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INTRODUCTION |
The glucocorticoid receptor
(GR)1 belongs to the family
of nuclear receptors (NRs) and selectively regulates a network of
hormone responsive genes. This includes both stimulation and repression of target genes. Activation of target genes relies in most cases on the
ability of the GR to bind to specific DNA sequences, termed glucocorticoid responsive elements (GREs) (1-4). These GREs act as
hormone-regulated enhancer elements. Following the interaction of the
GR with the GREs and in vivo most likely with other
transcription factors and cofactors, it is thought that the GR directly
or indirectly contacts the proteins present in the basal transcription
machinery (4).
In some cases glucocorticoids stimulate gene expression independent of
GR binding to the GREs. Instead, this effect is mediated through a
protein-protein interaction between the GR and a second transcription
factor (5). However, the outcome of the response, i.e.
stimulation or repression of gene expression, is dependent on the
composition of the transcription factor complex targeted by the GR. The
best characterized example of this is the effect GR confers on AP-1
controlled target genes. In the case of a c-Jun homodimer binding to
the AP-1 site, the interaction with the GR leads to stimulation of AP-1
mediated transcription (6). In contrast, a c-Jun/c-Fos heterodimeric
AP-1 complex is repressed by the interaction with the GR (6-8). In
addition, glucocorticoids enhance prolactin-stimulated transcription of
the
-casein gene through the interaction of the GR with STAT5 (9).
The GR can also stimulate STAT3 and C/EBP
controlled transcription
without contacting the DNA (10, 11). Furthermore, we have recently described potentiation of retinoic acid (RA) induced transcription through the Hoxb-1 promoter autoregulatory element b1-ARE,
where this potentiation seems to involve a protein-protein interaction between the GR and the RA-induced Pbx1 and HOXB1 proteins binding to
the b1-ARE.2
However, the role for GR interacting
cofactors in this type of synergy between GR and other transcription
factors is not known.
Glucocorticoids also repress transcription. In principle, this involves
either binding of the GR to negative GREs (nGREs) leading to
displacement of transcription factors or interference with their
transcriptional activation, or alternatively binding of the GR to a
second transcription factor independent of GR binding to the DNA (12,
13). Examples of nGREs are the PRL3 element in the bovine prolactin
promoter and the human osteocalcin nGRE overlapping the TATA box
(14-16). Examples of repression of target genes independent of GR
binding to the DNA are the suppression of NF-
B signaling (17, 18)
and AP-1 activity if the AP-1 complex is composed of a c-Jun/c-Fos
heterodimer (see above).
The GR contains a major transcriptional activation domain called
activator function-1 (AF-1 or
1) localized in the less
conserved N-terminal region of the receptor (Ref. 19 and references
therein). A second activation domain, the highly conserved
ligand-dependent AF-2, is localized in the multifunctional
ligand-binding domain (LBD) of the GR (20-22). During the past few
years, so called coactivators and corepressors have been identified
that modulate the transcriptional activity of NRs. With regard to the
GR, several coactivators have been shown to interact with the GR and
stimulate its transcriptional activity, including GRIP1/TIF2 (23, 24),
SRC-1 (25), CBP/p300 (26), and TIF1
(27). These coactivators are
thought to act as bridging factors between the receptors and the basal
transcription machinery. In addition, some of the coactivators have
been shown to contain intrinsic histone acetyltransferase activity,
thus linking gene activation by NRs to histone modification and
chromatin alterations (28, 29). Several of the recently identified
coactivators have been shown to interact with the AF-2 of the NRs (Ref.
3 and references therein). In most cases this interaction requires the
binding of an agonist to the receptor (30-32). This correlates to a
conformational change in the LBD and the ability of the NRs to
transactivate (Ref. 3 and references therein).
One cofactor identified is the receptor interacting protein 140 (RIP140). This cofactor was first identified by in vitro
protein-protein interaction assays using the hormone-binding domain of
the estrogen receptor (ER) as a bait (32). The interactions of RIP140
with ER, retinoic acid receptor, and thyroid hormone receptor (TR) are
all induced by their respective ligands and RIP140 weakly enhances
in vivo transcription of these NRs (33). RIP140 has also
been shown to act as a coactivator for the rat androgen receptor (34).
