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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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ONOO− mediates d-flow–induced PKCζ activation, p53 SUMOylation, and EC apoptosis. (A) ONOO− mediates d-flow–induced PKCζ activation and p53 SUMOylation. HUVECs were pretreated by 5 µM ebselen, 20 µM L-NAME, and 10 µM Mn-TBAP for 30 min and exposed to d-flow for 3 h. PKCζ phosphorylation at Thr560 and p53 SUMOylation were determined as described in Materials and methods. (B and C) Densitometry analyses of p53 SUMOylation (B) and PKCζ phosphorylation (C) were performed as described in Fig. 1. **, P < 0.01 compared with the vehicle control in static condition, and #, P < 0.01 compared with the vehicle control in d-flow stimulation for 3 h. (D and E) HUVECs were pretreated by each inhibitor for 30 min and exposed to d-flow for 36 h followed by TUNEL staining as described in Materials and methods (D), and quantification of apoptosis is shown as the percentage of TUNEL-positive cells (E). Bars, 30 µm. Data are from three separate experiments using two or more different EC preparations (**, P < 0.01 compared with the vehicle control in static condition, and #, P<0.05 and ##, P<0.01 compared with the vehicle control in d-flow stimulation for 36 h). Error bars show means ± SD. Molecular masses are given in kilodaltons. IB, immunoblot. IP, immunoprecipitation.
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fig5: ONOO− mediates d-flow–induced PKCζ activation, p53 SUMOylation, and EC apoptosis. (A) ONOO− mediates d-flow–induced PKCζ activation and p53 SUMOylation. HUVECs were pretreated by 5 µM ebselen, 20 µM L-NAME, and 10 µM Mn-TBAP for 30 min and exposed to d-flow for 3 h. PKCζ phosphorylation at Thr560 and p53 SUMOylation were determined as described in Materials and methods. (B and C) Densitometry analyses of p53 SUMOylation (B) and PKCζ phosphorylation (C) were performed as described in Fig. 1. **, P < 0.01 compared with the vehicle control in static condition, and #, P < 0.01 compared with the vehicle control in d-flow stimulation for 3 h. (D and E) HUVECs were pretreated by each inhibitor for 30 min and exposed to d-flow for 36 h followed by TUNEL staining as described in Materials and methods (D), and quantification of apoptosis is shown as the percentage of TUNEL-positive cells (E). Bars, 30 µm. Data are from three separate experiments using two or more different EC preparations (**, P < 0.01 compared with the vehicle control in static condition, and #, P<0.05 and ##, P<0.01 compared with the vehicle control in d-flow stimulation for 36 h). Error bars show means ± SD. Molecular masses are given in kilodaltons. IB, immunoblot. IP, immunoprecipitation.

Mentions: We investigated the contribution of ONOO− production on the d-flow–mediated PKCζ activation and p53 SUMOylation and found that an ONOO− scavenger, ebselen, a nonselective inhibitor of NO synthesis, N-nitro-l-arginine methyl ester (L-NAME), superoxide dismutase mimetic, and an ONOO− scavenger, Mn (III)tetrakis(4-benzoic acid)porphyrin chloride (Mn-TBAP), significantly inhibited d-flow–mediated PKCζ activation as well as p53 SUMOylation (Fig. 5, A–C). Moreover, these compounds strongly inhibited d-flow–induced EC apoptosis (Fig. 5, D and E), strongly suggesting a critical role of ONOO− production in the d-flow–mediated signaling and subsequent apoptosis.


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)

ONOO− mediates d-flow–induced PKCζ activation, p53 SUMOylation, and EC apoptosis. (A) ONOO− mediates d-flow–induced PKCζ activation and p53 SUMOylation. HUVECs were pretreated by 5 µM ebselen, 20 µM L-NAME, and 10 µM Mn-TBAP for 30 min and exposed to d-flow for 3 h. PKCζ phosphorylation at Thr560 and p53 SUMOylation were determined as described in Materials and methods. (B and C) Densitometry analyses of p53 SUMOylation (B) and PKCζ phosphorylation (C) were performed as described in Fig. 1. **, P < 0.01 compared with the vehicle control in static condition, and #, P < 0.01 compared with the vehicle control in d-flow stimulation for 3 h. (D and E) HUVECs were pretreated by each inhibitor for 30 min and exposed to d-flow for 36 h followed by TUNEL staining as described in Materials and methods (D), and quantification of apoptosis is shown as the percentage of TUNEL-positive cells (E). Bars, 30 µm. Data are from three separate experiments using two or more different EC preparations (**, P < 0.01 compared with the vehicle control in static condition, and #, P<0.05 and ##, P<0.01 compared with the vehicle control in d-flow stimulation for 36 h). Error bars show means ± SD. Molecular masses are given in kilodaltons. IB, immunoblot. IP, immunoprecipitation.
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Related In: Results  -  Collection

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fig5: ONOO− mediates d-flow–induced PKCζ activation, p53 SUMOylation, and EC apoptosis. (A) ONOO− mediates d-flow–induced PKCζ activation and p53 SUMOylation. HUVECs were pretreated by 5 µM ebselen, 20 µM L-NAME, and 10 µM Mn-TBAP for 30 min and exposed to d-flow for 3 h. PKCζ phosphorylation at Thr560 and p53 SUMOylation were determined as described in Materials and methods. (B and C) Densitometry analyses of p53 SUMOylation (B) and PKCζ phosphorylation (C) were performed as described in Fig. 1. **, P < 0.01 compared with the vehicle control in static condition, and #, P < 0.01 compared with the vehicle control in d-flow stimulation for 3 h. (D and E) HUVECs were pretreated by each inhibitor for 30 min and exposed to d-flow for 36 h followed by TUNEL staining as described in Materials and methods (D), and quantification of apoptosis is shown as the percentage of TUNEL-positive cells (E). Bars, 30 µm. Data are from three separate experiments using two or more different EC preparations (**, P < 0.01 compared with the vehicle control in static condition, and #, P<0.05 and ##, P<0.01 compared with the vehicle control in d-flow stimulation for 36 h). Error bars show means ± SD. Molecular masses are given in kilodaltons. IB, immunoblot. IP, immunoprecipitation.
Mentions: We investigated the contribution of ONOO− production on the d-flow–mediated PKCζ activation and p53 SUMOylation and found that an ONOO− scavenger, ebselen, a nonselective inhibitor of NO synthesis, N-nitro-l-arginine methyl ester (L-NAME), superoxide dismutase mimetic, and an ONOO− scavenger, Mn (III)tetrakis(4-benzoic acid)porphyrin chloride (Mn-TBAP), significantly inhibited d-flow–mediated PKCζ activation as well as p53 SUMOylation (Fig. 5, A–C). Moreover, these compounds strongly inhibited d-flow–induced EC apoptosis (Fig. 5, D and E), strongly suggesting a critical role of ONOO− production in the d-flow–mediated signaling and subsequent apoptosis.

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