Signal Transducer and Activator of Transcription-1 and Heat Shock
Factor-1 Interact and Activate the Transcription of the Hsp-70 and
Hsp-90
Gene Promoters*
Anastasis
Stephanou
,
David A.
Isenberg§,
Koichi
Nakajima¶, and
David S.
Latchman
From the Departments of Molecular Pathology and
§ Medicine, Windyer Institute of Medical Sciences,
University College London, 46 Cleveland Street,
London W1P 6DB, United Kingdom and the ¶ Department of
Molecular Oncology, Biomedical Research Center, Osaka University
Medical School, Osaka 565, Japan
 |
ABSTRACT |
We have previously demonstrated that
interleukin-6 (IL-6) increases the levels of the heat shock protein 90 (Hsp-90) and activates the Hsp-90
promoter via the IL-6-activated
transcription factors NF-IL6 and signal transducer and activator of
transcription-3 (STAT-3). Here, we show that interferon-
(IFN-
)
treatment increases the levels of Hsp-70 and Hsp-90 and also enhances
the activity of the Hsp-70 and Hsp-90
promoters with these effects
being dependent on activation of the STAT-1 transcription factor by
IFN-
. These effects were not seen in a STAT-1-deficient cell line,
indicating that IFN-
modulates Hsp induction via a
STAT-1-dependent pathway. The effect of IFN-
/STAT-1 was
mediated via a short region of the Hsp-70/Hsp-90 promoters, which also
mediates the effects of NF-IL6 and STAT-3 and can bind STAT-1. This
region also contains a binding site for the stress-activated
transcription factor HSF-1. We show that STAT-1 and HSF-1 interact with
one another via a protein-protein interaction and produce a strong
activation of transcription, which is in contrast to our previous
finding that STAT-3 and HSF-1 antagonize one another. To our knowledge
this is the first report of HSF-1 interacting directly via a
protein-protein interaction with another transcription factor. Such
protein-protein interactions and the binding of a number of different
stress and cytokine-activated transcription factors to a short region
of the Hsp-90 and Hsp-70 gene promoters are likely to play a very important role in Hsp gene activation by non-stressful stimuli and the
integration of these responses with the stress response of these genes.
 |
INTRODUCTION |
The heat shock proteins (Hsps) are a group of proteins that were
originally identified on the basis of their increased synthesis in
cells exposed to elevated temperatures and subsequently were shown to
be similarly induced by exposure of cells to a variety of stresses (1,
2). The induction of Hsps in response to various stresses is dependent
on the activation of a specific transcription factor, the heat shock
factor (HSF-1),1 which binds
to the heat shock element (HSE) in the promoters of Hsp genes (3). In
addition, many Hsps are also expressed in unstressed cells, and their
levels are regulated in response to a wide variety of biological
processes such as T lymphocyte activation (4) and monocyte to
macrophage differentiation (5). In general, however, the stimuli that
induce such alteration in Hsp gene expression under non-stress
conditions have been poorly characterized, and the mechanisms by which
they act are unclear.
We have recently shown, however, that treatment of human peripheral
blood lymphocytes with interleukin-6 (IL-6) results in enhanced
expression of the Hsp-90
gene (6). IL-6 is a multifunctional cytokine with pleiotropic activities on a variety of cell types (7).
This property of IL-6 is dependent on the IL-6 receptor, which includes
the glycoprotein 130 subunit that is shared among the other cytokine
receptors belonging to the IL-6 receptor superfamily (leukemia
inhibitory factor, IL-11, oncostatin M, and cardiotrophin-1) as well as
a receptor chain, which is unique to the IL-6 receptor (8). Binding of
IL-6 to its receptor is known to stimulate two distinct signaling
pathways, resulting in the activation of two distinct transcription
factors NF-IL6 (C/EBP
) and STAT-3 (9). Both these factors have been
shown to activate the Hsp-90
promoter and have a strong synergistic
effect on its transcription resulting in its observed activation by
IL-6. Interestingly, activation of the Hsp promoters by these factors
is mediated via NF-IL6 and STAT-like binding sites that are located
close to the HSE (10). Moreover, the two transcription factors interact
differently with HSF-1 and heat shock stress (10). Thus activation of
STAT-3 reduced the stimulatory effect of HSF-1 or heat stress, whereas activation of NF-IL6 enhanced it. These results are of interest because
they demonstrate that a specific stimulus such as IL-6 can enhance the
expression of the Hsp in non-heat-stressed cells in an
HSF-1-independent manner and also indicate that others can functionally
interact with HSF-1 and play a role in Hsp gene regulation.
