(Received for publication, December 2, 1996)
From the Departments of Human Oncology and § Medicine, University of Wisconsin Medical School, Madison, Wisconsin 53792
Glutathione (GSH) is an abundant cellular
non-protein sulfhydryl that functions as an important protectant
against reactive oxygen species and electrophiles, is involved in the
detoxification of xenobiotics, and contributes to the maintenance of
cellular redox balance. The rate-limiting enzyme in the de
novo synthesis of glutathione is -glutamylcysteine synthetase
(GCS), a heterodimer consisting of heavy and light subunits expressing
catalytic and regulatory functions, respectively. Exposure of HepG2
cells to
-naphthoflavone (
-NF) resulted in a time- and
dose-dependent increase in the steady-state mRNA levels
for both subunits. In order to identify sequences mediating the
constitutive and induced expression of the heavy subunit gene, a series
of deletion mutants created from the 5
-flanking region (
3802 to
+465) were cloned into a luciferase reporter vector (pGL3-Basic) and
transfected into HepG2 cells. Constitutive expression was maximally
directed by sequences between
202 and +22 as well as by elements
between
3802 to
2752. The former sequence contains a consensus TATA box. Increased luciferase expression following exposure to 10 µM
-NF was only detected in cells transfected with a
reporter vector containing the full-length
3802:+465 fragment. Hence, elements directing constitutive and induced expression of the GCS heavy
subunit are present in the distal portion of the 5
-flanking region,
between positions
3802 and
2752. Sequence analysis revealed the
presence of several putative consensus response elements in this
region, including two potential antioxidant response elements (ARE3 and
ARE4), separated by 34 base pairs. When cloned into the thymidine
kinase-luciferase vector, pT81-luciferase, and transfected into HepG2
cells, both ARE3 and ARE4 increased basal luciferase expression
approximately 20-fold. When cloned in tandem in their native
arrangement the increase in luciferase activity was in excess of
100-fold, suggesting a strong interaction between the two sequences.
Luciferase expression was elevated in
-NF-treated cells transfected
with the ARE4-tk-luciferase vector and all DNA fragments
containing ARE4. In contrast, ARE3 did not direct increased luciferase
expression in response to
-NF nor did it significantly modify the
magnitude of induction directed by ARE4. The influence of the ARE4
oligonucleotide on constitutive and induced expression was eliminated
by introduction of a single base mutation, converting the core ARE
sequence in ARE4 from 5
-G
GACTCAGCG-3
to
5
-G
GACTCAGCG-3
. When introduced into the full-length
3802:+465 segment, the same single base mutation also eliminated both
functions. Collectively the data indicate that the constitutive and
-NF-induced expression of the human GCS heavy subunit gene is
mediated by a distal ARE sequence containing an
embedded
tetradecanoylphorbol-13-acetate-responsive element.
Glutathione
(L--glutamyl-L-cysteinyl-glycine,
GSH),1 a non-protein sulfhydryl compound
present in millimolar concentrations in virtually all cells, serves a
myriad of cellular functions and plays a prominent role as an
intracellular protectant (1, 2). GSH is an effective oxygen radical
scavenger and serves as a critical co-factor in peroxide detoxification
via a reaction catalyzed by glutathione peroxidase. Furthermore,
conjugation with GSH is an integral step in the detoxification and
elimination of diverse classes of toxic chemical compounds. The
formation of hydrophilic glutathionyl conjugates is catalyzed by
glutathione S-transferases, a family of isozymes that
mediate the conjugation reaction in a substrate-dependent
fashion (3). Long the object of interest from a toxicology perspective,
the protective properties of GSH have assumed even further significance
since GSH not only plays a critical role in protection of normal cells,
but it has recently been implicated in protection of neoplastic cells
from a number of chemotherapeutic agents that exert their cytotoxic effects via generation of reactive oxygen species or production of
electrophilic intermediates (4, 5). The augmentation of GSH and
GSH-related detoxification systems has also engendered considerable
interest as a possible approach for the chemoprevention of cancer. Many
chemical chemopreventive agents have been shown to exert an effect on
GSH homeostasis or on other elements of GSH detoxification pathways
(6-8).
