Synergistic Activation of the Serotonin-1A Receptor by Nuclear Factor-
B and Estrogen
Sacha Wissink,
Bart van der Burg,
Benita S. Katzenellenbogen and
Paul T. van der Saag
Hubrecht Laboratory (S.W., B.v.d.B., P.T.v.d.S.) Netherlands
Institute for Developmental Biology 3584 CT Utrecht, The
Netherlands Departments of Molecular and Integrative
Physiology (B.S.K.) University of Illinois Urbana, Illinois
61801-3704
 |
ABSTRACT
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Estrogen exerts profound effects on mood and
mental state. The ability of estrogen to modulate serotonergic function
raises the possibility that it may play a role in the mechanism
associated with depression and its treatment. A cellular mechanism for
estrogen to influence mood might be through the regulation of genes
involved at various levels of the serotonin system. Here we report that
estrogen can up-regulate the expression of the serotonin-1A receptor
via a new mechanism involving synergistic activation by nuclear
factor-
B (NF-
B) with estrogen receptor
. Interestingly, we
observed that only estrogen receptor-
, and not -ß, was able to
mediate this effect of estrogens. The partial antiestrogen,
4-hydroxytamoxifen, had the same effect as estrogen. In addition,
mutation analysis showed that both the transactivation function of p65
and activation function 1 of estrogen receptor-
were essential for
this synergistic regulation. Therefore, we propose that NF-
B
complexes cooperate with estrogen receptor-
to recruit cofactors
into the complex and thereby synergistically activate the serotonin-1A
receptor promoter through nonclassical estrogen response elements by a
mechanism that does not involve direct receptor binding to DNA.
 |
INTRODUCTION
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Estrogen and other gonadal steroids have profound effects on the
central nervous system (1). Specifically, the ability of estrogen to
modulate the brain serotonin system suggests that estrogens may play a
role in the mechanism associated with depression and its treatment (2, 3). Serotonin (5-hydroxytryptamine, 5-HT) is involved in the control of
a variety of behavioral processes (4). Dysregulation of the serotonin
system is thought to play an important role in neuropsychiatric
disorders, such as depression and anxiety (5, 6). The complex action of
serotonin is mediated by a large family of related receptors (7).
Particular attention has focused on the 5-HT1A receptor, which is a G
protein-coupled receptor that negatively regulates adenylate cyclase
(8). The 5-HT1A receptor is expressed in a restricted pattern in the
brain, and high levels of receptors were observed in the limbic areas,
cerebral cortex, and raphe nuclei of the brain (9, 10, 11). Studies in rats
have shown that ovariectomy caused decreases in 5-HT binding and 5-HT
transporter binding sites and that estrogen replacement reversed this
decline (12, 13, 14), suggesting possible estrogen regulation of serotonin
receptor expression.
The effects of estrogen are now known to be mediated by two estrogen
receptors (ER
and ß), that belong to the superfamily of nuclear
hormone receptors (15, 16, 17, 18). The two ERs share a well conserved modular
structure. While the DNA-binding domain is highly conserved between
ER
and ß (96% identity) and the ligand-binding domain is
relatively well conserved (58% identity), the A/B region is poorly
conserved between the two receptors (20% identity). Upon ligand
binding, the activated receptor dimerizes and interacts with specific
DNA sequences, termed estrogen response elements (EREs), located in the
regulatory region of target genes. The DNA-bound receptor can then
regulate transcription either positively or negatively. It is known for
ER
that the regulation of transcription is mediated by two
transactivation regions: AF-1 located in the A/B domain and AF-2
located in the ligand-binding domain. The two transactivation regions
may function independently or cooperate, depending on cell and promoter
context (19, 20). Several other mechanisms have been discovered
recently by which estrogen regulates target genes. These include genes
that utilize nonclassical EREs as the target sequence of ER action (21)
or genes that are regulated by ER through interaction with other
transcription factors bound to their respective DNA-binding sites, such
as AP-1, Sp 1, and nuclear factor-
B (NF-
B) (22, 23, 24).
