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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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Increases in phosphorylated and total PKCζ and nonnuclear p53 expression within the d-flow regions (HP areas) and decreased apoptosis in ECs of p53−/− mice. (A) A representative epifluorescence image of the whole specimen. Fixed aortas of wild-type mice were cut longitudinally, and the arch region was further cut into two halves. Areas of d-flow (HP area; lesser curvature) are outlined in red, and neighboring areas of s-flow (LP area) are lined in blue. a, artery. (B and C) En face preparations were double stained with anti–VE-cadherin (VE-cad; used as an EC marker) and an anti–total PKCζ antibody (B) or phospho-PKCζ T560 antibody (C). X-y axis images were collected at 0.5-µm increments so that a z stack of ∼4-µm thickness from the luminal surface was obtained. From each image background, fluorescence intensity was subtracted, and the pixel number of the stained region per unit area of the endothelium in HP and LP area within the aortic arch was determined (n = 3). Areas of d-flow (HP areas; lesser curvature) show both increased total and phospho-PKCζ expression compared with the neighboring areas of s-flow (LP area). Bars, 20 µm. Bar graphs show quantification of total (B) and phospho (C)-PKCζ in HP and LP areas. Data are shown as means ± SEM; *, P < 0.05. (D and E) Increased cytoplasmic p53 localization in HP area ECs. Aortic arches were immunostained for endothelial p53 (green). Nuclei were stained using TO-PRO3 (red). From initial stacked x-y axis images (top), a narrow rectangular area crossing an EC was selected for x-y-z scanning at 0.1-µm increments. Images below the clipped images show rectangular z-axis images. Two representative sets of images (i and ii) are shown for LP (D) and HP (E) areas. Bars, 10 µm. (F) Quantification of nuclear p53. The pixel number of nuclear and nonnuclear regions per cell was determined, and the ratio of nuclear/total intensity was calculated from 60 cells from each HP and LP area (four cells/field, five fields/mouse, and a total of three mice). (G) The number of d-flow–mediated annexin V–positive cells (red) in the HP area is decreased in the mice deficient for p53 (p53 knockout) compared with the wild-type mouse aorta. Anti–VE-cadherin staining (green) was used as a marker for ECs. Bars, 100 µm. (H, left) Quantification of apoptotic cells. Percentages of annexin V–positive cells in LP and HP areas determined from 7-wk wild-type and p53-deficient mice (n = 3 each) are shown. In HP area, the number of annexin V–positive cells are significantly decreased in the p53 knockout compared with the wild type. **, P < 0.01. (right) Deletion of p53 was confirmed by Western blotting with anti-p53 using a lung protein lysate. Molecular masses are given in kilodaltons. Data are shown as means ± SEM. WT, wild type.
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fig10: Increases in phosphorylated and total PKCζ and nonnuclear p53 expression within the d-flow regions (HP areas) and decreased apoptosis in ECs of p53−/− mice. (A) A representative epifluorescence image of the whole specimen. Fixed aortas of wild-type mice were cut longitudinally, and the arch region was further cut into two halves. Areas of d-flow (HP area; lesser curvature) are outlined in red, and neighboring areas of s-flow (LP area) are lined in blue. a, artery. (B and C) En face preparations were double stained with anti–VE-cadherin (VE-cad; used as an EC marker) and an anti–total PKCζ antibody (B) or phospho-PKCζ T560 antibody (C). X-y axis images were collected at 0.5-µm increments so that a z stack of ∼4-µm thickness from the luminal surface was obtained. From each image background, fluorescence intensity was subtracted, and the pixel number of the stained region per unit area of the endothelium in HP and LP area within the aortic arch was determined (n = 3). Areas of d-flow (HP areas; lesser curvature) show both increased total and phospho-PKCζ expression compared with the neighboring areas of s-flow (LP area). Bars, 20 µm. Bar graphs show quantification of total (B) and phospho (C)-PKCζ in HP and LP areas. Data are shown as means ± SEM; *, P < 0.05. (D and E) Increased cytoplasmic p53 localization in HP area ECs. Aortic arches were immunostained for endothelial p53 (green). Nuclei were stained using TO-PRO3 (red). From initial stacked x-y axis images (top), a narrow rectangular area crossing an EC was selected for x-y-z scanning at 0.1-µm increments. Images below the clipped images show rectangular z-axis images. Two representative sets of images (i and ii) are shown for LP (D) and HP (E) areas. Bars, 10 µm. (F) Quantification of nuclear p53. The pixel number of nuclear and nonnuclear regions per cell was determined, and the ratio of nuclear/total intensity was calculated from 60 cells from each HP and LP area (four cells/field, five fields/mouse, and a total of three mice). (G) The number of d-flow–mediated annexin V–positive cells (red) in the HP area is decreased in the mice deficient for p53 (p53 knockout) compared with the wild-type mouse aorta. Anti–VE-cadherin staining (green) was used as a marker for ECs. Bars, 100 µm. (H, left) Quantification of apoptotic cells. Percentages of annexin V–positive cells in LP and HP areas determined from 7-wk wild-type and p53-deficient mice (n = 3 each) are shown. In HP area, the number of annexin V–positive cells are significantly decreased in the p53 knockout compared with the wild type. **, P < 0.01. (right) Deletion of p53 was confirmed by Western blotting with anti-p53 using a lung protein lysate. Molecular masses are given in kilodaltons. Data are shown as means ± SEM. WT, wild type.

