Skip to main content

Advertisement

SpringerLink
Log in

Peroxidase Enzymes Regulate Collagen Biosynthesis and Matrix Mineralization by Cultured Human Osteoblasts

  • Original Research
  • Published:
Calcified Tissue International Aims and scope Submit manuscript

Abstract

The early recruitment of inflammatory cells to sites of bone fracture and trauma is a critical determinant in successful fracture healing. Released by infiltrating inflammatory cells, myeloperoxidase (MPO) and eosinophil peroxidase (EPO) are heme-containing enzymes, whose functional involvement in bone repair has mainly been studied in the context of providing a mechanism for oxidative defense against invading microorganisms. We report here novel findings that show peroxidase enzymes have the capacity to stimulate osteoblastic cells to secrete collagen I protein and generate a mineralized extracellular matrix in vitro. Mechanistic studies conducted using cultured osteoblasts show that peroxidase enzymes stimulate collagen biosynthesis at a post-translational level in a prolyl hydroxylase-dependent manner, which does not require ascorbic acid. Our studies demonstrate that osteoblasts rapidly bind and internalize both MPO and EPO, and the catalytic activity of these peroxidase enzymes is essential to support collagen I biosynthesis and subsequent release of collagen by osteoblasts. We show that EPO is capable of regulating osteogenic gene expression and matrix mineralization in culture, suggesting that peroxidase enzymes may play an important role not only in normal bone repair, but also in the progression of pathological states where infiltrating inflammatory cells are known to deposit peroxidases.

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
Fig. 7

Similar content being viewed by others

References

  1. Claes L, Recknagel S, Ignatius A (2012) Fracture healing under healthy and inflammatory conditions. Nat Rev Rheumatol 8:133–143

    Article  CAS  PubMed  Google Scholar 

  2. Prasad GC, Udupa KN (1972) Studies on ultrastructural patter of osteogenic cells during bone repair. Acta Orthop Scand 43:163–175

    Article  CAS  PubMed  Google Scholar 

  3. Andrew JG, Andrew SM, Freemont AJ, Marsh DR (1994) Inflammatory cells in normal human fracture healing. Acta Orthop Scand 65:462–466

    Article  CAS  PubMed  Google Scholar 

  4. Bastian O, Pillay J, Alblas L, Leenen L, Koenderman L, Blokhuis T (2011) Systemic inflammation and fracture healing. J Leukoc Biol 89:669–673

    Article  CAS  PubMed  Google Scholar 

  5. Dimitriou R, Tsiridis E, Giannoudis PV (2005) Current concepts of molecular aspects of bone healing. Injury 36:1392–1404

    Article  PubMed  Google Scholar 

  6. Einhorn TA, Gerstenfeld LC (2015) Fracture healing: mechanisms and interventions. Nat Rev Rheumatol 11:45–54

    Article  PubMed Central  PubMed  Google Scholar 

  7. Grundnes O, Reikeras O (1993) The importance of the hematoma for fracture healing in rats. Acta Orthop Scand 64:340–342

    Article  CAS  PubMed  Google Scholar 

  8. Kolar P, Schmidt-Bleek K, Schell H et al (2010) The early fracture hematoma and its potential role in fracture healing. Tissue Eng Part B 16:427–434

    Article  Google Scholar 

  9. Davies MJ, Hawkins CL, Pattison DI, Rees M (2008) Mammalian heme peroxidases: from molecular mechanisms to health implications. Antioxid Redox Signal 10:1199–1234

    Article  CAS  PubMed  Google Scholar 

  10. Van der Veen BS, de Winther MPJ, Heeringa P (2009) Myeloperoxidase: molecular mechanisms of action and their relevance to human health and disease. Antioxid Redox Signal 11:2899–2937

    Article  PubMed  Google Scholar 

  11. Acharya KR, Ackerman SJ (2014) Eosinophil granule proteins: form and function. J Biol Chem 289:17406–17415

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  12. Klebanoff SJ (2005) Myeloperoxidase: friend or foe. J Leukoc Biol 77:598–625

    Article  CAS  PubMed  Google Scholar 

  13. DeNichilo MO, Panagopoulos V, Rayner TE et al (2015) Peroxidase enzymes regulate collagen extracellular matrix biosynthesis. Am J Pathol 185:1372–1384

    Article  CAS  PubMed  Google Scholar 

  14. Panagopoulos V, Zinonos I, Leach DA et al (2015) Uncovering a new role for peroxidase enzymes as drivers of angiogenesis. Int J Biochem Cell Biol 68:128–138

