Monocyte Pathogenesis: Regulation of NADPH Oxidase

描述（由申请人提供）：活化的单核细胞和中性粒细胞产生超氧阴离子（O2-）是宿主防御和炎症的基本要素。02-是通过酶复合物NADPH氧化酶产生的。除了其在杀死微生物中的作用之外，O2-产生有助于脂质的氧化。由单核细胞NADPH氧化酶介导的脂质氧化被认为是动脉粥样硬化形成的重要因素。对单核细胞NADPH氧化酶和02-产生的调节和功能的理解是有限的。我们发现单核细胞NADPH氧化酶的调节方式与中性粒细胞酶复合体非常不同。由于中性粒细胞不存在于动脉壁，无论是在早期或晚期病变的发展，了解单核细胞中这种酶复合物的活性调节是至关重要的。NADPH氧化酶在动脉粥样硬化形成中的特别重要性来自于最近的研究，表明动脉粥样硬化的发展在这种酶缺乏的动物中较少。NADPH氧化酶由几种组分组成。在静息单核细胞中，一些组分是膜结合的，而另一些是胞质的。后者必须与其他氧化酶组分形成膜缔合复合物以允许酶活性。NADPH氧化酶的三个关键胞质组分易位形成活性酶复合物是本申请的重点。它们是p47 phox、p67 phox和Racl。我们实验室先前的研究已经描述了一种激活诱导途径，涉及细胞内钙离子增加、PKCalpha易位、PKCalpha依赖性磷酸化和cPLA 2激活、花生四烯酸（AA）生成和NADPH氧化酶激活。在目的1中，我们提出了确定关键的，PKCa依赖的磷酸化位点的cPLA 2和检查他们对cPLA 2活性的影响。将在体外产生的和单核细胞介导的磷酸化位点之间进行比较。目的2中的研究探索cPLA 2衍生的AA对NADPH氧化酶活性的调节以及AA如何调节NADPH氧化酶组分的易位。我们有新的证据表明PKC δ参与p47 phox的磷酸化。因此，在第三个目标中，我们提出研究p47 phox上的PKC δ依赖性磷酸化位点，并确定它们对p47 phox与其他NADPH氧化酶组分相互作用的影响。我们还将启动研究，以确定负责磷酸化p67 phox的激酶。目的4中的研究将检验Racl对于介导p47 phox和p67 phox相互作用和活性NADPH氧化酶复合物的形成是关键的假设。由于我们已经开发了探测单核细胞中信号转导通路的工具，我们处于独特的位置，可以特异性地识别这些通路，并发现影响已发展为致病过程的炎症反应的新方法。

DESCRIPTION (provided by applicant): The production of superoxide anion (02-) by activated monocytes and neutrophils is an essential element of host defense and inflammation. 02- is generated via the enzyme complex, NADPH oxidase. In addition to its role in killing microorganisms, 02- production contributes to the oxidation of lipids. Lipid oxidation, mediated by monocyte NADPH oxidase, is believed to significantly contribute to atherogenesis. Understanding of the regulation and function of the monocyte NADPH oxidase and 02- production is limited. We are finding that the monocyte NADPH oxidase is regulated very differently than the neutrophil enzyme complex. Since neutrophils are not present in the artery wall, either in early or late lesion development, understanding the regulation of the activity of this enzyme complex in monocytes is paramount. Particular importance of NADPH oxidase in atherogenesis is derived from recent studies showing that the development of atherosclerosis is less in animals deficient in this enzyme. NADPH oxidase is comprised of several components. In resting monocytes some components are membrane-bound while others are cytosolic. The latter must form a membrane-associated complex with other oxidase components to allow enzyme activity. Three critical cytosolic components of NADPH oxidase that translocate to form the active enzyme complex are the focus of this application. They are p47phox, p67phox and Racl. Prior studies from our laboratory have delineated an activation-induced pathway involving increased intracellular calcium, PKCalpha translocation, PKCalpha-dependent phosphorylation and activation of cPLA2, generation of arachidonic acid (AA) and activation of NADPH oxidase. In Aim 1 we propose to identify the critical, PKCalpha-dependent phosphorylation sites on cPLA2 and examine their effects on cPLA2 activity. Comparisons will be made between in vitro generated and monocyte-mediated phosphorylation sites. Studies in Aim 2 explore regulation of NADPH oxidase activity by cPLA2-derived AA and how AA can regulate translocation of NADPH oxidase components. We have new evidence that PKCdelta is involved in the phosphorylation of p47phox. Therefore in the third aim we propose studies to examine the PKCdelta-dependent phosphorylation sites on p47phox and determine their effects on p47phox interaction with other NADPH oxidase components. We will also initiate studies to identify the kinase responsible for phosphorylating p67phox. Studies in Aim 4 will test the hypothesis that Racl is critical for mediating p47phox and p67phox interaction and formation of the active NADPH oxidase complex. Due to the tools that we have developed to probe signal transduction pathways in monocytes, we are in a unique position to specifically identify these pathways and discover novel approaches for influencing inflammatory responses that have developed into pathogenic processes.