Institut National de la Santé et de la Recherche Médicale U33 Lab Hormones 94276 Le Kremlin Bicêtre Cedex, France
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ABSTRACT |
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INTRODUCTION |
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ER can be recovered from the hormone-free target cell cytosols in the inactive non-DNA binding 89S heterooligomeric form that includes, among others, the 90-kDa heat shock protein (Hsp90), the only protein of the complex that has the intrinsic ability for selective receptor binding (7, 8, 9, 10). The Hsp90 molecular chaperone (11), required for viability in eukaryotes (12), is found in all tissues or cells as an abundant dimer, essentially localized in the cytoplasm but also present at low levels in the nucleus (13, 14, 15). Biochemical, cell biological, genetic, and pharmacological approaches have been extensively used to study the interactions of Hsp90 and steroid receptors and their consequences for receptor function upon ligand binding. Results strongly support a dual role of Hsp90 in regulating steroid receptor activity: Hsp90 helps to maintain steroid receptors inactive in the absence of hormone and to ensure efficient hormonal responses (10, 16).
Concerning the in vivo interaction between Hsp90 and steroid receptors, a nuclear cotranslocation assay demonstrated that, in the absence of hormone, nuclear-targeted Hsp90 was able to shift cytoplasmic glucocorticoid receptor or progesterone receptor mutants into the nucleus (17). Likewise, the interaction between wild-type Hsp90 and two nuclear steroid receptors, progesterone receptor and ER, shifted Hsp90 into the nucleus (15, 17), implying that nuclear steroid receptors are, at least in part, complexed with Hsp90, which dissociates from the receptors upon ligand binding. Moreover, the transcriptional properties and subcellular localization of human or mouse ER and their mutants in the presence of E2 and mixed agonist-antagonists such as tamoxifen or pure antiestrogens like ICI 164 384 and ICI 182 780 have been determined. While tamoxifen only abolished the activating function of the LBD (AF-2), without interfering with the nuclear localization of ER, the ICI compounds were also able to inhibit AF-1 (located in the N-terminal domain of ER); ER bound to pure antiestrogens, although possessing DNA-binding capacity in vitro, was incapable of nucleo-cytoplasmic shuttling (6, 18, 19).
In the search for specific estrogen antagonists for ER-positive breast cancer therapy (20), RU 58668 (thereafter called RU) which, in vitro, displays affinities for human and murine ERs equivalent to those of E2, was selected for its complete antiestrogenic properties, in vivo, on the uterotrophy induced by E2 in mice as well as for its unique ability to induce a long-term regression of MCF-7 tumors implanted in nude mice (21, 22).
In view of these results, we investigated the effect of E2 and RU on the in vivo interaction between ER and Hsp90 using the nuclear translocation assay. In vivo dissociation of Hsp90 from ER was found after agonist or antagonist treatment. In addition, upon RU treatment, ER was segregated into cytoplasmic clusters that appeared and were maintained only in the presence of protein synthesis. Moreover, RU was unable to stimulate transcription of an estrogen-dependent reporter and completely repressed the estrogen-induced activity. These findings led us to propose that the antiestrogenic effects of this compound are primarily due to a protein synthesis-dependent mislocalization of ER concomitant to a disruption of its constitutive and ligand-induced nuclear localization functions.
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RESULTS |
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Hsp90 and ER, when expressed alone, were predominantly cytoplasmic and
nuclear, respectively (4, 5, 17). With the plasmid
pSVK390-HEGO, we confirmed our previous results (15),
demonstrating that, in the absence of hormone, Hsp90 was in part or
completely shifted to the nuclear compartment as a consequence of its
interaction with ER (Fig. 1, a and b).
The effects of the agonist, E2 (10 nM) (Fig. 1
, c and d), and the antagonist, RU (100 nM) (Fig. 1
, eh),
which were added to Cos-7 cells 24 h after transfection and
incubated for a further 24 h, were demonstrated by the disruption
of the in vivo interaction between ER and Hsp90 in 70% of
cells expressing both proteins, Hsp90 showing a predominant diffuse
cytoplasmic staining. Moreover, in the presence of RU, ER, which is
nuclear localized in the absence and in the presence of E2,
was found to form perinuclear cytoplasmic clusters of various size.
