Rxrα Represses NRF2-Mediated Transcription 1

Author Manuscript Published OnlineFirst on April 23, 2013; DOI: 10.1158/0008-5472.CAN-12-3386 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

RXRα represses NRF2-mediated transcription

Title: RXRα Inhibits the NRF2-ARE Signalling Pathway Through A Direct

Interaction With the Neh7 domain of NRF2

Running title: RXRα represses NRF2-mediated transcription

1,2 2 1 2 1 1 Hongyan Wang , Kaihua Liu , Miao Geng , Peng Gao , Xiaoyuan Wu , Yan Hai ,

Yangxia Li1, Yulong Li1, Lin Luo2, John D. Hayes3, Xiu Jun Wang2† and Xiuwen

Tang1†

1Department of Biochemistry and Genetics, 2Department of Pharmacology, School of

Medicine, Zhejiang University, Hangzhou, PR China, 310058, 3Division of Cancer

Research, Medical Research Institute, Ninewells Hospital & Medical School,

University of Dundee, Dundee DD1 9SY, Scotland, United Kingdom

Key words: RXRα, repressor, NRF2, ARE, drug resistance

†To whom correspondence should be addressed: PO BOX 18, The School of

Medicine, Zhejiang University, Hangzhou, PR China 310058. Tel: +0086-571-

88208266; Fax: +0086-571-88208266; E-mail: [email protected] (Xiu Jun Wang)

or [email protected] (Xiuwen Tang)

Word count: 4993

Total number of figures and tables: 6

The authors disclose no potential conflicts of interest.

Abbreviations: AKR1C, aldo-keto reductases 1C1 and 1C2; ARE, antioxidant response element; ATF3, activating transcription factor 3; BHA, butylated hydroxyanisole; CAR, constitutive androstane receptor; CBP, CREB, cAMP response element binding protein, binding protein; ChIP, chromatin immunoprecipitation; DAPI, 4’,6-diamidino-2-phenylindole; DBD, DNA-binding domain; DMSO, dimethyl sulfoxide; ERRβ, estrogen-related receptor beta; GAPDH, glyceraldehyde- 3-phosphate dehydrogenase; GFP, green fluorescent protein; GSH, reduced glutathione; GSK-3, glycogen synthase kinase-3; GST, glutathione S-transferase; HO- 1, heme oxygenase 1; KEAP1, Kelch-like ECH -associated protein 1; LBD, ligand- binding domain; Neh, NRF2-ECH homology; NQO1, NAD(P)H:quinone oxidoreductase 1; LXR, liver X receptor; NRF2, NF-E2 p45-related factor 2; NSCLC, non-small cell lung cancer; PBS, phosphate buffered saline; PXR, pregnane X receptor; RFP, red fluorescent protein; RNA Pol II, RNA polymerase II; ROS,

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2013 American Association for Cancer Research. Author Manuscript Published OnlineFirst on April 23, 2013; DOI: 10.1158/0008-5472.CAN-12-3386 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

RXRα represses NRF2-mediated transcription

reactive oxygen species; RT-PCR, real-time quantitative PCR; RAR, retinoic acid receptor; RXRα, retinoid X receptor alpha; siRNA, small interfering RNA; tBHQ, tert-butylhydroquinone.

RXRα represses NRF2-mediated transcription

ABSTRACT

The transcription factor NRF2 (NFE2L2) is a pivotal activator of genes

encoding cytoprotective and detoxifying enzymes that limit the action of cytotoxic

therapies in cancer. NRF2 acts by binding antioxidant response elements (AREs) in

its target genes, but there is relatively limited knowledge about how it is negatively

controlled. Here we report that retinoic X receptor alpha (RXRα) is a hitherto

unrecognized repressor of NRF2. RNAi-mediated attenuation of RXRα increased

basal ARE-driven gene expression and induction of ARE-driven genes by the NRF2

activator tert-butylhydroquinone (tBHQ). Conversely, over expression of RXRα

attenuated ARE-driven gene expression. Biochemical investigations showed that

RXRα interacts physically with NRF2 in cancer cells and in murine small intestine

and liver tissues. Further, RXRα bound to ARE sequences in the promoters of NRF2-

regulated genes. RXRα loading onto AREs was concomitant with the presence of

NRF2, supporting the hypothesis that a direct interaction between the two proteins on

gene promoters accounts for the antagonism of ARE-driven gene expression.

Mutation analyses revealed that interaction between the two transcription factors

involves the DNA-binding domain of RXRα and a region comprising amino acids

209-316 in human NRF2 that had not been defined functionally, but that we now

designate as the NRF2-ECH homology (Neh7) domain. In non-small cell lung cancer

cells where NRF2 levels are elevated, RXRα expression downregulated NRF2 and

sensitized cells to the cytotoxic effects of therapeutic drugs. In summary, our findings

demonstrate that RXRα downmodulates cytoprotection by NRF2 by binding directly

to the newly defined Neh7 domain in NRF2.

RXRα represses NRF2-mediated transcription

INTRODUCTION

The human body is continuously threatened by reactive oxygen species (ROS)

and electrophiles that are generated by metabolism and by environmental agents. The

NF-E2 p45-related factor 2 (NRF2) is a cap’n’collar (CNC) basic-region leucine

zipper (bZIP) transcription factor which plays a major role in protecting cells from

prooxidants and electrophiles because it regulates basal and inducible expression of

genes that contain antioxidant response element (ARE) sequences in their promoter

regions. NRF2-target genes include those encoding antioxidant and detoxication

enzymes such as aldo-keto reductase (AKR), heme oxygenase 1 (HO-1), glutathione

S-transferase (GST), glutamate-cysteine ligase, and NADP(H):quinone

oxidoreductase-1 (NQO1) (1-3).

The ubiquitin ligase substrate adaptor kelch-like ECH-associated protein 1

(KEAP1) is a major repressor of NRF2. Under normal conditions, NRF2 is constantly

degraded via the ubiquitin-proteasome pathway in a KEAP1-dependent manner.

Under stressed conditions, ROS or eletrophiles modify cysteine residues in KEAP1

causing loss of its adaptor activity, and in turn failure to ubiquitylate NRF2. Upon

inactivation of KEAP1, NRF2 accumulates in the nucleus where it heterodimerizes

with small Maf proteins and activates ARE-driven genes (4, 5). Recent studies have

demonstrated that KEAP1-dependent ubiquitylation of NRF2 can be prevented by

protein-protein interactions: these include the binding of p21 to NRF2 or the binding

of p62/sequestosome-1 to KEAP1 (6, 7).