Exposure of cells to a number of xenobiotic agents has been
demonstrated to result in an increase in the total intracellular GSH
content. In several cases (9-16) where it has been examined, the
increase in GSH has been attributed to an increase in the activity of
-glutamylcysteine synthetase (GCS, EC 6.3.2.2), the rate-limiting
enzyme in its de novo synthesis (17). The GCS holoenzyme is
a heterodimer that can be dissociated under nondenaturing conditions
into light (GCSl) and heavy (GCSh) subunits of
28,000 Da and 73,000 Da, respectively (18). Catalytic activity resides
entirely with the heavy subunit, but it has recently been demonstrated
that the kinetic properties of the heavy subunit can be profoundly
influenced by association with the light, or regulatory, subunit and
the redox state of a single disulfide linkage between the two subunits
(19, 20). The heavy and light subunits are encoded for by two distinct
genes located on chromosomes 1 and 6, respectively (21-23). The
cDNAs for each subunit of human GCS has been cloned and sequenced
(24, 25), as has the 5
-flanking sequence of the heavy subunit gene
(26). Northern analyses reveal that xenobiotic-induced GCS enzyme
activity is frequently accompanied by an increase in the steady-state
levels of GCSh-specific mRNA transcripts (10, 12-14,
16). Similar analyses for the light subunit have not yet been reported
as cloning of the corresponding cDNA has only recently been
completed. Exposure to the antioxidant butylated hydroxyanisole (16),
tert-butyl hydroquinone (14), and methyl mercury (10)
results in transcriptional up-regulation of the heavy subunit gene,
suggesting that GCSh gene expression in response to these
toxic insults is regulated via as yet unidentified specific
cis- and trans-acting factors.
Since several treatments that induce expression of key phase II
detoxifying enzymes also result in elevated GCS activity as well as
increased intracellular GSH levels, we hypothesized that the
transcriptional regulation of the GCSh subunit gene and
genes in the phase II battery are mediated by common regulatory
elements. Regulation of several phase II enzymes in response to a wide
array of chemically distinct classes of agents has been demonstrated to
be mediated, at least in part, by the presence of an antioxidant response element (ARE) within the 5-flanking region of the gene (27-31). Recent evidence suggests that other cis-acting
elements might also participate in concert with the ARE in
transcriptional regulation of these genes (30). We identified numerous
potential response elements and/or enhancer sequences, including a
putative ARE, in the 5
-flanking sequence of the human GCSh
gene (26). However, the involvement of any specific elements in
constitutive or induced expression of the GCSh gene has not
been established by functional analyses.
In the present study, we utilized a deletion mutagenesis analysis
strategy to identify those sequences that modulate constitutive expression of the human GCSh gene and to begin to decipher
mechanisms involved in the induced expression of the gene. To this end,
HepG2 cells transfected with a series of deletion mutant/reporter
fusion genes were exposed to -naphthoflavone, an inducing agent
which activates phase II gene expression via AREs (31, 32) and which we
demonstrate also induces expression of both the endogenous heavy and
light subunit genes. The results indicate that both constitutive and
induced expression of the heavy subunit gene is mediated via a distal
consensus ARE sequence.
Genomic DNA containing the GCS heavy subunit gene was isolated by screening a human foreskin fibroblast P1 library (Genome Systems) as described previously (26). Sequencing was performed using Sequenase (U. S. Biochemical Corp.) and synthetic oligonucleotide (20-mers) primers corresponding to internal sequences. All sequence information was verified by multiple bi-directional sequencing reactions.
