Visualization of bisphosphonate-induced caspase-3 activity in apoptotic osteoclasts in vitro

Bisphosphonates inhibit osteoclast-mediated bone resorption by mechanisms t hat have only recently become clear. Whereas nitrogen-containing bisphospho nates affect osteoclast function by preventing protein prenylation (especia lly geranylgeranylation), non-nitrogen-containing bisphosphonates have a di fferent molecular mechanism of action. In this study, we demonstrate that n itrogen-containing bisphosphonates (risedronate, alendronate, pamidronate, and zoledronic acid) and non-nitrogen-containing bisphosphonates (clodronat e and etidronate) cause apoptosis of rabbit osteoclasts, human osteoclastom a-derived osteoclasts, and human osteoclast-like cells generated in culture s of bone marrow in vitro. Osteoclast apoptosis was shown to involve charac teristic morphological changes, loss of mitochondrial membrane potential, a nd the activation of caspase-3-like proteases capable of cleaving peptide s ubstrates with the sequence DEVD, Caspase-3-like activity could be visualiz ed in unfixed, dying osteoclasts and osteoclast-like cells using a cell-per meable, fluorogenic substrate, Bisphosphonate-induced osteoclast apoptosis was dependent on caspase activation, because apoptosis resulting from alend ronate, clodronate, or zoledronic acid treatment was suppressed by zVAD-fmk , a broad-range caspase inhibitor, or by SB-281277, a specific isatin sulfo namide inhibitor of caspase-3/-7, Furthermore, caspase-3 (but not caspase-6 or caspase-7) activity could be detected and quantitated in lysates from p urified rabbit osteoclasts, whereas the p17 fragment of active caspase-3 co uld be detected in human osteoclast-like cells by immunofluorescence staini ng, Caspase-3, therefore, appears to be the major effector caspase activate d in osteoclasts by bisphosphonate treatment, Caspase activation and apopto sis induced by nitrogen-containing bisphosphonates are likely to be the con sequence of the loss of geranylgeranylated rather than farnesylated protein s, because the ability to cause apoptosis and caspase activation was mimick ed by GGTI-298, a specific inhibitor of protein geranylgeranylation, wherea s FTI-277, a specific inhibitor of protein farnesylation, had no effect on apoptosis or caspase activity. (C) 2001 by Elsevier Science Inc. All rights reserved.