[Show description][Hide description] DESCRIPTION: 2014 - Honolulu, HI - Joint Meeting of the Society for Glycobiology and the Japanese Society for Carbohydrate Research Poster:
Modulation of malignant properties of cancer cells by binding of a sialic acid-recognizing lectin Siglec-9 via calpain-mediated degradation of focal adhesion kinase and related proteins
[Show abstract][Hide abstract] ABSTRACT: Gangliosides are widely involved in the regulation of cells and organs. However, little is known about their roles in leptin secretion from adipose tissues. Genetic deletion of b-series gangliosides resulted in the marked reduction of serum leptin. Expression analysis of leptin revealed that leptin accumulated in the adipose tissues of GD3 synthase-knockout (GD3S KO) mice. Analysis of primary cultured stromal vascular fractions (SVF) derived from GD3S KO mice revealed that leptin secretion was reduced, although leptin amounts in cells were increased compared with those of wild type. Interestingly, addition of b-series gangliosides to the culture medium of differentiated SVF resulted in the restoration of leptin secretion. Results of methyl-β-cyclodextrin treatment of differentiated 3T3-L1 cells as well as immunocytostaining of leptin and caveolin-1 suggested that b-series gangliosides regulate the leptin secretion from adipose tissues in lipid rafts.
Biochemical and Biophysical Research Communications 02/2015; 459(2). DOI:10.1016/j.bbrc.2015.01.143 · 2.30 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Gangliosides, sialic acid-containing glycosphingolipids, have been considered to be involved in the development and functions of the nervous system. Recent progress in the genetic analysis of gangliosides in cultured cells and experimental animals revealed their roles in the maintenance of integrity of nervous tissues and neuroregeneration. The fact that ganglioside-deficient mice exhibited milder abnormalities than expected suggests that compensatory actions of remaining glycolipids might be present, and complex knockout of multiple glycosyltransferase genes might be useful to clarify essential roles of gangliosides.
[Show abstract][Hide abstract] ABSTRACT: Sialic acid-containing glycosphingolipids, gangliosides are expressed in various tissues and cells in our bodies. However, some relatively simple gangliosides are expressed in a tumor-specific manner in neuroectoderm-derived cancers and T-cell leukemias. They are also expressed in small cell lung cancers and osteosarcomas. Not only as tumor markers but as functional molecules on the cell surface membrane, they have been of interest, and indeed their roles in cancer cells have gradually been clarified. Recently, disialyl gangliosides and monosialyl gangliosides have been demonstrated to have opposite functions in the regulation of cancer properties. In particular, ganglioside GM1 showed suppressive effects on cell proliferation, invasion, and cancer metastasis in contrast with cancer-associated disialyl gangliosides such as GD3 and GD2. Based on the gene profiling with DNA array, it was demonstrated that the reduction of GM1 levels resulted in the increased expression of ppGalNAc-T13 and caused increased integrin functions, leading to enhanced metastatic potential of Lewis lung cancers. Trimeric Tn structure on syndecan-1 seems to be a key molecule to cause high metastasis. Both enhancing and suppressing actions of gangliosides on cancer properties have been shown to take place in membrane microdomains named lipid rafts. Therefore, regulatory functions of individual gangliosides in lipid rafts exerted by interacting with membrane molecules should be topics to be investigated now.
[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 08/2014; 9:307-320. DOI:10.1007/978-1-4939-1154-7_14
[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.
[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. DOI:10.1186/1742-2094-11-61 · 5.41 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; 445(2). DOI:10.1016/j.bbrc.2014.02.038 · 2.30 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.
The Journal of Neuroscience : The Official Journal of the Society for Neuroscience 01/2014; 34(3):880-91. DOI:10.1523/JNEUROSCI.3996-13.2014 · 6.34 Impact Factor
[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; 105(1). DOI:10.1111/cas.12310 · 3.52 Impact Factor
[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.
[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
[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.
[Show abstract][Hide abstract] ABSTRACT: We demonstrated previously that ppGalNAc-T13 (T13), identified as an up-regulated gene with increased metastasis in a DNA
microarray, generated trimeric Tn (tTn) antigen (GalNAcα1-Ser/Thr)3 on Syndecan 1 in highly metastatic sublines of Lewis lung cancer. However, it is not known how tTn antigen regulates cancer
metastasis. Here, we analyzed the 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 matrix metalloproteinase 9 and that these molecules
shifted to a glycolipid-enriched microdomain/rafts along with increased metastatic potential in T13-transfectant cells. We
also identified a 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 suggest that high expression of the ppGalNAc-T13 gene generates tTn antigen on Syndecan 1 under reduced expression of GM1, leading to enhanced invasion and metastasis via
the formation of a molecular complex consisting of integrin α5β1, Syndecan 1, and MMP-9 in the glycolipid-enriched microdomain/rafts.
[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; 110(12). DOI:10.1073/pnas.1218508110 · 9.67 Impact Factor
[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.
[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.