Mentions: PKCζ activation in the ECs of the lesser curvature of the aortic arch in porcine aorta was recently reported (Magid and Davies, 2005). Because activation of this kinase is proatherogenic, we investigated the expression of PKCζ, phosphorylated PKCζ, a nitrotyrosine-containing protein, p53, and apoptotic ECs using en face aorta preparations and confocal microscopy. Aortas from male wild-type C57BL/6 mice (6–8 wk old) fed with normal chow were isolated after perfusion fixation and en face preparations were made. We focused on areas designated as high probability (HP) regions (lesser curvature of aortic arch) and low probability (LP) regions (greater curvature of aortic arch) for atherogenesis (Fig. 10 A) as described previously (Iiyama et al., 1999) and in the Materials and methods section. When the endothelium was double stained with anti-PKCζ or antiphospho-PKCζ together with anti–vascular endothelial (VE)-cadherin as an EC marker, we found that the expression of total PKCζ increased in the HP area (Fig. 10 B), and especially phospho-PKCζ was significantly higher in the HP area compared with the LP area (Fig. 10 C). Quantification of these data obtained from five mice supports this conclusion (Fig. 10, B and C, bar graph). These results confirm our in vitro results that show d-flow–dependent activation of PKCζ (Fig. 1).


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)

Increases in phosphorylated and total PKCζ and nonnuclear p53 expression within the d-flow regions (HP areas) and decreased apoptosis in ECs of p53−/− mice. (A) A representative epifluorescence image of the whole specimen. Fixed aortas of wild-type mice were cut longitudinally, and the arch region was further cut into two halves. Areas of d-flow (HP area; lesser curvature) are outlined in red, and neighboring areas of s-flow (LP area) are lined in blue. a, artery. (B and C) En face preparations were double stained with anti–VE-cadherin (VE-cad; used as an EC marker) and an anti–total PKCζ antibody (B) or phospho-PKCζ T560 antibody (C). X-y axis images were collected at 0.5-µm increments so that a z stack of ∼4-µm thickness from the luminal surface was obtained. From each image background, fluorescence intensity was subtracted, and the pixel number of the stained region per unit area of the endothelium in HP and LP area within the aortic arch was determined (n = 3). Areas of d-flow (HP areas; lesser curvature) show both increased total and phospho-PKCζ expression compared with the neighboring areas of s-flow (LP area). Bars, 20 µm. Bar graphs show quantification of total (B) and phospho (C)-PKCζ in HP and LP areas. Data are shown as means ± SEM; *, P < 0.05. (D and E) Increased cytoplasmic p53 localization in HP area ECs. Aortic arches were immunostained for endothelial p53 (green). Nuclei were stained using TO-PRO3 (red). From initial stacked x-y axis images (top), a narrow rectangular area crossing an EC was selected for x-y-z scanning at 0.1-µm increments. Images below the clipped images show rectangular z-axis images. Two representative sets of images (i and ii) are shown for LP (D) and HP (E) areas. Bars, 10 µm. (F) Quantification of nuclear p53. The pixel number of nuclear and nonnuclear regions per cell was determined, and the ratio of nuclear/total intensity was calculated from 60 cells from each HP and LP area (four cells/field, five fields/mouse, and a total of three mice). (G) The number of d-flow–mediated annexin V–positive cells (red) in the HP area is decreased in the mice deficient for p53 (p53 knockout) compared with the wild-type mouse aorta. Anti–VE-cadherin staining (green) was used as a marker for ECs. Bars, 100 µm. (H, left) Quantification of apoptotic cells. Percentages of annexin V–positive cells in LP and HP areas determined from 7-wk wild-type and p53-deficient mice (n = 3 each) are shown. In HP area, the number of annexin V–positive cells are significantly decreased in the p53 knockout compared with the wild type. **, P < 0.01. (right) Deletion of p53 was confirmed by Western blotting with anti-p53 using a lung protein lysate. Molecular masses are given in kilodaltons. Data are shown as means ± SEM. WT, wild type.