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（02-）是宿主防御和炎症的重要组成部分。02-通过酶复合物NADPH氧化酶产生。除了杀死微生物的作用外，02-的产生还有助于脂质氧化。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的重要因素。对单核细胞NADPH氧化酶和02-生成的调节和功能的了解是有限的。我们发现单核细胞NADPH氧化酶的调节与中性粒细胞酶复合物的调节非常不同。由于中性粒细胞不存在于动脉壁上，无论是在病变发展的早期还是晚期，了解单核细胞中这种酶复合物活性的调节是至关重要的。最近的研究表明，缺乏这种酶的动物动脉粥样硬化的发展较少，因此NADPH氧化酶在动脉粥样硬化发生中的重要性尤为突出。NADPH氧化酶由几种成分组成。在静止的单核细胞中，一些成分是膜结合的，而另一些是细胞质结合的。后者必须与其他氧化酶组分形成膜相关复合物，以使酶具有活性。NADPH氧化酶的三个关键的细胞质成分，易位形成活性酶复合物是本应用的重点。他们是p47phox， p67phox和Racl。我们实验室之前的研究已经描绘了一个激活诱导的途径，包括细胞内钙增加、PKCalpha易位、PKCalpha依赖的磷酸化和cPLA2的激活、花生四烯酸（AA）的产生和NADPH氧化酶的激活。在Aim 1中，我们建议确定cPLA2上关键的pkcalpha依赖性磷酸化位点，并检查它们对cPLA2活性的影响。将比较体外产生的和单核细胞介导的磷酸化位点。Aim 2的研究探讨了cpla2衍生的AA对NADPH氧化酶活性的调节，以及AA如何调节NADPH氧化酶成分的易位。我们有新的证据表明PKCdelta参与了p47phox的磷酸化。因此，在第三个目标中，我们提出研究p47phox上pkcdelta依赖的磷酸化位点，并确定它们对p47phox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p67phox的激酶。Aim 4的研究将验证Racl在介导p47phox和p67phox相互作用以及活性NADPH氧化酶复合物形成中起关键作用的假设。由于我们已经开发了探测单核细胞信号转导途径的工具，我们处于一个独特的位置，可以专门识别这些途径，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.