However, more recent studies question a general coactivator role for
RIP140 as RIP140 inhibited Pit-1/ER and Pit-1/TR synergistic transactivation from the rat prolactin and growth hormone promoter, respectively (35). RIP140 also represses transcriptional activation of
the orphan nuclear receptor TR2 (36). Furthermore, RIP140 may act as a
coregulator of NRs rather than a coactivator as it antagonized the
function of the coactivator SRC-1 on peroxisome proliferator-activated
receptor
(PPAR
) (37).
In this report we have investigated the effect of RIP140 on
glucocorticoid responsive genes. This includes activation mediated through a classical GRE, activation through cross-talk with AP-1 and
homeodomain proteins as well as glucocorticoid-regulated repression via
an nGRE and NF-
B (RelA). We show that all glucocorticoid responses
investigated are deregulated by RIP140 and that this most likely is
mediated through an interaction between the GR and RIP140, abrogating
GR function. The repressive activity of RIP140 could be explained by
the ability of RIP140 to prevent binding of a "true" coactivator to
the GR.
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EXPERIMENTAL PROCEDURES |
Plasmid Constructs--
The (GRE)2-tk-Luc reporter
gene and the GR expression plasmid pSVGR1 have been described
previously (38). The constitutively active rat pSVGR1
LBD (amino
acids 1-525) was kindly provided by K. R. Yamamoto (39). The
luciferase reporter constructs pAdMLARE, 3xNF-
B(IC)tk-Luc, and
73ColA-Luc have been described elsewhere (8, 17, 40). The CAT
reporter plasmid PRL3CAT has also been described previously (41). The
expression and in vitro transcription and translation
plasmid pSG5-RIP140 contains the full coding sequence for the RIP140
protein (37). The in vitro transcription and translation
vector pGEM3-rGR was obtained by cloning the rat GR from SVGR1
(BamHI fragment) into the vector pGEM3Zf(+) (Promega). The
TIF2 expression plasmids pCMV5-TIF-2 and GST-TIF2 (IAD wt) were from J. Leers (24). Expression plasmids for GST (pGEX2T) and GST-GR fusion
protein (pGST2T-GR) were kindly provided by S. Wissink (42).
Cell Cultures and Transfections--
HeLa tk
cells, COS-7 cells, GH3 cells, and P19 embryonal carcinoma
(EC) cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 2 mM L-glutamine,
100 IU/ml penicillin, and 100 µg/ml streptomycin (all from Life
Technologies, Inc.) at 37 °C in 10% CO2. Cells were
plated at approximately 30% confluence in 6-well plates and the
following day cells were transfected by LipofectinTM with 1 µg/well of reporter plasmid and 250 ng of the GR expression plasmid
following the protocol of the manufacturer (Life Technologies, Inc.).
When indicated, 100 ng of RIP140 expression plasmid was co-transfected.
After a 6-h incubation at 37 °C in 10% CO2, the medium
containing the DNA-Lipofectin mixture was removed and fresh complete
medium was added containing either the vehicle ethanol (final
concentration 0.01%), 1 µM dexamethasone (Dex), or 100 nM RU486. In some experiments 1 µM
trans-RA or 100 ng/ml TPA (all from Sigma) dissolved
in dimethyl sulfoxide was added. In experiments when the synergy
between Dex and TPA was analyzed, medium containing 0.5% serum was
used. Following incubation for 16-24 h, cells were lysed and
luciferase or CAT (chloramphenicol acetyltransferase) activity was
determined as described (15, 17). Each experimental condition was
measured in triplicate and the values given represent the mean ± S.D from two to three experimental occasions.
