Involvement of nucleotides in glial growth following scratch injury in avian retinal cell monolayer cultures

Abstract

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 ADPβS 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.

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Acknowledgments

We would like to thank Maria Leite Eduardo and Sarah A. Rodrigues for technical assistance. This work was supported by grants from Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), Coordenação de Aperfeiçoamento de Pessoal do Ensino Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Pró-reitoria de Pesquisa, Pós-graduação e Inovação (Proppi-UFF). I.M.O is the recipient of post-doctoral fellowship from PNPD-CAPES. Transitin (EAP3) and vinculin (VN 3-24) monoclonal antibodies developed respectively by G.J. Cole and Shinsuke Saga were obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology, Iowa City, IA 52242. H.U. acknowledges grant support from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) and CNPq, Brazil.

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I declare that all authors have no conflict of interest.

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Correspondence to Ana Lucia Marques Ventura.

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Glial cell division and migration in control scratched cultures. Cultures at E8C7 were scratched and after 3 days, medium was changed to MEM buffered with 25 mM HEPES (pH 7.4) plus serum and antibiotics and cultures mounted on a Leica SP5 confocal microscope. Time-lapse live imaging were performed by photographing cultures every 5 min during 6 h under differential interference contrast illumination using a 20x objective plus 1.5 x zoom. Bar = 75 μm. (MPG 2150 kb)

Glial cell activity at the edge of the scratch in apyrase-treated cultures. Cultures at E8C7 were scratched and treated with 2.5 U/mL apyrase. After 3 days, medium was changed to MEM buffered with 25 mM HEPES (pH 7.4) plus apyrase, serum and antibiotics and cultures mounted on a Leica SP5 confocal microscope. Time-lapse live imaging were performed by photographing cultures every 5 min during ~8 h under differential interference contrast illumination using a 20x objective plus 1.5 x zoom. (MPG 3014 kb)

Online Resource 2

Effect of P2Y1 receptor antagonist and ADPβS on the incorporation of [ 3 H]-thymidine in retinal cell cultures. Retinal cultures were treated for 24 h with MRS2179 or ADPβS and the incorporation of [3H]-thymidine determined as described in the section of methods. (A) Retinal cultures at E7C1 were incubated with 500 μM ADP in the absence or presence of 5, 25 or 50 μM MRS 2179. (B) Retinal cultures at E8C8 that were scratched or not (control) were incubated with 50 μM ADPβS. Data represent the mean ± SEM of 3 to 5 independent experiments performed in duplicate. *** p < 0.001, compared to control cultures. ## p < 0.01 and ### p < 0.001, compared to ADP-treated cultures. (PPTX 67 kb)

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Silva, T.M., França, G.R., Ornelas, I.M. et al. Involvement of nucleotides in glial growth following scratch injury in avian retinal cell monolayer cultures. Purinergic Signalling 11, 183–201 (2015). https://doi.org/10.1007/s11302-015-9444-9

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Keywords

  • Müller glia
  • Nucleotides
  • P2Y receptors
  • Adhesion
  • Migration
  • Cytoskeleton