Skip to main content

Advertisement

SpringerLink
Log in

Characterization of the Bone Phenotype in ClC-7-Deficient Mice

  • Published:
Calcified Tissue International Aims and scope Submit manuscript

Abstract

Mice deficient in the chloride channel ClC-7, which is likely involved in acidification of the resorption lacuna, display severe osteopetrosis. To fully characterize the osteopetrotic phenotype, the phenotypes of osteoclasts and osteoblasts were evaluated. ClC-7−/− mice and their corresponding wild-type littermates were killed at 4–5 weeks of age. Biochemical markers of bone resorption (CTX-I), osteoclast number (TRAP5b), and osteoblast activity (ALP) were evaluated in serum. Splenocytes were differentiated into osteoclasts using M-CSF and RANKL. Mature osteoclasts were seeded on calcified or decalcified bone slices, and CTX-I, Ca2+, and TRAP were measured. Acidification rates in membrane vesicles from bone cells were measured using acridine orange. Osteoblastogenesis and nodule formation in vitro were investigated using calvarial osteoblasts. ClC-7−/− osteoclasts were unable to resorb calcified bone in vitro. However, osteoclasts were able to degrade decalcified bone. Acid influx in bone membrane vesicles was reduced by 70% in ClC-7−/− mice. Serum ALP was increased by 30% and TRAP5b was increased by 250% in ClC-7−/− mice, whereas the CTX/TRAP5b ratio was reduced to 50% of the wild-type level. Finally, evaluation of calvarial ClC-7−/− osteoblasts showed normal osteoblastogenesis. In summary, we present evidence supporting a pivotal role for ClC-7 in acidification of the resorption lacuna and evidence indicating that bone formation and bone resorption are no longer balanced in ClC-7−/− mice.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  1. Martin TJ (1993) Hormones in the coupling of bone resorption and formation. Osteoporos Int 3(Suppl 1):121–125

    Article  PubMed  Google Scholar 

  2. Martin TJ, Rodan GA (2001) Coupling of bone resorption and formation during bone remodeling. In: Marcus R, Feldman D, Kelsey J (eds) Osteoporosis, 2nd edn. Academic Press, San Diego, pp 361–370

    Google Scholar 

  3. Goltzman D (2002) Discoveries, drugs and skeletal disorders. Nat Rev Drug Discov 1:784–796

    Article  PubMed  CAS  Google Scholar 

  4. Teitelbaum SL, Ross FP (2003) Genetic regulation of osteoclast development and function. Nat Rev Genet 4:638–649

    Article  PubMed  CAS  Google Scholar 

  5. Roodman GD (1999) Cell biology of the osteoclast. Exp Hematol 27:1229–1241

    Article  PubMed  CAS  Google Scholar 

  6. Henriksen K, Sorensen MG, Nielsen RH, Gram J, Schaller S, Dziegiel MH, Everts V, Bollerslev J, Karsdal MA (2006) Degradation of the organic phase of bone by osteoclasts: a secondary role for lysosomal acidification. J Bone Miner Res 21:58–66

    Article  PubMed  CAS  Google Scholar 

  7. Baron R, Neff L, Louvard D, Courtoy PJ (1985) Cell-mediated extracellular acidification and bone resorption: evidence for a low pH in resorbing lacunae and localization of a 100-kD lysosomal membrane protein at the osteoclast ruffled border. J Cell Biol 101:2210–2222

    Article  PubMed  CAS  Google Scholar 

  8. Blair HC, Teitelbaum SL, Ghiselli R, Gluck S (1989) Osteoclastic bone resorption by a polarized vacuolar proton pump. Science 245:855–857

    Article  PubMed  CAS  Google Scholar 

  9. Frattini A, Orchard PJ, Sobacchi C, Giliani S, Abinun M, Mattsson JP, Keeling DJ, Andersson AK, Wallbrandt P, Zecca L, Notarangelo LD, Vezzoni P, Villa A (2000) Defects in TCIRG1 subunit of the vacuolar proton pump are responsible for a subset of human autosomal recessive osteopetrosis. Nat Genet 25:343–346

