Skip to main content
SpringerLink
Log in

Identification of novel regulators of osteoblast matrix mineralization by time series transcriptional profiling

Journal of Bone and Mineral Metabolism volume 32, pages 240–251 (2014)Cite this article

Abstract

Bone mineralization is a carefully orchestrated process, regulated by a number of promoters and inhibitors that function to ensure effective hydroxyapatite formation. Here we sought to identify new regulators of this process through a time series microarray analysis of mineralising primary osteoblast cultures over a 27 day culture period. To our knowledge this is the first microarray study investigating murine calvarial osteoblasts cultured under conditions that permit both physiological extracellular matrix mineralization through the formation of discrete nodules and the terminal differentiation of osteoblasts into osteocytes. RT-qPCR was used to validate and expand the microarray findings. We demonstrate the significant up-regulation of >6,000 genes during the osteoblast mineralization process, the highest-ranked differentially expressed genes of which were those dominated by members of the PPAR-γ signalling pathway, namely Adipoq, Cd36 and Fabp4. Furthermore, we show that the inhibition of this signalling pathway promotes matrix mineralisation in these primary osteoblast cultures. We also identify Cilp, Phex, Trb3, Sox11, and Psat1 as novel regulators of matrix mineralization. Further studies examining the precise function of the identified genes and their interactions will advance our understanding of the mechanisms underpinning biomineralization.

This is a preview of subscription content, access via your institution.

Access options

Buy single article

Instant access to the full article PDF.

USD 39.95

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

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

References

  1. Anderson HC (2003) Matrix vesicles and calcification. Curr Rheumatol Rep 5:222–226

    Article  PubMed  Google Scholar 

  2. Johnson K, Goding J, Van Etten D, Sali A, Hu SI, Farley D, Krug H, Hessle L, Millan JL, Terkeltaub R (2003) Linked deficiencies in extracellular PPi and osteopontin mediate pathologic calcification associated with defective PC-1 and ANK expression. J Bone Miner Res 18:994–1004

    Article  CAS  PubMed  Google Scholar 

  3. Murshed M, Schinke T, McKee MD, Karsenty G (2004) Extracellular matrix mineralization is regulated locally; different roles of two gla-containing proteins. J Cell Biol 165:625–630

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  4. Mackenzie NC, Huesa C, Rutsch F, MacRae VE (2012) New insights into NPP1 function: lessons from clinical and animal studies. Bone 51:961–968

    Article  CAS  PubMed  Google Scholar 

  5. Meyer JL (1984) Can biological calcification occur in the presence of pyrophosphate? Arch Biochem Biophys 231:1–8

    Article  CAS  PubMed  Google Scholar 

  6. Addison WN, Azari F, Sorensen ES, Kaartinen MT, McKee MD (2007) Pyrophosphate inhibits mineralization of osteoblast cultures by binding to mineral, up-regulating osteopontin, and inhibiting alkaline phosphatase activity. J Biol Chem 282:15872–15883

    Article  CAS  PubMed  Google Scholar 

  7. Nurnberg P, Thiele H, Chandler D, Hohne W, Cunningham ML, Ritter H, Leschik G, Uhlmann K, Mischung C, Harrop K, Goldblatt J, Borochowitz ZU, Kotzot D, Westermann F, Mundlos S, Braun HS, Laing N, Tinschert S (2001) Heterozygous mutations in ANKH, the human ortholog of the mouse progressive ankylosis gene, result in craniometaphyseal dysplasia. Nat Genet 28:37–41

    CAS  PubMed  Google Scholar 

  8. Johnson K, Vaingankar S, Chen Y, Moffa A, Goldring MB, Sano K, Jin-Hua P, Sali A, Goding J, Terkeltaub R (1999) Differential mechanisms of inorganic pyrophosphate production by plasma cell membrane glycoprotein-1 and B10 in chondrocytes. Arthritis Rheum 42:1986–1997

    Article  CAS  PubMed  Google Scholar 

  9. Roberts S, Narisawa S, Harmey D, Millan JL, Farquharson C (2007) Functional involvement of PHOSPHO1 in matrix vesicle-mediated skeletal mineralization. J Bone Miner Res 22:617–627

    Article  CAS  PubMed  Google Scholar 

  10. Stewart AJ, Roberts SJ, Seawright E, Davey MG, Fleming RH, Farquharson C (2006) The presence of PHOSPHO1 in matrix vesicles and its developmental expression prior to skeletal mineralization. Bone 39:1000–1007

    Article  CAS  PubMed  Google Scholar 

  11. Macrae VE, Davey MG, McTeir L, Narisawa S, Yadav MC, Millan JL, Farquharson C (2010) Inhibition of PHOSPHO1 activity results in impaired skeletal mineralization during limb development of the chick. Bone 46:1146–1155

