 |
INTRODUCTION |
Nuclear receptors comprise the largest superfamily of eucaryotic
transcription factors with more than 150 proteins identified (for
review, see Refs. 1-3). The capability of many family members to bind
structurally diverse hydrophobic ligands is a crucial regulatory
element for the transmission of extracellular signals into
intracellular transcriptional responses. However, alternative ligand-independent regulation mechanisms have been identified (4-6),
and ligands may not exist for all receptors. Nuclear receptors usually
modulate transcription of their target genes by binding to cognate
promoter response elements. Moreover, cross-talk mechanisms, which do
not require DNA binding, may allow nuclear receptors to influence the
activity of other transcription factors (7, 8).
Nuclear receptors are modular transcription factors containing a
variable N-terminal domain often exhibiting a constitutive transcription activation function
(AF-1),1 a highly conserved
zinc finger type DNA-binding domain (DBD), a variable linker region
(hinge), and a multifunctional C-terminal domain responsible for ligand
binding (LBD), dimerization, and ligand-regulated transcriptional
activation (AF-2). Nuclear receptors can be subdivided into steroid
receptors that mainly form homodimers and a large diverse subfamily of
nonsteroid receptors including receptors for thyroid hormone,
retinoids, and vitamin D as well as many orphan receptors, for which
natural ligands have not been identified. Whereas certain nonsteroid
receptors are monomeric or form homodimers, the majority
heterodimerizes with the retinoid X receptor (RXR). These heterodimers
have been recognized to function as very dynamic transcription factors
in which both subunits influence the other's capability to interact
with ligands and cofactors (9-13). Functional and structural analysis
(Refs. 14-17 and references therein) identified a common dimerization
surface within the LBDs which primarily is formed by helices 10/11
(dimerization helix).
Estrogen receptors (ERs) are unique steroid receptors because they
exist as two different paralogues (encoded by two separate genes),
ER
and ER
(Refs. 18 and 19 and references therein). Both ERs
display quite similar ligand binding characteristics (19) and
comparable agonist-dependent transcriptional activities on
"classical" estrogen response elements (EREs), consistent with the
conservation of LBD/AF-2 regions implicated to be critical for these
functions. However, differences have been reported with regard to their
antagonist-dependent activation properties at AP1 sites (8)
which might be related to the lack of conservation in their N termini.
These functional differences, together with their distinct expression
pattern in tissues, strongly suggest that ER
and ER
may play
different roles in gene regulation. They have been demonstrated to form
stable homo- and heterodimers with each other in solution and on DNA
(20, 21) indicating that heterodimerization is not a unique feature of
nonsteroid receptors forming RXR heterodimers.
Nuclear receptors including ERs function in concert with
transcriptional cofactors including basal transcription factors, chromatin-modifying complexes, corepressors, and coactivators (for
review see Refs. 22 and 23). Although various receptors in the absence
of agonistic ligands bind to corepressors (24, 25), ligand activation
is associated with structural rearrangements within the LBD/AF-2
domain, permitting the recruitment of coactivators. The predicted AF-2
coactivator-binding surface includes two highly conserved regions,
namely the C-terminal helix 12 (AF-2 core) as well as N-terminal
helices 3-5 including the "signature region" (Refs. 15-17 and
references therein). In particular, the precise positional
rearrangement of helix 12 upon binding of agonistic ligands seems to be
a prerequisite for coactivator binding. Conversely, antagonistic
ligands such as anti-estrogens are thought to induce a different
conformation that prevents the association of coactivators (15). Common
coactivators, which have been demonstrated to be relevant also for ERs,
include, for example, p300/CBP (7, 26-28) and members of the
p160/SRC-1 coactivator family (e.g. SRC-1, TIF2, AIB1; see
Refs. 29-38). The detection of intrinsic transcriptional activity and
histone-acetyltransferase activity in these proteins functionally
connects them to both the basal transcription machinery and to
chromatin (34, 39-41). In agreement with the structural conservation
of the putative coactivator interaction surface, many AF-2 cofactors
contain short conserved LXXLL interaction motifs, referred
to as NR box (30, 32, 35, 42). Biological evidence for the involvement
of p160/SRC-1 coactivators in, for example, ER-mediated gene expression
comes from the discovery of AIB1 gene amplification in ER-positive
breast and ovarian cancer cells (36) as well as from recent SRC-1
knock-out studies (43).
In searching for novel proteins interacting with the LBD/AF-2 domain of
nuclear receptors, an unusual orphan receptor has been isolated that
obviously lacks a nuclear receptor-type DBD but contains a putative LBD
(44, 45). Based on its small size and its ability to interact, like
RXR, with various nonsteroid receptors, this orphan receptor has been
designated SHP (short heterodimer partner). Its closest relative within
the superfamily is the orphan receptor DAX-1, which contains a new type
of DBD, interacts with the orphan receptor SF-1, and plays important
roles in both adrenal and gonadal function (Refs. 46-49 and references therein). Although it is unknown whether SHP has ligands and can bind
DNA, SHP has been suggested to act as a negative regulator of nuclear
receptor signaling pathways by competition with RXR for
heterodimerization (44). Furthermore, because SHP has been shown to
exert intrinsic repressor activity, active repression mechanisms may
contribute to its inhibitory effect (50).
