Laboratory of Receptor Biology and Gene Expression National Cancer Institute National Institutes of Health Bethesda, Maryland 20892
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ABSTRACT |
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INTRODUCTION |
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This classic model became considerably more complex with the discovery that some steroid receptors can be activated by other hormones and signal transduction pathways in the absence of their specific ligands (reviewed in Ref. 2 ). While the receptors for glucocorticoids (GR) and mineralocorticoids (MR) are generally refractory to activation by agents other than their specific ligands, receptors for progesterone (PR), estrogen (ER), and androgens (AR) can be activated through several other signaling pathways. These receptors have been shown to be activated by some inducers of cAMP signaling such as 8-Br-cAMP (3 4 5 6 ) and dopamine (7 ), and also by peptide growth factors such as epidermal growth factor (4 8 ), insulin growth factor (6 9 ), and keratinocyte growth factor (10 ). The mechanism of ligand-independent activation may not be uniform. While ligand-independent activation of the ER is correlated with changes in receptor phosphorylation (6 11 ), such activation of the chicken PR is independent of receptor phosphorylation (12 ).
Activation of steroid receptors can lead to changes in the chromatin structure of some target genes in vivo, as manifested by the appearance of hormone-inducible nuclease hypersensitive sites (13 14 15 16 17 ). Recently, a number of chromatin modifying factors have been identified that functionally interact with steroid receptors. Various members of large multi-subunit complexes known as SWI/SNF have been shown to be necessary for GR activation of transcription in both yeast and mammalian cells (18 19 20 21 ). These complexes have been shown to disrupt reconstituted nucleosomes and thereby facilitate the interaction of various transcription factors with their DNA binding sites (22 23 24 ).
Steroid receptors also interact with histone acetyltransferases (HATs).
Acetylation of lysine residues in the tails of core histones is thought
to reduce the electrostatic interactions between DNA and the very basic
histone tails, thereby loosening contacts between the DNA and the core
histones and increasing access of transcription factors (25 26 ).
Several HATs, including CREB-binding protein (CBP)/p300, p300/CBP
associated factor (PCAF), activator of thyroid and retinoic acid
receptors (ACTR), and steroid receptor coactivator 1 (SRC-1) (27 28 29 30 31 )
can interact with each other as well as with the carboxy-terminal
activation domains [activation function 2 (AF-2)] of various
steroid receptors. In addition, the amino-terminal transcriptional
activation domain of the GR (AF1 or 1), can interact with the factor
Ada2 both physically and functionally (32 ). Ada2 forms a complex with
at least two other proteins, Ada3 and Gcn5 (33 34 ), which can
acetylate histones in nucleosome cores (35 ).
The mouse mammary tumor virus (MMTV) promoter has been used as a model for GR activation mechanisms in chromatin. When this promoter exists in cells in a stably replicating form, it has a highly ordered nucleoprotein structure consisting of six nonrandomly positioned nucleosome families (36 37 ). Upon activation of the GR a region of the promoter containing its binding sites undergoes a chromatin remodeling event characterized by the formation of a nuclease-hypersensitive site (36 ). As a result, other transcription factors previously excluded from their sites are able to gain access to the promoter (38 39 40 ). The GR, PR, AR, and MR are members of a nuclear receptor subgroup that bind to the same DNA sequences. However, the mechanisms by which target gene specificity is achieved in vivo are not clearly understood. We have previously shown that the ability of the PR to activate the MMTV promoter in replicating chromatin is conditional (41 ). When it is transiently expressed (less than 48 h) in transfected cells, it is a very poor activator of the MMTV template in chromatin because it fails to induce the remodeling event in the promoter (42 ). Transiently transfected GR activates this template efficiently, indicating that the GR and PR have different requirements for productive interactions with chromatin. In contrast, both receptors efficiently activate transiently transfected MMTV reporter constructs that do not have a repressed nucleoprotein conformation and thus do not require remodeling (43 ). However, the PR gains the ability to activate the replicating MMTV template upon constitutive expression in stably transfected cells (41 ).