Recombinant PlasmidsRecombinant expression vectors were
created by cloning restriction fragments isolated from the 5-flanking
sequence of the GCSh gene into pGL3-Basic (Promega) for
determination of promoter activity or by introducing GCSh
DNA or synthetic oligonucleotides into pT81 (ATCC 37584) for
determination of enhancer activity. A 4.2-kb genomic DNA fragment was
isolated from the 5
-flanking region of GCSh by
HindIII/NcoI restriction digestion and cloned into the HindIII/NcoI sites of pGL3-Basic
creating the recombinant plasmid
3802/GCSh5
-luc (Fig. 3). This plasmid was
subjected to digestion with additional restriction enzymes to generate
a series of deletion mutants as detailed in Fig. 3. The
enhancer/reporter transgene,
3491:
2438/GCSh5
-luc, was created by cloning
an ~1-kb PpuMI fragment (
3491:
2438) containing AREs 2, 3, and 4 into pT81. Digestion of
3491:
2438/GCSh5
-luc with SacI
generated two additional constructs, containing either ARE2
(
2754:
2438) or ARE3 and ARE4 (
3491:
2755). In addition, a series
of enhancer/reporter transgenes was created by cloning specific
oligonucleotides (Fig. 5) corresponding to ARE1, ARE3, ARE4, ARE3,4,
NF-
B, and NQO1hARE sequences into pT81. To prepare the
oligonucleotides, complementary oligonucleotides were synthesized,
annealed, phosphorylated, and then cloned into pT81. Oligonucleotides
were dissolved in 1 × Sequenase reaction buffer and annealed by
heating to 90-100 °C, cooled slowly to 35 °C, extracted once
with phenol/chloroform/isoamyl alcohol and once with chloroform/isoamyl
alcohol, ethanol-precipitated, and finally resuspended in water. These
double-stranded fragments were then phosphorylated, extracted, and
precipitated as before and resuspended in water and ligated into
pT81.
Cell Culture and Transfection
HepG2 cells were maintained
in Dulbecco's modified Eagle's medium supplemented with 10% fetal
bovine serum and 50 µg/ml gentamicin (complete medium). Cells were
transfected with recombinant plasmids using a standard calcium
phosphate-glycerol shock procedure. HepG2 cells were plated at 1 × 106 cells/35-mm dish on day 0. On day 1, medium was
replaced with fresh complete medium. Two to four hours later the cells
were transfected by the addition of appropriate DNA expression vectors. Equimolar concentrations of plasmid DNA were used to compensate for
variations in plasmid size. To correct for transfection efficiency, 1.25 µg of the reporter plasmid pCMV (33) containing the
lacZ gene encoding
-galactosidase under the control of
the human cytomegalovirus immediate early promoter/enhancer was
cotransfected with each recombinant plasmid. Four hours after addition
of DNA, cells were shocked with media containing 10% glycerol for 3 min at room temperature and then maintained at 37 °C for an
additional 24 h. At the conclusion of this incubation period, the
medium was once again replaced with fresh complete medium containing
Me2SO (0.1%) or 10 µM
-NF (Sigma)
dissolved at 1000 × in Me2SO. Sixteen hours later
cells were harvested and prepared for determination of luciferase,
-galactosidase activity, and protein content. For cell harvest,
transfectants were washed twice with phosphate-buffered saline
(Mg2+- and Ca2+-free) and incubated at room
temperature for 15 min in 250 µl of reporter/lysis buffer (Promega).
Cells were then scraped from the plates and the resulting lysates spun
at top speed in a microcentrifuge for 2 min at 4 °C. The resultant
supernatants were transferred to Eppendorf tubes and stored on ice
pending assay.
Total (oxidized and reduced) glutathione content was determined using the technique described by Tietze (34) as modified by Bump et al. (35).
GCS activity was determined as described previously using a
modification (36) of a high performance liquid chromatography technique
described by Nardi (37). The rate of -glutamylcysteine formation was
quantitated by comparison to
-glutamylcysteine standards and
normalized on the basis of protein concentration.
-Galactosidase activity was quantified as described by Rosenthal
(38). Briefly, this assay monitors cleavage of
o-nitrophenyl-
-D-galactopyranoside and yields
-galactosidase units as (A420 × 380)/t where t = time in min at 37 °C and
380 is a conversion factor to convert absorbance to µmol of
o-nitrophenyl-
-D-galactopyranoside.
To initiate the luciferase assay, cell lysate (1-5 µl) was added to
reaction buffer (14 mM MgCl2, 14 mM
glycylglycine, 0.1 mg/ml bovine serum albumin, 18 mg/ml ATP, pH 7.8),
vortexed, and placed in an Analytical Luminescence Laboratory
luminometer (400-µl total volume). Following injection of 100 µl of
luciferin (4 mg/ml in 10 mM Na2CO3,
pH 6.0), luminescence was recorded as relative light units. Luciferase
activity was normalized for transfection efficiency on the basis of
-galactosidase activity and protein content of the lysate yielding a
final value of (relative light units/
-galactosidase)/µg
protein.
Protein content was determined by the Bradford method (39) using bovine serum albumin as a standard.
