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of Rhododendron brachycarpum via Reactive Oxygen Species (ROS) Regulation. 이자복 , 강상모 , 박성아. Journal of Investigative Cosmetology...
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Tetrachlorobenzoquinone Activates Nrf2 Signaling by Keap1 CrossLinking and Ubiquitin Translocation but Not Keap1-Cullin3 Complex Dissociation Chuanyang Su, Pu Zhang, Xiufang Song, Qiong Shi, Juanli Fu, Xiaomin Xia, Huiyuan Bai, Lihua Hu, Demei Xu, Erqun Song, and Yang Song* Key Laboratory of Luminescence and Real-Time Analytical Chemistry (Southwest University), Ministry of Education, College of Pharmaceutical Sciences, Southwest University, Chongqing 400715, People’s Republic of China ABSTRACT: Tetrachlorobenzoquinone (TCBQ), a metabolite of industrial herbicide pentachlorophenol, showed hepatotoxicity and genotoxicity through reactive oxygen species (ROS) mechanism in vivo and in vitro models. Nuclear factor erythroid-derived 2-like 2 (Nrf2) is a cellular sensor of oxidative or electrophilic stress, which controls the expression of detoxifying enzymes and antioxidant proteins. Using the human hepatoma HepG2 cell line as an in vitro model, we demonstrated a significant induction of Nrf2 but not its negative regulator Kelch-like ECH-associated protein 1 (Keap1), following exposure to TCBQ. Also, our results clearly demonstrated the translocation of cytosolic Nrf2 into the nucleus. After translocation, Nrf2 subsequently binds to the antioxidant response element (ARE), up-regulated heme oxygenase-1 (HO-1), and NADH quinone oxidoreductase subunit 1 (NQO1), which may be considered as an antioxidative response to TCBQ-intoxication. The luciferase reporter assay confirmed the formation of the Nrf2-ARE complex. Furthermore, mechanism studies proposed that TCBQ promoted the formation of the Keap1 cross-linking dimer, a ubiquitination switch from Nrf2 to Keap1 but not the dissociation of the Keap1-Cullin3 (Cul3) complex.

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INTRODUCTION Pentachlorophenol (PCP) is a chlorinated hydrocarbon insecticide and fungicide, which was used primarily as a wood preservative to protect wood from fungal rot and wood-boring insects.1 Although it is relatively stable, PCP biodegradation has been extensively reported. For example, several strains of Sphingobium chlorophenolicum isolated from PCP-contaminated soil and water have the ability to degrade PCP.2 It is commonly accepted that PCP-4-monooxygenase (PcpB), the first and ratelimiting enzyme, catalyzes PCP into tetrachlorobenzoquinone (TCBQ) and a new enzyme, tetrachlorobenzoquinone reductase (PcpD), the reduction of TCBQ into tetrachlorohydroquinone (TCHQ),3−5 which represents the typical PCP metabolic pathway. TCHQ and TCBQ are present in a redoxequilibrium in aqueous solutions, and this equilibrium can be easily shifted by reducing or oxidizing enzymes.4 Thus, it is practically impossible to distinguish or separate TCHQ from TCBQ unambiguously from biological samples. Because of the widespread distribution of PCP in the environment and its known toxicities, it is important to understand the biological behaviors of PCP and its reactive and presumably toxic metabolites, i.e., TCHQ and TCBQ, in mammalian systems. The in vitro toxic effects of PCP and TCHQ were reported extensively, and a mechanism study proposed the involvement of reactive oxygen species (ROS).6−11 Our recent in vivo studies demonstrated that TCBQ induces hepatic oxidative © XXXX American Chemical Society

damage and inflammatory response, which can be attenuated by antioxidant administration.12 However, there are large unknowns regarding the deleterious effects of TCBQ. For instance, we found that TCBQ activates heme oxygenase 1 (HO-1) and NAD(P)H quionone oxidoreductase 1 (NQO1) expressions in mice, which implied the activation of nuclear factor erythroid-derived 2-like 2 (Nrf2) signal pathway.13 However, its molecular mechanisms need further investigation. The cap’n’collar bZip transcription factor Nrf2 regulates oxidative and electrophilic stress responses by promoting the expressions of antioxidant response element (ARE)-mediated antioxidative or detoxifying enzymes.14 Under normal conditions, Kelch-like ECH-associated protein 1 (Keap1) regulates Nrf2 negatively, which prevents Nrf2 from activating target genes, such as HO-1 and NQO1. Keap1 associates with Cullin3 (Cul3) and Rbx1 to form an E3 ubiquitin ligase ubiquitination complex and anchors in the cytosol, then the 26S proteasome rapidly degrades cytosolic Nrf2 to maintain low Nrf2 levels.15 When cells are under oxidative or electrophilic stress conditions, Nrf2 is released from Keap1 “kidnapping” and accumulates in the nucleus and targets ARE, which lead to transcriptional activation of antioxidative or detoxifying genes. Received: December 12, 2014