In view of the effect of the STAT-3 transcription factor on the
Hsp-90
promoter, we wished to investigate whether the closely related STAT-1 factor would also have an effect. To do this we tested
the effect of IFN-
on Hsp gene expression. Like IL-6, IFN-
is
also a multifunctional cytokine that is known to have antiviral and
antitumor properties by inducing specific IFN-
-responsive genes (11,
12). In contrast to IL-6, however, IFN-
specifically activates the
STAT-1 signaling pathway. In the present study, we report that IFN-
treatment induced the expression of Hsp-70 and Hsp-90 in a
STAT-1-dependent manner. In addition, overexpression of
STAT-1 enhanced the activities of the Hsp-70 and Hsp-90
promoters. Interestingly, STAT-1 and HSF-1 were shown to have an additive effect
in activating the Hsp-70 and Hsp-90
promoters and to directly interact via a protein-protein interaction. These studies have identified a composite response element that integrates the
HSF-mediated heat shock response with IL-6 and IFN-
signaling to
mediate the differential regulation of Hsps.
 |
MATERIALS AND METHODS |
Cell Culture and Reagents--
HepG2 hepatoma cells were
obtained from the American Type Culture Collection (Rockville, MD) and
maintained in 10% Dulbecco's modified Eagle's medium. U3A and
U3A-ST1 cells were kindly provided by Ian Kerr (Imperial Cancer
Research Fund, London, UK). Recombinant IFN-
was purchased from
Autogen Bioclear, Wiltshire, UK. Antibodies to STAT-1 were purchased
from Santa Cruz Biotechnology, Inc., Santa Cruz, CA, and antibodies for
Hsp-70, Hsp-90, and HSF-1 were purchased from StessGen (Victoria, BC, Canada).
Plasmid Constructs and DNA Transfection--
The 5' Hsp-90
CAT reporter constructs A (
1044 to +36) and C (
299 to +36) were
kindly provided by Neil Rebbe (Washington University School of
Medicine, St. Louis, MO). The Hsp-70 CAT reporter constructs LSN (
188
to +1) and LSNP (
100 to +1) were kindly provided by Richard Morimoto
(Northwestern University, Evanston, IL). The HSE/STAT-90 CAT was
constructed by ligating the
643 to
623 fragment
(5'-GCCTGGAAACTGCTGGAAAT-3') of the Hsp-90
promoter into the
heterologous reporter construct pBLCAT2, and the HSE/STAT-70 CAT was
also constructed by ligating the
122 to
90 fragment of the
Hsp-70 promoter
(
122GATCCGGCGAAACCCCTGGAATATTCCCCGACCT
90)
into the heterologous reporter construct pBLCAT2. The expression vector
for HSF-1 was kindly provided by Carl Wu (NIH, Bethesda, MD). The
expression plasmid-encoding STAT-1 was in pCAGGSneo-HAStat1.
Transfection of reporter constructs was performed by the calcium
phosphate method by using 10 µg of the reporter plasmid and 5 µg of
the STAT-1 or the HSF-1 expression vectors. To assess and normalize for
transfection efficiency, a
-galactosidase expression vector was also
co-transfected. Protein concentration from cell lysates was also
determined to equalize for protein content.
