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Inhibition of neuronal cell death after retinoic acid-induced down-regulation of P2X7 nucleotide receptor expression

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Abstract

Apoptosis is a major mechanism for cell death in the nervous system during development. P2X7 nucleotide receptors are ionotropic ATP receptors that mediate cell death under pathological conditions. We developed an in vitro protocol to investigate the expression and functional responses of P2X7 nucleotide receptors during retinoic acid (RA)-induced neuronal differentiation of human SH-SY5Y neuroblastoma cells. Neuronal differentiation was examined measuring cellular growth arrest and neuritic processes elongation. We found that SH-SY5Y cells treated for 5 days with RA under low serum content exhibited a neuron-like phenotype with neurites extending more than twice the length of the cell body and cell growth arrest. Concurrently, we detected the abolishment of intracellular-free calcium mobilization and the down-regulation of P2X7 nucleotide receptor protein expression that protected differentiated cells from neuronal cell death and reduced caspase-3 cleavage-induced by P2X7 nucleotide receptor agonist. The role of P2X7 nucleotide receptors in neuronal death was established by selectively antagonizing the receptor with KN-62 prior to its activation. We assessed the involvement of protein kinases and found that p38 signaling was activated in undifferentiated after nucleotide stimulation, but abolished by the differentiating RA pretreatment. Importantly, P2X7 receptor-induced caspase-3 cleavage was blocked by the p38 protein kinase specific inhibitor PD169316. Taken together, our results suggest that RA treatment of human SH-SY5Y cells leads to decreased P2X7 nucleotide receptor protein expression thus protecting differentiated cells from extracellular nucleotide-induced neuronal death, and p38 signaling pathway is critically involved in this protection of RA-differentiated cells.

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References

  1. Deshmukh M, Johnson EM Jr (1997) Programmed cell death in neurons: focus on the pathway of nerve growth factor deprivation-induced death of sympathetic neurons. Mol Pharmacol 51:897–906

    CAS  PubMed  Google Scholar 

  2. Gao L, Abu Kwaik Y (2000) Hijacking of apoptotic pathways by bacterial pathogens. Microbes Infect 2:1705–1719

    Article  CAS  PubMed  Google Scholar 

  3. Encinas M, Iglesias M, Liu Y, Wang H, Muhaisen A, Cena V, Gallego C, Comella JX (2000) Sequential treatment of SH-SY5Y cells with retinoic acid and brain-derived neurotrophic factor gives rise to fully differentiated, neurotrophic factor-dependent, human neuron-like cells. J Neurochem 75:991–1003

    Article  CAS  PubMed  Google Scholar 

  4. Oppenheim RW (1991) Cell death during development of the nervous system. Annu Rev Neurosci 14:453–501

    Article  CAS  PubMed  Google Scholar 

  5. Prince JA, Oreland L (1997) Staurosporine differentiated human SH-SY5Y neuroblastoma cultures exhibit transient apoptosis and trophic factor independence. Brain Res Bull 43:515–523

    Article  CAS  PubMed  Google Scholar 

  6. Arthur DB, Akassoglou K, Insel PA (2005) P2Y2 receptor activates nerve growth factor/TrkA signaling to enhance neuronal differentiation. Proc Natl Acad Sci USA 102:19138–19143

    Article  CAS  PubMed  Google Scholar 

  7. Arthur DB, Georgi S, Akassoglou K, Insel PA (2006) Inhibition of apoptosis by P2Y2 receptor activation: novel pathways for neuronal survival. J Neurosci 26:3798–3804

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  9. Chorna NE, Santiago-Perez LI, Erb L, Seye CI, Neary JT, Sun GY, Weisman GA, Gonzalez FA (2004) P2Y receptors activate neuroprotective mechanisms in astrocytic cells. J Neurochem 91:119–132

    Article  CAS  PubMed  Google Scholar 

  10. Fumagalli M, Brambilla R, D’Ambrosi N, Volonte C, Matteoli M, Verderio C, Abbracchio MP (2003) Nucleotide-mediated calcium signaling in rat cortical astrocytes: role of P2X and P2Y receptors. Glia 43:218–230

    Article  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  13. North RA (2002) Molecular physiology of P2X receptors. Physiol Rev 82:1013–1067

