©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ARE- and TRE-mediated Regulation of Gene Expression
RESPONSE TO XENOBIOTICS AND ANTIOXIDANTS (*)

(Received for publication, June 22, 1994; and in revised form, January 4, 1995)

Tao Xie Martin Belinsky (§) Yuehang Xu Anil K. Jaiswal (¶)

From the Department of Pharmacology, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Antioxidant response elements (AREs) containing 12-O-tetradecanoylphorbol-13-acetate response element (TRE) (perfect AP1) and TRE-like (imperfect AP1) elements mediate high basal transcription of the NAD(P)H:quinone oxidoreductase(1) (NQO(1)) and glutathione S-transferase Ya genes in tumor cells and its induction in response to xenobiotics and antioxidants. Mutations in the human NQO(1) gene ARE (hARE) revealed the requirement for two TRE or TRE-like elements arranged in inverse orientation at the interval of three base pairs and a GC box for optimal expression and beta-naphthoflavone induction of the NQO(1) gene. A single TRE element from the human collagenase gene failed to respond to beta-naphthoflavone. These results demonstrate that ARE (2 times TRE or TRE-like elements)-containing detoxifying enzyme genes and not genes that contain 1 times TRE are responsive to xenobiotics and antioxidants. Bandshift assays showed shifting of a complex of more or less similar mobility with hARE and TRE that could be competed by each other. Mutations in the 3`-TRE of the NQO(1) gene hARE eliminated binding of nuclear proteins to the hARE and resulted in the loss of basal and induced expression, indicating that 3`-TRE is the most important element within the hARE. 5`-TRE-like element within the NQO(1) gene hARE is required for xenobiotic response but may not bind to the nuclear proteins by itself. The GC box located immmediately following the 3`-TRE is required for optimal expression and induction of the NQO(1) gene. The comparison of AREs from several different genes indicated the requirement for specific arrangement and spacing of two TRE and TRE-like elements within the AREs.


INTRODUCTION

Antioxidant response element (ARE) (^1)also referred to as electrophile response element has been found in the 5`-flanking regions of the human and rat NAD(P)H:quinone oxidoreductase(1) (NQO(1)/DT-diaphorase) genes, rat glutathione S-transferase P (GST P) gene, and rat and mouse glutathione S-transferase Ya subunit (GST Ya) genes(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) . AREs (designated as hARE in human genes) are known to mediate high basal expression of the NQO(1) and GST genes in tumor cells as compared with normal cells of the same origin and their induction in response to a variety of xenobiotics and antioxidants (e.g. beta-naphthoflavone (beta-NF), 3-methylcholentherene, tert-butylhydroquinone, and 2(3)-tert-butyl-4-hydroxyanisole)(1, 2, 10) . Increased activities of NQO(1), GSTs, and other phase II enzymes are known to provide protection against neoplastic, mutagenic, and other toxic effects of many carcinogens(13) . Enhanced detoxification and consequent elimination from the body by way of modulating the activities of detoxifying enzymes mediated through ARE is by far the best approach for chemoprevention(13, 14, 15) . Various AREs contain two or more TRE and TRE-like elements in a short stretch of 40-45 nucleotides of the DNA(2, 12, 16) . The TRE (TGACTCA), present in the promoter regions of several eukaryotic genes including human metallothionein and collagenase genes, is known to increase the transcription of these genes in response to 12-O-tetradecanoylphorbol-13-acetate (TPA)(17) . Recent studies have indicated increasing complexity in the assortment of proteins that can bind to the TRE. Both Jun (c-Jun, Jun-D, Jun-B) and Fos (c-Fos, Fos-B, Fra-1, Fra-2) have been expanded (18, 19, 20) . The products of the proto-oncogenes Jun and Fos have also been shown to bind to the ARE and mediate signal transduction from xenobiotics and antioxidants(2, 12) .