Scatter diagram analysis revealed that ER and Hsp90 did not colocalize
after treatment with E2 or RU. Upon treatment with
tamoxifen, a mixed agonist-antagonist ligand, the subcellular
localization of ER and Hsp90 was similar to that obtained by
E2, while ICI 164384, another pure antiestrogen, produced
the same effect as RU (not shown).
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The confocal laser microscopic analysis of untreated ERC3
cells revealed a partial nuclear cotranslocation of Hsp90 with ER,
confirming an in vivo interaction between the endogenous
Hsp90 and ER (Fig. 2, a and b). The
subcellular localization of Hsp90 in the parental Chinese hamster ovary
(CHO) cell line, with no detectable endogenous ER, in the absence or
presence of hormone, was essentially cytoplasmic as shown in Fig. 2k
.
The treatment of ERC3 cells with 10 nM
E2 (Fig. 2
, c and d) or 100 nM RU (Fig. 2
, e
and f) disrupted the Hsp90-ER interaction, the nucleus being almost
devoid of Hsp90. In the presence of cycloheximide (CHX, 50 µg/ml),
the Hsp90-ER interaction was still detectable, Hsp90 being found in
part in the nucleus (Fig. 2
, g and h) and was disrupted by
E2 (Fig. 2
, i and j), indicating that the loss of nuclear
localization of Hsp90 was actually a consequence of its dissociation
from ER and not due to new Hsp90 synthesis. Moreover, after RU
treatment, the ER was localized in cytoplasmic clusters as already
observed in transfected Cos-7 cells (Fig. 1
, f and h). Again, scatter
diagram analysis did not reveal any preferential colocalization of
Hsp90 and ER in the presence of E2 and RU.
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RU-Dependent ER Mislocalization Is Specific and Requires Protein
Synthesis
To investigate the mechanism by which RU acted on ER localization
and to avoid differences in subcellular distribution due to new
receptor synthesis, transfected Cos-7 cells were treated with RU in the
presence of CHX. As shown in Fig. 3, the
cytoplasmic ER clusters, observed in the presence of RU (Fig. 3c
), were
not detected in the presence of RU + CHX (Fig. 3d
), the receptor being
found in the nucleus as in control (Fig. 3a
) or in
E2-treated cells (Fig. 3b
). This result demonstrated that
RU was able to mislocalize the receptor only in the presence of
continuous protein synthesis, indicating that its clustering effect is
exerted on newly synthesized receptor and/or that it is dependent on
the synthesis of other protein(s).
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Kinetics of Appearance and Reversal of RU-Dependent ER
Mislocalization
Even though the clustering effect was detectable at 10
nM RU, transfection of Cos-7 cells and RU treatment at 100
nM were used for easy detection and classification of the
immunofluorescent cells, to study the kinetics of appearance (type I
experiments) and reversal (type II experiments) of RU effect on ER.
In the type I experiments (Fig. 4), the
antiestrogen was added 24 h after transfection for times varying
from 124 h. In the type II experiments (Fig. 5
), RU was added just after transfection,
when cells were plated, and the kinetics of reversal were analyzed
24 h later for times varying from 124 h. For each experiment,
the control is represented by the analysis of the cells 24 h after
transfection and pretreatment (Fig. 4
). At least three experiments were
performed for all the studied conditions, and for each condition, 200
transfected cells were counted and classified. The subcellular receptor
distribution followed four major patterns and was defined as N, when
the staining was exclusively nuclear; N>C, when nuclear staining was
stronger than the cytoplasmic one; N<C, when the cytoplasmic staining
was stronger than the nuclear one; and C, when the staining was
exclusively or almost exclusively cytoplasmic. The cytoplasmic ER
immunofluorescence due to RU was always speckled and clustered. The N =
C category was not considered because it represented approximately 3%
of the transfected cells, and it did not significantly vary in
different experimental conditions. Some speckled nuclear fluorescence
of ER was also detectable at intermediary times of RU treatment (616
h).