Northern Analysis and RNase ProtectionTotal cellular RNA was isolated using TRI Reagent (Molecular Research Center, Inc.) according to the procedure recommended by the manufacturer. For Northern analysis 25 µg of total RNA was size-fractionated in formaldehyde-agarose gels, transferred to a charged nylon membrane (Hybond N+, Amersham Corp.), and hybridized with a 764-bp or a 736-bp radiolabeled probe specific for GCSh or GCSl transcripts, respectively (24, 25). A radiolabeled probe specific for 36B4 (40) was used to normalize for RNA loading. RNA quantitation was accomplished using the RPAII (Ambion) ribonuclease protection assay. To generate single-stranded radiolabeled probes, the 764-bp PstI fragment isolated from the GCSh cDNA was cloned into the pAlter vector (Promega) and digested with NcoI. The GCSl-specific probe was prepared by cloning a 305-bp HindII fragment from GCSl cDNA into pSKII Bluescript (Stratagene) followed by digestion with DdeI. Using T7 RNA polymerase and [32P]UTP, these vectors yield 301- and 210-bp radiolabeled run-off transcripts and 250- and 179-bp protected fragments for GCSh and GCSl, respectively. To normalize for RNA content glyceraldehyde-3-phosphate dehydrogenase was used as an internal standard. pTRI-GAPDH-Human (Ambion) yields a 404-bp transcript and a 316-bp protected fragment. Following hybridization with total cellular RNA and incubation with RNase, fragments were separated on a 5% acrylamide, 8 M urea gel, visualized, and quantitated by using the PhosphorImager system (Molecular Dynamics).
To examine
regulation of GCSh induction, we first elected to
investigate the effect of exposure of HepG2 hepatocarcinoma cells to
-NF, a potent inducer of several phase II enzymes and an agent that
exerts its effects, at least in part, through activation of AREs. The
effect of
-NF exposure on GCS activity in exposed HepG2 cells is
illustrated in Fig. 1A. There was a gradual
decrease in GCS activity over the 24-h incubation period in untreated
and Me2SO-treated HepG2 cells. In contrast, exposure to 10 µM
-NF resulted in a progressive increase in GCS
activity, culminating in a moderate, although significant
(p < 0.05), increase in enzyme activity by 24 h
after addition of
-NF to the culture medium (Fig. 1A).
Induction of GCS expression occurred sooner and was greater following
exposure to 25 µM
-NF.
These changes in GCS expression were accompanied by a progressive
increase in intracellular GSH levels at each -NF dose over the 24-h
observation period (Fig. 1B). By 24 h GSH levels had increased significantly, reaching in excess of 2- and 4-fold over controls for 10 and 25 µM exposures, respectively. In the
case of 25 µM exposure, a significant increase was
evident as early as 12 h after addition of
-NF.
Exposure of HepG2 cells to increasing concentrations of
-NF also resulted in a dose-dependent increase in
steady-state mRNA levels for both subunits of the GCS holoenzyme
(Fig. 2, A and B). In all cases,
the two heavy subunit transcripts increased proportionally (Fig.
2A). Changes in the relative abundance of the transcripts
for the light and heavy subunits as a function of time after addition
of
-NF or Me2SO was quantitated by a ribonuclease protection assay engineered to permit quantitation of both transcripts simultaneously. A representative radiograph is shown in Fig.
2C. Quantitation by nuclease protection assay was
particularly important in the case of the light subunit transcripts
that are typically present at low abundance. Quantitative and temporal
changes in transcript abundance in
-NF-treated cells are illustrated
in Fig. 2, D and E. These data confirm the
dose-dependent increase in mRNA levels observed by
Northern analysis. The
-NF-induced increase in steady-state mRNA
levels for the GCSh and GCSl subunits displayed
similar kinetic patterns, reaching a maximum at approximately 12 h
after initiation of exposure. Interestingly, the increase in
GCSl transcripts levels was consistently higher than that
observed in the case of the GCSh mRNA.
Regulation of Constitutive Expression
We recently reported
(26) the cloning and partial sequence (1460:+547) of a genomic clone,
designated P1-GCS5
, containing approximately 4.7 kb of 5
-flanking
sequence and 0.5 kb of exon I of the human GCS catalytic subunit gene.
