Shimoyama Atsuko, Wada Masahiro, Ikeda Fumiyo, Hata Kenji, Matsubara Takuma, Nifuji Akira, Noda Masaki, Amano Katsuhiko, Yamaguchi Akira, Nishimura Riko, Yoneda Toshiyuki
The Department of Molecular and Cellular Biochemistry, Graduate School of Dentistry, Osaka University, 1-8 Yamadaoka, Suita, Osaka 565-0871, Japan.
Mol Biol Cell. 2007 Jul;18(7):2411-8. doi: 10.1091/mbc.e06-08-0743. Epub 2007 Apr 18.
Genetic and cell biological studies have indicated that Indian hedgehog (Ihh) plays an important role in bone development and osteoblast differentiation. However, the molecular mechanism by which Ihh regulates osteoblast differentiation is complex and remains to be fully elucidated. In this study, we investigated the role of Ihh signaling in osteoblast differentiation using mesenchymal cells and primary osteoblasts. We observed that Ihh stimulated alkaline phosphatase (ALP) activity, osteocalcin expression, and calcification. Overexpression of Gli2- but not Gli3-induced ALP, osteocalcin expression, and calcification of these cells. In contrast, dominant-negative Gli2 markedly inhibited Ihh-dependent osteoblast differentiation. Ihh treatment or Gli2 overexpression also up-regulated the expression of Runx2, an essential transcription factor for osteoblastogenesis, and enhanced the transcriptional activity and osteogenic action of Runx2. Coimmunoprecipitation analysis demonstrated a physical interaction between Gli2 and Runx2. Moreover, Ihh or Gli2 overexpression failed to increase ALP activity in Runx2-deficient mesenchymal cells. Collectively, these results suggest that Ihh regulates osteoblast differentiation of mesenchymal cells through up-regulation of the expression and function of Runx2 by Gli2.
遗传学和细胞生物学研究表明，印度刺猬因子（Ihh）在骨骼发育和成骨细胞分化中发挥重要作用。然而，Ihh调节成骨细胞分化的分子机制较为复杂，仍有待充分阐明。在本研究中，我们利用间充质细胞和原代成骨细胞研究了Ihh信号在成骨细胞分化中的作用。我们观察到，Ihh刺激碱性磷酸酶（ALP）活性、骨钙素表达和钙化。Gli2过表达而非Gli3过表达可诱导这些细胞的ALP、骨钙素表达和钙化。相反，显性负性Gli2显著抑制Ihh依赖的成骨细胞分化。Ihh处理或Gli2过表达还上调了Runx2的表达，Runx2是成骨细胞生成所必需的转录因子，并增强了Runx2的转录活性和成骨作用。免疫共沉淀分析表明Gli2与Runx2之间存在物理相互作用。此外，Ihh或Gli2过表达未能增加Runx2缺陷间充质细胞中的ALP活性。总的来说，这些结果表明，Ihh通过Gli2上调Runx2的表达和功能来调节间充质细胞的成骨细胞分化。
