Keiko Furukawa

Chubu University, Касугай, Aichi, Japan

Are you Keiko Furukawa?

Claim your profile

Publications (215)878.07 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: In this study, we used GM2/GD2 synthase knockout (GM2/GD2−/−) mice to examine the influence of deficiency in ganglioside “a-pathway” and “b-pathway” on cognitive performances and hippocampal synaptic plasticity. Eight-week-old GM2/GD2−/− male mice showed a longer escape-latency in Morris water maze test and a shorter latency in step-down inhibitory avoidance task than wild-type (WT) mice. Schaffer collateral-CA1 synapses in the hippocampal slices from GM2/GD2−/− mice showed an increase in the slope of EPSPs with reduced paired-pulse facilitation, indicating an enhancement of their presynaptic glutamate release. In GM2/GD2−/− mice, NMDA receptor (NMDAr)-dependent LTP could not be induced by high-frequency (100–200 Hz) tetanus or θ-burst conditioning stimulation (CS), whereas NMDAr-independent LTP was induced by medium-frequency CS (20–50 Hz). The application of mono-sialoganglioside GM1 in the slice from GM2/GD2−/− mice, to specifically recover the a-pathway, prevented the increased presynaptic glutamate release and 20 Hz-LTP induction, whereas it could not rescue the impaired NMDAr-dependent LTP. These findings suggest that b-pathway deficiency impairs cognitive function probably through suppression of NMDAr-dependent LTP, while a-pathway deficiency may facilitate NMDAr-independent LTP through enhancing presynaptic glutamate release. As both of the NMDAr-independent LTP and increased presynaptic glutamate release were sensitive to the blockade of L-type voltage-gated Ca2+ channels (L-VGCC), a-pathway deficiency may affect presynaptic L-VGCC.
    Hippocampus 04/2014; 24(4):369-82. · 5.49 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Gangliosides, sialic acid-containing glycosphingolipids, are highly expressed in nervous systems of vertebrates and have been considered to be involved in the development, differentiation, and function of nervous tissues. Recent studies with gene-engineered animals have revealed that they play roles in the maintenance and repair of nervous tissues. In particular, knockout (KO) mice of various ganglioside synthase genes have exhibited progressive neurodegeneration with aging. However, neurological disorders and pathological changes in the spinal cord of these KO mice have not been reported to date. Therefore, we examined neurodegeneration in double knockout (DKO) mice of ganglioside GM2/GD2 synthase (B4GANLT1) and GD3 synthase (ST8SIA1) genes to clarify roles of gangliosides in the spinal cord. Motor neuron function was examined by gait analysis, and sensory function was analyzed by von Frey test. Pathological changes were analyzed by staining tissue sections with Kluver-Barrera staining and by immunohistochemistry with F4/80 and glial fibrillary acidic protein (GFAP). Gene expression profiles were examined by using DNA micro-array of RNAs from the spinal cord of mice. Triple knockout mice were generated by mating DKO and complement component 3 (C3)-KO mice. Gene expression of the complement system and cytokines was examined by reverse transcription-polymerase chain reaction (RT-PCR) as a function of age. DKO mice showed progressive deterioration with aging. Correspondingly, they exhibited shrunk spinal cord, reduced thickness of spinal lamina II and III, and reduced neuronal numbers in spinal lamina IX, spinal lamina II, and spinal lamina I. Complement-related genes were upregulated in DKO spinal cord. Moreover, complement activation and inflammatory reactions were detected by GFAP-active astrocyte, microglial accumulation, and increased inflammatory cytokines such as tumor necrosis factor-alpha (TNFalpha) and interleukin-1-beta (IL-1beta). Triple knockout mice showed restoration of reduced neuron numbers in the spinal cord of DKO mice, getting close to levels of wild-type mice. Disruption in the architecture of lipid rafts in the spinal cord was not so prominent, suggesting that mechanisms distinct from those reported might be involved in the complement activation in the spinal cord of DKO mice. Gene profiling revealed that inflammation and neurodegeneration in the spinal cord of DKO mice are, at least partly, dependent on complement activation.
