Skip to main content
SpringerLink
Log in

Dual Role of P2 Receptors during Osteoblast Differentiation

  • Original Paper
  • Published:
Cell Biochemistry and Biophysics Aims and scope Submit manuscript

Abstract

P2 receptors activated by ATP are expressed in the skeletal system. However, the role of P2 receptors in osteoblast differentiation remains unclear. Methods: Participation of P2 receptors in differentiation was investigated in the preosteoblast MC3T3-M1 cell line. Preosteoblasts were stimulated for 7 or 14 days in the presence of osteogenic medium containing ATP and its analogs, and then alkaline phosphatase (ALP) activity, gene expression analyses, and protein expression were assessed. Results: We observed that ATP and its analogs promoted increased ALP activity after 7 days of treatment. In contrast, these agonists promoted reductions in ALP activity after 14 days. Some antagonists, such as PPADS (P2 antagonist), MRS2179 (P2Y1 antagonist), MRS2578 (P2Y6 antagonist), and AZ11645373 (P2X7 antagonist) reduced the increases in ALP activity after 7 days. However, only AZ11645373 inhibited the reduction in ALP activity after 14 days. The expression of the P2Y2, P2Y6, P2X4, and P2X7 receptors was observed. Furthermore, treatment with ATP modulated the expression of P2 receptors, increasing P2X4 expression and reducing P2Y6 and P2X7 expression. Similar results were observed after 14 days. In addition, ATP treatment for 7 days increased the expression of transcription factors associated with osteoblast differentiation, such as Runx2, SP7, and Dix5, whereas SP7 and Dix5 expression was reduced at 14 days. These results suggest that P2 receptor activation modulates the differentiation of osteoblasts and is dependent upon the stage of differentiation. These results also suggest that several P2 receptors are involved in this process.

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

Similar content being viewed by others

References

  1. Alvarenga, E. C., Rodrigues, R., Caricati-Neto, A., Silva-Filho, F. C., Paredes-Gamero, E. J., & Ferreira, A. T. (2010). Low-intensity pulsed ultrasound-dependent osteoblast proliferation occurs by via activation of the P2Y receptor: role of the P2Y1 receptor. Bone, 46(2), 355–362.

    Article  CAS  PubMed  Google Scholar 

  2. Rumney, R. M., Sunters, A., Reilly, G. C., & Gartland, A. (2012). Application of multiple forms of mechanical loading to human osteoblasts reveals increased ATP release in response to fluid flow in 3D cultures and differential regulation of immediate early genes. Journal of Biomechanics, 45(3), 549–554.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  3. Romanello, M., Codognotto, A., Bicego, M., Pines, A., Tell, G., & D’andrea, P. (2005). Autocrine/paracrine stimulation of purinergic receptors in osteoblasts: contribution of vesicular ATP release. Biochem Biophys Res Commun, 331(4), 1429–1438.

    Article  CAS  PubMed  Google Scholar 

  4. Bowler, W. B., Buckley, K. A., Gartland, A., Hipskind, R. A., Bilbe, G., & Gallagher, J. A. (2001). Extracellular nucleotide signaling: a mechanism for integrating local and systemic responses in the activation of bone remodeling. Bone, 28(5), 507–512.

    Article  CAS  PubMed  Google Scholar 

  5. Burnstock, G. (2009). Purinergic signalling: past, present and future. Brazilian Journal of Medical and Biological Research, 42(1), 3–8.

    Article  CAS  PubMed  Google Scholar 

  6. Webb, T. E., Simon, J., Krishek, B. J., Bateson, A. N., Smart, T. G., King, B. F., et al. (1993). Cloning and functional expression of a brain G-protein-coupled ATP receptor. FEBS Letters, 324(2), 219–225.

    Article  CAS  PubMed  Google Scholar 

  7. Nicholas, R. A., Lazarowski, E. R., Watt, W. C., Li, Q., Boyer, J., & Harden, T. K. (1996). Pharmacological and second messenger signalling selectivities of cloned P2Y receptors. Journal of Autonomic Pharmacology, 16(6), 319–323.

    Article  CAS  PubMed  Google Scholar 

  8. Von Kugelgen, I., & Wetter, A. (2000). Molecular pharmacology of P2Y-receptors. Naunyn Schmiedebergs Arch Pharmacol, 362(4–5), 310–323.

    Article  Google Scholar 

  9. Erb, L., Lustig, K. D., Sullivan, D. M., Turner, J. T., & Weisman, G. A. (1993). Functional expression and photoaffinity labeling of a cloned P2U purinergic receptor. Proc Natl Acad Sci U S A, 90(22), 10449–10453.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  10. Communi, D., Parmentier, M., & Boeynaems, J. M. (1996). Cloning, functional expression and tissue distribution of the human P2Y6 receptor. Biochem Biophys Res Commun, 222(2), 303–308.

