Active caspase-3 is required for osteoclast differentiation.
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-kappaB 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-kappaB in procaspase-3 silenced cells. We found that RANKL treatment prevented activation and nuclear translocation of NF-kappaB. Together these findings provide direct support for the hypothesis that caspase-3 activity is required for osteoclast differentiation.
Szymczyk
K H
KH
Department of Orthopaedic Surgery, Thomas Jefferson University, 1015 Walnut Street, Philadelphia, PA 19107, USA.
Freeman
T A
TA
Adams
C S
CS
Srinivas
V
V
Steinbeck
M J
MJ
D000818:Animals; D017209:Apoptosis / Q000502:physiology; D053148:Caspase 3 / Q000235:genetics / Q000378:metabolism*; D061945:Caspase Inhibitors; D002454:Cell Differentiation / Q000502:physiology*; D002478:Cells, Cultured; D004789:Enzyme Activation; D021962:Membrane Microdomains / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D016328:NF-kappa B / Q000378:metabolism; D010010:Osteoclasts / Q000166:cytology / Q000502:physiology*; D010641:Phenotype; D011498:Protein Precursors / Q000378:metabolism; D053245:RANK Ligand / Q000235:genetics / Q000378:metabolism
D061945:Caspase Inhibitors; D016328:NF-kappa B; D011498:Protein Precursors; D053245:RANK Ligand; C505689:Tnfsf11 protein, mouse; D053148:Caspase 3