Department of Orthopedics, First Affiliated Hospital, Shantou University Medical College, 57 Changping Road, Shantou, Guangdong 515041, China.
J Biol Chem. 2012 Jul 27;287(31):26200-12. doi: 10.1074/jbc.M112.349811. Epub 2012 May 31.
To search for factors promoting bone fracture repair, we investigated the effects of extracorporeal shock wave (ESW) on the adhesion, spreading, and migration of osteoblasts and its specific underlying cellular mechanisms. After a single period of stimulation by 10 kV (500 impulses) of shock wave (SW), the adhesion rate was increased as compared with the vehicle control. The data from both wound healing and transwell tests confirmed an acceleration in the migration of osteoblasts by SW treatment. RT-PCR, flow cytometry, and Western blotting showed that SW rapidly increased the surface expression of α5 and β1 subunit integrins, indicating that integrin β1 acted as an early signal for ESW-induced osteoblast adhesion and migration. It has also been found that a significant elevation occurred in the expression of phosphorylated β-catenin and focal adhesion kinase (FAK) at the site of tyrosine 397 in response to SW stimulation after the increasing expression of the integrin β1 molecule. When siRNAs of integrin α5 and β1 subunit were added, the level of FAK phosphorylation elevated by SW declined. Interestingly, the adhesion and migration of osteoblasts were decreased when these siRNA reagents as well as the ERK1/2 signaling pathway inhibitors, U0126 and PD98059, were present. Further studies demonstrated that U0126 could inhibit the downstream integrin-dependent signaling pathways, such as the FAK signaling pathway, whereas it had no influence on the synthesis of integrin β1 molecule. In conclusion, these data suggest that ESW promotes the adhesion and migration of osteoblasts via integrin β1-mediated expression of phosphorylated FAK at the Tyr-397 site; in addition, ERK1/2 are also important for osteoblast adhesion, spreading, migration, and integrin expression.
为了研究促进骨骨折修复的因素，我们研究了体外冲击波（ESW）对成骨细胞黏附、铺展和迁移的影响及其具体的细胞内机制。单次 10kV（500 次冲击）冲击波刺激后，与载体对照组相比，黏附率增加。划痕愈合和 Transwell 实验的数据均证实 SW 处理可加速成骨细胞的迁移。RT-PCR、流式细胞术和 Western blot 显示，SW 可迅速增加α5 和β1 亚基整联蛋白的表面表达，表明整联蛋白β1 作为 ESW 诱导成骨细胞黏附和迁移的早期信号。还发现，SW 刺激后，整合素β1 分子表达增加的同时，酪氨酸 397 处磷酸化 β-连环蛋白和粘着斑激酶（FAK）的表达也显著升高。当添加整合素α5 和β1 亚基的 siRNA 时，SW 升高的 FAK 磷酸化水平下降。有趣的是，当存在这些 siRNA 试剂以及 ERK1/2 信号通路抑制剂 U0126 和 PD98059 时，成骨细胞的黏附和迁移减少。进一步的研究表明，U0126 可以抑制整合素依赖性信号通路，如 FAK 信号通路，而对整合素β1 分子的合成没有影响。总之，这些数据表明，ESW 通过整合素β1 介导的 Tyr-397 位点磷酸化 FAK 的表达促进成骨细胞的黏附和迁移；此外，ERK1/2 对于成骨细胞的黏附、铺展、迁移和整合素表达也很重要。
