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J Biol Chem, Vol. 274, Issue 34, 23887-23892, August 20, 1999
From the Department of Molecular Biology and Research Center for
Cell Differentiation, Seoul National University,
Seoul 151-742, Korea
Copper/zinc superoxide dismutase
(SOD1) protects cells against oxidative hazards by the
dismutation of superoxide radicals. The promoter activity of the
SOD1 gene was increased 3-5-fold by hydrogen peroxide,
paraquat (PQ) and heat shock. Functional analyses of the regulatory
region of the SOD1 gene by deletions, mutations, and
heterologous promoter systems confirmed the induction of the
SOD1 gene by H2O2 through the
hydrogen peroxide-responsive element (HRE) (between nucleotides Copper/zinc superoxide dismutase
(SOD1)1 is one of the major
cellular defense enzymes that perform a vital role in protecting cells
against the toxic effect of superoxide radicals. It catalyzes the
dismutation of superoxide radicals (O Stress conditions (i.e. oxidative stresses, heat shock,
osmotic stress, and toxic metals) are deleterious to normal cellular function. To survive those environmental and physiological stresses, all organisms possess specific defense systems to protect themselves from various stresses. Aerobic organisms are continuously exposed to
oxygen, which renders them prone to damage generated by oxygen-derived free radicals. Oxidative stress is largely mediated by reactive oxygen
species, including superoxide anion (O Cells employ a number of defense mechanisms to sense and respond
appropriately to oxidative stress. Enzymes such as superoxide dismutases and catalases play critical roles in oxidative stress protection through catalyzing the conversion of reactive oxygen species
to less harmful products (12, 14). As has been demonstrated in yeast,
cells lacking a functional gene encoding SOD1 are highly sensitive to
dioxygen and redox-cycling drugs, fail to grow on respiratory carbon
sources, and exhibit increased spontaneous mutagenesis rates and a
number of other phenotypes during aerobic growth (15). Free radical
scavenging activities are also exacerbated by small antioxidant
molecules, including glutathione, thioredoxin, and ascorbic acid (12).
In addition to the prevention of oxidative damage, repair mechanisms
are employed by cells to remove or repair damaged cellular components,
including exo- and endonucleases for DNA damage repair, proteolytic
enzymes for degradation of severely damaged proteins, and
phospholipases glutathione peroxide/transferase/reductase for
degradation and repair of damaged lipids (16).
Superoxide dismutase plays a key role in protection from the damage
caused by oxygen radicals. The SOD1 expression is highly induced during environmental stresses. However, little is known about
how the environmental signal is transmitted to a transcriptional regulator in cells during oxidative stress such as hydrogen peroxide, paraquat (PQ) and heat shock. In this study, the induction of the
SOD1 gene by hydrogen peroxide, PQ, and heat shock was
investigated by the transfection of CAT expression vectors containing
the rat SOD1 promoter. The deletion, mutation, and
heterologous promoter systems were used to identify
cis-elements that respond to hydrogen peroxide, PQ, and heat
shock for minimizing hazardous effects. A specific interaction of the
hydrogen peroxide-responsive element (HRE) binding protein and heat
shock element (HSE) binding protein (heat shock factor) was also
investigated by gel mobility shift assays. The results demonstrated
that the SOD1 was inducible by H2O2
through the HRE and by PQ and heat shock through the same HSE to
protect cells from oxidative hazards.
