From the Department of Human Oncology, University of Wisconsin Medical School, Madison, Wisconsin 53792
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ABSTRACT |
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Exposure of HepG2 cells to -naphthoflavone
(
-NF) results in time- and dose-dependent increase in
the steady-state mRNA levels for both the catalytic
(GCSh) and regulatory (GCS1) subunits of
-glutamylcysteine synthetase (GCS) which catalyzes the rate-limiting step in the de novo synthesis of the cellular antioxidant
glutathione (GSH) (Mulcahy, R. T., Wartman, M. A., Bailey,
H. B., and Gipp, J. J. (1997) J. Biol. Chem.
272, 7445-7454). Cloning and sequencing of the GCS1
promoter region is reported. Regulatory sequences mediating basal and
-NF induced expression of the GCSl gene were identified
using a series of promoter/reporter fusion genes transfected into HepG2
cells. Sequences directing basal and
-NF induced expression were
localized between nucleotides
344 and
242 (numbered relative to the
translation start site).
Mutational analyses indicate that basal expression of the
GCSl gene is directed by a consensus AP-1-binding site
located 33 base pairs upstream of a consensus electrophile responsive
element (EpRE) sequence; both cis-elements are capable of
supporting -NF inducibility. Elimination of the inducible response
requires simultaneous mutation of both sequences, however, in the
presence of an intact EpRE the upstream AP-1 site is irrelevant to
induction. Regulation of expression of both human GCS subunit genes in
response to
-NF is therefore mediated by cis-elements
satisfying the consensus core EpRE motif.
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INTRODUCTION |
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Glutathione (L--glutamyl-cysteinyl-glycine;
GSH)1 is the prominent
cellular non-protein thiol, typically present in millimolar concentrations in most cell types (1, 2). GSH is a predominant cellular
antioxidant and as such serves critical functions in the maintenance of
cellular redox balance, provides protection against reactive oxygen
species, and is involved in the detoxification of xenobiotics either
through direct conjugation with reactive species or through enzymatic
reactions catalyzed by glutathione S-transferases (3). GSH
is frequently elevated in normal cells upon exposure to xenobiotics and
has been implicated in tumor cell resistance to alkylating agents, Pt
compounds, and anthracyclines (4, 5). Recent evidence (6-13) suggests
that increase in steady-state GSH levels in stressed cells is related
to increased activity of
-glutamylcysteine synthetase (GCS), which
catalyzes the rate-limiting step in the de novo synthesis of
GSH from its constituent amino acids (14).
In vivo the functional GCS holoenzyme exists as a heterodimer consisting of catalytic (heavy, Mr = 73,000) and regulatory (light, Mr = 27,700) subunits which can be dissociated under nondenaturing conditions (15). Studies by Meister (14) have demonstrated that all the catalytic activity and the site of GSH feedback inhibition reside with the heavy subunit. However, the kinetic properties of the heavy subunit under physiological conditions are greatly influenced by association with the light subunit, presumably mediated by a redox-sensitive disulfide bond between the two subunits (16). The influence of the regulatory subunit on the kinetic properties of the catalytic subunit is so profound that Huang et al. (16) hypothesized that the monomeric catalytic subunit would be nonfunctional at the substrate (glutamate) and inhibitor (GSH) concentrations typically existing in cells. Our laboratory has cloned the cDNAs for the human liver GCSh (17) and GCSl (18) subunits and recently reported the cloning and sequencing of the GCSh gene (19). We now report cloning and sequencing of the promoter and 5'-flanking sequence of the GCS light subunit gene.
Steady-state levels of mRNA corresponding to the heavy and the
light subunits of GCS have been reported to be elevated after exposure
of cells to various xenobiotics such as -NF (19), methyl mercury
(20), tert-butyl hydroquinone (11), and butylated hydroxyanisole (13). Since several of these treatments also induce the
expression of key Phase II detoxifying enzymes (21), we hypothesized
that the transcriptional up-regulation of the GCS subunit genes and
genes of the Phase II battery may be mediated by common regulatory
elements. Regulation of several Phase II enzymes in response to a wide
variety of inducing agents, including several of those which induce GCS
expression, is mediated, at least in part, by the presence of
electrophile responsive elements (EpRE)2 within the
5'-flanking region of the gene (21-28). Our laboratory has recently
demonstrated that basal and
-NF-inducible expression of the
GCSh gene is mediated by a consensus EpRE sequence located in the distal portion of the promoter of the GCS heavy subunit gene
(19). We have also identified numerous potential regulatory elements,
including a putative EpRE in the 5'-flanking sequence of the
GCSl gene, suggesting the possibility that the expression of the regulatory subunit gene may similarly be mediated by one or more
EpRE or EpRE-like sequences. We have also demonstrated that exposure of
HepG2 cells to
-NF, a planar aromatic compound capable of inducing
gene expression via EpREs, results in increased expression of the
GCSl gene, as well as the GCSh subunit gene (19). Although this observation is consistent with the hypothesis, the
involvement of any specific cis-acting elements in
constitutive and/or induced expression of the GCSl gene has
not been established.