Since all endogenous genes exist in the context of complex nucleoprotein structure, it is important to understand how steroid receptors function in this environment. We are interested in defining the requirements for PR interaction with chromatin. One possible mechanism by which the PR becomes competent to activate the MMTV promoter in ordered chromatin is that it alters the nucleoprotein structure of the template. In the course of multiple rounds of DNA replication, when nucleosomal restraints are temporarily removed, the PR might access its binding sites and influence the reassembly of the chromatin. In fact, the PR has been shown to maintain an integrated MMTV promoter in a constitutively remodeled state in a human mammary adenocarcinoma cell line (44 45 ). An alternative mechanism is that some feature of intracellular PR status may change over time. In this model the PR would be processed by the cell to make it a fully functional transcription factor, able to activate target genes that require the type of chromatin remodeling associated with the formation of nuclease-hypersensitive sites, as well as those, such as the transient MMTV template, that do not require this type of remodeling.
To distinguish between these possibilities, we have characterized various aspects of PR function when it is transiently or constitutively expressed. We find no major structural alterations in the replicating MMTV template in the presence of constitutively expressed PR. In fact, this receptor appears to activate the template by a basic mechanism highly similar to that used by the GR. In contrast, we observed major differences in the functional activity of the PR, indicating that some type of time-dependent PR processing takes place, which changes its response to extracellular signals and its ability to interact productively with chromatin. This work provides new insights into the regulation of PR function and has important implications for the way in which we study steroid receptor function in vivo.
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RESULTS |
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We compared the mechanism of GR and PR activation of the MMTV template
in chromatin by several criteria. The most obvious question is whether
the proximal promoter region containing the hormone response elements
(HREs) is in a constitutively open state in 3017.1 cells, or whether it
undergoes remodeling in response to hormone treatment as it does in the
parental 1470.2 cells. The remodeling event can be measured by
SacI cleavage of the MMTV promoter in nuclei from treated
cells (43 ). SacI cleaves the promoter in the area of the
proximal set of HREs. The extent of cleavage changes approximately
1020% upon dexamethasone (Dex) treatment in a variety of cell lines
with replicating forms of the MMTV long terminal repeat (LTR) (51 ).
Figure 2A shows the results of the
SacI cleavage assay in both 1470.2 and 3017.1 cells. In
1470.2 cells Dex treatment induces a change in SacI cleavage
of 11.6%, which is nearly identical to that induced in 3017.1 cells,
as is shown graphically in Fig. 2B
. Because the GR is clearly able to
induce hypersensitivity in 3017.1 cells and the change in
SacI cleavage observed in the two cell lines is of the same
magnitude, we think it unlikely that MMTV chromatin in 3017.1 cells
exists in a constitutively remodeled state. As shown in Fig. 2A
, R5020
treatment also results in the induction of SacI cleavage in
3017.1 cells. The change in cleavage averages to 6% (Fig. 2B
).
Remarkably, the ratio of R5020- to Dex-induced cleavage is virtually
identical to the same ratio applied to induction of RNA as shown in
Fig. 1C
, 0.53 to 0.52, respectively. This strongly suggests that the GR
and PR activate transcription of the replicating MMTV template to the
extent to which they can remodel the chromatin.
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Activation of MMTV transcription by the GR is transient, peaking at
1 h posttreatment and declining to near basal levels by 812 h
even in the continued presence of hormone. This change in transcription
correlates with a reversion of the nucleoprotein structure to a
repressive state (52 53 ). This is a chromatin-specific effect because
transiently transfected MMTV reporter constructs, which do not undergo
hormone-induced remodeling, remain activated in the continued presence
of hormone (39 ). We asked whether PR, after it initially remodels the
chromatin and activates transcription, keeps the promoter active for an
extended period of time. Figure 3 shows
the levels of MMTV mRNA generated from the replicating template for
short-term and long-term hormone treatment of 3017.1 cells. It is clear
that both receptors activate the template strongly over the short term,
but after 24 h of hormone treatment, RNA levels have declined
significantly. Thus, the constitutively expressed PR appears to follow
the same transient kinetics of MMTV promoter activation as the
endogenous GR.
