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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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Specific binding of p53 to Apaf1 promoter elements in neuronal extracts. (A) Comparison of p53 consensus binding sequence (p53 CBS; el-Deiry et al., 1992), with two putative p53 recognition sequences located in the Apaf1 promoter (Apaf1 BS1 and BS2). The sequence of the corresponding mutated versions of these oligonucleotides (Apaf1 BS1-mut and BS2-mut) used in the electrophoretic mobility shift assays are also indicated. (B) Protein was extracted from neurons 48 h after infection with Ad-p53 or Ad-p53-173L and p53 binding activity to the Apaf1 promoter elements was assayed by electrophoretic mobility shift assay. Binding reactions were carried out with neuronal extracts (10 μg protein) and the indicated oligonucleotides in the presence of p53 antibody (pAb1). (C) Cell extracts (20 μg protein) obtained from untreated neurons or neurons exposed to camptothecin (10 μM) for 12 h were tested for p53 binding activity to the Apaf1 promoter elements as described above. (D) Specificity of p53 binding activity to the Apaf1 promoter was examined in p53+/+ and p53−/− neurons treated with camptothecin. Supershifts with two antibodies directed against p53 were carried out on p53+/+ neurons to further confirm the presence of p53 binding to the Apaf1 promoter.
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fig4: Specific binding of p53 to Apaf1 promoter elements in neuronal extracts. (A) Comparison of p53 consensus binding sequence (p53 CBS; el-Deiry et al., 1992), with two putative p53 recognition sequences located in the Apaf1 promoter (Apaf1 BS1 and BS2). The sequence of the corresponding mutated versions of these oligonucleotides (Apaf1 BS1-mut and BS2-mut) used in the electrophoretic mobility shift assays are also indicated. (B) Protein was extracted from neurons 48 h after infection with Ad-p53 or Ad-p53-173L and p53 binding activity to the Apaf1 promoter elements was assayed by electrophoretic mobility shift assay. Binding reactions were carried out with neuronal extracts (10 μg protein) and the indicated oligonucleotides in the presence of p53 antibody (pAb1). (C) Cell extracts (20 μg protein) obtained from untreated neurons or neurons exposed to camptothecin (10 μM) for 12 h were tested for p53 binding activity to the Apaf1 promoter elements as described above. (D) Specificity of p53 binding activity to the Apaf1 promoter was examined in p53+/+ and p53−/− neurons treated with camptothecin. Supershifts with two antibodies directed against p53 were carried out on p53+/+ neurons to further confirm the presence of p53 binding to the Apaf1 promoter.

Mentions: To determine whether Apaf1 is a direct transcriptional target for p53 in neuronal apoptosis, we examined the Apaf1 promoter recently characterized in the Helin laboratory (Moroni et al., 2001). Analysis of the Apaf1 promoter sequence revealed the existence of two putative p53 consensus binding sites located at −572 to −604 (p53 BS1) and −739 to −765 (p53 BS2) relative to the transcription initiation site (Fig. 4 A). To determine whether p53 can interact with these consensus elements, oligonucleotides derived from these proposed binding sites were synthesized and used in electrophoretic mobility shift assays (EMSAs). Protein extracts were examined from neurons undergoing p53-induced apoptosis, including: (a) treatment with camptothecin and (b) direct adenovirus-mediated p53 gene delivery. 48 h after infection with an adenovirus vector carrying wild-type p53 or the DNA-binding mutant p53-173L, protein was extracted and examined by EMSA. EMSA demonstrates that neuronal extracts exhibit p53–DNA binding activity at both putative binding sites, although greater activity was found on BS1 relative to the BS2 (Fig. 4 B). Furthermore, specific DNA binding activity to BS1 was observed with cell extracts prepared from camptothecin-treated neurons, suggesting that endogenous p53 can also interact with these binding sites (Fig. 4 C). The specificity of p53 binding from neuronal extracts is supported by several control experiments: (a) the p53 DNA binding mutant (p53-173L) did not exhibit binding to either recognition site; (b) mutation of four key residues within each proposed p53 binding sites on the Apaf1 promoter (Fig. 4 A) abolished p53 binding activity; (c) DNA binding activity could be competed out by incubation with excess unlabeled probes; (d) the bands were supershifted by the addition of two different p53-specific antibodies (Fig. 4 D); and (e) no DNA binding activity was seen in extracts obtained from p53-deficient neurons treated under identical conditions (Fig. 4 D).


