Linze XIA, Fumiya Kano, Noboru Hashimoto, Cheng DING, Yang XU, Hideharu HIBI, Tomonori Iwasaki, Eiji Tanaka and Akihito Yamamoto : Conditioned Medium from Stem Cells of Human Exfoliated Deciduous Teeth Partially Alters the Expression of Inflammation-associated Molecules of Mouse Condylar Chondrocytes via Secreted Frizzled-related Protein 1, Journal of Oral Health and Biosciences, Vol.35, No.2, 52-60, 2023.
Fumiya Kano, Noboru Hashimoto, Yao Liu, Linze Xia, Takaaki Nishihara, Wakana Oki, Keita Kawarabayashi, Noriko Mizusawa, Keiko Aota, Takayoshi Sakai, Masayuki Azuma, Hideharu Hibi, Tomonori Iwasaki, Tsutomu Iwamoto, Nobuyasu Horimai and Akihito Yamamoto : Therapeutic benefits of factors derived from stem cells from human exfoliated deciduous teeth for radiation-induced mouse xerostomia, Scientific Reports, Vol.13, No.1, 2706-2719, 2023.
(要約)
Radiation therapy for head and neck cancers is frequently associated with adverse effects on the surrounding normal tissue. Irreversible damage to radiation-sensitive acinar cells in the salivary gland (SG) causes severe radiation-induced xerostomia (RIX). Currently, there are no effective drugs for treating RIX. We investigated the efficacy of treatment with conditioned medium derived from stem cells from human exfoliated deciduous teeth (SHED-CM) in a mouse RIX model. Intravenous administration of SHED-CM, but not fibroblast-CM (Fibro-CM), prevented radiation-induced cutaneous ulcer formation (p < 0.0001) and maintained SG function (p < 0.0001). SHED-CM treatment enhanced the expression of multiple antioxidant genes in mouse RIX and human acinar cells and strongly suppressed radiation-induced oxidative stress. The therapeutic effects of SHED-CM were abolished by the superoxide dismutase inhibitor diethyldithiocarbamate (p < 0.0001). Notably, quantitative liquid chromatography-tandem mass spectrometry shotgun proteomics of SHED-CM and Fibro-CM identified eight proteins activating the endogenous antioxidant system, which were more abundant in SHED-CM than in Fibro-CM (p < 0.0001). Neutralizing antibodies against those activators reduced antioxidant activity of SHED-CM (anti-PDGF-D; p = 0.0001, anti-HGF; p = 0.003). Our results suggest that SHED-CM may provide substantial therapeutic benefits for RIX primarily through the activation of multiple antioxidant enzyme genes in the target tissue.
YAO LIU, Fumiya Kano, Noboru Hashimoto, LINZE XIA, Qiao Zhou, Xingmei Feng, Hideharu Hibi, Aya Miyazaki, Tsutomu Iwamoto, Yoshizo Matsuka, Zhijun Zhang, Eiji Tanaka and Akihito Yamamoto : Conditioned Medium From the Stem Cells of Human Exfoliated Deciduous Teeth Ameliorates Neuropathic Pain in a Partial Sciatic Nerve Ligation Model., Frontiers in Pharmacology, Vol.13, 745020, 2022.
(要約)
In neuropathic pain (NP), injury or diseases of the somatosensory system often result in highly debilitating chronic pain. Currently, there is no effective drug for the complete and definitive treatment of NP. We investigated the therapeutic potential of conditioned medium (CM) derived from stem cells from human exfoliated deciduous teeth (SHED-CM) against NP using a mouse partial sciatic nerve ligation (PSL) model. Abnormal pain sensation, such as tactile allodynia and hyperalgesia, can be caused by PSL. In the behavioral test, intravenous administration of SHED-CM greatly improved the PSL-induced hypersensitivity. We found that treatment with SHED-CM resulted in the recruitment of M2 macrophages in the injured sciatic nerve and ipsilateral L4/L5 dorsal root ganglion and suppressed microglial activation in the spinal cord. Notably, specific depletion of the anti-inflammatory M2 macrophages by mannosylated-Clodrosome markedly reduced the antinociceptive effect of SHED-CM. Intravenous administration of CM from M2 induced by SHED-CM (M2-CM) ameliorated the PSL-induced hypersensitivity. We found that M2-CM directly suppressed the expression of nociceptive receptors as well as proinflammatory mediators in Schwann cells. Taken together, our data suggest that SHED-CM ameliorates NP through the induction of the analgesic anti-inflammatory M2 macrophages. Thus, SHED-CM may be a novel therapeutic candidate for NP.