GST Pull-down Assays--
The GR and RIP140 proteins were
transcribed and translated in vitro using the plasmids
pGEM3-rGR and pSG5-RIP140, respectively, in rabbit reticulocyte lysate
in the presence or absence of [35S]methionine as
instructed by the manufacturer (Promega). The expression and
purification of the GST, GST-GR, and GST-TIF2 fusion proteins was
carried out as described previously (24, 37). The translated
35S-labeled RIP140 or GR was incubated with GST, GST-GR, or
GST-TIF2 fusion protein loaded on glutathione-Sepharose beads in a
binding buffer containing 0.2 M NaCl (37). In the case of
GST-TIF2-35S-GR interaction, an increasing amount of
in vitro translated unlabeled RIP140 was added. Non-primed
reticulocyte lysate was supplemented so that all incubations contained
the same amount of lysate. After washing the beads, pelleted proteins
were analyzed by SDS-PAGE (10%) and the amount of
35S-labeled RIP140 or GR was detected by autoradiography.
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RESULTS |
RIP140 Antagonizes Glucocorticoid Receptor-mediated induction of a
GRE Controlled Reporter Gene--
In order to investigate the effect
of RIP140 on the ability of the GR to transactivate through GREs, a
luciferase reporter gene ((GRE)2-tk-Luc) controlled by a
heterologous promoter (the thymidine kinase promoter (tk)
105 to +52)
and two GREs was co-transfected with a GR expression vector (SVGR1)
with or without an expression vector for RIP140 (pSG-RIP140) into P19
EC cells. In the absence of RIP140, the synthetic glucocorticoid
dexamethasone (Dex) induced reporter gene activity 12-fold (Fig.
1, lane 2). In contrast, when
RIP140 was expressed in the cells, Dex-induced luciferase activity was
abolished (Fig. 1A, lane 4). This suggests that RIP140 somehow interferes with the transcriptional activity of GR.

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Fig. 1.
RIP140 antagonizes wild type GR function but
not the LBD deleted GR. A, the reporter gene
(GRE)2-tk-Luc (1 µg) was co-transfected with an
expression vector for the wild type GR (pSVGR1, 250 ng) in the absence
(lanes 1 and 2) or presence (lanes 3 and 4) of the expression vector for RIP140 (pSG-RIP140, 100 ng) into P19 EC cells as described under "Experimental Procedures."
Following transfection, cells were stimulated with or without 1 µM Dex for 16 h as indicated in the figure.
B, (GRE)2-tk-Luc was co-transfected into P19 EC
cells with (lanes 2 and 3) or without (lane
1) an expression vector for a GR lacking LBD (pSVGR1 LBD) in the
presence (lane 3) or absence (lane 2) of
pSG-RIP140 as described above. Following transfection, cells were refed
with fresh medium. 16 h later cells were lysed and analyzed for
luciferase activity. All luciferase activities were related to the
activity for (GRE)2-tk-Luc in the absence of hormone
(A) or in the absence of receptor (B). Each
bar gives the mean ± S.D. from three
experiments.
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As it has been previously demonstrated that RIP140 interacts with the
AF-2 domain located in the C-terminal LBD of NRs, we tested the ability
of RIP140 to interfere with a GR that lacked its LBD. A GR devoid of
its LBD harbors a constitutive ligand independent transactivation
capacity due to the presence of a constitutive transactivation domain
(
1 or AF-1) in the N-terminal domain (39). Fig.
1B, lane 2, shows that the GR devoid of its LBD enhanced the
reporter gene activity by about 5-fold in the absence of co-transfected
RIP140, and no effect on the constitutive transcriptional activity was
obtained in the presence of co-transfected RIP140 (compare lanes
2 and 3). This demonstrates that RIP140 is only able to
antagonize the GR activity when it contains its LBD that includes the
cofactor interacting AF-2 domain. Furthermore, this experiment showed
that the ability of RIP140 to repress the activity of the wild type GR
is not due to a general squelching of transcriptional activity, because
the constitutively transactivating GR devoid of its LBD was unaffected.
