Hydrogen Peroxide and Asbestosis

项目概述/摘要：H2O2是参与肺细胞信号传递的最重要的活性氧。PMA已被用于研究H2O2介导的信号传导，但石棉肺是探索H2O2在细胞信号传导中的作用的一个很好的模型，因为石棉具有生物学相关性，并且已知会诱导高水平的H2O2。石棉肺患者肺泡巨噬细胞释放tnf - α在该疾病的发展中至关重要；然而，将H2O2生成与tnf - α产生联系起来的机制尚不清楚。石棉肺的另一个特征是患者肺泡巨噬细胞类似单核细胞。我们的数据表明，单核细胞在体外用石棉刺激时产生tnf - α，与肺泡巨噬细胞不同。因此，人类单核细胞为探索tnf - α基因表达提供了一个很好的模型，因为它与H2O2的产生有关。在这方面，我们的初步数据表明H2O2是单核细胞中tnf - α的最佳生成所必需的，H2O2的来源主要来自Cu，Zn-SOD。我们的初步数据还表明，Cu，Zn-SOD在石棉沉滞患者肺泡巨噬细胞中高度表达。由于我们的数据显示石棉沉滞患者的肺泡巨噬细胞具有高水平的p38 MAP激酶活性和缺乏ERK活性，并且H2O2激活p38并抑制ERK，我们假设H2O2在差异调节MAP激酶活性中起关键作用，而Cu，Zn-SOD增强了人类单核细胞的差异MAP激酶激活和tnf - α的产生。我们将在最初的研究中使用PMA来了解H2O2在细胞信号传导中的作用，此外还将使用石棉进行研究，因为石棉在生物学上更相关。在Aim 1中，我们将确定Cu， zn - sod诱导的H2O2是否调节MAP激酶的激活。由于MAP激酶的活性通常由磷酸酶控制，我们将研究H2O2是否氧化磷酸酶MKP-1，并诱导PP2A的表达，从而分别导致p38激活和ERK失活。比较野生型和Cu，Zn- SOD KO小鼠的H2O2生成和MAP激活情况将提供生物学相关性。在Aim 2中，我们将确定Cu、zn - sod诱导的H2O2是否会增加单核细胞中tnf - α的产生，以及tnf - α的最佳表达是否取决于不同的MAP激酶活性。我们将比较取自石棉沉滞症患者肺泡巨噬细胞与正常巨噬细胞的MAP激酶、Cu、Zn-SOD活性和H2O2生成。通过研究野生型和Cu，Zn- SOD KO小鼠间质纤维化的发展，将提供额外的生物学相关性。项目说明：接触石棉和其他环境因素，如柴油废气和颗粒物，会增加肺部过氧化氢的生成。该项目的目标是了解过氧化氢导致肺损伤和纤维化的机制。虽然本提案中的研究与石棉沉滞有关，但它们也适用于其他炎症性和纤维化性肺部疾病。

DESCRIPTION (provided by applicant): Project Summary/Abstract: H2O2 is the most important ROS involved in cell signaling in the lung. PMA has been used to study H2O2-mediated signaling, but asbestosis is a good model to explore the role of H2O2 in cell signaling because asbestos is biologically relevant and is known to induce high levels of H2O2. The release of TNF-alpha by alveolar macrophages in asbestosis patients is critical in the development of the disease; however, the mechanism linking H2O2 generation to TNF-alpha production is not known. Another feature of asbestosis is that alveolar macrophages from patients resemble monocytes. Our data show that monocytes produce TNF-alpha when stimulated, in vitro, with asbestos unlike alveolar macrophages. Thus, human monocytes provide a good model to explore TNF-alpha gene expression, as it relates to H2O2 generation. In this regard, our preliminary data show that H2O2 is necessary for optimal production of TNF-alpha in monocytes and that the source of H2O2 is primarily from Cu,Zn-SOD. Our preliminary data also demonstrate that Cu,Zn-SOD is highly expressed in alveolar macrophages obtained from patients with asbestosis. Since our data shows that alveolar macrophages from asbestosis patients have a high level p38 MAP kinase activity and absent ERK activity and that H2O2 activates p38 and inhibits ERK, we hypothesize that H2O2 plays a pivotal role in differentially modulating MAP kinase activity, and Cu,Zn-SOD enhances differential MAP kinase activation and TNF-alpha production in human monocytes. We will use PMA in the initial studies to understand the role of H2O2 in cell signaling in addition to performing studies using asbestos, which is more biologically relevant. In Aim 1 we will determine if Cu,Zn-SOD-induced H2O2 modulates MAP kinase activation. Since MAP kinase activity is often controlled by phosphatases, we will investigate if H2O2 is oxidizing a phosphatase, MKP-1, and inducing expression of PP2A, which results in p38 activation and ERK inactivation, respectively. The comparison of H2O2 generation and MAP activation in wild-type and Cu,Zn- SOD KO mice will provide biological relevance. In Aim 2 we will determine if Cu,Zn-SOD-induced H2O2 increases TNF-alpha production in monocytes and if optimal TNF-alpha expression depends on differential MAP kinase activity. We will compare MAP kinase and Cu,Zn-SOD activity and H2O2 generation in alveolar macrophages obtained from asbestosis patients to normal macrophages. Additional biological relevance will be provided by investigating the development of interstitial fibrosis in wild-type and Cu,Zn- SOD KO mice. Project Narrative: Exposure to asbestos and other environmental agents, such as diesel exhaust and particulate matter, increase hydrogen peroxide generation in the lung. The goal of the project is to understand the mechanisms by which hydrogen peroxide causes lung injury and fibrosis. Although the studies in this proposal relate to asbestosis, they will also apply to other inflammatory and fibrotic lung diseases.