Inhibition of Heat Shock Transcription Factor by GR
Subhagya A. Wadekar,
Dapei Li,
Sumudra Periyasamy and
Edwin R. Sánchez
Department of Pharmacology, Medical College of Ohio, Toledo, Ohio
43614
Address all correspondence and request for reprints to: Dr. Edwin R. Sanchez, Department of Pharmacology, Medical College of Ohio, 3035 Arlington Avenue, Toledo, Ohio 43614. E-mail: esanchez{at}mco.edu
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ABSTRACT
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The GR is a hormone-activated transcription factor that
acts to regulate specific gene expression. In the absence of hormone,
the GR and other steroid receptors have been shown to form complexes
with several mammalian heat shock proteins. As heat shock proteins are
produced by cells as an adaptive response to stress, speculation has
existed that communication between the heat shock and glucocorticoid
hormone signal pathways must exist. Only recently has evidence to
support this hypothesis been reported. In almost all cases, the
evidence has been of an ability of heat shock to cause a potentiation
of the glucocorticoid hormone response. In this proposal, evidence is
now presented that heat shock signaling can, in turn, be regulated by
glucocorticoids. In mouse L929 cells stably expressing a
chloramphenicol acetyltransferase reporter controlled by the human heat
shock protein70 promoter and containing known binding sites for heat
shock transcription factor 1 treatment with glucocorticoid agonist
(dexamethasone) results in a dose-dependent decrease of stress-induced
chloramphenicol acetyltransferase gene expression. In these cells,
inhibition of heat shock protein70 promoter activity by dexamethasone
was completely blocked by GR antagonist (RU486). Similar treatment of
L929 cells stably expressing a chloramphenicol acetyltransferase
reporter under the control of the constitutively active SV40 promoter
showed no such inhibition by dexamethasone. More importantly,
dexamethasone was also found to inhibit heat shock-induced expression
of the major heat shock proteinsheat shock proteins70, 90, and 110.
Thus, the inhibitory effect of dexamethasone appears to apply to most,
if not all, heat shock transcription factor 1-regulated genes. Although
dexamethasone did not prevent the DNA-binding function of heat
shock-activated heat shock transcription factor 1, it did inhibit a
constitutively active mutant of human heat shock transcription factor 1
under nonstress conditions, suggesting that dexamethasone repression of
heat shock transcription factor 1 was primarily through an inhibition
of heat shock transcription factor 1 transcription enhancement
activity. To more accurately characterize the stage of GR signaling
responsible for inhibition of heat shock transcription factor 1, a
series of Chinese hamster ovary cells containing either no GR,
wild-type mouse GR, or single-point mutations of GR were employed.
Dexamethasone inhibition of heat shock-induced heat shock transcription
factor 1 activity was observed in the presence of wild-type GR, but not
in Chinese hamster ovary cells lacking GR, suggesting that signaling
cascades other than GR were not involved in this effect of
dexamethasone. Consistent with this conclusion was the observation that
dexamethasone had no effect on activity of the MAPKs (ERK1, ERK2, or
c-jun N-terminal kinase), which are known to
negatively regulate heat shock transcription factor 1. Dexamethasone
inhibition of heat shock transcription factor 1 was not seen in Chinese
hamster ovary cells expressing GR defective for DNA-binding function.
Moreover, dissociation of GR/Hsp90/Hsp70 complexes was observed in
response to hormone for both the wild-type and DNA binding-defective
forms of GR, demonstrating that release of Hsp90 or Hsp70 (both of
which are known to keep heat shock transcription factor 1 in its
inactive state) could be ruled out as a potential mechanism. Thus, it
appears that GR-mediated transactivation or transrepression is required
for the inhibitory effect of dexamethasone on heat shock transcription
factor 1 activity. Taken as a whole, these results provide evidence for
a novel mechanism of cross-talk in which signaling by the GR can
attenuate the heat shock response in cells through an inhibition
of the transcription enhancement activity of HSF1.
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INTRODUCTION
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THE GR IS A steroid-activated
transcription factor involved in physiological responses that serve to
protect organisms against stress (1, 2). The heat shock
response is a well known cellular adaptation to stress that is
mediated by heat shock transcription factors (HSFs), principally HSF1
(3, 4). Recently, it has become clear that both GR and
HSF1 are maintained in their respective inactive states in the
cytoplasm by association with a heat shock protein (HSP)-based
chaperone complex containing Hsp70 and Hsp90
(5, 6, 7, 8, 9, 10). Because of these similarities, a
variety of investigations have tested the notion that the heat shock
(HS) and steroid receptor signaling pathways are interrelated. In
most of these studies, a dramatic effect of heat shock on the function
of steroid receptors has been found. For example, heat shock-induced
translocation to the nucleus of unliganded GR has been observed for the
endogenous GR of L929 cells (11), mouse GR expressed in
Chinese hamster ovary (CHO) cells (12), and human GR
expressed in COS cells (13). Partial activation of
transcription enhancement activity of hormone-free GR has also been
shown in response to heat shock (12). It has also been
shown that GR-dependent transrepression of the collagenase promoter, a
response involving GR interaction with AP-1, can be induced in the
absence of hormone by heat shock treatment in both COS-7 and Hela cells
(13). Similarly, heat shock has been found to mimic the
ability of dexamethasone (Dex) to modulate Fc receptor expression in
murine macrophages (14).
When heat shock is combined with hormone treatment, dramatic increases
in steroid receptor activation have been documented. Combined hormone
and stress treatment of T47D breast cancer cells was found to produce a
level of PR-mediated transcription activity much higher than that seen
in response to hormone alone (15). We have found that heat
shock can increase Dex-induced GR-mediated gene expression above that
seen with maximal concentrations of hormone (16, 17). Our
recent data suggest a role for HSF1 in this response, as specific
modulation of HSF1 by quercetin, sodium vanadate, or wortmannin results
in a corresponding modulation of the stress potentiation of GR
(18, 19).
In contrast to the effects of heat shock on steroid receptor function,
evidence for control of the heat shock response by steroids is much
less common. Most early attempts to uncover such a relationship
tested the effects of steroids on the levels of HSP expression.
In our laboratory, we have not seen a change in HSP levels in response
to glucocorticoid treatment alone, although we have not measured Dex
effects on HSP synthesis under heat shock conditions. Yet, reports of
the induction of low molecular weight HSPs in Drosophila by
the insect steroid hormone
-ecdysterone are available
(20), as is a report by Fisher et al.
(21) in which Dex treatment of CHO cells induces a state
of thermotolerance similar to that obtained in response to mild heat
shock. In the latter report, however, Dex-induced thermotolerance was
not the result of any measurable increase in Hsp70 or Hsp90 protein
content. In contrast, Dex treatment of plasmacytoma cells was found to
actually prevent induction of Hsp70 in response to heat shock
(22).