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purchased from Aladdin Reagent Database Inc. (Shanghai, China). A stock solution of TCBQ (50 mM) was prepared in DMSO before use. Docosahexaenoic acid (DHA, CAS number: 6217-54-5) was purchased from Sigma-Aldrich Inc. (Shanghai, China). 1-Chloro-2,4dinitrobenzene (CDNB) was purchased from Xiya Reagent (Chengdu, China). Dulbecco’s modified Eagle’s medium (DMEM) was obtained from Keygen Biotech (Nanjing, China). 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI), pARE-luciferase vector, Protein A agarose beads, and western stripping buffer were supplied by Beyotime Institute of Biotechnology (Nanjing, China). Peroxidase-conjugated AffiniPure goat antirabbit IgG (H+L) antibody, EasyBlot ECL kit, and nuclear/cytosol fractionation kit were purchased from Sangon Biotech Co., Ltd. (Shanghai, China). Keap1, Lamin B, and Cul3 polyclonal antibodies were obtained from Beijing Biosynthesis Biotechnology Co., Ltd. (Beijing, China). Nrf2, HO-1, NQO1, fluorescein (FITC)conjugated AffiniPure Goat antirabbit IgG (H+L) and β-actin polyclonal antibodies were purchased from Proteintech group, Inc. (Wuhan, China). The ubiquitin antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). N-Acetyl-L-cysteine (NAC), Goldview nucleic acid stain, and RT-qPCR amplification primers of Nrf2, Keap1, and GAPDH were synthesized by Dingguo Biotechnology Co., Ltd. (Beijing, China). Lipofectamine 2000 transfection reagent, dual-luciferase reporter assay system kit, and PRL-SV40 vector were obtained from Promega (Madison, WI). All other chemicals used were of the highest commercial grade. Cell Culture. Human hepatoma HepG2 cells were obtained from Third Military Medical University, Chongqing, China. Cells were maintained in DMEM supplemented with 10% fetal bovine serum (HyClone, UT), penicillin (100 U/mL), and streptomycin (100 mg/ mL). Exponential growth phase cells were transferred onto 6-well culture plates and permitted to adhere overnight at 37 °C and 5% CO2. Immunofluorescence Staining. HepG2 cells were allowed to attach on sterile glass coverslips overnight at 37 °C and 5% CO2, then cells were treated with TCBQ for the indicated concentrations and periods. Cells were washed briefly with PBS and fixed with 4% paraformaldehyde for 30 min, permeabilized with 0.1% Triton-X 100, and blocked in milk based blocking buffer (10% nonfat dry milk) at 4 °C for 12 h. Cells were incubated with Nrf2 polyclonal antibody at 1:150 dilution in the antibody dilution buffer for 2 h at room temperature, then incubated with fluorescein (FITC)-conjugated AffiniPure goat antirabbit IgG (H+L) secondary antibody (1:150 dilution) for 1 h at room temperature. After 3 × 5 min rinsing with PBS, the sections were counterstained with DAPI for 10 min. Finally, cells were analyzed by a fluorescence microscope (OLYMPUS IX71). Nuclear and Cytosolic Proteins Extraction. Nuclear and cytosolic proteins were extracted by a nuclear/cytosol fractionation kit according to the manufacturer’s instructions and our previous publication.23 The cytosolic and nuclear fractions were stored at −20 °C and −80 °C, respectively. RNA Extraction and Reverse Transcription qPCR. HepG2 cells were precultured in 6-well culture plates for 24 h and then treated with TCBQ (25 and 50 μM) in 0.1% DMSO or vehicle (0.1% DMSO) as a control for 6 h. Total RNA was extracted with an Innu-PREP Micro RNA kit (AJ Innuscreen GmbH) as described in the manufacturer’s manual. RNA was reversely transcribed into cDNA (Toyobo Co., Ltd., Osaka, Japan) to perform quantitative real-time PCR analysis using LightCycler 96 instrument protocol with Faststart Essential DNA Green Master (Roche, Switzerland). The oligonucleotide primers, forward, 5′-GAC GGT ATG CAA CAG GAC ATT GAG-3′, and reverse, 5′-AAC TTC TGT CAG TTT GGC TTC TGGA-3′, were used to amplify human Nrf2. The primers, forward, 5′-GGA AAC AGA GAC GTG GAC TTT CGTA-3′, and reverse, 5′-TCC AGG AAC GTG TGA CCA TCA TA-3′, were used to amplify human Keap1, and the primers forward, 5′-GCA CCG TCA AGG CTG AGA AC-3′, and reverse, 5′-TGG TGA AGA CGC CAG TGGA-3′, were used to amplify human GAPDH as a housekeeping gene. These primers were designed based on the human gene sequences, according to Li et al.23 Three independent replicates were analyzed per sample,