    Journal of Neuroinflammation 03/2014; 11(1):61. · 4.35 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Although expression of gangliosides and their synthetic enzyme genes in malignant melanomas has been well studied, that in normal melanocytes has been scarcely analyzed. In particular, changes in expression levels of glycosyltransferase genes responsible for ganglioside synthesis during evolution of melanomas from melanocytes are very important to understand roles of gangliosides in melanomas. Here, expression of glycosyltransferase genes related to the ganglioside synthesis was analyzed using RNAs from cultured melanocytes and melanoma cell lines. Quantitative RT-PCR revealed that melanomas expressed high levels of mRNA of GD3 synthase and GM2/GD2 synthase genes and low levels of GM1/GD1b synthase genes compared with melanocytes. As a representative exogenous stimulation, effects of ultraviolet B (UVB) on the expression levels of 3 major ganglioside synthase genes in melanocytes were analyzed. Although direct UVB irradiation of melanocytes caused no marked changes, culture supernatants of UVB-irradiated keratinocytes (HaCaT cells) induced definite up-regulation of GD3 synthase and GM2/GD2 synthase genes. Detailed examination of the supernatants revealed that inflammatory cytokines such as TNFα and IL-6 enhanced GD3 synthase gene expression. These results suggest that inflammatory cytokines secreted from UVB-irradiated keratinocytes induced melanoma-associated ganglioside synthase genes, proposing roles of skin microenvironment in the promotion of melanoma-like ganglioside profiles in melanocytes.
    Biochemical and Biophysical Research Communications 03/2014; · 2.28 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Gangliosides are widely expressed sialylated glycosphingolipids with multifunctional properties in different cell types and organs. In the nervous system, they are highly enriched in both glial and neuronal membranes. Mice lacking complex gangliosides attributable to targeted ablation of the B4galnt1 gene that encodes β-1,4-N-acetylegalactosaminyltransferase 1 (GalNAc-transferase; GalNAcT(-/-)) develop normally before exhibiting an age-dependent neurodegenerative phenotype characterized by marked behavioral abnormalities, central and peripheral axonal degeneration, reduced myelin volume, and loss of axo-glial junction integrity. The cell biological substrates underlying this neurodegeneration and the relative contribution of either glial or neuronal gangliosides to the process are unknown. To address this, we generated neuron-specific and glial-specific GalNAcT rescue mice crossed on the global GalNAcT(-/-) background [GalNAcT(-/-)-Tg(neuronal) and GalNAcT(-/-)-Tg(glial)] and analyzed their behavioral, morphological, and electrophysiological phenotype. Complex gangliosides, as assessed by thin-layer chromatography, mass spectrometry, GalNAcT enzyme activity, and anti-ganglioside antibody (AgAb) immunohistology, were restored in both neuronal and glial GalNAcT rescue mice. Behaviorally, GalNAcT(-/-)-Tg(neuronal) retained a normal "wild-type" (WT) phenotype throughout life, whereas GalNAcT(-/-)-Tg(glial) resembled GalNAcT(-/-) mice, exhibiting progressive tremor, weakness, and ataxia with aging. Quantitative electron microscopy demonstrated that GalNAcT(-/-) and GalNAcT(-/-)-Tg(glial) nerves had significantly increased rates of axon degeneration and reduced myelin volume, whereas GalNAcT(-/-)-Tg(neuronal) and WT appeared normal. The increased invasion of the paranode with juxtaparanodal Kv1.1, characteristically seen in GalNAcT(-/-) and attributed to a breakdown of the axo-glial junction, was normalized in GalNAcT(-/-)-Tg(neuronal) but remained present in GalNAcT(-/-)-Tg(glial) mice. These results indicate that neuronal rather than glial gangliosides are critical to the age-related maintenance of nervous system integrity.