A 4.2-kb fragment from this clone, spanning the sequence
3802 to
+465, was cloned into the pGL3-luciferase reporter vector to create the
GCSh/luciferase fusion gene,
3802
GCSh5
-luc (Fig. 3). A series of
deletion mutants was then generated from this parent chimeric gene and
used to identify functional response elements directing constitutive
and
-NF-induced expression of the GCSh gene. The various
reporter constructs shown in Fig. 3 were transfected into HepG2 cells
and luciferase expression examined 48 h later. Transfection of
3802/GCSh5
-luc increased the luciferase
expression approximately 35-fold relative to the expression detected in
cells transfected with pGL3-luciferase alone. Progressive deletions
from
3802 to
510 resulted in significant reductions in luciferase
expression when compared with that produced by
3802/GCSh5
-luc. However, the differences in
luciferase expression among these three intermediate constructs were
not statistically significant (p > 0.05) from each
other. Transfection of
202/GCSh5
-luc resulted
in significantly increased luciferase activity, attaining levels
comparable with those observed following transfection with the
full-length fusion gene. Deletion of sequences between
202 and +22
resulted in a significant reduction in luciferase expression, whereas
further deletion to +358 completely eliminated promoter activity.
Collectively these results suggest that the fragment between
202 and
+22 contains a positive regulatory sequence(s) capable of directing
constitutive expression of the gene in HepG2 cells; negative regulatory
sequences exist between
202 and
814, and one or more positive
regulators exists between
2752 and
3802. Further analyses revealed
that the enhanced activity associated with inclusion of sequences from
202 to +22 corresponded to the presence of the TATA sequence
previously identified in this region of the promoter (data not
shown).
To
determine whether the increase in steady-state mRNA levels observed
following -NF exposure was the result of increased transcription and
to identify response elements that might mediate
-NF-induced
GCSh gene expression, HepG2 cells were transiently transfected with the GCSh promoter/reporter constructs and
then exposed to 10 µM
-NF for 16 h. As shown in
Fig. 3,
-NF failed to increase luciferase expression in cells
transfected with any of the recombinant reporter genes other than the
full-length
3802/GCSh5
-luc. The magnitude of
induction was approximately 2-3-fold, comparable with the magnitude of
increase in endogenous GCSh mRNA expression following
similar
-NF exposure. Hence, the response element(s) mediating
GCSh gene induction in response to
-NF is present
between
2752 and
3802.
Transfection experiments
therefore suggest that the DNA sequence from 2752:
3802 contains
elements that influence both constitutive and
-NF-induced expression
of GCSh. This region is beyond the 5
-distal end of the
sequence we had previously reported, and therefore we extended our
original sequencing to include an additional 2342 bp of this 5
-distal
fragment in order to identify potential enhancer sequences. The
complete sequence of
3802:+547/GCSh5
is shown in Fig.
4. In addition to the potential regulatory sequences identified previously, the distal genomic fragment includes a consensus
NF-
B binding site, a consensus AP-1 site, Sp-1 sites, and three
additional potential AREs.
In order to approximate the position of elements or enhancers that
contribute to the constitutive and induced expression of the heavy
subunit gene, a series of enhancer/reporter constructs were developed
by cloning various restriction fragments or synthetic oligomers (Fig.
5) into the reporter vector, pT81, in which luciferase expression is under the control of the herpes simplex thymidine kinase
(tk) minimal promoter. Fig. 6A
illustrates the structure of the individual enhancer vectors and their
effect on constitutive luciferase expression. The fragment
3491:
2438, representing the majority of the distal region of
GCSh5
responsible for increased constitutive expression,
increased luciferase expression 5-fold relative to that observed in
pT81 transfected cells. When this 1-kb fragment was subdivided by
SacI digestion, the fragment corresponding to the distal 0.7 kb (
3491:
2755) maintained enhanced luciferase expression, whereas
the proximal 0.3-kb fragment (
2754:
2438) failed to do so.
As shown in Figs. 3 and 4, 3491:
2755/GCSh5
contains
two putative AREs (3 and 4). A third (ARE2) is present in the
2754:
2438/GCSh5
fragment. Individually, ARE3 and ARE4
each significantly enhance pT81 luciferase expression, attaining levels
of expression (~20-fold) comparable with that produced by the
ARE-positive control vector, NQO1hARE-tk-luc
(32), containing the ARE from the human NQO1 gene (Fig. 6A).