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig10: Increases in phosphorylated and total PKCζ and nonnuclear p53 expression within the d-flow regions (HP areas) and decreased apoptosis in ECs of p53−/− mice. (A) A representative epifluorescence image of the whole specimen. Fixed aortas of wild-type mice were cut longitudinally, and the arch region was further cut into two halves. Areas of d-flow (HP area; lesser curvature) are outlined in red, and neighboring areas of s-flow (LP area) are lined in blue. a, artery. (B and C) En face preparations were double stained with anti–VE-cadherin (VE-cad; used as an EC marker) and an anti–total PKCζ antibody (B) or phospho-PKCζ T560 antibody (C). X-y axis images were collected at 0.5-µm increments so that a z stack of ∼4-µm thickness from the luminal surface was obtained. From each image background, fluorescence intensity was subtracted, and the pixel number of the stained region per unit area of the endothelium in HP and LP area within the aortic arch was determined (n = 3). Areas of d-flow (HP areas; lesser curvature) show both increased total and phospho-PKCζ expression compared with the neighboring areas of s-flow (LP area). Bars, 20 µm. Bar graphs show quantification of total (B) and phospho (C)-PKCζ in HP and LP areas. Data are shown as means ± SEM; *, P < 0.05. (D and E) Increased cytoplasmic p53 localization in HP area ECs. Aortic arches were immunostained for endothelial p53 (green). Nuclei were stained using TO-PRO3 (red). From initial stacked x-y axis images (top), a narrow rectangular area crossing an EC was selected for x-y-z scanning at 0.1-µm increments. Images below the clipped images show rectangular z-axis images. Two representative sets of images (i and ii) are shown for LP (D) and HP (E) areas. Bars, 10 µm. (F) Quantification of nuclear p53. The pixel number of nuclear and nonnuclear regions per cell was determined, and the ratio of nuclear/total intensity was calculated from 60 cells from each HP and LP area (four cells/field, five fields/mouse, and a total of three mice). (G) The number of d-flow–mediated annexin V–positive cells (red) in the HP area is decreased in the mice deficient for p53 (p53 knockout) compared with the wild-type mouse aorta. Anti–VE-cadherin staining (green) was used as a marker for ECs. Bars, 100 µm. (H, left) Quantification of apoptotic cells. Percentages of annexin V–positive cells in LP and HP areas determined from 7-wk wild-type and p53-deficient mice (n = 3 each) are shown. In HP area, the number of annexin V–positive cells are significantly decreased in the p53 knockout compared with the wild type. **, P < 0.01. (right) Deletion of p53 was confirmed by Western blotting with anti-p53 using a lung protein lysate. Molecular masses are given in kilodaltons. Data are shown as means ± SEM. WT, wild type.
Mentions: PKCζ activation in the ECs of the lesser curvature of the aortic arch in porcine aorta was recently reported (Magid and Davies, 2005). Because activation of this kinase is proatherogenic, we investigated the expression of PKCζ, phosphorylated PKCζ, a nitrotyrosine-containing protein, p53, and apoptotic ECs using en face aorta preparations and confocal microscopy. Aortas from male wild-type C57BL/6 mice (6–8 wk old) fed with normal chow were isolated after perfusion fixation and en face preparations were made. We focused on areas designated as high probability (HP) regions (lesser curvature of aortic arch) and low probability (LP) regions (greater curvature of aortic arch) for atherogenesis (Fig. 10 A) as described previously (Iiyama et al., 1999) and in the Materials and methods section. When the endothelium was double stained with anti-PKCζ or antiphospho-PKCζ together with anti–vascular endothelial (VE)-cadherin as an EC marker, we found that the expression of total PKCζ increased in the HP area (Fig. 10 B), and especially phospho-PKCζ was significantly higher in the HP area compared with the LP area (Fig. 10 C). Quantification of these data obtained from five mice supports this conclusion (Fig. 10, B and C, bar graph). These results confirm our in vitro results that show d-flow–dependent activation of PKCζ (Fig. 1).

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