Chromatin Recycling of Glucocorticoid Receptors: Implications for Multiple Roles of Heat Shock Protein 90
Jimin Liu and
Donald B. DeFranco
Departments of Biological Sciences (J.L., D.B.D.), Neuroscience
(D.B.D.), and Pharmacology (D.B.D.) University of Pittsburgh
Pittsburgh, Pennsylvania 15260
 |
ABSTRACT
|
---|
Unliganded glucocorticoid receptors (GRs) released
from chromatin after hormone withdrawal remain associated with the
nucleus within a novel subnuclear compartment that serves as a nuclear
export staging area. We set out to examine whether unliganded nuclear
receptors cycle between distinct subnuclear compartments or require
cytoplasmic transit to regain hormone and chromatin-binding capacity.
Hormone-withdrawn rat GrH2 hepatoma cells were permeabilized with
digitonin to deplete cytoplasmic factors, and then hormone-binding and
chromatin-binding properties of the recycled nuclear GRs were measured.
We found that recycled nuclear GRs do not require cytosolic factors or
ATP to rebind hormone. Nuclear GRs that rebind hormone in permeabilized
cells target to high-affinity chromatin-binding sites at 30 C, but not
0 C, in the presence of ATP. Since geldanamycin, a heat shock
protein-90 (hsp90)-binding drug, inhibits hormone binding to recycled
nuclear GRs, hsp90 may be required to reassemble the receptor into a
form capable of productive interactions with hormone. Geldanamycin also
inhibits GR release from chromatin during hormone withdrawal,
suggesting that hsp90 chaperone function may play multiple roles to
facilitate chromatin recycling of GR.
 |
INTRODUCTION
|
---|
The transduction of steroid hormone signals is mediated by cognate
receptor proteins, which belong to a large superfamily of nuclear
receptors (1, 2, 3). These receptors play an important role in many
metabolic, developmental, proliferative, and behavioral responses and
exert their effects primarily via their regulation of gene expression
(1, 4). Members of the nuclear receptor superfamily have been
subdivided into distinct classes based primarily on their sequence
recognition elements and dimerization properties. For example, steroid
hormone receptors interact primarily as homodimers with inverted repeat
DNA elements containing an internal spacing of 3 bp, while retinoid,
thyroid hormone, and vitamin D receptors bind primarily as heterodimers
to direct repeats of variable spacing (3). In this case, the extent of
spacing between direct repeats influences the selection of nuclear
receptor heterodimer (5, 6). While these DNA-binding characteristics
are useful for the placement of various nuclear receptors within
discrete subclasses, in many natural contexts, hormone response
elements may vary considerably from these idealized cases.
Steroid receptor proteins are also distinguished from other members of
the nuclear receptor family by their maturation and intracellular
trafficking properties. Unliganded steroid receptor proteins are
generally inert, although steroid receptor function can be uncovered
not only by hormone binding, but also upon the activation of
nonsteroidal signaling pathways (7, 8, 9, 10). Specific molecular chaperones
and other proteins that are associated with unliganded receptors (11, 12) supply a major impediment to steroid receptor function. Proteins
associated with fully mature, unliganded steroid receptors include the
90-kDa heat shock protein (hsp 90), a 23-kDa acidic protein, and one of
three immunophilins (FKBP52, FKBP51, or CyP-40) (11, 13). Thyroid
hormone receptors, and perhaps other members of the nuclear receptor
superfamily exclusive of steroid hormone receptors, are not maintained
in an inactive state when unliganded by associated proteins. In fact,
the nonsteroid nuclear receptors often function as transcriptional
repressors when unliganded through their association with
transcriptional corepressors (14, 15).
The assembly of steroid receptors into a heteromeric complex is a
multistep process that has been dissected in elegant in
vitro reconstitution experiments by the Pratt, Smith, and Toft
laboratories (16, 17, 18). Intermediate steroid receptor complexes may
contain other molecular chaperones including the 70-kDa heat shock
protein, hsp70, and utilize chaperone partners to facilitate the
ordered assembly of receptor heteromeric complexes (17, 19, 20). For
glucocorticoid and progesterone receptors (GRs and PRs), the formation
of a fully mature heteromeric complex is required for the receptors to
attain hormone binding competence (16, 21).
Although steroid receptor heteromeric complexes can be isolated from
whole cell extracts prepared in low-salt buffer, these complexes are
quite dynamic. For example, PR heteromeric complexes have been found to
turn over in vitro with a t1/2 of approximately
10 min (16). The dynamic nature of steroid receptor heteromeric
complexes has been postulated to play an important role in the
intracellular trafficking of steroid receptors (12, 22). PR (23, 24),
GR (25), and estrogen receptor (ER, Ref. 26) are shuttling proteins
that exchange between the nuclear and cytoplasmic compartments.
Disruption of the dynamic exchange between steroid receptors and their
associated proteins using pharmacological approaches disrupts receptor
shuttling and confines receptors to the cytoplasm (26, 27). The fact
that unliganded PR and ER are predominantly localized within the
nucleus (28, 29) suggests that their nuclear import is not limited by
dynamic interactions with hsp90 or other proteins.
In contrast to PR and ER, unliganded GRs are mainly localized within
the cytoplasm (30, 31, 32, 33). It has been postulated that the association of
hsp90, and perhaps other proteins, may be limiting the import of
unliganded GRs (12, 22). Accordingly, hormone binding would be expected
to stimulate the release of GR-associated proteins and expose the
receptor to the nuclear import machinery. While hormone-bound GRs still
maintain the capacity to shuttle between the nuclear and cytoplasmic
compartments (25), their nuclear import rate greatly exceeds their rate
of nuclear export, leading to the predominant localization of the
ligand-bound GRs within nuclei (25). Thus, it appears that the
limitation in GR nuclear export may not be mediated by a slow passage
across the nuclear pore complex, but more likely is influenced by the
retention of the receptor within distinct subnuclear compartments (34, 35).
Once GRs arrive within the nucleus, they rapidly locate and bind
to high-affinity target sites within native chromatin and alter the
transcriptional activity of linked promoters (1, 36). GR binding to
chromatin appears to be strictly hormone dependent (37). The withdrawal
of hormone leads to the release of receptors from high-affinity
chromatin-binding sites (38, 39). While the kinetics of GR chromatin
release appear to correlate with the kinetics of hormone dissociation
(34, 40), the nuclear export of unliganded receptors remains relatively
slow. Thus, nuclear export of GRs is not restricted solely by their
interactions with chromatin.
