Role of Protein Kinase C in Macrophage Activation

描述（由申请人提供）：巨噬细胞吞噬是抵抗感染的第一道防线。通常,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信号的失调有助于慢性炎症性疾病，如类风湿关节炎和动脉粥样硬化。因此，了解巨噬细胞吞噬的分子机制对于发现慢性炎症调节的新靶点至关重要。缺乏蛋白激酶C-epsilon （PKC-e）的小鼠对细菌感染高度敏感，无法产生有效的炎症反应。我们已经证明PKC-e是高效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并具有催化活性。然而，PKC-e如何被激活以及吞噬和基因诱导的信号尚不清楚。在功能上，我们已经证明PKC-e参与假足延伸，这一过程需要细胞内囊泡融合到吞噬体中。在结构上，我们已经确定了假底物结构域对PKC-e易位至关重要，并表明它优先与多磷酸肌苷相互作用。这一新发现对PKC-e激活的机制有影响，目前尚不清楚。根据我们和其他人的研究，我们提出催化活性PKC-e对于囊泡进入形成吞噬体的局部递送是必要的。PKC-e被二酰基甘油和磷酸肌醇单磷酸（PIP）激活。活性PKC-e磷酸化参与膜传递和假足延伸所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。该模型将使用野生型和PKC-e缺失小鼠骨髓来源的巨噬细胞进行测试。具体来说，我们将I)确定PKC-e易位到吞噬体所需的PIP， II)确定PKC-e在囊泡运输和融合中的作用，III)使用传统和可追溯的PKC技术来鉴定PKC-e底物，IV)使用无偏的qPCR阵列方法来鉴定PKC-e调节基因。定义PKC-e转导Fc的机制？吞噬和基因诱导中的r启动信号对我们理解宿主防御和导致慢性炎症性疾病的缺陷至关重要。提出的研究在原代巨噬细胞中使用尖端技术，结合经典方法。我们将严格检验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。所获得的信息将有助于深入了解PKC-e在Fc？r介导的信号转导及其丢失如何影响先天免疫。公共卫生相关性：igg介导的吞噬是先天免疫系统的基本功能。失调会导致慢性炎症性疾病，包括风湿性关节炎和动脉粥样硬化。吞噬作用需要将胞内膜集中递送到目标结合部位。这是如何发生的，以及是什么引导了焦点传递尚不清楚。我们有证据表明，蛋白激酶C-epsilon在吞噬过程中的膜动力学中起主要作用，但其发生的机制尚不清楚。将吞噬作用作为一种模型系统来理解膜重构将促进我们对先天免疫系统的认识，并可能适用于其他细胞过程，包括肿瘤转移和伤口愈合。

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.