    Article  CAS  PubMed  Google Scholar 

  11. Troadec, J. D., Thirion, S., Nicaise, G., Lemos, J. R. & Dayanithi, G. (1998) ATP-evoked increases in [Ca2+]i and peptide release from rat isolated neurohypophysial terminals via a P2X2 purinoceptor. J Physiol, 511(Pt 1), 89–103.

  12. Buell, G., Michel, A. D., Lewis, C., Collo, G., Humphrey, P. P., & Surprenant, A. (1996). P2X1 receptor activation in HL60 cells. Blood, 87(7), 2659–2664.

    CAS  PubMed  Google Scholar 

  13. Gever, J. R., Cockayne, D. A., Dillon, M. P., Burnstock, G., & Ford, A. P. (2006). Pharmacology of P2X channels. Pflugers Archiv. European Journal of Physiology, 452(5), 513–537.

    Article  CAS  PubMed  Google Scholar 

  14. Wiley, J. S., Chen, J. R., Snook, M. B., & Jamieson, G. P. (1994). The P2Z-purinoceptor of human lymphocytes: actions of nucleotide agonists and irreversible inhibition by oxidized ATP. British Journal of Pharmacology, 112(3), 946–950.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  15. Jin, J., Tomlinson, W., Kirk, I. P., Kim, Y. B., Humphries, R. G., & Kunapuli, S. P. (2001). The C6-2B glioma cell P2Y(AC) receptor is pharmacologically and molecularly identical to the platelet P2Y(12) receptor. British Journal of Pharmacology, 133(4), 521–528.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  16. Communi, D., Gonzalez, N. S., Detheux, M., Brezillon, S., Lannoy, V., Parmentier, M., et al. (2001). Identification of a novel human ADP receptor coupled to G(i). Journal of Biological Chemistry, 276(44), 41479–41485.

    Article  CAS  PubMed  Google Scholar 

  17. Morrison, M. S., Turin, L., King, B. F., Burnstock, G. & Arnett, T. R. (1998) ATP is a potent stimulator of the activation and formation of rodent osteoclasts. J Physiol, 511(Pt 2), 495–500.

  18. Bowler, W. B., Birch, M. A., Gallagher, J. A., & Bilbe, G. (1995). Identification and cloning of human P2U purinoceptor present in osteoclastoma, bone, and osteoblasts. Journal of Bone and Mineral Research, 10(7), 1137–1145.

    Article  CAS  PubMed  Google Scholar 

  19. Bowler, W. B., Littlewood-Evans, A., Bilbe, G., Gallagher, J. A., & Dixon, C. J. (1998). P2Y2 receptors are expressed by human osteoclasts of giant cell tumor but do not mediate ATP-induced bone resorption. Bone, 22(3), 195–200.

    Article  CAS  PubMed  Google Scholar 

  20. Nakamura, E., Uezono, Y., Narusawa, K., Shibuya, I., Oishi, Y., Tanaka, M., et al. (2000). ATP activates DNA synthesis by acting on P2X receptors in human osteoblast-like MG-63 cells. American Journal of Physiology. Cell Physiology, 279(2), C510–C519.

    CAS  PubMed  Google Scholar 

  21. Hoebertz, A., Mahendran, S., Burnstock, G., & Arnett, T. R. (2002). ATP and UTP at low concentrations strongly inhibit bone formation by osteoblasts: a novel role for the P2Y2 receptor in bone remodeling. Journal of Cellular Biochemistry, 86(3), 413–419.

    Article  CAS  PubMed  Google Scholar 

  22. Orriss, I. R., Utting, J. C., Brandao-Burch, A., Colston, K., Grubb, B. R., Burnstock, G., et al. (2007). Extracellular nucleotides block bone mineralization in vitro: evidence for dual inhibitory mechanisms involving both P2Y2 receptors and pyrophosphate. Endocrinology, 148(9), 4208–4216.

    Article  CAS  PubMed  Google Scholar 

  23. Dixon, C. J., Bowler, W. B., Walsh, C. A., & Gallagher, J. A. (1997). Effects of extracellular nucleotides on single cells and populations of human osteoblasts: contribution of cell heterogeneity to relative potencies. British Journal of Pharmacology, 120(5), 777–780.

    Article  CAS  PubMed  Google Scholar 

  24. Hoebertz, A., Townsend-Nicholson, A., Glass, R., Burnstock, G., & Arnett, T. R. (2000). Expression of P2 receptors in bone and cultured bone cells. Bone, 27(4), 503–510.