In this report, we have continued our analysis of the subnuclear
trafficking of GRs, focusing exclusively on the properties of
unliganded receptors that are released from chromatin. Our results
suggest that hsp90 chaperone function may play multiple roles to
facilitate chromatin recycling of GR.
 |
RESULTS
|
---|
Unliganded Nuclear GRs in Permeabilized GrH2 Cells Do Not Require
ATP or Cytosolic Factors to Rebind Hormone
The dissociation of hormone from liganded nuclear GRs is quite
rapid (40) but not associated with a corresponding rapid rate of
nuclear export (25, 34). Previously, we found that hormone withdrawal
leads to a rapid dissociation of GR from chromatin (34). These
unliganded nuclear receptors accumulate within a novel subnuclear
compartment and are competent to rebind chromatin in vitro
if exposed to hormone in the presence of ATP and cytosolic factors
(34).
In vitro reconstitution studies established that
hormone-binding competence of PR and GR is only accomplished after a
multistep maturation process that results in the assembly of mature
aporeceptor oligomeric complexes (13, 16). Is such a maturation process
required for chromatin-released (i.e. recycled) nuclear
receptors to regain hormone-binding capacity? We set out to address
this question using a digitonin-permeabilized cell system (41, 42) in
which the hormone-binding properties of GR could be manipulated by a
regimen of hormone withdrawal and reexposure. We initially examined the
role of ATP and cytosolic factors in the maturation of recycled nuclear
GR. As shown in Fig. 1A
, neither ATP nor
cytosol was required for unliganded nuclear GRs to rebind hormone
in digitonin-permeabilized GrH2 cells. In experiments assessing the ATP
dependence of hormone rebinding, permeabilized GrH2 cells were
pretreated with apyrase to eliminate residual ATP. The effectiveness of
the apyrase incubation in reducing ATP levels was confirmed by direct
measurements of ATP (not shown).

View larger version (23K):
[in this window]
[in a new window]
|
Figure 1. Unliganded Nuclear GRs in Permeabilized GrH2 Cells
Do Not Require Cytosolic Factors or ATP to Rebind Hormone
A, Hormone-withdrawn GrH2 cells were permeabilized with digitonin
and treated with apyrase to remove ATP where indicated (-ATP). B,
Hormone-withdrawn GrH2 cells were permeabilized with digitonin and
pretreated with 0.2 mg/ml WGA for 10 min on ice. Permeabilized cells
were then incubated in transport buffer with [3H]Dex
± HeLa cell cytosol, for 20 min at 30 C. In panel A, ATP was either
added or omitted, while in panel B, ATP was added and WGA was either
added or omitted during incubation. Nuclei were then collected and
radioactivity (in cpm) measured by scintillation counting.
Background radioactivity (in cpm) obtained from control samples
containing unlabeled Dex has been subtracted from values shown. Data
shown are the average of at least two separate experiments performed
with duplicate samples.
|
|
Although digitonin-permeabilized GrH2 cells are not competent to
support nuclear import in the absence of cytosolic factors (41, 42),
import of residual cytosolic factors might contribute to hormone
binding of nuclear GRs. To more convincingly eliminate this
possibility, we included the general nuclear transport inhibitor, wheat
germ agglutinin (WGA), in our in vitro hormone-binding
experiments to further impede the nuclear import capacity of
permeabilized cells. We had previously shown that WGA treatment of
permeabilized GrH2 cells effectively blocked hormone-dependent import
of GR (42). As shown in Fig. 1B
, WGA treatment of
digitonin-permeabilized GrH2 cells had no effect on the in
vitro hormone-binding activity of unliganded nuclear GR, either in
the presence or absence of cytosol. Thus, recycled, unliganded nuclear
GRs in permeabilized GrH2 cells do not require cytosolic factors to
rebind hormone.
Chromatin Rebinding, but Not Hormone Rebinding, of Nuclear GRs Is
Temperature Dependent in Vitro
Once steroid receptors have assembled into mature heteromeric
complexes, hormone binding can proceed even at lower temperatures. As
shown in Fig. 2A
, the binding of
dexamethasone (Dex) to unliganded nuclear GRs in permeabilized GrH2
cells is as efficient at 0 C as it is at 30 C. Longer incubation times
at 0 C did not significantly enhance hormone binding (not shown). Thus,
receptor maturation, which should not proceed at 0 C, most likely
occurred during the 30-min period of in vivo hormone
withdrawal of GrH2 cells.

View larger version (27K):
[in this window]
[in a new window]
|
Figure 2. Chromatin Rebinding, but Not Hormone Rebinding, of
Nuclear GRs Is Temperature Dependent in Vitro
Hormone-withdrawn GrH2 cells were permeabilized with digitonin and
incubated in transport buffer containing ATP and [3H]Dex
for 40 min at 0 C (lanes 13) or 30 C (lanes 46). In panel A, cells
were collected for scintillation counting to measure
[3H]Dex binding. Background radioactivity (in cpm)
obtained from control samples containing unlabeled Dex has been
subtracted from values shown. In panel B, cells were either unextracted
(lanes 1 and 4) or extracted with hypotonic (lanes 2 and 5) or CK
(lanes 3 and 6) buffer, and subjected to Western blot analysis to
visualize GR remaining within nuclei. The nuclear protein, NuMa,
provides an internal control for the relative amount of nuclear protein
recovery between samples.
|
|
To ascertain whether these hormone-bound GRs in permeabilized GrH2
cells were associated with chromatin, a differential extraction
paradigm was used that distinguishes the chromatin-binding state of the
receptor (34). Briefly, receptors that are bound with high affinity to
chromatin are resistant to a low-salt, hypotonic extraction, but
sensitive to high-salt cytoskeletal (CK) extraction (34). As
shown in Fig. 2B
, hormone-bound GRs in permeabilized GrH2 cells were
resistant to hypotonic extraction (compare lanes 4 and 5), and thus
associated with chromatin, if subjected to a 40-min incubation at 30 C.