Recently, Xiao and DeFranco (23) showed that
overexpression of GR in COS-1 cells by transient transfection results
in the stress-free activation of HSF1. As this response could be mapped
to the steroid-binding domain of the GR (also the HSP-binding region),
and as this response was abolished when GR was transfected in the
presence of Dex, the authors concluded that activation of HSF1 was
occurring due to sequestration of HSPs from HSF1 to the newly forming
GR/HSP complexes. As concurrent overexpression of Hsp70 prevented HSF1
activation by GR expression, it was also concluded that Hsp70 was the
likely negative regulator of HSF1 sequestered in this way. From our
perspective, these observations by Xiao and DeFranco were important
evidence that the HSP-based chaperone complexes of intact cells can be
readily exchanged between GR and HSF1. In the present study, we have
examined the effect of GR signaling on HSF1 by use of cells containing
naturally or stably expressed GR. In contrast to transiently expressed
GR, we find that hormone activation of GR in these cells results in
repression of stress-induced HSF1 activity, by a mechanism that does
not involve HSPs released from the GR complex. Instead, repression of
HSF1 requires the DNA-binding and the transrepression or
transactivation functions of GR. Our data have therefore provided the
first direct evidence for cross-talk between GR and HSF1, by
demonstrating that GR repression of HSF1 occurs through the genomic
actions of naturally expressed receptora mechanism that may have
important endocrine and therapeutic consequences.
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RESULTS
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Inhibition of Human Hsp70 Promoter Activity and of Endogenous
HSP Expression by Glucocorticoid Agonist
We have stably transfected the GR-containing mouse L929 cell line
with a chloramphenicol acetyltransferase (CAT) reporter gene
(p2500-CAT) under the control of the human Hsp70 promoter (LHSE cells).
Heat shock-induced activation of this promoter has been shown to
require binding by HSF1 to consensus heat shock elements (HSEs) present
between -65 and -52 bp of the transcription start site
(24). As an initial test of the effects of GR signaling on
the heat shock response, we performed the experiments of Fig. 1
in which LHSE cells were subjected to a
variety of hormone and stress conditions. In Fig. 1A
, a
Dex-concentration dependence was performed. Maximal inhibition of heat
shock-induced Hsp70 promoter activity was observed at approximately 1
µM Dex. This concentration of Dex was then used to
compare the effects of hormone on two forms of stress. The results show
that although heat shock (43 C, 2 h) and chemical shock (200
µM sodium arsenite, 2 h) will activate transcription
from the Hsp70 promoter to different levels (Fig. 1B
), 1
µM Dex caused about the same level of inhibition of CAT
gene expression induced by heat or chemical stress (70% and 75%,
respectively). In all of these experiments, Dex was added to the cells
4 h before the stress event and was maintained in the media during
the 20-h recovery period before assay for CAT. However, similar results
have been obtained when Dex is not present during the recovery period,
or when Dex is added for only 4 h during recovery (data not
shown).

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Figure 1. Dex Inhibits Stress-Induced, HSF1-Mediated
CAT Gene Expression in LHSE Cells
A, Mouse L929 cells were stably-transfected with a CAT reporter
construct driven by the human Hsp70 promoter to generate LHSE cells.
Replicate flasks of LHSE cells were treated with increasing
concentrations of Dex, as indicated, followed by heat shock (43 C,
2 h) and recovery for 20 h in the continued presence of
hormone. Lysates were prepared and assayed for CAT activity. The
results represent means ± SEM of six independent
experiments. B, Replicate flasks of LHSE cells were subjected to the
hormone and stress conditions indicated, followed by recovery for
20 h and assay for CAT. The results represent means ±
SEM of 36 independent experiments. C, No treatment; HS,
43 C for 2 h; DHS, Dex (1 µM) for 4 h followed
by HS; CS, 200 µM sodium arsenite, 2 h; DCS, Dex (1
µM) for 4 h followed by CS.
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The specificity of this response was tested in two ways. First, the
effect of RU486 antagonist was determined (Fig. 2
). The results show that Dex inhibition
of heat shock-induced CAT activity in the LHSE cells can be completely
blocked by RU486 (Fig. 2A
). That RU486 is actually acting as a GR
antagonist in these cells was determined by use of L929 cells stably
transfected with the GR-responsive mouse mammary tumor virus (MMTV)-CAT
reporter (LMCAT cells). In this case, RU486 did not by itself cause
activation of GR but did effectively block Dex activation of the
receptor (Fig. 2B
). To ensure that Dex inhibition of CAT expression
from the Hsp70 promoter was not due to a generalized increase in
transcription or to alterations in turnover for CAT mRNA, the effect of
Dex was measured on CAT expression controlled by the constitutively
active SV40 promoter (LSV2CAT cells). The results of Fig. 3
show that Dex alone or combinations of
heat shock, arsenite, and hormone have essentially no effect on
CAT enzyme levels in these cells, even after 24 h of treatment
with hormone. Thus, by these initial criteria, it appears that
inhibition of Hsp70 promoter activity in our system is mediated by
agonist-activated GR.

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Figure 2. The GR Antagonist RU486 Blocks Dex-Induced
Inhibition of HSF1 Activity in Heat-Shocked LHSE Cells
A, Effects of RU486 on Dex inhibition of HSF1 activity. Replicate
flasks of LHSE cells subjected to the indicated hormone and stress
treatments were allowed to recover for 20 h in the continued
presence of hormone. Lysates were prepared and assayed for CAT
activity. The results represent means ± SEM of three
to six independent experiments. C, No treatment; HS, 43 C for 2 h;
DHS, Dex (1 µM) plus 0, 1, or 10 µM RU486
for 4 h followed by HS; RHS, RU486 (1 µM) for 4
h followed by HS. B, Effects of RU486 on Dex-induced GR-mediated CAT
gene expression. L929 cells were stably transfected with an MMTV-CAT
reporter to generate LMCAT cells. Replicate flasks of LMCAT were
subjected to the indicated hormone treatments. Lysates were prepared
and assayed for CAT activity. The results represent means ±
SEM of six independent experiments. C, No treatment; D, Dex
(1 µM) for 20 h; DR, Dex (1 µM) and
RU486 (10 µM) for 20 h; R, RU486 (10
µM) for 20 h.
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Figure 3. Effects of Dex and Stress on CAT Gene
Expression Controlled by the SV40 Promoter
Mouse L929 cells were stably transfected with a CAT reporter construct
driven by the constitutively active SV40 promoter to generate LSV2
cells. Replicate flasks of LSV2 cells were subjected to a variety of
hormone and stress conditions and were allowed to recover for 20 h
in the continued presence of hormone until lysates were prepared and
assayed for CAT activity. The results represent means ±
SEM of nine independent experiments. C, No treatment; D,
Dex (1 µM) for 24 h; HS, 43 C, 2 h; DHS, Dex (1
µM) for 4 h, followed by HS; CS, 200
µM sodium arsenite, 2 h; DCS, Dex (1
µM) for 4 h followed by CS.
