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【关键词】 Functional
Human carbonyl reductase 1 (CBR1) metabolizes a variety of substrates, including the anticancer doxorubicin and the antipsychotic haloperidol. The transcriptional regulation of CBR1 has been largely unexplored. Therefore, we first investigated the promoter activities of progressive gene-reporter constructs encompassing up to 2.4 kilobases upstream of the translation start site of CBR1. Next, we investigated whether CBR1 mRNA levels were altered in cells incubated with prototypical receptor activators (e.g., dexamethasone and rifampicin). CBR1 mRNA levels were significantly induced (5-fold) by the ligand of the aryl hydrocarbon receptor (AHR) -naphthoflavone. DNA sequence analysis revealed two xenobiotic response elements (–122XRE and –5783XRE) with potential regulatory functions. CBR1 promoter constructs lacking the –122XRE showed diminished (9-fold) promoter activity in AHR-proficient cells incubated with -naphthoflavone. Fusion of –5783XRE to the –2485CBR1 reporter construct enhanced its promoter activity after incubations with -naphthoflavone by 5-fold. Furthermore, we tested whether the potent AHR ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induced Cbr1 expression in Ahr+/– and Ahr–/– mice. TCDD induced hepatic Cbr1 mRNA (TCDD, 2-fold) and Cbr1 protein levels (TCDD, 2-fold) in Ahr+/– mice compared with vehicle-injected controls. In contrast, no significant Cbr1 mRNA and Cbr1 protein induction was detected in livers from Ahr–/– mice treated with TCDD. These studies provide the first insights on the functional characteristics of the human CBR1 gene promoter. Our data indicate that the AHR pathway contributes to the transcriptional regulation of CBR1.
Human carbonyl reductase 1 (CBR1) catalyzes the NADPH-dependent reduction of a variety of xenobiotic compounds including smoke derived carcinogens and many relevant pharmacological agents. For example, CBR1 catalyzes the two-electron reduction of the C-13 carbonyl group of the anticancer anthracyclines doxorubicin and daunorubicin to generate their corresponding alcohol metabolites (doxorubicinol and daunorubicinol) (Forrest and Gonzalez, 2000). Anthracycline C-13 alcohol metabolites are cardiotoxic, have diminished tumor cell killing activities, circulate in plasma at various levels, and contribute to the unpredictable pharmacology of anthracycline drugs (Frost et al., 2002; Minotti et al., 2004). Significant interindividual variability in carbonyl reductase activity (CBR) has been documented in liver, erythrocytes, and in breast and lung tumors (Iwata et al., 1993; Wong et al., 1993; Rady-Pentek et al., 1997; Lopez de Cerain et al., 1999). We observed wide ranges of CBR activities in liver cytosols from black (range, 4.1–21.5 nmol/min · mg) and white donors (range, <0.1–28.0 nmol/min · mg) (Covarrubias et al., 2006). However, the molecular basis of such disparities and its potential impact on CBR-mediated drug metabolism remain to be elucidated. We hypothesize that interindividual differences in CBR activity may in part reflect variable rates of CBR1 gene transcription. CBR1 spans approximately 3.2 kb on chromosome 21 (21q22.13), contains three exons, and encodes for a monomeric 277 amino acid protein with a molecular weight of 30,375 (Wermuth et al., 1988). It is noteworthy that despite the major role of CBR1 in the biotransformation of xenobiotics, there is a paucity of reports focused on the functional characterization of the human CBR1 gene promoter. Therefore, the first aim of our study was to investigate the potential promoter activities of progressive DNA deletion constructs encompassing up to 2485 base pairs (bp) of genomic sequence 5' upstream the translation start site of CBR1 by using gene-reporter assays.
Our second aim was to test whether CBR1 mRNA levels were induced in cell cultures incubated with prototypical activators of the nuclear glucocorticoid receptor, the constitutive androstane receptor, the pregnane X receptor, and the aryl hydrocarbon receptor (AHR), respectively. We detected significant induction of CBR1 mRNA expression in HepG2 and MCF-7 cells treated with the AHR ligand -naphthoflavone. AHR is a ligand-activated basic helix-loop-helix transcription factor that participates in the regulation of several key mammalian genes involved in the metabolism of xenobiotics (e.g., CYP1A1 and CYP1B1). After ligand binding, AHR translocates from the cytoplasm into the nucleus to form a complex with aryl hydrocarbon receptor nuclear translocator. The resulting ligand/AHR/aryl hydrocarbon receptor nuclear translocator complex interacts with specific DNA sequences termed xenobiotic responsive elements (XREs) to induce the transcription of target genes (Nebert et al., 2000; Nioi and Hayes, 2004). The consensus XRE sequence (5'-T/GNGCGTG-3') contains the substitution intolerant XRE core motif (5'-GCGTG-3') (Lusska et al., 1993). It is interesting that we identified two perfect XRE motifs located at 122 bp and 5783 bp upstream the translation start site of CBR1 (–122XRE, and –5783XRE). Therefore, our third aim was to investigate the functional impact of the proximal (–122XRE), and distal (–5783XRE) XRE motifs by performing gene reporter assays with engineered CBR1 promoter constructs.
