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.

Involvement of hydrogen peroxide in the differentiation and apoptosis of preosteoclastic cells exposed to arsenite.

Long-term exposure to sodium arsenite (AsO(2)) promotes the development of various cancers. Paradoxically, arsenic also induces pro-myelomonocytic leukemia cell differentiation, and at higher concentrations, apoptosis. The present study investigated the effects of AsO(2) on preosteoclasts. When treated with 2.5-5microM AsO(2), RAW264.7 cells underwent osteoclast differentiation as evidenced by an increase in the number of multinucleate cells expressing tartrate resistant acid phosphatase (TRAP). The appearance of these phenotypic markers was preceded by a low level increase in extracellular production of H(2)O(2) and was prevented by the addition of catalase (4.5microg/ml), an enzyme that removes H(2)O(2). Only at high concentrations (10-25microM) of AsO(2) was a significant loss of cell viability and a high level increase in H(2)O(2) production (1.5microM) observed. Apoptosis was blocked by pretreatment with diphenylene iodonium chloride (2microM), a NAD(P)H-flavoprotein inhibitor, suggesting the involvement of NADPH-oxidase. The data show that AsO(2), dose-dependently, stimulates increasing amounts of H(2)O(2) production. Moreover, at concentrations found in tissues of individuals exposed to geochemical AsO(2), osteoclasts underwent an H(2)O(2)-dependent differentiation. Therefore, chronic exposure to low-level amounts of AsO(2) could result in increased bone resorption and contribute to bone related pathologies.

Ionizing radiation sensitizes bone cells to apoptosis.

Osteoradionecrosis is a common sequelae of radiation therapy for head and neck cancer. To test the hypothesis that radiation induces osteoradionecrosis by induction of bone cell apoptosis, we exposed MC3T3-E1 osteoblast-like cells to gamma-radiation and evaluated cell viability. Twenty-four hours postirradiation, measurement of osteoblast dehydrogenase activity suggested that there was a small decrease in cell viability. However, TUNEL and flow cytometric analysis indicated that the viability loss was caused by inhibition of cell proliferation and not by induction of apoptosis. The effect of irradiation on osteoblast function was examined by Western blot and flow cytometric analysis. It was found that irradiated osteoblasts underwent G2 cell cycle arrest. In addition, we observed changes in expression of molecules that regulate the cell cycle. Thus, there was an increase in p53 transcription, a raised level of MDM2 dephosphorylation, and elevation in p21 and GADD153 protein levels. Since these proteins are concerned with the regulation of the cell cycle, the observed changes in expression would be expected to disturb cyclin activity and cause G2M arrest. The arrested cells displayed a dramatic increase in sensitivity to specific apoptogens. Thus, when irradiated, and then treated with Ca2+Pi or staurosporine, agents that cause mitochondrial dysfunction, more osteoblasts underwent apoptosis than with the apoptogen alone. In contrast, irradiated cells treated with anti-Fas antibody showed no change in apoptotic sensitivity; apoptosis was inhibited when osteoblasts were treated with etoposide. Similar alterations in sensitivity were observed when cells were arrested in G2/M by pretreatment with colchicine and then challenged with apoptogens. It was concluded that activation of radiation-induced G2 arrest sensitizes osteoblasts to agents that mediate apoptosis through a mitochondrial-dependent death pathway.