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. Author manuscript; available in PMC: 2014 Nov 15.
Published in final edited form as: J Immunol. 2014 Apr 14;192(10):4913–4920. doi: 10.4049/jimmunol.1301984

Bromodomain and Extra-Terminal (BET) proteins suppress nuclear factor E2-related factor 2 (Nrf2) -mediated antioxidant gene expression

Charalambos Michaeloudes *,#, Nicolas Mercado *,#, Colin Clarke *, Pankaj K Bhavsar *, Ian M Adcock *, Peter J Barnes *, Kian Fan Chung *
PMCID: PMC4011694  EMSID: EMS57649  PMID: 24733848

Abstract

Oxidative stress, a pathogenetic factor in many conditions including chronic obstructive pulmonary disease (COPD) arises due to accumulation of reactive oxygen species (ROS) and defective antioxidant defences in the lungs. The latter is due, at least in part, to impaired activation of nuclear factor E2-related factor 2 (Nrf2), a transcription factor involved in the activation of antioxidant and cytoprotective genes. The bromodomain and extra-terminal (BET) proteins, Brd2, Brd3, Brd4 and BrdT, bind to acetylated lysine residues on histone or non-histone proteins recruiting transcriptional regulators and thus activating or repressing gene transcription. We investigated whether BET proteins modulate the regulation of Nrf2-dependent gene expression in primary human airway smooth muscle cells (ASMCs) and the human monocytic cell line, THP-1. Inhibition of BET protein bromodomains using the inhibitor JQ1+, or attenuation of Brd2 and Brd4 expression using siRNA led to activation of Nrf2-dependent transcription and expression of the antioxidant proteins heme oxygenase (HO)-1, NADPH quinone oxidoreductase 1 (NQO1) and glutamate-cysteine ligase catalytic subunit (GCLC). Also, JQ1+ prevented hydrogen peroxide (H2O2)-induced intracellular ROS production. By co-immunoprecipitation, BET proteins were found to be complexed with Nrf2, whilst chromatin-immunoprecipitation studies indicated recruitment of Brd2 and Brd4 to Nrf2-binding sites on the promoters of HO-1 and NQO1. BET proteins, particularly Brd2 and Brd4, may play a key role in the regulation of Nrf2-dependent antioxidant gene transcription and are hence an important target for augmenting antioxidant responses in oxidative stress-mediated diseases.

Introduction

Cellular oxidative stress which results from an imbalance between the production of reactive oxygen species (ROS) and their removal by antioxidant defence mechanisms is pivotal in the pathogenesis of a number of diseases, including chronic obstructive pulmonary disease (COPD). In COPD, cigarette smoke inhalation and the resulting chronic inflammatory response lead to release of ROS in the airways. Defective antioxidant mechanisms in COPD lungs could lead to accumulation of ROS, resulting in activation of redox-sensitive transcription factors, epigenetic changes and oxidative damage in immune and structural cells (1). NADPH oxidase-mediated ROS trigger airway smooth muscle cell (ASMC) proliferation and hypertrophy (2). Exogenous oxidative stress stimuli reduce histone deacetylase 2 (HDAC2) activity in alveolar macrophages leading to increased expression of inflammatory genes (3). Moreover, cigarette smoke-mediated ROS reduce sirtuin 1 (SIRT1) activity leading to up-regulation of matrix metalloproteinase-9 (MMP-9) in monocytes and sputum macrophages (4). As a result, there is amplification of the inflammatory response and induction of pathogenic processes such as lung parenchymal destruction and small airway remodelling (5,6).

Cells are normally protected from ROS by the concerted action of a large array of endogenous antioxidant proteins. The transcription factor nuclear factor E2-related factor 2 (Nrf2) plays a pivotal role in cellular antioxidant defences by activating a wide range of antioxidant genes, including heme oxygenase (HO)-1, NADPH quinone oxidoreductase 1 (NQO1) and glutamate-cysteine ligase catalytic subunit (GCLC) (7). Under normal conditions, Nrf2 is found in a complex with its inhibitor Kelch-like ECH-associated protein (Keap1) and the ubiquitin-ligase Cul3, where it is constantly targeted for proteosomal degradation. In the presence of oxidative stress, Nrf2 dissociates from the complex leading to an increase in its protein levels and subsequent activation of antioxidant genes by binding to common regulatory elements, termed antioxidant response elements (AREs), in their promoter regions (8). Impaired Nrf2 function has been linked to defective antioxidant defences in several age-related diseases, including COPD (9-12). Nrf2 expression has been shown to be reduced in lung tissue and alveolar macrophages of patients with COPD (9,13,14). Moreover, we have reported that reduced HDAC activity in monocyte-derived macrophages leads to decreased Nrf2 protein stability through increased acetylation, implicating epigenetic mechanisms in impaired Nrf2 activation in COPD (15). Identification of molecular targets, particularly epigenetic effectors, for augmenting Nrf2 activity would therefore be crucial for the development of new antioxidant therapies for COPD and other oxidative stress-dependent diseases.