Plasmid Constructions--
The plasmids, containing the upstream
region of the rat SOD1 gene (17) and the chloramphenicol
acetyltransferase structural gene (CAT), were constructed as
described previously (3). For the construction of pHREtk and pHSEtk,
the oligonucleotide of the HRE (between nucleotides Cell Culture and Transfection--
Human HepG2 hepatoma cells
were grown in Dulbecco's modified Eagle's medium/10% fetal calf
serum/penicillin G sodium at 100 units/ml/streptomycin sulfate at 100 µg/ml/amphotericin B at 250 ng/ml. Cells were seeded into 60-mm
plastic culture dishes (30-50% confluence) for 24 h prior to
transfection. An equal amount (3.0 pmol) of the various constructs was
transfected to the cells by the calcium phosphate DNA coprecipitation
method (19). 5 µg of pRSV Chemical and Heat Shock Treatment and Nuclear Extract
Preparation--
Chemicals were added to and heat shock treatment was
carried out on the culture medium at 36 h after transfection, and
the cells were maintained for appropriate times. The various chemicals were evaluated for cytotoxicity over a concentration range of 1-500
µM by monitoring cell death. Nuclear extracts were
prepared by a modified procedure of Andrews and Faller (21).
CAT Assay--
The CAT assay was performed as described
previously (22). The transfected cells were washed twice with
phosphate-buffered saline and harvested. The pelleted cells were
resuspended in 100 µl of 0.25 M Tris-Cl (pH 7.9) and
lysed by three cycles of freezing and thawing. After removing cell
debris by centrifugation, cell extracts were first assayed for
Mobility Shift Assay--
The oligonucleotides for the HRE and
HSE sites were synthesized and labeled with [ Activation of the SOD1 Promoter by Hydrogen Peroxide, PQ, and Heat
Shock--
SOD1 is one of the major cellular defense enzymes
protecting cells against oxidative stress. It was hypothesized that
hydrogen peroxide, PQ, and heat shock are involved in the activation of the SOD1 promoter. H2O2 was used as
a membrane-permeable reagent, which allows studies of the effects of
oxygen radicals in living cells. If hydrogen peroxide is inefficiently
removed or if an excess is present, it can react with superoxide to
generate the highly toxic hydroxyl radical. PQ is a redox-active
compound that is photoreduced and subsequently reoxidized by transfer
of its electrons to oxygen, generating superoxide (O cis-Element Responsible for the Activation of SOD1 Promoter by
H2O2--
When the plasmids pRSP-576 and
pRSP-412 were transfected into HepG2 cells and exposed to
H2O2, the induction with
H2O2 was observed only in the case of
transfection with pRSP-576 (Fig. 3C). However, the induction
with PQ was observed in both cases (data not shown). These results
might suggest that H2O2 and PQ regulate the
SOD1 gene by different regulatory elements and factors. The
region between Heat Shock and PQ Activation of the SOD1 Promoter through the Same
HSE--
The activation of the SOD1 gene by heat shock and
PQ was due to the DNA sequences between HRE Binding Protein and Increased Binding Activity Because of
H2O2--
To determine the nuclear factors
that interact with the HRE sequence, gel mobility shift assays were
carried out by using a double-stranded oligonucleotide corresponding to
HRE as a probe (Fig. 6). When a nuclear
extract prepared from HepG2 cultures was incubated with a
32P-labeled SOD1 HRE probe, one prominent DNA-protein
complex was observed. An HRE oligonucleotide-protein complex
disappeared when unlabeled oligonucleotide (50×, 100×) was added to
the reaction mixture (Fig. 6A, lanes 3 and
4). The formation of the protein-DNA complex was not
inhibited with a mutated HRE oligonucleotide, indicating the
specificity of this protein-DNA interaction. The binding of Elk1 to the
HRE was further confirmed by the result that the protein-DNA complex
was supershifted by the addition of the anti-Elk1 antibody (Fig.
6A, lane 6). These results imply that Elk1 is the
binding protein of HRE. When nuclear proteins prepared from the
H2O2-treated HepG2 cells (2 h) were used, the intensity of the specific complex increased (Fig. 6B). The
binding activity of the HRE-binding factor was also investigated in the cell-free system (Fig. 6C). Nuclear extract prepared from
nontreated HepG2 cells was incubated with H2O2
(200 µM) to examine the variation of its binding activity
to the HRE site. The incubation of the nuclear extract with
H2O2 resulted in increasing the intensity of
the retarded complex (Fig. 6C, lane 3) and
disappearance by competition with the cold probe (Fig. 6C,
lane 4). Because the intensity of the specific HRE-protein
complex was increased by H2O2 treatment in the
cell-free system (Fig. 6C, lane 3), it was also
assumed that hydrogen peroxide treatment increased the binding activity
of the HRE binding protein.
