Studies on molecular mechanisms involved in differentiation of chondrocyte/osteoblast by Notch

• Project/Area Number: 15390557
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Morphological basic dentistry
• Research Institution: Tokyo University of Science
• Principal Investigator: HOZUMI Nobumichi Tokyo University of Science, Research Institute for Biological Sciences, Professor, 生命科学研究所, 教授 (60051744)
• Co-Investigator(Kenkyū-buntansha): SUZUKI Mitsuhiro Tokyo University of Science, Research Institute for Biological Sciences, Research Associate, 助手 (00321662) ; MORIMURA Naoko RIKEN, The Institute for Physical Chemical Research, Researcher, 比較神経発生研究チーム, 研究員 (00349044) ; 香山 雅子 東京理科大学, 生命科学研究所, 助手 (80318229)
• Project Period (FY): 2003 – 2005
• Project Status: Completed (Fiscal Year 2005)
• Budget Amount: ¥11,800,000 (Direct Cost: ¥11,800,000); Fiscal Year 2005: ¥3,000,000 (Direct Cost: ¥3,000,000); Fiscal Year 2004: ¥4,400,000 (Direct Cost: ¥4,400,000); Fiscal Year 2003: ¥4,400,000 (Direct Cost: ¥4,400,000)
• Keywords: Notch / Limb bud micromass culture / SST-REX method / ATDC5 / Chondrogenesis / Ossification / Limb Bud Micromass Culture / DAPT / マウス肢芽 / 間葉系幹細胞 / 細胞凝集 / 内軟骨性骨化 / 遺伝子発現 / 転写因子 / Conditional deletion mutantマウス / Notch1 / 軟骨基質 / 骨芽細胞

Research Abstract

In endochondral ossification occurring in longitudinal bone, cartilagineous tissue derived from mesenchymal cells is replaced with bone within ossification centers. Thus, endochondral ossification precedes ossification osteoblasts. Therefore it is prerequisite to elucidate the molecular mechanisms involved in chondrogenesis for better understanding of skeltogenesis. We have shown that Notch signaling stimulates the differentiation of osteoblasts. The current research projects are aimed at elucidation of mechanisms in bone formation regulated by Notch.
Notch1 was localized starting from the mesenchymal condensation stage of embryonic mouse forelimbs. Interestingly, although localization could not be detected in the proliferating chondrocytes, obvious immuno-reactivity indicating its expression was retained in the perichondral region. To assess the effect of Notch activation, we transferred an active form of Notch (NIC) into the cell line by an adenovirus vector. The differentiation of ch ondrocytes was inhibited. We utilized limb bud micromass culture (LBMC) for further analysis. LBMC is an established model that recapitulates mesenchymal condensation and chondrocyte differentiation. RT-PCR showed that Notch and its related genes were expressed in such cultures at day 1 and day 5, when cell condensation and nodule formation were initiated. Immuno-histochiemical experiments revealed that the expression of Notch1 was initially localized within the nodules and shifted to their peripheral region as the cell differentiation progressed. We disrupted Notch signaling by using a gamma-secretase inhibitor, DAPT, to analyze the function of Notch signaling in the culture system. Blocking Notch signaling by DAPT apparently promoted the initiation of prechondrogenic condensation and fusion of the nodules, and such an effect was reversed by exogenous expression of the Notch cytoplasmic domain. These observations imply that the Notch signal may have an important role in chondrogenic differentiation by negatively regulating the initiation of prechondrogenic condensation and nodule formation.
We constructed a cDNA library from the mRNA fraction derived from a chondrogenic lell line (ATDC5) at the condensation stage by using the signal sequence trap by retrovirus mediated expression (SST-REX) method. We obtained 486 factor (IL-3)-independent clones by screening 5.7 x 10^3 clones. DNA sequencing analysis of the clones identified genes encoding 157 known proteins and 4 novel proteins. A series of the analyses demonstrate that the SST-REX method is a useful experimental system to identify genes involved in the complicated mechanisms of bone formation.