The bromodomain and extra-terminal (BET) proteins act as “readers” of protein acetylation by binding to acetylated lysine residues through two highly conserved N-terminal bromodomain modules to regulate gene expression (16). Specifically, BET proteins interact with transcription factors and chromatin remodelling complexes via their extra terminal (ET) and C-terminal domains, and recruiting them to the gene promoter to either activate or repress transcription of genes involved in inflammation, cell cycle and differentiation (17-20). The human BET protein family comprises of Brd2, Brd3, Brd4, and BrdT, the latter being solely expressed in the testis (21,22). Small molecule bromodomain inhibitors, developed in recent years, are important tools for the study of BET proteins. The prototype compound, JQ1, is a thieno-triazolo-1,4-diazepine that specifically interferes with the binding of BET bromodomains to acetylated lysines (23). BET proteins have been shown to be implicated in diseases where oxidative stress and compromised antioxidant protection play an important pathogenic role, such as acute myeloid leukaemia (24), heart failure (25), and obesity (26,27). However, it is currently unknown whether BET proteins are directly involved in the regulation of antioxidant gene expression and hence the protection against ROS-mediated disease pathogenesis. We hypothesised that BET proteins are involved in the regulation of antioxidant genes in both structural and immune cells and are thus important targets for augmenting antioxidant defences in COPD. We therefore examined the effect of pharmacological and molecular inhibitors of BET protein activity on antioxidant gene expression in primary human ASMCs and the human monocytic cell line, THP-1. Furthermore, we investigated the effect of BET protein inhibition on the Nrf2 pathway, the interaction of BET proteins with Nrf2 and their recruitment to antioxidant gene promoters.

Materials and Methods

Reagents

The active JQ1 enantiomer (+)- JQ1 (JQ1+) and the inactive enantiomer (−)- JQ1 (JQ1−) were purchased from Cayman (Cambridge, UK). MG132 was purchased from Sigma-Aldrich (Poole, UK).

Cell culture and stimulation

THP-1 cells (human monocytic cell line) were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in RPMI 1640 medium (Cayman) containing 10% heat-inactivated fetal bovine serum (FBS) and 2 mM L-glutamine at 37°C, 5% CO2 and humidified atmosphere. For stimulation, THP-1 cells were seeded (0.3 × 106 cells/ml) in starvation medium containing RPMI-1640 supplemented with 1% FBS and 15 mM L-glutamine.

ASMCs were dissected from tracheas or bronchi of transplant donor lungs and were cultured in Dulbecco’s Modified Eagles Medium (DMEM) supplemented with 4 mM L-glutamine, 20 U/l of penicillin, 20 μg/ml streptomycin, 2.5 μg/ml amphotericin B and 10% FBS. Presence of ASMCs was confirmed by identifying the characteristic “hill and valley” morphology using light microscopy. Cell stocks were kept in 150 cm2 flasks at 37°C, 5% CO2 and humidified atmosphere. Cells between passages 3 and 7 were used for experiments. Before treatment, cells were incubated in serum free medium containing phenol-free DMEM supplemented with 1 mM sodium pyruvate, 4 mM L-glutamine, non-essential amino acids, 1% insulin-transferrin-selenium-X supplement, 0.1% bovine serum albumin (BSA) and antibiotics as described above.

Peripheral blood mononuclear cells (PBMCs) were isolated from the peripheral blood of three healthy volunteers, using AccuSPIN columns (Sigma-Aldrich). Monocytes were isolated by adherence to tissue culture plates for 90 mins. This study was approved by the local ethics committee of Royal Brompton and Harefield NHS Trust and written informed consent was obtained from each volunteer. PBMCs were cultured in RPMI 1640 medium containing 10% FBS and 2 mM L-glutamine at 37°C, 5% CO2 and humidified atmosphere.

siRNA transfection

THP-1 cells were transfected with 300nM of ON-TARGETplus SMARTpool small interfering (siRNA) against Nrf2 (cat no: L-003755-00-0005), Brd2 (cat no: L-004935-00-0005), Brd3 (cat no: L-004936-00-0005) or Brd4 (cat no: L-004937-00-0005) (Thermo Scientific, Epsom, UK), or a random oligonucleotide control siRNA (Qiagen, Crawley, UK) for 48 hrs using HiPerfect transfection reagent (Qiagen) following the manufacturer’s instructions. ASMCs were transfected, with the same siRNA sequences (300nM) for 48 hrs using Amaxa nucleofection (Lonza AG, Cologne, Germany) following the manufacturer’s instructions.