In the present report, we have made several mutations in the hARE to demonstrate that two TRE elements and a GC box are required for optimal basal expression and beta-NF induction of the NQO(1) gene expression. A single TRE element from the human collagenase gene did not show a beta-NF response. However, bandshift assays indicated shifting of a complex of more or less similar mobility with hARE and TRE. Both oligonucleotides (hARE and TRE) competed with each other for binding to the nuclear proteins. The binding of nuclear proteins in the hARE required the presence of the 3`-TRE (perfect AP1 consensus). Bandshift and competition assays also revealed that the 5`-TRE-like element of the NQO(1) gene hARE, though required for xenobiotic response, may not bind to the nuclear protein(s) by itself. The comparison of AREs from several genes revealed specific arrangements and spacing of the TRE and TRE-like elements, which may be required for ARE functions.


EXPERIMENTAL PROCEDURES

Construction of hARE- and TRE-tk-CAT Plasmids

Both strands of the human NQO(1) gene hARE1 (region between -476 to -437) with BamHI ends were synthesized, annealed, kinased, and cloned at the BamHI site of the pBLCAT2 to produce pNQO(1)hARE1tkCAT. Purine to pyrimidine (and vice versa) changes were incorporated to make mutant hARE1 (3`-TRE-like element mutated), mutant-1 hARE1 (middle TRE element mutated), and mutant-2 hARE1 (5`-TRE-like element mutated (Fig. 1, Table 1). In another construct, the extreme 3`-TRE sequence was deleted to generate hARE-tk-CAT. It is noteworthy that hARE1 and hARE differed in containing three or two TRE and TRE-like elements (Table 1, Fig. 1and Fig. 2). TRE and TRE-like elements and GC box were mutated in the hARE, one at a time, to make mutant hARE and mutants 1-4 hARE-tk-CAT (Table 1, Fig. 2). Mutants 3 and 4 hARE contained mutations in the first four and last three base pairs of the 3`-TRE, respectively (Table 1, Fig. 2). In addition, 12 base pairs of the oligonucleotides from the human collagenase gene containing a single copy of the TRE followed by GC box were synthesized, annealed, and cloned in pBLCAT2 to generate pCollagenaseTRE-tk-CAT plasmid. Purine to pyrimidine (and vice versa) changes in TRE sequences or GC box were made for mutant and mutant-1 TRE (Table 1, Fig. 2). In each case, two strands with BamHI ends were synthesized, annealed, kinased, and cloned in pBLCAT2. The clones containing a single copy of the inserted sequence in 5`3` orientation with respect to the tk-CAT in pBLCAT2 were selected by sequencing with universal M13 primer. The nucleotide sequences of normal and mutated hARE1, hARE, and TRE are shown in Table 1. The mutated sequences are underlined.


Figure 1: Nucleotide sequence analysis of the human antioxidant response element-1 (hARE1). The TRE and TRE-like elements and GC box are shown. TRE is a perfect consensus sequence for binding to Jun and Fos proteins. TRE-like elements are imperfect binding sites for Jun and Fos proteins because of 2-3 base pair variations in their 3`-end as compared with the perfect TRE consensus sequence. 25 base pairs of the hARE containing the first two TRE and TRE-like elements, as indicated between the upwardarrows, were the initial elements characterized by deletion mutations in the human NQO(1) gene promoter(1, 15) .






Figure 2: Mutational analysis of hARE and TRE. Normal and mutated hARE1, hARE, and TRE were synthesized with BamHI ends, annealed, kinased, and cloned in pBLCAT2. Purine to pyrimidine and vice versa changes were made to generate mutations in TRE, TRE-like, and GC elements within the hARE1, hARE, and TRE as indicated by times. In all of these cases, complete binding sites were mutated. Specific base pair mutations are indicated by an asterisk. Various recombinant plasmids were co-transfected with RSV-beta-galactosidase plasmid in Hepa-1 cells by calcium phosphate procedure in separate experiments. The transfected cells were treated with either Me(2)SO (control) or beta-NF (50 µM for 16 h) or TPA (60 ng/ml for 16 h). Cells were scrapped, homogenized, and analyzed for beta-galactosidase normalized CAT activity.