Research Products

• [Journal Article] Use of the SST-REX method for identification of genes espressed at the condensation stage of chondrogenic cell line ATDC5 (2006)
  • Author(s): Noguchi A, et al.
  • Journal Title: J.Bone Miner.Metab. 24 Pages: 153-157
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Site-specific DNA methylation by a complex of PU.I and Dumt3a/b (2006)
  • Author(s): Suzuki M, et al.
  • Journal Title: Oncogene 25 Pages: 2477-2488
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Involvement of Notch signaling in initiation of prechondrogenic condensation and formation of nodules in limb bud micromass culture (2006)
  • Author(s): Fujimaki R, et al.
  • Journal Title: J.Bone Miner.Metab. 24 Pages: 191-198
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] use of the SST-REX method for the identification of genes expressed at the condensation, stage of chondrogenic cell line ATDC5 (2006)
  • Author(s): Noguchi, A., Watanabe, N., Fujimaki, R., Kitamura, T., Hayashizaki, Y., Miyaki, S., Tezuka, K., Hozumi, N.
  • Journal Title: J.Bone Miner.Metab. 24 Pages: 153-157
  • NAID: 10018194449
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Site specific DNA methylatlion by a complex of PU.1 and Dnmt3a/b (2006)
  • Author(s): Suzuki, M., Yamada, F., Kihara-Negishi, F., Sakurai, T., Hara, E., Tenen, D.G., Hozumi, N., Oikawa, T.
  • Journal Title: Oncogene 25 Pages: 2477-2488
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Involvement of Notch signaling in initiation of prechondrogenic condensation and formation of nodules in limb bud micromass cultures (2006)
  • Author(s): Fujimaki, R., Toyama, Y., Hozumi, N., Tezuka, K.
  • Journal Title: J.Bone Miner.Metab. 24 Pages: 191-198
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Site-specific DNA methlation by a comples of PU.1 and Dnmt3a/b (2006)
  • Author(s): Suzuki, M., et al.
  • Journal Title: Oncogene (in press)
• [Journal Article] Involvement of Notch signaling in initiation of prech ondrogenic condensation and formation of nodules in limb bud micromass cultures (2006)
  • Author(s): Fujimaki, R., et al.
  • Journal Title: J.Bone Miner.Metab. (in press)
• [Journal Article] Expression of Cre rcombinase in the mouse developing chondrocytes driven by the mouse a2(XI)collagen promoter (2005)
  • Author(s): Fujimaki R, et al.
  • Journal Title: J.Bone Miner.Metab. 90 Pages: 270-273
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Expression of Cre recombinase in the mouse developing chondrocytes driven by the mouse a2(XI) collagen promoter. (2005)
  • Author(s): Fujimak, R., Hayashi, K., Watanabe, N., Yamada, T., Toyama, Y., Tezuka, K., Hozumi, N.
  • Journal Title: J.Bone Miner.Metab. 23 Pages: 270-273
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Expression of Cre recombinase in the mouse developing chondrocytes driven by the mouse α2 (XI) collagen promoter (2005)
  • Author(s): Fujimaki, R., et al.
  • Journal Title: J.Bone Miner.Metab. 23 Pages: 270-273
• [Journal Article] Dual regulation of BCR-mediated growth inhibition signaling by CD72 (2005)
  • Author(s): Baba, T., et al.
  • Journal Title: Eur.J.Immunol. 35 Pages: 1634-1642
• [Journal Article] 1'-acetoxychavicol acetate is a novel nuclear factor κB inhibitor with significant activity against multiple myeloma in vitro and in vivo (2005)
  • Author(s): Ito, K., et al.
  • Journal Title: Cancer Res. 65 Pages: 4417-4424
• [Journal Article] Use of the SST-REX method for the identification of genes expressed at the condensation stage of chondrogenic cell line ATDC5 (2005)
  • Author(s): Noguchi, A., et al.
  • Journal Title: J.Bone Miner.Metab. 24 Pages: 153-157
• [Journal Article] Expression of Cre recombinase in the mouse developing chondrocytes driven by the mouse α2(XI) collagen promoter (2005)
  • Author(s): Fujimaki, R.et al.
  • Journal Title: J.Bone Miner.Metab. (in press)
• [Journal Article] Inducible costimulator-dependent IL-10 production by regulatory Tcells specific for self-antigen (2004)
  • Author(s): Kohyama M, et al.
  • Journal Title: Proc.Natl.Acad.Sci.USA 101 Pages: 4192-4197
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Inducible costimulator-dependent IL-10 production by regulatory T cells specific for self-antigen. (2004)
  • Author(s): Kohyama, M., Sugahara, D., Sugiyama, S., Yagita, H., Okumura, K., Hozumi, N.
  • Journal Title: Proc.Natl.Acad.Sci.USA 101 Pages: 4192-4197
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Tumor angiogenesis in the bone marrow of multiple myeloma patients and its alteration by thalidomide treatment (2004)
  • Author(s): Du, W.et al.
  • Journal Title: Pathol Int. 54 Pages: 285-294
• [Journal Article] Hepatitis C virus infection in human liver tissue engrafted in mice with an infectious molecular clone (2004)
  • Author(s): Maeda, N.et al.
  • Journal Title: Liver Int. 24 Pages: 259-267
• [Journal Article] Suooerssion of differentiation and proliferation of early chondrogenic cells by Notch (2003)
  • Author(s): Watanebe N, et a;
  • Journal Title: J.Bone Miner.Metab. 21 Pages: 344-352
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Suppression of differentiation and proliferation of early chondrogenic cells by Notch. (2003)
  • Author(s): Watanabe, N., Tezuka, Y., Matsuno, K., Miyatani, S., Morimura, N., Yasuda, M., Fujimaki, R., Kuroda, K., Hiraki, Y., Hozumi, N., Tezuka, K.
  • Journal Title: J.Bone Miner.Metab. 21 Pages: 344-352
  • NAID: 10014445248
  • Description: 「研究成果報告書概要(欧文)」より
• [Book] Recent development in Immunology(分担執筆) (2004)
  • Author(s): Hozumi, N., Kohyama, M.
  • Total Pages: 371
  • Publisher: Research Signpost
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Watanabe, N., Tezuka, Y.et al.: "Suppression of differentiation and proliferation of early chondrogenic cells By Notch"J.Bone Miner.Metab.. 21. 344-352 (2003)
• [Publications] Baba, T., Fusaki, N.et al.: "Myosin is an in vivo substrate of the protein tyrosine phospatase (SHP-1) after mlgM cross-linking"Biochem.Biophys.Res.Comm.. 304. 67-72 (2003)
• [Publications] Kohyama, M., Sugahara, D.et al.: "Inducible costimulator-dependent IL-10 production by regulatory T cells specific for self-antigen"Proc.Natl.Acad.Sci.USA. (in press). (2004)