Cell Biology International ISSN 1065-6995 doi: 10.1002/cbin.10450
RESEARCH ARTICLE
Identification of a functional antioxidant responsive element in the promoter of the Chinese hamster carbonyl reductase 3 (Chcr3) gene Takeshi Miura1,2*, Ayako Taketomi1, Toshikatsu Nakabayashi2,3, Toru Nishinaka1 and Tomoyuki Terada1 1 Laboratory of Biochemistry, Faculty of Pharmacy, Osaka Ohtani University, 3–11-1 Nishikiori-kita, Tondabayashi, Osaka 584–8540, Japan 2 Pharmaceutical Education Support Center, School of Pharmacy and Pharmaceutical Sciences, Mukogawa Women’s University, 11–68 Koshien, 9-Bancho, Nishinomiya, Hyogo 663–8179, Japan 3 First Department of Biochemistry, School of Pharmacy and Pharmaceutical Sciences, Mukogawa Women’s University, 11–68 Koshien, 9-Bancho, Nishinomiya, Hyogo 663–8179, Japan
Abstract CHCR3, a member of the short-chain dehydrogenase/reductase superfamily, is a carbonyl reductase 3 enzyme in Chinese hamsters. Carbonyl reductase 3 in humans has been believed to involve the metabolism and/or pharmacokinetics of anthracycline drugs, and the mechanism underlying the gene regulation has been investigated. In this study, the nucleotide sequence of the Chcr3 promoter was originally determined, and its promoter activity was characterised. The proximal promoter region is TATA-less and GC-rich, similar to the promoter region of human carbonyl reductase 3. Cobalt stimulated the transcriptional activity of the Chcr3 gene. The results of a luciferase gene reporter assay demonstrated that cobalt-induced stimulation required an antioxidant responsive element. Forced expression of Nrf2, the transcription factor that binds to antioxidant responsive elements, enhanced the transcriptional activity of the Chcr3 gene. These results suggest that cobalt induces the expression of the Chcr3 gene via the Nrf2-antioxidant responsive element pathway. Keywords: carbonyl reductase; Chinese hamster; genome walk; SDR21C2; short-chain dehydrogenase/reductase Introduction The short-chain dehydrogenase/reductase (SDR) superfamily is a large family of enzymes that are catalytically active against endogenous and xenobiotic compounds, including steroids, prostaglandins, and anti-cancer drugs (Matsunaga et al., 2006; Malátkoivá et al., 2010). The SDR superfamily includes two monomeric carbonyl-reducing enzymes, carbonyl reductase 1 (CBR1) (Wermuth et al., 1998) and carbonyl reductase 3 (CBR3) (Watanabe et al., 1998). In humans, these enzymes are homologous at the nucleotide and amino acid sequence levels (77% and 72%, respectively). They reduce endogenous and xenobiotic carbonyl compounds using NADPH as a coenzyme (Miura et al., 2008). Despite their amino acid sequence similarity, the physiological roles of CBR1 and CBR3 are different, as evidenced by in vitro enzymatic analyses (Miura et al., 2008, 2009a; El-Hawari et al., 2009; Pilka et al., 2009). The CBR1 and CBR3 enzymes metabolize anthracycline anti-cancer drugs such as daunorubicin and doxorubicin.
The existence of a genetic variant of the CBR3 gene affects the cardiotoxicity and pharmacokinetics of the drugs, and the enzymatic activity of CBR3 may relate to the development of the cardiotoxicity (Blanco et al., 2008, 2012; Fan et al., 2008; Lal et al., 2008; Volkan-Salanci et al., 2012). The CBR3 gene was first identified by Watanabe et al. (1998); it lies near the CBR1 gene in the human genome. The enzymatic characteristics of human CBR3 were described in 2008 (Miura et al., 2008). CBR3 is involved not only in the metabolism of anthracyclines, but also in some physiological regulations. Treatment with 9-cis-retinoic acid induces CBR3 gene expression, and the induced enzyme suppresses cell growth and migration potential in oral squamous cell carcinomas (Ohkura-Hada et al., 2008). Furthermore, increased expression of the CBR3 gene in human adipose tissues is negatively associated with fasting insulin and systemic insulin resistance, suggesting that CBR3 modulates the diabetic state (Chang et al., 2012). These findings indicate that regulation of CBR3 gene expression is related to physiological functions. Therefore, it is important to
Corresponding author: e-mail: [email protected] or [email protected]
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clarify the physiological roles of CBR3 and elucidate the mechanism underlying the regulation of CBR3 gene expression. We originally identified and characterised a Chinese hamster ortholog of CBR3, named CHCR3 (Terada et al., 2001, 2003; Miura et al., 2009b). The transcriptional regulation of Chcr3 remains unclear, although the human CBR3 gene is regulated by the transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2) (Cheng et al., 2012; Ebert et al., 2010) and pro-inflammatory stimuli (Malátková et al., 2012). In the present study, we determined the nucleotide sequence of the Chcr3 promoter region and investigated whether Nrf2 regulated Chcr3 gene expression. Materials and methods
Materials Restriction enzymes were obtained from New England BioLabs (Beverly, MA, USA). The pGL3-basic vector and pRL-TK vector were from Promega (Madison, WI, USA). The PCR primers were purchased from Invitrogen (Carlsbad, CA, USA). All the other reagents were of the highest grade commercially available.
Genomic DNA extraction from Chinese hamster livers Genomic DNA was extracted from the livers of Chinese hamsters. The liver was placed in 500 mL of DNA extract buffer (50 mM Tris-HCl, pH 8.0, 100 mM EDTA, pH 8.0, 100 mM NaCl, and 1% SDS) with 20 mL of 20 mg/mL proteinase K and was rotated overnight at 55 C, after which 25 mL of 1 mg/mL RNase A was added. After 1 h of incubation, the solution was mixed with 500 mL of phenol. The aqueous phase was added and extracted twice with 500 mL of phenol/chloroform/isoamyl alcohol (25:24:1 [v/v]). Genomic DNA was isolated by adding 500 mL of isopropanol.
Cell culture HEK293 cells and Chinese hamster ovary (CHO)-K1 cells were maintained in Dulbecco’s modified Eagle’s medium (Nissui Pharmaceutical Co. Ltd., Tokyo, Japan) and Ham’s F-12 medium (N6658; Sigma–Aldrich, St. Louis, MO, USA), respectively, supplemented with 10% fetal bovine serum (BioWest, Nuaille, France) at 37 C under an atmosphere of 95% air and 5% CO2.