Transient Transfection and Expression of hARE and TRE-tk-CAT Recombinant Plasmids

10 µg of the CAT plasmids were co-transfected with 5 µg of pRSV-beta-galactosidase plasmid into mouse hepatoma (Hepa-1) cells as described(2, 21) . The transfected cells were treated with either Me(2)SO (control) or beta-NF (50 µM) or TPA (60 ng/ml) for 16 h prior to harvesting. 48 h after transfection, the cells were scraped, homogenized by sonication in 0.2 M Tris buffer (pH 7.4), and analyzed for CAT gene expression by measuring CAT activity(22) . The hARE-tk-CAT and TRE-tk-CAT plasmids were also transfected in mouse embryonal carcinoma (F9) cells. The CAT activities are presented as pmoles of [^14C]chloramphenicol acetylated/min/unit of beta-galactosidase activity.

Nuclear Extract Preparation and Bandshift Assays

The nuclear extracts from Hepa-1 cells were made according to the procedure of Dignam et al.(23) modified by Kadonaga and Tjian(24) . Normal and mutated hARE1, hARE, and TRE were end labeled with [-P]ATP in separate experiments by T4 polynucleotide kinase. 20,000-30,000 cpm (<2 ng) of the end-labeled probes were mixed with 4 µg of poly(dI-dC)bulletpoly(dI-dC) and 15-30 µg of nuclear extract proteins in the presence of 25 mM Hepes (K), pH 7.8, 12.5 mM MgCl(2), 1 mM dithiothreitol, 20% glycerol (v/v), 0.1% Nonidet P-40, and 0.1 M KCl and incubated at room temperature for 20 min(2) . The samples were electrophoresed at 25 mA, gel-dried under vacuum, and autoradiographed. In several experiments, normal and mutant hARE, TRE, and nonspecific oligonucleotides (AP2 and repeat-2) were used as cold competitors. repeat-2 was an unrelated oligonucleotide selected from the NQO(1) gene promoter. The nucleotide sequences of the AP2 and repeat-2 are shown in Table 1.

Supershift Assays

The Hepa-1 nuclear extract was incubated with specific antibody at 4 °C for 2 h prior to bandshift assays as described above. The antibodies used in the present study were against specific peptides from c-Jun, Jun-D, c-Fos, NF-kB (p65) and v-Maf. The c-Jun, Jun-D, and c-Fos antibodies were obtained from Dr. Kevin Ryder (Fox Chase Cancer Center, Philadelphia) and are described(20) . The antibodies against NF-kB were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). v-Maf antibodies were a gift from Dr. Makoto Nishizawa (Cancer Institute, Japan)(26) . The observation of a supershifted band representing a antibody-protein-DNA complex indicated presence of the respective protein.


RESULTS

The nucleotide sequences of the human NQO(1) gene hARE1 and collagenase gene TRE are shown in Fig. 1. The human NQO(1) gene hARE1 contains three copies of the TRE and TRE-like elements and nucleotides GCA (GC box). The first two TRE elements (5` and 3`) in the hARE1 are arranged in inverse orientation separated by three nucleotides. The last TRE-like (extreme 3`) element is in the same orientation as the middle TRE and separated by eight base pairs.

The hARE1 when attached to the thymidine kinase promoter hooked to the CAT gene expressed high levels of the CAT activity upon transfection in Hepa-1 cells. hARE1-tk-CAT was 17.5- and 3.3-fold higher than pBLCAT2 (vector alone) and collagenase gene TRE-tk-CAT plasmid, respectively (Fig. 2). Higher levels of hARE1-mediated CAT gene expression as compared with the TRE was also observed in human hepatoblastoma (Hep-G2) cells (data not shown). Mutations or deletions in the extreme 3`-TRE-like element from the hARE1 did not show any major changes in basal or beta-NF- and TPA-induced expression of the CAT gene (Fig. 2). Mutations in the middle TRE element abolished both basal and inducible expression. Mutations in the 5`-TRE resulted in the loss of most of the induction and a minor decrease in the basal expression. These results revealed that the extreme 3`-TRE-like element in the hARE1 is not essential for induced expression. However, in the absence of 5`-TRE-like element, the extreme 3`-TRE-like element may be able to sustain basal expression. The above results indicate that the hARE containing the first two TRE elements in reverse orientation are sufficient to mediate high basal expression of the NQO(1) gene and its induction in response to xenobiotics and antioxidants. The results also indicate that extreme 3`-TRE-like element could not completely substitute for the 5`-TRE-like element. Therefore, from here onward, only data related to the hARE containing the first two TRE/TRE-like elements will be discussed.

