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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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Activation of the Apaf1 promoter by p53 in a neuronal cell line. (A) p53 responsiveness of the Apaf1 promoter was tested using luciferase reporter constructs (pGL3b; Promega) consisting of the luciferase gene fused to an Apaf1 promoter fragment containing both p53 binding sites (BS1 and BS2), or truncated promoter fragments deleted for one or both p53 recognition sequences. (B) SN48 cells were cotransfected with the indicated luciferase reporter construct, a CMV–β-gal reporter construct, and an expression plasmid for either wild-type p53, DNA binding–defective p53-173L, or empty vector as control. Luciferase activity was measured in cell lysates obtained 48 h after transfection and normalized to β-galactosidase activity. Fold increase indicates the ratio of normalized luciferase activity of each Apaf1 promoter construct in the presence of p53 expression vector versus empty vector control. Data represent the mean and standard error of triplicate samples from three independent experiments.
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fig5: Activation of the Apaf1 promoter by p53 in a neuronal cell line. (A) p53 responsiveness of the Apaf1 promoter was tested using luciferase reporter constructs (pGL3b; Promega) consisting of the luciferase gene fused to an Apaf1 promoter fragment containing both p53 binding sites (BS1 and BS2), or truncated promoter fragments deleted for one or both p53 recognition sequences. (B) SN48 cells were cotransfected with the indicated luciferase reporter construct, a CMV–β-gal reporter construct, and an expression plasmid for either wild-type p53, DNA binding–defective p53-173L, or empty vector as control. Luciferase activity was measured in cell lysates obtained 48 h after transfection and normalized to β-galactosidase activity. Fold increase indicates the ratio of normalized luciferase activity of each Apaf1 promoter construct in the presence of p53 expression vector versus empty vector control. Data represent the mean and standard error of triplicate samples from three independent experiments.

Mentions: The ability of p53 to activate the Apaf1 promoter in neurons was further examined by using a luciferase reporter assay regulated by the Apaf1 promoter. Three different Apaf1-luc reporter constructs were tested, including the full length Apaf1 promoter (−871 to +208), a truncated promoter missing one p53 recognition sequence (−715 to +208), and a truncated Apaf1 promoter deleted for both proposed p53 recognition sequences (−396 to +208; Fig. 5 A). Cultured neuronal cell lines cotransfected with the intact Apaf1 promoter construct and wild-type p53 exhibited a fourfold increase in luciferase activity (Fig. 5 B). In contrast, no induction of luciferase activity occurred upon cotransfection with the DNA binding mutant p53-173L. Deletion of BS2 from the Apaf1 promoter resulted in an ∼25% decrease in p53-induced luciferase activity, and deletion of both p53 recognition sites essentially abolished all p53-induced promoter activity. Together, our EMSA results demonstrating DNA binding activity at p53 consensus sites in extracts derived from neurons undergoing p53-induced apoptosis, as well as the direct activation of the Apaf1 promoter by p53 show that Apaf1 is a direct target for p53 in the regulation of neuronal cell death.


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)

Activation of the Apaf1 promoter by p53 in a neuronal cell line. (A) p53 responsiveness of the Apaf1 promoter was tested using luciferase reporter constructs (pGL3b; Promega) consisting of the luciferase gene fused to an Apaf1 promoter fragment containing both p53 binding sites (BS1 and BS2), or truncated promoter fragments deleted for one or both p53 recognition sequences. (B) SN48 cells were cotransfected with the indicated luciferase reporter construct, a CMV–β-gal reporter construct, and an expression plasmid for either wild-type p53, DNA binding–defective p53-173L, or empty vector as control. Luciferase activity was measured in cell lysates obtained 48 h after transfection and normalized to β-galactosidase activity. Fold increase indicates the ratio of normalized luciferase activity of each Apaf1 promoter construct in the presence of p53 expression vector versus empty vector control. Data represent the mean and standard error of triplicate samples from three independent experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2198828&req=5

fig5: Activation of the Apaf1 promoter by p53 in a neuronal cell line. (A) p53 responsiveness of the Apaf1 promoter was tested using luciferase reporter constructs (pGL3b; Promega) consisting of the luciferase gene fused to an Apaf1 promoter fragment containing both p53 binding sites (BS1 and BS2), or truncated promoter fragments deleted for one or both p53 recognition sequences. (B) SN48 cells were cotransfected with the indicated luciferase reporter construct, a CMV–β-gal reporter construct, and an expression plasmid for either wild-type p53, DNA binding–defective p53-173L, or empty vector as control. Luciferase activity was measured in cell lysates obtained 48 h after transfection and normalized to β-galactosidase activity. Fold increase indicates the ratio of normalized luciferase activity of each Apaf1 promoter construct in the presence of p53 expression vector versus empty vector control. Data represent the mean and standard error of triplicate samples from three independent experiments.
Mentions: The ability of p53 to activate the Apaf1 promoter in neurons was further examined by using a luciferase reporter assay regulated by the Apaf1 promoter. Three different Apaf1-luc reporter constructs were tested, including the full length Apaf1 promoter (−871 to +208), a truncated promoter missing one p53 recognition sequence (−715 to +208), and a truncated Apaf1 promoter deleted for both proposed p53 recognition sequences (−396 to +208; Fig. 5 A). Cultured neuronal cell lines cotransfected with the intact Apaf1 promoter construct and wild-type p53 exhibited a fourfold increase in luciferase activity (Fig. 5 B). In contrast, no induction of luciferase activity occurred upon cotransfection with the DNA binding mutant p53-173L. Deletion of BS2 from the Apaf1 promoter resulted in an ∼25% decrease in p53-induced luciferase activity, and deletion of both p53 recognition sites essentially abolished all p53-induced promoter activity. Together, our EMSA results demonstrating DNA binding activity at p53 consensus sites in extracts derived from neurons undergoing p53-induced apoptosis, as well as the direct activation of the Apaf1 promoter by p53 show that Apaf1 is a direct target for p53 in the regulation of neuronal cell death.

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