Fukuda Akira, Hikita Atsuhiko, Wakeyama Hidetoshi, Akiyama Toru, Oda Hiromi, Nakamura Kozo, Tanaka Sakae
Department of Orthopaedic Surgery, Faculty of Medicine, The University of Tokyo, Japan.
J Bone Miner Res. 2005 Dec;20(12):2245-53. doi: 10.1359/JBMR.050816. Epub 2005 Aug 22.
The role of Rac1 in osteoclast survival and bone-resorbing activity was examined using adenovirus vector expression systems. Rac1 is critically involved in M-CSF receptor signaling and mediates survival signaling primarily through PI3K/Akt pathways. Rac1 also plays a significant role in bone resorptive activity, probably by regulating the motility of osteoclasts.
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 osteoclast survival and function are not fully elucidated.
We constructed the adenovirus vector carrying cDNA of either the dominant negative Rac1 (Rac1(DN)) 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 assays, survival assays, and Western blotting, including an 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 quantify membrane movement before and after macrophage colony-stimulating factor (M-CSF) treatment, OCLs expressing either enhanced green fluorescent protein (EGFP) or Rac1(DN) were recorded with a time-lapse video microscope.
Adenovirus vector-mediated dominant negative Rac1 (Rac1(DN)) expression significantly reduced pit formation, and promoted their apoptosis. M-CSF rapidly activated Rac1, and the prosurvival 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, whereas a Mek inhibitor had only partial effect. Rac1(DN) also partially blocked the activation of Akt induced by the overexpressing catalytic subunit of PI3K. Using time-lapse video microscopy, we found that Rac1(DN) expression reduced membrane ruffling and the spreading of OCLs in response to M-CSF.
Small guanosine triphosphatase (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 the bone resorptive activity of cells, probably by regulating the motility of osteoclasts.
使用腺病毒载体表达系统研究了Rac1在破骨细胞存活和骨吸收活性中的作用。Rac1在M-CSF受体信号传导中起关键作用，主要通过PI3K/Akt途径介导存活信号。Rac1在骨吸收活性中也起重要作用，可能是通过调节破骨细胞的运动性。
Rac1是Rho家族小G蛋白的成员，最近的研究表明它在某些类型的细胞中介导抗凋亡信号。据报道，Rac1是破骨细胞细胞骨架组织和骨吸收活性所必需的，但其在破骨细胞存活和功能中的作用尚未完全阐明。
我们构建了携带显性负性Rac1（Rac1(DN)）或组成型活性Rac1（Rac1(CA)）基因cDNA的腺病毒载体，并用这些病毒感染在小鼠共培养系统中产生的破骨细胞样细胞（OCLs）。为了研究Rac1在破骨细胞存活和功能中的作用，我们进行了蚀斑形成试验、存活试验和蛋白质印迹分析，包括使用腺病毒感染的OCLs进行的活化Rac1下拉试验。为了进一步阐明Rac1调节破骨细胞存活的机制，使用了一些信号转导分子的特异性抑制剂和腺病毒载体。为了量化巨噬细胞集落刺激因子（M-CSF）处理前后的膜运动，用延时视频显微镜记录表达增强型绿色荧光蛋白（EGFP）或Rac1(DN)的OCLs。
腺病毒载体介导的显性负性Rac1（Rac1(DN)）表达显著减少蚀斑形成，并促进其凋亡。M-CSF迅速激活Rac1，Rac1(DN)过表达消除了M-CSF对OCLs的促存活作用。组成型活性Rac1增强了OCLs的存活，磷脂酰肌醇3'-激酶（PI3K）抑制剂完全抑制了这种作用，而Mek抑制剂只有部分作用。Rac1(DN)也部分阻断了PI3K催化亚基过表达诱导的Akt激活。使用延时视频显微镜，我们发现Rac1(DN)表达减少了OCLs对M-CSF的膜皱襞和铺展。
小GTP酶（GTPase）Rac1在M-CSF受体信号传导中起关键作用，主要通过调节PI3K/Akt途径介导破骨细胞的存活信号。Rac1在细胞的骨吸收活性中也起重要作用，可能是通过调节破骨细胞的运动性。