    Journal of Neuroscience 01/2014; 34(3):880-91. · 6.91 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The highest expression of gangliosides, sialic acid-containing glycosphingolipids (GSLs), is found in the nervous tissue of vertebrates. Changes in the profiles of gangliosides during the development of nervous tissues indicate that they are involved in the regulation of neurogenesis and synaptogenesis. Their distinct distribution patterns support the suggestion that they are involved in both the differentiation and function of neural cells. In addition to results of studies of GSLs done using biochemical, histopathological, and cell biological approaches, recent progress in the genetic engineering of glycosyltransferase genes has resulted in novel findings and concepts about their roles in the nervous system. Roles of GSLs in the regulation of signaling that determine cell fates in membrane microdomains such as lipid rafts have been extensively studied. In particular, gene targeting of glycosyltransferases in mice has enabled investigation of the in vivo functions of GSLs. The majority of abnormal phenotypes exhibited by knockout (KO) mice may reflect an abnormal structure and a resultant altered function of lipid rafts caused by alterations in their GSL composition. Generally speaking, abnormal phenotypes found in most KO mice were milder than expected, suggesting that the remaining GSLs compensate for the functions of those lost. There are also functions that cannot be replaced by the remaining GSLs. Thus, there may be two modes of function of GSLs: one is nonspecific and can be carried out by multiple GSLs, the second mode is that in which the function of the missing GSL(s) cannot be compensated by others. Identification of natural ligands for individual GSLs is crucial in order to clarify the functions of each structure.
    Advances in neurobiology. 01/2014; 9:307-320.
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Ganglioside GD3 is highly expressed in human melanomas and enhances malignant properties of melanomas, such as cell proliferation and invasion activity. In this study, we analyzed the effects of GD3 expression on cell signals triggered by hepatocyte growth factor (HGF)/Met interaction and by adhesion to collagen type I (CL-I). Although stimulation of melanoma N1 cells (GD3+ and GD3-) with either HGF or adhesion to CL-I did not show marked differences in the phosphorylation levels of Akt at Ser473 and Thr308 between two types of cells, simultaneous treatment resulted in definite and markedly increased activation of Akt in GD3+ cells. Similar increases were also shown in Erk1/2 phosphorylation levels with the costimulation in GD3+ cells. When resistance to induced apoptosis by H2 O2 was examined, only GD3+ cells treated with both HGF and adhesion to CL-I showed clearly low percentages of dead cells compared with GD3- cells or GD3+ cells treated with either one of the stimulants. Cell growth measured by 5-ethynyl-2' deoxyuridine uptake also showed synergistic effects in GD3+ cells. These results suggested that GD3 plays a crucial role in the convergence of multiple signals, leading to the synergistic effects of those signals on malignant properties of melanomas.
    Cancer Science 10/2013; · 3.48 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Although regulatory mechanisms for immune cells with inhibitory signals via immunoreceptor tyrosine-based inhibitory motifs (ITIM) have been well known, signals transduced via interaction between Siglecs and sialyl-compounds on their counter receptors into target cells are not reported to date. In this study, we found that an astrocytoma cell line, AS showed detachment from culture plates when co-cultured with Siglec-9-expressing cells and/or soluble Siglec-9. Moreover, detached AS cells re-grew as co-cultured cells with Siglec-9-deficient cells. They also showed increased motility and invasiveness upon Siglec-9 binding. In immunoblotting, rapid degradation of focal adhesion kinase (FAK) and related signaling molecules such as Akt, paxillin and p130Cas was observed immediately after the co-culture. Despite of degradation of these molecules, increased p-Akt was found at the front region of cytoplasm, probably reflecting increased cell motility. Calpain was considered to be a responsible protease for the protein degradation by the inhibition experiments. These results suggest that protein degradation of FAK and related molecules was induced by Siglec-9 binding to its counter receptors via sialylglycoconjugates, leading to the modulation of adhesion kinetics of cancer cells. Thus, this might be a mechanism by which cancer cells utilize Siglec-9-derived signals to escape from immuno- surveillance.
