title: Acetylcholine-induced neuronal differentiation: muscarinic receptor activation regulates EGR-1 and REST expression in neuroblastoma cells.
abstract: Neurotransmitters are considered part of the signaling system active in nervous system development and we have previously reported that acetylcholine (ACh) is capable of enhancing neuronal differentiation in cultures of sensory neurons and N18TG2 neuroblastoma cells. To study the mechanism of ACh action, in this study, we demonstrate the ability of choline acetyltransferase-transfected N18TG2 clones (e.g. 2/4 clone) to release ACh. Analysis of muscarinic receptors showed the presence of M1-M4 subtypes and the activation of both IP(3) and cAMP signal transduction pathways. Muscarinic receptor activation increases early growth response factor-1 (EGR-1) levels and treatments with agonists, antagonists, and signal transduction enzyme inhibitors suggest a role for M3 subtype in EGR-1 induction. The role of EGR-1 in the enhancement of differentiation was investigated transfecting in N18TG2 cells a construct for EGR-1. EGR-1 clones show increased neurite extension and a decrease in Repressor Element-1 silencing transcription factor (REST) expression: both these features have also been observed for the 2/4 clone. Transfection of this latter with EGR zinc-finger domain, a dominant negative inhibitor of EGR-1 action, increases REST expression, and decreases fiber outgrowth. The data reported suggest that progression of the clone 2/4 in the developmental program is dependent on ACh release and the ensuing activation of muscarinic receptors, which in turn modulate the level of EGR-1 and REST transcription factors.
authors.affiliation: Dipartimento di Biologia Cellulare e dello Sviluppo, Unità di Ricerca di Neurobiologia e Centro di Ricerca in Neurobiologia Daniel Bovet, Università La Sapienza, Piazzale Aldo Moro, Roma, Italy.
authors.forename: Monica
authors.initials: M
authors.lastname: Salani
authors.forename: Tonino
authors.initials: T
authors.lastname: Anelli
authors.forename: Gabriella Augusti
authors.initials: GA
authors.lastname: Tocco
authors.forename: Elena
authors.initials: E
authors.lastname: Lucarini
authors.forename: Chiara
authors.initials: C
authors.lastname: Mozzetta
authors.forename: Giancarlo
authors.initials: G
authors.lastname: Poiana
authors.forename: Ada Maria
authors.initials: AM
authors.lastname: Tata
authors.forename: Stefano
authors.initials: S
authors.lastname: Biagioni
mesh_terms: D000109:Acetylcholine / Q000378:metabolism / Q000494:pharmacology* / Q000502:physiology; D001667:Binding, Competitive / Q000187:drug effects; D015153:Blotting, Western; D001932:Brain Neoplasms / Q000378:metabolism* / Q000473:pathology; D002454:Cell Differentiation / Q000187:drug effects*; D045744:Cell Line, Tumor; D048429:Cell Size; D000242:Cyclic AMP / Q000378:metabolism; D051766:Early Growth Response Protein 1 / Q000096:biosynthesis* / Q000235:genetics; D006801:Humans; D018721:Muscarinic Agonists; D009447:Neuroblastoma / Q000378:metabolism* / Q000473:pathology; D009474:Neurons / Q000187:drug effects*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011813:Quinuclidinyl Benzilate / Q000378:metabolism; D011976:Receptors, Muscarinic / Q000187:drug effects*; D012097:Repressor Proteins / Q000096:biosynthesis* / Q000235:genetics; D015398:Signal Transduction / Q000187:drug effects; D014162:Transfection; D000099229:RE1-Silencing Transcription Factor
chemical_list: D000109:Acetylcholine; D000242:Cyclic AMP; D051766:Early Growth Response Protein 1; D018721:Muscarinic Agonists; D047428:Protein Kinase Inhibitors; D011813:Quinuclidinyl Benzilate; D011976:Receptors, Muscarinic; D012097:Repressor Proteins; D000099229:RE1-Silencing Transcription Factor