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Functional Comparison of Transiently and Constitutively Expressed
PR
Another possible model for the change in the ability of the PR to
activate the replicating MMTV template over time is based on
intracellular receptor status rather than template structure. The PR
may undergo a change in processing over time, which may have functional
outcomes. This might be reflected by functional differences in PR
activity at the transient MMTV template, which poses no barriers to
activation. Therefore, we measured various aspects of PR function on a
transiently transfected MMTV reporter construct in 3017.1 cells and
also in 1470.2 cells cotransfected with the PR expression vector,
pcPRO.
Typically, experiments done on cells transfected with various steroid
receptor expression vectors are carried out in the presence of
charcoal-stripped FBS since untreated serum contains levels of steroids
that might result in activation of the receptors. However, our
experience with 3017.1 cells and other PR-expressing lines
indicated that untreated serum does not activate the PR. We therefore
designed a set of experiments to systematically determine whether the
transiently and constitutively expressed forms of the PR respond
differently to serum. We transfected 1470.2 cells with pLTRluc, in the
presence or absence of the PR expression vector, and then cultured the
cells in medium containing charcoal-stripped serum or regular,
untreated serum. The 3017.1 cells were transfected with pLTRluc alone
and cultured in the same fashion. To ensure that equal levels of PR
were being expressed in 3017.1 and in transfected 1470.2 cells grown in
the two types of serum, we did a parallel experiment in which we
included the IL-2 receptor expression vector and isolated the
transfected 1470.2 cell population by magnetic affinity. Cytosols
isolated from 3017.1 cells and sorted 1470.2 cells were examined for PR
expression by immunoblotting. As shown in Fig. 4A, both PR isoforms, A and B, are
expressed at similar levels in transfected 1470.2 and in 3017.1 cells,
regardless of the type of serum in which they were cultured. Therefore,
any observed differences in PR activity are not due to altered PR
expression levels.
Figure 4B shows the results of analysis in 1470.2 cells transfected
with pLTRluc in the presence or absence of the PR expression vector.
The data are expressed as fold inductions relative to the untreated
control grown in charcoal-stripped serum. In the absence of pcPRO,
there was a relatively strong induction of luciferase activity by Dex
treatment and, as expected, no induction by R5020. Growth of the
transfected cells in untreated serum slightly boosted the level of
luciferase activity generated under all conditions. In the presence of
the PR expression vector, both Dex and R5020 treatment resulted in the
induction of luciferase levels when the cells were grown in
charcoal-stripped serum. However, in untreated serum the basal level of
luciferase activity was elevated 5-fold relative to basal levels in
stripped serum. Treatment with Dex or R5020 resulted in greater amounts
of transcriptional activity but the inductions were small, around
2-fold. These observations indicate that growth of the transfected
cells in untreated serum leads to the partial activation of the
transfected PR without the addition of ligand. In contrast, neither the
basal level of luciferase activity nor the hormone inductions were
affected by growth of transfected 3017.1 cells in charcoal-stripped
vs. untreated serum (Fig. 4C
). Unlike the transiently
expressed PR, the constitutively expressed PR is refractory to
activation by serum.
There are two possible explanations for serum-induced activation of the transiently expressed PR. Either a signal transduction pathway is induced selectively by the untreated serum that leads to ligand-independent activation, or the concentration of progestins in the untreated serum is sufficient to cause partial activation of the PR. This latter possibility seems unlikely given that the constitutively expressed PR in 3017.1 cells was not activated at all by untreated serum. However, it has been shown recently that increasing amounts of transiently expressed GR can cause a left shift in the dose-response curve for activation of a glucocorticoid-inducible promoter (54 ), which indicates an increasing sensitivity to ligand.