Immunotherapy of malignant cancers is now becoming one of representative approaches to overcome cancers. To construct strategies for immunotherapy, presence of tumor-specific antigens should be a major promise. A number of cancer specific- or cancer-associated antigens have been reported based on various experimental sets and various animal systems. The most reasonable strategy to define tumor-specific antigens might be "autologous typing" performed by Old's group, proposing three classes of tumor-antigens recognized by host immune systems of cancer patients. Namely, class 1, individual antigens that is present only in the patient's sample analyzed; class 2, shared antigens that can be found only in some group of cancers in some patients, but not in normal cells and tissues; class 3, universal antigens that are present in some cancers but also in normal cells and tissues with different densities. Sen Hakomori reported there were novel carbohydrates in cancers that could not be detected in normal cells mainly by biochemical approaches. Consequently, many of class 2 cancer-specific antigens have been revealed to be carbohydrate antigens, and been used for cancer diagnosis and treatment. Not only as cancer markers, but roles of those cancer-associated carbohydrates have also been recognized as functional molecules in cancer cells. In particular, roles of complex carbohydrates in the regulation of cell signaling on the cell surface microdomains, glycolipid-enriched microdomain (GEM)/rafts have been reported by Hakomori and many other researchers including us. The processes and present status of these studies on cancer-associated glycolipids were summarized.
Yuki Ohkawa, Pu Zhang, Hiroyuki Momota, Akira Kato, Noboru Hashimoto, Yuhsuke Ohmi, Robiul H. Bhuiyan, Yesmin Farhana, Atsushi Natsume, Toshihiko Wakabayashi, Keiko Furukawa and Koichi Furukawa : Lack of GD3 synthase (St8sia1) attenuates malignant properties of gliomas in genetically engineered mouse model., Cancer Science, 2021.
(要約)
High expression of gangliosides GD3 and GD2 is observed in human gliomas. The functions of GD3 and GD2 in malignant properties have been reported in glioma cells in vitro, but those functions have not yet been investigated in vivo. In this study, we showed that deficiency of GD3 synthase (GD3S, St8sia1) attenuated glioma progression and clinical and pathological features in a platelet-derived growth factor B-driven murine glioma model. Lack of GD3S resulted in the prolonged lifespan of glioma-bearing mice and low-grade pathology in generated gliomas. Correspondingly, they showed reduced phosphorylation levels of Akt, Erks, and Src family kinases in glioma tissues. A DNA microarray study revealed marked alteration in the expression of various genes, particularly in MMP family genes, in GD3S-deficient gliomas. Re-expression of GD3S restored expression of MMP9 in primary-cultured glioma cells. We also identified a transcription factor, Ap2α, expressed in parallel with GD3S expression, and showed that Ap2α was critical for the induction of MMP9 by transfection of its cDNA and luciferase reporter genes, and a ChIP assay. These findings suggest that GD3S enhances the progression of gliomas by enhancement of the Ap2α-MMP9 axis. This is the first report to describe the tumor-enhancing functions of GD3S in vivo.
Naoko Ogasawara, Fumiya Kano, Noboru Hashimoto, Hiroki Mori, YAO LIU, LINZE XIA, Takuma Sakamaki, Hideharu Hibi, Tsutomu Iwamoto, Eiji Tanaka and Akihito Yamamoto : Factors Secreted from Dental Pulp Stem Cells Show Multifaceted Benefits for Treating Experimental Temporomandibular Joint Osteoarthritis, Osteoarthritis and Cartilage, Vol.28, No.6, 831-841, 2020.
(要約)
Temporomandibular joint osteoarthritis (TMJOA) is a degenerative disease characterized by progressive cartilage degeneration, abnormal bone remodeling, and chronic pain. In this study, we aimed to investigate effective therapies to reverse or suppress TMJOA progression. To this end, we performed intravenous administration of serum free conditioned media from human exfoliated deciduous teeth stem cells (SHED-CM) into a mechanical-stress induced murine TMJOA model. SHED-CM administration markedly suppressed temporal muscle inflammation, and improved bone integrity and surface smoothness of the destroyed condylar cartilage. Moreover, SHED-CM treatment decreased the number of IL-1β, iNOS, and MMP-13 expressing chondrocytes, whereas it specifically increased PCNA-positive cells in the multipotent polymorphic cell layer. Notably, the numbers of TUNEL-positive apoptotic chondrocytes in the SHED-CM treated condyles were significantly lower than in those treated with DMEM, whereas the proteoglycan positive area was restored to a level similar to that of the sham treated group, demonstrating that SHED-CM treatment regenerated the mechanical-stress injured condylar cartilage and subchondral bone. Secretome analysis revealed that SHED-CM contained multiple therapeutic factors that act in osteochondral regeneration. Our data demonstrated that SHED-CM treatment promoted the regeneration and repair of mechanical-stress induced mouse TMJOA. Our observations suggest that SHED-CM has potential to be a potent tissue-regenerating therapeutic agent for patients with severe TMJOA.
Koichi Furukawa, Yuhsuke Ohmi, Farhana Yesmin, Orie Tajima, Yuji Kondo, Pu Zhang, Noboru Hashimoto, Yuki Ohkawa, H. Robiul Bhuiyan and Keiko Furukawa : Novel Molecular Mechanisms of Gangliosides in the Nervous System Elucidated by Genetic Engineering, International Journal of Molecular Sciences, Vol.21, No.6, 1906, 2020.
