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. 2016 Dec 1;311(6):L1183-L1201.
doi: 10.1152/ajplung.00224.2016. Epub 2016 Oct 28.

BRD4 mediates NF-κB-dependent epithelial-mesenchymal transition and pulmonary fibrosis via transcriptional elongation

Bing Tian  1   2 , Yingxin Zhao  3   4   2 , Hong Sun  3 , Yueqing Zhang  3 , Jun Yang  3   2 , Allan R Brasier  3   4   2
Affiliations

BRD4 mediates NF-κB-dependent epithelial-mesenchymal transition and pulmonary fibrosis via transcriptional elongation

Bing Tian et al. Am J Physiol Lung Cell Mol Physiol. .

Abstract

Chronic epithelial injury triggers a TGF-β-mediated cellular transition from normal epithelium into a mesenchymal-like state that produces subepithelial fibrosis and airway remodeling. Here we examined how TGF-β induces the mesenchymal cell state and determined its mechanism. We observed that TGF-β stimulation activates an inflammatory gene program controlled by the NF-κB/RelA signaling pathway. In the mesenchymal state, NF-κB-dependent immediate-early genes accumulate euchromatin marks and processive RNA polymerase. This program of immediate-early genes is activated by enhanced expression, nuclear translocation, and activating phosphorylation of the NF-κB/RelA transcription factor on Ser276, mediated by a paracrine signal. Phospho-Ser276 RelA binds to the BRD4/CDK9 transcriptional elongation complex, activating the paused RNA Pol II by phosphorylation on Ser2 in its carboxy-terminal domain. RelA-initiated transcriptional elongation is required for expression of the core epithelial-mesenchymal transition transcriptional regulators SNAI1, TWIST1, and ZEB1 and mesenchymal genes. Finally, we observed that pharmacological inhibition of BRD4 can attenuate experimental lung fibrosis induced by repetitive TGF-β challenge in a mouse model. These data provide a detailed mechanism for how activated NF-κB and BRD4 control epithelial-mesenchymal transition initiation and transcriptional elongation in model airway epithelial cells in vitro and in a murine pulmonary fibrosis model in vivo. Our data validate BRD4 as an in vivo target for the treatment of pulmonary fibrosis associated with inflammation-coupled remodeling in chronic lung diseases.

Keywords: BRD4; airway epithelial cells; fibrosis; mesenchymal transition; nuclear factor-κB.