HSE Binding Protein (Heat Shock Factor) and Change of
Binding--
Gel mobility shift assays were used to detect
sequence-specific binding of factors to the SOD1 HSE sequence in
extracts from HepG2 cells. Nuclear extracts were prepared from cells
that had been heat treated at 42 °C for 1 h or maintained at
the control temperature of 37 °C. These extracts were incubated with
a 5' end-labeled oligonucleotide containing an HSE sequence (between sequence In transient expression experiments the activation of the
SOD1 promoter by H2O2 was mediated
through the HRE sequence. To analyze the target of transcriptional
activation of the SOD1 gene, CAT plasmids were constructed
that contain these elements in the upstream region of the thymidine
kinase promoter. From these results, it was shown that the HRE sequence
is one of the targets of transcriptional activation by
H2O2. Also, we have found that superoxides
could activate the SOD1 gene. Because the steady state
concentration of H2O2 in the cell is so low
(1 × 10 To mount an appropriate oxidative stress defense, cells must harbor
oxidative stress sensors. A number of antioxidant responses in bacteria
have been elegantly studied, and it has been established that reactive
oxygen species are directly sensed by key regulatory molecules that
activate the expression of genes encoding antioxidant proteins at the
level of transcription (32-34). Distinct defense mechanisms are
involved in H2O2 and O Heat shock proteins are known to protect cells from thermal and
oxidative injuries as well as other types of injuries (36). In this
study, we have found that the paraquat, O The results presented in this study showed that the rat SOD1
gene was inducible by hydrogen peroxide, paraquat, and heat shock. It
was shown that the HRE sequence is one of the targets of
transcriptional activation by H2O2. Also, it
was found that paraquat and heat shock induced the SOD1 gene
through the HSE. These results establish that heat and PQ are
communicated to HSF to activate gene transcription. The SOD1 enzyme
could function as an inducible protective response against oxidative
stresses. The SOD1 gene was induced by nontoxic ginsenoside
Rb2 through the AP-2 site (3). The diversity of SOD1 inducers implies that there are multiple regulatory
elements for the proper adjustment to various conditions.
*
This work was supported by a grant from Korea Science and
Engineering Foundation through the Research Center for Cell
Differentiation.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 abbreviations used are:
SOD1, copper/zinc
superoxide dismutase;
SOD1, SOD1 gene;
PQ, paraquat;
HRE, hydrogen peroxide-responsive element(s);
HSE, heat shock element(s);
HSF, heat shock factor(s);
CAT, chloramphenicol acetyltransferase;
CAT, CAT gene;
PRE, positive regulatory element.
The Activation of the Rat Copper/Zinc Superoxide Dismutase Gene
by Hydrogen Peroxide through the Hydrogen Peroxide-responsive Element
and by Paraquat and Heat Shock through the Same Heat Shock Element*
ABSTRACT
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INTRODUCTION
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533
and 520). Gel mobility shift assays showed that the existence of an
H2O2-inducible protein bound to the
oligonucleotide of the HRE. Similar analyses showed that the heat shock
activated the SOD1 promoter through the heat shock element
(HSE) (between nucleotides 185 and 171). A strong specific
far-shifted complex with the oligonucleotide of the HSE was observed by
the treatment of heat shock. When cells were treated with PQ, a strong
far-shifted complex with the HSE was observed and was competed out by
the cold HSE probe, indicating that PQ also activated the
SOD1 promoter through the same HSE site. It is very
interesting to note that chemical and physical stresses, such as PQ and
heat shock, respectively, activated the SOD1 promoter through the same cis-element HSE. These results indicate
that the SOD1 was inducible by H2O2
through the HRE and by PQ and heat shock through the same HSE to
protect cells from oxidative hazards.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2) to oxygen and hydrogen peroxide (1). SOD not only prevents the Fenton reaction and DNA nicking
in vitro but also protects against toxicity by
H2O2 in vivo without O2
being directly involved in the generation of DNA damage (2). The
production and/or removal of superoxides has been observed to play a
significant role in a variety of critical homeostatic mechanisms both
at the cellular and organismic levels. Because biological
macromolecules are targets for the damaging action of abundant oxygen
radicals, it is assumed that these increased superoxides should be
initially eliminated by SOD. Therefore, the regulation and induction
mechanism of the SOD1 gene would be of great interest (3).