To explore the molecular mechanism by which estrogen modulates the
serotonin system, we have investigated the effect of estrogen on the
5-HT1A receptor gene. In the present study, we show that ER
acts
synergistically with NF-
B to activate the 5-HT1A receptor promoter.
This activation already occurred in the absence of hormone and could be
further induced by the addition of either 17ß-estradiol
(E2) or 4- hydroxytamoxifen (OH-T), but not
by ICI 164384 (ICI). In contrast to ER
, ERß was hardly able to
mediate this effect, suggesting different roles in gene regulation for
the two receptors. We also found that this synergistic activation was
dependent both on the transactivation domains of the p65 subunit of
NF-
B and the A/B domain of ER
, containing AF-1. Our findings show
that estrogens may regulate the expression of the 5-HT1A receptor via a
new mechanism involving synergistic activation by NF-
B with
ER
.
 |
RESULTS
|
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Synergistic Activation of the 5-HT1A Receptor Promoter by NF-
B
and ER
To determine the effect of estrogens on 5-HT1A receptor promoter
activity, we transiently transfected COS-1 cells with a reporter
construct containing the 5-HT1A receptor promoter together with an
expression vector encoding ER
or ERß. As shown in Fig. 1A
, cotransfection of ER
or ERß and
treatment of the cells with E2 had a minimal
effect on 5-HT1A promoter activity. However, in addition to
direct regulation, ER target genes can also be regulated indirectly
through interaction of ER with other transcription factors. Putative
NF-
B binding sites were shown to be present in the -901luc 5-HT1A
receptor promoter construct (see Fig. 3A
), and transfection of this
reporter construct with expression vectors encoding the p50 and p65
subunit of NF-
B resulted in an 10-fold induction of the reporter.
Interestingly, cotransfection of ER
in combination with NF-
B now
resulted in a very strong induction of promoter activity, which could
be further increased by the addition of E2 (Fig. 1A
). In contrast to ER
, ERß showed only minimal induction when
cotransfected with NF-
B, and no effect of E2
could be observed (Fig. 1B
). Similar results were obtained in 293 cells
(results not shown), although the level of activation by ER
was less
high compared with COS-1 cells. These results indicate that the 5-HT1A
receptor promoter can be synergistically activated by NF-
B and
ER
.
In the past, several groups have reported an inhibitory effect of
estrogens on NF-
B activity (33, 34, 35). Therefore, we also studied the
effect of estrogen on a reporter construct containing four NF-
B
elements from the human immunodeficiency virus-long terminal repeat
(HIV-LTR) in front of the thymidine kinase promoter coupled to
luciferase in combination with expression constructs encoding ER
or
ERß and the p50 and p65 subunits of NF-
B. On this reporter
construct, cotransfection of ER
resulted in repression of the
transcriptional activity of NF-
B already in the absence of hormone,
while addition of hormone resulted in a further repression (Fig. 2B
). Cotransfection of ERß also showed
some repression of NF-
B activity. Similar results were obtained in
293 cells (results not shown). These results indicate that while ER
acts as a transcriptional repressor of NF-
B on an artificial NF-
B
reporter construct, ER
acts as a transcriptional activator with
NF-
B on the 5-HT1A receptor promoter.
Involvement of NF-
B Elements in 5-HT1A Receptor Promoter
Regulation by NF-
B and ER
To localize the effect of ER
on the 5-HT1A receptor promoter,
several promoter deletion constructs were used (Fig. 3A
). Mutation of both NF-
B elements
(-901 365/64Mluc) completely abolished the effect of NF-
B on the
5-HT1A receptor promoter (Fig. 3B
). However, ER
, only in combination
with NF-
B, was still able to induce promoter activity as efficient
as on the wild-type promoter (-901luc). Likewise, the promoter
construct -81luc could not be induced by NF-
B, although it still
contained one NF-
B element. However, also on this promoter
construct, the effect of ER
with NF-
B was maintained. When the
single NF-
B element present in the -81luc construct was mutated
(-81 64Mluc), the ER
effect was almost completely abolished (Fig. 3C
). Thus, synergistic activation of the 5-HT1A receptor promoter
involves NF-
B binding sites, although activation of the promoter by
NF-
B itself appears not to be required for the effect of ER
.