(要約)
Acidic glycosphingolipids, i.e., gangliosides, are predominantly and consistently expressed in nervous tissues of vertebrates at high levels. Therefore, they are considered to be involved in the development and function of nervous systems. Recent studies involving genetic engineering of glycosyltransferase genes have revealed novel aspects of the roles of gangliosides in the regulation of nervous tissues. In this review, novel findings regarding ganglioside functions and their modes of action elucidated mainly by studies of gene knockout mice are summarized. In particular, the roles of gangliosides in the regulation of lipid rafts to maintain the integrity of nervous systems are reported with a focus on the roles in the regulation of neuro-inflammation and neurodegeneration via complement systems. In addition, recent advances in studies of congenital neurological disorders due to genetic mutations of ganglioside synthase genes and also in the techniques for the analysis of ganglioside functions are introduced.
Noboru Hashimoto, Shizuka Ito, Akiko Tsuchida, H Robiul Bhuiyan, Tetsuya Okajima, Akihito Yamamoto, Keiko Furukawa, Yuhsuke Ohmi and Koichi Furukawa : The ceramide moiety of disialoganglioside (GD3) is essential for GD3 recognition by the sialic acid-binding lectin SIGLEC7 on the cell surface., The Journal of Biological Chemistry, Vol.294, No.28, 10833-10845, 2019.
(要約)
) gene, involved in phytoceramide synthesis, disclosing that DES2 inhibition confers SIGLEC7 binding. Furthermore, knocking out fatty acid 2-hydroxylase also resulted in the emergence of SIGLEC7 binding to the cell surface. To analyze the effects of binding between SIGLEC7 and various GD3 species on natural killer function, we investigated cytotoxicity of peripheral blood mononuclear cells from healthy donors toward GD3S-transfected DLD-1 (DLD-1-GD3S) cells and DLD-1-GD3S cells with modified ceramides. We found that cytotoxicity is suppressed in DLD-1-GD3S cells with dehydroxylated GD3s. These results indicate that the ceramide structures in glycosphingolipids affect SIGLEC7 binding and distribution on the cell surface and influence cell sensitivity to killing by SIGLEC7-expressing effector cells.
Robiul H. Bhuiyan, Yuhsuke Ohmi, Yuki Ohkawa, Pu Zhang, Maiko Takano, Noboru Hashimoto, Tetsuya Okajima, Keiko Furukawa and Koichi Furukawa : Loss of Enzyme Activity in Mutated B4GALNT1 Gene Products in Patients with Hereditary Spastic Paraplegia Results in Relatively Mild Neurological Disorders: Similarity with Phenotypes of B4galnt1 Knockout Mice., Neuroscience, Vol.397, 94-106, 2018.
(要約)
B4GALNT1 is an enzyme essential for the synthesis of complex gangliosides, whose absence leads to progressive neurodegeneration with aging in mice. Recently, eleven cases of hereditary spastic paraplegia with mutation in the coding region of B4GALNT1 were reported. However, changes in the enzymatic activity of their products have never been studied. We have constructed expression vectors for individual mutant cDNAs, and examined their activities by cell-free in vitro enzyme assays, and flow cytometry of cells transfected with their expression vectors. Among them, almost all mutant genes showed the complete loss of B4GALNT1 activity in both the in vitro enzyme assays and flow cytometry. Two mutants exceptionally showed weak activity. One of them, M4, had a mutation at amino acid 228 with a premature termination codon. Interestingly, the intensity of fluorescence of GM2 measured by flow cytometry was equivalent between the WT and M4 mutant, although the positive cell population was relatively small in M4. Western immunoblotting of cell lysates from transfectants with cDNA plasmids revealed 67-kDa bands except those containing premature termination codons or frame-shift mutation. Taken together with the clinical findings of patients, loss of enzyme activity may be responsible for the clinical features of hereditary spastic paraplegia, whereas the intensity of neurological disorders was relatively milder than expected. These clinical features of patients including those with male hypogonadism are very similar to the abnormal phenotypes detected in B4galnt1-deficient mice.
Koichi Furukawa, Yuhsuke Ohmi, Orie Tajima, Yuki Ohkawa, Yuji Kondo, Ji Shuting, Noboru Hashimoto and Keiko Furukawa : Gangliosides in Inflammation and Neurodegeneration., Progress in Molecular Biology and Translational Science, Vol.156, 265-287, 2018.
(要約)
Gangliosides play roles in the regulation of cell signaling that are mediated via membrane microdomains, lipid rafts. In this review, functions of gangliosides in the maintenance of nervous systems with a focus on regulation of inflammation and neurodegeneration are addressed. During analyses of various ganglioside-lacking mutant mice, we demonstrated that nervous tissues exhibited inflammatory reactions and subsequent neurodegeneration. Among inflammation-related genes, factors of the complement system showed up-regulation with aging. Analyses of architectures and compositions of lipid rafts in nervous tissues from these mutant mice revealed that dysfunctions of complement regulatory proteins based on disrupted lipid rafts were main factors to induce the inflammatory reactions resulting in neurodegeneration. Ganglioside changes in development and senescence, and implication of them in the integrity of cell membranes and cellular phenotypes in physiological and pathological conditions including Alzheimer disease have been summarized. Novel directions to further analyze mechanisms for ganglioside functions in membrane microdomains have been also addressed.
