Takeyama Kazuhiro, Chatani Masahiro, Inohaya Keiji, Kudo Akira
Department of Biological Information, Tokyo Institute of Technology, Yokohama 226-8501, Japan; Japan Society for the Promotion of Science, Tokyo 102-8472, Japan.
Department of Biological Information, Tokyo Institute of Technology, Yokohama 226-8501, Japan.
Bone. 2016 May;86:68-78. doi: 10.1016/j.bone.2016.03.001. Epub 2016 Mar 4.
TGFβ is known as a canonical coupling factor based on its effects on bone formation and bone resorption. There are 3 different isoforms of it related to bone metabolism in mammals. TGFβ function in vivo is complicated, and each isoform shows a different function. Since TGFβs are secreted during inflammation accompanied by the release of latent TGFβ from inside of the bones where they are stored in the extracellular matrix, TGFβ function is potentially related to fracture healing. Although a few reports examined the TGFβ expression during fracture healing, the function of TGFβ in this process is poorly understood. To investigate TGFβ function during fracture healing in vivo, we used the fracture healing model of the medaka fish, which enabled us to observe the behavior and function of living cells in response to a bone-specific injury. RNA in-situ hybridization analysis showed that only tgfβ-2 of the 4 TGFβ isoforms in medaka was expressed in the bone-forming region. To examine the TGFβ-2 function for bone formation by osteoblasts, we used a medaka transgenic line, Tg (type X collagen: GFP); and the results revealed that type X collagen-positive immature osteoblasts migrated to the fracture site and differentiated to osterix-positive osteoblasts. However, only a few type X collagen-positive osteoblasts exhibited BrdU incorporation after the fracture. Then we inhibited TGFβ signaling by using a chemical TGFβ receptor kinase inhibitor (SB431542), and demonstrated that inhibition of TGFβ strongly impaired osteoblast migration and differentiation. In addition, this TGFβ inhibitor reduced the RANKL expression and caused a delay of osteoclast differentiation. Our findings thus demonstrated that TGFβ-2 functioned specifically during fracture healing to stimulate the migration of osteoblasts as well as the differentiation of osteoblasts and osteoclasts.
基于其对骨形成和骨吸收的影响，转化生长因子β（TGFβ）被认为是一种典型的耦合因子。在哺乳动物中，有3种与骨代谢相关的不同亚型。TGFβ在体内的功能很复杂，每种亚型都表现出不同的功能。由于TGFβ在炎症期间分泌，同时伴有储存于细胞外基质中的潜伏TGFβ从骨内部释放，因此TGFβ的功能可能与骨折愈合有关。尽管有一些报告研究了骨折愈合过程中TGFβ的表达，但对TGFβ在此过程中的功能了解甚少。为了研究体内骨折愈合过程中TGFβ的功能，我们使用了青鳉鱼的骨折愈合模型，这使我们能够观察活细胞对骨特异性损伤的反应行为和功能。RNA原位杂交分析表明，青鳉鱼4种TGFβ亚型中只有tgfβ - 2在骨形成区域表达。为了研究TGFβ - 2对成骨细胞骨形成的功能，我们使用了青鳉转基因品系Tg（X型胶原蛋白：绿色荧光蛋白）；结果显示，X型胶原蛋白阳性的未成熟成骨细胞迁移到骨折部位并分化为osterix阳性的成骨细胞。然而，骨折后只有少数X型胶原蛋白阳性的成骨细胞表现出BrdU掺入。然后我们使用化学TGFβ受体激酶抑制剂（SB431542）抑制TGFβ信号传导，并证明抑制TGFβ会严重损害成骨细胞的迁移和分化。此外，这种TGFβ抑制剂降低了RANKL的表达并导致破骨细胞分化延迟。因此，我们的研究结果表明，TGFβ - 2在骨折愈合过程中具有特异性功能，可刺激成骨细胞的迁移以及成骨细胞和破骨细胞的分化。
