Calcified Tissue International

, Volume 82, Issue 3, pp 191–201 | Cite as

Aminobisphosphonates Cause Osteoblast Apoptosis and Inhibit Bone Nodule Formation In Vitro

  • Aymen I. Idris
  • Javier Rojas
  • Iain R. Greig
  • Rob J. van’t Hof
  • Stuart H. Ralston
Article

Abstract

Bisphosphonates are widely used for the treatment of bone diseases associated with increased osteoclastic bone resorption. Bisphosphonates are known to inhibit biochemical markers of bone formation in vivo, but it is unclear to what extent this is a consequence of osteoclast inhibition or a direct inhibitory effect on cells of the osteoblast lineage. In order to investigate this issue, we studied the effects of various bisphosphonates on osteoblast growth and differentiation in vitro. The aminobisphosphonates pamidronate and alendronate inhibited osteoblast growth, caused osteoblast apoptosis, and inhibited protein prenylation in osteoblasts in a dose-dependent manner over the concentration range 20−100 μM. Further studies showed that alendronate in a dose of 0.1 mg/kg inhibited protein prenylation in calvarial osteoblasts in vivo, indicating that alendronate can be taken up by osteoblasts in sufficient amounts to inhibit protein prenylation at clinically relevant doses. Pamidronate and alendronate inhibited bone nodule formation at concentrations 10-fold lower than those required to inhibit osteoblast growth. These effects were not observed with non-nitrogen-containing bisphosphonates or with other inhibitors of protein prenylation and were only partially reversed by cotreatment with a fourfold molar excess of ß-glycerol phosphate. We conclude that aminobisphosphonates cause osteoblast apoptosis in vitro at micromolar concentrations and inhibit osteoblast differentiation at nanomolar concentrations by mechanisms that are independent of effects on protein prenylation and may be due in part to inhibition of mineralization. While these results need to be interpreted with caution because of uncertainty about the concentrations of bisphosphonates that osteoblasts are exposed to in vivo, our studies clearly demonstrate that bisphosphonates exert strong inhibitory effects on cells of the osteoblast lineage at similar concentrations to those that cause osteoclast inhibition. This raises the possibility that inhibition of bone formation by bisphosphonates may be due in part to a direct inhibitory effect on cells of the osteoblast lineage.

Keywords

Aminobisphosphonate Osteoblast Apoptosis Bone nodule formation Bisphosphonate 

Notes

Acknowledgements

This work was supported by a grant from the Arthritis Research Campaign (UK). A. I. is supported by an ECTS-AMGEN award.