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Both in control and E2-pretreated cells, the presence of CHX inhibited the RU-dependent cytoplasmic mislocalization of ER, which remained nuclear, indicating that the RU-induced cytoplasmic clustering of ER is strictly dependent on protein synthesis.
Reversal of RU Effect
After 24 h of RU treatment, different conditions for reversal of
RU effect were used (Fig. 5). The RU clustering effect established
during the first 24 h after transfection was found to be
reversible in the presence of fresh medium, CHX, RU + CHX,
E2, and E2 + CHX (Fig. 5
, AE). ER cytoplasmic
clustering disappeared progressively during the time course of the
treatment substituting the RU. The recovery to N or N>C ER
localization was more efficient in short time courses in the
absence of CHX, probably on account of the neosynthesis of the
receptor. The experiments with E2 (Fig. 5D
) showed a more
efficient reversal to exclusive nuclear staining after 24 h
(75%), even in the presence of CHX (Fig. 5E
). The reversal of the RU
effect progressively consisted of more diffuse cytoplasmic ER
immunofluorescence and nuclear localization.
These results prove that the RU-dependent formation of cytoplasmic ER
clusters is not an irreversible phenomenon, the receptor being able to
recover, at least in part, during the RU withdrawal, the constitutive
nuclear localization function (pNLS), N and N>C categories
representing 20% and 60%, respectively, of the cells at 24 h in
Fig. 5, A, B, and C.
In the presence of E2, the N category reached 80% at
24 h (Fig. 5, D and E), compared with 2025% in the absence of
agonist ligand (Fig. 5
, A, B, and C). CHX did not interfere with the
recovery of the normal nuclear localization of ER in the absence or
presence of E2 (Fig. 5
, B and E), even though it inhibited
the establishment of RU-dependent ER cytoplasmic mislocalization (Fig. 4
, B and D). More interestingly, in the presence of CHX, RU was unable
to maintain the cytoplasmic clustering of ER synthesized in the
presence of RU alone (Fig. 5C
). Therefore, we favor the hypothesis that
clustering and mislocalization of ER by RU require the synthesis of
other protein(s): possibly RU provokes an abnormal receptor
conformation maintained and clustered in the cytoplasm via other
protein(s).
ER clusters under RU treatment, detected with the monoclonal H222 antibody directed against the LBD (28), was also recognized in fixed cells by the JS32/34 antibody (23), and by the H226 antibody (28), directed against the N-terminal region of the protein (data not shown), indicating that clustering of ER by RU did not modify at least three epitopes.
In the ERC3 cell line, the kinetics of appearance and
reversal of RU-dependent cytoplasmic ER clusters were faster, the 8-h
time lapse of each treatment being comparable to 16 h in Cos-7
cells. This was possibly due to a faster turnover of the receptor
and/or to its expression at a lower level. Indeed, we noted that, in
the presence of RU, the level of expression of ER decreased in
ERC3 cells and not in Cos-7 cells on the basis of
immunofluorescence studies. Western blot analyses and quantification of
ER expression level under control, E2, and RU treatment
demonstrated an increase of ER after E2 (Cos-7 cells: 2.7-
and 1.8-fold at 16 and 24 h treatment, respectively;
ERC3 cells: 2.1- and 1.2-fold at 8 and 16 h treatment,
respectively, compared with the control), while a decreased expression
after RU was evident only in ERC3 cells (2.2-fold decrease
at 16 h treatment) (Fig. 6). This
result, although ER is not controlled by its promoter in
ERC3 and Cos-7 cells, is in agreement with previous
findings showing different effects of ligands on ER stability,
according to cell line (29).
