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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 induces p53 SUMOylation and apoptosis via PIASy activation. (A) HUVECs were transfected with PIASy siRNA (si-PIASy) or control siRNA for 48 h and then stimulated with d-flow for the indicated times. p53 SUMOylation, expression of PIASy, p53, and SUMO2/3 were detected as described in Material and methods. Densitometric analyses of p53 SUMOylation were performed as described in Fig. 1. (B and C) HUVECs were transfected with PIASy or control siRNA for 48 h. After treatment with d-flow for 36 h, apoptotic nuclei were detected by TUNEL staining (B, bottom), and Western blotting with anti–cleaved caspase 3 (C, top) was performed. Immunoblots of PIASy conformed depletion of PIASy by the specific siRNA (B, top). Densitometry analysis of cleaved caspase 3 expression was performed as described in Fig. 2 C (bottom). The experiments were performed in triplicate using three different batches of d-flow–stimulated HUVECs. (D) HUVECs were transduced with an adenovirus vector containing p53, p53-K386R (KR; sumoylation defect mutant), or p53-ΔNES (L348,350A; NES mutant) for 24 h and then stimulated with d-flow for 36 h followed by TUNEL staining as described in Materials and methods. (E, top) Quantification of apoptosis shown as the percentage of TUNEL-positive cells. Bars, 30 µm. (bottom) Equal expressions of p53, p53-K386R, and p53-ΔNES were analyzed by Western blotting in ECs. Data are from three separate experiments using two or more different EC preparations. Error bars show means ± SD; *, P < 0.05; **, P < 0.01. Molecular masses are given in kilodaltons. IB, immunoblot. IP, immunoprecipitation. WT, wild type.
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fig7: D-flow induces p53 SUMOylation and apoptosis via PIASy activation. (A) HUVECs were transfected with PIASy siRNA (si-PIASy) or control siRNA for 48 h and then stimulated with d-flow for the indicated times. p53 SUMOylation, expression of PIASy, p53, and SUMO2/3 were detected as described in Material and methods. Densitometric analyses of p53 SUMOylation were performed as described in Fig. 1. (B and C) HUVECs were transfected with PIASy or control siRNA for 48 h. After treatment with d-flow for 36 h, apoptotic nuclei were detected by TUNEL staining (B, bottom), and Western blotting with anti–cleaved caspase 3 (C, top) was performed. Immunoblots of PIASy conformed depletion of PIASy by the specific siRNA (B, top). Densitometry analysis of cleaved caspase 3 expression was performed as described in Fig. 2 C (bottom). The experiments were performed in triplicate using three different batches of d-flow–stimulated HUVECs. (D) HUVECs were transduced with an adenovirus vector containing p53, p53-K386R (KR; sumoylation defect mutant), or p53-ΔNES (L348,350A; NES mutant) for 24 h and then stimulated with d-flow for 36 h followed by TUNEL staining as described in Materials and methods. (E, top) Quantification of apoptosis shown as the percentage of TUNEL-positive cells. Bars, 30 µm. (bottom) Equal expressions of p53, p53-K386R, and p53-ΔNES were analyzed by Western blotting in ECs. Data are from three separate experiments using two or more different EC preparations. Error bars show means ± SD; *, P < 0.05; **, P < 0.01. Molecular masses are given in kilodaltons. IB, immunoblot. IP, immunoprecipitation. WT, wild type.

Mentions: Because PIASy (SUMO E3 ligase) is involved in p53 SUMOylation in fibroblasts (Bischof et al., 2006), we wondered whether it was involved in the ONOO−- and d-flow–elicited p53 SUMOylation in HUVECs. When HUVECs were transfected with PIASy siRNA, the ONOO− and d-flow–dependent p53 SUMOylation was inhibited (Fig. 6 A and Fig. 7 A). Using the PIASy-depleted cells, we studied the role of PIASy in the ONOO−-elicited p53–Bcl-2 association and found that the PIASy depletion inhibited this interaction (Fig. 6 B), suggesting that PIASy, which SUMOylates p53, is involved in ONOO−-induced p53 nuclear export and subsequent p53–Bcl-2 binding in ECs. To see whether PIASy has a role in the ONOO− and d-flow–induced EC apoptosis, ECs were transfected with PIASy siRNA, challenged by ONOO− or d-flow, and assayed for apoptosis. The number of TUNEL-positive cells and expression of cleaved caspase 3 induced by ONOO− or d-flow were down-regulated by PIASy siRNA (Fig. 6 C and Fig. 7, B and C).


