of April 12, 2018.This information is current as

Antioxidant Gene ExpressionMediated−Related Factor 2−Suppress NF-E2

Bromodomain and Extraterminal Proteins

Fan ChungKianPankaj K. Bhavsar, Ian M. Adcock, Peter J. Barnes and

Charalambos Michaeloudes, Nicolas Mercado, Colin Clarke,

http://www.jimmunol.org/content/192/10/4913doi: 10.4049/jimmunol.1301984April 2014;

2014; 192:4913-4920; Prepublished online 14J Immunol 

MaterialSupplementary

4.DCSupplementalhttp://www.jimmunol.org/content/suppl/2014/04/14/jimmunol.130198

Referenceshttp://www.jimmunol.org/content/192/10/4913.full#ref-list-1

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists, Inc. All rights reserved.Copyright © 2014 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

is published twice each month byThe Journal of Immunology

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The Journal of Immunology

Bromodomain and Extraterminal Proteins SuppressNF-E2–Related Factor 2–Mediated Antioxidant GeneExpression

Charalambos Michaeloudes,1 Nicolas Mercado,1 Colin Clarke, Pankaj K. Bhavsar,

Ian M. Adcock, Peter J. Barnes, and Kian Fan Chung

Oxidative stress, a pathogenetic factor in many conditions, including chronic obstructive pulmonary disease, arises due to accu-

mulation of reactive oxygen species and defective antioxidant defenses in the lungs. The latter is due, at least in part, to impaired

activation of NF-E2–related factor 2 (Nrf2), a transcription factor involved in the activation of antioxidant and cytoprotective

genes. The bromodomain and extraterminal (BET) proteins, Brd2, Brd3, Brd4, and BrdT, bind to acetylated lysine residues on

histone or nonhistone 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 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 small interfering RNA led to activation of Nrf2-dependent transcription and

expression of the antioxidant proteins heme oxygenase-1, NADPH quinone oxidoreductase 1, and glutamate-cysteine ligase

catalytic subunit. Also, JQ1+ prevented H2O2-induced intracellular reactive oxygen species production. By coimmunoprecipita-

tion, BET proteins were found to be complexed with Nrf2, whereas chromatin–immunoprecipitation studies indicated recruitment

of Brd2 and Brd4 to Nrf2-binding sites on the promoters of heme oxygenase-1 and NADPH quinone oxidoreductase 1. 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. The Journal of

Immunology, 2014, 192: 4913–4920.

Cellular oxidative stress that results from an imbalance be-tween the production of reactive oxygen species (ROS) andtheir removal by antioxidant defense mechanisms is pivotal

in the pathogenesis of a number of diseases, including chronic ob-structive pulmonary disease (COPD). In COPD, cigarette smoke in-halation and the resulting chronic inflammatory response lead torelease of ROS in the airways. Defective antioxidant mechanisms inCOPD lungs could lead to accumulation of ROS, resulting in acti-vation of redox-sensitive transcription factors, epigenetic changes, andoxidative damage in immune and structural cells (1). NADPH oxi-

dase–mediated ROS trigger airway smooth muscle cell (ASMC)proliferation and hypertrophy (2). Exogenous oxidative stress stimulireduce histone deacetylase 2 activity in alveolar macrophages, lead-ing to increased expression of inflammatory genes (3). Moreover,cigarette smoke–mediated ROS reduce sirtuin 1 activity, leading toupregulation of matrix metalloproteinase-9 in monocytes and sputummacrophages (4). As a result, there is amplification of the inflam-matory response and induction of pathogenic processes, such as lungparenchymal destruction and small airway remodeling (5, 6).Cells are normally protected from ROS by the concerted action of

a large array of endogenous antioxidant proteins. The transcriptionfactor NF-E2–related factor 2 (Nrf2) plays a pivotal role in cellularantioxidant defenses 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 inhibitorKelch-like ECH-associated protein (Keap1) and the ubiquitin-ligaseCul3, where it is constantly targeted for proteosomal degradation.In the presence of oxidative stress, Nrf2 dissociates from the com-plex, leading to an increase in its protein levels and subsequentactivation of antioxidant genes by binding to common regulatoryelements, termed antioxidant response elements (AREs), in theirpromoter regions (8). Impaired Nrf2 function has been linked todefective antioxidant defenses in several age-related diseases, in-cluding COPD (9–12). Nrf2 expression has been shown to be re-duced in lung tissue and alveolar macrophages of patients withCOPD (9, 13, 14). Moreover, we have reported that reduced histonedeacetylase activity in monocyte-derived macrophages leads to de-creased Nrf2 protein stability through increased acetylation, impli-cating epigenetic mechanisms in impaired Nrf2 activation in COPD(15). Identification of molecular targets, particularly epigenetic

Airway Disease Section, National Heart and Lung Institute, Imperial College Londonand Biomedical Research Unit, Royal Brompton Hospital, London SW3 6LY, UnitedKingdom

1C.M. and N.M. contributed equally to this work.

