Functional decreases in P2X 7 receptors are associated with retinoic acid-induced neuronal differentiation of Neuro-2a neuroblastoma cells Pei-Yu Wu a , Yu-Chia Lin a , Chia-Ling Chang a , Hsing-Tsen Lu a , Chia-Hsuan Chin a , Tsan-Ting Hsu a , Dachen Chu b , Synthia H. Sun a,c, ⁎ a Institute of Neuroscience, National Yang-Ming University, Taipei, Taiwan, ROC b Department of Neurosurgery in Hoping Branch, Taipei, Taiwan, ROC c Department of Education and Research, Taipei City Hospital, Taiwan, ROC abstract article info Article history: Received 24 September 2008 Received in revised form 10 January 2009 Accepted 23 January 2009 Available online 31 January 2009 Keywords: ATP Basal [Ca 2+ ] i Neuronal differentiation Neuroblastoma cells P2X 7 receptor Retinoic acid Neuro-2a (N2a) cells are derived from spontaneous neuroblastoma of mouse and capable to differentiate into neuronal-like cells. Recently, P2X 7 receptor has been shown to sustain growth of human neuroblastoma cells but its role during neuronal differentiation remains unexamined. We characterized the role of P2X 7 receptors in the retinoic acid (RA)-differentiated N2a cells. RA induced N2a cells differentiation into neurite bearing and neuronal specific proteins, microtubule-associated protein 2 (MAP2) and neuronal specific nuclear protein (NeuN), expressing neuronal-like cells. Interestingly, the RA-induced neuronal differentiation was associated with decreases in the expression and function of P2X 7 receptors. Functional inhibition of P2X 7 receptors by P2X 7 receptor selective antagonists, 5′-triphosphate, periodate-oxidized 2′,3′-dialdehyde ATP (oATP), brilliant blue G (BBG) or A438079 induced neurite outgrowth. In addition, RA and oATP treatment stimulated the expression of neuron-specific class III beta-tubulin (TuJ1), and knockdown of P2X 7 receptor expression by siRNA induced neurite outgrowth. To elucidate the possible mechanism, we found the levels of basal intracellular Ca 2+ concentrations ([Ca 2+ ] i ) were decreased in either RA- or oATP-differentiated or P2X 7 receptor knockdown N2a cells. Simply cultured N2a cells in low Ca 2+ medium induced a 2-fold increase in neurite length. Treatment of N2a cells with ATP hydrolase apyrase and the P2X 7 receptors selective antagonist oATP or BBG decreased cell viability and cell number. Nevertheless, oATP but not BBG decreased cell proliferation and cell cycle progression. These results suggest for the first time that decreases in expression/ function of P2X 7 receptors are involved in neuronal differentiation. We provide additional evidence shown that the ATP release-activated P2X 7 receptor is important in maintaining cell survival of N2a neuroblastoma cells. © 2009 Elsevier Inc. All rights reserved. 1. Introduction ATP is a well recognized extracellular signaling molecule to mediate many physiological functions via two families, P2X and P2Y, of purinergic receptors. P2Y receptors are G-protein coupled receptors consisting of P2Y 1 , P2Y 2 , P2Y 4 , P2Y 6 , P2Y 11 , P2Y 12 , P2Y 13 and P2Y 14 eight subtypes of receptors. P2X receptors are ionotropic receptors consisting of P2X 1 , P2X 2 , P2X 3 , P2X 4 , P2X 5 , P2X 6 and P2X 7 seven subtypes of receptors. These receptors are widely distributed in the nervous system to modulate important physiological functions such as neurotransmission [1,2]. ATP signal transduction pathways have been found to induce proliferation of glial cells [3], human mesenchymal stem cells [4] and developing retinal cells [5]. In contrast, ATP is also recognized as a differentiation and survival factor to regulate cell growth in CNS [6]. Thus modification of P2 receptor expression may play a role in regulating neuronal differentiation. P2X 7 receptor has been cloned from rat brain [7] and known to induce cytolysis and cell death in various types of immunoresponsive cells [8–12]. An early study demonstrated that transfection of P2X 7 cDNA into the P2X 7 receptor lacking lymphoid cells sustained their proliferation in serum-free medium [13]. In addition, stimulation of P2X 7 receptors supports proliferation in human neuroblastoma cells [14]. However, P2X 7 receptors were found to trigger the death of retinal cholinergic cells thereby controlling the total number, the local density and spacing of neurons [15]. Thus, function of P2X 7 receptors may play a key role during neuronal differentiation. Activation of P2X 7 receptors have been shown to induce a sustain increase in intracellular Ca 2+ concentration ([Ca 2+ ] i ) through Ca 2+ influx in astrocytes [16–19]. Ca 2+ signal is well known to associate with cell growth and proliferation, thus P2X 7 receptor signaling may regulate neuroblastoma cells growth and proliferation via stimulating increases in [Ca 2+ ] i . However, expression and function of P2X 7 receptors during neuronal differentiation remains unknown. Therefore, we sought to Cellular Signalling 21 (2009) 881–891 ⁎ Corresponding author. Institute of Neuroscience, National Yang-Ming University, #155, Section 2, Li-Non Street, Taipei, 11221, Taiwan, ROC. Tel.: +886 2 2826 7103; fax: +886 2 2820 0259. E-mail address: shsun@ym.edu.tw (S.H. Sun). 0898-6568/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.cellsig.2009.01.036 Contents lists available at ScienceDirect Cellular Signalling journal homepage: www.elsevier.com/locate/cellsig