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 2

Brd4 inhibition blocks TGF-β1–induced Nox4 gene upregulation in human lung fibroblasts.

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Brd4 inhibition blocks TGF-β1–induced Nox4 gene upregulation in human lu...
(A–C) Normal human lung fibroblasts (IMR90) were transfected with siRNA Brd4 or NT and then treated with vehicle or TGF-β1 (2 ng/mL) for 48 hours. (A) The whole cell lysate were collected to examine Brd4 expression by Western blots. (B) Densitometry of Brd4-associated signals detected (ratio to β-actin) in A. *P < 0.05, Brd4 siRNA–transfected cells compared with NT control of the same cell line, by 2-tailed t test. (C) Treated as in A, cells were analyzed for Nox4 mRNA by real-time PCR (mean ± SD; n = 3 in each group). *P < 0.05, each group compared with vehicle only; #P < 0.05, TGF-β1–treated siRNA Brd4 vs. NT cells, by 2-tailed t test. (D) IMR90 fibroblasts were incubated overnight with 1% fetal bovine serum at 70% confluence and then treated with vehicle or various Brd4 inhibitors with the same concentration as in Figure 1 for 2 hours before stimulation with TGF-β1 (2 ng/mL) for 48 hours. Cells were analyzed for Nox4 mRNA by real-time PCR (mean ± SD; n = 3 in each group). *P < 0.05, each group compared with TGF-β1 with vehicle only (Vehl/TGF-β1), by 2-tailed t test. (E) IMR90 fibroblasts were pretreated with or without OTX015 for 2 hours and then with or without TGF-β1 for 48 hours. Cells were collected and subjected to SDS-PAGE and Western blot analysis for Nox4 and β-actin (loading control). (F) The densitometry of Nox4-associated signals detected (ratio to β-actin) in E. *P < 0.05, OTX015 pretreated cells with TGF-β1 compared with TGF-β1, by 2-tailed t test. (G) IMR90 fibroblasts stimulated with/without TGF-β1 (2 ng/mL for 24 hours) in the presence/absence of OTX015 (0.5 μM) were analyzed for extracellular H2O2 production, as a marker of Nox4 activity (mean ± SD; n = 6 in each group). *P < 0.05, compared with TGF-β1/OTX015, by 2-tailed t test.