    Article  CAS  PubMed  Google Scholar 

  15. Atkins GJ, Kostakis P, Pan B et al (2003) RANKL expression is related to the differentiation state of human osteoblasts. J Bone Miner Res 18:1088–1098

    Article  CAS  PubMed  Google Scholar 

  16. Kettle AJ, Gedye CA, Winterbourn CC (1997) Mechanism of inactivation of myeloperoxidase by 4-aminobenzoic acid hydrazide. Biochem J 321:503–508

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  17. Carinci F, Pezzetti F, Spina A et al (2005) Effect of Vitamin C on pre-osteoblast gene expression. Arch Oral Biol 50:481–496

    Article  CAS  PubMed  Google Scholar 

  18. Maehata Y, Takamizawa S, Ozawa S et al (2007) Type III collagen is essential for growth acceleration of human osteoblastic cells by ascorbic acid 2-phosphate, a long-acting vitamin C derivative. Matrix Biol 26:371–381

    Article  CAS  PubMed  Google Scholar 

  19. Kivirikko KI, Myllylä R, Pihlajaniemi T (1989) Protein hydroxylation: prolyl 4-hydroxylase, an enzyme with four cosubstrates and a multifunctional subunit. FASEB J 3:1609–1617

    CAS  PubMed  Google Scholar 

  20. Myllyharju J, Kivirikko KI (2001) Collagens and collagen-related diseases. Ann Med 33:7–21

    Article  CAS  PubMed  Google Scholar 

  21. Canty EG, Kadler KE (2005) Procollagen trafficking, processing and fibrillogenesis. J Cell Sci 118:1341–1353

    Article  CAS  PubMed  Google Scholar 

  22. Baader E, Tschank G, Baringhaus K, Burghard H, Günzler V (1994) Inhibition of prolyl 4-hydroxylase by oxalyl amino acid derivatives in vitro, in isolated microsomes and in embryonic chicken tissues. Biochem J 300:525–530

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  23. Rosen V (2009) BMP2 signaling in bone development and repair. Cytokine Growth Factor Rev 20:475–480

    Article  CAS  PubMed  Google Scholar 

  24. Harris NL, Rattray KR, Tye CE et al (2000) Functional analysis of bone sialoprotein: identification of the hydroxyapatite-nucleating and cell-binding domains by recombinant peptide expression and site-directed mutagenesis. Bone 27:795–802

    Article  CAS  PubMed  Google Scholar 

  25. Yamaguchi TP, Bradley A, McMahon AP, Jones S (1999) A Wnt5a pathway underlies outgrowth of multiple structures in the vertebrate embryo. Development 126:1211–1223

    CAS  PubMed  Google Scholar 

  26. Okamoto M, Udagawa N, Uehara S et al (2014) Noncanonical Wnt5a enhances Wnt/β-catenin signalling during osteoblastogenesis. Sci Rep 4:4493–4500

    PubMed Central  PubMed  Google Scholar 

  27. Cox G, Einhorn TA, Tzioupis C, Giannoudis PV (2010) Bone-turnover markers in fracture healing. J Bone Joint Surg 3:329–334

    Article  Google Scholar 

  28. Baldus S, Eiserich JP, Mani A et al (2001) Endothelial transcytosis of myeloperoxidase confers specificity to vascular ECM proteins as targets of tyrosine nitration. J Clin Invest 108:1759–1770

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  29. Genestra M (2007) Oxyl radicals, redox-sensitive signalling cascades and antioxidants. Cell Signal 19:1807–1819

    Article  CAS  PubMed  Google Scholar 

  30. Gerald D, Berra E, Frapart YM et al (2004) JunD reduces tumor angiogenesis by protecting cells from oxidative stress. Cell 118:781–794

    Article  CAS  PubMed  Google Scholar 

  31. Page EL, Chan DA, Giaccia AJ et al (2008) Hypoxia-inducible factor-1α stabilization in nonhypoxic conditions: role of oxidation and intracellular ascorbate depletion. Mol Biol Cell 19:86–94

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  32. Bai XC, Lu D, Bai J et al (2004) Oxidative stress inhibits osteoblastic differentiation of bone cells by ERK and NF-kappaB. Biochem Biophys Res Commun 314:197–207

    Article  CAS  PubMed  Google Scholar 

  33. Ramp WK, Arnold RR, Russell JE, Yancey JM (1987) Hydrogen peroxide inhibits glucose metabolism and collagen synthesis in bone. J Periodontol 58:340–344

    Article  CAS  PubMed  Google Scholar 

  34. Lecanda F, Avioli LV, Cheng S (1997) Regulation of bone matrix protein expression and induction of differentiation of human osteoblasts and human bone marrow stromal cells by bone morphogenetic protein-2. J Cell Biochem 67:386–398

