RESUMO
In the avian retina, ADP induces the proliferation of late developing glia progenitors. Here, we show that in serum-containing retinal cell cultures, ADP-induced increase in [3H]-thymidine incorporation can be prevented by the IGF-1 receptor antagonists AG1024 and I-OMe-Tyrphostin AG 538, suggesting the participation of IGF-1 in ADP-mediated progenitor proliferation. In contrast, no increase in [3H]-thymidine incorporation is observed in retinal cultures treated only with IGF-1. Under serum starvation, while no increase in cell proliferation is detected in cultures treated only with ADP or IGF-1, a significant increase in [3H]-thymidine incorporation and number of PCNA expressing cells is observed in cultures treated concomitantly with ADP plus IGF-1, suggesting that both molecules are required to induce proliferation of retinal progenitors. In serum-starved cultures, although an increase in cell viability is detected by MTT assays in IGF-1-treated cultures, no significant increase in viability of [3H]-thymidine labeled progenitors is observed, suggesting that IGF-1 may contribute to survival of postmitotic cells in culture. While only ADP increases intracellular calcium, only IGF-1 induces the phosphorylation of Akt in the retinal cultures. IGF-1 through the PI3K/Akt pathway induces a significant increase in the transcription and expression of CDK1 with a decrease in phospho-histone H3 expression that is concomitant with an increase in the expression of cyclins D1 and E and CDK2. These findings suggest that IGF-1 stimulates CDK-1 mRNA and protein expression that enable progenitors to progress through the cell cycle. However, signaling of ADP in the presence IGF-I seems to be required for DNA synthesis.
RESUMO
Development of progenitors in the embryonic retina is modulated by signaling molecules, and cannabinoid receptors are highly expressed in the early developing retina. Here, we investigated whether the CB1/CB2 receptor agonist WIN 5212-2 (WIN) modulated the proliferation, viability, and calcium responses in chick embryo retinal progenitors in culture. A decline in [3H]-thymidine incorporation was observed when cultures were incubated with 0.5-1.0 µM WIN, an effect that was mimicked by URB602 and URB597, inhibitors of the monoacylglycerol lipase and fatty acid amide hydrolase, respectively. A reduction in the number of proliferating cell nuclear antigen-positive nuclei was also noticed in WIN-treated cultures, suggesting that activation of cannabinoid receptors decreases the proliferation of cultured retinal progenitors. WIN (0.5-5.0 µM), but not capsaicin, decreased retinal cell viability, an effect that was blocked by CB1 and CB2 receptor antagonists and by the P2X7 receptor antagonist A438079, implicating this nucleotide receptor in the cannabinoid-mediated cell death. Treatment with WIN also induced an increase in mitochondrial superoxide and P2X7 receptor-mediated uptake of sulforhodamine B in the cultured cells. While a high proportion of cultured cells responded to glutamate, GABA, and 50 mM KCl with intracellular calcium shifts, very few cells responded to the activation of P2X7 receptors by ATP. Noteworthy, while decreasing the number of cells responding to glutamate, GABA, and KCl, treatment of the cultures with WIN induced a significant increase in the number of cells responding to 1 mM ATP, suggesting that activation of cannabinoid receptors primes P2X7 receptor calcium signaling in retinal progenitors in culture.
Assuntos
Apoptose/efeitos dos fármacos , Canabinoides/farmacologia , Neuroglia/citologia , Receptores Purinérgicos P2X7/metabolismo , Retina/citologia , Transdução de Sinais/efeitos dos fármacos , Células-Tronco/metabolismo , Animais , Benzoxazinas/farmacologia , Cálcio/metabolismo , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Embrião de Galinha , Corantes Fluorescentes/metabolismo , Morfolinas/farmacologia , Naftalenos/farmacologia , Nestina/metabolismo , Fenótipo , Receptor CB1 de Canabinoide/metabolismo , Receptor CB2 de Canabinoide/metabolismo , Células-Tronco/efeitos dos fármacosRESUMO
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+]i is observed in glial cells only when cultures maintained for 9days are used. In glia from cultures cultivated for only 2days, no increase in [Ca2+]i is detected with MRS2365 and no inhibition of ADP-mediated calcium response is observed with MRS2179. In contrast, MRS2211 attenuates ADP-mediated increase in [Ca2+]i in glial cells from cultures at both stages, suggesting the presence of P2Y13 receptors coupled to calcium mobilization in proliferating retinal glial progenitors in culture.
