Overexpression of Unliganded Steroid Receptors Activates Endogenous Heat Shock Factor
Nianqing Xiao and
Donald B. DeFranco
Department of Biological Sciences University of Pittsburgh
Pittsburgh, Pennsylvania 15260
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ABSTRACT
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The synthesis of a number of heat shock proteins
is induced in response to various environmental stresses. The resultant
induction of heat shock protein gene transcription is brought about by
the activation of specific transcription factors termed heat shock
factors (HSFs) that exist in a latent form in nonstressed cells.
Multiple mechanisms are likely to contribute to negative regulation of
HSF activity. One model, which remains controversial, proposes the
existence of a negative feedback loop by which one of the products of
HSF activation, the 70-kDa heat shock protein (hsp70), acts as one of
its negative regulators. Accordingly, HSF activation would proceed upon
sequestration of hsp70 by substrates (i.e. unfolded
proteins) that may accumulate to relatively high levels in stressed
cells. To examine whether putative native substrates of hsp70
(e.g. steroid receptors) could impact the regulation of HSF
activity, we have examined whether steroid receptors could activate
endogenous HSF. We have found that overexpression of androgen (AR),
glucocorticoid (GR), mineralocorticoid, and progesterone receptors in
transiently transfected COS-1 cells induced HSF activity. With the
exception of AR, which was competent to activate HSF when either
liganded or unliganded, all other steroid receptors tested only
activated HSF when unliganded. This activity was mapped to the
ligand-binding domain of rat GR, making it unlikely that HSF activation
results from the induction of a novel gene product by unliganded
receptors. As overexpression of hsp70 can eliminate HSF activation by
AR, GR, and progesterone receptors, we favor the view that HSF
activation can result from the sequestration, by steroid receptor
ligand-binding domains, of a negative regulator of HSF, such as hsp70
or an hsp70-associated protein.
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INTRODUCTION
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Unliganded steroid hormone receptors are poised to respond to
hormonal signals and alter the expression of unique target genes, often
in a tissue- or cell type-specific manner (1, 2, 3). Typically, the
binding of cognate hormone is required to unleash the transcriptional
regulatory activity of the receptor, although in some cases, receptors
can modulate transcription in response to the activation of other
signal transduction pathways (4, 5). Activation of the estrogen
receptor by epidermal growth factor is associated with direct
phosphorylation of the receptor by mitogen-activated protein kinase (6, 7). Whether steroid receptor phosphorylation accounts for
ligand-independent activation in other systems (8) has not been
established.
The carboxyl-terminal ligand-binding domain (LBD) of steroid receptors
is responsible for maintaining unliganded receptors in an inactive form
(9). The dominant inactivation function associated with unoccupied
steroid receptor LBDs is imparted upon linked receptor DNA-binding and
trans-activation domains (10), but can also impair the
activity of heterologous functional domains (10, 11). Upon hormone
binding, the LBD undergoes a conformational change (12) that not only
leads to the relief of its repressive effects on DNA-binding and
amino-terminal trans-activation domains, but also exposes a
dimerization interface (13) and a trans-activation domain
(14, 15, 16, 17) encoded within the LBD.
The LBD of steroid receptors also serves to assemble a heteromeric
complex comprised of various members of the heat shock and immunophilin
families of proteins (18, 19). Principal protein components associated
with glucocorticoid receptors (GRs) and progesterone receptors (PRs)
include the 90-kDa heat shock protein (hsp90), a 52-kDa immunophilin
(FKBP52), and a 23-kDa protein (p23) (18, 19). The association of these
proteins with GR and PR is required for the receptors to attain a high
affinity ligand-binding capacity (20, 21).
The assembly of GR and PR heteromeric complexes is a multistep process
that proceeds in a defined order in vitro (21, 22). GR and
PR associate with a 70-kDa heat shock protein (hsp70) in one of the
earliest steps in the formation of receptor-heteromeric complexes (21, 23). This binding appears to be only transient, as intermediate
complexes are then generated composed of a the 48-kDa Hip protein and
p60 (24). Hormone binding competence is finally acquired as mature
forms of GR and PR are generated possessing stably associated hsp90,
FKBP52, and p23 (21, 24).
