Actin-related protein 2/3 complex is required for actin ring formation.
Actin rings are vital for osteoclastic bone resorption, and actin-related protein 2/3 complex is a pivotal regulator of actin polymerization. Actin-related protein 2/3 complex was found in the podosomes of actin rings. A short interfering RNA knocked down expression of actin-related protein 2 in osteoclasts and disrupted actin rings, suggesting that the complex is crucial to actin ring formation.


BACKGROUND
To resorb bone, osteoclasts form an extracellular acidic compartment segregated by a sealing zone. This is dependent on an actin ring that is composed of filamentous actin organized into dynamic structures called podosomes. The actin-related protein 2/3 (Arp2/3) complex is a vital regulator of actin polymerization. We tested whether the Arp2/3 complex is a component of actin rings and is important for actin ring formation.


METHODS
Western blot analysis was used to determine levels of Arp2 and Arp3, two components of the Arp2/3 complex in osteoclast-like cells. Confocal microscopy studies using antibodies for immunocytochemistry demonstrated localization of Arp2/3 complex in osteoclasts. Short interfering RNA oligonucleotides (siRNAs) were made against Arp2 and used to knock down its expression.


RESULTS
A 3-fold increase in Arp2 and Arp3 was detected during RANKL-induced differentiation of RAW 264.7 cells into osteoclast-like cells. Arp2/3 complex was concentrated in actin rings and enriched near the sealing zone. Arp2/3 complex co-localized with cortactin, a component of podosomes, but not vinculin, which surrounds podosomes. siRNA against Arp2, transfected into RAW 264.7 cells 5 days after stimulation with RANKL, reduced Arp2 protein levels 70% compared with cells transfected with ineffective siRNAs. Cytochemical characterization of RAW 264.7 osteoclast-like cells and marrow osteoclasts in which Arp2 was knocked down revealed fewer podosomes and no actin rings, although many cells remained well spread.


CONCLUSIONS
These data show that Arp2/3 complex is a component of actin rings and that the presence of Arp2/3 complex is vital to the formation of actin rings. In addition, the results show the use of siRNAs for the study of RAW 264.7 osteoclast-like cells.
Hurst
I Rita
IR
Department of Orthodontics, University of Florida College of Dentistry, Gainesville, Florida 32610-0444, USA.
Zuo
Jian
J
Jiang
Jin
J
Holliday
L Shannon
LS
D008841:Actin Cytoskeleton / Q000648:ultrastructure*; D051377:Actin-Related Protein 2; D051378:Actin-Related Protein 3; D000199:Actins / Q000378:metabolism; D001483:Base Sequence; D001862:Bone Resorption; D003598:Cytoskeletal Proteins / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D008969:Molecular Sequence Data; D010010:Osteoclasts / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D015854:Up-Regulation
C495926:ACTR2 protein, human; C495928:ACTR3 protein, human; D051377:Actin-Related Protein 2; D051378:Actin-Related Protein 3; D000199:Actins; D003598:Cytoskeletal Proteins; D034741:RNA, Small Interfering