P2X7 nucleotide receptor signaling potentiates the Wnt/β-catenin pathway in cells of the osteoblast lineage Matthew W. Grol Patricia J. Brooks Alexey Pereverzev S. Jeffrey Dixon Email author Original Article First Online: 21 May 2016 Received: 25 July 2015 Accepted: 06 May 2016 DOI :
10.1007/s11302-016-9517-4
Cite this article as: Grol, M.W., Brooks, P.J., Pereverzev, A. et al. Purinergic Signalling (2016) 12: 509. doi:10.1007/s11302-016-9517-4
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Abstract The P2X7 and Wnt/β-catenin signaling pathways regulate osteoblast differentiation and are critical for the anabolic responses of bone to mechanical loading. However, whether these pathways interact to control osteoblast activity is unknown. The purpose of this study was to investigate the effects of P2X7 activation on Wnt/β-catenin signaling in osteoblasts. Using MC3T3-E1 cells, we found that combined treatment with Wnt3a and the P2X7 agonist 2′(3′)-O -(4-benzoylbenzoyl)adenosine 5′-triphosphate (BzATP) elicited more sustained β-catenin nuclear localization than that induced by Wnt3a alone. Wnt3a-induced increases in β-catenin transcriptional activity were also potentiated by treatment with BzATP. Consistent with involvement of P2X7, a high ATP concentration (1 mM) potentiated Wnt3a-induced β-catenin transcriptional activity, whereas a low concentration (10 μM) of ATP, adenosine 5′-diphosphate (ADP), or uridine 5′-triphosphate (UTP) failed to elicit a response. The potentiation of β-catenin transcriptional activity elicited by BzATP was also inhibited by two distinct P2X7 antagonists: A 438079 and A 740003. Furthermore, responses to Wnt3a in calvarial cells isolated from P2rx7 knockout mice were significantly less than in cells from wild-type controls. In MC3T3-E1 cells, BzATP increased inhibitory phosphorylation of glycogen synthase kinase 3β (GSK3β), a process that was blocked by A 438079 and diminished by inhibition of protein kinase C. Thus, P2X7 signaling may potentiate the canonical Wnt pathway through GSK3β inhibition. Taken together, we show that P2X7 activation prolongs and potentiates Wnt/β-catenin signaling. Consequently, cross-talk between P2X7 and Wnt/β-catenin pathways may modulate osteoblast activity in response to mechanical loading.
Keywords P2X7 receptors Wnt β-catenin Osteoblast Glycogen synthase kinase 3β
References 1.
Robling AG, Castillo AB, Turner CH (2006) Biomechanical and molecular regulation of bone remodeling. Annu Rev Biomed Eng 8:455–498. doi:
10.1146/annurev.bioeng.8.061505.095721 CrossRef PubMed Google Scholar 2.
Bonewald LF, Johnson ML (2008) Osteocytes, mechanosensing and Wnt signaling. Bone 42:606–615. doi:
10.1016/j.bone.2007.12.224 CrossRef PubMed PubMedCentral Google Scholar 3.
Baron R, Kneissel M (2013) WNT signaling in bone homeostasis and disease: from human mutations to treatments. Nat Med 19:179–192. doi:
10.1038/nm.3074 CrossRef PubMed Google Scholar 4.
Lin C, Jiang X, Dai Z et al (2009) Sclerostin mediates bone response to mechanical unloading through antagonizing Wnt/beta-catenin signaling. J Bone Miner Res 24:1651–1661. doi:
10.1359/jbmr.090411 CrossRef PubMed Google Scholar 5.
Tu X, Rhee Y, Condon KW et al (2012) Sost downregulation and local Wnt signaling are required for the osteogenic response to mechanical loading. Bone 50:209–217. doi:
10.1016/j.bone.2011.10.025 CrossRef PubMed Google Scholar 6.