The fact that receptors in these cells were sensitive to CK buffer
extraction (Fig. 2B
, lane 6) demonstrated they were chromatin bound and
not associated with the nuclear matrix (34). In contrast, incubation of
hormone-bound GRs in permeabilized cells at 0 C for 40 min (Fig. 2B
) or
longer (not shown) did not lead to the high-affinity binding of
receptors to chromatin (compare lanes 1 and 2). Thus, recycled,
ligand-bound nuclear GRs do not spontaneously associate with chromatin,
but require an additional temperature-dependent step for high- affinity
chromatin binding.
In previous studies, we found it difficult to assess the ATP dependence
of GR chromatin binding in permeabilized cells due to the increased
extent of receptor targeting to the matrix under ATP depletion
conditions (43) (J. Yang and D. B. DeFranco, unpublished
data). Nonetheless, we have found that appropriate chromatin rebinding
of recycled, nuclear GRs in permeabilized GrH2 cells has a strict
requirement for ATP hydrolysis. Thus, neither GTP, ADP, nor the
nonhydrolyzable ATP analog, AMP-PNP, could substitute for ATP to
generate nuclear GRs that were resistant to hypotonic buffer
extraction, but sensitive to high-salt buffer extraction (not
shown).
Geldanamycin, an hsp90-Binding Benzoquinoid Ansamycin, Inhibits
Hormone Rebinding of Unliganded Nuclear GRs in Permeabilized GrH2
Cells
Various molecular chaperones are involved in the maturation of GRs
and PRs in vitro (13). hsp90 participates in a number of
discrete steps in this pathway and is found not only associated with
mature, hormone-binding competent receptors, but also in intermediate
inactive aporeceptor complexes (13, 19). Establishing a role for hsp90
in steroid receptor activation has been greatly facilitated by the use
of the hsp90-binding benzoquinoid ansamycin, geldanamycin (GA). GA
occupies the nucleotide-binding site on hsp90 and prevents the switch
to its ATP-bound conformation (44, 45), which is required for
production of steroid-binding competent aporeceptor complexes (46). We
therefore used GA to ascertain whether hsp90 is involved in the
generation of hormone-binding competent nuclear GRs after their release
from chromatin.
As shown in Fig. 3A
, when GA is added to
permeabilized GrH2 cells, hormone binding to nuclear GRs is reduced by
50% (lane 2). If GA is added to GrH2 cells during the withdrawal
period, a more potent inhibitory effect on in vitro binding
of hormone to GR (i.e. a 90% decrease) was observed (Fig. 3A
, lane 3). Although prolonged GA treatment has been associated with
accelerated down-regulation of GR protein (47), the brief
(i.e. 20 min) exposure that we employed did not lead to any
dramatic change in steady state GR levels (Fig. 3
, B and C). Thus, the
effects of GA on in vitro hormone binding to GR reflect a
decrease in hormone-binding activity of the receptor. Including GA in
the in vitro binding assay as well as during the withdrawal
in vivo was no more effective than inclusion of GA during
the withdrawal period alone (Fig. 3A
, lanes 3 and 4). If cells are
maintained in the continuous presence of hormone (i.e. 90
min), GR hormone-binding activity is still reduced upon a 15- or 30-min
incubation with GA (not shown). Thus, as observed in reconstituted
cell-free systems (12), the association of hormone with GR is dynamic
and sensitive to disruptions in hsp90 function.

View larger version (25K):
[in this window]
[in a new window]
|
Figure 3. GA Inhibits Hormone Rebinding to Recycled Nuclear
GR
Hormone-treated GrH2 cells were withdrawn from hormone in the
absence (lanes 1 and 2) or presence (lanes 3 and 4) of 1 µg/ml GA and
then permeabilized with digitonin. Permeabilized cells were incubated
with (lanes 2 and 4) or without (lanes 1 and 3) GA in transport buffer
containing ATP and [3H]Dex for 20 min at 30 C. Nuclei
were collected and subjected to scintillation counting to measure
[3H]Dex binding (A) or Western blot analysis (B) to
reveal GR remaining within nuclei. In panel A, background radioactivity
(in cpm) obtained from control samples containing unlabeled Dex
has been subtracted from values shown. Data shown are the average of at
least two separate experiments performed with duplicate samples. In
panel C, relative GR levels are normalized to NuMa protein levels
detected on the costained Western blot (B).
|
|
GA Inhibits the Release of Unliganded GR from Chromatin
The effectiveness of GA, particularly when given during in
vivo hormone withdrawal, in preventing hormone binding to recycled
nuclear GRs in permeabilized GrH2 cells suggests that hsp90 may be
required for these receptors to reacquire hormone-binding competence.
As shown in Fig. 4
, whole-cell
hormone-binding assays confirmed that glucocorticoid hormone was
dissociated from GRs upon hormone withdrawal of GrH2 cells in the
presence of GA. Thus, nuclear GRs in GA-treated, hormone-withdrawn GrH2
cells were predominantly unliganded, yet incapable of effectively
interacting with hormone. This result also demonstrates that hsp90 is
unlikely to be required for hormone dissociation from nuclear
receptors. To identify the subnuclear compartment where these
unliganded nuclear receptors reside, we used differential extractions
both in situ for indirect immunofluorescence assays (Fig. 5A
) and with GrH2 cells in suspension for
Western blot analysis (Fig. 5
, B and C). As shown in Fig. 5
, the
inclusion of GA during the in vivo withdrawal of hormone
from GrH2 cells dramatically inhibited GR release from chromatin. As
shown previously, and confirmed in Fig. 5A
(panel b) and Figs. 5B
and 5C
(lane 5), GRs in hormone-withdrawn GrH2 cells were sensitive to a
hypotonic buffer extraction. The sensitivity of GR to hypotonic buffer
extraction was dramatically reduced when GA was included during hormone
withdrawal (Fig. 5A
, panel d; Figs. 5B
and 5C
, lane 8). Unliganded
nuclear receptors in GA-treated GrH2 cells were sensitive to extraction
by high-salt CK buffer (Fig. 5A
, panel e; Figs. 5B
and 5C
, lane 9) and
are therefore likely to remain associated with chromatin after hormone
dissociation. Thus, while hormone can be dissociated from
chromatin-bound GRs, their release from high- affinity interactions
with chromatin may require the chaperoning function of hsp90.

