceramides was determined using an LC/MS/MS.

    Muscle sphingosine-1-phosphate (S1P) level was markedly increased by exercise at both 55 and 85% of Wmax. In the latter case there was also an elevation in sphinganine content. Exercise did not affect venous plasma concentration of the examined sphingolipids in either working or resting leg. On the other hand, arterial plasma S1P level was increased by exercise at 25 and 55% of Wmax. Baseline S1P concentration in the venous plasma was ~29% higher as compared to the arterial one. However, the arteriovenous difference in S1P level in both working and resting leg tended to shift from a negative value before to a positive value after exercise. In addition, exercise at 85% of Wmax decreased the arteriovenous difference in sphingosine level to a negative value (from a positive one at rest) in both legs.

    Exercise increases muscle S1P level in a time- and intensity-dependent manner. However, blood drained from working muscles is not enriched in S1P. Nevertheless, our results suggest that during intense exercise muscles may release sphingosine to the circulation which could contribute to enhanced S1P synthesis in blood cells. 

  • Sphingosine-1-phosphate permanently enhances the contractile responsiveness of vascular smooth muscle via the S1P2 receptor – G12/13 – Rho-kinase signaling pathway.


    Éva Ruisancheza, Péter Dancsa, Dorottya Móréa, Stefan Offermannsb, Zoltán Benyóa

    aInstitute of Human Physiology and Clinical Experimental Research, Semmelweis University, Budapest,Hungary; bMax Planck Institute for Heart and Lung Research, Bad Nauheim, Germany

    We aimed to examine the effect of S1P on the vascular tone and to elucidate the underlying signaling pathways. Thoracic aorta segments were isolated from adult male wild type (WT), as well as S1P2, S1P3 receptor and Gα12/13 knock-out (KO) mice and investigated under isometric conditions with myography. The effect of S1P on the basal tone as well as the potential changes in K+- and phenylephrine-induced vasoconstrictions during and after the incubation with S1P were determined.

    S1P (10 µM) induced weak contraction in WT segments (9±1% compared to the effect of 124 mM K+, mean±SEM), which was abolished by the Rho-kinase inhibitor Y-27632 (2±1%). The contractile effect of S1P was similar in S1P3-KO (11±1%) but was absent in S1P2-KO (0±1%) and Gα12/13-KO (1±2%) vessels. Incubation with 10 µM S1P for 20 minutes enhanced the contractile effect of 20 mM K+ in WT segments (121±8% compared to the mean response before incubation). This potentiation was present in S1P3-KO (132±10%) but not in S1P2-KO (102±5%) and Gα12/13-KO (104±2%) vessels. Interestingly, K+-induced contractions remained elevated even 3 hours after the removal of S1P in WT (136±8%) and S1P3-KO rings (141±9%). This sustained potentiation was absent in S1P2-KO (105±8%) and Gα12/13-KO (108±2%) vessels. Co-incubation with Y-27632 prevented the augmenting effect of S1P on K+-induced vasoconstriction (100±4%) but the potentiation occurred surprisingly after the removal of S1P and Y-27632 (135±5% after 3 hours). Furthermore, the contractile effect of 0.1 μM phenylephrine was also enhanced in the presence of 10 μM S1P (119±7%) and further increased after its removal (158±7% at 3 hours).

    S1P enhances the contractile responsiveness of the vascular smooth muscle via S1P2 receptor – Gα12/13 – Rho-kinase signaling. This effect persists for hours after S1P exposition and may contribute to the development of vasospasm under conditions associated with enhanced S1P production.




  • Extracellular sphingosine-1-phosphate acts as a survival factor for human glioblastoma stem cells.


    Paola Giussani1, Elena Riccitelli1, Clara Di Vito1, Cristina Tringali1, Rossella Galli2, Paola Viani1, Laura Riboni1

    1Department of Medical Biotechnology and Translational Medicine, University of Milan, LITA Segrate, Via Fratelli Cervi 93, 20090 Segrate, Milan, Italy; 2Neural Stem Cell Biology Unit, Division of Regenerative Medicine Stem Cells and Gene Therapy, San Raffaele Scientific Institute, Milan, Italy

    Sphingosine-1-phosphate (S1P) is an oncopromoter molecule, favouring growth, invasion, and therapy-resistance of tumours, including glioblastomas (GBM), the most frequent and aggressive human brain tumours. Increasing evidence show that GBM stem-like cells (GSCs), a subpopulation of cells with the exclusive ability to self-renew and maintain the tumor, might contribute to GBM aggressiveness and resistance to therapy. We investigated S1P secretion by GSCs, and its possible role in cell survival. To this purpose we used GSCs isolated from the human U87 glioblastoma cell line (U-SC) and GSCs derived from a primary culture of human glioblastoma (L0627). We found that both GSC models efficiently form neurospheres in mitogen-defined medium, and express high levels of recognized neural-stem cell markers. Moreover, GSCs exhibited resistance to temozolomide, despite not expressing the DNA repair protein MGMT, a major contributor to temozolomide-resistance. Further analyses revealed the presence of S1P, not only inside the cells, but also in the culture medium from both GSCs and U87. Notably the extracellular S1P level was found much higher in GSC models than in U87 cells, and the ratio between extracellular and intracellular S1P was 1:10 and 1:1 in U87 and GSCs, respectively. Enzyme activity assays excluded SKs presence in GSC medium, implicating an efficient secretion of S1P in GSCs. Intriguingly, concomitant treatment with temozolomide and a SKs inhibitor made GSCs sensitive to drug toxicity. Furthermore, S1P administration promoted cell survival after this co-treatment.

    Altogether our data implicate GSCs as an important source of extracellular S1P, which might act as an autocrine signal contributing to the survival, stemness and chemoresistant properties of GSCs.

  • Serum deprivation-dependent modulation of sphingomyelin species in hn9.10 embryonic hippocampal cells.


    Mercedes Garcia-Gil1 ,Andrea Lazzarini2, Remo Lazzarini2, Emanuela Floridi2, Elisa Bartoccini2, Alessandro Floridi2, Elisabetta Albi2.

    1Department of Biology , University of Pisa, 56127, Pisa  (Italy); 2 Laboratory of Nuclear Lipid BioPathology, Research Center of Biochemical-Specialized Analyses, 06100 Perugia (Italy)

    Understanding the mechanism of apoptosis is necessary to improve the use of stem cells for the treatment of neurodegenerative disorders. In HN9 embryonic hippocampal cells, serum deprivation induces apoptosis preceded by sphingomyelinase (SMase) activation and  raise of ceramide levels. It is not known so far whether the SMase degrades all the species of sphingomyelins (SMs) in the same way and if it is the only enzyme that uses SM to produce ceramide. In order to begin to address this aspect still unclear about the relationship between SM and HN9.10 apoptosis, we carried out the analysis of the main species of SM and phosphatidylcholine (PC) with the idea that SM could be partly used as a source of phosphorylcholine to synthesize PC. To better investigate this possibility we measured the enzymatic activity of the reverse-SM synthase. In addition, since the  ceramides involved in starvation-induced cell death are unknown, we have studied the content of ceramide and sphingosine-1-phosphate species as metabolites of SM produced by both SMase and reverse-SMsynthase.

    Cells have been incubated with 10% (control) or 0.2% fetal calf serum for 48 h (deprived). Lipid species has been separated and measured by ultraperformance liquid chromatography tandem mass spectrometry. Reverse-SMsynthase activity was assayed by using 14C labelled SM.

    The results show that serum deprivation reduces total SM and in particular 18:1/16:0 SM whereas it increases total PC and in particular 18:1/18:0 PC. There is a 2-fold increase of ceramides 18:1/ 16:0 and 18:1/ 24:0 and of   sphingosine-1-phoshate 18:1. Following serum deprivation, the activity of Reverse-SM synthase remains constant for 8 h, increases afterwards up to 3.82 –fold compared to control at 48h. In conclusion, SMase activation might lead to decrease of SM 18:1/16:0, increase in ceramide 18:1/16:0 which can originate sphingosine-1-phoshate 18:1.  In addition SM could be a source for PC synthesis with fatty acid rearrangement.

    Individual ceramide species regulated by specific pathways in distinct subcellular compartments might execute distinct cellular functions.

  • Deficiencies of ceramide synthases 1 and 4 causing sarcopenia and hair loss.