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Figures

Fig. 1.
Fig. 1.
TGF-β activates the NF-κB signaling pathway. A: TGF-β increases total RelA abundance. hSAECs were incubated with TGF-β (10 ng/ml) for 15 days. Whole cell extracts of hSAECs were isolated and immunoprecipitated with primary anti-RelA Ab. RelA abundance was quantified by SID-SRM-MS. Shown is fold change abundance normalized to the input protein concentration. *P = 0.002 compared with mock treatment. B, top: TGF-β induces RelA nuclear translocation. hSAECs were incubated with a time series of TGF-β (10 ng/ml) for up to 15 days. One hundred fifty micrograms of nuclear extracts was processed for Western blot using anti-RelA Ab (top). Lamin B was detected as a loading control (bottom). Bottom: quantification of nuclear RelA. Shown is the fold change in protein abundance normalized to Lamin B. *P < 0.01 compared with mock treatment. C: TGF-β induces secretion of NF-κB-dependent cytokines/chemokines. hSAECs were incubated with a time series of TGF-β (10 ng/ml) up to 16 days. The conditioned medium was collected for cytokine determination by multiplex ELISA. Shown are the concentrations of IL-6, IL-8, TNF-α, IFN-γ, VEGF, and G-CSF. *P < 0.05 compared with control epithelial cells. D: conditioned medium from TGF-β-treated cells induces RelA nuclear translocation in naive hSAECs. Naive hSAECs were incubated for 1 h with 3-, 12-, or 15-day conditioned medium from TGF-β-treated hSAECs. Left: Western blot of 150 μg of nuclear extracts using anti-RelA Ab (top); the blot was probed with Lamin B as the loading control (bottom). Right: quantification of nuclear RelA. Shown is the fold change in protein abundance normalized to Lamin B. *P < 0.01 compared with mock treatment. All data shown are means ± SD from 3 independent experiments.
Fig. 2.
Fig. 2.
Enhanced innate immune responses in hSAECs with EMT. A: innate genes are hyperinducible in the mesenchymal state. hSAECs in the absence or presence of tonic TGF-β stimulation (15 days) were challenged with TNF-α, Sendai virus (SeV), respiratory syncytial virus (RSV), junin virus, poly(I:C), or LPS as shown. Expression of IL-6 and IL-8 mRNA was quantified by Q-RT-PCR. Shown is the fold change in mRNA abundance normalized to cyclophilin. *P < 0.001 compared with control (without TGF-β). Data are means ± SD from n = 3 experiments. B: accumulation of euchromatin marks and processive RNA polymerase in EMT. hSAECs were stimulated with TGF-β for 0 or 15 days, chromatin cross-linked, and immunoprecipitated with Abs specific for H3K4Me3, RelA, or phospho-Ser2 CTD RNA Pol II. Anti-rabbit IgG was used as the negative control. Binding of H3K4Me3, RelA, and phospho-Ser2 CTD RNA Pol II to the 5′ NF-κB site of the IL-6 (top) and IL-8 (bottom) promoters was determined by Q-gPCR. *P < 0.01 compared to without TGF-β; #P < 0.05 compared to without TNF-α. Data are means ± SD from 3 independent experiments.
Fig. 3.
Fig. 3.
Requirement of NF-κB signaling for the TGF-β-induced type II EMT. A: efficiency of RelA depletion. hSAECs stably expressing doxycycline (Doxy)-inducible RelA shRNA were cultured for 5 days ± Doxy (2 μg/ml) to silence RelA. Left: protein levels of RelA in whole cell lysate by Western blots. Wild-type (WT) or RelA-silenced hSAECs were treated ± TNF (25 ng/ml), and expression of IL-6 and IκBα mRNAs was measured by Q-RT-PCR. Data are mean ± SD normalized fold change from n = 3 experiments. *P < 0.001 compared to without Doxy treatment. B: RelA depletion blocks TGF-β-induced EMT gene expression. RNA from WT and RelA-silenced SAECs were treated ± TGF-β (10 ng/ml) for 15 days and were quantified for the expression of RelA, IL-6, SNAI1, ZEB1, TWIST1, VIM, Groβ, IL-8, and FN1 mRNAs. *P < 0.01 compared to control siRNA. Data are means ± SD from 3 independent experiments.