ARE reporter assay

ASMCs were transfected, using Amaxa nucleofection, with DNA plasmid constructs (2.5μg) expressing ARE-inducible firefly luciferase and constitutively active Renilla luciferase (SABiosciences, Frederick, MD, USA) for 24 hrs, and then incubated with the required treatments for a further 24 hrs. At the end of the incubation time cells were lysed and firefly and Renilla luciferase activities were determined by measuring luminescence. ARE-inducible luciferase activity was normalised to Renilla luciferase activity. The Nrf2 inducer sulforaphane (4μM) was used as a positive control in all experiments.

Western Blotting

Protein extracts were prepared using modified RIPA buffer (50mM Tris HCL pH 7.4, 0.5% NP-40, 0.5% w/v Na-deoxycholate, 150mM NaCl containing protease inhibitors (Roche, Welwyn Garden City, UK). Protein extracts were fractionated by SDS-PAGE on 3-8% Tris-Acetate or 4-12% Bis-Tris precast polyacrylamide gels (Invitrogen, Paisley, UK) and transferred to a nitrocellulose membrane (Invitrogen). Proteins were detected using anti-NQO1 (Sigma Aldrich); Nrf2 and HO-1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA); GCLC and β-actin (Abcam, Cambridge, UK); Brd2, Brd3 and Brd4 (Bethyl Laboratories, Cambridge, UK); Keap1 (Origene, Rockville, MD, USA). Bands were visualised by chemiluminescence (ECL Plus; GE Healthcare, Hatfield, UK) and protein expression levels were normalised to β-actin expression.

Real-time PCR

Total RNA was isolated using RNeasy Mini Kit (Qiagen) and reverse-transcribed using random primers and AMV reverse transcriptase (Promega, Southampton, UK). mRNA was quantified by real-time PCR (Rotor Gene 3000; Qiagen) using SYBR Green PCR Master Mix Reagent and QuantiTect primer assays (Qiagen) for HO-1 (cat no: QT00092645), NQO1 (cat no: QT00050281), GCLC (cat no: QT00037310), Nrf2 (cat no: QT00027384), Keap1 (cat no: QT00080220), MnSOD (cat no: QT01008693) and catalase (cat no: QT00079674), and normalised to 18S rRNA or GNB2L1 expression. GNB2L1 expression was also determined by a QuantiTect primer assay (cat no: QT01156610). For 18S rRNA the following primer sequences were used: 5′-CTTAGAGGGACAAGTGGCG-3′ and 5′-ACGCTGAGCCAGTCAGTGTA-3′.

Immunoprecipitation

Nrf2 was immunoprecipitated from 300-1000μg of cell lysate by incubating with 5μg anti-Nrf2 antibody (Santa Cruz Biotechnology) overnight at 4°C. Immunoprecipitates were captured with rabbit TrueBlot IP beads (eBioscience, Hatfield, UK). After extensive washing, bound proteins were released by boiling in SDS–PAGE sample buffer (Invitrogen, Paisley, UK). Immunoprecipitates were fractionated by SDS-PAGE and the presence of Brd2, Brd3 and Brd4 was determined by Western blot analysis, as described above, and normalised to the amount of immunoprecipitated Nrf2. Cells were treated with the protease inhibitor MG132 (2.5 μg/ml) for 1.5 hrs before immunoprecipitation to maximise the amount of Nrf2 protein immunoprecipitated.

Chromatin immunoprecipitation (ChIP)

Chromatin immunoprecipitation (ChIP) assays were performed with the Magna ChIP A/G kit (Millipore, Temecula, CA, USA). Cells were fixed in 1% formaldehyde for 10 minutes and DNA fragmented by means of sonication (five 10-second pulses). Samples were diluted in ChIP dilution buffer and incubated with 4μg of Brd2 or Brd4 antibody (Bethyl Laboratories) and protein A/G magnetic beads overnight. Antibody/DNA complexes were captured, washed, eluted, and reverse cross-linked. DNA was purified by means of spin columns containing activated silica membrane filters. The precipitated DNA was re-suspended, and real-time PCR was performed using primers spanning ARE sites on the HO-1 and NQO1 gene promoters. The primers used were: NQO1 F: 5′-CAGTGGCATGCACCCAGGGAA-3′, R: 5′-GCATGCCCCTTTTAGCCTTGGCA-3′ (28) and HO-1 F: 5′-CTGCCCAAACCACTTCTGTT-3′, R: 5′-ATAAGAAGGCCTCGGTGGAT-3′ (29). DNA expression was normalized to input DNA for each sample.

