Developpement, Evolution et Plasticité du System Nerveux Institut de Neurobiologie Alfred Fessard Centre Nationale de la Recherche Scientifique 91198 Gif-sur-Yvette cedex, France
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ABSTRACT |
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INTRODUCTION |
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For both ER and -ß, a number of variant transcripts have been
described, particularly in cancer cell lines or tumors (see Refs. 9, 10 for reviews). While several of these transcripts, created by
skipping internal exons, retain the same reading frame as the
full-length transcript, the corresponding variant proteins have been
rarely detected. In nontumoral tissue, only one ER
variant (TERP1;
Refs. 11, 12, 13, 14) and one ERß variant (15) have been shown to be
translated into functional proteins.
We have recently identified in the pituitary gland three naturally
occurring ER isoforms (
E3,
E4, and
E34), the expression
of which is specifically modulated during development (16).
E3
harbors a deletion of exon 3 encoding the second zinc finger of the
DNA-binding domain,
E4 lacks exon 4 that encodes a nuclear
localization signal (NLS) and part of the steroid binding domain, and
E34 lacks both exons 3 and 4 (Fig. 1
). At early stages of pituitary
development in the rat, these spliced isoforms are expressed
abundantly, at least 4 days earlier than full-length ER
. In
addition,
E3 and
E4 are found essentially in the pituitary
intermediate lobe and in the cytoplasm, in contrast to full-length
ER
, which is mainly present in the nucleus (16).
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The peculiar cellular localization of the E3,
E4, and
E34 ER
isoforms observed in fetal pituitary tissue, and the fact that they
lack certain functional regions, prompted us to investigate whether
they might play a specific regulatory function during pituitary
development. Using transiently transfected cells, we first studied
their precise subcellular distribution, in the presence or absence of
hormone. Then, we examined the ability of these short isoforms to
modulate transcription of estrogen-target genes, i.e.
whether they are similarly activated by the hormone, whether they act
as real transcription factors, and finally whether and how they affect
the transcriptional activity of full-length ER
.
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RESULTS |
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Estradiol Effect on the Association of ER Isoforms with the
Different Nuclear Constituents
It has been consistently shown that upon binding of an
agonist ligand, ER undergoes changes in its subnuclear distribution
and becomes tightly associated with nuclear matrix (NM) (20, 27, 28, 29, 30).
We thus qualitatively assessed whether the three deleted isoforms
showed the same hormone-induced tethering to the NM.
To identify the subnuclear compartments in which liganded or unliganded
ER isoforms would reside, serial extractions were performed on
transfected COS-7 cells treated or not with E2 (1
nM). Cytosolic fractions, as well as nuclear fractions
containing detergent-extractable proteins (Triton X-100-supernatant
or nucleoplasm), DNase I-extractable proteins (DNase I supernatant or
chromatin), and finally, proteins remaining after removal of DNA and
its associated proteins (NM), were analyzed by Western blotting using a
previously described anti-ER antiserum (16). As shown in Fig. 3
, LDH and histone, used as controls,
were only detected in the cytosolic and chromatin fractions,
respectively. Still, overexpression of proteins in transfected cells
may lead to nuclear leakage, as well as nuclear contamination with
perinuclear cytoplasm during cell fractionation procedures. This may
account, for instance, for the presence of
E34 in the nucleoplasm
fraction. The distribution of the three other isoforms in the absence
of hormone was nevertheless qualitatively similar to that described in
our cytological study, with large amounts of full-length ER and
E3
in the nucleoplasm, and
E4 being located both in the nucleoplasm
and the cytosol. The amount of full-length ER and
E3 resistant to
extractions was significantly increased after treatment of cells with
E2 for 2 h, thus showing their tight
association with the chromatin and NM fractions. By contrast,
E34
distribution was almost unaffected, and the nuclear
E4 remained
fully soluble in Triton X-100 (nucleoplasm), being unassociated
with chromatin or NM. Thus, for all ER
isoforms, the hormone
effect on their subcellular distribution reflected the changes in their
ability to tightly associate to the functional components of the
nucleus.
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Transfection of CHO cells with full-length ER and reporter
plasmid resulted in an approximately 4-fold increase in chloramphenicol
acetyltransferase (CAT) synthesis in response to
E2 (Fig. 4A
).
