ROLE OF CDK2 CELL CYCLE SIGNALING IN ISCHEMIC INJURY AND PROTECTION

首席调查员/项目主任(最后、第一、中间)：平，佩佩(麦克莱伦，项目4) 本计划项目申请的主题是了解信号转导途径 使用结合生物物理、生理学和心脏保护的多学科方法 蛋白质组学和遗传学。虽然项目1-3专注于两条先前已知的调节信号通路 缺血性损伤和保护(即PKCE和p38MAPK)，项目4重点关注最近发现的一条途径 在心肌缺血细胞周期调节蛋白领域的重要性，特别是它将重点放在三个方面 在这一途径中功能相互关联的分子：细胞周期蛋白依赖性激酶-2(CDK2)、 视网膜母细胞瘤基因产物(Rb)和调控细胞周期相关基因的转录因子(E2F) 条目包括CDK2的催化伙伴Cyclin A和Cyclin E。 项目4中提出的研究得到了1)最近证据的支持，这些证据表明，脑缺血损伤 心脏伴随着一些细胞周期调节蛋白的上调，包括CDK2；和2)通过 令人震惊的初步数据显示，Rb缺失心肌梗死面积增加。 缺血性损伤。相反，体内使用cdk2抑制剂的治疗阻止了cdk2活性的预期增加。 缺血性损伤，并导致梗塞面积缩小。这表明CDK2在脑缺血中起着关键作用。 损伤和Rb蛋白对损伤的心脏保护作用。尽管这些观察结果很耐人寻味，因为 这些效应背后的细胞机制尚不清楚。 项目4与项目1-3和核心合作，提出了3个目标。目标1将阐明 CDK2的S调节缺血损伤能力的机制；与心脏生物学核心合作，这 AIM将确定CDK2如何调节线粒体功能和细胞死亡途径；与 蛋白质组学核心，AIM 1将鉴定新的CDK2底物。目标2将研究Rb的分子基础 在心肌缺血损伤中的保护作用，它将确定P38A MAPK对Rb的依赖调节，并将定义 E2F家族(RB的主要靶点)在这些过程中的作用。最后，目标3将通过以下方式阐明机制 其中PKCE调控心肌保护中的CDK2活性。这些研究将与Project合作进行 2和心脏生物学核心，并将采用两种成熟的小鼠心脏保护模型：PKCE 转基因和一氧化氮供体诱导的预适应晚期。拟议的调查将 为理解Rb的心脏保护作用的分子基础和机制做出了重要贡献 CDK2‘S调节缺血损伤和细胞凋亡的能力。

Principal Investigator/ProgramDirector (Last, First, Middle): Ping, Peipei (MacLellan,Project 4) The theme of this Program Project application is to understand the signal transduction pathways mediating ischemic injury and cardioprotection using a multidisciplinary approach combining biophysics, physiology, proteomics and genetics. While Projects 1-3 are focused on two signaling pathways previously known to modulate ischemic injury and protection (i.e., PKCe and p38 MAPK), Project 4 focuses on a pathway with recently recognized importance in the field of myocardial ischemiacell cycle regulatory proteins, in particular, it will focus on three molecules whose functions are interrelated in this pathway: the cyclin-dependent kinase-2 (Cdk2), the retinoblastoma gene product (Rb), and the transcription factors (E2Fs) that regulate genes responsible for cell cycle entry including Cyclin A and E, the catalytic partners of Cdk2. Studies proposed in Project 4 are supported 1) by recent evidence demonstrating that ischemic injury to the heart are accompanied by the upregulation of a number of cell cycle regulatory proteins including Cdk2; and 2) by striking preliminary data demonstrating that infarct size was increased in Rb-null myocardium subjected to regional ischemic injury. Conversely, treatment with a Cdk2 inhibitor in vivo blocked the expected increase in Cdk2 activity with ischemic injury and led to a reduced infarct size formation. This suggests a critical role for Cdk2 in ischemic injury and a cardioprotective role of the Rb protein against injury. Although these observations are intriguing, as the cellular mechanisms underlying these effects are unknown. In collaboration with Projects 1-3 and the Cores, Project 4 proposes 3 Aims. Aim 1 will elucidate the mechanisms underlying Cdk2's ability to modulate ischemic injury; In collaboration with the Heart Biology Core, this aim will determine how Cdk2 modulates mitochondrial function and the cell death pathways; In collaboration with the Proteomic Core, Aim 1 will identify novel Cdk2 substrates. Aim 2 will examine the molecular basis forRb's cardioprotective role in ischemic injury, it will determine p38a MAPK dependent modulation of Rb, and will define the role of E2F family (theprimary targets of Rb) in these processes. Finally, Aim 3 will elucidate mechanisms by which PKCe regulates Cdk2 activity in cardioprotection. These studies will be performed in collaboration with Project 2 and the Heart Biology Core, and will employ two well-established murine models of cardioprotection: the PKCe transgenesis and the nitric oxide donor induced late phase of preconditioning. The proposed investigations will make key contributions to understanding the molecular basis for Rb's cardioprotective effect and the mechanism underlying Cdk2's ability to regulate ischemic damage and apoptotic cell death.