Nobutoshi Esaki, Yuki Ohkawa, Noboru Hashimoto, Yuhsuke Tsuda, Yuhsuke Ohmi, Robiul H. Bhuiyan, Norihiro Kotani, Koichi Honke, Atsushi Enomoto, Masahide Takahashi, Keiko Furukawa and Koichi Furukawa : ASC amino acid transporter 2, defined by enzyme-mediated activation of radical sources, enhances malignancy of GD2-positive small-cell lung cancer., Cancer Science, Vol.109, No.1, 141-153, 2018.
(要約)
Ganglioside GD2 is specifically expressed in small-cell lung cancer (SCLC) cells, leading to enhancement of malignant phenotypes, such as cell proliferation and migration. However, how GD2 promotes malignant phenotypes in SCLC cells is not well known. In this study, to reveal the mechanisms by which GD2 increases malignant phenotypes in SCLC cells, we used enzyme-mediated activation of radical sources combined with mass spectrometry in GD2 SCLC cells. Consequently, we identified ASC amino acid transporter 2 (ASCT2), a major glutamine transporter, which coordinately works with GD2. We showed that ASCT2 was highly expressed in glycolipid-enriched microdomain/rafts in GD2 SCLC cells, and colocalized with GD2 in both proximity ligation assay and immunocytostaining, and bound with GD2 in immunoprecipitation/TLC immunostaining. Malignant phenotypes of GD2 SCLC cells were enhanced by glutamine uptake, and were suppressed by L-γ-glutamyl-p-nitroanilide, a specific inhibitor of ASCT2, through reduced phosphorylation of p70 S6K1 and S6. These results suggested that ASCT2 enhances glutamine uptake in glycolipid-enriched microdomain/rafts in GD2 SCLC cells, leading to the enhancement of cell proliferation and migration through increased phosphorylation of the mTOR complex 1 signaling axis.
(キーワード)
Amino Acid Transport System ASC / Cell Line, Tumor / Cell Movement / Cell Proliferation / Gangliosides / Glutamine / Humans / Lung Neoplasms / Membrane Microdomains / Minor Histocompatibility Antigens / Small Cell Lung Carcinoma
Koichi Furukawa, Yuhsuke Ohmi, Shuting Ji, Pu Zhang, Robiul H. Bhuiyan, Yuki Ohkawa, Orie Tajima, Noboru Hashimoto and Keiko Furukawa : Glycolipids: Essential regulator of neuro-inflammation, metabolism and gliomagenesis., Biochimica et Biophysica Acta (BBA) - General Subjects, Vol.1861, No.10, 2479-2484, 2017.
(要約)
Gene knockout mice of glycosyltransferases have clearly showed roles of their products in the bodies, while there are examples where phenotype of knockout was much less severe than expected probably due to functional redundancy. The most striking novel finding obtained from ganglioside-deficient mice was that progressive inflammatory reaction took place, leading to neurodegeneration. In particular, dysfunction of complement-regulatory proteins due to deteriorated architecture of lipid rafts seemed to be essential mechanisms for the inflammation. Furthermore, roles of gangliosides in neurons were demonstrated by neuron-specific transgenic of B4galnt1 with genetic background of B4galnt1 deficiency. From study of gene knockout mice of St8sia1, new roles of b-series gangliosides in leptin secretion from adipocytes, and roles of a-series gangliosides in leptin receptor, ObR in hypothalamus were demonstrated, leading to apparent intact balance of energy. Essential roles of b-series gangliosides in malignant properties of gliomas were also shown, suggesting their roles in the regulation of inflammation and proliferation in nervous tissues. How to apply these findings for the control of newly discovered patients with ganglioside deficiency remains to be investigated. This article is part of a Special Issue entitled Neuro-glycoscience, edited by Kenji Kadomatsu and Hiroshi Kitagawa.
Kei Kaneko, Yuki Ohkawa, Noboru Hashimoto, Yuhsuke Ohmi, Norihiro Kotani, Koichi Honke, Mitsutaka Ogawa, Tetsuya Okajima, Keiko Furukawa and Koichi Furukawa : Neogenin, Defined as a GD3-associated Molecule by Enzyme-mediated Activation of Radical Sources, Confers Malignant Properties via Intracytoplasmic Domain in Melanoma Cells., The Journal of Biological Chemistry, Vol.291, No.32, 16630-16643, 2016.