It is generally accepted that the activation of the Nrf2 pathway and the up-regulations of antioxidative or detoxifying enzymes represent an adaptive response, which is regarded as a cell survival pathway and reduces the risk of cancer. For instance, Nrf2 deficient mice are more susceptible to chemical toxicants and carcinogen insults, which undoubtedly illustrate that Nrf2 plays in important role in chemoprevention.16,17 However, the pro-survival effect of Nrf2 may protect cancer cell against cell death and result in enhanced resistance of cancer cells to chemotherapeutic agents. In fact, the constitutive expressions of Nrf2 in different cancer cell lines were found, both in the laboratory and epidemiological studies.18,19 Therefore, the inhibition of Nrf2 may be a promising strategy against chemoresistance.20 Since there are “bright” and “dark” sides of Nrf2, it is interesting to investigate the molecular mechanism of TCBQinduced Nrf2 activation, which may provide further information on therapeutic intervention against TCBQ-induced toxicity. Previous studies indicated that a large number of Nrf2 inducers operate by alkylation and/or oxidation of Keap1 cysteine residues,21 and the modification of critical cysteine residues results in a conformational change in Keap1 that results in the dissociation of Keap1 from Cul3, thereby inhibiting Nrf2 ubiquitination.22 In the current study, we showed the evidence that TCBQ-induced Nrf2 activation involves Keap1 disulfide formation and ubiquitin translocation but not Keap1-Cul3 complex dissociation.

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MATERIALS AND METHODS

Materials and Reagents. Tetrachlorobenzoquinone (TCBQ, CAS number 118-75-2; the structure is shown in Figure 1A) was

Figure 1. (A) Chemical structure of TCBQ. TCBQ induces (B) Western blot analysis of TCBQ on HO-1, NQO1, and Nrf2 expressions at different time points and (C) Western blot analysis of TCBQ on HO-1, NQO1, and Nrf2 expressions at different concentrations. Data are representative of three or four separate experiments. B

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Figure 2. Effect of TCBQ on Nrf2 activation, detected by immunofluorescence analysis. DAPI staining was applied to indicate the location of the nucleus (blue), Nrf2 (green) expression was identified using anti-Nrf2 antibody and FITC-conjugated secondary antibody, and the merged image of TCBQ-treated cells indicates the nuclear location of Nrf2 protein. (A) Cells were exposed to TCBQ (25 and 50 μM) or DMSO controls for 6 h. (B) Cells were treated with 25 μM TCBQ for different time periods. The bar is equal to 20 μm. and the relative gene expression normalized to the internal control gene GAPDH was obtained by the 2−ΔΔCt method. Transient Transfection and Luciferase Reporter Gene Assay. HepG2 cells were plated onto 96-well plates and cultured for 24 h. Eight nanograms of pRL-SV plasmid and 0.4 μg of pARE-luciferase

cDNA were mixed together with OPTI-MEM and incubated at room temperature for 20 min to allow the formation of complexes. Then, lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA) was incubated in serum-free medium for 6 h followed by another 24 h of incubation in 10% serum medium. After incubation, cells were exposed to various C

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Figure 3. Effect of TCBQ on Nrf2 and Keap1 mRNA expressions. Cells were treated with indicated concentrations of TCBQ for 6 h. Total RNA was extracted, and the expressions of Nrf2 and Keap1 mRNA levels were detected by RT-qPCR. Data are representative of three separate experiments and are presented as the mean ± SD; *p < 0.05 and ***p < 0.001, significantly different as compared to the control. Statistical Analysis. Each data set was performed with at least three independent experiments. The data were analyzed using SPSS 18.0 software. A probability of p < 0.05 was considered to be significant.