hARE and not TRE-mediated CAT gene expression was induced in response to beta-NF (Fig. 2). However, both hARE- and TRE-mediated CAT gene expressions were induced by TPA. Mutations in the 3`-TRE of the hARE abolished basal and induced expression by beta-NF and TPA, indicating its absolute requirement for ARE functions (Fig. 2). Mutations in the GC box immediately following the 3`-TRE element of the hARE (mutant-1 hARE) resulted in decreased basal expression and beta-NF induction in Hepa-1 cells. Interestingly, mutations in the 5`-TRE-like element within the hARE (mutant-2 hARE) substantially reduced the basal expression and eliminated the induction by beta-NF. However, mutations in the GC box (mutant-1 hARE) and 5`-TRE-like element (mutant-2 hARE) did not affect TPA induction. Mutant-2 hARE was the closest to the human collagenase gene TRE in its structure and function. It was also interesting to observe that mutations in the first four and not the last three base pairs of the AP1 binding site in the 3`-TRE resulted in loss of basal and induced expression by beta-NF and TPA (compare mutant-3 and 4 in Fig. 2). Mutations in the nucleotides other than 5`- and 3`-TRE and GC box of the hARE did not interfere with basal and beta-NF-induced expression of the CAT gene (data not shown). It is noteworthy that replacement of beta-NF with 60 µM phenolic antioxidant (2(3)-tert-butyl-4-hydroxyanisole) produced similar effects as described above, except that optimum induction was only 2.5-fold in this case (data not shown).

Mutations in the AP1 binding region of the collagenase gene TRE (mutant TRE) resulted in complete loss of the basal and TPA-induced CAT gene expression (Fig. 2). In addition, the mutations in the GC region immediately following the AP1 binding site of the collagenase gene TRE also resulted in decreases in basal expression of the CAT gene. These results were similar to results obtained with mutant and mutant-1 hARE, respectively.

The hARE- and TRE-mediated CAT gene expression were much lower in F9 cells as compared with the Hepa-1 cells (Fig. 3). However, the hARE-mediated expression was more than 2-fold higher in F9 cells when compared with TRE-mediated CAT gene expression.


Figure 3: NQO(1) gene hARE and collagenase gene TRE-mediated CAT gene expression and induction in Hepa-1 and F9 cells. hARE and TRE-tk-CAT plasmids were co-transfected with RSV-beta-galactosidase plasmid in Hepa-1 and F9 cells in separate experiments. The CAT gene expression was monitored in absence and presence of beta-NF and TPA by measuring the beta-galactosidase normalized CAT activity. Errorbars represent ± S.E. of three independent experiments.



Bandshift assays with hARE and Hepa-1 nuclear extract revealed shifting of a complex that was specifically competed by cold hARE, collagenase gene TRE, mutant-1 hARE, and mutant-2 hARE but not competed by mutant hARE, AP2, and repeat-2 (Fig. 4). Repeat-2 was nonspecific oligonucleotide selected from the NQO(1) gene promoter. Mutant hARE did not show any detectable binding (Fig. 4). A complex of similar mobility as observed in the case of the hARE was also seen with mutant-1 and mutant-2 hARE (Fig. 5). The mutant-1 and mutant-2 complexes were competed away by cold hARE but not by unspecific repeat-2 oligonucleotide (Fig. 5). Interestingly, the shifted complex with collagenase gene TRE also showed more or less similar mobility as detected with hARE on the same gel (Fig. 5). The TRE-nuclear proteins complex was slightly slow moving as compared with the hARE complex in repeated experiments. However, the TRE-regulatory protein complex was specifically competed away with cold hARE but not with unspecific repeat-2 oligonucleotide. Supershift analysis with hARE and nuclear extract from Hepa-1 cells showed a supershifted band with Jun-D and c-Fos and not with c-Jun, NF-kB, and v-Maf (Fig. 6).