    Martin Krönke, Carola Pongratz, Susanne Brodesser, Franziska Peters, Carien Niessen, Jens Brüning

    Institute for Medical Microbiology, Immunology and Hygiene, Center for Molecular Medicine, Institute for Genetics, and Cluster of Excellence in Cellular Stress Responses in Aging-assoiated Diseases (CECAD), University of Cologne, Germany 

    Ceramide (Cer) forms the structural backbone of all sphingolipids and has been implicated in functionally important membrane processes. In mammals, Cer consists of a fatty acid of variable chain length linked by an amide bond to C-2 of the long-chain base, sphinganine, or sphingosine. N-acylation of the long-chain base is catalyzed by a family of six ceramide synthases (CerS), each of which uses a relatively restricted subset of acyl CoAs for N-acylation. The generation of mouse strains deficient for individual CerS genes made it possible to address fundamental questions about specific functions of ceramide species of distinct acyl chain lengths.  CerS1 knockout mice display an obvious phenotype as they are significantly smaller than their wildtype littermates and exhibit movement disorders. CerS1-/- mice show a 7-fold decrease of Cer C18:0 in skeletal muscle compared to wt littermates, which is similar to the values of aged mice. CerS1-/- mice displayed significantly smaller femoral muscle relative to whole body mass. Histochemical analysis revealed a loss of predominantly type 2 muscle fibers characteristic of sarcopenia, the most prominent age-related muscle disorder. Muscle cells from CerS1-/- mice spontaneously release TNF secondary to enhanced activity of the TNF-converting enzyme TACE. Mechanistically loss of Cer C18:0 leads to a disruption of membrane microdomains leading to a displacement of TACE in membrane sections where the tight regulation of its activity is unleashed.

    CerS4 deficient mice display epidermal thickening and hair loss as they age. Mass-spectrometry of the stratum corneum (the outermost epidermal layer) of CerS4- deficient mice revealed a most prominent reduction of C20:0 and C22:0 non-hydroxylated Cer and hydroxylated 22:0 Cer. These mice develop an increase in thickening of the interfollicular epidermis (IFE) due to altered interfollicular differentiation, whereas the hyper-keratinization of the epidermis is caused by hyper-proliferation. The advent of the LoxP Cre technology makes it possible to generate conditional CerS knock-out mice, which has become a valuable tool for the characterization of tissue- and time-specific functions of individual CerS.

  • Inhibition of angiogenesis by β-galactosylceramidase deficiency in globoid cell leukodystrophy.


    Mirella Belleri1, Roberto Ronca1, Daniela Coltrini1, Beatrice Nico2, Domenico Ribatti2, Pietro L. Poliani1, Arianna Giacomini1, Patrizia Alessi1, Sergio Marchesini1, Marta Santos3, Ernesto R. Bongarzone3,  and  Marco Presta1.  

    1Department of Molecular and Translational Medicine, School of Medicine, University of Brescia, Italy.2Department of Basic Biomedical Sciences, Unit of Human Anatomy and Histology, University of Bari, Italy.3Department of Anatomy and Cell Biology, University of Illinois, Chicago, IL, USA.

    Globoid cell leukodystrophy (Krabbe disease) is a neurological disorder of infants caused by genetic deficiency of the lysosomal enzyme β-galactosylceramidase leading to accumulation of the neurotoxic metabolite psychosine in the central nervous system. Angiogenesis plays a pivotal role in the physiology and pathology of the brain.

    Here, we demonstrate that psychosine has antiangiogenic properties by causing the disassembling of endothelial cell actin structures at micromolarconcentrations as found in the brain of globoid cell leukodystrophy patients. Accordingly, significant alterations of microvascular endothelium were observed in the post-natal brain of twitcher mice, an authentic model of globoid cell leukodystrophy. Also, twitcher endothelium showed a progressively reduced capacity to respond to pro-angiogenic factors that was restored by lentivirus-mediated murine β-galactosylceramidase cDNA transduction. Finally, RNA interference-mediated β-galactosylceramidase gene silencing causes psychosine accumulation in human endothelial cells and hampers their mitogenic and motogenic response to vascular endothelial growth factor. Accordingly, significant alterations were observed in human microvasculature from brain biopsy of a globoid cell leukodystrophy case.

    Together these data demonstrate that β-galactosylceramidase deficiency induces significant alterations in endothelial neovascular responses that may contribute to central nervous system and systemic damages that occur in globoid cell leukodystrophy.

  • Ceramide synthesis is activated by 17β-estradiol in human breast cancer cells.


    Marthe-Susanna Wegner, Ruth Anna Wanger, Daniela Hartmann, Nerea Ferreirós, Gerd Geisslinger, Sabine Grösch

    Pharmazentrum frankfurt/ ZAFES, Institut für Klinische Pharmakologie, Klinikum der Goethe-Universität Frankfurt, Theodor-Stern-Kai 7, 60590 Frankfurt/ Main, Germany.

    Previously, we could show that ceramide synthase 4 and 6 (CerS4, 6) are significantly upregulated in estrogen receptor (ER)-positive breast tumors in comparison to ER-negative breast tumors. Here we investigated the molecular mechanisms leading to CerS increase and its contribution to tumor growth.

    The expression of CerS2,4,5 and 6 is in MCF-7 (ERα/ERβ) and MDA-MB-231 (ERβ) cells increased after treatment with 17β-Estradiol. Promoter reportergene assays revealed that only the promoter of CerS4 and CerS5 are responsive to estradiol. Nevertheless, co-treatment of cells with estradiol and Fulvestrant (a selective ERα down-regulator) completely prevented the induction of CerS expression in MCF-7 cells whereas it has no effect in MDA-MB-231 cells. Also CerS activity is slightly affected by estradiol treatment within 4h in both cell lines. Interestingly, the proliferation promoting effect of estradiol in MCF-7 and MDA-MB-231 cells could be prevented by the non-selective CerS inhibitor Fumonisin and by the de novo sphingolipid synthesis inhibitor Myriocin but only in part by the acid shingomyelinase inhibitor Desipramine, indicating that the sphingolipid de novo synthesis is important for the tumor promoting effect of estradiol whereas the salvage pathway plays only a minor part.

    Our in vitro studies revealed that CerS expression is dependent on estradiol treatment in human breast cancer cells and that an enhancement of sphingolipid de novo synthesis is important for the proliferation promoting effect of estradiol.

  • Neutral sphingomyelinase-2 regulation of FoxO transcription factor.


    Aneta Dobierzewska, and Lihua Shi, and Mariana Nikolova-Karakashian

    University of Kentucky College of Medicine, Department of Physiology, Lexington  KY

    The members of the FoxO family of transcription factors play a central role in aging, cancer, type II diabetes, and other pathophysiological conditions. FoxO activity is regulated through reversible translocation between the nucleus and cytosol determined by a growth factors-driven, Akt-1 phosphorylation-dependent nuclear export.  The goal of this study was to test the possible role of neutral sphingomyelinase-2 (nSMase2), the bona fide signaling nSMase, in the FoxO1 nuclear translocation. 

    IL-1b stimulation of HEK293 cells stably overexpressing IL-1b receptor and primary hepatocytes leads to transient activation of nSMase2, followed by increased nuclear localization of FoxO1.   Silencing of nSMase2 using adenovirus-delivered siRNA abolishes the effects of IL-1b on FoxO1, indicating a mediatory role of nSMase2 in the process. Exogenous addition of ceramide, or overexpression of nSMase2 has the opposite effect.  As ceramide is known to inhibit Akt-1 pathway, we tested the possibility that IL-1b and nSMase2 induce nuclear accumulation of FoxO1 through interference with the Akt-1-dependent export.  However, several lines of evidence suggested that this was not the case: (i) IL1b and nSMase2 lead to FoxO1 nuclear accumulation in the absence of serum,  or other inducers of Akt-1, such as insulin; (ii) a mutant FoxO1 lacking all three Akt-1 phosphorylation sites was regulated similarly as the wild type FoxO1.   The IL-1b and nSMase2 effects also were not caused by redistribution or new protein synthesis of FoxO1 as changes in neither the cytosolic levels nor the mRNA content of FoxO1 were observed. Instead, PD 980059 and SP600125, inhibitors of ERK-1 and JNK respectively, efficiently prevented IL-1b and nSMase2 - induced FoxO1 nuclear accumulation. 

    This study delineate a novel , Akt - independent pathway for up-regulation of FoxO1.

