gp91 phox Function in VCAM-1-dependent Lung Eosinophilia

gp91 phox 在 VCAM-1 依赖性肺嗜酸性粒细胞增多中的功能

• 批准号: 6923320
• 负责人: JOAN M COOK-MILLS
• 金额: $ 34.54万
• 依托单位: UNIVERSITY OF CINCINNATI
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-04-01 至 2005-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6923320
• 关键词: NAD(P)H dehydrogenase; asthma; enzyme activity; eosinophilia; free radical oxygen; glycoproteins; laboratory mouse; pathologic process; protein structure function; receptor binding; receptor expression; vascular cell adhesion molecule; vascular endothelium

DESCRIPTION (provided by applicant): Asthmatic responses are induced by environmental oxidants and allergens. A component of the asthmatic response is lung eosinophilia. Eosinophilia with asthma is a risk factor for mortality from chronic obstructive pulmonary disease. Infiltration of eosinophils into the lung in experimental asthma is dependent on the adhesion molecule vascular cell adhesion molecule-1 (VCAM-1) on endothelial cells. We have discovered a signal transduction pathway for VCAM-1. VCAM-1 activates endothelial cell NADPH oxidase production of low levels of reactive oxygen species (ROS). These ROS are required for localized endothelial cell retraction during VCAM- 1-dependent leukocyte migration in vitro. Thus, VCAM-1 is not simply a scaffold for leukocyte migration but activates an endothelial cell 'gate-like' function. We propose to examine VCAM-1-dependent signals in vivo and structure/function analysis of the cytoplasmic domain of VCAM-1. To test whether these VCAM-1 signals function in vivo, VCAM-1-dependent eosinophilia in experimental asthma will be examined. We hypothesize that in experimental asthma, endothelial cell gp91 phox is required for lung eosinophilia. To study the pathogenesis of asthma, we will use a NADPH oxidase deficient model consisting of chimeric mice with wild type leukocytes and gp91 phox deficient nonhematopoietic cells. In specific aims 1-2, we will determine whether ovalbumin-challenged chimeric mice exhibit alterations in lung 1) leukocyte infiltration, 2) expression of adhesion molecules, cytokines and chemokines that regulate eosinophilia, and 3) airway hyperresponsiveness. It will be determined whether endothelial cell gp91 phox-mediated changes in the lung inflammation are rescued using transgenic and adoptive cell transfer approaches. To examine the structure/function of the cytoplasmic domain of VCAM-1, VCAM-1 signals will be examined in vitro using wild type and chimeric VCAM-1 molecules. The 13 amino acid cytoplasmic domain of VCAM-1 is identical in mice and humans, suggesting that it has an important function. This domain contains potential phosphorylation sites. We hypothesize that ligand binding to VCAM-1 activates phosphorylation of the VCAM-1 cytoplasmic domain for stimulation of endothelial cell ROS generation and endothelial cell actin restructuring. Biochemical, pharmacologic and genetic approaches will be used to address this hypothesis. In aim 3, it will be determined whether mutations in serines and tyrosine in the cytoplasmic domain of VCAM-1 alters VCAM-1 activation of NADPH oxidase. In aim 4, it will be determined whether ligand binding to VCAM-1 activates phosphorylation of serines and tyrosine in the cytoplasmic domain of VCAM-1. The identification of mechanisms for VCAM-1 modulation of lung eosinophilia will provide new insights into regulation of lung eosinophilia as well as provide a basis towards proposing interventions in the VCAM-1-dependent eosinophilia component of asthma.

描述（由申请人提供）：哮喘反应是由环境氧化剂和过敏原引起的。哮喘反应的一个组成部分是肺嗜酸性粒细胞增多。哮喘伴嗜酸性粒细胞增多是慢性阻塞性肺病死亡的危险因素。实验性哮喘中嗜酸性粒细胞向肺部的浸润依赖于内皮细胞上的粘附分子血管细胞粘附分子-1 (VCAM-1)。我们发现了 VCAM-1 的信号转导途径。 VCAM-1 激活内皮细胞 NADPH 氧化酶产生低水平的活性氧 (ROS)。这些 ROS 是 VCAM-1 依赖性白细胞体外迁移过程中局部内皮细胞收缩所必需的。因此，VCAM-1 不仅仅是白细胞迁移的支架，而且还激活内皮细胞的“门样”功能。我们建议检查体内 VCAM-1 依赖性信号以及 VCAM-1 细胞质结构域的结构/功能分析。为了测试这些 VCAM-1 信号在体内是否发挥作用，将检查实验性哮喘中 VCAM-1 依赖性嗜酸性粒细胞增多。我们假设在实验性哮喘中，内皮细胞 gp91 phox 是肺嗜酸性粒细胞增多所必需的。为了研究哮喘的发病机制，我们将使用 NADPH 氧化酶缺陷模型，该模型由具有野生型白细胞和 gp91 phox 缺陷非造血细胞的嵌合小鼠组成。在具体目标 1-2 中，我们将确定卵清蛋白攻击的嵌合小鼠是否表现出肺部变化：1) 白细胞浸润，2) 调节嗜酸性粒细胞增多的粘附分子、细胞因子和趋化因子的表达，以及 3) 气道高反应性。将确定内皮细胞 gp91 phox 介导的肺部炎症变化是否可以使用转基因和过继细胞转移方法来挽救。为了检查 VCAM-1 细胞质结构域的结构/功能，将使用野生型和嵌合 VCAM-1 分子在体外检查 VCAM-1 信号。 VCAM-1 的 13 个氨基酸胞质结构域在小鼠和人类中是相同的，表明它具有重要的功能。该结构域包含潜在的磷酸化位点。我们假设配体与 VCAM-1 结合激活 VCAM-1 胞质结构域的磷酸化，从而刺激内皮细胞 ROS 生成和内皮细胞肌动蛋白重组。生物化学、药理学和遗传学方法将用于解决这一假设。在目标 3 中，将确定 VCAM-1 胞质结构域中丝氨酸和酪氨酸的突变是否会改变 VCAM-1 对 NADPH 氧化酶的激活。在目标 4 中，将确定与 VCAM-1 结合的配体是否激活 VCAM-1 胞质结构域中丝氨酸和酪氨酸的磷酸化。确定 VCAM-1 调节肺嗜酸性粒细胞增多的机制将为肺嗜酸性粒细胞增多的调节提供新的见解，并为提出干预哮喘的 VCAM-1 依赖性嗜酸性粒细胞增多奠定基础。