Sen B, Styner M, Xie Z et al (2009) Mechanical loading regulates NFATc1 and beta-catenin signaling through a GSK3beta control node. J Biol Chem 284:34607–34617. doi:
10.1074/jbc.M109.039453 CrossRef PubMed PubMedCentral Google Scholar 7.
Case N, Sen B, Thomas JA et al (2011) Steady and oscillatory fluid flows produce a similar osteogenic phenotype. Calcif Tissue Int 88:189–197. doi:
10.1007/s00223-010-9448-y CrossRef PubMed Google Scholar 8.
Case N, Ma M, Sen B et al (2008) Beta-catenin levels influence rapid mechanical responses in osteoblasts. J Biol Chem 283:29196–29205. doi:
10.1074/jbc.M801907200 CrossRef PubMed PubMedCentral Google Scholar 9.
Burnstock G (2007) Purine and pyrimidine receptors. Cell Mol Life Sci 64:1471–1483. doi:
10.1007/s00018-007-6497-0 CrossRef PubMed Google Scholar 10.
Ke HZ, Qi H, Weidema AF et al (2003) Deletion of the P2X7 nucleotide receptor reveals its regulatory roles in bone formation and resorption. Mol Endocrinol 17:1356–1367. doi:
10.1210/me.2003-0021 CrossRef PubMed Google Scholar 11.
Li J, Liu D, Ke HZ et al (2005) The P2X7 nucleotide receptor mediates skeletal mechanotransduction. J Biol Chem 280:42952–42959. doi:
10.1074/jbc.M506415200 CrossRef PubMed Google Scholar 12.
Panupinthu N, Rogers JT, Zhao L et al (2008) P2X7 receptors on osteoblasts couple to production of lysophosphatidic acid: a signaling axis promoting osteogenesis. J Cell Biol 181:859–871. doi:
10.1083/jcb.200708037 CrossRef PubMed PubMedCentral Google Scholar 13.
Ortega F, Pérez-Sen R, Delicado EG, Miras-Portugal MT (2009) P2X7 nucleotide receptor is coupled to GSK-3 inhibition and neuroprotection in cerebellar granule neurons. Neurotox Res 15:193–204. doi:
10.1007/s12640-009-9020-6 CrossRef PubMed Google Scholar 14.
Solle M, Labasi J, Perregaux DG et al (2001) Altered cytokine production in mice lacking P2X(7) receptors. J Biol Chem 276:125–132. doi:
10.1074/jbc.M006781200 CrossRef PubMed Google Scholar 15.
Masin M, Young C, Lim K et al (2012) Expression, assembly and function of novel C-terminal truncated variants of the mouse P2X7 receptor: re-evaluation of P2X7 knockouts. Br J Pharmacol 165:978–993. doi:
10.1111/j.1476-5381.2011.01624.x CrossRef PubMed PubMedCentral Google Scholar 16.
Spencer GJ, Utting JC, Etheridge SL et al (2006) Wnt signalling in osteoblasts regulates expression of the receptor activator of NFkappaB ligand and inhibits osteoclastogenesis in vitro. J Cell Sci 119:1283–1296. doi:
10.1242/jcs.02883 CrossRef PubMed Google Scholar 17.
Grol MW, Zelner I, Dixon SJ (2012) P2X
7 -mediated calcium influx triggers a sustained, PI3K-dependent increase in metabolic acid production by osteoblast-like cells. Am J Physiol Endocrinol Metab 302:E561–75. doi:
10.1152/ajpendo.00209.2011 CrossRef PubMed Google Scholar 18.
Caverzasio J, Biver E, Thouverey C (2013) Predominant role of PDGF receptor transactivation in Wnt3a-induced osteoblastic cell proliferation. J Bone Miner Res 28:260–270. doi:
10.1002/jbmr.1748 CrossRef PubMed Google Scholar 19.
