破骨細胞を調節するシグナル伝達系と骨・歯周疾患における骨破壊機序の解明

• Project/Area Number: 12137209
• Research Category: Grant-in-Aid for Scientific Research on Priority Areas
• Allocation Type: Single-year Grants
• Review Section: Biological Sciences
• Research Institution: Matsumoto Dental University (2001-2004); Showa University (2000)
• Principal Investigator: 高橋 直之 松本歯科大学, 大学院・歯学独立研究科, 教授 (90119222)
• Co-Investigator(Kenkyū-buntansha): 宇田川 信之 松本歯科大学, 歯学部, 教授 (70245801) ; 平岡 行博 松本歯科大学, 大学院・歯学独立研究科, 教授 (20097512) ; 小澤 英浩 (小沢 英浩) 松本歯科大学, 大学院・歯学独立研究科, 教授 (60018413) ; 中村 美どり 松本歯科大学, 歯学部, 助手 (90278177) ; 小林 泰浩 松本歯科大学, 大学院・歯学独立研究科, 助教授 (20264252) ; 片桐 岳信 昭和大学, 歯学部, 助教授 (80245802) ; 自見 英治郎 昭和大学, 歯学部, 助手 (40276598)
• Project Period (FY): 2000 – 2004
• Project Status: Completed (Fiscal Year 2004)
• Budget Amount: ¥43,600,000 (Direct Cost: ¥43,600,000); Fiscal Year 2004: ¥9,800,000 (Direct Cost: ¥9,800,000); Fiscal Year 2003: ¥9,800,000 (Direct Cost: ¥9,800,000); Fiscal Year 2002: ¥12,000,000 (Direct Cost: ¥12,000,000); Fiscal Year 2001: ¥12,000,000 (Direct Cost: ¥12,000,000)
• Keywords: 破骨細胞 / 骨髄マクロファージ / PGE_2 / ヒトCD14陽性細胞 / TAK1 / 破骨細胞形成抑制因子 / RAW264細胞 / リポ多糖 / IL-1 / MyD88 / ムラミルジペプチド / Nod2 / 骨粗鬆症治療薬 / マクロファージ / ビスフォスフォネート / 骨形成 / 骨吸収 / RANKL / p38MAPK / 骨芽細胞 / 炎症性サイトカイン / p38MAPキナーゼ / BMP / Smad / SaOS-4 / 3細胞 / カルシウム / PKC / M-CSF / シグナル伝達

Research Abstract

破骨細胞の分化と機能を調節するシグナル伝達系の解析を目指してきた。PGE_2は、骨芽細胞の破骨細胞分化因子(RANKL)を誘導することは広く知られているが,破骨細胞前駆細胞に対しても強力に作用する。今回我々は,マウスとヒト破骨細胞形成におけるPGE_2の作用を解析し、以下の結果を得た。
(1)PGE_2は、RANKL駕誘導するマウス骨髄マクロファージとマクロファージ細胞株RAW264細胞の破骨細胞への分化を促進した。PGE_2による促進効果は、cAMP-PKAの活性化により誘導された。
(2)TGF-β activated kinase 1(TAK1)が、PKAがリン酸化するモチーフ(RRXS)を有することを見出した。TAK1はPGE_2刺激でリン酸化され、H89はそのリン酸化を抑制した。
(3)412番目のSerをA1aに置換したS412A-TAK1を発現させたRAW264細胞では、PGE_2の促進作用が消失した。
(4)PGE_2は、RANKLおよびM-CSFが誘導するヒトCD14陽性細胞から破骨細胞への分化を強力に抑制した。H89はPGE_2による抑制作用を解除した。
(5)CD14陽性細胞はPGE_2に反応して、強力な破骨細胞形成抑制因子を産生した。この抑制因子は、マウス破骨細胞形成も強力に抑制した。(6)CD14陽性細胞が産生する破骨細胞形成抑制因子は,破骨細胞形成を抑制することが知られている既知のサイトカイン(GM-CSF,IL-4,INF-γ)ではなく、新規のサイトカインである可能性が示された。
以上の知見より、マウスとヒトの破骨細胞形成に対して、PGE_2は異なる作用を有することが明らかにされた。また、ヒト破骨細胞前駆細胞はPGE_2に反応して、破骨細胞形成を抑制する新規のサイトカインを産生することが示された。

