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Lack of effect of adenosine on the function of rodent osteoblasts and osteoclasts in vitro

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Abstract

Extracellular ATP, signalling through P2 receptors, exerts well-documented effects on bone cells, inhibiting mineral deposition by osteoblasts and stimulating the formation and resorptive activity of osteoclasts. The aims of this study were to determine the potential osteotropic effects of adenosine, the hydrolysis product of ATP, on primary bone cells in vitro. We determined the effect of exogenous adenosine on (1) the growth, alkaline phosphatase (TNAP) activity and bone-forming ability of osteoblasts derived from the calvariae of neonatal rats and mice and the marrow of juvenile rats and (2) the formation and resorptive activity of osteoclasts from juvenile mouse marrow. Reverse transcription polymerase chain reaction (RT-PCR) analysis showed marked differences in the expression of P1 receptors in osteoblasts from different sources. Whilst mRNA for the A1 and A2B receptors was expressed by all primary osteoblasts, A2A receptor expression was limited to rat bone marrow and mouse calvarial osteoblasts and the A3 receptor to rat bone marrow osteoblasts. We found that adenosine had no detectable effects on cell growth, TNAP activity or bone formation by rodent osteoblasts in vitro. The analogue 2-chloroadenosine, which is hydrolysed more slowly than adenosine, had no effects on rat or mouse calvarial osteoblasts but increased TNAP activity and bone formation by rat bone marrow osteoblasts by 30–50 % at a concentration of 1 μM. Osteoclasts were found to express the A2A, A2B and A3 receptors; however, neither adenosine (≤100 μM) nor 2-chloroadenosine (≤10 μM) had any effect on the formation or resorptive activity of mouse osteoclasts in vitro. These results suggest that adenosine, unlike ATP, is not a major signalling molecule in the bone.

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References

  1. Burnstock G (1972) Purinergic nerves. Pharmacol Rev 24:509–581

    CAS  PubMed  Google Scholar 

  2. Abbracchio MP, Burnstock G (1994) Purinoceptors: are there families of P2X and P2Y purinoceptors? Pharmacol Ther 64:445–475

    Article  CAS  PubMed  Google Scholar 

  3. Burnstock G (2007) Purine and pyrimidine receptors. Cell Mol Life Sci 64:1471–1483

    Article  CAS  PubMed  Google Scholar 

  4. Burnstock G, Arnett TR, Orriss IR (2013) Purinergic signalling in the musculoskeletal system. Purinergic Signal 9:541–572

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  6. Bowler WB, Buckley KA, Gartland A, Hipskind RA, Bilbe G, Gallagher JA (2001) Extracellular nucleotide signaling: a mechanism for integrating local and systemic responses in the activation of bone remodeling. Bone 28:507–512

    Article  CAS  PubMed  Google Scholar 

  7. Orriss IR, Knight GE, Ranasinghe S, Burnstock G, Arnett TR (2006) Osteoblast responses to nucleotides increase during differentiation. Bone 39:300–309

    Article  CAS  PubMed  Google Scholar 

  8. Orriss IR, Key ML, Brandao-Burch A, Patel JJ, Burnstock G, Arnett TR (2012) The regulation of osteoblast function and bone mineralisation by extracellular nucleotides: the role of P2X receptors. Bone 51:389–400

    Article  CAS  PubMed  Google Scholar 

  9. Kumagai H, Sakamoto H, Guggino S, Filburn CR, Sacktor B (1989) Neurotransmitter regulation of cytosolic calcium in osteoblast-like bone cells. Calcif Tissue Int 45:251–254

    Article  CAS  PubMed  Google Scholar 

  10. Schofl C, Cuthbertson KS, Walsh CA, Mayne C, Cobbold P, von zur MA, Hesch RD, Gallagher JA (1992) Evidence for P2-purinoceptors on human osteoblast-like cells. J Bone Miner Res 7:485–491

    Article  CAS  PubMed  Google Scholar 

  11. Hoebertz A, Mahendran S, Burnstock G, Arnett TR (2002) ATP and UTP at low concentrations strongly inhibit bone formation by osteoblasts: a novel role for the P2Y2 receptor in bone remodeling. J Cell Biochem 86:413–419

    Article  CAS  PubMed  Google Scholar 

  12. 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 

  13. Orriss IR, Key ML, Hajjawi MO, Arnett TR (2013) Extracellular ATP released by osteoblasts is a key local inhibitor of bone mineralisation. PLoS One 8:e69057