Received for publication July 26, 2013. Accepted for publication March 10, 2014.

This work was supported by Wellcome Trust Grant 085935, an Asthma UK grant,and the National Institute for Health Research Respiratory Disease Biomedical Re-search Unit at the Royal Brompton and Harefield National Health Service FoundationTrust and Imperial College London.

Address correspondence and reprint requests to Prof. Kian Fan Chung, NationalHeart and Lung Institute, Airway Disease Section, Guy Scadding Building, Dove-house Street, London SW3 6LY, U.K. E-mail address: f.chung@imperial.ac.uk

The online version of this article contains supplemental material.

Abbreviations used in this article: ARE, antioxidant response element; ASMC, air-way smooth muscle cell; BET, bromodomain and extraterminal; ChIP, chromatinimmunoprecipitation; COPD, chronic obstructive pulmonary disease; CSE, cigarettesmoke extract; ET, extraterminal; GCLC, glutamate-cysteine ligase catalytic subunit;HO, heme oxygenase; NQO1, NADPH quinone oxidoreductase 1; Nrf2, NF-E2–related factor 2; PPAR, peroxisome proliferator-activated receptor; ROS, reactiveoxygen species; RXR, retinoic X receptor; siRNA, small interfering RNA; SMRT,silencing mediator for retinoid and thyroid hormone receptor.

Copyright� 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00

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effectors, for augmenting Nrf2 activity would therefore be cru-cial for the development of new antioxidant therapies for COPDand other oxidative stress–dependent diseases.The bromodomain and extraterminal (BET) proteins act as readers

of protein acetylation by binding to acetylated lysine residues throughtwo highly conserved N-terminal bromodomain modules to regulategene expression (16). Specifically, BET proteins interact with tran-scription factors and chromatin remodeling complexes via theirextraterminal (ET) and C-terminal domains, and recruiting them to thegene promoter to either activate or repress transcription of genes in-volved in inflammation, cell cycle, and differentiation (17–20). Thehuman BET protein family comprises Brd2, Brd3, Brd4, and BrdT,the latter being solely expressed in the testis (21, 22). Small moleculebromodomain inhibitors, developed in recent years, are importanttools for the study of BET proteins. The prototype compound, JQ1, isa thieno-triazolo-1,4-diazepine that specifically interferes with thebinding of BET bromodomains to acetylated lysines (23). BET pro-teins have been shown to be implicated in diseases in which oxidativestress and compromised antioxidant protection play an importantpathogenic role, such as acute myeloid leukemia (24), heart failure(25), and obesity (26, 27). However, it is currently unknown whetherBET proteins are directly involved in the regulation of antioxidantgene expression and hence the protection against ROS-mediated dis-ease pathogenesis. We hypothesized that BET proteins are involved inthe regulation of antioxidant genes in both structural and immune cellsand are thus important targets for augmenting antioxidant defenses inCOPD. We therefore examined the effect of pharmacological andmolecular inhibitors of BET protein activity on antioxidant gene ex-pression in primary human ASMCs and the human monocytic cellline, THP-1. Furthermore, we investigated the effect of BET proteininhibition on the Nrf2 pathway, the interaction of BET proteins withNrf2, and their recruitment to antioxidant gene promoters.

Materials and MethodsReagents

The active JQ1 enantiomer (+)2 JQ1 (JQ1+) and the inactive enantiomer(2)2 JQ1 (JQ12) were purchased from Cayman (Cambridge, U.K.).MG132 was purchased from Sigma- Aldrich (Poole, U.K.).

Cell culture and stimulation

THP-1 cells (human monocytic cell line) were obtained from the AmericanType Culture Collection (Manassas, VA) and cultured in RPMI 1640medium (Cayman) containing 10% heat-inactivated FBS and 2 mML-glutamine at 37˚C, 5% CO2, and humidified atmosphere. For stimulation,THP-1 cells were seeded (0.3 3 106 cells/ml) in starvation medium con-taining RPMI 1640 supplemented with 1% FBS and 15 mM L-glutamine.