Therefore, in this study we utilized a deletion mutagenesis strategy to
identify the regulatory elements required for -NF inducible
expression of the GCSl gene. Using a series of progressive promoter deletion/reporter transgenes transfected into HepG2 cells, we
have been able to discern that the EpRE sequence identified in the
GCSl promoter is required for maximal induction in response to
-NF and that a neighboring AP-1 site located 33 bp upstream of
the EpRE mediates constitutive expression of the gene and is also
capable of directing increased expression following
-NF exposure
should the core EpRE sequence be mutated.
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EXPERIMENTAL PROCEDURES |
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GCSl Genomic DNA and Sequencing-- Two clones containing genomic sequence corresponding to the light subunit of human GCS and its 5'-flanking region were isolated from a human foreskin fibroblast P1 library by Genome Systems, Inc. (St. Louis, MO) as described previously (29), utilizing polymerase chain reaction primers corresponding to specific sequences present in GCSl cDNA (18). A PstI fragment containing 63 nucleotides of GCSl coding sequence and approximately 6-kb of 5'-flanking sequence was subcloned into pSKII-Bluescript (CLONTECH) and subsequently sequenced. Sequencing was performed by the dideoxynucleotide method using Sequenase (U. S. Biochemical Corp.) and synthetic oligonucleotide (20-mers) primers corresponding to internal sequences. The nucleotide sequence was verified by multiple bidirectional sequencing reactions.
Recombinant Plasmids--
Expression vector constructs were
created by cloning restriction fragments isolated from the 5'-flanking
sequence of the GCSl gene into pGL3-Basic vector (Promega)
for determination of the promoter activity or by introducing
GCSl DNA fragments or synthetic oligonucleotides into pT81
(ATCC 37584) for determination of enhancer activity. A 6.0-kb genomic
DNA fragment was isolated from 5'-flanking region of GCSl
by XhoI restriction digestion and cloned into the XhoI site of pGL3-Basic creating the recombinant plasmid
6000/GCSl5'-luc. This plasmid was subjected to
digestion with additional restriction enzymes to generate a series of
deletion mutants as detailed in Fig. 2. A series of promoter/reporter
transgenes containing mutations in regions of interest within the
GCSl promoter (Fig. 4A) were prepared by
site-directed mutagenesis. The sequence of the final constructs were
verified by dideoxynucleotide sequencing.
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 4 × 105/35-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 amounts of plasmid DNA were used to compensate for variations in plasmid size. To correct for transfection efficiency, 1.5 µg of
the reporter plasmid pCMV (30) containing the lacZ gene encoding
-galactosidase under the control of the human
cytomegalovirus immediate-early promoter/enhancer was co-transfected
with each recombinant plasmid. Four hours after addition of DNA, cells
were shocked by the addition of media containing 10% glycerol for 3 min at room temperature and then maintained at 37 °C for an
additional 24 h in complete medium. At the conclusion of this
incubation period, the medium was once again replaced with the 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, plates 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 the top speed in a microcentrifuge for 2 min at 4 °C. The
resulting supernatants were transferred to Eppendorf tubes and stored
on ice pending assay.
Biochemical Assays--
-Galactosidase activity was
quantified as described by Rosenthal (31). Briefly, this assay monitors
cleavage of
o-nitrophenyl-
-D-galactopyranoside and yields
-galactosidase units as (OD420 × 380)/t,
where t = time in minutes at 37 °C and 380 converts
OD420 to micromoles of
o-nitrophenyl-
-D-galactopyranoside.
Statistics-- Differences between experimental groups were compared by analysis of variance using Fisher's protected least significant difference.
