Regulation of ischemic neuronal death by E2F4-p130 protein complexes

Abstract

Inappropriate activation of cell cycle proteins, in particular cyclin D/Cdk4, is implicated in neuronal death induced by various pathologic stresses, including DNA damage and ischemia. Key targets of Cdk4 in proliferating cells include members of the E2F transcription factors, which mediate the expression of cell cycle proteins as well as death-inducing genes. However, the presence of multiple E2F family members complicates our understanding of their role in death. We focused on whether E2F4, an E2F member believed to exhibit crucial control over the maintenance of a differentiated state of neurons, may be critical in ischemic neuronal death. We observed that, in contrast to E2F1 and E2F3, which sensitize to death, E2F4 plays a crucial protective role in neuronal death evoked by DNA damage, hypoxia, and global ischemic insult both in vitro and in vivo. E2F4 occupies promoter regions of proapoptotic factors, such as B-Myb, under basal conditions. Following stress exposure, E2F4-p130 complexes are lost rapidly along with the presence of E2F4 at E2F-containing B-Myb promoter sites. In contrast, the presence of E2F1 at B-Myb sites increases with stress. Furthermore, B-Myb and C-Myb expression increases with ischemic insult. Taken together, we propose a model by which E2F4 plays a protective role in neurons from ischemic insult by forming repressive complexes that prevent prodeath factors such as Myb from being expressed.

Keywords: Apoptosis; Cardiovascular Disease; Cell Biology; Cell Cycle; Cell Death; Cyclin-dependent Kinase (Cdk); E2F Transcription Factor; Hypoxia; Ischemia; Stroke.

© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Figures

FIGURE 1.

E2F family members play differential roles in neuronal death induced by genotoxic stress. E2F1 (A), E2F3 (B), and E2F4 (C) wild-type and knockout neurons were treated with 10 μm camptothecin (campto, a DNA-damaging agent) and evaluated for survival at 8 and 12 h following treatment. E2F1 (D), E2F3 (E), and E2F4 (F) containing a plasmid or vector control were transfected in cortical neuronal cultures 3 days after plating and treated with 10 μm camptothecin 24 h later. Survival was assessed 24 h following treatment. Results are expressed as percent of control ± S.E. **, p < 0.01.

FIGURE 2.

E2F1 and E2F4 have opposing roles in neuronal death induced by hypoxia.A, primary CGN cultures were infected with adenovirus expressing E2F1, E2F4, or GFP control at the time of plating and treated with hypoxia after a week in culture for 18 h, followed by reoxygenation for 24 h. B, Western blot analysis showing E2F4 knockdown in CGN cultures treated with E2F4 siRNA mixture. Ctrl, control. C, CGNs were transfected with E2F4 or control siRNA 5 days after plating. Cultures were treated with 18 h of hypoxia followed by 24 h of reoxygenation 2 days after transfection. D, CGNs from E2F4 KO and wild-type mice were similarly treated with 18 h of hypoxia and 24 h of reoxygenation. Neuronal survival was evaluated 24 h after hypoxia. Results are expressed as percent of control ± S.E. E, E2F1 protein levels are unaffected by E2F4 deficiency. CGNs from E2F4+/+ and E2F4−/− neurons were subjected to Western blot analysis and probed with anti-E2F1 antibody and anti-β-actin as a control. F, quantitation of E2F1 levels as in E. G, E2F3 protein levels are diminished in E2F4 knockout neurons. A Western blot analysis was conducted on E2F4+/+ and E2F4−/− CGNs. E2F3 was detected using anti-E2F3 antibody. β-actin was used as a loading control. H, quantitation of E2F3 protein levels as in G by densitometry. The protein levels of E2F1 (E) and E2F3 (G) were normalized to β-actin. Error bars represents the mean ± S.E. (n ≥ 3). *, p < 0.05; **, p < 0.01; ***, p < 0.001.

FIGURE 3.

p130 and E2F4 protein levels are down-regulated following hypoxia/reoxygenation of CGNs in culture.A, E2F4 forms a complex with p130 in CGNs. Total cell lysates from CGN cultures were subjected to reciprocal immunoprecipitation (IP) with anti-E2F4 and anti-p130 antibodies. Immune complexes were resolved by Western blot (WB) analysis and probed with anti-E2F4 and anti-p130 antibodies. B, Western blot analysis showing a loss of E2F4 protein in CGNs subjected to varying durations of hypoxia or 18 h of hypoxia with varying durations of reoxygenation. NH, no hypoxia. C, densitometry was performed on E2F4 blot as in B. E2F4 protein levels were normalized to the actin control. H, hypoxia; R, reoxygenation; R0, 18 h of hypoxia and 0 h of reoxygenation. D, Western blot analysis showing the loss of p130 following different durations of hypoxia or 18 h of hypoxia with varying reoxygenation times. E, densitometry was performed on p130 protein levels as in D and normalized to the β-actin control. F, basal p130 protein levels were diminished in E2F4−/− CGNs. A Western blot analysis was conducted on total protein lysate obtained from E2F4+/+ and E2F4−/− CGN cultures. The blots were probed with anti-p130 antibody and anti-β-actin as a control. G, densitometry was conducted on p130 blots as in F. p130 levels were normalized to β-actin. Error bars represent the mean ± S.E. (n ≥ 3). *, p < 0.05.

