Role of Protein Kinase C in Macrophage Activation

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。通常，Fc?R 介导的吞噬作用有效地清除病原体并向适应性免疫系统呈递抗原。然而，FcR 信号传导失调会导致类风湿性关节炎和动脉粥样硬化等慢性炎症性疾病。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) 的小鼠非常容易受到细菌感染，并且无法产生有效的炎症反应。我们已经证明 PKC-e 对于有效的 Fc?R 介导的吞噬作用和促炎基因的产生是必需的。 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 的激活方式以及吞噬作用和基因诱导的信号尚不清楚。从功能上讲，我们已经证明 PKC-e 参与伪足延伸，该过程需要细胞内囊泡融合到吞噬体中。在结构上，我们已经确定假底物结构域对于 PKC-e 易位至关重要，并表明它优先与多磷酸肌醇相互作用。这一新发现对目前未知的 PKC-e 激活机制具有重要意义。根据我们和其他人的研究，我们提出催化活性 PKC-e 对于将囊泡聚焦递送到形成的吞噬体中是必要的。 PKC-e 由二酰基甘油和磷酸肌醇单磷酸 (PIP) 激活。活性 PKC-e 磷酸化参与膜递送和伪足延伸所需的囊泡融合的蛋白质。 PKC-e 对于诱导消除感染所需的基因也是必需的。该模型将使用来自野生型和 PKC-e 缺失小鼠的骨髓来源的巨噬细胞进行测试。具体来说，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 在吞噬作用和基因诱导中转导 Fc?R 起始信号的机制对于我们了解宿主防御和导致慢性炎症性疾病的缺陷至关重要。拟议的研究在初级巨噬细胞中使用了尖端技术，并结合了经典方法。我们将严格测试由初步数据支持的假设，并超越当前的范式来识别 PKC-e 调节的基因和底物。获得的信息将有助于深入了解 PKC-e 在 Fc?R 介导的信号转导中的作用以及其丢失如何影响先天免疫。公共卫生相关性：IgG 介导的吞噬作用是先天免疫系统的一项重要功能。失调可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递送至靶标结合位点。这是如何发生的以及是什么指导了焦点传递尚不清楚。我们有证据表明蛋白激酶 C-ε 在吞噬过程中的膜动力学中发挥着重要作用，但其发生机制尚不清楚。使用吞噬作用作为模型系统来了解膜重塑将增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.