  • Use of the antiestrogen tamoxifen to regulate sphingolipid metabolism in cancer cells – a driving influence on the ceramide effect.


    Samy A. F. Morad, Jonathan C. Levin, Myles C. Cabot

    John Wayne Cancer Institute, Santa Monica, CA 90404

    Tamoxifen (tam), the gold-standard treatment for breast cancer, we show has a number of novel “off-target” actions centering on sphingolipid metabolism: inhibition of ceramide glycosylation and acid ceramidase (AC) and accelerated generation of long-chain ceramides (LCC) from C6-ceramide (C6-cer). Thus, tam is ideal to drive ceramide’s therapeutic benefits. We have therefore designed and tested a drug regimen consisting of C6-cer, selected because it is cell-permeable, and tam.

    Tam effectively inhibited the synthesis of glucosylceramide (GC) and synthesis of C6-GC from supplemented C6-cer in a myriad of cancer cell types. Whereas single agent tam and C6-cer were minimally cytotoxic, co-administration produced synergistic cytotoxicity that was accompanied by increased Annexin binding, caspase-3 activation, and enhanced DNA fragmentation. Use of a colon cancer cell line to assess C6-cer-tam-orchestrated signaling revealed upregulated expression of JNK and p53 and downregulation of Akt and survivin. Also, the mix produced cell cycle arrest at G1 and G2. New to the non-genomic activities of tam, we found potent inhibition of AC activity in cancer cells. Mechanism studies showed that tam elicited rapid lysosomal membrane permeability accompanied by dose- and time-dependent proteolytic degradation of AC by cathepsin B. Whereas tam was not cytotoxic, it retarded cancer cell migration. Tamoxifen inclusion also produced a mani-fold increase in production of LCC’s from C6-cer. Interestingly, L-C6-cer, a non-hydrolyzable racemate, yielded no LCC when delivered with tamoxifen, nor did an L-C6-cer-tam regimen produce DNA fragmentation.

    This innovative drug regimen allows for delivery of C6-cer along with a unique means to produce high amounts of intracellular LCC’s, tumor suppressor ceramides not otherwise deliverable due to solubility issues, and a mode to suppress AC activity. 

  • MicroRNA-515-5p is downregulated by the estrogen receptor leading to enhanced sphingosine kinase 1 mediated proliferation in breast cancer.


    Filipa G. Pinho1*, Jonathan Krell1, Adam E. Frampton2, Heba Alshaker1, Justin Stebbing3, Leandro Castellano1, Dmitri Pchejetski1

    1Division of Oncology, Dept. of Surgery & Cancer, Imperial Centre for Translational and Experimental Medicine (ICTEM), Imperial College, Hammersmith Hospital campus, Du Cane Road, London, W12 0NN, UK. 2HPB Surgical Unit, Dept. of Surgery & Cancer, Imperial College, Hammersmith Hospital campus, Du Cane Road, London, W12 0HS, UK.

    3Dept. of Oncology, Imperial College Healthcare NHS Trust, Charing Cross Hospital, 1st Floor, E Wing, Fulham Palace Road, London, W6 8RF, UK.

    Sphingosine Kinase 1 (SK1) plays an important role in estrogen-dependent breast tumorigenesis, but its regulation is poorly understood. A subset of microRNAs (miRNAs) is regulated by estrogen and contributes to proliferation and cancer progression. Here, we describe that miR-515-5p is transcriptionally repressed by ERα and functions as a tumor suppressor in breast cancer (BC). Its downregulation enhances cell proliferation, due to an estrogen-dependent increase in SK1 activity, mediated by a reduction of miR-515-p post-transcriptional repression. Enforced expression of miR-515-5p in BC cells causes a reduction in SK1 activity, resulting in reduced cell proliferation and the induction of caspase-dependent apoptosis. Conversely, opposing effects occur with miR-515-5p inhibition and by SK1 silencing. Notably, we show that estradiol (E2) treatment downregulates miR-515-5p levels, whilst the anti-estrogen tamoxifen (TAM) causes a decrease in SK1, which is rescued by silencing miR-515-5p. Analysis of ChIP-Seq data reveals that miR-515-5p suppression is mediated by a direct interaction of ERa within its promoter. The clinical implications of this novel regulatory system is demonstrated as miR-515-5p is significantly downregulated in ER-positive (n=146) compared to ER-negative (n=98) BCs. Overall we identify a new link between ERα, miR-515-5p and SK1 in BC tumorigenesis.

    In this study, we have elucidated a new miRNA-mediated mechanism by which estrogen up-regulates SK1 levels.  btmiR-515-5p replacement strategies may offer a novel therapeutic approach in hormone resistant ERα-positive BC and as an adjunct to currently available therapies.

  • Sphingosine 1-phosphate (S1P) from bone microenvironment promotes cell proliferation and survival of prostate cancer cells through S1P receptor 1 (S1P1).


    Leyre Brizuela 1,2,3, Claire Martin 1,2,3,,  Pauline Jeannot 1,2,3, Isabelle Ader 1,2,3,  Guillaume Andrieu 1,2,3, Magalie Bocquet 1,2,3, Bernard Malavaud 1,2,3,4, Roger A. Sabbadini 5, Olivier Cuvillier 1,2,3

    1 CNRS, Institut de Pharmacologie et de Biologie Structurale, Toulouse, France. 2 Université de Toulouse, UPS, IPBS, Toulouse, France. 3 Equipe Labellisée Ligue contre le Cancer 4 Hôpital Rangueil, Service d’Urologie et de Transplantation Rénale, Toulouse, France. 5 LPath Inc., San Diego, CA, USA

    Sphingosine 1-phosphate (S1P) plays important roles in cell proliferation, differentiation or survival mainly through its five surface G-protein-coupled receptors S1P1-5. Bone represents the major site of metastasis for prostate cancer cells, which rely on bone-derived factors to support their proliferation and resistance to therapeutics. Because of the osteoblastic nature of prostate cancer bone metastasis, we investigated whether S1P could act as a possible osteoblast-derived factor to stimulate proliferation and survival of prostate cancer cells . In the present work we have found that conditioned medium (CM) from the MC3T3 osteoblastic cell line or primary osteoblast-like cells, as well as co-culture with MC3T3 stimulate proliferation of bone metastastic PC-3 and C4-2B prostate cell lines in S1P-dependent manner. In addition, osteoblastic derived S1P induces resistance of metastatic prostate cancer cells to therapeutics including docetaxel-based chemotherapy and radiotherapy in vitro. When S1P release from osteoblastic cells is decreased (pharmacological inhibition of SphK1, knock-down of SphK1 or the S1P transporter, Spns2 by RNA interference) or secreted S1P neutralized with anti-S1P antibody (Sphingomab), the proliferative and survival  effects of osteoblasts on PC-3 and C4-2B cells are abolished. Because of the paracrine nature of the signaling, we studied the role of the S1P receptors expressed on prostate cancer cells in the dialogue with S1P secreted by osteoblasts. Strategies aimed at down-regulating S1P1, S1P2 or S1P3 (RNA interference, pharmacological antagonists), established the exclusive role of the S1P/S1P1 signaling between osteoblasts and tumor cells. Bone metastasis is a lethal form of prostate cancer and presents considerable challenges for treatment. Interfering with the interactions between prostate cancer cells and aspects of the bone microenvironment may hold the key to preventing the development of bone metastases or resensitize to therapeutics, and S1P may be a candidate as a target in the treatment of prostate cancer bone metastasis.

  • Sphingosine kinase 1 mediates JAK2/STAT3-independent breast cancer progression triggered by adipokines.


    Heba Alshaker1, Jonathan Krell1, Jonathan Waxman1,Ernesto Yagüe1, Dmitry Pshezhetskiy2,3

    1 Department of Surgery & Cancer,  Faculty of Medicine, Imperial College London; 2 Department of Medicine, Faculty of Medicine, Imperial College London 3 School of Medicine, University of East Anglia, Norwich

    The prevalence of obesity increases in the modern society. Obesity is a known risk factor in breast cancer patients and has been linked with cancer progression and chemoresistance. Adipose tissue secretes adipokines (e.g. leptin), which affect breast cancer growth, angiogenesis and chemoresistance. Sphingosine kinase 1 (SK1) is an oncogenic lipid kinase that promotes cancer cell growth and has been recently shown to be overexpressed in breast tumours and linked with poor prognosis.

