Role of Protein Kinase C in Macrophage Activation

巨噬细胞吞噬作用是抵抗感染的第一道防线。通常，Fc受体介导的 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信号传导的失调有助于慢性炎性疾病，例如 类风湿性关节炎和动脉粥样硬化。因此，了解巨噬细胞的分子机制 吞噬作用对于发现调节慢性炎症的新靶点是必不可少的。小鼠 缺乏蛋白激酶C-κ B（PKC-κ）的人对细菌感染高度敏感， 有效的炎症反应。我们已经证明，PKC-β是有效的Fc受体介导的 吞噬作用和促炎基因的产生。PKC的作用机制需要 定位于吞噬体和催化活性。然而，PKC-κ如何被激活并发出信号， 吞噬作用和基因诱导是未知的。在功能上，我们已经证明PKC-κ参与了 伪足延伸，这是一个需要细胞内囊泡融合到吞噬体中的过程。 在结构上，我们已经确定了pseudosubstrate结构域对PKC-κ易位至关重要，并显示 它优先与聚磷酸肌醇相互作用。这一新的发现对 PKC激活的机制，目前尚不清楚。根据我们和其他人的研究， 我们认为，催化活性的PKC-β是必要的，为重点交付的囊泡进入 形成吞噬体。PKC-κ被甘油二酯和磷酸肌醇激活 单磷酸（PIP）。活性PKC-<$磷酸化参与囊泡融合的蛋白质 这是膜递送和伪足延伸所必需的。PKC-<$也是必要的， 诱导解决感染所需的基因。该模型将使用骨骼进行测试 来自野生型和PKC-敲除小鼠的骨髓源性巨噬细胞。具体而言，我们将（I）确定 PKC-<$易位到吞噬体所需的PIP，II）确定PKC-<$在囊泡中的作用 贩运和融合，III）使用常规和可追踪的PKC技术来识别PKC- 底物，和IV）使用无偏的qPCR阵列方法来鉴定PKC-β调节的基因。 确定PKC-<$在吞噬作用和基因表达中转导Fc <$R起始信号的机制 诱导对于我们理解宿主防御和导致慢性炎症的缺陷至关重要。 炎症性疾病。拟议的研究使用尖端技术，加上经典的 方法，在初级巨噬细胞中。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-β调节的基因和底物。的信息 获得将提供深入了解PKC-<$在Fc <$R介导的信号转导中的作用，以及它的损失如何 影响先天免疫

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-¿) are highly susceptible to bacteria infections and fail to mount an effective inflammatory response. We have shown that PKC-¿ is necessary for efficient Fc¿R-mediated phagocytosis and for production of pro-inflammatory genes. The mechanism of PKC-¿ action requires localization to phagosomes and catalytic activity. However, how PKC-¿ is activated and signals for phagocytosis and gene induction are unknown. Functionally, we have shown that PKC-¿ 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-¿ translocation and shown that it preferentially interacts with polyphosphoinositides. This novel finding has implications for the mechanism of PKC-¿ activation, which is currently unknown. Based on our research and that of others, we propose that catalytically active PKC-¿ is necessary for the focal delivery of vesicles into the forming phagosome. PKC-¿ is activated by diacylglycerol and a phosphoinositide monophosphate (PIP). Active PKC-¿ phosphorylates proteins involved in the vesicle fusion necessary for membrane delivery and pseudopod extension. PKC-¿ 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-¿ null mice. Specifically, we will I) Identify the PIP required for PKC-¿ translocation to phagosomes, II) Determine the role of PKC-¿ in vesicle trafficking and fusion, III) Use conventional and traceable PKC techniques to identify PKC-¿ substrates, and IV) Use an unbiased, qPCR array approach to identify PKC-¿-regulated genes. Defining the mechanisms by which PKC-¿ 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-¿-regulated genes and substrates. The information gained will provide insight into the role of PKC-¿ in Fc¿R-mediated signal transduction and how its loss impacts innate immunity.