Skip to main content
NCBI home page
Annals of the Rheumatic Diseases logoLink to Annals of the Rheumatic Diseases
. 2004 Jan;63(1):67–70. doi: 10.1136/ard.2002.005256

Metabolic activation stimulates acid secretion and expression of matrix degrading proteases in human osteoblasts

M George 1, B Stein 1, O Muller 1, M Weis-Klemm 1, T Pap 1, W Parak 1, W Aicher 1
PMCID: PMC1754733  PMID: 14672894

Abstract

Background: Both cellular and matrix components of healthy bone are permanently renewed in a balanced homoeostasis. Osteoclastic bone resorption involves the expression of vacuolar-type ATPase proton pumps (vATPase) on the outer cell membrane and the secretion of matrix degrading proteases. Osteoblasts modulate the deposition of bone mineral components and secrete extracellular matrix proteins.

Objectives: To investigate the ability of osteoblasts and osteosarcoma to secrete acid and express matrix degrading proteases upon metabolic activation. To examine also the potential contribution of vATPases to proton secretion expressed on osteoblasts.

Methods: Osteoblasts were isolated from trabecular bone and characterised by reverse transcriptase-polymerase chain reaction and immunohistochemistry. Proton secretion was analysed by a cytosensor microphysiometer.

Results: Osteoblasts not only express matrix degrading proteases upon stimulation with tumour necrosis factor or with phorbol ester but they also secrete protons upon activation. Proton secretion by osteoblasts is associated partially with proton pump ATPases.

Conclusion: These data suggest that, in addition to monocyte derived osteoclasts, cytokine activated mesenchymal osteoblasts and osteosarcoma cells may contribute to the acidic milieu required for bone degradation.

Full Text

The Full Text of this article is available as a PDF (301.0 KB).

Figure 1 .

Figure 1

Open in a new tab

Activation of proton secretion (rmax/req in per cent) in osteoblasts upon stimulation by PMA or TNF. (A) Osteoblasts were preincubated for about 20 minutes in running medium to reach metabolic quiescence characterised by a low spontaneous acidification (r(t) = req, white zone). After 15 minutes of preincubation the fluctuations of the acidification value drop below 5%. Then cells are activated with PMA (grey zone), and the metabolic responses are recorded as a function of time. Each data point represents the mean value of n individual measurements of xi (n = 5–10) and the error bars represent the normalised standard deviations. Addition of 1 µg/ml PMA induced an immediate increase in acidification, reaching a plateau at about 115% after 20 minutes. Addition of 5 µg/ml PMA induced a higher response reaching more than 120% acidification. Addition of 10 µg/ml PMA induced an immediate acidification response and a plateau was not observed after 40 minutes of induction. (B) Osteoblasts were activated with TNF (grey zone) as described above (see (A)). Addition of 20 ng/ml TNF induced a slow increase in acidification reaching about 115% after 20 minutes. Addition of 50 ng/ml TNF induced a response reaching more than 120% acidification. Addition of 100 ng/ml TNFα at first reduced the acidification rate below the equilibrium level, then an acidification response was recorded reaching about 125% after 20 minutes

Figure 2 .

Figure 2

Open in a new tab

Blocking proton secretion by osteoblasts upon addition of Bafilomycin A1 or Amiloride. Osteoblast-like cells were incubated in medium containing 1 mM and 2 mM Bafilomycin A 1 or 500 mM and 10000 mM Amiloride. Pericellular acidification was recorded by cytosensor microphysiometer. Each data point represents the mean of two individual measurements and the error bars represent the normalised standard deviations. Both the vATPase proton pump blocker, Bafilomycin A1, and the blocker of Na+/H+ transmembrane exchange pathways, Amiloride, reduced proton secretion by 5–12%.

