Fluid Shear Stress Signal Transduction in Endothelium

描述（由申请人提供）：炎症有助于动脉粥样硬化的发展。动脉粥样硬化在与高剪切应力相关的稳定血流区域（称为s-flow）与受干扰和低血流区域（称为d-flow）相比减少。这一发现产生了s流是动脉粥样硬化保护和d流是动脉粥样硬化促进的概念。先前我们发现s-flow激活内皮细胞（EC）中的MEK5-ERK5，并抑制肿瘤坏死因子（TNF）信号传导。使用一种新的MEK5抑制剂，我们发现ERK5激活是s-flow介导的TNF信号抑制所必需的。从概念上讲，有两种方法可以改善EC功能障碍。最常见的方法是激活动脉粥样硬化保护机制，如增加全身一氧化氮。然而，我们认为一种更优雅的方法是抑制在d-flow区域独特发生的动脉粥样硬化机制。一些研究结果表明，pkczeta依赖性信号代表了一种独特的动脉粥样硬化促进机制。1) PKCzeta通过刺激NF: b依赖性ICAM-1的表达，在TNF激活时促进EC的粘附表型。2) PKCzeta活性在暴露于d-flow的EC中特异性增加。3) EC中tnf介导的JNK和caspase-3激活需要PKCzeta活性。4)令人兴奋的初步数据显示，TNF和ONOO-通过激活PKCzeta，增强促凋亡转录因子p53的SUMOylation。SUMO化途径与泛素化途径类似，但SUMO偶联涉及不同的酶，包括活化STAT蛋白抑制剂（PIASy）家族。我们的数据显示，PKCzeta激活PIASy SUMO E3连接酶活性，增加p53-SUMO，从而增加p53蛋白的稳定性，增强p53的凋亡功能（图）。我们提出的主要假设是PKCzeta在EC动脉粥样硬化区激活抑制ERK5依赖的转录活性并刺激p53-SUMOylation，从而促进EC炎症和凋亡。为了定义限制pkceta依赖信号事件的机制，我们提出了四个目标。目的1。显示PKC6结合并磷酸化ERK5，从而抑制ERK5的转录活性。目标2。基于活化STAT y蛋白抑制剂（PIASy； SUMO连接酶）与PKCzeta增加p53- SUMOylation、p53表达和EC凋亡相关的概念，明确PKCzeta作为p53激活剂的分子机制。目标3。基于d-flow刺激PKCzeta激活、p53核输出和EC凋亡的假设，表征s-flow和d-flow对PKCzeta、ERK5、PIASy和p53体外功能的影响。目标4。表明PKCzeta和PIASy通过抑制ERK5活性和刺激p53-SUMOylation而增加apoE-/-小鼠的动脉粥样硬化。这些研究应该为血流如何抑制血管炎症提供见解，并促进发展新的治疗方法来限制动脉粥样硬化。

DESCRIPTION (provided by applicant): 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.