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 5

p300 mediates Nox4 gene expression in lung fibroblasts.

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p300 mediates Nox4 gene expression in lung fibroblasts. (A–C) Primary IP...
(A–C) Primary IPF fibroblasts were transfected with nontargeting (NT) siRNA or p300 siRNA. (A) Representative Western blots of fibroblasts from 2 different individuals with IPF transfected with siRNA NT or p300 demonstrated the levels of p300 and β-actin. (B) The densitometry of p300-associated signals detected (ratio to β-actin) in A. *P < 0.05, compared with NT control of the same cell line, by 2-tailed t test. (C) The expression of Nox4 mRNA was analyzed by real-time quantitative PCR of the IPF cells transfected with siRNA NT or p300 (mean ± SD, n = 3 experimental replicates of a representative cell line). *P < 0.05 compared with NT control, by 2-tailed t test. (D–F) IMR90 cells were transfected with siRNA NT or p300, and then the cells were treated with TGF-β1 (2 ng/mL) for 48 hours and analyzed for p300 expression by Western blot and Nox4 mRNA expression by real-time PCR. (D) Representative Western blots of p300 and β-actin. (E) Densitometric analysis of p300-associated signals detected (ratio to β-actin) in D. *P < 0.05, TGF-β1–treated cells with p300 siRNA vs. NT siRNA, by 2-tailed t test. (F) The mRNA expression of Nox4 by real-time PCR (mean ± SD, n = 3 experimental replicates). *P < 0.05, compared with vehicle control; #P < 0.05, the transfected cells with TGF-β1 treatment, siRNA p300 vs. NT, by 2-tailed t test.