These results suggest that this synergistic promoter activation by
ER
is independent of DNA binding and involves protein-protein
interactions.
Effects of Antiestrogens on the 5-HT1A Receptor Promoter
Antiestrogens have been described to have differential effects
depending on promoter context and receptor subtype. In transactivation
experiments, tamoxifen inhibited transcription of genes regulated by a
classical ERE, while, like E2, it activated
transcription of genes that are under the control of an AP-1 element
with ER
(22). Moreover, only antiestrogens were transcriptional
activators with ERß at an AP-1 site (36). We examined the effect of
antiestrogens on the 5-HT1A receptor promoter using the partial
antagonist OH-T, which blocks AF-2, and the pure antagonist ICI, which
blocks AF-1 and AF-2. As shown in Fig. 4
, ICI treatment did not enhance the activity of the 5-HT1A receptor
promoter by ER
and NF-
B, while OH-T was as potent as
E2 in transcriptional activation. These data
indicate that the partial antagonist OH-T, still able to activate AF-1,
is as potent as E2 in synergistic activation of
the 5-HT1A receptor promoter by ER
.
Domains of NF-
B and ER
Involved in the Synergistic Activation
of the 5-HT1A Receptor Promoter
To determine the importance of the transactivation function of
NF-
B, we examined the effect of deleting the transactivation
domains, or impairing the DNA-binding function of the p65 subunit of
NF-
B, on its ability to synergistically activate the 5-HT1A receptor
promoter with ER
in a transient transfection assay. While
cotransfection of p50 and p65 or p65 alone strongly activated the
promoter in combination with ER
and E2,
cotransfection of p50 alone, which has no transactivation function, had
almost no effect (Fig. 5
).
Deletion of the transactivation domains of p65 resulted in a construct
containing only the Rel homology domain (p65RHD). P65RHD was still able
to bind to DNA (27), but was unable to activate the promoter both in
the absence or presence of ER
and E2. The
DNA-binding defective mutant (p65Nsi) still contained intact
transactivation domains, but was also unable to synergistically
activate the promoter. Taken together, these data show that both the
transactivation function as well as the DNA binding function of p65 are
essential for synergistic activation of the 5-HT1A receptor promoter by
ER
.
To identify the regions of ER
involved in activation of the 5-HT1A
receptor promoter, deletion constructs of mouse ER
that lack part of
the A/B region containing AF-1, or that lack part of the ligand binding
region containing AF-2, were used. While deletion of the A/B region
(ER
121599) inhibited the synergistic activation of the promoter,
deletion of the ligand-binding domain (ER
1339) resulted in a
receptor that was at least as active as wild-type ER
(Fig. 6A
). The DNA-binding defective mutant of
ER
(ER
C241/244A) was unable to activate the 5-HT1A receptor
promoter, possibly because a functional DNA-binding domain is needed
for interaction with NF-
B (34). Note that in contrast to human
ER
, no ligand dependency can be observed for mouse ER
in
synergistic activation of the 5-HT1A receptor promoter. This
synergistic activation of the promoter by ER
could be observed only
in combination with NF-
B, although in the absence of NF-
B a small
activation of the promoter could be found with ER
1339 (results
not shown). In a control experiment, ER
, ERß, and the deletion
mutants were cotransfected with a reporter construct containing three
copies of a consensus ERE and a TATA box coupled to luciferase to
determine their ability to activate transcription from a classical ERE.