In contrast to the wealth of knowledge about the activation of NRF2, far less is

known about the mechanisms by which cells turn off or down-regulate NRF2 once it

has been activated. Importantly, several transcriptional repressors of NRF2 have been

identified, such as Bach1, P53, activating transcription factor 3 (ATF3), and estrogen-

RXRα represses NRF2-mediated transcription

related receptor beta (ERRβ) (8-11), suggesting that NRF2 activity is strictly

regulated even when KEAP1 is inactivated. Nonetheless, constitutive up-regulation of

NRF2 has been observed in various tumors, including non-small cell lung cancer

(NSCLC), and those of breast, head and neck, and gallbladder (12-14). Indeed, de-

regulation of NRF2 has been shown to contribute to tumorgenesis and drug resistance

(14-18). Thus, NRF2 is emerging as a new molecular target for the treatment of

certain cancers. It is therefore important to understand the molecular mechanisms by

which NRF2 activity can be suppressed because this might provide novel strategies

for therapeutic intervention.

In a previous study, we reported that retinoic acid receptor alpha (RARα)

antagonises NRF2 activity (19). Retinoid X receptor (RXR) is the obligatory

heterodimerization partner for RARα (20, 21), but its role in regulating the function

of NRF2 has not been investigated to date. In the present study we discovered that

RXRα can inhibit the transcriptional activity of NRF2 through a physical interaction

between the two factors. A RXRα-binding region, which is located between the

NRF2-ECH homology (Neh) 5 and Neh6 domains of human NRF2, has been

identified. Mutation analyses have revealed that the DNA-binding domain (DBD) of

RXRα is required for the interaction with NRF2. Moreover, we have provided

evidence that RXRα is capable of interacting with NRF2 on ARE sites in gene

promoters, demonstrating a previously unrecognised mechanism by which the CNC-

bZIP factor can be inhibited. Down-regulation of NRF2 via forced expression of

RXRα in NSCLC A549 cells, where the CNC-bZIP factor is constitutively active,

increased sensitivity to therapeutic drugs. Thus our data demonstrate a novel

mechanism by which RXRα can suppress drug resistance.

RXRα represses NRF2-mediated transcription

MATERIALS AND METHODS

Chemicals and cell culture

Unless otherwise stated, all chemicals were from Sigma-Aldrich Co., Ltd.

(Shanghai, China), and all antibodies were from Santa Cruz Biotechnology (Shanghai,

China). Antibody against mouse Nrf2 (H300) (sc-13032, Santa Cruz) was used for

this study. Actin and β-tubulin antibodies were purchased from Sigma (China). Alexa

Fluor 488 goat anti-rabbit IgG(H+L) was obtained from Invitrogen (Shanghai, China).

HEK293 (human embryonic kidney-293), MCF7 (human breast carcinoma), Caco2

(human colon cancer) and NSCLC A549 cell lines were from ATCC (China).

Animals

Five-week-old male C57BL/6 male mice were used in this study. The mice (n

= 8) were given butylated hydroxyanisole (BHA) by intragastric gavage (i.g.) at 200

mg/kg daily for 3 days. The equivalent volume of corn oil (vehicle) was given to the

control mice (n = 8). Mice were sacrificed and tissues were processed as described

previously (22). All animal procedures were performed in accordance with the

approval of Laboratory Animals Ethics Committee of Zhejiang University.

Plasmids

Plasmids encoding mouse (m) Nrf2 were provided by Dr Mike McMahon

(Medical Research Institute, University of Dundee, UK): these included

pcDNA3.1/V5-mNrf2 (full-length), pcDNA3.1/V5-mNrf2∆ETGE (lacking amino acids

79-82) and pcDNA3.1/V5-mNrf2∆DIDLID (lacking amino acids 17-32) (23). pHyg-EF-

hNRF2 encoding EGFP tagged full-length human (h) NRF2 was kindly provided by

Dr. Masayuki Yamamoto and Dr. Ken Itoh (Institute of Basic Medical Sciences,

University of Tsukuba, Japan). pSG5-mRXRα encoding full-length mRNAα was

RXRα represses NRF2-mediated transcription

generously provided by Dr. Pierre Chambon (Institut de Génétique et de Biologie

Molèculaire et Cellulaire, CNRS/INSERM/ULP, France). We generated a series of

plasmids expressing tagged hNRF2, mNrf2 or mRXRα wild-type or mutants (see

Supplementary Methods): as shown in Figure 2C, 6 plasmids expressing GST-

tagged hNRF2 mutants and 7 plasmids encoding GFP-tagged hNRF2 or mNrf2

mutants were created; as shown in Figure 3A, 4 plasmids expressing GST-tagged

mRXRα mutants were generated. All plasmids were verified by DNA sequencing.

Transfections, luciferase reporter gene activity

Lipofectamine 2000 (Invitrogen, China) was used for transfection (24). The

small interfering RNA (siRNA) against hRXRα (RXRα-siRNA) or non-targeting

negative control siRNA (scrambled-siRNA) were synthesized by TaKaRa

Biotechnology (Dalian, China). The sequences for RXRα-siRNA were 5’-

GGAGAUGCAUCUAUUUUAATT-3’ (forward) and 5’-

UUAAAAUAGAUGCAUCUCCTG-3’ (reverse). Empty vectors were used as

negative controls for transfection experiments with plasmids. Twenty-four or 48 hr

post transfection, the transfected cells were treated with xenobiotics for 6 hr to 24 hr

before being harvested for further analysis. The ARE-luciferase reporter plasmid

pGL-GSTA2.41bp-ARE was used and the dual luciferase activities were determined

as described elsewhere (25). Stable cell lines A549-mRXRα and A549-EGFP over-

expressing GFP-mRXRα and GFP, were generated as described in Supplementary

Methods.

Real-time quantitative PCR (RT-PCR)

Total RNA isolation and RT-PCR was performed as described previously (24).

RXRα represses NRF2-mediated transcription

Western blot analysis, GST pull-down assay and immunoprecipitation

Preparation of protein samples, SDS-PAGE gels and immunoblotting was

carried out using standard protocols (24). Immunoblotting with antibody against actin

or β-tubulin was performed to confirm equal loading for whole-cell extracts and

nuclear extracts, respectively. GST pull-down assay and immunoprecipitation was

performed to detect the interaction between NRF2 and mRXRα mutant GST or GFP

fusion proteins. The procedures are provided in Supplementary Methods.