Thus, we carried out an R5020 dose-response experiment on 1470.2 cells
transfected with pcPRO and pLTRluc and also on 3017.1 cells transfected
with the latter. Transfected cells were grown in charcoal-stripped
serum to ensure maximal hormone inductions. As shown in Fig. 5A, the transiently expressed PR gave
rise to a significantly left-shifted dose-response curve when
compared with that generated by the constitutively expressed PR. In the
case of the latter, the dose response could be considered typical for
the PR, the EC50 (0.89 nM) being
similar to the Kd (0.58 nM, see Table 1
). For the transiently expressed PR, this was not the case. Its
EC50 (0.056 nM) was more than 1 order
of magnitude lower than its measured Kd of 0.4
nM (data not shown), which is in agreement with
observations made by others (48 55 ) (see Discussion).
Levels of PR were analyzed by Western blotting of whole-cell extracts
made after 0, 3, and 6 h of R5020 treatment (Fig. 5B
). Since
receptor levels were comparable between the two cell lines at all time
points tested, this cannot be the reason underlying the differences in
EC50. Therefore, the two forms of the PR have the
same affinity for R5020, but differ significantly in their sensitivity
to this ligand.
Based on the progesterone content of the untreated serum provided
by the manufacturer, we estimate that the progesterone concentration in
our complete media is approximately 0.020.03 nM.
According to the dose-response curves in Fig. 5, this amount of
progesterone may result in a 2- to 3-fold activation of the transfected
MMTV template by the transiently expressed PR but would not be
sufficient to activate the constitutively expressed receptor.
Transiently expressed PR has been shown to be partially activated by
EGF treatment (4 ). We do not know the epidermal growth factor (EGF)
content of our serums but serum mitogens are undoubtedly removed by
charcoal-dextran treatment since most of our cell lines grow at a
reduced rate in the stripped serum (C. L. Smith, unpublished
observations). It is possible that the PR-dependent, 5-fold induction
of the transfected MMTV promoter in untreated serum is due to a
combination of endogenous progesterone and higher levels of serum
mitogens.
In addition to EGF-induced signaling, it has been reported that the
chicken PR is activated very well by cAMP signaling in a
ligand-independent fashion (3 4 12 ). However, in each of these
studies, the PR had been transiently expressed. We therefore tested PR
activity in the presence of cAMP in our highly related cell lines. In
transfected 1470.2 cells treatment with 8-Br-cAMP caused a PR-dependent
induction of luciferase activity (Fig. 6A), which was, on average, greater than
that generated by either Dex or R5020 alone, an observation also made
in other studies. Once again, the behavior of the constitutively
expressed PR was different. There was very little induction of
luciferase activity by 8-Br-cAMP treatment in 3017.1 cells (Fig. 6B
).
The amount of induction observed is very similar to that induced by
cAMP treatment in 1470.2 cells in the absence of transiently expressed
PR. Analysis of PR expression levels in both cell lines indicates that
they are unchanged by treatment of cAMP (Fig. 6C
), which is in
agreement with other studies (12 56 ). These results led to the
conclusion that while the transiently expressed PR is easily activated
by cAMP signaling the constitutively expressed form of the PR is
refractory to this form of ligand-independent activation. Dopamine,
which can activate the cAMP signaling pathway, has also been shown to
activate PR in a ligand-independent fashion (7 ). Unfortunately, our
cell lines do not appear to express the D1 dopamine receptor because
they do not respond to dopamine at all (T. B. ONeill and C.
L. Smith, unpublished observations).
Localization and Nuclear Binding Properties of Transiently
and Constitutively Expressed PR
Taken together, our data imply that transiently expressed
and constitutively expressed PR respond differently to extracellular
signals, the latter being more refractory to activation than the
former. The dramatically enhanced sensitivity of the transiently
expressed PR to activation suggests that it has greater access to both
coactivators and its binding sites on the transfected MMTV template.
One possible explanation for this could be an altered partitioning
between the nucleus and cytoplasm relative to the constitutively
expressed PR. A predominantly nuclear localization in the absence of
ligand could result in greater accessibility to DNA and transcription
complexes once the receptor is activated. Therefore, we carried out
indirect immunofluorescence experiments on 3017.1 and transfected
1470.2 cells. Cells were either untreated or treated with 30
nM R5020 for 1 h before fixation. As shown in Fig. 7, the PR is predominantly localized to
the nucleus regardless of whether it is transiently or constitutively
expressed. In the absence of ligand there is a small fraction of
fluorescence in the cytoplasm that appears to move into the nucleus
upon treatment with ligand. These results are different from those
reported for the transiently expressed human PR(B) (57 ). Its
predominant localization pattern was a distribution between the nucleus
and cytoplasm. Since those experiments were carried out in a cell line
very similar to ours, the different localization pattern is probably
due to species-specific differences between the chicken and human
PR.