It has also been reported that SOD1 could prevent oncogenesis and tumor
promotion (4), reduce the cytotoxic and cardiotoxic effects of
anticancer drugs (5), and protect against reperfusion damage of
ischemic tissue (6). A recent report suggested that overexpression of
SOD1 and catalase could increase the average lifespan of the fly (7).
Lutropin, Ca2+, and reactive oxygen seemed to induce SOD1
in rats (8-10).
2),
H2O2, and hydroxyl radical (OH·), which
are intermediates of oxygen reduction generated by metal ion catalyzed
redox reactions, metabolism of chemicals, and normal physiological
activities, including respiration and inflammatory responses to
infection (11). The free radicals generated by these mechanisms cause
severe damage to critical cellular macromolecules, including nucleic
acids, proteins, and lipids (12). Oxidative damage has been strongly
correlated with aging and a number of diseases, including Parkinson's
disease, Lou Gehrig's disease (amyotrophic lateral sclerosis),
rheumatoid arthritis, and cancer (11, 13). Therefore, free radical
levels must be carefully monitored under both physiological conditions
and when generated by environmental stress.
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DISCUSSION
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533 and 520)
and HSE (between nucleotides 185 and 171) sequence of the
SOD1 promoter was cloned into the BamHI site of
pBLCAT2
, which is derived from pBLCAT2. The plasmid pBLCAT2 has a
minimal region (80 to +51) of the herpes simplex virus thymidine
kinase promoter (18). Three copies of the HRE and HSE oligonucleotides
were introduced. The plasmid pmHREtk and pmHSEtk are mutants of pHREtk
and pHSEtk with three copies of the mutated site. Their
sequences are as follows: SOD-HRE, 5'-GATCGGCTTGCCTAGGAAGCGCAAGG-3'/3'-CCGAACGGATCCTTCGCGTTCCCTAG-5'; SOD-mHRE,
5'-GATCGGCTTGCCTACTCTTAGCAAGG-3'/3'-CCGAACGGATGAGAATCGTTCCCTAG-5'; SOD-HSE,
5'-GATCTCGATTCTGGAACTTTCTCAG-3'/3'-AGCTAAGACCTTGAAAGAGTCCTAG-5'; SOD-mHSE,
5'-GATCTCGAGGATGTCCCTGGATCAG-3'/3'-AGCTCCTACAGGGACCTAGTCCTAG-5'. The inser- tion of the HRE and HSE consensus and mutant
sequences was confirmed by DNA sequencing.
-gal plasmid (20) was introduced in all
experiments to correct the variations of transfection efficiency.
-galactosidase activity (23). Equal quantities of proteins were
assayed for CAT activity on the basis of -galactosidase activity.
Extracts were incubated with 0.025 µCi of
[14C]chloramphenicol, 0.25 M Tris-Cl (pH
7.6), 0.4 mM acetyl coenzyme A for 1 h at 37 °C.
The enzyme assay was terminated by adding ethyl acetate. The organic
layer was analyzed by TLC with chloroform/methanol (95:5). After
autoradiography, both acetylated and unacetylated forms of
[14C]chloramphenicol were scraped from the plate, and the
conversion of chloramphenicol to acetylated form was calculated by
measuring radioactivities. The relative CAT activities were calculated
from the percentage conversion. Results are the average of three
independent experiments.
