Study of the role of small G proteins on the activation and apoptosis of the osteoclast.

简介：Rac1是Rho家族小g蛋白中的一员，最近的研究发现它在某些类型的细胞中介导抗凋亡信号。据报道，破骨细胞的细胞骨架组织和骨吸收活性需要Rac1，但其在破骨细胞存活和功能中的作用尚未完全阐明。材料和方法：构建了携带显性阴性Rac1 （Rac1DN）基因和组成型活性Rac1 （Rac1 CA）基因cDNA的腺病毒载体，并在小鼠共培养体系中生成破骨细胞样细胞（ocl）。为了研究Rac1在破骨细胞存活和功能中的作用，我们使用腺病毒感染的ocl进行了坑形成实验、存活实验和Western blotting，包括激活的Rac1拉下实验。为了进一步阐明Rac1调控破骨细胞存活的机制，我们使用了一些特异性抑制剂和信号转导分子的腺病毒载体。为了进一步量化M-CSF处理前后的膜运动，用延时视频显微镜记录表达EGFP或Rac1 DN的ocl。结果：腺病毒介导的显性阴性Rac1 （Rac1DN）表达可显著减少细胞坑形成，促进细胞凋亡。巨噬细胞集落刺激因子（M-CSF）可快速激活Rac1，并且M-CSF对oclc的促生存作用因Rac1 DN过表达而消失。组成型活性Rac1提高了OCL的存活，而PI3K抑制剂完全抑制了OCL的存活，而Mek抑制剂仅部分抑制了OCL的存活。Rac1DN还部分阻断了PI3K催化亚基过表达诱导的Akt活化。通过延时视频显微镜，我们发现Rac1 DN的表达减少了M-CSF对ocl膜褶皱和扩散的反应。结论：小GTPase Rac1参与M-CSF受体信号通路，并主要通过调节PI3K/Akt通路介导破骨细胞的存活信号。Rac1在细胞的骨吸收活性中也起着重要作用，可能是通过调节破骨细胞的运动。少

Introduction : Rac1 is a member of Rho family small G-proteins and recent studies have revealed that it mediates anti-apoptotic signals in some types of cells. Rac1 is reported to be required for the cytoskeletal organization and bone-resorbing activity of osteoclasts, but their roles in the osteoclast survival and function are not fully elucidated yet.Materials and methods : We constructed the adenovirus vector carrying cDNA of either dominant negative Rac1 (Rac1DN) or constitutively active Rac1 (Rac1 CA) gene, and osteoclast-like cells (OCLs) generated in mouse co-culture system were infected with these viruses. To examine the role of Rac1 in osteoclast survival and function, we performed pit formation assay, survival assay and Western blotting including activated-Rac1 pull down assay using adenovirus-infected OCLs. To further clarify the mechanism of Rac1 regulation in osteoclast survival, some specific inhibitors and adenovirus vectors of signal transduction molecules were used. To … More quantify membrane movement before and after M-CSF treatment, OCLs expressing either EGFP or Rac1 DN were recorded with a time-lapse video-microscope.Results : Adenovirus vector-mediated dominant negative Rac1 (Rac1DN) expression significantly reduced pit formation, and promoted their apoptosis. Macrophage colony-stimulating factor (M-CSF) rapidly activated Rac1, and the pro-survival effect of M-CSF for OCLs was abrogated by Rac1 DN overexpression. Constitutively active Rac1 enhanced OCL survival, which was completely suppressed by phosphatidylinositol 3'-kinase (PI3K) inhibitors, while a Mek inhibitor had only partial effect. Rac1DN also partially blocked the activation of Akt induced by overexpressing catalytic subunit of PI3K. Using time-lapse video-microscopy, we found that Rac1 DN expression reduced the membrane ruffling and spreading of OCLs in response to M-CSF.Conclusion : Small GTPase Rac1 is critically involved in M-CSF receptor signaling, and mediates survival signaling of osteoclasts primarily by modulating PI3K/Akt pathways. Rac1 also plays a significant role in bone resorptive activity of the cells, probably by regulating the motility of osteoclasts. Less