Hydrogen Peroxide and Asbestosis

描述(申请人提供)：项目摘要/摘要：过氧化氢是参与肺细胞信号转导的最重要的ROS。PMA已被用于研究过氧化氢介导的信号转导，但石棉肺是一个很好的模型来探索过氧化氢在细胞信号转导中的作用，因为石棉具有生物学意义，并且已知可以诱导高水平的过氧化氢。石棉肺患者肺泡巨噬细胞释放肿瘤坏死因子-α在疾病的发展过程中起着至关重要的作用；然而，将过氧化氢的产生与肿瘤坏死因子-α的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的肺泡巨噬细胞类似单核细胞。我们的数据显示，与肺泡巨噬细胞不同，单核细胞在体外用石棉刺激时会产生肿瘤坏死因子-α。因此，人类单核细胞提供了一个很好的模型来研究肿瘤坏死因子-α基因的表达，因为它与过氧化氢的产生有关。在这方面，我们的初步数据表明，在单核细胞中，H_2O_2是产生肿瘤坏死因子-α所必需的，并且H_2O_2的来源主要是铜，锌-超氧化物歧化酶。我们的初步数据还表明，在石棉肺患者的肺泡巨噬细胞中，铜、锌-超氧化物歧化酶高度表达。由于我们的数据显示石棉肺患者的肺泡巨噬细胞有高水平的p38 MAP激酶活性，而ERK活性缺失，而H_2O_2激活P38并抑制ERK，我们推测H_2O_2在差异性调节MAP激酶活性中起关键作用，而铜，锌-超氧化物歧化酶促进人单核细胞分化MAP K活性和肿瘤坏死因子-α的产生。除了使用更具生物学意义的石棉进行研究外，我们还将在最初的研究中使用PMA来了解过氧化氢在细胞信号传递中的作用。在目标1中，我们将确定铜，锌-超氧化物歧化酶诱导的过氧化氢是否调节MAP激酶的激活。由于MAP激酶活性通常由磷酸酶控制，我们将调查H_2O_2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。比较野生型和铜锌超氧化物歧化酶KO小鼠的过氧化氢生成和MAP活性，将提供生物学意义。在目标2中，我们将确定铜，锌-超氧化物歧化酶诱导的过氧化氢是否增加单核细胞中肿瘤坏死因子-α的产生，以及最佳的肿瘤坏死因子-α的表达是否取决于不同的MAPK活性。我们将比较石棉肺患者肺泡巨噬细胞和正常巨噬细胞的MAP激酶、铜、锌-超氧化物歧化酶活性和过氧化氢的产生。通过研究野生型和铜锌超氧化物歧化酶KO小鼠间质纤维化的发展，将提供更多的生物学相关性。项目简介：暴露在石棉和其他环境物质中，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。虽然这项建议中的研究与石棉肺有关，但它们也将适用于其他炎症性和纤维性肺部疾病。

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.