p66Shc Signaling Functions in Cardiomyocytes

描述(由申请人提供)：p66shc最近成为活性氧簇(ROS)产生和心血管氧化应激反应的主要调节者。在动物模型中，p66Shc作为一种能放大线粒体中ROS生成并导致可测量的寿命变化的“老化”基因，其作用令人信服，但关于p66Shc在心肌细胞中的功能几乎没有任何信息。这项应用的初步研究表明，p66Shc是涉及ROS和PKC的肾上腺素能受体途径的靶点，该途径定位于小窝，调节AKT-FOXO3a的磷酸化和抗氧化基因的表达。我们确定了依赖于G1q的肥大通路增加p66Shc表达的作用，并将p66Shc靶向线粒体，在那里它被预测增加ROS的产生。我们确定了蛋白激酶C-4(PKC4)和PKC5对p66Shc磷酸化和定位的影响。PKC4对p66shc的调控尤其值得注意，因为PKC4还参与控制ROS积累和细胞凋亡的线粒体事件。在这方面的研究将考虑p66Shc作为G1q诱导的基因产物与PKC合作影响心力衰竭表型演变的作用。具体目的I研究p66Shc在心肌细胞中的激活机制和信号功能。我们的目标是确定p66Shc是否参与了小窝和线粒体中影响生长和/或凋亡的ROS调节信号机制。大多数研究没有解决p66Shc与更小的p46/p52Shc异构体(不调节氧化还原信号)的信号功能。在特定目标II的研究中，将使用p66Shc基因敲除模型、siRNA基因沉默和腺病毒介导的过度表达策略来解决p66Shc(以及p66Shc蛋白结构中的关键决定因素)在调节ROS产生和心肌细胞生长/凋亡的机制中的特定功能。在特定目的III中的研究将集中在PKC4与p66Shc合作控制心肌细胞重构的作用。这些研究的基础是初步数据显示，PKC4是一个p66Shc结合伙伴，影响p66Shc-S36的磷酸化和p66Shc亚细胞靶向。最后，特定目的IV的研究将检验p66Shc基因沉默是否能防止在选定的心肌肥厚/衰竭模型中发生的结构重塑和功能恶化。有证据表明p66Shc参与了心功能障碍的病因和/或发病机制，这将为进一步研究p66Shc作为影响与氧化应激(缺血性、糖尿病、动脉粥样硬化、高血压和肥厚性心脏病)相关的心血管疾病演变的“衰老基因”提供基础。这类研究的长期目标将是开发新的治疗策略，以防止p66Shc的表达或阻断p66Shc的功能，从而提供心脏保护。

DESCRIPTION (provided by applicant): p66Shc has recently emerged as a master regulator of reactive oxygen species (ROS) production and cardiovascular oxidative stress responses. While a role for p66Shc as an 'aging' gene that amplifies ROS generation in mitochondria and leads to measurable changes in lifespan in animal models is convincing, there is almost no information on p66Shc functions in cardiomyocytes. Preliminary studies in this application implicate p66Shc as a target of an adrenergic receptor pathway involving ROS and PKC that is localized to caveolae and regulates AKT-FOXO3a phosphorylation and anti-oxidant gene expression. We identify an effect of the G1q-dependent hypertrophy pathway to increase p66Shc expression and target p66Shc to mitochondria where it is predicted to increase ROS generation. We identify a role for protein kinase C-4 (PKC4) and PKC5 to influence p66Shc phosphorylation and localization. p66Shc regulation by PKC4 is particularly noteworthy, as PKC4 also participates in mitochondrial events that control ROS accumulation and apoptosis. Studies in this application will consider the role of p66Shc as a G1q-induced gene product that cooperates with PKC to influence the evolution of cardiac failure phenotypes. Specific aim I will identify p66Shc activation mechanisms and p66Shc signaling functions in cardiomyocytes. The goal is to determine whether p66Shc participates in ROS-regulatory signaling mechanisms in caveolae and mitochondria that influence growth and/or apoptosis. Most studies do not resolve the signaling functions of p66Shc versus the smaller p46/p52Shc isoforms (that do not regulate Redox signaling). Studies in Specific Aim II will use p66Shc knockout models, siRNA gene silencing, and adenoviral mediated overexpression strategies to resolve the specific functions of p66Shc (and critical determinants within the p66Shc protein structure) in mechanisms that regulate ROS production and cardiomyocyte growth/apoptosis. Studies in Specific Aim III will focus on the role of PKC4 to cooperate with p66Shc to control cardiomyocyte remodeling. These studies are based upon preliminary data showing that PKC4 is a p66Shc binding partner that influences p66Shc-S36 phosphorylation and p66Shc subcellular targeting. Finally, studies in Specific Aim IV will examine whether p66Shc gene silencing prevents the structural remodeling and functional deterioration that develops in selected models of cardiac hypertrophy/failure. Evidence that p66Shc contributes to the etiology and/or pathogenesis of cardiac dysfunction would provide the basis for future research focusing on p66Shc as an 'aging gene' that influences the evolution of cardiovascular disorders associated with oxidative stress (ischemic, diabetic, atherosclerotic, hypertensive, and hypertrophic heart disease). The long-term goal of such studies would be to develop novel therapeutic strategies that prevent p66Shc expression or interdict p66Shc function that afford cardioprotection.