Brd4-p300 inhibition downregulates Nox4 and accelerates lung fibrosis resolution in aged mice
Yan Y. Sanders, … , Steven M. Rowe, Victor J. Thannickal
Yan Y. Sanders, … , Steven M. Rowe, Victor J. Thannickal
Published June 16, 2020
Citation Information: JCI Insight. 2020;5(14):e137127. https://doi.org/10.1172/jci.insight.137127.
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Brd4-p300 inhibition downregulates Nox4 and accelerates lung fibrosis resolution in aged mice

Abstract

Tissue regeneration capacity declines with aging in association with heightened oxidative stress. Expression of the oxidant-generating enzyme, NADPH oxidase 4 (Nox4), is elevated in aged mice with diminished capacity for fibrosis resolution. Bromodomain-containing protein 4 (Brd4) is a member of the bromodomain and extraterminal (BET) family of proteins that function as epigenetic “readers” of acetylated lysine groups on histones. In this study, we explored the role of Brd4 and its interaction with the p300 acetyltransferase in the regulation of Nox4 and the in vivo efficacy of a BET inhibitor to reverse established age-associated lung fibrosis. BET inhibition interferes with the association of Brd4, p300, and acetylated histone H4K16 with the Nox4 promoter in lung fibroblasts stimulated with the profibrotic cytokine, TGF-β1. A number of BET inhibitors, including I-BET-762, JQ1, and OTX015, downregulate Nox4 gene expression and activity. Aged mice with established and persistent lung fibrosis recover capacity for fibrosis resolution with OTX015 treatment. This study implicates epigenetic regulation of Nox4 by Brd4 and p300 and supports BET/Brd4 inhibition as an effective strategy for the treatment of age-related fibrotic lung disease.

Authors

Yan Y. Sanders, Xing Lyv, Q. Jennifer Zhou, Zheyi Xiang, Denise Stanford, Sandeep Bodduluri, Steven M. Rowe, Victor J. Thannickal

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Figure 7

The inhibitor OTX015 facilitates resolution of established lung fibrosis in aged mice.

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The inhibitor OTX015 facilitates resolution of established lung fibrosis...
(A) The schedule of lung fibrosis induced by bleomycin injury in 18-month-old mice. OTX015 was given twice daily orally from day 21–42 after bleomycin injury. All the mice were sacrificed at day 42 after bleomycin injury. (B) Whole lung histology by immunohistochemistry with H&E staining of mice with saline, bleomycin injury (bleo), or bleomycin injury with OTX015 treatment (Bleo/OTX). (C) Mouse whole lung tissue lysates from saline (n = 7), bleomycin (n = 7), and bleomycin with OTX015 treatment (n = 9) groups were subjected to SDS-PAGE and Western blots analysis for Nox4 and β-actin. (D) Densitometry of the Western blots in C for the ratio of Nox4 to β-actin (mean ± SD). (E) Primary lung fibroblasts were cultured from mouse lungs. Whole cell lysate from saline (n = 3), bleomycin (n = 3), and bleomycin with OTX015 treatment (n = 4) groups were collected at passage 1 and subjected to SDS-PAGE and Western blots analysis for Nox4 and β-actin. (F) Densitometry of the Western blots in E for the ratio of Nox4 to β-actin in the primary murine lung fibroblasts (mean ± SD). (G) Representative axial micro-CT images of mouse lungs 6 weeks after they were subjected to bleomycin injury with/without OTX015 treatment. Voxels of the lung field that are below –550 Hounsfield units are in green (representing aerated lung) and those between –550 and –350 Hounsfield units are in red (representing nonaerated lung). (H) Quantitation of lung density in uninjured (saline) and injured (Bleo) mice treated with/without OTX015. Whisker plots represent mean ± SD; n = 4–6 per group (saline, n = 6; bleo, n = 4; bleo/OTX015, n = 4). *P < 0.05, by 2-tailed t test. (I) Hydroxyproline content in lungs of mice after saline (n = 5), bleomycin (n = 6), or bleomycin with OTX015 treatment (n = 8) (mean ± SD). *P < 0.05, for comparisons of indicted groups as compared with the bleomycin group, by 2-tailed t test.