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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 depletion by siRNA and DN-PKCζ inhibits d-flow and ONOO−-induced apoptosis. (A) HUVECs were transfected with control or PKCζ siRNA for 48 h and then stimulated with d-flow for 36 h followed by TUNEL staining. Images were recorded as described in Materials and methods after counterstaining with DAPI to visualize nuclei (bottom). Apoptotic nuclei appear green (top). Bars, 25 µm. (right) Quantification of apoptosis is shown as the percentage of TUNEL-positive cells. (B) HUVECs were transfected with control or PKCζ siRNA for 48 h (left) or transduced with Ad-DN-PKCζ or Ad-LacZ as a control for 24 h (center). Cells were then treated with 100 µM ONOO− or vehicle for 8 h and assayed by TUNEL staining. (right) DN-PKCζ and reduced PKCζ expression were confirmed by Western blotting with anti-PKCζ. Data are from three separate experiments using two or more different EC preparations (**, P < 0.01). (C) After transduction of Ad-DN-PKCζ or Ad-LacZ for 24 h, HUVECs were treated with 100 µM ONOO− for 9 and 18 h, and Western blotting with anti–cleaved caspase 3 was performed. DN-PKCζ expression and protein loading were assessed by Western blotting with anti-PKCζ (middle) and antitubulin (bottom). (right) Quantification of cleaved caspase 3 is expressed as the relative ratio compared with tubulin expression. Results are expressed as the relative percentage of untreated cells in the LacZ control (100%). n = 3. **, P < 0.01 compared with each control. Molecular masses are given in kilodaltons. Error bars are means ± SD.
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fig2: PKCζ depletion by siRNA and DN-PKCζ inhibits d-flow and ONOO−-induced apoptosis. (A) HUVECs were transfected with control or PKCζ siRNA for 48 h and then stimulated with d-flow for 36 h followed by TUNEL staining. Images were recorded as described in Materials and methods after counterstaining with DAPI to visualize nuclei (bottom). Apoptotic nuclei appear green (top). Bars, 25 µm. (right) Quantification of apoptosis is shown as the percentage of TUNEL-positive cells. (B) HUVECs were transfected with control or PKCζ siRNA for 48 h (left) or transduced with Ad-DN-PKCζ or Ad-LacZ as a control for 24 h (center). Cells were then treated with 100 µM ONOO− or vehicle for 8 h and assayed by TUNEL staining. (right) DN-PKCζ and reduced PKCζ expression were confirmed by Western blotting with anti-PKCζ. Data are from three separate experiments using two or more different EC preparations (**, P < 0.01). (C) After transduction of Ad-DN-PKCζ or Ad-LacZ for 24 h, HUVECs were treated with 100 µM ONOO− for 9 and 18 h, and Western blotting with anti–cleaved caspase 3 was performed. DN-PKCζ expression and protein loading were assessed by Western blotting with anti-PKCζ (middle) and antitubulin (bottom). (right) Quantification of cleaved caspase 3 is expressed as the relative ratio compared with tubulin expression. Results are expressed as the relative percentage of untreated cells in the LacZ control (100%). n = 3. **, P < 0.01 compared with each control. Molecular masses are given in kilodaltons. Error bars are means ± SD.