(要約)
To investigate mechanisms for increased malignant properties in malignant melanomas by ganglioside GD3, enzyme-mediated activation of radical sources and subsequent mass spectrometry were performed using an anti-GD3 antibody and GD3-positive (GD3+) and GD3-negative (GD3-) melanoma cell lines. Neogenin, defined as a GD3-neighbored molecule, was largely localized in lipid/rafts in GD3+ cells. Silencing of neogenin resulted in the reduction of cell growth and invasion activity. Physical association between GD3 and neogenin was demonstrated by immunoblotting of the immunoprecipitates with anti-neogenin antibody from GD3+ cell lysates. The intracytoplasmic domain of neogenin (Ne-ICD) was detected in GD3+ cells at higher levels than in GD3- cells when cells were treated by a proteasome inhibitor but not when simultaneously treated with a γ-secretase inhibitor. Exogenous GD3 also induced increased Ne-ICD in GD3- cells. Overexpression of Ne-ICD in GD3- cells resulted in the increased cell growth and invasion activity, suggesting that Ne-ICD plays a role as a transcriptional factor to drive malignant properties of melanomas after cleavage with γ-secretase. γ-Secretase was found in lipid/rafts in GD3+ cells. Accordingly, immunocyto-staining revealed that GD3, neogenin, and γ-secretase were co-localized at the leading edge of GD3+ cells. All these results suggested that GD3 recruits γ-secretase to lipid/rafts, allowing efficient cleavage of neogenin. ChIP-sequencing was performed to identify candidates of target genes of Ne-ICD. Some of them actually showed increased expression after expression of Ne-ICD, probably exerting malignant phenotypes of melanomas under GD3 expression.
Hasan Robiul Bhuiyan, Yuji Kondo, Tokiaki Yamaguchi, Noriyo Tokuda, Yuki Ohkawa, Noboru Hashimoto, Yuhsuke Ohmi, Yoshio Yamauchi, Keiko Furukawa, Tetsuya Okajima and Koichi Furukawa : Expression analysis of 0-series gangliosides in human cancer cell lines with monoclonal antibodies generated using knockout mice of ganglioside synthase genes., Glycobiology, Vol.26, No.9, 984-998, 2016.
(要約)
Some gangliosides, sialic acid-containing glycosphingolipids, have been considered as tumor-associated antigens. GD1α or a GD1α synthase gene ST6GalNAc5 was reported to be involved in the metastasis of murine lymphomas or human breast cancers, respectively. But expression patterns of 0-series gangliosides GD1α and its precursor GM1b in human cancers have not yet been investigated mainly due to lack of specific antibodies. We established specific monoclonal antibodies (mAbs) reactive with GD1α or GM1b using gangliosides from brain tissues of GM3 synthase (St3gal5)-deficient mice as immunogens. We used GM2/GD2 synthase (B4galnt1)-deficient mice to immunize by liposomes embedded with GD1α or acidic glycolipid fractions from brain of St3gal5-deficient mice. Specificities of established mAbs as analyzed by enzyme-linked immunosorbent assay and thin-layer chromatography-immunostaining were very high among various gangliosides. Increased expression of GD1α and reduced GM1b in the St6galnac5 cDNA-transfected RAW117 cell line also substantiated the specificities of two mAbs. Then, we analyzed expression of GD1α and GM1b, and of relevant glycosyltransferase genes in various human cancer cell lines using generated anti-GD1α mAb 122 or anti-GM1b mAb MR155A-7. A few human cancer cell lines showed significant expression of these gangliosides with reasonable expression of relevant glycosyltransferase genes.
Reiko Ando, Noriyo Tokuda, Tokunori Yamamoto, Kazutaka Ikeda, Noboru Hashimoto, Ryo Taguchi, Xiaoen Fan, Keiko Furukawa, Yukio Niimura, Akemi Suzuki, Momokazu Goto and Koichi Furukawa : Immunization of A4galt-deficient mice with glycosphingolipids from renal cell cancers resulted in the generation of anti-sulfoglycolipid monoclonal antibodies., Glycoconjugate Journal, Vol.33, No.2, 169-180, 2016.
(要約)
In this study, we immunized Gb3/CD77 synthase gene (A4galt) knockout (KO) mice with glycosphingolipids (GSLs) extracted from 3 renal cell cancer (RCC) cell lines to raise monoclonal antibodies (mAbs) reactive with globo-series GSLs specifically expressed in RCCs. Although a number of mAbs reactive with globo-series GSLs were generated, they reacted with both RCC cell lines and normal kidney cells. When we analyzed recognized antigens by mAbs that were specifically reactive with RCC, but not with normal kidney cells at least on the cell surface, many of them turned out to be reactive with sulfoglycolipids. Eight out of 11 RCC-specific mAbs were reactive with SM2 alone, and the other 3 mAbs were more broadly reactive with sulfated glycolipids, i.e. SM3 and SM4 as well as SM2. In the immunohistochemistry, these anti-sulfoglycolipids mAbs showed RCC-specific reaction, with no or minimal reaction with adjacent normal tissues. Thus, immunization of A4galt KO mice with RCC-derived GSLs resulted in the generation of anti sulfated GSL mAbs, and these mAbs may be applicable for the therapeutics for RCC patients.