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 induces p53 SUMOylation and apoptosis via PIASy activation. (A) HUVECs were transfected with PIASy siRNA (si-PIASy) or control siRNA for 48 h and then stimulated with d-flow for the indicated times. p53 SUMOylation, expression of PIASy, p53, and SUMO2/3 were detected as described in Material and methods. Densitometric analyses of p53 SUMOylation were performed as described in Fig. 1. (B and C) HUVECs were transfected with PIASy or control siRNA for 48 h. After treatment with d-flow for 36 h, apoptotic nuclei were detected by TUNEL staining (B, bottom), and Western blotting with anti–cleaved caspase 3 (C, top) was performed. Immunoblots of PIASy conformed depletion of PIASy by the specific siRNA (B, top). Densitometry analysis of cleaved caspase 3 expression was performed as described in Fig. 2 C (bottom). The experiments were performed in triplicate using three different batches of d-flow–stimulated HUVECs. (D) HUVECs were transduced with an adenovirus vector containing p53, p53-K386R (KR; sumoylation defect mutant), or p53-ΔNES (L348,350A; NES mutant) for 24 h and then stimulated with d-flow for 36 h followed by TUNEL staining as described in Materials and methods. (E, top) Quantification of apoptosis shown as the percentage of TUNEL-positive cells. Bars, 30 µm. (bottom) Equal expressions of p53, p53-K386R, and p53-ΔNES were analyzed by Western blotting in ECs. Data are from three separate experiments using two or more different EC preparations. Error bars show means ± SD; *, P < 0.05; **, P < 0.01. Molecular masses are given in kilodaltons. IB, immunoblot. IP, immunoprecipitation. WT, wild type.
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fig7: D-flow induces p53 SUMOylation and apoptosis via PIASy activation. (A) HUVECs were transfected with PIASy siRNA (si-PIASy) or control siRNA for 48 h and then stimulated with d-flow for the indicated times. p53 SUMOylation, expression of PIASy, p53, and SUMO2/3 were detected as described in Material and methods. Densitometric analyses of p53 SUMOylation were performed as described in Fig. 1. (B and C) HUVECs were transfected with PIASy or control siRNA for 48 h. After treatment with d-flow for 36 h, apoptotic nuclei were detected by TUNEL staining (B, bottom), and Western blotting with anti–cleaved caspase 3 (C, top) was performed. Immunoblots of PIASy conformed depletion of PIASy by the specific siRNA (B, top). Densitometry analysis of cleaved caspase 3 expression was performed as described in Fig. 2 C (bottom). The experiments were performed in triplicate using three different batches of d-flow–stimulated HUVECs. (D) HUVECs were transduced with an adenovirus vector containing p53, p53-K386R (KR; sumoylation defect mutant), or p53-ΔNES (L348,350A; NES mutant) for 24 h and then stimulated with d-flow for 36 h followed by TUNEL staining as described in Materials and methods. (E, top) Quantification of apoptosis shown as the percentage of TUNEL-positive cells. Bars, 30 µm. (bottom) Equal expressions of p53, p53-K386R, and p53-ΔNES were analyzed by Western blotting in ECs. Data are from three separate experiments using two or more different EC preparations. Error bars show means ± SD; *, P < 0.05; **, P < 0.01. Molecular masses are given in kilodaltons. IB, immunoblot. IP, immunoprecipitation. WT, wild type.
Mentions: Because PIASy (SUMO E3 ligase) is involved in p53 SUMOylation in fibroblasts (Bischof et al., 2006), we wondered whether it was involved in the ONOO−- and d-flow–elicited p53 SUMOylation in HUVECs. When HUVECs were transfected with PIASy siRNA, the ONOO− and d-flow–dependent p53 SUMOylation was inhibited (Fig. 6 A and Fig. 7 A). Using the PIASy-depleted cells, we studied the role of PIASy in the ONOO−-elicited p53–Bcl-2 association and found that the PIASy depletion inhibited this interaction (Fig. 6 B), suggesting that PIASy, which SUMOylates p53, is involved in ONOO−-induced p53 nuclear export and subsequent p53–Bcl-2 binding in ECs. To see whether PIASy has a role in the ONOO− and d-flow–induced EC apoptosis, ECs were transfected with PIASy siRNA, challenged by ONOO− or d-flow, and assayed for apoptosis. The number of TUNEL-positive cells and expression of cleaved caspase 3 induced by ONOO− or d-flow were down-regulated by PIASy siRNA (Fig. 6 C and Fig. 7, B and C).

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