Visualization of bisphosphonate-induced caspase-3 activity in apoptotic osteoclasts in vitro.
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.
Benford
H L
HL
Department of Medicine and Therapeutics, University of Aberdeen Medical School, Foresterhill, Aberdeen, AB25 2ZD, UK.
McGowan
N W
NW
Helfrich
M H
MH
Nuttall
M E
ME
Rogers
M J
MJ
D000818:Animals; D000831:Animals, Newborn; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D001851:Bone Diseases, Metabolic / Q000188:drug therapy* / Q000201:enzymology / Q000503:physiopathology; D001842:Bone and Bones / Q000187:drug effects* / Q000201:enzymology / Q000503:physiopathology; D053148:Caspase 3; D053178:Caspase 6; D053179:Caspase 7; D020169:Caspases / Q000187:drug effects* / Q000378:metabolism; D004164:Diphosphonates / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000493:pharmacokinetics; D006801:Humans; D009584:Nitrogen / Q000378:metabolism; D010010:Osteoclasts / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology; D017368:Protein Prenylation / Q000187:drug effects / Q000502:physiology; D011817:Rabbits; D014407:Tumor Cells, Cultured / Q000166:cytology / Q000187:drug effects / Q000201:enzymology
D004164:Diphosphonates; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; C505407:CASP3 protein, human; C505514:CASP6 protein, human; C505520:CASP7 protein, human; D053148:Caspase 3; D053178:Caspase 6; D053179:Caspase 7; D020169:Caspases; D009584:Nitrogen