ASMCs were dissected from tracheas or bronchi of transplant donorlungs and were cultured in DMEM supplemented with 4 mM L-glutamine,20 U/L penicillin, 20 mg/ml streptomycin, 2.5 mg/ml amphotericin B, and10% FBS. Presence of ASMCs was confirmed by identifying the charac-teristic “hill and valley” morphology using light microscopy. Cell stockswere kept in 150-cm2 flasks at 37˚C, 5% CO2, and humidified atmosphere.Cells between passages 3 and 7 were used for experiments. Before treat-ment, cells were incubated in serum-free medium containing phenol-freeDMEM supplemented with 1 mM sodium pyruvate, 4 mM L-glutamine,nonessential amino acids, 1% insulin-transferrin-selenium-X supplement,0.1% BSA, and antibiotics, as described above.

PBMCs were isolated from the peripheral blood of three healthy volunteers,using AccuSPIN columns (Sigma-Aldrich). Monocytes were isolated by ad-herence to tissue culture plates for 90 min. This study was approved by thelocal ethics committee of Royal Brompton and Harefield National HealthService Trust, and written informed consent was obtained from each volunteer.PBMCs were cultured in RPMI 1640 medium containing 10% FBS and2 mM L-glutamine at 37˚C, 5% CO2, and humidified atmosphere.

Small interfering RNA transfection

THP-1 cells were transfected with 300 nM ON-TARGETplus SMARTpoolsmall interfering (siRNA) against Nrf2 (catalogue L-003755-00-0005),

Brd2 (catalogue L-004935-00-0005), Brd3 (catalogue L-004936-00-0005),or Brd4 (catalogue L-004937-00-0005) (Thermo Scientific, Epsom, U.K.),or a random oligonucleotide control siRNA (Qiagen, Crawley, U.K.) for 48 husing HiPerfect transfection reagent (Qiagen) following the manufacturer’sinstructions. ASMCs were transfected with the same siRNA sequences(300 nM) for 48 h using Amaxa nucleofection (Lonza AG, Cologne,Germany), following the manufacturer’s instructions.

ARE reporter assay

ASMCs were transfected, using Amaxa nucleofection, with DNA plasmidconstructs (2.5 mg) expressing ARE-inducible firefly luciferase and con-stitutively active Renilla luciferase (SABiosciences, Frederick, MD) for24 h, and then incubated with the required treatments for an additional24 h. At the end of the incubation time, cells were lysed and firefly andRenilla luciferase activities were determined by measuring luminescence.ARE-inducible luciferase activity was normalized to Renilla luciferaseactivity. The Nrf2 inducer sulforaphane (4 mM) was used as a positivecontrol in all experiments.

Western blotting

Protein extracts were prepared using modified radioimmunoprecipitationassay buffer (50 mM Tris HCL [pH 7.4], 0.5% Nonidet P-40, 0.5% w/v Na-deoxycholate, 150 mM NaCl containing protease inhibitors) (Roche, WelwynGarden City, U.K.). Protein extracts were fractionated by SDS-PAGE on 3–8%Tris-acetate or 4–12% Bis-Tris precast polyacrylamide gels (Invitrogen,Paisley, U.K.) 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); GCLC and b-actin (Abcam,Cambridge, U.K.); Brd2, Brd3, and Brd4 (Bethyl Laboratories, Cambridge,U.K.); and Keap1 (Origene, Rockville, MD). Bands were visualized bychemiluminescence (ECL Plus; GE Healthcare, Hatfield, U.K.), and proteinexpression levels were normalized to b-actin expression.

Real-time PCR

Total RNA was isolated using RNeasy Mini Kit (Qiagen) and reverse tran-scribed using random primers and avian myeloblastosis virus reverse tran-scriptase (Promega, Southampton, U.K.). mRNAwas quantified by real-timePCR (Rotor Gene 3000; Qiagen) using SYBRGreen PCRMasterMix Reagentand QuantiTect primer assays (Qiagen) for HO-1 (catalogue QT00092645),NQO1 (catalogue QT00050281), GCLC (catalogue QT00037310), Nrf2(catalogue QT00027384), Keap1 (catalogue QT00080220), MnSOD (cata-logue QT01008693), and catalase (catalogue QT00079674), and normalizedto 18S rRNA or GNB2L1 expression. GNB2L1 expression was also deter-mined by a QuantiTect primer assay (catalogue QT01156610). For 18S rRNA,the following primer sequences were used: 59-CTTAGAGGGACAAGT-GGCG-39 and 59-ACGCTGAGCCAGTCAGTGTA-39.