    Journal of Biological Chemistry 10/2013; · 4.65 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The Siglecs are a family of I-type lectins that specifically recognize sialic acids. Siglec-9 (sialic acid-binding Ig-like lectin-9) (CD329) is one of the Siglecs. Although regulatory mechanisms for immune cells with inhibitory signals via ITIM are well known, signals transduced via interaction between Siglecs and their counter receptors in the target cells are not reported to date. Here, we found that an astrocytoma cell line, AS showed detachment from culture plates when co-cultured with Siglec-9-expressing cells and/or soluble Siglec-9. Moreover, detached AS re-grew more rapidly than AS co-cultured with Siglec-9-deficient cells. They also showed increased motility upon Siglec-9 binding. In immunoblotting, rapid degradation of FAK and related signaling molecules such as Akt and paxillin were observed immediately after the co-culture. Calpain was considered to be a responsible protease for the protein degradation using the inhibitor. Consequently, this fact might indicate that Siglec-9-mediated signaling in cancer cells confers cancer cells potency to well migrate and survive by escaping from immune surveillance as well as the suppression of immune cells via ITIM.
    第72回日本癌学会学術総会 The 72th annual meeting of the Japanese Cencer association, Yokohama, Japan; 10/2013
  • [Show abstract] [Hide abstract]
    ABSTRACT: Siglecs-9 (CD329) is a member of I-type lectins (immunoglobulin superfamily proteins that bind to sugars) expressed at high or intermediate levels in monocytes, neutrophils, and a minor population of CD16+, CD56- cells. It specifically recognizes sialic acid-containing glycoconjugates on cancer cells. Among past studies on Siglecs-9 mediated signaling, ITIM-associated inhibitory signals in immune cells have been studies. In this study, we examined the binding of soluble Siglec-9Fc on the cell surface of cancer cells, showing majority of tested cells expressed its ligands and the binding was really sialic acid dependent. Here, we demonstrate, for the first time, that Siglec-9 transduced signals for cell motility with degradation of FAK, Akt and related molecules in cancer cells via interaction with its ligands. We found that an astrocytoma cell line, AS detached from culture plates when co-cultured with Siglec-9-expressing U937 (monocyte) cells and/or soluble Siglec-9Fc. Moreover, we observed that the detached AS cells were rescued more rapidly when co-cultured with Siglec-9-expressing U937. As for rapid degradation of FAK and related signaling molecules observed after the co-culture, calpain was considered to be a responsible protease by the inhibition experiments. These results suggest that protein degradation of FAK and related molecules was induced by the interaction between Siglec-9 and its counter receptors via sialyl-glycoconjugates. Consequently, this fact might suggest that Siglec-9-mediated signaling confers cancer cells potency to well migrate and survive by escaping from immune surveillance as well as the suppression of immune cells via ITIM.
    第86回日本生化学会大会 The 86th annual meeting of the Japanese Biochemical Society, Yokohama, Japan; 09/2013
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Glycosphingolipids are expressed on the cell membrane, and act as important factors in various events that occur across the plasma membrane. Lactosylceramide is synthesized from glucosylceramide and is a common precursor of various glycosphingolipids existing in whole body. Based on the enzyme purification, β1,4-galactosyltransferase 6 (B4galt6) cDNA was isolated as a lactosylcaramide synthase-coding gene in rat brain. We generated B4galt6 gene knockout (KO) mice and analyzed their phenotypes to examine roles of β4GalT6. β4galt6 KO mice were born and grew up apparently normal. Lactosylceramide synthase activity and composition of acidic glycosphingolipids in brain were almost equivalent or minimally different between wild type and KO mice. Studies by mouse embryonic fibroblasts revealed that silencing of B4galt5 gene resulted in the marked reduction of lactosylceramide synthase activity and this reduction was more severe in mouse embryonic fibroblasts derived from β4galt6 KO mice than those from wild type mice. These results suggested that β4GalT6 plays a role as a lactosylceramide synthase, while β4GalT5 acts as a main enzyme for lactosylceramide biosynthesis in these tissues and cells.
