(Received for publication, November 17, 1995; and in revised form, February 1, 1996)
From the
Nuclear orphan receptors belong to the superfamily of ligand-activated transcription factors that show a close structural relationship and sequence homology. Ligands and functions of most of the orphan receptors have not yet been identified. The first nuclear orphan receptors that were cloned displayed a high degree of amino acid identity with the human estrogen receptor and were termed estrogen receptor-related (ERR) 1 and 2. In the present study, we show that ERR2 functions as a potent repressor of transcriptional activity mediated by the glucocorticoid receptor (GR). Transient transfection of different cell lines with a steroid-responsive reporter plasmid and receptor expression plasmids revealed that transcriptional activity mediated by GR in response to agonists was strongly suppressed by coexpression of ERR2. The orphan receptor displayed no promoter activity when expressed without GR. The inhibitory activity of ERR2 is cell-specific and also receptor-specific because transactivation mediated by the progesterone receptor is unaffected by ERR2. Our observations provide evidence that the nuclear orphan receptor ERR2 acts as an endogenous modulator of GR transcriptional activity.
The nuclear receptor superfamily is composed of ligand-activated
transcription factors including receptors for glucocorticoids,
mineralocorticoids, gonadal steroids, retinoids, thyroid hormone, and
vitamin D. These receptors have a major influence on differentiation,
cell proliferation, development, and maintenance of homeostasis. The
action of nuclear receptors is mediated by binding of the
ligand-activated receptor to cis-acting sequences regulating the
expression of target genes in cooperation with proteins of the basic
transcriptional machinery or additional transcription
factors(1, 2, 3, 4) . Molecular cDNA
cloning and comparison of the amino acid sequences deduced have shown
that all members of the nuclear receptor superfamily are structured in
a similar way and organized into different domains. Moreover, they show
a particularly high degree of sequence homology, mainly in the
DNA-binding domain(1, 2, 3, 4) .
Based on structural similarities and sequence homology in comparison
with members of the nuclear receptor superfamily, a number of putative
receptors, designated nuclear orphan receptors, have been cloned, whose
functions and ligands have not yet been
identified(3, 4, 5) . Furthermore, the nature
of action of the orphan receptors that were first cloned and termed
estrogen receptor-related (ERR) ()1 and 2 remains unknown to
date(5) . Since interactions between members of the nuclear
receptor superfamily are not uncommon(4, 6) , we
investigated the functional consequence of coexpression of these orphan
receptors with the more traditional members of the nuclear receptor
superfamily with regard to transcriptional activity. Our findings
indicate that ERR2 acts as a cell-specific inhibitor of glucocorticoid
receptor (GR)-mediated gene expression.
CV-1 cells lacking endogenous GRs were transfected
transiently with MTV-LUC, a reporter plasmid encoding the mouse mammary
tumor virus promoter upstream of the luciferase gene (MTV-LUC)
and expression plasmids encoding the human GR and/or the human ERR2.
Stimulation of GR-transfected cells with agonists revealed a
remarkable, dose-dependent reporter gene expression (Fig. 1),
whereas the orphan receptor displayed no promoter activity (Fig. 1A). However, coexpression of ERR2 drastically
repressed transcriptional activity mediated by GR in response to a
physiological concentration of cortisol (Fig. 1A).
