Cytosolic Entry of Bisphosphonate Drugs Requires Acidification of Vesicles after Fluid-Phase Endocytosis
Open Access
- 24 February 2006
- journal article
- Published by American Society for Pharmacology & Experimental Therapeutics (ASPET) in Molecular Pharmacology
- Vol. 69 (5), 1624-1632
- https://doi.org/10.1124/mol.105.020776
Abstract
Bisphosphonates such as alendronate and zoledronate are blockbuster drugs used to inhibit osteoclast-mediated bone resorption. Although the molecular mechanisms by which bisphosphonates affect osteoclasts are now evident, the exact route by which they are internalized by cells is not known. To clarify this, we synthesized a novel, fluorescently labeled analog of alendronate (AF-ALN). AF-ALN was rapidly internalized into intracellular vesicles in J774 macrophages and rabbit osteoclasts; uptake of AF-ALN or [14C]zoledronate was stimulated by the presence of Ca2+ and Sr2+ and could be inhibited by addition of EGTA or clodronate, both of which chelate calcium ions. Both EGTA and clodronate also prevented the bisphosphonate-induced inhibition of Rap1A prenylation, an effect that was reversed by addition of Ca2+. In J774 cells and osteoclasts, vesicular AF-ALN colocalized with dextran (but not wheat germ agglutinin or transferrin), and uptake of AF-ALN or [14C]zoledronate was inhibited by dansylcadaverine, indicating that fluid-phase endocytosis is involved in the initial internalization of bisphosphonate into vesicles. Endosomal acidification then seems to be absolutely required for exit of bisphosphonate from vesicles and entry into the cytosol, because monensin and bafilomycin A1, both inhibitors of endosomal acidification, did not inhibit vesicular uptake of AF-ALN or internalization of [14C]zoledronate but prevented the inhibitory effect of alendronate or zoledronate on Rap1A prenylation. Taken together, these results demonstrate that cellular uptake of bisphosphonate drugs requires fluid-phase endocytosis and is enhanced by Ca2+ ions, whereas transfer from endocytic vesicles into the cytosol requires endosomal acidification.Keywords
This publication has 32 references indexed in Scilit:
- Identification of a Novel Phosphonocarboxylate Inhibitor of Rab Geranylgeranyl Transferase That Specifically Prevents Rab Prenylation in Osteoclasts and MacrophagesOnline Journal of Public Health Informatics, 2001
- The molecular mechanism of action of the antiresorptive and antiinflammatory drug clodronate: Evidence for the formation in vivo of a metabolite that inhibits bone resorption and causes osteoclast and macrophage apoptosisArthritis & Rheumatism, 2001
- Protein Geranylgeranylation Is Required for Osteoclast Formation, Function, and Survival: Inhibition by Bisphosphonates and GGTI-298Journal of Bone and Mineral Research, 2000
- Alendronate Is a Specific, Nanomolar Inhibitor of Farnesyl Diphosphate SynthaseArchives of Biochemistry and Biophysics, 2000
- Farnesol and Geranylgeraniol Prevent Activation of Caspases by Aminobisphosphonates: Biochemical Evidence for Two Distinct Pharmacological Classes of Bisphosphonate DrugsMolecular Pharmacology, 1999
- A New Method to Isolate Large Numbers of Rabbit Osteoclasts and Osteoclast-like Cells: Application to the Characterization of Serum Response Element Binding Proteins During Osteoclast DifferentiationJournal of Bone and Mineral Research, 1998
- Identification of adenine nucleotide-containing metabolites of bisphosphonate drugs using ion-pair liquid chromatography–electrospray mass spectrometryJournal of Chromatography B: Biomedical Sciences and Applications, 1997
- Alendronate distributed on bone surfaces inhibits osteoclastic bone resorption in vitro and in experimental hypercalcemia modelsBone, 1995
- Bafilomycins: a class of inhibitors of membrane ATPases from microorganisms, animal cells, and plant cells.Proceedings of the National Academy of Sciences of the United States of America, 1988
- Transglutaminase is essential in receptor-mediated endocytosis of α2-macroglobulin and polypeptide hormonesNature, 1980