RIP140 Antagonizes the Enhancement by Glucocorticoids of Phorbol
Ester and Retinoic Acid-induced Responses--
Since RIP140
antagonized GR-mediated gene activation through GREs, we were
interested to know whether this effect of RIP140 also occurred on
alternative glucocorticoid responsive genes whose expression are
stimulated by glucocorticoids independent of binding of the GR to GREs.
For this, we investigated the consequence of RIP140 expression on
glucocorticoid stimulation of phorbol ester (TPA)-induced expression
from a AP-1 responsive reporter gene. HeLa cells expressing endogenous
GR were transfected with a TPA inducible collagenase A promoter,
containing a TPA response element (TRE), coupled to a luciferase
reporter gene (
73ColA-Luc) with or without the RIP140 expression
vector. In the absence of transfected RIP140, TPA treatment of the
cells stimulated luciferase activity 15-17-fold (Fig.
2A). Under our conditions,
co-treatment with TPA and Dex caused a further enhancement of the
luciferase activity to 33-35-fold stimulation. Dex alone caused a
5-fold induction of reporter gene activity. This is probably due to a
small constitutive AP-1 activity in the cells.

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Fig. 2.
RIP140 prevents agonist but not antagonist
induced synergy. A, the TPA responsive reporter gene
73ColA-Luc (1 µg) was co-transfected without (lanes
1-4) or with pSG-RIP140 (lanes 5-8) into HeLa
tk cells. After transfection, cells were treated with or
without 1 µM Dex and/or 100 ng/ml TPA for 16 h as
indicated in the figure. B, pAdMLARE (1 µg) was
co-transfected with wild type pSVGR1 (250 ng), in the absence
(lanes 1-4 and 9-10) or presence (lanes
5-8 and 11-12) of pSG-RIP140 (100 ng) into P19 EC
cells. After 6 h of incubation, cells were stimulated with or
without 1 µM Dex and/or 1 µM RA or 100 nM RU486 and/or 1 µM RA for 16 h as
indicated in the figure. All luciferase activities were related to the
activity for 73ColA-Luc or pAdMLARE in the absence of hormone
treatment. Each bar gives the mean ± S.D. from two and
three experiments, respectively.
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Interestingly, RIP140 specifically inhibited the glucocorticoid-induced
synergistic enhancement of TPA stimulated activity, while it only
modestly reduced the TPA response (Fig. 2A). Also the small
Dex induced activation of the collagenase A-luciferase reporter gene in
the absence of TPA treatment was abolished by co-transfection with
RIP140. Similar results were obtained when the luciferase reporter gene
was controlled by a heterologous promoter (tk promoter
105 to +52)
and three TREs (data not shown). This demonstrates that RIP140 also
inhibits the cross-talk between GR and AP-1 transcription factors.
Glucocorticoids enhance transcription not only through AP-1
transcription factors but also via a second class of proteins, namely
homeodomain containing proteins. We have observed that glucocorticoids
enhance the RA-induced expression of a luciferase reporter gene
controlled by the Hoxb-1 gene promoter autoregulatory element b1-ARE.2 The b1-ARE is recognized by two
homeodomain containing proteins, Pbx1 and HOXB1, both induced by RA in
P19 embryonal carcinoma (P19 EC) cells. Results suggest that the GR
enhances the RA-induced transcription from the b1-ARE by directly
contacting the Pbx1 and HOXB1 proteins without binding to the DNA
itself. In order to investigate whether RIP140 will also interfere with
glucocorticoid enhancement of RA-induced expression from the b1-ARE,
the reporter gene pAdMLARE and the GR expression vector were
co-transfected into P19 EC cells with or without the expression vector
for RIP140. Treatment of the cells with RA induced b1-ARE luciferase
gene activity 20-fold (Fig. 2B, lane 2). Dex in itself had
no effect on the b1-ARE luciferase gene activity, but co-treatment with RA and Dex caused a synergistic activation (35-fold stimulation). Transfection with RIP140 abolished this Dex/RA mediated synergy on the
b1-ARE element (Fig. 2B, compare lanes 6 and
8). This demonstrates that a separate GR controlled
cross-talk pathway, in addition to AP-1, is affected by RIP140. To be
noted is that the RIP140 does not significantly affect the RA-induced
expression of the b1-ARE luciferase gene, demonstrating a specificity
in the inactivation function of RIP140.