Band Shift Assays--
HepG2 cells were treated with 10 ng/ml
IFN-
for 15 min or heat shocked for 30 min and harvested in cold
phosphate-buffered saline. Nuclear extracts were prepared and
examined for band shift as described previously (13) with HSE-70
(
122GATCCGGCGAAACCCCTGGAATATTCCCCGACCT
90),
HSE-90 (
643GCCTGGAAACTGCTGGAAAT
623), and a
mutant HSE-90M
(
643GCCcaGAAACTGCcaGAAAT
623,
where underlining indicates bases that have been changed from wild-type to mutant-type) DNA probe. A 23-base pair high affinity SIEm67 (5'-GATCTGATTACGGGAAATG-3') DNA probe that serves as a binding site for STAT-1 was used in competition studies. For supershift assays, nuclear extracts were incubated with anti-STAT-1 for 30 min
prior to incubation with the DNA probe. Complexes were separated by 4%
SDS-polyacrylamide gel electrophoresis and exposed to autoradiography.
Co-immunoprecipitation--
HepG2 cells (5 × 106) were lysed in radioimmune precipitation buffer (150 mM NaCl, 1.0% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS,
50 mM Tris, pH 8.0) and incubated with either anti-STAT-1 or anti-HSF-1 for 1 h at 4 °C on a shaking platform. Protein
A-Sepharose was added to each tube and incubated further for 1 h.
Complexes were washed three times with radioimmune precipitation
buffer, and 20 µl of sample buffer were added and boiled at 95 °C
for 3 min. Samples were then electrophoresed on a 8%
SDS-polyacrylamide gel, transferred onto nitrocellulose filters, and
subjected to Western blotting with either anti-STAT-1 or anti-HSF-1 antibodies.
 |
RESULTS |
To test whether IFN-
could stimulate the levels of Hsp in the
IFN-
-responsive HepG2 cell line, we treated these cells with IFN-
and measured the Hsp-70 and Hsp-90 levels by Western blotting. As shown
in Fig. 1, both Hsp-70 and Hsp-90 levels
were increased by IFN-
in a dose-dependent manner. 50 ng/ml IFN-
resulted in an 8- and 6-fold induction in the levels of
Hsp-70 and Hsp-90, respectively.

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Fig. 1.
Western blot showing the protein levels of
Hsp-70 and Hsp-90 in HepG2 cells treated with 0, 5, and 50 ng/ml
IFN- for 24 h. Actin level was also measured to assess for
protein loading.
|
|
To test whether this effect was mediated via a direct effect on the Hsp
gene promoters, we transfected HepG2 cells with Hsp-70 and Hsp-90
promoter reporter constructs and treated the cells again with IFN-
.
As indicated in Fig. 2, IFN-
stimulated the activities of the Hsp-70 and Hsp-90
promoters. These
results suggest that IFN-
is acting directly to increase Hsp-70 and
Hsp-90 levels by activating the corresponding gene promoters.

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Fig. 2.
Functional CAT activity of the Hsp-70 and
Hsp-90 reporter constructs in response to IFN- , overexpression of
STAT-1, or IFN- plus STAT-1 together in HepG2 cells. CAT
activity was assessed from a total of three experiments (mean ± S. D.).
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|
In other IFN-
-responsive cell types, the effects of IFN-
are
mediated by the activation and phosphorylation of the STAT-1 pathway.
We therefore determined whether overexpression of STAT-1 in HepG2 cells
could also activate the Hsp-70 and Hsp-90
promoters. As illustrated
in Fig. 2, co-transfection of an expression vector for STAT-1 resulted
in enhanced stimulation of both Hsp-70 and Hsp-90
promoters upon
addition of IFN-
. In contrast, overexpression of STAT-1 alone caused
only a modest stimulation of either promoter, suggesting that STAT-1
requires Janus kinase-STAT-1 activation and phosphorylation via IFN-
for maximal Hsp promoter activation.