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The scanning of the ER signal in Fig. 7
revealed that in control cells, cytosolic ER signal was greater
[integrated optical density (IOD) = 0.93] than in E2-
(IOD = 0.09) or RU- (IOD = 0.17) treated cells, due to
leakage from the nucleus during cell fractionation (28). Demonstration
of ER nuclear retention in control cells was the result of rapid
preparation of nuclear extract, the nuclear retention in
E2-treated cells being due to the tight interaction of the
receptor within the nucleus (27). After RU treatment, the ER signal of
nuclear extract was lower (IOD = 2.33) than in control (IOD =
5.81) and E2-treated (IOD = 10.74) cells: some nuclear
receptor after 16 h of RU treatment (see Fig. 4A
) and the possible
cofractionation of some ER clusters may account for this signal. The
presence of ER in cytosolic pellets of control (IOD = 2.49) and
E2-treated (IOD = 1.94) cells was possibly a result of
overexpression; the most important signal (IOD =6.83) was found in this
fraction after RU treatment, indicating that ER did lose its
solubility. This fraction generally contains particulate cytoplasmic
material such as plasma membrane, microsomes, lysosomes, peroxisomes,
and mitochondria. Moreover, we did not notice any preferential
subcellular copartitioning of the receptor with Hsp90 and Hsp70 by
Western blot analysis (data not shown).
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Coimmunolocalization of RU-dependent clusters and Grp78/94, specifically localized in endoplasmic reticulum and Golgi apparatus, or catalase, localized in peroxisomes, or Grp75, a specific mitochondrial marker, or cathepsin D, which is found in lysosomes, was investigated by double indirect immunofluorescence. A low level of colocalization was found with endoplasmic reticulum or peroxisome markers using scatter diagram analysis (data not shown), but it was only observed in a few percent of transfected RU-treated cells and not considered as significant. Lysosomal and mitochondrial fluorescence was completely independent from ER clusters fluorescence (data not shown). In addition, we tested the possible association between clustered ER and Hsp70. As with Hsp90, we did not find any preferential colocalization of the two proteins using the scatter diagrams (data not shown).
In parallel, using immunogold labeling, electron microscopy studies
were carried out on transfected control Cos-7 cells and after 16 h
of E2 or RU treatment. In control experiments, immunogold
decoration was found predominantly distributed in the nucleus (Fig. 8a), as after E2 treatment
(Fig. 8b
). The treatment with RU during 16 h led to immunogold
labeling of cytoplasmic clusters found near the cytoplasmic side of
nuclear pores (Fig. 8
, c and d) and in cytoplasmic regions rich in
ribosomes and polyribosomes (Fig. 8
, e and f). Despite the dramatic
decrease of nuclear decoration, some clustering of ER by RU was also
found in the nucleus.
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The LBD of ER Is Sufficient for RU-Dependent Cytoplasmic
Clustering
In transfected Cos-7 cells or in the ERC3 cell line,
the treatment with the antiestrogen RU elicited the mislocalization and
the clustering of ER in the cytoplasm (Fig. 1, f and h, and Fig. 2
f), indicating a loss of constitutive and ligand-dependent nuclear
localization functions.
To analyze the involvement of different domains of the ER in the
cytoplasmic clustering due to RU, we transfected Cos-7 cells with two
ER mutants. HE255G, a mutant deleted from amino acids (aa) 1274,
contains part of the hinge region and the LBD with the p-NLS1 located
between the two regions (aa 299303). HE241G is a mutant deleted from
aa 250303, a region containing pNLS1, pNLS2, and pNLS3. Both mutants
are unable to be efficiently nuclear localized, in the absence and
presence of E2 (5). As shown in Fig. 9, in the absence of hormone, HE255G
(Fig. 9a
) and HE241G (Fig. 9b
) were localized in the nucleus and in the
cytoplasm, showing a homogeneous staining. For both mutants, the
addition of RU (100 nM) led to the formation of cytoplasmic
clusters and to a disappearance of the nuclear staining (Fig. 9
, c and
d), indicating that the LBD alone bound to RU was sufficient for the
observed clustering while the N-terminal portion of the receptor
encompassing the AF-1 region plus the entire DNA-binding domain was
dispensable. The constitutive nuclear localization function contained
in the aa 250303 region, although completely repressed by RU binding,
was not necessary to determine ER mislocalization.
