Role of Protein Kinase C in Macrophage Activation

蛋白激酶 C 在巨噬细胞激活中的作用

• 批准号: 8051924
• 负责人: Michelle R Lennartz
• 金额: $ 9.62万
• 依托单位: ALBANY MEDICAL COLLEGE
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-05 至 2011-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8051924
• 关键词: 1,2-diacylglycerol; Address; Antigens; Atherosclerosis; Bacteria; Bacterial Infections; Binding; Biological Models; Bone Marrow; Cell physiology; Cells; Chronic; Confocal Microscopy; Coupled; Data; Defect; Detection; Diglycerides; Disease; Down-Regulation; Exocytosis; Fluorescence Resonance Energy Transfer; Gene Expression; Genes; Host Defense; Immune system; Immunoglobulin G; Infection; Inflammation; Inflammatory; Inflammatory Response; Intracellular Membranes; Knockout Mice; Knowledge; Lead; Ligation; Macrophage Activation; Mediating; Membrane; Membrane Fusion; Modeling; Molecular; Movement; Mus; Natural Immunity; Neoplasm Metastasis; Pathway interactions; Phagocytosis; Phagosomes; Phosphatidylinositol Phosphates; Phosphatidylinositols; Phospholipase C; Phosphotransferases; Play; Polylysine; Printing; Process; Production; Protein Kinase C; Proteins; Regulation; Research; Resolution; Rheumatoid Arthritis; Role; Signal Transduction; Site; Small Interfering RNA; Surface; System; Techniques; Technology; Testing; Vesicle; Wound Healing; base; gene induction; in vitro Assay; innovation; insight; macrophage; nano; novel; pathogen; protein kinase C epsilon; public health relevance; receptor; response; syntaxin 4; trafficking; tumor

DESCRIPTION (provided by applicant): Macrophage phagocytosis is the first line of defense against infection. Normally, Fc?R-mediated phagocytosis efficiently clears pathogens and presents antigen to the adaptive immune system. However, dysregulation of FcR signaling contributes to chronic inflammatory diseases such as rheumatoid arthritis and atherosclerosis. Thus, understanding the molecular mechanisms of macrophage phagocytosis is essential to the discovery of novel targets for regulation of chronic inflammation. Mice lacking protein kinase C-epsilon (PKC-e) are highly susceptible to bacterial infections and fail to mount an effective inflammatory response. We have shown that PKC-e is necessary for efficient Fc?R-mediated phagocytosis and for production of pro-inflammatory genes. The mechanism of PKC-e action requires localization to phagosomes and catalytic activity. However, how PKC-e is activated and signals for phagocytosis and gene induction are unknown. Functionally, we have shown that PKC-e is involved in pseudopod extension, a process that requires fusion of intracellular vesicles into the phagosome. Structurally, we have identified the pseudosubstrate domain as critical for PKC-e translocation and shown that it preferentially interacts with polyphosphoinositides. This novel finding has implications for the mechanism of PKC-e activation, which is currently unknown. Based on our research and that of others, we propose that catalytically active PKC-e is necessary for the focal delivery of vesicles into the forming phagosome. PKC-e is activated by diacylglycerol and a phosphoinositide monophosphate (PIP). Active PKC-e phosphorylates proteins involved in the vesicle fusion necessary for membrane delivery and pseudopod extension. PKC-e is also necessary for the induction of genes required for resolution of infection. This model will be tested using bone marrow-derived macrophages from wild type and PKC-e null mice. Specifically, we will I) Identify the PIP required for PKC-e translocation to phagosomes, II) Determine the role of PKC-e in vesicle trafficking and fusion, III) Use conventional and traceable PKC techniques to identify PKC-e substrates, and IV) Use an unbiased, qPCR array approach to identify PKC-e-regulated genes. Defining the mechanisms by which PKC-e transduces Fc?R-initiated signals in phagocytosis and gene induction is critical to our understanding of host defense and the defects that contribute to chronic inflammatory diseases. The proposed studies use cutting edge technologies, coupled with classical approaches, in primary macrophages. We will rigorously test hypotheses supported by preliminary data and reach beyond current paradigms to identify PKC-e-regulated genes and substrates. The information gained will provide insight into the role of PKC-e in Fc?R-mediated signal transduction and how its loss impacts innate immunity. PUBLIC HEALTH RELEVANCE: IgG-mediated phagocytosis is an essential function of the innate immune system. Dysregulation can lead to chronic inflammatory diseases including rheumatoid arthritis and atherosclerosis. Phagocytosis requires the focal delivery of intracellular membranes to the site of target binding. How this occurs and what directs that focal delivery is unknown. We have evidence that protein kinase C-epsilon plays a major role in membrane dynamics during phagocytosis but the mechanism by which that occurs is unknown. Understanding membrane remodeling using phagocytosis as a model system will advance our knowledge of the innate immune system and may be applicable to other cell processes, including tumor metastasis and wound healing.