To confirm that the effect of IFN-
on the Hsp genes was mediated via
STAT-1, we repeated the above experiments in the STAT-1-deficient cell
line U3A. As shown in Fig. 3, INF-
was
unable to significantly enhance the levels of Hsp-70 and Hsp-90 in U3A
cells. However, when STAT-1 was re-introduced into the STAT-1-deficient
cells (U3AST1), INF-
treatment enhanced the levels of both Hsp-70
and Hsp-90 (Fig. 3). Similar results were also obtained by transfection studies with the Hsp-70 and Hsp-90
promoters positively responding to IFN-
in U3AST1 cells but not in the U3A cells (data not shown). Hence, the induction of Hsp-70 and Hsp-90 expression by IFN-
is
dependent upon STAT-1.

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Fig. 3.
Western blot showing the protein levels of
Hsp-70 and Hsp-90 in untreated (C), IFN- -treated (50 ng/ml) STAT-1-deficient U3A cells, or U3A-ST1 cells in which a STAT-1
gene was reintroduced. The actin level was also measured to assess
for protein loading.
|
|
As described previously, STAT-3-like binding sites are present on the
Hsp-90
promoter in close proximity to the HSE (
643 to
623 base
pairs) (10). In addition, analysis of the Hsp-70 promoter also
indicated a similar STAT-3-like binding site close to the HSE (
110 to
90 base pairs) (Fig. 4). Because STAT-1
and -3 proteins recognize very similar binding sites, we investigated whether the IFN-
signaling effect observed above is mediated by this
same element by analyzing various Hsp-70 and Hsp-90
reporter constructs. As illustrated in Fig. 5 and
Table I, by deleting the region of the
Hsp-90
promoter from
1044 (construct A) down to
299 base pairs
(construct C) we abolished the activation of the promoter by IFN-
and STAT-1. In addition, a Hsp-70 construct that was deleted from
188
(construct LSN) to
100 (construct LSNP), resulting in the loss of the
HSE-containing region, was unresponsive to IFN-
and STAT-1 (Fig. 5
and Table I). Hence, in both promoters, stimulation by IFN-
is
eliminated by deletions that remove the region of the promoter
containing the HSE/STAT-like element, suggesting that this element is
necessary in both the Hsp-90
and Hsp-70 gene promoters for them to
respond to activated STAT-1 produced by IFN-
.

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Fig. 4.
Sequences of the 70- and 90-HSE/STAT
region of the Hsp-70 and Hsp-90 promoter. The
homologies for the consensus binding sites for HSF-1 (19) and STAT are
shown (12, 20).
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Fig. 5.
Functional CAT activity of the full-length
and truncated Hsp-70 and Hsp-90 reporter constructs, respectively,
containing or lacking the STAT/HSE region in response to either IFN-
or STAT-1 overexpression in HepG2 cells. CAT activity
was assessed from a total of three experiments (mean ± S.D.).
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Table I
Responses of Hsp promoter constructs to interferon
CAT activity is shown of the different length Hsp-70 and Hsp-90
reporter constructs and also the heterologous 70-HSE/STAT and
90-HSE/STAT reporter construct linked to the thymidine kinase promoter
vector pBLCAT2 either untreated ( ) or treated with 50 ng/ml IFN-
(+) in HepG2 cells. CAT activity was assessed from a total of three
experiments (mean + S.D.).
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|
To address this point further, oligonucleotides containing the HSE/STAT
site in the Hsp-70 and Hsp-90
promoters were coupled to the
heterologous thymidine kinase promoter and assessed for responsiveness
to IFN-
and STAT-1 activation. As shown in Table I, both
HSE/STAT-Hsp-70 and HSE/STAT-Hsp-90
constructs conferred responsiveness to STAT-1 activation by IFN-
to the heterologous promoter with similar induction being observed to that seen with the
intact Hsp promoters. Hence this short region of either promoter can
render a heterologous promoter inducible by IFN-
and STAT-1.