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DISCUSSION |
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ER Ligands Abolish ER-Hsp90 Interaction
Here we have extended a previous description of the in
vivo Hsp90-ER interaction, based on nuclear cotranslocation assay
of the two proteins overexpressed in Cos-7 cells (15), to a CHO-derived
cell line, ERC3, stably expressing ER.
Moreover, we found that the Hsp90 nuclear localization, due to its interaction with the karyophilic ER, decreased in the presence of the agonist ligand, E2, as well as in the presence of tamoxifen, a mixed agonist-antagonist, and RU, a fully antiestrogenic compound. All ER ligands tested allowed a diffuse cytoplasmic relocalization of transfected or endogenous Hsp90.
Both the ER-dependent nuclear translocation of Hsp90 in the absence of hormone and Hsp90 exit from the nucleus when ER is liganded and tightly bound to the nuclear structure are insensitive to CHX. It is worth noting that the hormone-independent, NLS-based, and the hormone-dependent nuclear localization functions of nuclear steroid receptors, as well as their nucleo-cytoplasmic shuttling, are also insensitive to CHX (6, 31, 32).
With respect to Hsp90 nuclear cotranslocation with unliganded ER, a piggy-back transport by the karyophilic interacting protein is the most plausible explanation, even though Hsp90 itself may possess a weak or regulated nuclear localization function (15). Exit of Hsp90 from the nucleus as a consequence of its dissociation from liganded ER seems not to be a rapid phenomenon (812 h are needed to visualize a decrease in the nuclear fluorescence in 80% of the cells). By contrast, the exit of nuclear receptors from the nucleus, in the presence or absence of hormone, needs only 34 h in order to be documented in experiments with heterokaryons (32).
The NLS-based mechanism of nuclear ER localization, involving - and
ß-importins and Ran GTP/GDP cycle, is, to a large degree, established
and may account for some coimport of Hsp90. However, steps intervening
in the exit of ER from the nucleus, a prerequisite for
nucleo-cytoplasmic shuttling, are largely unknown (32, 33, 34).
We suggest therefore that in the presence of ligand, the dynamic association between Hsp90 and ER being shifted toward a dissociated status, the export of Hsp90 from the nucleus, is mostly independent of that of ER. Whether Hsp90 possesses nuclear export signals (33) remains to be determined.
RU Induces Cytoplasmic Clustering of ER
When the pure antiestrogen RU was used, the subcellular
distribution of ER was profoundly modified in a dose- and
time-dependent manner. ER immunolocalization was found to be
cytoplasmic and speckled, clusters of specific immunofluorescence being
found in large perinuclear regions without significant colocalization
with Hsp90 and Hsp70. The localization of ER under RU treatment was
similar to that described after treatment with two pure antiestrogens,
ICI 164384 and ICI 182780 (6, 19, 35). Probably due to a lack of time
course experiments, the predominantly cytoplasmic clustered
localization of ER was observed only in a few percent of cells (4.5%)
after 1 h of ICI 184780 treatment (6). Similarly, a short time and
a low dose of antiestrogen treatment also failed to provide evidence
for the ICI 164384 effect on the subcellular mislocalization of ER (5).
The overall effect of RU was a dose- and time-dependent accumulation of
ER in cytoplasmic clusters concomitant with a progressive nuclear
depletion of ER, indicating the inefficiency of the constitutive and
ligand-dependent nuclear localization functions of ER under RU
treatment.
The kinetics of nuclear depletion of ER under RU seems to be the result
of a blocked nuclear import, which may be concomitant with a diminished
ER steady state level according to cell line. It will be of interest to
perform experiments to look for a lack of functional interaction
between the NLS of ER and importin under RU treatment and to
elucidate how the hormone-dependent nuclear localization function
located in the LBD of ER (5) is disrupted. The finding that some
transcription intermediary factors (TIFs) or coactivators interacting
with the AF-2 domain of the LBD in an agonist-dependent or -enhanced
manner, possess functional NLSs and are able, according to their
intracellular levels, to bring mutated "cytoplasmic" receptors to
the nucleus, gave some explanation for the hormone-dependent nuclear
localization function of the LBD and for the inactivation of this
function by RU (36, 37, 38, 39).