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RESULTS |
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Sequence Analysis--
Two genomic clones containing the
full-length human GCSl gene were obtained by polymerase
chain reaction screening of a human foreskin fibroblast P1 genomic
library (Genesystem, Inc.) using a pair of polymerase chain reaction
primers spanning a 300-bp region of the 3'-end of the GCSl
cDNA. A 6-kb PstI fragment from one clone was identified
by in-gel hybridization using three oligonucleotide probes
complimentary to sequences in the 5'-region of the GCSl cDNA, isolated from agarose gels, and subcloned into
pSKII-Bluescript. Sequence analysis revealed that this fragment
contained 316 bp corresponding to the 5'-end of the GCSl
cDNA and ~5.7-kb of 5'-flanking sequence. 3.3-kb of the 3'-half
of the fragment was sequenced (Fig. 1).
The 5'-flanking region of the GCS light subunit gene shares several
characteristics typical of many "housekeeping" genes, including a
large proportion of GC residues and several putative Sp-1 binding
sites. Multiple consensus AP-1, as well as several AP-1-like, sites
were also identified. No nuclear factor-B-binding sites and no core
sequence corresponding to the AhRE were identified. Sequence analysis
did, however, identify a core EpRE motif at
290 to
301. The
5'-flanking sequence of the GCS light subunit gene also contains a
consensus metal responsive element, which directs induction of some
genes, such as metallothionein-1, in response to exposure to heavy
metals and H2O2. Since the gene utilizes
multiple start sites which vary among cell lines examined and which are
not associated with consensus TATA sequences, we have adopted a
numbering convention which assigns the number +1 to the first base of
the translation start codon.
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Functional Analysis of the 5'-Flanking Region of the
GCSl Gene--
In previous experiments, we have
demonstrated that the steady-state mRNA levels for both the
GCSh and GCSl genes are increased in HepG2
cells after treatment with as little as 10 µM -NF
(19), reaching peak levels at approximately 12 h (about 3-fold
increase compared with the untreated cells) after the addition of
-NF. To localize regions of the GCSl 5'-flanking region
controlling basal and
-NF inducible expression of the
GCSl gene, a series of deletion mutant/luciferase reporter
fusion genes were generated by cloning various length restriction
fragments derived from the GCSl clone into the luciferase
reporter vector, pGL3-Basic (Fig. 2).
These constructs were transiently transfected into HepG2 cells and
luciferase activity was determined in the presence and absence of 10 µM
-NF.
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Analysis of Synthetic Oligonucleotides Containing the
GCSl EpRE--
Analysis of the sequence between 208 and
344 of the GCSl promoter revealed the presence of a
consensus (5'-(A/G)TGACNNGCA-3') EpRE core sequence between
290 and
301. Since EpREs have been demonstrated to enhance constitutive and
-NF-induced expression of Phase II enzymes and we previously
demonstrated that a distal EpRE was responsible for these activities in
the case of the human GCSh subunit gene (19), we considered
this element a candidate enhancer of GCSl gene expression.
In order to determine whether this potential responsive element
contributed to basal and
-NF induced expression of the
GCSl gene, an oligonucleotide (GCSl EpRE)
spanning nucleotides
308 to
281 of the GCSl promoter
was synthesized and subcloned into the SacI site of pT81, a
luciferase reporter vector in which luciferase expression is under
control of the herpes simplex I thymidine kinase (tk)
minimal promoter. A second oligonucleotide containing the functional
EpRE sequence (31) from the human NQO1 gene (NQO1hARE) was subcloned
into the same vector and used as a positive control. When transfected into HepG2 cells, vectors containing the GCSl EpRE sequence
in either orientation directed a significant increase in basal
luciferase activity relative to that detected in cells transfected with
pT81. The magnitude of the increase was comparable to that observed in
HepG2 cells transfected with the NQO1hARE-luc transgene
(12-15-fold higher than pT81 alone).
-NF treatment of HepG2 cells
transfected with these constructs resulted in a 2.5-fold induction of
luciferase activity. The pT81 vector alone did not respond to
-NF
(Fig. 3). Thus, the GCSl EpRE
oligo functioned like other EpREs, directing both increased basal and
inducible expression of the reporter gene in response to
-NF
treatment.
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Mutation of the Putative EpRE Within the Native Genomic 5'-Flanking
Sequence--
In an attempt to confirm that the GCSl EpRE
mediates GCSl gene expression in vivo, the same
GCSl EpRE point mutation described above was introduced
into 1927/GCSl5'-luc by site-directed
mutagenesis, generating the mutant m1 (Fig.
4A). The transgenes containing the wild-type (wt) and mutated genomic 5'-flanking sequences were then
transfected into HepG2 cells and their effects on basal and
-NF-induced expression of the reporter gene were compared. The introduction of the EpRE core-disrupting mutation into the genomic sequence failed to significantly (p < 0.05) alter
constitutive expression in comparison to that observed in HepG2 cells
transfected with the transgene containing the wild-type sequence (Fig.