To demonstrate that the effect of STAT-1 on gene transcription via the
HSE/STAT region was mediated via binding of STAT-1 to this region of
both the Hsp-90
and Hsp-70 promoter, we examined the ability of
IFN-
to induce DNA binding of STAT-1 to the labeled 70- and
90-HSE/STAT oligonucleotides in band shift assays. The IFN-
-activated site (GAS) m67SIE oligoprobe containing STAT-1 binding sites was used as a positive control. As shown in Fig. 6, a low mobility-retarded band was
obtained with both the 70- and 90- HSE/STAT probe, and the intensity of
this band was enhanced in response to IFN-
or heat shock. The
binding observed was sequence-specific because it was competed with
excess unlabeled probe but not with a nonspecific probe. In addition
the retarded band was also competed with the unlabeled m67SIE
oligonucleotide containing a consensus STAT-1 binding site, suggesting
that the band was likely to contain STAT-1. This was confirmed by
showing that the band was abolished by an addition of anti-STAT-1
antibody but not by an anti-STAT-3 antibody. Moreover, this band was
also abolished by the addition of an antibody to HSF-1, indicating that
it also contains HSF-1. Hence exposure to IFN-
and/or heat shock
results in the detection of a complex containing both STAT-1 and HSF-1,
which binds to the HSE/STAT element in the Hsp-70 and Hsp-90 gene
promoters. This complex was virtually abolished by mutation of the STAT
site in the 90-HSE/STAT element (Fig. 6C), indicating that
the DNA binding of the complex containing the two transcription factors is strongly reduced by mutating the binding site for one of the two
factors.

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Fig. 6.
DNA mobility shift assay using labeled
oligonucleotide containing the region of the 70-HSE/STAT sequence
(A) or the region of the 90-HSE/STAT (B)
sequence or an oligonucleotide containing the 90-HSE/STAT with a mutant
STAT site, HSE-90M (C). HepG2 nuclear extracts from
either untreated ( ) or IFN- -treated (+) or
heat-shocked (HS) cells were incubated with the labeled
probe in the presence of unlabeled competitor oligonucleotide
containing either the 70-HSE/STAT or the 90-HSE/STAT, the SIE consensus
binding site for STAT-1, an SP1 binding site or incubated with either
anti-STAT-1 antibody (AbST1), anti-STAT-3 antibody
(AbST3), or anti-HSF-1 antibody (AbHSF1) as
indicated. Arrows show the complex induced in response to
IFN- or heat shock. P, free probe; C,
control.
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|
In view of the finding that the response to IFN-
/STAT-1 was mediated
via binding sites adjacent to the HSF-1 region and formation of a DNA
binding complex containing both STAT-1 and HSF-1, we wished to
investigate whether the STAT-1 and HSF-1 pathways interacted with one
another. To do this, we performed co-transfection studies in the U3A
cell line (lacking endogenous STAT-1) with the Hsp reporter constructs
together with expression vectors for STAT-1 or HSF-1 alone or together.
Interestingly, STAT-1 and HSF-1 had an additive effect in activating
both the Hsp-70 and Hsp-90
constructs compared with the effect of
either factor alone (Fig. 7, A
and B). We also investigated whether a similar effect could
be observed by exposing the cells to a heat shock stress, which
activates HSF-1, alone or in combination with IFN-
, which activates
STAT-1. As shown in Fig. 8, A
and B, an additive effect was observed on both the Hsp-70
and Hsp-90 promoters when both HSF-1- and STAT-1-activating stimuli
were given together compared with the effect with either stimulus
alone. This suggests that although both factors are bound to the
promoter following exposure to either stimulus alone, maximal activation is only observed when both stimuli are present, presumably resulting in the post-translational activation of both factors, for
example by phosphorylation.

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Fig. 7.
Functional CAT activity of the Hsp-70
(A) and Hsp-90 (B) reporter constructs in
response to expression vectors encoding either HSF-1 (H) or
STAT-1 (S) alone or in combination in U3A cells.
IFN- was added to those cells transfected with STAT-1. CAT activity
was assessed from a total of three experiments (mean ± S.D.).
C, control.
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Fig. 8.
Functional CAT activity of the Hsp-70
(A) and Hsp-90 (B) reporter constructs in
response to either heat shock (HS) at 42 °C alone or HS
plus IFN- in U3A-ST1 cells. CAT activity assessed from a total
of three experiments (mean ± S.D.) is shown. C,
control.