Yuki Ohkawa, Hiroyuki Momota, Akira Kato, Noboru Hashimoto, Yusuke Tsuda, Norihiro Kotani, Koichi Honke, Akio Suzumura, Keiko Furukawa, Yuhsuke Ohmi, Atsushi Natsume, Toshihiko Wakabayashi and Koichi Furukawa : Ganglioside GD3 Enhances Invasiveness of Gliomas by Forming a Complex with Platelet-derived Growth Factor Receptor α and Yes Kinase., The Journal of Biological Chemistry, Vol.290, No.26, 16043-16058, 2015.
(要約)
There have been a few studies on the ganglioside expression in human glioma tissues. However, the role of these gangliosides such as GD3 and GD2 has not been well understood. In this study we employed a genetically engineered mouse model of glioma to clarify the functions of GD3 in gliomas. Forced expression of platelet-derived growth factor B in cultured astrocytes derived from p53-deficient mice resulted in the expression of GD3 and GD2. GD3-positive astrocytes exhibited increased cell growth and invasion activities along with elevated phosphorylation of Akt and Yes kinase. By enzyme-mediated activation of radical sources reaction and mass spectrometry, we identified PDGF receptor α (PDGFRα) as a GD3-associated molecule. GD3-positive astrocytes showed a significant amount of PDGFRα in glycolipid-enriched microdomains/rafts compared with GD3-negative cells. Src kinase family Yes was co-precipitated with PDGFRα, and its pivotal role in the increased cell invasion of GD3-positive astrocytes was demonstrated by silencing with anti-Yes siRNA. Direct association between PDGFRα and GD3 was also shown, suggesting that GD3 forms ternary complex with PDGFRα and Yes. The fact that GD3, PDGFRα, and activated Yes were colocalized in lamellipodia and the edge of tumors in cultured cells and glioma tissues, respectively, suggests that GD3 induced by platelet-derived growth factor B enhances PDGF signals in glycolipid-enriched microdomain/rafts, leading to the promotion of malignant phenotypes such as cell proliferation and invasion in gliomas.
Kohki Matsubara, Yoshihiro Matsushita, Kiyoshi Sakai, Fumiya Kano, Megumi Kondo, Mariko Noda, Noboru Hashimoto, Shiro Imagama, Naoki Ishiguro, Akio Suzumura, Minoru Ueda, Koichi Furukawa and Akihito Yamamoto : Secreted ectodomain of sialic acid-binding Ig-like lectin-9 and monocyte chemoattractant protein-1 promote recovery after rat spinal cord injury by altering macrophage polarity., The Journal of Neuroscience, Vol.35, No.6, 2452-2464, 2015.
(要約)
Engrafted mesenchymal stem cells from human deciduous dental pulp (SHEDs) support recovery from neural insults via paracrine mechanisms that are poorly understood. Here we show that the conditioned serum-free medium (CM) from SHEDs, administered intrathecally into rat injured spinal cord during the acute postinjury period, caused remarkable functional recovery. The ability of SHED-CM to induce recovery was associated with an immunoregulatory activity that induced anti-inflammatory M2-like macrophages. Secretome analysis of the SHED-CM revealed a previously unrecognized set of inducers for anti-inflammatory M2-like macrophages: monocyte chemoattractant protein-1 (MCP-1) and the secreted ectodomain of sialic acid-binding Ig-like lectin-9 (ED-Siglec-9). Depleting MCP-1 and ED-Siglec-9 from the SHED-CM prominently reduced its ability to induce M2-like macrophages and to promote functional recovery after spinal cord injury (SCI). The combination of MCP-1 and ED-Siglec-9 synergistically promoted the M2-like differentiation of bone marrow-derived macrophages in vitro, and this effect was abolished by a selective antagonist for CC chemokine receptor 2 (CCR2) or by the genetic knock-out of CCR2. Furthermore, MCP-1 and ED-Siglec-9 administration into the injured spinal cord induced M2-like macrophages and led to a marked recovery of hindlimb locomotor function after SCI. The inhibition of this M2 induction through the inactivation of CCR2 function abolished the therapeutic effects of both SHED-CM and MCP-1/ED-Siglec-9. Macrophages activated by MCP-1 and ED-Siglec-9 extended neurite and suppressed apoptosis of primary cerebellar granule neurons against the neurotoxic effects of chondroitin sulfate proteoglycans. Our data suggest that the unique combination of MCP-1 and ED-Siglec-9 repairs the SCI through anti-inflammatory M2-like macrophage induction.
Ilhamjan Sabit, Noboru Hashimoto, Yasuyuki Matsumoto, Toshiyuki Yamaji, Keiko Furukawa and Koichi Furukawa : Binding of a sialic acid-recognizing lectin Siglec-9 modulates adhesion dynamics of cancer cells via calpain-mediated protein degradation., The Journal of Biological Chemistry, Vol.288, No.49, 35417-35427, 2013.