    In this study, we explored the effect of leptin on SK1 and its mechanistic contribution to breast cancer progression using qRT-PCR and western blotting in ER-positive and triple negative breast cancer cells. Our findings show for the first time that leptin induces SK1 expression and activation in triple-negative breast cancer cell lines MDAMB-231 and BT-549. Using small molecule inhibitors and siRNA pools to inhibit kinases involved in leptin signalling we have shown that leptin-induced SK1 up-regulation is independent of the major pathways downstream of leptin receptor (LEPR) Janus kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3). Alternatively, leptin-induced SK1 expression is mediated by activation of extracellular signal-regulated kinases 1/2 (ERK1/2). Further investigation showed that in addition to ERK1/2, SK1 up-regulation is mediated by Src family kinase (SFK) pathway where Src-homology 2 domain-containing phosphatase 2 (SHP2) works as an adaptor protein between SFKs and LEPR. Interestingly, SK1 activation was accompanied with an increase in cancer cell proliferation, which was abrogated by SK1 specific siRNA. Human primary breast tumours and positive lymph node metastases exhibited a strong correlation between SK1 and LEPR expression (Pearson R=0.75 and R=0.72, respectively, p<0.001).

    Overall,our data show a new role for leptin-induced up-regulation of oncogenic SK1 highlighting a new pathway regulating obesity driven triple-negative breast cancer. Our findings point to the potential applicability of second generation pharmacological SK1 inhibitors as novel therapies for obesity-driven breast cancer.

  • Targeting de novo ceramide synthesis in inflammation and infection in human cells and mouse model of Cystic Fibrosis.


    Anna Caretti 1, Alessandra Bragonzi 2, Marcella Facchini2, Ida De Fino2, Camilla Riva2 , Paolo Gasco 3, Claudia Musicanti3 , Josefina Casas 4, Gemma Fabriàs 4, Riccardo Ghidoni 1 and Paola Signorelli 1.

    1, Department of Health Sciences, University of Milan, San Paolo Hospital, Italy; 2 Infections and Cystic Fibrosis Unit, San Raffaele Scientific Institute, Milan, Italy; 3, Nanovector S.r.l. Turin, Italy; 4, Research Unit on BioActive Molecules, Department of Biomedicinal Chemistry, Catalan Institute of Advanced Chemistry (IQAC/CSIC), Barcelona, Spain.

    Sphingolipids take part in immune response and can initiate and/or sustain inflammation. Various inflammatory diseases have been associated with increased ceramide content in mucosa, and pharmacological reduction of ceramide diminishes inflammation damage in vivo. Inflammation and susceptibility to microbial infection are two elements in a vicious circle. Recently, sphingolipid metabolism inhibitors were used to inhibit infection. Cystic Fibrosis (CF) is characterized by a hyper-inflammation of lung mucosa and a deregulated immune response. Innate immunity fails to eradicate acute infection and to evolve into adaptive immunity, allowing chronic infections, lung damage, and patient morbidity. Indeed, ceramide pulmonary content is higher in CF mouse models and in patients and a clinical trial with amitriptyline, an inhibitor of sphingomyelinase, reportedly reduced lung infection and inflammation. CF human respiratory epithelium cells were treated with myriocin, an inhibitor of de novo ceramide synthesis. This treatment resulted in reduced basal, as well as TNFα-stimulated, inflammation. Furthermore, myriocin-loaded nanocarrier treatment resulted in a significant reduction of lung infection by P. aeruginosa and a decreased release of host pulmonary inflammation mediators. We speculate that de novo sphingolipid synthesis is constitutively enhanced in CF mucosa and that it represents a pharmacological target for modulating inflammation and restoring effective innate immunity against acute infection. 

  • Identification of new compounds targeting the fungal sphingolipid pathway.


    Visesato Mor1, Antonella Rella1, Chiara Luberto2, Michael Linke3, Melanie T. Cushion3,4, Margaret S. Collins3,4,  Mitchell Mutz5, and Maurizio Del Poeta1

    1Department of Molecular Genetics and Microbiology, 2Department of Physiology and Biophysics, Stony Brook University, Stony Brook, NY 11794; 3VA Medical Center and 4Division of Infectious Diseases, University of Cincinnati, Cincinnati, OH 45267; and 5Amplyx Pharmaceuticals, Inc., San Diego, CA 92121. 

    Fungal glucosylceramide (GlcCer) is a sphingolipid required for the pathogenic fungus Cryptococcus neoformans (Cn) to cause a lethal meningo-encephalitis by inhibiting fungal replication in the host environments characterized by neutral/alkaline pH and 5% CO21-4. In addition to be required for Cn pathogenicity, fungal GlcCer is also important for the pathogenesis of other human pathogens, such as Candida albicans5-7 and Aspergillus fumigatus8, and to plant pathogens, such as Fusarium graminearum9. The synthesis of GlcCer seems to be important also during Pneumocystis pneumonia (PCP), as glucosylceramide synthase transcripts have been found to be abundant at the time of isolation of the fungus from fulminate lung infection10, and for infection caused by dimorphic fungi, as GlcCer is detected only in the lung infective form (yeast) and not in the environmental form (mold) 11-13. Taken together, these studies suggest that the GlcCer pathway is most likely a pan-fungal virulence pathway required in promoting fungal growth in neutral/alkaline and 5% CO2 environments (e.g. alveolar spaces).

    Therefore, we looked for inhibitors of GlcCer synthesis by screening a ChemBridge library for compounds that would inhibit Cn replication in an environment similar to the lung: neutral/alkaline pH, 37C and 5% CO2. We identified 2 inhibitory compounds (N’–(3-bromo-4-hydroxybenzylidene)-2-methylbenzohydrazide (BHBM) and its derivative D0) that significantly decreased the synthesis of GlcCer in Cn but not in mammalian cells. Importantly, 90% of mice daily treated with an intraperitoneal injection of 1.2 mg/kg of BHBM survived a lethal intranasal injection of Cn. The uninfected control mice treated with BHBM appeared to be normal: they maintained a normal weight and showed a normal physical activity throughout the observation period. Blood work showed a slight increase of liver AST (125 ± 75 U/L) in BHBM treated mice compared normal mice (50 ± 9 U/L) but all other blood parameters for liver and kidney functions were normal. Also normal were the number of erythrocytes, thrombocytes, and leucocytes in BHBM treated mice. Histological analysis of lungs, liver, and kidney revealed no difference between the BHBM treated and untreated/uninfected mice. These results suggest that BHBM is a potent antifungal and overall well tolerated in mice with no clinical or hematological/ histological signs.

  • Exosomes secreted by the mouse parasite Heligmosomoides polygyrus are enriched in sphingolipids and internalised by host cells.


    Simbari Fabio1, Ceroni Alessandro1, Lear Marissa1, Maizels Rick1 and Buck Amy1.

    1. University of Edinburgh, Institute of Infection and Immunology Research, King’s Buildings, West Mains Road, Edinburgh (UK) EH9 3JT.

    Exosomes are bioactive nanovesicles (50-100nm diameter) secreted by many cell types that are involved in communication between cells. They play diverse functions in important biological processes including host-pathogen interactions, modulation of the immune response as antigen presenting molecules, spreading of virus infection such as HIV. Recently, researchers have also investigated their possible use as therapeutic tools for delivery of small molecules and siRNAs. Different parasites have been shown to secrete exosome-like vesicles (Echinostoma caproni, Fasciola Hepatica,Leishmania). We have identified exosomes in the secretion products of the mouse parasite nematode Heligmosomoides polygyrus (H. polygyrus). These vesicles pellet upon ultracentrifugation and, as observed by Transmission Electron Microscopy, are round in shape with a diameter of ~ 100nm. Confocal microscopy studies together with FACS analysis suggest that they are actively uptaken by MODE-K cells (small intestinal epithelial mice cells). In order to better characterise the nature of these vesicles, we performed a comprehensive mass-spec analysis of their lipid content, compared to the rest of the secretion product (supernatant fraction) and the whole worm extract. As expected, the vesicle fraction is enriched in sphingolipids and phospholipids compared to the supernatant. Furthermore, the H. polygyrus derived vesicles show an enrichment in ceramide and sphingomyelin levels when compared with the total worm extract, whereas the phospholipid levels appear similar within the two groups. Taken together these results suggest that H. polygyrus secretes exosomes that are enriched in sphingolipids. Finally, the internalization assays strongly suggest that these vesicles are taken up by the host cells and may therefore play an important role in communication between pathogen and host.