Changes in gene expression were dependent on the expression of an
exogenous ER
and not the result of an overall increase in basal
transcription. Indeed, when CHO cells transfected with the reporter
gene alone were treated with E2, in no instance
was transcription of the pVit-TK-CAT target gene stimulated (Fig. 4A
, lanes 1and 2).
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As shown in Fig. 4B, transcription of the CAT target gene was
stimulated approximately 4-fold when GH4 C1 cells transfected with the
estrogen response element (ERE)-containing reporter plasmid alone were
treated with E2, confirming the presence of
active endogenous ERs. In the same conditions, the activity of the
minimal TK-CAT promoter was not stimulated. GH4 C1 cells were used to
further analyze the potential interactions between a distinct isoform
and the endogenous ER activity. In these cells, overexpression of
full-length ER
resulted in an approximately 7-fold increase in CAT
quantity in response to E2. Expression of either
E3 or
E34 did not stimulate CAT synthesis (
3-fold or 5-fold,
respectively) above the level obtained by the reporter plasmid alone
(VTC) in response to E2. By contrast, expression
of
E4 resulted in an approximately 12-fold increase in CAT quantity
in response to E2. Thus, in GH4 C1 cells,
E4
appeared to be able to enhance CAT reporter gene expression at least as
efficiently as full-length ER
.
Coimmunoprecipitation of Full-Length ER and ER Isoforms
To investigate whether the effects of ER isoforms on
transcription could involve protein-protein interactions, a series of
immunoprecipitations was performed in which the ability of the short
variants to form heterodimers with full-length ER
was examined. To
differentiate isoforms from full-length ER, we used a full-length ER
tagged with an hemagglutinin epitope (HA).
COS-7 cells were transfected with plasmids encoding HA-tagged
full-length ER alone or together with a plasmid encoding one isoform.
Western blots prepared from aliquots of cell extracts were used to
confirm that the proteins were expressed at equal levels. Blots were
reacted either with a monoclonal antibody against HA (data not shown)
or with the anti-ER antiserum (Fig. 5A
). The remainder of the extracts were
immunoprecipitated with the anti-HA monoclonal antibody, and Western
blots of the immunoprecipitated proteins were probed with the
anti-ER
polyclonal antiserum. The data presented in Fig. 5B
demonstrate that
E3 was efficiently coimmunoprecipitated with the
HA-tagged full-length ER
, in the presence or absence of hormone. In
contrast,
E4 and
E34 were unable to form stable complexes with
full-length ER
.
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Aliquots of WCEs containing individual receptors, prepared from cells
treated or not with forskolin/IBMX (isobutylmethylxanthine),
were incubated with a 32P-labeled double-stranded
ERE oligonucleotide and assayed by electrophoresis on a
nondenaturing polyacrylamide gel. As expected, specific ER-ERE
complexes were formed with full-length ER (in the basal, or
phosphorylated state), and the intensity of the band increased with the
amount of WCE (Fig. 6A
, lanes 3 to
6). It may be noticed that more than one complex has formed. However,
the lower band (marked with an asterisk) is likely to be
nonspecific, being present also in the control lane, which contains
extracts from COS-7 cells transfected with the empty expression
vector.
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Forskolin-Induced Change in E4-GFP Subcellular Localization
Since E4 expressed in COS cells that were treated with
forskolin/IBMX was able to bind a consensus ERE, we tested whether the
same treatment could also promote specific morphological changes in the
nucleus, suggestive of an activated state for
E4, and its tethering
to NM.
COS-1 cells transfected with expression plasmids encoding the
GFP-tagged ERs, and treated or not with forskolin/IBMX for 15 min, were
analyzed under confocal fluorescence microscopy. The distribution of
full-length ER-GFP, E3-GFP, or
E34-GFP was totally unaffected
by this treatment (data not shown). On the contrary, as shown in Fig. 7
, unlike E2
treatment (see Fig. 2
), forskolin/IBMX treatment appeared to change the
diffuse nucleoplasmic pattern of
E4-GFP distribution (left
image) into a partially speckled one and may even induce the
complete disappearance of
E4-GFP from the cytoplasm (right
image).
