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APAF1 is a key transcriptional target for p53 in the regulation of neuronal cell death.

Fortin A, Cregan SP, MacLaurin JG, Kushwaha N, Hickman ES, Thompson CS, Hakim A, Albert PR, Cecconi F, Helin K, Park DS, Slack RS - J. Cell Biol. (2001)

Bottom Line: Induction of neuronal cell death by camptothecin, a DNA-damaging agent that functions through a p53-dependent mechanism, resulted in increased Apaf1 mRNA in p53-positive, but not p53-deficient neurons.In transient transfections in a neuronal cell line with p53 and Apaf1 promoter-luciferase constructs, p53 directly activated the Apaf1 promoter via both p53 sites.Neurons treated with camptothecin were significantly protected in the absence of Apaf1 relative to those derived from wild-type littermates.

View Article: PubMed Central - PubMed

Affiliation: Ottawa Health Research Institute - Neuroscience, University of Ottawa, Ottawa, Ontario K1H-8M5, Canada.

ABSTRACT
p53 is a transcriptional activator which has been implicated as a key regulator of neuronal cell death after acute injury. We have shown previously that p53-mediated neuronal cell death involves a Bax-dependent activation of caspase 3; however, the transcriptional targets involved in the regulation of this process have not been identified. In the present study, we demonstrate that p53 directly upregulates Apaf1 transcription as a critical step in the induction of neuronal cell death. Using DNA microarray analysis of total RNA isolated from neurons undergoing p53-induced apoptosis a 5-6-fold upregulation of Apaf1 mRNA was detected. Induction of neuronal cell death by camptothecin, a DNA-damaging agent that functions through a p53-dependent mechanism, resulted in increased Apaf1 mRNA in p53-positive, but not p53-deficient neurons. In both in vitro and in vivo neuronal cell death processes of p53-induced cell death, Apaf1 protein levels were increased. We addressed whether p53 directly regulates Apaf1 transcription via the two p53 consensus binding sites in the Apaf1 promoter. Electrophoretic mobility shift assays demonstrated p53-DNA binding activity at both p53 consensus binding sequences in extracts obtained from neurons undergoing p53-induced cell death, but not in healthy control cultures or when p53 or the p53 binding sites were inactivated by mutation. In transient transfections in a neuronal cell line with p53 and Apaf1 promoter-luciferase constructs, p53 directly activated the Apaf1 promoter via both p53 sites. The importance of Apaf1 as a p53 target gene in neuronal cell death was evaluated by examining p53-induced apoptotic pathways in primary cultures of Apaf1-deficient neurons. Neurons treated with camptothecin were significantly protected in the absence of Apaf1 relative to those derived from wild-type littermates. Together, these results demonstrate that Apaf1 is a key transcriptional target for p53 that plays a pivotal role in the regulation of apoptosis after neuronal injury.

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p53-mediated induction of Apaf1 protein in neurons. (A) Protein was extracted from neurons 48 or 60 h after infection with Ad-p53 or Ad-p53-173L and assayed for Apaf1 levels by Western blot analysis. (B) Protein was extracted from wild-type or p53-deficient neurons at the indicated times after treatment with 10 μM camptothecin and assayed for Apaf1 levels by Western blot analysis. Specificity of the Apaf1 antibody is demonstrated by lack of immunoreactivity in extracts derived from Apaf1 knockout brain and loading is standardized with actin.
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fig2: p53-mediated induction of Apaf1 protein in neurons. (A) Protein was extracted from neurons 48 or 60 h after infection with Ad-p53 or Ad-p53-173L and assayed for Apaf1 levels by Western blot analysis. (B) Protein was extracted from wild-type or p53-deficient neurons at the indicated times after treatment with 10 μM camptothecin and assayed for Apaf1 levels by Western blot analysis. Specificity of the Apaf1 antibody is demonstrated by lack of immunoreactivity in extracts derived from Apaf1 knockout brain and loading is standardized with actin.

Mentions: To determine whether the increase in Apaf1 mRNA levels was accompanied by an increase in protein expression, we conducted Western analysis on neurons undergoing p53-induced apoptosis. Cell extracts were obtained from neurons infected at 20 MOI with Ad-p53 or the control vector Ad-LacZ and Apaf1 protein expression was examined. A significant increase in Apaf1 protein levels was evident at 48 and 60 h after infection with Ad-p53 relative to control (Fig. 2 A). We next examined Apaf1 protein levels in camptothecin-induced apoptosis that activates endogenous p53 using wild-type and p53-deficient cortical neurons. A time-dependent increase in Apaf1 protein levels was found in wild-type neurons treated with camptothecin, whereas Apaf1 protein levels did not increase in p53-deficient neurons treated under identical conditions (Fig. 2 B).