The development of Ahr-deficient mice (Ahr–/–) has contributed to the identification of a battery of genes regulated through the AHR pathway (Fernandez-Salguero et al., 1995; Zaher et al., 1998; Sugihara et al., 2001; Jiang et al., 2004). AHR mediates the induction of several key xenobiotic-metabolizing enzymes such as CYP1A1, CYP1B1, glutathione transferase, and NAD(P)H:quinone oxidoreductase (NQO1) (Nebert et al., 2000; Shimada et al., 2002). Thus, we extended our observations by testing whether the potent AHR ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induced Cbr1 mRNA and Cbr1 protein levels in livers from Ahr-proficient (Ahr+/–), and Ahr-deficient (Ahr–/–) mice. Together, our findings provide insights on the regulation of CBR1 and lay the foundation for future studies aimed toward the elucidation of the molecular bases that govern variable CBR activity in humans.
Cell Culture and Reagents. HepG2 (human hepatocarcinoma, HB-8065), and MCF-7 (human breast adenocarcinoma, HTB-22) cell lines were obtained from the American Type Culture Collection (Manassas, VA). Minimum essential medium, fetal bovine serum, and other cell culture reagents were purchased from Invitrogen (Carlsbad, CA). Cells were routinely cultured in 75-cm2 vented flasks using -minimum essential medium supplemented with 10% fetal bovine serum. Cultures were grown in an incubator at 37°C, 5% CO2, and 95% relative humidity. Cultures were maintained at low passage numbers (n <12) and were free of mycoplasma contamination.
Dexamethasone, clotrimazole, 1,4-bis-[2-(3,5-dichloropyridyloxy)]-benzene (TCPOBOP), and rifampicin were purchased from Sigma-Aldrich (St. Louis, MO). -Naphthoflavone was purchased from Indofine (Hillsborough, NJ).
Cloning of CBR1 Promoter Constructs. Approximately 5 kilobases of DNA sequence upstream from the translation start codon (ATG) of CBR1 were amplified from human DNA sample HD17030 (Coriell Institute for Medical Research, Camden, NJ) by using the Expand Long Template PCR system (Roche, Indianapolis, IN). PCR primers were 5'-CCCCTGACTGCCCTTTCTTA-3' (forward) and 5'-TCACCAGCGCTACATGGAT-3' (reverse). A derivative fragment of 2485 bp was cloned into a pGL3 basic luciferase vector (Promega, Fitchburg, WI) by using the following primers: 5'-GCTCTTACGCGTGCTAGCCCGAGCTCTGAATTATCCTGAGTGG-3' (forward), and 5'-CCGCGCGCCCCGTTCAGCCGAATTCATCTGCGATCTAAG-3 (reverse). Eight 5' progressive deletion constructs were made by PCR using primers listed below. The resulting products were cloned into pGL3 basic firefly luciferase reporter vectors. The identity of each construct and the absence of cloning artifacts were verified by direct sequencing with the dye-terminator method in a 3130XL Genetic Analyzer (Applied Biosystems, Foster City, CA).
The –122XRE substitution intolerant core (5'-GCGTG-3') was deleted from the –413CBR1 promoter construct using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) with the following primers: 5'-CCTGCGCGCTCAGCGGCCGGTAACCCACGGGTGCGCGCCC-3', and 5'-GGGCGCGCACCCGTGGGTTACCGGCCGCTGAGCGCGCAGG-3'. Deletion of –122XRE was confirmed by direct sequencing analysis.
A 12-bp sequence containing the distal –5783XRE element (5'-TTGCGTGCCTTG-3', bases –5790 to –5779) was added to the 5' end of the –2485CBR1 construct by using QuikChange with the following primers: 5'-CGCGTGCTAGCCTTGCGTGCCTTGGAGCTCTGAATTATCC-3', and 5'-GGATAATTCAGAGCTCCAAGGCACGCAAGGGCTAGCACGCG-3'. The addition of –5783XRE was verified by direct sequencing.
List of primers. The forward primers included –1847CBR1:5'-CCTAAATCTGTACTGCCCAATACGCGTACAGTGACCACTAACACATGC-3'; –1561CBR1:5'-GAGGGAGTCACTCTGTTGACGCGTCCCAGGCTGGAGTGCAG-3'; –1101CBR1:5'-CCAGACCCCTCACCTGCAACGCGTGCCTGATGCCTGTTGAC-3'; –746CBR1: 5'-GGTACATCCTAGAGTGTACGCGTTATTGTCCGTGTAAAATAGGG-3'; –600C- BR1: 5'-CTGGCTAAGTCAGTAGCACGCGTTTTGTTTTCATATACTTAGGGG-3'; –413CBR1: 5'-CACAACTAGGAATGAACGCGTTTGAACAGCTGGGAG-3'; –205CBR1:5'-GCTCCGCACCCCGGACGCGTGGTTCCGGTGG-3'; and –101CBR1:5'-GGGCGTGTAACCCACGCGTGCGCGCCCACG-3'; The reverse primer was RevCBR1:5'-CCGCGCGCCCCGTTCAGCCGAATTCATCTGCGATCTAAG-3'.
