Stomatological Hospital and State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing, China.
J Cell Physiol. 2010 Jun;223(3):610-22. doi: 10.1002/jcp.22052.
The objectives of this study were to investigate the early response to mechanical stress in neonatal rat mandibular chondrocytes by proteomic analysis. To evaluate its molecular mechanism, chondrocytes were isolated and cultured in vitro, then loaded mechanical stress by four-point bending system on different patterns. Morphological observation, flow cytometric analysis, and MTT assays indicated that 4,000 microstrain loading for 60 min was an appropriate mechanical stimulus for the following proteome analysis, which produced a transient but obvious inhibitory effect on the cell cycle. Therefore, we took a proteomic approach to identify significantly differential expression proteins in chondrocytes under this mechanical stress. Using 2-DE and MALDI-TOF, we identified seven differentially expressed proteins including the MAPK pathway inhibitor RKIP, cytoskeleton proteins, actin and vimentin, and other selected proteins. Some differentially expressed proteins were validated by both Western blot analysis and fluorescent staining of cytoskeleton at different loading times. The vimentin and RKIP responsive expression were also proven in vivo in oral orthopedic treatment rats, which was in line with the result in vitro. The histological changes in cartilage also showed the inhibition effect. Furthermore, the expressional level of phosphorylated ERK was increased, which demonstrates the changes in MAPK activity. Taken together, these data indicate that mechanical stress resulted in vimentin expression changes first and then led to the subsequent changes in actin expression, MAPK pathway regulated by RKIP and heat shock protein GRP75. All those changes contributed to the cytoskeleton remolding and cell cycle inhibition, finally led to condylar remodeling.
本研究旨在通过蛋白质组学分析探讨机械应力对新生大鼠下颌软骨细胞的早期应答。为了评估其分子机制，我们将软骨细胞分离并在体外培养，然后通过四点弯曲系统以不同模式加载机械应力。形态学观察、流式细胞分析和 MTT 测定表明，4000 微应变加载 60 分钟是进行后续蛋白质组分析的适当机械刺激，它对细胞周期产生短暂但明显的抑制作用。因此，我们采用蛋白质组学方法鉴定在这种机械应力下软骨细胞中差异表达的蛋白质。通过 2-DE 和 MALDI-TOF，我们鉴定了 7 种差异表达的蛋白质，包括 MAPK 通路抑制剂 RKIP、细胞骨架蛋白肌动蛋白和波形蛋白以及其他选定的蛋白质。一些差异表达的蛋白质通过 Western blot 分析和不同加载时间的细胞骨架荧光染色得到了验证。在口腔矫形治疗大鼠体内也证实了波形蛋白和 RKIP 的应答表达，与体外结果一致。软骨的组织学变化也显示出抑制作用。此外，磷酸化 ERK 的表达水平增加，表明 MAPK 活性发生变化。总之，这些数据表明，机械应力首先导致波形蛋白表达的变化，然后导致肌动蛋白表达的后续变化，受 RKIP 和热休克蛋白 GRP75 调节的 MAPK 通路。所有这些变化都导致细胞骨架重塑和细胞周期抑制，最终导致髁突重塑。