描述（由申请人提供）：巨噬细胞吞噬作用是抵抗感染的第一道防线。一般来说，FC？R介导的吞噬作用有效地清除病原体并将抗原呈递给适应性免疫系统。然而，FcR信号转导的失调有助于慢性炎性疾病，如类风湿性关节炎和动脉粥样硬化。因此，了解巨噬细胞吞噬作用的分子机制对于发现调节慢性炎症的新靶点至关重要。缺乏蛋白激酶C-ε（PKC-e）的小鼠对细菌感染高度敏感，并且无法产生有效的炎症反应。我们已经表明，PKC-e是必要的有效的Fc？R介导的吞噬作用和促炎基因的产生。PKC-e的作用机制需要定位于吞噬体和催化活性。然而，PKC-e如何被激活以及吞噬和基因诱导的信号是未知的。在功能上，我们已经表明，PKC-e参与伪足的延伸，一个过程，需要融合的细胞内囊泡到吞噬体。在结构上，我们已经确定了pseudosubstrate域作为关键的PKC-e易位，并表明它优先与polyphosphoinositides相互作用。这一新的发现对目前未知的PKC-e激活机制有影响。基于我们的研究和其他人的研究，我们提出，催化活性的PKC-ε是必要的，为重点交付的囊泡到形成吞噬体。PKC-e被甘油二酯和磷酸肌醇单磷酸（PIP）激活。活性PKC-e磷酸化参与膜递送和伪足延伸所必需的囊泡融合的蛋白质。PKC-e对于诱导解决感染所需的基因也是必需的。将使用来自野生型和PKC-e缺失小鼠的骨髓源性巨噬细胞测试该模型。具体而言，我们将I）鉴定PKC-e易位至吞噬体所需的PIP，II）确定PKC-e在囊泡运输和融合中的作用，III）使用常规和可追踪的PKC技术来鉴定PKC-e底物，以及IV）使用无偏的qPCR阵列方法来鉴定PKC-e调节的基因。确定PKC-e转导Fc的机制？R-启动信号的吞噬和基因诱导是至关重要的，我们了解宿主的防御和缺陷，有助于慢性炎症性疾病。拟议的研究在原代巨噬细胞中使用尖端技术，结合经典方法。我们将严格测试由初步数据支持的假设，并超越目前的范式，以确定PKC-e调节的基因和底物。所获得的信息将提供深入了解PKC-e在Fc？R介导的信号转导及其损失如何影响先天免疫。公共卫生相关性：IgG介导的吞噬作用是先天免疫系统的一项基本功能。失调可导致慢性炎性疾病，包括类风湿性关节炎和动脉粥样硬化。吞噬作用需要将细胞内膜集中递送至靶结合位点。这是如何发生的，以及是什么引导了这种局部递送是未知的。我们有证据表明，蛋白激酶C-β在吞噬过程中的膜动力学中起着重要作用，但发生的机制是未知的。使用吞噬作用作为模型系统来理解膜重塑将推进我们对先天免疫系统的认识，并且可能适用于其他细胞过程，包括肿瘤转移和伤口愈合。