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To determine whether the inhibitory effect of hormone was either
specific to the Hsp70 promoter or the result of a more general effect
on HSP expression, we measured the effects of Dex on the rates of
synthesis of the major HSPs. In the experiments of Fig. 4
, LHSE cells were subjected to chemical
shock using 200 µM arsenite followed by pulse labeling
with [35S]methionine during a time course of
recovery. Replicate flasks were treated with 1 µM Dex for
4 h before chemical shock, followed by recovery in the continued
presence of hormone. As expected, the results show that chemical shock
will dramatically increase the rates of synthesis for Hsp70, Hsp90, and
Hsp110, especially at the 8 h time point of recovery. These
results are consistent with our prior observations, in which maximal
induction of HSP synthesis following stress is observed between 8 and
12 h of recovery (18). With respect to "Hsp70,"
both the so-called constitutive (Hsc70) and inducible (Hsp70) forms of
this protein are up-regulated in response to arsenite (Fig. 4A
). More
importantly, Dex appeared to inhibit the rates of synthesis for all of
these HSPs (Fig. 4A
)a result that was confirmed for Hsc/Hsp70 and
Hsp90 by densitometric scanning of the autoradiograms (Fig. 4B
).
Interestingly, the level of inhibition by hormone (at 8 h of
recovery) was about the same (5055%) for both Hsp70 and Hsp90. Taken
as a whole, therefore, it appears that the inhibitory effect of Dex is
not limited to the Hsp70 promoter but, rather, is due to actions on
some factor common to the expression of HSPs in general. As the obvious
candidate in this regard is HSF1, we tested this possibility in the
following section.

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Figure 4. Dex Inhibits Heat Shock-Induced Expression of
the Major HSPsHsp70, Hsp90, and Hsp110
A, LHSE cells were subjected to chemical shock (CS) using 200
µM sodium arsenite (2 h) followed by washing and
recovery. Duplicate flasks were incubated with 1 µM Dex
for 4 h before chemical shock, followed by recovery in the
continued presence of Dex. At the indicated time intervals during
recovery, cells were pulse labeled with [35S]methionine
for 45 min, and whole cell extracts (equal protein) were analyzed by
gel electrophoresis and autoradiography. B, Quantitation of relative
expression levels for Hsp70 (Hsc70 and Hsp70) and Hsp90 was performed
by densitometric scanning of the autoradiograms. The results represent
means ± SEM of two independent experiments.
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Dex Repression of Heat Shock Signaling through Inhibition of HSF1
Transactivity
In the p2500-CAT reporter used in this study, expression of CAT is
controlled by a promoter derived from the inducible form of human Hsp70
(25). The promoter is 2,500 bp in length, of which
approximately 700 bp have been sequenced by Voellmy and co-workers
(24). Because of the large nature of this promoter, it is
highly likely that many trans-acting factors in addition to
HSF1 bind to this region, some of which may be regulated by stress.
Indeed, it has been shown that maximal response to heat shock by this
promoter requires more than merely binding by HSF1 (26).
For these reasons, it was reasonable to speculate that Dex inhibition
of stress-induced p2500-CAT activity was due to an effect of Dex on a
transcription factor(s) other than HSF1. To discriminate between these
two possibilities, we sought a means by which HSF1 transactivity could
be measured in isolation. This was achieved through use of a
constitutively active mutant of human HSF1 (hHSF1-E189) developed by
Voellmy and co-workers (27). hHSF1-E189 was generated by a
single-amino acid substitution at residue 189 (Fig. 5A
), which resides in one of three
hydrophobic LZ domains thought to be required for maintaining the
monomeric state of HSF1 and for interaction with HSP chaperones.
Mutation of this residue has therefore resulted in a form of HSF1 that
cannot be chaperoned and which, by default, is converted into active
HSF1 trimers under nonstress conditions (27, 28).

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Figure 5. The Constitutively Active E189 Mutant of
Human HSF1 Is Inhibited by Dex
A, Domain structure of hHSF1-E189. The 529-amino acid sequence of human
HSF1 contains a conserved DNA-binding domain (DBD) at the amino
terminus, as well as three conserved hydrophobic domains (LZ1LZ3)
responsible for maintaining the monomeric, inactive state of HSF1. In
E189, a single point mutation at amino acid 189 within LZ2 results in
HSF1, which undergoes trimerization and activation under nonstress
conditions. B, LHSE cells containing the p2500-CAT reporter were stably
transfected with a tetracycline-inducible construct controlling
expression of hHSF1-E189 to generate LHSE-E189 cells, as described in
Materials and Methods. Replicate flasks of LHSE-E189
cells were treated with or without doxycycline (Dox) antibiotic (10
µg/ml for 20 h), followed by Western blotting of whole cell
extracts using an antibody to human HSF1 as probe. C and D, Replicate
flasks of LHSE-E189 cells were treated with doxycycline or doxycycline
and Dex, as indicated, followed by assay for E189 protein by Western
blotting (panel C) or CAT activity (panel D). The results are
representative of three independent experiments (panel C) or depict the
means ± SEM of three independent experiments (panel
D). C, Control; Dox, 10 µg/ml doxycycline for 20 h; Dex+Dox, 1
µM dexamethasone + 10 µg/ml doxycycline for 20
h.
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To test hHSF1-E189 in our system, we placed the cDNA for hHSF1-E189
under the control of a tetracycline-inducible vector (pBI,
CLONTECH Laboratories, Inc., Palo, Alto, CA). This vector
was cotransfected into the p2500-CAT-containing LHSE cells, along with
the pUHD1721hygro vector expressing the "reverse tet"
transcriptional activator and hygromycin resistance genes, as
originally developed by Bujard and co-workers (29). After
selection with hygromycin antibiotic, the stably transfected LHSE-E189
cell line was established. In an initial test of these cells,
hHSF1-E189 expression in response to doxycycline antibiotic was
measured by Western blotting using an antibody specific to human HSF1
(Fig. 5B
). The results show a large increase in hHSF1-E189 protein
following 20 h of doxycycline treatment. Measurement of CAT
activity following doxycycline treatment showed an approximate 5-fold
increase compared with vehicle-treated controls (Fig. 5D
), indicating
that the expressed hHSF1-E189 can indeed stimulate Hsp70 promoter
activity in nonstressed cells. More importantly, concurrent treatment
of these cells with doxycycline and Dex showed a large decrease in CAT
activity relative to doxycycline alone (65% inhibition) with no
effect on the levels of hHSF1-E189 expression (Fig. 5C
)demonstrating that Dex hormone can inhibit the intrinsic activity
of hHSF1-E189 to the same degree as that seen for endogenous HSF1
(70%; see Fig. 1
). As parallel experiments in LHSE cells (p2500-CAT
only) showed no effect of doxycycline on the endogenous, wild-type HSF1
(data not shown), it is clear that the inhibition seen in the LHSE-E189
cells is due solely to an effect of hormone on mutant HSF1. Based on
these results, we conclude that the inhibitory effect of Dex on Hsp70
promoter activity under stress conditions is most likely not mediated
by unknown stress-activated transcription factors acting on this
promoter. Rather, it appears to be an effect on the intrinsic activity
of HSF1 alone.
To test whether the repression of HSF1 activity by Dex was due to an
inhibition of HSF1 binding to DNA, we performed EMSA assays using a
synthetic, 32P-labeled oligonucleotide containing
multiple, consensus HSEs (Fig. 6
). In
response to heat shock alone, activation of HSF1 DNA-binding function
can clearly be seen. Interestingly, pretreatment of cells with 1
µM Dex before heat shock had no effect on this function,
even after 24 h of hormone pretreatment. It appears, therefore,
that the inhibitory effect of Dex cannot be explained on this basis.