Research Products

• [Journal Article] Dolomite supplementation improves bone metabolism through modulation of calcium-regulating hormone secretion in ovariectomized rats. (2005)
  • Author(s): Mizoguchi T et al.
  • Journal Title: J Bone Miner Metab (in press)
• [Journal Article] Experimental spinal fusion with recombinant human bone morphogenetic protein-2 delivered by a synthetic polymer and beta-tricalcium phosphate in a rabbit model. (2005)
  • Author(s): Namikawa T et al.
  • Journal Title: Spine (in press)
• [Journal Article] Cyclic AMP/protein kinase A signals enhance osteoclastic differentiation through TAK1 in osteoclast precursors. (2005)
  • Author(s): Kobayashi Y et al.
  • Journal Title: J Biol Chem (in press)
• [Journal Article] Prostaglandin E_2 receptors EP2 and EP4 are down-regulated during differentiation of mouse osteoclasts from their precursors. (2005)
  • Author(s): Kobayashi Y et al.
  • Journal Title: J Biol Chem (in press)
• [Journal Article] Suppression of osteoprotegerin expression by PGE_2 is crucially involved in LPS-induced osteoclast formation1. (2004)
  • Author(s): Suda K et al.
  • Journal Title: J Immunol 172・4 Pages: 2504-2510
• [Journal Article] MyD88 is an essential molecule in osteoclastogenesis induced by lipopolysaccharide, diacyl lipopeptide and IL-1α, and MyD88 knockout mice exhibit a low turnover osteoporotic phenotype. (2004)
  • Author(s): Sato N et al.
  • Journal Title: J Exp Med 200・5 Pages: 601-611
• [Book] Arthritis Research : Methods and Protocols (2005)
  • Author(s): Takahashi N et al.
  • Publisher: Humana Press, Totowa, New Jersey (in press)
• [Publications] Suda K.et al.: "Suppression of osteoprotegerin expression by prostaglandin E_2 is crucially involved in LPS-induced osteoclast formation."Journal of Immunology. 172・4. 2504-2510 (2004)
• [Publications] Nakamura M.et al.: "Osteoprotegerin regulates bone formation through a coupling mechanism with bone resortion."Endocrinology. 144・12. 5441-5449 (2003)
• [Publications] Li X.et al.: "p38 MAPK is crucially involved in osteoclast differentiation but not in cytokine production, phagocytosis or dendritic cell differentiation of bone marrow macrophages."Endocrinology. 144・11. 4999-5005 (2003)
• [Publications] Itoh K.et al.: "LPS promotes the survival of osteoclasts via toll-like receptor 4,but cytokine production of osteoclasts in response to LPS is different from that of macrophages."Journal of Immunology. 170・7. 3688-3695 (2003)
• [Publications] Nakagawa H.et al.: "Destruxins, cyclodepsipeptides, block the formation of actin rings and prominent clear zones and ruffled borders in osteoclasts."Bone. 33・3. 443-455 (2003)
• [Publications] Takami M.et al.: "Involvement of vacuolar H+-ATPase in incorporation of risedronate into osteoclasts."Bone. 32・4. 341-349 (2003)
• [Publications] Takahashi N.et al.: "Bone Research Protocols"Humana Press Inc. 448 (2003)
• [Publications] Li X et al.: "p38 MAPK is crucially involved in osteoclast differentiation but not in cytokine production, phagocytosis or dendritic cell differentiation of bone marrow macrophages"Endocrinology. (in press). (2003)
• [Publications] Itoh K et al.: "Lipopolysaccharide promotes the survival of osteoclasts via toll-like receptor 4, but cytokine production of osteoclasts in response to lipopolysaccharide is different from that of macrophages"J Immunol. (in press). (2003)
• [Publications] Takami M et al.: "Involvement of vacuolar H^+ -ATPase in incorporation of risedronate into osteoclasts"Bone. (in press). (2003)