Cigarette smoke extract preparation

Cigarette smoke extract (CSE) was prepared by filtering one cigarette through a 50 ml syringe and passing the smoke through 10 ml of starvation media. The concentration of cigarette smoke extract (CSE) used was calculated as % v/v of this extract with respect to the total volume of media.

Detection of ROS using dihydroethidium (DHE) staining

THP-1 cells were seeded (0.3 × 106 cells/ml) in 96 well tissue culture plates in starvation medium and then incubated with the indicated treatments. Cells were then incubated with DHE (40μM) for 30 mins at 37°C and 5% CO2, washed using Hank’s Balanced Salt solution and re-seeded in 96 well tissue culture plates at a density of 0.3 × 106 cells/ml. Fluorescence was measured at excitation/emission wavelengths of 520/610nm using a fluorescence plate reader (Synergy HT Biotek, VT, USA).

Statistical analysis

Data are expressed as mean ± SEM. Results were analysed using one-way ANOVA for repeated measures, followed by Dunnet post-hoc test. Statistical analysis was performed using the GraphPad Prism 4 software (Prism, San Diego, CA, USA). p < 0.05 was considered statistically significant.

Results

JQ1+ increases antioxidant gene expression

In THP-1 cells, the BET bromodomain inhibitor JQ1+ (30-1000nM) augmented the mRNA expression of Nrf2-dependent genes HO-1 (maximum of ~10-fold), NQO1 (maximum of ~3-fold) and GCLC (maximum of ~2-fold) in a concentration-dependent manner, 24 hrs post-treatment (Figure 1A-C). On the other hand, its inactive enantiomer JQ1− did not modulate gene expression. JQ1+ (300nM) increased the mRNA of HO-1 (~8-fold), NQO1 (~2.5-fold) and GCLC (~4-fold) 8-24 hrs post-treatment (Figure 1D-F). These changes were accompanied by increased HO-1 (Figure 1G and H), NQO1 (Figure 1G and I) and GCLC (Figure G and J) protein levels 24-32 hrs post-treatment. Consistent with the increase in antioxidant protein expression, we observed that pre-treatment of THP-1 cells with JQ1+ for 32 hrs reduced both baseline and H2O2 (100μM)-induced intracellular ROS production, whilst JQ1− had no effect (Figure 1K).

Figure 1. Effect of JQ1+ and JQ1− on HO-1, NQO1 and GCLC expression in THP-1 cells.

Figure 1

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(A-C) mRNA expression of HO-1 (A), NQO1 (B) and GCLC (C) was determined in THP-1 cells after treatment with vehicle, JQ1− or JQ1+ (30-1000 nM) for 24 hrs and was normalised to GNB2L1 mRNA levels. Data are represented as fold-changes with respect to vehicle control. (D-F) The mRNA expression of HO-1 (D), NQO1 (E) and GCLC (F) was determined after treatment with vehicle, JQ1− or JQ1+ (300 nM) for 4, 8 and 24hrs and was normalised to GNB2L1 mRNA levels. Data are represented as fold-change with respect to vehicle control at each time-point (dotted line). (G-J) HO-1, NQO1 and GCLC protein expression was determined in whole cell protein extracts after treatment with vehicle, JQ1− or JQ1+ (300 nM) for 8, 24 and 32 hrs, and normalised to β-actin expression (G). Fold-changes in HO-1 (H), NQO1 (I) and GCLC (J) protein were determined with respect to vehicle control (dotted line) at each time-point. (K) THP-1 cells were pre-treated with JQ1+ for 32 hrs and then stimulated with H2O2 (100 μM) for 15 mins. Intracellular ROS levels were determined by staining with dihydroethidium (DHE) and measuring fluorescence at 520/610nm using a fluorescence plate reader. Data are represented as fold-change with respect to vehicle control. Results are representative of mean ± SEM of 3 independent experiments. * p<0.05, ** p<0.01 and *** p<0.001 vs vehicle.

We also assessed the effect of JQ1+ on antioxidant protein expression in THP-1 cells in the presence or absence of CSE, a potent source of oxidative stress. CSE increased Nrf2, HO-1 and NQO1 protein expression. JQ1+ increased CSE-induced HO-1 and NQO1 protein levels further, without affecting Nrf2 protein expression, suggesting that BET bromodomain inhibition can augment antioxidant gene expression even under conditions of oxidative stress (Supplementary data, Figure S1D).