Fluorescently tagged proteins and immunofluorescence

Cells were seeded on glass coverslips, and transiently transfected with the

indicated fluorescently labelled proteins. Forty-eight hr later, cells were fixed,

processed and examined as described previously (4, 5). Anti-RXRα antibody was

used to detect endogenous RXRα, followed by staining with Texas Red goat anti-

rabbit IgG(H+L). Counterstaining with 4’,6-diamidino-2-phenylindole (DAPI) was

used to verify the location and integrity of nuclei. The fluorescence images were

observed with a Zeiss LSM510 Meta laser-scanning confocal microscope (Carl Zeiss,

Inc, Germany).

Chromatin immunoprecipitation (ChIP) assay

ChIP assays were performed as described previously (24). The relative

binding of NRF2 or RXRα to ARE sites were calculated by quantification of band

® intensity with an Odyssey Infrared Imaging System (LI-COR Biosciences)

normalized to that of input.

Biotinylated ARE-binding assay

The preparation of nuclear extracts and the Bio-ARE pull-down assay was

performed as described previously (19). A double-stranded 5′-biotinylated ARE probe,

RXRα represses NRF2-mediated transcription

representing the 41 bp of nucleotides -682 to -722 in the rat GSTA2 gene promoter,

was synthesized by TaKaRa Biotechnology. NRF2 pre-depletion was performed by 1

hr incubation with antibody to NRF2 prior to the Bio-ARE pulldown procedures. The

pulled down mixture was analysed on SDS–PAGE followed by immunoblotting with

antibody against RXRα or NRF2.

Cytotoxicity assay

Cytotoxicity was determined as described previously (24). The IC50 and the

combination index for determining synergism were calculated as described elsewhere

(24).

Statistical analysis

Statistical comparisons were performed by unpaired Student’s t tests. A value

of p < 0.05 was considered statistically significant.

RXRα represses NRF2-mediated transcription

RESULTS

RXRα inhibits basal and inducible ARE-driven gene expression.

To evaluate the effect of RXRα on NRF2 and ARE-driven gene expression,

we transfected MCF7 cells with siRNA to knockdown RXRα; immunoblotting

confirmed successful knockdown of RXRα in these cells (Fig. 1A, upper panel). In

MCF7 cells cotransfected with the ARE-driven reporter plasmid pGL-GSTA2.41bp-

ARE, knockdown of RXRα was found to increase basal luciferase reporter activity

about 1.5-fold, and increased induction of the reporter gene activity by 20 μM tert-

butylhydroquinone (tBHQ) from 4-fold to 6-fold. To test whether this increase in

ARE-driven gene expression was a general effect, we also examined colon cancer

Caco2 cells (Fig. 1B, upper panel). Knockdown of RXRα in Caco2 cells resulted in a

2-fold increase in the basal levels of mRNA for endogenous AKR1C1 and HO-1, both

of which are NRF2-target genes. Treatment of Caco2 cells with 20 μM tBHQ in

which RXRα had been knocked down further increased AKR1C1 mRNA from 10-

fold to 14-fold, and HO-1 mRNA from 5-fold to 7-fold (Fig. 1B, lower panel). These

data indicate that loss of RXRα increases NRF2 activity

We next tested whether over-expression of RXRα might suppress ARE-driven

gene expression in Caco2 cells using the pEGFP-C1-mRXRα expression vector;

transient expression of exogenous GFP-mRXRα was confirmed by immunoblotting

with a specific antibody against RXRα (Fig. 1C). As anticipated, both basal and

inducible NRF2-target gene expression were inhibited by forced over-expression of

RXRα: the basal AKR1C1 and HO-1 mRNA levels were reduced by 20% and the

induction of AKR1C1 and HO-1 mRNA levels by tBHQ was decreased from 11-fold

RXRα represses NRF2-mediated transcription

to just 2- and 4-fold, respectively (Fig. 1D). These data demonstrate that NRF2-

regulated genes are the targets of RXRα-mediated repression, and RXRα can

suppress the expression of ARE-driven genes in a ligand-independent manner.

Antagonism of ARE-driven gene expression by RXRα is independent of KEAP1.

It is well established that KEAP1 is a major repressor of NRF2 activity (5). To

investigate whether KEAP1 plays any role in the antagonism of NRF2 by RXRα, we

carried out a further study in the A549 NSCLC cell line, which contains a loss-of-

function-mutation in KEAP1(14). RXRα-siRNA was transfected into A549 cells, and

knockdown of RXRα was confirmed by immuoblotting (Fig. 1E, left panel).

Consistent with our observations in MCF7 and Caco2 cells, knockdown of RXRα in

A549 cells increased AKR1C1 mRNA 2-fold and HO-1 mRNA 4-fold (Fig. 1E, right

panel). It also increased AKR1C1 and HO-1 protein levels significantly (Fig. 1E, left

panel). These findings suggest that RXRα inhibition of ARE-driven transcription

occurs independently of KEAP1.

RXRα and NRF2 physically interact in vitro.

To investigate the mechanism by which RXRα represses NRF2, we examined

the localization of the CNC-bZIP transcription factor and its abundance after over-

expression of RXRα. Using Caco2 and A549 cells, we found that RXRα altered

neither the nuclear accumulation of NRF2 nor its abundance (Fig. 1C & Fig. 6A). We

next considered whether RXRα-mediated repression of NRF2 might be a

consequence of a direct interaction between the two proteins. To test this possibility,

we generated a GST-tagged NRF2 construct, and performed GST-pulldown

experiments that tested its ability to interact with RXRα (Fig. 2A & B). The

recombinant full-length NRF2 (GST-hNRF2) interacted strongly with His-tagged

RXRα represses NRF2-mediated transcription

full-length RXRα protein (Fig. 2B, lane 1). In contrast, the GST control did not bind

specifically to RXRα (lane 2). An inverse GST-pulldown assay with recombinant

GST-RXRα and His-tagged NRF2 confirmed that the two proteins interact

specifically (Fig. 3B, lane 2). Thus, our data indicate that NRF2 and RXRα can form

a complex in vitro.

To determine the region of NRF2 that is required to interact with RXRα, a

series of NRF2 truncated proteins tagged with GST (see Fig. 2C) were expressed and

their abilities to interact with purified recombinant His-RXRα were tested by GST-

pulldown assay. We found RXRα interacted with the N-terminal NRF217-338 protein

(Fig. 2D, lane 2). In contrast, RXRα failed to interact with the C-terminal NRF2339-605

protein (Fig. 2D, lane 7), suggesting that the Neh6, Neh1 and Neh3 domains of NRF2

are not required for the interaction between the two factors. In addition, RXRα failed

to interact with the NRF217-110 or NRF2109-219 proteins, which contain Neh2 or Neh4

plus Neh5, respectively (Fig. 2D, lanes 4 and 5), suggesting that these individual

domains are not sufficient to enable NRF2 to bind RXRα. Remarkably, RXRα

interacted with NRF2109-338 and NRF2209-316 (lanes 3 and 6), whereas deletion of the

amino acids 209-316 completely abolished the interaction (Fig. S1A, lane 2 & 3).