    Article  PubMed  CAS  Google Scholar 

  10. Vaananen HK, Karhukorpi EK, Sundquist K, Wallmark B, Roininen I, Hentunen T, Tuukkanen J, Lakkakorpi P (1990) Evidence for the presence of a proton pump of the vacuolar H+-ATPase type in the ruffled borders of osteoclasts. J Cell Biol 111:1305–1311

    Article  PubMed  CAS  Google Scholar 

  11. Jentsch TJ (2007) Chloride and the endosomal–lysosomal pathway: emerging roles of CLC chloride transporters. J Physiol 578:633–640

    Article  PubMed  CAS  Google Scholar 

  12. Jentsch TJ, Stein V, Weinreich F, Zdebik AA (2002) Molecular structure and physiological function of chloride channels. Physiol Rev 82:503–568

    PubMed  CAS  Google Scholar 

  13. Kornak U, Kasper D, Bosl MR, Kaiser E, Schweizer M, Schulz A, Friedrich W, Delling G, Jentsch TJ (2001) Loss of the ClC–7 chloride channel leads to osteopetrosis in mice and man. Cell 104:205–215

    Article  PubMed  CAS  Google Scholar 

  14. Jentsch TJ, Neagoe I, Scheel O (2005) CLC chloride channels and transporters. Curr Opin Neurobiol 15:319–325

    Article  PubMed  CAS  Google Scholar 

  15. Accardi A, Miller C (2004) Secondary active transport mediated by a prokaryotic homologue of ClC Cl− channels. Nature 427:803–807

    Article  PubMed  CAS  Google Scholar 

  16. Picollo A, Pusch M (2005) Chloride/proton antiporter activity of mammalian CLC proteins ClC-4 and ClC-5. Nature 436:420–423

    Article  PubMed  CAS  Google Scholar 

  17. Scheel O, Zdebik AA, Lourdel S, Jentsch TJ (2005) Voltage-dependent electrogenic chloride/proton exchange by endosomal CLC proteins. Nature 436:424–427

    Article  PubMed  CAS  Google Scholar 

  18. Graves AR, Curran PK, Smith CL, Mindell JA (2008) The Cl−/H+ antiporter ClC-7 is the primary chloride permeation pathway in lysosomes. Nature 453:788–792

    Article  PubMed  CAS  Google Scholar 

  19. Lange PF, Wartosch L, Jentsch TJ, Fuhrmann JC (2006) ClC-7 requires Ostm1 as a beta-subunit to support bone resorption and lysosomal function. Nature 440:220–223

    Article  PubMed  CAS  Google Scholar 

  20. Balemans W, Van Wesenbeeck L, Van Hul W (2005) A clinical and molecular overview of the human osteopetroses. Calcif Tissue Int 77:263–274

    Article  PubMed  CAS  Google Scholar 

  21. Helfrich MH (2003) Osteoclast diseases. Microsc Res Tech 61:514–532

    Article  PubMed  Google Scholar 

  22. Ogbureke KU, Zhao Q, Li YP (2005) Human osteopetroses and the osteoclast V-H+-ATPase enzyme system. Front Biosci 10:2940–2954

    Article  PubMed  CAS  Google Scholar 

  23. Tolar J, Teitelbaum SL, Orchard PJ (2004) Osteopetrosis. N Engl J Med 351:2839–2849

    Article  PubMed  Google Scholar 

  24. Frattini A, Pangrazio A, Susani L, Sobacchi C, Mirolo M, Abinun M, Andolina M, Flanagan A, Horwitz EM, Mihci E, Notarangelo LD, Ramenghi U, Teti A, Van Hove J, Vujic D, Young T, Albertini A, Orchard PJ, Vezzoni P, Villa A (2003) Chloride channel ClCN7 mutations are responsible for severe recessive, dominant, and intermediate osteopetrosis. J Bone Miner Res 18:1740–1747

    Article  PubMed  CAS  Google Scholar 

  25. Alatalo SL, Ivaska KK, Waguespack SG, Econs MJ, Vaananen HK, Halleen JM (2004) Osteoclast-derived serum tartrate-resistant acid phosphatase 5b in Albers-Schonberg disease (type II autosomal dominant osteopetrosis). Clin Chem 50:883–890

