Masamichi Shiraga

Publications (10)64.08 Total impact

• Article: The tetraspanin CD9 modulates epidermal growth factor receptor signaling in cancer cells.

  Yoko Murayama, Yasuhisa Shinomura, Kenji Oritani, Jun-Ichiro Miyagawa, Hitoshi Yoshida, Makoto Nishida, Fumie Katsube, Masamichi Shiraga, Tamana Miyazaki, Taisei Nakamoto, Shusaku Tsutsui, Shinji Tamura, Shigeki Higashiyama, Iichirou Shimomura, Norio Hayashi
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  ABSTRACT: CD9 is a member of the tetraspanins, and has been shown to be involved in a variety of cellular activities such as migration, proliferation, and adhesion. In addition, it has been known that CD9 can associate with other proteins. Here we demonstrated the physical and functional association of CD9 with epidermal growth factor receptor (EGFR) on MKN-28 cells. Double-immunofluorescent staining and immunoprecipitation demonstrated the complex formation of CD9-EGFR and CD9-beta(1) integrin, and that both complexes are colocalized on the cell surface especially at the cell-cell contact site. Anti-CD9 monoclonal antibody ALB6 induced a dotted or patch-like aggregation pattern of both CD9-EGFR and CD9-beta(1) integrin. The internalization of EGFR after EGF-stimulation was significantly enhanced by the treatment with ALB6. CD9 can associate with EGFR in hepatocellular carcinoma cells (HepG2/CD9) and Chinese hamster ovary cancer cells (CHO-HER/CD9), which were transfected with pTJ/human EGFR/CD9. Furthermore expression of CD9 specifically attenuated EGFR signaling in CHO-HER/CD9 cells through the down regulation of surface expression of EGFR. These results suggest that CD9 might have an important role that attenuates EGFR signaling. Therefore, CD9 not only associates EGFR but also a new regulator, which may affect EGF-induced signaling in cancer cells.
  Journal of Cellular Physiology 08/2008; 216(1):135-43. · 3.87 Impact Factor
• Article: Expression of CD27 on peripheral CD4+ T-lymphocytes correlates with the development of severe acute graft-versus-host disease after allogeneic bone marrow transplantation.

  Hitoshi Yoshida, Tetsuo Maeda, Jun Ishikawa, Shinya Inoue, Hitomi Matsunaga, Satoru Kosugi, Masamichi Shiraga, Kenji Oritani, Yuzuru Kanakura, Yoshiaki Tomiyama
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  ABSTRACT: Allogeneic immune responses during hematopoietic reconstitution play central roles in beneficial and adverse effects after allogeneic bone marrow transplantation (allo-BMT). Appropriate regulation of the immune responses might improve the outcome of allo-BMT. However, a useful marker for monitoring allogeneic immune responses remains to be established. We enrolled 22 consecutive patients who underwent myeloablative allo-BMT between March 2002 and March 2006 and examined the relationship between CD27 expression on peripheral blood T-lymphocytes, a possible marker for naive/effector phenotypes, and clinical events, especially acute graft-versus-host disease (aGVHD). In 8 patients with aGVHD of grades II to IV, the CD27+/CD27- ratios of CD4+ (but not CD8+) T-lymphocytes were significantly higher after allo-BMT, even at day 21, than the ratios in patients with aGVHD of grade 0 or I and remained high after day 21. In contrast, the ratios were low after day 21 following allo-BMT in 14 patients with aGVHD of grade 0 or I. Moreover, the clinical analysis suggested a relationship between the ratio and aGVHD grade. Thus, we showed that the CD27+/CD27- ratio in CD4+ T-lymphocytes may have value in predicting the development of severe aGVHD and may correlate with clinical symptoms of aGVHD.
  International Journal of Hematology 12/2006; 84(4):367-76. · 1.27 Impact Factor
• Article: Erythropoietin enhances neovascularization of ischemic myocardium and improves left ventricular dysfunction after myocardial infarction in dogs.