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Orriss IR, Wang N, Burnstock G, Arnett TR, Gartland A, Robaye B, Boeynaems JM (2011) The P2Y6 receptor stimulates bone resorption by osteoclasts. Endocrinology 152:3706–3716

    Article  CAS  PubMed  Google Scholar 

  15. Reyes JP, Sims SM, Dixon SJ (2011) P2 receptor expression, signaling and function in osteoclasts. Front Biosci (Schol Ed) 3:1101–1118

    Article  Google Scholar 

  16. Morrison MS, Turin L, King BF, Burnstock G, Arnett TR (1998) ATP is a potent stimulator of the activation and formation of rodent osteoclasts. J Physiol 511:495–500

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Hoebertz A, Meghji S, Burnstock G, Arnett TR (2001) Extracellular ADP is a powerful osteolytic agent: evidence for signaling through the P2Y1 receptor on bone cells. FASEB J 15:1139–1148

    Article  CAS  PubMed  Google Scholar 

  18. Su X, Floyd DH, Hughes A, Xiang J, Schneider JG, Uluckan O, Heller E, Deng H, Zou W, Craft CS, Wu K, Hirbe AC, Grabowska D, Eagleton MC, Townsley S, Collins L, Piwnica-Worms D, Steinberg TH, Novack DV, Conley PB, Hurchla MA, Rogers M, Weilbaecher KN (2012) The ADP receptor P2RY12 regulates osteoclast function and pathologic bone remodeling. J Clin Invest 122:3579–3592

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Syberg S, Brandao-Burch A, Patel JJ, Hajjawi M, Arnett TR, Schwarz P, Jorgensen NR, Orriss IR (2012) Clopidogrel (Plavix(R)), a P2Y12 receptor antagonist, inhibits bone cell function in vitro and decreases trabecular bone in vivo. J Bone Miner Res 27:2373–2386

    Article  CAS  PubMed  Google Scholar 

  20. Martin C, Leone M, Viviand X, Ayem ML, Guieu R (2000) High adenosine plasma concentration as a prognostic index for outcome in patients with septic shock. Crit Care Med 28:3198–3202

    Article  CAS  PubMed  Google Scholar 

  21. Fredholm BB, Sollevi A (1981) The release of adenosine and inosine from canine subcutaneous adipose tissue by nerve stimulation and noradrenaline. J Physiol 313:351–367

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Fredholm BB APIJ, Jacobson KA, Linden J, Muller CE (2011) International Union of Basic and Clinical Pharmacology. LXXXI. Nomenclature and classification of adenosine receptors—an update. Pharmacol Rev 63:1–34

    Article  PubMed  PubMed Central  Google Scholar 

  23. Kara FM, Doty SB, Boskey A, Goldring S, Zaidi M, Fredholm BB, Cronstein BN (2010) Adenosine A(1) receptors regulate bone resorption in mice: adenosine A(1) receptor blockade or deletion increases bone density and prevents ovariectomy-induced bone loss in adenosine A(1) receptor-knockout mice. Arthritis Rheum 62:534–541

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Gharibi B, Abraham AA, Ham J, Evans BA (2011) Adenosine receptor subtype expression and activation influence the differentiation of mesenchymal stem cells to osteoblasts and adipocytes. J Bone Miner Res 26:2112–2124

    Article  CAS  PubMed  Google Scholar 

  25. Pellegatti P, Falzoni S, Donvito G, Lemaire I, Di Virgilio F (2011) P2X7 receptor drives osteoclast fusion by increasing the extracellular adenosine concentration. FASEB J 25:1264–1274

    Article  CAS  PubMed  Google Scholar 

  26. Vincenzi F, Targa M, Corciulo C, Gessi S, Merighi S, Setti S, Cadossi R, Goldring MB, Borea PA, Varani K (2013) Pulsed electromagnetic fields increased the anti-inflammatory effect of A(2)A and A(3) adenosine receptors in human T/C-28a2 chondrocytes and hFOB 1.19 osteoblasts. PLoS One 8:e65561

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Evans BA, Elford C, Pexa A, Francis K, Hughes AC, Deussen A, Ham J (2006) Human osteoblast precursors produce extracellular adenosine, which modulates their secretion of IL-6 and osteoprotegerin. J Bone Miner Res 21:228–236