Immunoprecipitation

Nrf2 was immunoprecipitated from 300 to 1000 mg cell lysate by incubatingwith 5 mg anti-Nrf2 Ab (Santa Cruz Biotechnology) overnight at 4˚C.Immunoprecipitates were captured with rabbit TrueBlot IP beads (eBio-science, Hatfield, U.K.). After extensive washing, bound proteins werereleased by boiling in SDS-PAGE sample buffer (Invitrogen). Immunoprecip-itates were fractionated by SDS-PAGE, and the presence of Brd2, Brd3, andBrd4 was determined by Western blot analysis, as described above, and nor-malized to the amount of immunoprecipitated Nrf2. Cells were treated with theprotease inhibitor MG132 (2.5 mg/ml) for 1.5 h before immunoprecipitation tomaximize the amount of Nrf2 protein immunoprecipitated.

Chromatin immunoprecipitation

Chromatin immunoprecipitation (ChIP) assays were performed with theMagna ChIP A/G kit (Millipore, Temecula, CA). Cells were fixed in 1%formaldehyde for 10 min, and DNAwas fragmented by means of sonication(five 10-s pulses). Samples were diluted in ChIP dilution buffer and in-cubated with 4 mg Brd2 or Brd4 Ab (Bethyl Laboratories) and protein A/Gmagnetic beads overnight. Ab/DNA complexes were captured, washed,eluted, and reverse cross-linked. DNA was purified by means of spincolumns containing activated silica membrane filters. The precipitatedDNA was resuspended, and real-time PCR was performed using primersspanning ARE sites on the HO-1 and NQO1 gene promoters. The primersused were as follows: NQO1 forward, 59-CAGTGGCATGCACCCAGG-GAA-39, and reverse, 59-GCATGCCCCTTTTAGCCTTGGCA-39 (28),and HO-1 forward, 59-CTGCCCAAACCACTTCTGTT-39, and reverse,59-ATAAGAAGGCCTCGGTGGAT-39 (29). DNA expression was nor-malized to input DNA for each sample.

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Cigarette smoke extract preparation

Cigarette smoke extract (CSE) was prepared by filtering one cigarettethrough a 50-ml syringe and passing the smoke through 10 ml starvationmedia. The concentration of CSE used was calculated as percentage v/v ofthis extract with respect to the total volume of media.

Detection of ROS using dihydroethidium staining

THP-1 cells were seeded (0.3 3 106 cells/ml) in 96-well tissue cultureplates in starvation medium and then incubated with the indicated treat-ments. Cells were then incubated with dihydroethidium (40 mM) for30 min at 37˚C and 5% CO2, washed using HBSS, and reseeded in 96-welltissue culture plates at a density of 0.3 3 106 cells/ml. Fluorescence wasmeasured at excitation/emission wavelengths of 520/610 nm using a fluo-rescence plate reader (Synergy HT Biotek).

Statistical analysis

Data are expressed as mean6 SEM. Results were analyzed using one-wayANOVA for repeated measures, followed by Dunnet post hoc test. Statisticalanalysis was performed using the GraphPad Prism 4 software (Prism, SanDiego, CA). A p value ,0.05 was considered statistically significant.

ResultsJQ1+ increases antioxidant gene expression

In THP-1 cells, the BET bromodomain inhibitor JQ1+ (30–1000nM) augmented the mRNA expression of Nrf2-dependent genesHO-1 (maximum of ∼10-fold), NQO1 (maximum of ∼3-fold), andGCLC (maximum of ∼2-fold) in a concentration-dependentmanner, 24 h posttreatment (Fig. 1A–C). In contrast, its inactiveenantiomer, JQ12, 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 h posttreatment (Fig. 1D–F). Thesechanges were accompanied by increased HO-1 (Fig. 1G, 1H),NQO1 (Fig. 1G, 1I), and GCLC (Fig. 1G, 1J) protein levels 24–32 hposttreatment. Consistent with the increase in antioxidant proteinexpression, we observed that pretreatment of THP-1 cells withJQ1+ for 32 h reduced both baseline and H2O2 (100 mM)-inducedintracellular ROS production, whereas JQ12 had no effect(Fig. 1K).

