Osteoclast-like multinucleated cells (OCLs) were prepared on collagen gels in a coculture system of mouse bone marrow cells and osteoblasts, and purified by collagenase and a subsequent pronase treatment. More than 80% of the purified OCLs were found to undergo apoptotic cell death by 48 h during the culture in a culture medium containing 10% fetal bovine serum (FBS). Withdrawal of FBS from the culture medium accelerated the cell death, which induced more than 80% of OCLs to undergo apoptotic cell death by as early as 18 h. Two peptide inhibitors of caspases (interleukin-1β-converting enzyme family proteases), benzyloxycarbonyl-Val-Ala-Asp (OMe)-fluoromethyl ketone (Z-VAD-FMK) and benzyloxycarhonyl-Asp-Glu-Val-Asp (OMe)-fluoromethyl ketone (Z-DEVD-FMK), extended the survival time of OCLs in the presence and absence of 10% FBS, but the effect was rather limited in the absence of FBS. Because interleukin-1α (IL-1α) and the macrophage colony stimulating factor (M-CSF) are known to promote the survival of osteoclasts, we examined the effect of the peptide inhibitors and these cytokines. Combinations of the peptide inhibitors and IL-1α, or the peptide inhibitors and M-CSF, were more effective than the inhibitors alone. When endogenous caspase activities of OCLs were analyzed using fluorescence peptide substrates, the activities, in particular, caspase-3 (CPP32)-like activity, were markedly increased in OCLs by the withdrawal of FBS from the culture medium, IL-1α and M-CSF suppressed the activation of the caspases. In addition, western blot analysis revealed that the expression of Bcl-2, which inhibits the activation of caspases, was very weak or even negligible in OCLs. Taken together, these results suggest that the caspases are involved in the regulation of survival and apoptotic cell death of osteoclasts.
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