The effect of JQ1+ on Nrf2 target genes was also investigated in primary blood monocytes from three healthy volunteers. In line with our findings in THP-1 cells, JQ1+ (300 nM) increased HO-1 and GCLC mRNA levels in the monocytes from all volunteers, 4 hrs post-treatment (Supplemental data, Figure S1E and F). However, JQ1+ increased NQO1 mRNA in the monocytes from only one volunteer suggesting patient-specific differences in the regulation of NQO1 gene expression (Supplemental data, Figure S1G).

In ASMCs cells, JQ1+ also increased HO-1 (maximum of ~5-fold) and NQO1 (maximum of ~6-fold) mRNA expression in a concentration-dependent manner, 24 hrs post-treatment (Figure 2A-B). JQ1+ (300nM) also increased HO-1 (~4-fold), NQO1 (~4-fold) and GCLC (~2-fold) mRNA 8-24 hrs post-treatment (Figure 2C-E). These changes were accompanied by increased HO-1 (Figure 2F-G), NQO1 (Figure 2H-I) and GCLC (Figure 2J-K) protein expression. Interestingly, the antioxidant genes MnSOD and catalase, which are not classical Nrf2 targets, were not modulated by JQ1+ in ASMCs (Supplemental data, Figure S1A-B). These data suggest an involvement of BET bromodomains in the inhibition of Nrf2-dependent antioxidant genes in both structural and immune cells.

Figure 2. Effect of JQ1+ and JQ1− on HO-1, NQO1 and GCLC expression in ASMCs.

Figure 2

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(A-B) The mRNA expression of HO-1 (A) and NQO1 (B) was determined in ASMCs after treatment with vehicle, JQ1− or JQ1+ (30-1000 nM) for 24 hrs and was normalised to 18S rRNA expression. (C-E) The mRNA expression of HO-1 (C), NQO1 (D) and GCLC (E) was determined after treatment with vehicle, JQ1− or JQ1+ (300 nM) for 4, 8 and 24 hrs and was normalised to 18S rRNA expression. Data are represented as fold-change with respect to vehicle control (dotted line) at each time-point. (F-K) HO-1 (F-G), NQO1 (H-I) and GCLC (J-K) protein expression was determined in whole cell protein extracts, after treatment with vehicle, JQ1− or JQ1+ (300 nM) for 8, 24 and 32 hrs, and was normalised to β-actin expression. Data are represented as fold-change with respect to vehicle control (dotted line) at each time-point. Results are representative of mean ± SEM of 3 ASMC (A-B) and 4-5 ASMC donors (C-K). * p<0.05, ** p<0.01 and *** p<0.001 vs vehicle.

siRNA-mediated inhibition of BET protein expression leads to increased antioxidant expression

The role of BET proteins in the regulation of antioxidant genes was confirmed by specific inhibition of Brd2, Brd3 and Brd4 expression using siRNA-mediated gene knock-down. In THP-1 and ASMCs cells, a strong reduction of Brd2, Brd3 and Brd4 protein levels was observed 48hrs after transfection with specific siRNA sequences (300nM) (Figures 3A-D and 3G-J). In THP-1 cells, HO-1 expression was increased by ~6-fold in response to Brd4 siRNA and by ~3-fold in response to Brd2 siRNA, whilst Brd3 siRNA had no effect (Figure 3E) 48 hours after transfection. On the other hand NQO1 expression was increased only by Brd2 siRNA (~3-fold) (Figure 3F). In ASMCs, HO-1 (~4-fold) and NQO1 (~3-fold) protein expression were increased in response to Brd2 siRNA, whilst it was not affected by Brd3 or Brd4 siRNA (Figure 3K-L). Thus, BET proteins are directly involved in the suppression of Nrf2-dependent antioxidant genes in both cell types.

Figure 3. Effect of BET protein siRNA knock-down on HO-1 and NQO1 protein expression.

Figure 3

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(A-F) THP-1 cells were transfected with random oligonucleotide control (Ct), Brd2 (B2), Brd3 (B3) or Brd4 (B4) siRNA (300 nM) for 48 hrs, and Brd2 (B), Brd3 (C), Brd4 (D), HO-1 (E) and NQO1 (F) protein expression was determined in whole cell extracts and normalised to β-actin protein expression. Representative blots are shown (A). Results are representative of mean ± SEM of 4 independent experiments. (G-L) ASMCs were transfected with random oligonucleotide control (Ct), Brd2 (B2), Brd3 (B3) or Brd4 (B4) siRNA (300 nM) for 48 hrs and Brd2 (H), Brd3 (I), Brd4 (J), HO-1 (K) and NQO1 (L) protein expression was determined in whole cell extracts and normalised to β-actin protein expression. Representative blots are shown (G). Results are representative of mean ± SEM of 5 ASMC donors. Data are expressed as fold-change with respect to random oligonucleotide control. * p<0.05, ** p<0.01 and *** p<0.001 vs random oligonucleotide control.