FIGURE 1. Effect of JQ1+ and JQ12 on HO-1,

NQO1, and GCLC expression in THP-1 cells. (A–C)

mRNA expression of HO-1 (A), NQO1 (B), and

GCLC (C) was determined in THP-1 cells after

treatment with vehicle, JQ12 or JQ1+ (30–1000

nM), for 24 h and was normalized 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, JQ12 or

JQ1+ (300 nM), for 4, 8, and 24 h and was normal-

ized 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,

JQ12 or JQ1+ (300 nM), for 8, 24, and 32 h and

normalized to b-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 h and then stimulated with

H2O2 (100 mM) for 15 min. Intracellular ROS levels

were determined by staining with dihydroethidium

and measuring fluorescence at 520/610 nm using

a fluorescence plate reader. Data are represented as

fold change with respect to vehicle control. Results

are representative of mean 6 SEM of three inde-

pendent experiments. *p , 0.05, **p , 0.01, and

***p , 0.001 versus vehicle.

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We also assessed the effect of JQ1+ on antioxidant proteinexpression in THP-1 cells in the presence or absence of CSE,a potent source of oxidative stress. CSE increased Nrf2, HO-1, andNQO1 protein expression. JQ1+ increased CSE-induced HO-1 andNQO1 protein levels further, without affecting Nrf2 protein ex-pression, suggesting that BET bromodomain inhibition can aug-ment antioxidant gene expression even under conditions ofoxidative stress (Supplemental Fig. 1D).The effect of JQ1+ on Nrf2 target genes was also investigated in

primary blood monocytes from three healthy volunteers. In linewith our findings in THP-1 cells, JQ1+ (300 nM) increased HO-1and GCLC mRNA levels in the monocytes from all volunteers, 4 hposttreatment (Supplemental Fig. 1E, 1F). However, JQ1+ in-creased NQO1 mRNA in the monocytes from only one volunteer,suggesting patient-specific differences in the regulation of NQO1gene expression (Supplemental Fig. 1G).In ASMCs cells, JQ1+ also increased HO-1 (maximum of ∼5-

fold) and NQO1 (maximum of ∼6-fold) mRNA expression ina concentration-dependent manner, 24 h posttreatment (Fig. 2A,2B). JQ1+ (300 nM) also increased HO-1 (∼4-fold), NQO1 (∼4-fold), and GCLC (∼2-fold) mRNA 8–24 h posttreatment(Fig. 2C–E). These changes were accompanied by increasedHO-1 (Fig. 2F, 2G), NQO1 (Fig. 2H, 2I), and GCLC (Fig. 2J,2K) protein expression. Interestingly, the antioxidant genesMnSOD and catalase, which are not classical Nrf2 targets, werenot modulated by JQ1+ in ASMCs (Supplemental Fig. 1A, 1B).These data suggest an involvement of BET bromodomains in theinhibition of Nrf2-dependent antioxidant genes in both structuraland immune cells.

siRNA-mediated inhibition of BET protein expression leads toincreased antioxidant expression

The role of BET proteins in the regulation of antioxidant genes wasconfirmed by specific inhibition of Brd2, Brd3, and Brd4 ex-pression using siRNA-mediated gene knockdown. In THP-1 andASMCs cells, a strong reduction of Brd2, Brd3, and Brd4 proteinlevels was observed 48 h after transfection with specific siRNAsequences (300 nM) (Fig. 3A–D, 3G–J). In THP-1 cells, HO-1expression was increased by ∼6-fold in response to Brd4 siRNAand by ∼3-fold in response to Brd2 siRNA, whereas Brd3 siRNAhad no effect (Fig. 3E) 48 h after transfection. In contrast, NQO1expression was increased only by Brd2 siRNA (∼3-fold) (Fig. 3F).In ASMCs, HO-1 (∼4-fold) and NQO1 (∼3-fold) protein ex-pression were increased in response to Brd2 siRNA, whereasit was not affected by Brd3 or Brd4 siRNA (Fig. 3K, 3L). Thus,BET proteins are directly involved in the suppression of Nrf2-dependent antioxidant genes in both cell types.

Effect of BET proteins on Nrf2 signaling

To determine whether the inhibitory effect of BET proteins onantioxidant gene expression occurs through suppression of Nrf2signaling, we determined the effect of BET bromodomain inhi-bition on Nrf2 transcriptional activity using an ARE-driven lu-ciferase reporter gene vector in ASMCs. JQ1+ (300–1000 nM)increased luciferase activity 24 h posttreatment (maximum of ∼2-fold), whereas JQ12 did not have any effect (Fig. 4A). Intrigu-ingly, the effect of JQ1+ was comparable to the effect of the Nrf2inducer sulforaphane at 4 mM (Supplemental Fig. 1C). In line withthese findings, knockdown of Nrf2 gene expression using siRNA

FIGURE 2. Effect of JQ1+ and JQ12 on

HO-1, NQO1, and GCLC expression in ASMCs.

