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-kB 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-kB in procaspase-3 silenced cells. We found that RANKL treatment prevented activation and nuclear translocation of NF-kB. 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.

Bone is a dynamic tissue that undergoes constant remodeling. The remodeling process is a delicate balance between bone formation by osteoblasts and bone resorption by osteoclasts (Chambers, 2000; Teitelbaum, 2000). Interference with this balance can affect bone integrity and function resulting in debilitating human diseases. Most commonly, disregulation of this balance is due to excess osteoclastic activity, leading to increased bone resorption. For this reason, the molecular signaling pathways that regulate osteoclast differentiation and function have been subjected to intense scrutiny. A number of TNF family members exert modulatory effects on osteoclast differentiation, function, survival, and/or apoptosis (Feng, 2005). Receptors for these ligands can be divided into two subfamilies: death domain-containing receptors such as TNFR1, Fas, and TRAIL-R1, 2 and receptors lacking this domain such as RANK, TNFR2, CD27, and CD40. Receptors in subfamily-1 recruit adapter proteins which lead to the activation of proteases of the caspase family that are involved in inducing apoptosis, referred to as the extrinsic (caspase-8) pathway (Solary et al., 1998; Peter et al., 1999). Caspases are a family of cysteinedependent proteases that are synthesized as inactive zymogens and are activated by dimer formation (eg, apoptotic initiator caspases-8,-9) or by cleavage (eg, apoptotic effector caspases-3)(Boatright et al., 2003). For both the extrinsic death receptor and the intrinsic stress response (caspase-9) apoptotic pathways, the cleavage of procaspase-3 to caspase-3 triggers the proteolysis of intracellular structural and regulatory proteins, such as poly (ADP-ribose) polymerase (PARP), needed for DNA repair. Receptors in subfamily-2 also associate with adaptor proteins. These proteins play a major role in regulating cellular signaling pathways that promote osteoclast differentiation, function, and survival. Of the TNF family members in subfamily-2, RANKL is considered the critical mediator by which osteoblastic/stromal cells stimulate osteoclast formation and resorption via the osteoclast RANK receptor (Lacey et al., 1998; Yasuda et al., 1998; Hsu et al., 1999; Wong et al., 1999; Suda et al., 2001). The initial binding of RANKL to RANK induces the recruitment of key