Plasmids To construct the luciferase gene reporter vector pGL3CHCR3–1663, which contained the promoter region (from Cell Biol Int 39 (2015) 808–815 © 2015 International Federation for Cell Biology
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1663 bp to 89 bp) of the Chcr3 gene, polymerase chain reaction (PCR) was performed using YSCR11f (50 -act gct ccc tgg agt tct gtt ca-30 ) and YSCRr25 (50 -ggg acc cgg cgc tgc aac tgc tgg ag-30 ) as primers and Chinese hamster genomic DNA as template. The amplified DNA fragment was digested with XhoI and HindIII and inserted into the pGL3-basic vector. The primers were designed based on the nucleotide sequence determined with the genome walking method. To generate Chcr3 promoter mutants, site-directed mutagenesis was performed according to the method of Higuchi et al. (1988). The primers were as follows: CHCR3ARE1mtf01 (50 -agc cgg atc cct act ccg ca-30 ) and CHCR3ARE1mtr01 (50 -tgc gga gta ggg atc cgg ct-30 ) for the mutation of ARE1, CHCR3ARE2mtf01 (50 -agc tga gtc ccc gtg agg gc-30 ) and CHCR3ARE2mtr01 (50 -gcc ctc acg ggg act cag ct-30 ) for the mutation of ARE2, and CHCR3ARE3mtf01 (50 -act gcg gaa cca gct cca cg-30 ) and CHCR3ARE3mtr01 (50 -cgt gga gct ggt tcc gca gt-30 ) for the mutation of ARE3. The nucleotide sequences of the PCR products were confirmed using an ABI PRISM 310 Genetic Analyzer (Applied Biosystems Japan, Ltd., Tokyo, Japan). The Nrf2 expression vector (pNrf2) was a gift from Dr. Cecil B. Picket (Schering-Plough Research Institute, Kenilworth, NJ, USA) (Nguyen et al., 2000). The dominant-negative Nrf2 expression vector (pNrf2-DN) was prepared by the method described previously (Nishinaka et al., 2005).
Genome walking To determine the nucleotide sequence of the 50 -upstream region of Chcr3, genome walking was performed. A summary of the experiments is shown in Supporting Information, Figure S1. Genomic DNA was digested with BamHI. The digested DNA fragment was ligated to a Sau3AI cassette (double-stranded DNA; 50 -gta cat att gtc gtt aga acg cgt aat acg cgt aat acg act cac tat agg ga-30 and 50 -gat ctc cct ata gtg agt cgt att acg cgt tct aac gac aat atg tac-30 ). Using the ligated DNA fragment as template, PCR was performed with a cassette 1 primer (50 -gta cat att gtc gtt aga acg cg-30 ) and a CHCR3specific primer (YSCRr12, 50 -tgg tcc gcg tgt ccc tc-30 ). Nested PCR with a cassette 2 primer (50 -taa tac gac tca cta tag gga ga30 ) and a CHCR3-specific nested primer (YSCRr14, 50 -agc cgg ctc tgc aag cc-30 ) was performed using the solutions from the first PCR as a template. Amplified DNA fragments were cloned into the pGEM-T Easy vector (Promega), and the nucleotide sequences were confirmed using an ABI PRISM 310 Genetic Analyzer (Applied Biosystems Japan, Ltd.). The length of the DNA fragment was 770 bp. To obtain further nucleotide sequence of the 50 -upstream region of the gene, two primers (YSCRr19, 50 -ggg caa aac cag acg ct-30 and YSCRr20, 50 -cta tgt aca tac tcg ggt ac-30 ) were designed based on the nucleotide sequence determined from the first genome walking experiment. A second genome 809
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walking experiment was carried out in the same way using the primers. The length of the DNA fragment was 905 bp.
50 -rapid amplification of cDNA ends (RACE) Using total RNA from CHO-K1 cells, 50 -RACE was performed with the 50 -RACE System for Rapid Amplification of cDNA Ends (Gibco, Tokyo, Japan). Total RNA prepared from CHO-K1 cells was reverse transcribed with an RT primer (YSCRr21, 50 -tag aaa tgt ctg caa tg-30 ) using reverse transcriptase. The cDNA was purified, and a poly-dA tail was added using the terminal transferase enzyme. The dA-tailed cDNA was amplified by PCR with an oligo dTanchor primer and gene-specific primer (GSP) 1 (YSCRr04, 50 -cgt gtg gat ccg ata tgt cgt cct g-30 ) for the first PCR, with a dT-anchor primer and GSP 2 (YSCRr12, 50 -tgg tcc gcg tgt ccc tc-30 ) for the second PCR, and with a dT-anchor primer and GSP 3 (YSCRr14, 50 -agc cgg ctc tgc aag cc-30 ) for the third PCR. The resulting DNA fragments were cloned and sequenced using an ABI PRISM 310 Genetic Analyzer (Applied Biosystems Japan, Ltd.).
Quantification of Chcr3 gene expression CHO-K1 cells were treated with or without CoCl2, and total RNA was prepared using a ReliaPrep RNA Cell Miniprep System (Promega) according to the manufacturer’s instructions. Using a ReverTra Ace qPCR RT kit (Toyobo, Osaka, Japan), cDNA was synthesized from 2 mg of total RNA. The primers used for the detection of Chcr3 gene expression were 50 -acc agg gct cca gga ctg tg-30 (forward) and 50 -cct cca ttt gga tcc ctc tc-30 (reverse). To normalize the quantities of cDNA, expression of the b-actin gene was also measured using the primers 50 -tgg cat cca cga aac tac at-30 (forward) and 50 -tgg tac cac cag aca gca ct-30 (reverse) (Han et al., 2012). Real-time PCR was performed using SYBR Green Realtime PCR Master Mix -Plus- v.2 (Toyobo) with an Opticon 2 real-time PCR apparatus (Bio-Rad Laboratories, Inc., Tokyo, Japan). Real-time PCR standard curves were calculated using cDNA from CHO-K1 cells as a template. The PCR program was as follows for the Chcr3 and b-actin genes: hot start, 96 C, 60 s; denature, 95 C, 15 s; annealing, 62 C, 15 s; and extension, 72 C, 60 s. The analyses of the melting curves revealed specific amplification of the Chcr3 and b-actin genes. The nucleotide sequences of the PCR products were confirmed using an ABI PRISM 310 Genetic Analyzer (Applied Biosystems Japan, Ltd.).
Protein extraction and western blotting CHO-K1 cells were washed twice with phosphate-buffered saline (PBS, pH 7.4; 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, and 1 mM KH2PO4), suspended in NP-40 lysis 810
buffer [50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5% [v/v] Nonidet P-40, 1 mM dithiothreitol, and protease inhibitor cocktail (Nacalai Tesque, Kyoto, Japan)], and centrifuged. The cleared lysates were stored at 30 C until SDSpolyacrylamide gel electrophoresis (SDS-PAGE) analysis. The protein concentration was determined with the Bradford method using bovine serum albumin as the standard (Bradford, 1976). Following SDS-PAGE (Laemmli. 1970), proteins were blotted onto a polyvinylidene difluoride membrane using a Trans-Blot Turbo Blotting System (Bio-Rad). The blotted membrane was washed with PBS containing 0.1% Tween (PBS-T) and blocked with Blocking One solution (Nacalai Tesque) for 1 h at room temperature. After washing with PBS-T, the membrane was probed using goat anti-CBR3 (Cterminal) polyclonal antibody (1:1000; Sigma–Aldrich) or mouse anti-b-actin monoclonal antibody (AC-15, 1:2000; Sigma–Aldrich) for 1 h at room temperature. Each membrane was then incubated for 1 h with an anti-goat (Sigma– Aldrich) or anti-mouse (GE Healthcare Co., Seattle, WA, USA) IgG polyclonal antibody (1:10,000) conjugated to horseradish peroxidase. Immunoreactive proteins were visualized with the enhanced chemiluminescence method using the ImmunoStar LD reagent (Wako, Kyoto, Japan), according to the manufacturer’s instructions.