Although PR and GR heteromeric complexes of various composition have
been visualized using in vitro assembly reactions (21, 25),
confirmation that such an ordered pathway operates in vivo
has proven difficult to obtain (26, 27). Coimmunoprecipitation
experiments have identified hsp90 (28, 29) and FKBP52 (30, 31) as
components of unliganded steroid receptor heteromeric complexes, but
the association of other proteins with unliganded steroid receptors
in vivo remains controversial (32). For example, there are
conflicting reports regarding the association of hsp70 with steroid
receptors such as GR and PR (28, 33, 34, 35, 36). As hsp70 does not appear to
be a component of hormone binding-competent steroid receptor
heteromeric complexes reconstituted in vitro (21, 24), it
remains to be established whether hsp70 association with steroid
receptors in vivo is functionally significant.
Heat shock proteins are involved in steroid receptor signal
transduction in nonstressed cells (9), indicating that constitutive
levels of these stress-activated proteins are sufficient to impact
receptor function. In response to various environmental stresses, the
levels of inducible forms of heat shock proteins increase dramatically
(37, 38). Does the elevation in hsp90 and hsp70 levels in stressed
cells alter steroid hormone signaling? An increased association of
hsp90 and hsp70 with PR was found to result from environmental stresses
that induced expression of heat shock proteins (39). Under these
conditions, steroid receptor-mediated trans-activation was
potentiated (39). Heat shock potentiation of GR
trans-activation has also been observed and is
representative of effects that result from other types of environmental
stress (40). Although the mechanistic basis for these effects remains
obscure, these results illustrate that steroid hormone signaling is
sensitive to conditions that activate particular cellular stress
responses.
The transcriptional activation of heat shock protein genes such as
hsp90 and hsp70 is regulated by a family of DNA-binding proteins termed
heat shock factors (HSFs) (41, 42). HSF-1 is the principal member of
this family that is thought to regulate hsp70 gene transcription (43, 44) via its binding, as a trimer, to a unique sequence within the hsp70
promoter, termed heat shock response elements (HSEs) (41, 42). HSF-1 is
present in nonstressed cells and is, therefore, maintained in a dormant
state until activated by the appropriate environmental stress (41, 42).
A specific regulatory domain has been identified within HSF-1 that is
responsible for negative regulation of is activity in nonstressed cells
(45, 46, 47). The activity of the human HSF-1 regulatory domain is
influenced by direct phosphorylation mediated via the
raf/ERK pathway (48). In addition to this level of control,
there is considerable evidence for the involvement of hsp70 in negative
regulation of HSF activity (44, 49). In this case, a negative feedback
loop may be established that limits the extent and duration of HSF
activity and perhaps partially contributes to the maintenance of
inactive HSF (50). Although results from in vivo studies in
yeast (51) and mammalian cells (52) lend support to this model, results
that contradict this model have also been obtained (53).
The association of hsp70 with distinct transcriptional regulators
(i.e. GR and HSF) implicates an involvement for this
stress-activated protein in signal transduction that may extend beyond
its well established chaperone functions. As hsp70 pools in
vivo are likely to be dynamic, dramatic alterations in the
bioavailability of hsp70 that are likely to accompany the mobilization
of a cellular stress response may have an impact on GR- or HSF-mediated
transcriptional responses. We, therefore, examined whether manipulation
of steroid receptor levels affects HSF activity in cultured cells. Our
observed activation of HSF activity by unliganded steroid receptors
provides evidence for an unique level of cross-talk between steroid
hormone signaling and cellular responses to environmental stresses.