Transient Transfections and Luciferase Activity Assays. Cells were plated 24 to 48 h before transfections in 12-well plates. Reporter gene constructs (firefly luciferase) and the SV40-driven Renilla reniformis luciferase pRL-SV40 plasmid (Promega) were cotransfected into 60 to 70% confluent cell cultures by using FuGENE 6 (Roche). Twenty-four hours after cotransfection, cultures were washed once with phosphate-buffered saline solution, and the cells were lysed with passive lysis buffer (250 µl/well) (Promega). Cell lysates were incubated at room temperature (15 min), mixed with a vortex blender (10 s), and centrifuged at 4°C (1500 rpm for 30 s). Luciferase reporter gene activities were determined with the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer's instructions. Light intensity was measured in a Synergy HT luminometer equipped with proprietary software for data analysis (BioTek, Winooski, VT). Light intensity values from cell cultures transfected with the promoterless (pGL3) vector were used to correct for background. Corrected firefly luciferase activities were normalized to R. reniformis luciferase activities and expressed as fold increases with respect to the values obtained with pGL3-basic empty vector. In all cases, three to five independent experiments were performed in duplicate to evaluate reproducibility. Unpaired Student's t tests (two groups) and analysis of variance (ANOVA, three or more groups) were used to compare experimental means. In all cases, differences were considered to be significant at p < 0.05. Computations were performed with Microsoft Excel 2000 version 9.0 (Microsoft, Redmond, WA) and SigmaPlot version 8.02 (SPSS Inc., Chicago, IL).
Quantification of CBR1 mRNA in Cell Cultures by Real-Time RT-PCR. Cell cultures (70–80% confluence) were treated for 24 h with dexamethasone (10 µM), rifampicin (10 µM), -naphthoflavone (10–50 µM), clotrimazole (20 µM), TCPOBOP (0.250 µM), or vehicle (DMSO). Total RNA was extracted with RNeasy Mini kit (QIAGEN, Valencia, CA) according to the manufacturer's instructions. RNA was eluted with molecular biology-grade water, and stored at –80°C until use. RNA concentrations were measured by spectrophotometric analysis at 260 nm in a Shimadzu UV-1601 PC spectrophotometer (Shimadzu, Kyoto, Japan). Total RNA (100 ng) was reverse-transcribed and amplified by using one-step QuantiTect SYBR Green RT-PCR kits (QIAGEN). RT-PCR reaction mixtures were incubated on a MX4000 engine thermal cycler equipped with proprietary software for data analysis (Stratagene). The comparative quantitation method was used to determine relative CBR1 mRNA levels in drug-treated samples. Vehicle-treated samples were used as references, and individual -actin mRNA levels were used as normalizers (Blanquicett et al., 2002; Bustin, 2002). CBR1 primers were: 5'-CTGATCCCACACCCTTTCAT-3' (forward), and 5'-TTAAGGGCTCTGACGCTCAT-3' (reverse); -actin primers, 5'-ACGGCTCCGGCATGTGCAAG-3' (forward), and 5'-TGACGATGCCGTGCTCGATG-3' (reverse). Cycling parameters for the amplifications in parallel of CBR1 and -actin mRNAs were: 50°C for 30 min (reverse transcription), 95°C for 10 min (Taq polymerase activation); 40 cycles of 95°C for 15 s (denaturation), 51°C for 30 s (annealing), 72°C for 30 s (extension), and 78°C for 30 s (fluorescence collection). Standard curves for CBR1 and -actin mRNA (10-fold dynamic range) were run in parallel to ensure accurate mRNA quantifications. In all cases, the regression coefficients (r) of the standard curves were r 0.9. Amplification efficiencies for CBR1 and -actin mRNAs were similar and ranged between 125 and 175%. In all cases, experimental samples and standards for calibration curves were analyzed in quadruplicate.
Animals and Treatments. Ahr+/– and Ahr–/– mice were procured from the laboratory of Dr. Christopher Bradfield (University of Wisconsin). The Institutional Animal Care and Use Committee approved the experimental protocol. Animals were housed in a temperature- and humidity-controlled room under a light cycle with free access to food and water. Mice (aged 81 ± 14 days) were treated with intraperitoneal injections of TCDD (50 µg/kg; AccuStandard Inc., New Haven, CT), or corn oil vehicle (200 µl), respectively. Animals were sacrificed by CO2 inhalation. Livers were removed, snap-frozen in liquid nitrogen, and stored at –80°C until use.