These results indicate residues 209-316 of NRF2 are sufficient to support an

interaction with RXRα in vitro.

To further confirm that amino acids 209-316 of NRF2 are required for the

interaction with RXRα, we created a series of expression vectors encoding GFP-

tagged NRF2 mutants (Fig. 2C). The analyses were performed using purified His-

RXRα to co-immunoprecipitate material from lysates of HEK293 cells that had been

previously transiently transfected with the various NRF2 mutant expression plasmids.

RXRα represses NRF2-mediated transcription

Consistent with the GST-pulldown assay results, an antibody against RXRα could

coimmunoprecipitate the mNrf2∆ETGE, NRF217-316, and NRF2109-316 mutant proteins,

all of which contain amino acids 209-316 of NRF2 (Fig. 2E, lanes 1, 2 and 6). The

deletion of the amino acids 209-316 abolished this interaction (Fig. S1B, lane 3).

Moreover, residues 201-329 of mNrf2, which are orthologous to residues 209-316 of

hNRF2, could also be immunoprecipitated from the cell extracts (Fig. 2E, lane 7). As

a further control, we tested HEK293 cell extracts that expressed GFP alone, and

demonstrated that RXRα could not co-immunoprecipitate GFP (Fig. 3D & E).

Collectively, these results indicate that amino acids 209-316 of human NRF2 are

necessary for its interaction with RXRα in vitro.

The RXRα DBD is required for interaction with NRF2.

RXRα comprises three major domains: the N-terminal AF-1 domain, the well-

conserved DNA-binding domain (DBD), and the ligand-binding domain (LBD) that is

responsible for dimerization. It also contains the relatively small AF-2 region with a

ligand-dependent transactivation function (26-28) (Fig. 3A). To delineate which

region of RXRα is necessary for interaction with NRF2, four recombinant GST-

RXRα mutants (GST-∆RXRα) were expressed and purified, and their abilities to

interact with purified His-NRF2 were tested by GST-pulldown assay (Fig. 3A).

Mutant RXRα230-467, which contains the C-terminal ligand binding and dimerization

domains, failed to interact with NRF2 (Fig. 3B, lane 4). Likewise, NRF2 was unable

to bind to RXRα1-139, which represents the AF-1 region (lane 6). In contrast,

mRXRα1-229, representing the N-terminal amino acid region of RXRα, interacted well

with NRF2 (lane 3). Importantly, the mutant RXRα140-205 protein that comprises the

DBD domain, interacted strongly with NRF2 (lane 5). Accordingly, the GST-luc and

RXRα represses NRF2-mediated transcription

GST controls did not bind NRF2 (lanes 1 and 7). Taken together, our data indicate

that the DBD is sufficient to interact with NRF2.

RXRα and NRF2 directly interact in vivo.

To demonstrate whether a physical interaction occurs between RXRα and

NRF2 in vivo, we examined whether both proteins co-localise in cells. Plasmids

encoding GFP-tagged NRF2 and RFP-tagged mRXRα were transfected into HEK293

cells. Images obtained by confocal laser scanning microscopy revealed that singly

expressed GFP-NRF2 and RFP-RXRα were predominately localized in the nucleus of

the transfected cells (Fig. 4A, image a-d). As a control, we monitored the cellular

localization of the GFP and RFP proteins, both of which were distributed uniformly in

the cytoplasm and the nucleus (Fig. S2). When GFP-NRF2 and RFP-RXRα were co-

expressed, a substantial portion of both proteins co-existed in small foci-like

structures within the nucleus (Fig. 4A, image e-g). To confirm these observations, we

next tested whether the ectopically expressed GFP-NRF2 could localize with

endogenous RXRα. After pHyg-GFP-hNrf2 was transiently transfected into A549

cells alone, the cellular localization of endogenous RXRα was examined using

indirect immunofluorescence. Confocal laser scanning microscopy demonstrated a

similar nuclear co-localization of the two proteins (Fig. 4B, image c). Thus, these

results support our contention that NRF2 interacts with RXRα in the nucleus.

Moreover, we performed co-immunoprecitation experiments with total lysates

prepared from COS7 cells transfected with the expression vector for V5-tagged

mouse Nrf2 with a deletion of residues 17-32 in its Neh2 domain (mNrf2∆DIDLID-V5).

When precipitated with an anti-RXRα antibody, a strong mNrf2 band was detected

RXRα represses NRF2-mediated transcription

that suggested both transcription factors coexist in an immunoprecitable complex (Fig.

S3, upper blot, lane 4).

To examine whether an interaction occurs between endogenous NRF2 and

endogenous RXRα, we exposed MCF7 cells to 20 μM tBHQ for 24 hr.

Immunoprecipitation using antibodies against NRF2 and RXRα revealed the presence

of an immuno-complex in the MCF7 cell lysates between endogenous NRF2 and

RXRα, which could be increased further by tBHQ treatment (Fig. 4Ca & 4Cb, lane 3

& 4). The abundance of NRF2 (Fig. 4Cc, lane 2) and the expression of its target gene

AKR1C (Fig. S4, lane 2) was increased as reported previously (25). We next

performed similar studies with cell extracts prepared from mouse tissues. Strikingly,

we found the two transcription factors interacted strongly in both small intestine and

liver (Fig. 4Da & 4Db, lane 3). When the mice were treated with BHA, expression of

the Nrf2-target genes Nqo1 and Gstm1 was increased in the small intestine and liver

(Fig. S5A & S5B, lane 2) as expected (1, 22, 29, 30). Again, the interaction between

Nrf2 and RXRα was increased by BHA (Fig. 4Da & 4Db, lane 4), suggesting that

RXRα is important in regulating the function of Nrf2 in these tissues. Taken together,

these results establish that RXRα and NRF2 interact directly in vivo.

RXRα is recruited to the ARE in an NRF2-dependent manner.

To assess whether the interaction between RXRα and NRF2 occurs when the

factors are bound to DNA, we performed a ChIP assay with antibody against RXRα

or NRF2. As expected, increased NRF2 bound to ARE sequences in the promoters of

HO-1 and AKR1C1 after MCF7 cells had been exposed to 20 μM tBHQ for 6 hr (Fig.