    Glycobiology 07/2013; · 3.54 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: We previously demonstrated that ppGalNAc-T13 (T13), identified as an up-regulated gene with increased metastasis in DNA microarray, generated trimeric Tn (tTn) antigen (GalNAcα1-Ser/Thr)3 on Syndecan-1 in high metastatic sublines of Lewis lung cancer. However, it is not known how tTn antigen regulates cancer metastasis. Here, we analyzed roles of tTn antigen in cancer properties. tTn antigen on Syndecan-1 increased cell adhesion to fibronectin in an integrin-dependent manner. Furthermore, cell adhesion to fibronectin induced phosphorylation of focal adhesion kinase and paxillin in T13-transfectant cells. In the search of Syndecan-1-interacting molecules, it was demonstrated that tTn antigen-carrying Syndecan-1 interacted with integrin α5β1 and MMP-9, and these molecules shifted to glycolipid-enriched microdomain (GEM)/rafts along with increased metastatic potential in T13-transfectant cells. We also identified tTn-substitution site on Syndecan-1, demonstrating that tTn on Syndecan-1 is essential for the interaction with integrin α5β1 as well as for the reaction with mAb MLS128. These data suggested that high expression of ppGalNAc-T13 gene generated tTn antigen on Syndecan-1 under reduced expression of GM1, leading to the enhanced invasion and metastasis via the formation of a molecular complex consisting of integrin α5β1, Syndecan-1 and MMP-9 in GEM/rafts.
    Journal of Biological Chemistry 06/2013; · 4.65 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Although endogenous ligands for Toll-like receptor (TLR)4-myeloid differentiation factor 2 (MD2) have not been well-understood, we here report that a globo-series glycosphingolipid, globotetraosylceramide (Gb4), attenuates the toxicity of lipopolysaccharides (LPSs) by binding to TLR4-MD-2. Because α1,4-galactosyltransferase (A4galt)-deficient mice lacking globo-series glycosphingolipids showed higher sensitivity to LPS than wild-type mice, we examined mechanisms by which globo-series glycosphingolipids attenuate LPS toxicity. Cultured endothelial cells lacking A4galt showed higher expression of LPS-inducible genes upon LPS treatment. In turn, introduction of A4galt cDNA resulted in the neo expression of Gb4, leading to the reduced expression of LPS-inducible genes. Exogenous Gb4 induced similar effects. As a mechanism for the suppressive effects of Gb4 on LPS signals, specific binding of Gb4 to the LPS receptor TLR4-MD-2 was demonstrated by coprecipitation of Gb4 with recombinant MD-2 and by native PAGE. A docking model also supported these data. Taken together with colocalization of TLR4-MD-2 with Gb4 in lipid rafts after LPS stimulation, it was suggested that Gb4 competes with LPS for binding to TLR4-MD-2. Finally, administration of Gb4 significantly protected mice from LPS-elicited mortality. These results suggest that Gb4 is an endogenous ligand for TLR4-MD-2 and is capable of attenuating LPS toxicity, indicating the possibility for its therapeutic application in endotoxin shock.
    Proceedings of the National Academy of Sciences 03/2013; · 9.81 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Expression and implication of carbohydrate antigens in squamous cell carcinomas (SCCs) in oral cavity was examined. In the cell lines, type 2H and Lewis y antigens were markedly expressed. In the tissues from SCC patients and benign disorders, type 2H was highly expressed in hyperplasia (96.4 %), displasia (92.9 %) and SCC (100 %). Lewis y was, in turn, expressed mainly in cancer tissues (91.3 %), suggesting that Lewis y is a cancer-associated antigen. Normal oral mucosa showed no expression of these blood group antigens. Surprisingly, Lewis y antigen disappeared in the invasion sites where Ki-67 was definitely stained. Over-expression of Lewis y with manipulation of a fucosyltransferase cDNA resulted in suppression of cell growth and invasion, and knockdown of Lewis y also brought about increased cell growth and invasion. In either situations, no changes in the expression of sialyl-Lewis x could be found. Lowered tumor growth and invasion into surrounding tissues were also shown in Lewis y-positive SCC grafts in nu/nu mice. All these results together with alternative staining between Lewis y and Ki-67 in cancer tissues and FUT1 transfectants suggested that loss of Lewis y is a crucial event for the late stage of SCCs.