As shown in Fig. 6B
, both ER
and ERß stimulate transcription from
3xERE-TATA-luc, although the transcriptional activity of ERß was
significantly less than that of ER
, a phenomenon that has been
described previously (28). Both deletion mutants, lacking either AF-1
or AF-2, stimulated transcription although much less efficiently than
wild-type ER
, indicating that the transactivation domains are able
to synergize on this promoter construct. Furthermore, it was shown that
ER
1339 was already maximally activated in the absence of ligand,
clearly demonstrating the ligand-independent activity of AF-1. As
expected, the DNA-binding defective mutant, ER
C241/244A, was unable
to activate this reporter construct.

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Figure 6. The Synergistic Activation of the 5-HT1A Receptor
Promoter by Mouse ER Occurs in an AF-2-Independent Fashion
A, COS-1 cells were transiently transfected with the -901luc reporter
construct together with empty expression vector or expression vectors
encoding the p50 and p65 subunits of NF- B in combination with
expression vectors encoding mouse ER , ERß, ER 121599, ER
1339, or ER C241/244A. Cells were either untreated (hatched
bars) or treated with 10-8
M E2 (black bars) for
24 h. Depicted is the induction of luciferase activity evoked by
NF- B over cells transfected with empty expression vector.
Bars represent the mean of at least three independent
experiments ± SD. B, COS-1 cells were
transiently transfected with 3xERE-TATAluc in combination with empty
expression vector or expression vectors encoding mouse ER , ERß,
ER 121599, ER 1339, or ER C241/244A. Cells were either
untreated (hatched bars) or treated with
10-8 M E2
(black bars) for 24 h. Depicted is the induction of
luciferase activity evoked by ER over cells transfected with empty
expression vector. Bars represent the mean of at least
three independent experiments ± SD.
|
|
Since ER
and not ERß was able to synergistically activate the
5-HT1A receptor promoter with NF-
B, chimeric constructs with ER
and ERß were used to further determine the region of ER
involved
in this activation. Replacement of the A/B region of ERß with the A/B
region of ER
(ER
/ß) resulted in a chimeric receptor that was
even more potent than wild-type ER
in activation of the 5-HT1A
receptor promoter (Fig. 7A
). However,
replacement of the A/B region of ER
with the A/B region of ERß
(ERß/
) totally abolished the ability of the receptor to
synergistically activate the promoter. Again this synergistic
activation of the promoter by ER
and ER
/ß could only be
observed in combination with NF-
B, although in the absence of
NF-
B a small ligand-independent activation of the promoter could be
seen with ER
/ß (results not shown). In a control experiment, both
chimeric constructs were able to activate transcription from
3xERE-TATA-luc as efficiently as wild-type ER
, while some
ligand-independent activity could be observed only with ER
/ß (Fig. 7B
). These results suggest that the synergistic activation of the
5-HT1A receptor promoter by ER
and NF-
B is dependent on the DNA
binding domain and the A/B region of ER
containing AF-1.
 |
DISCUSSION
|
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In the present study, we show that estrogen may regulate the
5-HT1A receptor promoter via a new mechanism involving synergistic
activation of NF-
B with ER
. The cis-regulatory region
of the 5-HT1A receptor contains two putative NF-
B binding sites, and
the presence of NF-
B proteins is critical for synergistic induction
by ER
. This suggests that NF-
B complexes cooperate with ER
to
synergistically regulate 5-HT1A receptor gene expression.
In most systems that have been examined the estrogen and NF-
B
signaling pathways are mutually antagonistic. For instance, regulation
of the IL-6 promoter has been extensively studied, and NF-
B-
induced activation of this gene could clearly be inhibited by
estrogen (33, 34). Consistent with these findings, we have shown that
on an artificial NF-
B reporter construct, estrogen inhibits NF-
B
activity. However, on the 5-HT1A receptor promoter, estrogen further
enhanced NF-
B-induced activity, indicating that positive or negative
regulation by estrogen is dependent on the promoter context. Similar
results have been described for both negative and positive regulation
of AP-1-dependent promoters by estrogens (36) and glucocorticoids (37, 38).
