Elucidation of role of beta ig-3 derived from osteoclasts in bone resorption and formation

• Project/Area Number: 15591946
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Morphological basic dentistry
• Research Institution: Meikai University
• Principal Investigator: HAKEDA Yoshiyuki Meikai University, School of Dentistry, Department of Oral Anatomy, Professor, 歯学部, 教授 (90164772)
• Co-Investigator(Kenkyū-buntansha): SATO Takuya Meikai University, School of Dentistry, Department of Oral Anatomy, Lecturer, 歯学部, 講師 (00316689) ; MASUHARA Masaaki Meikai University, School of Dentistry, Department of Oral Anatomy, Assistant Professor, 歯学部, 助手 (70372901) ; KANEDA Toshio Hoshi University, Faculty of Pharmaceutical Sciences, Department of Pathphysiology, Assistant Professor, 薬学部, 助手 (70339521)
• Project Period (FY): 2003 – 2004
• Project Status: Completed (Fiscal Year 2004)
• Budget Amount: ¥3,500,000 (Direct Cost: ¥3,500,000); Fiscal Year 2004: ¥1,300,000 (Direct Cost: ¥1,300,000); Fiscal Year 2003: ¥2,200,000 (Direct Cost: ¥2,200,000)
• Keywords: osteoclastogenesis / beta ig-h3 / osteoclast progenitors / RANKL / TGF-β / RGD sequence / integrin / osteopontin / 接着タンパク / 骨芽細胞 / 骨吸収

Research Abstract

Background : Osteoclastogenesis is promoted by a trigger molecule, receptor activator of NF-κB(RANKL). However, the molecular mechanism for RANKL-induced osteoclast formation and how the osteoclast progenitors are committed to differentiate into osteoclasts have remained to be investigated. Through our project to seek for master genes for osteoclast generation dependent of RANKL-signals, we identified beta ig-h3, which is induced to be expressed in osteoclast progenitors in response to RANKL. In this study, we attempted to elucidate the role of osteoclast progenitor-derived beta ig-h3 in bone resorption by osteoclasts and bone formation by osteoblasts.
Methods : We employed in vitro culture system of M-CSF dependent bone marrow cells-derived osteoclast progenitors for osteoclast formation. We searched for genes expressed in osteoclast progenitors that are dependent on RANKL-stimuli by fluorescent differential display.
Results and Discussion : In osteoclast progenitors, gene expression of beta ig-h3 was induced within early times after exposure to RANKL. The gene expression was also induced by TGF-β and further amplified by the simultaneous addition of RANKL and TGF-β. The induction occurred earlier than the expressions of osteoclast differentiation-related molecules such as tartrate-resistant acid phosphatase (TRAP), cathepsin K, and calcitonin receptors. Since other bone cells such as osteoblasts did not express beta ig-h3, the expression is likely to be specific for osteoclast lineage. Beta ig-h3 contains RGD sequence, and is considered to be an adherent protein that associates with some integrins. Among such adherent proteins, osteopontin is also well known to be expressed in osteoclasts. However, the osteopontin is also expressed in osteoblasts, and is not specific for osteoclasts. In addition, the osteopontin expression did not depended on RANKL. We cloned full-length gene of beta ig-h3, and confirmed from compelling expression in HEK293 cells that the adherent molecule was a secretion protein. Since beta ig-h3 interacts with some integrins, we examined the expression of integrins in osteoclast progenitors. Among these integrins, expression of a V and β3 integrins was linked to osteoclast formation as well as beta ig-h3. Neutralizing antibody against β3 blocked the osteoclast generation in a culture of osteoclast progenitors with M-CSF, TGF-β, and RANKL. Since the culture of osteoclast progenitors does not contain other cells, the molecule associated with β3 integrin is the progenitor-derived one. Taken together, beta ig-h3 would play a possible role in Osteoclastogenesis as a positive regulator.

Research Products

• [Journal Article] The active metabolite of lefluonomide, A771726,inhibits both the generation of and the bone-resorbing activity of osteoclasts by acting directly on cells of the osteoclast lineage. (2004)
  • Author(s): Kobayashi, Y., Ueyama, S., Arai, Y., Yoshida, Y., Kaneda, T., Sato, T., Shin, K., Kumegawa, M., Hakeda, Y.
  • Journal Title: J.Bone Miner.Metab. 22 Pages: 318-328
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] The active metabolite of leflunomide, A771726, inhibits both the generation of and the bone-resorbing activity of osteoclasts by acting directly on cells of the osteoclast lineage (2004)
  • Author(s): Kobayashi Y, Ueyama S, Arai Y, Yoshida Y, Kaneda T, Sato T, Shin K, Kumegawa M, Hakeda Y
  • Journal Title: J. Bone Miner. Metab. 22 Pages: 318-328
  • NAID: 10013606023
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Dexamethasone enhances osteoclast formation synergistically with transforming growth factor-b by stimulating the priming of osteolast progenitors for differentiation into osteoclasts. (2003)
  • Author(s): Takuma, A., Kaneda, T., Sato, T., Ninomiya, S., Kumegawa, M., Hakeda, Y.
  • Journal Title: J.Biol.Chem. 278 Pages: 44667-44674
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Dexamethasone enhances osteoclast formation synergistically with transforming growth factor-b by stimulating the priming of osteoclast progenitors for differentiation into osteoclasts (2003)
  • Author(s): Takuma A, Kaneda T, Sato T, Ninomiya S, Kumegawa M, Hakeda Y
  • Journal Title: J. Biol. Chem. 278 Pages: 44667-44674
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Takuma, A., Kaneda, T., Sato, T., Ninomiya, S., Kumegawa, M., Hakeda Y.: "Dexamethasone enhances osteoclast formation synergistically with transforming growth factor-β by stimulating the priming of osteoclast progenitors for differentiation into osteoclasts."J.Biol.Chem.. 278・45. 44667-44674 (2003)
• [Publications] Kobayashi, Y., Ueyama.S., Arai, Y., Yoshida, Y., Kaneda, T., Sato, T., Shin, K., Kumegawa, M., Hakeda, Y.: "The active metabolite of leflunomide, A771726, inhibits both generation and bone-resorbing activity of osteoclasts by acting directly on cells of the osteoclast lineage."J.Bone.Miner.Metab.. In press. (2004)