When cloned into pT81 in their normal tandem arrangement (GCShARE3,4), ARE3 and ARE4 increase luciferase expression
in excess of 100-fold. In contrast, ARE1, the putative ARE identified previously on the basis of sequence similarity, did not function as an
enhancer in this system. Although an oligomer corresponding to ARE2 was
not cloned into pT81, the 0.3-kb
2754:
2438/GCSh5
fragment, containing ARE2, did not increase luciferase activity, suggesting that ARE2 was likewise not functional as an enhancer of
constitutive expression. The consensus NF-
B sequence also failed to
influence luciferase expression.
When a similar series of experiments was conducted with transfected
HepG2 cells exposed to 10 µM -NF for 16 h,
GCSh ARE4, GCShARE3,4 (containing the tandem
combination of ARE3 and ARE4), and those larger fragments containing
them directed increased luciferase expression in treated cells (Fig.
6B). Luciferase expression in cells transfected with
NQO1hARE-tk-luc was increased approximately 3.5-fold
relative to that in untreated cells. A similar level of induction was
detected in cells transfected with ARE4-tk-luc. However, in
the presence of ARE3, which did not itself respond to
-NF induction,
the
-NF inducibility of ARE4 was diminished slightly. Nevertheless,
the induction of luciferase expression in
-NF-treated cells
transfected with any vector containing the combination of ARE3 and ARE4
was similar in magnitude to 1) the increase in endogenous
GCSh transcripts in HepG2 cells exposed to the same dose of
-NF, and 2) to the
-NF-induced increase in luciferase expression
in cells transfected with the full-length
3802/GCSh5
-luc. Other potential elements,
including NF-
B, ARE1, and ARE2, failed to direct a
-NF-induced
increase in luciferase expression.
Of the four putative ARE sequences identified in the 5-flanking
sequence of the GCSh gene, only ARE4 influenced both
constitutive and induced gene expression in a fashion typical of other
functional AREs. In contrast, ARE3 enhanced constitutive expression
only, although it did modulate both the constitutive and inducible
properties of ARE4. In order to confirm that ARE4 functions as a true
ARE, a synthetic oligonucleotide containing a single base mutation in
the consensus core ARE sequence of ARE4 (converting the sequence from
5
-G
GACTCAGC-3
to
5
-G
GACTCAGC-3
; Fig. 5) was synthesized and
cloned into pT81. The T in this position has been shown to be a
required element for the maintenance of both the constitutive and the
inducible properties of AREs. When this vector (GCShARE4m)
was transfected into HepG2 cells, constitutive expression was
significantly reduced (Fig. 6A), and
-NF induction was
eliminated (Fig. 6B) in cells transfected with the mutated ARE4 fusion gene.
To determine whether ARE4 functions in the same capacity when present
at its distal location in the genomic sequence of the GCSh
gene 3.1 kb upstream of the transcription start site, the same
single-base mutation was introduced into the full-length fusion
reporter gene 3802/GCSh5
-luc by site-directed
mutagenesis. The mutation of this single base within this 3.8-kb
GCSh fragment likewise reduced constitutive expression and
eliminated induction in response to
-NF
(
3802mA4/GCSh5
-luc in Fig. 3).
Diverse chemical compounds, including Michael reaction acceptors, quinones, diphenols, peroxides, isothiocyanates, mercaptans, heavy metals, arsenicals, and certain planar aromatic and metabolizable polycyclic aromatic hydrocarbons, induce the expression of phase II detoxifying enzymes (8, 30). The induction of several genes in the phase II battery, including the rat (31) and mouse (28) glutathione S-transferase-Ya genes, rat (41) and human (42) NAD(P)H quinone oxidoreductase genes, and the rat GST-P gene (43, 44), is mediated through an antioxidant responsive element (or its functional equivalent, an electrophile responsive element, EpRE), in the promoter region of the gene (27). It is hypothesized that these inducing agents share in common the ability to generate reactive oxygen intermediates directly or via redox cycling (28, 31) and result in activation of transcription factors, perhaps through alterations in their redox status. While this mechanism is favored by some, Talalay and colleagues (8) point out that all of these inducers are capable of generating electrophilic intermediates capable of reacting with sulfhydryl groups. They therefore suggest that a better designation for these responsive elements would be EpREs to reflect the nature of the active intermediates.