View larger version (24K):
[in this window]
[in a new window]
|
Figure 4. GA Does Not Affect the Dissociation of Hormone from
Nuclear GR in Hormone-Withdrawn GrH2 Cells
GrH2 cells were treated with 10-7 M
[3H]corticosterone for 1 h. Cells were then either
kept in hormone-containing medium (-withdrawal) or withdrawn from
hormone, in the absence or presence of 1 µg/ml GA, for 30 min. Each
sample was then collected in 200 µl lysis buffer, with 100 µl used
for scintillation counting to measure [3H]corticosterone
binding and 10 µl used for Western blot analysis (not shown). The
measurements shown have been normalized to relative GR content
determined from Western blots. Data shown are the average of at least
two separate experiments performed with duplicate samples.
|
|

View larger version (43K):
[in this window]
[in a new window]
|
Figure 5. Addition of GA during Hormone Withdrawal Inhibits
GR Release from Chromatin
GrH2 cells were treated with 10-7 M
corticosterone for 1 h, and then either untreated (panels B and C,
lanes 13) or withdrawn from hormone (panel A and panels B and C,
lanes 49) for 30 min. GA (1 µg/ml) was either omitted (A, panels a
and b; B and C, lanes 46) or included (A, panels c, d, and e; B and
C, lanes 79) during the hormone withdrawal. Cells were then extracted
with hypotonic (Hypo) or CK buffer before processing for
immunofluorescence staining (A) or Western blot analysis (B). In panel
C, average measurements of relative GR levels (±SD) from
two to three separate Western blots are shown.
|
|
 |
DISCUSSION
|
---|
Steroid receptor proteins can be reutilized and regain their
competence to respond to hormone when recycled from the nuclear to
cytoplasmic compartment (32, 48). Munck and co-workers (48) have also
hypothesized the existence of a nuclear bypass pathway that permits the
reutilization of nuclear GRs without an obligatory passage through the
cytoplasm. Although direct experimental evidence for recycling of
nuclear GRs has recently been provided in experiments utilizing
digitonin-permeabilized cells (34), fundamental questions regarding the
mechanism of GR recycling within the nucleus remain unanswered. It is
well established that hormone-binding competent cytoplasmic GRs exist
as heteromeric complexes that include molecular chaperones, such as
hsp90, immunophilins, and various chaperone partners (11, 13). How do
receptors that release hormone, yet remain associated with the nucleus,
regain the capacity to bind hormone? What are the requirements for
recycling of nuclear GRs?
Through the use of a permeabilized cell system, where receptor exchange
between the nuclear and cytoplasmic compartments can be minimized, we
have provided definitive evidence for nuclear recycling of GR. The
inhibition of nuclear GR hormone binding by GA suggests that hsp90 may
function in the nucleus to assemble recycled receptors into
hormone-binding competent heteromeric complexes (see model in Fig. 6
). Future studies directed toward the
characterization of such putative nuclear aporeceptor complexes will
reveal whether maturation of nuclear GR, or other steroid receptors, is
related to the cytoplasmic receptor maturation pathway established in
cell-free systems.

View larger version (23K):
[in this window]
[in a new window]
|
Figure 6. Recycling of Nuclear GR
A proposed model for the recycling of nuclear GRs is shown with hps90
implicated in chromatin release and reassembly of aporeceptor
heteromeric complexes. ATP may be required for hsp90 chaperone function
at the level of GR chromatin release and for maintaining appropriate
dynamic association of GR with the nuclear matrix.
|
|
As observed in various in vitro systems (16, 17, 18), the
binding of hormone to unliganded nuclear GRs in permeabilized cells is
temperature independent and does not require ATP. Thus once matured,
nuclear receptors are fully primed to respond to a hormonal signal and
do not require active cellular processes to do so. In contrast, the
subsequent high-affinity interaction of liganded receptors with
chromatin appears to be an active process. The ATP dependence of
in vitro GR chromatin binding was difficult to assess as
in vitro incubations of permeabilized cells at ambient
temperatures in the absence of ATP leads to increased nuclear matrix
binding of receptors. The in vitro binding of GR to
reconstituted nucleosomes does not require ATP (49, 50), suggesting
that ATP may be used in intact nuclei not necessarily to facilitate
appropriate receptor targeting to chromatin binding sites, but to
prevent inappropriate targeting of receptors to alternative subnuclear
compartments (i.e. the nuclear matrix). ATP hydrolysis
appears to be essential in our permeabilized cell system to restrict GR
interactions with the matrix. Furthermore, GTP can not substitute for
ATP, suggesting that Ran (51, 52, 53) and perhaps other small GTP-binding
proteins are not involved in directing GR to appropriate
chromatin-binding sites.
Recent work from our laboratory has established a role for hsp70 and
one of its chaperone partners, DnaJ, in the correction of protein
misfolding, or aggregation within the nucleus (54, 55). Such chaperone
systems may be required for the efficient subnuclear trafficking of
steroid receptors, as relatively high protein concentrations and
limited free diffusion space within the nucleus may favor inappropriate
protein-protein interactions that could lead to aggregation.
Our studies have also uncovered another potential function of molecular
chaperones in subnuclear trafficking of GR. Hormone withdrawal leads to
rapid chromatin release of both bulk GRs (Ref. 34 and this report) and
receptors associated with high-affinity target sites (39). However,
despite the fact that chromatin-associated GRs release their bound
hormone in the presence of GA, their subsequent release from chromatin
is dramatically inhibited. It should be noted that nuclear receptors in
GA-treated, hormone-withdrawn cells that resist low-salt extraction are
high-salt extractable, eliminating the possibility of increased nuclear
matrix association of the receptors under these conditions. We
therefore postulate that the chaperoning activity of hsp90 may be
needed to facilitate GR release from high-affinity chromatin-binding
sites upon hormone dissociation (see model, Fig. 6
). Since our studies
examined the chromatin-binding properties of bulk receptors, it is
unknown whether this hsp90 dependence applies to GR associated with
chromatin of specific target genes. However, the techniques used to
examine GR association with target gene chromatin (39) can be applied
in our permeabilized cell system to address this issue.