    CAS  PubMed  Google Scholar 

  14. White N, Burnstock G (2006) P2 receptors and cancer. Trends Pharmacol Sci 27:211–217

    Article  CAS  PubMed  Google Scholar 

  15. North RA, Verkhratsky A (2006) Purinergic transmission in the central nervous system. Pflugers Arch 452:479–485

    Article  CAS  PubMed  Google Scholar 

  16. Sperlagh B, Vizi ES, Wirkner K, Illes P (2006) P2X7 receptors in the nervous system. Prog Neurobiol 78:327–346

    Article  CAS  PubMed  Google Scholar 

  17. Zhang X, Zhang M, Laties AM, Mitchell CH (2005) Stimulation of P2X7 receptors elevates Ca2+ and kills retinal ganglion cells. Invest Ophthalmol Vis Sci 46:2183–2191

    Article  PubMed  Google Scholar 

  18. Burnstock G, Williams M (2000) P2 purinergic receptors: modulation of cell function and therapeutic potential. J Pharmacol Exp Ther 295:862–869

    CAS  PubMed  Google Scholar 

  19. Ballerini P, Giulinai P, Buccella S, Nargi E, Santavenere C, Scemens E, Rathbone MP, Caciagli F (2000) P2X7 ATP receptor-mediated modulation of astrocytes proliferation and coupling. Drug Dev Res 50:108

    Google Scholar 

  20. Franke H, Grosche J, Schadlich H, Krugel U, Allgaier C, Illes P (2001) P2X receptor expression on astrocytes in the nucleus accumbens of rats. Neuroscience 108:421–429

    Article  CAS  PubMed  Google Scholar 

  21. Wang X, Arcuino G, Takano T, Lin J, Peng WG, Wan P, Li P, Xu Q, Liu QS, Goldman SA, Nedergaard M (2004) P2X7 receptor inhibition improves recovery after spinal cord injury. Nat Med 10:821–827

    Article  CAS  PubMed  Google Scholar 

  22. Cheung KK, Chan WY, Burnstock G (2005) Expression of P2X purinoceptors during rat brain development and their inhibitory role on motor axon outgrowth in neural tube explant cultures. Neuroscience 133:937–945

    Article  CAS  PubMed  Google Scholar 

  23. Resende RR, Majumder P, Gomes KN, Britto LR, Ulrich H (2007) P19 embryonal carcinoma cells as in vitro model for studying purinergic receptor expression and modulation of N-methyl-d-aspartate-glutamate and acetylcholine receptors during neuronal differentiation. Neuroscience 146:1169–1181

    Article  CAS  PubMed  Google Scholar 

  24. Pahlman S, Hoehner JC, Nanberg E, Hedborg F, Fagerstrom S, Gestblom C, Johansson I, Larsson U, Lavenius E, Ortoft E et al (1995) Differentiation and survival influences of growth factors in human neuroblastoma. Eur J Cancer 31A:453–458

    Article  CAS  PubMed  Google Scholar 

  25. Singh US, Pan J, Kao YL, Joshi S, Young KL, Baker KM (2003) Tissue transglutaminase mediates activation of RhoA and MAP kinase pathways during retinoic acid-induced neuronal differentiation of SH-SY5Y cells. J Biol Chem 278:391–399

    Article  CAS  PubMed  Google Scholar 

  26. Larsson KP, Hansen AJ, Dissing S (2002) The human SH-SY5Y neuroblastoma cell-line expresses a functional P2X7 purinoceptor that modulates voltage-dependent Ca2+ channel function. J Neurochem 83:285–298

    Article  CAS  PubMed  Google Scholar 

  27. Tozaki-Saitoh H, Koizumi S, Sato Y, Tsuda M, Nagao T, Inoue K (2006) Retinoic acids increase P2X2 receptor expression through the 5′-flanking region of P2rx2 gene in rat phaeochromocytoma PC-12 cells. Mol Pharmacol 70:319–328

    CAS  PubMed  Google Scholar 

  28. Fujishita K, Koizumi S, Inoue K (2006) Upregulation of P2Y2 receptors by retinoids in normal human epidermal keratinocytes. Purinergic Signal 2:491–498

    Article  CAS  PubMed  Google Scholar 

  29. Gorodeski GI (2002) Expression, regulation, and function of P2X(4) purinergic receptor in human cervical epithelial cells. Am J Physiol Cell Physiol 282:C84–C93

    CAS  PubMed  Google Scholar 

  30. Oppenheimer O, Cheung NK, Gerald WL (2007) The RET oncogene is a critical component of transcriptional programs associated with retinoic acid-induced differentiation in neuroblastoma. Mol Cancer Ther 6:1300–1309