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|
The finding that the binding sites for HSF-1 and STAT-1 are close
together and the formation of a DNA binding complex containing both
factors suggest the possibility of a direct physical protein-protein interaction between HSF-1 and STAT-1. We therefore performed
co-immunoprecipitation experiments by immunoprecipitating with
anti-STAT-1 and then immunoblotting the precipitate with a specific
antibody to HSF-1. As illustrated in Fig.
9, a visible HSF-1-specific band was
observed when cell extracts were immunoprecipitated with an anti-STAT-1
antibody, and the immunoprecipitate was then probed by Western blotting with anti-HSF-1. No HSF-1 band was observed by Western blotting after
immunoprecipitating with an anti-STAT-3 antibody, although STAT-3
itself was readily detectable in the immunoprecipitate. Hence HSF-1
interacts specifically with STAT-1 but not with STAT-3.

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Fig. 9.
Co-immunoprecipitation of STAT-1 and
HSF-1. Endogenous STAT-1 or -3 was immunoprecipitated by
anti-STAT-1 or -3 polyclonal antibody from HepG2 cell extract. Samples
were analyzed by Western blotting (WB) with a specific
anti-HSF-1 polyclonal antibody (upper panel), an anti-STAT-1
antibody (middle panel), and an anti-STAT-3 antibody
(lower panel). The arrows indicate either IgG or
the specific band. IP, immunoprecipitate; NS,
nonspecific anti-rabbit serum.
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 |
DISCUSSION |
Although the mechanisms mediating the induction of Hsp genes by
heat stress have been intensively studied (3, 14, 16) and shown to
predominantly involve the HSF-1 transcription factor, much less
attention has been paid to elucidating the mechanism mediating Hsp gene
expression modulated by non-stressful stimuli such as immune
activation. Our previous reports (6, 10) together with our present
study have analyzed the molecular mechanisms regulating Hsp gene
expression by the cytokines IL-6 and IFN-
. Thus, IL-6 has been
demonstrated to induce the expression of Hsp-90 via signaling pathways
activating the transcription factors STAT-3 and NF-IL6 (10). Here we
have shown that IFN-
is also able to activate the promoters for
Hsp-70 and Hsp-90
via the STAT-1 signaling pathway. Furthermore, a
short sequence in both the Hsp-70 and Hsp-90
promoter containing the
HSE and also incorporating a STAT-like binding site is important for
mediating IL-6 and IFN-
signaling. Regulatory sequences of
eukaryotic genes commonly contain binding sites for multiple
transcription factors and provide a basis for combinatorial
interactions between different factors. These regions are usually
referred to as composite response elements that mediate the integration
of multiple regulatory signals. Our study provides another example of
such a composite response element on both the Hsp-70 and Hsp-90
promoters and identifies this element as critical for the responses to
cytokines such as IL-6 and IFN-
as well as to stressful stimuli such
as heat shock.
Our previous work demonstrated that the Hsp-90
promoter appears to
have a novel pattern of inducibility, which is dependent upon STAT-3
and NF-IL6 (10). This finding renders the promoter distinct from those
of the liver acute phase protein genes that appear to fall into two
separate classes and are regulated either by the MAPK/NF-IL6 signaling
pathways (7) or by the Janus kinase/STAT-3 pathway (12, 15). It is
possible that this difference may reflect the tissue-specific
expression of acute phase protein genes in the liver compared with
constitutive expression of Hsp-90 in all cell types. The complexity of
the composite response elements in both the Hsp-70 and Hsp-90
promoter is illustrated further by the observation that both STAT-1 and
-3 (10) cooperate with NF-IL6 to induce the activity of the promoters.
Moreover, STAT-1 and HSF-1 have an additive effect on Hsp-70 and
Hsp-90
promoters, and protein binding studies have shown that HSF-1
and STAT-1 interact and form a DNA binding complex, which binds to the
HSE/STAT element in both promoters.