Additional studies of GA effects illustrate the dynamic nature of
unliganded and liganded GR interactions with chromatin. For example,
GRs are resistant to hypotonic extraction, and therefore chromatin
associated, if GA is added to cells after a 30-min hormone withdrawal
(not shown). This observation suggests that unliganded nuclear
receptors may also have some limited capacity to interact with
chromatin. We hypothesize that as long as hsp90 function is not
compromised, the release of unliganded receptors from high-affinity
chromatin-binding sites is more favored than their association with
these sites. This could explain why unliganded nuclear receptors, while
not detected on chromatin, can be driven to high-affinity chromatin
binding sites by GA treatment after hormone withdrawal. Since there is
a minimal loss of chromatin-bound receptors when cells are incubated
with GA in the continuous presence of hormone (not shown), some
fraction of hormone-bound receptors may release from high-affinity
chromatin-binding sites. Once hormone dissociates from these
chromatin-released liganded receptors, their hormone and chromatin
rebinding would be inhibited by GA.
Despite the fact that hsp90 has always been considered to be an
abundant (i.e. 12%) cytoplasmic protein, a small but
significant fraction (
3%) of hsp90 is found within nuclei (56).
Thus, there appears to be sufficient nuclear pools of hsp90 to perform
biologically relevant functions. In vitro studies have
revealed a role for hsp90 in DNA binding of helix-loop-helix
transcription factors (57, 58). Interestingly, hsp90 has been found to
facilitate the release of GR (59) and ER (60) from DNA in
vitro. In these studies, the ATP dependence of hsp90 effects on
receptor release from DNA was not strictly addressed. GA inhibits ATP
binding to hsp90 through its occupancy of the ATP-binding site of hsp90
(61). Given the GA effects on GR chromatin binding that we observed in
our permeabilized cell system, it will be relevant to consider the ATP
requirement for hsp90 effects on steroid receptor release from
chromatin and DNA.
What property of steroid receptors could account for the putative hsp90
requirements for chromatin release? Upon hormone binding, steroid
receptor ligand-binding domains (LBDs) undergo a conformational change
(62) that is characterized by the movement of a particular exposed
-helix (i.e. helix 12) toward the hormone-binding pocket
(63, 64). As a result, hydrophobic segments of the LBD, as well as the
bound hormone itself, are no longer solvent exposed. Furthermore,
previously inaccessible LBD surfaces become exposed and available for
interactions with appropriate coactivators and other components of the
transcriptional machinery. It is unclear how the elaborately networked
LBD structure responds to the release of bound hormone. Does the LBD
simply "relax" to reaquire the conformation it possessed when
initially unliganded? In such a scenario, the exposure of hydrophobic
segments of the unliganded receptors hormone-binding pocket might
increase the propensity for receptor aggregation unless molecular
chaperones such as hsp90 are present to prevent such inappropriate
interactions. Unliganded nuclear receptors in GA-treated,
hormone-withdrawn cells remain salt extractable and thus are unlikely
to be significantly aggregated.
The results presented in this report establish that steroid receptor
encounters with molecular chaperones are not restricted solely to the
cytoplasm. As receptors traffic through distinct subnuclear
compartments, their individual domains may adopt various folded or
unfolded states. Thus, nuclear molecular chaperone complexes, which may
bear some resemblance to cytoplasmic complexes or possess unique
compositions, may be called upon to maintain nuclear receptors in
biologically active conformations.
 |
MATERIALS AND METHODS
|
---|
Cell Culture
The GrH2 rat hepatoma cell line, which expresses elevated levels
of GR (62), was maintained at 37 C in DMEM (Life Technologies,
Gaithersburg, MD) supplemented with 5% FBS (Irvine Scientific, Santa
Ana, CA).
Antibodies and Chemicals
The BuGR2 monoclonal antibody (63) was used to detect GR. Ab-1
is a mouse monoclonal antibody directed against the NuMA nuclear matrix
protein (Oncogene Science, Cambridge, MA). GA was provided by the Drug
Synthesis & Chemistry Branch, Developmental Therapeutics Program,
Division of Cancer Treatment, National Cancer Institute.
In Vivo Hormone Treatment and Hormone Withdrawal
For all hormone treatments, GrH2 cells were treated with
10-7 M corticosterone (Sigma Chemical Co., St.
Louis, MO) for 1 h. For hormone withdrawal, hormone-treated cells
were briefly rinsed three times with phenol red-free DMEM (Life
Technologies) plus 5% charcoal-stripped FBS and then incubated with
this medium at 37 C for 30 min, unless otherwise noted. GA (1 µg/ml)
was included in the withdrawal medium where indicated.
Cell Permeabilization
GrH2 cells were collected using PBS containing 2 mM
EDTA. Cells were rinsed with ice-cold transport buffer (20
mM HEPES, pH 7.3, 110 mM potassium acetate, 5
mM sodium acetate, 2 mM magnesium acetate, 1
mM EGTA, 2 mM dithiothreitol (DTT), and 1
µg/ml each of protease inhibitors, aprotinin, leupeptin, and
pepstatin A), and then permeabilized using ice-cold transport buffer
containing 40 µg/ml digitonin (Sigma Chemical Co.) for 5 min on ice.
Cell pellets were then rinsed with transport buffer before indicated
incubations with hormone.
In Vitro and in Vivo Hormone Binding
Permeabilized cells were incubated at 0 C or 30 C for 20 min in
50100 µl transport buffer containing 10-7
M [3H]Dex (41.0 Ci/mmol, Amersham Life
Science, Arlington Heights, IL), 10 µg/ml BSA, 5 mM
creatine phosphate, 20 U/ml creatine phosphokinase, in the presence or
absence of 2 mM ATP. To deplete ATP, permeabilized cells
were incubated with 35 U of apyrase (Sigma) in 100 µl of transport
buffer for 3 min on ice. In some experiments 25% (vol/vol) HeLa cell
cytosol (42) or 0.2 mg/ml WGA (Sigma) was also included. All in
vitro hormone binding experiments included a control in which a
1000-fold molar excess of unlabeled Dex was included to monitor
nonspecific binding of [3H]Dex, which typically
represented 1020% or lower of total binding.
Nuclei were recovered from hormone-treated permeabilized cells as
described previously (34). Intact nuclei isolated after centrifugation
at 400 x g were resuspended in 200 µl ethanol and
added to 5 ml ScintiSafe Solution (Fisher Scientific, Pittsburgh, PA)
for liquid scintillation counting. Specific binding of
[3H]Dex was determined after the subtraction of
nonspecific binding.