    Article  CAS  PubMed  Google Scholar 

  31. Santiago-Perez LI, Flores RV, Santos-Berrios C, Chorna NE, Krugh B, Garrad RC, Erb L, Weisman GA, Gonzalez FA (2001) P2Y(2) nucleotide receptor signaling in human monocytic cells: activation, desensitization and coupling to mitogen-activated protein kinases. J Cell Physiol 187:196–208

    Article  CAS  PubMed  Google Scholar 

  32. Velazquez B, Garrad RC, Weisman GA, Gonzalez FA (2000) Differential agonist-induced desensitization of P2Y2 nucleotide receptors by ATP and UTP. Mol Cell Biochem 206:75–89

    Article  CAS  PubMed  Google Scholar 

  33. Suh BC, Kim JS, Namgung U, Ha H, Kim KT (2001) P2X7 nucleotide receptor mediation of membrane pore formation and superoxide generation in human promyelocytes and neutrophils. J Immunol 166:6754–6763

    CAS  PubMed  Google Scholar 

  34. Wiley JS, Gargett CE, Zhang W, Snook MB, Jamieson GP (1998) Partial agonists and antagonists reveal a second permeability state of human lymphocyte P2Z/P2X7 channel. Am J Physiol 275:C1224–C1231

    CAS  PubMed  Google Scholar 

  35. Cheah FC, Hampton MB, Darlow BA, Winterbourn CC, Vissers MC (2005) Detection of apoptosis by caspase-3 activation in tracheal aspirate neutrophils from premature infants: relationship with NF-kappaB activation. J Leukoc Biol 77:432–437

    Article  CAS  PubMed  Google Scholar 

  36. Cavaliere F, Dinkel K, Reymann K (2005) Microglia response and P2 receptor participation in oxygen/glucose deprivation-induced cortical damage. Neuroscience 136:615–623

    Article  CAS  PubMed  Google Scholar 

  37. Preis PN, Saya H, Nadasdi L, Hochhaus G, Levin V, Sadee W (1988) Neuronal cell differentiation of human neuroblastoma cells by retinoic acid plus herbimycin A. Cancer Res 48:6530–6534

    CAS  PubMed  Google Scholar 

  38. Gever JR, Cockayne DA, Dillon MP, Burnstock G, Ford AP (2006) Pharmacology of P2X channels. Pflugers Arch 452:513–537

    Article  CAS  PubMed  Google Scholar 

  39. Chessell IP, Michel AD, Humphrey PP (1998) Effects of antagonists at the human recombinant P2X7 receptor. Br J Pharmacol 124:1314–1320

    Article  CAS  PubMed  Google Scholar 

  40. Humphreys BD, Virginio C, Surprenant A, Rice J, Dubyak GR (1998) Isoquinolines as antagonists of the P2X7 nucleotide receptor: high selectivity for the human versus rat receptor homologues. Mol Pharmacol 54:22–32

    CAS  PubMed  Google Scholar 

  41. Gudipaty L, Humphreys BD, Buell G, Dubyak GR (2001) Regulation of P2X(7) nucleotide receptor function in human monocytes by extracellular ions and receptor density. Am J Physiol Cell Physiol 280:C943–C953

    CAS  PubMed  Google Scholar 

  42. Burgos M, Neary JT, Gonzalez FA (2007) P2Y2 nucleotide receptors inhibit trauma-induced death of astrocytic cells. J Neurochem 103:1785–1800

    Article  CAS  PubMed  Google Scholar 

  43. Harms C, Lautenschlager M, Bergk A, Katchanov J, Freyer D, Kapinya K, Herwig U, Megow D, Dirnagl U, Weber JR, Hortnagl H (2001) Differential mechanisms of neuroprotection by 17 beta-estradiol in apoptotic versus necrotic neurodegeneration. J Neurosci 21:2600–2609

    CAS  PubMed  Google Scholar 

  44. Charles I, Khalyfa A, Kumar DM, Krishnamoorthy RR, Roque RS, Cooper N, Agarwal N (2005) Serum deprivation induces apoptotic cell death of transformed rat retinal ganglion cells via mitochondrial signaling pathways. Invest Ophthalmol Vis Sci 46:1330–1338

    Article  PubMed  Google Scholar 

  45. Kong Q, Wang M, Liao Z, Camden JM, Yu S, Simonyi A, Sun GY, Gonzalez FA, Erb L, Seye CI, Weisman GA (2005) P2X(7) nucleotide receptors mediate caspase-8/9/3-dependent apoptosis in rat primary cortical neurons. Purinergic Signal 1:337–347