concentrations of TCBQ in 0.1% DMSO or 0.1% DMSO alone as a control for 6 h. The activity of luciferase was measured in a fluorescence microplate reader with the dual-luciferase assay kit (Promega, Beijing) according to the supplier’s recommendations. Redox Western Blotting. Redox Western blotting analysis for Keap1 was performed according to a previously reported method.24 HepG2 cells were pretreated with CDNB (25 μM) or NAC (10 mM) for 60 min and then incubated with or without TCBQ (25 μM) for another 60 min. Cells were lysed with lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 0.25% sodium deoxycholate, 1 mM PMSF, EDTA, sodium orthovanadate, sodium fluoride, and leupeptin protease inhibitor). Then, lysate samples were centrifuged at 14,000 rpm at 4 °C for 10 min, after which the supernatant was collected as the redox whole cell extract. Half of the samples were diluted in sample buffer containing β-mercaptoethanol (βME). After heat denaturation, proteins were separated by 10% SDS−PAGE, and Keap1 was detected by Western blotting. Determination of GSH. The level of GSH in HepG2 cells was measured by a commercial kit according to its manufacturer’s instructions (Nanjing Jiancheng Institute of Biotechnology). GSH level was detected based on the colorimetric reaction of GSH reduced 5,5′-dithiobis-2-nitrobenzoic acid (DTNB) into 2-nitro-5-thiobenzoic acid (TNB), which was monitored at 405 nm spectrophotometrically. Immunoprecipitation. After treatment with TCBQ, HepG2 cells were lysed with RIPA lysis buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM PMSF, and proteinase inhibitor cocktail. The lysates were incubated on ice for 30 min and homogenized with an ultrasonicator for 7 min. The homogenates were centrifuged at 12,000 rpm for 10 min at 4 °C. The supernatants were collected, and the concentrations of proteins were determined by Bradford Protein Assay Kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). For immunoprecipitation, whole-cell lysates were precleared with Protein A Agarose beads for 10 min. Then, the solutions were incubated with 3 μg of anti-Cullin3 or anti-Keap1 antibody at 4 °C overnight and then mixed with 30 μL of Protein A agarose beads at 4 °C for 3 h. Then, immunoprecipitation solutions were centrifuged, and the pellets were washed 4 times with lysis buffer. The complexes were eluted with loading buffer, heated at 97 °C for 7 min, and subsequently analyzed by Western blotting. Western Blotting. Either the whole-cell lysates or the immunoprecipitation products were separated by 8% or 10% SDS− PAGE and transferred to the PVDF membrane. Membranes were incubated with HO-1, NQO1, Nrf2, ubiquitin, Keap1, Cul3, Lamin B, or β-actin polyclonal antibodies overnight at 4 °C and further incubated with peroxidase-conjugated goat antirabbit IgG (H+L) secondary antibody for 2 h. The proteins were detected using the ECL system or the HRP substrate DAB system. Representative blots were chosen from three separate experiments showing similar results.

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RESULTS TCBQ Induces Nrf2 Activation and the Expressions of HO-1 and NQO1 in HepG2 Cells. We first assessed whether TCBQ induces the expressions of HO-1 and NQO1 in HepG2 cells. Our results showed that TCBQ exposure induced the expressions of HO-1 and NQO1 protein in a time- and concentration-dependent manner (Figure 1B and C). Since the Nrf2/Keap1/ARE pathway is a major transcription factor that regulates HO-1 and NQO1 expression, we then examined whether Nrf2 is activated under the treatment of TCBQ. The up-regulation of Nrf2 upon TCBQ treatment was also verified by Western blot and immunofluorescence analysis. As shown in Figures 1 and 2, treatment with TCBQ (25 and 50 μM) for 6 h increased the expression of Nrf2. Interestingly, Nrf2 bands were shown both at ∼68 kDa and 100−110 kDa, which is consistent with previous reports.25 These two bands have the same pattern in our experiments; therefore, we reported both bands. Although the nature of the aberrant migration pattern of Nrf2 is still uncertain, different mechanisms were proposed. Kang et al. suggested that Nrf2 binds with actin and that the Nrf2-actin complex is translocated to the nucleus as a complex bound with a 42 kDa actin,26 while Li et al. deduced that four ubiquitins (about 8.6 kDa each) covalently bind Nrf2 yielding a complex with an approximate molecular weight of ∼100 kDa.27 These results indicated that TCBQ causes Nrf2 activation and the expression of HO-1 and NQO1 in HepG2 cells. The most effective time for Western blotting and the fluorescent signal was 6 h. The different time points for maximum Nrf2 expression and its downstream gene expressions may be due to the lag time from Nrf2 upregulation/translocation to ARE binding/downstream gene regulation. TCBQ Enhances the Nrf2 mRNA Level while Reducing the Keap1 mRNA Level. To identify whether TCBQ stimulates mRNA expression of Nrf2/Keap1, the mRNA level of Nrf2 and Keap1 were examined using reverse transcription RT-qPCR. TCBQ (25 and 50 μM) exposure induced the mRNA transcript level of Nrf2 in a concentration-dependent manner (Figure 3). A slight but not significant reduction of Keap1 mRNA level was found. D

