Role of Protein Kinase C in Macrophage Activation

描述(申请人提供)：巨噬细胞吞噬功能是抵抗感染的第一道防线。正常情况下，Fc？R介导的吞噬作用可有效清除病原体，并将抗原呈递给适应性免疫系统。然而，FCR信号的失调会导致慢性炎症性疾病，如类风湿性关节炎和动脉粥样硬化。因此，了解巨噬细胞吞噬的分子机制对于发现调控慢性炎症的新靶点至关重要。缺乏蛋白激酶C-epsilon(PKC-e)的小鼠对细菌感染高度敏感，无法启动有效的炎症反应。我们已经证明，PKC-e对于Fc？R介导的有效吞噬和产生促炎基因是必要的。PKC-e的作用机制需要定位于吞噬小体并具有催化活性。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参与了伪足延伸，这是一个需要细胞内小泡融合到吞噬小体中的过程。在结构上，我们已经确定假底物结构域是PKC-e转位的关键，并表明它优先与多聚磷脂酰肌醇相互作用。这一新发现对目前尚不清楚的PKC-e激活机制有一定的意义。基于我们的研究和其他人的研究，我们认为具有催化活性的PKC-e是囊泡向形成的吞噬小体集中输送所必需的。PKC-e被二酰甘油和磷脂酰肌醇单磷酸盐(PIP)激活。活性PKC-e磷酸化参与囊泡融合的蛋白，膜输送和伪足延伸是必需的。PKC-e对于诱导分解感染所需的基因也是必要的。这一模型将使用野生型和PKC-e缺失小鼠的骨髓来源的巨噬细胞进行测试。具体地说，我们将1)确定PKC-e转位到吞噬小体所需的PIP，2)确定PKC-e在囊泡运输和融合中的作用，3)使用常规的和可追踪的PKC技术来鉴定PKC-e底物，以及iv)使用无偏见的qPCR阵列方法来鉴定PKC-e调节的基因。明确PKC-e在吞噬和基因诱导中传递Fc？R启动信号的机制对于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。拟议的研究使用尖端技术，结合经典方法，对原代巨噬细胞进行研究。我们将严格测试由初步数据支持的假设，并超越目前的范式来识别PKC-e调节的基因和底物。所获得的信息将为深入了解PKC-e在Fc？R介导的信号转导中的作用以及它的缺失如何影响先天免疫。公共卫生相关性：免疫球蛋白介导的吞噬作用是先天免疫系统的一项基本功能。调节失调会导致慢性炎症性疾病，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输送到靶结合部位。这种情况是如何发生的，以及是什么引导了这种集中交付，目前尚不清楚。我们有证据表明，蛋白激酶C-epsilon在吞噬过程中在膜动力学中起主要作用，但其发生机制尚不清楚。以吞噬作用为模型系统了解细胞膜重塑将促进我们对先天免疫系统的了解，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.