The ER mislocalization was reversed by RU withdrawal, and, more efficiently, by 10 nM E2 treatment. Surprisingly, the cytoplasmic clustering and mislocalization of ER by RU was inhibited in the presence of CHX, suggesting that this RU effect was dependent on ongoing protein synthesis, possibly the synthesis of ER itself. However and most importantly, once the mislocalization of ER was established by RU, its reversal by CHX, even in the continuous presence of RU, strongly indicates the participation of protein(s) turning over faster than ER, in the maintenance of RU-dependent ER mislocalization. Such protein(s) may interact with ER in a RU-dependent or -enhanced manner.
Because a similar subcellular mislocalization of ER under ICI 164384
and 184780 treatment has been reported (6, 19), we suspect that RU and
both ICI antiestrogens act in a similar way. Indeed, the ICI and RU
antiestrogens possess a long side chain at the 7 and 11ß
positions, respectively, that confers a chiral symmetry between the two
steroidal structures (6, 21, 22). However, in contrast to the
indication of an impeded nucleo-cytoplasmic shuttling of ER in
heterokarions in the presence of ICI 184780 plus CHX (6), our
experiments clearly demonstrated that the nuclear import is restored in
the presence of RU plus CHX, pointing to a protein synthesis-dependent
step as a crucial event in the RU mechanism.
By testing for the ER domain responsible for the RU-dependent mislocalization, we found that the LBD alone was sufficient to observe ER cytoplasmic clustering and that the region bearing the constitutive NLS was dispensable.
The fact that a certain mutation in the AF-2 domain (H12 helix of ER) reverses the antiestrogenic properties of ICI 164384 to estrogenic properties (19, 40) is in agreement with the importance of this region in the functional and physical interaction with coactivators (37). The binding of antiestrogens seems to preclude these interactions (37, 38, 39, 41, 42). RU, in particular, may promote or enhance the interaction of the LBD, possibly the H12 helix, with a rapidly turning over protein that sequesters ER out of the nucleus.
Protein(s) Responsible for ER Clustering: a New Step in Pure
Antiestrogen Action
We propose that conformational changes in the LBD of ER provoked
by RU binding may include 1) incorrect positioning of the H12 helix of
the LBD, precluding interaction with transcription intermediary factors
that may also be involved in the hormone-dependent nuclear localization
of the receptor; 2) disruption of hormone-dependent as well as
constitutive nuclear localization functions of ER, thus impeding the
access of the receptor to specific ERE sequences; 3) interaction of the
LBD with protein(s) responsible for ER clustering. It is not excluded
that such a protein may interact transiently with ER in the nucleus, in
particular when RU was reversing the natural nuclear localization of
ER.
The sequence and the interdependency of these events cannot be inferred on the basis of the results presented here, opening the way to further investigations and to a new hypothesis to be tested in addition to the already proposed mechanisms for antiestrogens based on accelerated receptor degradation (35), inhibition of dimerization (43), and of interaction with coactivators (36, 37, 38). Interestingly, ER clustering by RU, concomitant to a decreased immunoreactivity, can be detected in target tissues such as uterus or MCF-7 derived tumors (F. Delahaye, unpublished observations).
Most importantly, because ER clustering seems to be the major determinant of the biocharacter of the antagonist, this may suggest rapid screening strategies for antiestrogenic and antitumoral properties of synthetic and natural compounds relevant for an overall long-term antiestrogenic effect required in cancer therapy. Such a screening may be complementary to testing for inhibition of ER AF-1 and AF-2 activity by antiestrogens.
We can indeed predict that antiestrogens inducing ER clustering, in addition to being pure antiestrogens, will also be effective in inhibiting the estrogen-independent transcription activation and growth promotion by ER (44) and, more generally, all effects of ER at nuclear level.
