Redox Factor-1 (Ref-1) Mediates the Activation of AP-1 in HeLa
and NIH 3T3 Cells in Response to Heat Shock*
David A.
Diamond
§,
Azemat
Parsian
,
Clayton R.
Hunt
,
Sam
Lofgren
,
Douglas R.
Spitz
,
Prabhat C.
Goswami
, and
David
Gius
§¶
From the
Section of Cancer Biology, Radiation
Oncology Center, Mallinckrodt Institute of Radiology and the
§ Washington University School of Medicine, St. Louis,
Missouri 63108
 |
ABSTRACT |
The early response genes, c-Fos and c-Jun, are
induced by environmental stress and are thought to modulate injury
processes via the induction of AP-1-dependent target genes.
AP-1 activation is thought to be regulated by changes in intracellular
oxidation/reduction reactions involving the redox factor-1 (Ref-1)
protein. In this study, NIH 3T3 and HeLa cells were used to determine
whether heat shock induces the AP-1 transcription factor via signaling
pathways involving Ref-1. Reverse transcriptase-polymerase chain
reaction analysis and immunoblotting demonstrated that c-Fos and c-Jun were induced 2-10 h following heat shock, and this induction was accompanied by an increase in AP-1 DNA binding. Electrophoretic mobility shift assay extracts immunodepleted of Ref-1 protein demonstrated that the increase in AP-1 DNA-binding activity following heating was dependent upon the presence of Ref-1 and that Ref-1 regulates inducible, but not basal, AP-1 DNA-binding activity. This was
confirmed by the restoration of heat-inducible DNA binding upon
addition of Ref-1 to immunodepleted extracts. The ability of Ref-1 from
heated cells to stimulate AP-1 DNA binding was abolished by chemical
oxidation and restored by chemical reduction. These results indicate
that heat shock activates c-Fos/c-Jun gene expression and AP-1 DNA
binding and suggests that redox-sensitive signal transduction pathways
involving Ref-1 may mediate heat-induced alterations in AP-1 activation.
 |
INTRODUCTION |
Most living cells are sensitive to sudden elevations in
temperature and respond to this environmental insult by activating the
heat shock response (1, 2). The heat shock response has been
intensively investigated and is primarily mediated at the
transcriptional level by pre-existing transcriptional activators, termed heat shock factors
(HSFs)1 (3). Once activated,
HSFs bind to regulatory heat shock elements present in the promoter
region of genes coding for heat shock proteins (HSPs) (1, 3). In
addition to HSFs, it appears that several additional signal
transduction cascades are also activated in response to heat including
p38/HOG1 kinase (4), Jun N-terminal kinase (5), MAPK1 (6), and protein
kinase C (7-9). These signaling pathways are also activated by a wide variety of additional environmental insults including oxidative stress.
Studies of cross-resistance induced in response to oxidative stress and
heat shock (10, 11) suggest that one aspect of the cellular response to
heat may be similar to cellular responses to oxidative stress.
The mechanism responsible for causing cell death after thermal injury
remains unclear. It has been suggested that one aspect of heat-induced
cellular injury results from mitochondrial damage that disrupts
intracellular oxidation/reduction reactions (10-17). Hence, heat shock
may result in altered generation of reactive oxygen species as well as
alterations in intracellular antioxidant capacity (13, 15, 17-20).
Mammalian cells and tissues exposed to heat shock have been shown: 1)
increased conversion of xanthine dehydrogenase to the
superoxide-generating enzyme xanthine oxidase, 2) alterations in thiol
metabolism leading to the increased synthesis of glutathione (GSH) and
increase in formation of oxidized glutathione (GSSG), 3) increased
sensitivity to heat-induced cell killing when GSH is depleted, and 4)
increased resistance to heat-induced cell killing in stable
H2O2-resistant cell types which overexpress cellular antioxidants (glutathione, catalase, superoxide dismutase, glutathione peroxidase, and glutathione transferase) (13, 15, 16, 21,
22). In addition, in Saccharomyces cerevisiae prooxidant production, as measured by the oxidation-sensitive fluorescent probe,
2',7'-dichlorofluoroscin diacetate, increased following heat shock
(17). Manipulations designed to reduce the production of reactive
oxygen species (over expression of catalase, superoxide dismutase,
cytochrome c peroxidase, and anaerobic conditions) protected
cells from the lethal effects of heat (17). These studies support the
hypothesis that heat shock induces an imbalance in intracellular
oxidation/reduction (redox) reactions resulting in increased
steady-state prooxidant production and oxidative stress that
contributes to the biological effects of heat shock.
