Pathophysiological Role of the Association between Metastasis-Associated Protein, S100A4 and Angiogenesis-Related Protein, MetAP2

2004 Fiscal Year Final Research Report Summary

• Project/Area Number: 15590331
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Experimental pathology
• Research Institution: The University of Tokyo
• Principal Investigator: SATOH Hitoshi The University of Tokyo, Graduate School of Frontier Sciences, Associate Professor, 大学院新領域創成科学研究科, 助教授 (70183829)
• Co-Investigator(Kenkyū-buntansha): ASAGIRI Masataka Tokyo Medical and Dental University, Graduate School, Assistant Professor for COE project, 大学院医歯学総合研究科, COE特任助手 (20372435) ; ENDO Hideya Totori University, School of Medicine, Emeritus Professor, 医学部, 名誉教授 (40037320)
• Project Period (FY): 2003 – 2004
• Keywords: Vascular endothelial cell / Neovasucularization / Calcium binding protein / Tumor / Metastasis

Research Abstract

S100A4 is an EF-hand type calcium-binding protein that regulates tumor metastasis and a variety of cellular processes via interaction with different target proteins. We have revealed that S100A4 physically interacts with methionine aminopeptidase 2 (MetAP2), the primary target for protein angiogenesis inhibitors such as fumagillin and ovalicin. By a yeast two-hybird screening system, S100A4 was found to interact with the N-terminal half of MetAP2. In vitro pull-down assays have showed S100A4 associates with MetAP2 in a calcium-dependent manner. The binding site of S100A4 was found to be located within the region between amino acid residures 170 and 229 of MetAP2. In vivo interaction of S100A4 with MetAP2 was verified by co-immunoprecipitation analysis. Immunofluorescent staining revealed that S100A4 and MetAP2 co-localized in both quiescent and basic fibroblast growth factor (bFGF)-treated murine endothelial MSS31 cells, in the latter of which a significant change of intracellular dist ribution of both proteins was observed. Although the binding of S100A4 did not affect the in vitro methionine aminopeptidase activity of MetAP2, the cytochemical observation suggests a possible involvement of S100A4 in the regulation of MetAP2 activity through changing its localization, thereby modulating the N-terminal methionine processing of nascent substrates. These results may offer an essential clue for understanding the functional role of S100A4 in regulating endothelial cell growth and tumor metastasis.
In order to verify whether the association of two proteins finally influence to form new blood vessels, we first performed an overexpression experiment of the binding domain of MetAP2 in bFGF-stimulated murine vascular endothelial MSS31 cells. Incorporation of BrdU at DNA synthesis phase of the cell cycle was suppressed by overexpression of amino acid residues 1-229 compare to the case of overexpression by amino acid residures 1-169, however, the oscillation rate of intra-cellular calcium ion was maintained as the same level. These results suggest that the activated S100A4 by intracellular calcium influx suppresses DNA synthesis of vascular endothelial cells and may regulate their proliferation through the specific binding to MetAP2. We further prepared four synthesized peptides (p38 ; involving 39 amino acids from 170 to 208 of MetAP2, p39 ; 38aa from 192 to 229, p8 ; 30aa from 175 to 204, and p9 ; 23aa from 170 to 192) to minimize the binding domain to S100A4 in calcium-dependent manner. By using BIACORE system for quantitative measurement of protein-protein interaction, it had shown that p38 has more than twice binding activity to GST-S100A4 compare to 60 as (amino acid residures from 170 to 229 of MetAP2) and p8, but neither of p39 and p9 had binding acitivity. These results indicate that the 23^<rd> or 24^<th> of amino acid residues from N-terminus of MetAP2 is crusial for maintaining the binding activity to S100A4 in calcium-dependent manner.
Our data demonstrate that the possibility of therapoietic suppression of neovascularization in tumor cell growth by treatment of small- sized peptides as a molecular target.