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  12. Huesa C, Yadav MC, Finnila MA, Goodyear SR, Robins SP, Tanner KE, Aspden RM, Millan JL, Farquharson C (2011) PHOSPHO1 is essential for mechanically competent mineralization and the avoidance of spontaneous fractures. Bone 48:1066–1074

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  13. Yadav MC, Simao AM, Narisawa S, Huesa C, McKee MD, Farquharson C, Millan JL (2011) Loss of skeletal mineralization by the simultaneous ablation of PHOSPHO1 and alkaline phosphatase function: a unified model of the mechanisms of initiation of skeletal calcification. J Bone Miner Res 26:286–297

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  14. Orriss IR, Utting JC, Brandao-Burch A, Colston K, Grubb BR, Burnstock G, Arnett TR (2007) Extracellular nucleotides block bone mineralization in vitro: evidence for dual inhibitory mechanisms involving both P2Y2 receptors and pyrophosphate. Endocrinology 148:4208–4216

    Article  CAS  PubMed  Google Scholar 

  15. Staines KA, Macrae VE, Farquharson C (2012) The importance of the SIBLING family of proteins on skeletal mineralisation and bone remodelling. J Endocrinol 214:241–255

    Article  CAS  PubMed  Google Scholar 

  16. Burger EH, Klein-Nulend J (1999) Mechanotransduction in bone–role of the lacuno-canalicular network. FASEB J 13:S101–S112

    CAS  PubMed  Google Scholar 

  17. Bakker AD, Huesa C, Hughes A, Aspden RM, Van’t Hof RJ, Klein-Nulend J, Helfrich MH (2012) Endothelial nitric oxide synthase is not essential for nitric oxide production by osteoblasts subjected to fluid shear stress in vitro. Calcif Tissue Int

  18. Zhu D, Mackenzie NC, Millan JL, Farquharson C, MacRae VE (2011) The appearance and modulation of osteocyte marker expression during calcification of vascular smooth muscle cells. PLoS One 6:e19595

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  19. Prideaux M, Loveridge N, Pitsillides AA, Farquharson C (2012) Extracellular matrix mineralization promotes E11/gp38 glycoprotein expression and drives osteocytic differentiation. PLoS One 7:e36786

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  20. Newton PT, Staines KA, Spevak L, Boskey AL, Teixeira CC, Macrae VE, Canfield AE, Farquharson C (2012) Chondrogenic ATDC5 cells: an optimised model for rapid and physiological matrix mineralisation. Int J Mol Med 30:1187–1193

    CAS  PubMed Central  PubMed  Google Scholar 

  21. Irizarry RA, Bolstad BM, Collin F, Cope LM, Hobbs B, Speed TP (2003) Summaries of Affymetrix GeneChip probe level data. Nucleic Acids Res 31:e15

    Article  PubMed Central  PubMed  Google Scholar 

  22. Kauffmann A, Gentleman R, Huber W (2009) ArrayQualityMetrics–a bioconductor package for quality assessment of microarray data. Bioinformatics 25:415–416

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  23. Staines KA, Mackenzie NC, Clarkin CE, Zelenchuk L, Rowe PS, MacRae VE, Farquharson C (2012) MEPE is a novel regulator of growth plate cartilage mineralization. Bone 51:418–430

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  24. Doi M, Nagano A, Nakamura Y (2002) Genome-wide screening by cDNA microarray of genes associated with matrix mineralization by human mesenchymal stem cells in vitro. Biochem Biophys Res Commun 290:381–390

    Article  CAS  PubMed  Google Scholar 

  25. Lencel P, Delplace S, Hardouin P, Magne D (2011) TNF-alpha stimulates alkaline phosphatase and mineralization through PPAR-γ inhibition in human osteoblasts. Bone 48:242–249

    Article  CAS  PubMed  Google Scholar 

  26. Monemdjou R, Vasheghani F, Fahmi H, Perez G, Blati M, Taniguchi N, Lotz M, St-Arnaud R, Pelletier JP, Martel-Pelletier J, Beier F, Kapoor M (2011) Association of cartilage-specific deletion of peroxisome proliferator-activated receptor gamma with abnormal endochondral ossification and impaired cartilage growth and development in a murine model. Arthritis Rheum 64:1551–1561

    Article  Google Scholar 

  27. Challa TD, Rais Y, Ornan EM (2010) Effect of adiponectin on ATDC5 proliferation, differentiation and signalling pathways. Mol Cell Endocrinol 323:282–291