Luciferase reporter gene assay CHO-K1 cells (5 104 cells) were seeded in 6-well plates and transfected 1 day later using TransIT LT1 transfection reagent (Mirus Bio, Madison, WI, USA) according to the manufacturer’s instructions. To normalize the transfection efficiency, the Renilla luciferase expression plasmid pRL-TK was co-transfected into the cells. The assay was performed following previously described methods (Nishinaka et al., 2011). Briefly, CHO-K1 cells were treated with CoCl2 after transfection and then harvested. Luciferase activity was measured using a dual-luciferase reporter assay system (Promega) and a Luminescensor PSN Luminometer (ATTO, Tokyo, Japan). Results
Characterization of the Chcr3 promoter region The nucleotide sequence of the promoter region of CHCR3 was determined with the genome walking method (Figures S1 and 1), and the transcriptional start site was determined with 50 -RACE (Figure 1). The proximal promoter region is TATA-less and GC-rich, similar to the promoter regions of human CBR3 (Zhang et al., 2009) and CBR1 (Miura et al., 2013), as well as CBR1 from Chinese hamsters (Miura et al., 2013). Cell Biol Int 39 (2015) 808–815 © 2015 International Federation for Cell Biology
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Figure 1 The nucleotide sequence of the promoter region of the Chcr3 gene. Boxes indicate the consensus sequences of transcription factor binding sites. The sites were found using the TFSEARCH (http://mbs.cbrc.jp/research/db/TFSEARCH.html) and TESS programs (http://www.cbil.upenn. edu/cgi-bin/tess/tess). Gray-colored sequences are the AREs investigated in this study. The transcription start site identified with 50 -RACE is indicated with each triangle. For the determination, five independent Escherichia coli clones were analysed. GR, glucocorticoid receptor; HIF-1, hypoxia-inducible factor-1; IRF, interferon regulatory factor; NF-E2, nuclear factor erythroid 2; XRE, xenobiotic response element; C/EBP, CCAAT/enhancer-binding protein.
The transcription factor Nrf2 is one of the key regulators of human CBR3 transcription. The protein binds to the antioxidant responsive element (ARE) at 2698 bp in the promoter region of human CBR3 (Cheng et al., 2012). Ebert et al. (2010) have suggested that it is unlikely that human Cell Biol Int 39 (2015) 808–815 © 2015 International Federation for Cell Biology
CBR3 transcription is regulated via aryl hydrocarbon receptor (AhR)-dependent pathways. The promoter region of Chcr3 contains potential sites for transcription factor binding, including three AREs and one xenobiotic response element (XRE) that contains the 811
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consensus sequence for AhR binding. To investigate the functionality of the XRE, CHO-K1 cells were treated for 48 h with 10 nM of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a ligand with a high affinity for AhR (Yoshioka et al., 2012). The results of real-time PCR analysis showed that TCDD did not alter Chcr3 gene expression (data not shown), suggesting that AhR does not regulate the transcription of the Chcr3 gene.
Effect of cobalt on Chcr3 gene expression Next, we investigated whether the three AREs were functional by treating cells with cobalt, an activator of Nrf2 in CHO cells (Gong et al., 2001). To analyze the effect of cobalt on Chcr3 gene expression, Chcr3 mRNA levels were measured with real-time PCR (Figure 2A). Treatment of CHO-K1 cells with cobalt for 24 h increased Chcr3 expression approximately 1.7-fold. Western blotting showed a 1.8-fold induction of CHCR3 protein after cobalt treatment (Figure 2B,C). The increase in CHCR3 mRNA was similar to the increase in CHCR3 protein, suggesting that the stimulatory effect of cobalt was due to transcriptional enhancement of gene expression.
Cobalt enhances the transcriptional activity of the Chcr3 gene To investigate the effect of cobalt on the transcriptional activity of the Chcr3 gene, a luciferase gene reporter assay was carried out using the promoter region (1605 bp, determined in this study) of the Chcr3 gene. The results showed that cobalt enhanced the transcriptional activity of the Chcr3 gene (Figure 3A, pGL3-CHCR3), indicating that the cobalt-induced increase in CHCR3 mRNA and protein was due to transcriptional activation. The results prompted us to identify the functional element(s) in the promoter region that mediated transcriptional activation by cobalt. The Chcr3 promoter region contains three AREs that might be stimulated by cobalt (Figure S2). Mutations were introduced into each element, and the transcriptional activity was analysed with luciferase gene reporter assays (Figure 3A). Only the ARE2 mutant was insensitive to cobalt. The results suggest that cobalt enhances the transcriptional activity of Chcr3 via ARE2.
Nrf2 upregulates Chcr3 gene expression Because cobalt stimulates the transcription factor Nrf2 in CHO cells (Gong et al., 2001), the role of Nrf2 in the upregulation of CHCR3 expression by cobalt was examined. In luciferase gene reporter assays, forced expression of Nrf2 enhanced the transcriptional activity of the Chcr3 gene (Figure 3B). Luciferase gene reporter assays with Chcr3 812
Figure 2 Increase in endogenous CHCR3 at the protein and mRNA levels. (A) CHO-K1 cells were treated with 100 mM CoCl2 or 100 mM MgCl2 (control) for 24 h. After treatment, RNA was extracted and reverse transcribed. Values relative to the intensity in the control sample are presented (mean S.D.; n ¼ 3) and statistically analyzed with Student’s ttest. *P < 0.05. (B) CHCR3 protein was detected by western blotting (n ¼ 3). (C) Densitometric analyses using ImageJ showed that CBR1 protein levels (mean S.D.) were higher in CoCl2-treated cells (1.87 0.35) than in control cells (1.0 0.29). Values relative to the intensity in the control sample are presented and statistically analysed with Student’s t-test. *P < 0.05.
promoters carrying a mutation in each ARE were also performed. As in the experiments with cobalt treatment (Figure 3A), enhanced transcriptional activity after overexpression of Nrf2 was not detected when ARE2 was Cell Biol Int 39 (2015) 808–815 © 2015 International Federation for Cell Biology
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mutated (Figure 3B). Furthermore, dominant negative Nrf2 suppressed the induction by forced expression of Nrf2 (Figure 3C). These results suggest that cobalt stimulates Nrf2 and that the transcription factor enhances the
A functional ARE in the promoter of Chcr3
transcriptional activity of Chcr3 via ARE2. Luciferase gene reporter assays were also performed in HEK293 cells, and similar results were obtained (data not shown). Discussion In the present study, we determined the nucleotide sequence of the promoter region of the Chcr3 gene, which encodes a CBR3 enzyme in Chinese hamsters. Chcr3 was transcriptionally upregulated after treatment with cobalt (Figure 2), and ARE2 was necessary for the increase in gene expression (Figure 3). Cobalt is an efficient stimulator of Nrf2 in CHO cells (Gong et al., 2001). Forced expression of Nrf2 enhanced the transcriptional activity of Chcr3 via ARE2 (Figure 3B). These results suggest that ARE2 is a novel functional element in the Chcr3 promoter and is dependent on Nrf2 activity. Recent clinical study revealed that a Nrf2 agonist improve chronic kidney disease, and the drugs have been developed (Choi et al., 2014). The results in the present study imply that anthracyclines chemotherapy in combination with such agonists modulate the probability of the cardiotoxicity onset, because anthracyclines metabolite by CBR3 has been believed to induce a cardiotoxicity (Blanco et al., 2008, 2012; Fan et al., 2008; Lal et al., 2008; Volkan-Salanci et al., 2012). Cobalt acts as a hypoxia-mimetic reagent by activating hypoxia-inducible factor (HIF) (Triantafyllou et al., 2006), and the effect was also found in CHO cells (Gong et al., 2001). However, cobalt was used to stimulate Nrf2 in the present study. The induction of the Chcr3 gene by cobalt required ARE2, as shown in luciferase gene reporter assays. The nucleotide sequence near ARE2 does not include the consensus sequence for HIF-1 binding (RCGTG) (Benita et al., 2009). This suggests that cobalt enhanced the transcriptional activity of Chcr3 mainly via the activation of Nrf2, although the authors can not rule out the possibility
Figure 3 (A) Increase in the transcriptional activity of the Chcr3 gene after treatment with CoCl2 and co-transfection of the Nrf2 expression vector (pNrf2). Luciferase expression vectors and a control vector (pRL-TK) were co-transfected into CHO-K1 cells. After incubation for 24 h, the cells were treated with 100 mM CoCl2 or 100 mM MgCl2 (control) for 24 h. Values relative to the intensity in the control sample without additional treatment are presented (mean S.D.; n ¼ 3) and statistically analysed with Student’s t-test. *P < 0.05. (B) The effect of forced expression of Nrf2 decreased with co-expression of dominantnegative Nrf2 expression vector (pNrf2-DN). Values relative to the intensity in the control sample without additional treatment are presented (mean S.D.; n ¼ 3) and statistically analysed with Dunnett’s test. *P < 0.05. (C) The effect of forced expression of Nrf2 decreased with co-expression of dominant-negative Nrf2 expression vector (pNrf2-DN). Values relative to the intensity in the control sample without additional treatment are presented (mean S.D.; n ¼ 3) and statistically analysed with Dunnett’s test. *P < 0.05.