Alterations in intracellular oxidation/reduction reactions have been
shown to activate signal transduction cascades that regulate as early
response genes. These genes are believed to function in a protective or
reparative capacity (23-25). The early response genes c-Fos and c-Jun
are members of a multigene family implicated in a number of
stress-induced signal transduction cascades and thus provide useful
models for investigating stress-invoked alterations in gene expression
(26). c-Fos and c-Jun proteins associate in homo- and/or heterodimers
to form the mammalian transcription factor AP-1. It has been suggested
that AP-1 may regulate expression of downstream target genes that are
known to be involved with cellular antioxidant defense mechanisms
(27-32). Stress-induced activation of these early response genes
appears to rely, at least in part, on changes in intracellular
oxidation/reduction (redox) (33-35). Hence, mammalian cells appear to
capitalize on inherent redox-sensitive signaling circuitry to respond
to certain forms of environmental stresses that perturb oxidative
metabolism (27, 36).
At least one mechanism regulating c-Fos/c-Jun DNA binding is mediated
by a conserved cysteine (Cys) located in the basic DNA-binding domain
of both proteins (27). In vitro these regulatory cysteines are not permissive for DNA binding under oxidized conditions, whereas
reduction to a sulfhydryl state promotes DNA binding (27, 35). As such,
these critical cysteines act as a redox-sensitive "sulfhydryl
switch" that reversibly modulates DNA binding (36). In the absence of
reducing agents, the redox factor-1 (Ref-1) protein regulates
c-Fos/c-Jun DNA binding via the same conserved cysteine. Ref-1 (also
designated APE, and HAP-1) is a DNA repair enzyme that also acts as a
signaling factor regulating the DNA binding of several redox-sensitive
transcription factors including NF-
B, Egr-1, and p53 (35, 36).
Interestingly, a wide range of cellular stress agents also induce these
transcription factors (23, 24). Thus, Ref-1 may act as a pivotal
signaling factor involved in the induction of early response genes,
such as c-Fos and c-Jun.
It has been suggested heat shock causes perturbations in cellular redox
status. In addition, several early response genes as well as signaling
cascades are activated by changes in cellular redox conditions. Hence,
it is reasonable to hypothesize that heat-induced increases in early
response gene expression (c-Fos and c-Jun) leading to increased
AP-1-DNA binding may encompass a mechanism involving Ref-1. To address
this hypothesis, the expression of c-Fos and c-Jun as well as the
activation of the AP-1 transcription factor was determined following
heat shock in human and rodent cell lines. When c-Fos and c-Jun
expression as well as AP-1 activation were found to increase following
heat shock, the role of Ref-1 in the activation of AP-1 binding was
determined. These results indicate the induction of the early response
genes c-Fos and c-Jun in response to heat shock is similar to that
observed for other types of environmental stress. Furthermore, the
redox-sensitive signal transduction protein, Ref-1, via heat-induced
alternations in the oxidation/reduction state of the protein appears to
regulate heat-inducible, but not basal, AP-1 DNA-binding activity.
These results suggest that early response genes may play a role in the cellular responses to heat shock and that redox-sensitive signal transduction pathways involving Ref-1 may represent a common mechanism for the induction of AP-1 binding activity in response to environmental stress.
 |
EXPERIMENTAL PROCEDURES |
Cell Culture and Heat Shock Conditions--
HeLa (human cervical
carcinoma) and NIH 3T3 (rodent fibroblasts) were grown in Dulbecco's
modified Eagle's medium supplemented with 10% heat-inactivated calf
serum with penicillin (50 units/ml) and streptomycin (50 units/ml) in a
humidified 5% CO2 atmosphere at 37 °C. For heat shock
experiments, cells were seeded at 2.5 × 106
cells/10-cm diameter plate the night before exposure. Cells were heated
at various time points by submerging parafilm sealed 10-cm plates in a
pre-warmed circulating water bath at 45 °C for 15 min. Cells were
immediately placed at 37 °C after heating and harvested at various
time points.
