G Protein Regulation of pMN NADPH Oxidase

中性粒细胞(PMN)是一种吞噬细胞，能产生宿主防御所需的超氧化物(O2-)，但在特定情况下，O2-的产生也会导致组织损伤，如急性肺损伤。我们目前对PMN烟酰胺腺嘌呤二核苷酸磷酸(NADPH)氧化酶的了解主要是基于对佛波酯的研究。N-甲酰-蛋氨酸-亮氨酸-苯丙氨酸(FMLF)等化学诱导剂诱导细胞产生O2- 然而，其基本机制仍然知之甚少。利用COS-PHOX细胞，开发了一种新的、基于细胞的重建系统，以系统地解决介导NADPH氧化酶激活的信号机制。这些研究将得到使用野生型PMN和来自基因缺失小鼠的PMN的实验的补充，以及关于特定信号通路的激活如何导致肺血管损伤或在其他情况下可能具有保护作用的实验。目的1是基于我们的初步发现，即PKcheeta足以重建FMLF诱导的COS-Phox细胞产生O2-。因此，我们将检验PKcheeta在FMLF激活NADPH氧化酶中起关键作用的假设，并确定涉及的磷酸化步骤。在目标2中，我们将扩展我们的发现，即p40Phox选择性地增强FMLF-而不是PMA诱导的O2- 并将检验p40Phox除了在NADPH氧化酶复合体的形成中发挥作用外，还在调节介导NADPH氧化酶激活的信号通路中发挥重要作用的假设。在目标3中，我们将检验这样一种假设，即G-α-Q介导的PLcheeta激活(由血小板激活因子的PMN启动作用诱导)通过异源三聚体G蛋白β-伽马亚基敏感于随后的激活，这些亚基在FMLF刺激其G蛋白偶联受体(GPCR)FPR时释放。在目的4中，我们将确定新型脂质介质溶血磷脂酰胆碱(LPC)在下调NADPH氧化酶中的作用，并探讨LPC在预防PMN介导的肺血管损伤中的作用。我们将检验LPC与其G-α-S偶联结合的假设 受体G2a导致细胞内cAMP升高，从而抑制PMN O2-的产生。通过将细胞和分子研究结合到小鼠肺微血管通透性的研究中，我们将更好地理解GPCRs激活NADPH氧化酶的机制以及NADPH氧化酶的激活是如何被调节的。有了这些洞察力，我们将能够开发针对PMN不当激活和PMN介导的肺血管损伤和水肿的新的治疗策略。

Neutrophils (PMNs) are phagocytic cells that produce superoxide (O2-) needed for host defense, but under specific circumstances O2- production can also result in tissue damage such as found in Acute Lung Injury. Our current understanding of the PMN nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is based primarily on studies using phorbol esters. Chemoattractants such as N-formyl-Met-Leu-Phe (fMLF) induce O2- generation in PMNs, yet the underlying mechanisms remain poorly understood. A novel, whole cell-based reconstitution system, using COS-phox cells, has been developed to address, in a systematic manner, the signaling mechanisms mediating NADPH oxidase activation. These studies will be complemented with experiments using wild-type PMNs and PMNs from mice with gene deletions, as well as experiments addressing how the activation of specific signaling pathways induces lung vascular injury, or may be protective in other instances. Aim 1 is based on our preliminary findings that PKCzeta is sufficient for reconstituting the fMLF-induced O2- production in COS-phox cells. Thus, we will test the hypothesis that PKCzeta plays a critical role in fMLF activation of NADPH oxidase and identify the phosphorylation steps involved. In Aim 2, we will extend our finding that p40phox selectively enhances fMLF- but not PMA-induced O2- generation, and will test the hypothesis that p40phox plays an important role in regulating the signaling pathways mediating NADPH oxidase activation, in addition to its role in NADPH oxidase complex formation. In Aim 3, we will test the hypothesis that G-alpha-q-mediated PLCzeta activation (as induced by the PMN priming action of platelet-activating factor) sensitizes the enzyme for subsequent activation by heterotrimeric G protein beta-gamma subunits, which are released upon fMLF-stimulation of its G protein coupled receptor (GPCR), FPR. In Aim 4, we will determine the function of the novel lipid mediator lysophosphatidylcholine (LPC) in down-regulating NADPH oxidase and address the role of LPC in preventing PMN-mediated lung vascular injury. We will test the hypothesis that LPC binding to its G-alpha-s-coupled receptor G2A leads to a rise in intracellular cAMP that thereby inhibits PMN O2- production. By incorporating cellular and molecular studies into the study of microvascular permeability in mouse lung models, we will gain a better understanding of the mechanisms of NADPH oxidase activation by GPCRs and how NADPH oxidase activation can be modulated. With the insights gained, we will be in a position to develop novel therapeutic strategies targeting inappropriate PMN activation and PMN-mediated lung vascular injury and edema.