• [Publications] Nakagawa H et al.: "Novel anti-resorptive reagents, destruxins reversibly block poralization of osteoclasts"Bone. (in press). (2003)
• [Publications] Li X et al.: "p38 MAP kinase-mediated signals are required for inducing osteoclast differentiation but not for osteoclast function"Endocrinology. 143. 3105-3113 (2002)
• [Publications] Udagawa N et al.: "The molecular mechanism of osteoclastogenesis in rheumatoid arthritis"Arthritis Res. 4. 281-289 (2002)
• [Publications] Takahashi N et al.: "Principles of Bone Biology, Second Edition, Volume 1"Academic press. 882 (2002)
• [Publications] Kotake S, Udagawa N, Hakoda M, Mogi M, Yano K, Tsuda E, Takahashi K, Furuya T, Ishiyama S, Kim KJ, Saito S, Nishikawa T, Takahashi N, Togari A, Tomatsu T, Suda T, Kamatani N: "Activated human T cells directly induce osteoclastogenesis from human monocytes : possible role of T cells in bone destruction in rheumatoid arthritis patients"Arthritis Rheum. 44. 1003-1012 (2001)
• [Publications] Itoh K, Udagawa N, Katagiri T, Iemura S, Ueno N, Yasuda H, Higashio K, Quinn JMW, Gillespie MT, Martin TJ, Suda T, Takahashi N: "Bone morphogenetic protein 2 stimulates osteoclast differentiation and survival supported by receptor activator of nuclear factor-kB ligand"Endocrinology. 142. 3656-3662 (2001)
• [Publications] Quinn JM, Itoh K, Udagawa N, Hausler K, Yasuda H, Shima N, Mizuno A, Higashio K, Takahashi N, Suda T, Martin TJ, Gillespie MT: "Transforming growth factor b effects on osteoclast differentiation via direct and indirect actions"J. Bone Miner. Res.. 16. 1787-1794 (2001)
• [Publications] Yanai T, Katagiri T, Akiyama S, Imada M, Yamashita T, Chiba H, Takahashi N, Suda T: "Expression of mouse osteocalcin transcripts, OG1 and OG2, is differently regulated in bone tissues and osteoblast cultures"J. Bone Mineral Metab.. 19. 345-351 (2001)
• [Publications] Katagiri T, Takahashi N: "Regulatory mechanisms of osteoblast and osteoclast differentiation"Oral Diseases. (in press). (2002)
• [Publications] Takahashi N, Udagawa N, Takami N, Suda T: "Principles of Bone Biology, ed by Raisz LG, Rodan GA, Bilezikian JP [Cells of bone : Osteoclast generation. pp109-126]"Academic Press. 1695 (2002)
• [Publications] Itoh K et al.: "Importance of membrane- or matrix-associated forms of M-CSF and RANKL/ODF in osteoclastogenesis supported by SaOS-4/3 cells expressing recombinant PTH/PTHrP Receptors."J.Bone Miner.Res.. 15. 1766-1775 (2000)
• [Publications] Takami M et al.: "Intracellular calcium and protein kinase C mediate expression of receptor activator of NF-κB ligand (RANKL) and osteoprotegerin in osteoblasts."Endocrinology. 141. 4711-4719 (2000)
• [Publications] Udagawa N, et al.: "Osteoprotegerin produced by osteoblasts in an important regulator in osteoclast development and function."Endocrinology. 141. 3478-3484 (2000)
• [Publications] Tsurukai T, et al.: "The Essential role of macrophage-colony stimulating factor and osteoclast differentiation factor in osteoclastogenesis."J.Bone Miner.Metab.. 18. 177-184 (2000)
• [Publications] Kobayashi K, et al.: "Tumor necrosis factor α stimulates osteoclast differentiation by a mechanism independent of the ODF/RANKL-RANK interaction."J.Exp.Med.. 191. 275-286, (2000)
• [Publications] Kotake S et al.: "Activated human T cells directly induce osteoclastogenesis from human monocytes : possible role of T cells in bone destruction in rheumatoid arthritis patients."Arthritis Rheum. (in press). (2001)
• [Publications] 高橋直之(共著): "歯科医学2 歯周病-新しい治療を求めて"先端医療技術研究所. 536 (2000)