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Programmed cell death 5 mediates HDAC3 decay to promote genotoxic stress response.

Choi HK, Choi Y, Park ES, Park SY, Lee SH, Seo J, Jeong MH, Jeong JW, Jeong JH, Lee PC, Choi KC, Yoon HG - Nat Commun (2015)

Bottom Line: Restoration of PDCD5(WT) in PDCD5(-/-) MEFs restores ET-induced HDAC3 cleavage.Reduction of both PDCD5 and p53, but not reduction of either protein alone, significantly enhances in vivo tumorigenicity of AGS gastric cancer cells and correlates with poor prognosis in gastric cancer patients.Our results define a mechanism for p53 activation via PDCD5-dependent HDAC3 decay under genotoxic stress conditions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Brain Korea 21 PLUS Project for Medical Sciences, Yonsei University College of Medicine, Seoul 120-752, Korea.

ABSTRACT
The inhibition of p53 activity by histone deacetylase 3 (HDAC3) has been reported, but the precise molecular mechanism is unknown. Here we show that programmed cell death 5 (PDCD5) selectively mediates HDAC3 dissociation from p53, which induces HDAC3 cleavage and ubiquitin-dependent proteasomal degradation. Casein kinase 2 alpha phosphorylates PDCD5 at Ser-119 to enhance its stability and importin 13-mediated nuclear translocation of PDCD5. Genetic deletion of PDCD5 abrogates etoposide (ET)-induced p53 stabilization and HDAC3 cleavage, indicating an essential role of PDCD5 in p53 activation. Restoration of PDCD5(WT) in PDCD5(-/-) MEFs restores ET-induced HDAC3 cleavage. Reduction of both PDCD5 and p53, but not reduction of either protein alone, significantly enhances in vivo tumorigenicity of AGS gastric cancer cells and correlates with poor prognosis in gastric cancer patients. Our results define a mechanism for p53 activation via PDCD5-dependent HDAC3 decay under genotoxic stress conditions.

No MeSH data available.


Related in: MedlinePlus

PDCD5 is required for genotoxic stress-induced HDAC3 cleavage and p53 activation.(a) Depletion of PDCD5 abrogated the ET-induced HDAC3 cleavage and p53 activation. MEFs were infected with Ad-Cre or Ad-GFP and then electroporated with Myc-p53 or treated with ET (50 μM, 8 h). Whole-cell lysates were immunoblotted with indicated antibodies. Arrow indicates cleaved HDAC3. (b) PDCD5 promotes ET-induced p53 activation and HDAC3 cleavage. PDCD5−/− MEFs were electroporated with indicated plasmids, treated with ET, lysed and then analysed by immunoblotting. (c) Restoration of PDCD5 into PDCD5−/− MEFs induces the recruitment of the p53–p300 complex to the promoter region of Bax. ChIP assays were performed with the indicated antibodies. Error bars, s.d. (n=3). *P<0.05, **P<0.01. (d) Both PDCD5 and p53 are mutually required for ET-induced activation of apoptosis. Stable shPDCD5-expressing p53−/− MEFs were electroporated with indicated plasmids and treated with ET. (e) Knockdown of HDAC3 rescues the suppression of p53 caused by depletion of PDCD5. PDCD5−/− MEFs were electroporated with indicated plasmids and/or shHDAC3, and cell lysates were analysed by immunoblotting. (f) Negative effect of cleavage at Asp-391 on the anti-apoptotic function of HDAC3. Cell lysates were analysed by immunoblotting. (g) Ablation of PDCD5 abolishes the genotoxic stress response in vivo. Mice were injected with ET (10 mg kg−1) for the indicated days. Adenovirus expressing GFP or Cre recombinase was injected in mice 6 days before ET injection, as indicated. Tissues from individual livers were harvested and processed for western blotting. Total RNA was isolated from individual livers, and qRT–PCR was performed for the indicated genes. Error bars, s.d. (n=8). *P<0.05 versus without ET; #P<0.05 versus ET (2 days).
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f6: PDCD5 is required for genotoxic stress-induced HDAC3 cleavage and p53 activation.(a) Depletion of PDCD5 abrogated the ET-induced HDAC3 cleavage and p53 activation. MEFs were infected with Ad-Cre or Ad-GFP and then electroporated with Myc-p53 or treated with ET (50 μM, 8 h). Whole-cell lysates were immunoblotted with indicated antibodies. Arrow indicates cleaved HDAC3. (b) PDCD5 promotes ET-induced p53 activation and HDAC3 cleavage. PDCD5−/− MEFs were electroporated with indicated plasmids, treated with ET, lysed and then analysed by immunoblotting. (c) Restoration of PDCD5 into PDCD5−/− MEFs induces the recruitment of the p53–p300 complex to the promoter region of Bax. ChIP assays were performed with the indicated antibodies. Error bars, s.d. (n=3). *P<0.05, **P<0.01. (d) Both PDCD5 and p53 are mutually required for ET-induced activation of apoptosis. Stable shPDCD5-expressing p53−/− MEFs were electroporated with indicated plasmids and treated with ET. (e) Knockdown of HDAC3 rescues the suppression of p53 caused by depletion of PDCD5. PDCD5−/− MEFs were electroporated with indicated plasmids and/or shHDAC3, and cell lysates were analysed by immunoblotting. (f) Negative effect of cleavage at Asp-391 on the anti-apoptotic function of HDAC3. Cell lysates were analysed by immunoblotting. (g) Ablation of PDCD5 abolishes the genotoxic stress response in vivo. Mice were injected with ET (10 mg kg−1) for the indicated days. Adenovirus expressing GFP or Cre recombinase was injected in mice 6 days before ET injection, as indicated. Tissues from individual livers were harvested and processed for western blotting. Total RNA was isolated from individual livers, and qRT–PCR was performed for the indicated genes. Error bars, s.d. (n=8). *P<0.05 versus without ET; #P<0.05 versus ET (2 days).