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that there is some effect of HIF-1 for affecting the regulation through cross-talk pathway, like a physical interaction between Nrf2 and HIF-1. Nrf2 is a transcriptional regulator of human CBR3 gene expression. The upstream region of human CBR3 contains a distal ARE (2698 bp) (Cheng et al., 2012). By contrast, ARE2 is located proximal to the Chcr3 gene in Chinese hamsters. On the other hand, conserved AREs are located at proximal sites in the promoters of human and Chinese hamster CBR1 (Miura et al., 2013). Because Nrf2 is a transacting factor, the location of the ARE within the promoter might not affect the transcriptional activity of Nrf2. However, the methylation status should affect Nrf2 transcriptional regulation. Different CBR3 expression patterns in humans and Chinese hamsters have been reported. The expression of CHCR3 is higher than that of human CBR3 in most tissues (Miura et al., 2009b). The same methylation pattern in the promoters of human and Chinese hamster CBR3 has different effects on the Nrf2 binding sites in each species. Therefore, variations in the location of the AREs might explain the different levels of expression in humans and Chinese hamsters, although the involvement of Nrf2 with the basal promoter activity of Chcr3 requires further elucidation. In conclusion, the promoter sequence of the Chcr3 gene was identified, and a novel functional site for transcriptional regulation was found. The transcriptional element is conserved in the human CBR3 and Chinese hamster Chcr3 genes, although the location within the promoters differs. The difference may explain the different levels of CBR3/CHCR3 expression in humans and Chinese hamsters. Acknowledgements and funding The authors thank Ms. Y. Sugihara and Ms. K. Nakamura for their participation in the initial stage of the study. This work was supported in part by a grant-in-aid for young scientists (B) from the Ministry of Education, Culture, Sports, Science and Technology, Japan (TM). References Benita Y, Kikuchi H, Smith AD, Zhang MQ, Chung DC, Xavier RJ (2009) An integrative genomics approach identifies hypoxia inducible factor-1 (HIF-1)-target genes that form the core response to hypoxia. Nucleic Acids Res 37:4587–602. Blanco JG, Sun CL, Landier W, Chen L, Esparza-Duran D, Leisenring W, Mays A, Friedman DL, Ginsberg JP, Hudson MM, Neglia JP, Oeffinger KC, Ritchey AK, Villaluna D, Relling MV, Bhatia S (2012) Anthracycline-related cardiomyopathy after childhood cancer: Role of polymorphisms in carbonyl reductase genes—a report from the Children’s Oncology Group. J. Clin Oncol 30:1415–21.
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Nishinaka T, Miura T, Okumura M, Nakao F, Nakamura H, Terada T (2011) Regulation of aldo-keto reductase AKR1B10 gene expression: involvement of transcription factor Nrf2. Chem Biol Interact 191:185–91. Nishinaka T, Ichijo Y, Ito M, Kimura M, Katsuyama M, Iwata K, Miura T, Terada T, Yabe-Nishimura C (2007) Curcumin activates human glutathione S-transferase P1 expression through antioxidant response element. Toxicol Lett 170: 238–47. Nishinaka T, Yabe-Nishimura C (2005) Transcription factor Nrf2 regulates promoter activity of mouse aldose reductase (AKR1B3) gene. J Pharmacol Sci 97:43–51. Malátková P, Ebert B, Ws ol V, Maser E (2012) Expression of human carbonyl reductase 3 (CBR3; SDR21C2) is inducible by pro-inflammatory stimuli. Biochem Biophys Res Commun 420:368–73. Malátkoivá P, Maser E, Ws ol V (2010) Human carbonyl reductases. Curr Drug Metab 11:639–58. Matsunaga T, Shintani S, Hara A (2006) Multipulicity of mammalian reductases for xenobiotic carbonyl compounds. Drug Metab Pharmacokinet 21:1–18. Miura T, Taketomi A, Nishinaka T, Terada T (2013) Regulation of human carbonyl reductase 1 (CBR1, SDR21C1) gene by transcription factor Nrf2. Chem Biol Interact 202:126–35. Miura T, Nishinaka T, Terada T (2009a) Importance of the substrate-binding loop region of human monomeric carbonyl reductases in catalysis and coenzyme binding. Life Sci 85: 303–8. Miura T, Nishinaka T, Takama M, Murakami M, Terada T (2009b) Chinese hamster monomeric carbonyl reductases of the short-chain dehydrogenase/reductase superfamily. Chem Biol Interact 178:110–6. Miura T, Nishinaka T, Terada T (2008) Different functions between human monomeric carbonyl reductase 3 and carbonyl reductase 1. Mol. Cell Biochem 315:113–21. Ohkura-Hada S, Kondoh N, Hada A, Arai M, Yamazaki Y, Shindoh M, Kitagawa Y, Takahashi M, Ando T, Sato Y, Yamamoto M (2008) Carbonyl reductase 3 (CBR3) mediates 9-cis-retinoic acid-induced cytostatis and is a potential prognostic marker for oral malignancy. Open Dent J 2: 78–8. Pilka ES, Niesen FH, Lee WH, El-Hawari Y, Dunford JE, Kochan G, Wsol V, Martin HJ, Maser E, Oppermann U (2009) Structural basis for substrate specificity in human monomeric carbonyl reductases. PLoS ONE 4:e7113. Terada T, Sugihara Y, Nakamura K, Mizobuchi H, Maeda M (2003) Further characterization of Chinese hamster carbonyl reductases (CHCRs). Chem Biol Interact 143:373–81. Terada T, Sugihara Y, Nakamura K, Sato R, Sakuma S, Fujimoto Y, Fujita T, Inazu N, Maeda M (2001) Characterization of multiple Chinese hamster carbonyl reductases. Chem Biol Interact 130:847–61.