Moreover, it can also be concluded that all earlier stages in the HSF1
signal pathway are also not targets for the actions of hormone. Thus,
the inhibitory effect of Dex on HSF1 activation is most likely due to
an effect on the transcription enhancement function of this factora
process that, at present, is poorly understood.

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Figure 6. Dex Activation of Wild-Type GR Has No Effect
on HSF1 DNA-Binding Function
A, WHSE cells were subjected to a time course of treatment with Dex, as
indicated, followed by heat shock (43 C, 2 h). Cells were
harvested immediately after stress and EMSAs for HSF1 were performed,
as described in Materials and Methods. *, Lanes marked
with asterisk represent extracts from cells allowed to
culture an additional 8 or 24 h without Dex and
then subjected to heat shock, to account for any effect of prolonged
culture on HSF1 DNA-binding activity. B, Quantitation of HSF1
DNA-binding function was performed by densitometric scanning of
DNA-bound HSF1 and normalization to the HS-only control. The results
represent means ± SEM of 12 experiments. C, Control;
HS, 43 C for 2 h; D1, 1 µM Dex for 1 h followed
by HS; D24, 1 µM Dex for 24 h followed by HS.
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In work by our laboratory, we have shown that induction of MAPKs (ERK1
and ERK2) will lead to an inactivation of HSF1 activity in stressed
cells as measured by the p2500-CAT reporter (19), an
observation that is consistent with other reports demonstrating
negative regulation of HSF1 by MAPK family members (30, 31). In our prior work, activation of ERK1/2 was achieved by
treatment of cells with sodium vanadate, a known tyrosine phosphatase
inhibitor, resulting in the attenuation of the transcriptional
enhancement activity of HSF1 (19). Based on these
observations, we reasoned that the inhibitory effect of Dex on HSF1
could result from an effect of hormone to increase ERK1/2 activity. We
therefore tested the effect of Dex on these kinases by use of an
antibody specific to the active, phosphorylated forms of ERK1/2 (Fig. 7A
). As expected, the results show
activation of ERK1/2 by sodium vanadate. However, ERK1/2 activity was
affected neither by short- nor long-term treatment of cells with
Dex. Because more recent reports (32, 33, 34) have also
demonstrated a similar inhibition of HSF1 by c-jun
N-terminal kinase (JNK), we also tested the effects of in
vivo Dex treatment on the activity of this MAPK family member
(Fig. 7B
). The results show activation of JNK activity by vanadate
treatment but no such activation by hormone. Based on these results, it
appears that targeting of these members of the MAPK family cannot
explain the actions of hormone on HSF1.

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Figure 7. Dex Treatment Does Not Activate the MAPKs
ERK1/2 or JNK
Replicate flasks of WHSE cells were treated with 1 µM Dex
for increasing amounts of time, as indicated. Cytosolic fractions were
prepared and aliquots containing equal protein were resolved by
SDS-PAGE and immunoblotting with the SC-7383 antibody recognizing the
active forms of ERK1 and ERK2 (panel A) or the SC-6254 antibody against
active JNK (panel B). As positive controls, flasks of WHSE cells were
treated with the tyrosine phosphatase inhibitor sodium vanadate (SV;
200 µM) for 2 h to activate ERK or JNK. In prior
studies, this level of ERK activation by vanadate has been shown to
cause 70% inhibition of HSF1 activity (19 ).
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Inhibition of HSF1 Requires GR Transactivity
Our results to this point have provided evidence that hormonal
inhibition of HSF1 requires agonist-bound receptor. As a first step to
determining the exact stage of GR signaling responsible for cross-talk
with HSF1, we reasoned that this inhibition could occur through one of
two general stages: 1) hormone-induced release of GR-associated HSPs
(Hsp70 and Hsp90), which negatively regulate HSF1 (dissociation model),
or 2) inhibition of HSF1 through GR-mediated transactivation or
transrepression (genomic model). To discriminate between these
alternatives, we have used a series of Chinese hamster ovary (CHO) cell
lines that stably express either no GR (CHOd cells), wild-type mouse GR
(WCL2 cells), DNA-binding-defective GR (NB cells), or hormone-binding-
defective GR (NA cells). These cells were originally developed by
Ringold and co-workers (35) and were further characterized
by us (36). For the present work, we stably transfected
these cells with the p2500-CAT reporter to generate CHSE, WHSE, NBHSE,
and NAHSE cells, respectively. The various properties of the receptors
expressed in these cells, and in the LHSE cell line described
above, can be seen in Fig. 8
.
Panel A of this figure shows the relative amounts of GR protein present
in each cell line, along with relative values for hormone-binding and
Dex-induced transactivation functions. The results show that wild-type
GR (WHSE cells) can bind hormone and activate transcription, while the
GR of NAHSE cells cannot effectively perform either function, as would
be expected of receptor with a functional mutation in the
hormone-binding pocket. The GR of NBHSE cells showed the
highest level of hormone-binding function, in keeping with its greater
level of expression, but, as expected, this mutant GR did not exhibit
appreciable transactivation function. Analysis of untransformed GR/HSP
complexes in the WHSE and NBHSE cells (Fig. 7B
) showed association of
both Hsp90 and Hsp70 to the GR, a result that is in agreement with
prior observations (36). A similar pattern of GR binding
to Hsp90 and Hsp70 was seen for the GR of NAHSE cells (data not shown);
while the GR of LHSE cells was found to associate only with Hsp90 (data
not shown), as previously documented for GR present in the parental
L929 cells (36).

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Figure 8. Properties of Wild-Type and Mutant Mouse GR
Expressed in L929 and CHO Cells
A, Relative levels of GR protein, steroid-binding capacity, and
transactivation function. LHSE cells were generated as previously
described. CHO cells stably transfected with the same HSF1-responsive
CAT reporter and expressing no GR (CHSE), wild-type GR (WHSE),
DNA-binding-defective GR (NBHSE), or hormone-binding-defective GR
(NAHSE) were generated as described in Materials and
Methods. Western blot analysis of GR from each cell line was
performed using the BuGR2 monoclonal antibody as probe on aliquots of
cytosol containing equal protein (400 µg). Steroid-binding capacities
(dpm/µg protein) were measured by incubation of cytosols with 50
nM [3H]triamcinolone acetonide, as previously
described (55 ). To measure Dex-induced MMTV-CAT activity,
the parental CHOd, WCL2, NB, and NA cell lines were transiently
cotransfected with MMTV-CAT and ß-galactosidase reporters,
followed by treatment with or without 1 µM Dex for
24 h. CAT assays were performed, and the disintegrations per min
were first corrected for transfection efficiency based on galactosidase
activity. This was followed by normalization to basal activities for
each cell line to yield fold-induction values. ND, Not determined.
Panel B, Western blot analysis of GR/HSP complexes. Aliquots of cytosol
from WHSE and NBHSE cells were immunoadsorbed with the FiGR monoclonal
antibody against GR (F) or with nonimmune mouse IgG (NI). Samples were
resolved by SDS-PAGE and immunoblotted using BuGR2, anti-Hsp90, and
anti-Hsp70 antibodies as probes.