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RESULTS
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Induction of HSF Activity by Unliganded GR
Activated HSFs bind to heat shock elements (HSEs) as trimers and
stimulate the transcription of linked promoters (42). HSF-induced
transcription can be easily monitored in cells transfected with
HSE-containing reporter plasmids. As shown in Fig. 1
, a 1-h heat shock (i.e. 43
C) of COS-1 cells activated endogenous HSF activity, leading to the
robust induction of chloramphenicol acetyltransferase (CAT) activity
from a reporter that possess a minimal human hsp70 gene promoter with
an intact HSE (54). A CAT reporter lacking the HSE sequences was
unaffected by an analogous heat shock of transfected COS-1 cells (data
not shown). Interestingly, cotransfection with a rat GR expression
vector (i.e. 6RGR) also led to induced transcription from
the HSE-CAT reporter plasmid (Fig. 1
) in the absence of heat shock.
This activation of HSF activity was dependent upon the presence of the
HSE, as no change in basal transcription from a CAT reporter lacking
the HSE was noted upon cotransfection with the GR expression vector
(Fig. 1
). GR maintained its property as a hormone-dependent
trans-activator under our transfection conditions, as
revealed by the hormone-dependent activation from a cotransfected
glucocorticoid response element (GRE)-linked CAT reporter (Fig. 1
).

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Figure 1. Overexpression of Unliganded GR Activates HSF
COS-1 cells were transfected with 2 µg CAT reporter plasmids that
either lacked upstream regulatory elements (CAT) or possessed an
upstream GRE (GRE-CAT) or HSE (HSE-CAT). Where indicated (GR), 4 µg
of the rat GR expression plasmid p6RGR were cotransfected. In all
cases, 0.2 µg of a cytomegalovirus-ßgal reporter plasmid was also
cotransfected to provide a standard to normalize transfection
efficiency. Total DNA was constant in all transfections. Transfected
cells were grown in the presence or absence of 1 µM Dex
or RU486 as indicated for 36 h and then harvested, lysed, and
assayed for CAT activity. Some transfected cells (HS) were heat shocked
at 43 C for 1 h and then recovered for 3 h before harvesting.
Results are an average of at least three independent experiments.
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As unliganded GR did not appear to function as a
trans-activator in transfected COS-1 cells (Fig. 1
), it was
unlikely that GR induction of HSE-CAT activity resulted from
receptor-dependent synthesis of a gene product that activated HSF
activity. In fact, in the presence of dexamethasone, GR-dependent
induction of HSE-CAT promoter activity was suppressed (Fig. 1
). As will
be shown below (Fig. 8B
), GR levels were not dramatically reduced upon
dexamethasone treatment of transfected COS-1 cells, indicating that the
hormone-dependent relief of GR-dependent activation of HSF was not due
to decreased synthesis of the receptor. The glucocorticoid antagonist
RU486 is also effective in relieving GR-dependent HSE-CAT promoter
activation (Fig. 1
), although the suppression was somewhat less than
that observed with dexamethasone.

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Figure 8. GR Expression in Dexamethasone (Dex)-treated and
hsp70 Cotransfected Cells
COS-1 cells were cotransfected with p6RGR DNA, HSE-CAT, and, where
indicated, an hsp70 expression plasmid (pßact-Hsp70). Cells (either
untreated or treated with 1 µM Dex) were harvested after
36 h, and equivalent amounts of total protein in cell-free
extracts were separated by SDS-PAGE. GR was visualized by Western blot
analysis.
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To determine the relative effectiveness of GR effects on GRE-
vs. HSE-linked promoters, varying amounts of p6RGR DNA were
independently cotransfected into COS-1 cells with either a GRE-linked
or a HSE-linked CAT reporter. Although the induction of GRE-linked
promoter activity by ligand-bound GR reached its maximum at relatively
low amounts of 6RGR plasmid (i.e. 12 µg/transfection;
Fig. 2
), robust induction of HSE-linked
promoter activity required at least 5-fold higher amounts of
transfected p6RGR DNA (i.e. 8 µg/transfection; Fig. 2
).