In this report, we have identified ERs as novel receptor targets for
SHP. We provide evidence for the existence of a novel mechanism by
which SHP inhibits nuclear receptor activation, and we suggest opposing
regulatory functions of SHP and AF-2 coactivators in estrogen signaling.
 |
EXPERIMENTAL PROCEDURES |
Plasmids
All plasmids were generated using standard cloning procedures
and verified by restriction enzyme analysis and DNA sequencing.
Yeast Expression Plasmids--
The Gal4 DNA-binding domain
fusion constructs Gal-ER
LBD/AF-2 (aa 249-595) and
Gal-ER
LBD/AF-2 (aa 168-485) were constructed by inserting
PCR-generated fragments of the corresponding human ER cDNAs (8)
into the BamHI site of AS2-1
(CLONTECH). Gal-PPAR
LBD and Gal-RXR
LBD have
been described previously (51). The Gal4 activation domain constructs
GAD-SHP WT, D1, and D2 (see Fig. 1) were constructed by inserting
PCR-generated fragments of the SHP cDNA into the EcoRI
site (WT) or into the EcoRI/XhoI sites (D1 and
D2) of GADGH (CLONTECH).
GST/His Fusion Constructs--
GST-ER
(aa 249-595) was made
similar to the Gal4 construct by cloning into the BamHI site
of pGEX-2TK (Amersham Pharmacia Biotech). GST-SHPWT was made by cloning
a SalI fragment from the original yeast two-hybrid library
clone (GAD10-SHP, CLONTECH) into the
XhoI site of pGEX4T-3 (Amersham Pharmacia Biotech). GST-SHP D1 and GST-SHP D2 were constructed by using the same PCR fragment as
for the yeast vectors inserted into the
EcoRI/SalI sites of pGEX4T-1 (Amersham Pharmacia
Biotech). GST-TIF2 (aa 594-766) was made by PCR cloning of a TIF2
fragment into the EcoRI/XhoI sites of pGEX4T-1.
His-SHP WT (aa 1-260) was constructed by inserting a PCR-generated
fragment into the NdeI/SalI site of pET15b
(Novagen). His-RIP140 (aa 747-1158) has been described previously
(51).
Plasmids for in Vitro Translation--
pT7ER
(aa 1-595) was
as described previously (8). pT3ER
(aa 1-485) was made by recloning
the human ER
cDNA into the XhoI/NotI sites
of pBKCMV HA (51). T3-TIF2 (aa 1-1465) was made by cloning the
corresponding cDNA into the EcoRI/Xho sites
of pBK-CMV (Stratagene). T3-RIP140 (aa 1-1158) has been described previously (51).
Mammalian Expression Constructs--
The following plasmids have
been described previously: pSG5-based expression vectors for ER
and
ER
(Ref. 8 and references therein), ER reporter constructs
2xERE-tk-luc (20) and 3xERE-TATA-luc (30), pCMXGaL4ER
LBD (aa
247-599) (20), and the Gal4 reporter construct UAS-tk-luc (9). pSG5
rSHPWT was made by cloning an EcoRI insert from the library
cDNA clone (see above) into the EcoRI site of
pSG5(Stratagene). pSG5-TIF2/GRIP1 was described previously (33).
pSHP-GFP was constructed by PCR cloning of full-length SHP (aa 1-260)
into the EcoRI/BamHI sites of pEGFP-N3 (CLONTECH).
Yeast Two-hybrid Screening and Interaction Assay
SHP was isolated in a yeast two-hybrid screening for proteins
interacting with the rat PPAR
LBD/AF-2 (aa 166-468) as described previously for the isolation of hRIP140 (51), except that a rat liver
cDNA library (CLONTECH) was used. For the yeast
two-hybrid analysis, HF7c (MAT
) transformed with Gal4 DBD plasmids
was mated with Y187(MAT
) transformed with GAD plasmids. Diploid
strains were selected for the presence of both plasmids. Interactions were monitored as growth on selective
His plates using different dilutions of yeast cells in the absence or presence of 1 µM 17
-estradiol or 1 µM 4-OH tamoxifen, respectively.
GST Pull-down Assay
Interaction studies were performed essentially as described
(51). Briefly, 35S-labeled proteins, generated by in
vitro transcription/translation of either plasmids or PCR products
containing a 5'-T3 promoter using a TNT kit (Promega), were incubated
with approximately 1 µg of GST fusion protein in the absence
(Me2SO) or presence of 1 µM 17
-estradiol
or 1 µM 4-OH tamoxifen. The proteins were incubated for
2-3 h at 4 °C. For the competition assay, either purified His-SHP,
His-RIP140, or TIF2 (which was generated by thrombin cleavage of the
purified GST-TIF2 protein) was added to the binding reaction. Protein
interactions were analyzed by SDS-polyacrylamide gel electrophoresis
followed by autoradiography.