Biechele TL, Moon RT (2008) Assaying beta-catenin/TCF transcription with beta-catenin/TCF transcription-based reporter constructs. Methods Mol Biol 468:99–110. doi:
10.1007/978-1-59745-249-6_8 CrossRef PubMed Google Scholar 20.
MacDonald BT, Tamai K, He X (2009) Wnt/beta-catenin signaling: components, mechanisms, and diseases. Dev Cell 17:9–26. doi:
10.1016/j.devcel.2009.06.016 CrossRef PubMed PubMedCentral Google Scholar 21.
North RA (2002) Molecular physiology of P2X receptors. Physiol Rev 82:1013–1067. doi:
10.1152/physrev.00015.2002 CrossRef PubMed Google Scholar 22.
Ortega F, Pérez-Sen R, Morente V et al (2010) P2X7, NMDA and BDNF receptors converge on GSK3 phosphorylation and cooperate to promote survival in cerebellar granule neurons. Cell Mol Life Sci 67:1723–1733. doi:
10.1007/s00018-010-0278-x CrossRef PubMed PubMedCentral Google Scholar 23.
Grol MW, Pereverzev A, Sims SM, Dixon SJ (2013) P2 receptor networks regulate signaling duration over a wide dynamic range of ATP concentrations. J Cell Sci 126:3615–3626. doi:
10.1242/jcs.122705 CrossRef PubMed Google Scholar 24.
Bowler WB, Dixon CJ, Halleux C et al (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 274:14315–14324
CrossRef PubMed Google Scholar 25.
Nakamura E, Uezono Y, Narusawa K et al (2000) ATP activates DNA synthesis by acting on P2X receptors in human osteoblast-like MG-63 cells. Am J Physiol Cell Physiol 279:C510–9
PubMed Google Scholar 26.
Ortega F, Pérez-Sen R, Miras-Portugal MT (2008) Gi-coupled P2Y-ADP receptor mediates GSK-3 phosphorylation and beta-catenin nuclear translocation in granule neurons. J Neurochem 104:62–73. doi:
10.1111/j.1471-4159.2007.05021.x PubMed Google Scholar 27.
Orriss IR, Burnstock G, Arnett TR (2010) Purinergic signalling and bone remodelling. Curr Opin Pharmacol 10:322–330. doi:
10.1016/j.coph.2010.01.003 CrossRef PubMed Google Scholar 28.
Suzuki A, Ozono K, Kubota T et al (2008) PTH/cAMP/PKA signaling facilitates canonical Wnt signaling via inactivation of glycogen synthase kinase-3beta in osteoblastic Saos-2 cells. J Cell Biochem 104:304–317. doi:
10.1002/jcb.21626 CrossRef PubMed Google Scholar 29.
Nelson WJ, Nusse R (2004) Convergence of Wnt, beta-catenin, and cadherin pathways. Science 303:1483–1487. doi:
10.1126/science.1094291 CrossRef PubMed PubMedCentral Google Scholar 30.
Panupinthu N, Zhao L, Possmayer F et al (2007) P2X7 nucleotide receptors mediate blebbing in osteoblasts through a pathway involving lysophosphatidic acid. J Biol Chem 282:3403–3412. doi:
10.1074/jbc.M605620200 CrossRef PubMed Google Scholar 31.
Noronha-Matos JB, Coimbra J, Sá-e-Sousa A et al (2014) P2X7-induced zeiosis promotes osteogenic differentiation and mineralization of postmenopausal bone marrow-derived mesenchymal stem cells. FASEB J 28:5208–5222. doi:
10.1096/fj.14-257923 CrossRef PubMed Google Scholar 32.
Zimmermann H, Zebisch M, Sträter N (2012) Cellular function and molecular structure of ecto-nucleotidases. Purinergic Signal 8:437–502. doi:
10.1007/s11302-012-9309-4 CrossRef PubMed PubMedCentral Google Scholar 33.