This nonlinear dose-response curve of GR-dependent induction of
HSE-linked promoter activity resembles that obtained when induction
from an HSE-linked reporter was analyzed after microinjection of
varying amounts of denatured proteins into Xenopus laevis
oocytes (55). As higher amounts of p6RGR DNA reduced transfection
efficiency and had adverse affects on cell survival (data not shown),
we did not examine induction of HSE-CAT activity at higher amounts of
transfected p6RGR DNA. As it is difficult to definitively measure GR
levels in transient transfection experiments, particularly at the level
of individual cells, we cannot relate the increased amount of
transfected p6RGR DNA with absolute GR levels. Nonetheless, it appears
likely that effective activation of HSF activity required GR levels
exceeding those required for effective induction from a GRE-linked
promoter.

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Figure 2. Dose-Response Analysis of Transfected p6RGR DNA
Effects on HSE- and GRE-Linked Promoters
COS-1 cells were transfected with different amounts of p6RGR DNA, as
indicated, 2 µg HSE-CAT or GRE-CAT, and 0.2 µg
cytomegalovirus-ßgal. Cells were grown in the presence or absence of
Dex for 36 h and then harvested, lysed, and assayed for CAT
activity.
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The GR LBD Is Required for Activation of HSF Activity
To reveal whether a unique domain within the rat GR is responsible
for activation of HSF activity, receptor deletion mutants were
cotransfected into COS-1 cells with the HSE-CAT reporter. As shown in
Fig. 3A
, deletion of the
carboxyl-terminal LBD (i.e. GR mutant VAN525) dramatically
reduced the ability of GR to activate HSF, whereas deletion of the
amino-terminal 407 amino acids (i.e. VA407C) actually led to
an increase in HSF activation. For both full-length wild type GR (VARO)
and VA407C, dexamethasone treatment abolished HSF activation (Fig. 3A
).
The inability of VAN525 to activate HSF activity was not due to
differences in its expression, as Western blot analysis showed that
VARO, VA407C, and VAN525 were expressed equivalently in transfected
COS-1 cells (Fig. 3B
). To confirm that the GR LBD was primarily
responsible for HSF activation, this domain of the receptor was
expressed separately as a ß-galactosidase (ßgal) chimera. As shown
in Fig. 3C
, the GR LBD-ßgal chimera (i.e. VAL-HBD) was as
effective as the full-length GR-ßgal chimera (i.e. VARL)
in activating HSF activity in transfected COS-1 cells. The ßgal
protein (i.e. VAL) did not activate HSF (Fig. 3C
),
establishing that the LBD portion of VAL-HBD was responsible for this
activity. The LBD-ßgal chimera lacks a DNA-binding domain and, thus,
is unable to activate target gene transcription through DNA binding.
Thus, these results support our contention that activation of HSF does
not result from the induction of a gene product by unliganded GR.

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Figure 3. Mapping of the GR Domain Responsible for Activation
of HSF
A, COS-1 cells were cotransfected with 4 µg of plasmids expressing
full-length GR (VARO), amino-terminal-truncated (VA407C) or
carboxyl-terminal-truncated GR (VAN525), 2 µg HSE-CAT, and 0.2 µg
cytomegalovirus-ßgal. Cells were harvested after 36 h and then
lysed and assayed for CAT activity. Dexamethasone (Dex; 1
µM) was added, where indicated, during the 36-h
incubation. Results are an average of at least three independent
experiments. B, Cell-free extracts were prepared from COS-1 cells
transfected as described in A, and total protein was separated by
SDS-PAGE. Full-length GR as well as 407C and N525 deletion derivatives
were visualized by Western blot analysis. C, COS-1 cells were
cotransfected with HSE-CAT and expression plasmids encoding the ßgal
gene alone (VAL), a full-length GR-ßgal chimera (VARL), or a GR
LBD-ßgal chimera (VAL-HBD). Cells were harvested after 36 h,
lysed, and assayed for CAT activity. Results are an average of at least
three independent experiments.
