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.

Introduction: 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.

Materials and 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.