  • A computational framework for the investigation of functional connections between Snf1 kinase complex and sphingolipid metabolism in S. cerevisiae.


    E. Yücel, K. Ö. Ülgen

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

    Sphingolipids are one of the subclasses of lipid components of eukaryotic membranes, serving not only as building blocks of membranes and lipoproteins, but also as regulators of various signaling pathways of the cell.

    In the present study, a computational framework was recruited for the reconstruction of functional interaction network of sphingolipid metabolism in Baker’s yeast. The Selective Permissibility Algorithm (SPA) was employed to integrate GO annotations with functional interaction data and to reconstruct a protein interaction network (SSN) that has the potential for predicting signal transduction pathways regulating and/or being regulated by sphingolipid biosynthesis in yeast. The reconstructed network was then served as a basis for a case study involving Snf1 kinase complex. By mapping the transcriptome data available in literature onto SSN, the impact and specific action mechanisms of Snf1p on the sphingolipid metabolism were investigated.

    The present study emphasizes the important role of sphingolipids as secondary messengers more thoroughly by presenting a detailed map of physical and functional connections that link the sphingolipid metabolism and signaling to other relevant signal transduction networks in S. cerevisiae.

  • Effects of methylation and hydroxylation of sphingomyelins on their biophysical properties and interactions with cholesterol.


    Shishir Jaikishan

    Biochemistry, Department of Biosciences, Åbo Akademi University

    Sphingomyelins (SMs) are important sphingolipid molecules found in the exoplasmic leaflet of the cell membrane of animal cells. SMs are rare in plants and microorganisms. Thudicum derived the term ‘sphingomyelin’ from the Greek word ‘sphinx’, when he named the enigmatic molecule he isolated from brain tissue in the late 19th century. The membranous myelin sheath in nerve tissues is particularly rich in SMs, where they take part in the mechanical and electrical insulation of the nerve cell axons. Many physiological abnormalities and associated diseases arise from malfunctions in the metabolism of SMs, for example Niemann-Pick Disease, a fatal inherited metabolic disorder. Recent studies have reported that SM and cholesterol, along with specific proteins, form raft like regions in otherwise fluid cell membranes. Lipid rafts play major roles in many biological processes such as transmembrane signal transduction, membrane trafficking, protein sorting during endocytosis and exocytosis, apoptosis, cell adhesion, migration and synaptic transmission. It is of utmost interest to know why SMs and cholesterol are included into lipid rafts and how these molecules interact with each other. This thesis focuses on the interaction between SMs and cholesterol. To study the membrane biophysical properties of SMs and their interactions with cholesterol, we synthesized novel SM analogs by methylating and hydroxylating SM at different positions. All the SM analogs involved in the study are biologically relevant. We reported that the hydroxyl and amide groups present in SM are important for hydrogen bonding between SM-SM and SM-cholesterol. The substitutions of methyl groups in the N-linked acyl chain or interfacial region of SMs significantly decreased the stability of SM bilayer, and weakened/abolished SM interactions with cholesterol. The stability of SM bilayer and SM-cholesterol interactions with the hydroxyl groups in the N-linked acyl chain of SM depend on the position and configuration of the hydroxyl group. The presence of an additional hydroxyl group in the saturated long chain base of SM stabilized SM bilayers as well as SM-cholesterol interactions. 

  • Is there a role between sub-cellular localisation of sphingosine kinase 1 and clinical outcome in colorectal cancer?


    S. Cochrane1, S. Moug2, J. Edwards2, N.J. Pyne1, D. MacMillan3, P. Horgan3, R. Bittman4 and S. Pyne1

    1. Cell Biology, SIPBS, University of Strathclyde, Glasgow, G4 0RE 2. Unit of Experimental Therapeutics, University of Glasgow, Glasgow G11 6NT 3. Academic Unit of Surgery, University of Glasgow, Glasgow G31 2ER, 4. Department of Chemistry and Biochemistry, Queens College of the City University of New York, Flushing, USA

    Colorectal cancer remains the third most common killer in the UK. There is growing evidence that sphingosine 1-phosphate and the enzymes responsible for its synthesis, sphingosine kinase 1/2, have a role in cancer. For example, SK1 mRNA and protein expression is increased in 75% of azoxymethane-induced tumours (a rat model of CRC) compared to the normal mucosa. Additionally, data suggests that SK1 expression is associated with disease progression and metastatic potential when analysed in ~60 human colorectal samples.

    In this study, using a cohort of 277 patients, we assessed the sub-cellular localisation of SK1 (membrane, cytoplasmic and nuclear) against patient cancer-specific survival. Patients were stratified based on lymph node status, stage of disease and plasma C-reactive protein (CRP) levels (a systemic inflammatory marker associated with poor prognosis). Unexpectedly, high cytoplasmic SK1 was associated with increased cancer-specific survival in Stage III disease. Interestingly; this association was lost in patients with node positive disease. In contrast, nuclear SK1 was associated with reduced survival in Stage IV disease. In vitro studies were conducted using SW480 and SW620 cell lines (models of node-negative and node positive disease, respectively) and sphingosine kinase isoform selective inhibitors. Using [3H]-thymidine, we found that pre-treatment of cells with SKi, reduced thymidine incorporation (p<0.05), whereas (R)-FTY720 O-methyl ether (ROMe) had no effect.

    Here we show that high cytoplasmic SK1 expression is associated with protection against death in Stage III disease. Conversely, high expression of nuclear SK1 is detrimental Stage IV disease. In vitro studies of colorectal cell lines indicate that SK1 promotes DNA synthesis and SK2 does not.

  • Cytotossic effects of perifosine in human glioblastoma cells are associated to an altered ceramide metabolism.


    Alessandra Cinque, Loredana Brioschi, Paola Giussani, Laura Riboni, Paola Viani

    Department of Medical Biotechnology and Translational Medicine, University of Milano, LITA Segrate, Via Fratelli Cervi, 93, 20090 Segrate (MI) Italy

    Glioblastoma multiforme (GBM) is the most malignant brain tumor and, despite aggressive therapy, prognosis for patients is poor. Temozolomide (TMZ), the chosen drug in GBM therapy, exerts cytotoxic effects also through an increase of ceramide levels. Perifosine (PF) is currently being tested for treatment of major human cancers, but little is known about its efficacy in gliomas. The aim of this work is to evaluate in GMB cell lines if PF is able to regulate sphingolipid metabolism and to enhance the cytotoxic effect of TMZ.

    The results obtained demonstrated that in different GBM cell lines, the treatment with PF led to a dose-dependent decrease in cell viability. In all GBM cells lines, PF promoted a time-dependent decrease of p-Akt levels, with an about 50% decrease after 4 hour treatment. In these conditions, metabolic studies showed that treatment with PF significantly modified sphingolipid (SL) metabolism by inhibiting sphingomyelin (SM) and, in a lesser extent, glycosphingolipid (GSL) biosynthesis with a consequent accumulation of ceramide (Cer). In addition, PF weakly reduced sphingosine-1-phosphate (S1P) biosynthesis. The treatment with LY294002, in conditions giving an almost complete inhibition of the PI3K/Akt pathway, only partly mimicked the effects of PF on SL metabolism, suggesting that PF could regulate SL metabolism also through a mechanism other than Akt inhibition. In the same GBM cell lines, the treatment of PF in combination with TMZ had an additive, but not synergistic cytotoxic effect. More of interest, a TMZ resistant cell line, that is characterized by an altered SL profile, a high capacity to release S1P and in which TMZ is unable to accumulate Cer, was still sensitive to PF.

    These data suggest that PF can exert its anticancer activity through a marked modulation of sphingolipid metabolism with increased Cer/S1P ratio, where Cer acts as a pro-death and S1P as a survival factor. PF in combination with TMZ could thus improve the treatment efficacy in GBM.

  • Lipidomic and immunohistochemical analysis of sphingosine-1-phosphate and other sphingolipids following spinal cord injury.


    Del Águila Angela, Nieto-Díaz Manuel, Casas Josefina, Fabriàs Gemma, Reigada David, Munoz-Galdeano Teresa, Navarro-Ruiz Rosa, Caballero-Lopez Marcos, Maza Martinez.