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Chemical Research in Toxicology TCBQ Facilitates Nuclear Translocation of Nrf2 and Activates ARE-Regulated Luciferase Activity. It is well known that Nrf2 associates with Keap1 through a constitutive proteasome-dependent pathway in cytoplasm under normal conditions. When stimuli occur, Nrf2 dissociates from Keap1 and translocates into the nucleus. Here, we detected whether TCBQ triggers the translocation of Nrf2. Our results demonstrated the increased nuclear localization of Nrf2 with corresponding decreased Nrf2 in cytoplasm in TCBQ treated cells (Figure 4). Keap1 expression showed a small decrease in

Figure 5. Effects of TCBQ on ARE activation. Cells were transfected with the ARE-luciferase vector. After transfection, cells were treated with TCBQ (25 and 50 μM) or DMSO controls for 24 h, and the ARE-luciferase activity was measured by an analyzer enzyme fluorescent assay. The data are from four independent experiments and are presented as the mean ± SD; ***p < 0.001, significantly different as compared to the control.

sulfhydryl by analyzing the redox state of Keap1 ectopically expressed in HepG2 cells, using H2O2 as the positive control. After treatment with H2O2 (200 μM) for 5 min, two variable bands with an apparent molecular mass of 120 kDa (OxIR1) and 115 kDa (OxIR2) were detected under nonreducing conditions (Figure 6A). However, OxIR1 and OxIR2 were absent with the treatment of βME, indicating that they probably result from disulfide formation, i.e., the formation of the Keap1 dimer via cysteine−cysteine cross-linking. Along with the disappearance of OxIR1/2, there is another band with faster mobility (OxIM, right under the band of the reduced Keap1) that vanished under reducing conditions. According to a previous publication, OxIM was identified as intramolecular oxidized Keap1, while OxIR1/2 was in the intermolecular oxidized form.24 Similar result was observed in the treatment of TCBQ, which implied a shared mechanism of the activation of TCBQ and H2O2. As shown in Figure 6B, the intensity of the 120 kDa band was increased over time, when HepG2 cells were treated with 25 μM TCBQ for 5 to 60 min under nonreducing conditions. However, OxIR2 has not been observed, while there is a weak intensity of OxIM. Under reducing conditions, only the 69 kDa band was detected, regardless of the TCBQ treatment duration. CDNB, a GSH-depleting agent, induces the formation of the Keap1 dimer, and coexposure of TCBQ enhanced this effect (Figure 6C, lanes 1 and 2), suggesting that this effect is GSH-dependent. Indeed, TCBQ-induced timedependent GSH depletion has been verified (Figure 6D). The antioxidant effect of NAC has been extensively examined, and our study demonstrated that the pretreatment of NAC significantly inhibited TCBQ-induced ROS in HepG2 cells.28 Another study also indicated that NAC ameliorates the loss of cellular GSH induced by xenobiotics.29 However, NAC does not inhibit the formation of the Keap1 dimer (Figure 6C, lane 3). These results suggested the disulfide formation of Keap1 in cells treated with TCBQ. TCBQ Does Not Induce the Dissociation of Keap1 from Cul3. Ubiquitination of Nrf2 is regulated by the Keap1Cul3 E3 ubiquitin ligase complex; thus, the association of Keap1 with Cul3 is quite important in controlling the steady level of Nrf2. Therefore, it is rational to investigate whether the dissociation of Keap1 from Cul3 is responsible for TCBQinduced Nrf2 activation. We then assessed the effect of TCBQ on the dissociation of Keap1 from Cul3 by performing immunoprecipitated experiments with anti-Cul3 and antiKeap1 antibodies. DHA, the positive control, has been shown

Figure 4. Effect of TCBQ on Nrf2 accumulation in the nucleus. HepG2 cells were treated with TCBQ (25 and 50 μM) or DMSO controls for 6 h, and then cell lysates were subjected to Western blot analysis. Lamin B and β-actin were used as loading controls in both nuclear and cytosolic fractions. (A) Nuclear fraction. (B) Cytosolic fraction. Data are representative of three separate experiments showing similar results.

nucleic fraction and no obvious change in cytosolic extracts. Furthermore, HO-1 and NQO1 were up-regulated both in the nucleus and cytoplasm fractions. After Nrf2 translocates into the nucleus, and it binds to ARE sequences that are located in the promoter region of the genes encoding cytoprotective proteins. Then, we examined the effect of TCBQ on ARE-reporter gene activity. As shown in Figure 5, TCBQ treatment increased ARE-luciferase activity in a concentration-dependent manner. These results demonstrated that TCBQ induced nuclear translocation of Nrf2 and activated ARE-regulated luciferase activity. TCBQ Induces the Formation of the Keap1 Dimer. A previous study demonstrated that cysteine sulfhydryl groups are subject to modification by ROS or electrophiles.24 Modified Keap1 alters its conformation and no longer holds Nrf2, which inhibits Keap1-mediated Nrf2 ubiquitination. Thus, we evaluated whether TCBQ could oxidize Keap1 at cysteine E