Interestingly, exposure to several of the agents that induce expression
of the ARE-containing phase II genes results in an elevation in
intracellular GSH levels as well. These increases are frequently
coupled to an increase in the activity of GCS, which led us to
hypothesize that the increased GSH levels might result from
transcriptional up-regulation of GCS expression mediated by ARE-like
elements in the 5-flanking region of the GCS catalytic subunit and/or
light subunit genes. Previous cloning efforts identified an ARE-like
sequence in the promoter of the GCSh gene (26), and three
additional potential ARE sequences were found further upstream in the
present studies. In addition to sequence data, this hypothesis is
supported by several other recent lines of evidence. Based on the
demonstration that GCS expression in rat lung epithelial cells is
inducible by exposure to the pro-oxidant, 2,3-dimethoxy-1,4-naphthoquinone, Shi et al. (13) suggested the possibility that transcription of the rat GCSh gene is
regulated by an ARE-like element. Similarly, Liu et al. (9)
predicted the existence of an EpRE in the murine GCS gene. They
demonstrated that exposure to the synthetic indolic antioxidant,
5,10-dihydroindeno[1,2-b]indole, increased GCS activity
and GSH levels in mouse hepatoma cells. Borroz et al. (16)
also postulated the existence of an ARE in the GCSh gene of
the rat after demonstrating transcriptional up-regulation of the
GCSh subunit in murine liver following dietary
treatment with the antioxidant
2(3)tert-butyl-4-hydroxyanisole.
Functional AREs display two cardinal properties; they enhance basal
expression and are inducible by phase II enzyme inducers (27). Using
these criteria, the current study provides the first direct evidence
that a functional ARE is present in the 5-flanking region of the human
GCSh gene. Despite the fact that four ARE-like sequences
were identified on the basis of sequence similarity to the ARE
consensus core sequence, only two, ARE3 and ARE4, contributed to basal
expression, each augmenting expression of luciferase in the presence of
the tk minimal promoter by ~20-fold. Interestingly, when
cloned into pT81 in tandem in their native configuration, separated
only by 34 bp, these two sequences enhance basal expression greater
than 100-fold. However, this potent enhancer activity is dampened by
the inclusion of additional DNA, such that in the full-length
GCSh5
sequence, presence of the native context ARE3/ARE4 tandem only doubles basal expression (i.e.
3802/GCSh5
-luc versus
2752/GCSh5
-luc).
Both ARE3 and ARE4 match the consensus 11-nucleotide ARE motif
described by Pickett et al. (31, 45), yet only the ARE4 oligonucleotide directed enhanced expression following exposure to
-NF. Since these two enhancers only differ by two nucleotides, in
the region of the core (NNN) previously demonstrated to be of no
consequence in influencing either basal or
-NF induction (31, 45),
it is not immediately obvious why ARE3 failed to respond to
-NF
induction. Analyses of functional AREs in other genes have revealed
that the organization of these enhancers may be more complex than the
simple linear arrangement of the "core" nucleotides, often
involving proximal nucleotides flanking either side of the core motif
itself. Furthermore, functionality of specific AREs may be dependent on
the spatial arrangement of these multiple partners. As illustrated in
Fig. 7, AREs in the rat GSTP (43) and GST-Ya (31) genes,
the murine GST-Ya (28) gene, and rat (41) and human (42) NQO1 genes
include an adjacent pair of TRE or TRE-like elements. Favreau and
Pickett (45) divided the 31-base pair rat NQO1 gene ARE into three
regions as follows: proximal and distal half-sites of a 13-base pair
palindromic sequence and a 3
-flanking region containing 4 adenines.
Mutations in these various regions influenced basal and induced
expression in different ways supporting the conclusion that the two
half-sites are not functionally equivalent. While only the proximal
half-site was required for binding of nucleoproteins to the ARE, both
half-sites contribute to full basal gene expression. However, only
three nucleotides in the proximal site were absolutely required for maximal basal and induced transcription (45). Considering this complexity, it is possible that selection of the 20 base pairs used to
create the ARE3 oligonucleotide might have omitted additional neighboring elements that are required for inducibility in
situ. However, no evidence for comparable elements around ARE3 was
found. Even though ARE3 doesn't appear to function as an independent ARE and despite the fact that mutation of ARE4 within the ARE3/ARE4 tandem eliminated basal and
-NF-induced expression by
3802/GCSh5
, the current data are insufficient to
preclude the possibility that an interaction between ARE3 and ARE4 is
required for regulation of GCSh gene expression in
situ. Further mutational analysis of the ARE3/ARE4 tandem is being
conducted to clarify the relationship between these two sequences.