(要約)
Although regulatory mechanisms for immune cells with inhibitory signals via immunoreceptor tyrosine-based inhibitory motifs are well known, signals transduced via interaction between Siglecs and sialyl compounds on their counterreceptors into target cells have not been 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 regrew 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 degradation of these molecules, increased p-Akt was found at the front region of the 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 counterreceptors 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 immunosurveillance.
Noriyo Tokuda, Shinichiro Numata, Xiaojin Li, Tomoko Nomura, Minoru Takizawa, Yuji Kondo, Yoriko Yamashita, Noboru Hashimoto, Tohru Kiyono, Takeshi Urano, Keiko Furukawa and Koichi Furukawa : β4GalT6 is involved in the synthesis of lactosylceramide with less intensity than β4GalT5., Glycobiology, Vol.23, No.10, 1175-1183, 2013.
(要約)
Glycosphingolipids are expressed on the cell membrane and act as important factors in various events that occur across the plasma membrane. Lactosylceramide (LacCer) 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 LacCer synthase-coding gene in the rat brain. We generated B4galt6 gene knockout (KO) mice and analyzed their phenotypes to examine roles of β4GalT6. B4galt6 KO mice were born and grew up apparently normal. LacCer synthase activity and the composition of acidic glycosphingolipids in the brain were almost equivalent or minimally different between wild-type and KO mice. Studies by mouse embryonic fibroblasts (MEFs) revealed that the silencing of B4galt5 gene resulted in the marked reduction in LacCer synthase activity and this reduction was more severe in MEFs derived from B4galt6 KO mice than those from wild-type mice. These results suggested that β4GalT6 plays a role as a LacCer synthase, whereas β4GalT5 acts as a main enzyme for LacCer biosynthesis in these tissues and cells.
Hiroshi Hotta, Kazunori Hamamura, Kyoko Yamashita, Hidenobu Shibuya, Noriyo Tokuda, Noboru Hashimoto, Keiko Furukawa, Noriyuki Yamamoto, Hisashi Hattori, Shinya Toyokuni, Minoru Ueda and Koichi Furukawa : Lewis y antigen is expressed in oral squamous cell carcinoma cell lines and tissues, but disappears in the invasive regions leading to the enhanced malignant properties irrespective of sialyl-Lewis x., Glycoconjugate Journal, Vol.30, No.6, 585-597, 2012.
(要約)
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.
(キーワード)
Animals / Carcinoma, Squamous Cell / Cell Line, Tumor / Fucosyltransferases / Gene Expression Regulation, Neoplastic / Humans / Ki-67 Antigen / Lewis Blood Group Antigens / Mice, Inbred BALB C / Mouth Mucosa / Mouth Neoplasms / Neoplasm Invasiveness / Neoplasm Transplantation / Oligosaccharides / Organ Specificity / Sialyl Lewis X Antigen
Maiko Kurosawa, D Anand Jeyasekharan, Eva-Maria Surmann, Noboru Hashimoto, Vignesh Venkatraman, Gene Kurosawa, Koichi Furukawa, R Ashok Venkitaraman and Yoshikazu Kurosawa : Expression of LY6D is induced at the surface of MCF10A cells by X-ray irradiation., The FEBS Journal, Vol.279, No.24, 4479-4491, 2012.
(要約)
In order to identify membrane proteins whose expression is induced by X-ray irradiation, we developed an antibody (Ab)-directed strategy using a phage Ab library. X-Ray-irradiated cells were screened with a phage Ab library in the presence of a large excess of polyclonal Abs prepared against membrane proteins that are commonly present at the surface of both X-ray-irradiated and nonirradiated cells. After isolation of Ab that bound only to X-ray-irradiated cells, the antigen was identified using MS. Using this approach, we found that expression of LY6D is induced in MCF10A cells by X-ray irradiation. The induction of LY6D expression is triggered through a pathway regulated by ATM, CHK2 and p53. This method is a new Ab-directed proteomic strategy for analysis of membrane proteins, and is applicable to various biological phenomena in situations in which both target molecule-expressing cells and nonexpressing cells are available.
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.
Kazunori Hamamura, Momoko Tsuji, Hiroshi Hotta, Yuki Ohkawa, Masataka Takahashi, Hidenobu Shibuya, Hideyuki Nakashima, Yoshio Yamauchi, Noboru Hashimoto, Hisashi Hattori, Minoru Ueda, Keiko Furukawa and Koichi Furukawa : Functional activation of Src family kinase yes protein is essential for the enhanced malignant properties of human melanoma cells expressing ganglioside GD3., The Journal of Biological Chemistry, Vol.286, No.21, 18526-18537, 2011.
