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

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.

• Authors: H L Benford, N W McGowan, M H Helfrich, M E Nuttall, M J Rogers
• Journal: Bone (Bone) Vol. 28 Issue 5 Pg. 465-73 (May 2001) ISSN: 8756-3282 [Print] United States
• PMID: 11344045 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Diphosphonates
• Enzyme Inhibitors
• Fluorescent Dyes
• CASP3 protein, human
• CASP6 protein, human
• CASP7 protein, human
• Caspase 3
• Caspase 6
• Caspase 7
• Caspases
• Nitrogen

Topics

• Animals
• Animals, Newborn
• Apoptosis (drug effects, physiology)
• Bone Diseases, Metabolic (drug therapy, enzymology, physiopathology)
• Bone and Bones (drug effects, enzymology, physiopathology)
• Caspase 3
• Caspase 6
• Caspase 7
• Caspases (drug effects, metabolism)
• Diphosphonates (pharmacology)
• Enzyme Inhibitors (pharmacology)
• Fluorescent Dyes (pharmacokinetics)
• Humans
• Nitrogen (metabolism)
• Osteoclasts (cytology, drug effects, enzymology)
• Protein Prenylation (drug effects, physiology)
• Rabbits
• Tumor Cells, Cultured (cytology, drug effects, enzymology)