Purinergic Signalling

, Volume 13, Issue 4, pp 545–557 | Cite as

Bone turnover is altered in transgenic rats overexpressing the P2Y2 purinergic receptor

  • Maria Ellegaard
  • Cansu Agca
  • Solveig Petersen
  • Ankita Agrawal
  • Lars Schack Kruse
  • Ning Wang
  • Alison Gartland
  • Jens-Erik Beck Jensen
  • Niklas Rye JørgensenEmail author
  • Yuksel AgcaEmail author
Original Article


It is now widely recognized that purinergic signaling plays an important role in the regulation of bone remodeling. One receptor subtype, which has been suggested to be involved in this regulation, is the P2Y2 receptor (P2Y2R). In the present study, we investigated the effect of P2Y2R overexpression on bone status and bone cell function using a transgenic rat. Three-month-old female transgenic Sprague Dawley rats overexpressing P2Y2R (P2Y2R-Tg) showed higher bone strength of the femoral neck. Histomorphometry showed increase in resorptive surfaces and reduction in mineralizing surfaces. Both mineral apposition rate and thickness of the endocortical osteoid layer were higher in the P2Y2R-Tg rats. μCT analysis showed reduced trabecular thickness and structural model index in P2Y2R-Tg rats. Femoral length was increased in the P2Y2R-Tg rats compared to Wt rats. In vitro, there was an increased formation of osteoclasts, but no change in total resorption in cultures from P2Y2R-Tg rats. The formation of mineralized nodules was significantly reduced in the osteoblastic cultures from P2Y2R-Tg rats. In conclusion, our study suggests that P2Y2R is involved in regulation of bone turnover, due to the effects on both osteoblasts and osteoclasts and that these effects might be relevant in the regulation of bone growth.


Genetic animal models Osteoblasts Osteoclasts Cell/tissue signaling P2Y2 purinergic receptors 



This work was partially supported by the start-up funds from University of Missouri, National Institute of Health (grant no. P40 OD011062) and by the European Commission under the 7th Framework Programme (proposal no. 202231) performed as a collaborative project among the members of the ATPBone Consortium (Copenhagen University, University College London, University of Maastricht, University of Ferrara, University of Liverpool, University of Sheffield, and Université Libre de Bruxelles), and is a substudy under the main study “Fighting osteoporosis by blocking nucleotides: purinergic signalling in bone formation and homeostasis.”

Author contributions

Concept and design of the study: YA, NRJ, CA, AG. Conducting experiments: CA, YA, LSK, NW. Acquiring data: CA, YA, NRJ, SP, ME, AA, LSK, NW, AG, JEBJ. Analyzing data: NRJ, YA, CA, ME, SP, LSK, NW, AA, AG. Providing reagents and animal model: YA, CA. Writing, critically reviewing and approving the manuscript: NRJ, YA, CA, ME, SP, LSK, NW, AA, AG, JEBJ.

Compliance with ethical standards

Ethical approval

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. All procedures performed in studies involving animals were in accordance with the ethical standards of the institution at which the studies were conducted.

Conflict of interest

Maria Ellegaard declares that she has no conflict of interest.

Cansu Agca declares that she has no conflict of interest.

Solveig Petersen declares that she has no conflict of interest.

Ankita Agrawal declares that she has no conflict of interest.

Lars Schack Kruse declares that he has no conflict of interest.

Ning Wang declares that he/she has no conflict of interest.

Alison Gartland declares that she has no conflict of interest.

Jens-Erik Beck Jensen declares that he has no conflict of interest.

Niklas Rye Jørgensen declares that he has no conflict of interest.

Yuksel Agca declares that he has no conflict of interest.