5A, lanes 3 and 4). Interestingly, the ChIP assays revealed that RXRα was also able

to associate with ARE sites (Fig. 5A, lanes 5 and 6), though it was observed that the

RXRα represses NRF2-mediated transcription

DNA bands representing RXRα associated with ARE sequences were much weaker

than those representing NRF2. Upon exposure to tBHQ, the amount of RXRα that

associated with ARE sequences increased ~1.5-fold (Fig. 5A, lanes 5 and 6),

correlating with the increase in NRF2 on ARE sites (Fig. 5A, lane 3 & 4); this

occurred in spite of the fact that the abundance of RXRα protein remained unchanged,

as seen in MCF7 cells (Fig. 5B, lower blot, lanes 1 and 2). These results are specific

to ARE-containing gene promoters; a DNA sequence in the glyceraldehyde-3-

phosphate dehydrogenase (GAPDH) promoter was not detected in complexes

immunoprecipitated with either antibody (Fig. 5A). Our findings suggest that the

association of RXRα with ARE sequences possibly requires the presence of NRF2,

which in turn is able to recruit RXRα to the cis-element.

To further evaluate whether RXRα requires NRF2 in order to recognise AREs,

we carried out a biotinylated-ARE (Bio-ARE) oligonucleotide pull-down assay on

nuclear extracts from MCF7 cells treated with 20 μM tBHQ. Although tBHQ did not

influence the nuclear levels of RXRα (Fig. 5B, lower blot, lanes 1 and 2), only RXRα

from tBHQ-treated MCF7 cells bound strongly to Bio-ARE beads (Fig. 5B, lower

blot, lane 8). Such binding was concomitant with nuclear accumulation of NRF2 and

its increased binding to Bio-ARE beads (Fig. 5B, upper blot, lane 2 & 8). Importantly,

depletion of NRF2 from the nuclear extracts after pre-incubation with an antibody

against NRF2 (Fig. 5C, lane 3), not only abolished the binding of NRF2 to Bio-ARE

as expected (Fig. 5C, upper blot, lanes 8 and 9), but also significantly reduced the

binding of RXRα to Bio-ARE (Fig. 5C, lower blot, lanes 8 and 9). As a negative

control, an unrelated biotin-labelled double-stranded oligonucleotide failed to pull

down NRF2 or RXRα from the nuclear extracts (Fig. 5B, lane 16; 5C, lane 7). These

RXRα represses NRF2-mediated transcription

results suggest that RXRα can be tethered onto DNA by forming a heteromeric

protein-protein complex with NRF2.

In order to examine whether RXRα might form inactive complexes with

NRF2 on ARE sequences, we generated a stable MCF7 cell line, designated MCF7-

RXRα, which over-expresses RXRα from a pEGFP-mRXRα plasmid.

Immunoprecipitation confirmed that both the endogenous RXRα and exogenously

derived GFP-mRXRα associated with NRF2 in these stably-transfected cells (Fig. S6,

lane 4). In agreement with the observation following transient transfection of RXRα

(Fig. 1D), induction of HO-1 by tBHQ (20 μM) was nearly completely blocked in

MCF7-RXRα cells (Fig. 5D, lanes 3 and 4). We found comparable levels of nuclear

NRF2 and its binding to ARE sequences stimulated by tBHQ (Fig. 5D; Fig. S7, lanes

5 - 8). However, a ChIP assay revealed that the loading of CREB (cAMP Response

Element Binding protein) Binding protein (CBP) and RNA Polymerase II (RNA Pol

II) onto ARE-sites upon tBHQ treatment were markedly attenuated in MCF7-mRXRα

cells (Fig. 5E, lanes 7, 8, 11 and 12). This is in contrast to the situation in MCF7 cells,

where the levels of CBP and RNA Pol II on ARE site were dramatically increased

under the same conditions (Fig. 5E, lanes 5, 6, 9 and 10). Taken together, our data

suggest that the lack of transcriptional activity of NRF2 caused by RXRα over-

expression is likely due to direct negative interference by RXRα on ARE-sites,

leading to the disruption of the recruitment of CBP and RNA Pol II to the promoters.

Over-expression of RXRα in A549 cells down-regulates NRF2 and increases

sensitivity to anticancer drugs.

Recent studies have provided evidence that high constitutive expression of

NRF2 occurs in many cancer cells (31), and RNAi knockdown of the CNC-bZIP

RXRα represses NRF2-mediated transcription

factor can sensitize such cells to chemotherapeutic drugs (24, 32, 33). To test whether

the repression of NRF2 by RXRα has similar biological consequences, we generated

a cell line named A549-RXRα in which RXRα is stably expressed. As A549 cells

carry a somatic KEAP1 mutation, it contains supranormal levels of NRF2 and its

target genes are constitutively over-expressed, which in turn increases cell

proliferation and resistance to anticancer drugs (14, 32). Immunoblotting confirmed

transgene expression (Fig. 6A, lane 2). As expected, expression of the NRF2-target

gene HO-1 was diminished in A549-RXRα cells. Cytotoxicity revealed that the IC50

of A549-RXRα to the anticancer drug oxaliplatin was about 40 μmol/l, compared

with an IC50 of 100 μmol/l in A549 cells that stably expressed GFP, named A549-

GFP (Fig. 6B). The A549-RXRα cells also displayed increased sensitivity to

doxorubicin (Fig. 6C). Our results therefore suggest that the down-regulation of

cytoprotective genes regulated by NRF2 contributes to the increased sensitivity of

A549 cells by over-expression of RXRα.

RXRα represses NRF2-mediated transcription

DISCUSSION

Regulation of NRF2 by KEAP1 has been a subject of intense study and it is

relatively well characterized. However, little is known about other mechanisms by

which NRF2 activity is controlled. Herein, we have presented the first evidence that

RXRα functions as a repressor of ARE-driven gene expression. We found RXRα-

mediated repression of ARE-driven genes is ligand- and redox-independent, and

requires the presence of NRF2 to recruit RXRα to the promoters of target genes.

Importantly, we have discovered that amino acids 209-316 of human NRF2 are

necessary for its interaction with RXRα, and as this region has not previously been

shown to possess functional importance we have designated it the Neh7 domain.

Repression of NRF2 by RXRα was observed in five different cell types, as well as

mouse small intestine and liver, thereby indicating its general significance. To our

knowledge, it has not been reported previously that RXRα attenuates ARE-driven

gene transcription by directly targeting NRF2.