Assuntos
Difosfato de Adenosina/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Receptores Purinérgicos P2Y1/genética , Receptores Purinérgicos P2/genética , Retina/metabolismo , Animais , Cálcio/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Embrião de Galinha , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/antagonistas & inibidores , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Naftóis/administração & dosagem , Neuroglia/efeitos dos fármacos , Neuroglia/metabolismo , Organofosfatos/administração & dosagem , Fosfatidilinositol 3-Quinases/genética , Fosforilação/efeitos dos fármacos , Agonistas do Receptor Purinérgico P2/administração & dosagem , Antagonistas do Receptor Purinérgico P2Y/administração & dosagem , Fosfato de Piridoxal/administração & dosagem , Fosfato de Piridoxal/análogos & derivados , Retina/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Xantinas/administração & dosagemRESUMO
When retinal cell cultures were mechanically scratched, cell growth over the empty area was observed. Only dividing and migrating, 2 M6-positive glial cells were detected. Incubation of cultures with apyrase (APY), suramin, or Reactive Blue 2 (RB-2), but not MRS 2179, significantly attenuated the growth of glial cells, suggesting that nucleotide receptors other than P2Y1 are involved in the growth of glial cells. UTPγS but not ADPßS antagonized apyrase-induced growth inhibition in scratched cultures, suggesting the participation of UTP-sensitive receptors. No decrease in proliferating cell nuclear antigen (PCNA(+)) cells was observed at the border of the scratch in apyrase-treated cultures, suggesting that glial proliferation was not affected. In apyrase-treated cultures, glial cytoplasm protrusions were smaller and unstable. Actin filaments were less organized and alfa-tubulin-labeled microtubules were mainly parallel to scratch. In contrast to control cultures, very few vinculin-labeled adhesion sites could be noticed in these cultures. Increased Akt and ERK phosphorylation was observed in UTP-treated cultures, effect that was inhibited by SRC inhibitor 1 and PI3K blocker LY294002. These inhibitors and the FAK inhibitor PF573228 also decreased glial growth over the scratch, suggesting participation of SRC, PI3K, and FAK in UTP-induced growth of glial cells in scratched cultures. RB-2 decreased dissociated glial cell attachment to fibronectin-coated dishes and migration through transwell membranes, suggesting that nucleotides regulated adhesion and migration of glial cells. In conclusion, mechanical scratch of retinal cell cultures induces growth of glial cells over the empty area through a mechanism that is dependent on activation of UTP-sensitive receptors, SRC, PI3K, and FAK.
Assuntos
Movimento Celular/efeitos dos fármacos , Neuroglia/citologia , Nucleotídeos/metabolismo , Retina/efeitos dos fármacos , Animais , Apirase/farmacologia , Movimento Celular/fisiologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Galinhas , Cromonas/farmacologia , Morfolinas/farmacologia , Neurogênese/efeitos dos fármacos , Neuroglia/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Quinolonas/farmacologia , Retina/lesões , Transdução de Sinais/efeitos dos fármacos , Sulfonas/farmacologia , Suramina/farmacologiaRESUMO
PI3K/Akt is an important pathway implicated in the proliferation and survival of cells in the CNS. Here we investigated the participation of the PI3K/Akt signal pathway in cell cycle of developing retinal progenitors. Immunofluorescence assays performed in cultures of chick embryo retinal cells and intact tissues revealed the presence of phosphorylated Akt and 4E-BP1 in cells with typical mitotic profiles. Blockade of PI3K activity with the chemical inhibitor LY 294002 (LY) in retinal explants blocked the progression of proliferating cells through G2/M transition, indicated by an expressive increase in the number of cells labeled for phosphorylated histone H3 in the ventricular margin of the retina. No significant level of cell death could be detected at this region. Retinal explants treated with LY for 24 h also showed a significant decrease in the expression of phospho-Akt, phospho-GSK-3 and the hyperphosphorylated form of 4E-BP1. Although no change in the expression of cyclin B1 was detected, a significant decrease in CDK1 expression was noticed after 24 h of LY treatment both in retinal explants and monolayer cultures. Our results suggest that PI3K/Akt is an active pathway during proliferation of retinal progenitors and its activity appears to be required for proper CDK1 expression levels and mitosis progression of these cells.