For in vivo measurements of hormone binding, GrH2 cells,
grown in DMEM containing charcoal-stripped serum, were incubated with
20 nM [3H]corticosterone (77.0 Ci/mmol,
Amersham Life Science) for 1 h. Nonspecific binding was assessed
by the inclusion of 1000-fold excess of unlabeled corticosterone
(Sigma). Where indicated, cells were withdrawn from hormone for various
lengths of time by incubation in withdrawal medium. Cells were then
extensively washed with culture medium and lysed by sonication in
high-salt lysis buffer [10 mM HEPES, pH 7.0, 450
mM NaCl, 5 mM EDTA, 0.05% SDS, 1% Triton
X-100, 2 mM DTT, and protease inhibitors, (42)]. One half
of the lysate was added to ScintiSafe Solution to measure
[3H]corticosterone binding, while the other half was used
to determine relative GR levels by Western blot analysis (42).
Biochemical Extractions
For low-salt, hypotonic extraction, nuclei isolated from
digitonin-permeabilized GrH2 cells were treated with 150 µl hypotonic
(Hypo) buffer [10 mM HEPES, pH 7.9, 10 mM KCl,
1.5 mM MgCl2, 0.5 mM DTT, 0.1%
Triton X-100, and protease inhibitors (43)] for 3 min on ice. For
high-salt extraction, nuclei were treated for 3 min on ice with 150
µl of CK buffer [10 mM
piperazine-N,N'-bis[2-ethanesulfonic
acid], pH 6.8, 100 mM NaCl, 300 mM
sucrose, 3 mM MgCl2, 1 mM EGTA,
0.5% Triton X-100, and protease inhibitors, (43)]. The Hypo or CK
buffer-extracted nuclei, as well as an aliquot of unextracted nuclei,
were dissolved in 1x SDS sample buffer (132 mM Tris-HCl,
pH 6.8, 20% glycerol, 10% SDS, 10.4% ß-mercaptoethanol, 0.02%
pyronin Y), boiled for 5 min, and then subjected to SDS-PAGE.
Occasionally, whole cells were directly lysed on culture plates using
high-salt lysis buffer.
Western Blot Analysis
GRs in intact or extracted nuclei were detected by Western blot
analysis using the BuGR2 antibody as previously described (42). To
provide an internal control for gel loading and transfer efficiency,
NuMA (nuclear matrix protein) was also detected on the same blots using
the Ab-1 antibody. GR or NuMA on Western blots was visualized using the
enhanced chemiluminescence (ECL) (Amersham International, Little
Chalfont, Buckinghamshire, U.K.) detection system.
Indirect Immunofluorescence
GrH2 cells were fixed with -20 C methanol for 5 min at room
temperature. Fixed cells were incubated with the BuGR2 anti-GR
antibody. An fluorescein isothiocyanate-coupled antimouse IgG
antibody (Boehringer Mannheim Biochemicals, Indianapolis, IN) was used
as secondary antibody to detect GR. Stained cells were observed by
fluorescence microscopy through an Optiphot-2 microscope (Nikon Inc.,
Garden City, NY) and photographed with T-Max 400 film (Eastman-Kodak
Co., Rochester, NY).
 |
ACKNOWLEDGMENTS
|
---|
Geldanamycin was provided by the Drug Synthesis and Chemistry
Branch, Developmental Therapeutics Program, Division of Cancer
Treatment, National Cancer Institute. We thank Drs. David Toft and
Péter Csmerley for helpful discussions.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Donald B. DeFranco, Department of Biological Sciences, University of Pittsburgh, A234 Langley Hall, Fifth and Ruskin Streets, Pittsburgh, Pennysylvania 15260. E-mail:
dod1{at}vms.cis.pitt.edu
This work was supported by NIH Grant CA-43037 (to D.B.D.).
Received for publication October 1, 1998.
Revision received December 2, 1998.
Accepted for publication December 16, 1998.
 |
REFERENCES
|
---|
-
Yamamoto KR 1985 Steroid receptor regulated transcription
of genes and gene networks. Annu Rev Genet 19:209252[CrossRef][Medline]
-
Evans RM 1988 The steroid and thyroid hormone receptor
superfamily. Science 240:889895[Medline]
-
Mangelsdorf DJ, Thummel C, Beato M, Herrlich P, Schütz
G, Umesono K, Blumberg B, Kastner P, Mark L, Chambon P, Evans RM 1995 The nuclear receptor superfamily: the second decade. Cell 83:835839[Medline]
-
Tsai MJ, OMalley BW 1994 Molecular mechanisms of action of
steroid/thyroid receptor superfamily members. Annu Rev Biochem 63:451486[CrossRef][Medline]
-
Näär AM, Boutin J-M, Lipkin SM, Yu VC, Holloway
JM, Glass CK, Rosenfeld MG 1991 The orientation and spacing of core
DNA-binding motifs dictate selective transcriptional responses to three
nuclear receptors. Cell 65:12671279[Medline]
-
Umesono K, Murakami KK, Thompson CC, Evans RM 1991 Direct
repeats as selective response elements for the thyroid hormone retinoic
acid and vitamin D3 receptors. Cell 65:12551266[Medline]
-
Denner LA, Weigel NL, Maxwell BL, Schrader WT, OMalley BW 1990 Regulation of progesterone receptor-mediated transcription by
phosphorylation. Science 250:17401743[Medline]
-
Power RF, Mani SK, Codina J, Conneely OM, OMalley BW 1991 Dopaminergic and ligand-independent activation of steroid hormone
receptors. Science 254:16361639[Medline]
-
Somers JP, DeFranco DB 1992 Effects of okadaic acid, a
protein phosphatase inhibitor, on glucocorticoid receptor mediated
enhancement. Mol Endocrinol 6:2634[Abstract]
-
Zhang Y, Bai W, Allgood VE, Weigel NL 1994 Multiple signaling
pathways activate the chicken progesterone receptor. Mol Endocrinol 8:577584[Abstract]
-
Smith DF, Toft DO 1993 Steroid receptors and their associated
proteins. Mol Endocrinol 7:411[Medline]
-
Pratt WB 1993 The role of heat shock proteins in regulating
the function, folding, and trafficking of the glucocorticoid receptor.