RXRs play an essential role in the regulation of multiple nuclear hormone-

signaling pathways through their ability to dimerize with other nuclear receptors.

RXRs mediate retinoid signalling through forming a heterodimer with RAR and by

forming a homodimer (21, 34). In addition, RXRs form heterodimers with many other

members of the subfamily of nuclear receptors, including peroxisome proliferator-

activated receptor, liver X receptor (LXR), pregnane X receptor (PXR) and

constitutive androstane receptor (CAR) (21, 34). Heterodimerization of RXR with its

partners dramatically enhances its DNA-binding activity (21, 34). Upon binding DNA,

some nuclear receptors repress transcription of target genes through their interaction

with transcriptional corepressors in the absence of ligands. Furthermore, ligand

binding by a transcriptional agonist causes conformational changes in corepressors,

RXRα represses NRF2-mediated transcription

allowing dissociation of transcriptional corepressors and association of transcriptional

coactivators (35). Herein we found that RXRα did not influence nuclear translocation

of NRF2 or its binding to ARE sequences. Instead, RXRα associated with ARE-

bound NRF2, suggesting that inhibition of NRF2 by RXRα is likely due to the direct

interference of recruitment by the CNC-bZIP factor of coactivators to gene promoters.

Significantly, we found that an RXRα mutant lacking its ligand binding and

heterodimerization domains was as efficient at interacting with NRF2 as the wild-type

protein, demonstrating that the nuclear receptor is able to repress NRF2 in a ligand-

independent manner.

Previously we reported that RARα mediates inhibition of NRF2 by all trans

retinoic acid through an undefined protein-protein interaction (19). Herein we have

described physical and functional interactions between RXRα and NRF2. Although

RXRα and RARα can heterodimerize, our in vitro GST-pull down study indicates

that RXRα binds NRF2 directly and that RARα is not necessary for the interaction

between RXRα and NRF2. Specifically, the physical interaction involves the DBD of

RXRα and the Neh7 domain of NRF2, and we failed to detect any interaction

between RXRα and the CNC-bZIP Neh1 domain of NRF2. This is distinct from other

interactions between nuclear hormone receptors and bZIP proteins such as jun and

BZLF1 (36, 37) in which the zinc finger DNA-binding domain of the receptor

interacts with the bZIP domain of the transcription factor. Thus our studies

demonstrate that both RARα and RXRα are repressors of NRF2 activity and

presumably act via distinct mechanisms.

Previous work has shown NRF2 contains six functional domains, named

Neh1-Neh6 (5). It is well known that the stability of NRF2 is controlled through

protein-protein interactions between its Neh2 domain and KEAP1. The stability of

RXRα represses NRF2-mediated transcription

NRF2 is also controlled by its Neh6 domain, and recently it has been found that this

involves β-TrCP-mediated ubiquitylation and phosphorylation of residues in Neh6 by

GSK-3 (23, 38). In contrast, the Neh4 and Neh5 regions, which lie adjacent to each

other, were found by Katoh et al to act together as a transactivation domain (39);

Neh4 and Neh5, both individually and cooperatively, bind CBP, and are indispensable

for maximal NRF2 transactivation. Subsequently, Zhang et al (40) reported that the

Neh4 and Neh5 domains of NRF2 recruit BRG1, a catalytic subunit of SWI2/SNF2-

like chromatin-remodeling complexes, to HO-1 enhancers for transcription initiation.

In the present study we identified a RXRα interaction domain in NRF2 (now called

Neh7), which abuts the Neh5 domain of NRF2. We found over-expression of RXRα

reduced the loading of CBP and RNA Pol II onto ARE sites, suggesting that the

binding of RXRα may disrupt the binding of CBP to the Neh4 and Neh5 domains of

NRF2, thereby suppressing transcriptional initiation. We therefore propose a model in

which protein-protein contact between RXRα and NRF2 prevents a productive

interaction between the transactivation domains of NRF2 and the basal transcription

machinery.

In adult mammalian liver, RXRα is the most abundant among the three RXR

isoforms (i.e. RXRα, -β, and -γ) and is an obligatory partner of two major xenobiotic

receptors, CAR and PXR (41). Previous studies have revealed that Nrf2 is also highly

expressed in the liver (22), and plays a key role in regulating the expression of phase

II drug-metabolizing enzymes and drug-efflux pumps (30, 33, 42, 43). In the present

study, we demonstrated that Nrf2 and RXRα interact in vivo. In agreement with our

observation, Dai et al (44) reported that in hepatocyte-specific RXRα knockout mice,

the expression of Gsta1 and/or Gsta2, Gstm1 and/or Gstm3, Gstm2 and Gstm4, all of

which are regulated by NRF2 (29, 30, 44), were increased compared to their levels in

RXRα represses NRF2-mediated transcription

the liver of wild-type mice, presumably due to loss of NRF2 suppression. Moreover,

the expression of these Gst subunits from hepatocyte-specific RXRα knockout mice

was further enhanced by acetaminophen, whose hepatotoxic metabolite was able to

directly activate the KEAP1-NRF2 pathway (45), demonstrating RXRα represses

NRF2/ARE signalling pathway in vivo. Taken together, we hypothesize that RXRα

plays a key role in linking xenobiotic metabolism with the NRF2-ARE cytoprotective

signaling pathway. Furthermore, several nuclear receptors besides RXRα repress

NRF2 (11, 46, 47). It is interesting to speculate that antagonism of NRF2 by RXRα

occurs because at some level the CNC-bZIP protein and nuclear receptors are

functionally incompatible, and that it might be advantageous to attenuate NRF2

activity.

Recent studies have revealed that NRF2 exhibits abnormal increased activity

in several types of tumor due to oncogene activation, or somatic mutation of KEAP1

or NRF2 (15, 17). The over-activation of NRF2-ARE signaling in cancer cells

promotes drug resistance and cell proliferation (31, 48). It has been reported that

RXRα is down-regulated in many tumors (49-51). Based on our study, it seems

plausible that a reduction of RXRα will up-regulate the NRF2-ARE signalling

pathway, thereby contributing to tumourgenesis and drug resistance. In this study, we

have shown that forced-expression of RXRα in NSCLC A549 cells down-regulated

the NRF2/ARE cytoprotective pathway and sensitized them to anticancer drugs,

suggesting that transcriptional repression by RXRα may be used as a mechanism to

attain an appropriate level of gene expression in NRF2 overactive cell types. Here we

describe the Neh7 domain in Nrf2 as a potential target by which the CNC-bZIP factor

can be inhibited.