References

  1. 1.
    Frith JC, Monkkonen J, Auriola S, Monkkonen H, Rogers MJ (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 apoptosis. Arthritis Rheum 44:2201–2210PubMedCrossRefGoogle Scholar
  2. 2.
    Frith JC, Monkkonen J, Blackburn GM, Russell RG, Rogers MJ (1997) Clodronate and liposome-encapsulated clodronate are metabolized to a toxic ATP analog, adenosine 5’-(beta, gamma-dichloromethylene) triphosphate, by mammalian cells in vitro. J Bone Miner Res 12:1358–1367PubMedCrossRefGoogle Scholar
  3. 3.
    van Beek E, Pieterman E, Cohen L, Lowik C, Papapoulos S (1999) Farnesyl pyrophosphate synthase is the molecular target of nitrogen- containing bisphosphonates. Biochem Biophys Res Commun 264:108–111PubMedCrossRefGoogle Scholar
  4. 4.
    Dunford JE, Thompson K, Coxon FP, Luckman SP, Hahn FM, Poulter CD, Ebetino FH, Rogers MJ (2001) 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 296:235–242PubMedGoogle Scholar
  5. 5.
    Luckman SP, Hughes DE, Coxon FP, Graham R, Russell G, Rogers MJ (1998) Nitrogen-containing bisphosphonates inhibit the mevalonate pathway and prevent post-translational prenylation of GTP-binding proteins, including Ras. J Bone Miner Res 13:581–589PubMedCrossRefGoogle Scholar
  6. 6.
    Dunford JE, Rogers MJ, Ebetino FH, Phipps RJ, Coxon FP (2006) Inhibition of protein prenylation by bisphosphonates causes sustained activation of Rac, Cdc42, and Rho GTPases. J Bone Miner Res 21:684–694PubMedCrossRefGoogle Scholar
  7. 7.
    Nancollas GH, Tang R, Phipps RJ, Henneman Z, Gulde S, Wu W, Mangood A, Russell RG, Ebetino FH (2006) Novel insights into actions of bisphosphonates on bone: differences in interactions with hydroxyapatite. Bone 38:617–627PubMedCrossRefGoogle Scholar
  8. 8.
    Plotkin LI, Weinstein RS, Parfitt AM, Roberson PK, Manolagas SC, Bellido T (1999) Prevention of osteocyte and osteoblast apoptosis by bisphosphonates and calcitonin. J Clin Invest 104:1363–1374PubMedCrossRefGoogle Scholar
  9. 9.
    Plotkin LI, Manolagas SC, Bellido T (2006) Dissociation of the pro-apoptotic effects of bisphosphonates on osteoclasts from their anti-apoptotic effects on osteoblasts/osteocytes with novel analogs. Bone 39:443–452PubMedCrossRefGoogle Scholar
  10. 10.
    Giuliani N, Pedrazzoni M, Negri G, Passeri G, Impicciatore M, Girasole G (1998) Bisphosphonates stimulate formation of osteoblast precursors and mineralized nodules in murine and human bone marrow cultures in vitro and promote early osteoblastogenesis in young and aged mice in vivo. Bone 22:455–461PubMedCrossRefGoogle Scholar
  11. 11.
    Duque G, Rivas D (2007) Alendronate has an anabolic effect on bone through the differentiation of mesenchymal stem cells. J Bone Miner Res 10:1603–1611Google Scholar
  12. 12.
    Tobias JH, Chow JW, Chambers TJ (1993) 3-Amino-1-hydroxypropylidine-1-bisphosphonate (AHPrBP) suppresses not only the induction of new, but also the persistence of existing bone-forming surfaces in rat cancellous bone. Bone 14:619–623PubMedCrossRefGoogle Scholar
  13. 13.
    Delmas PD, Vergnaud P, Arlot ME, Pastoureau P, Meunier PJ, Nilssen MH (1995) The anabolic effect of human PTH (1–34) on bone formation is blunted when bone resorption is inhibited by the bisphosphonate tiludronate—is activated resorption a prerequisite for the in vivo effect of PTH on formation in a remodeling system? Bone 16:603–610PubMedCrossRefGoogle Scholar
  14. 14.
    Black DM, Bilezikian JP, Ensrud KE, Greenspan SL, Palermo L, Hue T, Lang TF, McGowan JA, Rosen CJ (2005) One year of alendronate after one year of parathyroid hormone (1–84) for osteoporosis. N Engl J Med 353:555–565PubMedCrossRefGoogle Scholar
  15. 15.
    Ettinger B, San Martin J, Crans G, Pavo I (2004) Differential effects of teriparatide on BMD after treatment with raloxifene or alendronate. J Bone Miner Res 19:745–751PubMedCrossRefGoogle Scholar
  16. 16.