Mammalian Cell Transfections
293 cells were maintained in a 1:1 mixture of F-12 medium with
glutamine and Dulbecco's modified Eagle's medium supplemented with
10% FCS, 100 µl/ml penicillin, and 100 µl/ml streptomycin (Life
Technologies, Inc.). MCF-7 cells were maintained in RPMI 1640 (Life
Technologies, Inc.) media supplemented with 10% FCS, 1% non-essential
amino acids (Life Technologies, Inc.), 100 µl/ml penicillin, and 100 µl/ml streptomycin. Both 293 cells and MCF-7 cells were plated onto
6-well plates in phenol red-free modified Eagle's medium (Life
Technologies, Inc.) supplemented with 10% charcoal-stripped FCS, 100 µl/ml penicillin, and 100 µl/ml streptomycin. Twenty-four hours
later, cells were transfected with plasmid constructs using Lipofectin
(MCF-7 cells and 293 cells transfected with Gal-ER
LBD) as instructed
by the manufacturer (Life Technologies, Inc.) or by using DOTAP (293 cells transfected with ER
or ER
) as instructed by the
manufacturer (Boehringer Mannheim). Transfections were performed using
1.0 µg of ERE-tk-luciferase reporter or 0.8 µg of ERE-TATA-luc
reporter, together with 0.2 µg of either ER
or ER
or 50 ng of
Gal-ER
LBD. Empty expression vectors were added to equalize total
transfected plasmid DNA concentrations. After 12 h, the medium was
changed, and fresh medium (without phenol red) containing
Me2SO or 10 nM 17
-estradiol was added.
30 h after changing media, the cells were harvested. Cell extracts
were analyzed for luciferase activity as described (20, 51).
RT-PCR for mRNA Expression Analysis
Total RNA isolation and total cDNA preparation have been
described previously (52). For the PCR reaction, 1 µl of the
synthesized cDNA was added to the reaction mix and amplified,
starting with a preincubation at 94 °C for 2 min, followed by 25 cycles at 94 °C for 20 s, 56 °C for 30 s, and 72 °C
for 90 s in a PCR 9600 thermocycler (Perkin-Elmer). The
oligonucleotides R1051 5'-AGGAACAAGACACAGACCATGAGCT-3' and R1031
5'-AGTCCTTGGACGGCAGGAAGACGG-3' were used for amplification of a
258-base pair fragment of SHP. The oligonucleotides used for
amplification of actin were as described previously (52). The PCR for
actin was performed with an annealing temperature of 50 °C. After
agarose gel electrophoresis and blotting to nitrocellulose filters, the
PCR products were hybridized to the labeled 258-base pair SHP
oligonucleotide prepared from the original SHP cDNA clone, using
the same primers as for the RT-PCR. The actin PCR products were
hybridized to an internal actin primer, according to previously described protocol (52). Hybridization of SHP probe was performed at
65 °C for 1 h in ExpressHyb hybridization solution
(CLONTECH) followed by four 20-min washes in 2×
SSC and 1% SDS at 65 °C and finally two 20-min washes in 0.1× SSC
and 0.5% SDS. Hybridization of actin oligonucleotide was performed at
37 °C for 1 h in ExpressHyb hybridization solution followed by
four 10-min washes in 2× SSC and 0.05% SDS at room temperature and
finally two 20-min washes in 0.1× SSC and 0.1% SDS.
Analysis of Subcellular Localization of GFP-tagged SHP
293-cells were plated on 6-well plates containing glass
coverslips in Dulbecco's modified Eagle's medium supplemented with 10% FCS, 100 µl/ml penicillin, and 100 µl/ml streptomycin.
Twenty-four hours later, cells were transfected with 2 µg of SHP-GFP
or with 2 µg of GFP alone, using Lipofectin as instructed by the
manufacturer (Life Technologies, Inc.). Cellular localization was
visualized 36 h after transfection. The cells were fixed with 4%
formaldehyde in phosphate-buffered saline for 15 min at room
temperature and stained with 0.001 mg/ml Hoechst 33342 for 10 min at
room temperature. The coverslips were mounted on micro slides with
FluorSave Reagent (Calbiochem) followed by viewing in a Zeiss Axiophot
epifluorescence microscope. Photographs were recorded on T-max 400 film (Kodak).
 |
RESULTS |
Cloning of SHP and Interaction with ERs--
The yeast two-hybrid
approach was used to identify novel proteins that interact with the LBD
of PPAR
, beyond the known dimerization partner RXR (for details see
Ref. 51). Screening of an activation domain-tagged rat liver cDNA
library led to the isolation of independent partial clones encoding
RXR
, dUTPase (a previously characterized PPAR-interacting protein,
Ref. 53), and one full-length clone encoding the orphan nuclear
receptor SHP (44). The rat SHP is highly conserved compared with its
mouse and human orthologues and consists of 260 aa with a predicted
molecular mass of 29 kDa, in concordance with recently published data
(45). RACE experiments failed to detect extended 5'-coding regions
(data not shown), supporting the view that SHP lacks a nuclear
receptor-type DNA-binding domain. Structural features of SHP are
illustrated in Fig. 1A.

View larger version (23K):
[in this window]
[in a new window]
|
Fig. 1.
Interaction of SHP with ER and ER .
A, schematic representation of SHP wild-type and two
C-terminal deletions. Highlighted are some regions conserved between
SHP and nuclear receptors. The black box corresponds to the
putative dimerization helix 10/11, the hatched box the
signature sequence, and the gray box represents the AF-2
core, helix 12. The cross-hatched box represents the part of
SHP that is identical to DAX-1. B, pull-down assay using
35S-labeled wild-type SHP and purified GST-ER (aa
249-595) or GST alone in the absence (NH) or presence of 1 µM 17 -estradiol (E2) or 1 µM
4-OH tamoxifen (OHT). The approximate size of SHP is 29 kDa.