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Activation of HSF Activity by Other Steroid Receptors
The LBDs of steroid receptors, although sharing some degree of
homology, possesses multiple amino acid determinants that permit
discrimination between closely related steroidal ligands (56). It was,
therefore, of interest to reveal whether activation of HSF activity was
unique to unliganded GR or shared by other steroid receptors. As shown
in Fig. 4
, in addition to GR, unliganded
PR, mineralocorticoid receptor (MR), and androgen receptor (AR)
activated HSF activity in transfected COS-1 cells. HSF activity was not
activated by ligand-bound GR, PR, or MR, but curiously, AR maintained
its ability to activate HSF activity when ligand bound (Fig. 4
). To
confirm this result, a DNA titration was performed for both AR and GR
expression plasmids, and HSF activation was monitored in both the
presence and absence of hormone. In these transfections, a
HRE-luciferase (luc) reporter was also cotransfected into cells with
the HSE-CAT reporter so that hormone-dependent transcriptional
induction mediated by receptor binding to its cognate HRE could be
monitored in the same population of transfected cells as HSE activation
by HSF. As shown in Fig. 5
, AD,
activation of HSF by both AR and GR required higher amounts of
transfected DNA than hormone-dependent receptor induction from the
HRE-linked reporter. This experiment provided an independent
confirmation of our previous experiment with GR (Fig. 2
) that was
performed with separate transfected cell populations. Likewise, no
induction of HSF activity was noted for ligand-bound GR regardless of
the amount of GR expression plasmid transfected (Fig. 5C
). In contrast,
the hormone-binding state of AR had little effect on its ability to
activate HSF activity at all DNA concentrations examined (Fig. 5D
).
Thus, although AR, GR, MR, and PR have the capacity to activate HSF
activity when unliganded, only AR maintained this activity when ligand
bound.

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Figure 4. Activation of HSF by Other Steroid Receptors
COS-1 cells were cotransfected with GR, PR, MR, and AR expression
plasmids along with either an HSE-CAT or a HRE-CAT reporter gene. GR-,
PR-, MR-, and AR-transfected cells were grown either untreated or
treated (+H) with 1 µM dexamethasone (Dex), R5020,
aldosterone, or DHT, respectively. Cells were harvested after 36
h, lysed, and assayed for CAT activity. Results are an average of at
least three independent experiments.
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Figure 5. Dose-Response Analysis of Transfected p6RGR and
p6RAR DNA Effects on HSE- and HRE-Linked Promoters
COS-1 cells were cotransfected with varying amounts of GR (A and C) or
AR (B and D) expression plasmids along with HSE-CAT and HRE-luciferase
reporters. Cells were grown in the presence or absence of 1
µM Dex (A and C) or DHT (B and D) for 36 h. A and B
show results from hormone-treated cells. Cells were then harvested and
assayed for both CAT and luciferase activities. A, B, and D show the
results of a typical experiment; C shows the average results from three
independent experiments.
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Activation of HSF Activity by Steroid Receptors Is Overcome upon
Coexpression of hsp70
HSFs are present in nonstressed cells, but are maintained in an
inactive state (41, 42). A specific regulatory domain has been
characterized within HSF-1 that is responsible for negative regulation
of its activity (45, 46, 47). It has been postulated that hsp70, one of the
products of HSF activation, participates in a negative feedback loop to
inactivate HSFs and thereby limits the extent of heat shock protein
induction (50). Members of the hsp70 family may also play a role in
nonstressed cells to inactivate HSFs through direct interactions with
their regulatory domain (50). According to this view, HSF activation
might be initiated once hsp70 is released and becomes recruited to
other targets (i.e. misfolded or aggregated proteins) that
are generated after various environmental stresses. Interactions
between steroid receptors and hsp70 have been detected in
vivo, but not in all studies (33, 34, 36). Could overexpression of
unliganded steroid receptors initiate activation of HSFs through a
sequestration of hsp70? As shown by the indirect immunofluorescence
micrographs in Fig. 6
, expression of GR
in transfected COS-1 cells led to an increased accumulation of
endogenous hsp70, which was predicted from the activation of HSF
activity as measured using a model cotransfected HSE-linked reporter
(see Fig. 1
). Interestingly, the subcellular localization of induced
hsp70 in these cases coincided with the compartmentalization of GR.
Thus, in transfected cells in which GR was cytoplasmic, hsp70
accumulated predominantly within the cytoplasm (Fig. 6
), whereas in
cells with unliganded nuclear GR, hsp70 correspondingly accumulated
within the nucleus. Thus, it seems likely that in transiently
transfected COS-1 cells, GR may stably associate with hsp70 and
sequester it into either the cytoplasmic or the nuclear
compartment.

