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Cell Biochemistry and Biophysics

, Volume 71, Issue 2, pp 1225–1233 | Cite as

Dual Role of P2 Receptors during Osteoblast Differentiation

  • Renata Rodrigues-Ribeiro
  • Érika Costa Alvarenga
  • Michele Longoni Calio
  • Edgar Julian Paredes-Gamero
  • Alice Teixeira Ferreira
Original Paper

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.

Keywords

Osteoblasts Purinergic receptors Differentiation 

Notes

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.

Supplementary material

12013_2014_332_MOESM1_ESM.pdf (217 kb)
Supplementary material 1 (PDF 217 kb)

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Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Renata Rodrigues-Ribeiro
    • 1
  • Érika Costa Alvarenga
    • 1
  • Michele Longoni Calio
    • 1
  • Edgar Julian Paredes-Gamero
    • 2
    • 3
  • Alice Teixeira Ferreira
    • 1
  1. 1.Departamento de BiofísicaUniversidade Federal de São PauloSão PauloBrazil
  2. 2.Departamento de BioquímicaUniversidade Federal de São PauloSão Paulo, SPBrazil
  3. 3.Centro Interdisciplinar de Investigação BioquímicaUniversidade de Mogi das CruzesMogi Das CruzesBrazil

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