    Article  CAS  PubMed  Google Scholar 

  25. Alqallaf, S. M., Evans, B. A., & Kidd, E. J. (2009). Atypical P2X receptor pharmacology in two human osteoblast-like cell lines. British Journal of Pharmacology, 156(7), 1124–1135.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  26. Orriss, I. R., Key, M. L., Brandao-Burch, A., Patel, J. J., Burnstock, G., & Arnett, T. R. (2012). The regulation of osteoblast function and bone mineralisation by extracellular nucleotides: The role of p2x receptors. Bone, 51(3), 389–400.

    Article  CAS  PubMed  Google Scholar 

  27. Orriss, I. R., Knight, G. E., Ranasinghe, S., Burnstock, G., & Arnett, T. R. (2006). Osteoblast responses to nucleotides increase during differentiation. Bone, 39(2), 300–309.

    Article  CAS  PubMed  Google Scholar 

  28. Chaves Neto, A. H., Yano, C. L., Paredes-Gamero, E. J., Machado, D., Justo, G. Z., Peppelenbosch, M. P., et al. (2010). Riboflavin and photoproducts in MC3T3-E1 differentiation. Toxicology in Vitro, 24(7), 1911–1919.

    Article  CAS  PubMed  Google Scholar 

  29. Siggelkow, H., Rebenstorff, K., Kurre, W., Niedhart, C., Engel, I., Schulz, H., et al. (1999). Development of the osteoblast phenotype in primary human osteoblasts in culture: comparison with rat calvarial cells in osteoblast differentiation. Journal of Cellular Biochemistry, 75(1), 22–35.

    Article  CAS  PubMed  Google Scholar 

  30. Korcok, J., Raimundo, L. N., Ke, H. Z., Sims, S. M., & Dixon, S. J. (2004). Extracellular nucleotides act through P2X7 receptors to activate NF-kappaB in osteoclasts. Journal of Bone and Mineral Research, 19(4), 642–651.

    Article  CAS  PubMed  Google Scholar 

  31. Virginio, C., North, R. A. & Surprenant, A. (1998) Calcium permeability and block at homomeric and heteromeric P2X2 and P2X3 receptors, and P2X receptors in rat nodose neurones. J Physiol, 510(Pt 1), 27–35.

  32. Vassort, G. (2001). Adenosine 5′-triphosphate: a P2-purinergic agonist in the myocardium. Physiological Reviews, 81(2), 767–806.

    CAS  PubMed  Google Scholar 

  33. Abbracchio, M. P., & Ceruti, S. (2007). P1 receptors and cytokine secretion. Purinergic Signal, 3(1–2), 13–25.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  34. Costa, M. A., Barbosa, A., Neto, E., Sa-E-Sousa, A., Freitas, R., Neves, J. M., et al. (2011). On the role of subtype selective adenosine receptor agonists during proliferation and osteogenic differentiation of human primary bone marrow stromal cells. Journal of Cellular Physiology, 226(5), 1353–1366.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This work was supported by INFAR/UNIFESP Confocal and Flow Cytometry Facility, “Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)”, “Conselho nacional de desenvolvimento científico e Tecnológico” (CNPq) and “Fundação de Amparo à Pesquisa do Estado de São Paulo” (FAPESP). RR-R was supported by a master fellowship from the FAPESP (2009/02775-4).

Conflict of interest

All of the authors state that they have no conflicts of interest.

Author information

Authors and Affiliations

  1. Departamento de Biofísica, Universidade Federal de São Paulo, R. Botucatu, 862, São Paulo, 04062-023, Brazil

    Renata Rodrigues-Ribeiro, Érika Costa Alvarenga, Michele Longoni Calio & Alice Teixeira Ferreira

  2. Departamento de Bioquímica, Universidade Federal de São Paulo, R. Pedro de Toledo, 669 - 9 andar, São Paulo, SP, CEP 04039-032, Brazil

    Edgar Julian Paredes-Gamero

  3. Centro Interdisciplinar de Investigação Bioquímica, Universidade de Mogi das Cruzes, Prédio I, Av. Dr. Candido X. de Almeida Souza 200, Mogi Das Cruzes, SP, 08780-911, Brazil

    Edgar Julian Paredes-Gamero

Authors
  1. Renata Rodrigues-Ribeiro
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Érika Costa Alvarenga
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Michele Longoni Calio
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Edgar Julian Paredes-Gamero
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Alice Teixeira Ferreira
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Edgar Julian Paredes-Gamero.

Electronic Supplementary Material

Below is the link to the electronic supplementary material.

Supplementary material 1 (PDF 217 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Rodrigues-Ribeiro, R., Alvarenga, É.C., Calio, M.L. et al. Dual Role of P2 Receptors during Osteoblast Differentiation. Cell Biochem Biophys 71, 1225–1233 (2015). https://doi.org/10.1007/s12013-014-0332-7

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12013-014-0332-7

Keywords

Search

Navigation