mRNA Isolation and Reverse Transcriptase-Polymerase Chain
Reaction Analysis--
Total cellular RNA was isolated by using TRI
reagent (Molecular Research, Cincinnati, OH) following the protocol of
the manufacturer. The RNA was checked for purity and used in a
quantitative RT-PCR analysis method that has been previously described
(37). Primer sequences for c-Fos, c-Jun, and
glyceraldehyde-3'-phosphate dehydrogenase were selected on separate
exons to distinguish amplified products of mRNA from possible
contaminating DNA or precursor RNA. The primer sequences and PCR
product sizes have been previously described (37). Five microliters of
the PCR amplified products were analyzed by electrophoresis on a 1.5%
agarose gel. Gels were stained with ethidium bromide, photographed,
dried and exposed to phosphoimager screen for quantitation of
incorporated radioactivity in each individual band. Quantitation of
results was obtained using a STORM 840 Phosphoimager. All results are
presented as -fold increase above base-line control, unheated cells.
SDS-Polyacrylamide Gel Electrophoresis and Western Blot
Analysis--
Whole cell extracts were prepared at 2, 4, 6, 8, 10, and
24 h after heating as described (38). Protein samples were
separated on SDS-polyacrylamide gels and transferred to a
nitrocellulose filter using a semi-dry transfer apparatus (Owl, Inc).
Western blotting analysis was done with polyclonal antibodies to c-Fos (Oncogene Science, AP1), c-Jun (Oncogene Science, AP2), and Ref-1 (Santa Cruz Biotech, Inc). Antibodies were diluted 1:1000 in a 2.5%
milk, phosphate-buffered saline, 0.01% Tween 20 solution as described
previously and hybridized overnight (39). The blots were developed with
an enhanced chemiluminescence method (Amersham Pharmacia Biotech).
Electromobility Shift Assays--
EMSAs were performed as
described previously utilizing a 32P-radiolabeled
oligonucleotide corresponding to the consensus AP-1 DNA-binding site
(38). Briefly, whole cell extracts (10 µg) were incubated with
poly(dI-dC) for 10 min on ice, followed by addition of radiolabeled
oligonucleotide (200,000 cpm of radiolabeled probe per reaction) and
incubated at 25 °C for 20 min. Supershift experiments were performed
by the addition of either anti-Fos or anti-Jun antibody to the whole
cell protein extract-poly(dI-dC), radiolabeled oligonucleotide followed
by incubation for 20 min at 25 °C. For the cold AP-1 oligomer
competition experiment, 1 µg of unlabeled AP-1 oligomer was added to
the whole cell protein extract-poly(dI-dC) complex, kept on ice for 10 min followed by addition of radiolabeled oligonucleotide and incubation
for 20 min at 25 °C. Samples were run on a 4.5% nondenaturing
polyacrylamide gel electrophoresis, dried, and exposed to the
phosphoimager screen for quantitation using a STORM 840 PhosphorImager
from Molecular Dynamics.
Immunodepletion EMSAs for Ref-1 were performed immediately after the
extracts were prepared by adding 5 µl of anti-Ref-1 and 25 µl of
protein A to 20 µl of EMSA control and heated extracts as described
(40). After 2 h of gentle shaking at 4 °C, the tubes were spun
at 12,000 rpm for 1 min and washed. The protein levels in the remaining
extracts were determined and EMSAs were performed. Chemical
modification of immunoprecipitated Ref-1 was performed by adding either
diamide (80 µM), N-ethylmaleimide (NEM) (50 µM), or dithiothreitol (DTT) (8 mM) to
immunoprecipitation/protein A pellet for 30 min. To remove the free
residual chemicals from the immunoprecipitation reaction, the pellets
were washed eight times before EMSA using non-heated Ref-1
immunodepleted extracts.
 |
RESULTS |
Induction of Fos and Jun in Response to Heat--
Previous
investigations have shown that HeLa cell c-Jun mRNA levels increase
in response to heat (41). As a first step, therefore, we sought to
confirm and expand upon this finding by examining c-Jun and c-Fos
mRNA levels following heating. Experiments were performed with NIH
3T3 and HeLa cells, representing immortalized and fully transformed
cell lines, respectively. Asynchronous cycling NIH 3T3 and HeLa cells
were heated (45 °C for 15 min) and then incubated at 37 °C until
RNA isolation at 2, 4, 6, 8, 10, and 24 h. c-Fos and c-Jun
mRNA levels were determined using quantitative RT-PCR (37). In NIH
3T3 cells (Fig. 1A), c-Fos and
c-Jun expression increased 6- and 3-fold, respectively, when compared
with control, unheated cells. The induction was initially apparent
2 h following heating and returned to basal levels by 10 h.