The input represents 20% of the amount of labeled protein used in each
pulldown. C and D, pull-down assay using
35S-labeled wild-type ER (C) or ER
(D) and purified GST-SHP fusions as indicated, or GST alone
in the absence (NH) or presence of 1 µM
17 -estradiol (E2) or 1 µM 4-OH tamoxifen
(OHT). The approximate sizes of ER and ER are 67 and
60 kDa, respectively. The input represents 20% of the amount of
labeled protein used in each pull down.
|
|
To identify the nuclear receptors with which SHP can interact, we
performed two-hybrid interaction assays using GAL4-LBD fusion proteins
and the activation domain-tagged SHP (GAD-SHP). We found that SHP
interacted with all non-steroid receptors tested, namely PPAR
, TR
, RXR
, and HNF4 (Table I and data
not shown), but surprisingly, SHP also interacted with steroid
receptors, namely the two ER subtypes ER
and ER
(Table I). The
interaction with the ER
LBD was verified in vitro using
the GST pull-down assay (Fig. 1B) and appeared to depend on
agonistic ligands, i.e. estradiol, whereas antagonistic
ligands, i.e. the anti-estrogen tamoxifen, did not promote
interaction of the proteins. The interaction with non-steroid receptors
is in agreement with previously reported data (44, 45, 50) and supports
the hypothesis that SHP, like RXR, is a heterodimerization partner for
nuclear receptors (44). In contrast to RXR, however, SHP also interacts
with ERs, indicating major differences between SHP and RXR with regard
to receptor specificity and mode of interaction with the LBD.
View this table:
[in this window]
[in a new window]
|
Table I
Yeast two-hybrid interaction of SHP with ER and ER
Interaction was measured as growth on plates lacking histidine and
studied in the absence (NH) or presence of 17 -estradiol (E2) or 4-OH
tamoxifen (OHT). +represents growth after 3 days; represents no
growth after 3 days.
|
|
SHP C Terminus Including the Putative Nuclear Receptor Dimerization
Helix Is Not Required for ER Interaction--
Previous functional and
structural studies have established a requirement of the LBD helices
10/11 for nuclear receptor dimerization (14-16). To investigate the
involvement of the predicted SHP helix 10/11 in the interaction with
ERs we made two C-terminal SHP deletions (D1 and D2, see Fig.
1A) and tested them for interaction both in vitro
and in vivo. First, we expressed and purified SHP WT, D1,
and D2 as GST fusion proteins, and we performed pull-down assays using
35S-radiolabeled in vitro translated wild-type
ER
and ER
, respectively. As seen in Fig. 1C and D, SHP
WT and D1 interacted with comparable efficacy with both ERs, whereas
further deletion up to aa 113 (D2) significantly decreased the
interaction. In contrast to the pull-down assay using GST-ER
LBD
(Fig. 1B), in vitro translated ERs displayed some
ligand-independent interaction with SHP, causing only a weak
enhancement in the presence of estradiol. However, the
"ligand-independent" interaction was decreased in the presence of
antagonist (tamoxifen), suggesting that translated ERs were partially
activated. Therefore, differences with respect to the folding and
activation status between the Escherichia coli expressed LBD, and the in vitro translated wild-type ERs cannot be excluded.
The in vitro results could be further supported in
vivo using the yeast two-hybrid assay (Table I); SHP WT and D1
interacted with both ERs, whereas SHP D2 only interacted with ER
but
not with ER
, perhaps illustrating structural differences between their LBDs. Interestingly, both SHP deletions efficiently interacted with RXR and PPAR, respectively, even in the absence of added ligands,
whereas the in vivo interaction with the ERs was strictly dependent on the presence of estradiol, and no interaction was seen
without hormone or in the presence of tamoxifen. In summary, we
conclude the following: 1) that the predicted SHP helix 10/11 does not
appear to be involved in the ER interaction, 2) that an N-terminal
region encompassing aa 113-209 is required for efficient interaction
with ERs, and 3) that agonistic, but not antagonistic, ER ligands
significantly enhance the interaction of SHP with the ER LBD.
SHP Inhibits Ligand Activation of ERs in Mammalian Cells--
SHP
has previously been suggested to act as a negative regulator of
retinoid receptor signaling in mammalian cells (44). To determine
whether SHP also interferes with ER-mediated transcriptional activation
in response to estradiol, we performed transient cotransfection studies
in 293 cells using expression vectors for the wild-type receptors and
an ER-responsive reporter plasmid. 293 cells (derived from human
embryonal kidney cells) were selected as a test system because these
cells do not express detectable levels of endogenous ER (data not
shown). Therefore, reporter gene activation strictly depends on the
expression of exogenous ERs. As shown in Fig.
2, A and B,
coexpression of increasing amounts of SHP inhibited the ligand-induced
transcriptional activity of both ER
and ER
, respectively. The
inhibition was approximately 80% of the ligand-induced activity in the
absence of SHP in case of ER
and almost complete in case of ER
.
The residual activity of ER
is probably due to transcriptional activity mediated by AF-1, which apparently is not present in ER
,2 although we do not
exclude other explanations, e.g. different expression levels
of the two ERs. To confirm these results in a cell line expressing
endogenous ERs, we cotransfected MCF7 breast cancer cells with the
ER-responsive reporter construct and increasing amounts of SHP (Fig.
2C). Under these conditions, SHP was able to down-regulate
the estradiol-dependent activity approximately 50%.