4B). More importantly, luciferase expression in cells
transfected with the mutant transgene was still significantly
(p = 0.04) induced following incubation with 10 µM
-NF, although the level of expression was only
~60% of that detected in
-NF-treated cells transfected with the
wild-type
1927/GCSl 5' sequence. Hence,
cis-acting elements within the
344:
208 region of the
gene, other than the core EpRE, apparently contribute to
-NF
inducibility.
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DISCUSSION |
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Constitutive expression of the human GCSl gene is
mediated by at least two distinct cis-elements or groups of
elements, one within the 344:
242 fragment and a second within the
712:
344 fragment. The former fragment includes a consensus EpRE
motif, GCSl EpRE, at
301:
291 and a consensus AP-1
binding motif positioned 32 bp upstream, at position
340:
334. The
upstream AP-1 site at
340:
334 was identified as the sole
cis-element within the
344:
242 fragment responsible for
the basal enhancing properties attributable to this fragment.
Additional deletion analyses are required to definitively establish the
identity of specific elements responsible for the enhancing properties
associated with the
712:
344 fragment. These elements are sufficient
to account for the residual basal activity observed in cells
transfected with transgenes containing the mutations of the
340:
334
AP-1 site (i.e. all m4 series mutants).
The identification of a functional role for AP-1 in regulation of the constitutive expression of the GCSl subunit gene is consistent with recent evidence from other groups which likewise suggest that AP-1 is an important component in the regulation of GCS gene expression. Transcriptional regulation of the GSH1 gene, the yeast GCS homolog, has been shown to be mediated by yAP-1 (34), the biochemical homolog of mammalian AP-1. In fact, basal and hydrogen peroxide induced expression of the yeast GSH1 gene has been shown to be yAP-1 dependent (35). Sekhar et al. (36) recently suggested a similar AP-1 dependence for expression of the human GCS heavy subunit gene based on the observation that the constitutive expression of the GCSh gene and steady-state GSH levels were reduced in fibroblasts derived from c-Jun null mice by comparison to wild-type murine fibroblasts. Yao et al. (37) reported that overexpression of the GCS heavy subunit mRNA in a cisplatinum-resistant ovarian carcinoma cell line was secondary to constitutive overexpression of c-Jun and a concomitant increase in AP-1 binding activity. Similarly, mutational analyses and preliminary gel shift studies performed in our laboratory also suggest a role for AP-1 family members in regulation of basal expression of both GCS genes.4 It is therefore likely that the basal and induced expression of both GCS subunit genes involves AP-1 transcription factors, acting alone, or more likely, in concert with other trans-acting factors.
Although evaluation of basal regulation was necessary to establish
baseline expression levels, the major thrust of the current studies was
to define those cis-elements which function to control -NF induced expression of the GCSl subunit gene. By
analogy to the GCSh subunit promoter, the EpRE at
301:
291 of the GCSl 5'-flanking sequence was considered
a strong candidate for this activity. This possibility was supported by
the pT81 enhancer studies which clearly demonstrated that the EpRE
sequence could indeed support induction following
-NF exposure.
However, mutation of this EpRE sequence in the 1.9-kb 5'-promoter
fragment isolated from the GCSl gene resulted in a
reduction in
-NF responsiveness, but it did not completely eliminate
induction as was true in the case of the EpRE in the heavy subunit
promoter (19). The use of individual and tandem mutants unequivocally
demonstrated that the residual inducibility characteristic of the
GCSl EpRE-disrupting mutants, m1 and m2, was attributable
to the presence of the upstream AP-1 site. Addition of a mutation to
the upstream AP-1 site to these single mutants (i.e. m1m4,
m2m4) abolished the
-NF response. Ablation of the
-NF
inducibility therefore required simultaneous mutation of both of these
cis-elements. Hence, individually in either the
GCSl EpRE or the upstream AP-1 site is sufficient to direct
the induced response, and only one of them is absolutely necessary.
The magnitude of induction following -NF exposure in cells
transfected with either the m4 or m3m4 transgenes was comparable to
that observed in cells transfected with fusion genes containing both
intact elements (wt, m3), suggesting that the presence of the EpRE
sequence is dominant and able to produce the full induced response
regardless of the status of the upstream AP-1 site. It is therefore
difficult to ascertain the role that the upstream AP-1 element might
play in
-NF responsiveness in the presence of an intact EpRE
in situ. However, the apparent regulatory redundancy provided by this arrangement of tandem cis-responsive
elements may provide an important safeguard against the potentially
catastrophic effects of mutations to the regulatory region of this
vitally important gene. Although regulation of their expression does
not appear to be entirely analogous, the data for the two GCS subunit genes clearly indicate that the induction of gene expression provoked by
-NF exposure is mediated by similar cis-elements
present in the promoter regions of the two subunit genes.