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Figure 6. Effect of TCBQ and H2O2 on the formation of the Keap1 cross-linking dimer. (A) HepG2 cells were treated with H2O2 (200 μM) for the indicated time periods. Keap1 protein was revealed by Western blot under nonreducing (−βME) or reducing (+βME) conditions. βME, βmercaptoethanol. (B) HepG2 cells were exposed to TCBQ (25 μM) for the indicated time and then detected by Western blot under nonreducing (−βME) or reducing (+βME) conditions. (C) HepG2 cells were pretreated with CDNB (25 μM) or NAC (10 mM) for 60 min and then incubated with or without TCBQ (25 μM) for another 60 min. Western blots were performed under nonreducing (−βME) or reducing (+βME) conditions. (D) TCBQ (25 μM) decreases the cellular GSH level in a time-dependent manner. All data are representative of three separate experiments showing similar results.

to destabilizing the association between Keap1 and Cul3.30 However, immunoprecipitation with the Cul3 antibody followed by immunoblotting with the Keap1 antibody demonstrated that Keap1 and Cul3 associated in a complex with TCBQ-treatment, which is in contrast with the response to DHA shown in Figure 7A. Furthermore, immunoprecipita-

tion with the anti-Keap1 antibody and immunoblot with the Cul3 antibody showed a similar effect (Figure 7B). These results demonstrated that the up-regulation of Nrf2 by TCBQ was not due to the dissociation of Keap1 from Cul3. TCBQ Reduces the Ubiquitination of Nrf2 and Enhanced the Ubiquitination of Keap1. Previous studies demonstrated that oxidative stress could inhibit steady-state levels of Keap1 through increasing ubiquitination of Keap1, resulting in an enhancing of Nrf2 correspondingly.31 To determine whether the up-regulation of Nrf2 by TCBQ is due to the inhibition of Nrf2 ubiquitination and the enhancement of Keap1 ubiquitination, we detected the ubiquitination of Keap1 and Nrf2 by immunoprecipitation. Because ubiquitinated proteins are rapidly degraded by the 26S proteasome, a 26S proteasome-specific inhibitor, MG132, was added to cells prior to cell lysis to enable the detection of ubiquitinated proteins. As shown in Figure 8A, degradation of ubiquitin was blocked by treatment of cells with MG132 prior to cell lysis as expected (lanes 3 and 4 vs lanes 1 and 2); meanwhile, TCBQ showed no effect on the levels of ubiquitin conjugation (lane 3 vs lane 4). MG132, by inhibiting the proteasome, prevents the degradation of Nrf2, confirming the ability of Keap1 to target Nrf2 for proteasome-mediated degradation in HepG2 cells. However, after immunoprecipitation with anti-Keap1 or anti-Nrf2 antibodies, a marked increase of ubiquitination of Keap1 (Figure 8B) and a significant reduction of ubiquitination of Nrf2 (Figure 8C) were detected in HepG2 cells cotreated with TCBQ and MG132, respectively. These results indicated that

Figure 7. TCBQ does not induce the disassociation of Keap1 from Cul3. HepG2 cells lysates were immunoprecipitated with (A) Cul3 antibody or (B) Keap1 antibody after treatment with TCBQ (25 μM) or DMSO controls for 6 h. DHA, an inducer which can initiates the disassociation of Keap1 from Cul3 as a positive control. Precipitated proteins were analyzed by SDS−PAGE with Keap1 or Cullin3 antibody. Results are representative of three experiments. F

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Figure 8. Effects of TCBQ on the ubiquitination of Keap1 and Nrf2. (A) Western blot analysis of endogenous Keap1, Nrf2, and ubiquitin. HepG2 cells were treated with MG132 (10 μM) for 1 h and then treated with or without 25 μM TCBQ for 6 h. Whole-cell lysates were used to detect Keap1, Nrf2, and ubiquitin with their antibodies. Effects of TCBQ on the ubiquitination of (B) Keap1 and (C) Nrf2. Cell lysates were immunoprecipitated with Keap1 antibody or Nrf2 antibody, and precipitated proteins were analyzed by SDS−PAGE with the ubiquitin antibody. All data represent three independent experiments.