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DISCUSSION |
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ERs are known to undergo a continuous nucleocytoplasmic shuttling (22, 32, 33). Nevertheless, at any given time, a major fraction of the
protein is present in the nucleus even in the absence of hormone (18, 19). The unliganded receptor with a sedimentation coefficient of 9S is
loosely associated to nuclear components, and hormone binding causes
activation of the receptor, i.e. a biochemical
transformation to a complex with a sedimentation coefficient of 5S that
associates more tightly with the nucleus (20, 28, 34). Recently, using
GFP tagging, two groups provided visual descriptions of the unoccupied
and the ligand-bound ER. The majority of unliganded GFP-ER appears to
be evenly distributed throughout the nucleus. Addition of
E2 dramatically changes the diffuse nucleoplasmic
pattern of GFP-ER to a punctate or hyperspeckled pattern (27, 30).
This early intranuclear redistribution of GFP-ER
corresponds to the
formation of NM-bound foci of ER
. These foci are probably similar to
the so-called matrix-attachment regions (MARs) that involve 14% of
the genomic DNA, occur at DNase hypersensitive sites near active genes,
and contain many transcription factors (35, 36). The NM-bound foci are
quite numerous as compared with real sites of transcription starts, and
most of the receptor attachment sites are not actively involved in
transcription (27, 37). In fact, it is possible that both constitutive
and regulatory MARs exist, and that only the latter are
transiently attached to transcriptionally active genes and involved in
their regulation (38). The formation of these NM-bound foci of ER
is
not sufficient for transcription to occur, as both agonist and
antagonist ligands may elicit such an event (27). Nevertheless, this
early intranuclear redistribution of the receptor and association with
NM is a mandatory step to transcription activation that, provided it is
induced by an agonist, enables ERs to additionally recruit coactivators
and finally trigger transcription (27).
In full agreement with these results, our present studies in COS cells
transfected with full-length ER, fused or not to GFP, showed that,
in the absence of ligand, the protein was predominantly present in the
nucleus and exhibited a diffuse nucleoplasmic pattern. In a nuclear
fractionation study, unliganded full-length ER
was found most
prominently in the Triton X-100 extract. After E2
treatment, receptor distribution was restricted to the nucleus where it
displayed a characteristic speckled pattern, and a substantial amount
of this protein was strongly associated with chromatin and NM. It has
been pointed out by Stenoien et al. (27) that transient
expression of ERs in heterologous cells may lead to saturation of the
intracellular transport mechanisms, and hence to a misrepresentation of
soluble vs. NM-bound forms of ER. While this may explain why
even after ligand binding, a certain amount of full-length ER
remained in the Triton X-100 soluble fraction, our observations
concerning the ligand-induced changes in compartmentalization are in
agreement with those described for cells naturally expressing ERs.
In addition, our present study on ER variants revealed marked
differences between full-length receptor and its isoforms with regard
to their subcellular localization, solubility partitioning, activation,
and biological actions.
E3: An Inhibitor of Estrogen-Target Gene Transcription
In the absence of ligand, the E3 protein was present both in
the cytoplasm and the nucleus of transfected cells, as determined
either by fluorescence studies employing GFP tagging or by Western blot
analysis of subcellular/subnuclear fractions. This isoform retains the
ligand binding domain and the NLSs, allowing for hormone binding and
translocation to the nucleus. Accordingly, we observed that
E2 treatment clearly altered the partitioning of
E3 between cytosol and nucleus, and its intranuclear distribution:
in the presence of hormone,
E3 was found exclusively in the nucleus,
showed a punctate distribution, and was found tightly associated with
chromatin and NM, like full-length ER
. Yet,
E3 lacks the second
zinc finger of the DNA-binding domain, and, as anticipated, our current
gel mobility shift assay data show that this protein was unable to bind
to a canonical ERE in vitro or to activate transcription of
a CAT reporter gene containing an ERE in vivo. In fact, when
cotransfected with full-length ER
,
E3 repressed the
estrogen-dependent increase in CAT synthesis induced by full-length
ER
in vivo. Thus, in agreement with previous results
obtained in the MCF-7 breast cancer cell line (39),
E3 inhibits
transcriptional activation by full-length ER
in a dominant negative
fashion. This finding is probably related to the fact that
E3
inhibits full-length ER binding to a canonical ERE in vitro,
as demonstrated by Wang and Miksicek (39).