Quantification of Hepatic Cbr1 mRNA by Real-Time RT-PCR. Liver RNA was extracted with RNeasy Mini kits (QIAGEN). RNA samples (100 ng) were subjected to one-step quantitative real-time RT-PCR using QuantiTect SYBR green RT-PCR kit (QIAGEN). Mouse Cbr1 primers were: 5'-ATCACTCGTGACCTGTGTCG-3' (forward), and 5'-GGTGTCGTCATTGACCTTGA-3'(reverse); -actin primers: 5'-GACCCAGATCATGTTTGAGACCTTC-3' (forward), and 5'-GGAGTCCATCACAATGCCAGTG-3' (reverse). Amplification conditions for murine Cbr1 and -actin mRNAs were: 50°C for 30 min (reverse transcription), 95°C for 10 min (Taq polymerase activation); 40 cycles of 95°C for 15 s (denaturation), 52°C for 30 s (annealing), 72°C for 30 s (extension), and 78°C for 30 s (fluorescence collection). Standard curves (10-fold dynamic range) for Cbr1 and -actin mRNA were run in parallel. Relative Cbr1 mRNA levels were calculated by using the comparative quantitation method as described above. Samples were analyzed in quadruplicate.
Detection of Hepatic Cbr1 by Immunoblotting. Fragments of frozen mouse liver were homogenized in three volumes of ice-cold lysis buffer (Promega). The homogenates were centrifuged at 13,000g for 20 min at 4°C. The resulting supernatants (100 µg) were separated on 4 to 20% precast polyacrylamide gels (Pierce, Rockford, IL) and transferred onto Hybond ECL nitrocellulose membranes (GE Healthcare, Chalfont St. Giles, Buckinghamshire, UK). Membranes were first incubated with a monoclonal anti-human CBR1 antibody (1:1000 dilution) that cross-react with murine Cbr1 (Abnova Corporation, Taipei City, Taiwan) and with a secondary anti-mouse IgG conjugated with horseradish peroxidase (1:1000 dilution; GE Healthcare). The membranes were also probed with anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (1:10,000 dilution; Chemicon International, Temecula, CA) to correct for differences in protein loading. Immunoreactive bands were visualized with the ECL Plus Western blotting detection system (GE Healthcare) and quantified by using a ChemiDoc XRS gel documentation system equipped with Quantity One software (Bio-Rad Laboratories, Hercules, CA).
CBR Activity. Maximal CBR activity was measured in cellular lysates, and in mice liver cytosols by using the specific NQO1 inhibitor dicoumarol in the presence of the substrate menadione and the NADPH cofactor (Wermuth et al., 1986; Bello et al., 2004; Covarrubias et al., 2006). Typical incubation mixtures (1 ml) contained sodium phosphate buffer (0.1 M), pH 7.4, 200 µM NADPH (Sigma-Aldrich), 200 µM menadione (Sigma-Aldrich), and 5 µM dicoumarol. Mixtures were equilibrated for 2 min at 37°C after the addition of cytosols (200 µg). The rates of NADPH oxidation were recorded for 4 min at 37°C in a Cary Varian Bio 300 UV-visible spectrophotometer (Palo Alto, CA). Enzymatic velocities were automatically calculated by linear regression of the abs/time points (2400 readings) and expressed as micromoles per minutes per milligram. Protein concentrations were determined with the Bradford assay (Bio-Rad).
Cloning and Functional Analysis of CBR1 Promoter Constructs. First, we cloned a 2485-bp DNA fragment from the 5'-flanking region of CBR1 to perform functional characterization studies. Sequencing of the insert revealed 100% identity with a segment of nucleotide sequence from locus AP001724 (Homo sapiens genomic DNA, chromosome 21q, section 68/105; Entrez Nucleotide Database, available at http://www.ncbi.nlm.nih.gov/sites/entrez?db=Nucleotide). According to the Data Base of Transcriptional Start Sites (DBTSS), CBR1 has a predominant transcription start site (TSS, 123/146 cDNA clones) located 92 bp upstream of the ATG codon (Fig. 1). Analysis of the core promoter sequence of CBR1 revealed the presence of a typical initiator element containing the TSS [Inr, Py-Py(C)-A+1-N-T/A-Py-Py]. The CBR1 core promoter region has no downstream core promoter element (A/G+28-G-A/T-C/T-G/A/C), no TATA box, and no CAAT box. The core promoter is embedded in a CpG island of approximately 0.65 kb that encompasses –273 bp of 5'-flanking sequence, and extends 369 bp downstream of the ATG codon. There are two contiguous GC boxes located at –165 and –152 bp, respectively. In addition, there is a proximal SP1 motif at –53 bp and a relatively more distal SP1 motif embedded in the –152 bp GC box. Together, these findings indicate that the core promoter of CBR1 has the configuration of a typical CpG island promoter (Butler and Kadonaga, 2002).
Fig. 1. Annotated sequence from the 5'-flanking region of human CBR1. The transcription start site (–92 bp, DBTSS) is indicated with a solid arrow, and the Inr element is underlined. The different fragments corresponding to the series of deletion promoter constructs are indicated with dotted arrows. The proximal XRE motif (–122XRE) is indicated in a gray box, and putative transcription factor binding sites are indicated in clear boxes. AP1, activator protein 1; HINF A, histone nuclear factor A; SF1, steroidogenic factor 1; NF-B, nuclear factor B; Oct 1, octamerbinding transcription factor; CAC, CACCC binding protein; IK-2, Ikaros 2 protein; GATA, GATA or GATAA sequence; HNF 3, hepatic nuclear factor 3; YY1, Yin Yang 1.