The synergistic activity between Pbx1/Hoxb1 and GR on the b1-ARE is
also observed when the GR is bound to the glucocorticoid antagonist
RU486 (Fig. 2B, lane 10). In contrast to the ability of
RIP140 to abolish the Pbx1/Hoxb1 and GR synergy when the GR was bound
to an agonist (Dex), RIP140 was unable to abolish the synergy when the
GR was bound to the antagonist RU486 (lane 12). This
suggests that RIP140, like several other cofactors, only interacts with
NRs bound to agonists (30, 32). This also supports the previous
conclusion that RIP140 does not act as a general inhibitor of transcription.
Repression of Gene Transcription Conferred by the GR Is Also
Inhibited by RIP140--
In order to explore whether the inhibitory
activity of RIP140 also applied to other pathways repressed by the GR,
two negatively regulated response elements were investigated. In the
first case we examined the effect of RIP140 on gene repression by the
GR via the interaction of the GR with a negative GRE (nGRE) from the
bovine prolactin gene (PRL3 nGRE (43)). We have previously shown that
repression via this nGRE requires the interaction of the GR with the
PRL3 nGRE (15). Binding of the GR to the PRL3 nGRE involves
displacement in pituitary cells of the pituitary-specific transcription
factor Pit-1 and a ubiquitously expressed Pbx protein (14, 15). When
the PRL3nGRE-CAT reporter gene was transfected into pituitary
GH3 cells, Dex repressed transcription by about 40% (Fig.
3A). However, when RIP140 was
co-transfected, GR was no longer able to repress the transcriptional
activity from the PRL3 element. Interestingly, RIP140 slightly but
significantly (p < 0.05) increased the basal activity
from the PRL3 element in the absence of glucocorticoid treatment.
Identical results were observed in COS-7 cells, in which cells GR also
represses transcription from the PRL3 nGRE (data not shown). In
non-pituitary cells Oct-1 replaces the function of Pit-1 (14).

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Fig. 3.
Repression by GR is inhibited in the presence
of RIP140. A, the reporter gene containing an nGRE from
the bovine prolactin promoter, PRL3CAT (1 µg), was co-transfected
with pSVGR1 (250 ng) in the absence (lanes 1 and
2) or presence of pSG-RIP140 (100 ng) into GH3
cells. After transfection, cells were stimulated with or without 1 µM Dex for 24 h as indicated in the figure. Cells
were lysed and CAT activity was determined as described under
"Experimental Procedures." B, the NF- B controlled
reporter gene 3xNF- B(IC)-tk-Luc (1 µg) was co-transfected with
pSVGR1 (250 ng) and the expression vector CMV-RelA (250 ng) in the
absence (lanes 1 and 2) or presence (lanes
3 and 4) of pSG-RIP140 (100 ng) into COS-7 cells as
indicated in the figure. Following transfection, cells were stimulated
with or without 1 µM Dex for 16 h as indicated in
the figure. All CAT or luciferase activities were related to the
activity of PRL3CAT or 3xNF- B(IC)-tk-Luc in the absence of hormone
treatment. Each bar gives the mean ± S.D. from three
experiments.
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Glucocorticoids also repress NF-
B controlled target genes by the
ability of GR to interfere with the transcriptional activity of NF-
B
proteins (17). This mainly occurs via a protein-protein interaction
between the GR and NF-
B proteins independent of the ability of GR to
interact with the DNA. To assess whether repression of NF-
B
signaling by glucocorticoid is relieved by RIP140, we transfected a
luciferase reporter gene containing three NF-
B sites from the human
intercellular adhesion molecule-1 promoter and a minimal (
105 to +52)
tk promoter together with expression vectors for the GR and the NF-
B
family member RelA with or without the expression vector for RIP140
into COS-7 cells. Transfection with RelA stimulated the transcription
from the 3xNF-
B(IC)tk-luciferase gene 10-fold (data not shown) and
treatment with Dex repressed this activity by 50% (Fig.
