title: Ca2+/calmodulin-dependent kinase II signalling cascade mediates P2X7 receptor-dependent inhibition of neuritogenesis in neuroblastoma cells.
abstract: ATP, via purinergic P2X receptors, acts as a neurotransmitter and modulator in both the central and peripheral nervous systems, and is also involved in many biological processes, including cell proliferation, differentiation and apoptosis. Previously, we have reported that P2X7 receptor inhibition promotes axonal growth and branching in cultured hippocampal neurons. In this article, we demonstrate that the P2X7 receptor negatively regulates neurite formation in mouse Neuro-2a neuroblastoma cells through a Ca2+/calmodulin-dependent kinase II-related mechanism. Using both molecular and immunocytochemical techniques, we characterized the presence of endogenous P2X1, P2X3, P2X4 and P2X7 subunits in these cells. Of these, the P2X7 receptor was the only functional receptor, as its activation induced intracellular calcium increments similar to those observed in primary neuronal cultures, exhibiting pharmacological properties characteristic of homomeric P2X7 receptors. Patch-clamp experiments were also conducted to fully demonstrate that ionotropic P2X7 receptors mediate nonselective cation currents in this cell line. Pharmacological inhibition of the P2X7 receptor and its knockdown by small hairpin RNA interference resulted in increased neuritogenesis in cells cultured in low serum-containing medium, whereas P2X7 overexpression significantly reduced the formation of neurites. Interestingly, P2X7 receptor inhibition also modified the phosphorylation state of focal adhesion kinase, Akt and glycogen synthase kinase 3, protein kinases that participate in the Ca2+/calmodulin-dependent kinase II signalling cascade and that have been related to neuronal differentiation and axonal growth. Taken together, our results provide the first mechanistic insight into P2X7 receptor-triggered signalling pathways that regulate neurite formation in neuroblastoma cells.
authors.affiliation: Departamento de Bioquímica y Biología Molecular, Facultad de Veterinaria, Universidad Complutense de Madrid, Spain.
authors.forename: Rosa
authors.initials: R
authors.lastname: Gómez-Villafuertes
authors.forename: Ana
authors.initials: A
authors.lastname: del Puerto
authors.forename: Miguel
authors.initials: M
authors.lastname: Díaz-Hernández
authors.forename: Diego
authors.initials: D
authors.lastname: Bustillo
authors.forename: Juan I
authors.initials: JI
authors.lastname: Díaz-Hernández
authors.forename: Paula G
authors.initials: PG
authors.lastname: Huerta
authors.forename: Antonio R
authors.initials: AR
authors.lastname: Artalejo
authors.forename: Juan J
authors.initials: JJ
authors.lastname: Garrido
authors.forename: Ma Teresa
authors.initials: MT
authors.lastname: Miras-Portugal
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000502:physiology*; D051416:Focal Adhesion Protein-Tyrosine Kinases / Q000502:physiology; D006801:Humans; D008274:Magnesium / Q000494:pharmacology; D016501:Neurites / Q000502:physiology; D009447:Neuroblastoma / Q000503:physiopathology*; D055495:Neurogenesis; D019869:Phosphatidylinositol 3-Kinases / Q000502:physiology; D018048:Receptors, Purinergic P2 / Q000502:physiology*; D058486:Receptors, Purinergic P2X7; D015398:Signal Transduction / Q000502:physiology*; D014407:Tumor Cells, Cultured
chemical_list: C546468:P2RX7 protein, human; C546469:P2rx7 protein, mouse; D018048:Receptors, Purinergic P2; D058486:Receptors, Purinergic P2X7; C033901:3'-O-(4-benzoyl)benzoyladenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D019869:Phosphatidylinositol 3-Kinases; D051416:Focal Adhesion Protein-Tyrosine Kinases; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D008274:Magnesium