Regulation of Osteoclast Apoptosis and Motility by Small GTPase Binding Protein Rac1

• Published in: Journal of bone and mineral research Vol. 20; no. 12; pp. 2245 - 2253
• Main Authors: Fukuda, Akira, Hikita, Atsuhiko, Wakeyama, Hidetoshi, Akiyama, Toru, Oda, Hiromi, Nakamura, Kozo, Tanaka, Sakae
• Format: Journal Article
• Language: English
• Published: Washington, DC John Wiley and Sons and The American Society for Bone and Mineral Research (ASBMR) 01.12.2005; American Society for Bone and Mineral Research
• Subjects: Actins - metabolism; Adenoviridae - genetics; Adenovirus; Akt; Androstadienes - pharmacology; Animals; Animals, Newborn; apoptosis; Apoptosis - physiology; Biological and medical sciences; Bone Marrow Cells - cytology; Bone Resorption - metabolism; cdc42 GTP-Binding Protein - genetics; cdc42 GTP-Binding Protein - metabolism; Cell Movement - physiology; Cell Survival - drug effects; Cell Survival - genetics; Cells, Cultured; Chromones - pharmacology; Coculture Techniques; Enzyme Inhibitors - pharmacology; Fundamental and applied biological sciences. Psychology; Green Fluorescent Proteins - genetics; Green Fluorescent Proteins - metabolism; Macrophage Colony-Stimulating Factor - pharmacology; macrophage colony‐stimulating factor; Male; Mice; Morpholines - pharmacology; Neuropeptides - genetics; Neuropeptides - metabolism; Neuropeptides - physiology; osteoclast; Osteoclasts - cytology; Osteoclasts - drug effects; Osteoclasts - physiology; Phosphatidylinositol 3-Kinases - antagonists & inhibitors; Phosphorylation - drug effects; Proto-Oncogene Proteins c-akt - genetics; Proto-Oncogene Proteins c-akt - metabolism; rac GTP-Binding Proteins - genetics; rac GTP-Binding Proteins - metabolism; rac GTP-Binding Proteins - physiology; Rac1; rac1 GTP-Binding Protein; Receptor, Macrophage Colony-Stimulating Factor - physiology; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; rhoA GTP-Binding Protein - genetics; rhoA GTP-Binding Protein - metabolism; Signal Transduction - drug effects; Signal Transduction - physiology; Skeleton and joints; Skull - cytology; Transfection; Vertebrates: osteoarticular system, musculoskeletal system; Motility; Enzyme; Triphosphoric monoester hydrolases; Rodentia; Resorption; dGTPase; Esterases; Osteoclast; Akt; Osteoarticular system; Binding protein; Vertebrata; Mammalia; Mouse; Hydrolases; Bone; Macrophage colony stimulating factor; Rac1; macrophage colony-stimulating factor; Apoptosis
• ISSN: 0884-0431; 1523-4681
• DOI: 10.1359/JBMR.050816

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. 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 osteoclast survival and function are not fully elucidated. Materials and Methods: We constructed the adenovirus vector carrying cDNA of either the dominant negative Rac1 (Rac1DN) or constitutively active Rac1 (Rac1CA) 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 Rac1DN 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. M‐CSF rapidly activated Rac1, and the prosurvival effect of M‐CSF for OCLs was abrogated by Rac1DN 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. Rac1DN also partially blocked the activation of Akt induced by the overexpressing catalytic subunit of PI3K. Using time‐lapse video microscopy, we found that Rac1DN expression reduced membrane ruffling and the spreading of OCLs in response to M‐CSF. Conclusions: 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.