PKR-dependent CHOP induction limits hyperoxia-induced lung injury

Abstract

Supplemental O(2) is commonly employed in patients with respiratory failure; however, hyperoxia is also a potential contributor to lung injury. In animal models, hyperoxia causes oxidative stress in the lungs, resulting in increased inflammation, edema, and permeability. We hypothesized that oxidative stress from prolonged hyperoxia leads to endoplasmic reticulum (ER) stress, resulting in activation of the unfolded protein response (UPR) and induction of CCAAT enhancer-binding protein homologous protein (CHOP), a transcription factor associated with cell death in the setting of persistent ER stress. To test this hypothesis, we exposed the mouse lung epithelial cell line MLE-12 to 95% O(2) for 8-24 h and evaluated for evidence of UPR induction and CHOP induction. Hyperoxia caused increased CHOP expression without other evidence of UPR activation. Because CHOP expression is preceded by phosphorylation of the α-subunit of the eukaryotic initiation factor-2 (eIF2α), we evaluated the role of double-stranded RNA-activated protein kinase (PKR), a non-UPR-associated eIF2α kinase. Hyperoxia caused PKR phosphorylation, and RNA interference knockdown of PKR attenuated hyperoxia-induced CHOP expression. In vivo, hyperoxia induced PKR phosphorylation and CHOP expression in the lungs without other biochemical evidence for ER stress. Additionally, Ddit3(-/-) (CHOP-null) mice had increased lung edema and permeability, indicating a previously unknown protective role for CHOP after prolonged hyperoxia. We conclude that hyperoxia increases CHOP expression via an ER stress-independent, PKR-dependent pathway and that increased CHOP expression protects against hyperoxia-induced lung injury.

Fig. 1.

Hyperoxia induces CCAAT enhancer-binding protein homologous protein (CHOP) in vitro. A: fold change in CHOP and activating transcription factor (ATF)-4 mRNA normalized to hypoxanthine ribosyltransferase (HPRT1) from murine alveolar epithelial (MLE-12) cells exposed to 95% O₂-5% CO₂ for up to 24 h. B: representative immunoblots of whole cell lysates for phosphorylated eukaryotic initiation factor (eIF)-2α (p-eIF2α), total eIF2α, CHOP, and actin from MLE-12 cells exposed to 95% O₂-5% CO₂ for up to 24 h. tm, Lysate from cells exposed to 5 μM tunicamycin for 24 h to induce ER stress. C: fold change in CHOP mRNA normalized to HPRT1 from primary mouse lung type II epithelial cells exposed to 95% O₂-5% CO₂ for up to 72 h. Quantitative PCR data summarize 3 separate experiments for each time. *P < 0.05 vs. 0 h.

Fig. 2.

Hyperoxia does not cause general activation of the unfolded protein response (UPR). A: representative agarose gel electrophoresis of X-box binding protein (XBP1) PCR product demonstrating different-sized cDNA corresponding to spliced and unspliced XBP1 mRNA. mRNA collected from MLE-12 cells was exposed to 95% O₂-5% CO₂ for up to 24 h or 5 μM tunicamycin (tm) for 6 h. B: fold change in binding protein (BiP) mRNA normalized to HPRT1 from MLE-12 cells exposed to 95% O₂-5% CO₂ for up to 24 h. Data summarize 3 separate experiments for each time. C: representative immunoblots of whole cell lysates for BiP, phosphorylated protein kinase RNA-like endoplasmic reticulum kinase (p-PERK), total PERK, and actin from MLE-12 cells exposed to 95% O₂-5% CO₂ for up to 24 h or 5 μM tunicamycin for 24 h.

Fig. 3.

Double-stranded RNA-dependent protein kinase (PKR) is activated by hyperoxia and induces downstream CHOP expression. A: representative immunoblots of whole cell lysates for phosphorylated PKR (p-PKR) and total PKR from MLE-12 cells exposed to 95% O₂-5% CO₂ for up to 24 h or 5 μM tunicamycin for 24 h. B–D: PKR, ATF4, and CHOP mRNA expression normalized to HPRT1 in MLE-12 cells exposed to transfection reagent alone (untreated), PKR small interfering RNA (siRNA), or control (cyclophilin B) siRNA (25 nM for 72 h). Cells were exposed to normoxia (21% O₂) or hyperoxia (95% O₂) for the final 24 h. Data summarize 3 independent experiments. *P < 0.05.

Fig. 4.

Hyperoxia increases CHOP expression and PKR phosphorylation but does not cause general activation of endoplasmic reticulum (ER) stress responses. A: ATF4 and CHOP mRNA normalized to HPRT1 from lungs of mice exposed to 95% O₂ for up to 72 h (n = 5/group). *P < 0.05. B: representative immunoblots of phosphorylated and total eIF2α, CHOP, BiP, and actin from whole lung lysates of mice exposed to 95% O₂ for up to 72 h. tm, MLE-12 cells treated with 5 μM tunicamycin for 24 h. C: XBP1 PCR product demonstrating different-sized cDNA corresponding to spliced and unspliced XBP1 mRNA. mRNA collected from lungs of mice was exposed to 95% O₂ for up to 72 h. tm, MLE-12 cells treated with 5 μM tunicamycin for 6 h. D: immunoblots of phosphorylated PKR and actin from lung lysates of mice exposed to 95% O₂ for up to 72 h.

Fig. 5.

Mice lacking CHOP have increased lung permeability in response to hyperoxia. A–D: bronchoalveolar lavage fluid (BALF) total protein concentration, BALF IgM concentration, lung weight normalized to starting body weight, and BALF polymorphonuclear (neutrophil) cell count in Ddit3^−/− and wild-type (WT) mice exposed to 72 h of normoxia (21% O₂) or hyperoxia (95% O₂). E: survival of Ddit3^−/− and WT mice exposed to 80% O₂ for up to 10 days. *P < 0.05 vs. 21% O₂.

Fig. 6.

Mice lacking CHOP have greater alveolar septal thickening and intra-alveolar exudate than WT mice following hyperoxia exposure. A–D: hematoxylin-eosin-stained sections of lungs from WT and Ddit3^−/− mice following 76 h of exposure to 95% O₂. Original magnification: ×100 (A and B) and ×400 (C and D).