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To use the above cells to discriminate between the
dissociation and genomic models, we first measured the effects of
stress and hormone treatment on CAT gene expression (Fig. 9
). In the WHSE cells, we observed a
strong inhibitory effect of Dex on heat shock-induced CAT gene
expression (80%) that could be completely blocked by RU486 antagonist
(Fig. 9B
). In contrast, no inhibitory effect of Dex was seen in the
CHSE cells (Fig. 9A
). Based on this comparison, we can now eliminate
all signal pathways that do not involve GR as mechanisms to explain
this inhibitory property of hormone. There was no inhibitory effect of
Dex on HSF1 activity in the NAHSE cells, as was expected of a receptor
in which hormone cannot activate any step in its signal pathway (Fig. 9C
). More interesting were the results obtained in the NBHSE cells
(Fig. 9D
). In this case, Dex had little or no effect on heat
shock-induced HSF1 activity, demonstrating that DNA-binding function is
required for GR-mediated repression of HSF1. In addition, these data
also provide evidence against the dissociation model, since the NB GR
is perfectly capable of binding hormone (see Fig. 8
), which would
presumably lead to transformation of the GR/HSP complex. Thus, it is
likely that hormone-induced release of Hsp70 and/or Hsp90 cannot
account for the inhibitory effect observed with the wild-type receptor
(WHSE cells). However, it has yet to be shown that hormone-induced
transformation of the NB GR can indeed take place. Although the NB GR
is a single-point mutation in the DNA-binding domain (35),
and not in the region of GR responsible for Hsp90 interaction, it
remained remotely possible that this substitution could alter the
ability of hormone to cause release of this HSP. For these reasons, we
performed the experiments of Fig. 10
to
directly measure NB GR transformation under in vivo
conditions. The results demonstrate that the NB GR is found in the
cytosolic fraction in the absence of hormone as a complex with both
Hsp90 and Hsp70 (Fig. 10A
). However, in response to hormone treatment,
a large shift of NB GR to the nuclear pellet fraction can be seen that
coincides with a decrease in the amount of receptor-associated Hsp90
and Hsp70. To corroborate these results, we have analyzed GR/HSP
transformation in LHSE cells in response to both Dex and RU486 (Fig. 10B
). The results show that both Dex and RU486 will cause nuclear
translocation of GR and dissociation of the GR/Hsp90 complex,
demonstrating that RU486 is an antagonist solely at the level of GR
transactivation function. As RU486 treatment of these cells does not
inhibit HSF1 activity (Fig. 2
), it can be concluded that dissociation
of the GR/HSP complex cannot be the mechanism by which HSF1 inhibition
is achieved. Taken as a whole, our results are consistent with a model
in which GR-mediated inhibition of HSF1 is a "nuclear" event,
requiring the DNA-binding function of the receptor and, most likely,
transcription enhancement activity by the GR.

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Figure 9. Dex Inhibition of HSF1 Requires GR
DNA-Binding Function
Replicate flasks of CHO cells stably transfected with the
HSF1-responsive CAT reporter and expressing no GR (CHSE), wild-type GR
(WHSE), nonhormone-binding GR (NAHSE), and non-DNA-binding GR (NBHSE)
were subjected to the indicated hormone and heat shock conditions.
After recovery for 20 h, lysates were prepared and assayed for CAT
activity. These results represent means ± SEM of six
independent experiments. C, No treatment; HS, 43 C, 2 h; DHS, Dex
(1 µM) for 4 h followed by HS; DRHS, Dex (1
µM) plus RU486 (10 µM) for 4 h
followed by HS.
|
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Figure 10. Dex- and RU486-Induced Dissociation of
GR/HSP Complexes in NBHSE and LHSE Cells
Panel A, Replicate flasks (175 cm2) of NBHSE cells were
treated with vehicle or 1 µM Dex for 2 h followed by
Dounce homogenization and preparation of cytosolic (C) and nuclear
pellet (N) fractions, as described in Materials and
Methods. Fractions were immunoadsorbed with the FiGR monoclonal
antibody against GR (F) or with nonimmune mouse IgG (NI). Each sample
was split in half followed by SDS-PAGE and immunoblotting using BuGR2
(upper blot), or anti-Hsp90 and anti-Hsp70 antibodies
(lower blot), as probes. Panel B, Replicate flasks (175
cm2) of LHSE cells were treated with vehicle, 1
µM Dex, or 1 µM RU486 for 2 h followed
by preparation of cytosolic (C) and nuclear (N) fractions. Fractions
were immunoadsorbed with FiGR antibody against GR, followed by
immunoblotting with BuGR2 (upper blots) or antibody to
Hsp90 (lower blots).
|
|
 |
DISCUSSION
|
---|
Using cells stably transected with the human Hsp70 promoter, we
have provided evidence for negative regulation of HSF1 by the
agonist-activated GR. Dex treatment of these cells resulted in a
dose-dependent inhibition of Hsp70 promoter activity as induced by heat
or chemical shock (Fig. 1
). Inhibition of Hsp70 promoter activity by GR
was shown to be at the level of HSF1 transcriptional activity based on
the fact that Dex treatment of cells caused inhibition of a
constitutively active form of HSF1 under nonstress conditions (Fig. 5
)
and that heat shock-induced binding of HSF1 to DNA was not prevented by
Dex (Fig. 6
). Moreover, the inhibitory effect of Dex on HSF1-mediated
transactivation was not limited to the Hsp70 promoter, as hormone
treatment before heat shock reduced the rates of synthesis of several
endogenous HSPs, including Hsp70 (both constitutive and inducible
forms), Hsp90, and Hsp110 (Fig. 4
).
As mentioned above, we initially postulated that antagonism of HSF1 by
GR could result from one of two overall stages of GR signaling: 1)
hormone-induced release of Hsp70 and/or Hsp90 from the GR complex, or
2) genomic actions on the part of GR. Before our studies, evidence to
support the dissociation model could be found. First and foremost was
the existence of strong evidence for an Hsp70/Hsp90-based chaperone
complex that serves to keep HSF1 in an inactive state
(6, 7, 8, 9). In addition, Xiao and DeFranco (23)
had recently shown that transient overexpression of GR in COS-1 cells
caused stress-free activation of HSF1, by a mechanism involving
temporary loss of Hsp70 from inactive HSF1 complexes to newly forming
GR/HSP complexes. Thus, if HSPs could move from HSF1 to GR, why not
from GR to HSF1? However, it is clear from the present results that
this mechanism of action is not the means by which
hormone-activated GR is causing repression of HSF1, as hormone-induced
dissociation of the GR/HSP complex was observed under conditions in
which there was no inhibition of Hsp70 promoter activity (Figs. 2
, 8
, and 9
). Instead, we propose that inhibition of HSF1 by GR is through a
genomic mechanism of action. Evidence to support this conclusion is as
follows: 1) inhibition of HSF1 requires GR DNA-binding function (Fig. 9D
); and 2) inhibition of HSF1 is blocked by RU486, which acts as a GR
antagonist, not by preventing transformation of the GR/HSP complex
(Fig. 10
), but by preventing GR transactivation (Fig. 2
).