RXRα represses NRF2-mediated transcription

In summary, in this report we describe the interaction of NRF2 with RXRα

introducing a new dimension to our understanding of the transcriptional hierarchy in

ARE-driven gene regulation. Our studies, therefore, suggest a novel, and as yet

unrecognized, function of RXRα as a putative transcriptional co-repressor of NRF2.

ACKNOWLEDGEMENTS

We are grateful to Professor Roland Wolf for his generosity in providing

reagents. We thank Ms Ai Xin for her technical assistance. This work was supported

by NSFC (30970581, 31170743 and 81172230), ZJKJT (2010C33156, 2010R50046

and 20112308) and ZJNSFC (LZ12H16001).

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Figure legend

Figure 1 RXRα inhibits ARE-driven gene expression. (A) Knock-down of RXRα

in MCF7 cells enhanced both the basal and tBHQ-induced ARE-luciferase activity.

MCF7 cells were transiently transfected with RXRα-siRNA, the ARE reporter

plasmid pGL-GSTA2.41bp-ARE and the plasmid pRL-TK. Forty-eight hr later the

cells were treated with 20 μM tBHQ for 6 hr before they were harvested. The level of

RXRα protein in the lysed cells was determined by immunoblotting (upper panel).

Actin was used as a loading control. The relative luciferase activity in lysates was

determined as described in Materials and Methods (lower panel). The value for cells

RXRα represses NRF2-mediated transcription

transfected with scrambled siRNA and treated with DMSO, was used as control and

set at 1. (B) Knock-down of RXRα enhanced both the basal and tBHQ-induced

AKR1C1 and HO-1 mRNA levels in Caco2 cells. The Caco2 cells were transfected

with RXRα-siRNA. Forty-eight hr later cells were treated with 20 μM tBHQ, and

after 6 hr incubation they were harvested. The AKR1C1 and HO-1 mRNA levels

were determined by RT-PCR. The level of 18S rRNA was used as an internal

standard. The value for the same gene from the cells that had been transfected with

scrambled siRNA and treated with DMSO, was used as a control and set at 1. (C) &

(D) Over-expression of RXRα reduced AKR1C1 and HO-1 mRNA levels in Caco2

cells. The Caco2 cells were transiently transfected with pEGFP-mRXRα. After 24 hr

recovery, the cells were treated for 6 hr with 20 μM tBHQ. After lysis, the whole cell

extracts were subjected to SDS-PAGE analysis, and the protein levels of GFP-RXRα

and the endogenous RXRα were measured by Western blotting with antibody against

RXRα. The level of NRF2 protein was measured by Western blotting. Actin was used

as a loading control. Also, AKR1C1 and HO-1 mRNA levels were measured by RT-

PCR (D). The value for the same gene from cells that had been transfected with

pEGFP and treated with DMSO, was used as control and set at 1. (E) Knock-down of

RXRα in A549 cells increased the expression of AKR1C1 and HO-1. Forty-eight hr

after A549 cells were transfected with RXRα-siRNA, they were harvested. The levels

of RXRα, HO-1, AKR1C proteins were determined by immunoblotting with

individual antibodies. Actin was used as a loading control (left panel). The AKR1C1

and HO-1 mRNA levels were determined by RT-PCR (right panel). The amount of

18S rRNA was used as an internal standard. The value for the same gene from the

cells transfected with scrambled siRNA was used as control and set at 1. Values

RXRα represses NRF2-mediated transcription

shown are mean ± SD. *p < 0.05, **p < 0.005. The results are from three separate

experiments.

Figure 2 Mapping of the domain in NRF2 required for its interaction with

RXRα. (A) NRF2 was expressed in E. coli as a GST fusion protein, and purified on

glutathione (GSH)-Sepharose beads. The purified proteins were resolved on a 10%

SDS-PAGE gel and visualized by staining with Coomassie brilliant blue. (B) NRF2

and RXRα physically interact in vitro. Association of GST-NRF2 fusion protein with

His-RXRα was evaluated in a GST-pulldown assay. The same amounts of GST

protein or GST-NRF2 fusion protein, shown in (A), were incubated with his-RXRα.

The proteins bound to GSH-Sepharose were eluted, separated by SDS-PAGE and

subjected to immunoblotting using antibody against either RXRα or GST. The input

control represents 10% of the total amount used for GST-pulldown. (C) Schematic

illustration of the GST- or GFP-tagged NRF2 mutants and their interactions with

RXRα. In the cartoon, the regions within NRF2 that are of interest are indicated by

bars, and the amino acid residues involved are indicated by the polypeptide

designations. (D) Pull-down of his-RXRα with the indicated mutant hNRF2 proteins

fused at their N-termini with GST. The GSH-Sepharose-bound proteins were

separated by SDS-PAGE and subjected to immunoblotting using antibodies against

either RXRα or GST; both GST and GST-luc were used as negative controls. The

input represents 10% of the total amount of his-RXRα used for the GST pull-down

assay. (E) HEK293T cells were transfected with various pEGFPC1 plasmids

encoding GFP tagged to truncated forms of NRF2 as depicted in (C). Twenty-four hr

post-transfection, the whole cell extracts were incubated with purified His-RXRα.

The mixture was subjected to immunoprecipitation using antibody specific to RXRα.

RXRα represses NRF2-mediated transcription

The immunoprecipitates were separated by SDS-PAGE and immunoblotted using

antibodies against GFP or RXRα. The input represents 10% of the total amount of

cell lysate used for immunoprecipitation. Abbreviation: GST-luc, GST-luciferase.

NRF2 mutant proteins are indicated by * respectively. The molecular mass in kD is

indicated. IB, immunoblot. n.s., nonspecific bands. The results presented are typical

examples from at least three separate experiments.

Figure 3 Interaction of RXRα with NRF2 in vitro. (A) Schematic of GST-tagged

mRXRα mutants and their interactions with NRF2. In the cartoon, the regions within

RXRα that are of interest are indicated by bars, and the specific amino acid residues

are indicated by the polypeptide designations. (B) GST pull-down assay of his-NRF2

with GST-ΔRXRα fusion proteins. GST-ΔRXRα fusion proteins, as depicted in (A),

were incubated with GSH-Sepharose beads. The bound GST-ΔRXRα (b) was

incubated with purified His-NRF2 (c). The bound NRF2 was detected by

immunoblotting with antibody against NRF2 (a). The input represents 10% of the

total amount of his-NRF2 used for the GST pull-down assay. GST and GST-luc were

used as negative controls. Abbreviation: GST-luc, GST-luciferase. RXRα mutant

proteins are indicated by * respectively. The molecular mass in kD is indicated. IB,

immunoblot. The results shown are from at least three separate experiments.