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MATERIALS AND METHODS |
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Plasmids
Wild-type ER (HEGO), and mutants HE255G and HE241G cDNA cloned
in the expression vector pSG5 (Stratagene, La Jolla, CA)
have been previously described (5).
To obtain the highest coexpression of ER and Hsp90, the SalI-SalI fragment from pSG5 HEGO containing ER cDNA was subcloned into the plasmid pSVK3 90 containing the Hsp90 cDNA (17) to give the plasmid pSVK3 90-HEGO. In this construct, the cDNAs of Hsp90 and ER are under the control of separate promoters.
Plasmid ERE-TK-LUC containing ERE with the reporter gene luciferase has been described previously (30).
Cell Culture and DNA Transfection
Cos-7 cells were grown in DMEM (Sigma Chemical Co., St. Louis,
MO) supplemented with 10% FCS (Life Technologies, Gaithersburg, MD).
For transient transfection, DMEM without phenol red (Sigma) was
supplemented with 10% of twice charcoal-stripped FCS.
DNA transfections were performed using an Easy Ject Plus electroporator (Eurogentec, Seraing, Belgium) under the following conditions: voltage = 230 V and capacity = 1650 µFarads. The resulting pulse times varied between 30 and 35 msec.
The ERC3 cell line was a generous gift from P. Kushner (24). Cells were cultured in Hams F-12/DMEM (1:1) without phenol red (Sigma). The medium was supplemented with 10% of iron-supplemented newborn calf serum (Sigma).
Transactivation Assays
CHO cells were grown in Hams F12/DMEM (1:1) (Sigma).
Transfections were performed in the same medium, without phenol red and
with 10% of twice charcoal-stripped FCS (Life Technologies), by
electroporation using 320 V and 1800 µFarads. The resulting pulse
times varied between 35 and 40 msec. For each dish (6 cm diameter), 150
ng HEGO, 1 µg ERE-TK-LUC, 3 µg pCH110 (ß-Gal internal standard
plasmid), and 10 µg salmon sperm DNA were transfected. Each
experimental point was done in triplicate. Hormone or anti-hormone was
added when cells were plated just after electroporation and for 48
h. Cells were harvested and lysed to perform ß-Gal and luciferase
assays as previously described (45).
Antibodies and Indirect Immunofluorescence
Indirect simple and double immunofluorescence studies were
performed as described (15, 17). Monoclonal rat BF4 antiavian Hsp90
antibody (7), which did not recognize endogenous Cos-7 cells Hsp90, was
used. The anti-Hsp90 polyclonal antibody used to detect endogenous
Hsp90 in ERC3 cell line was a generous gift from Y. Miyata
(26).
Human ER and the mutants HE255G and HE241G were revealed with rat H222 or H226 monoclonal antibodies (25) or with mouse JS 3432 monoclonal antibody (23).
Confocal Laser Scanning Microscopy
Analyses were performed at Service de Microscopie Confocale de
lIFR 21 (Hormones et Génétique, Kremlin-Bicêtre,
France) using a Zeiss Axiovert 135 M with 63/1.4 and 100/1.4 Oil
plan-Apochromat objectives (Carl Zeiss, Iena, Germany). Selected images
were treated by the Zeiss LSM 410 confocal microscopy system with
software version 3.92 beta. An AR laser (exitation wave length 488 nm)
was used for fluorescein isothiocyanate (FITC) detection and a HeNe
laser (excitation wave length, 543 nm) for tetramethyl rhodamine
isothiocyonate (TRITC) detection. Double staining microfluorometry
analysis was performed as previously described (46).