Compared with the results obtained using 293 cells, the inhibitory effect of SHP appears to be less pronounced in MCF-7 cells for several
reasons as follows. (i) The transfection efficiency differed between
the two cell lines (data not shown). (ii) The ratio between ERs and SHP
might have been different. (iii) MCF-7 cell are known to contain
unusual high levels of the ER coactivator AIB1 (36). Therefore, higher
levels of SHP might be required for inhibition. Irrespective of these
differences, the interaction of SHP with ligand-bound ERs significantly
inhibited their ligand-dependent transcriptional activity
in both human cell lines.

View larger version (12K):
[in this window]
[in a new window]
|
Fig. 2.
Inhibition of ligand-induced ER activity by
SHP. 293 cells were cotransfected with the ERE-TATA-luc reporter
plasmid and the expression plasmids for either wild-type ER
(A) or wild-type ER (B), together with
increasing amounts of the expression vector for SHP, either in the
absence (Me2SO) or presence of 17 - estradiol. All values
represent the mean of duplicate samples, and similar results were
obtained in at least three independent experiments. C, MCF-7
cells were transfected with the ERE-tk-luc reporter plasmid together
with increasing amounts of SHP expression plasmid in the absence or
presence of 17 -estradiol. The values shown are the mean of two
independent experiments. The luciferase activity observed with the
endogenous ER in the presence of 17 -estradiol (E2),
but no added SHP, was set to 100%.
|
|
SHP Inhibits ER
AF-2 Activity and Antagonizes TIF2-mediated
Coactivation in Mammalian Cells--
To elucidate regulatory
mechanisms by which SHP antagonizes ER activation, we wanted to
distinguish between negative effects of SHP at the level of DNA binding
versus DNA-independent inhibition mechanisms,
i.e. at the transcriptional level. Because the ER LBD/AF-2
domain was sufficient for interaction with SHP, we made constructs
expressing GAL4-ER
LBD/AF-2 fusion proteins and analyzed them in a
GAL4-responsive reporter system in 293 cells. As seen in Fig.
3A, coexpression of increasing
amounts of SHP clearly inhibited the ligand-dependent
activation function AF-2 of ER
, similar to the inhibition observed
above with wild-type ER
(see Fig. 2A). This strongly
suggests that SHP is able to down-regulate ER activity by inhibiting
its ligand-dependent AF-2, without affecting the capacity
of ER to bind to DNA.

View larger version (12K):
[in this window]
[in a new window]
|
Fig. 3.
SHP inhibits ER AF-2 activity and
antagonizes TIF2 coactivation in vivo. A,
293 cells were cotransfected with the UAS-tk-luc reporter plasmid
together with the expression plasmid for Gal-ER and increasing
amounts of SHP expression plasmid in the absence (Me2SO) or
presence of 17 -estradiol. The values shown are the mean of two
independent experiments. The luciferase activity observed with
Gal-ER in the presence of 17 -estradiol (E2), but no
added SHP, was set to 100%. B, 293 cells were cotransfected
with the UAS-tk-luc reporter, Gal-ER and TIF2 expression plasmid
together with increasing amounts of the expression plasmid for SHP in
the absence (Me2SO) or presence of 17 -estradiol. One
representative experiment is shown. All values represent the mean of
duplicate samples, and similar results were obtained in at least three
independent experiments and also by using other expression plasmids.
C, 293 cells were cotransfected with the UAS-tk-luc
reporter, Gal-ER and SHP expression plasmid together with increasing
amounts of the expression plasmid for TIF2 in the absence
(Me2SO) or presence of 17 -estradiol. One
representative experiment is shown. All values represent the mean of duplicate
samples, and similar results were obtained in at least three
independent experiments.
|
|
Considering the interaction characteristics of SHP and its effect on
AF-2 activity in mammalian cells, striking similarities are apparent
between the action of SHP and negative AF-2 coregulators, for example
RIP140 (51) or dominant-negative fragments of AF-2 coactivators such as
SRC-1 (29, 30) or TIF2 (32). Thus, SHP might act at the same level as
AF-2 coactivators. To test this hypothesis, we asked whether SHP could
antagonize the coactivation mediated by one distinct AF-2 coactivator.
As illustrated in Fig. 3B, coexpression of SHP together with
TIF2/GRIP-1, a member of the p160/SRC-1 family of coactivators
previously demonstrated to function as a coactivator for ER
(32, 33,
54), indeed resulted in inhibition of TIF2-mediated coactivation in an
SHP concentration-dependent manner. We further reasoned
that if SHP is acting as a dominant-negative AF-2 inhibitor, then
increasing amounts of TIF2 should overcome SHP-mediated inhibition. The
experiment shown in Fig. 3C clearly confirms this assumption.
SHP Competes for Direct Binding of TIF2 to the ER
AF-2
Domain--
Our transient transfection studies suggested that SHP and
AF-2 coactivators, respectively, may exert antagonistic functions on
the ER AF-2. Since SHP has been demonstrated to contain repression domains (50) (DAX-1 homology box, see Fig. 1A),
it may utilize dominant repression mechanisms to inhibit AF-2 even in
the presence of AF-2 coactivators. Alternatively, a competition model,
as recently proposed for RIP140 (51), could explain the
dominant-negative effect of SHP in mammalian cells. To investigate
whether binding of SHP to the LBD/AF-2 domain occurs simultaneously or
competitively with other LBD/AF-2 cofactors, we performed in
vitro competition studies based on the GST pull-down assay.