    Article  PubMed  CAS  Google Scholar 

  26. Bollerslev J, Steiniche T, Melsen F, Mosekilde L (1989) Structural and histomorphometric studies of iliac crest trabecular and cortical bone in autosomal dominant osteopetrosis: a study of two radiological types. Bone 10:19–24

    Article  PubMed  CAS  Google Scholar 

  27. Del Fattore A, Peruzzi B, Rucci N, Recchia I, Cappariello A, Longo M, Fortunati D, Ballanti P, Iacobini M, Luciani M, Devito R, Pinto R, Caniglia M, Lanino E, Messina C, Cesaro S, Letizia C, Bianchini G, Fryssira H, Grabowski P, Shaw N, Bishop N, Hughes D, Kapur RP, Datta HK, Taranta A, Fornari R, Migliaccio S, Teti A (2006) Clinical, genetic, and cellular analysis of 49 osteopetrotic patients: implications for diagnosis and treatment. J Med Genet 43:315–325

    Article  PubMed  CAS  Google Scholar 

  28. Karsdal MA, Martin TJ, Bollerslev J, Christiansen C, Henriksen K (2007) Are nonresorbing osteoclasts sources of bone anabolic activity? J Bone Miner Res 22:487–494

    Article  PubMed  CAS  Google Scholar 

  29. Karsdal MA, Henriksen K, Sorensen MG, Gram J, Schaller S, Dziegiel MH, Heegaard AM, Christophersen P, Martin TJ, Christiansen C, Bollerslev J (2005) Acidification of the osteoclastic resorption compartment provides insight into the coupling of bone formation to bone resorption. Am J Pathol 166:467–476

    PubMed  CAS  Google Scholar 

  30. Karsdal MA, Neutzsky-Wulff AV, Dziegiel MH, Christiansen C, Henriksen K (2008) Osteoclasts secrete non-bone derived signals that induce bone formation. Biochem Biophys Res Commun 366:483–488

    Article  PubMed  CAS  Google Scholar 

  31. Martin TJ, Sims NA (2005) Osteoclast-derived activity in the coupling of bone formation to resorption. Trends Mol Med 11:76–81

    Article  PubMed  CAS  Google Scholar 

  32. Kasper D, Planells-Cases R, Fuhrmann JC, Scheel O, Zeitz O, Ruether K, Schmitt A, Poet M, Steinfeld R, Schweizer M, Kornak U, Jentsch TJ (2005) Loss of the chloride channel ClC-7 leads to lysosomal storage disease and neurodegeneration. EMBO J 24:1079–1091

    Article  PubMed  CAS  Google Scholar 

  33. Winding B, NicAmhlaoibh R, Misander H, Hoegh-Andersen P, Andersen TL, Holst-Hansen C, Heegaard AM, Foged NT, Brunner N, Delaisse JM (2002) Synthetic matrix metalloproteinase inhibitors inhibit growth of established breast cancer osteolytic lesions and prolong survival in mice. Clin Cancer Res 8:1932–1939

    PubMed  CAS  Google Scholar 

  34. Ridnour LA, Windhausen AN, Isenberg JS, Yeung N, Thomas DD, Vitek MP, Roberts DD, Wink DA (2007) Nitric oxide regulates matrix metalloproteinase-9 activity by guanylyl-cyclase-dependent and -independent pathways. Proc Natl Acad Sci USA 104:16898–16903

    Article  PubMed  CAS  Google Scholar 

  35. Sondergaard BC, Henriksen K, Wulf H, Oestergaard S, Schurigt U, Brauer R, Danielsen I, Christiansen C, Qvist P, Karsdal MA (2006) Relative contribution of matrix metalloprotease and cysteine protease activities to cytokine-stimulated articular cartilage degradation. Osteoarthritis Cartilage 14:738–748

    Article  PubMed  CAS  Google Scholar 

  36. Edwards JC, Cohen C, Xu W, Schlesinger PH (2006) c-Src control of chloride channel support for osteoclast HCl transport and bone resorption. J Biol Chem 281:28011–28022