-32P]ATP
and polynucleotide kinase (23). An equal amount (10 µg) of nuclear
extract from each sample was mixed with labeled oligonucleotide for 20 min at 20 °C in a 15-µl solution containing 10 mM
HEPES, 100 mM KCl, 5 mM MgCl2, 1 mM dithiothreitol, 1 mM EDTA, 10% glycerol, and 2 µg of poly(dI-dC). The binding reaction mixtures were
electrophoresed in 6% acrylamide gels in 0.5× TBE (44 mM
Tris, 44 mM boric acid, and 1 mM EDTA). After
electrophoresis, the gels were dried and exposed to x-ray film. For
competition assays, a binding reaction was performed with an excess of
cold probe or competitor DNA. In the supershift assay, the specific
polyclonal antibody against Elk1 (Santa Cruz Biotech. Inc) was added to
the reaction mixture after the binding reaction.
RESULTS
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ABSTRACT
INTRODUCTION
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DISCUSSION
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2). HepG2
cells were transfected with plasmid pRSP-1633, containing the rat
SOD1 upstream region fused to the bacterial CAT. 36 h
after transfection, hydrogen peroxide, PQ, and heat shock were
separately applied to the transfected cells. The transfected cells were
incubated in the presence of 200 µM
H2O2, 50 µM PQ and at a
temperature of 42 °C, respectively. After 2 h, the CAT activity
of each transfected cell was determined (Fig.
1B). These treatments
increased the expression of the SOD1 promoter about 3-fold.
H2O2 and PQ at concentrations of up to 300 and
100 µM, respectively, did not affect cell viability. As
shown in Fig. 2, the
time-dependent activation of the SOD1 promoter
by H2O2, PQ, and heat shock was observed. The
level of CAT activity reached a maximum at 2 h when the cells
transfected with pRSP-576 were treated with
H2O2. Maximum activity appeared at 1 h
with the treatment of PQ and heat shock. It may be possible that the
peak of SOD1 activation occurred earlier than 1 h. When the cells transfected with pRSP-576 were treated with
H2O2, a 3-fold induction of CAT activity was
observed. No induction was observed in cells bearing the plasmid
pRSP-412 (Fig. 3C). These results mean that the activation of the SOD1 gene by
H2O2 may be due to the existence of DNA
elements between 576 and 412. The induction by PQ was observed in
both transfection experiments with pRSP-576 and pRSP-412 (data not
shown). It seems that the cis-element corresponding to
induction by PQ was located downstream of 412 of the SOD1
gene. When the SOD-CAT construct (pRSP-305) was transiently expressed
in HepG2 cells, 4- and 3-fold induction of CAT activity by heat shock
(42 °C) and PQ was observed, respectively (Fig. 3D). No
induction was observed in cells bearing the plasmid pRSP-55 (Fig.
3D). These results mean that the activation of the SOD1 promoter by heat shock and PQ may be due to the
existence of DNA elements between 305 and 56.
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Fig. 1.
Schematic diagram and the effect of
H2O2, PQ, and heat shock on the SOD1
gene expression. A, schematic representation of
the putative transcription factor binding sites with approximate
locations in the SOD1 promoter. CCAAT, CAAT box;
TATA, TATA box. The numbers represent the left
end of the consensus sequence of each transcription factor binding
site. B, activation of the SOD1 promoter
(pRSP-1633) by H2O2, PQ, and heat shock. The
transfected cells were treated with factors
(H2O2, 200 µM for
2 h; HS, heat shock, 42 °C for 1 h;
PQ, paraquat, 50 µM for 1 h). The results
are the means of three experiments.
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Fig. 2.