Mentions: Our results illustrating the p53-dependent action of PDCD5 in genotoxic stress responses led us to further investigate whether PDCD5 is critical for the stabilization and activation of p53. For this, we generated PDCD5flox/flox mice (Supplementary Fig. 14). MEFs generated from these mice were subjected to a Cre recombinase (Ad-Cre)-expressing adenovirus to delete PDCD5, generating a PDCD5−/− MEF line. Ad-Cre recombinase treatment depleted most PDCD5 protein in MEFs derived from PDCD5f/f mice (Fig. 6a). Depletion of PDCD5 strongly reduced HDAC3 cleavage and p53 activation in response to ET treatment and p53 overexpression (Fig. 6a; Supplementary Fig. 15a). Consistently, a Mdm2 inhibitor, nutlin3a treatments had negligible effects on HDAC3 cleavage and p53 activation in PDCD5−/− MEFs (Supplementary Fig. 15b). However, rescue of PDCD5−/− MEFs with haemagglutinin (HA)-tagged PDCD5WT, but not with PDCD5E16D, markedly increased p53 activity, HDAC3 cleavage and recruitment of the p53–p300 complex to the p53-RE of Bax and Puma, indicating that PDCD5 is required for p53 activation via HDAC3 cleavage (Fig. 6b,c; Supplementary Figs 13b and 15b,c). Importantly, rescue of PDCD5-knockdown p53−/− MEFs with p53 and wild-type PDCD5WT, but not mutant PDCD5E16D, resulted in regained p53 action on ET-induced HDAC3 cleavage, target gene expression and apoptosis (Fig. 6d; Supplementary Fig. 15d). These results again verify that PDCD5 and p53 are mutually required for genotoxic stress-induced apoptosis and HDAC3 cleavage. We observed that HDAC3 knockdown rescued p53 suppression caused by PDCD5 depletion (Fig. 6e; lane 3 versus 4). Restoration of PDCD5WT, but not PDCD5E16D, further enhanced the effect of HDAC3 knockdown on p53 acetylation and activation (lane 2 versus 5), again confirming the crucial role of PDCD5 in acetylation-induced p53 stabilization (Fig. 6e; Supplementary Fig. 15e). Notably, overexpression of uncleaved mutant HDAC3D391A markedly suppressed ET-induced p53 acetylation and activation, as well as apoptosis, when compared with wild-type HDAC3WT. As expected, the cleaved form of HDAC3 (HDAC31–391) had negligible effects on both p53 activation and apoptosis, supporting our notion that HDAC3 function is negatively regulated during genotoxic stress responses (Fig. 6f; Supplementary Figs 13c and 15f).