Remarkable inhibition of GR activity by ERR2 was also obtained when
different amounts of GR expression plasmid were transfected and the
GR/ERR2 ratio varied between 2 and 0.5. ()ERR2-mediated
repression of glucocorticoid-induced transcriptional activity was
obtained when transfected cells were stimulated with low to high
nanomolar concentrations of cortisol or dexamethasone. However, the
dose-response curve of GR-activated reporter gene expression in
response to increasing concentrations of endogenous or synthetic
glucocorticoids was not only suppressed but also right-shifted when
ERR2 was coexpressed (Fig. 1, B and C). The
concentration of cortisol and dexamethasone needed to obtain
half-maximal response was 21 and 2 nM, respectively, when GR
was expressed alone and 104 and 11 nM, respectively, when ERR2
was coexpressed. Antagonism toward glucocorticoid activity by ERR2 was
also obtained when SK-N-MC cells (Fig. 2) or COS-1 cells (Fig. 6A) were transfected, whereas transcriptional
activity of cotransfected as well as endogenous GR in HeLa cells was
nearly unaffected by ERR2 (Fig. 3). Therefore, the inhibitory
activity of the orphan receptor depends on the cell type. We next
investigated whether the orphan receptor is a general repressor of
nuclear receptor transcriptional activity. As shown in Fig. 4,
coexpression of ERR2 in CV-1 cells does not inhibit transcriptional
activity mediated by the human PR in response to progesterone. Similar
results were obtained when SK-N-MC cells were transfected.
These data indicate that repression of GR-activated reporter gene
expression is not mediated via an influence of ERR2 on the basic
transcriptional or translational machinery. It also makes clear that
the orphan receptor does not suppress transcriptional activity by
competing for functionally limiting factors generally needed for
steroid receptor activity(18) . To exclude the possibility that
repression of GR transcriptional activity is caused by a decrease in GR
expression, we tested whether coexpression of ERR2 has an effect on the
promoter that regulates GR expression. As shown in Fig. 5,
expression of pRSV-LUC is unaffected by ERR2, which indicates that the
Rous sarcoma virus promoter is not influenced by the orphan receptor.
Western blot analysis also confirmed that ERR2 has no effect on the
expression of cotransfected GR.
Investigation of DNA
binding of ERR2 expressed in COS-1 cells using an electrophoretic
mobility shift assay showed that the orphan receptor was not able to
bind to a labeled GRE under these conditions (Fig. 6B, lane 2), whereas the GR was able to bind (Fig. 6B, lane 3) as has been shown
previously(6, 19) . The specificity of this DNA
binding complex was confirmed by supershift with a polyclonal GR
antibody (Fig. 6B, lane 4). Next, we asked
whether the orphan receptor affects DNA binding of GR. As shown in Fig. 6B (lane 5), coexpression of GR and ERR2
in COS-1 cells does not influence binding of the GR to the GRE. These
data are also another proof that ERR2 has no influence on the
expression of GR and provide evidence that repression of GR activity by
ERR2 is not caused by a competition for response element binding.
Figure 1: Transactivation properties of GR, ERR2, and coexpressed GR and ERR2 in CV-1 cells. A, CV-1 cells were transfected with expression vector for either GR (2 µg) or ERR2 (4 µg), or 2 µg of GR expression plasmid was transfected in combination with 1, 2, or 4 µg of ERR2 expression plasmid. Transfected cells were incubated with 10 nM cortisol (gray bars) or with vehicle (black bars). B and C, CV-1 cells were transfected with either 2 µg of GR expression plasmid (circles) alone or with 2 µg of GR and 4 µg of ERR2 expression plasmid together (squares). After transfection, cells were incubated with cortisol (B) or dexamethasone (C) concentrations as indicated.
Figure 2: Transactivation properties of GR, ERR2, and coexpressed GR and ERR2 in SK-N-MC cells. SK-N-MC cells were transfected with expression vector for either GR (2 µg) or ERR2 (4 µg), or 2 µg of GR expression plasmid was transfected in combination with 1, 2, or 4 µg of ERR2 expression plasmid. Transfected cells were incubated with 10 nM cortisol (gray bars) or with vehicle (black bars).
Figure 6: Transactivation and DNA binding properties of GR, ERR2, and coexpressed GR and ERR2 in COS-1 cells. A, COS-1 cells were transfected with expression vector for either GR (5 µg) or ERR2 (5 µg), or 5 µg of GR expression plasmid was transfected in combination with 5 µg of ERR2 expression plasmid. Transfected cells were incubated with 10 nM cortisol (gray bars) or with vehicle (black bars). B, extracts of COS-1 cells transfected with pRSV-CAT either alone (lane 1) or together with expression plasmid for ERR2 (lanes 2 and 5) and/or GR (lanes 3, 4, and 5) were incubated with labeled GRE, and electrophoresis was performed on a native polyacrylamide gel. Anti-GR polyclonal antibody was included in the reaction as indicated (lane 4).