  1. 1.
    Ralevic V, Burnstock G (1998) Receptors for purines and pyrimidines. Pharmacol Rev 50:413–492PubMedGoogle Scholar
  2. 2.
    Burnstock G, Arnett TR, Orriss IR (2013) Purinergic signalling in the musculoskeletal system. Purinergic Signal 9:541–572CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    James G, Butt AM (2001) P2X and P2Y purinoreceptors mediate ATP-evoked calcium signalling in optic nerve glia in situ. Cell Calcium 30:251–259CrossRefPubMedGoogle Scholar
  4. 4.
    Bowler WB, Dixon CJ, Halleux C, Maier R, Bilbe G, Fraser WD, Gallagher JA, Hipskind RA (1999) Signaling in human osteoblasts by extracellular nucleotides. Their weak induction of the c-fos proto-oncogene via Ca2+ mobilization is strongly potentiated by a parathyroid hormone/cAMP-dependent protein kinase pathway independently of mitogen-activated protein kinase. J Biol Chem 274:14315–14324CrossRefPubMedGoogle Scholar
  5. 5.
    Schofl C, Cuthbertson KS, Walsh CA, Mayne C, Cobbold P, von-zur Mühlen A, Hesch RD, Gallagher JA (1992) Evidence for P2-purinoceptors on human osteoblast-like cells. J Bone Miner Res 7:485–491CrossRefPubMedGoogle Scholar
  6. 6.
    Orriss IR, Knight GE, Ranasinghe S, Burnstock G, Arnett TR (2006) Osteoblast responses to nucleotides increase during differentiation. Bone 39:300–309CrossRefPubMedGoogle Scholar
  7. 7.
    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 148:4208–4216CrossRefPubMedGoogle Scholar
  8. 8.
    Ayala-Pena VB, Scolaro LA, Santillan GE (2013) ATP and UTP stimulate bone morphogenetic protein-2,-4 and -5 gene expression and mineralization by rat primary osteoblasts involving PI3K/AKT pathway. Exp Cell Res 319:2028–2036CrossRefPubMedGoogle Scholar
  9. 9.
    Bowler WB, Littlewood-Evans A, Bilbe G, Gallagher JA, Dixon CJ (1998) P2Y2 receptors are expressed by human osteoclasts of giant cell tumor but do not mediate ATP-induced bone resorption. Bone 22:195–200CrossRefPubMedGoogle Scholar
  10. 10.
    Jorgensen NR, Geist ST, Civitelli R, Steinberg TH (1997) ATP- and gap junction-dependent intercellular calcium signaling in osteoblastic cells. J Cell Biol 139:497–506CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Jorgensen NR, Henriksen Z, Sorensen OH, Eriksen EF, Civitelli R, Steinberg TH (2002) Intercellular calcium signaling occurs between human osteoblasts and osteoclasts and requires activation of osteoclast P2X7 receptors. J Biol Chem 277:7574–7580CrossRefPubMedGoogle Scholar
  12. 12.
    Jorgensen NR, Henriksen Z, Brot C, Eriksen EF, Sorensen OH, Civitelli R, Steinberg TH (2000) Human osteoblastic cells propagate intercellular calcium signals by two different mechanisms. J Bone Miner Res 15:1024–1032CrossRefPubMedGoogle Scholar
  13. 13.
    Gardinier J, Yang W, Madden GR, Kronbergs A, Gangadharan V, Adams E, Czymmek K, Duncan RL (2014) P2Y2 receptors regulate osteoblast mechanosensitivity during fluid flow. Am J Phys Cell Phys 306:C1058–C1067CrossRefGoogle Scholar
  14. 14.
    Xing Y, Gu Y, Bresnahan JJ, Paul EM, Donahue HJ, You J (2014) The roles of P2Y2 purinergic receptors in osteoblasts and mechanotransduction. PLoS One 9:e108417CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Buscher R, Hoerning A, Patel HH, Zhang S, Arthur DB, Grasemann H, Ratjen F, Insel PA (2006) P2Y2 receptor polymorphisms and haplotypes in cystic fibrosis and their impact on Ca2+ influx. Pharmacogenet Genomics 16:199–205PubMedGoogle Scholar