APAF1 is a key transcriptional target for p53 in the regulation of neuronal cell death.

Fortin A, Cregan SP, MacLaurin JG, Kushwaha N, Hickman ES, Thompson CS, Hakim A, Albert PR, Cecconi F, Helin K, Park DS, Slack RS - J. Cell Biol. (2001)

p53-mediated induction of Apaf1 protein in neurons. (A) Protein was extracted from neurons 48 or 60 h after infection with Ad-p53 or Ad-p53-173L and assayed for Apaf1 levels by Western blot analysis. (B) Protein was extracted from wild-type or p53-deficient neurons at the indicated times after treatment with 10 μM camptothecin and assayed for Apaf1 levels by Western blot analysis. Specificity of the Apaf1 antibody is demonstrated by lack of immunoreactivity in extracts derived from Apaf1 knockout brain and loading is standardized with actin.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2198828&req=5

fig2: p53-mediated induction of Apaf1 protein in neurons. (A) Protein was extracted from neurons 48 or 60 h after infection with Ad-p53 or Ad-p53-173L and assayed for Apaf1 levels by Western blot analysis. (B) Protein was extracted from wild-type or p53-deficient neurons at the indicated times after treatment with 10 μM camptothecin and assayed for Apaf1 levels by Western blot analysis. Specificity of the Apaf1 antibody is demonstrated by lack of immunoreactivity in extracts derived from Apaf1 knockout brain and loading is standardized with actin.
Mentions: To determine whether the increase in Apaf1 mRNA levels was accompanied by an increase in protein expression, we conducted Western analysis on neurons undergoing p53-induced apoptosis. Cell extracts were obtained from neurons infected at 20 MOI with Ad-p53 or the control vector Ad-LacZ and Apaf1 protein expression was examined. A significant increase in Apaf1 protein levels was evident at 48 and 60 h after infection with Ad-p53 relative to control (Fig. 2 A). We next examined Apaf1 protein levels in camptothecin-induced apoptosis that activates endogenous p53 using wild-type and p53-deficient cortical neurons. A time-dependent increase in Apaf1 protein levels was found in wild-type neurons treated with camptothecin, whereas Apaf1 protein levels did not increase in p53-deficient neurons treated under identical conditions (Fig. 2 B).

Bottom Line: Induction of neuronal cell death by camptothecin, a DNA-damaging agent that functions through a p53-dependent mechanism, resulted in increased Apaf1 mRNA in p53-positive, but not p53-deficient neurons.In transient transfections in a neuronal cell line with p53 and Apaf1 promoter-luciferase constructs, p53 directly activated the Apaf1 promoter via both p53 sites.Neurons treated with camptothecin were significantly protected in the absence of Apaf1 relative to those derived from wild-type littermates.

View Article: PubMed Central - PubMed

Affiliation: Ottawa Health Research Institute - Neuroscience, University of Ottawa, Ottawa, Ontario K1H-8M5, Canada.

ABSTRACT
p53 is a transcriptional activator which has been implicated as a key regulator of neuronal cell death after acute injury. We have shown previously that p53-mediated neuronal cell death involves a Bax-dependent activation of caspase 3; however, the transcriptional targets involved in the regulation of this process have not been identified. In the present study, we demonstrate that p53 directly upregulates Apaf1 transcription as a critical step in the induction of neuronal cell death. Using DNA microarray analysis of total RNA isolated from neurons undergoing p53-induced apoptosis a 5-6-fold upregulation of Apaf1 mRNA was detected. Induction of neuronal cell death by camptothecin, a DNA-damaging agent that functions through a p53-dependent mechanism, resulted in increased Apaf1 mRNA in p53-positive, but not p53-deficient neurons. In both in vitro and in vivo neuronal cell death processes of p53-induced cell death, Apaf1 protein levels were increased. We addressed whether p53 directly regulates Apaf1 transcription via the two p53 consensus binding sites in the Apaf1 promoter. Electrophoretic mobility shift assays demonstrated p53-DNA binding activity at both p53 consensus binding sequences in extracts obtained from neurons undergoing p53-induced cell death, but not in healthy control cultures or when p53 or the p53 binding sites were inactivated by mutation. In transient transfections in a neuronal cell line with p53 and Apaf1 promoter-luciferase constructs, p53 directly activated the Apaf1 promoter via both p53 sites. The importance of Apaf1 as a p53 target gene in neuronal cell death was evaluated by examining p53-induced apoptotic pathways in primary cultures of Apaf1-deficient neurons. Neurons treated with camptothecin were significantly protected in the absence of Apaf1 relative to those derived from wild-type littermates. Together, these results demonstrate that Apaf1 is a key transcriptional target for p53 that plays a pivotal role in the regulation of apoptosis after neuronal injury.

Show MeSH
Related in: MedlinePlus