The E3 isoform is thus likely to modulate the activity of
full-length ER
by protein-protein interactions. For this inhibition,
at least two mechanisms that are not mutually exclusive seem possible.
First,
E3, which still contains a strong hormone-inducible
dimerization motif within the ligand-binding domain, could form
inactive heterodimers with full-length ER
. In support of this model,
we showed herein that
E3, in the presence or absence of
E2, can be coimmunoprecipitated with full-length
ER-HA, implying a direct physical association between
E3 and
full-length ER
. Such heterodimers could then sterically hinder
binding of functional receptors. A second mechanism would involve
interactions of
E3, as a homo- or heterodimer, with a limiting
factor required either for receptor function or transcriptional
activation. This type of factor, which interact both with enhancer
binding proteins and basal transcription factors, can, under some
circumstances, be readily depleted via binding to other transcription
factors. In this respect, Bollig and Miksicek (40) showed that in the
presence of E2,
E3 can bind the nuclear
receptor coactivator SRC-1, as does full-length ER
. The coactivators
are limiting factors for which
E3 and full-length ER-
are
probably competing. Such interaction with an adaptor molecule may also
account for the tight association of
E3 with chromatin or NM that we
observed upon addition of E2.
It has been shown that the amount of E3 transcript is dramatically
reduced in primary breast cancers and cancer cell lines, while
transfection of
E3 into MCF-7 cells leads to a suppression of the
transformed phenotype (41). In the light of these latter results and
our data, it seems likely that
E3 expression in normal tissue may
provide a means of decreasing or blocking estrogen responsiveness.
E4: An Estrogen-Independent Activator of Transcription
The E4 isoform lacks amino acids 255366, corresponding mainly
to the hinge region that contains a constitutive NLS. Yet, the present
study shows that, in the absence of hormone, a certain amount of this
protein was found in the nucleus of transiently transfected COS cells.
Lacking also the first 48 amino acids of the ligand binding domain,
E4 was expected to be insensitive to estrogens. Accordingly,
E2 promoted no change in the subcellular
distribution of
E4: it was unable to induce a speckled nuclear
distribution pattern for
E4-GFP, or a tight association of
E4 to
NM.
Nevertheless, E4 has an intact DNA-binding domain. Since the
DNA-binding domain alone was shown to be sufficient for transcriptional
activity, at least for certain nuclear receptors (42, 43), it has been
suggested that
E4 might play a role as constitutively active or
estrogen-independent transcription factor. In support of this
hypothesis, we showed that this protein indeed retained some activity
as a transcription factor, and that this activity was independent of
the hormonal ligand:
E4 protein exhibited an estrogen-independent
transactivation capacity when expressed in an ER-deficient cell line
and enhanced estrogen-dependent transcription activity when transfected
into cells naturally expressing full-length ER. Thus,
E4 is an
effective transcriptional activator of estrogen target genes. Since it
failed to coimmunoprecipitate with full-length ER
, a
heterodimerization with full-length ER
will not be involved in this
ligand-independent transcriptional activation. In fact,
E4 was able,
by its own, to bind a consensus ERE in vitro, as it formed a
complex with the ERE probe in a gel mobility shift assay. This complex
was clearly different from that formed between full-length ER
and
the ERE probe. First, it was of an obviously smaller size. Second,
anti-ER
antiserum did not retard its migration, but inhibited its
formation in a dose-dependent manner. According to a recent study from
Tyulmenkov and Klinge (44), who compared the impact of various ER
and ERß antibodies on the interaction of ERs with a consensus ERE,
both supershift and inhibition of ER-ERE interaction with a specific
antibody are equally reliable as means of detecting an ER isoform in a
gel mobility shift assay. Therefore, the observed differences between
E4-ERE and full-length ER
-ERE complexes are suggestive of a
difference in the action mechanism of these two protein isoforms. In
the classical model, the ERs bind, in response to their ligand
(E2), as homodimers to specific estrogen response
elements (EREs) within target genes. In fact, we and others have shown
that formation of ER homodimers is not the only way for ER to regulate
gene transcription. Heterodimers could also be formed and modulate
transcription. For instance, heterodimeric complexes can form between
ER
and ERß in vitro, with retained DNA-binding ability
and specificity (45).