Specifically, the binding of radiolabeled in vitro
translated cofactors and/or dimerization partners (TIF2, RIP140, SHP,
and ER
) to purified GST-ER
LBD/AF-2 fusion protein was assessed
in the absence or presence of purified histidine-tagged SHP WT protein,
respectively (Fig. 4A-D). The following results were observed: 1) the functionality of the assay is
demonstrated in Fig. 4A, as purified SHP almost completely eliminated
binding of SHP generated by in vitro translation; 2) purified SHP apparently competed for binding of the
ligand-dependent AF-2 cofactors TIF2 or RIP140 (Fig. 4,
B and C); 3) purified SHP did not compete for
binding of the ligand-independent homodimerization partner ER
(Fig.
4D), indicating that the competition seen with TIF2 and
RIP140 was not due to nonspecific protein effects and, more
importantly, suggesting that SHP does not interfere with LBD-mediated
ER
homodimerization; 4) the binding of purified SHP to purified
GST-ER
could be confirmed using Western blot analysis (data not
shown) and supported the direct character of the interaction between
the two proteins.

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 4.
SHP competes for binding of TIF2 or RIP140 to
ER AF-2 in vitro. A-D, purified SHP
displaces in vitro translated SHP, TIF2, or RIP140 but not
in vitro translated ER . GST-ER (aa 249-595) was
incubated with in vitro translated proteins as indicated in
the absence or presence of His-tagged SHP (aa 1-260) and in the
absence (Me2SO) or presence of 17 -estradiol.
E and F, purified RIP140 or TIF2, respectively,
displaces in vitro translated SHP but not in
vitro translated ER . GST-ER was incubated with in
vitro translated proteins in the absence or presence of His-tagged
RIP140 (aa 747-1158) or TIF2 (aa 594-766), respectively, and in the
absence (Me2SO) or presence of 17 -estradiol
(E2). The input represents 20% of the amount of labeled
protein used in each pull down.
|
|
For verification, the competition assay was additionally performed in
the reciprocal arrangement using purified receptor interaction domains
of TIF2 or RIP140, respectively, as competitor and using in
vitro translated SHP, or for control, ER
. As demonstrated in
Fig. 4, E and F, binding of in vitro
translated SHP, but not ER
, to GST-ER
LBD/AF-2 was abolished in
the presence of either TIF2 or RIP140, confirming the results observed
earlier using purified SHP. Importantly, for all competition assays
shown in Fig. 4, equal amounts of GST-ER
protein were used, as
judged from staining of the SDS gels, and no interaction with purified GST protein alone was seen with any of the ER-interacting proteins (data not shown).
SHP mRNA Is Expressed in ER Target Tissues--
Both human and
rat SHP have been isolated originally from liver two-hybrid cDNA
libraries, and subsequent Northern hybridizations have suggested that
SHP mRNA is highly expressed only in liver and, in case of the rat
mRNA, also in heart (44, 45). Although these tissues contain high
levels of nuclear receptors that may serve as relevant SHP targets (for
example PPAR
or HNF4), only low levels of ER
mRNA, and no
ER
, are detectable in liver or heart (19). This raised the question
whether the interaction of SHP with ERs is biologically relevant with
regard to their coexpression in tissues. In this context it is worth
considering that current expression data for SHP are derived from
Northern blots testing only a limited number of tissues. To analyze SHP mRNA tissue distribution in rat more extensively and, compared with
the Northern approach, more sensitively, we prepared mRNA from 19 different rat tissues and performed RT-PCR using primers specific for
the unique SHP N terminus. Unexpectedly, by using that approach we
could detect SHP mRNA in most of the analyzed rat tissues (Fig.
5), strongly indicating that SHP is much
more widely expressed than previously thought. This might, at least in
part, be explained by the differences in sensitivity between the RT-PCR
and Northern approach.

View larger version (25K):
[in this window]
[in a new window]
|
Fig. 5.
Rat tissue distribution of SHP mRNA.
RT-PCR from the indicated rat tissues was performed as described under
"Experimental Procedures." Shown are the autoradiographs of
Southern blots after hybridization with a cDNA probe specific for
SHP (top) or a oligonucleotide probe specific for actin
(bottom).
|
|
Although our assay was only semi-quantitative, comparison to the actin
signal allowed some estimations about the relative SHP expression in
different tissues. SHP appeared to be highly expressed in uterus, lung,
liver, heart, adrenals, epididymis, olfactory lobes, and cerebellum;
moderate levels could be detected in prostate, small intestine,
stomach, thymus, and spinal cord, and low levels seemed to exist in
testis, colon, and spleen. SHP could not be detected in kidney and
pituitary, thus at the same time serving as a negative control for the
RT-PCR. Importantly, these expression data not only demonstrate that
SHP mRNA is expressed ubiquitously in rat, they also indicate the
possibility for coexpression of SHP with at least one of the two
differentially expressed ER subtypes (19) in ER target tissues such as
uterus, prostate, and testis but also in bladder, lung, adrenal, and in
certain brain regions.
SHP Is Localized in the Nucleus--
Our data indicate that SHP
inhibits ER activity in transfected cells, suggesting competition for
binding of AF-2 coactivators as one possible explanation for this
effect. To function as a negative regulator of ER activation, SHP
should be coexpressed with ERs also at the subcellular level. ERs and
coactivators such as TIF2 are believed to exist mainly in the nucleus,
consistent with the direct function of these proteins as
transcriptional (co-) factors and further consistent with the presence
of nuclear localization signals (NLS) in these proteins. In most
nuclear receptors, NLS sequences are located at the C-terminal end of the DBD and within the hinge region. However, in case of SHP, which
lacks a nuclear receptor-type DBD and apparently also any conventional
(basic) NLS-like sequence, the subcellular localization has been
uncertain and needed to be determined. To address this issue, we
constructed an expression vector for wild-type SHP (aa 1-260) fused to
the N terminus of green fluorescent protein (GFP) and transiently
transfected this construct into 293 cells. As seen in the photograph of
one representative transfected cell (Fig.