    Samadfam R, Xia Q, Goltzman D (2007) Pretreatment with anticatabolic agents blunts but does not eliminate the skeletal anabolic response to parathyroid hormone in oophorectomized mice. Endocrinology 148:2778–2787PubMedCrossRefGoogle Scholar
  17. 17.
    Finkelstein JS, Hayes A, Hunzelman JL, Wyland JJ, Lee H, Neer RM (2003) The effects of parathyroid hormone, alendronate, or both in men with osteoporosis. N Engl J Med 349:1216–1226PubMedCrossRefGoogle Scholar
  18. 18.
    Gasser JA, Kneissel M, Thomsen JS, Mosekilde L (2000) PTH and interactions with bisphosphonates. J Musculoskelet Neuronal Interact 1:53–56PubMedGoogle Scholar
  19. 19.
    van’t Hof RJ, von Lindern M, Nijweide PJ, Beug H (1997) Stem cell factor stimulates chicken osteoclast activity in vitro. FASEB J 11:287–293PubMedGoogle Scholar
  20. 20.
    van’t Hof RJ (2003) Osteoclast formation in the mouse coculture assay. In: Helfrich MH, Ralston SH (eds) Bone research protocols. Humana Press, Totowa, NJ, pp 145–152CrossRefGoogle Scholar
  21. 21.
    van’t Hof RJ, Idris AI, Ridge SA, Dunford J, Greig IR, Ralston SH (2004) Identification of biphenylcarboxylic acid derivatives as a novel class of bone resorption inhibitors. J Bone Miner Res 19:1651–1660CrossRefGoogle Scholar
  22. 22.
    Stewart TL, Roschger P, Misof BM, Mann V, Fratzl P, Klaushofer K, Aspden RM, Ralston SH (2005) Association of COLIA1 Sp1 alleles with defective bone nodule formation in vitro and abnormal bone mineralisation in vivo. Calcif Tissue Int 77:113–118PubMedCrossRefGoogle Scholar
  23. 23.
    Black DM, Greenspan SL, Ensrud KE, Palermo L, McGowan JA, Lang TF, Garnero P, Bouxsein ML, Bilezikian JP, Rosen CJ (2003) The effects of parathyroid hormone and alendronate alone or in combination in postmenopausal osteoporosis. N Engl J Med 349:1207–1215PubMedCrossRefGoogle Scholar
  24. 24.
    Twiss IM, Pas O, Ramp-Koopmanschap W, den Hartigh J, Vermeij P (1999) The effects of nitrogen-containing bisphosphonates on human epithelial (Caco-2) cells, an in vitro model for intestinal epithelium. J Bone Miner Res 14:784–791PubMedCrossRefGoogle Scholar
  25. 25.
    Suri S, Monkkonen J, Taskinen M, Pesonen J, Blank MA, Phipps RJ, Rogers MJ (2001) Nitrogen-containing bisphosphonates induce apoptosis of Caco-2 cells in vitro by inhibiting the mevalonate pathway: a model of bisphosphonate-induced gastrointestinal toxicity. Bone 29:336–343PubMedCrossRefGoogle Scholar
  26. 26.
    Shipman CM, Rogers MJ, Apperley JF, Russell RG, Croucher PI (1997) Bisphosphonates induce apoptosis in human myeloma cell lines: a novel anti-tumour activity. Br J Haematol 98:665–672PubMedCrossRefGoogle Scholar
  27. 27.
    Reszka AA, Halasy-Nagy J, Rodan GA (2001) Nitrogen-bisphosphonates block retinoblastoma phosphorylation and cell growth by inhibiting the cholesterol biosynthetic pathway in a keratinocyte model for esophageal irritation. Mol Pharmacol 59:193–202PubMedGoogle Scholar
  28. 28.
    Plotkin LI, Bellido T (2001) Bisphosphonate-induced, hemichannel-mediated, anti-apoptosis through the Src/ERK pathway: a gap junction-independent action of connexin43. Cell Commun Adhes 8:377–382PubMedCrossRefGoogle Scholar
  29. 29.
    Sato M, Grasser W, Endo N, Akins R, Simmons H, Thompson DD, Golub E, Rodan GA (1991) Bisphosphonate action. Alendronate localization in rat bone and effects on osteoclast ultrastructure. J Clin Invest 88:2095–2105PubMedCrossRefGoogle Scholar
  30. 30.
    Nesbitt SA, Horton MA (1997) Trafficking of matrix collagens through bone-resorbing osteoclasts. Science 276:266–269PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2008

Authors and Affiliations

  • Aymen I. Idris
    • 1
  • Javier Rojas
    • 1
  • Iain R. Greig
    • 2
  • Rob J. van’t Hof
    • 1
  • Stuart H. Ralston
    • 1
  1. 1.Rheumatic Diseases Unit, Molecular Medicine CentreUniversity of Edinburgh, General Western HospitalEdinburghUK
  2. 2.Department of Medicine and TherapeuticsInstitute of Medical Sciences, University of AberdeenForesterhillUK

Personalised recommendations