Time-dependent activation of the
SOD1 promoter by H2O2, PQ, and
heat shock. Cells transfected with pRSP-576 were treated with
H2O2 (A), PQ (B), and
heat shock (HS; C) for 0-4 h, and then CAT
assays were carried out. The results are the means of three
experiments. Relative CAT activity is expressed as compared with CAT
activity obtained in untreated cells.
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Fig. 3.
Effect of H2O2, PQ,
and heat shock on the activation of the SOD1
promoter. A, schematic diagram of the
SOD1 promoter from nucleotides 576 to +85 (pRSP-576) and
from nucleotides 412 to +85 (pRSP-412) attached to CAT. pRSP-576
contains the HRE site, but pRSP-412 does not. B, schematic
diagram of the SOD1 promoter from nucleotides 305 to +85
(pRSP-305) and from nucleotides 55 to +85 (pRSP-55) attached to CAT.
pRSP-305 contains the HSE site, but pRSP-55 does not. The transfected
cells were treated with factors in the same conditions as Fig.
1B. The numbers in the names of plasmids
represent the deletion points of SOD1 5'-flanking sequence.
The results are the means of three experiments. Relative CAT activities
with (+) and without () stress are indicated by the solid
and open bars, respectively.
576 and 413 was previously identified as a positive
regulatory element (PRE) (24). As an initial trial to identify the
transcriptional enhancer element that was involved in
H2O2 induction of the SOD1 gene,
transient assays using CAT constructs were carried out. The plasmid
pPREtk, which has the PRE linked to a herpes simplex virus-thymidine
kinase promoter connected to the CAT gene, and pRSP-576 as a positive
control were introduced into HepG2 cells, and the cells were treated
with H2O2 for 2 h. The CAT activity was
measured, and typical results are shown in Fig.
4B. The results clearly
indicate that the CAT activity of pPREtk increased about 5-fold with
H2O2. The PRE was thought to be a major target
for H2O2 stimuli to induce the SOD1 gene. From the sequence analysis of transcription factor binding sites
in the PRE, the sequence of Elk1, which is the serum response factor
accessory protein (25), was located between 533 and 520 of the
SOD1 gene (24). Induction by H2O2 of
c-fos and possibly other early response genes was mediated
through the activation of the serum response element for the binding
site of serum response factor (26). The 533 to 520 sequence named
as the HRE was tested for induction ability by
H2O2. Double-stranded oligonucleotide containing the HRE sequence was inserted upstream of the thymidine kinase promoter connected to the CAT gene (Fig.
5A). CAT activity was induced
by H2O2 in cells transfected with pHREtk by
3-fold, but CAT activity was not induced by
H2O2 when pmHREtk containing a mutated HRE site
was transfected (Fig. 5C). These results confirmed that the
HRE is responsible for the activation of the SOD1 promoter by H2O2.
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Fig. 4.
Detection of the H2O2
related cis-element. A, schematic
diagram of pRSP-576 and DNA fragment of PRE in the heterologous
promoter (tk) attached to CAT (pPREtk). B, effect
of H2O2 on the SOD1 promoter and PRE
in the heterologous promoter. The results are the means of three
experiments. Relative CAT activity is expressed as compared with CAT
activity obtained in untreated cells.
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Fig. 5.
Transcriptional activation of the
SOD1 promoter by H2O2, heat
shock, and PQ through HRE and HSE. A, schematic diagram
of synthetic HRE (pHREtk) and mutated HRE oligonucleotide
(pmHREtk) in the heterologous promoter (tk)
attached to CAT. B, schematic diagram of synthetic HSE
(pHSEtk) and mutated HSE oligonucleotide
(pmHSEtk) in the heterologous promoter (tk)
attached to CAT. C, effect of H2O2
on the synthetic HRE and mutated HRE in the heterologous promoter.
D, effect of heat shock and PQ on the synthetic HSE and
mutated HSE in the heterologous promoter. The transfected cells were
treated by heat shock (42 °C) for 1 h and PQ (50 µM) for 1 h. Relative CAT activity is expressed as
compared with CAT activity obtained in untreated cells.