Figure 4: Bandshift assay. hARE and mutant hARE were end labeled with [-P]ATP. 20,000 cpm of the labeled hARE was incubated with 30 µg of the Hepa-1 nuclear extract in absence (control) and presence of cold competitors as shown on the top of each lane. In competition experiments, the nuclear extract was pre-incubated with the competing oligonucleotides for 5 min before bandshift assays. 100 ng of the competing DNA was used in each case. Repeat-2 was an unspecific oligonucleotide selected from the human NQO(1) gene promoter. 20,000 cpm of the labeled mutant hARE was also incubated with 30 µg of the Hepa-1 nuclear extract. Binding of nuclear proteins to the mutant hARE was not detected in repeated bandshift assays.




Figure 5: Bandshift assay. Mutant-1 and -2 hARE and TRE oligonucleotides were end labeled with [-P]ATP. 20,000 cpm of the end-labeled probes were incubated with 30 µg of nuclear extract from Hepa-1 cells in absence or presence of cold competitors. 100 ng of the normal hARE and an unspecific oligonucleotide (repeat-2) selected from the NQO(1) gene promoter were used as cold competitors. The reaction mixture was run on 5% non-denaturing polyacrylamide gel, dried under vacuum, and exposed for 24 h.




Figure 6: Supershift assay. Nuclear extract from Hepa-1 cells was incubated with either pre-immune serum or antisera against c-Jun, Jun-D, c-Fos, NF-kB, and v-Maf peptide at 4 °C for 24 h before bandshift assay with hARE. The supershifted bands (SSB) representing the antibody-protein-DNA complex are observed with Jun-D and c-Fos and not with c-Jun, NF-kB, and v-Maf antibodies. The absence of the supershifted band with c-Jun may be because the c-Jun gene is expressed at undetectable levels in Hepa-1 cells. SB, shifted band representing protein-DNA complex.




DISCUSSION

AREs present in the upstream regions of the NQO(1) and GST genes are known to mediate high levels of expression of the NQO(1) and GSTs and their induction in response to xenobiotics and antioxidants(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) . GSTs promote conjugation of hydrophobic electrophiles with glutathione(27, 28) , and NQO(1) catalyzes obligatory two-electron reduction of quinones and its derivatives and prevents their participation in redox cycling and oxidative stress(29, 30) . In addition to the NQO(1) and GSTs, the ARE sequences are also expected to be present in epoxide hydrolase, UDP-glucuronosyl transferase, and many other phase II detoxifying enzymes. These observations strongly suggest the significance of ARE in activation of detoxifying enzymes and chemoprevention, which has attracted much attention recently primarily because of an increase in the incidence of chemical carcinogenesis due to the almost unavoidable exposure to several potential carcinogenic chemicals present in food, water, and the environment(14, 15) . ARE-mediated regulation in the eukaryotic cells may be similar to the oxyR-and soxRS-mediated induction of more than a dozen genes in bacteria in response to oxidative stress(31, 32) . At present, there are more questions than answers in regard to minimum sequence needed for optimal activity of the ARE, different kinds of AREs, proteins binding to the ARE, and mechanism of signal transduction through ARE.

Various AREs have been shown to contain two or more copies of the TRE and TRE-like elements(2, 12, 16) . The products of the proto-oncogenes Jun and Fos have been shown to bind to the human NQO(1) and mouse GST gene AREs(2, 12) . However, Jun and Fos binding to the rat NQO(1) and GST Ya subunit gene AREs has not been confirmed (4, 33) . The differences in Jun and Fos binding to the human and rat NQO(1) gene ARE may be attributed to the species differences. It is noteworthy that human ARE but not rat ARE contains a perfect AP1 binding site(2) . These studies raised important questions about increases in expression of a number of eukaryotic genes containing a single TRE element such as collagenase and metallothionein genes with no apparent role in drug metabolism and detoxification. In addition, it was important to determine the role of each of the TRE and TRE-like elements and the GC box in ARE-mediated regulation of gene expression and induction by beta-NF and TPA. In the present report, we mutated various TRE and TRE-like elements contained within the human NQO(1) gene ARE (hARE) and human collagenase gene TRE to address the question of similarities and differences in hARE- and TRE-mediated regulation of the eukaryotic gene expression with special emphasis on their response to xenobiotics and antioxidants. Our results clearly demonstrated that hARE containing a minimum of two copies of the TRE and/or TRE-like sequences responded to beta-NF treatment and increased the CAT gene expression in Hepa-1 cells. However, 1 times TRE sequence from the human collagenase gene failed to increase the CAT activity in presence of beta-NF. These results suggested that ARE (2 times TRE) but not the 1 times TRE-containing genes are induced in response to xenobiotics and antioxidants. This is reasonable given the fact that activation of detoxifying enzyme genes rather than collagenase and metallothionein genes are required to reduce the risk of oxidative stress due to exposure to xenobiotics and carcinogens.