3B). Interestingly, co-transfection with RIP140 not only
derepressed the GR-dependent inhibition but also slightly
but significantly (p < 0.05) enhanced the
RelA-mediated transactivation of the luciferase reporter gene. These
results show that in addition to inhibiting GR-mediated enhancement of transcription, RIP140 also blocks GR-mediated repression, both from
target genes which involves binding of the GR to nGREs and through
mechanisms that involve a protein-protein interaction between the GR
and other transcription factors. The effect is specific for the GR,
since RIP140 showed a slight stimulatory effect on the activity of RelA
and Pit-1/Pbx in the absence of glucocorticoids.
RIP140 Interacts with the Glucocorticoid Receptor in
Vitro--
Our transfection data demonstrating the ability of RIP140
to inhibit all tested types of GR-mediated responses in several cell
lines while leaving other responses unaffected or only slightly affected, suggest that RIP140 mediates it effects by acting directly on
the GR. This may be explained by a direct interaction between RIP140
and GR. In order to find support for such a protein-protein interaction, we performed a pull-down assay in which the GR fused to
GST or GST alone were incubated with 35S-labeled RIP140.
The presence of 35S-labeled RIP140 in the pellet was
analyzed by SDS-PAGE and the amount of 35S-labeled RIP140
was detected by autoradiography. As can be seen in Fig.
4, RIP140 strongly interacted with the
GR-GST fusion protein. In contrast, only a weak interaction between
RIP140 and the GST alone was observed, demonstrating that RIP140
physically interacts with the GR in vitro.

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Fig. 4.
GR physically interacts with RIP140 in
vitro. In vitro translated
35S-labeled RIP140 was incubated with GST or GST-GR fusion
protein bound to glutathione-Sepharose beads. After washing the beads,
precipitated proteins were analyzed by SDS-PAGE and
35S-RIP140 was visualized by autoradiography. Lane
1 represents the input of the labeled RIP140.
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The Repressive Activity of RIP140 Is Partially Relieved by
Overexpression of the Coactivator TIF2 Due to Competition with RIP140
for Binding to the GR--
One possibility for the repressive action
of RIP140 is that it interacts with the GR and prevents a GR
interaction with coactivators. We investigated this possibility by
co-transfecting the (GRE)2-tk-Luc reporter gene and the GR
and RIP140 expression vectors with or without an expression vector for
the coactivator TIF2. As observed before, GR in the presence of Dex
activated the (GRE)2-tk-Luc reporter gene and this
activation was completely abolished by RIP140 (Fig.
5A). Transfection of TIF2 in
the absence of RIP140 potentiated GR activation of the reporter gene
approximately 2.5-fold. Interestingly, co-transfection with TIF2
partially restored the ability of the GR to activate the
(GRE)2-tk-Luc reporter gene in the presence of RIP140. This
suggested a model where RIP140 prevents the interaction of GR with
coactivators by competing for binding to the GR. This possibility was
further investigated by a GST competitive pull-down assay. GST-TIF2 was
incubated with in vitro translated 35S-labeled
GR with or without in vitro translated unlabeled RIP140. As
can be seen from Fig. 5B, increasing the amount of RIP140
reduced binding of the GR to TIF2 (compare lanes 4-5 to
lane 3). This decrease in GR binding was specific and not
due to the presence of an increasing amount of reticulocyte lysate, as
all incubations contained the same amount of lysate. These experiments
support a model in which RIP140 inhibits GR activity by preventing
binding to the GR of a coactivator.

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Fig. 5.