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DISCUSSION |
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We have shown previously that, when the PR is transiently expressed, it
is a poor activator of the MMTV promoter, having an ordered and
repressive chromatin structure (41 ). This deficiency in activation
results from the inability of the PR to induce the chromatin remodeling
event (42 ), which is an integral step in the transactivation of the
MMTV promoter by GR (43 ). This form of the PR is not generally
deficient in transcriptional activity, because it efficiently activates
a transiently transfected form of the MMTV promoter (42 ), which does
not have an ordered nucleoprotein structure (43 ). The action of the
transiently expressed PR may therefore be restricted to endogenous
target genes that are not in a repressed conformation and do not
require a remodeling event characterized by the formation of a
nuclease-hypersensitive site. Upon prolonged expression in our cell
lines, the PR undergoes a dramatic increase in its ability to activate
the stably replicating form of the MMTV promoter. Although the
constitutively expressed PR is present at a level slightly higher than
that of the endogenous GR, it is only about half as efficient as the GR
in activating transcription from this promoter. This difference in
transactivation potential is not observed at the transient MMTV
template, which is activated by the constitutively expressed PR to
approximately the same extent as by the endogenous GR (see Figs. 4B and 6B
). This observation implies that PR-induced activation is limited by
a feature of its interaction with the ordered chromatin, possibly
specific rate-limiting cofactors that help PR bind to and/or remodel
chromatin. In fact, the ability of the PR to induce the chromatin
remodeling event in the MMTV promoter region is tightly linked to its
transactivation potential, again being only half as effective as the
endogenous GR. Therefore, the PR activates the promoter to the extent
to which it can open the chromatin.
In this study we show that the PR does not appear to gain the ability to activate the MMTV promoter in ordered chromatin through major alterations in promoter structure. Such a change might be necessary if the PR activates the MMTV promoter by a different mechanism than the GR, as was implied by the studies of Archer and colleagues (44 45 ). In a human mammary adenocarcinoma cell line containing integrated MMTV templates and expressing endogenous PR, they found that the structure of the promoter was in a constitutively hypersensitive state and that NF1 binding was no longer hormone-dependent. In our PR-expressing cell line this is clearly not the case. The MMTV promoter does not exhibit constitutive hypersensitivity or NF1 binding; both events remain hormone dependent. This discrepancy may be due to cell type differences or to the fact that PR levels in 3017.1 cells are much lower than in the cells described above. When we examined various characteristics of GR induction of the stably replicating MMTV template, we did not find that the constitutively expressed PR behaved differently. It induced the same changes in nucleoprotein structure and demonstrated the same transient activation kinetics as the GR. Therefore, if constitutively expressed PR does cause changes in template structure, they must be more subtle and not necessarily related to a PR-specific mechanism of action.
Our previous work indicated that the difference in PR function between the transiently and constitutively expressed states occurred in the presence of ligand. Our current study extends those differences to the unliganded state, strongly indicating that the PR goes from one functional state to the other through intracellular processing rather than template remodeling. We demonstrate dramatic changes in PR function at a promoter which, unlike the stably replicating MMTV template, provides no barriers to activation. In the transiently expressed state, the PR can be activated in a ligand-independent fashion by cAMP signaling and possibly other signal transduction pathways induced by exposure to serum. It also has an altered sensitivity to its own ligand, becoming maximally active at doses at least 1 order of magnitude lower than would be expected based on its dissociation constant. In fact, these doses fall at the low end of the physiological range (62 ). Thus, in vivo, this form of the receptor would have the potential to be constitutively active. In sharp contrast, the constitutively expressed form of the PR is refractory to activation by ligand-independent mechanisms and has an appropriate sensitivity to progestins. In fact, the constitutively expressed PR functions in accordance with the classical model for steroid receptor action in that it is activated only in the presence of physiological doses of its specific ligand.