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Figure 6. Visualization of GR and hsp70 Subcellular
Localization in Transfected COS-1 Cells by Indirect Immunofluorescence
COS-1 cells were transfected with p6RGR DNA, fixed with methanol, and
processed for indirect immunofluorescence to visualize transiently
expressed GR and endogenous hsp70. Cells were grown in the absence or
presence of Dex for 36 h.
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If sequestration of hsp70 by unliganded steroid receptors leads to the
induction of HSF activity, we might expect to overcome this induction
upon elevation of hsp70 levels. As shown in Fig. 7
, cotransfection of an hsp70 expression
vector along with either GR, PR, or AR expression vectors led to a
dramatic reduction in the extent of HSF activation from the HSE-linked
reporter in COS-1 cells that were not treated with hormone. In
addition, the induction of HSF activity by ligand-bound AR was overcome
upon cotransfection with an hsp70 expression plasmid. As Western blot
analysis demonstrated that GR expression was not affected by either
hormone treatment or hsp70 DNA cotransfection of COS-1 cells (Fig. 8
), the alleviation of unliganded
GR-dependent HSF activation was not due to a reduction in receptor
levels.

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Figure 7. Suppression of Steroid Receptor-Induced HSF
Activation by Coexpression of hsp70
COS-1 cells were cotransfected with GR, PR, or AR expression plasmids;
HSE-CAT; cytomegalovirus-ßgal; and an hsp70 expression plasmid
(pßact-Hsp70). Cells were harvested after 36 h, lysed, and
assayed for CAT activity. Where indicated, cells were treated with 1
µM dihydrotestosterone (DHT). Results are an average of
at least three independent experiments.
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DISCUSSION
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The HSF family of transcription factors regulates heat shock
protein gene expression and is activated in response to a variety of
environmental stresses (41, 42). The accumulation of unfolded proteins
within stressed cells may provide one of the intracellular signals that
mediate HSF activation (55). As chaperone proteins, such as members of
the hsp70 family, are recruited to unfolded protein substrates in
stressed cells (37), HSFs may be released from their association within
an inhibitory complex, which could include hsp70 (44, 49). This
putative mechanism is most likely not solely responsible for HSF
activation, as alterations in HSF phosphorylation (48) or association
with other regulatory factors (42) may also contribute to the
regulation of HSF activity.
In this report we demonstrate that overexpression of steroid receptors
in transiently transfected cells can lead to activation of HSF
activity. The extent of receptor overexpression required to elicit this
effect is difficult to assess on a single cell basis. However, DNA
titration experiments suggest that the extent of receptor
overexpression to generate measurable HSF activation probably exceeds
that required to generate maximum hormonal induction from HRE-linked
promoters by approximately 5-fold. This overexpression does not
dramatically dampen the ability of functional receptors to maximally
activate transcription, indicating that receptors and HSF are not
competing for limiting cofactors or coactivators. Importantly, at the
same relative level of receptor overexpression, HSF is not activated if
receptors are ligand bound, with one notable exception (i.e.
AR). Thus, the spare receptors that may be responsible for bringing
about HSF activation are maintained in a conformation competent to bind
hormone and are not grossly misfolded. Curiously, activation of HSF
that results from overexpression of unliganded steroid receptors or the
accumulation of unfolded proteins (55) appears to be mediated by a
common mechanism (see below). Under our transfection conditions, the
unliganded steroid receptors were ineffective as
trans-activators from HRE-driven reporters, making it
unlikely that HSF activation was brought about by the action of a
receptor-induced gene product. This contention was supported by the
observation that overexpression of the unliganded GR LBD as a ßgal
chimera was sufficient to effectively activate HSF.
