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 3

BET inhibitor OTX015 blocks the Nox4 promoter association with Brd4, H4K16ac, and p300 in IPF fibroblasts.

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BET inhibitor OTX015 blocks the Nox4 promoter association with Brd4, H4K...
(A) Schematic of the relative locations of 3 PCR primer sets used to examine the association of transcriptional complex proteins with Nox4 (ENSG00000086991) by ChIP assays. (B–D) Primary IPF lung fibroblasts were treated with vehicle or OTX015 (0.5 μM) for 48 hours before the cells were subjected to ChIP assays. The Nox4 promoter region association with Brd4, H4K16Ac, and p300 was analyzed by ChIP assays, with primer sets indicated in A. DNA was immunoprecipitated with specific antibodies against Brd4 (B), H4K16Ac (C), and p300 (D). The relative levels of the PCR product are represented in the bar graphs as Nox4 promoter region association with Brd4, H4K16ac, or p300. Negative control represents IgG pull-down. qPCR data were analyzed by the 2-ΔΔCt method and were normalized to input DNA, expressed as fold change relative to primer set FcRc or FaRa. The values are expressed as mean ± SD from average of 3 independent experiments of 1 representative cell line. *P < 0.05, OTX015-treated compared with control (vehicle only), by 2-tailed t test.