FIGURE 4.

Reduction of E2F4 and induction of E2F1 binding at the B-Myb promoter following hypoxia and reoxygenation.A, ChIP was conducted with E2F4 and E2F1 antibody on CGNs treated with hypoxia (hyp) for 18 h, followed by reoxygenation (reoxy) for 16 h. B, densitometry of the B-Myb signal following ChIP performed with anti-E2F4 antibody as shown in A. Loss of E2F4 was observed at the B-Myb promoter following hypoxia and reoxygenation. C, quantitation of the B-Myb signal following ChIP with anti-E2F1 antibody. D, ChIP was performed with anti-p130 antibody. p130 binding at the B-Myb promoter was lost following hypoxia and reoxygenation. E, densitometry of the B-Myb signal following ChIP as shown in D. F, E2F activity increased after hypoxia and reoxygenation in CGNs. Cells were infected with AAV expressing B-Myb-promoter-luciferase with wild-type E2F or a mutated E2F site and β-galactosidase. Cells were treated with 18 h of hypoxia followed by reoxygenation for up to 8 h. Luciferase activity and β-galactosidase were measured at the indicated times. Data represent values of luciferase/β-galactosidase activity. G, a luciferase assay was conducted in E2F4 wild-type and knockout CGNs treated with 18 h of hypoxia followed by 4 h of reoxygenation. Error bars represent mean ± S.E. (n ≥ 3). *, p < 0.05; **, p < 0.01; ***, p < 0.005.

FIGURE 5.

E2F4 expression protects CA1 neurons from global cerebral ischemia.A, immunofluorescence staining showing E2F4 overexpression in hippocampal CA1 neurons of rats injected with AAV expressing E2F4. B, hematoxylin and eosin-stained sections of CA1 neurons in sham- and 4VO-operated rats injected with GFP or E2F4 expressing AAV. C, quantitation of live CA1 neurons following 4VO in GFP and E2F4 injected rats. Rats were injected with AAV expressing E2F4 or a GFP control and subjected to sham surgery of 10 min of 4VO. Brains were extracted 4 days following reperfusion, sectioned, and stained with hematoxylin and eosin. H&E-stained coronal sections were evaluated for cell survival in the hippocampal CA1 (n ≥ 4/group, data are mean ± S.E). *, p < 0.05 compared with GFP control. D, Western blot analysis of E2F4 expression in the hippocampus of GFP and E2F4 injected rats.

FIGURE 6.

p130 and E2F4 protein levels are decreased following global cerebral ischemia in rats.A, Western blot analyses of p130 and E2F4 expression following 4VO in rats. Animals were subjected to 10 min of 4VO and sacrificed at the indicated times following reperfusion. Hippocampal tissues were extracted and subjected to Western blot analysis. Densitometry was performed on p130 protein levels (B) as well as E2F4 protein levels (C) and normalized to the actin control. D, Western blot analysis of p130 phosphorylation following 4VO. Rats were treated as in A and subjected to analysis using antibody directed at p130 phosphorylated at Ser-952. E, densitometry of Ser-952-phosphorylated p130. Expression levels were normalized to the actin control (n ≥ 3). *, p < 0.05 compared with sham.

FIGURE 7.

B- and C-Myb mRNA transcript levels are increased following global cerebral ischemia.A, semiquantitative RT-PCR of the B-Myb message following global ischemia. Rats were subjected to 10 min of 4VO followed by 12 and 24 h of reperfusion. Hippocampal tissues were extracted and analyzed by semiquantitative RT-PCR. GAPDH is shown as a control. B, densitometry of B-Myb levels. Data are expressed as fold over sham-operated control. C, semiquantitative RT-PCR of the C-Myb message following 4VO as described in A. E, semiquantitative RT-PCR showing siRNA mediated knockdown of the C-Myb message 24 h following transfection. S 12 is shown as a loading control. F, C-Myb knockdown protects CGNs from hypoxia/reoxygenation insult. Neurons were transfected with C-Myb siRNA and subjected to 18 h of hypoxia followed by 16 h of reoxygenation. Error bars represent the mean ± S.E. (n ≥ 3). *, p < 0.05.