TCBQ also yielded hydrogen peroxide and the hydroxyl radical as its downstream ROS during the futile recycling of the quinone/semiquinone/hydroquinone triad.43 Thus, TCBQ may serve as an alkylating agent and oxidant in the current study and is anticipated to modify cysteine residues by both ways. Thus, it is of interest to us to investigate which is the predominant mechanism. Time course analysis revealed that the peak time of H2O2-inducd Keap1 modification is 5 min and that Keap1 returned to the redox homeostatic status, which implied that the oxidative modification of Keap1 is transient.24 However, our results showed that TCBQ-induced oxidative Keap1 bands (OxIR1 and OxIM) were sustained up to 60 min, which suggested that alkylation may play a major role in the formation of the Keap1 dimer. Another evidence of alkylation is that NAC pretreatment cannot inhibit the formation of the OxIR1 band. In addition, our results showed that βME treatment inhibited the formation of oxidative Keap1 bands, which proposed that βME reversed the alkylation of Keap1 by removing TCBQ from Keap1 via S-transarylation.44 Our previous study indicated that all of the chlorine on the quinone ring of TCBQ is replaceable,43 which strongly supported the formation of intramolecular (OxIR1) and intermolecular (OxIM) cross-linking bands of Keap1. Although TCBQ can produce H2O2 in aqueous solution, the OxIR2 band was not seen upon cell exposure to TCBQ treatment, which is different from H2O2 incubation. This result may due to the different activities of cysteine residues since a higher concentration of H2O2 (200 μM) was used directly. However, modifying specific cysteines of Keap1 protein is insufficient to disrupt binding to the Nrf2 domain Neh2,45 which did not lead to the stabilization of Nrf2.46 Therefore, other mechanisms may apply to TCBQinduced Nrf2 activation. TCBQ Induces a Ubiquitination Switch from Nrf2 to Keap1. Keap1 is a BTB-Kelch protein, which is considered as a substrate adaptor protein for a Cul3-dependent E3 ubiquitin ligase complex. Once the E3 ubiquitin ligase complex is assembled, ubiquitin transferred from a conserved cysteine residue to lysine residues in the substrate protein. Under homeostatic conditions, Nrf2 is maintained at low levels because of Cul3-dependent E3-ubiquitin ligase-mediated Nrf2 ubiquitination and subsequent degradation by the 26S proteasome.47 Inhibition of Keap1-dependent ubiquitination

TCBQ activated the Nrf2 pathway, maybe partially due to an inhibitory Keap1-dependent ubiquitination of Nrf2.

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DISCUSSION TCBQ Induces Nrf2 Activation on Its Transcriptional and Post-transcriptional Levels. Quinones are important moieties that range from drugs, 32 naturally occurring phytochemicals,33 to environmental pollutants.34 Quinones exhibit electrophilic characteristics and redox properties, which resulted in protein S-alkylation and oxidative stress in cells. Growing evidence indicates that the Keap1/Nrf2/ARE system plays an important role in protecting cells from electrophile insults or oxidative stress through the upregulation of cytoprotective proteins.35 Thus, the first aim of the present study is to investigate whether TCBQ has an effect on Nrf2 activation in HepG2 cells. In line with previous studies on quinones,36,37 TCBQ resulted in the activation of Nrf2 on its transcriptional and post-transcriptional levels. TCBQ Induces Keap1 Dimer Formation via Alkylation (C−S Bond) and the Formation of Disulfide Protein (S−S Bond). Mechanism studies indicated that electrophilic compounds activate Nrf2 primarily by inhibiting Keap1dependent Nrf2 degradation through the alkylation of cysteines on Keap1.38 For instance, previous studies demonstrated that 2tert-butyl-1,4-benzoquinone39 and 1,2-naphthoquinone37 covalently bind to Keap1 yielding quinone-Keap1 conjugates, through the formation of a C−S bond. Keap1 contains 27 cysteine residues within human homologues, and the sensitivities of cysteine on Keap1 to electrophile assault are different, e.g., cysteine 151 on the BTB domain and cysteine 288 on the IVR domain came out as the most reactive residues; however, most of the cysteines were identified as modifiable.40,41 Another type of cysteine modification is involved in oxidative stress, which refers to the situation of temporally decreasing GSH and increasing of its disulfide GSSG, in turn resulting in the glutathionylation of cellular proteins and the formation of the protein disulfide bond.42 Here, the decreased GSH level was found in the TCBQ-treated cell, indicating the increased oxidative stress. Interestingly, our previous study illustrated that TCBQ can directly react with GSH (cysteine residue) via a nonenzymatic, nucleophilic chlorine displacement reaction yielding a glutathionylated conjugated quinone. G