What mechanism is responsible for activation of HSF by unliganded
steroid receptors? The LBD, which mediates HSF induction by GR, is
known to associate with many proteins, particularly in the unliganded
state (19). Thus, analogous to the mechanism thought to be responsible
for HSF activation by unfolded proteins (50), this domain may sequester
a negative regulator of HSF. Hsp70 has been postulated to be at least
one such negative regulator of HSF activity, as activation of HSF
brought about by the accumulation of unfolded proteins is abrogated
upon overexpression of hsp70 (57). Our experiments also support a role
for hsp70 in negative regulation of HSF activity, as activation by
unliganded steroid receptors was likewise abrogated by overexpression
of hsp70.
The implication from our findings is that HSF activation may result
from the sequestration of members of the hsp70 family by unliganded
steroid receptors. Although members of the hsp70 family of chaperone
proteins have been found to be associated with steroid receptors in
some in vivo studies (33, 35, 36), the specificity of such
receptor/hsp70 complexes has been questioned (34). In vitro,
the association between hsp70 and steroid receptors appears to be only
transient and involved principally in the earliest stages of receptor
maturation (21, 23). We would hypothesize that upon overexpression of
unliganded receptors in transfected cells, such normally transient
steroid receptor/hsp70 complexes may be stabilized to a certain extent.
If this results in a significant depletion of available hsp70 pools,
negative regulation of HSF may be overcome.
Regardless of the mechanism responsible for steroid receptor-mediated
activation of HSF, we feel that caution must be applied toward the
interpretation of any results regarding receptor function that are
generated in cells that overexpress receptors. Such cells may be
mobilizing a stress response, particularly if unliganded receptors are
allowed to accumulate in excess of physiologically relevant levels. We
would not expect that this level of receptor overexpression
(i.e.
45 fold) sequesters all of the cellular hsp70
pools. However, given the multitude of protein folding and trafficking
pathways that use the chaperone activity of hsp70 (37), any significant
departure from the putative equilibrium compartmentalization of these
chaperones may exert profound effects on hsp70-regulated events.
A unique aspect of our manipulation of HSF activity was the fact that
steroid receptor-dependent activation of HSF was reversible and
completely abolished in all but one case by the binding of ligand. We,
therefore, speculate that ligand binding might facilitate the release
of GR-bound hsp70, which would then become available to participate in
negative regulation of HSF activity. AR was unique in its ability, when
overexpressed, to activate HSF activity in both the absence and
presence of hormone. Both hormone-independent and -dependent
activations of HSF activity by AR were reduced upon cotransfection with
hsp70 DNA, suggesting that both unliganded and ligand-bound AR function
analogously to sequester available hsp70. The physiological
significance of this unique behavior of AR is unknown, but may become
apparent once the role of molecular chaperones in the maturation of AR
is established.
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MATERIALS AND METHODS
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Plasmids
The rat GR, AR, and MR complementary DNA (cDNA) expression
plasmids (provided by K. R. Yamamoto, University of California-San
Francisco; R. Miesfeld, University of Arizona, Tucson, AZ; and D.
Pearce, University of California-San Francisco, respectively) were in
the same plasmid backbone (i.e. p6R) that possesses the
adenovirus major late promoter to drive receptor expression (58). The
chicken PRA cDNA (provided by B. OMalley, Baylor College
of Medicine, Houston, TX) (59) was cloned into the p6R vector. Plasmids
encoding full-length and truncated GR, including VARO, VAN525, and
VA407C, and the ßgal gene fused to full-length (VARL) and the HBD
(VAL-HBD) of GR (provided by K. R. Yamamoto and D. Picard, University
of Geneva, Geneva, Switzerland) have been described previously (60, 61). HSE-CAT (R. Kingston, Harvard University, Boston, MA) encodes a
CAT reporter gene driven by a segment of the human HSP70 gene promoter
(i.e. -34 to -1) that contains a potent HSE (54). G140CAT
(provided by R. Kingston) is from the same parental plasmid as HSE-CAT,
except it contains nucleotides -40 to -1 of the HSP70 promoter with a
Gal4-binding site in place of the HSE (61A ). MMTV-CAT contains a CAT
gene linked to MMTV LTR (62), whereas the pLC-Luc (provided by J.