Although enhancement of transcription is the most commonly accepted
genomic effect of GR, a variety of other genomic mechanisms exist that
could explain antagonism of HSF1 by GR. These include repression of
gene expression by GR bound to so-called negative glucocorticoid
response elements (nGREs); direct or indirect inactivation by GR of
stress-activated, trans-acting factors, such as HSF1 itself
or factors cooperatively binding to the Hsp70 promoter; or competition
for a common coactivator that mediates both HSF1 and GR
transactivation. With respect to the common coactivator model, data
from our laboratory and others suggest that this mechanism is not
likely to be operating in our system, as reciprocal inhibition of GR by
HSF1 does not seem to occur. Rather, activity of both GR
(16) and PR (15) have been shown to greatly
increase in cells subjected to heat shock and other forms of stressin
a manner that appears to require intrinsic HSF1 activity (18, 19). In fact, under the same conditions of hormone treatment and
heat shock used in the present study, response of GR in the L929 cells
can be increased several fold by the stress event, as measured by a CAT
reporter gene driven by a minimal GRE promoter (16). It is
conceivable, however, that competition between GR and HSF1 for a common
coregulator may not be reciprocal, and for this reason further studies
into this potential mechanism are warranted. Such studies would require
some indication of coregulators that mediate HSF1 signaling. Yet, to
our knowledge, no such observations have been reported.
The present data also suggest that a mechanism by which GR binds
or otherwise inactivates stress-induced transcription factors,
including HSF1, is not likely to be operating. First, Dex was found to
inhibit the constitutive activity of the HSF1-E189 mutant (Fig. 5
). As
this inhibition occurred under nonstress conditions, it is clear that
the GR cannot be repressing other stress-activated transcription
factors; even though several candidates exist (e.g. AP-1,
CCAAT binding factor) for which cognate binding elements have been
found within the human Hsp70 promoter (our personal
observation). Second, results of the EMSA experiments (Fig. 6
)
showed no effect of Dex on the amounts or relative sizes of the
HSF1/DNA complexes, suggesting that GR is neither preventing the HSF1
DNA-binding event nor participating in it. Yet, it could be argued
that, if the putative GR/HSF1 interaction is relatively weak, GR that
is tethered to the HSF1/DNA complex could dissociate during
electrophoresis and would thus go undetected. Conceivably, our data
with the NB mutant (Fig. 9
) could be taken as evidence that the GR
DNA-binding domain may be the site for interaction with HSF1 or that a
mutation in this domain alters a distal HSF1-interaction surface.
Although this mechanism remains a possibility, our data with RU486 do
not support it. In this case, RU486 was found to effectively block
Dex-induced inhibition of HSF1 (Figs. 2
and 9
), while at the same time
causing translocation of the GR and tight binding to nuclei (Fig. 10
).
Thus, the RU486-bound GR appears to have a fully-functional DNA-binding
domain but is incapable of HSF1 inhibition.
Based on the above, we propose that GR-mediated inhibition of HSF1 can
occur either through the transrepression or transactivation
functions of the agonist-bound receptor (Fig. 11
). Transrepression by the GR is
typically thought to occur by direct binding of GR to nGREs in the
promoter region of genes. With this in mind, we have screened the
published sequence (729 bp) of the human Hsp70 promoter used in this
study and have found several consensus or near-consensus nGRE sequences
known to be sites of GR transrepression in a variety of genes
(37, 38, 39, 40). By these criteria, therefore, the actions of GR
as a repressor remain a plausible mechanism. Further experimentation to
test this potential mechanism will require mutagenesis/deletion studies
of the Hsp70 promoter or use of a minimal promoter comprised of
synthetic HSE elements.

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Figure 11. Model for Repression of HSF1 Transactivation
by Agonist-Activated GR
We have shown that glucocorticoid agonist (Dex) but not antagonist
(RU486) results in the inhibition of HSF1 transactivity following
stress. This response is not the result of HSPs (e.g.
Hsp90) released from the untransformed GR complex. Rather, it is a
process requiring the DNA-binding function of GR. Based on these
results, we propose that hormone-activated GR can inhibit the
transcriptional activity of HSF1 by one of two processes: 1) by direct
binding of GR to nGREs present in the hsp70 promoter, or 2) by the
actions of a GR-induced gene product (X) that directly or indirectly
inhibits the activity of DNA-bound HSF1 (see Discussion
for further details).
|
|
Our data are also consistent with a model in which GR transactivation
function is responsible for inhibition of HSF1. As depicted in Fig. 11
, inhibition of HSF1 could result from the production of a GR-regulated
gene product (X) that directly or indirectly affects the activity of
DNA-bound HSF1. Yet, at present, there is no obvious, Dex-induced gene
that could serve this role. However, a variety of proteins known to act
as negative regulators of HSF1 have been identified. These include:
heat shock factor binding protein 1 (41), DNA-dependent
protein kinase (19, 42) and MAPK members ERK1 and 2
(30, 31); as well as Hsp90, Hsp70, and Cyp40 (6, 7, 9). Of these, Hsp90, Hsp70, and Cyp40 are thought to principally
act by chaperoning HSF1 into its inactive state in the cytoplasm. As
such, the HSPs and Cyp40 would not likely act on DNA-bound HSF1,
although reports do exist that Hsp70 will cause release of HSF1 from
DNA (43). Moreover, no reports exist demonstrating
glucocorticoid-induced expression of Hsp90, Hsp70, or Cyp40, and, in
our laboratory, no such effect of Dex has been observed on overall
cellular levels of Hsp90 and Hsp70 (our unpublished
observations). In contrast, heat shock factor binding protein 1, DNA
protein kinase, and ERK1/2 fit the criteria of being able to act on the
DNA-bound form of HSF1. Thus, any effect of glucocorticoid hormones on
amounts or activities of these factors would do much to explain the
actions of hormone on HSF1. Indeed, evidence for a rapid activation of
MAPK in MCF-7 cells by 17ß-estradiol has recently been reported
(44). For these reasons, we have tested for an effect of
Dex on two members of the MAPK family. The results show that Dex
treatment of cells for up to 24 h does not increase activity of
ERK1/2 or of JNK, as measured by an antibody specific to the
phosphorylated forms of these kinases (Fig. 7
). While ruling out a
fast-acting, nongenomic effect of Dex on ERK1/2 and JNK signaling in
our system, this result does not bring us any closer to identifying the
GR-regulated gene product(s) responsible for the actions of hormone on
heat shock signaling. Future approaches to solve this problem may
require the use of screening methods, such as DNA arrays, as a way by
which to identify one or more genes potentially involved in this
response.
A working hypothesis of our laboratory is that heat shock and
glucocorticoid hormone responses are coordinated to ensure survival of
cells following stress. We base this hypothesis on the following
observations. First, it is clear that HSPs can protect cells through
their ability to act as chaperones that prevent denaturation of
proteins in response to stress (45). Second, it has been
long known that glucocorticoids are required for physiological
adaptation to stress. In this case, stress (e.g. infection,
surgery, trauma), acting through the hypothalamus-pituitary axis, will
lead to increased secretion of cortisol from the adrenal [see Munck
and colleagues (1, 46) for excellent reviews of this
topic]. For these reasons, we have not found it surprising that heat
shock leads to increased activation of the GR, and that these results
have recently been corroborated for GR-regulated genes in certain
tissues (13, 14).