  Akio Hirata, Tetsuo Minamino, Hiroshi Asanuma, Masashi Fujita, Masakatsu Wakeno, Masafumi Myoishi, Osamu Tsukamoto, Ken-ichiro Okada, Hidekazu Koyama, Kazuo Komamura, Seiji Takashima, Yoshiro Shinozaki, Hidezo Mori, Masamichi Shiraga, Masafumi Kitakaze, Masatsugu Hori
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  ABSTRACT: We investigated the effects of erythropoietin (EPO) on neovascularization and cardiac function after myocardial infarction (MI). Erythropoietin exerts antiapoptotic effects and mobilizes endothelial progenitor cells (EPCs). We intravenously administered EPO (1,000 IU/kg) immediately [EPO(0) group], 6 h [EPO(6h) group], or 1 week [EPO(1wk) group] after the permanent ligation of the coronary artery in dogs. Control animals received saline immediately after the ligation. The infarct size 6 h after MI was significantly smaller in the EPO(0) group than in the control group (61.5 +/- 6.0% vs. 22.9 +/- 2.2%). One week after MI, the circulating CD34-positive mononuclear cell numbers in both the EPO(0) and the EPO(6h) groups were significantly higher than in the control group. In the ischemic region, the capillary density and myocardial blood flow 4 weeks after MI was significantly higher in both the EPO(0) and the EPO(6h) groups than in the control group. Four weeks after MI, left ventricular (LV) ejection fraction in the EPO(6h) (48.6 +/- 1.9%) group was significantly higher than that in either the control (41.9 +/- 0.9%) or the EPO(1wk) (42.6 +/- 1.2%) group but significantly lower than that in the EPO(0) group (56.1 +/- 2.3%). The LV end-diastolic pressure 4 weeks after MI in both the EPO(0) and the EPO(6h) groups was significantly lower than either the control or the EPO(1wk) group. Hematologic parameters did not differ among the groups. In addition to its acute infarct size-limiting effect, EPO enhances neovascularization, likely via EPC mobilization, and improves cardiac dysfunction in the chronic phase, although it has time-window limitations.
  Journal of the American College of Cardiology 08/2006; 48(1):176-84. · 14.16 Impact Factor
• Article: Adiponectin acts as an endogenous antithrombotic factor.

  Hisashi Kato, Hirokazu Kashiwagi, Masamichi Shiraga, Seiji Tadokoro, Tsuyoshi Kamae, Hidetoshi Ujiie, Shigenori Honda, Shigeki Miyata, Yoshinobu Ijiri, Junichiro Yamamoto, Norikazu Maeda, Tohru Funahashi, Yoshiyuki Kurata, Iichiro Shimomura, Yoshiaki Tomiyama, Yuzuru Kanakura
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  ABSTRACT: Obesity is a common risk factor in insulin resistance and cardiovascular diseases. Although hypoadiponectinemia is associated with obesity-related metabolic and vascular diseases, the role of adiponectin in thrombosis remains elusive. We investigated platelet thrombus formation in adiponectin knockout (APN-KO) male mice (8 to 12 weeks old) fed on a normal diet. There was no significant difference in platelet counts or coagulation parameters between wild-type (WT) and APN-KO mice. However, APN-KO mice showed an accelerated thrombus formation on carotid arterial injury with a He-Ne laser (total thrombus volume: 13.36+/-4.25 x 10(7) arbitrary units for APN-KO and 6.74+/-2.87x10(7) arbitrary units for WT; n=10; P<0.01). Adenovirus-mediated supplementation of adiponectin attenuated the enhanced thrombus formation. In vitro thrombus formation on a type I collagen at a shear rate of 250 s(-1), as well as platelet aggregation induced by low concentrations of agonists, was enhanced in APN-KO mice, and recombinant adiponectin inhibited the enhanced platelet aggregation. In WT mice, adenovirus-mediated overexpression of adiponectin additionally attenuated thrombus formation. Adiponectin deficiency leads to enhanced thrombus formation and platelet aggregation. The present study reveals a new role of adiponectin as an endogenous antithrombotic factor.
  Arteriosclerosis Thrombosis and Vascular Biology 01/2006; 26(1):224-30. · 6.37 Impact Factor
• Article: Negative regulation of platelet function by a secreted cell repulsive protein, semaphorin 3A.