    Article  CAS  PubMed  Google Scholar 

  46. Franke H, Illes P (2006) Involvement of P2 receptors in the growth and survival of neurons in the CNS. Pharmacol Ther 109:297–324

    Article  CAS  PubMed  Google Scholar 

  47. Neary JT, Rathbone MP, Cattabeni F, Abbracchio MP, Burnstock G (1996) Trophic actions of extracellular nucleotides and nucleosides on glial and neuronal cells. Trends Neurosci 19:13–18

    Article  CAS  PubMed  Google Scholar 

  48. Noguchi T, Ishii K, Fukutomi H, Naguro I, Matsuzawa A, Takeda K, Ichijo H (2008) Requirement of reactive oxygen species-dependent activation of ASK1-p38 MAPK pathway for extracellular ATP-induced apoptosis in macrophage. J Biol Chem 283:7657–7665

    Article  CAS  PubMed  Google Scholar 

  49. Parvathenani LK, Tertyshnikova S, Greco CR, Roberts SB, Robertson B, Posmantur R (2003) P2X7 mediates superoxide production in primary microglia and is up-regulated in a transgenic mouse model of Alzheimer’s disease. J Biol Chem 278:13309–13317

    Article  CAS  PubMed  Google Scholar 

  50. Reed JC (2000) Mechanisms of apoptosis. Am J Pathol 157:1415–1430

    CAS  PubMed  Google Scholar 

  51. Zile MH (2001) Function of vitamin A in vertebrate embryonic development. J Nutr 131:705–708

    CAS  PubMed  Google Scholar 

  52. Carraro-Lacroix LR, Ramirez MA, Zorn TM, Reboucas NA, Malnic G (2006) Increased NHE1 expression is associated with serum deprivation-induced differentiation in immortalized rat proximal tubule cells. Am J Physiol Renal Physiol 291:F129–F139

    Article  CAS  PubMed  Google Scholar 

  53. De Chiara G, Marcocci ME, Torcia M, Lucibello M, Rosini P, Bonini P, Higashimoto Y, Damonte G, Armirotti A, Amodei S, Palamara AT, Russo T, Garaci E, Cozzolino F (2006) Bcl-2 Phosphorylation by p38 MAPK: identification of target sites and biologic consequences. J Biol Chem 281:21353–21361

    Article  PubMed  Google Scholar 

  54. Balmer JE, Blomhoff R (2002) Gene expression regulation by retinoic acid. J Lipid Res 43:1773–1808

    Article  CAS  PubMed  Google Scholar 

  55. Wu PY, Lin YC, Chang CL, Lu HT, Chin CH, Hsu TT, Chu D, Sun SH (2009) Functional decreases in P2X7 receptors are associated with retinoic acid-induced neuronal differentiation of Neuro-2a neuroblastoma cells. Cell Signal 21:881–891

    Article  CAS  PubMed  Google Scholar 

  56. Raymond VW, Grisham JW, Earp HS (1990) Characterization of epidermal growth factor receptor induction by retinoic acid in a chemically transformed rat liver cell line. Cell Growth Differ 1:393–399

    CAS  PubMed  Google Scholar 

  57. Wang L, Feng YH, Gorodeski GI (2005) Epidermal growth factor facilitates epinephrine inhibition of P2X7-receptor-mediated pore formation and apoptosis: a novel signaling network. Endocrinology 146:164–174

    Article  CAS  PubMed  Google Scholar 

  58. Michaelis M, Bliss J, Arnold SC, Hinsch N, Rothweiler F, Deubzer HE, Witt O, Langer K, Doerr HW, Wels WS, Cinatl J Jr (2008) Cisplatin-resistant neuroblastoma cells express enhanced levels of epidermal growth factor receptor (EGFR) and are sensitive to treatment with EGFR-specific toxins. Clin Cancer Res 14:6531–6537

    Article  CAS  PubMed  Google Scholar 

  59. Resta V, Novelli E, Di Virgilio F, Galli-Resta L (2005) Neuronal death induced by endogenous extracellular ATP in retinal cholinergic neuron density control. Development 132:2873–2882

    Article  CAS  PubMed  Google Scholar 

  60. Aga M, Johnson CJ, Hart AP, Guadarrama AG, Suresh M, Svaren J, Bertics PJ, Darien BJ (2002) Modulation of monocyte signaling and pore formation in response to agonists of the nucleotide receptor P2X(7). J Leukoc Biol 72:222–232