A, TIF2 partially rescues
RIP140-mediated repression. The reporter gene (GRE)2-tk-Luc
(1 µg) was co-transfected with an expression vector for wild type GR
(pSVGR1, 100 ng) and TIF2 (pCMV5-TIF2, amounts indicated in the figure)
in the absence (lanes 1-4) or presence (lanes
5-14) of the expression vector for RIP140 (pSG-RIP140, 50 ng)
into COS-7 cells as described under "Experimental Procedures."
Following transfection, cells were stimulated with or without 1 µM Dex for 16 h as indicated in the figure. All
luciferase activities were related to the activity for
(GRE)2-tk-Luc in the absence of hormone. Each
bar gives the mean ± S.D. from two experiments.
B, RIP140 prevents binding of the GR to TIF2 in
vitro. GST-TIF2 (lanes 3-5) or GST (lane 2)
bound to glutathione-Sepharose beads were incubated with in
vitro translated 35S-labeled GR without (lanes
2 and 3) or with 2 µl (lane 4) or 4 µl
(lane 5) of in vitro translated unlabeled RIP140.
Non-primed reticulocyte lysate was added to all incubations to obtain
an equal volume of lysate. PhosphorImager analysis revealed that the
formation of GST-TIF2-GR complexes were reduced by 44 (lane
4) or 88% (lane 5) as compared with complex formation
in the absence of RIP140 (lane 3) when nonspecific GR
binding to GST alone (lane 2) was deducted. Lane
1 shows 50% of the input of 35S-labeled GR.
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DISCUSSION |
Recent results have revealed that the function of the ubiquitously
expressed receptor interacting protein RIP140 in NR transactivation might be more complex than initially described. RIP140 has been shown
to be a weak coactivator of ER, retinoic acid receptor, and TR, binding
to the AF-2 domain of these NRs (32, 33). RIP140 also stimulates
transcription of the AR (34). RIP140 interacts with additional NRs,
including PPAR and the orphan nuclear receptor TR2 (36, 37). However,
in contrast to other coactivators, RIP140 does not stimulate
transcriptional activation of the PPAR, rather it counteracts
coactivators that are able to enhance PPAR transcription (37). RIP140
also acts as a corepressor of TR2 activity (36). In addition, it was
recently demonstrated that RIP140 can both repress or stimulate
Pit-1/NR transcriptional synergy, depending on the absence or presence
of defined domains of the Pit-1 protein in a promoter dependent way
(35). Thus, the consequence of RIP140 interaction with NRs is complex
in that it can act both as a coactivator and an inhibitor of transcription.
We demonstrate that RIP140 acts as an inhibitor of all tested GR
mediated activities, which include positive regulation through classical GREs, synergy through cross-talk with AP-1 and Pbx1/HOXB1, and negative regulation via nGRE and cross-talk with RelA. These inhibitory effects are apparently independent of promoter context and
cell context as inhibition was seen in four different cell lines. No or
only minor effects were seen on the transactivating activities of AP-1,
RelA, Pit-1/Pbx, and Pbx1/HOXB1, demonstrating that RIP140 is not a
general squelcher of transcriptional activities. In fact, RIP140 may
act as a week coactivator of RelA or the Pit-1·Pbx complex (Fig. 3,
A and B). Furthermore, RIP140 did not
significantly influence retinoic acid receptor induction of
Pbx1/HOXB1-mediated transcription (Fig. 2B), suggesting that
RIP140 is not an inhibitor of all NRs in P19 EC cells.
An explanation for the repressive effect on all types of glucocorticoid
responses is that RIP140 interacts with the GR, preventing true
coactivators to bind to the GR. Support for this assumption comes from
our transfection and pull-down experiments (Figs. 4 and 5). The
competition model is further substantiated by our previous observation
where we demonstrated that RIP140 competed with SRC-1 for binding to
the AF-2 domain of PPAR
(37). The same mechanism has been proposed
for RIP140 inhibition of Pit-1/ER and Pit-1/TR synergy and TR2 activity
(35, 36). The competition model for cofactor binding to a common site
in the GR can also explain the ability of RIP140 to inhibit negative
gene regulation by the GR. It has been suggested that repression of
gene activity by the GR involves competition for a limiting coactivator
(CBP) common to the GR and a second positively acting transcription factor complex, e.g. AP-1 or NF-
B (31, 44). Thus, binding of GR to RIP140 would then prevent GR to titrate out CBP. An
alternative explanation for the ability of RIP140 to prevent GR
mediated responses is that RIP140 functions as a steric obstacle
hampering GR-DNA or protein-protein interaction, both being important
in positive and negative gene regulation by the GR (12). Interestingly, it was recently reported that another cofactor, RAP46, is a negative regulator of GR transcriptional activity and acts by preventing GR
binding to a GRE (45).
