title: Nucleotide P2Y13-stimulated phosphorylation of CREB is required for ADP-induced proliferation of late developing retinal glial progenitors in culture.
abstract: Nucleotides stimulate phosphorylation of CREB to induce cell proliferation and survival in diverse cell types. We report here that ADP induces the phosphorylation of CREB in a time- and concentration-dependent manner in chick embryo retinal progenitors in culture. ADP-induced increase in phospho-CREB is mediated by P2 receptors as it is blocked by PPADS but not by the adenosine antagonists DPCPX or ZM241385. Incubation of the cultures with the CREB inhibitor KG-501 prevents ADP-induced incorporation of [3H]-thymidine, indicating that CREB is involved in retinal cell proliferation. No effect of this compound is observed on the viability of retinal progenitors. While no significant increase in CREB phosphorylation is observed with the P2Y1 receptor agonist MRS2365, ADP-induced phosphorylation of CREB is blocked by the P2Y13 receptor selective antagonist MRS2211, but not by MRS2179 or PSB0739, two antagonists of the P2Y1 and P2Y12 receptors, respectively, suggesting that ADP-induced CREB phosphorylation is mediated by P2Y13 receptors. ADP-induced increase in phospho-CREB is attenuated by the PI3K inhibitor LY294002 and completely prevented by the MEK inhibitor U0126, suggesting that at least ERK is involved in ADP-induced CREB phosphorylation. A pharmacological profile similar to the activation and inhibition of CREB phosphorylation is observed in the phosphorylation of ERK, suggesting that P2Y13 receptors mediate ADP induced ERK/CREB pathway in the cultures. While no increase in [3H]-thymidine incorporation is observed with the P2Y1 receptor agonist MRS2365, both MRS2179 and MRS2211 prevent ADP-mediated increase in [3H]-thymidine incorporation, but not progenitor's survival, suggesting that both P2Y1 and P2Y13 receptor subtypes are involved in ADP-induced cell proliferation. P2Y1 receptor-mediated increase in [Ca2+]iis observed in glial cells only when cultures maintained for 9days are used. In glia from cultures cultivated for only 2days, no increase in [Ca2+]iis detected with MRS2365 and no inhibition of ADP-mediated calcium response is observed with MRS2179. In contrast, MRS2211 attenuates ADP-mediated increase in [Ca2+]iin glial cells from cultures at both stages, suggesting the presence of P2Y13 receptors coupled to calcium mobilization in proliferating retinal glial progenitors in culture.
authors.affiliation: Department of Neurobiology, Neuroscience Program, Fluminense Federal University, Outeiro de São João Batista s/n, Centro, Niterói, Rio de Janeiro CEP 24020-141, Brazil.
authors.forename: Flavia Jesus
authors.initials: FJ
authors.lastname: Jacques
authors.affiliation: Department of Neurobiology, Neuroscience Program, Fluminense Federal University, Outeiro de São João Batista s/n, Centro, Niterói, Rio de Janeiro CEP 24020-141, Brazil.
authors.forename: Thayane Martins
authors.initials: TM
authors.lastname: Silva
authors.affiliation: Department of Neurobiology, Neuroscience Program, Fluminense Federal University, Outeiro de São João Batista s/n, Centro, Niterói, Rio de Janeiro CEP 24020-141, Brazil.
authors.forename: Flavia Emenegilda
authors.initials: FE
authors.lastname: da Silva
authors.affiliation: Department of Neurobiology, Neuroscience Program, Fluminense Federal University, Outeiro de São João Batista s/n, Centro, Niterói, Rio de Janeiro CEP 24020-141, Brazil.
authors.forename: Isis Moraes
authors.initials: IM
authors.lastname: Ornelas
authors.affiliation: Department of Neurobiology, Neuroscience Program, Fluminense Federal University, Outeiro de São João Batista s/n, Centro, Niterói, Rio de Janeiro CEP 24020-141, Brazil. Electronic address: almv@vm.uff.br.
authors.forename: Ana Lucia Marques
authors.initials: ALM
authors.lastname: Ventura
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D002642:Chick Embryo; D017362:Cyclic AMP Response Element-Binding Protein / Q000037:antagonists & inhibitors / Q000378:metabolism*; D020935:MAP Kinase Signaling System / Q000187:drug effects; D009284:Naphthols / Q000008:administration & dosage; D009457:Neuroglia / Q000187:drug effects / Q000378:metabolism; D010755:Organophosphates / Q000008:administration & dosage; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics; D010766:Phosphorylation / Q000187:drug effects; D058910:Purinergic P2 Receptor Agonists / Q000008:administration & dosage; D058921:Purinergic P2Y Receptor Antagonists / Q000008:administration & dosage; D011732:Pyridoxal Phosphate / Q000008:administration & dosage / Q000031:analogs & derivatives; D018048:Receptors, Purinergic P2 / Q000235:genetics*; D058472:Receptors, Purinergic P2Y1 / Q000235:genetics*; D012160:Retina / Q000187:drug effects / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics; D014970:Xanthines / Q000008:administration & dosage
chemical_list: D017362:Cyclic AMP Response Element-Binding Protein; D009284:Naphthols; D010755:Organophosphates; C440036:P2RY13 protein, human; D058910:Purinergic P2 Receptor Agonists; D058921:Purinergic P2Y Receptor Antagonists; D018048:Receptors, Purinergic P2; D058472:Receptors, Purinergic P2Y1; D014970:Xanthines; C495860:naphthol AS-E phosphate; C077792:pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid; D011732:Pyridoxal Phosphate; D000244:Adenosine Diphosphate; C051360:1,3-dipropyl-8-cyclopentylxanthine; D019869:Phosphatidylinositol 3-Kinases; D002118:Calcium
keywords: ADP; Calcium; P2Y13 receptor; Phospho-CREB; Proliferation; Retinal progenitors