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，但是在特定情况下，O2-产生也会导致组织损伤，例如在急性肺损伤中发现。我们目前对PMN烟酰胺腺嘌呤二核苷酸磷酸（NADPH）氧化酶的理解主要基于使用佛波酯的研究。诸如N-甲基甲基 - leu-phe（FMLF）等化学吸引剂诱导o2-生成 PMN，但潜在的机制仍然很少理解。使用COS-PHOX细胞的一种新型的，全细胞的重建系统已开发出以系统的方式解决NADPH氧化酶激活的信号传导机制。这些研究将与使用基因缺失的小鼠的野生型PMN和PMN进行实验相辅相成，以及解决特定信号通路激活如何诱导肺血管损伤的实验，或者在其他情况下可能具有保护性。 AIM 1基于我们的初步发现，即PKCZETA足以重新建立FMLF诱导的Cos-Phox细胞中的O2-产生。因此，我们将检验以下假设：PKCZETA在NADPH氧化酶的FMLF激活中起关键作用并确定所涉及的磷酸化步骤。在AIM 2中，我们将扩展发现P40Phox有选择地增强FMLF-但不为PMA诱导的O2-- 生成，并将检验以下假设：p40phox在调节介导NADPH氧化酶活性的信号传导途径中起重要作用，此外其在NADPH氧化酶复合物的形成中的作用。在AIM 3中，我们将检验以下假说：G-Alpha-Q介导的plczeta激活（由血小板激活因子的PMN启动作用引起）使酶敏感以随后创三聚聚合物G蛋白β-GAMMA亚基的后续激活，这些β-GAMMAβ-GAMMA亚基是基于FMLF蛋白的FMLF蛋白（Gppr）fprpr）fprpr）couppr ppr）coupr ppreper couppre couppre cou couppre cou cou cou cou cou cou cou cou cou cou coupred a insepimeric overimeric G蛋白β-GAMMANits。在AIM 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氧化酶中的功能，并解决LPC在防止PMN介导的肺血管损伤中的作用。我们将测试LPC与其G-Alpha-S耦合结合的假设 受体G2A导致细胞内cAMP的增加，从而抑制PMN O2的产生。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中，我们将更好地了解GPCR的NADPH氧化酶活性的机制以及如何调节NADPH氧化酶活性。通过获得的见解，我们将有能力开发针对不适当的PMN激活和PMN介导的肺血管损伤和水肿的新型治疗策略。