Abstract

Bisphosphonates (BPs), such as clodronate and pamidronate, are inhibitors of bone resorption and are used on a widespread basis in the treatment of hyper-resorptive bone diseases. At the cellular level, BPs inhibit osteoclasts, but the precise molecular mechanisms are unclear. BPs have also been shown to affect the survival of macrophages, cells ontogenetically related to osteoclasts. We show that both clodronate and pamidronate induce apoptosis in isolated osteoclasts. Clodronate, when administered in liposomes, also induced apoptosis in rat peritoneal macrophages in vitro and in liver macrophages of mice in vivo but not in murine macrophage-like RAW-264 cells. The subcellular localization and staining intensity of Bcl-2, an anti-apoptotic protein known to protect several cell types against drug-induced apoptosis, were similar in RAW-264 and peritoneal macrophage cells, as revealed by immunofluorescence. The clodronate-induced apoptotic pathway was further characterized in isolated osteoclasts cultured on glass coverslips through the use of clodronate-containing liposomes and several inhibitors of the apoptotic cascade. None of the agents tested could totally prevent clodronate-induced osteoclast death. Partial protection was, however, obtained by the addition of staurosporine or homocysteine. The results suggest that primarily cytoplasmic, protein kinase C-activated mechanisms are involved in the execution of clodronate-induced apoptosis of osteoclasts.