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PKCζ mediates disturbed flow-induced endothelial apoptosis via p53 SUMOylation.

Heo KS, Lee H, Nigro P, Thomas T, Le NT, Chang E, McClain C, Reinhart-King CA, King MR, Berk BC, Fujiwara K, Woo CH, Abe J - J. Cell Biol. (2011)

Bottom Line: Atherosclerosis is readily observed in regions of blood vessels where disturbed blood flow (d-flow) is known to occur.En face confocal microscopy revealed increases in nonnuclear p53 expression, nitrotyrosine staining, and apoptosis in aortic EC located in d-flow areas in wild-type mice, but these effects were significantly decreased in p53(-/-) mice.We propose a novel mechanism for p53 SUMOylation mediated by the PKCζ-PIASy interaction during d-flow-mediated EC apoptosis, which has potential relevance to early events of atherosclerosis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Aab Cardiovascular Research Institute, University of Rochester, Rochester, NY 14642, USA.

ABSTRACT
Atherosclerosis is readily observed in regions of blood vessels where disturbed blood flow (d-flow) is known to occur. A positive correlation between protein kinase C ζ (PKCζ) activation and d-flow has been reported, but the exact role of d-flow-mediated PKCζ activation in atherosclerosis remains unclear. We tested the hypothesis that PKCζ activation by d-flow induces endothelial cell (EC) apoptosis by regulating p53. We found that d-flow-mediated peroxynitrite (ONOO(-)) increased PKCζ activation, which subsequently induced p53 SUMOylation, p53-Bcl-2 binding, and EC apoptosis. Both d-flow and ONOO(-) increased the association of PKCζ with protein inhibitor of activated STATy (PIASy) via the Siz/PIAS-RING domain (amino acids 301-410) of PIASy, and overexpression of this domain of PIASy disrupted the PKCζ-PIASy interaction and PKCζ-mediated p53 SUMOylation. En face confocal microscopy revealed increases in nonnuclear p53 expression, nitrotyrosine staining, and apoptosis in aortic EC located in d-flow areas in wild-type mice, but these effects were significantly decreased in p53(-/-) mice. We propose a novel mechanism for p53 SUMOylation mediated by the PKCζ-PIASy interaction during d-flow-mediated EC apoptosis, which has potential relevance to early events of atherosclerosis.

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PKCζ mediates ONOO−-induced p53 nuclear export and p53–Bcl-2 binding instead of the regulation of p53 transcriptional activity. (A and B) HUVECs were transfected with the p53-Luc reporter and Renilla luciferase–encoding plasmid (pRL- thymidine kinase) used as an internal control reporter together with p53–wild type or vector alone (pcDNA3.1; A). Some cells were further transfected with or without pcDNA3.1-CATζ (B). Transcriptional activity was determined by a reporter plasmid encoding 13 copies of the p53-binding sequence (p53-Luc reporter; Kern et al., 1992). After 24 h of transfection, p53 transcriptional activity was assayed using the dual-luciferase kit (B), or the cells were further treated with 10 or 50 µM ONOO− for 8 h as indicated, and luciferase activity was assayed (A). Data are representative of triplicates using two or more different preparations of ECs. *, P < 0.05; **, P < 0.01. (C) HUVECs were transduced with Ad-DN-PKCζ or Ad-LacZ as a control for 24 h, treated with vehicle or 100 µM ONOO− for 4 h, and immunostained with anti-p53 followed by DAPI counter staining for nuclei. Bar, 5 µm. (D, top) HUVECs were transduced with Ad-DN-PKCζ or Ad-LacZ for 24 h and stimulated with 100 µM ONOO– for the indicated times. p53–Bcl-2 binding was determined by coimmunoprecipitation with anti-p53 followed by immunoblotting with anti–Bcl-2. p53, PKCζ, and Bcl-2 in total cell lysates were detected by Western blotting with each specific antibody. (bottom) Quantification of p53–Bcl-2 binding expressed as the relative band intensity ratio between coimmunoprecipitated versus total Bcl-2. Results were normalized as described in Fig. 1. n = 3. *, P < 0.05 and **, P < 0.01 compared with the vehicle control, and #, P < 0.05 and ##, P < 0.01 compared with the LacZ control at each time point. Molecular masses are given in kilodaltons. Error bars indicate means ± SD. IB, immunoblot. IP, immunoprecipitation.
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fig3: PKCζ mediates ONOO−-induced p53 nuclear export and p53–Bcl-2 binding instead of the regulation of p53 transcriptional activity. (A and B) HUVECs were transfected with the p53-Luc reporter and Renilla luciferase–encoding plasmid (pRL- thymidine kinase) used as an internal control reporter together with p53–wild type or vector alone (pcDNA3.1; A). Some cells were further transfected with or without pcDNA3.1-CATζ (B). Transcriptional activity was determined by a reporter plasmid encoding 13 copies of the p53-binding sequence (p53-Luc reporter; Kern et al., 1992). After 24 h of transfection, p53 transcriptional activity was assayed using the dual-luciferase kit (B), or the cells were further treated with 10 or 50 µM ONOO− for 8 h as indicated, and luciferase activity was assayed (A). Data are representative of triplicates using two or more different preparations of ECs. *, P < 0.05; **, P < 0.01. (C) HUVECs were transduced with Ad-DN-PKCζ or Ad-LacZ as a control for 24 h, treated with vehicle or 100 µM ONOO− for 4 h, and immunostained with anti-p53 followed by DAPI counter staining for nuclei. Bar, 5 µm. (D, top) HUVECs were transduced with Ad-DN-PKCζ or Ad-LacZ for 24 h and stimulated with 100 µM ONOO– for the indicated times. p53–Bcl-2 binding was determined by coimmunoprecipitation with anti-p53 followed by immunoblotting with anti–Bcl-2. p53, PKCζ, and Bcl-2 in total cell lysates were detected by Western blotting with each specific antibody. (bottom) Quantification of p53–Bcl-2 binding expressed as the relative band intensity ratio between coimmunoprecipitated versus total Bcl-2. Results were normalized as described in Fig. 1. n = 3. *, P < 0.05 and **, P < 0.01 compared with the vehicle control, and #, P < 0.05 and ##, P < 0.01 compared with the LacZ control at each time point. Molecular masses are given in kilodaltons. Error bars indicate means ± SD. IB, immunoblot. IP, immunoprecipitation.