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Barrett M. G., Belinsky G. S., Tashjian A. H., Jr A new action of parathyroid hormone. receptor-mediated stimulation of extracellular acidification in human osteoblast-like SaOS-2 cells. J Biol Chem. 1997 Oct 17;272(42):26346–26353. doi: 10.1074/jbc.272.42.26346. [DOI] [PubMed] [Google Scholar]
  2. Belinsky G. S., Tashjian A. H., Jr Direct measurement of hormone-induced acidification in intact bone. J Bone Miner Res. 2000 Mar;15(3):550–556. doi: 10.1359/jbmr.2000.15.3.550. [DOI] [PubMed] [Google Scholar]
  3. Blair H. C., Sidonio R. F., Friedberg R. C., Khan N. N., Dong S. S. Proteinase expression during differentiation of human osteoclasts in vitro. J Cell Biochem. 2000 Jun 12;78(4):627–637. doi: 10.1002/1097-4644(20000915)78:4<627::aid-jcb12>3.0.co;2-3. [DOI] [PubMed] [Google Scholar]
  4. Chen J. J., Jin H., Ranly D. M., Sodek J., Boyan B. D. Altered expression of bone sialoproteins in vitamin D-deficient rBSP2.7Luc transgenic mice. J Bone Miner Res. 1999 Feb;14(2):221–229. doi: 10.1359/jbmr.1999.14.2.221. [DOI] [PubMed] [Google Scholar]
  5. Clohisy D. R., Ogilvie C. M., Carpenter R. J., Ramnaraine M. L. Localized, tumor-associated osteolysis involves the recruitment and activation of osteoclasts. J Orthop Res. 1996 Jan;14(1):2–6. doi: 10.1002/jor.1100140103. [DOI] [PubMed] [Google Scholar]
  6. Clohisy D. R., Ramnaraine M. L. Osteoclasts are required for bone tumors to grow and destroy bone. J Orthop Res. 1998 Nov;16(6):660–666. doi: 10.1002/jor.1100160606. [DOI] [PubMed] [Google Scholar]
  7. Ducy P., Schinke T., Karsenty G. The osteoblast: a sophisticated fibroblast under central surveillance. Science. 2000 Sep 1;289(5484):1501–1504. doi: 10.1126/science.289.5484.1501. [DOI] [PubMed] [Google Scholar]
  8. Dørup J., Scholz H., Maunsbach A. B., Kaissling B. Effects of inhibition of the vacuolar-type proton pump on nephron ultrastructure and acidification in the isolated perfused rat kidney. Exp Nephrol. 1995 May-Jun;3(3):180–192. [PubMed] [Google Scholar]
  9. Heinemann T., Bulwin G. C., Randall J., Schnieders B., Sandhoff K., Volk H. D., Milford E., Gullans S. R., Utku N. Genomic organization of the gene coding for TIRC7, a novel membrane protein essential for T cell activation. Genomics. 1999 May 1;57(3):398–406. doi: 10.1006/geno.1999.5751. [DOI] [PubMed] [Google Scholar]
  10. Hughes D. E., Dai A., Tiffee J. C., Li H. H., Mundy G. R., Boyce B. F. Estrogen promotes apoptosis of murine osteoclasts mediated by TGF-beta. Nat Med. 1996 Oct;2(10):1132–1136. doi: 10.1038/nm1096-1132. [DOI] [PubMed] [Google Scholar]
  11. Karet F. E., Finberg K. E., Nelson R. D., Nayir A., Mocan H., Sanjad S. A., Rodriguez-Soriano J., Santos F., Cremers C. W., Di Pietro A. Mutations in the gene encoding B1 subunit of H+-ATPase cause renal tubular acidosis with sensorineural deafness. Nat Genet. 1999 Jan;21(1):84–90. doi: 10.1038/5022. [DOI] [PubMed] [Google Scholar]
  12. Kobayashi K., Takahashi N., Jimi E., Udagawa N., Takami M., Kotake S., Nakagawa N., Kinosaki M., Yamaguchi K., Shima N. Tumor necrosis factor alpha stimulates osteoclast differentiation by a mechanism independent of the ODF/RANKL-RANK interaction. J Exp Med. 2000 Jan 17;191(2):275–286. doi: 10.1084/jem.191.2.275. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kornak U., Schulz A., Friedrich W., Uhlhaas S., Kremens B., Voit T., Hasan C., Bode U., Jentsch T. J., Kubisch C. Mutations in the a3 subunit of the vacuolar H(+)-ATPase cause infantile malignant osteopetrosis. Hum Mol Genet. 2000 Aug 12;9(13):2059–2063. doi: 10.1093/hmg/9.13.2059. [DOI] [PubMed] [Google Scholar]
  14. Lee B. S., Holliday L. S., Krits I., Gluck S. L. Vacuolar H+-ATPase activity and expression in mouse bone marrow cultures. J Bone Miner Res. 1999 Dec;14(12):2127–2136. doi: 10.1359/jbmr.1999.14.12.2127. [DOI] [PubMed] [Google Scholar]