Research Products

• [Journal Article] Effects of PU.1-induced mouse calcium-calmodulin-dependent kinase I-like kinase (CKLiK) on apoptosis of murine erythroleukemia cells. (2004)
  • Author(s): Yamada T, Suzuki M, Satoh H, Kihara NF, Nakano H, Oikawa T
  • Journal Title: Exp Cell Res 294 Pages: 39-50
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Altered expression of ARPP protein in skeletal muscles of patients with muscular dystrophy, congenital myopathy and spinal muscular atrophy. (2004)
  • Author(s): Nakada C, Tsukamoto Y, Oka A, Nonaka I, Sato K, Mori S, Ito H, Moriyama M
  • Journal Title: Pathobiology 71 Pages: 43-51
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Japanese multicenter phase II and pharmacokinetic study of rituximab in relapsed or refractory patients with aggressive B-cell lymphoma. (2004)
  • Author(s): Tobinai K, Igarashi T, Itoh K, Kobayashi Y, Taniwaki M, Ogura M, Kinoshita T, Hotta T, Aikawa K, Tsushita K, Hiraoka A, Matsuno Y, Nakamura S, Mori S, Ohashi Y
  • Journal Title: Annals Oncology 15 Pages: 821-830
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Assignment of the murine ankyrin-repeated protein gene (Ankrd2) to mouse chromosome 19C3→D1 and rat chromosome 1q51→q53 by fluorescence in situ hybridization. (2004)
  • Author(s): Fujiwara M, Tsukamoto Y, Miyazaki A, Moriyama M, Satoh H
  • Journal Title: Cytogenet Genome Res 105 Pages: 159A
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Characterization of a newly established nonproducer lymphoma cell line for feline leukemia virus. (2004)
  • Author(s): Fujino Y, Ma Zhiyong, Satoh H, Mizuno T, Hisasue M, Baba K, Masuda K, Ohno K, Onishi T, Tsujimoto H
  • Journal Title: Vet Immunol Immunopathol 102 Pages: 429-439
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Effects of PU.1-induced mouse calcium-calmodulin-dependent kinase I-like kinase (CKLiK) on apoptosis of murine erythroleukemia cells. (2004)
  • Author(s): Yamada T, Suzuki M, Satoh H, Kihara NF, Nakano H, Oikawa T.
  • Journal Title: Exp Cell Res 294 Pages: 39-50
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Altered expression of ARPP protein in skeletal muscles of patients with muscular dystrophy, congenital myopathy and spinal muscular atrophy. (2004)
  • Author(s): Nakada C, Tsukamoto Y, Oka A, Nonaka I, Sato K, Mori S, Ito H, Moriyama M.
  • Journal Title: Pathobiology 71 Pages: 43-51
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Japanese multicenter phase II and pharmacokinetic study of rituximab in relapsed or refractory patients with aggressive B-cell lymphoma. (2004)
  • Author(s): Tobinai K, Igarashi T, Itoh K, Kobayashi Y, Taniwaki M, Ogura M, Kinoshita T, Hotta T, Aikawa K, Tsushita K, Hiraoka A, Matsuno Y, Nakamura S, Mori S, and Ohashi Y.
  • Journal Title: Annals Oncology 15 Pages: 821-830
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Assignment of the murine ankyrin-repeated protein gene (Ankrd2) to mouse chromosome 19C3 → D1 and rat chromosome 1q51 → q53 by fluorescence in situ hybridization. (2004)
  • Author(s): Fujiwara M, Tsukamoto Y, Miyazaki A, Moriyama M, Satoh H.
  • Journal Title: Cytogenet Genome Res 105 Pages: 159A
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Characterization of a newly established nonproducer lymphoma cell line for feline leukemia virus. (2004)
  • Author(s): Fujino Y, Ma Zhiyong, Satoh H, Muno T, Hisasue M, Baba K, Masuda K, Ohno K, Onishi T, Tsujimoto H.
  • Journal Title: Vet Immunol Immunopathol 102 Pages: 429-439
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Cardiac restricted ankyrin repeated protein is differentially induced in Duchenne and congenital muscular dystrophy. (2003)
  • Author(s): Nakada C, Tsukamoto Y, Oka A, Nonaka I, Takeda S, Sato K, Mori S, Ito H, Moriyama M
  • Journal Title: Lab Invest 83 Pages: 711-719
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] IL-17 production from activated T cells is required for the spontaneous development of destructive arthritis in mice deficient in IL-1 receptor antagonist. (2003)
  • Author(s): Nakae S, Saijo S, Horai R, Sudo K, Mori S, Iwakura Y
  • Journal Title: Proc Natl Acad Sci USA 100 Pages: 5986-5990
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Detection of germinal center B-cell lymphoma in archival specimens : critical evaluation of Bcl-6 protein expression in diffuse large B-cell lymphoma of the tonsil. (2003)
  • Author(s): Ree HJ, Ohsima K, Aozasa K, Takeuchi K, Kim CW, Yang WI, Huh JY, Lee SS, Ko YH, Kwon MS, Cho EY, Choi YL, Rhee JC, Kikuchi M, Mori S
  • Journal Title: Human Pathology 34 Pages: 610-616
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Mice lacking a transcriptional corepressor Tob are predisposed to cancer. (2003)