    Article  CAS  PubMed  Google Scholar 

  28. Lerner UH, Sahlberg K, Fredholm BB (1987) Characterization of adenosine receptors in bone. Studies on the effect of adenosine analogues on cyclic AMP formation and bone resorption in cultured mouse calvaria. Acta Physiol Scand 131:287–296

    Article  CAS  PubMed  Google Scholar 

  29. Jones SJ, Gray C, Boyde A, Burnstock G (1997) Purinergic transmitters inhibit bone formation by cultured osteoblasts. Bone 21:393–399

    Article  CAS  PubMed  Google Scholar 

  30. Carroll SH, Wigner NA, Kulkarni N, Johnston-Cox H, Gerstenfeld LC, Ravid K (2012) A2B adenosine receptor promotes mesenchymal stem cell differentiation to osteoblasts and bone formation in vivo. J Biol Chem 287:15718–15727

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Mediero A, Wilder T, Perez-Aso M, Cronstein BN (2015) Direct or indirect stimulation of adenosine A2A receptors enhances bone regeneration as well as bone morphogenetic protein-2. FASEB J 29:1577–1590

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Korcok J, Raimundo LN, Ke HZ, Sims SM, Dixon SJ (2004) Extracellular nucleotides act through P2X7 receptors to activate NF-kappaB in osteoclasts. J Bone Miner Res 19:642–651

    Article  CAS  PubMed  Google Scholar 

  33. Mediero A, Kara FM, Wilder T, Cronstein BN (2012) Adenosine A(2A) receptor ligation inhibits osteoclast formation. Am J Pathol 180:775–786

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. He W, Cronstein BN (2012) Adenosine A1 receptor regulates osteoclast formation by altering TRAF6/TAK1 signaling. Purinergic Signal 8:327–337

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Orriss IR, Taylor SE, Arnett TR (2012) Rat osteoblast cultures. Methods Mol Biol 816:31–41

    Article  CAS  PubMed  Google Scholar 

  36. Orriss IR, Hajjawi MO, Huesa C, MacRae VE, Arnett TR (2014) Optimisation of the differing conditions required for bone formation in vitro by primary osteoblasts from mice and rats. Int J Mol Med 3:1201–1208

    Google Scholar 

  37. Taylor SE, Shah M, Orriss IR (2014) Generation of rodent and human osteoblasts. BoneKEy Rep 3:585

    Article  PubMed  PubMed Central  Google Scholar 

  38. Orriss IR, Arnett TR (2012) Rodent osteoclast cultures. Methods Mol Biol 816:103–117

    Article  CAS  PubMed  Google Scholar 

  39. Evans BA (2012) Does adenosine play a role in bone formation, resorption and repair? Purinergic Signal 8:177–180

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Costa MA, Barbosa A, Neto E, Sa-e-Sousa A, Freitas R, Neves JM, Magalhaes-Cardoso T, Ferreirinha F, Correia-de-Sa P (2011) On the role of subtype selective adenosine receptor agonists during proliferation and osteogenic differentiation of human primary bone marrow stromal cells. J Cell Physiol 226:1353–1366

    Article  CAS  PubMed  Google Scholar 

  41. Gharibi B, Abraham AA, Ham J, Evans BA (2012) Contrasting effects of A1 and A2b adenosine receptors on adipogenesis. Int J Obes 36:397–406

    Article  CAS  Google Scholar 

  42. Daly JW, Padgett WL, Secunda SI, Thompson RD, Olsson RA (1993) Structure-activity relationships for 2-substituted adenosines at A1 and A2 adenosine receptors. Pharmacology 46:91–100

    Article  CAS  PubMed  Google Scholar 

  43. Bellows CG, Wang YH, Heersche JN, Aubin JE (1994) 1,25-dihydroxyvitamin D3 stimulates adipocyte differentiation in cultures of fetal rat calvaria cells: comparison with the effects of dexamethasone. Endocrinology 134:2221–2229

    CAS  PubMed  Google Scholar 

  44. Orriss IR, Knight GE, Utting JC, Taylor SE, Burnstock G, Arnett TR (2009) Hypoxia stimulates vesicular ATP release from rat osteoblasts. J Cell Physiol 220:155–162. doi:10.1002/jcp.21745