(A and B) The mRNA expression of HO-1 (A)

and NQO1 (B) was determined in ASMCs after

treatment with vehicle, JQ12 or JQ1+ (30–

1000 nM), for 24 h and was normalized to 18S

rRNA expression. (C–E) The mRNA expression

of HO-1 (C), NQO1 (D), and GCLC (E) was

determined after treatment with vehicle, JQ12or JQ1+ (300 nM), for 4, 8, and 24 h and was

normalized to 18S rRNA expression. Data are

represented as fold change with respect to ve-

hicle control (dotted line) at each time point.

(F–K) HO-1 (F and G), NQO1 (H and I), and

GCLC (J and K) protein expression was deter-

mined in whole-cell protein extracts, after

treatment with vehicle, JQ12 or JQ1+ (300

nM), for 8, 24, and 32 h and was normalized to

b-actin expression. Data are represented as fold

change with respect to vehicle control (dotted

line) at each time point. Results are represen-

tative of mean 6 SEM of three ASMC (A and

B) and four to five ASMC donors (C–K). *p ,0.05, **p , 0.01, and ***p , 0.001 versus

vehicle.

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abrogated the upregulation of HO-1 and NQO1 protein by JQ1+ inboth ASMCs (Fig. 4B) and THP-1 cells (Fig. 4C), confirming thatthe inhibitory effect of BET proteins on antioxidant genes occursthrough inhibition of Nrf2 signaling.To better understand the mechanism behind the inhibition of

Nrf2-mediated transcription by BET proteins, we determined theeffect of JQ1 on the expression of Nrf2 andKeap1. In ASMCs, JQ1+(300 nM) increased Nrf2 protein (Supplemental Fig. 2A, 2B) andmRNA (Supplemental Fig. 2C), 8–32 h posttreatment. At the sametime, JQ1+ reduced Keap1 protein levels 24–48 h posttreatment(Supplemental Fig. 2A, 2D), without significantly affecting Keap1mRNA (Supplemental Fig. 2E). This change in the balance be-tween Nrf2 and Keap1 occurs concurrently with the increase inantioxidant genes, suggesting that it is not the mechanism behindthe activation of Nrf2 but probably a secondary effect that leads tosustained Nrf2 activation in response to BET protein inhibition.Intriguingly, although a reduction in Keap1 protein was also ob-served in JQ1+ (300 nM)-treated THP-1 cells (Supplemental Fig.2F, 2I), Nrf2 protein was also strongly reduced 24–32 h post-treatment (Supplemental Fig. 2F, 2G). 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 BETproteins is a result of direct protein–protein interaction, we per-formed coimmunoprecipitation of Nrf2 with BET proteins in

THP-1 cells. Immunoprecipitation of Nrf2, followed by detectionof Brd2, Brd3, and Brd4 protein by Western blot analysis, revealedinteraction of Nrf2 with all three BET proteins. Treatment withJQ1+ (300 nM) did not attenuate the association between Nrf2 andBET proteins, suggesting that the interaction is bromodomainindependent (Fig. 5A).

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

We investigated the presence of Brd2 and Brd4 at ARE sites onthe 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 (Fig. 5B, 5C). There was alsoincreased recruitment of Brd2 (∼3-fold) and Brd4 (∼2-fold) to theHO-1 promoter (Fig. 5D, 5E), suggesting that BET proteins areconstitutively associated with the promoters of these genes. Base-line recruitment of Nrf2 to these sites was also observed (data notshown). Unexpectedly, the inactive enantiomer JQ12 led to anincrease in the recruitment of Brd2 and Brd4 to both promoterscompared with vehicle control (Fig. 5B–E). This is possiblya nonspecific effect of the compound that does not have a func-tional consequence, as JQ12 did not modulate Nrf2-dependenttranscription. Moreover, JQ1+ did not significantly affect the re-cruitment of Brd2 or Brd4 to the antioxidant gene promoterscompared with JQ12, indicating that recruitment of BET proteinsis also bromodomain independent.

FIGURE 3. Effect of BET protein

siRNA knockdown on HO-1 and

NQO1 protein expression. (A–F)

THP-1 cells were transfected with

random oligonucleotide control (Ct),

Brd2 (B2), Brd3 (B3), or Brd4 (B4)

siRNA (300 nM) for 48 h, and Brd2

(B), Brd3 (C), Brd4 (D), HO-1 (E),

and NQO1 (F) protein expression was

determined in whole-cell extracts and

normalized to b-actin protein ex-

pression. Representative blots are

shown (A). Results are representative

of mean 6 SEM of four independent

experiments. (G–L) ASMCs were

transfected with random oligonucle-

otide control (Ct), Brd2 (B2), Brd3

(B3), or Brd4 (B4) siRNA (300 nM)

for 48 h, and Brd2 (H), Brd3 (I), Brd4

(J), HO-1 (K), and NQO1 (L) pro-

tein expression was determined in

whole-cell extracts and normalized

to b-actin protein expression. Rep-

resentative blots are shown (G). Re-

sults are representative of mean 6SEM of five ASMC donors. Data are

expressed as fold change with re-

spect to random oligonucleotide

control. *p , 0.05, **p , 0.01, and

***p , 0.001 versus random oli-

gonucleotide control.