    Article  PubMed  CAS  Google Scholar 

  37. Sorensen MG, Henriksen K, Neutzsky-Wulff AV, Dziegiel MH, Karsdal MA (2007) Diphyllin, a novel and naturally potent V-ATPase inhibitor, abrogates acidification of the osteoclastic resorption lacunae and bone resorption. J Bone Miner Res 22:1640–1648

    Article  PubMed  Google Scholar 

  38. Henriksen K, Karsdal M, Delaisse JM, Engsig MT (2003) RANKL and vascular endothelial growth factor (VEGF) induce osteoclast chemotaxis through an ERK1/2-dependent mechanism. J Biol Chem 278:48745–48753

    Article  PubMed  CAS  Google Scholar 

  39. Karsdal MA, Hjorth P, Henriksen K, Kirkegaard T, Nielsen KL, Lou H, Delaisse JM, Foged NT (2003) Transforming growth factor-beta controls human osteoclastogenesis through the p38 MAPK and regulation of RANK expression. J Biol Chem 278:44975–44987

    Article  PubMed  CAS  Google Scholar 

  40. Henriksen K, Gram J, Høegh-Andersen P, Jemtland R, Ueland T, Dziegiel MH, Schaller S, Bollerslev J, Karsdal MA (2005) Osteoclasts from patients with autosomal dominant osteopetrosis type I caused by a T253I mutation in low-density lipoprotein receptor-related protein 5 are normal in vitro, but have decreased resorption capacity in vivo. Am J Pathol 167:1341–1348

    PubMed  CAS  Google Scholar 

  41. Henriksen K, Tanko LB, Qvist P, Delmas PD, Christiansen C, Karsdal MA (2007) Assessment of osteoclast number and function: application in the development of new and improved treatment modalities for bone diseases. Osteoporos Int 18:681–685

    Article  PubMed  CAS  Google Scholar 

  42. Cleiren E, Benichou O, Van Hul E, Gram J, Bollerslev J, Singer FR, Beaverson K, Aledo A, Whyte MP, Yoneyama T, De Vernejoul MC, Van Hul W (2001) Albers-Schonberg disease (autosomal dominant osteopetrosis, type II) results from mutations in the ClCN7 chloride channel gene. Hum Mol Genet 10:2861–2867

    Article  PubMed  CAS  Google Scholar 

  43. Waguespack SG, Koller DL, White KE, Fishburn T, Carn G, Buckwalter KA, Johnson M, Kocisko M, Evans WE, Foroud T, Econs MJ (2003) Chloride channel 7 (ClCN7) gene mutations and autosomal dominant osteopetrosis, type II. J Bone Miner Res 18:1513–1518

    Article  PubMed  CAS  Google Scholar 

  44. Henriksen K, Gram J, Schaller S, Dahl BH, Dziegiel MH, Bollerslev J, Karsdal MA (2004) Characterization of osteoclasts from patients harboring a G215R mutation in ClC-7 causing autosomal dominant osteopetrosis type II. Am J Pathol 164:1537–1545

    PubMed  CAS  Google Scholar 

  45. Chu K, Snyder R, Econs MJ (2006) Disease status in autosomal dominant osteopetrosis type 2 is determined by osteoclastic properties. J Bone Miner Res 21:1089–1097

    Article  PubMed  CAS  Google Scholar 

  46. Blair HC, Borysenko CW, Villa A, Schlesinger PH, Kalla SE, Yaroslavskiy BB, Garcia-Palacios V, Oakley JI, Orchard PJ (2004) In vitro differentiation of CD14 cells from osteopetrotic subjects: contrasting phenotypes with TCIRG1, CLCN7, and attachment defects. J Bone Miner Res 19:1329–1338

    Article  PubMed  Google Scholar 

  47. Letizia C, Taranta A, Migliaccio S, Caliumi C, Diacinti D, Delfini E, D’Erasmo E, Iacobini M, Roggini M, Albagha OM, Ralston SH, Teti A (2004) Type II benign osteopetrosis (Albers-Schonberg disease) caused by a novel mutation in CLCN7 presenting with unusual clinical manifestations. Calcif Tissue Int 74:42–46

