Abstract
Based on our earlier observation that caspase‐3 is present in osteoclasts that are not undergoing apoptosis, we investigated the role of this protein in the differentiation of primary osteoclasts and RAW264.7 cells (Szymczyk KH, et al., 2005, Caspase‐3 activity is necessary for RANKL‐induced osteoclast differentiation. The Proceedings of the 8th ICCBMT). We noted that osteoclast numbers are decreased in long bones of procaspase‐3 knockout mice and that receptor activator of NF‐κB ligand (RANKL) does not promote differentiation of isolated preosteoclasts. In addition, after treatment with inhibitors of caspase‐3 activity, neither the wild‐type primary nor the RAW264.7 cells express TRAP or became multinucleated. We found that immediately following RANKL treatment, procaspase‐3 is cleaved and the activated protein is localized to lipid regions of the plasma membrane and the cytosol. We developed RAW264.7 procaspase‐3 knockdown clonal cell lines using RNAi technology. Again, treatment with RANKL fails to induce TRAP activity or multinucleation. Finally, we evaluated NF‐κB in procaspase‐3 silenced cells. We found that RANKL treatment prevented activation and nuclear translocation of NF‐κB. Together these findings provide direct support for the hypothesis that caspase‐3 activity is required for osteoclast differentiation. J. Cell. Physiol. 209: 836–844, 2006. © 2006 Wiley‐Liss, Inc.