Computer-assisted searches for additional cis-acting elements using the TESS and TRANSFAC databases pinpointed potential consensus motifs for a number of transcription factors including hepatic nuclear factor-3, Ikaros 2 protein, and octamer-binding transcription factor (Fig. 1). We identified one proximal sequence motif for a xenobiotic response element (–122XRE; Fig. 1). Further analysis of up to 6 kb upstream of the ATG codon revealed a distal XRE motif containing the substitution-intolerant core sequence 5'-GCGTG-3' at position –5783 (–5783XRE).
Next, we generated a series of progressive 5'-deletion constructs and performed gene reporter assays in HepG2 and MCF-7 cells (Fig. 2, A and B). Results from both cell lines suggested the presence of a negative regulatory element in the –2485/–1847 region, because deletion of the 653-bp segment resulted in significant increases in luciferase activities (HepG2, Student's t test, p < 0.05; MCF-7, Student's t test, p < 0.05). In both cell lines, further 5' truncation of up to 746 bp resulted in no significant changes in the promoter activities of constructs –1847CBR1, –1561CBR1, and –1101CBR1, respectively (HepG2, ANOVA, p = 0.75; MCF-7, ANOVA, p = 0.83). In HepG2 cells, the –413CBR1 construct exerted the highest promoter activity from the series suggesting that the –600/–413 region may harbor an element whose regulatory role depends on the cellular context. Further deletion of 208 bp (–205CBR1) decreased the promoter activity in HepG2 (2.7-fold) and MCF-7 cells (2.4-fold). Data from both cell lines showed that the –205/–101 region contains cis-acting elements that are crucial to sustain gene transcription because deletion of 104 bp resulted in substantial decreases in the promoter activities by 22-fold (HepG2, Student's t test, p < 0.01) and 41-fold (MCF-7, Student's t test, p < 0.001), respectively. The –205/–101 segment contains two GC boxes and the proximal –122XRE. Thus, it is likely that the removal of these elements resulted in a construct (–101CBR1) with diminished promoter activity (Figs. 1 and 2). In both cell lines, the –101CBR1 showed minimal although significant increases in transcriptional activity compared with the pGL3-Basic vector (HepG2 = 3-fold, Student's t test, p < 0.05; MCF-7 = 8-fold, Student's t test, p < 0.05). It is possible that the Inr element (–93 bp) and the proximal SP1 site (–53 bp) dictate the minimal promoter activity of the –101CBR1 construct.
Fig. 2. Functional analysis of human CBR1 promoter constructs in HepG2 cells (A) and MCF-7 cells (B). Panels show schematic representations of each CBR1 promoter construct (left) and its corresponding luciferase activity from gene reporter experiments (right). Luciferase activities were measured as described under Materials and Methods. Light intensity values from transfections with the promoterless vector were used to correct for background. Corrected luciferase activity values were normalized to R. reniformis luciferase activity and expressed as fold increases with respect to the values obtained with pGL3-basic empty vector. Each value represents the mean ± S.D. of four independent experiments performed in duplicate.
Induction of CBR1 mRNA and CBR Activity by a Ligand of the Aryl Hydrocarbon Receptor. To pinpoint pathways potentially involved in the transcriptional regulation of CBR1, we analyzed the effect of different receptor activators on CBR1 mRNA levels. Cultures of HepG2 cells were incubated with different receptor activators at concentrations known to affect the regulation of other drug-metabolizing enzymes (Schuetz et al., 1993; Zhang et al., 2003; Hempel et al., 2004). CBR1 and -actin (normalizer) mRNA levels were determined simultaneously by quantitative real-time RT-PCR (see Materials and Methods). We detected no changes in CBR1 mRNA levels after incubations with the glucocorticoid receptor agonist dexamethasone. Likewise, incubations with activators of constitutive androstane receptor (clotrimazole and TCPOBOP) and pregnane X receptor (rifampicin) did not significantly affect CBR1 mRNA levels. In contrast, incubations with the prototypical AHR ligand -naphthoflavone (50 µM, 24 h) induced CBR1 mRNA levels by 5.5-fold (Student's t test, p < 0.005) compared with controls (Fig. 3). In MCF-7 cells, -naphthoflavone exerted moderate cytotoxicity (20–30%) at the 50 µM concentration, whereas incubations with 10 µM resulted in negligible cytotoxicity ( 5%) and induced CBR1 mRNA by 2.5-fold (Student's t test, p < 0.05; Fig. 3). The increase in CBR1 mRNA levels in MCF-7 cells treated with -naphthoflavone was paralleled by a 3-fold increase in maximal cytosolic CBR activity (CBRcontrols (DMSO):50 ± 13 pmol/min · mg versus CBR-naphthoflavone: 140 ± 2 pmol/min · mg).