HSFs (HSF-1 to -4) have been cloned from a number of organisms and
their roles have been characterized. Only HSF-1 has been shown to be
involved in regulating Hsp in response to heat stress, whereas HSF-2,
-3, and -4 may be involved in Hsp gene regulation under non-heat stress
conditions, for example during cellular differentiation (16, 17). HSF-1
is present in the cytoplasm as a monomer and is maintained in a non-DNA
binding state in unstressed cells. In response to heat shock, HSF-1
undergoes conformational changes and forms a trimer that is able to
bind to the HSEs on Hsp promoters. HSF-1 has also been recently
demonstrated to regulate the IL-1 promoter under non-heat stress
conditions (18). Our present results have also shown that HSF-1
interacts with other transcription factors to modulate Hsp promoter
activity under non-heat stress conditions. These studies suggest that
HSF-1 is able to modulate promoter activity in the absence of
physiological stress. A number of studies have demonstrated that
transcriptional activity is attained by protein-protein interaction of
transcription factors on DNA response elements. Recently HSF-3 and
c-Myb have been reported to interact physically, and such interaction
may be important in the regulation of Hsp gene expression during
cellular proliferation (19).
Our results are the first demonstration of HSF-1 forming a direct
protein-protein interaction with another transcription factor, STAT-1,
and suggest that the effect of HSF-1 and STAT-1 on the Hsp promoters
may require such physical interaction. Thus, in contrast to the
additive effect of STAT-1 and HSF-1, STAT-3 and HSF-1 appear to have a
mutually antagonistic effect. Although STAT-3 and HSF-1 can
individually activate the Hsp-90
promoter, such activation is
greatly reduced in the presence of both factors together (10).
Interestingly, unlike STAT-1 and HSF-1, STAT-3 and HSF-1 do not
interact with one another in the same conditions where a STAT-1/HSF-1
interaction can be demonstrated (Fig. 8). It is possible therefore that
HSF-1 and STAT-3 compete for binding to the short Hsp promoter region
that contains both of these binding sites resulting in an antagonistic
effect. In contrast, the protein-protein interaction between STAT-1 and
HSF-1 may facilitate binding of both factors to the promoter leading to
enhanced activation of transcription. Although the sequence of the
STAT-like binding site located around the HSE region of both the Hsp-70
and Hsp-90
promoter does not fully match the previously
characterized STAT-1 binding site ATTNNNNAAT (12), there is evidence
that the consensus STAT binding site on other genes characterized
recently may not match fully, for example CTGGNAA, which is found in
the promoter of the CD14 binding protein gene (20). The ability of
STAT-1 to bind to the sequence in the Hsp-70 and Hsp-90 promoters has been directly confirmed in our experiments where the protein binding to
this element in IFN-
-treated cells was identified as STAT-1 on the
basis of its reactivity with anti-STAT-1 antibody and its removal with
competitor oligonucleotide containing a consensus STAT-1 binding site.
From our previous and present data, it is clear therefore that the
Hsp-70 and Hsp-90
promoters contain a composite response element
that is able to integrate multiple regulatory signals and allow
different transcription factors to interact with each other to have
either a stimulatory or inhibitory effect on transcriptional activation. Preliminary analysis of other Hsp promoters in different species shows similar sequences with potential binding sites for STATs
and NF-IL6 around the HSE and suggests that such a region has been
conserved during evolution. This conserved element may thus be of
critical importance in regulating the activation of Hsp genes by
non-stressful stimuli and integrate the responses with their responses
to stressful stimuli.
 |
ACKNOWLEDGEMENTS |
We thank N. Rebbe for the Hsp-90
promoter
constructs and R. Morimoto for the Hsp-70 promoter constructs. We also
thank C. Wu for the HSF-1 expression vector.
 |
FOOTNOTES |
*
This work was supported by the Arthritis and Rheumatism
Council and also by the British Heart Foundation.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.
The abbreviations used are:
HSF, heat shock
factor; IL, interleukin; IFN, interferon; HSE, heat shock element; CAT, chloramphenicol acetyltransferase; STAT, signal transducer and
activator of transcription.
 |
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