Based on several different approaches, synergistic activation of the
5-HT1A receptor promoter by NF-
B and ER
was found to be dependent
on the N-terminal region of ER
, containing AF-1. First, in contrast
to ER
, ERß was unable to mediate this synergistic effect. The two
receptors show a high degree of homology in the DNA-binding domain and
moderate homology in the ligand-binding domain; however, the A/B region
is poorly conserved between the two receptors. This already suggested
the importance of the A/B region of ER
in the synergistic
activation. Second, synergistic activation by ER
1339, an AF-2
defective mutant, was comparable to wild-type ER
, while ER
121599, an AF-1-defective mutant, was unable to mediate this effect,
comparable to wild-type ERß. Third, replacement of the A/B region of
ERß with the A/B region of ER
(ER
/ß) resulted in a chimeric
receptor that was even more potent than wild-type ER
in activation
of the 5-HT1A receptor promoter. However, replacement of the A/B region
of ER
with the A/B region of ERß (ERß/
) totally abolished the
ability of the receptor to synergistically activate the promoter.
Fourth, the partial antiestrogen OH-T, which blocks only AF-2, was as
potent as E2 in activation of the 5-HT1A receptor
promoter. The AF-1-mediated agonistic effect of antiestrogens has
recently been reported to be mediated via the A/B region of ER
but
not by the A/B region of ERß (28). These differences between ER
and ERß suggest different regulatory functions for the two ER
subtypes. Finally, the fact that the ER
effect is mostly
estrogen-independent also indicates the involvement of AF-1, which is
the hormone-independent activation function that resides in the N
terminus of ER
. Taken together, these data clearly show the
involvement of ER
AF-1 in 5-HT1A receptor promoter regulation.
Although the regulation by ER of AP-1-dependent promoters and the
NF-
B-dependent 5-HT1A receptor promoter shares several features,
clear differences are also present. Estrogen-induced transcription from
an AP-1-dependent promoter requires both ER
and AP-1 transcription
factors (22). Similarly, estrogen- induced transcription from an
NF-
B-dependent promoter requires ER
and NF-
B. Both pathways
appear to require the amino terminus of ER
, containing AF-1. While
both tamoxifen and ICI activate transcription via AP-1 sites, only
tamoxifen induces synergistic activation of the 5-HT1A receptor
promoter with NF-
B. This lack of activity of ICI and its apparent
capacity to decrease the ability of ER
to activate the 5-HT1A
receptor promoter could be explained by differences in receptor
conformation, but may also be due to enhanced receptor turnover (39, 40). Furthermore, while with ERß, E2 inhibited
AP-1-dependent transcription, antiestrogens stimulated AP-1-dependent
transcription (36). In contrast to this, E2 was
not a very potent activator of the 5-HT1A receptor promoter with ERß.
These findings highlight the unique pharmacology of estrogen
receptor-regulated transcription at different gene sites.
Our findings clearly show a crucial role for NF-
B complexes and
NF-
B binding sites in the synergistic activation of the 5-HT1A
receptor promoter by ER
. However, mutation of the two NF-
B
elements in the -901luc construct abolished the NF-
B effect while
the ER
effect was maintained. One explanation could be that NF-
B
proteins bind as monomers to these mutated
B elements. This
NF-
B-DNA complex, unable to activate transcription in this
conformation, might be stabilized by ER
and consequently result in
activation. Furthermore, it is evident from the use of ER
mutants
and chimeras that, although a functional DNA binding domain of ER
is
required, there is no clear correlation between ER
activity on the
ERE reporter and the 5-HT1A receptor promoter. Therefore, the most
likely explanation for the synergistic activation of the 5-HT1A
receptor promoter is that ER
activates this promoter not via direct
binding to DNA but via protein-protein interactions. This model is
supported by the fact that both the DNA-binding domain of ER
and an
intact RHD of p65 are required for the synergistic activation, since
ER
has been described to directly interact with p65 involving the
DNA-binding domain of ER
and the RHD of p65 (34). In addition, ER
could also interact with other transcription factors present in COS-1
cells or with other components of the transcription machinery involved
in promoter regulation. Furthermore, the fact that both the
transactivation domains of p65 and AF-1 of ER
are essential for this
response clearly indicates the involvement of cofactors. Therefore, we
propose that NF-
B complexes cooperate with ER
to recruit
coactivators into the complex via AF-1 and thereby synergistically
activate the 5-HT1A receptor promoter. An alternative explanation could
be that the 5-HT1A receptor promoter contains a cryptic site that
directly binds ER
but requires functional cooperation with NF-
B,
bound to nearby DNA binding sites. Since our data cannot rule out
direct binding of ER
to this promoter, it could be possible that the
5-HT1A receptor promoter contains a composite element that
simultaneously binds ER
and NF-
B.