Fig. 3. Induction of CBR1 mRNA in HepG2 cells (A) and MCF-7 cells (B) by prototypical receptor activators. HepG2 cells were incubated with vehicle (DMSO, 0.01%), dexamethasone (10 µM), -naphthoflavone (50 µM), clotrimazole (20 µM), TCPOBOP (0.250 µM), and rifampicin (10 µM) for 24 h. MCF-7 cells were incubated with vehicle (DMSO, 0.01%) and -naphthoflavone (10 µM) for 24 h. The expression of CBR1 mRNA was analyzed by quantitative real-time RT-PCR using specific primers as described under Materials and Methods. Each value represents the mean ± S.D. from three independent experiments analyzed in quadruplicate. Asterisks indicate significant differences from the CBR1 mRNA levels from vehicle treated cells (*, p < 0.005; **, p < 0.05).
Transcriptional Activation of CBR1 Promoter Constructs by -Naphthoflavone. Next, we tested whether -naphthoflavone induced the gene reporter activities of different CBR1 promoter constructs encompassing up to 1561 bp of the 5' flanking region. In all cases, incubations with -naphthoflavone (10 µM, 48 h) or vehicle (DMSO) were performed 24 h after the cotransfections with reporter constructs (see Materials and Methods). On average, -naphthoflavone induced the luciferase activities of the constructs by 11-fold (–1561CBR1, p < 0.05), 5-fold (–600CBR1, p < 0.05), and 15-fold (–413CBR1, p < 0.001) in MCF-7 cells compared with vehicle-treated controls (Fig. 4).
Fig. 4. Effect of the AHR ligand -naphthoflavone on the gene reporter activities of CBR1 promoter constructs. Cultures of MCF-7 cells were cotransfected with CBR1 reporter constructs (–1561CBR1, –600CBR1, and –413CBR1) and the normalizer plasmid pRL-SV40. Twenty-four hours after cotransfections, cells were treated with -naphthoflavone (10 µM) or vehicle (DMSO, 0.01%) for 48 h. Luciferase activities were measured as described under Materials and Methods. For each construct, normalized luciferase activities were expressed as fold increases with respect to the values from control incubations, which were set arbitrarily at 1. Data represent the mean ± S.D. from three independent experiments performed in duplicate. Asterisks indicate significant difference from vehicle-treated cells (*, p < 0.05; **, p < 0.05; ***, p < 0.001).
Fig. 5. Effect of -naphthoflavone on the gene reporter activities of the promoter constructs –413CBR1 and –413CBR1-–122 XRE. Cotransfections included the normalizer construct (pRL-SV40) and either the intact –413CBR1 construct or the engineered –413CBR1-–122XRE construct (without –122XRE motif). Twenty-four hours after cotransfections, cells were treated with -naphthoflavone (10 µM) or vehicle (DMSO, 0.01%) for 48 h. Luciferase activities were measured as described under Materials and Methods. For each construct, normalized luciferase activities were expressed as fold increases with respect to the values from control incubations (DMSO), which were set arbitrarily at 1. Data represent the mean ± S.D. from three independent experiments performed in duplicate. The asterisk indicates significant difference from the luciferase activity exerted by the –413CBR1 construct in the presence of -naphthoflavone (*, p < 0.001).
Functional XRE motifs in the promoters of drug-metabolizing enzymes are necessary to activate gene transcription in response to AHR ligands (Nioi and Hayes, 2004). Thus, we first we tested whether the –122XRE motif was necessary to induce luciferase reporter gene expression in the presence of the ligand -naphthoflavone. The removal of –122XRE decreased the -naphthoflavone response by 9-fold in MCF-7 cells (Student's t test, p < 0.001; Fig. 5).
In another set of experiments, we evaluated the ability of the distal –5783XRE to augment reporter gene activity in response to -naphthoflavone. To achieve this end, a 12-bp sequence (bases, –5790 to –5779) containing the –5783XRE was fused into the –2485CBR1 reporter construct. Treatment with -naphthoflavone increased the reporter activity of –2485CBR1 by 4-fold compared with incubations with the vehicle DMSO (Student's t test, p < 0.05). Fusion of the distal –5783XRE to –2485CBR1 further enhanced the -naphthoflavone response by 5-fold (Student's t test, p < 0.0001; Fig. 6).
Fig. 6. Effect of -naphthoflavone on the gene reporter activities of –2485CBR1 and –2485CBR1 + –5783XRE. Both constructs are schematized at the top of the graph. Cotransfections included the normalizer construct (pRL-SV40) and either the intact –2485CBR1 construct or the engineered –2485CBR1 + –5783XRE construct. Twenty-four hours after cotransfections, cells were treated with -naphthoflavone (10 µM) or vehicle (DMSO, 0.01%) for 48 h. Luciferase activities were measured as described under Materials and Methods. For each construct, normalized luciferase activities were expressed as fold increases with respect to the values from control incubations, which were set arbitrarily at 1. Data represent the mean ± S.D. from three independent experiments performed in duplicate. Asterisks indicate significant difference from vehicle treated cells (*, p < 0.05; **, p < 0.0001).