  Hirokazu Kashiwagi, Masamichi Shiraga, Hisashi Kato, Tsuyoshi Kamae, Naoko Yamamoto, Seiji Tadokoro, Yoshiyuki Kurata, Yoshiaki Tomiyama, Yuzuru Kanakura
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  ABSTRACT: Semaphorin 3A (Sema3A) is a secreted disulfide-bound homodimeric molecule that induces growth cone collapse and repulsion of axon growth in the nervous system. Recently, it has been demonstrated that Sema3A is produced by endothelial cells and inhibits integrin function in an autocrine fashion. In this study, we investigated the effects of Sema3A on platelet function by using 2 distinct human Sema3A chimera proteins. We detected expression of functional Sema3A receptors in platelets and dose-dependent and saturable binding of Sema3A to platelets. Sema3A dose-dependently inhibited activation of integrin alphaIIbbeta3 by all agonists examined including adenosine diphosphate (ADP), thrombin, convulxin, phorbol 12-myristate 13-acetate, and A23187. Sema3A inhibited not only platelet aggregation induced by thrombin or collagen but also platelet adhesion and spreading on immobilized fibrinogen. Moreover, Sema3A impaired alphaIIbbeta3-independent spreading on glass coverslips and aggregation-independent granular secretion. Sema3A inhibited agonist-induced elevation of filamentous action (F-actin) contents, phosphorylation of cofilin, and Rac1 activation. In contrast, Sema3A did not affect the levels of cyclic nucleotides or agonist-induced increase of intracellular Ca2+ concentrations. Thus, the extensive inhibition of platelet function by Sema3A appears to be mediated, at least in part, through impairment of agonist-induced Rac1-dependent actin rearrangement.
  Blood 09/2005; 106(3):913-21. · 9.90 Impact Factor
• Article: SHPS-1 negatively regulates integrin alphaIIbbeta3 function through CD47 without disturbing FAK phosphorylation.

  Hisashi Kato, Shigenori Honda, Hitoshi Yoshida, Hirokazu Kashiwagi, Masamichi Shiraga, Nakayuki Honma, Yoshiyuki Kurata, Yoshiaki Tomiyama
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  ABSTRACT: CD47 (integrin-associated protein) serves as a receptor for thrombospondin-1 (TSP-1) and Src homology 2 domain-containing protein tyrosine phosphatase substrate-1 (SHPS-1), and the TSP-1/CD47 interaction has been believed to augment integrin-mediated platelet function. Here, employing SHPS-1-immunoglobulin (Ig) as a ligand, we have newly demonstrated that CD47 acts as an inhibitory receptor for platelet function. The binding of SHPS-1-Ig was solely mediated by CD47, because CD47-deficient platelets failed to bind murine SHPS-1-Ig. The human SHPS-1/CD47 interaction inhibited the platelet aggregation induced by several kinds of agonists at a low concentration. Moreover, human SHPS-1 expressed on the cell surface as well as soluble SHPS-1-Ig markedly inhibited the platelet spreading on, but not initial adhesion to, immobilized fibrinogen. Again, neither murine SHPS-1 expressed on the cell surface nor murine SHPS-1-Ig inhibited the spreading of CD47-deficient platelets. We further investigated the tyrosine phosphorylation of signaling proteins during platelet spreading on immobilized fibrinogen. Unexpectedly, SHPS-1 inhibited alpha(IIb)beta(3)-mediated platelet spreading without disturbing focal adhesion kinase (FAK) tyrosine phosphorylation. Further examination revealed that SHPS-1 inhibited the tyrosine phosphorylation of alpha-actinin, a downstream effector of FAK, but not of cortactin. Thus, it is likely that the SHPS-1/CD47 interaction inhibits alpha(IIb)beta(3)-mediated outside-in signaling by interfering with the downstream pathway of FAK. Taken together, our data suggest that SHPS-1 negatively regulates platelet function via CD47, especially alpha(IIb)beta(3)-mediated outside-in signaling.
  Journal of Thrombosis and Haemostasis 05/2005; 3(4):763-74. · 5.73 Impact Factor
• Article: Differential effects of a novel IFN-zeta/limitin and IFN-alpha on signals for Daxx induction and Crk phosphorylation that couple with growth control of megakaryocytes.