    Article  CAS  PubMed  Google Scholar 

  35. Robubi A, Berger C, Schmid M, Huber KR, Engel A, Krugluger W (2014) Gene expression profiles induced by growth factors in in vitro cultured osteoblasts. Bone Joint Res 3:236–240

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  36. Furtmüller PG, Zederbauer M, Jantschko W et al (2006) Active site structural and catalytic mechanisms of human peroxidases. Arch Biochem Biophys 445:199–213

    Article  PubMed  Google Scholar 

  37. Mehta V, Campeau NG, Kita H, Hagan JB (2008) Blood and sputum eosinophil levels in asthma and their relationships to sinus computed tomographic findings. Mayo Clin Proc 83:671–678

    Article  PubMed Central  PubMed  Google Scholar 

  38. Snidvongs K, McLachlan R, Chin D et al (2012) Osteitic bone: a surrogate marker of eosinophilia in chronic rhinosinusitis. Rhinology 50:299–305

    CAS  PubMed  Google Scholar 

  39. Macias MP, Fitzpatrick LA, Brenneise I, McGarry MP, Lee JJ, Lee NA (2001) Expression of IL-5 alters bone metabolism and induces ossification of the spleen in transgenic mice. J Clin Invest 107:949–959

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  40. Abu-Ghazaleh RI, Dunnette SL, Loegering DA et al (1992) Eosinophil granule proteins in peripheral blood granulocytes. J Leukoc Biol 52:611–618

    CAS  PubMed  Google Scholar 

  41. Levi-Schaffer F, Garbuzenko E, Rubin A et al (1999) Human eosinophils regulate lung- and skin-derived fibroblast properties in vitro: a role for transforming growth factor β (TGF-β). Proc Natl Acad Sci USA 96:9660–9665

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  42. Evans KN, Forsberg JA, Potter BK et al (2012) Inflammatory cytokine and chemokine expression is associated with heterotopic ossification in high-energy penetrating war injuries. Orthop Trauma 26:204–212

    Google Scholar 

  43. Convente MR, Wang H, Pignolo RJ, Kaplan FS, Shore EM (2015) The immunological contribution to heterotopic ossification disorders. Curr Osteoporos Rep 13:116–124

    Article  PubMed  Google Scholar 

Download references

Acknowledgments

This work was supported in part by The Hospital Research Foundation and the National Health and Medical Research Council (Career Development Fellowship/627015; Project Grant/1050694).

Author information

Authors and Affiliations

  1. Breast Cancer Research Unit, Discipline of Surgery, The University of Adelaide, Adelaide, Australia

    Mark O. DeNichilo, Alexandra J. Shoubridge, Vasilios Panagopoulos, Vasilios Liapis, Aneta Zysk, Irene Zinonos, Shelley Hay & Andreas Evdokiou

  2. Discipline of Orthopaedics and Trauma, The University of Adelaide, Adelaide, Australia

    Gerald J. Atkins & David M. Findlay

  3. Centre for Personalized Cancer Medicine, The University of Adelaide, Adelaide, Australia

    Andreas Evdokiou

  4. TQEH, Basil Hetzel Research Institute, 28 Woodville Road, Woodville, SA, 5011, Australia

    Mark O. DeNichilo

Authors
  1. Mark O. DeNichilo
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Alexandra J. Shoubridge
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Vasilios Panagopoulos
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Vasilios Liapis
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Aneta Zysk
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Irene Zinonos
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Shelley Hay
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Gerald J. Atkins
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. David M. Findlay
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Andreas Evdokiou
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Mark O. DeNichilo.

Ethics declarations

Conflict of Interest

Mark O. DeNichilo, Alexandra J. Shoubridge, Vasilios Panagopoulos, Vasilios Liapis, Aneta Zysk, Irene Zinonos, Shelley Hay, Gerald J. Atkins, David M. Findlay, and Andreas Evdokiou declare that they have no conflict of interest.

Human and Animal Rights and Informed Consent

The use of all normal human donor-derived bone tissue was approved by the human ethics committees of the Royal Adelaide Hospital/University of Adelaide (Approval No. RAH130114). Human bone samples were obtained with informed written donor consent, as required and approved by the ethics committee.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

DeNichilo, M.O., Shoubridge, A.J., Panagopoulos, V. et al. Peroxidase Enzymes Regulate Collagen Biosynthesis and Matrix Mineralization by Cultured Human Osteoblasts. Calcif Tissue Int 98, 294–305 (2016). https://doi.org/10.1007/s00223-015-0090-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00223-015-0090-6

Keywords

Search

Navigation