Induction of Hepatic Cbr1 by TCDD Treatment in Ahr+/– and Ahr–/– Mice. We extended our observations by evaluating whether the administration of the potent AHR ligand TCDD affected the expression of Cbr1 in livers from Ahr+/– and Ahr–/– mice. First, TCDD (50 µg/kg) was administered by a single intraperitoneal injection, and the expressions of Cbr1 mRNA and protein were analyzed from livers collected 72 h after treatments. In heterozygous Ahr+/– animals, TCDD treatment resulted in a 2-fold induction of Cbr1 mRNA levels compared with vehicle-treated heterozygous controls. In contrast, TCDD treatment failed to induce the expression of hepatic Cbr1 mRNA in homozygous null (Ahr–/–) mice (Fig. 7, A and B). It is noteworthy that the induction of hepatic Cbr1 mRNA in heterozygous Ahr+/– animals treated with TCDD was paralleled by a 2-fold increase in Cbr1 protein levels as determined by semiquantitative immunoblotting (Fig. 7, C–F). In line, hepatic Cbr activity increased by 40% in TCDD-treated mice with one active Ahr allele (Ahr+/–), whereas the levels of Cbr activity remained essentially unchanged in the livers of TCDD-treated Ahr–/– mice (data not shown). Moreover, Cbr activity was induced by 5-fold in livers of Ahr+/– mice treated with three consecutive doses of TCDD (50 µg/kg/day for 3 days) compared with vehicle-treated animals (p < 0.05; Fig. 8A). Identical TCDD treatments failed to induce hepatic Cbr activity in Ahr–/– mice (p = 0.32; Fig. 8B).
Fig. 7. Effect of TCDD on Cbr1 mRNA and Cbr1 protein expression in livers from Ahr+/– and Ahr–/– mice. Hepatic Cbr1 mRNA levels in Ahr+/– (A) and Ahr–/– (B) mice treated with vehicle (n = 2) or TCDD (n = 2), respectively. The expression of Cbr1 mRNA was analyzed by using specific primers as described under Materials and Methods. Bars represent the mean ± S.D. from two quantifications performed in quadruplicate for each animal. Immunodetection of hepatic Cbr1 in Ahr+/– (C) and Ahr–/– mice (E). Hepatic Cbr1 and GAPDH were detected with specific antibodies as described under Materials and Methods. Immunoreactive bands were visualized in a ChemiDoc XRS gel documentation system. The intensities from the immunoreactive GAPDH bands were used to correct for differences in protein loading during densitometric analyses. Densitometric analyses of Cbr1 in livers from Ahr+/– (D) and Ahr–/– mice (F). Each bar represent the level of Cbr1 expressed as fold induction with respect to the average intensity value obtained from vehicle-treated animals.
Fig. 8. Effect of TCDD on hepatic Cbr activity in heterozygous Ahr+/– (A) and homozygous Ahr–/– (B) mice. Each bar represents the average from two measurements performed in duplicate. Insets, hepatic Cbr activity expressed as fold induction with respect to the average activity value obtained from vehicle-treated animals.
The first aim of our study was to perform the functional characterization of the promoter of human CBR1. Our sequence annotation showed that the core promoter of CBR1 has the features of a prototypical CpG promoter including two GC boxes, proximal SP1 sites, and the absence of TATA and downstream core promoter element elements (Fig. 1). In humans, approximately half of the promoter regions are located in CpG islands, and gene transcription may occur at different start sites (Butler and Kadonaga, 2002). In line, CBR1 has a predominant start site at –92 bp, and 16% of the clones reported in DBTSS showed alternative start sites (e.g., –101 and –125 bp). Our results from gene reporter experiments in HepG2 and MCF-7 cells demonstrated the presence of regulatory regions that seem to be relevant to promote transcription under basal conditions in both cell types. For example, deletion of the segment that contains the two GC boxes and the proximal –122XRE (–205/–101) significantly reduced the reporter gene activity of the –205CBR1 construct compared with the –413CBR1 construct in HepG2 (22-fold) and MCF-7 (41-fold) cells. It has been demonstrated that SP1 binding sites together with an Inr motif can activate transcription in CpG promoters (Smale and Baltimore, 1989; Butler and Kadonaga, 2002). Consequently, the CBR1 –101/+1 region harboring both SP1 and Inr consensus displayed minimal although significant promoter activities in both cell lines. Functional mutagenesis analysis within the context of the minimal CBR1 promoter will provide further evidence on the role of the Inr and SP1 elements.
The second aim of this study was to evaluate the ability of prototypical receptor activators to induce the expression of CBR1 mRNA. In agreement with the seminal observation by Forrest et al. (1990), the AHR ligand -naphthoflavone was the only compound that significantly induced CBR1 mRNA levels in HepG2 and MCF-7 cells. Furthermore, our data with engineered reporter constructs suggest that –122XRE, and –5697XRE may act as bona fide functional elements to activate AHR-mediated gene transcription in the presence of AHR ligands.
