Inhibition of PI3K/Akt pathway impairs G2/M transition of cell cycle in late developing progenitors of the avian embryo retina

Abstract

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.

Figure 1. Phospho-Akt expression in mitotic cells in retinal monolayer cultures and intact embryo retina.

(A–D) Retinal cell cultures from 7-day-old chick embryos maintained for 1 day (E7C1) were fixed and assayed for immunofluorescence against phospho-Akt (A) and phospho-histone H3 (B). Nuclei were stained with DAPI (C). Merged figures in D. (E–H) Retinal sections from 8-day-old embryo retinas were assayed for immunofluorescence against phospho-Akt (E) and phospho-histone H3 (F). DAPI staining of nuclei (G). Merged figures in (H). Arrows point to double labeled mitotic cells. Scale bar  = 20 µm in A–D and 30 µm in E–H.

Figure 2. Phospho-Akt is expressed in BrdU positive cells and over centrosomes of dividing cells in retinal monolayer cultures.

(A–C) Cultures at E7C1 were incubated with BrdU for 6 h, washed and cultivated for additional 15–16 h in fresh medium. Immunoassays were performed against phospho-Akt (A) and BrdU (B). Merged figures in C. Immunoassays of cultures at E7C1 performed against phospho-Akt (D) and gamma-tubulin (E). Merged figures in F. (G–J) High magnification micrographs showing double labeling against phospho-Akt and gamma-tubulin in dividing cells at prophase (G), metaphase (H), anaphase (I) and telophase (J). Nuclei were stained with DAPI in F–J. Arrow points to double labeled pAkt, γ-tubulin positive cells. Scale bar  = 10 µm in A–C and G, H, I, J and 20 µm in D–F.

Figure 3. Inhibition of PI3K increases phospho-histone H3 expression in retinal explants and monolayer cultures.

(A) Retinal explants from 7-day-old chick embryos were treated for 22 h with 25 µM LY294002, fixed and labeled with DAPI (blue) or antiserum against phospho-histone H3 (P-H3) (red). Control explants were cultured for the same period without inhibitor. Note the increase in the number of p-H3-positive cells at the border of the treated retina. (B) Explants of retinas from embryo at E7 were treated with 25 µM LY 294002 for 12 or 24 h and processed for P-H3 detection by western blot. (C) Retinal monolayer cultures at E7C1 were treated for 24 h with 10 µM or 25 µM LY 294002 and processed for P-H3 detection. Protein gel loading was assessed with anti-α-tubulin and anti-ERK antisera, respectively. Representative blots from at least 3 independent experiments are shown. (D) Quantification of blots represented in B and C. Data represent the mean ± S.E.M. in arbitrary units (A.U.) of 5–6 experiments performed in duplicate or triplicate. **p<0.01 and *p<0.05, compared to control cultures without LY treatment. Ct  =  control cultures or explants cultivated without drugs. NBL  =  Neuroblastic Layer. GCL  =  Prospective Ganglion Cell Layer. Scale bar  = 20 µm.

Figure 4. PI3K inhibition decreases phospho-Akt and phospho-GSK-3 levels in retinal cells.

Retinal explants from 7-day-old chick embryos were treated with 25 µM LY 294002 for 4, 8 or 24 h and protein extracts processed for detection of phospho-Akt and phospho-GSK3 expression. Gel loading was assessed with anti-Akt antiserum. Blots were quantified by densitometry and data are expressed as the mean ± S.E.M. of arbitrary units. **p<0.01 and ***p<0.001 relative to control. In both cases, representative blots from at least 3 independent experiments are shown. Ct  =  control explants cultivated without drug.

Figure 5. Phospho-4E-BP1 can be easily visualized in mitotic cells of retinal monolayer cultures or intact embryonic retina.

(A–D) Retinal cell cultures at E7C1 were fixed and assayed by immunocytochemistry for phospho-4E-BP1 (A) and phospho-histone H3 (B). DNA was stained with DAPI (C) and merged figures are shown in (D). Arrows point to double labeled cells. (E-G) Phospho-4E-BP1 labeling in mitotic cells of 8-day-old chick embryo retinas. Retinal sections were stained with anti-phospho-4E-BP1 (E) and anti-phospho-histone H3 (F). Merged figures are shown in (G). Phospho-4E-BP1 labeled cells were confined to the ventricular margin of the retina. (H) Detection of phospho-4E-BP1 in extracts of retinal cultures at E7C1 without any treatment. The phosphorylated forms γ and β of the protein are indicated by arrows, respectively. (I) Expression of phospho-4E-BP1 in extracts of retinal explants treated with 25 µM LY 294002 for 4, 8 or 12 h. Representative blots from at least 3 independent experiments are shown. Ct  =  control explants cultivated without drug. NBL  =  Neuroblastic Layer. Scale bar  = 20 µm.

Figure 6. Inhibition of PI3K/Akt pathway modulates expression of cell cycle proteins in retinal explants (A, B) and monolayer cultures (C, D).

Retinal explants from 7-day-old chick embryos (A, B) were treated with 25 µM LY 294002 for 12 or 24 h and processed for the detection of cyclin B1 (A), phospho-CDK1 and CDK 1 (B). Monolayer cultures at E7C1 were treated with 10 µM or 25 µM LY 294002 for 24 h and processed for detection of cyclin B1 (C), phospho-CDK 1 and CDK1 (D). Representative blots from at least 3 independent experiments are shown. Gel loading was assessed with anti-α-tubulin antiserum. Blots were quantified by densitometry and data represent the mean ± S.E.M. (% control). *p<0.05 and **p<0.01 relative to control.

Figure 7. Inhibition of PI3K with LY 294002 does not alter the pattern of apoptotic cells in the outer retina.

(A) Retinal explants from 7-day-old chick embryos were treated for 22 h with 25 µM LY 294002, fixed and sections processed by TUNEL assays as described in methods. Representative micrographs of TUNEL labeling (green) are shown. DAPI (blue) was used to label nuclei of all cells and explants were oriented with their outer, ventricular margin at the upper side of the micrographs. Data are representative of three experiments. (B) Retinal explants were treated for 22 h with 25 µM LY 294002, fixed and sections processed for immunocytochemistry against cleaved caspase-3 (green) and phospho-histone H3 (red). (C) Quantification of phospho-histone H3 and cleaved caspase 3 positive cells in retinal explants. Positive cells were counted in 10 transverse sections of retinal explants with 150 µm of linear extent parallel to their surface that were divided in 3 segments with the same width (o  =  outer segment; m  =  medium segment; I  =  inner segment). Data represent the mean ± S.E.M. of cell counts/segment from four independent experiments. Scale bar  = 20 µm.

Figure 8. Effect of LY294002 removal on the expression of cell cycle regulatory proteins and development markers.

(A) Monolayer cultures of retinal cells from E7 were established as described. After 2 h, LY294002 to a final concentration of 25 µM was added. After 24 h, medium was removed, fresh medium was added and cells cultivated for an additional 24 h period. At the end of incubations, protein extracts were analyzed for the indicated proteins by western blotting. Representative blots are shown. (B) Blots were quantified by densitometry and data are expressed as the mean ± S.E.M. (% control) of 3 or 4 experiments. ***p<0.001 relative to control without treatment. ^##p<0.01 relative to cultures treated on the second day or during the entire period. (C) Expression of transitin in treated explants. Retinal explants from 7-day-old chick embryos were treated for 24 h with 25 µM LY294002, fixed and labeled with anti-pH3 (green) or antiserum against transitin (red). Arrows point to double labeled cells. Control explants were cultured for the same period without inhibitor. Scale bar  = 10 µm.