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

简介：Rac 1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Rac 1是破骨细胞的细胞骨架组织和骨吸收活性所必需的，但它们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：我们构建了携带显性失活Rac 1（Rac 1DN）和组成型活性Rac 1（Rac 1CA）基因cDNA的腺病毒载体，用这些病毒感染小鼠共培养系统中产生的破骨细胞样细胞（OCL）。为了研究Rac 1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进行了陷窝形成测定、存活测定和蛋白质印迹法，包括活化的Rac 1下拉测定。为了进一步阐明Rac 1调控破骨细胞存活的机制，我们使用了一些特异性的抑制剂和信号转导分子的腺病毒载体。到 ...更多信息 结果：腺病毒载体介导的显性负性Rac 1（Rac 1DN）表达可显著减少空泡形成，促进空泡凋亡。巨噬细胞集落刺激因子（M-CSF）迅速激活Rac 1，M-CSF对OCL的促生存作用被Rac 1 DN过表达所废除。组成型活性Rac 1增强OCL存活，这被磷脂酰肌醇3 '-激酶（PI 3 K）抑制剂完全抑制，而Mek抑制剂仅具有部分作用。Rac 1DN还部分阻断过表达PI 3 K催化亚基诱导的Akt活化。结论：小G蛋白Rac 1参与了M-CSF受体信号转导，主要通过调节PI 3 K/Akt信号通路介导破骨细胞的生存信号。Rac 1可能通过调节破骨细胞的运动性，在细胞的骨吸收活性中也起重要作用。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