  • Author(s): Yoshida Y, Nakamura T, Komoda M, Satoh H, Suzuki T, Tsuzuku KJ, Miyasaka T, Yoshida HE, Umemori H, Kunisaki KR, Tani K, Ishii S, Mori S, Suganuma M, Noda T, Yamamoto T
  • Journal Title: Genes ＆ Development 17 Pages: 1201-1206
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] A tetraspanin-family protein, T-cell acute lymphoblastic leukemia-associated antigen 1, is induced by the Ewing's sarcoma-Wilms' tumor 1 fusion protein of desmoplastic small round-cell tumor. (2003)
  • Author(s): Ito E, Honma R, Imai J, Azuma S, Kanno T, Mori S, Yoshie O, Nishio J, Iwasaki H, Yoshida K, Gohda J, Inoue J, Watanabe S, Semba K
  • Journal Title: American J Pathology 163 Pages: 2165-2172
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Detection of the integrated feline leukemia viruses in a cat lymphoid tumor cell line by fluorescence in situ hybridization. (2003)
  • Author(s): Fujino Y, Satoh H, Hisasue M, Masuda K, Ohno K, Tsujimoto H
  • Journal Title: J Hered 94 Pages: 251-255
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Comparative assignments of the TSLC1 and TSLC1-like tumor suppressor genes, TSLL1 and TSLL2, to rat chromosomes by fluorescence in situ hybridization. (2003)
  • Author(s): Satoh H, Fujiwara M, Fukami T, Fukuhara H, Murakami Y
  • Journal Title: Bull Nippon Vet Anim Sci Univ 52 Pages: 24-28
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Isolation of the mouse Tsll1 and Tsll2 genes, orthologues of the human TSLC1-like genes 1 and 2 (TSLL1 and TSLL2). (2003)
  • Author(s): Fukami T, Satoh H, Williams NY, Masuda M, Fukuhara H, Maruyama T, Yageta M, Kuramochi M, Takamoto S, Murakami Y
  • Journal Title: Gene 323 Pages: 11-18
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Cardiac restricted ankyrin repeated protein is differentially induced in Duchenne and congenital muscular dystrophy. (2003)
  • Author(s): Nakada C, Tsukamoto Y, Oka A, Nonaka I, Takeda S, Sato K, Mod S, Ito H, Moriyama M.
  • Journal Title: Lab Invest 83 Pages: 711-719
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] IL-17 production from activated T cells is required for the spontaneous development of destructive arthritis in mice deficient in IL-1 receptor antagonist. (2003)
  • Author(s): Nakae S, Saijo S, Horai R, Sudo K, Mori S, Iwakura Y.
  • Journal Title: Proc Natl Acad Sci USA 100 Pages: 5986-5990
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Detection of germinal center B-cell lymphoma in archival specimens : critical evaluation of Bcl-6 protein expression in diffuse large B-cell lymphoma of the tonsil. (2003)
  • Author(s): Ree HJ, Ohsima K, Aozasa K, Takeuchi K, Kim CW, Yang WI, Huh JY, Lee SS, Ko YH, Kwon MS, Cho EY, Choi YL, Rhee JC, Kikuchi M, Mori S.
  • Journal Title: Human Pathology 34 Pages: 610-616
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Mice lacking a transcriptional corepressor Tob are predisposed to cancer. (2003)
  • Author(s): Yoshida Y, Nakamura T, Komoda M, Satoh H, Suzuki T, Tsuzuku KJ, Miyasaka T, Yoshida HE, Umemori H, Kunisaki KR, Tani K, Ishii S, Mori S, Suganuma M, Noda T, Yamamoto T.
  • Journal Title: Genes ＆ Development 17 Pages: 1201-1206
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] A tetraspanin-family protein, T-cell acute lymphoblastic leukemia-associated antigen 1, is induced by the Ewing's sarcoma-Wilms' tumor 1 fusion protein of desmoplastic small round-cell tumor. (2003)
  • Author(s): Ito E, Honma R, Imai J, Azuma S, Kanno T, Mori S, Yoshie O, Nishio J, Iwasaki H, Yoshida K, Gohda J, Inoue J, Watanabe S, Semba K.
  • Journal Title: American J Pathology 163 Pages: 2165-2172
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Detection of the integrated feline leukemia viruses in a cat lymphoid tumor cell line by fluorescence in situ hybridization. (2003)
  • Author(s): Fujino Y, Satoh H, Hisasue M, Masuda K, Ohno K, Tsujimoto H.
  • Journal Title: J Hered 94 Pages: 251-255
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Comparative assignments of the TSLC1 and TSLC1-like tumor suppressor genes, TSLL1 and TSLL2, to rat chromosomes by fluorescence in situ hybridization. (2003)
  • Author(s): Satoh H, Fujiwara M, Fukami T, Fukuhara H, Murakami Y.
  • Journal Title: Bull Nippon Vet Anim Sci Univ 52 Pages: 24-28
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Isolation of the mouse Tsll1 and Tsll2 genes, orthologues of the human TSLC1-like genes 1 and 2 (TSLL1 and TSLL2). (2003)
  • Author(s): Fukami T, Satoh H, Williams NY, Masuda M, Fukuhara H, Maruyama T, Yageta M, Kuramochi M, Takamoto S, Murakami Y.
  • Journal Title: Gene 323 Pages: 11-18
  • Description: 「研究成果報告書概要(欧文)」より