305 and 56 (Fig.
3D). From the sequence analysis of transcription factor
binding sites in this region, the heat shock element (NGAAN) (27), was
found to be located between 185 and 171 of the SOD1 gene
(Fig. 1A). Heat shock treatment of murine macrophage
resulted in an enhanced capacity to release superoxide anion
(O2) (28). Also, Omar and Pappolla (29) showed that heat shock
protein synthesis was induced by high levels of superoxide anion.
Therefore, it was assumed that the activation of the SOD1
promoter by heat shock and PQ may be mediated through the heat shock
element. To confirm this possibility, the heat shock element was
synthesized and ligated to the herpes simplex virus minimal thymidine
kinase promoter proximal to the CAT gene (Fig. 5B). The CAT
activity of this construct was analyzed after exposure of cells to
either heat shock or PQ (Fig. 5D). When pHSEtk was
transfected into HepG2 cells, CAT activity was induced by heat shock or
PQ by about a 5-fold. But the CAT activity was not induced by heat
shock or PQ when pmHSEtk containing the mutated HSE site was
transfected. These findings indicate that the HSE is sufficient to
confer the PQ response and that this inducibility is mediated by HSF.
These results establish that the HSF play some kind of role in
eukaryotic oxidative stress protection and suggest that heat and
oxidative stress signals communicate with the HSF to activate gene transcription.
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Fig. 6.
HRE binding protein and increased binding
activity because of H2O2. A,
identification of HRE binding protein. Gel mobility shift assay was
performed using nuclear extracts prepared from untreated HepG2 cells.
The probe corresponding to HRE was labeled at its 5' end. The
DNA-protein complex disappeared by cold competitor (lane 3 and 4) but did not disappear by mutated HRE
(mHRE) and was supershifted by anti-Elk1 antibody
(lanes 5 and 6). B, increased binding
activity of HRE binding protein in vivo. A gel mobility
shift assay was performed using nuclear extracts prepared from
H2O2-treated HepG2 cells (lane 2).
C, nuclear extracts prepared from HepG2 cells grown in the
absence of H2O2 were incubated with
(lanes 3 and 4)
H2O2 in vitro for 2 h.
185 and 171 of the SOD1 gene). In reactions
with extracts from the control cells, two sets of retarded bands were
observed (Fig. 7). Bands produced by
sequence-specific factor binding were identified by competition assays
with unlabeled DNA fragments. Unlabeled HSE fragments competed
efficiently with the formation of a faster-migrating protein-DNA
complex (Fig. 7, lanes 3 and 4). No such
competition was observed with the mHSE oligonucleotide (Fig. 7,
lane 5). The formation of the more slowly migrating complex was not in competition with the HSE fragment. Thus, the latter complexes did not result from sequence-specific factor binding to the
HSE sequences. When nuclear proteins prepared from the heat-treated
cells were used, a strong specific far-shifted complex was observed.
This strong new complex disappeared by self-competition (Fig. 7,
lanes 9 and 10). The HSF in eukaryotes acquires
high affinity DNA binding activity upon heat shock activation by
conversion from a monomer to homotrimer (30). Therefore, the new
far-shifted complex may be a homotrimer HSF-DNA complex. Analogous
results were obtained from experiments with nuclear extracts from cells treated with PQ for 1 h (Fig.
8A). A far-shifted complex
disappeared by self-competition, demonstrating a specific interaction
(Fig. 8A, lane 5). These results indicate that
the SOD1 gene protects cells from PQ toxicity in an
HSF-dependent manner and that heat and oxidative stress
signals communicate with HSF to activate gene transcription. The
far-shifted protein complex disappeared after reequilibration to
37 °C for 1 h following a heat shock (Fig. 8B,
lane 4). These results also showed that the transcription of
the SOD1 gene is induced by heat shock through the same
binding of the HSF to the HSE site.