J Biol Chem 268:2145521458[Free Full Text]
-
Pratt WB, Toft DO 1997 Steroid receptor interactions with heat
shock protein and immunophilin chaperones. Endocr Rev 18:306360[Abstract/Free Full Text]
-
Chen JD, Evans RM 1995 A transcriptional co-repressor that
interacts with nuclear hormone receptors. Nature 377:454457[CrossRef][Medline]
-
Horlein AJ, Naar AM, TH, Torchia J, Gloss B, Kurokawa R, Ryan
A, Kamei Y, Soderstrom M, Glass CK, Rosenfeld MG 1995 Ligand-independent repression by the thyroid hormone receptor mediated
by a nuclear receptor co-repressor. Nature 377:397404[CrossRef][Medline]
-
Smith DF 1993 Dynamics of heat shock protein 90-progesterone
receptor binding and the disactivation loop model for steroid receptor
complexes. Mol Endocrinol 7:14181429[Abstract]
-
Johnson JL, Toft DO 1995 Binding of p23 and hsp90 during
assembly with the progesterone receptor. Mol Endocrinol 9:670678[Abstract]
-
Dittmar KD, Hutchinson KA, Owens-Grillo JK, Pratt WB 1996 Reconstitution of the steroid receptor-hsp90 heterocomplex assembly
system of rabbit reticulocyte lysate. J Biol Chem 271:1283312839[Abstract/Free Full Text]
-
Smith DF, Whitesell L, Nair SC, Chen S, Prapapanich V,
Rimerman RA 1995 Progesterone receptor structure and function altered
by geldanamycin, an hsp90-binding agent. Mol Cell Biol 15:68046812[Abstract]
-
Prapapanich V, Chen S, Nair SC, Rimerman RA, Smith DF 1996 Molecular cloning of human p48, a transient component of progesterone
receptor complexes and an hsp70-binding protein. Mol Endocrinol 10:420431[Abstract]
-
Bresnick EH, Dalman FC, Sanchez ER, Pratt WB 1989 Evidence
that the 90-kDa heat shock protein is necessary for the steroid binding
conformation of the L cell glucocorticoid receptor. J Biol Chem 264:49924997[Abstract/Free Full Text]
-
DeFranco DB, Madan AP, Tang Y, Chandran UR, Xiao N, Yang J 1995 Nucleocytoplasmic shuttling of steroid receptors. In: Litwack G
(ed) Vitamins and Hormones. Academic Press, New York, vol 51:315338
-
Guiochon-Mantel A, Lescop P, Christin-Maitre S, Loosefelt H,
Perrot-Applanat M, Milgrom E 1991 Nucleocytoplasmic shuttling of the
progesterone receptor. EMBO J 10:38513859[Abstract]
-
Chandran UR, DeFranco DB 1992 Internuclear migration of
chicken progesterone receptor, but not simian virus-40 large tumor
antigen, in transient heterokaryons. Mol Endocrinol 6:837844[Abstract]
-
Madan AP, DeFranco DB 1993 Bidirectional transport of
glucocorticoid receptors across the nuclear envelope. Proc Natl Acad
Sci USA 90:35883592[Abstract]
-
Dauvois S, White R, Parker MG 1993 The antiestrogen ICI 182780
disrupts estrogen receptor nucleocytoplasmic shuttling. J Cell Sci 106:13771388[Abstract/Free Full Text]
-
Yang J, DeFranco DB 1996 Assessment of glucocorticoid
receptor-heat shock protein 90 interactions in vivo during
nucleocytoplasmic trafficking. Mol Endocrinol 10:313[Abstract]
-
Guiochon-Mantel A, Loosfelt H, Lescop P, Sar S, Atger M,
Perrot-Applanat M, Milgrom E 1989 Mechanisms of nuclear localization of
the progesterone receptor: evidence for interactions between monomers.
Cell 57:11471154[Medline]
-
Ylikomi T, Bocquel MT, Berry M, Gronemeyer H, Chambon P 1992 Cooperation of proto-signals for nuclear accumulation of estrogen and
progesterone receptors. EMBO J 11:36813694[Abstract]
-
Wikström A-C, Bakke O, Okret S, Bronnegard M, Gustafsson
J-A 1987 Intracellular localization of the glucocorticoid receptor:
evidence for cytoplasmic and nuclear localization. Endocrinology 120:12321242[Abstract]
-
Picard D, Kumar V, Chambon P, Yamamoto KR 1990 Signal
transduction by steroid hormones: nuclear localization is
differentially regulated in estrogen and progesterone receptors. Cell
Regul 1:291299[Medline]
-
Qi M, Hamilton BJ, DeFranco D 1989 v-mos
Oncoproteins affect the nuclear retention and reutilization of
glucocorticoid receptors. Mol Endocrinol 3:12791288[Abstract]
-
Htun H, Barsony J, Renyi I, Gould DL, Hager GL 1996 Visualization of glucocorticoid receptor translocation and intranuclear
organization in living cells with a green fluorescent protein chimera.
Proc Natl Acad Sci USA 93:48454850[Abstract/Free Full Text]
-
Yang J, Liu J, DeFranco DB 1997 Subnuclear trafficking of
glucocorticoid receptors in vitro: chromatin recycling and
nuclear export. J Cell Biol 137:523538[Abstract/Free Full Text]
-
Sackey FNA, Hache RJG, Reich T, Kwast-Welfeld J, Lefebvre YA 1996 Determinants of subcellular distribution of the glucocorticoid
receptor. Mol Endocrinol 10:11911205[Abstract]
-
Ucker DS, Ross SR, Yamamoto KR 1981 Mammary tumor virus DNA
contains sequences required for its hormone-regulated transcription.
Cell 27:257266[Medline]
-
Becker PB, Gloss B, Schmid W, Strähle U, Schütz G 1986 In vivo protein-DNA interactions in a glucocorticoid
response element require the presence of hormone. Nature 324:686688[Medline]
-
Zaret KS, Yamamoto KR 1984 Hormonally induced alterations of
chromatin structure in the polyadenylation and transcription
termination regions of the chicken ovalbumin gene. Cell 38:2938[Medline]
-
Reik A, Schütz G, Stewart AF 1991 Glucocorticoids are
required for establishment and maintenance of an alteration in
chromatin structure:induction leads to a reversible disruption of
nucleosomes over an enhancer. EMBO J 10:25692576[Abstract]
-
Munck A, Foley R 1976 Kinetics of glucocorticoid-receptor
complexes in rat thymus cells. J Steroid Biochem 7:11171122[CrossRef][Medline]
-
Adam SA, Sterne Marr R, Gerace L 1990 Nuclear protein import
in permeabilized mammalian cells requires soluble cytoplasmic factors.
