(Received for publication, June 22, 1994; and in revised form, January 4, 1995)
From the
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
(NQO
) and glutathione S-transferase Ya genes in
tumor cells and its induction in response to xenobiotics and
antioxidants. Mutations in the human NQO
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
-naphthoflavone induction of the
NQO
gene. A single TRE element from the human collagenase
gene failed to respond to
-naphthoflavone. These results
demonstrate that ARE (2
TRE or TRE-like elements)-containing
detoxifying enzyme genes and not genes that contain 1
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
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
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
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.
Antioxidant response element (ARE) ()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
(NQO
/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
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.
-naphthoflavone (
-NF), 3-methylcholentherene, tert-butylhydroquinone, and
2(3)-tert-butyl-4-hydroxyanisole)(1, 2, 10) .
Increased activities of NQO
, 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
-NF induction of the NQO
gene expression. A single TRE
element from the human collagenase gene did not show a
-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
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.
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 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 . 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-
-galactosidase plasmid in Hepa-1 cells by calcium
phosphate procedure in separate experiments. The transfected cells were
treated with either Me
SO (control) or
-NF (50
µM for 16 h) or TPA (60 ng/ml for 16 h). Cells were
scrapped, homogenized, and analyzed for
-galactosidase normalized
CAT activity.
The nucleotide sequences of the human NQO gene
hARE1 and collagenase gene TRE are shown in Fig. 1. The human
NQO
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 -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
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 -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
-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
-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
-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
-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
-NF-induced expression of the CAT gene (data not shown). It is
noteworthy that replacement of
-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 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-
-galactosidase plasmid in Hepa-1 and F9 cells in separate
experiments. The CAT gene expression was monitored in absence and
presence of
-NF and TPA by measuring the
-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 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
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
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.
AREs present in the upstream regions of the NQO and GST genes are known to mediate high levels of expression of
the NQO
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
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
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 and mouse GST gene AREs(2, 12) .
However, Jun and Fos binding to the rat NQO
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
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
-NF and TPA. In the present report, we
mutated various TRE and TRE-like elements contained within the human
NQO
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
-NF treatment and
increased the CAT gene expression in Hepa-1 cells. However, 1
TRE sequence from the human collagenase gene failed to increase the CAT
activity in presence of
-NF. These results suggested that ARE (2
TRE) but not the 1
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 -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
-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
-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 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
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
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
gene(12) ; RNQO
, rat
NQO
/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
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
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 gene hARE reduced the basal expression and did not result in the
loss of induction by
-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
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 TRE) but not 1
TRE mediate xenobiotic and
antioxidant induction of the gene expression. Therefore, ARE-containing
detoxifying enzyme genes and not 1
TRE-containing genes may be
activated upon exposure to xenobiotics and antioxidants. Mutational
studies with human NQO
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
gene.
A second TRE like element in the human NQO
gene hARE is
required for optimal expression and xenobiotic induction of the
NQO
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
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.