Our observations of altered sensitivity to ligand are supported by other studies on steroid receptors. Simons and colleagues (54 ) recently addressed this issue in a study in which they examined activity of GR transiently expressed in Hela cells. Although interpretation of their results is more complex because Hela cells also express GR endogenously, they observed leftward shifts in the dose-response curves with increasing amounts of GR expression vector transfected. Weigel and colleagues (48 55 ) have also observed a difference between the dissociation constant and EC50 of transfected chicken PR (48 55 ). The affinity of the transiently expressed PR for ligand is in the same range as that of the constitutively expressed PR, but its EC50 for transcriptional activation is about 10 times lower. This means that the transiently expressed PR is a much more effective transactivator in that far fewer occupied receptors (relative to the constitutively expressed state) are necessary for achieving the maximal transcriptional response.
One interpretation of this phenomenon would be that the transiently expressed PR has a greater affinity for coactivators than the constitutively expressed PR, perhaps by virtue of its access to those factors, since the two receptors are identical in amino acid sequence. Although the transiently and constitutively expressed forms of the PR are predominantly localized to the nucleus, their nuclear binding properties are significantly different. In the unoccupied state, a significant fraction of the transiently expressed receptor is tightly bound to the nucleus. Upon activation with ligand, there is little change in the partitioning between the cytosol and nuclear extracts. In contrast, the majority of constitutively expressed PR is not bound tightly to the nucleus and partitions with the cytosolic extract. Upon activation with ligand, there is a significant increase in the amount of both isoforms that become tightly bound to the nucleus. These results strongly suggest that the PR can be localized to two different nuclear compartments. The transiently expressed receptor, given the fact that it is tightly bound to the nucleus even in the unoccupied state, may be constitutively associated with target genes. This contention is supported by the observation that the nuclear binding properties of the transiently expressed PR in the absence of ligand closely resemble those of the constitutively expressed PR in the presence of ligand. The constitutively expressed receptor may be targeted to a different compartment of the nucleus where it is refractory to activation by other signaling pathways. It must then relocate to its target sites when activated by ligand. In contrast, the transiently expressed PR, if already associated with target genes in the absence of ligand, may be in an environment rich in transcriptional coactivators, prepoised for activation. This would provide an explanation for its enhanced sensitivity to ligand and its ability to be activated by other signaling pathways. In this sense the localization of the unoccupied PR in the nucleus may have important effects on its function.
In the absence of ligand, steroid receptors are thought to exist in complexes with various heat shock proteins and immunophilins even if they are localized to the nucleus (63 64 ). These complexes are thought to keep the receptor in a conformation able to bind ligand but unable to bind DNA or coactivators (65 ). The majority of studies done on PR processing have been carried out on in vitro assembled receptor complexes or on receptor complexes from tissues or cells in which the expression of the receptor is endogenous. It is possible that atypical receptor/chaperone complexes may be generated in a transfected cell that is suddenly flooded with a receptor it does not express. These atypical complexes may result in a receptor that is partially transformed and localized to accessible target genes in the nucleus. Association with hsp90 and p23 is necessary to confer efficient and stable hormone binding ability on GR (66 ) and PR (67 68 ). Since affinity for ligand is the same for both forms of the PR, these associations probably occur, but may be unstable or altered in the case of the transiently expressed PR, since it behaves like it is partially transformed. In addition to hsp90 and p23, mature receptor complexes also contain one of several large immunophilins, notably FKBP52, FKBP51/54, and Cyp40, which bind directly to hsp90 (65 ). The role of such proteins in these complexes has not been firmly established, but they are reported to be involved in stability of the untransformed receptor complex (69 70 ) and trafficking of the receptor between the cytoplasm and the nucleus (71 ). Thus, the immunophilins might play a role in receptor processing (folding, posttranslational modification, etc.) and localization.
