Molecular Mechanisms of Apoptosis in Monocytes

单核细胞凋亡的分子机制

基本信息

批准号：
7044522
负责人：
ANDREA DOSEFF
金额：
$ 29.9万
依托单位：
OHIO STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2006
资助国家：
美国
起止时间：
2006-02-09 至 2011-01-31
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant): Inflammation is implicated in the pathogenesis of many diseases. In the lung, some of the examples include asthma, bronchitis, sarcoidosis, and a variety of disorders that ultimately lead to the loss of lung function. Monocytes are important components during the initiation and resolution of the innate immune response. Circulating monocytes normally live 24-48 hrs and in the absence of a survival signal undergo spontaneous apoptosis. In the presence of stimulatory factors, monocytes survive and differentiate into macrophages, which live up to 3 months. Thus, the understanding of the molecular mechanisms that control the apoptotic proteins is essential to manipulate the accumulation of monocyte/macrophages at inflammatory sites. Caspase-3 is a key executioner of the apoptotic pathway in monocytes. We demonstrated that stimulatory stimulus, such as lipopolysaccharide (LPS), induces monocyte survival by blocking the activation of caspase-3. Our preliminary studies demonstrate that caspase-3 is a phosphoprotein that associates and is phosphorylated by a member of the protein kinase C family (PKC). Our results suggest that phosphorylation induces caspase-3 activity and promotes apoptosis. The caspase-3-associated-kinase activity is modulated during monocyte life span and by inflammatory stimuli. The goal of this proposal is to understand the mechanisms that regulate apoptosis in monocytes. Here, we propose in Aim 1 to determine the biological role of caspase-3 phosphorylation. For this purpose, we will determine first the number and location of the phosphorylated sites on caspase-3 in vivo and in vitro. Next, using in vivo labeled caspase-3 and caspase-3 mutants we will elucidate whether phosphorylation affects the activation or the activity of caspase-3. Next, we will determine the role the caspase-3 phosphorylation in apoptosis. In Aim 2, we propose to identify the kinase/s that are responsible for caspase-3 phosphorylation in vivo using siRNA and animal models. We propose in Aim 3, to investigate whether the phosphorylation changes the localization of caspase-3 and to study whether the localization changes during apoptosis. Finally, in Aim 4 we propose to determine the mechanisms by which caspase-3 phosphorylation and the caspase-3-associated-kinases regulate stimulation, differentiation, and life span of monocytes. Altogether, these studies should advance our understanding of the basic mechanisms that regulate apoptosis and help identifying new therapeutic targets to selectively induced apoptosis of inflammatory cells. Furthermore, it is our goal that the studies proposed here will contribute to improve the treatments for inflammation.

描述（由申请人提供）：炎症与许多疾病的发病机理有关。在肺中，一些例子包括哮喘，支气管炎，结节病以及最终导致肺功能丧失的多种疾病。单核细胞是先天免疫反应的启动和解决过程中的重要组成部分。循环的单核细胞通常生活在24-48小时，而在没有生存信号的情况下会自发凋亡。在存在刺激因素的情况下，单核细胞生存并分化为巨噬细胞，这些巨噬细胞寿命长达3个月。因此，对控制凋亡蛋白的分子机制的理解对于操纵炎症部位的单核细胞/巨噬细胞的积累至关重要。 caspase-3是单核细胞中凋亡途径的关键执行者。我们证明了刺激性刺激，例如脂多糖（LPS），通过阻断caspase-3的激活来诱导单核细胞存活。我们的初步研究表明，caspase-3是一种磷蛋白，由蛋白激酶C家族（PKC）的成员磷酸化并磷酸化。我们的结果表明，磷酸化诱导caspase-3活性并促进凋亡。 caspase-3相关 - 激酶活性在单核细胞寿命和炎症性刺激期间进行调节。该提案的目的是了解调节单核细胞凋亡的机制。在这里，我们在AIM 1中提出，以确定caspase-3磷酸化的生物学作用。为此，我们将首先确定caspase-3在体内和体外上磷酸化位点的数量和位置。接下来，使用体内标记为caspase-3和caspase-3突变体，我们将阐明磷酸化是否会影响caspase-3的激活或活性。接下来，我们将确定caspase-3磷酸化在凋亡中的作用。在AIM 2中，我们建议鉴定使用siRNA和动物模型在体内负责caspase-3磷酸化的激酶。我们建议在AIM 3中研究磷酸化是否会改变caspase-3的定位并研究凋亡过程中的定位是否改变。最后，在AIM 4中，我们建议确定caspase-3磷酸化和caspase-3相关 - 激酶调节单核细胞的刺激，分化和寿命的机制。总的来说，这些研究应提高我们对调节细胞凋亡的基本机制的理解，并有助于确定新的治疗靶标，以选择性诱导炎症细胞的凋亡。此外，我们的目标是，这里提出的研究将有助于改善炎症的治疗方法。