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Bone. 2001 May;28(5):465-73.

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

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  • 1Department of Medicine and Therapeutics, University of Aberdeen Medical School, Foresterhill, Aberdeen, AB25 2ZD, UK.

Abstract

Bisphosphonates inhibit osteoclast-mediated bone resorption by mechanisms that have only recently become clear. Whereas nitrogen-containing bisphosphonates affect osteoclast function by preventing protein prenylation (especially geranylgeranylation), non-nitrogen-containing bisphosphonates have a different molecular mechanism of action. In this study, we demonstrate that nitrogen-containing bisphosphonates (risedronate, alendronate, pamidronate, and zoledronic acid) and non-nitrogen-containing bisphosphonates (clodronate and etidronate) cause apoptosis of rabbit osteoclasts, human osteoclastoma-derived osteoclasts, and human osteoclast-like cells generated in cultures of bone marrow in vitro. Osteoclast apoptosis was shown to involve characteristic morphological changes, loss of mitochondrial membrane potential, and the activation of caspase-3-like proteases capable of cleaving peptide substrates with the sequence DEVD. Caspase-3-like activity could be visualized in unfixed, dying osteoclasts and osteoclast-like cells using a cell-permeable, fluorogenic substrate. Bisphosphonate-induced osteoclast apoptosis was dependent on caspase activation, because apoptosis resulting from alendronate, clodronate, or zoledronic acid treatment was suppressed by zVAD-fmk, a broad-range caspase inhibitor, or by SB-281277, a specific isatin sulfonamide inhibitor of caspase-3/-7. Furthermore, caspase-3 (but not caspase-6 or caspase-7) activity could be detected and quantitated in lysates from purified rabbit osteoclasts, whereas the p17 fragment of active caspase-3 could be detected in human osteoclast-like cells by immunofluorescence staining. Caspase-3, therefore, appears to be the major effector caspase activated in osteoclasts by bisphosphonate treatment. Caspase activation and apoptosis induced by nitrogen-containing bisphosphonates are likely to be the consequence of the loss of geranylgeranylated rather than farnesylated proteins, because the ability to cause apoptosis and caspase activation was mimicked by GGTI-298, a specific inhibitor of protein geranylgeranylation, whereas FTI-277, a specific inhibitor of protein farnesylation, had no effect on apoptosis or caspase activity.

PMID:
11344045
[PubMed - indexed for MEDLINE]
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