Inflammation contributes to development of atherosclerosis. Atherosclerosis is decreased in regions of steady flow associated with high shear stress (termed s-flow), compared to regions of disturbed and low flow (termed d-flow). This finding has yielded the concept that s-flow is atheroprotective and d-flow is atheropromoting. Previously we showed that s-flow activated MEK5-ERK5 in endothelial cells (EC), and inhibited tumor necrosis factor (TNF) signaling. Using a novel MEK5 inhibitor we showed that ERK5 activation was required for s-flow- mediated inhibition of TNF signaling. Conceptually there are two ways to improve EC dysfunction. The most common approach has been to activate atheroprotective mechanisms such as increasing systemic nitric oxide. However, we believe that a more elegant approach is to inhibit the atheroprone mechanisms that occur uniquely in areas of d-flow. Several findings indicate that PKCzeta-dependent signaling represents a unique atheropromoting mechanism. 1) PKCzeta promotes the adhesive phenotype of EC when activated by TNF, via stimulation of NF:B-dependent ICAM-1 expression. 2) PKCzeta activity is specifically increased in EC exposed to d-flow. 3) PKCzeta activity is required for TNF-mediated JNK and caspase-3 activation in EC. 4) Exciting preliminary data show that TNF and ONOO-, via activation of PKCzeta, enhance SUMOylation of the pro- apoptotic transcription factor p53. The SUMOylation pathway is analogous to that of ubiquitination, but SUMO conjugation involves a different enzymes including the protein inhibitor of activated STAT (PIASy) family. Our data show that PKCzeta activates PIASy SUMO E3 ligase activity, increases p53-SUMO, which increases p53 protein stability and enhances the apoptotic function of p53 (Figure). The major hypothesis of our proposal is that PKCzeta activation in EC at atheroprone areas inhibits ERK5- dependent transcriptional activity and stimulates p53-SUMOylation, thereby promoting EC inflammation and apoptosis. To define mechanisms that limit PKCzeta-dependent signal events we propose four aims.
Aim 1. Show that PKC6 binds and phosphorylates ERK5, thereby inhibiting ERK5 transcriptional activity.
Aim 2. Define the molecular mechanisms by which PKCzeta functions as an activator for p53 based on the concept that protein inhibitor of activated STAT y (PIASy;SUMO ligase) associates with PKCzeta increasing p53- SUMOylation, p53 expression and EC apoptosis.
Aim 3. Characterize the effects of s-flow and d-flow on PKCzeta, ERK5, PIASy, and p53 function in vitro based on the hypothesis that d-flow stimulates PKCzeta activation, p53 nuclear export, and EC apoptosis.
Aim 4. Show that PKCzeta and PIASy augment atherosclerosis in the apoE-/- mouse by inhibiting ERK5 activity and stimulating p53-SUMOylation. These studies should provide insight on how flow inhibits vascular inflammation and facilitate development of new therapeutic approaches to limit atherosclerosis.

Public Health Relevance

Strokes and heart attacks are the leading cause of death in the US. Interventions such as bypass surgery and angioplasty treat acute events, but there are limited therapies to prevent the underlying disease termed atherosclerosis. We have found that an enzyme - protein kinase C-zeta - is powerfully activated in blood vessels at sites where atherosclerotic plaques develop. Here we will focus on novel approaches to inhibit the function of this enzyme. Elucidating the specific pathways by which protein kinase C-zeta modulates vessel function would provide the basis to develop new therapies to prevent atherosclerosis.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Atherosclerosis and Inflammation of the Cardiovascular System Study Section (AICS)
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Goldberg, Suzanne H
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University of Rochester
Internal Medicine/Medicine
Schools of Dentistry
United States
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Le, Nhat-Tu; Heo, Kyung-Sun; Takei, Yuichiro et al. (2013) A crucial role for p90RSK-mediated reduction of ERK5 transcriptional activity in endothelial dysfunction and atherosclerosis. Circulation 127:486-99
Heo, Kyung-Sun; Chang, Eugene; Takei, Yuichiro et al. (2013) Phosphorylation of protein inhibitor of activated STAT1 (PIAS1) by MAPK-activated protein kinase-2 inhibits endothelial inflammation via increasing both PIAS1 transrepression and SUMO E3 ligase activity. Arterioscler Thromb Vasc Biol 33:321-9
Abe, Jun-ichi; Berk, Bradford C (2013) Atheroprone flow activation of the sterol regulatory element binding protein 2 and nod-like receptor protein 3 inflammasome mediates focal atherosclerosis. Circulation 128:579-82

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