Similarly, in HeLa cells (Fig. 1B), c-Fos and c-Jun
expression was induced 5- and 3-fold, respectively, following heating.
Thus, in NIH 3T3 and HeLa cells the relative abundance of c-Fos and
c-Jun mRNA is increased in response to heat shock. Furthermore, the
accumulation of c-Fos and c-Jun mRNA following heat shock is
similar in both magnitude and temporal expression.

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Fig. 1.
Increased expression of c-Fos and c-Jun
mRNA in response to heat. RT-PCR analysis of RNA isolated from
NIH 3T3 (A) and HeLa (B) cells subjected to heat
stress. HeLa or NIH 3T3 cells were plated at 2.5 × 106 cells/10-cm diameter plate, heated to 45 °C for 15 min, and then incubated at 37 °C until harvested. RNA was isolated
from non-heated or heated cells at 2, 4, 6, 8, 10, and 24 h
following heat shock. Relative mRNA level of each target gene was
first normalized to the corresponding glyceraldehyde-3'-phosphate
dehydrogenase mRNA level in individual samples, and the fold
increase was determined relative to non-heated cells. C,
control.
|
|
To determine whether the increased abundance of c-Fos and c-Jun
mRNA in response to heating is accompanied by increased
accumulation of immunoreactive protein, total cellular protein was
isolated from both cell lines at 2, 4, 6, 8, 10, and 24 h
following heat shock. Following heat shock in NIH 3T3 cells, an
increase in c-Fos and c-Jun immunoreactive protein was apparent at 4-6
h (Fig. 2A), reached a peak at
8 h (5- and 6-fold, respectively, as compared with control
unheated cells), and returned to base line by 24 h. Similarly, in
HeLa cells an increase in c-Fos and c-Jun immunoreactive protein was
demonstrated 4-6 h following heat shock and returned to base line by
24 h (Fig. 2B). Thus, increased accumulation of c-Jun
and c-Fos mRNA following heat shock correlated with an increase in
protein levels of similar magnitude and temporal expression. Furthermore, these experiments demonstrate that heat, like other types
of environmental stress, increases early response gene expression.

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Fig. 2.
Increased expression of c-Fos and c-Jun
protein in response to heat. Western blot analysis of whole cell
extracts from cells subjected to heat stress. Total cellular protein
was isolated from asynchronously cycling NIH 3T3 (A) and
HeLa (B) cells from control, non-heated (lane 1)
and heated cells at 2, 4, 6, 8, 10, and 24 h. Cells were heated at
45 °C for 15 min. 10 µg of cellular protein was separated by
SDS-PAGE, transferred onto nitrocellulose, and processed for
immunoblotting with rabbit polyclonal antibodies to c-Fos or c-Jun
(Oncogene Products Research). All results are presented as -fold
increase above base-line control, unheated cells. Equal protein loading
was determined using a Bradford protein assay. C,
control.
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Induction of AP-1 DNA-binding Activity following Heating--
The
Fos and Jun protein families form an array of heterodimeric protein
complexes that bind to specific cis-acting DNA regulatory elements,
referred to as AP-1 sites, to activate the expression of downstream
target genes (24, 42). AP-1 sites are present in the promoter region of
many genes, including c-Fos and c-Jun, as well as in other genes
involved in the cellular response to environmental stress (34). Since
c-Fos and c-Jun comprise the AP-1 DNA-binding complex and both c-Fos
and c-Jun expression increase in response to heat, we sought to
determine whether AP-1 DNA-binding activity increased following heat exposure.
AP-1 DNA binding was measured by performing EMSA with extracts from NIH
3T3 and HeLa cells. Cells were heated to 45 °C for 15 min and then
incubated for 2, 4, 6, 8, 10, and 24 h at 37 °C until
harvested. NIH 3T3 cells demonstrated no difference in AP-1 DNA-binding
activity between unheated controls and cells harvested at 2 and 4 h after heating (Fig. 3A,
lanes 1-4). In contrast, a 2-fold increase in DNA-binding
activity was noted at 6 h (lane 5), reached a peak
induction of 3-fold at 8 and 10 h (lanes 6 and 7), and
returned to base line at 24 h (lane 8). These
experiments were repeated in HeLa cells (Fig. 3B) with a
similar induction of AP-1 binding at 8-10 h (2.5-fold) after heat
shock. Thus, the increased accumulation of c-Fos and c-Jun RNA and
immunoreactive protein following heat shock temporally parallels an
increase in AP-1 DNA-binding activity.