  16. 16.
    Dasari VR, Sandhu AK, Mills DC, Athwal RS, Kunapuli SP (1996) Mapping of the P2U purinergic receptor gene to human chromosome 11q 13.5–14.1. Somat Cell Mol Genet 22:75–79CrossRefPubMedGoogle Scholar
  17. 17.
    Janssens R, Paindavoine P, Parmentier M, Boeynaems JM (1999) Human P2Y2 receptor polymorphism: identification and pharmacological characterization of two allelic variants. Br J Pharmacol 127:709–716CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Wesselius A, Bours MJ, Henriksen Z, Syberg S, Petersen S, Schwarz P, Jorgensen NR, van Helden S, Dagnelie PC (2013) Association of P2Y(2) receptor SNPs with bone mineral density and osteoporosis risk in a cohort of Dutch fracture patients. Purinergic Signal 9:41–49CrossRefPubMedGoogle Scholar
  19. 19.
    Wesselius A, Bours MJ, Agrawal A, Gartland A, Dagnelie PC, Schwarz P, Jorgensen NR (2011) Role of purinergic receptor polymorphisms in human bone. Front Biosci (Landmark Ed) 16:2572–2585CrossRefGoogle Scholar
  20. 20.
    Agca C, Seye C, Kashuba Benson CM, Rikka S, Chan AW, Weisman GA, Agca Y (2009) Development of a novel transgenic rat overexpressing the P2Y(2) nucleotide receptor using a lentiviral vector. J Vasc Res 46:447–458CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Ellegaard M, Kringelbach T, Syberg S, Petersen S, Beck Jensen JE, Bruel A, Jorgensen NR, Schwarz P (2013) The effect of PTH(1–34) on fracture healing during different loading conditions. J Bone Miner Res 28:2145–2155CrossRefPubMedGoogle Scholar
  22. 22.
    Syberg S, Schwarz P, Petersen S, Steinberg TH, Jensen JE, Teilmann J, Jorgensen NR (2012) Association between P2X7 receptor polymorphisms and bone status in mice. J Osteoporos 2012:637986PubMedPubMedCentralGoogle Scholar
  23. 23.
    Orriss IR, Taylor SE, Arnett TR (2012) Rat osteoblast cultures. Methods Mol Biol 816:31–41CrossRefPubMedGoogle Scholar
  24. 24.
    Dahl M, Syberg S, Jorgensen NR, Pinholt EM (2013) Adipose derived mesenchymal stem cells—their osteogenicity and osteoblast in vitro mineralization on titanium granule carriers. J Craniomaxillofac Surg 41:e213–e220CrossRefPubMedGoogle Scholar
  25. 25.
    Goodyear SR, Gibson IR, Skakle JM, Wells RP, Aspden RM (2009) A comparison of cortical and trabecular bone from C57 Black 6 mice using Raman spectroscopy. Bone 44:899–907CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2017

Authors and Affiliations

  • Maria Ellegaard
    • 1
  • Cansu Agca
    • 2
  • Solveig Petersen
    • 1
  • Ankita Agrawal
    • 1
    • 3
  • Lars Schack Kruse
    • 1
  • Ning Wang
    • 3
  • Alison Gartland
    • 3
  • Jens-Erik Beck Jensen
    • 4
  • Niklas Rye Jørgensen
    • 1
    • 5
    Email author
  • Yuksel Agca
    • 2
    Email author
  1. 1.Department of Clinical BiochemistryRigshospitaletCopenhagenDenmark
  2. 2.College of Veterinary MedicineUniversity of MissouriColumbiaUSA
  3. 3.Mellanby Centre for Bone ResearchUniversity of SheffieldSheffieldUK
  4. 4.Osteoporosis and Bone Metabolic Unit, Department of EndocrinologyCopenhagen University Hospital HvidovreHvidovreDenmark
  5. 5.OPEN, Odense Patient data Explorative Network, Odense University Hospital/Institute of Clinical ResearchUniversity of Southern DenmarkOdenseDenmark

Personalised recommendations