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Triantafyllou S, Liakos P, Tsakalof A, Georgatsou E, Simos G, Bonanou S (2006) Cobalt induces hypoxia-inducible factor-1a (HIF-1a) in HeLa cells by an iron-independent, but ROS-, PI-3Kand MAPK-dependent mechanism. Free Radic Res 40:847–56. Watanabe K, Sugawara C, Ono A, Fukumizu Y, Itakura S, Yamazaki M, Tashiro H, Osoegawa K, Soeda E, Nomura T (1998) Mapping of a novel human carbonyl reductase, CBR3 , and ribosomal pseudogenes to human chromosome 21q22.2. Genomics 51:95–100. Volkan-Salanci B, Aksoy H, Kiratli PO, Tülümen E, Güler N, Öksüzoglu B, Tokgözoglu L, Erbaş B, Alikaşifoglu M (2012) The relationship between changes in functional cardiac parameters following anthracycline therapy and carbonyl reductase 3 and glutathione S transferase Pi polymorphisms. J Chemother 24:285–91. Wermuth B, Bohren KM, Heinemann G, von Wartburg JP, Gabbay KH (1998) Human carbonyl reductase. Nucleotide sequence analysis of a cDNA and amino acid sequence of the encoded protein. J Biol Chem 263:16185–8. Yoshioka H, Hiromori Y, Aoki A, Kimura T, Fujii-Kuriyama Y, Nagase H, Nakanishi T (2012) Possible aryl hydrocarbon receptor-independent pathway of 2,3,7,8-tetrachlorodibenzop-dioxin-induced antiproliferative response in human breast cancer cells. Toxicol Lett 211:257–65. Zhang J, Blanco JG (2009) Identification of the promoter of human carbonyl reductase 3 (CBR3) and impact of common promoter polymorphisms on hepatic CBR3 mRNA expression. Pharmacol Res 26:2209–15. Received 16 August 2014; revised 22 January 2015; accepted 24 January 2015. Final version published online 23 February 2015.
Supporting Information Additional Supporting Information may be found in the online version of this article at the publisher’s web-site. Figure S1. Overview of genome walking for determining the promoter region of the Chcr3 gene. The genomic DNA fragment digested by BamHI was ligated to a Sau3AI cassette, and PCR was performed using the ligated DNA fragment as template. The nucleotide sequences of YSCRr12 and YSCRr14 were designed based on the Chcr3 gene sequence, and those of YSCRr19 and YSCRr20 were based on the sequence of the promoter region determined in the first genome walking experiment. Figure S2. ARE sequences in the promoter region of Chcr3 are shown. Functional AREs previously reported are also shown (Nishinaka et al. 2005, 2007; Cheng et al. 2012; Miura et al. 2013). The underlined “T“ in each ARE was mutated to “G“ in the mutation analyses.
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RESEARCH ARTICLE
Identification of a functional antioxidant responsive element in the promoter of the Chinese hamster carbonyl reductase 3 (Chcr3) gene Takeshi Miura1,2*, Ayako Taketomi1, Toshikatsu Nakabayashi2,3, Toru Nishinaka1 and Tomoyuki Terada1 1 Laboratory of Biochemistry, Faculty of Pharmacy, Osaka Ohtani University, 3–11-1 Nishikiori-kita, Tondabayashi, Osaka 584–8540, Japan 2 Pharmaceutical Education Support Center, School of Pharmacy and Pharmaceutical Sciences, Mukogawa Women’s University, 11–68 Koshien, 9-Bancho, Nishinomiya, Hyogo 663–8179, Japan 3 First Department of Biochemistry, School of Pharmacy and Pharmaceutical Sciences, Mukogawa Women’s University, 11–68 Koshien, 9-Bancho, Nishinomiya, Hyogo 663–8179, Japan
Abstract CHCR3, a member of the short-chain dehydrogenase/reductase superfamily, is a carbonyl reductase 3 enzyme in Chinese hamsters. Carbonyl reductase 3 in humans has been believed to involve the metabolism and/or pharmacokinetics of anthracycline drugs, and the mechanism underlying the gene regulation has been investigated. In this study, the nucleotide sequence of the Chcr3 promoter was originally determined, and its promoter activity was characterised. The proximal promoter region is TATA-less and GC-rich, similar to the promoter region of human carbonyl reductase 3. Cobalt stimulated the transcriptional activity of the Chcr3 gene. The results of a luciferase gene reporter assay demonstrated that cobalt-induced stimulation required an antioxidant responsive element. Forced expression of Nrf2, the transcription factor that binds to antioxidant responsive elements, enhanced the transcriptional activity of the Chcr3 gene. These results suggest that cobalt induces the expression of the Chcr3 gene via the Nrf2-antioxidant responsive element pathway. Keywords: carbonyl reductase; Chinese hamster; genome walk; SDR21C2; short-chain dehydrogenase/reductase Introduction The short-chain dehydrogenase/reductase (SDR) superfamily is a large family of enzymes that are catalytically active against endogenous and xenobiotic compounds, including steroids, prostaglandins, and anti-cancer drugs (Matsunaga et al., 2006; Malátkoivá et al., 2010). The SDR superfamily includes two monomeric carbonyl-reducing enzymes, carbonyl reductase 1 (CBR1) (Wermuth et al., 1998) and carbonyl reductase 3 (CBR3) (Watanabe et al., 1998). In humans, these enzymes are homologous at the nucleotide and amino acid sequence levels (77% and 72%, respectively). They reduce endogenous and xenobiotic carbonyl compounds using NADPH as a coenzyme (Miura et al., 2008). Despite their amino acid sequence similarity, the physiological roles of CBR1 and CBR3 are different, as evidenced by in vitro enzymatic analyses (Miura et al., 2008, 2009a; El-Hawari et al., 2009; Pilka et al., 2009). The CBR1 and CBR3 enzymes metabolize anthracycline anti-cancer drugs such as daunorubicin and doxorubicin.
The existence of a genetic variant of the CBR3 gene affects the cardiotoxicity and pharmacokinetics of the drugs, and the enzymatic activity of CBR3 may relate to the development of the cardiotoxicity (Blanco et al., 2008, 2012; Fan et al., 2008; Lal et al., 2008; Volkan-Salanci et al., 2012). The CBR3 gene was first identified by Watanabe et al. (1998); it lies near the CBR1 gene in the human genome. The enzymatic characteristics of human CBR3 were described in 2008 (Miura et al., 2008). CBR3 is involved not only in the metabolism of anthracyclines, but also in some physiological regulations. Treatment with 9-cis-retinoic acid induces CBR3 gene expression, and the induced enzyme suppresses cell growth and migration potential in oral squamous cell carcinomas (Ohkura-Hada et al., 2008). Furthermore, increased expression of the CBR3 gene in human adipose tissues is negatively associated with fasting insulin and systemic insulin resistance, suggesting that CBR3 modulates the diabetic state (Chang et al., 2012). These findings indicate that regulation of CBR3 gene expression is related to physiological functions. Therefore, it is important to
Corresponding author: e-mail: [email protected] or [email protected]
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clarify the physiological roles of CBR3 and elucidate the mechanism underlying the regulation of CBR3 gene expression. We originally identified and characterised a Chinese hamster ortholog of CBR3, named CHCR3 (Terada et al., 2001, 2003; Miura et al., 2009b). The transcriptional regulation of Chcr3 remains unclear, although the human CBR3 gene is regulated by the transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2) (Cheng et al., 2012; Ebert et al., 2010) and pro-inflammatory stimuli (Malátková et al., 2012). In the present study, we determined the nucleotide sequence of the Chcr3 promoter region and investigated whether Nrf2 regulated Chcr3 gene expression. Materials and methods
Materials Restriction enzymes were obtained from New England BioLabs (Beverly, MA, USA). The pGL3-basic vector and pRL-TK vector were from Promega (Madison, WI, USA). The PCR primers were purchased from Invitrogen (Carlsbad, CA, USA). All the other reagents were of the highest grade commercially available.