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Chemical Research in Toxicology Funding

of Nrf2 increases steady-state levels of Nrf2 and enables the activation of cytoprotective Nrf2-dependent genes. To determine if ubiquitination of Nrf2 and Keap1 is altered in TCBQ-induced cells, levels of ubiquitin conjugation onto both Keap1 and Nrf2 were measured following exposure to TCBQ. Consistent with a previous quinone study, TCBQ inhibits Keap1-dependent ubiquitination of Nrf2, while ubiquitination of Keap1 is increased;46,48 however, another Nrf2 inducer, sulforaphane, has no effect on the ubiquitination of Keap1.31 A mechanism study revealed that although chemical modifications of Keap1 on cysteine 151 both occurred in quinone and sulforaphane treatment, oxidative modification may play a central role in quinone-induced inhibition of Keap1, which modified Keap1 not only on cysteine 151 but also on other lysine residues. These modifications may discompose the Keap1-Cul3-Rbx1 E3 ubiquitin ligase complex, resulting in the ubiquitination switch from Nrf2 to Keap1. According to a previous study, the ubiquitination switch between Nrf2 and Keap1 is specific to quinone.31 TCBQ Does Not Induce the Dissociation of the Keap1Cul3 Complex. The Keap1 function requires the association with Cul3 that targets Nrf2 for ubiquitination.48 However, electrophiles have been shown to dissociate the Keap1−Cul3 interaction by modifying cysteine 151.48,49 Consequently, the ubiquitination of Nrf2 was inhibited. It was suggested that cysteine 151 is essential for binding of Keap1 to Cul3, while other cysteines (273 and 288) are less important. Keap1 with mutations on cysteines 273 and 288 has no effect on the ability of ectopically expressed Keap1 to bind Cul3 or inhibit the association between Keap1 and Nrf2.45,50 Surprisingly, two critical cysteine residues C273 and C288 on Keap1 are required for Keap1-dependent ubiquitination of Nrf2.50,51 Interestingly, arsenic showed a unique Nrf2 activation mechanism by enhancing the interaction between Keap1 and Cul3, which retard the assembly and disassembly process of the E3 ubiquitin ligase and decreased its ligase activity.52 Moreover, the increased association of Keap1 and Cul3 promotes the ubiquitination of Keap1. On the basis of these discoveries and our results, it is likely that TCBQ-induced activation of Nrf2 is independent of cysteine 151; still, further evidence is needed for clarifying whether cysteine 273 and 288 modification or the increased association of Keap1 and Cul3 applies in TCBQ-induced Nrf2 activation.

This work is supported by National Natural Science Foundation of China (21477098 and 21035005), Science and Technology Talent Cultivation Project of Chongqing (cstc2014kjrc-qnrc00001), Scientific Research Foundation for the Returned Overseas Chinese Scholars from State Education Ministry (2011[508]), and Fundamental Research Funds for the Central Universities (XDJK2014A020 and XDJK2013B009). Notes

The authors declare no competing financial interest.

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ABBREVIATIONS ARE, antioxidant response element; βME, β-mercaptoethanol; CDNB, 1-chloro-2,4-dinitrobenzene; Cul3, Cullin 3; DAPI, 4′,6-diamidino-2-phenylindole; DHA, docosahexaenoic acid; HO-1, heme oxygenase 1; Keap1, Kelch-like ECH-associated protein 1; NAC, N-acetyl-L-cysteine; NQO1, NADH quinone oxidoreductase subunit 1; Nrf2, nuclear factor erythroid-derived 2-like 2; PCP, pentachlorophenol; ROS, reactive oxygen species; RT-qPCR, reverse-transcription quantitative polymerase chain reaction; TCBQ, tetrachlorobenzoquinone; TCHQ, tetrachlorohydroquinone

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CONCLUSIONS Nrf2 activation is a multiple control mechanism. Here, our results clearly demonstrated TCBQ-induced Nrf2 activation via the formation of the Keap1 cross-linking dimer and the switch of ubiquitin from Nrf2 to Keap1 but not Keap1-Cullin3 complex dissociation. These mechanisms are Keap1-dependent; however, growing evidence suggests some alternative Keap1independent mechanisms of Nrf2 activation, including transcriptional regulation, post-transcriptional regulation, protein stability, post-translational modification, and subcellular localization.53

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REFERENCES

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Corresponding Author

*College of Pharmaceutical Sciences, Southwest University, Beibei, Chongqing, 400715, P R China. Tel: +86-23-68251503. Fax: +86-23-68251225. E-mail: [email protected]. H

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DOI: 10.1021/tx500513v Chem. Res. Toxicol. XXXX, XXX, XXX−XXX