The PERK/ATF4 pathway is required for metabolic reprogramming and progressive lung fibrosis
Jyotsana Pandey, Jennifer L. Larson-Casey, Mallikarjun H. Patil, Chao He, Nisarat Pinthong, A. Brent Carter
Jyotsana Pandey, Jennifer L. Larson-Casey, Mallikarjun H. Patil, Chao He, Nisarat Pinthong, A. Brent Carter
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Research Article Immunology Pulmonology

The PERK/ATF4 pathway is required for metabolic reprogramming and progressive lung fibrosis

Abstract

Asbestosis is a prototypical type of fibrosis that is progressive and does not resolve. ER stress is increased in multiple cell types that contribute to fibrosis; however, the mechanism(s) by which ER stress in lung macrophages contributes to fibrosis is poorly understood. Here, we show that ER stress resulted in protein kinase RNA-like ER kinase (PERK; Eif2ak3) activation in humans with asbestosis. Similar results were seen in asbestos-injured mice. Mice harboring a conditional deletion of Eif2ak3 were protected from fibrosis. Lung macrophages from asbestosis individuals had evidence of metabolic reprogramming to fatty acid oxidation (FAO). Eif2ak3fl/fl mice had increased oxygen consumption rate (OCR), whereas OCR in Eif2ak3–/– Lyz2-cre mice was reduced to control levels. PERK increased activating transcription factor 4 (Atf4) expression, and ATF4 bound to the Ppargc1a promoter to increase its expression. GSK2656157, a PERK-specific inhibitor, reduced FAO, Ppargc1a, and Aft4 in lung macrophages and reversed established fibrosis in mice. These observations suggest that PERK is a therapeutic target to reverse established fibrosis.

Authors

Jyotsana Pandey, Jennifer L. Larson-Casey, Mallikarjun H. Patil, Chao He, Nisarat Pinthong, A. Brent Carter

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Figure 4

Asbestos-induced PERK activation mediates metabolic reprogramming.

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Asbestos-induced PERK activation mediates metabolic reprogramming. Lung ...
Lung macrophages were obtained by BAL from normal and asbestosis humans. (A) Acetyl-CoA concentration measured by fluorometry in humans (n = 5). (B) TCA metabolites measured by mass spectrometry in humans and shown as fold-change (n = 4–5). (C) Ratio of NAD+/NADH in humans measured by fluorometry (n = 4). (D) Immunoblot analysis of CPT1A and (E) densitometry of CPT1A in BAL cell mitochondrial fractions from humans (n = 4). WT mice were exposed to MMVF or asbestos (100 μg i.t.). VDAC, voltage-dependent anion channel. (F) Lung macrophages from exposed mice were subjected to mass spectrometry. l-carnitine was normalized to total ion chromatography (TIC) (n = 3–4). (G) Hydroxyproline assay in lung tissues at the designated time points (n = 5). Cpt1a mRNA expression in (H) BAL cells isolated at day 21 from exposed Eif2ak3fl/fl and Eif2ak3–/– Lyz2-cre mice (n = 4) and (I) FACS-sorted RAMs and MDMs isolated from BAL at day 21 from Eif2ak3fl/fl and Eif2ak3–/– Cx3cr1creER mice (n = 3). (J) OCR kinetics in BAL cells isolated at day 21 from exposed Eif2ak3fl/fl and Eif2ak3–/– Lyz2-cre mice (n = 3). Oligo, oligomycin; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; Rot/Anti A, rotenone/antimycin A; min-μg, minute/μg protein. Data shown as mean ± SEM. Two-tailed Student’s t test in A, C, and E. One-way ANOVA with Tukey’s post hoc comparison in F–I. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001. (See also Supplemental Figure 3.)