Effect of BET proteins on Nrf2 signalling

In order to determine whether the inhibitory effect of BET proteins on antioxidant gene expression occurs through suppression of Nrf2 signalling, we determined the effect of BET bromodomain inhibition on Nrf2 transcriptional activity using an ARE-driven luciferase reporter gene vector in ASMCs. JQ1+ (300-1000nM) increased luciferase activity 24 hrs post-treatment (maximum of ~2-fold), whilst JQ1− did not have any effect (Figure 4A). Intriguingly, the effect of JQ1+ was comparable to the effect of the Nrf2 inducer sulforaphane at 4μM (Supplemental data, Figure S1C). In line with these findings, knock-down of Nrf2 gene expression using siRNA abrogated the upregulation of HO-1 and NQO1 protein by JQ1+ in both ASMCs (Figure 4B) and THP-1 cells (Figure 4C), confirming that the inhibitory effect of BET proteins on antioxidant genes occurs through inhibition of Nrf2 signalling.

Figure 4. Effect of JQ1+ and JQ1− on Nrf2-dependent transcription.

Figure 4

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(A) ASMCs were transfected with an ARE-driven luciferase reporter vector (2.5 μg) for 24 hrs, and then treated with vehicle, JQ1− or JQ1+ (300 and 1000nM) for 24hrs. ARE-driven transcriptional activity was determined by measuring firefly luciferase activity and normalising to Renilla luciferase activity. Data are expressed as fold-change with respect to vehicle control. Results are representative of mean ± SEM of 5 ASMC donors. ** p<0.01 and *** p<0.001 vs vehicle. (B-C) ASMCs (B) and THP-1 cells (C) were transfected with random oligonucleotide control (Ct) or Nrf2 siRNA (300 nM) for 24 hours and then treated with vehicle, JQ1− or JQ1+ (300 nM) for 32 hrs. Nrf2, NQO1, HO-1 and β-actin protein expression was determined by western blotting. Blots are representative of 1 experiment from each cell type.

To better understand the mechanism behind the inhibition of Nrf2-mediated transcription by BET proteins, we determined the effect of JQ1 on the expression of Nrf2 and Keap1. In ASMCs, JQ1+ (300nM) increased Nrf2 protein (Supplemental data, Figure S2A-B) and mRNA (Supplemental data, Figure S2C), 8-32 hrs post-treatment. At the same time, JQ1+ reduced Keap1 protein levels 24-48 hrs post-treatment (Supplemental data, Figure S2A and D), without significantly affecting Keap1 mRNA (Supplemental data, Figure S2E). This change in the balance between Nrf2 and Keap1 occurs concurrently with the increase in antioxidant genes suggesting that it is not the mechanism behind the activation of Nrf2 but probably a secondary effect which leads to sustained Nrf2 activation in response to BET protein inhibition. Intriguingly, although a reduction in Keap1 protein was also observed in JQ1+ (300nM)-treated THP-1 cells (Supplemental data, Figure S2F and I), Nrf2 protein was also strongly reduced 24-32 hrs post-treatment (Supplemental data, Figure S2F-G). These findings suggest cell-specific differences in Nrf2 homeostasis.

Study of in vitro BET protein-Nrf2 interaction

To determine whether the suppression of Nrf2 activity by BET proteins is a result of direct protein-protein interaction we performed co-immunoprecipitation of Nrf2 with BET proteins in THP-1 cells. Immunoprecipitation of Nrf2 followed by detection of Brd2, Brd3 and Brd4 protein by western blot analysis revealed interaction of Nrf2 with all three BET proteins. Treatment with JQ1+ (300nM) did not attenuate the association between Nrf2 and BET proteins, suggesting that the interaction is bromodomain-independent (Figure 5A).

Figure 5. Determination of Nrf2 - BET protein interaction and recruitment of BET proteins to NQO1 and HO-1 gene promoters.

