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Regulation of Intracellular Ceramide Content in B16 Melanoma Cells

BIOLOGICAL IMPLICATIONS OF CERAMIDE GLYCOSYLATION*
  • Hironobu Komori
    Affiliations
    From the Laboratory of Marine Biochemistry, Faculty of Agriculture, Kyushu University, Fukuoka 812-8581, Japan
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  • Shinichi Ichikawa
    Affiliations
    Laboratory for Cellular Glycobiology, Frontier Research Program, The Institute of Physical and Chemical Research (RIKEN), Saitama 351-0198, Japan
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  • Yoshio Hirabayashi
    Affiliations
    Laboratory for Cellular Glycobiology, Frontier Research Program, The Institute of Physical and Chemical Research (RIKEN), Saitama 351-0198, Japan
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  • Makoto Ito
    Correspondence
    To whom correspondence should be addressed: Laboratory of Marine Biochemistry, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan. Tel.: 81-92-642-2900; Fax: 81-92-642-2907;
    Affiliations
    From the Laboratory of Marine Biochemistry, Faculty of Agriculture, Kyushu University, Fukuoka 812-8581, Japan
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  • Author Footnotes
    * This work was supported in part by Grants-in-aid for Scientific Research 09460051 (to M. I.), 05274106 (to Y. H.), and 09780586 (to S. I.) from the Ministry of Education, Science and Culture of Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Open AccessPublished:March 26, 1999DOI:https://doi.org/10.1074/jbc.274.13.8981
      We previously reported that ceramide released from glycosphingolipids (GSLs) by endoglycoceramidase was directly metabolized to GSLs, and thus the content of GSLs was constantly maintained in B16 melanoma cells (Ito, M., and Komori, H. (1996)J. Biol. Chem.271, 12655–12660). In this study, the metabolism of ceramide released from sphingomyelin (SM) by bacterial sphingomyelinase (SMase) was examined using B16 cells and their GSL-deficient mutant counterpart GM95 cells. Treatment of B16 melanoma cells with bacterial SMase effectively hydrolyzed SM on the plasma membrane. Under these conditions, NeuAcα2,3Galβ1,4Glcβ1,1ceramide was significantly increased. Interestingly, UDP-glucose:ceramide glucosyltransferase-1 (GlcT-1) activity and GSL synthesis, but not SM synthesis or sphingosine generation, were found to be up-regulated by SMase treatment. The up-regulation of GSL synthesis seemed to occur at both the transcriptional and post-translational steps of GlcT-1 synthesis. Accumulation of ceramide by bacterial SMase was much higher in GM95 cells than in the parental cells. When the enzyme was removed from the culture medium, the intracellular ceramide level in B16 cells, but not that in the mutant cells, normalized. No rapid restoration of SM in either of the cell lines was observed after removal of the enzyme. SMase treatment strongly inhibited DNA synthesis in GM95 cells but not that in B16 cells. In the presence ofd-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol, an inhibitor of GlcT-1, SMase treatment markedly increased the ceramide content and thus inhibited DNA synthesis in B16 cells. Our study provides the first evidence that GlcT-1 functions to regulate the level of intracellular ceramide by glycosylation of the ceramide when it is present in excess.
      Glycosphingolipids (GSLs)
      The abbreviations used are:GSL, glycosphingolipid; C6-ceramide, N-hexanoylsphingosine; CMH, ceramide monohexoside; GlcT-1, UDP-glucose:ceramide glucosyltransferase-1; GM3, NeuAcα2,3Galβ1,4Glcβ1,1ceramide; MEM, minimum essential medium; PBS, phosphate-buffered saline; PDMP, d-threo-1-phenyl-2-decanoylamino3-morpholino-1-propanol; SM, sphingomyelin; SMase, sphingomyelinase; TLC, thin layer chromatography; FBS, fetal bovine serum.
      1The abbreviations used are:GSL, glycosphingolipid; C6-ceramide, N-hexanoylsphingosine; CMH, ceramide monohexoside; GlcT-1, UDP-glucose:ceramide glucosyltransferase-1; GM3, NeuAcα2,3Galβ1,4Glcβ1,1ceramide; MEM, minimum essential medium; PBS, phosphate-buffered saline; PDMP, d-threo-1-phenyl-2-decanoylamino3-morpholino-1-propanol; SM, sphingomyelin; SMase, sphingomyelinase; TLC, thin layer chromatography; FBS, fetal bovine serum.