Genomic DNA extraction from Chinese hamster livers Genomic DNA was extracted from the livers of Chinese hamsters. The liver was placed in 500 mL of DNA extract buffer (50 mM Tris-HCl, pH 8.0, 100 mM EDTA, pH 8.0, 100 mM NaCl, and 1% SDS) with 20 mL of 20 mg/mL proteinase K and was rotated overnight at 55 C, after which 25 mL of 1 mg/mL RNase A was added. After 1 h of incubation, the solution was mixed with 500 mL of phenol. The aqueous phase was added and extracted twice with 500 mL of phenol/chloroform/isoamyl alcohol (25:24:1 [v/v]). Genomic DNA was isolated by adding 500 mL of isopropanol.
Cell culture HEK293 cells and Chinese hamster ovary (CHO)-K1 cells were maintained in Dulbecco’s modified Eagle’s medium (Nissui Pharmaceutical Co. Ltd., Tokyo, Japan) and Ham’s F-12 medium (N6658; Sigma–Aldrich, St. Louis, MO, USA), respectively, supplemented with 10% fetal bovine serum (BioWest, Nuaille, France) at 37 C under an atmosphere of 95% air and 5% CO2.
Plasmids To construct the luciferase gene reporter vector pGL3CHCR3–1663, which contained the promoter region (from Cell Biol Int 39 (2015) 808–815 © 2015 International Federation for Cell Biology
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1663 bp to 89 bp) of the Chcr3 gene, polymerase chain reaction (PCR) was performed using YSCR11f (50 -act gct ccc tgg agt tct gtt ca-30 ) and YSCRr25 (50 -ggg acc cgg cgc tgc aac tgc tgg ag-30 ) as primers and Chinese hamster genomic DNA as template. The amplified DNA fragment was digested with XhoI and HindIII and inserted into the pGL3-basic vector. The primers were designed based on the nucleotide sequence determined with the genome walking method. To generate Chcr3 promoter mutants, site-directed mutagenesis was performed according to the method of Higuchi et al. (1988). The primers were as follows: CHCR3ARE1mtf01 (50 -agc cgg atc cct act ccg ca-30 ) and CHCR3ARE1mtr01 (50 -tgc gga gta ggg atc cgg ct-30 ) for the mutation of ARE1, CHCR3ARE2mtf01 (50 -agc tga gtc ccc gtg agg gc-30 ) and CHCR3ARE2mtr01 (50 -gcc ctc acg ggg act cag ct-30 ) for the mutation of ARE2, and CHCR3ARE3mtf01 (50 -act gcg gaa cca gct cca cg-30 ) and CHCR3ARE3mtr01 (50 -cgt gga gct ggt tcc gca gt-30 ) for the mutation of ARE3. The nucleotide sequences of the PCR products were confirmed using an ABI PRISM 310 Genetic Analyzer (Applied Biosystems Japan, Ltd., Tokyo, Japan). The Nrf2 expression vector (pNrf2) was a gift from Dr. Cecil B. Picket (Schering-Plough Research Institute, Kenilworth, NJ, USA) (Nguyen et al., 2000). The dominant-negative Nrf2 expression vector (pNrf2-DN) was prepared by the method described previously (Nishinaka et al., 2005).
Genome walking To determine the nucleotide sequence of the 50 -upstream region of Chcr3, genome walking was performed. A summary of the experiments is shown in Supporting Information, Figure S1. Genomic DNA was digested with BamHI. The digested DNA fragment was ligated to a Sau3AI cassette (double-stranded DNA; 50 -gta cat att gtc gtt aga acg cgt aat acg cgt aat acg act cac tat agg ga-30 and 50 -gat ctc cct ata gtg agt cgt att acg cgt tct aac gac aat atg tac-30 ). Using the ligated DNA fragment as template, PCR was performed with a cassette 1 primer (50 -gta cat att gtc gtt aga acg cg-30 ) and a CHCR3specific primer (YSCRr12, 50 -tgg tcc gcg tgt ccc tc-30 ). Nested PCR with a cassette 2 primer (50 -taa tac gac tca cta tag gga ga30 ) and a CHCR3-specific nested primer (YSCRr14, 50 -agc cgg ctc tgc aag cc-30 ) was performed using the solutions from the first PCR as a template. Amplified DNA fragments were cloned into the pGEM-T Easy vector (Promega), and the nucleotide sequences were confirmed using an ABI PRISM 310 Genetic Analyzer (Applied Biosystems Japan, Ltd.). The length of the DNA fragment was 770 bp. To obtain further nucleotide sequence of the 50 -upstream region of the gene, two primers (YSCRr19, 50 -ggg caa aac cag acg ct-30 and YSCRr20, 50 -cta tgt aca tac tcg ggt ac-30 ) were designed based on the nucleotide sequence determined from the first genome walking experiment. A second genome 809
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walking experiment was carried out in the same way using the primers. The length of the DNA fragment was 905 bp.
50 -rapid amplification of cDNA ends (RACE) Using total RNA from CHO-K1 cells, 50 -RACE was performed with the 50 -RACE System for Rapid Amplification of cDNA Ends (Gibco, Tokyo, Japan). Total RNA prepared from CHO-K1 cells was reverse transcribed with an RT primer (YSCRr21, 50 -tag aaa tgt ctg caa tg-30 ) using reverse transcriptase. The cDNA was purified, and a poly-dA tail was added using the terminal transferase enzyme. The dA-tailed cDNA was amplified by PCR with an oligo dTanchor primer and gene-specific primer (GSP) 1 (YSCRr04, 50 -cgt gtg gat ccg ata tgt cgt cct g-30 ) for the first PCR, with a dT-anchor primer and GSP 2 (YSCRr12, 50 -tgg tcc gcg tgt ccc tc-30 ) for the second PCR, and with a dT-anchor primer and GSP 3 (YSCRr14, 50 -agc cgg ctc tgc aag cc-30 ) for the third PCR. The resulting DNA fragments were cloned and sequenced using an ABI PRISM 310 Genetic Analyzer (Applied Biosystems Japan, Ltd.).
Quantification of Chcr3 gene expression CHO-K1 cells were treated with or without CoCl2, and total RNA was prepared using a ReliaPrep RNA Cell Miniprep System (Promega) according to the manufacturer’s instructions. Using a ReverTra Ace qPCR RT kit (Toyobo, Osaka, Japan), cDNA was synthesized from 2 mg of total RNA. The primers used for the detection of Chcr3 gene expression were 50 -acc agg gct cca gga ctg tg-30 (forward) and 50 -cct cca ttt gga tcc ctc tc-30 (reverse). To normalize the quantities of cDNA, expression of the b-actin gene was also measured using the primers 50 -tgg cat cca cga aac tac at-30 (forward) and 50 -tgg tac cac cag aca gca ct-30 (reverse) (Han et al., 2012). Real-time PCR was performed using SYBR Green Realtime PCR Master Mix -Plus- v.2 (Toyobo) with an Opticon 2 real-time PCR apparatus (Bio-Rad Laboratories, Inc., Tokyo, Japan). Real-time PCR standard curves were calculated using cDNA from CHO-K1 cells as a template. The PCR program was as follows for the Chcr3 and b-actin genes: hot start, 96 C, 60 s; denature, 95 C, 15 s; annealing, 62 C, 15 s; and extension, 72 C, 60 s. The analyses of the melting curves revealed specific amplification of the Chcr3 and b-actin genes. The nucleotide sequences of the PCR products were confirmed using an ABI PRISM 310 Genetic Analyzer (Applied Biosystems Japan, Ltd.).