    Article  CAS  PubMed  Google Scholar 

  28. Wilson R, Norris EL, Brachvogel B, Angelucci C, Zivkovic S, Gordon L, Bernardo BC, Stermann J, Sekiguchi K, Gorman JJ, Bateman JF (2012) Changes in the chondrocyte and extracellular matrix proteome during post-natal mouse cartilage development. Mol Cell Proteomics 11:M111.014159

    Google Scholar 

  29. Woods A, James CG, Wang G, Dupuis H, Beier F (2009) Control of chondrocyte gene expression by actin dynamics: a novel role of cholesterol/Ror-alpha signalling in endochondral bone growth. J Cell Mol Med 13:3497–3516

    Article  PubMed  Google Scholar 

  30. Cecil DL, Appleton CT, Polewski MD, Mort JS, Schmidt AM, Bendele A, Beier F, Terkeltaub R (2009) The pattern recognition receptor CD36 is a chondrocyte hypertrophy marker associated with suppression of catabolic responses and promotion of repair responses to inflammatory stimuli. J Immunol 182:5024–5031

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  31. Campbell SE, Tandon NN, Woldegiorgis G, Luiken JJ, Glatz JF, Bonen A (2004) A novel function for fatty acid translocase (FAT)/CD36: involvement in long chain fatty acid transfer into the mitochondria. J Biol Chem 279:36235–36241

    Article  CAS  PubMed  Google Scholar 

  32. Furuhashi M, Fucho R, Gorgun CZ, Tuncman G, Cao H, Hotamisligil GS (2008) Adipocyte/macrophage fatty acid-binding proteins contribute to metabolic deterioration through actions in both macrophages and adipocytes in mice. J Clin Invest 118:2640–2650

    CAS  PubMed Central  PubMed  Google Scholar 

  33. Lee HW, Kim SY, Kim AY, Lee EJ, Choi JY, Kim JB (2009) Adiponectin stimulates osteoblast differentiation through induction of COX2 in mesenchymal progenitor cells. Stem Cells 27:2254–2262

    Article  CAS  PubMed  Google Scholar 

  34. Garcia T, Roman–Roman S, Jackson A, Theilhaber J, Connolly T, Spinella-Jaegle S, Kawai S, Courtois B, Bushnell S, Auberval M, Call K, Baron R (2002) Behavior of osteoblast, adipocyte, and myoblast markers in genome-wide expression analysis of mouse calvaria primary osteoblasts in vitro. Bone 31:205–211

    Article  CAS  PubMed  Google Scholar 

  35. Hasegawa T, Oizumi K, Yoshiko Y, Tanne K, Maeda N, Aubin JE (2008) The PPAR-γ-selective ligand BRL-49653 differentially regulates the fate choices of rat calvaria versus rat bone marrow stromal cell populations. BMC Dev Biol 8:71

    Article  PubMed Central  PubMed  Google Scholar 

  36. Yoshiko Y, Oizumi K, Hasegawa T, Minamizaki T, Tanne K, Maeda N, Aubin JE (2010) A subset of osteoblasts expressing high endogenous levels of PPAR-γ switches fate to adipocytes in the rat calvaria cell culture model. PLoS One 5:e11782

    Article  PubMed Central  PubMed  Google Scholar 

  37. Holm IA, Nelson AE, Robinson BG, Mason RS, Marsh DJ, Cowell CT, Carpenter TO (2001) Mutational analysis and genotype-phenotype correlation of the PHEX gene in X-linked hypophosphatemic rickets. J Clin Endocrinol Metab 86:3889–3899

    Article  CAS  PubMed  Google Scholar 

  38. Du L, Desbarats M, Viel J, Glorieux FH, Cawthorn C, Ecarot B (1996) cDNA cloning of the murine Pex gene implicated in X-linked hypophosphatemia and evidence for expression in bone. Genomics 36:22–28

    Article  CAS  PubMed  Google Scholar 

  39. Ruchon AF, Marcinkiewicz M, Siegfried G, Tenenhouse HS, DesGroseillers L, Crine P, Boileau G (1998) Pex mRNA is localized in developing mouse osteoblasts and odontoblasts. J Histochem Cytochem 46:459–468

    Article  CAS  PubMed  Google Scholar 

  40. Thompson DL, Sabbagh Y, Tenenhouse HS, Roche PC, Drezner MK, Salisbury JL, Grande JP, Poeschla EM, Kumar R (2002) Ontogeny of Phex/PHEX protein expression in mouse embryo and subcellular localization in osteoblasts. J Bone Miner Res 17:311–320

    Article  CAS  PubMed  Google Scholar 

  41. Bresler D, Bruder J, Mohnike K, Fraser WD, Rowe PS (2004) Serum MEPE-ASARM-peptides are elevated in X-linked rickets (HYP): implications for phosphaturia and rickets. J Endocrinol 183:R1–R9