Programmed cell death 5 mediates HDAC3 decay to promote genotoxic stress response.

Choi HK, Choi Y, Park ES, Park SY, Lee SH, Seo J, Jeong MH, Jeong JW, Jeong JH, Lee PC, Choi KC, Yoon HG - Nat Commun (2015)

PDCD5 is required for genotoxic stress-induced HDAC3 cleavage and p53 activation.(a) Depletion of PDCD5 abrogated the ET-induced HDAC3 cleavage and p53 activation. MEFs were infected with Ad-Cre or Ad-GFP and then electroporated with Myc-p53 or treated with ET (50 μM, 8 h). Whole-cell lysates were immunoblotted with indicated antibodies. Arrow indicates cleaved HDAC3. (b) PDCD5 promotes ET-induced p53 activation and HDAC3 cleavage. PDCD5−/− MEFs were electroporated with indicated plasmids, treated with ET, lysed and then analysed by immunoblotting. (c) Restoration of PDCD5 into PDCD5−/− MEFs induces the recruitment of the p53–p300 complex to the promoter region of Bax. ChIP assays were performed with the indicated antibodies. Error bars, s.d. (n=3). *P<0.05, **P<0.01. (d) Both PDCD5 and p53 are mutually required for ET-induced activation of apoptosis. Stable shPDCD5-expressing p53−/− MEFs were electroporated with indicated plasmids and treated with ET. (e) Knockdown of HDAC3 rescues the suppression of p53 caused by depletion of PDCD5. PDCD5−/− MEFs were electroporated with indicated plasmids and/or shHDAC3, and cell lysates were analysed by immunoblotting. (f) Negative effect of cleavage at Asp-391 on the anti-apoptotic function of HDAC3. Cell lysates were analysed by immunoblotting. (g) Ablation of PDCD5 abolishes the genotoxic stress response in vivo. Mice were injected with ET (10 mg kg−1) for the indicated days. Adenovirus expressing GFP or Cre recombinase was injected in mice 6 days before ET injection, as indicated. Tissues from individual livers were harvested and processed for western blotting. Total RNA was isolated from individual livers, and qRT–PCR was performed for the indicated genes. Error bars, s.d. (n=8). *P<0.05 versus without ET; #P<0.05 versus ET (2 days).
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f6: PDCD5 is required for genotoxic stress-induced HDAC3 cleavage and p53 activation.(a) Depletion of PDCD5 abrogated the ET-induced HDAC3 cleavage and p53 activation. MEFs were infected with Ad-Cre or Ad-GFP and then electroporated with Myc-p53 or treated with ET (50 μM, 8 h). Whole-cell lysates were immunoblotted with indicated antibodies. Arrow indicates cleaved HDAC3. (b) PDCD5 promotes ET-induced p53 activation and HDAC3 cleavage. PDCD5−/− MEFs were electroporated with indicated plasmids, treated with ET, lysed and then analysed by immunoblotting. (c) Restoration of PDCD5 into PDCD5−/− MEFs induces the recruitment of the p53–p300 complex to the promoter region of Bax. ChIP assays were performed with the indicated antibodies. Error bars, s.d. (n=3). *P<0.05, **P<0.01. (d) Both PDCD5 and p53 are mutually required for ET-induced activation of apoptosis. Stable shPDCD5-expressing p53−/− MEFs were electroporated with indicated plasmids and treated with ET. (e) Knockdown of HDAC3 rescues the suppression of p53 caused by depletion of PDCD5. PDCD5−/− MEFs were electroporated with indicated plasmids and/or shHDAC3, and cell lysates were analysed by immunoblotting. (f) Negative effect of cleavage at Asp-391 on the anti-apoptotic function of HDAC3. Cell lysates were analysed by immunoblotting. (g) Ablation of PDCD5 abolishes the genotoxic stress response in vivo. Mice were injected with ET (10 mg kg−1) for the indicated days. Adenovirus expressing GFP or Cre recombinase was injected in mice 6 days before ET injection, as indicated. Tissues from individual livers were harvested and processed for western blotting. Total RNA was isolated from individual livers, and qRT–PCR was performed for the indicated genes. Error bars, s.d. (n=8). *P<0.05 versus without ET; #P<0.05 versus ET (2 days).
Mentions: Our results illustrating the p53-dependent action of PDCD5 in genotoxic stress responses led us to further investigate whether PDCD5 is critical for the stabilization and activation of p53. For this, we generated PDCD5flox/flox mice (Supplementary Fig. 14). MEFs generated from these mice were subjected to a Cre recombinase (Ad-Cre)-expressing adenovirus to delete PDCD5, generating a PDCD5−/− MEF line. Ad-Cre recombinase treatment depleted most PDCD5 protein in MEFs derived from PDCD5f/f mice (Fig. 6a). Depletion of PDCD5 strongly reduced HDAC3 cleavage and p53 activation in response to ET treatment and p53 overexpression (Fig. 6a; Supplementary Fig. 15a). Consistently, a Mdm2 inhibitor, nutlin3a treatments had negligible effects on HDAC3 cleavage and p53 activation in PDCD5−/− MEFs (Supplementary Fig. 15b). However, rescue of PDCD5−/− MEFs with haemagglutinin (HA)-tagged PDCD5WT, but not with PDCD5E16D, markedly increased p53 activity, HDAC3 cleavage and recruitment of the p53–p300 complex to the p53-RE of Bax and Puma, indicating that PDCD5 is required for p53 activation via HDAC3 cleavage (Fig. 6b,c; Supplementary Figs 13b and 15b,c). Importantly, rescue of PDCD5-knockdown p53−/− MEFs with p53 and wild-type PDCD5WT, but not mutant PDCD5E16D, resulted in regained p53 action on ET-induced HDAC3 cleavage, target gene expression and apoptosis (Fig. 6d; Supplementary Fig. 15d). These results again verify that PDCD5 and p53 are mutually required for genotoxic stress-induced apoptosis and HDAC3 cleavage. We observed that HDAC3 knockdown rescued p53 suppression caused by PDCD5 depletion (Fig. 6e; lane 3 versus 4). Restoration of PDCD5WT, but not PDCD5E16D, further enhanced the effect of HDAC3 knockdown on p53 acetylation and activation (lane 2 versus 5), again confirming the crucial role of PDCD5 in acetylation-induced p53 stabilization (Fig. 6e; Supplementary Fig. 15e). Notably, overexpression of uncleaved mutant HDAC3D391A markedly suppressed ET-induced p53 acetylation and activation, as well as apoptosis, when compared with wild-type HDAC3WT. As expected, the cleaved form of HDAC3 (HDAC31–391) had negligible effects on both p53 activation and apoptosis, supporting our notion that HDAC3 function is negatively regulated during genotoxic stress responses (Fig. 6f; Supplementary Figs 13c and 15f).

