Elsevier

Bone

Volume 29, Issue 6, December 2001, Pages 553-559
Bone

Inhibition of bone resorption by alendronate and risedronate does not require osteoclast apoptosis

https://doi.org/10.1016/S8756-3282(01)00615-9Get rights and content

Abstract

Bisphosphonate inhibition of bone resorption was proposed to be due to osteoclast apoptosis. We tested this hypothesis for both the N-containing bisphosphonates alendronate and risedronate, which inhibit farnesyldiphosphate synthase and thus protein isoprenylation, and for clodronate and etidronate, which are metabolized to adenosine triphosphate (ATP) analogs. We found, in dose-response studies, that alendronate and risedronate inhibit bone resorption (in pit assays) at doses tenfold lower than those reducing osteoclast number. At an N-bisphosphonate dose that inhibited resorption and induced apoptosis, the antiapoptotic caspase inhibitor, Z-VAD-FMK, maintained osteoclast (Oc) number but did not prevent inhibition of resorption. Furthermore, when cells were treated with either alendronate alone or in combination with Z-VAD-FMK for 24 or 48 h, subsequent addition of geranylgeraniol, which restores geranylgeranylation, returned bone resorption to control levels. On the other hand, Z-VAD-FMK did block etidronate and clodronate inhibition of resorption. Moreover, in cells treated with etidronate, but not alendronate or risedronate, Z-VAD-FMK also prevented actin disruption, an early sign of osteoclast inhibition by bisphosphonates. These observations indicate that, whereas induction of apoptosis plays a major role in etidronate and clodronate inhibition of resorption, alendronate and risedronate suppression of bone resorption is independent of their effects on apoptosis.

Introduction

Bisphosphonates (BPs) are the most potent and effective inhibitors of bone resorption in clinical use. Their P-C-P backbone structure: O3P— R2|C|R1—PO3 resembles pyrophosphate. BPs bind tightly to the bone mineral, and binding is enhanced when R1 (above) is a hydroxyl.33, 36 The R2 side chain defines individual BPs in this family and determines pharmacological activity. Based on their structure-dependent mode of action, BPs can be separated into two classes. The nitrogen-containing BPs (N-BPs), such as alendronate (ALN) and risedronate (RIS), contain one or more nitrogen atoms within R2. Non-N-BPs, such as etidronate (EHDP) or clodronate (CL2), have CH3 or Cl, respectively, at R2 or a nitrogen-free ring structure (tiludronate). Recent studies have shown that these features, in addition to the P-C-P backbone, confer specificity in intracellular action.

CL2, and to a lesser extent EHDP and tiludronate, but not N-BPs, are metabolized by macrophages into toxic adenosine triphosphate (ATP) analogs.11 Tiludronate also inhibits the vacuolar ATPase.7 N-BPs inhibit the mevalonate to cholesterol pathway enzyme farnesyldiphosphate (FPP) synthase and, consequently, geranylgeranylation.3, 8, 35 This action was shown to mediate N-BPs inhibition of osteoclast (Oc) formation and bone resorption and induction of apoptosis.2, 6, 10, 16, 26, 34 Recent data have also demonstrated in vivo inhibition of the mevalonate pathway selectively in the Oc by the N-BPs ALN, RIS, and ibandronate, but not by CL2 and EHDP.9

This study examines the relationship between Oc apoptosis and inhibition of bone resorption induced by N-BPs (ALN and RIS) and non-N-BPs (CL2 and EHDP). The findings suggest that, while inhibition of FPP synthase by N-BPs is responsible for suppression of bone resorption, as well as induction of osteoclast apoptosis and disruption of actin structure, apoptosis itself is not required for the other two inhibitory actions and may not occur at the lowest effective doses. In contrast, the non-N-BPs failed to inhibit bone resorption or disrupt the cytoskeleton when apoptosis was blocked. Therefore, it seems that apoptosis is required for inhibition of osteoclastic bone resorption by EHDP and CL2 but not by ALN or RIS.

Section snippets

Rabbit Oc bone resorption assay

Bone resorption assays were performed as previously described.26 Briefly, tibiae and femora were aseptically isolated and minced in α-MEM containing HEPES (10 mmol/L, pH 7.1) and penicillin/streptomycin (100 IU and 100 mg/mL, respectively). Cells in supernatant were isolated, supplemented with 10% fetal bovine serum (FBS), and plated onto 0.25-mm-thick slices of bovine bone. After 1 h, test compounds were added (at concentrations noted in the text) into triplicate wells in a volume of 200 μL

Alendronate and risedronate inhibit bone resorption in the absence of Oc cell death

In a previous study, ALN-induced Oc death and inhibition of bone resorption changed in the same direction, suggesting a possible link.16 However, in vivo N-BP treatment was shown to increase Oc number, at least initially, when inhibition of bone resorption clearly occurs.4, 10, 32 To investigate more closely the correlation between Oc death and inhibition of bone resorption, we examined the effects of ALN or RIS on bone resorption and cell number, across five doses between 0.06 and 6 nmol

Discussion

The findings of this study further support the separation of BPs into two classes based on their mode of action. The difference resides not only in their immediate intracellular targets, but, as indicated in this study, also in the events responsible for inhibition of bone resorption.

All BPs tested here and previously can clearly induce Oc apoptosis.2, 6, 13, 16, 26 Furthermore, Oc apoptosis would certainly reduce bone resorption and may occur as a result of treatment with any of the BPs.

References (38)

  • A.J. Ridley et al.

    The small GTP-binding protein rac regulates growth factor-induced membrane ruffling

    Cell

    (1992)
  • E. van Beek et al.

    Farnesyl pyrophosphate synthase is the molecular target of nitrogen-containing bisphosphonates

    Biochem Biophys Res Commun

    (1999)
  • E.R. van Beek et al.

    Binding and antiresorptive properties of heterocycle-containing bisphosphonate analogsStructure-activity relationships

    Bone

    (1998)
  • H. Asha et al.

    The Rap1 GTPase functions as a regulator of morphogenesis in vivo

    Eur Med Biol Org J

    (1999)
  • H.L. Benford et al.

    Farnesol and geranylgeraniol prevent activation of caspases by aminobisphosphonatesBiochemical evidence for two distinct pharmacological classes of bisphosphonate drugs

    Mol Pharmacol

    (1999)
  • D.D. Bikle et al.

    Alendronate increases skeletal mass of growing rats during unloading by inhibiting resorption of calcified cartilage

    J Bone Miner Res

    (1994)
  • F.P. Coxon et al.

    Protein geranylgeranylation is required for osteoclast formation, function, and survivalInhibition by bisphosphonates and GGTI-298

    J Bone Miner Res

    (2000)
  • P. David et al.

    The bisphosphonate tiludronate is a potent inhibitor of the osteoclast vacuolar H(+)-ATPase

    J Bone Miner Res

    (1996)
  • J.E. Dunford et al.

    Structure-activity relationships for inhibition of farnesyl diphosphate synthase in vitro and inhibition of bone resorption in vivo by nitrogen-containing bisphosphonates

    J Pharmacol Exp Ther

    (2001)
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