E3 and full-length ER
were herein shown to
form inactive heterodimers. Furthermore, some other nuclear receptors,
such as orphan receptors of the Nur type, have been shown to bind DNA
as monomers (46). This may also be the case for
E4, which lacks
sequences involved in dimerization (47) and was shown here to form a
complex with the consensus ERE much smaller than the one formed between
full-length ER
and ERE.
E4 appears not to require
E2 to activate transcription of a reporter gene
but needs to be phosphorylated to bind a consensus ERE in
vitro. In support of this idea, a partially speckled pattern of
E4-GFP distribution was observed in the nucleus, after treatment of
the transfected cells with forskolin/IBMX. In fact, many studies have
shown that, in addition to the conventional hormone-dependent
regulation of ER activity, there is substantial cross-talk between
signal transduction pathways and steroid receptors. In a number of
cases, a modulation of kinase/phosphatase activity in cells leads to
activation of steroid receptors in the absence of hormone (48). Thus,
an altered phosphorylation of the receptor and/or associated proteins
is likely to be a key event in the ligand-independent activation of
ERs.
Transcriptional regulation by ERs involves two activation functions
(AFs) that reside on opposite ends of the receptors. AF-1 is located at
the amino terminus and is constitutively active, whereas AF-2 is
situated at the carboxy-terminal end of the ligand- binding domain (49, 50) and is strictly hormone regulated. Although both AF-1 and AF-2 are
required to achieve maximal transcriptional activity, the two
transactivating regions may either function independently or cooperate,
depending on the target gene promoter and the presence of
tissue-specific factors (51). When steroid receptors, including ERs,
are activated by nonsteroid agents such as growth factors, protein
kinase A, phorbol ester, and dopamine (for reviews, see Refs. 52, 53), which all induce protein phosphorylation, their AF-1 domain
appears to be the phosphorylation target. Moreover, it has been shown
that the MAP kinase-mediated phosphorylation of the AF-1 domain of
ERß and of SF-1 promotes ligand-independent recruitment of nuclear
receptor coactivators (54, 55). In the case of E4, it seems possible
that phosphorylation of its AF-1 domain would allow for a recruitment
of specific coactivators, and a functional interaction with the
transcription machinery, independently of the ligand.
E34: A Silent Variant
The third ER isoform that we had detected in the developing
pituitary, E34, appeared to have no biological activity: In
transfected cells, it was found predominantly in the cytoplasm, it
exhibited no real ability to enter the nucleus and to associate tightly
with NM upon ligand binding, nor did it bind a consensus ERE in
vitro, dimerize with full-length ER
, or modulate transcription
of estrogen-dependent genes.
Conclusions
In summary, our results demonstrate that E3 and
E4, two
alternatively spliced isoforms of ER
mRNA, may antagonize, or mimic,
respectively, the function of the full-length gene product. The
functional properties of these variants, as well as their expression
time course and relative abundance, suggest that they may well play a
physiological role as negative and positive regulators of transcription
in the pituitary, at least before birth. The strong expression of
E3, which antagonizes full-length ER
, in male embryos during the
critical period of development (16), could represent for the
pituitary an additional protection mechanism against the potentially
deleterious effects of maternal estrogens. Conversely, the
E4
isoform, which is early and abundantly expressed in the female
embryonic pituitary (16), could represent a hormone-independent,
phosphorylation-activated transcription factor, allowing for some
intrinsic ER activity regardless of the circulating estrogen
levels. The coordinated action during development of differentially
active transcription factors created from a single ER gene probably
contributes to the differentiation of pituitary cells first in the
absence, and then in the presence, of high concentrations of
estrogens.
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MATERIALS AND METHODS |
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The coding sequence of the ER (gift of Professor Muramatsu) in
pUC118 (56) was subcloned in pCDNA3 (Invitrogen). To
obtain the complete sequence of the ER
isoforms, the corresponding
PCR products inserted in pCRII and ER
in pCDNA3 were digested with
BsmI and BglI and separated on agarose gel. The
isoform-specific fragments and the remaining ER
sequence in pCDNA3
were ligated together to form
ER-pCDNA3 plasmids. All the constructs
were checked by full-length sequencing.
