Regulation of osteoclast apoptosis and motility by small GTPase binding protein Rac1.
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.


BACKGROUND
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.


METHODS
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.


RESULTS
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.


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.
Fukuda
Akira
A
Department of Orthopaedic Surgery, Faculty of Medicine, The University of Tokyo, Japan.
Hikita
Atsuhiko
A
Wakeyama
Hidetoshi
H
Akiyama
Toru
T
Oda
Hiromi
H
Nakamura
Kozo
K
Tanaka
Sakae
S
D000199:Actins / Q000378:metabolism; D000256:Adenoviridae / Q000235:genetics; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D000831:Animals, Newborn; D017209:Apoptosis / Q000502:physiology*; D001854:Bone Marrow Cells / Q000166:cytology; D001862:Bone Resorption / Q000378:metabolism; D002465:Cell Movement / Q000502:physiology*; D002470:Cell Survival / Q000187:drug effects / Q000235:genetics; D002478:Cells, Cultured; D002867:Chromones / Q000494:pharmacology; D018920:Coculture Techniques; D004791:Enzyme Inhibitors / Q000494:pharmacology; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D016173:Macrophage Colony-Stimulating Factor / Q000494:pharmacology; D008297:Male; D051379:Mice; D009025:Morpholines / Q000494:pharmacology; D009479:Neuropeptides / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D010010:Osteoclasts / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation / Q000187:drug effects; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism; D016186:Receptor, Macrophage Colony-Stimulating Factor / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D012886:Skull / Q000166:cytology; D014162:Transfection; D000077191:Wortmannin; D020764:cdc42 GTP-Binding Protein / Q000235:genetics / Q000378:metabolism; D020744:rac GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D020830:rac1 GTP-Binding Protein; D020742:rhoA GTP-Binding Protein / Q000235:genetics / Q000378:metabolism
D000199:Actins; D000730:Androstadienes; D002867:Chromones; D004791:Enzyme Inhibitors; D009025:Morpholines; D009479:Neuropeptides; D000081082:Phosphoinositide-3 Kinase Inhibitors; C484602:Rac1 protein, mouse; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D016173:Macrophage Colony-Stimulating Factor; D016186:Receptor, Macrophage Colony-Stimulating Factor; D051057:Proto-Oncogene Proteins c-akt; D020764:cdc42 GTP-Binding Protein; D020744:rac GTP-Binding Proteins; D020830:rac1 GTP-Binding Protein; D020742:rhoA GTP-Binding Protein; D000077191:Wortmannin