Figure 5

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(A) THP-1 cells were treated with vehicle, JQ1− or JQ1+ (300 nM) for 5 hrs. For the final 1.5 hrs cells were treated with the protease inhibitor MG132 (2.5 μg/ml). Nrf2 immunoprecipitation was performed in whole cell lysates or non-lysate control (NL). The immune complexes and whole cell extracts (WCE) from the same samples were separated by SDS-PAGE, and Brd2, Brd3, Brd4 and Nrf2 protein expression was determined by western blotting. Blots are from 1 representative experiment of 3 independent experiments. (B-E) THP-1 cells were treated with vehicle, JQ1− or JQ1+ (300 nM) for 5 hrs. Recruitment of Brd2 and Brd4 to ARE sites on the promoters of HO-1 and NQO1 was determined by ChIP. Data are expressed as fold-change with respect to IgG isotype control. Results are representative of mean ± SEM of 4 independent experiments. * p<0.05, ** p<0.01 and *** p<0.001 vs IgG isotype control. ## p<0.01 vs vehicle control.

Recruitment of BET proteins to ARE elements of NQO1 and HO-1 gene promoters

We investigated the presence of Brd2 and Brd4 at ARE sites on the promoters of HO-1 and NQO1 genes by ChIP in THP-1 cells. We observed increased recruitment of Brd2 (~4-fold) and Brd4 (~3-fold) to the NQO1 promoter (Figure 5B-C). There was also increased recruitment of Brd2 (~3-fold) and Brd4 (~2-fold) to the HO-1 promoter (Figure 5D-E), suggesting that BET proteins are constitutively associated with the promoters of these genes. Baseline recruitment of Nrf2 to these sites was also observed (data not shown). Unexpectedly, the inactive enantiomer JQ1− led to an increase in the recruitment of Brd2 and Brd4 to both promoters compared to vehicle control (Figure 5B-E). This is possibly a non-specific effect of the compound that does not have a functional consequence, as JQ1− did not modulate Nrf2-dependent transcription. Moreover, JQ1+ did not significantly affect the recruitment of Brd2 or Brd4 to the antioxidant gene promoters compared to JQ1−, indicating that recruitment of BET proteins is also bromodomain-independent.

Discussion

BET proteins act as “readers” of protein acetylation by binding to acetylated lysine residues and providing a scaffold for transcriptional activators or repressors (16). We demonstrate for the first time that BET proteins are constitutively associated with Nrf2 on ARE sites of antioxidant gene promoters. Reduction of BET protein expression using siRNA or inhibition of their binding to acetylated lysine residues, using the pharmacological inhibitor JQ1+, leads to activation of Nrf2-mediated transcription and increased expression of the antioxidant genes HO-1, NQO1 and GCLC. Our findings demonstrate that BET proteins act as inhibitors of Nrf2 activity and are thus putative molecular targets for enhancing endogenous antioxidant defences and thus protection from oxidative stress.

BET proteins are primarily known as activators of gene transcription. Brd2 and Brd4 in particular are involved in cell cycle progression by recruiting co-activators, such as E2F transcription factor 1 (E2F1) and the positive transcription elongation factor complex (P-TEFb) to acetylated histone at the promoters of proliferative genes (21,22,30). Brd4 has also been shown to directly interact with acetylated nuclear factor (NF)-κB leading to activation of inflammatory gene transcription (18). Intriguingly, BET proteins can also act as transcriptional repressors. Brd2 has been shown to interact with peroxisome proliferator-activated receptor (PPAR)-γ leading to suppression of its transcriptional activity (27). We show that inhibition of BET bromodomain using the inhibitor JQ1+ or siRNA-mediated knock-down of Brd2 and Brd4 genes leads to activation of Nrf2-dependent antioxidant genes in ASMCs and THP-1 cells, indicating an inhibitory effect of BET proteins on Nrf2 activity. In line with these findings, JQ1+ had a protective effect against oxidative stress-induced intracellular ROS production, highlighting the potential of BET proteins as therapeutic targets for augmenting antioxidant responses. This protective effect of BET protein inhibition was further corroborated by our findings that JQ1+ can up-regulate Nrf2-dependent antioxidant proteins even in cells exposed to CSE, an important instigator of oxidative stress in COPD (31). Moreover, we show that this mechanism also occurs in primary monocytes which are a major source of ROS in the airways (32). Our study identifies Nrf2 as a new target of the inhibitory effect of BET proteins providing a novel insight into the mechanisms regulating Nrf2 activity and thus antioxidant protection.