It has been shown that interaction with an agonist induces a
conformational change in the LBD of NRs leading to the formation of an
AF-2 domain capable of interacting with cofactors, including RIP140
(32, 33, 46). Like the case for other NRs, the interaction between
RIP140 and GR is also likely to take place through the conserved AF-2
domain present in the LBD of the GR (20, 21). In line with this
assumption, RIP140 was unable to inhibit the transcriptional activity
of a GR devoid of its LBD (Fig. 1B). Opposite to the
situation when NRs are bound to agonists, the AF-2 domain is not able
to interact with cofactors when NRs bind antagonists (46). In our
experiments, RIP140 did not inhibit the GR synergy with Pbx1/HOXB1 when
the GR was bound to the glucocorticoid antagonist RU486 (Fig.
2B). This is in agreement with the suggestion that the
agonistic activity of a mixed antagonist, like RU486, when bound to the
receptor is mediated through the AF1 domain of the receptor, while
RIP140 binds and interferes with the function of the AF-2 domain (47).
This would explain the lack of inhibitory activity of RIP140 on
RU486-GR-Pbx1/HOXB1 synergy.
When HeLa cells that had been transfected with TRE-controlled reporter
genes were treated with a combination of TPA and Dex, a Dex stimulated
enhancement of the TPA effect was observed (Fig. 2A). Most
previous reports have demonstrated a Dex-mediated repression in
response to TPA (48). The reason for this discrepancy is unknown but
could be due to different lines of HeLa cells. A synergistic effect of
Dex and TPA can be obtained if our HeLa cells contain AP-1 complexes
consisting mainly of c-Jun homodimers following TPA treatment. It has
previously been demonstrated that the activity of c-Jun homodimers is
activated by GR in contrast to the repression observed with c-Jun/c-Fos
heterodimers (6).
Interestingly, we observed a small up-regulation of RelA and Pit-1/Pbx
activities following co-transfection with RIP140 (Fig. 3, A
and B). The mechanism behind this effect is unknown but it may be that RIP140 acts as a weak coactivator for these transcription factors, although it has so far not been reported that RIP140 can
interact with transcription factors other than NRs. Alternatively, it
may reflect a small constitutive GR repression of RelA and Pit-1/Pbx
activities, which is relieved upon transfection by RIP140 (49).
There are several observations to suggest that RIP140 differs from true
coactivators. RIP140 is generally less efficient in enhancing the AF-2
activity of NRs in mammalian cells compared with other coactivators
(25, 33, 50). Most of the other coactivators function as bridging
proteins between the basal transcriptional apparatus and the nuclear
receptors but in contrast, the RIP140 has not been demonstrated to
interact with members of the basal transcription machinery (31, 32).
Furthermore, the lack of a histone acetyltransferase domain in RIP140,
distances it from the CBP/p300, SRC-1, and P/CAF (51, 52). Most of the
coactivators, but not RIP140, interact with histone acetyltransferase
domain containing CBP/p300 to give a concerted activation function (51, 52). In summary, although RIP140 may act both as a coactivator and
corepressor on different NRs or in different contexts, its main effect
on glucocorticoid mediated responses is inhibitory. A dual role for
cofactors is not unique as both the receptor interacting factors
TIF1
and NSD1 exhibit characteristics of both corepressors and
coactivators (27, 53, 54). Thus, the primary role for RIP140 may be to
act as a coregulator fine tuning a complex network of genes.