Our study suggests that the processing of the PR can have important consequences for its ability to productively interact with target genes in repressed chromatin. Even though the transiently expressed PR is promiscuously activated by other signaling pathways and has increased sensitivity to ligand, it is a poor activator of the MMTV promoter in ordered, replicating chromatin. The complexes formed by the PR and its localization in the nucleus may be important for efficient interactions with chromatin remodeling machinery in vivo. In addition, achievement of a more repressed, unoccupied state allows the PR to ignore other intracellular signals and respond to the appropriate extracellular stimuli. Since the majority of studies on steroid receptor function are carried out with receptors that are transiently expressed, it is important to know whether regulation of these receptors is representative of that which would occur in vivo. Further studies will be directed at addressing this important issue.
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MATERIALS AND METHODS |
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Hormone Binding Analysis
GR and PR were extracted from either 3017.1 cells or sorted
1470.2 cells by the following method. Pelleted cells were resuspended
in HEDM buffer (10 mM HEPES, pH 7.4, 1 mM EDTA,
2 mM dithiothreitol, 10 mM sodium molybdate)
and lysed by Dounce homogenization (A pestle). Glycerol was then added
to the lysate to a final concentration of 10%. Cytosols were isolated
by subjecting the lysate to centrifugation at 100,000 x
g. Before use, they were maintained in liquid nitrogen. For
hormone binding analysis, cytosols were divided into aliquots
containing equal quantities of total protein (250300 µg) and
allowed to incubate for 90 min with various concentrations of either
[3H]promegestone (NEN Life Science Products, Boston, MA) for analysis of the PR or
[3H]triamcinolone acetonide (NEN Life Science Products) for analysis of the GR, in the presence or
absence of a 500-fold excess of the appropriate unlabeled ligand. Free
steroid was removed by exposure of the cytosols to dextran-coated
charcoal [3% Norit-A (ICN Biochemicals, Inc., Cleveland,
OH), 0.6% Dextran T-70 (Sigma, St. Louis, MO) in 10
mM HEPES, pH 7.3], and bound steroid was
assessed by liquid scintillation counting. Hormone binding data was
then analyzed by the method of Scatchard.
Analysis of RNA and Luciferase
Total RNA was isolated from 3017.1 cells or sorted 1470.2 cells
and subjected to S1 nuclease analysis as described previously (46 ).
Probes used were specific for detection of MMTV-CAT and ß-actin mRNA.
S1 digestion products were separated on 8% denaturing gels that were
dried and exposed to phosphorimaging screens. Quantitation was carried
out using Imagequant software (Molecular Dynamics, Inc.,
Sunnyvale, CA). For analysis of luciferase activity, transfected cells
were harvested by scraping, pelleted, and resuspended in 0.1
M potassium phosphate pH 7.8, 1 mM
dithiothreitol. Cells were lysed by three cycles of freezing and
thawing; cellular debris was removed by centrifugation. After protein
analysis, 510 µg extract protein were analyzed for luciferase
activity. Luciferase values were normalized to total protein in each
sample.
Analysis of Nucleoprotein Structure
Nuclei were isolated from 3017.1 and 1470.2 cells after hormone
treatment for 1 h as described previously (42 ). SacI
digestion of nuclei was carried out at a concentration of 5 U per µg
DNA for 15 min at 30 C in buffer containing 50 mM
NaCl, 50 mM Tris pH 8.0, 1
mM MgCl2, 1
mM ß-mercaptoethanol, and 2.5% glycerol.
Reactions were stopped by the addition of 5 volumes 10
mM Tris, pH 7.5, 10 mM
EDTA, 0.5% SDS, and 100 µg/ml proteinase K. DNA was purified and
subjected to digestion with DpnII. For exonuclease analysis,
nuclei were digested at 37 C for 15 min with HaeIII (1000
U/ml) in the presence or absence of exonuclease (100200 U/ml). An
antisense oligonucleotide containing MMTV sequences from +27 to +1 bp
was end-labeled with
-32P-ATP and
polynucleotide kinase and used in multiple rounds of linear
amplification with Taq polymerase (Stratagene,
La Jolla, CA) to detect specific digestion products, which were
subsequently separated on 8% denaturing gels. Quantitation of
radiolabeled digestion products was carried out using a Phosphorimager
and Imagequant software (Molecular Dynamics, Inc.).