Generation of ER-GFP Fusion Plasmid
The plasmid pEGFP-N1 (CLONTECH Laboratories, Inc.,
Palo Alto, CA) was modified by mutation of the translation start codon
of the GFP to a valine codon, to generate pEGFPµ plasmid. To fuse the
C terminus of the various ER sequences to the mutated EGFP,
the
ER-pCDNA3 and ER
-pCDNA3 plasmids were modified by mutation of
the stop codon to a glycine codon and digested by EcoRI and
KpnI. The fragments coding for full-length ER
and
ERs
were isolated on agarose gel and subcloned in pEGFPµ in the Bsm and
BglII sites to generate the ER-pEGFP and
ER-pEGFP fusion
plasmids (Fig. 1B
). The integrity of the fusion was checked by DNA
sequencing and direct observation of the fluorescence in transfected
cells. Functionality of full-length ER-GFP as a ligand-dependent
transcription factor was assayed on an ERE-containing reporter gene in
transient transfections experiments using CHOk1 cells, as described in
Transcriptional Activity. The activity of full-length ER-GFP
was 130 ± 15% that of the native receptor.
Subcellular Localization of GFP-Tagged ER Isoforms in Transfected
Cells
COS-1 cells were grown in DMEM (Life Technologies, Inc.) supplemented with 10% FCS. Twenty four hours before
transfections, DMEM without phenol red was supplemented with 10% twice
charcoal/dextran-stripped FCS. Cells were transfected with 8 µg
expression vector ERs-pEGFP, using an electroporator (Bio-Rad Laboratories, Inc., Hercules, CA), and plated on glass
coverslips for 30 h in serum-free medium. They were then treated
or not with E2 (1 nM) for 2 h.
Cells were washed twice in ice-cold PBS and fixed for 30 min at 4 C in
PBS-paraformaldehyde 3%. Before mounting in Mowiol
(Polysciences, Inc., Warrington, PA), coverglasses were washed
twice for 5 min with PBS and cells were observed under a fluorescence
microscope, with a laser scanning confocal imaging system.
Sequential Fractionation of the Nuclear Constituents
Forty-eight hours after electroporation, COS-7 cells transfected
with individual isoform and treated or not with 1 nM
E2 as described above, were washed twice with
ice-cold PBS, scraped into PBS, and pelleted by centrifugation at
700 x g for 5 min at 4 C. They were then gently
resuspended in 5 vol. of lysis buffer (Tris, pH 7.5, 10
mM NP-40 0.05%, 3 mM
MgCl2, 100 mM NaCl, 1
mM EGTA, aprotinin, 20 µg/ml, 1
mM orthovanadate, 1 mM
4-[2-aminoethyl]-benzenosulfonyl fluoride (AEBSF), and
leupeptin, 10 µg/ml) and centrifuged at 350 x g for
5 min at 4 C: the supernatant, corresponding to the cytosol, was
collected. It represented approximately 80% of the total cell
proteins, as assayed using the Coomassie assay reagent (Pierce Chemical Co., Bezons, France).
The crude nuclear pellet was washed once in lysis buffer and twice in
ice-cold CSK buffer (10 mM
piperazine-N,N'-bis(2-ethanesulfonic acid), pH 6.8, 300
mM sucrose, 3 mM
MgCl2, 100 mM NaCl, 1
mM EGTA, aprotinin, 20 µg/ml, 1
mM orthovanadate, 1 mM
AEBSF, and leupeptin, 10 µg/ml) by gentle resuspension in 5 vol
buffer and centrifugation at 350 x g for 5 min at 4 C.