Mentions: We examined whether PKCζ activation was also required for d-flow and ONOO−-mediated EC apoptosis using two different approaches. PKCζ activity was down-regulated by inhibiting its expression by siRNA and also by expressing an adenoviral dominant-negative (DN) kinase-dead form of PKCζ (Ad-DN-PKCζ). Although d-flow and ONOO− treatment increased apoptosis in control cells, a significant reduction in the number of TUNEL-positive cells was noted in PKCζ-depleted HUVECs (Fig. 2, A and B, left). When HUVECs were infected with Ad-DN-PKCζ (mutating Lys281to Met), the ONOO−-induced increase in TUNEL-positive cells and the expression of cleaved caspase 3 fragments (17/19 kD) were reduced compared with control cells (Fig. 2, B [center] and C). These data suggest the critical role of PKCζ kinase activity in d-flow and ONOO−-mediated EC 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)

PKCζ depletion by siRNA and DN-PKCζ inhibits d-flow and ONOO−-induced apoptosis. (A) HUVECs were transfected with control or PKCζ siRNA for 48 h and then stimulated with d-flow for 36 h followed by TUNEL staining. Images were recorded as described in Materials and methods after counterstaining with DAPI to visualize nuclei (bottom). Apoptotic nuclei appear green (top). Bars, 25 µm. (right) Quantification of apoptosis is shown as the percentage of TUNEL-positive cells. (B) HUVECs were transfected with control or PKCζ siRNA for 48 h (left) or transduced with Ad-DN-PKCζ or Ad-LacZ as a control for 24 h (center). Cells were then treated with 100 µM ONOO− or vehicle for 8 h and assayed by TUNEL staining. (right) DN-PKCζ and reduced PKCζ expression were confirmed by Western blotting with anti-PKCζ. Data are from three separate experiments using two or more different EC preparations (**, P < 0.01). (C) After transduction of Ad-DN-PKCζ or Ad-LacZ for 24 h, HUVECs were treated with 100 µM ONOO− for 9 and 18 h, and Western blotting with anti–cleaved caspase 3 was performed. DN-PKCζ expression and protein loading were assessed by Western blotting with anti-PKCζ (middle) and antitubulin (bottom). (right) Quantification of cleaved caspase 3 is expressed as the relative ratio compared with tubulin expression. Results are expressed as the relative percentage of untreated cells in the LacZ control (100%). n = 3. **, P < 0.01 compared with each control. Molecular masses are given in kilodaltons. Error bars are means ± SD.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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fig2: PKCζ depletion by siRNA and DN-PKCζ inhibits d-flow and ONOO−-induced apoptosis. (A) HUVECs were transfected with control or PKCζ siRNA for 48 h and then stimulated with d-flow for 36 h followed by TUNEL staining. Images were recorded as described in Materials and methods after counterstaining with DAPI to visualize nuclei (bottom). Apoptotic nuclei appear green (top). Bars, 25 µm. (right) Quantification of apoptosis is shown as the percentage of TUNEL-positive cells. (B) HUVECs were transfected with control or PKCζ siRNA for 48 h (left) or transduced with Ad-DN-PKCζ or Ad-LacZ as a control for 24 h (center). Cells were then treated with 100 µM ONOO− or vehicle for 8 h and assayed by TUNEL staining. (right) DN-PKCζ and reduced PKCζ expression were confirmed by Western blotting with anti-PKCζ. Data are from three separate experiments using two or more different EC preparations (**, P < 0.01). (C) After transduction of Ad-DN-PKCζ or Ad-LacZ for 24 h, HUVECs were treated with 100 µM ONOO− for 9 and 18 h, and Western blotting with anti–cleaved caspase 3 was performed. DN-PKCζ expression and protein loading were assessed by Western blotting with anti-PKCζ (middle) and antitubulin (bottom). (right) Quantification of cleaved caspase 3 is expressed as the relative ratio compared with tubulin expression. Results are expressed as the relative percentage of untreated cells in the LacZ control (100%). n = 3. **, P < 0.01 compared with each control. Molecular masses are given in kilodaltons. Error bars are means ± SD.
Mentions: We examined whether PKCζ activation was also required for d-flow and ONOO−-mediated EC apoptosis using two different approaches. PKCζ activity was down-regulated by inhibiting its expression by siRNA and also by expressing an adenoviral dominant-negative (DN) kinase-dead form of PKCζ (Ad-DN-PKCζ). Although d-flow and ONOO− treatment increased apoptosis in control cells, a significant reduction in the number of TUNEL-positive cells was noted in PKCζ-depleted HUVECs (Fig. 2, A and B, left). When HUVECs were infected with Ad-DN-PKCζ (mutating Lys281to Met), the ONOO−-induced increase in TUNEL-positive cells and the expression of cleaved caspase 3 fragments (17/19 kD) were reduced compared with control cells (Fig. 2, B [center] and C). These data suggest the critical role of PKCζ kinase activity in d-flow and ONOO−-mediated EC 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