Extract Preparation and Immunoblotting
For comparison of PR levels in transfected, sorted 1470.2 cells
vs. 3017.1 cells, whole-cell extracts were made as follows.
Cells were harvested, washed with PBS, and resuspended in HEGDM buffer
(10 mM HEPES, pH 7.4, 1 mM
EDTA, 2 mM dithiothreitol, 10% glycerol, and 10
mM sodium molybdate) containing 250
mM NaCl and 0.1% NP40. After a 5-min lysis
period on ice, cellular debris was removed by centrifugation at
12,000 x g for 5 min at 4 C. For comparison of PR
partitioning, cytosols and nuclear extracts were made as follows.
Harvested cells were resuspended in HEDM buffer (10
mM HEPES, pH 7.4, 1 mM
EDTA, 2 mM dithiothreitol, 10
mM sodium molybdate) and lysed by Dounce
homogenization (A pestle). Glycerol was then added to the lysate to a
final concentration of 10%. Nuclei were pelleted by low-speed
centrifugation for 5 min and the supernatant (cytosol) was removed. The
nuclei were gently resuspended in 510 volumes in HEGDM buffer and
centrifuged again. The supernatants were combined and subjected to
centrifugation at 100,000 x g for 1 h to yield
the cytosolic fraction. The nuclei were resuspended again in HEGDM
buffer to which NaCl was added to a final concentration of 250
mM. After incubation on ice for 30 min, the
samples were subjected to centrifugation at 30,000 x g
for 20 min to generate nuclear extracts. All buffers used in making
extracts from R5020-treated cells contained 30 nM
R5020.
Extracted proteins (2040 µg) were subjected to SDS-PAGE (3% stack, 7.5% separating) and transferred to nitrocellulose membranes (ECL, Amersham Pharmacia Biotech, Arlington Heights, IL) in Tris glycine buffer containing 20% methanol. Before immunoblotting, membranes were blocked in Tris-buffered saline (TBS)/2% nonfat dry milk. The PR was detected using antibody PR22 (kindly provided by D. Toft) as described previously (42 ). Detection of bound antibody was carried out using chemiluminescence kits (Amersham Pharmacia Biotech or Pierce Chemical Co., Rockford, IL).
Indirect Immunofluorescence.
Cells were plated in 35-mm wells containing glass coverslips.
After the appropriate treatments, cells were fixed with 3.5%
paraformaldehyde in PBS (without Ca+2 and
Mg+2) and permeabilized with PBS (without
Ca+2 and Mg+2) containing
0.5% Triton X-100. Coverslips were incubated with primary antibody
(0.2 µg/ml) diluted in PBS/0.1% Tween-20/10% calf serum overnight
at 4 C. After several washes in PBS/0.1% Tween-20/10% calf serum,
coverslips were exposed to secondary antibody (1:500) for 12 h at
room temperature. After several washes in PBS/0.1% Tween-20/10% calf
serum, cells were rinsed in dH2O and mounted on
slides. Images were collected using a TCS SP confocal microscope
(Leica Corp., Deerfield, IL). The primary antibody was
directed against the both isoforms of the PR (aPR-22, Affinity BioReagents, Inc., Golden, CO). The secondary antibody was
Texas Red-conjugated goat antimouse (Calbiochem, La Jolla,
CA).
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Congress: 37th SEMDSA (Society for Endocrinology, Metabolism and Diabetes of South Africa) and 10th Bone & Mineral Metabolism |
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Venue: Holiday Inn Crowne Plaza, Sandton
Contact: Shelley Harris, SEMDSA (Congress Organiser) PO BOX 783155 SANDTON 2146 SOUTH AFRICA email: rsh@novo.dk phone: +27 11 8070794 fax: +27 11 8077989
Registration Fees: US$300.000 if before January 24, 2001 US$400.00 if after January 24, 2001
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication November 16, 1999. Revision received February 16, 2000. Accepted for publication March 16, 2000.
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REFERENCES |
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