      and sphingomyelin (SM) are characteristic components of vertebrate plasma membranes and have the same hydrophobic residue, ceramide, which consists of a sphingosine and a fatty acid. GSLs have been defined as tumor antigens, receptors for microbes and their toxins, and possible modulators of cell proliferation, differentiation, and cell-cell interactions (
      • Hakomori S.
      ,
      • Karlsson K.-A.
      ,
      • Iwabuchi K.
      • Yamamura S.
      • Prinetti A.
      • Handa K.
      • Hakomori S.
      ). Recently, ceramide has emerged as a novel second messenger for intracellular signaling pathways responding to various cytokines and stress (
      • Hannun Y.A.
      ). Several lines of evidence indicate that a signaling ceramide is produced from SM by the action of endogenous neutral (
      • Okazaki T.
      • Bell R.M.
      • Hannun Y.A.
      ) and acid sphingomyelinase (SMase; EC3.1.4.12) (
      • Schwandner R.
      • Wiegmann K.
      • Bernardo K.
      • Kreder D.
      • Kronke M.
      ), or by de novo synthesis (
      • Bose B.
      • Verheij M.
      • Haimovitz-Friedman A.
      • Scotto K.
      • Fuks Z.
      • Kolesnick R.
      ). However, little is known about the mechanism of regulation of intracellular level of ceramide, which should be strictly regulated within cells.
      Endoglycoceramidase (EC3.2.1.123) is a GSL-specific enzyme fromRhodococcus sp. that hydrolyzes the glycosidic linkage of ceramide and sugar chains of various GSLs (
      • Ito M.
      • Yamagata T.
      ). The cell surface GSLs of various erythrocytes (
      • Ito M.
      • Ikegami Y.
      • Tai T.
      • Yamagata T.
      ) and cultured mammalian cells (
      • Ji L.
      • Ito M.
      • Zhang G.
      • Yamagata T.
      ) were hydrolyzed by the purified rhodococcal endoglycoceramidase (
      • Ito M.
      • Yamagata T.
      ) with the assistance of its protein activator (
      • Ito M.
      • Ikegami Y.
      • Yamagata T.
      ). We found that treatment of B16 melanoma cells with a microbial endoglycoceramidase activated GSL synthesis via transient up-regulation of UDP-glucose:ceramide glucosyltransferase-1 (GlcT-1, glucosylceramide synthase; EC2.4.1.80) (
      • Ito M.
      • Komori H.
      ). As a result, cell surface NeuAcα2,3Galβ1,4Glcβ1,1ceramide (GM3), the end product of GSL synthesis in B16 cells, was restored quickly when the enzyme was removed from the culture medium (
      • Ito M.
      • Komori H.
      ).
      In this study, we examined the effects of bacterial SMase on the metabolism of GSLs and SM using B16 cells and their GSL-deficient mutant counterpart GM95 cells, which lack GlcT-1 (
      • Ichikawa S.
      • Nakajo N.
      • Sakiyama H.
      • Hirabayashi Y.
      ). Although GSLs were quickly restored after endoglycoceramidase treatment, restoration of SM was not observed after treatment with bacterial SMase in B16 melanoma cells. Interestingly, ceramides generated from not only GSLs but also SM by the microbial enzymes were primarily glucosylated by GlcT-1, metabolized to GM3, and then transported to the plasma membrane. Ceramide was accumulated during SMase treatment in GM95 or B16 cells in the presence ofd-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) (
      • Radin N.S.
      • Shayman J.A.
      • Inokuchi J.
      ) or N-butyldeoxynojirimycin (
      • Platt F.M.
      • Neises G.R.
      • Dwek R.A.
      • Butters T.D.
      ), potent inhibitors of GlcT-1. These results suggest a biological role of GlcT-1 in the regulation of intracellular ceramide content.
      Because excess generation of ceramide is toxic to cells, GlcT-1 seems to function for expulsion of ceramide from the cell. This regulation, regarded as a putative detoxification mechanism, may function as a defense against an unexpected increase of ceramide, which could be caused by various forms of stress, e.g. infections with pathogenic microorganisms that produce SMase or endoglycoceramidase. This paper also indicates the biological role of ceramide as a modulator of the overall synthesis of GSLs by regulating GlcT-1 at both the transcriptional and post-translational levels.

