Circadian clock component REV-ERBα controls homeostatic regulation of pulmonary inflammation
Marie Pariollaud, … , Andrew S.I. Loudon, David W. Ray
Marie Pariollaud, … , Andrew S.I. Loudon, David W. Ray
Published March 13, 2018
Citation Information: J Clin Invest. 2018;128(6):2281-2296. https://doi.org/10.1172/JCI93910.
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Research Article Inflammation Pulmonology

Circadian clock component REV-ERBα controls homeostatic regulation of pulmonary inflammation

Abstract

Recent studies reveal that airway epithelial cells are critical pulmonary circadian pacemaker cells, mediating rhythmic inflammatory responses. Using mouse models, we now identify the rhythmic circadian repressor REV-ERBα as essential to the mechanism coupling the pulmonary clock to innate immunity, involving both myeloid and bronchial epithelial cells in temporal gating and determining amplitude of response to inhaled endotoxin. Dual mutation of REV-ERBα and its paralog REV-ERBβ in bronchial epithelia further augmented inflammatory responses and chemokine activation, but also initiated a basal inflammatory state, revealing a critical homeostatic role for REV-ERB proteins in the suppression of the endogenous proinflammatory mechanism in unchallenged cells. However, REV-ERBα plays the dominant role, as deletion of REV-ERBβ alone had no impact on inflammatory responses. In turn, inflammatory challenges cause striking changes in stability and degradation of REV-ERBα protein, driven by SUMOylation and ubiquitination. We developed a novel selective oxazole-based inverse agonist of REV-ERB, which protects REV-ERBα protein from degradation, and used this to reveal how proinflammatory cytokines trigger rapid degradation of REV-ERBα in the elaboration of an inflammatory response. Thus, dynamic changes in stability of REV-ERBα protein couple the core clock to innate immunity.

Authors

Marie Pariollaud, Julie E. Gibbs, Thomas W. Hopwood, Sheila Brown, Nicola Begley, Ryan Vonslow, Toryn Poolman, Baoqiang Guo, Ben Saer, D. Heulyn Jones, James P. Tellam, Stefano Bresciani, Nicholas C.O. Tomkinson, Justyna Wojno-Picon, Anthony W.J. Cooper, Dion A. Daniels, Ryan P. Trump, Daniel Grant, William Zuercher, Timothy M. Willson, Andrew S. MacDonald, Brian Bolognese, Patricia L. Podolin, Yolanda Sanchez, Andrew S.I. Loudon, David W. Ray

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Figure 5

REV-ERBα ligand GSK1362 represses inflammatory genes in macrophages and epithelial cells and stabilizes REV-ERBα protein.

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REV-ERBα ligand GSK1362 represses inflammatory genes in macrophages and ...
(A) Chemical structure of GSK1362. (B) Effect of GSK1362 and GSK4112 on peptide fragment recruitment to REV-ERBα. (C) Cotransfection of HEK293 cells with HA–Rev-Erbα and Bmal1-Luc reporter. Cells were treated with GSK1362 or GSK4112 at different concentrations for 24 hours before luciferase assay. Values plotted relatively to 0.1% DMSO; error bars indicate mean ± SD. Data representative of n = 3. (D) Models showing GSK1362 docked in REV-ERBα ligand-binding domain. (E) qPCR analysis of LXR target genes in peritoneal exudate cells treated ex vivo with GSK1362 at 10 μM or GW3965, a standard LXR agonist, at 2 μM for 4 hours. Data presented as mean ± SD; n = 3, ***P < 0.001 (1-way ANOVA, post hoc Bonferroni). (F) qPCR analysis of cytokine mRNA in alveolar macrophages collected at ZT8, seeded into plates and directly treated with GSK1362 at 10 μM in the presence or absence of LPS at 100 ng/ml for 4 hours. Data presented as mean ± SD; n = 3, *P < 0.05, **P < 0.01 (1-way ANOVA, post hoc Bonferroni). (G) qPCR analysis of Cxcl5 in LA-4 cells synchronized by serum shock and treated 16 hours later with ligands at 10 μM, followed 2 hours later by IL-1β at 1 ng/ml for 2 additional hours. Data normalized to unstimulated control cells and presented as mean ± SD; representative of n = 3, **P < 0.01, ***P < 0.001 (2-way ANOVA, post hoc Bonferroni). (H) REV-ERBα protein in LA-4 cells synchronized by serum shock and treated 16 hours later with ligands at 10 μM for 4 hours. Representative of n = 3. (I) REV-ERBα protein in NHBE cells synchronized by serum shock and treated 16 or 28 hours later with ligands at 10 μM for 4 hours. Representative of n = 3.