ARE4 is the only element to fulfill the functional requirements of an
ARE in terms of both basal and inducible expression. Identification of
ARE4 as a functional element was confirmed by mutational analysis in
which conversion of the sequence
5-G
GACTCAGCA-3
to
5
-G
GACTCAGCA-3
in the ARE4 oligonucleotide
and in
3802/GCSh5
-luc effectively eliminated
constitutive and
-NF-induced activity in both cases, as demonstrated
in the functional characterization of other AREs (31, 45). Abolishment
of the basal and induced expression directed by
3802/GCSh-5
by introduction of this mutation provides
strong evidence that this sequence is involved in the regulation of
expression of the endogenous GCSh subunit gene in situ. Hence, all or part of ARE4 is necessary for basal and
-NF-induced expression of the gene.
Although ARE4 contains a consensus ARE core sequence, this element also
includes a canonical TRE motif (5-TGA(C/G)T(C/A)A-3
), an arrangement
also found in AREs from the human NQO1 (42) and murine heme oxygenase
(46) genes (Fig. 7). Since the mutation we introduced in ARE4 corrupted
both the ARE and TRE sequences, we have not as yet distinguished
experimentally which of these responsive elements is involved in the
expression of the GCSh gene. Evidence from other ARE
analyses favors the involvement of the ARE core, however. In analyzing
the elemental requirements of the ARE in the human NQO1 gene, Xie
et al. (32) reported that the sequence
5
-GATGAGTCAGCC-3
, containing a single TRE motif (in
boldface), but not an ARE failed to increase expression in transfected
HepG2 cells in the response to
-NF. Similarly, Prestera et
al. (46) demonstrated that maintenance of the ARE consensus was
required for the induction of murine HO-1 gene expression in response
to a battery of phase II inducers. Mutation of the TRE sequences had no
influence on inducibility, provided the ARE core sequence was retained.
In a recent characterization of the ARE sequence in the rat NQO1 gene,
Favreau and Pickett (46) mutated (underlined) the existing sequence,
5
-GTGACT
GCA-3
, to one containing a TRE motif,
5
-GTGACT
GCA-3
. When the mutant oligonucleotide was used
in electrophoretic band shift assays, unique nuclear proteins
recognizing either the ARE or the TRE sequence were detected in HepG2
nuclear extracts. Since this mutated sequence matches the core sequence
of ARE4 with the exception of an A to G substitution at the 3
-terminal
nucleotide, a substitution that does not influence binding or ARE
function (45), these data provide evidence that proteins capable of
binding the ARE4 sequence are present in HepG2 cells. In preliminary
gel shift experiments,2 binding activity to
a probe corresponding to ARE4 is elevated in nuclear extracts from
-NF-treated HepG2 cells; however, an increase in TRE-binding
activity is not detected. This ARE4 binding activity is effectively
competed by unlabeled ARE4 probe but not by a probe containing a
consensus TRE sequence, providing further evidence to support the
importance of the ARE sequence in mediating basal and
-NF-induced
expression of GCSh. Definitive identification requires
completion of additional mutational analyses currently in progress.
Finally, the location of a functional ARE in a position 3.1 kb distal to the transcription start site is atypical by comparison to the position of most other AREs, which have been typically identified proximal to the transcription origin in phase II enzymes. However, Prestera et al. (46) recently identified a functional ARE 4.1 kb upstream in the human heme oxygenase-1 gene, capable of regulating constitutive and xenobiotic induced expression of this important stress response gene. Collectively these observations in two different genes indicate that AREs can exert an effect on gene transcription over a long expanse of intervening sequence.
In summary, constitutive and -NF-induced expression of the GCS heavy
subunit gene, at least in part, is mediated by a distal sequence
including a consensus ARE motif containing an embedded TRE. Experiments
designed to determine specific sequences involved in GCSh
expression as well as to identify additional elements that may
contribute to the composite regulation of this important gene are in
progress.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) L39773[GenBank].