J Cell Biol 111:807816[Abstract]
-
Yang J, DeFranco DB 1994 Differential roles of heat shock
protein 70 in the in vitro nuclear import of glucocorticoid
receptor and simian virus 40 large tumor antigen. Mol Cell Biol 14:50885098[Abstract]
-
Tang Y, DeFranco DB 1996 ATP-dependent release of
glucocorticoid receptors from the nuclear matrix. Mol Cell Biol 16:19892001[Abstract]
-
Sullivan W, Stensgard B, Caucutt G, Bartha B, McMahon N,
Alnemri ES, Litwack G, Toft D 1997 Nucleotides and Two Functional
States of hsp90. J Biol Chem 272:80078012[Abstract/Free Full Text]
-
Grenert JP, Sullivan WP, Fadden P, Haystead TAJ, Clark J,
Mimnaugh E, Krutzsch H, Ochel HJ, Schulte TW, Sausville E, Neckers
LM, Toft DO 1997 The amino-terminal domain of heat shock protein 90
(hsp90) that binds geldanamycin is an ATP/ADP switch domain that
regulates hsp90 conformation J Biol Chem 272: 2384323850
-
Dittmar KD, Demady DR, Stancato LF, Krishna P, Pratt WB 1997 Folding of the glucocorticoid receptor by the heat shock protein (hsp)
90-based chaperone machinery. J Biol Chem 272:2121321220[Abstract/Free Full Text]
-
Whitesell L, Cook P 1996 Stable and specific binding of heat
shock protein 90 by geldanamycin disrupts glucocorticoid receptor
function in intact cells. Mol Endocrinol 10:705712[Abstract]
-
Orti E, Mendel DB, Smith LI, Bodwell JE, Munck A 1989 A
dynamic model of glucocorticoid receptor phosphorylation and recycling
in intact cells. J Steroid Biochem 34:8596[CrossRef][Medline]
-
Pina B, Brüggenmeier U, Beato M 1990 Nucleosome
positioning modulates accessibility of regulatory proteins to the mouse
mammary tumor virus promoter. Cell 60:719731[Medline]
-
Archer TK, Cordingley MG, Wolford RG, Hager GL 1991 Transcription factor access is mediated by accurately positioned
nucleosomes on the mouse mammary tumor virus promoter. Mol Cell Biol 11:688698[Medline]
-
Melchior F, Paschal B, Evans J, Gerace L 1993 Inhibition of
nuclear protein import by nonhydrolyzable analogues of GTP and
identification of the small GTPase Ran/TC4 as an essential nuclear
factor. J Cell Biol 123:16491659[Abstract]
-
Moore MS, Blobel G 1993 The GTP-binding protein Ran/TC4 is
required for protein import into the nucleus. Nature 365:661663[CrossRef][Medline]
-
Moroianu J 1997 Molecular mechanisms of nuclear protein
transport. Crit Rev Eukaryot Gene Expr 7:6172[Medline]
-
Tang Y, Ramakrishnan C, Thomas J, DeFranco DB 1997 A role for
HDJ-2/HSDJ in correcting subnuclear trafficking, transactivation and
transrepression defects of a glucocorticoid receptor zinc finger
mutant. Mol Biol Cell 8:795809[Abstract]
-
Cummings CJ, Mancini MA, Antalffy B, DeFranco DB, Orr HT,
Zoghbi HY 1998 Chaperone suppression of aggregation and altered
subcellular proteasome localization imply protein misfolding in SCA1.
Nat Genet 19:148154[CrossRef][Medline]
-
Csermely P, Schnaider T, Soti C, Prohaszka Z, Nardai G 1998 The 90 kDa molecular chaperone family: structure, function, and
clinical applications. A comprehensive review. Pharmacol Ther 79:129168[CrossRef][Medline]
-
Shaknovich R, Shue G, Kohtz S 1992 Conformational activation
of a basic helix-loop-helix protein (MyoD1) by the C-terminal region of
murine HSP90 (HSP84). Mol Cell Biol 12:50595068[Abstract]
-
Shue G, Kohtz DS 1994 Structural and functional aspects of
basic helix-loop-helix protein folding by heat shock protein 90. J
Biol Chem 269:27072711[Abstract/Free Full Text]
-
Scherrer LC, Dalman FC, Massa E, Meshinchi S, Pratt WB 1990 Structural and functional reconstitution of the glucocorticoid
receptor-hsp90 complex. J Biol Chem 265:2139721400[Abstract/Free Full Text]
-
Sabbah M, Radanyi, C, Redeuilh, G, Baulieu, E 1996 The 90-KDa
heat-shock protein (hsp90) modulates the binding of the oestrogen
receptor to its cognate DNA. Biochem J 314:205213[Medline]
-
Stebbins CE, Russo AA, Schneider C, Rosen N, Hartl FU, PPN 1997 Crystal structure of an hsp90-geldanamycin complex: targeting of a
protein chaperone by an antitumor agent. Cell 89:230250
-
Beekman JM, Allan GF, Tsai SY, Tsai M-J, OMalley BW 1993 Transcriptional activation by the estrogen receptor requires a
conformational change in the ligand binding domain. Mol Endocrinol 7:12661274[Abstract]
-
Brzozowski AM, Pike ACW, ZD, Hubbard RE, Bonn T, Engstron O,
Ohman L, Greene GL, Gustafsson J-A, Carlquist M 1997 Molecular
basis of agonism and antagonism in the oestrogen receptor. Nature 389:753758[CrossRef][Medline]
-
Tanenbaum DM, Wang Y, Williams SP, Sigler PB 1998 Crystallographic comparison of the estrogen and progesterone
receptors ligand binding domain. Proc Natl Acad Sci USA 95:59986003[Abstract/Free Full Text]
-
Howard KJ, Holley SJ, Yamamoto KR, Distelhorst CW 1990 Mapping
the HSP90 binding region of the glucocorticoid receptor. J Biol
Chem 265:1192811935[Abstract/Free Full Text]
-
Gametchu B, Harrison RW 1984 Characterization of a monoclonal
antibody to the rat liver glucocorticoid receptor. Endocrinology 114:274288[Abstract]