Molecular Mechanisms of Apoptosis in Monocytes

单核细胞凋亡的分子机制

• 批准号: 7841052
• 负责人: ANDREA DOSEFF
• 金额: $ 21.47万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-01 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7841052
• 关键词: Affect; Animal Model; Apoptosis; Apoptotic; Asthma; Biological; Biological Process; Bronchitis; Cells; Complex; Data; Development; Disease; Effector Cell; Family; Goals; Immune response; Immune system; In Vitro; Inflammation; Inflammatory; Label; Lead; Life; Lipopolysaccharides; Location; Longevity; Lung; Maps; Modeling; Molecular; Pathogenesis; Pathway interactions; Phosphoproteins; Phosphorylation; Phosphorylation Site; Phosphotransferases; Play; Positioning Attribute; Protein Kinase C; Proteins; Recombinant Proteins; Regulation; Resolution; Respiratory physiology; Role; Sarcoidosis; Signal Transduction; Site; Small Interfering RNA; Stimulus; base; caspase-3; improved; in vivo; macrophage; member; monocyte; mutant; new therapeutic target

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 understandingof 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.

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，结节病和多种疾病，最终导致肺功能丧失。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。循环单核细胞 通常生活24-48小时，在没有生存信号的情况下会自发凋亡。在场 刺激因子，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， 了解控制凋亡蛋白的分子机制对于操纵 单核细胞/巨噬细胞在炎症部位的积累。 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是一种磷酸蛋白，与蛋白质成员相关并磷酸化 激酶C家族（PKC）。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。这 该建议的目标是了解调节单核细胞凋亡的机制。在这里，我们在目标1中提出建议 确定caspase-3磷酸化的生物学作用。为此，我们将首先确定数字和 caspase-3在体内和体外的磷酸化位点的位置。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影响caspase-3的活化或活性。下一个， 我们将确定caspase-3磷酸化在凋亡中的作用。在AIM 2中，我们建议识别激酶/S 使用siRNA和动物模型在体内负责caspase-3磷酸化。我们在AIM 3中提议 研究磷酸化是否改变了caspase-3的定位，并研究了该定位 凋亡过程中的变化。最后，在目标4中，我们建议确定caspase-3的机制 磷酸化和caspase-3相关 - 激酶调节单核细胞的刺激，分化和寿命。 总之，这些研究应提高我们对调节细胞凋亡的基本机制的了解并有助于 鉴定新的治疗靶标，以选择性诱导炎症细胞的凋亡。此外，这是我们的目标 这里提出的研究将有助于改善炎症的治疗方法。