With this in mind, how do we explain our current results, in which
glucocorticoid hormone inhibits the heat shock response? One possible
answer, of course, is that the inhibition of HSF1 we have observed here
is not limited to glucocorticoids, but, rather, can also be achieved in
response to other classes of steroid hormones. In this case, the
physiological relevance of steroid inhibition of the heat shock
response is less clear. On the other hand, should the inhibitory effect
of steroids on HSF1 be unique to glucocorticoids, or to a limited set
of related hormones, then an attractive hypothesis would be that the
heat shock response is fast acting compared with that of the GR, in
which more time is needed for serum cortisol levels to rise in response
to a stress event. Once haven risen, however, one role of
glucocorticoids may be to attenuate the heat shock response, perhaps to
prevent overstimulation by this pathway. In this sense, glucocorticoid
actions on the heat shock response may be analogous to their
antiinflammatory properties in the lymph system, in which damage by
prolonged or excessive inflammation is effectively mitigated by these
hormones.
 |
MATERIALS AND METHODS
|
---|
Materials
[3H]Triamcinolone acetonide (42. 8
Ci/mmol), [3H]acetate (10.3 µCi/mmol),
[35S]methionine ("Tanslabel"; 1,175
Ci/mmol), and [125I]conjugates of goat
antimouse IgG (11.8 µCi/µg) and goat antirabbit IgG (9.0
µCi/µg) were obtained from ICN Biochemicals, Inc.
(Cleveland, OH). Sodium vanadate, ATP, dimethylsulfoxide, sodium
arsenite, Dex, G418 (geneticin) antibiotic, hygromycin, acetyl
coenzyme A (CoA) synthetase, acetyl CoA, Tris, HEPES, EDTA, protein
A-Sepharose, DMEM-powdered medium, and horseradish peroxidase
conjugates of goat antimouse and goat antirabbit IgG were from
Sigma (St. Louis, MO). Autoradiography enhancer (Amplify)
was obtained from Amersham Pharmacia Biotech
(Arlington Heights, IL). The steroidal antagonist RU486 was
obtained from Roussel-Uclaf (Paris, France). Iron-supplemented newborn
calf serum was from HyClone Laboratories, Inc. (Logan,
UT); Immobilon polyvinylidenefluoride membranes were obtained
from Millipore Corp. (Bedford, MA) GenePorter transfection
reagent was obtained from Gene Therapy Systems, Inc. (San Diego, CA).
The SC-7383 monoclonal antibody against phosphorylated (active) ERK and
the SC-6254 antibody against phosphorylated (active) JNK were obtained
from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The
BuGR2 monoclonal antibody against GR (47) was purchased
from Affinity BioReagents, Inc. (Golden, CO); FiGR
monoclonal antibody against GR (48) was a gift from Jack
Bodwell (Dartmouth Medical School, Hanover, NH). The SPA-901 antibody
against human HSF1 and the SPA-820 antibody against human Hsp70 were
purchased from StressGen Biotechnologies (Victoria, British Columbia,
Canada). Monoclonal antibody against Hsp90 was obtained from
Transduction Laboratories, Inc. (Lexington, KY).
In the p2500-CAT reporter used in this study, expression of CAT is
controlled by the human Hsp70 promoter containing consensus HSEs known
to be activated by the binding of HSF1 (25). The pMMTV-CAT
plasmid contains the complete mouse mammary tumor virus (MMTV)-long
terminal repeat promoter (MMTV-LTR) upstream of CAT (49).
Hormonally driven expression of CAT by this reporter is controlled by
GREs residing within the long terminal repeat region
(50). The pBI-EGFP vector was obtained from CLONTECH Laboratories, Inc. In this vector, tetracycline-induced
expression is controlled by a tetracycline response element and two
minimal cytomegalovirus promoters in opposite orientations. The
pUHD1721hygro vector (29) expressing the "reverse
tet" transactivator and hygromycin resistance genes was obtained from
Hermann Bujard (Universitat Heidelberg, Heidelberg, Germany). The cDNA
for the E189 mutant of human HSF1 (27) was the generous
gift of Richard Voellmy (University of Miami, Coral Gables, FL).
Transfection of Cell Lines
The LHSE and LMCAT2 cell lines were established as previously
described (16, 18). Briefly, mouse L929 cells were
cotransfected with pSV2neo and a 2-fold excess of p2500-CAT (LHSECAT
cells) or pMMTV-CAT (LMCAT2 cells) using lipofectin as carrier. This
was followed by selection for stably transfected, cloned cell lines
using G418 (Geneticin) antibiotic at 0.4 mg/ml. Once established, both
cell lines were grown in an atmosphere of 5% CO2 at 37 C in DMEM
containing 0.2 mg/ml G418 and 10% iron-supplemented NCS.
The tetracycline-inducible LHSF1-E189 cells were made by stably
transfecting LHSE cells with the pUHD1721hygro plasmid and a 7-fold
excess of pBI-E189 plasmid, followed by selection and cloning using 0.4
µg/ml hygromycin. The pBI-E189 construct was made by excising the
cDNA for the constitutively-active hHSF1-E189 mutant from the pGEM-E189
vector originally developed by Voellmy and co-workers
(27). This cDNA was then inserted into the multiple
cloning site of the pBI-EGFP vector (CLONTECH Laboratories, Inc.).
The CHSE, WHSE, NBHSE, and NAHSE cells used in this study were
generated by cotransfecting p2500-CAT and pSV2neo plasmids into CHO
cells that contain either no GR (CHOd cells), wild-type mouse GR (WCL2
cells), DNA-binding defective mouse GR (NB cells), or hormone-binding
defective mouse GR (NA cells), respectively. This was followed by
selection of cloned-resistant cell lines using G418 antibiotic. The
GR-expressing CHO cells were originally developed by Gordon Ringold and
co-workers (35) using methotrexate-based selection and
amplification. The various properties of these GRs have been further
characterized by us (36) and others.
Stress and Hormone Treatment of Cell Lines
For all experiments, the NCS was stripped of endogenous steroids
by extraction with dextran-coated charcoal. Most stress experiments
were performed on cells that were at or near confluence, although
similar results were obtained with subconfluent cultures. Heat shock
treatment was achieved by shifting replicate flasks to a second 5%
CO2 incubator set at 43 C. Typical duration of
heat shock treatment was 2 h. Cells were also subjected to
chemical shock by addition of 200 µM sodium arsenite to
the medium. In the chemical shock experiments, the arsenite-treated and
nontreated cells were incubated at 37 C for 2 h and were then
washed with DMEM and allowed to recover.