Fig. 4.
Fig. 4.
Requirement of RelA Ser276 phosphorylation for TGF-β-induced mesenchymal transition. A: TGF-β induced changes in RelA, BRD4, and phospho-Ser276 RelA. Both WT and RelA-silenced hSAECs were treated ± TGF-β (10 ng/ml) for 15 days. Cells were fixed, stained with Alexa Fluor 568-conjugated phalloidin (red) and DAPI (blue), and imaged by confocal microscopy. Separate coverslips were incubated with primary anti-RelA, BRD4, or phospho-Ser276 RelA Abs and stained with Alexa Fluor-conjugated secondary Abs (shown in green, red, and deep red, respectively). Cells were counterstained with DAPI (blue) and imaged via confocal fluorescence microscopy. B: induction of phospho-Ser276 RelA and total RelA in response to TGF-β stimulation. WCEs from WT or RelA-silenced hSAECs treated ± TGF-β (10 ng/ml) for 15 days were obtained. Equal amounts of WCE were immunoprecipitated with pan-anti-RelA Ab and subjected to SID-SRM-MS analysis for total (top) and phospho (p; bottom)-Ser276 RelA. Shown are fold changes in relative abundance relative to control cells. *P < 0.01 compared to without TGF-β; #P < 0.001 compared to RelA WT. C: RelA Ser276 phosphorylation is required for TGF-β-induced EMT gene program. RelA−/− MEFs stably transfected with either FLAG-EGFP-tagged RelA WT or a FLAG-EGFP-tagged nonphosphorylatable RelA with a Ser276-to-Ala mutation (RelA Ser276Ala) were treated with TGF-β for 3 days. The effects of TGF-β on mSNAI1, TWIST1, mZEB1, and mIL-6 gene expression were compared by Q-RT-PCR. Data are mean ± SD fold change from n = 3 experiments. *P < 0.05 compared with RelA WT.
Fig. 5.
Fig. 5.
NF-κB binding is required for TGF-β-induced recruitment of the CDK9/BRD4 complex to EMT genes. Cross-linked chromatin from WT or RelA-silenced hSAECs treated ± TGF-β (10 ng/ml) was subjected to IP with Abs specific for RelA, CDK9, BRD4, or phospho-Ser2 CTD Pol II. Anti-rabbit IgG was used as the negative control. The recruitment of RelA, CDK9, BRD4, and pSer2 CTD RNA Pol II to the 5′ NF-κB site of the SNAI1 (A), ZEB1 (B), IL-6 (C), and VIM (D) promoters was determined by Q-gPCR using gene-specific primers. *P < 0.01 compared to without doxycycline (Dox); #P < 0.01 compared to mock treatment. Data are from n = 3 independent experiments.
Fig. 6.
Fig. 6.
TGF-β induces RelA/BRD4/CDK9 nuclear complex formation. Nuclear extracts of hSAECs with or without TGF-β stimulation were enriched by IP with Abs to RelA or BRD4, and the abundance of RelA, CDK9, and BRD4 proteins was determined by SID-SRM-MS. Data are normalized by the input protein concentration and plotted as fold change over control. Controls represent samples immunoprecipitated with IgG. A: SID-SRM-MS analysis of nuclear RelA complexes. RelA, CDK9, and BRD4 protein levels were determined in the samples immunoprecipitated with anti-RelA or control IgG. *P < 0.01 compared with regular hSAECs IPed with anti-RelA. B: SID-SRM-MS analysis of nuclear BRD4 complexes. BRD4, RelA, and CDK9 protein levels were determined in the samples immunoprecipitated with anti-BRD4 or control IgG. *P < 0.01 compared with regular hSAECs immunoprecipitated with anti-BRD4. Data are means ± SD from n = 3 experiments.
Fig. 7.
Fig. 7.
Requirement of BRD4 in mesenchymal transition. A: BRD4 inhibitor JQ1 blocks mesenchymal transition. hSAECs treated with vehicle (Mock), TGF-β (10 ng/ml, 15 days) or TGF-β + JQ1 (10 μM, 15 days) were fixed and stained with Alexa Fluor 568-conjugated phalloidin and DAPI. Cells were imaged by confocal microscopy. Separate coverslips were subjected to immunofluorescence by incubation with primary Abs for SNAI1, VIM, CDH1, and H3K122ac, then stained with Alexa Fluor-conjugated secondary Abs (green or red), counterstained with DAPI (blue), and imaged via confocal fluorescence microscopy. B: JQ1 blocks TGF-β-induced EMT gene expression program. hSAECs were pretreated with JQ1 (10 μM) ± TGF-β (10 ng/ml) for 15 days. Expression of SNAI1, CDH1, VIM, ZEB1, FN1, IL-6, IL-8, and Groβ mRNA was measured by Q-RT-PCR. *P < 0.01 compared with TGF-β only. Data are means ± SD from n = 3 experiments.
