Cu,Zn-SOD, MMP-9, and Asbestosis

描述(由申请人提供)： 石棉肺是导致肺纤维化的重要原因。异常的细胞外基质沉积是石棉诱导的肺纤维化的一个特征，其特征是基质沉积和降解之间的失衡。石棉纤维在肺中停留的时间较长，导致肺泡巨噬细胞释放包括过氧化氢在内的活性氧物种(ROS)。肺泡巨噬细胞释放H_2O_2在石棉肺异常基质沉积的发病机制中起着不可或缺的作用。我们的初步数据表明，过氧化氢主要产生于巨噬细胞的线粒体中，过氧化氢的来源主要来自过氧化物酶-铜，锌-超氧化物歧化酶，该酶定位于石棉暴露后的线粒体膜间隙(IMS)。一个新兴的信号分子靶点，如过氧化氢，是调节纤维化表型的基质金属蛋白酶(MMPs)家族。在这方面，我们的初步数据还表明，与正常对照组相比，石棉肺患者肺泡巨噬细胞的铜、锌-超氧化物歧化酶活性增加了10倍，产生的过氧化氢增加了10倍，基质金属蛋白酶-9的表达减少了20倍。此外，这种表达的减少与BAL液中MMP9活性的降低和BAL液中成纤维细胞胶原分泌的增加有关。然而，关于过氧化物酶如铜、锌-超氧化物歧化酶在肺纤维化中对基质金属蛋白酶基因转录和活性的分子调控的研究资料有限。我们的初步数据还表明，铜，锌-超氧化物歧化酶的过表达显著抑制了基质金属蛋白酶-9的启动子活性和基质金属蛋白酶-9的活性，而siRNA抑制铜，锌-超氧化物歧化酶的表达增强了成纤维细胞的基质金属蛋白酶-9的活性，减少了成纤维细胞的胶原分泌。连接H_2O_2和基质金属蛋白酶-9基因表达的信号通路涉及到丝裂原活化蛋白激酶。除了其他的MAP激酶外，ERK MAP激酶已经被证明可以调节MMP9基因的表达。我们的数据表明，与WT小鼠相比，铜，锌-SOD-/-小鼠具有更高水平的激活ERK。这些新的初步数据表明，铜、锌-超氧化物歧化酶和过氧化氢通过ERK的调控，对与基质金属蛋白酶-9活性相关的基质金属蛋白酶-9基因的转录调控起抑制作用，并诱导肺纤维化的发生。我们推测线粒体铜锌超氧化物歧化酶通过调节胶原沉积而诱导肺纤维化，其机制涉及过氧化氢、ERK和基质金属蛋白酶-9。我们将用三个具体目标来评估这一假说。目的1将确定铜，锌-超氧化物歧化酶在IMS中移位和激活的机制，并增加H_2O_2的稳态水平。我们还将确定铜伴侣对铜，锌-超氧化物歧化酶(CCS)在调节移位和激活中的作用。目的2确定铜锌超氧化物歧化酶在调节基质金属蛋白酶-9基因表达和活性中的作用及其抑制ERK的机制。目的3将利用石棉肺的体内模型提供生物学相关性，以了解肺泡巨噬细胞和成纤维细胞胶原沉积之间的相互作用。虽然这些研究与石棉肺有关，但它们将为了解炎症性和纤维性肺部疾病提供重要线索，并可能为减缓肺纤维化的发展提供潜在的靶点。\

DESCRIPTION (provided by applicant): Asbestosis is an important cause of pulmonary fibrosis. Aberrant extracellular matrix deposition is a hallmark of asbestos-induced pulmonary fibrosis and is characterized by an imbalance between matrix deposition and degradation. Asbestos fibers remain in the lung for extended periods of time, which leads to the release of reactive oxygen species (ROS), including H2O2, from alveolar macrophages. The release of H2O2 by alveolar macrophages plays an integral role in the pathogenesis of aberrant matrix deposition in asbestosis. Our preliminary data show that H2O2 is primarily generated in the mitochondria in macrophages and that the source of H2O2 is predominantly derived from the peroxide-generating enzyme, Cu,Zn-SOD, which is localized in the mitochondrial intermembrane space (IMS) after asbestos exposure. An emerging target for signaling molecules, such as H2O2, are the family of matrix metalloproteinases (MMPs) which regulate the fibrotic phenotype. In this regard, our preliminary data also demonstrate that alveolar macrophages obtained from patients with asbestosis have increased Cu,Zn-SOD activity, generate 10-fold more H2O2, and express 20-fold less MMP-9 compared to normal subjects. Moreover, this decrease in expression was associated with decreased MMP-9 activity in BAL fluid and increased collagen secretion by fibroblasts exposed to BAL fluid. There is limited data, however, on the molecular regulation of MMP gene transcription and MMP activity by peroxide-inducing enzymes, such as Cu,Zn-SOD, in pulmonary fibrosis. Our preliminary data also demonstrate that over expression of Cu,Zn-SOD significantly inhibits MMP-9 promoter activity and MMP-9 activity, and knockdown of Cu,Zn-SOD by siRNA enhances MMP-9 activity and reduces collagen secretion by fibroblasts. The signaling pathway linking H2O2 to MMP-9 gene expression involves MAP kinases. In addition to other MAP kinases, the ERK MAP kinase has been shown to regulate MMP-9 gene expression. Our data demonstrates that Cu,Zn-SOD-/- mice have high levels of activated ERK compared to WT mice. These novel preliminary data suggest that Cu,Zn-SOD and H2O2, via control of ERK, act as inhibitory inputs to the transcriptional regulation of the MMP-9 gene, which correlates with MMP-9 activity, and induces the development of pulmonary fibrosis. We postulate that mitochondrial Cu,Zn-SOD induces pulmonary fibrosis by modulating collagen deposition by a mechanism involving H2O2, ERK, and MMP-9. We will evaluate this hypothesis with three Specific Aims. Aim 1 will determine the mechanism by which Cu,Zn-SOD translocates and is activated in the IMS and increases the steady-state levels of H2O2. We will also determine the role of copper chaperone for Cu,Zn-SOD (CCS) in regulating translocation and activation. Aim 2 will determine the role of Cu,Zn-SOD in regulating MMP-9 gene expression and MMP-9 activity and the mechanism by which Cu,Zn-SOD inhibits ERK. Aim 3 will provide biological relevance utilizing an in vivo model of asbestosis to understand the interaction between alveolar macrophages and fibroblast collagen deposition. Although these studies are related to asbestosis, they will provide important clues in understanding inflammatory and fibrotic lung diseases and may provide potential targets to attenuate the development of pulmonary fibrosis. \