G Protein Regulation of pMN NADPH Oxidase

pMN NADPH 氧化酶的 G 蛋白调节

• 批准号: 7312599
• 负责人: RICHARD D YE
• 金额: $ 33.71万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-08-01 至 2010-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7312599
• 关键词: G protein; NAD(P)H dehydrogenase; biological signal transduction; cell cell interaction; cell surface receptors; chemoattractants; cyclic AMP; edema; gene targeting; genetically modified animals; immunomodulators; inflammation; laboratory mouse; leukocyte activation /transformation; lung injury; lysolecithins; neutrophil; phosphorylation; platelet activating factor; protein isoforms; protein kinase C; respiratory circulation disorder; transfection

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.

中性粒细胞（PMN）是产生宿主防御所需的超氧化物（O2-）的吞噬细胞，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）氧化酶的了解主要基于使用佛波酯的研究。化学引诱剂如N-甲酰基-Met-Leu-Phe（fMLF）诱导O2-产生， PMNs，但其潜在的机制仍然知之甚少。一种新的，全细胞为基础的重建系统，使用COS-phox细胞，已开发解决，在一个系统的方式，信号转导机制介导的NADPH氧化酶激活。这些研究将与使用野生型PMNs和来自基因缺失小鼠的PMNs的实验以及解决特定信号通路的激活如何诱导肺血管损伤或在其他情况下可能具有保护作用的实验相补充。目的1是基于我们的初步发现，PKCzeta足以重建COS-phox细胞中fMLF诱导的O2-产生。因此，我们将检验PKCzeta在NADPH氧化酶的fMLF激活中起关键作用的假设，并确定所涉及的磷酸化步骤。在目标2中，我们将扩展我们的发现，即p40 phox选择性地增强fMLF-而不是PMA-诱导的O2-。 的产生，并将测试的假设，p40 phox起着重要的作用，在调节信号通路介导的NADPH氧化酶活化，除了它的作用，在NADPH氧化酶复合物的形成。在目的3中，我们将检验以下假设：G-α-q介导的PLCzeta活化（由血小板活化因子的PMN引发作用诱导）使酶对随后由异源三聚体G蛋白β-γ亚基活化敏感，所述异源三聚体G蛋白β-γ亚基在其G蛋白偶联受体（GPCR）FPR的fMLF刺激后释放。在目的4中，我们将确定新的脂质介质溶血磷脂酰胆碱（LPC）在下调NADPH氧化酶的功能，并解决LPC在防止中性粒细胞介导的肺血管损伤的作用。我们将检验LPC与其G-α-S-偶联的假设， 受体G2 A导致细胞内cAMP升高，从而抑制PMN O2-产生。通过将细胞和分子研究纳入小鼠肺模型中微血管通透性的研究，我们将更好地了解GPCR激活NADPH氧化酶的机制以及如何调节NADPH氧化酶的激活。有了这些见解，我们将能够开发新的治疗策略，靶向不适当的PMN激活和PMN介导的肺血管损伤和水肿。