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Fig. 3.
Analysis of AP-1 DNA-binding activity in
response to heat by electrophoretic mobility shift analysis.
Asynchronously cycling NIH 3T3 (A) and HeLa (B)
cells were plated at 2.5 × 106 cells/10-cm diameter
plate, exposed to heat for 45 °C for 15 min, and then incubated at
37 °C until harvested at indicated time periods. 10 µg of whole
cell extracts were analyzed by gel mobility shift assay using an
AP-1-specific 32P-labeled oligonucleotide.
Arrows indicate the AP-1 complex and free unbound AP-1
oligonucleotide. C, control.
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EMSA Supershift Assay of Heat-induced AP-1 DNA binding Using
Anti-Fos and Anti-Jun--
To determine whether heat-induced
activation of AP-1 DNA binding results from an increase in the DNA
binding of complexes, which contain c-Fos and/or c-Jun, supershift
experiments were performed using cell extracts from HeLa cells 8 h
following heat shock (Fig. 4, lane
1). Anti-Fos (Fig. 4, lane 2), anti-Jun antibody (Fig.
4, lane 3), or cold competitor DNA containing the AP-1
consensus motif (Fig. 4, lane 4) were added to cell extracts
10 min prior to addition of radiolabeled AP-1 oligomer. Fig. 4,
lanes 2 and 3 demonstrate that the activated AP-1
complexes from heated cells contain immunoreactive c-Fos and c-Jun
proteins. These results indicate that c-Fos and c-Jun proteins are
present in the supershifted AP-1 complexes and suggest that the
accumulation of c-Fos and c-Jun proteins (seen in Fig. 2) in response
to heat contributes to the formation of heat-induced activated AP-1
complexes.

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Fig. 4.
Supershift EMSA using extracts from HeLa
cells 8 h following heat shock. Cell extracts from heated
cells (lane 1), with addition of anti-Fos (lane
2) or anti-Jun (lane 3) antibody, or with cold
competitor AP-1 oligomer (lane 4), were subjected to EMSA as
described previously. Arrows indicate the position of the
AP-1 complex, nonspecific DNA binding (NS), and supershifted
AP-1 complexes.
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Immunodepletion of Ref-1 Impairs Inducible, but Not Basal, AP-1
DNA-binding Activity--
Because AP-1 DNA-binding activity is
increased by heat and Ref-1 is known to regulate AP-1 activity through
a redox-sensitive mechanism (35, 36, 38), we next sought to assess
whether Ref-1 is involved with the regulation of heat-induced increases in AP-1 DNA binding. These experiments were accomplished using a Ref-1
antibody to remove Ref-1 protein from the extracts that were used to
determine the increase in AP-1 DNA-binding activity in response to
heat. Briefly, NIH 3T3 and HeLa cells extracts from the experiments
shown in Fig. 3 were treated with the addition of protein A and
anti-Ref-1 antibody, followed by gentle shaking at 4 °C for 2 h
and spinning at 12,000 rpm for 1 min to pellet out the Ref-1 antibody
complex. Western blot analysis confirmed the removal of Ref-1 protein
from the gel-shift extracts as well as the presence of Ref-1 in the
immunoprecipitated complex (data not shown). Western blotting was also
performed to confirm the continued presence of c-Fos and c-Jun proteins
in the Ref-1-depleted extracts as well as the lack of c-Fos and c-Jun
protein in the immunoprecipitated complex (data not shown). Protein
concentrations were determined and EMSA was performed using the
Ref-1-depleted extracts.
AP-1 DNA binding in unheated NIH 3T3 (Fig.
5A, lane 1) and
HeLa cells (Fig. 5B, lane 1) with Ref-1 present
(immunoprecipitation was performed with protein A only) are shown as
controls. The same extracts were immunodepleted of Ref-1 (Fig. 5,
A and B, lanes 3), and when compared
with the DNA binding in lane 1, no difference is observed.
These results suggest that the presence of Ref-1 in the extracts was
not required for AP-1 DNA-binding activity in the unstressed condition.