Noronha-Matos JB, Costa MA, Magalhães-Cardoso MT et al (2012) Role of ecto-NTPDases on UDP-sensitive P2Y(6) receptor activation during osteogenic differentiation of primary bone marrow stromal cells from postmenopausal women. J Cell Physiol 227:2694–2709. doi:
10.1002/jcp.23014 CrossRef PubMed Google Scholar 34.
Bellido T, Ali AA, Gubrij I et al (2005) Chronic elevation of parathyroid hormone in mice reduces expression of sclerostin by osteocytes: a novel mechanism for hormonal control of osteoblastogenesis. Endocrinology 146:4577–4583. doi:
10.1210/en.2005-0239 CrossRef PubMed Google Scholar 35.
Yao G-Q, Wu J-J, Troiano N, Insogna K (2011) Targeted overexpression of Dkk1 in osteoblasts reduces bone mass but does not impair the anabolic response to intermittent PTH treatment in mice. J Bone Miner Metab 29:141–148. doi:
10.1007/s00774-010-0202-3 CrossRef PubMed Google Scholar 36.
Chen Y, Whetstone HC, Youn A et al (2007) Beta-catenin signaling pathway is crucial for bone morphogenetic protein 2 to induce new bone formation. J Biol Chem 282:526–533. doi:
10.1074/jbc.M602700200 CrossRef PubMed Google Scholar 37.
Fromigué O, Haÿ E, Barbara A, Marie PJ (2010) Essential role of nuclear factor of activated T cells (NFAT)-mediated Wnt signaling in osteoblast differentiation induced by strontium ranelate. J Biol Chem 285:25251–25258. doi:
10.1074/jbc.M110.110502 CrossRef PubMed PubMedCentral Google Scholar 38.
Kitase Y, Barragan L, Qing H et al (2010) Mechanical induction of PGE2 in osteocytes blocks glucocorticoid-induced apoptosis through both the β-catenin and PKA pathways. J Bone Miner Res 25:2657–2668. doi:
10.1002/jbmr.168 CrossRef PubMed PubMedCentral Google Scholar 39.
Armstrong S, Pereverzev A, Dixon SJ, Sims SM (2009) Activation of P2X7 receptors causes isoform-specific translocation of protein kinase C in osteoclasts. J Cell Sci 122:136–144. doi:
10.1242/jcs.031534 CrossRef PubMed Google Scholar 40.
Sawakami K, Robling AG, Ai M et al (2006) The Wnt co-receptor LRP5 is essential for skeletal mechanotransduction but not for the anabolic bone response to parathyroid hormone treatment. J Biol Chem 281:23698–23711. doi:
10.1074/jbc.M601000200 CrossRef PubMed Google Scholar 41.
Zhao L, Shim JW, Dodge TR et al (2013) Inactivation of Lrp5 in osteocytes reduces Young’s modulus and responsiveness to the mechanical loading. Bone 54:35–43. doi:
10.1016/j.bone.2013.01.033 CrossRef PubMed PubMedCentral Google Scholar 42.
Robinson JA, Chatterjee-Kishore M, Yaworsky PJ et al (2006) Wnt/beta-catenin signaling is a normal physiological response to mechanical loading in bone. J Biol Chem 281:31720–31728. doi:
10.1074/jbc.M602308200 CrossRef PubMed Google Scholar © Springer Science+Business Media Dordrecht 2016
Authors and Affiliations Matthew W. Grol Patricia J. Brooks Alexey Pereverzev S. Jeffrey Dixon Email author 1. Department of Anatomy and Cell Biology The University of Western Ontario London Canada 2. Department of Molecular and Human Genetics Baylor College of Medicine Houston USA 3. Schulich Dentistry The University of Western Ontario London Canada 4. Faculty of Dentistry University of Toronto Toronto Canada 5. Department of Physiology and Pharmacology The University of Western Ontario London Canada 6. Bone and Joint Institute The University of Western Ontario London Canada