Nrf2 activity is primarily regulated through changes in its protein stability as a result of its association or dissociation from the Keap1/Cul3 complex (8). Nrf2 nuclear translocation and DNA binding can also be modulated by post-translational modifications, whilst its transcriptional activity is regulated through interaction with co-activators or repressors (33-36). We show that JQ1+ did not modulate Nrf2 or Keap1 levels at early time-points suggesting that BET proteins do not affect Nrf2 protein stability (data not shown). In ASMCs JQ1+ led to a delayed increase, 8 hrs post-treatment, in Nrf2 mRNA and protein expression, possibly a result of positive feedback activation of the Nrf2 gene itself, which is known to contain an ARE in its promoter (37). Intriguingly, this effect was not observed in THP-1 cells suggesting cell-specific differences in this mechanism. Moreover, JQ1+ reduced Keap1 protein expression in both ASMCs and THP-1 cells. However, as this occurs concurrently with the activation of the antioxidant genes, it is possibly not related to the mechanism of Nrf2 activation by JQ1+, but may constitute a feedback-loop activation mechanism leading to prolonged Nrf2 activation.

Our data indicate that BET proteins interact with Nrf2 and are found at the ARE sites of antioxidant gene promoters under baseline conditions, suggesting that BET proteins may be constitutively present in the Nrf2 transcriptional complex. Depletion of BET proteins, particularly Brd2 and Brd4, using siRNA leads to upregulation of Nrf2 target proteins indicating that the presence of BET proteins in the complex leads to repression of Nrf2-mediated transcription. Treatment with JQ1+ also leads to activation of Nrf2-dependent antioxidant genes and an ARE-driven luciferase gene, indicating that the bromodomains are directly involved in the inhibition of Nrf2-mediated transcription by BET proteins. On the other hand, the interaction of BET proteins with Nrf2 and their recruitment to the antioxidant gene promoters are not prevented by JQ1+, suggesting that BET proteins do not directly bind to acetylated lysine residues on either histones or Nrf2. The interaction of BET proteins with Nrf2 thus possibly occurs through their ET or C-terminal domains either directly, or indirectly through other proteins or protein complexes (21,38,39). These interactions need to be investigated in more depth in the future.

Inhibition of Nrf2-dependent transcription by BET proteins may occur through the recruitment of a repressive complex to the Nrf2-regulated gene promoter. Belkina et al suggest that Brd2 inhibits PPAR-γ-mediated transcription by recruiting a repressor complex containing the silencing mediator for retinoid and thyroid hormone receptor (SMRT), through association with retinoic X receptor (RXR), which is found in a heterodimer with PPAR-γ (40). Both RXR and SMRT have also been shown to interact with Nrf2 and inhibit its transcriptional activity (36,41). Thus, a mechanism involving the recruitment of RXR and SMRT could also be implicated in the inhibition of Nrf2 by BET proteins; this will be explored in future studies.

Another interesting observation arising from our study is that BET family members show cell-type and gene specificity. Although all three BET proteins were found to interact with Nrf2, the siRNA knock-down experiments revealed that Brd2 and Brd4 were predominantly involved in the inhibition of antioxidant gene expression. Furthermore, in THP-1 cells, Brd2 was found to be more important in the inhibition of NQO1 whilst Brd4 in the inhibition of HO-1. On the other hand, in ASMCs Brd2 is the main isoform involved in the inhibition of both HO-1 and NQO1. These data suggest that although all isoforms can interact with Nrf2, they are likely to recruit different proteins due to differences in their protein-protein interaction motifs. Indeed, although the bromodomains and ET domains are highly similar between BET family members (>80% similarity), their C-terminal domains show different lengths and structures, which may account for the differences in the proteins they interact with (38,40). Furthermore, the differences in gene specificity may be a result of differences in the local environment of gene promoters in each cell type. This disparity between BET protein isoform specificities is reflected by the divergence observed in the phenotypes of Brd2 and Brd4 knockout mice (42). Further work looking at other Nrf2-dependent cytoprotective genes should shed more light on the roles of Brd2, Brd3 and Brd4.

Nrf2 is an important mechanism through which cells are protected from oxidative stress and thus the molecular mechanisms regulating its activity are of great interest, especially in diseases involving oxidative stress like COPD (7,9,13). Our study suggests that BET proteins may be a key player in the regulation of Nrf2 activity and thus an important target for augmenting antioxidant responses in COPD and other oxidative stress-mediated diseases. Moreover, it raises the possibility that perturbed BET protein signalling in disease could result in gene specific repression of Nrf2-dependent genes contributing to defective anti-oxidant defence. Our findings highlight an important role of BET protein inhibitors as novel antioxidant treatments.

Supplementary Material

Supplementary Figures 1-2

Acknowledgments

This work was supported by the Wellcome Trust Grant 085935, Asthma UK Grant and by the NIHR Respiratory Disease Biomedical Research Unit at the Royal Brompton and Harefield NHS Foundation Trust and Imperial College London.

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Supplementary Materials

Supplementary Figures 1-2
NIHMS57649-supplement-1.pdf (296.3KB, pdf)

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