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  42. Lorenzo P, Bayliss MT, Heinegard D (1998) A novel cartilage protein (CILP) present in the mid-zone of human articular cartilage increases with age. J Biol Chem 273:23463–23468

    Article  CAS  PubMed  Google Scholar 

  43. Hirose J, Masuda I, Ryan LM (2000) Expression of cartilage intermediate layer protein/nucleotide pyrophosphohydrolase parallels the production of extracellular inorganic pyrophosphate in response to growth factors and with aging. Arthritis Rheum 43:2703–2711

    Article  CAS  PubMed  Google Scholar 

  44. Hargrave M, Wright E, Kun J, Emery J, Cooper L, Koopman P (1997) Expression of the Sox11 gene in mouse embryos suggests roles in neuronal maturation and epithelio-mesenchymal induction. Dev Dyn 210:79–86

    Article  CAS  PubMed  Google Scholar 

  45. Cameron DA, Pennimpede T, Petkovich M (2009) Tulp3 is a critical repressor of mouse hedgehog signalling. Dev Dyn 238:1140–1149

    Article  CAS  PubMed  Google Scholar 

  46. Sock E, Rettig SD, Enderich J, Bosl MR, Tamm ER, Wegner M (2004) Gene targeting reveals a widespread role for the high-mobility-group transcription factor Sox11 in tissue remodeling. Mol Cell Biol 24:6635–6644

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  47. Baek JY, Jun DY, Taub D, Kim YH (2003) Characterization of human phosphoserine aminotransferase involved in the phosphorylated pathway of l-serine biosynthesis. Biochem J 373:191–200

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  48. Choi HD, Noh WC, Park JW, Lee JM, Suh JY (2011) Analysis of gene expression during mineralization of cultured human periodontal ligament cells. J Periodontal Implant Sci 41:30–43

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  49. Chang J, Wang Z, Tang E, Fan Z, McCauley L, Franceschi R, Guan K, Krebsbach PH, Wang CY (2009) Inhibition of osteoblastic bone formation by nuclear factor-kappaB. Nat Med 15:682–689

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  50. Chan MC, Nguyen PH, Davis BN, Ohoka N, Hayashi H, Du K, Lagna G, Hata A (2007) A novel regulatory mechanism of the bone morphogenetic protein (BMP) signalling pathway involving the carboxyl-terminal tail domain of BMP type II receptor. Mol Cell Biol 27:5776–5789

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  51. Zhang R, Oyajobi BO, Harris S, Chen D, Tsao C, Deng HW, Zhao M (2013) Wnt/beta-catenin signalling activates bone morphogenetic protein 2 expression in osteoblasts. Bone 52:145–156

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  52. Winkler DG, Sutherland MK, Geoghegan JC, Yu C, Hayes T, Skonier JE, Shpektor D, Jonas M, Kovacevich BR, Staehling-Hampton K, Appleby M, Brunkow ME, Latham JA (2003) Osteocyte control of bone formation via sclerostin, a novel BMP antagonist. EMBO J 22:6267–6276

    Article  CAS  PubMed Central  PubMed  Google Scholar 

Download references

Acknowledgments

The authors wish to acknowledge Alison Downing and Richard Talbot (Ark Genomics, The Roslin Institute, University of Edinburgh) and Al Ivens (Fios Genomics Ltd, University of Edinburgh) for the completion and analysis of the microarray. They would also like to acknowledge the Biotechnology and Biological Sciences Research Council (BBSRC; www.bbsrc.ac.uk) for an Institute Strategic Programme Grant and Institute Career Path Fellowship funding.

Conflict of interest

The authors declare that they have no conflicts of interest.

Author information

Authors and Affiliations

  1. The Roslin Institute and Royal (Dick) School of Veterinary Studies, The University of Edinburgh, Easter Bush, Midlothian, EH25 9RG, UK

    Katherine Ann Staines, Dongxing Zhu, Colin Farquharson & Vicky Elizabeth MacRae

  2. The Royal Veterinary College, University of London, Royal College Street, London, NW1 0TU, UK

    Katherine Ann Staines

Authors
  1. Katherine Ann Staines
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Dongxing Zhu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Colin Farquharson
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Vicky Elizabeth MacRae
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Katherine Ann Staines.

About this article

Cite this article

Staines, K.A., Zhu, D., Farquharson, C. et al. Identification of novel regulators of osteoblast matrix mineralization by time series transcriptional profiling. J Bone Miner Metab 32, 240–251 (2014). https://doi.org/10.1007/s00774-013-0493-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00774-013-0493-2

Keywords

search

Navigation