Protein extraction and western blotting CHO-K1 cells were washed twice with phosphate-buffered saline (PBS, pH 7.4; 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, and 1 mM KH2PO4), suspended in NP-40 lysis 810
buffer [50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5% [v/v] Nonidet P-40, 1 mM dithiothreitol, and protease inhibitor cocktail (Nacalai Tesque, Kyoto, Japan)], and centrifuged. The cleared lysates were stored at 30 C until SDSpolyacrylamide gel electrophoresis (SDS-PAGE) analysis. The protein concentration was determined with the Bradford method using bovine serum albumin as the standard (Bradford, 1976). Following SDS-PAGE (Laemmli. 1970), proteins were blotted onto a polyvinylidene difluoride membrane using a Trans-Blot Turbo Blotting System (Bio-Rad). The blotted membrane was washed with PBS containing 0.1% Tween (PBS-T) and blocked with Blocking One solution (Nacalai Tesque) for 1 h at room temperature. After washing with PBS-T, the membrane was probed using goat anti-CBR3 (Cterminal) polyclonal antibody (1:1000; Sigma–Aldrich) or mouse anti-b-actin monoclonal antibody (AC-15, 1:2000; Sigma–Aldrich) for 1 h at room temperature. Each membrane was then incubated for 1 h with an anti-goat (Sigma– Aldrich) or anti-mouse (GE Healthcare Co., Seattle, WA, USA) IgG polyclonal antibody (1:10,000) conjugated to horseradish peroxidase. Immunoreactive proteins were visualized with the enhanced chemiluminescence method using the ImmunoStar LD reagent (Wako, Kyoto, Japan), according to the manufacturer’s instructions.
Luciferase reporter gene assay CHO-K1 cells (5 104 cells) were seeded in 6-well plates and transfected 1 day later using TransIT LT1 transfection reagent (Mirus Bio, Madison, WI, USA) according to the manufacturer’s instructions. To normalize the transfection efficiency, the Renilla luciferase expression plasmid pRL-TK was co-transfected into the cells. The assay was performed following previously described methods (Nishinaka et al., 2011). Briefly, CHO-K1 cells were treated with CoCl2 after transfection and then harvested. Luciferase activity was measured using a dual-luciferase reporter assay system (Promega) and a Luminescensor PSN Luminometer (ATTO, Tokyo, Japan). Results
Characterization of the Chcr3 promoter region The nucleotide sequence of the promoter region of CHCR3 was determined with the genome walking method (Figures S1 and 1), and the transcriptional start site was determined with 50 -RACE (Figure 1). The proximal promoter region is TATA-less and GC-rich, similar to the promoter regions of human CBR3 (Zhang et al., 2009) and CBR1 (Miura et al., 2013), as well as CBR1 from Chinese hamsters (Miura et al., 2013). Cell Biol Int 39 (2015) 808–815 © 2015 International Federation for Cell Biology
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Figure 1 The nucleotide sequence of the promoter region of the Chcr3 gene. Boxes indicate the consensus sequences of transcription factor binding sites. The sites were found using the TFSEARCH (http://mbs.cbrc.jp/research/db/TFSEARCH.html) and TESS programs (http://www.cbil.upenn. edu/cgi-bin/tess/tess). Gray-colored sequences are the AREs investigated in this study. The transcription start site identified with 50 -RACE is indicated with each triangle. For the determination, five independent Escherichia coli clones were analysed. GR, glucocorticoid receptor; HIF-1, hypoxia-inducible factor-1; IRF, interferon regulatory factor; NF-E2, nuclear factor erythroid 2; XRE, xenobiotic response element; C/EBP, CCAAT/enhancer-binding protein.
The transcription factor Nrf2 is one of the key regulators of human CBR3 transcription. The protein binds to the antioxidant responsive element (ARE) at 2698 bp in the promoter region of human CBR3 (Cheng et al., 2012). Ebert et al. (2010) have suggested that it is unlikely that human Cell Biol Int 39 (2015) 808–815 © 2015 International Federation for Cell Biology
CBR3 transcription is regulated via aryl hydrocarbon receptor (AhR)-dependent pathways. The promoter region of Chcr3 contains potential sites for transcription factor binding, including three AREs and one xenobiotic response element (XRE) that contains the 811
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consensus sequence for AhR binding. To investigate the functionality of the XRE, CHO-K1 cells were treated for 48 h with 10 nM of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a ligand with a high affinity for AhR (Yoshioka et al., 2012). The results of real-time PCR analysis showed that TCDD did not alter Chcr3 gene expression (data not shown), suggesting that AhR does not regulate the transcription of the Chcr3 gene.
Effect of cobalt on Chcr3 gene expression Next, we investigated whether the three AREs were functional by treating cells with cobalt, an activator of Nrf2 in CHO cells (Gong et al., 2001). To analyze the effect of cobalt on Chcr3 gene expression, Chcr3 mRNA levels were measured with real-time PCR (Figure 2A). Treatment of CHO-K1 cells with cobalt for 24 h increased Chcr3 expression approximately 1.7-fold. Western blotting showed a 1.8-fold induction of CHCR3 protein after cobalt treatment (Figure 2B,C). The increase in CHCR3 mRNA was similar to the increase in CHCR3 protein, suggesting that the stimulatory effect of cobalt was due to transcriptional enhancement of gene expression.
Cobalt enhances the transcriptional activity of the Chcr3 gene To investigate the effect of cobalt on the transcriptional activity of the Chcr3 gene, a luciferase gene reporter assay was carried out using the promoter region (1605 bp, determined in this study) of the Chcr3 gene. The results showed that cobalt enhanced the transcriptional activity of the Chcr3 gene (Figure 3A, pGL3-CHCR3), indicating that the cobalt-induced increase in CHCR3 mRNA and protein was due to transcriptional activation. The results prompted us to identify the functional element(s) in the promoter region that mediated transcriptional activation by cobalt. The Chcr3 promoter region contains three AREs that might be stimulated by cobalt (Figure S2). Mutations were introduced into each element, and the transcriptional activity was analysed with luciferase gene reporter assays (Figure 3A). Only the ARE2 mutant was insensitive to cobalt. The results suggest that cobalt enhances the transcriptional activity of Chcr3 via ARE2.