Despite its identity with the core EpRE consensus sequence and its EpRE-like functional properties evident in enhancer studies, it is possible that the GCSl EpRE, unlike its GCSh counterpart, does not function as an EpRE in situ. This is suggested by the observation that this element is not involved in the regulation of constitutive expression of the GCSl gene when cloned into the promoter/reporter vectors as part of a large fragment of genomic DNA. This activity is consistently observed as a functional property of "true" EpREs described in other genes. In this regard, it is interesting to note that the GCSl EpRE sequence is very similar to the recently defined TRE-type Maf recognition (38, 39) element (T-MARE, 5'-TGCTGACTCAGCA-3'). This motif supports binding of homo- and heterodimers of small Maf proteins. The sequence also resembles an NF-E2-binding site (5'-TGCTGACTCAC-3') which is recognized by the NF-E2 family of transcription factors, including Nrf1 and Nrf2 (40, 41). The small Maf proteins form homodimers which exert negative regulatory influences when bound to their cognate binding sequence (42, 43). On the other hand, small Maf proteins also contribute to positive regulation of gene expression by forming heterodimers with numerous other bZIP transcription factors, including members of the AP-1 superfamily as well as Nrf1 and Nrf2 (38, 39, 42, 43).
It is conceivable, therefore, that regulation of the GCSl
gene might involve binding of Maf and Nrf factors to the sequence we
have referred to as the GCSl EpRE. In recent supershift
assays, we have been able to demonstrate the increased binding of Nrf-2 to the GCSl EpRE sequence in response to -NF
treatment,5 strengthening
this supposition. Similar hypotheses have recently been proposed for
other genes containing EpRE-related sequences, including the human NQO1
(44) and murine heme-oxygenase 1 genes (45). Itoh et al.
(46) recently demonstrated that regulation of several glutathione
S-transferase isozymes in the mouse is dependent on the
Nrf2 and MafK expression and binding of heterodimers to the GST
EpRE sequence, providing the most direct evidence that these factors
might contribute to the constitutive and induced expression of genes
containing EpRE-related motifs. Perhaps what has been referred to as
EpREs are in fact members or subsets of the MARE or NF-E2 family of
binding sites.
In summary, basal expression of the GCSl gene is influenced
by a consensus AP-1-binding site located 33 bp upstream of a consensus EpRE sequence in the proximal region of the promoter. The latter element is not required for maximal basal expression. Both of these
cis-elements are capable of supporting induction following -NF exposure, although the EpRE element appears to the be more potent of the two. Elimination of the inducible response requires simultaneous mutation of both sequences. The current studies provide a
solid foundation to support current efforts to identify specific trans-acting factors involved in regulation of GCS subunit
gene expression and the signaling cascades responsible for their
activation. Finally, it is possible that members of the Maf, Nrf, and
AP-1 family of proteins are involved in regulation of these important genes.
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FOOTNOTES |
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* This work supported by National Institutes of Health Grant R01-CA57549.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U72210.
To whom all correspondence should be addressed: University of
Wisconsin-Madison, Dept. of Human Oncology, K4/316, 600 Highland Ave.,
Madison, WI 53792. Tel.: 608-263-3695; Fax: 608-263-9947; E-mail:
mulcahy{at}mail.bascom.wisc.edu.
1
The abbreviations used are: GSH, glutathione;
AhRE, aromatic hydrocarbon responsive element; AP-1, activation
protein-1; -NF,
-naphthoflavone; EpRE, electrophile responsive
element; GCS,
-glutamylcysteine synthetase; GCSh, GCS
heavy subunit; GCSl, GCS light subunit; bp, base pair(s);
kb, kilobase pair(s); NQO1, human NAD(P)H quinone oxidoreductase.
2 In this report we use the designation EpRE to indicate elements matching the consensus sequence 5'-(A/G)TGACNNNGCA-3'. These elements have also been referred to as antioxidant responsive elements (AREs).
3 K. S. Chen and M. N. Gould, unpublished data.
4 A. C. Wild and R. T. Mulcahy, unpublished data.
5 H. R. Moinova and R. T. Mulcahy, unpublished data.
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REFERENCES |
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