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)

Specific binding of p53 to Apaf1 promoter elements in neuronal extracts. (A) Comparison of p53 consensus binding sequence (p53 CBS; el-Deiry et al., 1992), with two putative p53 recognition sequences located in the Apaf1 promoter (Apaf1 BS1 and BS2). The sequence of the corresponding mutated versions of these oligonucleotides (Apaf1 BS1-mut and BS2-mut) used in the electrophoretic mobility shift assays are also indicated. (B) Protein was extracted from neurons 48 h after infection with Ad-p53 or Ad-p53-173L and p53 binding activity to the Apaf1 promoter elements was assayed by electrophoretic mobility shift assay. Binding reactions were carried out with neuronal extracts (10 μg protein) and the indicated oligonucleotides in the presence of p53 antibody (pAb1). (C) Cell extracts (20 μg protein) obtained from untreated neurons or neurons exposed to camptothecin (10 μM) for 12 h were tested for p53 binding activity to the Apaf1 promoter elements as described above. (D) Specificity of p53 binding activity to the Apaf1 promoter was examined in p53+/+ and p53−/− neurons treated with camptothecin. Supershifts with two antibodies directed against p53 were carried out on p53+/+ neurons to further confirm the presence of p53 binding to the Apaf1 promoter.
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Related In: Results  -  Collection

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fig4: Specific binding of p53 to Apaf1 promoter elements in neuronal extracts. (A) Comparison of p53 consensus binding sequence (p53 CBS; el-Deiry et al., 1992), with two putative p53 recognition sequences located in the Apaf1 promoter (Apaf1 BS1 and BS2). The sequence of the corresponding mutated versions of these oligonucleotides (Apaf1 BS1-mut and BS2-mut) used in the electrophoretic mobility shift assays are also indicated. (B) Protein was extracted from neurons 48 h after infection with Ad-p53 or Ad-p53-173L and p53 binding activity to the Apaf1 promoter elements was assayed by electrophoretic mobility shift assay. Binding reactions were carried out with neuronal extracts (10 μg protein) and the indicated oligonucleotides in the presence of p53 antibody (pAb1). (C) Cell extracts (20 μg protein) obtained from untreated neurons or neurons exposed to camptothecin (10 μM) for 12 h were tested for p53 binding activity to the Apaf1 promoter elements as described above. (D) Specificity of p53 binding activity to the Apaf1 promoter was examined in p53+/+ and p53−/− neurons treated with camptothecin. Supershifts with two antibodies directed against p53 were carried out on p53+/+ neurons to further confirm the presence of p53 binding to the Apaf1 promoter.
Mentions: To determine whether Apaf1 is a direct transcriptional target for p53 in neuronal apoptosis, we examined the Apaf1 promoter recently characterized in the Helin laboratory (Moroni et al., 2001). Analysis of the Apaf1 promoter sequence revealed the existence of two putative p53 consensus binding sites located at −572 to −604 (p53 BS1) and −739 to −765 (p53 BS2) relative to the transcription initiation site (Fig. 4 A). To determine whether p53 can interact with these consensus elements, oligonucleotides derived from these proposed binding sites were synthesized and used in electrophoretic mobility shift assays (EMSAs). Protein extracts were examined from neurons undergoing p53-induced apoptosis, including: (a) treatment with camptothecin and (b) direct adenovirus-mediated p53 gene delivery. 48 h after infection with an adenovirus vector carrying wild-type p53 or the DNA-binding mutant p53-173L, protein was extracted and examined by EMSA. EMSA demonstrates that neuronal extracts exhibit p53–DNA binding activity at both putative binding sites, although greater activity was found on BS1 relative to the BS2 (Fig. 4 B). Furthermore, specific DNA binding activity to BS1 was observed with cell extracts prepared from camptothecin-treated neurons, suggesting that endogenous p53 can also interact with these binding sites (Fig. 4 C). The specificity of p53 binding from neuronal extracts is supported by several control experiments: (a) the p53 DNA binding mutant (p53-173L) did not exhibit binding to either recognition site; (b) mutation of four key residues within each proposed p53 binding sites on the Apaf1 promoter (Fig. 4 A) abolished p53 binding activity; (c) DNA binding activity could be competed out by incubation with excess unlabeled probes; (d) the bands were supershifted by the addition of two different p53-specific antibodies (Fig. 4 D); and (e) no DNA binding activity was seen in extracts obtained from p53-deficient neurons treated under identical conditions (Fig. 4 D).

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