  Naoko Ishida, Kenji Oritani, Masamichi Shiraga, Hitoshi Yoshida, Sin-Ichiro Kawamoto, Hidetoshi Ujiie, Hiroaki Masaie, Michiko Ichii, Yoshiaki Tomiyama, Yuzuru Kanakura
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  ABSTRACT: Although a novel IFN-zeta/limitin uses IFN-alpha/beta receptor, it lacks some common activities of type I IFNs. We compared effects on megakaryocyte proliferation and differentiation as well as signals for their biological activities. Recombinant IFN-zeta/limitin and IFN-alpha titrated with a cytopathic effect dye binding assay, were used in this study. Colony assays and serum-free suspension cultures for megakaryocytes were performed to compare their growth inhibitory effects. To analyze signals, megakaryocytes cultured in serum-free suspension cultures were stimulated and Western blotted with the indicated antibody. Both IFN-zeta/limitin and IFN-alpha suppressed the proliferation of megakaryocyte progenitors without influencing their differentiation. However, much higher concentrations of IFN-zeta/limitin were required for the growth inhibition than IFN-alpha. The growth inhibition by IFN-zeta/limitin and IFN-alpha was significantly reduced when either Tyk2 or STAT1 was disrupted. In addition, the antisense oligonucleotides against Crk and Daxx, downstream molecules of Tyk2, greatly rescued the IFN-zeta/limitin- and IFN-alpha-induced reduction of megakaryocyte colony numbers. In cultured megakaryocytes, IFN-zeta/limitin induced the expression of SOCS-1 as strongly as IFN-alpha. However, IFN-zeta/limitin induced weaker phosphorylation of Crk and lower induction of Daxx than IFN-alpha. Weaker signals for Crk and Daxx may participate in less megakaryocyte suppressive activity of IFN-zeta/limitin and may distinguish IFN-zeta/limitin from IFN-alpha in megakaryocytes. Our results extend the understanding about thrombocytopenia in patients with IFN-alpha treatment as well as the possibility for the clinical application of human homologue of IFN-zeta/limitin or an engineered cytokine with useful features of the IFN-zeta/limitin structure.
  Experimental Hematology 05/2005; 33(4):495-503. · 2.90 Impact Factor
• Article: Amino acid mutagenesis within ligand-binding loops in alpha v confers loss-of-function or gain-of-function phenotype on integrin alpha v beta 3.

  Shigenori Honda, Hirokazu Kashiwagi, Teruo Kiyoi, Hisashi Kato, Satoru Kosugi, Masamichi Shiraga, Yoshiyuki Kurata, Yoshiaki Tomiyama
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  ABSTRACT: The crystal structure of alpha(v)beta(3) in complex with a cyclic RGD-containing ligand has recently been demonstrated. However, the functional significance of each residue within ligand binding loops has not been fully elucidated. Here, by employing alanine-scanning mutagenesis, we have examined the functional role of ligand contact residues in alpha(v). Tyr178 --> Ala substitution (Tyr178Ala) and Asp218Ala abolished a monovalent ligand, WOW-1 Fab binding as well as soluble fibrinogen binding, which is in perfect agreement with the crystallography. However, Asp150Ala showed no or only a modest inhibition of ligand binding. In contrast, Tyr substitution at Ala215 (Ala215Tyr) increased WOW-1 Fab binding, suggesting that the substitution increased the integrin affinity. The adhesion assay to immobilized fibrinogen showed essentially the same data as obtained using soluble ligands. Our present data indicate that Tyr178 and Asp218, but not Asp150 in alpha(v) is critically involved in ligand-binding and that Ala215 could regulate the affinity of alpha(v)beta(3).
  Thrombosis and Haemostasis 11/2004; 92(5):1092-8. · 5.04 Impact Factor
• Article: Missense mutations in the beta(3) subunit have a different impact on the expression and function between alpha(IIb)beta(3) and alpha(v)beta(3).