Dudley, University of Texas, Austin, TX) plasmid has the luciferase
gene attached to mouse mammary tumor virus long terminal repeat (63).
Unless otherwise noted, the mouse mammary tumor virus-CAT and pLC-Luc
plasmids are referred to in the text as GRE (or HRE)-CAT and HRE-Luc,
respectively. The hsp70 expression plasmid pßacHSP70 (provided by R.
Morimoto, Northwestern University, Evanston, IL) was generated by
cloning the human hsp70 cDNA fragment downstream of the human ß-actin
promoter (64).
Cell Culture, Transfection, and Heat Shock Treatment
COS-1 monkey kidney fibroblasts were grown at 37 C in DMEM (Life
Technologies, Bethesda, MD) supplemented with 10% FBS (Irving
Scientific, Santa Ana, CA). Transient transfections were performed
using the calcium phosphate precipitation method (65). For all
transfections, a ßgal expression plasmid was cotransfected with CAT
and Luc expression plasmids, so that transfection efficiency could be
normalized from the comparison of ßgal activity (65). Each
transfection was performed at least three or four times, and
averages ± SD are indicated in all figures. Cells
were incubated with the DNA-calcium phosphate precipitate for 6 h
and then treated with 15% glycerol for 30 sec before feeding with DMEM
supplemented with 10% charcoal-stripped FBS (Sigma Chemical Co., St.
Louis, MO). Transfected cells were typically harvested after an
additional 36- to 48-h incubation at 37 C. Dexamethasone, R5020, or
dihydrotestosterone (Sigma) was added to a final concentration of 1
µM. For heat shock, cells were incubated at 43 C for
1 h, 36 h after transfection, and then recovered at 37 C for
3 h before harvesting.
CAT, Luciferase, and ßgal Assays
Thirty-six to 48 h after transfection, cells were harvested
and resuspended in 0.25 M Tris-HCl, pH 7.8. Cells were then
sonicated for 5 sec and centrifuged at 14,000 rpm for 5 min in a
microfuge. The supernatant was used for enzyme activity assays. CAT
assays were performed as described previously (66), and the resultant
CAT activity was quantified using a FUJIX BS 200 PhosphoImager Analyzer
(Fuji Photo Film, Tokyo, Japan). Luciferase and ßgal assays were
performed as described previously (65).
Western Blotting Analysis
Western blot analysis was performed as described previously
(36). Briefly, equivalent amounts of total protein in cell-free
extracts were separated by SDS-PAGE and transferred to Immobilon-P
membranes (Millipore Corp., Bedford, MA). Wild type and deletion mutant
derivatives of GR were then detected using the BuGR2 anti-GR monoclonal
antibody (67) and visualized using an enhanced chemiluminescence system
(Amersham International, Aylesbury, UK).
Indirect Immunofluorescence
Cells grown on glass coverslips were fixed with cold methanol
36 h after transfection and processed for indirect
immunofluorescence as described previously (68). GR and hsp70 were
detected with a rabbit polyclonal anti-GR antibody (Affinity
Bioreagents, Neshanic, NJ) and the SPA810 anti-hsp70 monoclonal
antibody (Stressgen, Victoria, Canada), respectively. SPA810
specifically recognizes hsp70 and does not recognize hsc70. A
tetramethyl rhodamine isothiocyanate-coupled antirabbit IgG (Boehringer
Mannheim, Indianapolis, IN) and fluorescein isothiocyanate-coupled
antimouse IgG antibody (Boehringer Mannheim) were used to visualize GR
and hsp70, respectively.
 |
ACKNOWLEDGMENTS
|
---|
Drs. J. Dudley, R. Kingston, R. Miesfeld, R. Morimoto, B. W.
OMalley, D. Pearce, D. Picard, and K. R. Yamamoto are thanked for
their kind gifts of DNA. R. Morimoto is also thanked for his critical
review of this manuscript.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Donald B. DeFranco, Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260.
This work was supported by USPHS Grant CA-43037 from the NIH.
Received for publication March 28, 1997.
Revision received May 8, 1997.
Accepted for publication May 10, 1997.
 |
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