In addition to the classical hormone activation pathway, other signal
transduction pathways have been described to regulate a number of
steroid receptors, including ER
, independently of hormonal ligands.
Nuclear receptors have been shown to be activated by nonsteroidal
agents, such as dopamine, growth factors, and PKA activators, via
phosphorylation (41). Phosphorylation of ER
was shown to enhance
receptor activity and major phosphorylation sites are located in the
A/B region of the receptor (42, 43). Recently it was demonstrated that
phosphorylation of ERß AF-1 regulates cofactor recruitment and gene
activation by nonsteroidal activators (44), whereas phosphorylation of
the A/B domain of peroxisome proliferator-activated receptor
decreased its transcriptional activity (45). The presence of several
kinase sites within the A/B region of ER
and ERß suggests that
differential phosphorylation of the AF-1 domain may result in diverse
responses of the receptors by different activators. The existence of
this additional pathway emphasizes the importance of AF-1 in
hormone-independent receptor activation.
These studies were all performed in nonneuronal cells, and it would be
interesting to determine whether the same effects can be observed in a
serotonergic neuronal environment. Additional neuronal-specific
transcription factors might regulate the 5-HT1A receptor promoter in
the same way or different from nonneuronal cells. However, several
lines of evidence suggest that estrogen also regulates 5-HT1A receptor
expression in the central nervous system (CNS). For instance, the
decline in estrogen before parturition and at the onset of menopause
has been correlated with negative affect (46), while estrogen
replacement therapy can, in some cases, alleviate depression or anxiety
in women (47, 48). Moreover, ovariectomy caused decreases in 5-HT
binding and 5-HT transporter binding sites (12, 13, 14), while replacement
of estrogen to ovariectomized rats reversed this decline. Both ER
and ERß have been identified in multiple regions of the brain,
including the cortex, hippocampus, and raphe nuclei (49). In addition,
NF-
B has also been described to be active in the brain, particularly
in the cortex and hippocampus (50). At the same time evidence is
emerging that NF-
B not only functions in immune cells, but also has
unique roles in processes such as neuronal plasticity,
neurodegeneration, and neuronal development (51). Thus, these
transcription factors and pathways may play an important role in
regulation of 5-HT1A receptor gene expression in the brain.
Furthermore, in addition to the direct mechanism described above,
estrogen may also have an indirect effect in the CNS. Estrogen may
induce the formation of an intermediate protein which might be able to
further induce 5-HT1A receptor expression. In conclusion, the ability
of estrogen to modulate serotonergic receptor function may underlie, at
least in part, the profound effects of this hormone on mood and mental
state.
 |
MATERIALS AND METHODS
|
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Special Reagents
E2 was obtained from Sigma
(St Louis, MO). 4-Hydroxytamoxifen and ICI 164384 were kind gifts from
Dr. A. Wakeling (Zeneca Pharmaceuticals,
Macclesfield, UK).
