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Molecular biological studies on the bioactive substances related to bone inflammation

引地, 尚子 東京大学 DOI:10.15083/0002006122

2023.03.20

概要

論文審査の結果の要旨
氏名

引地

尚子

骨は硬組織であるが静的な組織ではなく、動的代謝を行っている組織で、
常にリモデリングを繰り返している。論文提出者は、骨代謝研究のうち特に
骨の炎症機構の解明に取り組み、新たな研究成果を得た。本論文は3章から
なる。
第1章では、一酸化窒素(NO)の骨芽細胞分化と骨炎症における役割につ
いて解析した。マウス初代骨芽細胞において、炎症性サイトカイン TNF-α と
IL-1β は、構成的に発現する血管内皮細胞一酸化窒素(NO)合成酵素遺伝子
発現に影響しなかったが、誘導 NO 合成酵素遺伝子発現を増加した。しかし、
骨芽細胞分化指標であるアルカリホスファターゼ(ALPase)活性の減少は
NOS 阻害剤 L-NMMA によって回復しなかった。さらに、NO ドナーの投与
により ALPase 活性と同じく骨芽細胞分化指標である osteocalcin 遺伝子発現
は増加した。以上より、NO が骨芽細胞の分化を直接促進すること、サイト
カインによる ALPase 活性の抑制は NO 以外のメカニズムによることが示さ
れた。本研究で初めて、NO そのものは骨芽細胞の分化、すなわち骨形成作
用につながる現象を引き起こすことが示された。その後、マウス骨芽細胞様

細胞(MC3T3-E1)を用いて、iNOS 誘導に起因する炎症性変化は、産生され
た NO そのものによるものではなく、NO と炎症の際に産生されるラジカル
であるスーパーオキサイドとの相互作用により最終的に産生されるパーオ
キシナイトライトによるものであることが示された。以上より、NO は生理
的な骨代謝にも関与し、骨の炎症にも関与していることが示された。
第2章では、骨と生理活性脂質の関係について解析した。骨代謝領域にお
いては、PGE2 以外の生理活性脂質はほとんどその機能が検討されていなか
った。そこで、ロイコトリエン B4(LTB4)について検討し、LTB4 が骨吸収
機構に大きく関与していることを明らかにした。代表的 LTB4 レセプターで
ある BLT1 の遺伝子欠損マウスを用いて骨吸収モデルを作成し、骨吸収にお
ける BLT1 の役割を検討した。骨密度、骨塩量、骨形態計測パラメーターの
測定により、BLT1 遺伝子欠損マウスでは骨吸収が軽減されることが示され
た。また、BLT1 遺伝子欠損マウス由来の破骨細胞は、野生型マウス由来破
骨細胞と比較してカルシウム吸収活性が減弱していることも示された。なお
野生型マウス由来破骨細胞では別の LTB4 レセプターBLT2 は検知できなか
ったが、BLT1 遺伝子は発現しており、オートクリン/パラクリンに LTB4 を
産生していた。さらに、LTB4 は、BLT1-Gi タンパク質-Rac1 シグナリング経
路を通じて、破骨細胞の細胞形態を変化させた。破骨細胞形態と骨吸収活性

の間に関係があるとすれば、破骨細胞から生じたオートクリン/パラクリン
LTB4 が BLT1-Gi タンパク質-Rac1 シグナリング経路を通じて骨吸収活性を
増加することが示唆された。
第3章では骨と G タンパク質共役受容体(GPCR)の関係について調べた。
生理活性脂質の受容体の多くは GPCR である。骨代謝において破骨細胞が産
生した酸が骨基質を溶解することはよく知られているが、骨代謝のプロトン
感知受容体の機能は十分に検討されていない。T-cell death-associated gene 8

(TDAG8)は GPCR でプロトン感知受容体である。そこで、骨吸収過程で
TDAG8 がプロトン感知受容体として関与するか否かを検討した。すなわち
TDAG8 遺伝子ノックダウン(KD)マウスの骨塩量および骨形態計測パラメ
ーターの測定により、病的骨吸収はより増加することが示された。また、KD
マウス由来破骨細胞は野生型マウス由来破骨細胞よりカルシウム吸収活性
が増強していることも示された。酸性環境で破骨細胞形成は通常阻害される
が、KD マウス由来破骨細胞では阻害されなかった。酸性環境は、TDAG8Rho signaling 経路によりその細胞形態を変化させた。以上より、TDAG8 は
骨吸収を抑制して、骨代謝を維持するのに役立っていることが示唆された。
論文提出者は骨炎症機構に関わる分子の研究を行い、骨代謝における役割
を明らかにした。骨以外の組織でその重要性が知られている分子でも、骨代

謝学の領域ではあまり検討されていないことも多い。これらの骨炎症の機構
の解明は、将来的に骨炎症性疾患の治療にも寄与すると思われる。
なお、本論文第1章は、申偉秀、豊岡照彦らとの、第2章と第3章は清水
孝雄、石井聡らとの共同研究であるが、論文提出者が主体となって分析及び
検証を行ったもので、論文提出者の寄与が十分であると判断する。したがっ
て、博士(理学)の学位を授与できると認める。

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