    Glycoconjugate Journal 12/2012; · 1.88 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Ganglioside GD3 is specifically expressed in human melanomas, and plays a role in the enhancement of malignant phenotypes of melanoma cells. To analyze the mechanisms by which GD3 enhances malignant properties and signals in melanomas, it is essential to clarify how GD3 interacts with membrane molecules on the cell membrane. In this study, we performed proteomics analysis of glycolipid-enriched microdomains (GEM) with current sucrose density gradient ultracentrifugation of Triton X-100 extracts and MS. We also examined GD3-associated molecules using enzyme-mediated activation of radical sources (EMARS) reaction combined with MS. Comparison of molecules identified as residents in GEM/rafts and those detected by EMARS reaction using an anti-GD3 antibody revealed that a relatively low number of molecules is recruited around GD3, while a number of membrane and secreted molecules was defined in GEM/rafts. These results suggested that EMARS reaction is useful to identify actually interacting molecules with gangliosides such as GD3 on the cell membrane, and many other microdomains than GD3-associating rafts exist. Representative examples of GD3-associated molecules such as neogenin and MCAM were shown.
    Proteomics 08/2012; · 4.43 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Clostridium perfringens alpha-toxin is the major virulence factor in the pathogenesis of gas gangrene. Alpha-toxin is a 43-kDa protein with two structural domains; the N-domain contains the catalytic site and coordinates the divalent metal ions, and the C-domain is a membrane-binding site. The role of the exposed loop region (72-93 residues) in the N-domain, however, has been unclear. Here we show that this loop contains a ganglioside binding motif (H … SXWY … G) that is the same motif seen in botulinum neurotoxin and directly binds to a specific conformation of the ganglioside Neu5Acα2-3(Galβ1-3GalNAcβ1-4)Galβ1-4Glcβ1Cer (GM1a) through a carbohydrate moiety. Confocal microscopy analysis using fluorescently labeled BODIPY-GM1a revealed that the toxin colocalized with GM1a and induced clustering of GM1a on the cell membranes. Alpha-toxin was only slightly toxic in β1,4-N-acetylgalactosaminyltransferase knock-out mice, which lack the a-series gangliosides that contain GM1a, but was highly toxic in α2,8-sialyltransferase knock-out mice, which lack both b-series and c-series gangliosides, similar to the control mice. Moreover, experiments with site-directed mutants indicated that Trp-84 and Tyr-85 in the exposed alpha-toxin loop play an important role in the interaction with GM1a and subsequent activation of TrkA. These results suggest that binding of alpha-toxin to GM1a facilitates the activation of the TrkA receptor and induces a signal transduction cascade that promotes the release of chemokines. Therefore, we conclude that GM1a is the primary cellular receptor for alpha-toxin, which can be a potential target for drug developed against this pathogen.
    Journal of Biological Chemistry 07/2012; 287(39):33070-9. · 4.65 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Sialic acid-containing glycosphingolipids, gangliosides are highly expressed in human cancer cells and regulate cell signals transduced via membrane microdomains. Generally, disialyl gangliosides enhance tumor phenotypes, while monosialyl gangliosides suppress them. In particular, gangliosides GD3 and GD2 are highly expressed in melanomas and small cell lung cancer cells, and their expression cause increased cell growth and invasion. In osteosarcomas, expression of GD3 and GD2 also enhanced cell invasion and motility, and caused increased phosphorylation of focal adhesion kinase and paxillin. In addition to focal adhesion kinase, Lyn kinase was also activated by GD3/GD2 expression, leading to the phosphorylation of paxillin. In contrast with melanoma cells, osteosarcomas showed reduced cell adhesion with increased phosphorylation of paxillin. Thus, increased expression of GD3/GD2 caused enhanced activation of signaling molecules, leading to distinct phenotypes between melanomas and osteosarcomas, i.e. increased and decreased adhesion activity. Thus, whole features of glycolipid-enriched microdomain/rafts formed in the individual cancer types seem to determine the main signaling pathway and biological outcome.