    Article  CAS  PubMed  Google Scholar 

  45. Genetos DC, Geist DJ, Liu D, Donahue HJ, Duncan RL (2005) Fluid shear-induced ATP secretion mediates prostaglandin release in MC3T3-E1 osteoblasts. J Bone Miner Res 20(1):41–49

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Ciruela F, Casado V, Mallol J, Canela EI, Lluis C, Franco R (1995) Immunological identification of A1 adenosine receptors in brain cortex. J Neurosci Res 42:818–828

    Article  CAS  PubMed  Google Scholar 

  47. Canals M, Burgueno J, Marcellino D, Cabello N, Canela EI, Mallol J, Agnati L, Ferre S, Bouvier M, Fuxe K, Ciruela F, Lluis C, Franco R (2004) Homodimerization of adenosine A2A receptors: qualitative and quantitative assessment by fluorescence and bioluminescence energy transfer. J Neurochemi 88:726–734

    Article  CAS  Google Scholar 

  48. Ciruela F, Ferre S, Casado V, Cortes A, Cunha RA, Lluis C, Franco R (2006) Heterodimeric adenosine receptors: a device to regulate neurotransmitter release. Cell Mol Life Sci 63:2427–2431

    Article  CAS  PubMed  Google Scholar 

  49. Yoshioka K, Saitoh O, Nakata H (2001) Heteromeric association creates a P2Y-like adenosine receptor. Proc Natl Acad Sci U S A 98:7617–7622

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Suzuki T, Namba K, Tsuga H, Nakata H (2006) Regulation of pharmacology by hetero-oligomerization between A1 adenosine receptor and P2Y2 receptor. Biochem Biophys Res Commun 351:559–565

    Article  CAS  PubMed  Google Scholar 

  51. Kara FM, Chitu V, Sloane J, Axelrod M, Fredholm BB, Stanley ER, Cronstein BN (2010) Adenosine A1 receptors (A1Rs) play a critical role in osteoclast formation and function. FASEB J 24:2325–2333

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Salvatore CA, Tilley SL, Latour AM, Fletcher DS, Koller BH, Jacobson MA (2000) Disruption of the A(3) adenosine receptor gene in mice and its effect on stimulated inflammatory cells. J Biol Chem 275:4429–4434

    Article  CAS  PubMed  Google Scholar 

  53. Morrison RR, Talukder MA, Ledent C, Mustafa SJ (2002) Cardiac effects of adenosine in A(2A) receptor knockout hearts: uncovering A(2B) receptors. Am J Physiol Heart Circ Physiol 282:H437–H444

    Article  CAS  PubMed  Google Scholar 

  54. Ramanathan M, Pinhal-Enfield G, Hao I, Leibovich SJ (2007) Synergistic up-regulation of vascular endothelial growth factor (VEGF) expression in macrophages by adenosine A2A receptor agonists and endotoxin involves transcriptional regulation via the hypoxia response element in the VEGF promoter. Mol Biol Cell 18:14–23

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Ernens I, Leonard F, Vausort M, Rolland-Turner M, Devaux Y, Wagner DR (2010) Adenosine up-regulates vascular endothelial growth factor in human macrophages. Biochem Biophys Res Commun 392:351–356

    Article  CAS  PubMed  Google Scholar 

  56. Burnstock G (2007) Physiology and pathophysiology of purinergic neurotransmission. Physiol Rev 87:659–797

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

The authors are grateful for the funding from Arthritis Research UK (Career Developmental Fellowship, 19205, IRO).

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Authors and Affiliations

  1. Department of Cell and Developmental Biology, University College London, Anatomy Building, Gower Street, WC1E 6BT, London, UK

    Mark O. R. Hajjawi, Michelangelo Corcelli, Timothy R. Arnett & Isabel R. Orriss

  2. Department of Comparative Biomedical Sciences, Royal Veterinary College, Royal College Street, London, NW1 0TU, UK

    Jessal J. Patel & Isabel R. Orriss

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  1. Mark O. R. Hajjawi
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  2. Jessal J. Patel
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  3. Michelangelo Corcelli
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Correspondence to Timothy R. Arnett.

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Hajjawi, M.O.R., Patel, J.J., Corcelli, M. et al. Lack of effect of adenosine on the function of rodent osteoblasts and osteoclasts in vitro. Purinergic Signalling 12, 247–258 (2016). https://doi.org/10.1007/s11302-016-9499-2

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