Cytosolic entry of bisphosphonate drugs requires acidification of vesicles after fluid-phase endocytosis

Keith Thompson, Michael John Rogers, Fraser Coxon, Julie Clare Crockett

Research output: Contribution to journalArticle

155 Citations (Scopus)

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 [C-14]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 [C-14] 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 [C-14] 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.

Original languageEnglish
Pages (from-to)1624-1632
Number of pages9
JournalMolecular Pharmacology
Volume69
Issue number5
Early online date24 Feb 2006
DOIs
Publication statusPublished - May 2006

Keywords

  • nitrogen-containing bisphosphonates
  • receptor-mediated endocytosis
  • farnesyl diphosphates synthase
  • vacuolar H+-ATPASE
  • bone-resorption
  • in-vitro
  • rabbit osteoclasts
  • binding proteins
  • macrophages
  • alendronate

Cite this

Cytosolic entry of bisphosphonate drugs requires acidification of vesicles after fluid-phase endocytosis. / Thompson, Keith; Rogers, Michael John; Coxon, Fraser; Crockett, Julie Clare.

In: Molecular Pharmacology, Vol. 69, No. 5, 05.2006, p. 1624-1632.

Research output: Contribution to journalArticle

Thompson, Keith ; Rogers, Michael John ; Coxon, Fraser ; Crockett, Julie Clare. / Cytosolic entry of bisphosphonate drugs requires acidification of vesicles after fluid-phase endocytosis. In: Molecular Pharmacology. 2006 ; Vol. 69, No. 5. pp. 1624-1632.
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T1 - Cytosolic entry of bisphosphonate drugs requires acidification of vesicles after fluid-phase endocytosis

AU - Thompson, Keith

AU - Rogers, Michael John

AU - Coxon, Fraser

AU - Crockett, Julie Clare

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N2 - 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 [C-14]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 [C-14] 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 [C-14] 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.

AB - 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 [C-14]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 [C-14] 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 [C-14] 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.

KW - nitrogen-containing bisphosphonates

KW - receptor-mediated endocytosis

KW - farnesyl diphosphates synthase

KW - vacuolar H+-ATPASE

KW - bone-resorption

KW - in-vitro

KW - rabbit osteoclasts

KW - binding proteins

KW - macrophages

KW - alendronate

U2 - 10.1124/mol.105.020776

DO - 10.1124/mol.105.020776

M3 - Article

VL - 69

SP - 1624

EP - 1632

JO - Molecular Pharmacology

JF - Molecular Pharmacology

SN - 0026-895X

IS - 5

ER -