6A), the SHP-GFP fusion
protein was expressed in and localized to discrete regions of the
nucleus (for identification of the nucleus, see Hoechst stain in Fig.
6B), causing a characteristic dot pattern, which was
observed in different cell lines (data not shown). Because GFP alone,
as predicted, was not specifically localized to any subcellular
compartment in transfected cells (Fig. 6C), the nuclear
localization of the SHP-GFP fusion protein clearly indicates that SHP
is a nuclear protein.

View larger version (110K):
[in this window]
[in a new window]
|
Fig. 6.
Nuclear localization of SHP. 293 cells
were transfected with 2.0 µg of expression vector for either GFP-SHP
(A and B) or GFP alone (C and
D). 36 h after transfection, cells were fixed, and the
nuclei were stained with Hoechst 33342 (B and D).
The GFP expression was visualized by fluorescence microscopy
(A and C). Photographs in A and
B or C and D, respectively, show the
same cell.
|
|
 |
DISCUSSION |
SHP Represents a New ER-interacting Protein and Putative AF-2
Inhibitor--
The interaction of SHP with ERs represents, to our
knowledge, the first example of an orphan nuclear receptor directly
interacting with and negatively influencing the transcriptional
activity of a steroid receptor. The inhibition mechanism we propose
ascribes a novel putative function to SHP, which is not related to
conventional nuclear receptor functions (see below). The physical and
functional interaction of SHP with ERs may be biologically relevant,
because we were able to detect SHP mRNA in many target tissues
expressing at least one of the two ERs, although these expression data
clearly have to be complemented by analysis at the protein level. In
support of our RT-PCR based expression data, SHP mRNA was recently
detected in several mouse tissues such as adrenal, ovary, and testis
(55). Furthermore, since we have demonstrated that SHP is a nuclear protein, the possibility for physical interaction with ERs is given
also at the subcellular level. Because SHP lacks any obvious conventional NLS sequence, it will be important to determine which parts of SHP are responsible for its nuclear localization and whether
SHP either utilizes unconventional NLS sequences for direct transport
to the nucleus or, alternatively, indirect cotransport mechanisms.
Irrespective of the inhibition mechanism (see below), SHP represents a
novel negative coregulator for ERs, which might be able to attenuate
agonist-dependent transcriptional activation. The envisaged
inhibitory function of SHP on ER activation has implications for
feedback control mechanisms and for potential therapeutical
applications. For example, in ER-positive breast and ovarian cancer
cells, SHP would be expected to antagonize dominant ER coactivators
such as AIB1 (36). Thus, it might be important to determine whether
different SHP expression levels can account for different estrogen
responses in cells expressing ERs. Although mechanistically different
and only applicable to agonist-mediated effects,
SHP-dependent actions may complement recent models
suggesting a regulatory involvement of corepressors in certain aspects
of ER-dependent carcinogenesis (56, 57).
A Novel Mechanism by Which SHP Inhibits Nuclear Receptor
Activity--
Previous studies from Moore and co-workers (44) have
suggested that SHP may inhibit nuclear receptor signaling by two
alternative mechanisms as follows: indirectly by interfering with DNA
binding of nuclear receptor dimers, and directly via active repression mechanisms, i.e. recruiting (as yet unknown) corepressors to
ligand-activated receptors (50). In this study we have provided
evidence for a third mechanism, in which SHP is proposed to interfere
directly with AF-2 coactivator function. Although it is uncertain which of the three mechanisms accounts for the inhibition of wild-type ERs,
we have demonstrated that SHP is able to directly inhibit AF-2 activity
by competition for binding with the coactivator TIF2. This suggests
that SHP and AF-2 coactivators may contact a common surface on the
LBD/AF-2 or, alternatively, that binding of SHP to the LBD may induce
conformational changes leading to the dissociation of AF-2
coactivators. Interestingly, SHP may act similarly to RIP140, an
AF-2-binding protein of previously unknown function, which we have
recently suggested to act as a negative coregulator by competing for
coactivator binding (51). However, unlike RIP140, SHP further contains
intrinsic repression activity (50). We are currently investigating
whether the putative SHP repression domain is required or whether
competition alone is sufficient for ER inhibition by SHP in
vivo. In light of previous observations that SHP was able to
inhibit VP16-dependent transcriptional activity in
cis, but not in trans (50), it is uncertain
whether SHP will repress in the presence of simultaneously bound AF-2 coactivators. Thus, the competition we suggest may allow SHP more easily to exert its repressive function.
Similar Interaction Characteristics for SHP and AF-2
Cofactors--
Based on the evidence presented here for ERs and in
previous studies for RXR (44, 50), we noticed that SHP exhibits certain interaction characteristics similar to those expected for
ligand-dependent AF-2 cofactors, including coactivators,
but different from those expected for a conventional nuclear receptor
dimerization partner. First, SHP interacts efficiently only with the
liganded LBD/AF-2 domain. Although dimerization occasionally is
enhanced in the presence of ligands (58, 59), both ER and RXR dimers
usually associate stably also in the absence of ligands. It is
interesting to note that the RXR ligand 9-cis-retinoic acid
exerts a negative effect on dimerization of RXR with its heterodimer
partners (58, 59) but enhances the interaction with SHP (44, 50).