    Molecular Neuroprotection Group. Hospital Nacional de Parapléjicos (SESCAM). Toledo, Spain.Research Unit on Bioactive Molecules (RUBAM). Institut de Química Avançada de Catalunya (IQAC-CSIC). Barcelona, Spain.


    Spinal cord injury (SCI) triggers a complex cascade of biological events that determine its functional outcome. Understanding the molecular bases underlying these events is basic to design therapeutical tools. Sphingolipids are bioactive lipids involved in many of these events, such as astrogliosis, cell death, or pain, among others. Therefore, we here describe the variations of sphingolipid levels in a mice model of SCI, with special attention to the pleiotropic sphingosine-1-phosphate (S1P).

    We have used UPLC-MS to analyze the sphingolipid levels in spinal cord, blood, and blood serum from five groups of mice: non-injured and 1, 3, 7, and 21 days post injury (dpi). The results show that spinal cord presents a characteristic global sphingolipid pattern, which is preserved following SCI. However, total sphingolipid concentration increases after injury, and 9 species significantly change their levels, mostly at 3dpi, although S1P shows a maximum at 7dpi. Blood samples also have a stable, but different and characteristic general pattern. Nevertheless, levels of 21 sphingolipid species significantly change after SCI -mainly at 1dpi, including S1P-. Variations in sphingolipids levels of spinal cord and blood seem to be unrelated.

    Considering the importance of S1P, we have used inmmunohistochemstry (IHC) to characterize its spatial and temporal distribution in the spinal cord. Non-injured mice show immunoreactivity (IR) in motor and interneurons, astrocytes and oligodendrocytes. After injury, neural cell IR shows important temporal changes. Neurons increase and decrease their labeling at different times after SCI, whereas oligodendrocytes lose their IR progressively.

  • Spisulosine and 4,5-dehydrospisulosine stereoisomers as probes for profiling ceramide synthase activities in intact cells.


    José Luis Abad,1 Ingrid Nieves,1 Pedro Rayo,1 Josefina Casas,1 Gemma Fabrias,1 and Antonio Delgado1,2

    1Consejo Superior de Investigaciones Científicas (CSIC), Institut de Química Avançada de Catalunya (IQAC-CSIC), Research Unit on Bioactive Molecules (RUBAM), Barcelona, Spain. 2Universidad de Barcelona (UB), Facultad de Farmacia, Unidad de Química Farmacéutica (Unidad Asociada al CSIC), Barcelona, Spain.

    Spisulosines are a group of 1-deoxysphingolipids (1-DSLs) of marine origin with antiproliferative activity. In addition, recent evidences of the presence of 1-DSLs as natural metabolites in mammalian cells have been postulated from studies of a mutation in the SPTLC1 gene that is found in human sensory neuropathy type 1 (HSN1). Interestingly, the presence of several 1-DSLs in plasma has also been proposed as a novel class of biomarkers for the metabolic syndrome.

    A stereoselective synthesis of spisulosine (ES285) and 4,5-dehydrospisulosine stereoisomers is presented. We report on the design of efficient synthetic protocols for this type of 1-DSLs, with special attention to the stereochemical control at C2 and C3 positions and the presence of a C4-C5 unsaturation as part of the sphingoid backbone. The increasing interest in CerS activity, compartmentalization and function underscores the need for appropriate CerS probes. Characterization of (dihydro)ceramidomes is not a suitable means to profile CerS activities in intact cells, since (dihydro)ceramide populations are the result of the overall activities of ceramide metabolism enzymes. The compounds described here were envisaged as useful tools in CerS activity profiling, as they are inert towards enzymes of 1-O-functionalization. Among the sphingoid bases described in this work, spisulosine (ES285), RBM1-77 and RBM1-73 were the most suitable ones due to their highest acylation rates.

    In conclusion, some of the 1-DSLs synthesized should prove useful to study the role of the different ceramide synthases and the resulting N-acyl (dihydro)ceramides in cell fate.

  • The equilibrium between long and very long chain ceramides is important for the fate of the cell and can be influenced by co-expression of cers.


    Daniela Hartmann, Marthe-Susanna Wegner, Ruth Anna Wanger, Nerea Ferreirós, Yannick Schreiber, Jessica Lucks, Susanne Schiffmann, Gerd Geisslinger and Sabine Grösch.

    Fharmazentrum frankfurt/ ZAFES, Institut für Klinische Pharmakologie, Klinikum der Goethe-Universität Frankfurt, Theodor-Stern-Kai 7, 60590 Frankfurt/ Main, Germany.

    Ceramides are synthesized by six different ceramide synthases (CerS1-6), which differ in their specificity to produce ceramides of distinct chain length. We could show that over-expression of CerS4 and CerS6, which was accompanied by an increase in C18:0- and C16:0-Cer, respectively, had anti-proliferative effects in human breast and colon cancer cells. Now we wanted to investigate the impact of CerS-co-transfection on the physiological outcome and ceramide production in HCT-116 cells.

    Over-expression of CerS2 had no effect on the level of very long chain ceramide C24:0- and C24:1-Cer. Instead over-expression of CerS2 together with CerS4 or CerS6 increased the activity of CerS2 against very-long-chain ceramides about twofold. In contrast, co-expression of CerS4 with CerS6 inhibited slightly the production of C20:0-ceramide in comparison to cells over-expressing CerS4 alone, whereas the activity of CerS6 seemed not to be affected by other CerS. The production of very long chain ceramides was also affected by down-regulation of the fatty acid elongase ELOVL1, which is one of overall seven fatty acid elongase subtypes (ELOVL 1-7) in humans and mainly responsible for the elongation of C20- to C22-C26-fatty acid. The physiological effect of CerS2/CerS4/CerS6 co-expression became apparent as the anti-proliferative and pro-apoptotic effect of long chain ceramides was impeded. Even though we observed a twofold increase in total ceramide levels after co-expression of CerS2 and CerS4/CerS6, we detected no effect on cell proliferation.

    These data indicate that an increase in ceramide production per se is not critical for cell survival, but the equilibrium between long and very long chain ceramides and possibly protein/protein interactions determine the fate of the cell.

  • Regulation of GAP43-calmodulin interaction by the lipid mediators sphingosine and lysophophatidic acid.


    Juhász Tünde1; Kabai Monika1; Harmat Veronika2; Kardos Jozsef3; Szeltner Zoltan1; Liliom Karoly1

    1Institute of Enzymology, RCNS, Hungarian Academy of Sciences, Budapest; 2Institute of Chemistry, Eötvös University, Budapest; 3Department of Biochemistry, Eötvös University, Budapest 

    The brain-specific growth-associated protein 43 (GAP43) participates in the regulation of neurite formation, synaptic plasticity, and neuroregeneration. Its funtion is sensitive to intracellular Ca2+ level and has been shown to interact with calmodulin (CaM), the ubiquitous eukariotic effector protein for the second messenger Ca2+, both in vitro and in vivo. It is assumed that their complex is bound to the plasma membrane but the functional outcomes of these interactions are not well clarified.

    We have identified selective binding between GAP43 and lysophosphatidic acid (LPA), as well as CaM and sphingosine (SPH), utilizing fluorescence spectroscopy, isothermal titration calorimetry and quartz crystal microbalance. In both interactions, the lipids must be in an associated form to produce high affinity binding. In functional assays SPH binding to CaM inhibited the protein's ability to activate its effectors. We determined the crystal-structure of CaM-SPH complex, in which the lipids occupy the same place as the protein's target peptides. Chemical cross-linking, dynamic light scattering, and CD spectroscopy revealed that LPA binding induced the oligomerization and partial structural organization of GAP43, an intrinsically unstructured protein. The phosphorylation of GAP43 modifies its functions. We mimicked this effect by the Ser41Asp mutant, which showed weaker binding to CaM compared to the wild-type protein. In both the absence or presence of Ca2+, the lipids interact with CaM or GAP43 at a higher affinity than the proteins bind to each other, allowing their displacement by each lipid.

    The lipid mediators SPH and LPA, via selective high-affinity bindings to CaM and GAP43, respectively, may regulate the complex formation between the proteins, thereby modifying their function in the growth cone and synapses, possibly interconnecting this subsystem with other signaling processes.

  • Regulation of GAP43-calmodulin interaction by the lipid mediators sphingolipid metabolism is required for the toxic effects elicited by the environmental compound PCB153 on Cx43-formed gap junctions in liver stem-like cells.


