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) 是吞噬细胞，可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，例如急性肺损伤。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。 N-甲酰基-Met-Leu-Phe (fMLF) 等化学引诱剂可诱导 O2 生成 PMN，但其潜在机制仍知之甚少。一种使用 COS-phox 细胞的新型全细胞重建系统已被开发出来，以系统的方式解决介导 NADPH 氧化酶激活的信号机制。这些研究将与使用野生型中性粒细胞和来自基因缺失小鼠的中性粒细胞的实验相补充，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。目标 1 基于我们的初步发现，即 PKCzeta 足以在 COS-phox 细胞中重建 fMLF 诱导的 O2 产生。因此，我们将检验 PKCzeta 在 NADPH 氧化酶的 fMLF 激活中发挥关键作用的假设，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 p40phox 选择性增强 fMLF- 而不是 PMA 诱导的 O2- 生成，并将检验 p40phox 除了在 NADPH 氧化酶复合物形成中的作用之外，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，我们将测试以下假设：G-α-q 介导的 PLCzeta 激活（由血小板激活因子的 PMN 启动作用诱导）使酶敏感，以便随后被异源三聚体 G 蛋白 β-γ 亚基激活，这些亚基是在 fMLF 刺激其 G 蛋白偶联受体 (GPCR)、FPR 后释放。在目标 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-alpha-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纳入小鼠肺模型微血管通透性的研究中，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。凭借所获得的见解，我们将能够针对不适当的中性粒细胞激活和中性粒细胞介导的肺血管损伤和水肿开发新的治疗策略。