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DiscussionBET proteins act as readers of protein acetylation by binding toacetylated lysine residues and providing a scaffold for transcrip-tional activators or repressors (16). To our knowledge, we dem-onstrate for the first time that BET proteins are constitutivelyassociated with Nrf2 on ARE sites of antioxidant gene promoters.Reduction of BET protein expression using siRNA or inhibition oftheir binding to acetylated lysine residues, using the pharmaco-logical inhibitor JQ1+, leads to activation of Nrf2-mediatedtranscription and increased expression of the antioxidant genes

HO-1, NQO1, and GCLC. Our findings demonstrate that BETproteins act as inhibitors of Nrf2 activity and are thus putativemolecular targets for enhancing endogenous antioxidant defensesand thus protection from oxidative stress.BET proteins are primarily known as activators of gene tran-

scription. Brd2 and Brd4 in particular are involved in cell cycleprogression by recruiting coactivators, such as E2F transcriptionfactor 1 and the positive transcription elongation factor complex, toacetylated histone at the promoters of proliferative genes (21, 22,30). Brd4 has also been shown to directly interact with acetylatedNF-kB, leading to activation of inflammatory gene transcription(18). Intriguingly, BET proteins can also act as transcriptionalrepressors. Brd2 has been shown to interact with peroxisomeproliferator-activated receptor (PPAR)-g, leading to suppressionof its transcriptional activity (27). We show that inhibition ofBET bromodomain using the inhibitor JQ1+ or siRNA-mediatedknockdown of Brd2 and Brd4 genes leads to activation of Nrf2-dependent antioxidant genes in ASMCs and THP-1 cells, indi-cating an inhibitory effect of BET proteins on Nrf2 activity. Inline with these findings, JQ1+ had a protective effect againstoxidative stress–induced intracellular ROS production, high-lighting the potential of BET proteins as therapeutic targets foraugmenting antioxidant responses. This protective effect of BETprotein inhibition was further corroborated by our findings thatJQ1+ can upregulate Nrf2-dependent antioxidant proteins even incells exposed to CSE, an important instigator of oxidative stress inCOPD (31). Moreover, we show that this mechanism also occursin primary monocytes that are a major source of ROS in the air-ways (32). Our study identifies Nrf2 as a new target of the in-hibitory effect of BET proteins, providing a novel insight intothe mechanisms regulating Nrf2 activity and thus antioxidantprotection.Nrf2 activity is primarily regulated through changes in its pro-

tein stability as a result of its association or dissociation fromthe Keap1/Cul3 complex (8). Nrf2 nuclear translocation and DNAbinding can also be modulated by posttranslational modifications,whereas its transcriptional activity is regulated through interactionwith coactivators or repressors (33–36). We show that JQ1+ didnot modulate Nrf2 or Keap1 levels at early time points, suggestingthat BET proteins do not affect Nrf2 protein stability (data notshown). In ASMCs JQ1+ led to a delayed increase, 8 h post-treatment, in Nrf2 mRNA and protein expression, possibly a resultof positive feedback activation of the Nrf2 gene itself, which isknown to contain an ARE in its promoter (37). Intriguingly, thiseffect was not observed in THP-1 cells, suggesting cell-specificdifferences in this mechanism. Moreover, JQ1+ reduced Keap1protein expression in both ASMCs and THP-1 cells. However, asthis occurs concurrently with the activation of the antioxidantgenes, it is possibly not related to the mechanism of Nrf2 acti-vation by JQ1+, but may constitute a feedback-loop activationmechanism 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 underbaseline conditions, suggesting that BET proteins may be con-stitutively present in the Nrf2 transcriptional complex. Deple-tion of BET proteins, particularly Brd2 and Brd4, using siRNAleads to upregulation of Nrf2 target proteins, indicating that thepresence of BET proteins in the complex leads to repression ofNrf2-mediated transcription. Treatment with JQ1+ also leads toactivation of Nrf2-dependent antioxidant genes and an ARE-driven luciferase gene, indicating that the bromodomains are di-rectly involved in the inhibition of Nrf2-mediated transcription byBET proteins. In contrast, the interaction of BET proteins with Nrf2and their recruitment to the antioxidant gene promoters are not

FIGURE 4. Effect of JQ1+ and JQ12 on Nrf2-dependent transcription.