Figure 4 NRF2 and RXRα interact in vivo. (A) Ectopic NRF2 and RXRα co-

localise in HEK293T cells. The HEK293T cells were transfected with pDsRed-

mRXRα together with phyg-EF-hNRF2, both of which encode fluorescently tagged

proteins. Forty-eight hr after transfection, the cells were fixed and examined. EGFP-

NRF2 is shown in green, DsReD-RXRα in red. Nuclei were stained by DAPI (blue).

RXRα represses NRF2-mediated transcription

Scale bar, 10 μm. (B) Ectopic NRF2 co-localises with endogenous RXRα in A549

cells. The A549 cells were grown on cover-slips and transfected with the expression

vector phyg-EF-hNRF2 for fluorescently-tagged NRF2 protein. Forty-eight hr post

transfection, the cells were fixed. Indirect immunofluorescence staining was

performed to visualize endogenous RXRα using a primary rabbit antibody against

RXRα, and followed by Texas Red goat anti-rabbit secondary antibody. The

endogenous RXRα signal is shown in red. Cell nuclei were stained with DAPI (blue).

The scale bars represent 10 μm. (C) Endogenous RXRα interacts with endogenous

NRF2 in MCF7 cells. The MCF7 cells were exposed to tBHQ (20 μM) for 24 hr (lane

2 & 4) before being harvested and lysed. Cell lysate was immunoprecipitated with

antibody specific to RXRα (a) or Nrf2 (b). IgG was used as negative control (lane 1

& 2). After washing, the immunoprecipitates were analysed by immunoblotting with

antibody specific to NRF2 or RXRα. Input, 5% of the cell lysate used for

immunoprecipitation; Beads, immunoprecipitates after the washing procedure. IB,

immunoblot. The results presented have been replicated over three separate

experiments. (D) Endogenous RXRα interacts with endogenous Nrf2 in mouse small

intestine and liver. WT mice were given BHA i.g. daily at a dose of 200 mg/kg for 3

days (lane 2 & 4), and corn oil (vehicle) was administered as negative control (lane 1

and 3). a, Each lane shows results from a pooled sample of soluble extract from the

small intestine of 2-3 mice. b, Each lane shows results from a sample of soluble liver

extract from a single mouse. The data represent immunoprecipitation results from

three separate experiments (n = 8).

RXRα represses NRF2-mediated transcription

Figure 5 RXRα can associate with ARE sequences. (A) ChIP analysis shows

RXRα can be recruited to ARE sequences in the promoter regions of AKR1C1 and

HO-1. MCF7 cells were treated with 20 μM tBHQ for 6 hr. The abilities of RXRα

and NRF2 to bind to ARE sites in the promoters of AKR1C1 and HO-1 were explored

by ChIP analysis (Upper panel). The relative abilities of NRF2 and RXRα to bind to

ARE sites were determined as described by Materials and Methods (Lower panel).

The value of NRF2 or RXRα from cells treated with DMSO was set at 1. GAPDH

was used as negative control. PCR reactions were not saturated. (B) Treatment with

tBHQ increases the binding of RXRα to immobilised Bio-ARE. MCF7 cells were

exposed to 20 μM tBHQ for 24 hr. The nuclear extracts were pre-cleared with protein

G-Sepharose, then incubated with a double stranded 5′-biotinylated ARE probe (Bio-

ARE) (lane 1-8), or a 5′-biotinylated negative control DNA probe (Bio-control) (lane

9-16), in the presence of streptavidin–agarose beads as described in Materials and

Methods. After washing, the pull-down mixture was analysed by immunoblotting

with specific antibodies against NRF2 or RXRα. (C) Depletion of NRF2 from cell

lysates abolishes RXRα binding to Bio-ARE. Pre-depletion of NRF2 protein from

cell lysates was performed by 1 hr incubation with antibody against NRF2 prior to the

Bio-ARE pulldown procedure, as described above. The lanes are labelled as follows:

Input, pre-cleared nuclear extract after incubating with protein G sepharose only; SN,

the supernatant after the pre-cleared nuclear extract mixed with the Bio-ARE probe or

Bio-negative control DNA and streptavidin–agarose beads; Wash, the supernatant

solution after the immunoprecipitation and the four washes of the beads; Beads,

immunoprecipitates after the washing procedure. (D) & (E) RXRα over-expression

inhibits CBP and RNA Pol II binding to the ARE site in the HO-1 promoter. A stable

RXRα represses NRF2-mediated transcription

cell line, designated MCF7-RXRα, was generated by over-expressing pEGF-mRXRα

as described in Materials and Methods. The MCF7-RXRα cells were treated with 20

μM tBHQ for 6 hr. The HO-1 and nuclear NRF2 levels were determined by Western

immunoblotting with specific antibodies against individual proteins. Tubulin and

actin were used as loading control for the nuclear and whole cell extracts, respectively.

(E) The binding of CBP and RNA Pol II to the ARE sites in the promoters of HO-1

was detected by ChIP analysis in MCF7-RXRα cells. GAPDH was used as a negative

control. PCR reactions were not saturated. Results show typical blots from at least

three separate experiments.

Figure 6 Over-expression of RXRα increased the sensitivity of A549 cells to

oxaliplatin and doxorubicin. The A549-mRXRα and A549-EGFP cell lines were

generated after stable transfection with pEGFP-mRXRα and pEGFP, respectively, as

described in Materials and Methods. (A) The levels of exogenous and endogenous

RXRα were determined by Western blotting with antibody specific to RXRα. The

levels of NRF2 and HO-1 were determined by immunoblotting with antibody against

NRF2 or HO-1. Actin was used as a loading control. A549-mRXRα or A549-EGFP

cells were exposed to oxaliplatin (1 - 100 μM) (B) or doxorubicin (0.1 – 2 μM) (C)

for 48 hr. The cell viability was determined with an MTS Cell Proliferating Assay Kit.

*p < 0.05, **p < 0.005. Results are from three separate experiments.

RXRα Inhibits the NRF2-ARE Signalling Pathway Through A Direct Interaction With the Neh7 domain of NRF2

Hongyan Wang, Kaihua Liu, Miao Geng, et al.

Cancer Res Published OnlineFirst April 23, 2013.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-12-3386

Supplementary Access the most recent supplemental material at: Material http://cancerres.aacrjournals.org/content/suppl/2013/04/23/0008-5472.CAN-12-3386.DC1

Author Author manuscripts have been peer reviewed and accepted for publication but have not yet been Manuscript edited.

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