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 5

PERK activates PGC-1α by increasing ATF4 in lung macrophages.

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PERK activates PGC-1α by increasing ATF4 in lung macrophages. (A) Schema...
(A) Schematic representation of PERK pathway activation and downstream signaling molecules. CHOP, C/EBP homologous protein. (B) Lung macrophages from normal and asbestosis humans were obtained by BAL. Densitometry of immunoblot. Inset, immunoblot analysis of PGC-1α (n = 3). (C) Macrophages were cotransfected with renilla luciferase plasmid, pGL3-Ppargc1a luciferase promoter, and empty, PERKWT, or PERKDN and exposed to asbestos (24 hours). Ppargc1a promoter activity was determined by measuring firefly and renilla luciferase (n = 3). (D) ATF3 (n = 3), (E) ATF4 (n = 3), and (F) PPARGC1A mRNA expression (n = 3) in transfected macrophages exposed to asbestos. (G) Macrophages were exposed to control or asbestos and subjected to ChIP with antibodies against ATF3 or ATF4 followed by real-time PCR to determine Ppargc1a promoter binding (n = 3–9). (H) Lung macrophages were obtained from normal and asbestosis humans by BAL. Densitometry of immunoblot. Inset, immunoblot analysis of ATF4 (n = 4). (I) Macrophages were cotransfected with pGL3-Ppargc1a luciferase promoter combined with scramble or ATF4 siRNA, and empty or PERKWT, and exposed to asbestos. Ppargc1a promoter activity (n = 3–4). Inset, immunoblot analysis for ATF4. (J) Schematic illustration of ATF4 binding site on Ppargc1a promoter in the cAMP response element (CRE) domain and mutation sites on specific residues. (K) Ppargc1a promoter luciferase activity in macrophages transfected with empty, PERKWT, or pGL3-Ppargc1a luciferase mutant and exposed to asbestos (n = 3). (L) Ppargc1a mRNA expression in BAL isolated at day 21 from exposed Eif2ak3fl/fl and Eif2ak3–/– Lyz2-cre mice (n = 4). (M) Ppargc1a mRNA expression in FACS-sorted RAMs and MDMs isolated at day 21 from exposed Eif2ak3fl/fl and Eif2ak3–/– Cx3cr1creER mice (n = 3). Data shown as mean ± SEM. Two-tailed Student’s t test in B and H. One-way ANOVA with Tukey’s post hoc comparison in C–G, I, and K–M. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001. (See also Supplemental Figure 4.)