Mentions: p53 promotes transcription of several proapoptotic genes (Mihara et al., 2003; Mercer et al., 2005; Bischof et al., 2006; Garner et al., 2007). To test whether it plays a role in the ONOO−-induced EC apoptosis, we first examined the effect of ONOO− on p53 transcriptional activity in HUVECs that were overexpressing p53 with or without coexpression of a constitutively active PKCζ (catalytic domain of PKCζ [CATζ]; Garin et al., 2007). To our surprise, ONOO− inhibited p53 transcriptional activity in control cells and also in those overexpressing p53 (Fig. 3 A). Consistent with these results, cells transfected with CATζ also showed a decreased p53 transcriptional activity (Fig. 3 B). These results strongly suggest that ONOO−-mediated EC apoptosis is not caused by increased p53 transcriptional activation of proapoptotic genes.


PKCζ mediates disturbed flow-induced endothelial apoptosis via p53 SUMOylation.

Heo KS, Lee H, Nigro P, Thomas T, Le NT, Chang E, McClain C, Reinhart-King CA, King MR, Berk BC, Fujiwara K, Woo CH, Abe J - J. Cell Biol. (2011)

PKCζ mediates ONOO−-induced p53 nuclear export and p53–Bcl-2 binding instead of the regulation of p53 transcriptional activity. (A and B) HUVECs were transfected with the p53-Luc reporter and Renilla luciferase–encoding plasmid (pRL- thymidine kinase) used as an internal control reporter together with p53–wild type or vector alone (pcDNA3.1; A). Some cells were further transfected with or without pcDNA3.1-CATζ (B). Transcriptional activity was determined by a reporter plasmid encoding 13 copies of the p53-binding sequence (p53-Luc reporter; Kern et al., 1992). After 24 h of transfection, p53 transcriptional activity was assayed using the dual-luciferase kit (B), or the cells were further treated with 10 or 50 µM ONOO− for 8 h as indicated, and luciferase activity was assayed (A). Data are representative of triplicates using two or more different preparations of ECs. *, P < 0.05; **, P < 0.01. (C) HUVECs were transduced with Ad-DN-PKCζ or Ad-LacZ as a control for 24 h, treated with vehicle or 100 µM ONOO− for 4 h, and immunostained with anti-p53 followed by DAPI counter staining for nuclei. Bar, 5 µm. (D, top) HUVECs were transduced with Ad-DN-PKCζ or Ad-LacZ for 24 h and stimulated with 100 µM ONOO– for the indicated times. p53–Bcl-2 binding was determined by coimmunoprecipitation with anti-p53 followed by immunoblotting with anti–Bcl-2. p53, PKCζ, and Bcl-2 in total cell lysates were detected by Western blotting with each specific antibody. (bottom) Quantification of p53–Bcl-2 binding expressed as the relative band intensity ratio between coimmunoprecipitated versus total Bcl-2. Results were normalized as described in Fig. 1. n = 3. *, P < 0.05 and **, P < 0.01 compared with the vehicle control, and #, P < 0.05 and ##, P < 0.01 compared with the LacZ control at each time point. Molecular masses are given in kilodaltons. Error bars indicate means ± SD. IB, immunoblot. IP, immunoprecipitation.
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Related In: Results  -  Collection