Cell Culture
Monkey COS-1 cells and human 293 embryonal kidney cells were
obtained from American Type Culture Collection
(ATCC; Manassas, VA) and were cultured in a 1:1 mixture of
DMEM and Hams F-12 medium (DF; Life Technologies, Inc.,
Gaithersburg, MD), buffered with bicarbonate and supplemented with
7.5% FCS. Dextran-coated charcoal (DCC)-FCS was prepared by treatment
of FCS with DCC to remove steroids, as described previously (25).
Plasmids
-901luc was created by partial digestion of -1,588luc (a kind
gift from Dr. O. Meijer, Leiden, The Netherlands), with
StyI, filling-in and ligation into pGL3 digested with
SmaI, redigestion with HindIII, and religation;
-81luc was created by digestion of -1,588luc with StyI,
filling-in and digestion with BglII, and ligation into pGL3
digested with SmaI/BglII; -901 365Mluc and -901
64Mluc were constructed by introducing point mutations into the
original promoter constructs by site-directed mutagenesis using the
oligonucleotides
5'-gagccgaattctacagactaa-3' and
5'-aactgcaaggagatctacatcgcccctcg-3',
respectively. -901 365/64Mluc was created by digestion of -901 64Mluc
with SacII/HindIII and ligation into -901
365Mluc digested with SacII/HindIII; -81 64Mluc
was made by partial digestion of -901 64Mluc with StyI and
religation. The CMV4 expression vectors containing full-length cDNAs
encoding human p65 (RelA), p50 (NF-
B1) and p65RHD (1305), and
p65Nsi (1551E39I) have been described previously (26, 27). The
expression vectors encoding human ER
(pSG5-HEGO) and human ERß
(pSG5- ERß530) were kind gifts of Dr. Chambon (Strasbourg, France)
and Dr. Gustafsson (Stockholm, Sweden), respectively. Chimeric human
ER
/ERß and ERß/ER
receptors were described previously (28)
and contained the A/B domain of ER
and domain C, D, E, and F of
ERß in the ER
/ERß chimera and the A/B domain of ERß and domain
C, D, E, and F of ER
in the ERß/ER
chimera. Mouse ER
(pMT2MOR), ER
1339, ER
121599, and ER
C241/244A (29) were
kindly provided by Dr. Parker (London, UK). The reporter plasmids used,
4xNF-
B(HIV)tkluc and 3xERE-TATA-luc, have been described previously
(30, 31).
Transient Transfections
For transient transfections, COS-1 cells and 293 cells were
cultured in 24-well plates in phenol red-free DF supplemented with 5%
DCC-FCS. Cells were transfected using calcium-phosphate coprecipitation
with 0.4 µg of luciferase reporter, 0.6 µg of PDMlacZ, and 0.2 µg
of the indicated expression plasmids. pBluescript
SK- was added to obtain a total amount of 1.8
µg of DNA/well. After 16 h, the medium was refreshed and when
indicated hormone was added. Cells were harvested 24 h later and
assayed for luciferase activity using the Luclite luciferase reporter
gene assay kit (Packard Instruments, Meriden, CT) according to the
manufacturers protocol and the Topcount liquid scintillation counter
(Packard Instruments). Values were corrected for transfection
efficiency by measuring ß-galactosidase activity (32).
 |
ACKNOWLEDGMENTS
|
---|
We thank Drs. M. Parker, P. Chambon, and J.-Å Gustafsson for ER
cDNAs. We thank Dr. A. Wakeling for providing us with
4-hydroxytamoxifen and ICI 164384. We thank J. Heinen and F.
Vervoordeldonk for photographic reproductions.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Paul T. van der Saag, Hubrecht Laboratory, Netherlands Institute for Developmental Biology, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands. E-mail:
paul{at}niob.knaw.nl
This work was supported by grants from the Netherlands Organization for
Scientific Research (STIGO project no. 01480-005) and NV
Organon, Oss, The Netherlands (to S.W.) and NIH Grant
CA-18119 (to B.S.K.).
Received for publication April 28, 2000.
Revision received December 4, 2000.
Accepted for publication January 10, 2001.
 |
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