Nrf2 upregulates Chcr3 gene expression Because cobalt stimulates the transcription factor Nrf2 in CHO cells (Gong et al., 2001), the role of Nrf2 in the upregulation of CHCR3 expression by cobalt was examined. In luciferase gene reporter assays, forced expression of Nrf2 enhanced the transcriptional activity of the Chcr3 gene (Figure 3B). Luciferase gene reporter assays with Chcr3 812
Figure 2 Increase in endogenous CHCR3 at the protein and mRNA levels. (A) CHO-K1 cells were treated with 100 mM CoCl2 or 100 mM MgCl2 (control) for 24 h. After treatment, RNA was extracted and reverse transcribed. Values relative to the intensity in the control sample are presented (mean S.D.; n ¼ 3) and statistically analyzed with Student’s ttest. *P < 0.05. (B) CHCR3 protein was detected by western blotting (n ¼ 3). (C) Densitometric analyses using ImageJ showed that CBR1 protein levels (mean S.D.) were higher in CoCl2-treated cells (1.87 0.35) than in control cells (1.0 0.29). Values relative to the intensity in the control sample are presented and statistically analysed with Student’s t-test. *P < 0.05.
promoters carrying a mutation in each ARE were also performed. As in the experiments with cobalt treatment (Figure 3A), enhanced transcriptional activity after overexpression of Nrf2 was not detected when ARE2 was Cell Biol Int 39 (2015) 808–815 © 2015 International Federation for Cell Biology
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mutated (Figure 3B). Furthermore, dominant negative Nrf2 suppressed the induction by forced expression of Nrf2 (Figure 3C). These results suggest that cobalt stimulates Nrf2 and that the transcription factor enhances the
A functional ARE in the promoter of Chcr3
transcriptional activity of Chcr3 via ARE2. Luciferase gene reporter assays were also performed in HEK293 cells, and similar results were obtained (data not shown). Discussion In the present study, we determined the nucleotide sequence of the promoter region of the Chcr3 gene, which encodes a CBR3 enzyme in Chinese hamsters. Chcr3 was transcriptionally upregulated after treatment with cobalt (Figure 2), and ARE2 was necessary for the increase in gene expression (Figure 3). Cobalt is an efficient stimulator of Nrf2 in CHO cells (Gong et al., 2001). Forced expression of Nrf2 enhanced the transcriptional activity of Chcr3 via ARE2 (Figure 3B). These results suggest that ARE2 is a novel functional element in the Chcr3 promoter and is dependent on Nrf2 activity. Recent clinical study revealed that a Nrf2 agonist improve chronic kidney disease, and the drugs have been developed (Choi et al., 2014). The results in the present study imply that anthracyclines chemotherapy in combination with such agonists modulate the probability of the cardiotoxicity onset, because anthracyclines metabolite by CBR3 has been believed to induce a cardiotoxicity (Blanco et al., 2008, 2012; Fan et al., 2008; Lal et al., 2008; Volkan-Salanci et al., 2012). Cobalt acts as a hypoxia-mimetic reagent by activating hypoxia-inducible factor (HIF) (Triantafyllou et al., 2006), and the effect was also found in CHO cells (Gong et al., 2001). However, cobalt was used to stimulate Nrf2 in the present study. The induction of the Chcr3 gene by cobalt required ARE2, as shown in luciferase gene reporter assays. The nucleotide sequence near ARE2 does not include the consensus sequence for HIF-1 binding (RCGTG) (Benita et al., 2009). This suggests that cobalt enhanced the transcriptional activity of Chcr3 mainly via the activation of Nrf2, although the authors can not rule out the possibility
Figure 3 (A) Increase in the transcriptional activity of the Chcr3 gene after treatment with CoCl2 and co-transfection of the Nrf2 expression vector (pNrf2). Luciferase expression vectors and a control vector (pRL-TK) were co-transfected into CHO-K1 cells. After incubation for 24 h, the cells were treated with 100 mM CoCl2 or 100 mM MgCl2 (control) for 24 h. Values relative to the intensity in the control sample without additional treatment are presented (mean S.D.; n ¼ 3) and statistically analysed with Student’s t-test. *P < 0.05. (B) The effect of forced expression of Nrf2 decreased with co-expression of dominantnegative Nrf2 expression vector (pNrf2-DN). Values relative to the intensity in the control sample without additional treatment are presented (mean S.D.; n ¼ 3) and statistically analysed with Dunnett’s test. *P < 0.05. (C) The effect of forced expression of Nrf2 decreased with co-expression of dominant-negative Nrf2 expression vector (pNrf2-DN). Values relative to the intensity in the control sample without additional treatment are presented (mean S.D.; n ¼ 3) and statistically analysed with Dunnett’s test. *P < 0.05.
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that there is some effect of HIF-1 for affecting the regulation through cross-talk pathway, like a physical interaction between Nrf2 and HIF-1. Nrf2 is a transcriptional regulator of human CBR3 gene expression. The upstream region of human CBR3 contains a distal ARE (2698 bp) (Cheng et al., 2012). By contrast, ARE2 is located proximal to the Chcr3 gene in Chinese hamsters. On the other hand, conserved AREs are located at proximal sites in the promoters of human and Chinese hamster CBR1 (Miura et al., 2013). Because Nrf2 is a transacting factor, the location of the ARE within the promoter might not affect the transcriptional activity of Nrf2. However, the methylation status should affect Nrf2 transcriptional regulation. Different CBR3 expression patterns in humans and Chinese hamsters have been reported. The expression of CHCR3 is higher than that of human CBR3 in most tissues (Miura et al., 2009b). The same methylation pattern in the promoters of human and Chinese hamster CBR3 has different effects on the Nrf2 binding sites in each species. Therefore, variations in the location of the AREs might explain the different levels of expression in humans and Chinese hamsters, although the involvement of Nrf2 with the basal promoter activity of Chcr3 requires further elucidation. In conclusion, the promoter sequence of the Chcr3 gene was identified, and a novel functional site for transcriptional regulation was found. The transcriptional element is conserved in the human CBR3 and Chinese hamster Chcr3 genes, although the location within the promoters differs. The difference may explain the different levels of CBR3/CHCR3 expression in humans and Chinese hamsters. Acknowledgements and funding The authors thank Ms. Y. Sugihara and Ms. K. Nakamura for their participation in the initial stage of the study. This work was supported in part by a grant-in-aid for young scientists (B) from the Ministry of Education, Culture, Sports, Science and Technology, Japan (TM). References Benita Y, Kikuchi H, Smith AD, Zhang MQ, Chung DC, Xavier RJ (2009) An integrative genomics approach identifies hypoxia inducible factor-1 (HIF-1)-target genes that form the core response to hypoxia. Nucleic Acids Res 37:4587–602. Blanco JG, Sun CL, Landier W, Chen L, Esparza-Duran D, Leisenring W, Mays A, Friedman DL, Ginsberg JP, Hudson MM, Neglia JP, Oeffinger KC, Ritchey AK, Villaluna D, Relling MV, Bhatia S (2012) Anthracycline-related cardiomyopathy after childhood cancer: Role of polymorphisms in carbonyl reductase genes—a report from the Children’s Oncology Group. J. Clin Oncol 30:1415–21.
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A functional ARE in the promoter of Chcr3
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Supporting Information Additional Supporting Information may be found in the online version of this article at the publisher’s web-site. Figure S1. Overview of genome walking for determining the promoter region of the Chcr3 gene. The genomic DNA fragment digested by BamHI was ligated to a Sau3AI cassette, and PCR was performed using the ligated DNA fragment as template. The nucleotide sequences of YSCRr12 and YSCRr14 were designed based on the Chcr3 gene sequence, and those of YSCRr19 and YSCRr20 were based on the sequence of the promoter region determined in the first genome walking experiment. Figure S2. ARE sequences in the promoter region of Chcr3 are shown. Functional AREs previously reported are also shown (Nishinaka et al. 2005, 2007; Cheng et al. 2012; Miura et al. 2013). The underlined “T“ in each ARE was mutated to “G“ in the mutation analyses.
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