As a positive control, the extracts from heated cells harvested at
8 h were immunoprecipitated with protein A only (Fig. 5,
A and B, lanes 2). Similar to the
results in Fig. 3, lanes 5 and 6, these positive
controls demonstrated a 2.5-fold increase in DNA-binding activity (Fig.
5, A and B, lanes 2). In the Ref-1
depleted extracts from heated cells isolated at 2, 4, 6, 8, 10, and
24 h, there was no increase in AP-1 DNA-binding activity, relative
to the unheated extracts (Fig. 5, A and B, lanes 4-9). These results indicate that the presence of
Ref-1 in the extracts from heated cells was required for the
heat-inducible increase in AP-1 DNA-binding activity.

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Fig. 5.
Impact of immunodepletion of Ref-1 upon AP-1
DNA-binding activity in response to heat. NIH 3T3 (A)
and HeLa (B) cell extracts from the experiment shown in Fig.
3 were immunodepleted for Ref-1 via the addition of protein A and
anti-Ref-1 antibody, followed by gentle shaking at 4 °C for 2 h. Cell extracts were spun at 12,000 rpm for 1 min to pellet out the
Ref-1/antibody complex. 10 µg of Ref-1-immunodepleted whole cell
extracts were analyzed by EMSA as described previously. Minus
signs indicate Ref-1-immunodepleted extracts, and plus
signs indicate that Ref-1 was not depleted. C,
control.
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Restoration of Heat-induced AP-1 DNA-binding Activity following
Addition of Ref-1 Immunoprecipitates to Immunodepleted
Extracts--
To confirm that Ref-1 from heated cells must be present
in the NIH 3T3 cell extracts to observe heat-induced increases in AP-1
DNA binding, Ref-1-containing immunoprecipitates from heated and
unheated cells were added back to the Ref-1-immunodepleted extracts
from heated cells harvested 8 h following heat shock (Fig.
6A). When Ref-1
immunoprecipitate from heated cells was added back to Ref-1 minus
extracts from heated cells (Fig. 6, lane 3), the
heat-induced increase in AP-1 DNA binding was restored (compare
lane 3 to lane 2). When Ref-1 immunoprecipitate
from unheated cells was added to Ref-1 minus heated cell extracts
(lane 4), no increase in AP-1 DNA binding was observed
(compare lane 4 to lane 2). Interestingly, when
Ref-1 immunoprecipitate from heated cells (8 h) was added to Ref-1
minus extracts from non-heated cells (lane 5), AP-1 DNA
binding also increased (compare lane 5 to lane
1). Equal amounts of immunoprecipitated Ref-1 from control and
heated cells were confirmed by Western analysis to rule out the
possibility that the induction of AP-1 DNA binding was because of
increased Ref-1 immunoprecipitated protein (data not shown). These
results confirm that the presence of Ref-1 from heated cells is
required to activate AP-1 DNA binding

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Fig. 6.
AP-1 DNA-binding activity following
re-addition of Ref-1 to immunodepleted extracts in NIH 3T3 cells and
redox regulation of Ref-1. A, immunoprecipitated
Ref-1/protein A pellets from control (unheated) cells and heated cells
(8 h) were added back to immunodepleted (Ref-1-minus) cell extracts
from control, non-heated (lane 4) or heated (8 h)
(lanes 3 and 5) cells. The immunoprecipitated
Ref-1 and immunodepleted extracts were incubated on ice for 45 min
followed by analysis via EMSA as described. B, either
diamide (80 µM), NEM (50 µM), or DTT (8 mM) were added to the immunoprecipitated Ref-1/protein A
complex from heated cells after pelleting (using NIH 3T3 cells). The
chemicals were removed from the immunoprecipitation reaction
prior to EMSA by washing the complex eight times (41) before addition
to non-heated immunodepleted extracts. EMSA for AP-1 was performed as
described. Arrows in panels A and B
indicate the AP-1 complex and free unbound AP-1 oligonucleotide.
IP, immunoprecipitated.