Figure 3: Transactivation properties of GR and coexpressed GR and ERR2 in response to cortisol in HeLa cells. HeLa cells were transfected with 2 µg of GR expression vector either alone or in combination with 1, 2, or 4 µg of ERR2 expression plasmid. To investigate the influence of ERR2 on transcriptional activity of endogenous GR, cells were transfected with 4 µg of ERR2 expression plasmid without GR expression plasmid. Transfected cells were incubated with 10 nM cortisol (gray bars), 100 nM cortisol (white bars), or with vehicle (black bars).
Figure 4: Transactivation properties of PR, ERR2, and coexpressed PR and ERR2 in response to progesterone in CV-1 cells. CV-1 cells were transfected with expression vector for either human PR (2 µg) or ERR2 (4 µg), or 2 µg of PR expression plasmid was transfected in combination with 1, 2, or 4 µg of ERR2 expression plasmid. Transfected cells were incubated with 10 nM progesterone (gray bars) or with vehicle (black bars).
Figure 5: Coexpression of ERR2 and luciferase expression vectors in CV-1 cells. CV-1 cells were transfected with 2 µg of expression vector for luciferase alone or in combination with 1, 2, or 4 µg of ERR2 expression plasmid.
Our findings indicate that the nuclear orphan receptor ERR2 acts as
a cell-specific repressor of transcriptional activity mediated by GR.
The inhibitory activity of ERR2 appears to be constitutive because
cotransfection of ERR2 expression plasmid is sufficient to suppress GR
activity. Therefore, either no ligand is needed by the orphan to
inhibit transcription or an endogenous, activating ligand is present in
a sufficient amount. The possibility that ERR2 acts via an influence on
the basic transcriptional or translational machinery can be excluded
because expression of luciferase is unaltered by coexpression of ERR2
when the luciferase gene is under control of a constitutively active (Fig. 5) or PR-regulated promoter (Fig. 4). Moreover, the
fact that PR-regulated gene expression is not influenced by ERR2
provides evidence that the suppressive activity does not refer to all
members of the steroid receptor family. This excludes that the orphan
receptor acts via competing for a basic factor needed by steroid
receptors to regulate expression of genes(18) . Because ERR2 is
not able to bind to a GRE (Fig. 6B) the suppressive
action does not involve competition for response element binding, which
may be the mechanism of action of GR-mediated repression of GR
activity(20) . The inhibitory effect of the orphan receptor is
also not caused by the formation of an ERR2
GR complex that
displays altered DNA binding properties, as shown for other inhibitors
of GR action such as c-Jun(21, 22) ,
calreticulin(23, 24) , and the p65 subunit of
NF-
B(25) . This can be excluded because GRE binding of GR
is completely unaffected by coexpression of ERR2 (Fig. 6B).
The exact mechanism of ERR2-mediated
suppression of GR transcriptional activity remains unknown, but our
data correspond to a model of ERR2 action in which the orphan receptor
acts as a trans-repressor of glucocorticoid activity by competing with
high affinity for a factor specifically required by the GR to act as an
activator of transcription. This factor may be expressed or be
functionally available in limited amounts in some cell types. A similar
mode of action has been described for the inhibitory action of the A
form of the PR on the activity of the PR form or the
mineralocorticoid receptor(26, 27) .
Responsiveness and sensitivity to glucocorticoids differ widely in distinct tissues and cell types. Expression or activation of endogenous antagonists of glucocorticoid activity, such as ERR2 in glucocorticoid target tissues, may partially provide the mechanistic basis for the variance of GR activity in different tissues and represent another level of regulating glucocorticoid response. In this respect it is interesting that in a study recently published (28) it could be demonstrated that rat ERR2 is expressed in tissues that are also known to be enriched with GR, e.g. the pituitary and the paraventricular nucleus.