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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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D-flow–induced PKCζ–PIASy association in nuclei and p53–Bcl-2 binding in the cytosol. (A and B) HUVECs were stimulated with either static or d-flow for 3 h and immunoassayed with antibodies of mouse anti-PKCζ and rabbit anti-PIASy (A) or mouse anti-p53 and rabbit anti–Bcl-2 (B). After d-flow stimulation, yellow in the merged images represent colocalization between PKCζ and PIASy in nuclei or p53 and Bcl-2 in cytosol. Images were recorded using a confocal microscope equipped with a Plapon 60× 1.42 NA oil lens objective. Shown are representative images from cells analyzed from three independent experiments in which ≥30 cells were analyzed per experiment. Bars, 10 µm.
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fig9: D-flow–induced PKCζ–PIASy association in nuclei and p53–Bcl-2 binding in the cytosol. (A and B) HUVECs were stimulated with either static or d-flow for 3 h and immunoassayed with antibodies of mouse anti-PKCζ and rabbit anti-PIASy (A) or mouse anti-p53 and rabbit anti–Bcl-2 (B). After d-flow stimulation, yellow in the merged images represent colocalization between PKCζ and PIASy in nuclei or p53 and Bcl-2 in cytosol. Images were recorded using a confocal microscope equipped with a Plapon 60× 1.42 NA oil lens objective. Shown are representative images from cells analyzed from three independent experiments in which ≥30 cells were analyzed per experiment. Bars, 10 µm.

Mentions: Morphological evidence for the PKCζ–PIASy and p53–Bcl-2 association was provided by confocal microscopy of coimmunostained ECs with or without flow stimulation (Fig. 9 A). Cells cultured without flow expressed PKCζ mainly in the cytosol and PIASy in the nucleus as reported previously (Sachdev et al., 2001; Li et al., 2004). However, after d-flow stimulation, PKCζ and PIASy were colocalized in the nucleus. As for the p53 and Bcl-2 colocalization, p53 was localized in the nucleus of unstimulated cells, whereas Bcl-2 was mainly outside the nucleus as previously reported (Zhong et al., 1993; Ghosh et al., 2004). D-flow stimulation caused significant nuclear export of p53 and colocalization with Bcl-2. These data support d-flow–induced association of PKCζ–PIASy and p53–Bcl-2 in ECs.


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)

D-flow–induced PKCζ–PIASy association in nuclei and p53–Bcl-2 binding in the cytosol. (A and B) HUVECs were stimulated with either static or d-flow for 3 h and immunoassayed with antibodies of mouse anti-PKCζ and rabbit anti-PIASy (A) or mouse anti-p53 and rabbit anti–Bcl-2 (B). After d-flow stimulation, yellow in the merged images represent colocalization between PKCζ and PIASy in nuclei or p53 and Bcl-2 in cytosol. Images were recorded using a confocal microscope equipped with a Plapon 60× 1.42 NA oil lens objective. Shown are representative images from cells analyzed from three independent experiments in which ≥30 cells were analyzed per experiment. Bars, 10 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3105539&req=5

fig9: D-flow–induced PKCζ–PIASy association in nuclei and p53–Bcl-2 binding in the cytosol. (A and B) HUVECs were stimulated with either static or d-flow for 3 h and immunoassayed with antibodies of mouse anti-PKCζ and rabbit anti-PIASy (A) or mouse anti-p53 and rabbit anti–Bcl-2 (B). After d-flow stimulation, yellow in the merged images represent colocalization between PKCζ and PIASy in nuclei or p53 and Bcl-2 in cytosol. Images were recorded using a confocal microscope equipped with a Plapon 60× 1.42 NA oil lens objective. Shown are representative images from cells analyzed from three independent experiments in which ≥30 cells were analyzed per experiment. Bars, 10 µm.
Mentions: Morphological evidence for the PKCζ–PIASy and p53–Bcl-2 association was provided by confocal microscopy of coimmunostained ECs with or without flow stimulation (Fig. 9 A). Cells cultured without flow expressed PKCζ mainly in the cytosol and PIASy in the nucleus as reported previously (Sachdev et al., 2001; Li et al., 2004). However, after d-flow stimulation, PKCζ and PIASy were colocalized in the nucleus. As for the p53 and Bcl-2 colocalization, p53 was localized in the nucleus of unstimulated cells, whereas Bcl-2 was mainly outside the nucleus as previously reported (Zhong et al., 1993; Ghosh et al., 2004). D-flow stimulation caused significant nuclear export of p53 and colocalization with Bcl-2. These data support d-flow–induced association of PKCζ–PIASy and p53–Bcl-2 in ECs.

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