    Meacci Elisabetta1, Squecco Roberta2, Frati Alessia1, Machala Miroslav3, Francini Fabio2, Vicenti Catia1, Hofmanova Jirina3, Anderloni Giulia1

    1 Dipartimento di Scienze Biomediche, Sperimentali e Cliniche, Università di Firenze-Italy; 2 Dipartimento  di Biomedicina, Università di Firenze-Italy; 3Veterinary Research Institute, Brno, Czech Republic

    Abundant non-dioxin-like organic toxicant PCB153 were found to induce alterations of membrane proteins associated with inhibition of gap junction intercellular communication (GJIC) in rat liver progenitor cells, WB-F344. Since the molecular mechanisms triggered by this a highly lipophilic toxicant is largely unknown, on the notion that GJIC and connexin-43 (Cx43) expression are modulated by sphingosine 1-phosphate (S1P) in skeletal muscle cells, we hypothesized that the impairment of Cx43-formed GJIC elicited by PCB153 could involve the activation of sphingoid-mediated-signalling pathways.

    PCB153 treatment determined changes in the biophysical properties of gap junctional currents, as judged by dual whole cell voltage-clamp, and, in parallel, a time-dependent regulation of sphingosine kinase (SphK) activity, that leaded to an increase of S1P at 1 h and 24h and a decrease of the bioactive lipid at 3h after compound exposure. Similarly, ceramide kinase activity was significantly affected by the toxicant resulting in an increase of ceramide 1-phosphate  (C1P) and a marked decrease of ceramide after 1-3 h. Interestingly, pharmacological inhibition of SphK and PP2A phosphatase and their down-regulation by specific siRNA treatment affected GJIC and prevented GJIC impairment and changes in the Cx43 phosphorylation/expression elicited by PCB153.

    Altogether, these findings represent the first evidence of the role played by bioactive sphingolipids in the regulation of GJIC in liver stem-like cells and in the biological action of the environmental toxicant in these progenitor cells. Since malfunctioned GJIC has been thought associated with malignant transformation of normal cells, the finding of the role played by sphingolipids can lead to consider these bioactive molecules as potential targets to prevent the tumorigenic action of PCB153 and other dioxin and  non-dioxin like persistent environmental pollutants.

  • Characterization of acid sphingomyelinase activity in human cerebrospinal fluid.


    Christiane Mühle1, Hagen B. Huttner2, Silke Walter3, Martin Reichel1, Fabio Canneva4, Piotr Lewczuk1, Erich Gulbins5, Johannes Kornhuber1

    1Department of Psychiatry and Psychotherapy, Friedrich-Alexander-University of Erlangen-Nuremberg, Erlangen, Germany; 2Department of Neurology, Friedrich-Alexander-University of Erlangen-Nuremberg, Erlangen, Germany; 3Department of Neurology, University of the Saarland, Homburg/Saar, Germany; 4Experimental Therapy, Franz-Penzoldt-Center, Friedrich-Alexander-University of Erlangen-Nuremberg, Erlangen, Germany; 5Department of Molecular Biology, University of Duisburg-Essen, Essen, Germany

    As a key enzyme in sphingolipid metabolism, acid sphingomyelinase (ASM) is involved in the regulation of cell fate and signaling via the hydrolysis of sphingomyelin to form ceramide. While increased activity of the lysosomal form has been associated with various pathological conditions, there are few studies on secretory ASM limited only to cell models, plasma or serum.

    We have established a sensitive quantitative assay based on a fluorescent substrate to measure the ASM activity in cerebrospinal fluid (CSF) and characterized the enzyme’s properties. CSF localized ASM resembles plasmatic ASM with respect to protein stability and Zn2+-dependence. However, the assays differ considerably in the optimal detergent concentration. The optimized assay was applied to CSF samples collected from mice and from 42 patients who were classified as controls based on normal routine CSF values. Significantly increased activities in the CSF of ASM transgenic mice and undetectable levels in ASM knock-out mice prove that the measured ASM activity originates from the ASM-encoding gene SMPD1. In humans, CSF localized ASM activities were comparable to corresponding serum ASM levels at their respective optimal reaction conditions, but no correlation was observed. The large variance in ASM activity was independent of sex, age or analyzed routine CSF parameters.

    Human and mouse CSF contain detectable levels of secretory ASM, which are unrelated to serum ASM activities. Further investigations will help to elucidate the role of this enzyme in human disease and to assess its value as a potential biomarker for disease type, progress or therapeutic success.

  • Administration of glucosylceramide ameliorated the memory decline in aged mice.


    Yeonju Lee, Sergiy Oliynyk, Seikwan Oh

    Department of Neuroscience and TIDRC, School of Medicine, Ewha Womans University, Seoul 158-710, Korea

    The function and the role of glucosylceramide have not been well studied in the central nervous system. This study was aimed to investigate the possible roles of glucosylceramide in memory function in aged mice.

    Glucosylceramide (50 mg/kg, p.o.) showed memory enhancing activity after 3-month treatment in the aged mice (C56BL/6, 18-20 month-old) through Y-maze, novel objective test and Morris water maze test. Long-term treatment of glucosylceramide decreased the expression of iNOS and COX-2 in the brain of aged mice. The LPS-induced mRNA level of iNOS, COX-2, IL-1bband TNF-aawas reduced by the acute treatment with glucosylceramide in adult mice.

    These results suggest that glucosylceramide plays an important role in anti-inflammatory and memory enhancement, and it could be a potential new therapeutic agent for the treatment of neurodegenerative diseases such as Alzheimer’s disease.

  • Sphıngolıpıdomıcs ın sphıngolıpıd storage dıseases: ıdentıfıcatıon of dıfferencıated sphıngolıpıds that have role ın dıfferent clınıcal courses.


    Adem ÖZKAN1, Funda YILDIRIM2, Mehmet KAYILI2, Ömür ÇELİKBIÇAK2, Erdal COŞGUN3, Fatma Müjgan SÖNMEZ4, Meral TOPÇU5, Bekir SALİH2, Hatice Asuman ÖZKARA1

    1Hacettepe University Faculty of Medicine, Department of Medical Biochemistry, 06100 Ankara, Turkey; 2Hacettepe University Faculty of Science, Department of Chemistry, Ankara, Turkey;
    3Hacettepe University Faculty of Medicine, Department of Biostatistics, 06100 Ankara, Turkey;
    4Fatih University Faculty of Medicine, Department of Pediatrics, Ankara, Turkey;
    5Hacettepe University Faculty of Medicine, Department of Pediatrics, Neurology unit,06100 Ankara, Turkey

     Sphingolipids are ubiquitous elements of the plasma membrane. They are synthesized in ER and Golgi. Their degradation are made by lysosomal enzymes in lysosome. Defective lysosomal enzymes cause lysosomal storage diseases. Accumulation of undegraded substrates in the nervous system leads to severe impairment of neurological function with a fatal outcome. The aim of this study is to find out sphingolipid/s for predicting clinical phenotype and rate of disease progression.

    In this study, sphingolipids were extracted from serums of patients with gangliosidoses and Metachromatic Leukodystrophy which are the most common sphingolipid storage diseases in our country. Their mass spectrums are taken by MALDI-ToF-MS. Sphingolipids that make difference between groups and clinical findings were determined by cluster analysis. Internal standards were used for quantitative analysis by LC-ESI-MS/MS. After evaluation by cluster analysis of the obtained sphingolipid profiles, sphingosine and sphinganine 1-P in gangliosidoses group, sialic acid and phosphatidylcholine in Metachromatic leukodystrophy group were found as differentiated molecules. Phosphatidylcholine and sialic acid in bradykinesia, sphinganine 1-P and amino group containing fatty acids in spastic tetraparesis were found as molecules that make difference in gangliosidoses. Results of quantitative analysis supported the statistical analysis. In addition increased ceramide and ceramide 1-P were detected in gangliosidoses group.

  • Validation of a new epigenetic-based prognostic blood test to predict prostate cancer aggressiveness.


    Emily Corfield1, Heba Alshaker2, Constantine Alifrangis2, Jane Mellor3, Yoshiaki Kawano4, Masatoshi Eto4,Philip Jordan1, Aroul Ramadass1, Alexandre Akoulitchev1 and Dmitri Pchejetski2

    1. Oxford BioDynamics Limited, 26 Beaumont Street, Oxford, Oxfordshire, OX1 2NP, UK; 2. Department of Medicine, Imperial College, London, UK; 3. Department of Biochemistry, University of Oxford, Oxford, UK; 4. Department of Urology, Faculty of Life Sciences, Kumamoto University, Japan

    Sphingosine kinase 1 (SK1) plays has been reported to be elevated in many cancers including prostate, but its regulation is poorly understood. Chromosome conformation patterns represent early changes in epigenetic regulation during tumourigenesis.

