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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 activation by d-flow and ONOO−. (A and B) Generation of s-flow and d-flow using a cone and plate flow chamber. Tracks of fluorescent beads in a cone and plate flow chamber. S-flow was generated using a nongrooved cone (A), and d-flow was generated using a grooved cone (B). Note straight tracks with s-flow and short tracks with different orientations with d-flow, which are caused by beads going out of focus. Both cones were rotated at the same speed. Color tracks indicate time x (red), time x + 10 s (green), and time x + 20 s (blue). Bright dots are beads adhered to the dish. Exposure time is 0.4 s. Bars, 100 µm. (C and D) HUVECs were stimulated by s- or d-flow (C) or ONOO− (D) for the indicated times, and PKCζ phosphorylation was determined by Western blotting. The level of PKCζ phosphorylation was determined by taking the ratio of optical densities between antiphospho-PKCζ and anti-PKCζ bands (bar graphs) as described in Materials and methods. The experiments were performed in triplicate using three different batches of s- or d-flow or ONOO−-stimulated HUVECs (means ± SD; n = 3; *, P < 0.05; and **, P < 0.01 compared with control). Molecular masses are given in kilodaltons. IB, immunoblot.
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fig1: PKCζ activation by d-flow and ONOO−. (A and B) Generation of s-flow and d-flow using a cone and plate flow chamber. Tracks of fluorescent beads in a cone and plate flow chamber. S-flow was generated using a nongrooved cone (A), and d-flow was generated using a grooved cone (B). Note straight tracks with s-flow and short tracks with different orientations with d-flow, which are caused by beads going out of focus. Both cones were rotated at the same speed. Color tracks indicate time x (red), time x + 10 s (green), and time x + 20 s (blue). Bright dots are beads adhered to the dish. Exposure time is 0.4 s. Bars, 100 µm. (C and D) HUVECs were stimulated by s- or d-flow (C) or ONOO− (D) for the indicated times, and PKCζ phosphorylation was determined by Western blotting. The level of PKCζ phosphorylation was determined by taking the ratio of optical densities between antiphospho-PKCζ and anti-PKCζ bands (bar graphs) as described in Materials and methods. The experiments were performed in triplicate using three different batches of s- or d-flow or ONOO−-stimulated HUVECs (means ± SD; n = 3; *, P < 0.05; and **, P < 0.01 compared with control). Molecular masses are given in kilodaltons. IB, immunoblot.

Mentions: We first verified the potential role of shear stress in PKCζ activation in cultured ECs using a cone and plate type of flow apparatuses as described previously (Reinhart-King et al., 2008). To generate d-flow, we used cones with radial grooves that were 1-mm deep. Fig. 1 (A and B) shows tracks of fluorescent beads suspended in culture media when grooved and nongrooved cones were rotated at the same speed. Although the nongrooved cone created straight unidirectional tracks indicating s-flow, tracks made by the grooved cone were short and not oriented in the same direction, indicating nonlaminar movement of the media in the dish. Using these cones, we determined the effect of s- and d-flow on PKCζ activation in human umbilical vein ECs (HUVECs). D-flow increased PKCζ phosphorylation at both Thr410 and Thr560 after 10 min of stimulation (Fig. 1 C), whereas s-flow failed to activate PKCζ, although ERK5 was activated by s-flow as we reported previously (not depicted; Woo et al., 2008). PKCζ phosphorylation at Thr560 was sustained longer than phosphorylation at Thr410. These data obtained from our in vitro system are consistent with in vivo data, which showed increased PKCζ activity in d-flow area compared with s-flow area in porcine arteries (Magid and Davies, 2005), supporting the physiological relevance of our in vitro system.


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 activation by d-flow and ONOO−. (A and B) Generation of s-flow and d-flow using a cone and plate flow chamber. Tracks of fluorescent beads in a cone and plate flow chamber. S-flow was generated using a nongrooved cone (A), and d-flow was generated using a grooved cone (B). Note straight tracks with s-flow and short tracks with different orientations with d-flow, which are caused by beads going out of focus. Both cones were rotated at the same speed. Color tracks indicate time x (red), time x + 10 s (green), and time x + 20 s (blue). Bright dots are beads adhered to the dish. Exposure time is 0.4 s. Bars, 100 µm. (C and D) HUVECs were stimulated by s- or d-flow (C) or ONOO− (D) for the indicated times, and PKCζ phosphorylation was determined by Western blotting. The level of PKCζ phosphorylation was determined by taking the ratio of optical densities between antiphospho-PKCζ and anti-PKCζ bands (bar graphs) as described in Materials and methods. The experiments were performed in triplicate using three different batches of s- or d-flow or ONOO−-stimulated HUVECs (means ± SD; n = 3; *, P < 0.05; and **, P < 0.01 compared with control). Molecular masses are given in kilodaltons. IB, immunoblot.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig1: PKCζ activation by d-flow and ONOO−. (A and B) Generation of s-flow and d-flow using a cone and plate flow chamber. Tracks of fluorescent beads in a cone and plate flow chamber. S-flow was generated using a nongrooved cone (A), and d-flow was generated using a grooved cone (B). Note straight tracks with s-flow and short tracks with different orientations with d-flow, which are caused by beads going out of focus. Both cones were rotated at the same speed. Color tracks indicate time x (red), time x + 10 s (green), and time x + 20 s (blue). Bright dots are beads adhered to the dish. Exposure time is 0.4 s. Bars, 100 µm. (C and D) HUVECs were stimulated by s- or d-flow (C) or ONOO− (D) for the indicated times, and PKCζ phosphorylation was determined by Western blotting. The level of PKCζ phosphorylation was determined by taking the ratio of optical densities between antiphospho-PKCζ and anti-PKCζ bands (bar graphs) as described in Materials and methods. The experiments were performed in triplicate using three different batches of s- or d-flow or ONOO−-stimulated HUVECs (means ± SD; n = 3; *, P < 0.05; and **, P < 0.01 compared with control). Molecular masses are given in kilodaltons. IB, immunoblot.
Mentions: We first verified the potential role of shear stress in PKCζ activation in cultured ECs using a cone and plate type of flow apparatuses as described previously (Reinhart-King et al., 2008). To generate d-flow, we used cones with radial grooves that were 1-mm deep. Fig. 1 (A and B) shows tracks of fluorescent beads suspended in culture media when grooved and nongrooved cones were rotated at the same speed. Although the nongrooved cone created straight unidirectional tracks indicating s-flow, tracks made by the grooved cone were short and not oriented in the same direction, indicating nonlaminar movement of the media in the dish. Using these cones, we determined the effect of s- and d-flow on PKCζ activation in human umbilical vein ECs (HUVECs). D-flow increased PKCζ phosphorylation at both Thr410 and Thr560 after 10 min of stimulation (Fig. 1 C), whereas s-flow failed to activate PKCζ, although ERK5 was activated by s-flow as we reported previously (not depicted; Woo et al., 2008). PKCζ phosphorylation at Thr560 was sustained longer than phosphorylation at Thr410. These data obtained from our in vitro system are consistent with in vivo data, which showed increased PKCζ activity in d-flow area compared with s-flow area in porcine arteries (Magid and Davies, 2005), supporting the physiological relevance of our in vitro system.

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