    Article  PubMed  CAS  Google Scholar 

  48. Nielsen RH, Karsdal MA, Sorensen MG, Dziegiel MH, Henriksen K (2007) Dissolution of the inorganic phase of bone leading to release of calcium regulates osteoclast survival. Biochem Biophys Res Commun 360:834–839

    Article  PubMed  CAS  Google Scholar 

  49. Bollerslev J, Marks SC Jr, Pockwinse S, Kassem M, Brixen K, Steiniche T, Mosekilde L (1993) Ultrastructural investigations of bone resorptive cells in two types of autosomal dominant osteopetrosis. Bone 14:865–869

    Article  PubMed  CAS  Google Scholar 

  50. Blair HC, Teitelbaum SL, Tan HL, Koziol CM, Schlesinger PH (1991) Passive chloride permeability charge coupled to H+-ATPase of avian osteoclast ruffled membrane. Am J Physiol Cell Physiol 260:C1315–C1324

    CAS  Google Scholar 

  51. Schaller S, Henriksen K, Sveigaard C, Heegaard AM, Helix N, Stahlhut M, Ovejero MC, Johansen JV, Solberg H, Andersen TL, Hougaard D, Berryman M, Shiodt CB, Sorensen BH, Lichtenberg J, Christophersen P, Foged NT, Delaisse JM, Engsig MT, Karsdal MA (2004) The chloride channel inhibitor n53736 prevents bone resorption in ovariectomized rats without changing bone formation. J Bone Miner Res 19:1144–1153

    Article  PubMed  CAS  Google Scholar 

  52. Graves AR, Curran PK, Smith CL, Mindell JA (2008) The Cl−/H+ antiporter ClC-7 is the primary chloride permeation pathway in lysosomes. Nature 453:788–792

    Article  PubMed  CAS  Google Scholar 

  53. Garnero P, Ferreras M, Karsdal MA, NicAmhlaoibh R, Risteli J, Borel O, Qvist P, Delmas PD, Foged NT, Delaisse JM (2003) The type I collagen fragments ICTP and CTX reveal distinct enzymatic pathways of bone collagen degradation. J Bone Miner Res 18:859–867

    Article  PubMed  CAS  Google Scholar 

  54. Kiviranta R, Morko J, Alatalo SL, NicAmhlaoibh R, Risteli J, Laitala-Leinonen T, Vuorio E (2005) Impaired bone resorption in cathepsin K-deficient mice is partially compensated for by enhanced osteoclastogenesis and increased expression of other proteases via an increased RANKL/OPG ratio. Bone 36:159–172

    Article  PubMed  CAS  Google Scholar 

  55. Delaisse JM, Engsig MT, Everts V, del Carmen OM, Ferreras M, Lund L, Vu TH, Werb Z, Winding B, Lochter A, Karsdal MA, Troen T, Kirkegaard T, Lenhard T, Heegaard AM, Neff L, Baron R, Foged NT (2000) Proteinases in bone resorption: obvious and less obvious roles. Clin Chim Acta 291:223–234

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

We thank Thomas J. Jentsch and Jens Fuhrmann (FMP/MDC, Berlin, Germany) for providing the genetically engineered ClC-7 mouse strain and for critical reading and valuable discussions. We also thank Uwe Kornak (Max Planck Institute for Molecular Genetics, Charité University Hospital, Berlin, Germany) for critical reading and valuable discussions.

Author information

Authors and Affiliations

  1. Nordic Bioscience A/S, Herlev Hovedgade 207, 2730, Herlev, Denmark

    Anita Vibsig Neutzsky-Wulff, Morten A. Karsdal & Kim Henriksen

Authors
  1. Anita Vibsig Neutzsky-Wulff
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Morten A. Karsdal
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Kim Henriksen
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Kim Henriksen.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Neutzsky-Wulff, A.V., Karsdal, M.A. & Henriksen, K. Characterization of the Bone Phenotype in ClC-7-Deficient Mice. Calcif Tissue Int 83, 425–437 (2008). https://doi.org/10.1007/s00223-008-9185-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00223-008-9185-7

Keywords

Search

Navigation