Cbfal遺伝子を用いた骨・軟骨再生の為の骨髄由来間葉系幹細胞の開発

2001 Fiscal Year Annual Research Report

• Project/Area Number: 12557123
• Research Institution: Tokyo Dental and Medical University
• Principal Investigator: 野田 政樹 東京医科歯科大学, 難治疾患研究所, 教授 (50231725)
• Co-Investigator(Kenkyū-buntansha): 辻 邦和 東京医科歯科大学, 難治疾患研究所, 助手 (20323694) ; 二藤 彰 東京医科歯科大学, 難治疾患研究所, 助教授 (00240747)
• Keywords: Cbfa1 / 骨芽細胞 / プロモーター / CIZ / 転写 / 細胞接着 / 骨再生

Research Abstract

本年においては、Runx2のプロモーター領域の活性について骨芽細胞における機能解析を行った。骨・軟骨再生のためには、細胞の接着性が重要であり、基質の上で骨芽細胞が接着に際して必要とする接着班の形成に重要な役割をするCrkの基質であるp130Casに対する結合分子としてクローニングされたCIZ(CASインタラクティングジンクシンガープロテイン)の機能解析を行い、CIZが単独で骨芽細胞の分化決定因子のRunx2(Cbfa1)mRNAの発現を促進すること。またこの活性がRunx2のCbfa1の1.8kbのプロモーターに対する促進活性によっていることを明らかにした。次にCbfa1のプロモーター上に存在するSmadの応答配列について検討し、BMPがSmadを介して骨芽細胞の分化を制御する際にSmadがCbfa1プロモーターに結合して、その転写活性を抑制することを明らかにした。BMPはCbfa1mRNAの発現を抑制し、このことはCbfa1プロモーターに対するBMPの抑制活性に基づくと考えられた。更にノーザンブロットレベルでのCbfa1mRNAレベルをBMPが抑制する活性に対し、CIZの共発現がこれを更に阻止することを明らかにした。また、プロモーター活性の検討からBMPのSmadを介する転写促進活性に対してCbfa1が更にこれを抑止する活性を持つことを明らかにした。以上の結果からCbfa1プロモーターが骨・軟骨再生の為の細胞の活性に際し、主要な役割をすること、更に新しい細胞接着に関わる分子、CIZがCbfaのプロモーター活性の制御に当たることを明らかにし、今後の再生のための間葉系細胞の開発の上でこれらの細胞接着に関わる機能の分子機構が明らかとなった。

Research Products

• [Publications] Ishijima M: "Enhancement of osteoclastic bone resorption and suppression of osteoblastic bone formation in response to reduced mechanical stress do not occur in the absence of osteopontin"Journal of Experimental Medicine. 19. 399-404 (2001)
• [Publications] Ihara H: "Enomoto S, Nifuji A, Rittling SR, and Noda M Parathyroid hormone- induced bone resorption does not occur in the absence of osteopontin"Journal of Biological Chemistry. 276. 3065-3071 (2001)
• [Publications] Tsuji K: "Transient suppression of core-binding factor alpha 1 expression by basic fibroblast growth factor in rat osteoblast-like osteosarcoma ROS17/2.8 cells"Journal of Bone Miner Metabolism. 19. 213-219 (2000)
• [Publications] Tsuji K: "Vitamin D_3 enhances the expression of l-mfa, an inhibitor of the MyoD family, in osteoblasts"Biochemica et Biophysica Acta. 1539. 122-130 (2001)
• [Publications] Asou Y: "Osteopontin facilitates angiogenesis, accumulation of osteoclasts and resorption in ectopic bone"Endocrinology. 142. 1325-1332 (2001)
• [Publications] Nemoto H: "Osteopontin deficiency reduces experimental tumor cell metastasis to bone and soft tissues"Journal of Bone and Mineral Research. 6. 652-659 (2001)
• [Publications] Yumoto K: "Osteopontin deficiency protects joints against destruction in anti-type II collagen antibody-induced arthritis in mice"Proceeding of the National Academy of Sciences USA. (in press). (2002)
• [Publications] Usui M.: "Enhancing effect of Tob deficiency on bone formation is specific to BMP-induced osteogenesis"Journal of Bone and Mineral Research. (in press). (2002)
• [Publications] Ishijima M.: "Resistance to unloading-induced three-dimensional bone loss in osteopontin-deficient mice"Journal of Bone and Mineral Research. (in press). (2002)
• [Publications] Yamashita T: "Klotho-deficient mice are resistant to bone loss induced by unloading due to sciatic neurectomy"Journal of Endocrinology.
• [Publications] Kawaguchi N: "Spontaneous mutation in Mitf gene causes osteopetrosis in silver homozygote quail"Developmental Dynamics. 220. 133-140 (2001)
• [Publications] Sekiya I: "Transcriptional suppression of SOX9 expression by retinoic acid in chondrocytes"Journal of Cellular Biochemistry. S36. 71-78 (2001)
• [Publications] Sekiya I: "Dexamethasone enhances Sox9 expression in chondrocytes"Journal of Endocrinology. 169. 573-579 (2001)
• [Publications] Denhardt, DT: "Osteopontin as a means to cope with environmental insults: regulation of inflammation, tissue remodeling, and cell survival"Journal of Clinical Investigation. 104. 1055-1061 (2001)
• [Publications] Yoshida Y: "Negative regulation of BMP/Smad signaling by Tob in osteoblasts"Cell. 103. 1085-1097 (2000)
• [Publications] Yamashita T: "Retardation in bone resorption after bone marrow ablation in klotho mutant mice"Endocrinology. 141. 438-445 (2000)
• [Publications] Sekiya I: "OX9 enhances aggrecan gene promoter/enhancer activity and is upregulated by Retinoic Acid in a Cartilage-Derived Cell Line, TC6"Journal of Biological Chemistry. 275. 10738-10744 (2000)
• [Publications] Yamashita T: "High-resolution uCT (micro-computed tomography) analyses of the abnormal trabecular bone structures in klotho gene mutant mice"Journal of Endocrinology. 164. 239-245 (2000)
• [Publications] Murakami S: "Indian hedgehog during fracture healing in adult rat femora"Calcified Tissue International. 66. 272-276 (2000)
• [Publications] Takazawa Y: "CBFA1, is constitutively expressed in chondrocytic cell line, TC6, and its expression is upregulated by BMP2"Journal of Endocrinology. 162. 579-586 (2000)
• [Publications] Tsuji K: "Identification and Expression of a novel 3´-exon of mouse Runx1/Pebp2 α B/Cbfa2/AML1 gene"Biochemical and Biophysical Research Communications. 274. 171-176 (2000)
• [Publications] Kawaguchi N: "Mitf is expressed in osteoclast progenitors in vitro"Experimental Cell Research. 260. 284-291 (2000)
• [Publications] Furuya K: "Overexpression of Cas-interactiong zinc finger protein (CIZ) suppresses proliferation and enhances expression of type I collagen gene in osteoblast-like MC3T3E1 cells"Experimental Cell Research. 261. 329-335 (2000)
• [Publications] Shiraki-Iida T: "Improvement of multiple pathophysiological phenotypes of klotho (kl/kl) mice by adenovirus-mediated expression of the klotho gene"Journal of Gene Med. 2. 233-242 (2000)