  Seiji Tadokoro, Yoshiaki Tomiyama, Shigenori Honda, Hirokazu Kashiwagi, Satoru Kosugi, Masamichi Shiraga, Teruo Kiyoi, Yoshiyuki Kurata, Yuji Matsuzawa
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  ABSTRACT: Alpha(IIb)beta(3) and alpha(v)beta(3) belong to the beta(3) integrin subfamily. Although the beta(3) subunit is a key regulator for the biosynthesis of beta(3) integrins, it remains obscure whether missense mutations in beta(3) may induce the same defects in both alpha(IIb)beta(3) and alpha(v)beta(3). In this study, it is revealed that thrombasthenic platelets with a His280Pro mutation in beta(3), which is prevalent in Japanese patients with Glanzmann thrombasthenia, did contain significant amounts of alpha(v)beta(3) (about 50% of control) using sensitive enzyme-linked immunosorbent assay. Expression studies showed that the His280Probeta(3) mutation impaired alpha(IIb)beta(3) expression but not alpha(v)beta(3) expression in 293 cells. To extend these findings, the effects of several beta(3) missense mutations leading to an impaired alpha(IIb)beta(3) expression on alpha(v)beta(3) function as well as expression was examined: Leu117Trp, Ser162Leu, Arg216Gln, Cys374Tyr, and a newly created Arg216Gln/Leu292Ser mutation. Leu117Trp and Cys374Tyr beta(3) mutations did impair alpha(v)beta(3) expression, while Ser162Leu, Arg216Gln, and Arg216Gln/Leu292Ser mutations did not. With regard to ligand binding function, Ser162Leu mutation induced especially distinct effects between 2 beta(3) integrins: it markedly impaired ligand binding to alpha(IIb)beta(3) but not to alpha(v)beta(3) at all. These data clearly demonstrate that the biosynthesis and the ligand binding function of alpha(IIb)beta(3) and those of alpha(v)beta(3) are regulated in part by different mechanisms. Present data would be a clue to elucidate the regulatory mechanism of expression and function of beta(3) integrins.
  Blood 03/2002; 99(3):931-8. · 9.90 Impact Factor
• Article: Demonstration of a marked reduction in the amount of GPIIb in most type II patients with Glanzmann's thrombasthenia

  Yoshiaki Tomiyama, Hirokazu Kashiwagi, Satoru Kosugi, Masamichi Shiraga, Seiji Kinoshita, Yoshio Kanayama, Yoshiyuki Kurata, Yuji Matsuzawa
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  ABSTRACT: In this study employing a sensitive immunoblot assay, we have characterized GPIIb and GPIIIa in thrombasthenic platelets from seven type II and four type I patients from 10 unrelated families. The amounts of GPIIb and GPIIIa were both markedly reduced in all these patients, and abnormal molecular weight GPIIb or GPIIIa was not detected. In all of four type I patients the amount of GPIIb was much lower than that of GPIIIa. In this study, however, we found that the amount of GPIIb was also lower even in six out of seven type II patients. Immunodepletion of patients’platelets with AP2 (a monoclonal antibody specific for the GPIIb-IIIa complex), AP3 (specific for GPIIIa) or AMF7 (specific for v) further confirmed that GPIIIa existed in excess, and demonstrated that excess GPIIIa were mostly in free form and not associated with GPIIb or v. The reduction of GPIIb may represent an abnormality in GPIIb processing in these type II and type I thrombasthenic platelets. It remains unclear whether these two subgroups represent distinct categories.
  British Journal of Haematology 04/1994; 87(1):119 - 124. · 4.94 Impact Factor