CAT Assay
Measurement of CAT enzyme activity was performed according to
the method of Nordeen et al. (51) with minor
modifications. In this assay, a reaction mixture containing acetyl CoA
synthetase, [3H]sodium acetate, CoA, and ATP
was briefly preincubated to enzymatically generate labeled acetyl CoA
from CoA and labeled acetate. Acetylation of chloramphenicol was then
initiated by adding cell lysate containing CAT enzyme. The reaction was
stopped by extraction with cold benzene, and 75% of the organic phase
was counted. Cell lysates were prepared by sequential freezing and
thawing in 0.25 M Tris, 5
mM EDTA (pH 7.5), and centrifugation at 14,000x
g. Aliquots of lysate containing equal protein content were
added to the enzymatic reaction mixtures. As the GRE- and
HSE-containing promoters employed in this study have distinct basal and
inducible activities, all data are represented as percent of control,
maximum, or the equivalent. In this way, the relative inhibitory or
stimulatory effects of each treatment can be readily seen.
Analysis of HSP Synthesis by Labeling with
[35S]Methionine
In the experiment of Fig. 4
, LHSE cells were shocked by
incubation with 200 µM sodium arsenite in the presence or
absence of 1 µM Dex. At indicated intervals during
recovery, the cells were pulse labeled with
[35S]methionine for 45 min by removing the
medium and replacing it with methionine-free medium containing 10%
dialyzed calf serum and [35S]methionine at a
final concentration of 10 µCi/ml. All subsequent steps were carried
out on ice (04 C). Cells were washed three times by pelleting and
resuspension in HBSS, followed by three cycles of freezing and thawing
in 0.25 M Tris, 5 mM EDTA (pH 7.5) and
centrifugation at 100,000 x g. The protein content of
these whole cell extracts were determined by the BCA procedure of
Pierce Chemical Co. (Rockford, IL), and the extracts were
used immediately or were frozen at -80 C. Samples were resolved by
electrophoresis in 7% polyacrylamide SDS gels as described by Laemmli
(52). Following impregnation of the gels with an
autoradiography enhancer (Amplify), the gels were dried under vacuum
and mild heating (70 C) and exposed to Kodak XAR-5 x-ray
film (Eastman Kodak Co., Rochester, NY) with an
intensifying screen at -80 C. The relative amounts of newly
synthesized hsp70 and hsp90 were calculated by densitometric scanning
using Molecular Analyst software (Bio-Rad Laboratories, Inc., Hercules, CA).
Lysate Preparation, Immune Purification of GR Complexes, and
Western Blotting
In the experiments of Figs. 4
and 6
, whole cell extracts were
prepared by freeze/thaw in WCE buffer [20 mM HEPES, 25%
glycerol, 0.42 M NaCl, 1.5 mM MgCl2, 0.2
mM EDTA, 0.5 mM pregnant mares serum, and 0.5
mM dithiothreitol (DTT), pH 7.9] and centrifugation at
14,000 x g for 30 min. In the experiments of Fig. 8
, cytosols were prepared by Dounce A homogenization of cells in hypotonic
buffer (10 mM HEPES, 3 mM
EDTA, 10 mM sodium molybdate, pH 7.4), followed
by centrifugation at 14,000 x g. In the experiments of
Fig. 10
, cells were fractionated into cytosolic and nuclear portions by
Dounce A homogenization in hypotonic buffer, followed by centrifugation
at 1,000x g. The cytosolic fractions were saved and the
nuclear pellets were washed two times by resuspension and pelleting in
hypotonic buffer. Hypotonic buffer containing 0.5
M NaCl was then added to the pellet fractions and
incubated on ice with occasional vortexing for 1 h. After salt
extraction, the nuclear pellets were centrifuged at 14,000 x
g and the supernatants saved. For the experiments of Figs. 7
and 9
, BuGR2 anti-GR monoclonal antibody (1.5 µg) was added to the
regular cytosols, or to the cytosolic and nuclear fractions, and each
sample was then adsorbed in batch to protein A-Sepharose, followed by
washing with TEG buffer (10 mM
N-Tris[hydroxymethyl]methyl-2-aminoethane-sulfonic acid, 1
mM EDTA, 10% glycerol, 50
mM NaCl, 10 mM sodium
molybdate, pH 7.6) and elution with 2x SDS sample buffer.
All samples were resolved by electrophoresis in 7% polyacrylamide SDS
gels, followed by transfer to Immobilon polyvinylidenefluoride
membranes. The relative amounts of hHSF1-E189, ERK 1/2, GR, or
GR-associated HSPs were determined via a Western blotting technique
previously described (53), employing primary antibody and
both peroxidase- and 125I-conjugated counter
antibodies. After color development, the blots were exposed to
Kodak XAR-5 film with an intensifying screen at -70
C.
HSF1 EMSA
EMSAs for HSF1 were performed according to the protocol of
Mosser et al. (54), with minor modifications.
Briefly, single 75-cm2 flasks of LHSECAT cells
were subjected to the indicated stress and hormone conditions. Cells
were harvested, centrifuged, and rapidly frozen at -80 C. The frozen
pellets were resuspended in WCE buffer (20 mM
HEPES, 25% glycerol, 0.42 M NaCl, 1.5
mM MgCl2, 0.2 mM EDTA, 0.5
mM pregnant mares serum, and 0.5
mM DTT, pH 7.9) and centrifuged at 100,000
x g for 10 min. The supernatants were either stored at -80
C or used immediately. EMSA assays were performed by mixing 10 µg of
whole cell extract with 0.1 ng (50,000 cpm) of
[32P]-labeled HSE oligonucleotide
(5'-GAT,CTC,GGC,TGG,AAT,ATT,CCC,GAC, CTG,GCA,GCC,GA-3') and 1.0 µg of
poly (dI-dC) in 10 mM Tris (pH 7.8), 50
mM NaCl, 1 mM EDTA, 0.5
mM DTT, 5% glycerol in a final volume of 25
µl. For competition experiments, the binding reactions contained 0.1
ng of the [32P]HSE and a 100-fold molar excess
of unlabeled HSE. Reactions were incubated at 25 C for 30 min and
loaded onto 4% polyacrylamide gels in 0.5x Tris-borate-EDTA.
The gels were run at room temperature for 1.5 h at 150 V and were
exposed to Kodak XAR-5 film with an intensifying screen at
-80 C. The relative amounts of probe-bound HSF1 were then measured by
densitometric scanning of the film using the Bio-Rad Laboratories, Inc. Molecular Analyst system.
 |
ACKNOWLEDGMENTS
|
---|
The authors wish to thank Dr. Richard Voellmy for his generous
gift of cDNA for the human HSF1-E189 mutant and for the p2500-CAT
reporter. We are also grateful to Dr. Daniel Philibert for his gift of
RU486, Dr. Hermann Bujard for the pUHD1721 vector, and Dr. Jack
Bodwell for the FiGR antibody.
 |
FOOTNOTES
|
---|
This work was supported by grants to E.R.S. by the National Institutes
of Health (DK-43867) and the National Science Foundation
(MCB-9905117).
Abbreviations: CAT, chloramphenicol acetyltransferase; CoA,
coenzyme A; Dex, dexamethasone; DTT, dithiothreitol; GRE,
glucocorticoid response element; HSE, heat shock element; HSF,
heat shock transcription factor; HSP, heat shock protein; JNK,
c-jun N-terminal kinase; MMTV, mouse mammary tumor
virus; nGRE, negative glucocorticoid response element.
Received for publication October 20, 2000.
Accepted for publication April 10, 2001.
 |
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