An In-vitro Investigation of Glutathione Transferases in Idiopathic Pulmonary Fibrosis
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Liberti, Eileen
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Abstract
Idiopathic pulmonary fibrosis (IPF) is a fatal interstitial disease of the lung. While pathogenic mechanisms are uncertain, IPF is described as a disease of dysregulated wound repair and is characterized by alveolar epithelial cell (AEC) death and excessive accumulation of activated fibroblasts. As part of the unchecked wound repair response, persistent IPF fibroblasts produce excessive extracellular matrix that destroys lung architecture and impairs gas exchange, eventually leading to death. Lung tissue by its natural function experiences high oxidative stress. Additionally, IPF lung fibroblasts are known to excrete hydrogen peroxide. If IPF fibroblasts possess the ability to better metabolize and manage reactive oxygen species (ROS), it will contribute to their survival in the IPF lung. Management of this oxidative stress is mediated via superoxide dismutase, catalase and the highly polymorphic glutathione transferases. This study examines the role that glutathione transferases (GSTs) and GST𝜋 (GSTP) play in the management of oxidative stress and survival of the IPF fibroblasts. Using an in vitro model of IPF this study was conducted using IPF and normal primary culture fibroblast cells (n = 3), human lung epithelial A549 cells and human small airway epithelial cells. Cells were challenged with H2O2 to simulate oxidative stress and survival was assessed with and without the GSTP inhibitor, TLK199. Gene expression was assessed by means of quantitative real time polymerase chain reaction (qPCR). GST enzymatic activity was evaluated using a fluorescent substrate reporter for total GST activity. Increased survival in the presence of H2O2 was observed in IPF fibroblasts compared to normal fibroblasts. This increased survival correlated with both increased GST gene expression and increased GST enzyme activity in IPF fibroblasts. This is in contrast to normal fibroblast activity. Exposure to the GSTP inhibitor TLK199 in the presence of H2O2 decreased total GST enzyme activity and cell survival bringing IPF fibroblasts in line with normal fibroblasts. Our data also shows differential expression of GST isoforms in IPF fibroblasts with disease-specific upregulation of GSTA4, GSTK1 and GSTP1 in IPF fibroblasts in the presence of H2O2, demonstrating a more robust response to oxidative stress. In this study we focus on the role played by GSTs as they relate to the metabolism of ROS in IPF. This differential metabolism may be instrumental in IPF fibroblast survival in the hostile environment of the IPF lung.
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Idiopathic Pulmonary Fibrosis, Glutathione Transferase, JNK/c-Jun, I.P.F., GSTP