(要約)
The possible roles of Src family kinases in the enhanced malignant properties of melanomas related to GD3 expression were analyzed. Among Src family kinases only Yes, not Fyn or Src, was functionally involved in the increased cell proliferation and invasion of GD3-expressing transfectant cells (GD3+). Yes was located upstream of p130Cas and paxillin and at an equivalent level to focal adhesion kinase. Yes underwent autophosphorylation even before serum treatment and showed stronger kinase activity in GD3+ cells than in GD3- cells following serum treatment. Coimmunoprecipitation experiments revealed that Yes bound to focal adhesion kinase or p130Cas more strongly in GD3+ cells than in GD3- cells. As a possible mechanism for the enhancing effects of GD3 on cellular phenotypes, it was shown that majority of Yes was localized in glycolipid-enriched microdomain/rafts in GD3+ cells even before serum treatment, whereas it was scarcely detected in glycolipid-enriched microdomain/rafts in GD3- cells. An in vitro kinase assay of Yes revealed that coexistence of GD3 with Yes in membranous environments enhances the kinase activity of GD3- cell-derived Yes toward enolase, p125, and Yes itself. Knockdown of GD3 synthase resulted in the alleviation of tumor phenotypes and reduced activation levels of Yes. Taken together, these results suggest a role of GD3 in the regulation of Src family kinases.
Tomita Makoto, Noboru Hashimoto and Tanaka Yutaka : Association study for the relationship between a haplotype or haplotype set and multiple quantitative responses, Computational Statistics & Data Analysis, Vol.55, No.6, 2104-2113, 2011.
Eiji Tanaka, YAO LIU, LINZE XIA, Naoko Ogasawara, Takuma Sakamaki, Fumiya Kano, Noboru Hashimoto, Xingmei Feng and Akihito Yamamoto : Effectiveness of low-intensity pulsed ultrasound on osteoarthritis of the temporomandibular joint: A review., Annals of Biomedical Engineering, Vol.48, No.8, 2158-2170, Jun. 2020.
(要約)
Loading is indispensable for the growth, development, and maintenance of joint tissues, including mandibular condylar cartilage, but excessive loading or reduced host adaptive capacity can considerably damage the temporomandibular joint (TMJ), leading to temporomandibular joint osteoarthritis (TMJ-OA). TMJ-OA, associated with other pathological conditions and aging processes, is a highly degenerative disease affecting the articular cartilage. Many treatment modalities for TMJ-OA have been developed. Traditional clinical treatment includes mainly nonsurgical options, such as occlusal splints. However, non-invasive therapy does not achieve joint tissue repair and regeneration. Growing evidence suggests that low-intensity pulsed ultrasound (LIPUS) accelerates bone fracture healing and regeneration, as well as having extraordinary effects in terms of soft tissue repair and regeneration. The latter have received much attention, and various studies have been performed to evaluate the potential role of LIPUS in tissue regeneration including that applied to articular cartilage. The present article provides an overview of the status of LIPUS stimulation used to prevent the onset and progression of TMJ-OA and enhance the tissue regeneration of mandibular condylar cartilage. The etiology and management of TMJ-OA are explained briefly, animal models of TMJ-OA are described, and the effectiveness of LIPUS on cell metabolism and tissue regeneration in the TMJ is discussed.
Koichi Furukawa, Yuhsuke Ohmi, Yuki Ohkawa, Robiul H. Bhuiyan, Pu Zhang, Orie Tajima, Noboru Hashimoto, Kazunori Hamamura and Keiko Furukawa : New era of research on cancer-associated glycosphingolipids., Cancer Science, Vol.110, No.5, 1544-1551, Apr. 2019.
(要約)
Cancer-associated glycosphingolipids have been used as markers for diagnosis and targets for immunotherapy of malignant tumors. Recent progress in the analysis of their implications in the malignant properties of cancer cells revealed that cancer-associated glycosphingolipids are not only tumor markers, but also functional molecules regulating various signals introduced by membrane microdomains, lipid rafts. In particular, a novel approach, enzyme-mediated activation of radical sources combined with mass spectrometry, has enabled us to clarify the mechanisms by which cancer-associated glycosphingolipids regulate cell signals based on the interaction with membrane molecules and formation of molecular complexes on the cell surface. Novel findings obtained from these approaches are now providing us with insights into the development of new anticancer therapies targeting membrane molecular complexes consisting of cancer-associated glycolipids and their associated membrane molecules. Thus, a new era of cancer-associated glycosphingolipids has now begun.
(キーワード)
Animals / Biomarkers, Tumor / Cell Membrane / Glycosphingolipids / Humans / Mass Spectrometry / Neoplasms / Signal Transduction
Noboru Hashimoto, Ito Shizuka, Ikeda Kazutaka, Tsuchida Akiko, Crocker R. Paul, Furukawa Keiko, Taguchi Ryo and Furukawa Koichi : Structural specificity in molecular recognition of Siglec-7 to ganglioside GD3, Gordon Reserch Conference Glycolipid &Sphingolipid Biology, Lucca (Barga), Italy, Mar. 2016.
LINZE XIA, Fumiya Kano, Noboru Hashimoto and Akihito Yamamoto : Conditioned Medium from the Stem Cells of Human Exfoliated Deciduous Teeth Ameliorates Experimental Temporomandibular Joint Osteoarthritis by Inducing M2 Phenotype of Macrophages, 蔵本免疫懇話会, Nov. 2021.