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Fig. 7.
Detection of HSE binding protein. A gel
mobility shift assay was performed using nuclear extracts prepared from
untreated HepG2 cells (lanes 2-6) and from heat-treated
HepG2 cells (lanes 8-12). The DNA-protein complex
disappeared by cold competitor (lanes 3, 4,
9, and 10) but did not disappear with mutated HRE
(mHRE) (lanes 5 and 11) and
nonspecific competitor (lanes 6 and 12). Using
nuclear extracts prepared from heat-treated cells, a far-shifted
DNA-protein complex appeared. The location of specific DNA-protein
complex is indicated by an arrowhead. Note that the induced
strong bands disappeared with the cold probe.
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Fig. 8.
The change of binding of HSE binding protein
(HSF) by PQ and heat shock. A, a gel mobility shift
assay was performed using nuclear extracts prepared from untreated and
PQ-treated HepG2 cells. The DNA-protein complex disappeared by cold
competitor (lane 3). When nuclear extracts from the cells
treated with PQ (50 µM) for 1 h were used, a
far-shifted band appeared (lane 4). B, the
induction of a far-shifted band by heat shock in vitro. The
+/ represents that reequilibration to 37 °C following a heat shock
(42 °C). The location of specific DNA-protein complex is indicated
by an arrowhead.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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8 M) and specific enzymes
(i.e. catalase, glutathione peroxidase, and myeloperoxidase)
actively convert H2O2 to H2O in a
fast speed, the resulting hydrogen peroxide by SOD1 is thought not to
activate the SOD1 gene by the positive feedback mechanism.
In this experiment, the promoter activation was observed at the
concentration around 100 µM of
H2O2. Active oxygens are suggested to be
involved in inflammatory responses; the activations by
H2O2 of c-fos, c-jun, and
egr-1, which activate the expression of cellular genes, are probably essential for these responses. DNA damaging agents have been
reported to activate c-fos and AP1-inducible genes such as those for collagenase and metallothionein (31). Active oxygens such as
O2 or H2O2 are produced in
mitochondria during oxidative electron transport and in the endoplasmic
reticulum, peroxisomes, and nuclear and plasma membranes. The active
oxygens generated in these locations may regulate certain cellular
genes by inducing early response genes.
2 detoxification
in Escherichia coli through the OxyR and
SoxRS regulons, respectively. OxyR directly senses oxidative
stress to activate the expression of
H2O2-inducible genes, including those encoding
catalase and alkyl hydroperoxide reductase (34). The SoxRS
regulon is controlled in a two-stage process. First an iron-sulfur
protein, SoxR, is activated by increases in intracellular superoxide
anion levels and triggers transcription of the soxS gene.
The SoxS protein in turn induces transcription of other genes of the
regulon, including those encoding manganese superoxide dismutase, the
DNA repair endonuclease IV, and glucose-6-phosphate dehydrogenese (32,
33, 35). The oxidative stress response and its relationship to heat
shock phenomena are also being intensely investigated in eukaryotic
systems. Unlike the bacterial systems described earlier, little is
known about how eukaryotic cells co-ordinate gene expression in
response to oxidative stress (34).
2-generating agent,
could activate the SOD1 gene through the heat shock element. Heat shock may cause activation of the membrane-associated oxidase system directly or indirectly through HSP, resulting in increased O2 production. Oxygen free radicals induce heat shock protein synthesis in cultured human neuroblastoma cells (29). These findings
suggest a common mechanism by which various forms of injury, such as
hyperthermis, cause HSP induction, that is, via oxidative stress or
increased production of oxygen free radicals.
FOOTNOTES
To whom correspondence should be addressed: Dept. of Molecular
Biology, Seoul National University, Seoul 151-742, Korea. Tel.: 82-2-880-6688; Fax: 82-2-888-7784; E-mail:
hyunerho@plaza.snu.ac.kr.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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