    Results: To investigate epigenetic regulation of SK1 and other cancer-associated genes, 80 treatment naive patients with localised, locally advanced or metastatic PCa were recruited into this prospective study. Upon diagnosis blood samples were collected and chromosomal conformation patterns were determined. Multivariate analysis using relevance vector machine based algorithm was used to identify the epigenetic signatures.

    We identified 28 chromosomal conformation markers, in the loci of several PCa-related genes, including SK1, MYC, PSMA, PTEN, SMAD4, and SAM68, and correlated them with Gleason score, age or disease stage as predictors of survival probability and PCa aggressiveness. The test was trained on 64 samples and cross-validated on 16 independent samples. The final EpiSwitchTM test can accurately predict PCa aggressiveness status in both training and validation sets, with a combined accuracy of 98.6% (95% CI, 93% - 99%).

    In Addition to stratification of PCa patients this prognostic, non-invasive test has the potential to be used for making treatment decisions, monitoring and predicating for PCa mortality. The EpiSwitchTM platform technology can be extended to address other clinical questions in diagnostics and theranostics.

  • Characterization of the acid sphingomyelinase signal peptide polymorphism.


     Cosima Rhein, Christiane Mühle, Martin Reichel, Johannes Kornhuber

    Department of Psychiatry and Psychotherapy, Friedrich-Alexander-University Erlangen-Nuremberg, Germany

    Acid sphingomyelinase (ASM) catalyses the hydrolysis of sphingomyelin into ceramide, which acts as a lipid messenger regulating important cellular functions, ranging from differentiation to apoptosis. Dysregulated ASM activity was reported for different neuro-psychiatric disorders. ASM features an exceptional polymorphic part located in the region coding for its signal peptide, which is characterized by a three- to eight-fold repeat of the hexanucleotide GCTGGC and associated with the SNP c.107T>C. We investigated the role of these polymorphisms for intact enzymatic functioning.

    In our in vivo study investigating healthy individuals, alleles with 5 and 6 repeats were found with a much higher prevalence compared to 4 and 8 repeats. ANOVA revealed a significant impact of genotypic repeat configuration on the secretory (S-ASM) but not on the lysosomal (L-ASM) enzyme activity in peripheral mononuclear cells: ASM activity in plasma was significantly lower in individuals homozygous for 4 repeats compared to those homozygous for 5 or 6 repeats. To further test the significance of the varying repeat genotype, we performed overexpression studies in cultured cells. Plasmids carrying ASM cDNA varying from 1 to 9 repeats combined with either allele of the c.107T>C polymorphism were generated by site-directed mutagenesis. Our in vitro results showed that the ratio of S- to L-ASM activity was significantly decreased for the extreme repeats 1, 2 and 8, 9 compared to the reference variant with 6 repeats, indicating that the length of the polymorphic region is critical for the proper secretion of ASM. The SNP c.107T>C, on the contrary, led to a significantly increased ratio of S- to L-ASM activity. Interestingly, the natural occurring ASM variants feature a specific composition of repeat length and SNP manifestation that varied only modestly regarding their S- to L-ASM activity. Thus, the combination of the two genetic variations seem to be subjected to evolutionary selection to balance their impact on ASM trafficking.

    We conclude that the signal peptide polymorphisms significantly impact the trafficking of ASM. Since secreted and lysosomal localized ASM enzymes seem to differ in their function and their correlation with pathologic states, this genetic effect should be considered when treating associated diseases. 

  • Sphingomyelinase-induced changes of the vascular tone.


    Éva Ruisanchez, Zsuzsa Straky, Dávid Korda, Adrienn Párkányi, Levente Kiss, Zoltán Benyó

    Institute of Human Physiology and Clinical Experimental Research, Semmelweis University, Budapest, Hungary

    The first step of sphingolipid biosynthesis from sphingomyelin is catalyzed by sphingomyelinase (SMase) enzymes which are reportedly upregulated in certain cardiovascular and metabolic disorders. We aimed to analyze the effects of neutral SMase on the regulation of the vascular tone in wild type (WT), as well as inknock-out (KO) mice for sphingosine-1-phosphate receptors S1P2 or S1P3, endothelial nitric oxide synthase (eNOS), cyclooxygenase-1 (COX1) or thromboxane-receptor (TP).

    Thoracic aorta segments were isolated from adult male mice and investigated after precontraction with phenylephrine under isometric conditions in myographs. SMase evoked a biphasic vascular reaction in WT vessels with an initial contraction and subsequent relaxation. These effects were dose-dependent and submaximal responses were reached by 0.2 U/ml SMase. S1P2- and S1P3-KO vessels showed both the contractile and relaxant components of the SMase response. In eNOS-KO vessels, however, enhanced contractile effects were observed while the vasorelaxation disappeared. In contrast, COX1-KO vessels showed diminished contractions and enhanced relaxations. Neither eNOS-KO vessels treated with the COX-inhibitor indomethacin, nor COX1-KO vessels treated with the NOS-inhibitor NG-nitro-L-arginine methyl ester (L-NAME) responded with significant changes of the vascular tension upon administration of SMase. Interestingly, TP-KO vessels showed similar reactions to those observed in COX1-KOs: the contractile effect was diminished while the relaxant enhanced. In TP-KO vessels treated with L-NAME SMase failed to induce any significant vasoactive effects.

    SMase induces biphasic changes of the vascular tone with an initial contraction and subsequent relaxation. The contractile effect is mediated by COX1-dependent production of thromboxane A2 (or a related prostanoid), while the relaxation is due to endothelial nitric oxide release.

  • Altered calcium kinetics and mitochondrial oxidative stress in psychosine induced cell death: implications for globoid cell leucodistrophy.


    Vladimir Voccoli*, Ilaria Tonazzini*, Chiara Cerri§, Manuela Allegra§°, Matteo Caleo§ and Marco Cecchini*

    *NEST, Istituto Nanoscienze-CNR and Scuola Normale Superiore, § Istituto di Neuroscienze del CNR, ° Scuola Normale Superiore di Pisa.

    Globoid cell LeukoDystrophy (Krabbe disease) is a childhood leukodystrophy triggered by deficit of galattosyl-ceramidase (GALC) activity. GALC is a lysosomal enzyme which catalyzes the intracellular degradation of galactosyl-ceramide and galactosyl-sphingosine (psychosine, PSY). GALC loss of activity causes PSY accumulation and cell death. Little is known about the intracellular pathways affected by PSY accumulation. Here, we address the role of exogenous PSY in intracellular Ca2+ dynamics during cell death.

    MO3.13 cells (human oligodendrocyte model) were treated for 24h with PSY in 0%FBS. As expected, PSY induced apoptosis, as revealed by chromatin fragmentation. In order to evaluate if PSY affects Ca2+ kinetics, cells were loaded with Fluo3 and the mitochondrial dye, TMRM. A fast Ca2+ increase occurred after PSY administration, followed by a slower one preceding mitochondrial permeabilization. PSY was found to induce also necrosis: indeed in the 12,6% of dying cells a strong Ca2+ increase occurred coupled to fast cell death. To understand if Ca2+ increase involves mitochondria, cells were transfected with the mtcD2CPV Ca2+ probe and stained with TMRM. A rapid mitochondrial Ca2+ peak was detected after treatment, followed by a later increase preceding mitochondrial depolarization. Then, we treated cells with PSY in presence of EDTA: this had no effect on the intracellular Ca2+ increase but extended cell survival and abolished necrosis. Finally, cells were loaded with Fluo3 and MitoTrackerROS, an indicator of mitochondrial oxidative stress. Upon PSY administration, ROS production increased subsequent to the first Ca2+ increase while its maximum corresponded to the second Ca2+ increase.

    PSY induces Ca2+ mediated cell death involving mitochondria. It induces increase of mitochondrial Ca2+ and ROS, and their depolarization. Blocking Ca2+ influx by EDTA can extend cell life but does not block Ca2+ kinetics, leading to hypothesize a role of PSY on Endoplasmic Reticulum Ca2+ kinetics.