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A6207 - C/EBP Homologous Protein Mediates ER Stress-Induced Lung Fibrosis: A Potential Role for Localized Hypoxia
Author Block: A. Burman1, J. A. Kropski2, C. L. Calvi2, W. Lawson2, T. S. Blackwell2, H. Tanjore2; 1Vanderbilt University, Nashville, TN, United States, 2Vanderbilt University Medical Center, Nashville, TN, United States.
Rationale: Endoplasmic reticulum (ER) stress in type II alveolar epithelial cells (AECs) is prominent in idiopathic pulmonary fibrosis (IPF); however, mechanisms linking ER stress and fibrosis are poorly understood. We hypothesized that C/EBP homologous protein (CHOP) mediates the impact of ER stress on lung fibrosis.
Methods: Transgenic mice expressing a mutant version of surfactant protein C (L188Q SFTPC) associated with human IPF that results in ER stress in type II AECs were crossed with CHOP deficient mice. Mice were treated with intratracheal bleomycin (0.08 units) and harvested 3 weeks later. In separate experiments, wild type (WT) and CHOP-/- mice were treated with 6 repetitive doses of bleomycin (0.04 units) at intervals of 2 weeks. Mice in this experiment were injected with pimonidazole (hypoxia probe) 3 hours before harvest. Evaluations for cellular hypoxia, ER stress, AEC apoptosis and fibrosis were performed. Mouse lung epithelial cells (MLE12) were used for in vitro studies and hypoxia (1.5% O2) was used to induce ER stress. Human IPF lung sections were stained for hypoxia markers and CHOP.
Results: Following a single intratracheal injection of bleomycin, L188Q SFTPC mice had increased lung fibrosis compared to WT controls; however, this increase in fibrosis was eliminated in L188Q SFTPC/CHOP-/- mice. In contrast, no difference in lung fibrosis was observed between CHOP-/- and WT mice, where ER stress was minimally induced. In the repetitive bleomycin model, we detected robust induction of cellular hypoxia (pimonidazole staining) and ER stress markers, including CHOP, in type II AECs around areas of fibrosis. Compared to WT mice, CHOP-/- mice had significantly reduced fibrosis, as well as decreased AEC apoptosis, following repetitive bleomycin. Treatment of MLE12 cells with siRNA targeting CHOP showed that CHOP regulates AEC survival in hypoxia. An apoptosis array evaluating 84 genes revealed CHOP-regulated targets that were further validated by qPCR in vitro and in vivo. Top targets included activating transcription factor (ATF5), growth arrest DNA-damage-inducible 45 alpha (GADD45a), and BCL2 interacting protein 3 like (BNIP3L). In human IPF lung sections, robust induction of CHOP and hypoxia markers (pyruvate kinase and carbonic anhydrase) was observed in type II AECs.
Conclusion: CHOP regulates fibrosis following repetitive bleomycin treatment and in other models where substantial ER stress is induced in AECs. Localized hypoxia in areas of injured lung is a potential etiological factor in ER stress-induced CHOP expression. CHOP and its downstream mediators could prove to be potential therapeutic targets in lung fibrosis.