Preparation of Cytosols and Nuclear Extracts, Electrophoresis,
and Western Blot Analysis
Cytosols and nuclear extracts of transfected Cos-7 cells
were prepared as follows: cells were harvested 48 h after
transfection and centrifuged, and the pellet was resuspended in a
hypotonic buffer (20 mM Tris-HCl, pH 7.4; 15% glycerol; 1
mM EDTA; 30 mM natrium molybdate) containing
protease inhibitors (0.2 mM phenylmethylsulfonyl fluoride;
5 µg/ml leupeptin; 5 µg/ml antipaïn; 5 µg/ml aprotinin;
and 1 mM dithioerythritol). The cells were transferred to a
glass Dounce homogenizer and homogenized with 40 up-and-down strokes
using a type B pestle. The homogenate (total cell extract) was
centrifuged 10 min at 800 x g. The supernatant was
centrifuged 45 min at 100,000 x g to obtain the
cytosolic fraction. The 100,000 x g pellet was
resuspended in the cytosol buffer. The 800 x g nuclear
pellet was washed and resuspended in high ionic strength buffer (20
mM HEPES, pH 7.9; 20% glycerol; 0.55 M KCl;
1.5 mM MgCl2; 0.2 mM EDTA; 2
mM dithiothreitol) containing protease inhibitors as
described above. Nuclei were lysed in a glass Dounce homogenizer with
10 up-and-down strokes using a type B pestle, and then centrifuged 30
min at 15,000 x g. The supernatant was kept as nuclear
extract.
LDH was assayed in all fractions using the kit Enzyline LDH/HBDH (bio Mérieux, Marcy lEtoile, France).
The cytosols, the resuspended 100,000 x g pellets, and the nuclear extracts (40 µg of each fraction) were directly submitted to 10% SDS-PAGE analysis followed by Western blot.
Human ER was then detected with the monoclonal rat H222 antibody and avidin-biotin Vectastain reagents kit (Vector Laboratories Burlingame, CA), with revelation by enhanced chemiluminescence (ECL) reactions (Amersham, Arlington Heights, IL). The signal corresponding to ER was analyzed utilzing BioImage Software on BioImage Station (Millipore, Bedford, MA) and was expressed as IOD. The range of IODs between 0.01 and 10 was linear when using increasing amounts of loaded proteins.
Preparation of Total Extracts
After transfection cells were harvested and collected by
centrifugation. The pellet was frozen at -80 C and then resuspended in
high salt buffer (25 mM Tris/HPO4, pH 7.8; 0.55
M KCl; 10 mM MgCl2; 1% Triton
X-100; 15% glycerol; 1 mM EDTA; 1 mM
dithiothreitol), vortexed, and passed five times through a needle. The
suspensions were then centrifuged 15 min at 15,000 x g
to remove debris. The supernatants, called total extracts, were
submitted to 10% SDS-PAGE (50 µg of proteins), and the signals were
analyzed as described above.
Electron Microscopy
Cells were fixed in situ sequentially by
glutaraldehyde and osmic acid (47). Thin sections of 50 nM
were cut tangently to the cell layer with an Ultratome III (LKB) and
mounted on 200-mesh nickel grids coated with parlodion film. They were
pretreated with saturated sodium metaperiodate (Merck) to restore
antigenicity of osmium fixed tissues (47) and washed thoroughly in
distilled water before processing for immunological labeling. Sections
were first incubated with 3% normal goat serum in PBS to reduce
background. The grids were floated on a drop of monoclonal rat H222
antibody at a final concentration of 5 µg/ml, then incubated with
gold-conjugated secondary antibody (Biocell), washed in PBS containing
2% Tween, floated on 2% glutaraldehyde and finally stained with
alcoholic uranyl acetate and Reynolds reagent. A control test was made
by substitution of the primary antibody against ER by an irrelevant one
or by PBS. Grids were analyzed on a Siemens Elmiskop 101 at 80 KV.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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This work was supported by Institut National de la Santé et de la Recherche Médicale, Association pour la Recherche sur le Cancer, Ligue contre le Cancer, Fondation pour la Recherche Médicale and Ministère de la Défense (Direction de la Recherche et de la Technologie). X. Meng is a fellow of the Lalor Foundation.
Received for publication August 13, 1997. Revision received February 18, 1998. Accepted for publication February 20, 1998.
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REFERENCES |
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