Fig. 8.
Fig. 8.
XChIP analysis of NF-κB binding sites of EMT genes under BRD4 inhibition. hSAECs were treated with TGF-β for 0 or 15 days ± JQ1 and chromatin cross-linked and subjected to IP with Abs specific for RelA, CDK9, BRD4, or phospho-Ser2 CTD RNA Pol II. Anti-rabbit IgG was used as the negative control. The recruitment of RelA, CDK9, BRD4, and phospho-Ser2 CTD RNA Pol II to the SNAI1 (A), ZEB1 (B), IL-6 (C), and VIM (D) promoters was determined by XChIP using gene-specific primers in Q-gPCR. *P < 0.01 compared to without JQ1; #P < 0.01 compared to without TGF-β. Data are means ± SD from n = 3 independent experiments.
Fig. 9.
Fig. 9.
BRD4 mediates TGF-β-induced pulmonary fibrosis in mice. Eighteen-week-old C57BL/6 mice were pretreated ± JQ1 (50 mg/kg body wt ip; n = 5) and given repetitive intranasal challenges with TGF-β (1 μg/mouse every other day for 30 days). A: morphological changes. Masson trichrome staining of lungs from C57BL/6 mice chronically treated in the absence (AA and AC) or presence (AB and AD) of TGF-β; 2 groups of mice were treated with JQ1 (50 mg/kg, AC and AD). Images were taken at magnifications of ×10 (AA–AD), ×20 (AE–AH), and ×40 (AI–AL). B: additional histology. Images from different regions of lungs from mice chronically treated in the absence (BA) or presence (BB–BD) of TGF-β. Mice were also treated with JQ1 (50 mg/kg) in the presence of TGF-β (BD). All images were taken at ×10 magnification. C: Ashcroft scoring. Levels of lung fibrosis with assessed with the Ashcroft scoring method. *P = 0.002 compared to without JQ1 and #P < 0.001 compared to without TGF-β from n = 5 experiments. D: total collagen content. Whole lung hydroxyproline content was determined in all treatment groups. *P = 0.026 compared to without JQ1 and #P = 0.007 compared to without TGF-β from n = 5 experiments.
Fig. 10.
Fig. 10.
BRD4 controls TGF-β-induced pulmonary fibrotic program in vivo. Eighteen-week-old C57B6 mice were pretreated ± JQ1 (50 mg/kg body wt ip) and given repetitive intranasal challenges with TGF-β (1 μg/mouse every other day for 30 days). A: activation of the fibrotic program. Q-RT-PCR for mCol1A1, mFN1, mSNAI1, mVIM, mαSMA, and mIL-6 mRNAs was performed in total lung RNA. *P < 0.01 compared to without JQ1 from n = 5 experiments. B: expression of the mesenchymal transition. Paraffin-embedded lung sections were immunostained for mesenchymal markers SNAI1, VIM, FN1, and COL1A as well as BRD4 activation marker H3K122ac, detected with secondary Alexa Fluor 488 (green)- or 568 (red)-conjugated goat anti-rabbit IgG respectively, counterstained with DAPI, and imaged via confocal fluorescence microscopy. Images are at ×63 magnification. Data representative of n = 5 experiments.
Fig. 11.
Fig. 11.
BRD4 mediates NF-κB-dependent EMT and pulmonary fibrosis via transcriptional elongation. TGF-β stimulation activates an NF-κB-dependent inflammatory gene program by enhanced expression, nuclear translocation, and phosphorylation of the NF-κB/RelA transcription factor on Ser276. Phospho-Ser276-activated RelA binds to the BRD4/CDK9 transcriptional elongation complex, targeting it to core mesenchymal transcriptional regulators activating phosphorylation of the paused RNA Pol II carboxy-terminal domain, whose actions are required for initiation of type II EMT and its fibrotic program. NF-κB-dependent BRD4 recruitment is a major determinant of the type II mesenchymal transition of airway epithelium and of pulmonary fibrosis.
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