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Redox Alterations in the Ref-1 Protein from Heated Cells Regulates
AP-1 DNA-binding Activity--
It has been shown that the ability of
Ref-1 protein to regulate AP-1 DNA binding in vitro is
abolished by chemical oxidants and induced by chemical reductants (27,
36). To determine whether the redox status of Ref-1 protein from heated
cells regulates AP-1 DNA-binding activity, Ref-1 from heated cells was
immunoprecipitated and treated with chemicals that alter
oxidation/reduction status. Following a vigorous washing step, the
immunoprecipitated Ref-1 was then added to non-heated
Ref-1-immunodepleted cell extracts, and AP-1 DNA-binding activity was
elevated. Ref-1 from heated cells was immunoprecipitated and treated in
the presence of either diamide (a sulfhydryl oxidizing agent), NEM (a
sulfhydryl-specific alkylating agent), or DTT (a sulfhydryl-reducing
agent), and washed eight times to remove any free chemical. Ref-1
immunoprecipitated from heated NIH 3T3 cells without chemicals is shown
as a control (Fig. 6B, lane 1). The addition of
diamide or NEM to immunoprecipitated Ref-1 from heated cells
(lanes 2 and 3) decreased AP-1 DNA binding. Ref-1
immunoprecipitated from heated cells and treated with DTT increased
AP-1 DNA binding 2-fold as compared with Ref-1 immunoprecipitated from
heated cells and not treated with chemicals (lane 1 versus 4). Finally, immunoprecipitated Ref-1 from
heated cells treated with diamide were washed eight times to remove any
free chemical from the reaction and subsequently treated with DTT.
Treatment with DTT after exposure to diamide completely restored the
ability of Ref-1 from heated cells to induce AP-1 DNA binding
(lanes 1 versus 5). These results
suggest that alterations in the oxidation/reduction (redox) status of
the Ref-1 protein from heated cells play a central role in the
enhancement of AP-1 DNA-binding activity following heat shock.
 |
DISCUSSION |
The heat shock response is a rapid, complex, highly regulated
process that involves coordinate control of multiple signal transduction pathways. The specific signaling pathways activated by
elevated temperature, which result in the development of the heat shock
response, are not completely understood. Interestingly, several
components of the signaling pathways leading to the heat shock response
are also stimulated by environmental stresses other than elevated
temperature. These include exposure to hypoxia, heavy metals, amino
acid analogues, and viral infections (3, 5, 43), all of which appear to
cause perturbations in cellular redox status. These results have been
interpreted to suggest that heat, perhaps via alterations in cellular
metabolism, produces a condition of oxidative stress similar to that
seen with other forms of cellular stress (10-17). If this were true,
it might be expected that cellular pathways that sense changes in
intracellular redox might also respond to heat shock. Therefore, given
that several early response genes appear to play a role in the cellular response to oxidative stress, we questioned whether these same signal
transduction pathways could be activated in the cellular response to
heat. The central findings of our work show that heat shock leads to
the induction of the early response genes, c-Fos and c-Jun, as well as
increasing AP-1 DNA-binding activity by a mechanism that appears to
involve the redox-sensitive signaling protein, Ref-1.
To investigate the role of early response genes in cellular responses
to heat shock, NIH 3T3 and HeLa cells, representing immortalized and
malignant cell lines, respectively, were utilized. These experiments
demonstrate: 1) increased accumulation of c-Fos and c-Jun mRNA,
protein, and AP-1 DNA-binding activity in response to heating in a
temporally consistent manner; 2) that the induction of AP-1 DNA-binding
activity is dependent upon Ref-1; 3) that Ref-1 appears to regulate
heat-inducible, but not basal, AP-1 DNA-binding activity; and 4) that
Ref-1 signaling in response to heat is redox-regulated. Interestingly,
the kinetics of induction of these changes in early response genes were
somewhat delayed following heat shock (2-10 h) as compared with other
forms of environmental stress that perturb cellular redox more
immediately, such as phorbol esters and ionizing radiation. This may
reflect an inherent difference in the mechanism by which heat perturbs intracellular oxidation/reduction reactions as compared with other agents.
Immunodepletion experiments and re-addition of immunoprecipitated Ref-1
to immunodepleted EMSA cell extracts demonstrated that Ref-1 from
heated cells must be physically present in the binding reaction for the
heat-induced induction of AP-1 DNA binding to be observed.