    CAS  PubMed  Google Scholar 

  61. Solle M, Labasi J, Perregaux DG, Stam E, Petrushova N, Koller BH, Griffiths RJ, Gabel CA (2001) Altered cytokine production in mice lacking P2X(7) receptors. J Biol Chem 276:125–132

    Article  CAS  PubMed  Google Scholar 

  62. Erb L, Liao Z, Seye CI, Weisman GA (2006) P2 receptors: intracellular signaling. Pflugers Arch 452:552–562

    Article  CAS  PubMed  Google Scholar 

  63. Donnelly-Roberts DL, Namovic MT, Faltynek CR, Jarvis MF (2004) Mitogen-activated protein kinase and caspase signaling pathways are required for P2X7 receptor (P2X7R)-induced pore formation in human THP-1 cells. J Pharmacol Exp Ther 308:1053–1061

    Article  CAS  PubMed  Google Scholar 

  64. Limke TL, Bearss JJ, Atchison WD (2004) Acute exposure to methylmercury causes Ca2+ dysregulation and neuronal death in rat cerebellar granule cells through an M3 muscarinic receptor-linked pathway. Toxicol Sci 80:60–68

    Article  CAS  PubMed  Google Scholar 

  65. Volonte C, Ciotti MT, D’Ambrosi N, Lockhart B, Spedding M (1999) Neuroprotective effects of modulators of P2 receptors in primary culture of CNS neurones. Neuropharmacology 38:1335–1342

    Article  CAS  PubMed  Google Scholar 

  66. Hogg RC, Chipperfield H, Whyte KA, Stafford MR, Hansen MA, Cool SM, Nurcombe V, Adams DJ (2004) Functional maturation of isolated neural progenitor cells from the adult rat hippocampus. Eur J Neurosci 19:2410–2420

    Article  PubMed  Google Scholar 

  67. da Silva JS, Dotti CG (2002) Breaking the neuronal sphere: regulation of the actin cytoskeleton in neuritogenesis. Nat Rev Neurosci 3:694–704

    Article  CAS  PubMed  Google Scholar 

  68. Diaz-Hernandez M, Del Puerto A, Diaz-Hernandez JI, Diez-Zaera M, Lucas JJ, Garrido JJ, Miras-Portugal MT (2008) Inhibition of the ATP-gated P2X7 receptor promotes axonal growth and branching in cultured hippocampal neurons. J Cell Sci 121:3717–3728

    Article  CAS  PubMed  Google Scholar 

  69. Chambon P (1994) The retinoid signaling pathway: molecular and genetic analyses. Semin Cell Biol 5:115–125

    Article  CAS  PubMed  Google Scholar 

  70. Werner EA, Deluca HF (2002) Retinoic acid is detected at relatively high levels in the CNS of adult rats. Am J Physiol Endocrinol Metab 282:E672–E678

    CAS  PubMed  Google Scholar 

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Acknowledgments

The authors are grateful to Dr. Michelle Burgos (BD, Biosciences) for useful discussion and for critical reading of the manuscript. We appreciate the excellent technical assistance of Cydmarie Cruz Quintana. The project described was supported by Grant P20 RR016470 from the National Center for Research Resources. The content is solely the responsibility of the authors and does not necessarily represent the official view of the National Center for Research Resources or National Institutes of Health. Elsie A. Orellano has a pre-doctoral fellowship from Puerto Rico Industrial Development Company (PRIDCO).

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  1. Department of Biology, University of Puerto Rico, Río Piedras Campus, San Juan, PR, USA

    Elsie A. Orellano

  2. Department of Biochemistry, School of Medicine, University of Puerto Rico, San Juan, PR, USA

    Elsie A. Orellano, Migdalia Chevres & Fernando A. González

  3. Department of Chemistry, University of Puerto Rico, Río Piedras Campus, PO Box 23346, San Juan, PR, 00931-3346, USA

    Omayra J. Rivera, Nataliya E. Chorna & Fernando A. González

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  1. Elsie A. Orellano
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Correspondence to Fernando A. González.

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Orellano, E.A., Rivera, O.J., Chevres, M. et al. Inhibition of neuronal cell death after retinoic acid-induced down-regulation of P2X7 nucleotide receptor expression. Mol Cell Biochem 337, 83–99 (2010). https://doi.org/10.1007/s11010-009-0288-x

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