Nuclei were then resuspended in CSK buffer containing 1
M sucrose and centrifuged at 1,200 x
g for 10 min at 4 C. From this purified nuclei pellet,
soluble proteins (nucleoplasm; 20% of nuclear proteins) were
extracted by treatment for 5 min at 4 C with CSK buffer containing
0.5% Triton X-100, followed by a centrifugation at 700 x
g for 5 min at 4 C. Chromatin was then digested with DNase I
(700 U/ml) in CSK buffer containing 50 mM NaCl,
for 60 min at 4 C. The (digested) chromatin-associated proteins were
eluted by slowly adding ammonium sulfate in CSK buffer to a final
concentration of 0.25 M. The NM (
20% of
nuclear proteins) was pelleted at 1,000 x g for 5 min
and the chromatin fraction (
60% of nuclear proteins) was recovered
in the supernatant. All four fractions (cytosol, Triton X-100-soluble
fraction, i.e. nucleoplasm-, DNase I supernatant, i.e.chromatin- and NM), were prepared in Laemmli buffer and
subjected to SDS-PAGE and Western blot analysis.
Western Blot Analysis of ER Isoforms in Transfected COS Cells
Quantitation of Expression Level of ER Isoforms.
Extracts (5 or 8 µg protein) prepared from COS-1 cells transfected
with expression plasmids encoding individual isoforms were compared
with a scale of extracts (530 µg protein) from ER-naturally
expressing GH4C1 cells. All the extracts were thus loaded and run on a
10% SDS-PAGE. Corresponding blots were reacted with a previously
described (16) antiserum directed against ER
(1:1,000, Santa Cruz Biotechnology, Inc., Santa Cruz, CA), followed by an
HRP-coupled secondary antibody (The Jackson Laboratory,
Bar Harbor, ME) and enhanced chemiluminescence detection
(ECL, Amersham Pharmacia Biotech, Arlington Heights, IL).
As shown in Fig. 8
, the level of ER
isoforms expression in transfected COS cells is between 1.5 and 3
times that found in GH4C1 cells which naturally express ER
.
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Transcriptional Activity
ER-negative CHO k1 cells were grown in DMEM/F12 (1:1) medium
supplemented with 10% FCS. Twenty-four hours before transfection,
cells were plated in phenol red-free DMEM/F12 medium supplemented with
10% of twice charcoal-stripped FCS.
ER-positive GH4 C1 cells were maintained in MEM supplemented with 2.5% FCS and 10% horse serum. Twenty-four hours before transfection, cells were grown in phenol red-free DMEM supplemented with 2.5 and 10% twice charcoal-stripped FCS and horse serum, respectively, containing RU 58668 (50 nM), an ER antagonist.
Transcriptional effects of individual isoforms were tested using an
artificial promoter containing consensus Vit EREs inserted upstream of
the thymidine kinase (TK) promoter which drives the expression of the
CAT reporter gene. Cells were cotransfected with 10 µg expression
vector encoding the different forms of ER and 10 µg of the CAT
reporter plasmid, pVit-TK-CAT (VTC), with or without (10 µg)
full-length ER, by electroporation. Estradiol (1 nM) was
added immediately after transfection. Twelve hours (for GH4 C1 line),
or 48 h (for CHO k1 line) thereafter, cells were harvested, and
CAT quantity was measured in cell extracts. The CAT assays were
performed by a colorimetric enzyme immunoassay (CAT-ELISA kit,
Roche Molecular Biochemicals, Meylan, France) and
were all normalized to protein concentration. In some experiments, CAT
values were also standardized to the ß-galactosidase (expression
driven by the Rous sarcoma virus promoter) activity, without any
qualitative difference.
Immunoprecipitation
COS-7 cells were transiently transfected with an expression
plasmid encoding a full-length ER tagged with a HA epitope
(
68 kDa; HA-ER f.l.) alone or together with expression vector
encoding the different (nontagged) isoforms of ER
and grown for
48 h in a medium containing or not E2.
Functionality of HA-ER
as a ligand dependent-transcription factor
was assayed using CHOk1 cells, as described above (see
Transcriptional Activity). The activity of HA-ER
was
found to be 135 ± 20% that of the native receptor. To determine
whether all isoforms were expressed at equal levels in the cells,
aliquots of the cellular extracts were analyzed by Western blotting.
Blots were reacted either with a monoclonal antibody directed against
HA epitope (12CA5, Roche Molecular Biochemicals), or with
the antiserum directed against ER
(Santa Cruz Biotechnology, Inc.).