Interestingly, when immunoprecipitated Ref-1 from heated cells
harvested at 8 h was added to Ref-1-immunodepleted EMSA cell
extracts from non-heated cells, an increase in AP-1 DNA binding was
observed. These results suggest that Ref-1 is altered in a specific way
following heating such that it participates in the activation of AP-1
DNA binding even in extracts from non-heated cells. Furthermore, these
results support the conclusion that Ref-1 from heated cells must be
physically present in the EMSA reactions to pass a "signal" to the
AP-1 transcription factor complexes, resulting in an increase in DNA
binding. This observation is in agreement with the results published by
Yao et al. (40) where increased AP-1 DNA-binding activity in
response to hypoxia/reoxygenation were abolished by immunodepletion of
Ref-1 protein. Overall, our results using heat shock as well as the
work of others using hypoxia/reoxygenation (40) suggest that
environmental stress generates a signal that is passed through the
Ref-1 protein to activate the DNA binding capability of the AP-1
complex
Ref-1 is a bifunctional protein that acts as a DNA repair enzyme as
well as a stimulator of DNA binding of transcriptional factors by a
redox-dependent mechanism (35, 36, 40). Ref-1 protein
purified from HeLa cells increases the DNA-binding activity of c-Fos
and c-Jun through critical cysteine residues (Fos Cys-154 and Jun
Cys-272) shown to be sensitive to chemical oxidation-reduction experiments done in vitro (27). These critical cysteine
residues are flanked by the basic amino acids lysine and arginine
(KCR), creating a motif that is sensitive to changes in cellular
oxidation/reduction status that appear to alter DNA-binding activity
(35). The oxidation/reduction status of this motif appears to be
biologically significant (44) and is conserved in all of the c-Fos- and
c-Jun-related proteins (24). Genetic analysis of Ref-1 has identified a
cysteine at position 65 in the redox domain that is critical for the
redox-sensitive activation of c-Fos/c-Jun DNA binding. This critical
cysteine residue (reduced) is required for a direct interaction between Ref-1 and c-Jun in vitro, suggesting that Ref-1 may act as a
redox-sensitive sulfhydryl switch that activates AP-1 DNA binding (36,
45). The results from Fig. 6B suggest that heat shock
perturbs intracellular oxidation/reduction reactions such that a change
in Ref-1 protein redox state enables Ref-1 to alter AP-1 DNA binding.
It has been suggested that heat, perhaps via alterations in
mitochondrial function, thiol metabolism, and lipid peroxidation, can
cause oxidative stress (13, 22). If this were true, it might be
expected that previously identified biochemical pathways that respond
to protect cells against the detrimental effects of oxidative stress
would also respond to heat shock. c-Fos, c-Jun, and AP-1 complexes are
thought to regulate genes that control pathways responsible for the
detoxification of reactive oxygen species and resistance to oxidative
stress (i.e. heme oxygenase,
-glutamylcysteine synthase,
glutathione S-transferase, and NAD(P)H:quinone reductase)
(29-32). Some of these proteins, most notably heme-oxygenase (HSP-32)
are induced by heat shock. Therefore, our results support the
speculation that accumulation of c-Fos and c-Jun and activation of AP-1
complexes may play a role in regulating cellular defense against
oxidative stress following heat shock.
In summary, the central findings of our work are that heat shock leads
to the increased expression of early response genes, c-Fos and c-Jun,
as well as the activation of AP-1 DNA-binding activity by a mechanism
that appears to involve the redox factor-1 (Ref-1) protein. Taken
together with previous investigations (40), these results support the
concept that a common central pathway mediating cellular responses to
heat shock or other types of environmental or metabolic oxidative
stress may involve redox-sensitive signaling pathways leading to AP-1
activation via the Ref-1 protein.
 |
ACKNOWLEDGEMENTS |
We thank Drs. Joseph L. Roti Roti and Andrei
Laszlo for critical comments on the manuscript. We also thank Carla
Thuman and Kathy Bles for assistance with preparation of this manuscript.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grants PO1 CA75556 (to D. R. S. and C. R. H.), CA69593 (to P. G.), and K08 CA72602 (to D. G.).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.
¶
To whom correspondence should be addressed: Section of Cancer
Biology, Radiation Oncology Center, Mallinckrodt Institute of Radiology, Washington University School of Medicine, 4511 Forest Park
Blvd., Suite 411, St. Louis, MO 63108. Tel.: 314-362-9771; Fax:
314-362-9790; E-mail: davidg{at}radonc.wustl.edu.
 |
ABBREVIATIONS |
The abbreviations used are:
HSF, heat shock
factor;
RT-PCR, reverse transcriptase-polymerase chain reaction;
NEM, N-ethylmaleimide;
DTT, dithiothreitol;
EMSA, electrophoretic
mobility shift assay.
 |
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