(A) ASMCs were transfected with an ARE-driven luciferase reporter

vector (2.5 mg) for 24 h and then treated with vehicle, JQ12 or JQ1+ (300

and 1000 nM), for 24 h. ARE-driven transcriptional activity was deter-

mined by measuring firefly luciferase activity and normalizing to Renilla

luciferase activity. Data are expressed as fold change with respect to ve-

hicle control. Results are representative of mean 6 SEM of five ASMC

donors. **p , 0.01 and ***p , 0.001 versus vehicle. (B and C) ASMCs

(B) and THP-1 cells (C) were transfected with random oligonucleotide

control (Ct) or Nrf2 siRNA (300 nM) for 24 h and then treated with ve-

hicle, JQ12 or JQ1+ (300 nM), for 32 h. Nrf2, NQO1, HO-1, and b-actin

protein expression was determined by Western blotting. Blots are repre-

sentative of one experiment from each cell type.

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prevented by JQ1+, suggesting that BET proteins do not directlybind to acetylated lysine residues on either histones or Nrf2. Theinteraction of BET proteins with Nrf2 thus possibly occurs throughtheir ET or C-terminal domains either directly or indirectly throughother proteins or protein complexes (21, 38, 39). These interactionsneed 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 theNrf2-regulated gene promoter. Belkina and Denis (40) suggestthat Brd2 inhibits PPAR-g–mediated transcription by recruitinga repressor complex containing the silencing mediator for reti-noid and thyroid hormone receptor (SMRT), through associationwith retinoic X receptor (RXR), which is found in a heterodimerwith PPAR-g. Both RXR and SMRT have also been shown tointeract with Nrf2 and inhibit its transcriptional activity (36, 41).Thus, a mechanism involving the recruitment of RXR and SMRTcould also be implicated in the inhibition of Nrf2 by BET pro-teins; 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. Al-though all three BET proteins were found to interact with Nrf2, thesiRNA knockdown experiments revealed that Brd2 and Brd4 werepredominantly involved in the inhibition of antioxidant gene ex-pression. Furthermore, in THP-1 cells, Brd2 was found to be moreimportant in the inhibition of NQO1, whereas Brd4 in the inhibitionof HO-1. In contrast, in ASMCs, Brd2 is the main isoform involvedin 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–proteininteraction motifs. Indeed, although the bromodomains and ETdomains are highly similar between BET family members (.80%similarity), their C-terminal domains show different lengths andstructures, which may account for the differences in the proteinswith which they interact (38, 40). Furthermore, the differences ingene specificity may be a result of differences in the local envi-ronment of gene promoters in each cell type. This disparity be-tween BET protein isoform specificities is reflected by thedivergence observed in the phenotypes of Brd2 and Brd4 knockoutmice (42). Further work looking at other Nrf2-dependent cyto-protective genes should shed more light on the roles of Brd2,Brd3, and Brd4.Nrf2 is an important mechanism through which cells are pro-

tected from oxidative stress, and thus the molecular mechanismsregulating its activity are of great interest, especially in diseasesinvolving oxidative stress like COPD (7, 9, 13). Our study suggeststhat BET proteins may be a key player in the regulation of Nrf2activity and thus an important target for augmenting antioxidantresponses in COPD and other oxidative stress–mediated diseases.Moreover, it raises the possibility that perturbed BET proteinsignaling in disease could result in gene-specific repression ofNrf2-dependent genes, contributing to defective antioxidant de-fense. Our findings highlight an important role of BET proteininhibitors as novel antioxidant treatments.

DisclosuresThe authors have no financial conflicts of interest.

FIGURE 5. Determination of Nrf2–BET protein in-

teraction and recruitment of BET proteins to NQO1

and HO-1 gene promoters. (A) THP-1 cells were

treated with vehicle, JQ12 or JQ1+ (300 nM), for 5 h.

For the final 1.5 h, cells were treated with the protease

inhibitor MG132 (2.5 mg/ml). Nrf2 immunoprecipita-

tion was performed in whole-cell lysates or nonlysate

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 one representative experiment of three inde-

pendent experiments. (B–E) THP-1 cells were treated

with vehicle, JQ12 or JQ1+ (300 nM), for 5 h. Re-

cruitment of Brd2 and Brd4 to ARE sites on the pro-

moters 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 6SEM of four independent experiments. *p , 0.05,

**p , 0.01, and ***p , 0.001 versus IgG isotype

control. ##p , 0.01 versus vehicle control.

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