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fig3: PKCζ mediates ONOO−-induced p53 nuclear export and p53–Bcl-2 binding instead of the regulation of p53 transcriptional activity. (A and B) HUVECs were transfected with the p53-Luc reporter and Renilla luciferase–encoding plasmid (pRL- thymidine kinase) used as an internal control reporter together with p53–wild type or vector alone (pcDNA3.1; A). Some cells were further transfected with or without pcDNA3.1-CATζ (B). Transcriptional activity was determined by a reporter plasmid encoding 13 copies of the p53-binding sequence (p53-Luc reporter; Kern et al., 1992). After 24 h of transfection, p53 transcriptional activity was assayed using the dual-luciferase kit (B), or the cells were further treated with 10 or 50 µM ONOO− for 8 h as indicated, and luciferase activity was assayed (A). Data are representative of triplicates using two or more different preparations of ECs. *, P < 0.05; **, P < 0.01. (C) HUVECs were transduced with Ad-DN-PKCζ or Ad-LacZ as a control for 24 h, treated with vehicle or 100 µM ONOO− for 4 h, and immunostained with anti-p53 followed by DAPI counter staining for nuclei. Bar, 5 µm. (D, top) HUVECs were transduced with Ad-DN-PKCζ or Ad-LacZ for 24 h and stimulated with 100 µM ONOO– for the indicated times. p53–Bcl-2 binding was determined by coimmunoprecipitation with anti-p53 followed by immunoblotting with anti–Bcl-2. p53, PKCζ, and Bcl-2 in total cell lysates were detected by Western blotting with each specific antibody. (bottom) Quantification of p53–Bcl-2 binding expressed as the relative band intensity ratio between coimmunoprecipitated versus total Bcl-2. Results were normalized as described in Fig. 1. n = 3. *, P < 0.05 and **, P < 0.01 compared with the vehicle control, and #, P < 0.05 and ##, P < 0.01 compared with the LacZ control at each time point. Molecular masses are given in kilodaltons. Error bars indicate means ± SD. IB, immunoblot. IP, immunoprecipitation.
Mentions: p53 promotes transcription of several proapoptotic genes (Mihara et al., 2003; Mercer et al., 2005; Bischof et al., 2006; Garner et al., 2007). To test whether it plays a role in the ONOO−-induced EC apoptosis, we first examined the effect of ONOO− on p53 transcriptional activity in HUVECs that were overexpressing p53 with or without coexpression of a constitutively active PKCζ (catalytic domain of PKCζ [CATζ]; Garin et al., 2007). To our surprise, ONOO− inhibited p53 transcriptional activity in control cells and also in those overexpressing p53 (Fig. 3 A). Consistent with these results, cells transfected with CATζ also showed a decreased p53 transcriptional activity (Fig. 3 B). These results strongly suggest that ONOO−-mediated EC apoptosis is not caused by increased p53 transcriptional activation of proapoptotic genes.

Bottom Line: Atherosclerosis is readily observed in regions of blood vessels where disturbed blood flow (d-flow) is known to occur.En face confocal microscopy revealed increases in nonnuclear p53 expression, nitrotyrosine staining, and apoptosis in aortic EC located in d-flow areas in wild-type mice, but these effects were significantly decreased in p53(-/-) mice.We propose a novel mechanism for p53 SUMOylation mediated by the PKCζ-PIASy interaction during d-flow-mediated EC apoptosis, which has potential relevance to early events of atherosclerosis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Aab Cardiovascular Research Institute, University of Rochester, Rochester, NY 14642, USA.

ABSTRACT
Atherosclerosis is readily observed in regions of blood vessels where disturbed blood flow (d-flow) is known to occur. A positive correlation between protein kinase C ζ (PKCζ) activation and d-flow has been reported, but the exact role of d-flow-mediated PKCζ activation in atherosclerosis remains unclear. We tested the hypothesis that PKCζ activation by d-flow induces endothelial cell (EC) apoptosis by regulating p53. We found that d-flow-mediated peroxynitrite (ONOO(-)) increased PKCζ activation, which subsequently induced p53 SUMOylation, p53-Bcl-2 binding, and EC apoptosis. Both d-flow and ONOO(-) increased the association of PKCζ with protein inhibitor of activated STATy (PIASy) via the Siz/PIAS-RING domain (amino acids 301-410) of PIASy, and overexpression of this domain of PIASy disrupted the PKCζ-PIASy interaction and PKCζ-mediated p53 SUMOylation. En face confocal microscopy revealed increases in nonnuclear p53 expression, nitrotyrosine staining, and apoptosis in aortic EC located in d-flow areas in wild-type mice, but these effects were significantly decreased in p53(-/-) mice. We propose a novel mechanism for p53 SUMOylation mediated by the PKCζ-PIASy interaction during d-flow-mediated EC apoptosis, which has potential relevance to early events of atherosclerosis.

Show MeSH
Related in: MedlinePlus