    Glycoconjugate Journal 07/2012; 29(8-9):579-84. · 1.88 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The expression and implications of gangliosides in human osteosarcomas have not been systematically analyzed. In this study, we showed that gangliosides GD3 and GD2 are highly expressed in the majority of human osteosarcoma cell lines derived from oral cavity regions. Introduction of GD3 synthase cDNA into a GD3/GD2-negative (GD3/GD2-) human osteosarcoma subline resulted in the establishment of GD3/GD2+ transfectant cells. They showed increased cell migration and invasion activities in wound healing and Boyden chamber invasion assays, respectively, compared to the control cells. When treated with serum, GD3/GD2+ cells showed stronger tyrosine phosphorylation of p130Cas, focal adhesion kinase, and paxillin than GD3/GD2- cells. In particular, paxillin underwent much stronger phosphorylation, suggesting its role in cell motility. Furthermore, we tried to dissect the roles of GD3 and GD2 in the malignant properties of the transfectant cells by establishing single ganglioside-expressing cells, that is, either GD3 or GD2. Although GD3/GD2+ cells showed the most malignant properties, GD2+ cells showed almost equivalent levels to GD3/GD2+ cells in invasion and migration activities, and in the intensities of tyrosine phosphorylation of paxillin. Among Src family kinases, Lyn was expressed predominantly, and was involved in the invasion and motility of GD3- and/or GD2-expressing transfectants. Furthermore, it was elucidated by gene silencing that Lyn was located in a different pathway from that of FAK to eventually lead paxillin activation. These results suggested that GD2/GD3 are responsible for the enhancement of the malignant features of osteosarcomas, and might be candidate targets in molecular-targeted therapy.
    Cancer Science 05/2012; 103(9):1656-64. · 3.48 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Carbohydrates on the glycoproteins and glycosphingolipids expressed on the cell surface membrane play crucial roles in the determination of cell fates by being involved in the fine tuning of cell signalling as reaction molecules in the front line to various extrinsic stimulants. In glycoproteins, modification of proteins is performed by substitution of sugar chains to one or multiple sites of individual proteins, leading to quantitative and qualitative changes of receptor functions in the cell membrane. As for glycosphingolipids, majority of them consist of two moieties, i.e. carbohydrates and ceramides, and are localized in the microdomains such as lipid rafts or detergent-resistant microdomains. They generate and/or modulate cell signals to determine the cell fates by interacting with various carbohydrate-recognizing proteins. Modes of glycosylation and mechanisms by which glycosylation is involved in the regulation of cell signals are now hot subjects in glycobiology.
    Journal of Biochemistry 05/2012; 151(6):573-8. · 3.07 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Gangliosides are considered to be involved in the maintenance and repair of nervous tissues. Recently, novel roles of gangliosides in the regulation of complement system were reported. Here we summarized roles of gangliosides in the formation and maintenance of membrane microdomains in brain tissues by comparing complement activation, inflammatory reaction and disruption of glycolipid-enriched microdomain (GEM)/rafts among several mutant mice of ganglioside synthases. Depending on the defects in ganglioside compositions, corresponding up-regulation of complement-related genes, proliferation of astrocytes and infiltration of microglia were found with gradual severity. Immunoblotting of fractions separated by sucrose density gradient ultracentrifugation revealed that DAF and NCAM having GPI-anchors tended to disappear from the raft fraction with intensities of DKO > GM2/GD2 synthase KO > GD3 synthase KO > WT. The lipid raft markers tended to disperse from the raft fractions with similar intensities. Phospholipids and cholesterol also tended to decrease in GEM/rafts in GM2/GD2 synthase KO and DKO, although total amounts were almost equivalent. All these results indicate that GEM/rafts architecture is destroyed by ganglioside deficiency with gradual intensity depending on the degree of defects of their compositions. Implication of inflammation caused by deficiency of gangliosides in various neurodegenerative diseases was discussed.
