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.
We have found that exposure to environmental agents, such as asbestos, increases hydrogen peroxide generation in inflammatory cells in the lung. The source of this increase is from the mitochondria. Copper, zinc superoxide dismutase is an enzyme that can increase the production of hydrogen peroxide. Exposure to asbestos can also result in fibrosis, or scarring, in the lung. This application will determine if mechanism and source of hydrogen peroxide generation and its role in mediating pulmonary fibrosis.
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|Larson-Casey, Jennifer L; Carter, A Brent (2016) Determination of H2O2 Generation by pHPA Assay. Bio Protoc 6:|
|Larson-Casey, Jennifer L; Carter, A Brent (2016) Assay to evaluate BAL Fluid regulation of Fibroblast ?-SMA Expression. Bio Protoc 6:|
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|Ryan, Alan J; Larson-Casey, Jennifer L; He, Chao et al. (2014) Asbestos-induced disruption of calcium homeostasis induces endoplasmic reticulum stress in macrophages. J Biol Chem 289:33391-403|
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