  15. Mobasheri A., Golding S., Pagakis S. N., Corkey K., Pocock A. E., Fermor B., O'BRIEN M. J., Wilkins R. J., Ellory J. C., Francis M. J. Expression of cation exchanger NHE and anion exchanger AE isoforms in primary human bone-derived osteoblasts. Cell Biol Int. 1998;22(7-8):551–562. doi: 10.1006/cbir.1998.0299. [DOI] [PubMed] [Google Scholar]
  16. Nordström T., Grinstein S., Brisseau G. F., Manolson M. F., Rotstein O. D. Protein kinase C activation accelerates proton extrusion by vacuolar-type H(+)-ATPases in murine peritoneal macrophages. FEBS Lett. 1994 Aug 15;350(1):82–86. doi: 10.1016/0014-5793(94)00738-1. [DOI] [PubMed] [Google Scholar]
  17. Pap Thomas, Claus Anja, Ohtsu Susumu, Hummel Klaus M., Schwartz Peter, Drynda Susanne, Pap Géza, Machner Andreas, Stein Bernhard, George Michael. Osteoclast-independent bone resorption by fibroblast-like cells. Arthritis Res Ther. 2003 Mar 26;5(3):R163–R173. doi: 10.1186/ar752. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Parak W. J., Dannöhl S., George M., Schuler M. K., Schaumburger J., Gaub H. E., Müller O., Aicher W. K. Metabolic activation stimulates acid production in synovial fibroblasts. J Rheumatol. 2000 Oct;27(10):2312–2322. [PubMed] [Google Scholar]
  19. Rifas L., Fausto A., Scott M. J., Avioli L. V., Welgus H. G. Expression of metalloproteinases and tissue inhibitors of metalloproteinases in human osteoblast-like cells: differentiation is associated with repression of metalloproteinase biosynthesis. Endocrinology. 1994 Jan;134(1):213–221. doi: 10.1210/endo.134.1.8275936. [DOI] [PubMed] [Google Scholar]
  20. Rousselle A-V, Heymann D. Osteoclastic acidification pathways during bone resorption. Bone. 2002 Apr;30(4):533–540. doi: 10.1016/s8756-3282(02)00672-5. [DOI] [PubMed] [Google Scholar]
  21. Sato T., Foged N. T., Delaissé J. M. The migration of purified osteoclasts through collagen is inhibited by matrix metalloproteinase inhibitors. J Bone Miner Res. 1998 Jan;13(1):59–66. doi: 10.1359/jbmr.1998.13.1.59. [DOI] [PubMed] [Google Scholar]
  22. Sato T., del Carmen Ovejero M., Hou P., Heegaard A. M., Kumegawa M., Foged N. T., Delaissé J. M. Identification of the membrane-type matrix metalloproteinase MT1-MMP in osteoclasts. J Cell Sci. 1997 Mar;110(Pt 5):589–596. doi: 10.1242/jcs.110.5.589. [DOI] [PubMed] [Google Scholar]
  23. Scimeca J. C., Franchi A., Trojani C., Parrinello H., Grosgeorge J., Robert C., Jaillon O., Poirier C., Gaudray P., Carle G. F. The gene encoding the mouse homologue of the human osteoclast-specific 116-kDa V-ATPase subunit bears a deletion in osteosclerotic (oc/oc) mutants. Bone. 2000 Mar;26(3):207–213. doi: 10.1016/s8756-3282(99)00278-1. [DOI] [PubMed] [Google Scholar]
  24. Smith A. N., Skaug J., Choate K. A., Nayir A., Bakkaloglu A., Ozen S., Hulton S. A., Sanjad S. A., Al-Sabban E. A., Lifton R. P. Mutations in ATP6N1B, encoding a new kidney vacuolar proton pump 116-kD subunit, cause recessive distal renal tubular acidosis with preserved hearing. Nat Genet. 2000 Sep;26(1):71–75. doi: 10.1038/79208. [DOI] [PubMed] [Google Scholar]
  25. Teitelbaum S. L. Bone resorption by osteoclasts. Science. 2000 Sep 1;289(5484):1504–1508. doi: 10.1126/science.289.5484.1504. [DOI] [PubMed] [Google Scholar]
  26. Varghese S., Rydziel S., Canalis E. Basic fibroblast growth factor stimulates collagenase-3 promoter activity in osteoblasts through an activator protein-1-binding site. Endocrinology. 2000 Jun;141(6):2185–2191. doi: 10.1210/endo.141.6.7504. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

[Web only figure and tables]
annrheumd_63_1_67__index.html (6.5KB, html)
annrheumd_63_1_67__1.pdf (4.6KB, pdf)
annrheumd_63_1_67__2.pdf (5.9KB, pdf)
annrheumd_63_1_67__3.pdf (1.3MB, pdf)
annrheumd_63_1_67__4.pdf (47.5KB, pdf)
annrheumd_63_1_67__5.pdf (10.7KB, pdf)
annrheumd_63_1_67__6.pdf (106.6KB, pdf)
93294a93775c263b684228e80b534dd1_F1.html (2.5KB, html)
fc62903f8169aad462ba75d6ed8daf7b_ar5256.f1.jpeg (96.3KB, jpeg)
cded4bd645dc2a9af78337aa95162abe_ar5256.f1.gif (24.5KB, gif)
annrheumd_63_1_67__7.pdf (7.5KB, pdf)
annrheumd_63_1_67__8.pdf (6.8KB, pdf)

Articles from Annals of the Rheumatic Diseases are provided here courtesy of BMJ Publishing Group

RESOURCES