    Neurochemical Research 04/2012; 37(6):1185-91. · 2.13 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: In order to analyze the mechanisms for cancer metastasis, high metastatic sublines (H7-A, H7-Lu, H7-O, C4-sc, and C4-ly) were obtained by repeated injection of mouse Lewis lung cancer sublines H7 and C4 into C57BL/6 mice. These sublines exhibited increased proliferation and invasion activity in vitro. Ganglioside profiles exhibited lower expression of GM1 in high metastatic sublines than the parent lines. Then, we established GM1-Si-1 and GM1-Si-2 by stable silencing of GM1 synthase in H7 cells. These GM1-knockdown clones exhibited increased proliferation and invasion. Then, we explored genes that markedly altered in the expression levels by DNA microarray in the combination of C4 vs. C4-ly or H7 vs. H7 (GM1-Si). Consequently, pp-GalNAc-T13 gene was identified as up-regulated genes in the high metastatic sublines. Stable transfection of pp-GalNAc-T13 cDNA into C4 (T13-TF) resulted in increased invasion and motility. Then, immunoblotting and flow cytometry using various antibodies and lectins were performed. Only anti-trimeric Tn antibody (mAb MLS128), showed increased expression levels of trimeric Tn antigen in T13-TF clones. Moreover, immunoprecipitation/immunoblotting was performed by mAb MLS128, leading to the identification of an 80 kDa band carrying trimeric Tn antigen, i.e. Syndecan-1. Stable silencing of endogenous pp-GalNAc-T13 in C4-sc (T13-KD) revealed that primary tumors generated by subcutaneous injection of T13-KD clones showed lower coalescence to fascia and peritoneum, and significantly reduced lung metastasis than control clones. These data suggested that high expression of pp-GalNAc-T13 gene generated trimeric Tn antigen on Syndecan-1, leading to the enhanced metastasis.
    Biochemical and Biophysical Research Communications 03/2012; 419(1):7-13. · 2.28 Impact Factor

Publication Stats

4k Citations
878.07 Total Impact Points


  • 2008–2014
    • Chubu University
      Касугай, Aichi, Japan
  • 1998–2012
    • Nagoya University
      • • Graduate School of Medicine
      • • Graduate School of Bio-Agricultural Sciences
      Nagoya-shi, Aichi-ken, Japan
    • Nagoya Second Red Cross Hospital
      Nagoya, Aichi, Japan
  • 2009
    • University of Glasgow
      • BHF Glasgow Cardiovascular Research Centre
      Glasgow, SCT, United Kingdom
    • Pacific Northwest Diabetes Research Institute
      Seattle, Washington, United States
  • 2005
    • National Institute of Health Sciences, Japan
      • Division of Biomedical Food Research
      Edo, Tōkyō, Japan
  • 2002
    • Leiden University Medical Centre
      • Department of Neurology
      Leiden, South Holland, Netherlands
  • 1992–2002
    • Nagasaki University
      • • Department of Pediatrics
      • • Department of Oral and Maxillofacial Surgery
      • • School of Dentistry
      • • School of Medicine
      • • Department of Periodontology
      Nagasaki-shi, Nagasaki-ken, Japan
  • 2001
    • Johns Hopkins University
      Baltimore, Maryland, United States
    • Japanese Red Cross
      Edo, Tōkyō, Japan
  • 1992–2000
    • Nagasaki University Hospital
      Nagasaki, Nagasaki, Japan
  • 1999
    • National Institute of Infectious Diseases, Tokyo
      Edo, Tōkyō, Japan
  • 1986–1998
    • Memorial Sloan-Kettering Cancer Center
      • Department of Medicine
      New York City, New York, United States
    • Aichi Cancer Center
      Ōsaka, Ōsaka, Japan
  • 1997
    • Mie University
      Tu, Mie, Japan
  • 1994
    • Kyoto University
      Kioto, Kyōto, Japan
  • 1988
    • Tokyo Metropolitan Institute of Medical Science
      Edo, Tōkyō, Japan