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

描述（由申请人提供）： 石棉肺是引起肺纤维化的重要原因。细胞外基质异常沉积是石棉诱导的肺纤维化的标志，其特征在于基质沉积和降解之间的不平衡。天冬氨酸纤维在肺中保留较长时间，这导致活性氧（ROS）（包括H2 O2）从肺泡巨噬细胞释放。肺泡巨噬细胞释放H2 O2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，H2 O2主要是在巨噬细胞的线粒体中产生的，H2 O2的来源主要是来自过氧化物生成酶，铜，锌-SOD，这是本地化的线粒体膜间隙（IMS）石棉暴露后。信号分子如H2 O2的新兴靶点是调节纤维化表型的基质金属蛋白酶（MMP）家族。在这方面，我们的初步数据还表明，从石棉肺患者获得的肺泡巨噬细胞具有增加的Cu，Zn-SOD活性，产生10倍以上的H2 O2，和表达20倍以下的MMP-9相比，正常受试者。此外，这种表达的降低与BAL液中MMP-9活性降低和暴露于BAL液的成纤维细胞胶原分泌增加相关。然而，在肺纤维化中，过氧化物诱导酶如Cu，Zn-SOD对MMP基因转录和MMP活性的分子调控的数据有限。我们的初步数据还表明，过表达Cu，Zn-SOD显著抑制MMP-9启动子活性和MMP-9活性，并且通过siRNA敲低Cu，Zn-SOD增强MMP-9活性并减少成纤维细胞的胶原分泌。连接H2 O2与MMP-9基因表达的信号通路涉及MAP激酶。除了其他MAP激酶外，ERK MAP激酶已显示调节MMP-9基因表达。我们的数据表明，与WT小鼠相比，Cu，Zn-SOD-/-小鼠具有高水平的活化ERK。这些新的初步数据表明，Cu，Zn-SOD和H2 O2，通过ERK的控制，作为抑制性输入的MMP-9基因的转录调节，这与MMP-9的活性，并诱导肺纤维化的发展。我们推测线粒体Cu，Zn-SOD通过调节胶原沉积诱导肺纤维化，其机制涉及H2 O2，ERK和MMP-9。我们将用三个具体目标来评估这个假设。目的1将确定Cu，Zn-SOD在IMS中移位和激活并增加H2 O2稳态水平的机制。我们还将确定铜分子伴侣对Cu，Zn-SOD（CCS）在调节转运和激活中的作用。目的2探讨Cu，Zn-SOD对MMP-9基因表达和MMP-9活性的调节作用及Cu，Zn-SOD抑制ERK的机制。目的3将提供生物学相关性，利用石棉肺的体内模型来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，但它们将为理解炎症和纤维化肺部疾病提供重要线索，并可能提供潜在的靶点以减轻肺纤维化的发展。\

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. \