It was not surprising to observe that both hARE- and TRE-mediated expressions were induced by TPA in Hepa-1 cells. In the past, TPA-stimulated increases in the expression of the TRE-containing genes have been extensively studied(17, 19) . TPA-activated protein kinase C phosphorylates Jun and Fos, which form heterodimers and bind to the TRE, resulting in increased expression of the target genes. The increases in the hARE-mediated expression of the CAT gene in response to TPA may be due to presence of a perfect TRE sequence, which may act independent of the other TRE-like element in the hARE.

The nuclear protein(s) binding to the hARE is competed by TRE (and vice versa), indicating interaction of similar or closely related regulatory proteins with these elements. This is supported by reports of binding of Jun and Fos proteins to the hARE and TRE (Refs. 2, 12, 18, 19, and Fig. 6). The differences in xenobiotic and antioxidant response between hARE and TRE may be due to preference for a particular Jun and/or Fos proteins binding to the hARE or involvement of Maf(26) , NF-kB (p65) (34) , NF-E2(25) , and/or other as yet unknown protein(s). The Maf and NF-E2 proteins have recently been discovered and are known to form heterodimers with Jun and Fos proteins. Preliminary experiments using antibodies against NF-kB and v-Maf in supershift assays with the hARE and Hepa-1 nuclear extract did not reveal their presence in the hARE-nuclear protein complex observed in bandshift assays (Fig. 6). It is noteworthy that our experiments with v-Maf did not completely rule out the possibility of presence of Maf protein in the hARE-Hepa-1 nuclear protein complex because antibodies against v-Maf may not cross-react with rodent (Hepa-1) protein. It may be interesting to note that binding of Jun-D but not c-Jun to the hARE from Hepa-1 cells was detected in supershift assays(2) . In addition, hARE-mediated CAT gene expression was severalfold higher than TRE-mediated expression in F9 cells, which contain Jun-D but no c-Jun. Complete identification of all of the regulatory proteins in the hARE-nuclear protein complex may provide a more satisfactory answer to this question. Differences at the level of protein-protein interaction stimulated by xenobiotics and antioxidants cannot be ruled out. Interestingly, bandshift assays with mutant hARE (3`-TRE mutated) did not show any binding with nuclear extract from Hepa-1 cells even though it had a normal 5`-TRE-like element, which was needed for beta-NF-induced expression. Similarly, mutant-2 (5`-TRE-like element mutated) did not compete with hARE binding of nuclear proteins. These experiments suggested that a 5`-TRE-like element in the hARE may either be required for increased affinity of proteins at the 3`-TRE or bind to nuclear proteins only in presence of 3`-TRE within the hARE. It may be noteworthy that beta-NF and TPA both in separate experiments increased binding of proteins at the hARE (data not shown). A detailed analysis of the identity of proteins binding to the hARE in absence and presence of beta-NF and TPA will be required to fully understand the mechanism of signal transduction from inducers to the hARE.

The alignment of various AREs from NQO(1) and GST P and Ya subunit genes (Fig. 7) revealed specific arrangement of TRE and TRE-like elements. The arrangement and sequence of TRE elements within the hARE was found highly conserved between human and rat NQO(1) genes. The rat GST P gene ARE contains two TRE-like elements arranged in a manner similar to the elements in the human and rat NQO(1) genes. The rat and mouse GST Ya subunit genes also contain two copies of the TRE-like elements arranged as direct repeats at the interval of eight base pairs. To summarize this, the AREs in detoxifying genes usually contain two TRE or TRE-like elements arranged in varying orientations separated by either three (inverse repeat) or eight (direct repeat) nucleotides. The orientations and spacing between the two TRE elements could be important in determining the basal and induced expression of detoxifying enzyme genes.