      DISCUSSION

      Recently, Zhang et al. (
      • Zhang P.
      • Liu B.
      • Jenkins G.M.
      • Hannun Y.A.
      • Obeid L.M.
      ) reported the expression of a recombinant B. cereus SMase in Molt-4 leukemia cells. After the transfection of the gene and stable expression of the SMase, intracellular ceramide content increased, resulting in induction of apoptosis. However, exogenously added B. cereus SMase, despite causing a greater elevation of ceramide level, did not induce apoptosis in Molt-4 cells (
      • Zhang P.
      • Liu B.
      • Jenkins G.M.
      • Hannun Y.A.
      • Obeid L.M.
      ). This result suggested the existence of two distinct SM pools, one of which is accessible by endogenous SMase and the other by exogenous SMase. The former seems to be responsible for transduction of the apoptotic signal, and the turnovers of these two pools might be somewhat different. On the other hand, some reports indicated that exogenous bacterial SMase had biological effects on cells. For example, the Streptomyces SMase enhanced the action of subthreshold vitamin D3 in inducing HL60 cell differentiation (
      • Okazaki T.
      • Bielawska A.
      • Bell R.M.
      • Hannun Y.A.
      ), and the enzyme from S. aureus induced apoptosis of U937 cells (
      • Cuvillier O.
      • Pirianov G.
      • Kleuser B.
      • Vanek P.G.
      • Coso O.A.
      • Gutkind J.S.
      • Spiegel S.
      ). In the present study, treatment of B16 cells with bacterial SMase inhibited DNA synthesis under conditions of genetic or pharmacological blockade of GlcT-1. These results suggest that the localization and topology of SM and their susceptibility to SMase differ according to cell type.
      Luberto and Hannun (
      • Luberto C.
      • Hannun Y.A.
      ) reported that treatment of human lung fibroblast WI38 cells with bacterial SMase resulted in a decrease in SM level and concomitant generation of ceramide. This ceramide level decreased very slowly in the cells, but there was little restoration of SM content. In contrast, SV40-transformed cells, in which the activity of SM synthase (phosphatidylcholine-specific phospholipase C) was found to be 3-fold higher than that in parental cells, cleared ceramide much more rapidly and regenerated SM. The authors argued the potential significance of SM synthase for regulation of intracellular ceramide levels in the fibroblasts. We showed, on the other hand, that in B16 melanoma cells the ceramides generated from SM as well as GSLs (
      • Ito M.
      • Komori H.
      ) by microbial enzymes were primarily glucosylated by GlcT-1 and metabolized to GM3. This discrepancy may be attributable to the balance between GlcT-1 and SM synthase activities in cells, which is genetically defined depending on the origin of cells or their phenotype and might be affected by other environmental factors.
      The present study revealed that ceramide generated on the outer leaflet of the plasma membrane by bacterial SMase was directly, but not via the sphingoid-base salvage pathway (
      • Gillard B.K.
      • Clement R.
      • Colucci-Guyon E.
      • Babinet C.
      • Schwarzmann G.
      • Taki T.
      • Kasama T.
      • Marcus D.M.
      ), metabolized to GSLs in B16 melanoma cells. Because the catalytic domain of GlcT-1 is located on the cytosolic side of the Golgi membrane (
      • Jeckel D.
      • Karrenbauer A.
      • Burger K.N.J.
      • van Meer G.
      • Wieland F.
      ), the generated ceramide must be translocated to the outer leaflet of the Golgi membrane before it becomes accessible to the enzyme. Although the transportation of ceramide to the Golgi membrane remains unclear, our findings suggest that the transport of ceramide in protein-directed (
      • Kok J.W.
      • Nikolova-Karakashian M.
      • Klappe K.
      • Alexander C.
      • Merrill Jr., A.H.
      ) and vesicle-independent manners (
      • Kok J.W.
      • Babia T.
      • Klappe K.
      • Hoekstra D.
      ) is significant.
      Many pathogenic and opportunistic microbes produce SMases, some of which have been identified as hemolysins and cytotoxins (
      • Titball R.W.
      ). These observations indicate that cell surface SM of vertebrates might be exposed to the action of microbial SMase, which may result in the elevation of the intracellular ceramide level. Because the excess generation of ceramide must be toxic for the cell, the exclusion of ceramide from the cell by glycosylation can be regarded as a mechanism of defense against infection by SMase-producing pathogens. It is interesting to note that many opportunistic pathogens can also produce endoglycoceramidase extracellularly (
      • Ito M.
      • Komori H.
      ).
      Lavie et al. (
      • Lavie Y.
      • Cao H.
      • Bursten S.L.
      • Giuliano A.E.
      • Cabot M.C.
      ,
      • Lavie Y.
      • Cao H.
      • Volner A.
      • Lucci A.
      • Han T.
      • Geffen V.
      • Giuliano A.E.
      • Cabot M.C.
      ) reported that multidrug-resistant human breast cancer cells exhibited marked accumulation of glucosylceramide compared with the parental cells. The reverse multidrug resistance drug tamoxifen was found to inhibit GlcT-1, resulting in a decrease in the level of glucosylceramide and an increase in that of ceramide. This drug as well as 1-phenyl2-palmitoyoamino-3-morpholino-1-propanol, an inhibitor of GlcT-1, sensitized the multidrug-resistant cells to some anticancer drugs. These results suggested that GlcT-1 is involved in regulation of ceramide levels, which may affect the sensitivity of cancer cells to anticancer drugs.
      We conclude that GlcT-1, distributed ubiquitously in vertebrate cells, functions to regulate the level of intracellular ceramide by glycosylation of the ceramide when it is present in excess.

      ACKNOWLEDGEMENTS

      We thank Dr. Takashi Nakamura of Kyushu University and Dr. Tatsuya Yamagata of Japan Institute of Leather Research for encouragement throughout this study. We are also grateful to Dr. Toshiro Okazaki of Kyoto University for the gift ofsn-1,2-diacylglycerol kinase.

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