Localized hypoxia links ER stress to lung fibrosis through induction of C/EBP homologous protein
Ankita Burman, … , Timothy S. Blackwell, Harikrishna Tanjore
Ankita Burman, … , Timothy S. Blackwell, Harikrishna Tanjore
Published August 23, 2018
Citation Information: JCI Insight. 2018;3(16):e99543. https://doi.org/10.1172/jci.insight.99543.
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Research Article Pulmonology

Localized hypoxia links ER stress to lung fibrosis through induction of C/EBP homologous protein

Abstract

ER stress in type II alveolar epithelial cells (AECs) is common in idiopathic pulmonary fibrosis (IPF), but the contribution of ER stress to lung fibrosis is poorly understood. We found that mice deficient in C/EBP homologous protein (CHOP), an ER stress–regulated transcription factor, were protected from lung fibrosis and AEC apoptosis in 3 separate models where substantial ER stress was identified. In mice treated with repetitive intratracheal bleomycin, we identified localized hypoxia in type II AECs as a potential mechanism explaining ER stress. To test the role of hypoxia in lung fibrosis, we treated mice with bleomycin, followed by exposure to 14% O2, which exacerbated ER stress and lung fibrosis. Under these experimental conditions, CHOP–/– mice, but not mice with epithelial HIF (HIF1/HIF2) deletion, were protected from AEC apoptosis and fibrosis. In vitro studies revealed that CHOP regulates hypoxia-induced apoptosis in AECs via the inositol-requiring enzyme 1α (IRE1α) and the PKR-like ER kinase (PERK) pathways. In human IPF lungs, CHOP and hypoxia markers were both upregulated in type II AECs, supporting a conclusion that localized hypoxia results in ER stress–induced CHOP expression, thereby augmenting type II AEC apoptosis and potentiating lung fibrosis.

Authors

Ankita Burman, Jonathan A. Kropski, Carla L. Calvi, Ana P. Serezani, Bruno D. Pascoalino, Wei Han, Taylor Sherrill, Linda Gleaves, William E. Lawson, Lisa R. Young, Timothy S. Blackwell, Harikrishna Tanjore

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Figure 4

Exposure to hypoxia following bleomycin treatment increases ER stress and worsens lung fibrosis.

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Exposure to hypoxia following bleomycin treatment increases ER stress an...
(A) Schematic representation of experimental design in which a single dose of bleomycin (0.04 units) was injected intratracheally, followed 7 days later by exposure to normobaric hypoxia or normoxia (21% O2) for an additional 14 days. (B) Kaplan-Meier survival plot for mice treated with bleomycin and exposed to 21%, 14%, 12%, or 10% O2. n = 48 (in 21% O2), 35 (in 14% O2), 16 (in 12% O2), and 12 (in 10% O2). (C) Representative immunostaining for CHOP expression and pimonidazole adducts in mice injected with pimonidazole (60 μg/g) at 3 hours before lung harvest. Mice were treated with bleomycin, followed 7 days later by exposure to 14% O2 for an additional 14 days or maintenance in 21% O2. Additional controls were only exposed to 14% O2 for 2 weeks or were maintained in 21% O2. Scale bars: 800 μm (low magnification): 60 μm (high magnification). (D) Western blots for ER stress markers CHOP, PDI, ATF4, and XBP1s in lung tissue lysates. β-Actin was used as a loading control. (E) Quantification of CHOP+ AECs per high-power field (HPF) on lung sections. (F) Masson’s trichrome staining and (G) morphometric evaluation of lung fibrosis. (H) Total soluble collagen in right lower lobe (RLL) by Sircol assay. Comparisons between groups were made using unpaired, 2-tailed Student’s t test (G) or 1-way ANOVA with Tukey’s post hoc test (E and H). *P < 0.05 compared with single bleo + 21% O2, **P < 0.05 compared with untreated + 21% O2.