Bottom Line: Restoration of PDCD5(WT) in PDCD5(-/-) MEFs restores ET-induced HDAC3 cleavage.Reduction of both PDCD5 and p53, but not reduction of either protein alone, significantly enhances in vivo tumorigenicity of AGS gastric cancer cells and correlates with poor prognosis in gastric cancer patients.Our results define a mechanism for p53 activation via PDCD5-dependent HDAC3 decay under genotoxic stress conditions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Brain Korea 21 PLUS Project for Medical Sciences, Yonsei University College of Medicine, Seoul 120-752, Korea.

ABSTRACT
The inhibition of p53 activity by histone deacetylase 3 (HDAC3) has been reported, but the precise molecular mechanism is unknown. Here we show that programmed cell death 5 (PDCD5) selectively mediates HDAC3 dissociation from p53, which induces HDAC3 cleavage and ubiquitin-dependent proteasomal degradation. Casein kinase 2 alpha phosphorylates PDCD5 at Ser-119 to enhance its stability and importin 13-mediated nuclear translocation of PDCD5. Genetic deletion of PDCD5 abrogates etoposide (ET)-induced p53 stabilization and HDAC3 cleavage, indicating an essential role of PDCD5 in p53 activation. Restoration of PDCD5(WT) in PDCD5(-/-) MEFs restores ET-induced HDAC3 cleavage. Reduction of both PDCD5 and p53, but not reduction of either protein alone, significantly enhances in vivo tumorigenicity of AGS gastric cancer cells and correlates with poor prognosis in gastric cancer patients. Our results define a mechanism for p53 activation via PDCD5-dependent HDAC3 decay under genotoxic stress conditions.

No MeSH data available.


Related in: MedlinePlus