Figure 7: Alignment of AREs from several detoxifying enzyme genes. HNQO, human NQO(1) gene(12) ; RNQO, rat NQO(1)/QR gene(5) ; RGSTP, rat GST-P gene(28) ; RGSTYa, rat GST Ya subunit gene(20, 24, 26) ; MGSTYa, mouse GST Ya subunit gene (8, 22) . The sequences in italics are additional sequences from respective genes for better alignment. The TRE and TRE-like elements and their orientations are shown by arrows. The middle TRE in the human NQO(1) gene ARE is a perfect consensus sequence for Jun and Fos binding and has been separated from TRE-like (imperfect consensus) elements in all other genes. The last TRE-like element in the human and rat NQO(1) gene was not conserved in GST genes. The GC box and ETS binding sites are also shown in boxes.



Additional DNA elements contained within the ARE were shown to contribute to optimal expression and induction of the GST Ya subunit genes(9, 10) . It has been reported that two nucleotides (GC) immediately following the 3`-TRE-like element within the rat GST Ya gene ARE are not required for basal expression but are essential for induction by xenobiotics and antioxidants(10) . These two nucleotides are part of the three nucleotides, GCA, that are highly conserved in all kinds of the AREs (Fig. 7). In repeated experiments, mutations in these nucleotides in the human NQO(1) gene hARE reduced the basal expression and did not result in the loss of induction by beta-NF as seen in case of the rat ARE. These differences between rat and human ARE may be due to species differences and remain to be further investigated. Interestingly, the GC box was also found conserved and immediately followed the AP1 binding sites in most of 1 times TRE-containing genes, including collagenase, metallothionein, salivary statherin, and thyroglobulin genes. Mutations in the GC box of the collagenase gene also decreased the amount of CAT gene expression, indicating that the GC box is also required for optimal TRE-mediated expression.

In summary, we have shown that ARE (2 times TRE) but not 1 times TRE mediate xenobiotic and antioxidant induction of the gene expression. Therefore, ARE-containing detoxifying enzyme genes and not 1 times TRE-containing genes may be activated upon exposure to xenobiotics and antioxidants. Mutational studies with human NQO(1) gene hARE indicated that a TRE with perfect consensus sequence for binding to the AP1 proteins is the most essential element required for binding to the regulatory proteins, basal expression, and xenobiotic induction of the NQO(1) gene. A second TRE like element in the human NQO(1) gene hARE is required for optimal expression and xenobiotic induction of the NQO(1) gene. However, this second TRE element does not bind nuclear proteins by itself. The GC box is required for optimal gene expression mediated by hARE as well as 1 times TRE. Further studies will be required to completely understand the mechanism of signal transduction from xenobiotics and antioxidants to the hARE for increased transcription of the detoxifying enzyme genes.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant GM 47466. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by Institute Training Grant CA-09035.

To whom correspondence should be addressed: Dept. of Pharmacology, Fox Chase Cancer Center, 7701 Burholme Ave., Philadelphia, PA 19111. Tel.: 215-728-5282; Fax: 215-728-4333.

(^1)
The abbreviations used are: ARE, antioxidant response element; beta-NF, beta-naphthoflavone; TPA, 12-O-tetradecanoylphorbol-13-acetate; NQO(1), NAD(P)H:quinone oxidoreductase(1) also known as quinone reductase (QR), quinone:(acceptor) oxidoreductase (QAO), and DT-diaphorase (EC 1.6.99.2); GST, glutathione S-transferase; TRE, TPA response element with perfect consensus sequence for AP1 binding; TRE-like elements, imperfect AP1 binding consensus containing 2-3 base pair variations at their 3`-end; CAT, chloramphenicol acetyltransferase.


ACKNOWLEDGEMENTS

We thank our colleagues for helpful discussions.


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