The remainder of the cell extracts (containing 200400 µg total
protein) were incubated with 4 µl monoclonal antibody directed
against HA epitope and 20 µl pansorbin for 2 h at 4 C. After
four washes with cell lysis buffer, immunoprecipitates were pelleted by
centrifugation, released into 15 µl SDS loading buffer by boiling for
5 min, and subjected to electrophoresis on a 12% acrylamide gel. Blots
were reacted with an antiserum directed against ER, followed by the
appropriate horseradish peroxidase-coupled secondary antibody and
enhanced chemiluminescence revelation.
Gel Mobility Shift Assays for in Vitro DNA
Binding
Preparation of WCEs.
WCEs were prepared from COS-7 cells transfected with expression vectors
encoding individual isoforms of ER. Forty-eight hours after
electroporation, cells were lysed at 4 C in 500 µl of lysis buffer
[20 mM HEPES (pH 7.9), 50 mM NaCl, 5
mM MgCl2, 12% glycerol, 0.2
mM EDTA, 0.1% NP-40, 5 mM DTT, 1
mM AEBSF, 0.5 µg/ml leupeptin, 1 µg/ml chymostatin, 1
µg/ml pepstatin, 1 µg/ml aprotinin]. Extracts were then
centrifuged at 15,000 x g for 10 min at 4 C, and
supernatants were collected, aliquoted, and stored at -70 C. In some
experiments, 15 min before lysis and preparation of WCEs, the cells
were treated with forskolin (10 µM) and IBMX (1
µM) to activate the PKA pathway via an increase
in intracellular cAMP levels.
To determine whether the isoforms of ER are faithfully and equally
produced by COS-7 cells, aliquots of extracts were analyzed by Western
blotting using a polyclonal anti-ER
antiserum.
DNA Probe.
A synthetic oligonucleotide (27-mer) containing a 13-bp perfect
palindromic ERE and corresponding to the Vit A2 ERE:
5'-GATCCTAGAGGTCACAGTGACCTACGA -3' was mixed with an equal
molar quantity of the complementary strand and annealed in water by
heating at 95 C and cooling slowly to room temperature (4 h). Fifty
nanograms of this 27-bp double-stranded fragment was end-labeled
with 200 µCi [-32P]-dATP using 58 U T4
PNK in 20 µl. The radiolabeled DNA was purified on a S200 column
(Pharmacia Biotech).
In Vitro DNA Binding.
In vitro DNA binding was performed as previously described
(57). Briefly, aliquots of WCEs containing the receptors were incubated
with 1 µg poly(dI.dC) in a reaction buffer (final concentration,10
mM Tris-HCl, pH 7.5, 1 mM
EDTA, 80 mM KCl, 5% glycerol) at 4 C for 20 min.
Purified 32P-labeled double-stranded ERE
oligonucleotide probe (5 x 104 cpm) was
added, and the reaction was performed at room temperature for 30 min.
(For competition experiments, 50-fold excess radioinert
oligonucleotides were added before radiolabeled probe.) The resulting
20 µl reaction mixture was analyzed on a 56% nondenaturing
polyacrylamide gel. Electrophoresis was carried out at 150 V in
0.5 x Tris-borate-EDTA (TBE) for 2 h. The gel was dried and
exposed for autoradiography.
Forskolin-Induced Change in E4-GFP Subcellular
Localization
COS-1 cells were electroporated with 8 µg expression plasmid
encoding the GFP-tagged ERs, cultured on glass coverslips for 30 h
in DMEM without phenol red, supplemented with 10% FCS that had been
twice stripped by charcoal/dextran. They were finally treated or
not (0) either with estradiol (E2) for 2 h,
or with forskolin-IBMX (Fk) for 15 min. Cells were washed in ice-cold
PBS and fixed for 30 min at 4 C in PBS- 3% PFA. Coverslips
were then washed twice for 5 min with PBS, mounted in Mowiol, and
analyzed under confocal fluorescence microscopy.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Work in our laboratory was supported by Centre National de la Recherche Scientifique, University Paris-Sud, and ARC.
1 Both authors contributed equally to this work.
Received for publication July 17, 2000. Revision received February 26, 2001. Accepted for publication February 27, 2001.
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REFERENCES |
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