The overall identity between the human CBR1 proximal promoter region (600 bp) and the mouse Cbr1 putative promoter region is 33% (global alignment analysis). Similar overall identity values (36%) were obtained when comparisons were extended up to 2500 bp. Further analysis by using the sequence comparison tool from DBTSS pinpointed 3 DNA fragments (size range, 38–42 bp) with relatively high sequence identity values (average, 72%). In addition, we identified a proximal XRE and a GC box element on the murine sequence that correspond with similar motifs on the human CBR1 promoter (Fig. 9). Sun et al. (2004) analyzed the positional conservation of XRE core motifs between several murine and human genes and found that only 39% of the human-mouse orthologs contain positionally conserved XREs. Thus, the positional conservation of the substitution-intolerant XRE core in both murine and human CBR1 sequences is interesting and supports the notion that the transcription of CBR1 in both species is controlled by similar key regulatory factors (e.g., AHR). Furthermore, our experiments with heterozygous Ahr+/– and homozygous Ahr–/– mice clearly showed that Ahr plays a pivotal role in mediating Cbr1 induction in vivo. It is noteworthy that the presence of one active Ahr allele was essential to induce Cbr1 mRNA, Cbr1 protein, and Cbr activity in Ahr+/– mice treated with the AHR ligand TCDD. In contrast, TCDD treatment failed to induce Cbr1 expression in homozygous null animals (Ahr–/–).
Fig. 9. DNA sequence alignment of the human and mice CBR1 5'-flanking regions. Sequences were aligned by using the global positioning alignment algorithm. XRE core motifs and GC boxes are highlighted in gray.
The reduction of carbonyl moieties catalyzed by CBR1 is an important step in the metabolism of a wide variety of clinically relevant drugs such as the anticancer daunorubicin, the antipsychotic haloperidol, and the antidiabetic acetohexamide (Ohara et al., 1995; Forrest and Gonzalez, 2000; Rosemond and Walsh, 2004). CBR1 also catalyzes the reduction of toxins such as the potent tobacco carcinogen 4-methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK). In humans, NNK is detoxified through two-electron reductions catalyzed mainly by cytosolic CBR1 and microsomal 11-hydroxysteroid dehydrogenase type I. The resulting alcohol metabolite 4-methylnitrosamino-1-(3-pyridyl)-1-butanol can be further subjected to glucuronidation to form 4-methylnitrosamino-1-(3-pyridyl)-1-butanol-glucuronide, which is excreted in urine (Maser et al., 2000). Variable CBR1 mRNA expression has been described in human lung, and a recent study on 59 patients with non–small-cell lung carcinoma reported higher postoperative survival rates among patients having tumors containing "high" CBR1 mRNA expression compared with those with tumors presenting "low" CBR1 mRNA expression (5-year survival CBR1-high, 68.3%, versus 5-year survival CBR1-low, 36.5%; p = 0.03) (Finckh et al., 2001; Takenaka et al., 2005). The polycyclic aromatic hydrocarbon benzo-(a)pyrene (BP) is one of the best-characterized carcinogens in cigarette smoke and is also a powerful AHR ligand (Denison and Nagy, 2003). Moreover, BP induces Cbr1 expression significantly in Ahr-proficient mice but fails to induce Cbr1 in Ahr-deficient animals (S. S. Lakhman, E. G. Schuetz, and J. G. Blanco, unpublished observations). Thus, it is reasonable to hypothesize that BP may modulate CBR1 expression in the lungs of smokers via the AHR pathway, which in turn has an impact on the CBR1-mediated detoxification of other smoke carcinogens relevant to the pathogenesis of lung cancer such as NNK. In conclusion, our results describe the first functional characterization of the promoter of human CBR1 and indicate that AHR is a key mediator in dictating variable CBR activity.
Acknowledgements
We gratefully acknowledge the excellent assistance of Dr. Lubin Lan.
ABBREVIATIONS: CBR1, human carbonyl reductase 1; AHR, aryl hydrocarbon receptor; CBR, carbonyl reductase activity; XRE, xenobiotic response element; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; TCPOBOP, 1,4-bis-[2-(3,5-dichloropyridyloxy)]-benzene; kb, kilobase; bp, base pair; PCR, polymerase chain reaction; ANOVA, analysis of variance; RT-PCR, reverse transcription-polymerase chain reaction; DMSO, dimethyl sulfoxide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; DBTSS, Data Base of Transcriptional Start Sites; SP1, specificity protein 1; NNK, 4-methylnitrosamino-1-(3-pyridyl)-1-butanone; BP, benzo(a)pyrene.
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作者单位：Department of Pharmaceutical Sciences, the State University of New York at Buffalo, Buffalo, New York (S.S.L., X.C., V.G.-C., J.G.B.); Department of Pharmaceutical Sciences, St. Jude Children Research Hospital, Memphis, Tennessee (E.G.S.)