Furthermore, in the case of ERs, we observed significant differences
between SHP association in the presence of agonistic or antagonistic ER ligands, respectively, which were not seen for ER dimers (Refs. 15, 20,
and 21 and references therein). In addition, SHP is unique with respect
to its large number of putative nuclear receptor targets, including
nonsteroid receptors such as RXR and its heterodimer partners as well
as steroid receptors such as ERs. Significantly, RXR and all other
nonsteroid receptors do not directly interact with steroid receptors
(and vice versa). Furthermore, the nonrequirement of the SHP
C terminus including its putative dimerization helix 10/11 for
interaction with other receptors clearly points to the existence of
different interaction mechanisms for SHP, compared with conventional
nuclear receptor homo- and heterodimerization. Although the interaction
of other AF-2 cofactors appears to depend on functional
LXXLL (NR-box) motifs (30, 32, 35, 42), the central
interaction domain of SHP (aa 92-148, 50) does not contain such
motifs. Curiously, the SHP-specific N terminus, which contains an
LXXLL motif, apparently did not interact with RXR or TR
(50). These preliminary data are not necessarily contradictory
considering that certain cofactors also display NR box independent
interactions with the LBD/AF-2 domain (60, 61). Finally, although the
SHP interaction surface on the nuclear receptor LBD/AF-2 has not been
mapped yet, our own preliminary results2 suggest that the
conserved AF-2 helix 12 is necessary for interaction with SHP. This is
in agreement with the inability of SHP to interact with the
antagonist-bound ER
, in which the positional rearrangement of helix
12 is thought to interfere with coactivator binding but not with
dimerization (15), and it is further consistent with the notion that
all SHP-interacting receptors identified so far possess a conserved
helix 12 motif.
SHP and DAX-1 Represent a New Category of Nuclear Receptor
Coregulators--
Based on structural and functional parallels, we
suggest that SHP and its closest relative, the orphan receptor DAX-1,
represent a new category of negative coregulators for liganded nuclear
receptors. Recent evidence suggests that DAX-1 and the orphan receptor
SF-1 cooperate in development of steroidogenic tissues (46-48).
Interestingly, the non-conserved N terminus of DAX-1 has been
demonstrated to interact directly with SF-1 (47). Thus, as for the
interaction of SHP with other nuclear receptors, the interaction of
DAX-1 with SF-1 does not require the predicted dimerization helix
within the DAX-1 LBD. Strongly supporting our findings about SHP and TIF2, there are new indications for the existence of an overlapping interaction surface for DAX-1 and the TIF2-related SRC-1 on SF-1 (46).
Furthermore, both SHP and DAX-1 exhibit intrinsic transcriptional repression activity, in agreement with the presence of a conserved putative repression domain (see Fig. 1A) and the recent
observation that at least DAX-1 interacts with the nuclear receptor
corepressor N-CoR (46). Thus, both SHP and DAX (in the absence of
hypothetical ligands, see below) might function as negative
coregulators by recruiting conventional corepressors, which usually
bind unliganded receptors, to their transcriptionally active receptor
targets. Recently, DAX-1 has been found to bind, although not
sequence-specifically, to hairpin secondary structures in the promoter
region of putative target genes such as the StAR gene (49).
Such a DNA-binding function has yet to be established for the
SHP-specific N-terminal domain, which is different from the DAX-1 DBD
but with its approximately 50 aa is large enough to act as a separate
DNA-binding domain. If this domain functions as DBD, the intriguing
possibility exists that a putative SHP-nuclear receptor complex may
bind to, as yet unknown, novel binding sites. Additionally, in a
hypothetical situation, in which SHP would act like DAX-1 as a
DNA-bound receptor, a redirection of ligand (e.g. estrogen)
signaling to these novel binding sites could take place. Since the AF-2
domain of the interacting nuclear receptor (e.g. ER),
according to our view, may be occupied by SHP and not be available for
binding of coactivators, transcriptional activity in such a system
would depend on other activation domains, for example on the N-terminal
AF-1. This is particularly interesting in case of the ERs, since their
N termini are non-conserved and perhaps account for functional
differences of ER
and ER
on AP1 sites (8).
Despite the presence of an LBD-like domain in both SHP and DAX-1, it
has yet to be established that these proteins act as ligand-regulated
nuclear receptors. For homology reasons though, the existence of
ligands cannot be excluded, and a new cascade of events would be
expected upon binding of putative ligands. For example, a
ligand-induced conformational change within the LBD may cause
dissociation of corepressors converting the former repressor into a
transcriptional activator. This is most likely to be the case for
DAX-1, because it contains a consensus AF-2 helix 12 motif, whereas in
the predicted SHP helix 12 a glutamic acid, which is conserved in
all ligand-activable nuclear receptors (17), is changed to an aspartic
acid (see Fig. 1A). Thus, even in the presence of
hypothetical ligands, DAX-1 and SHP may exert opposing effects on their
gene or receptor targets. This might also be of biological
significance, considering the coexpression of DAX-1 and SHP with SF-1
and ERs in some steroidogenic tissues (Refs. 19, 48, and 55 and
references therein). In light of our new findings, we would finally
like to propose that a liganded SHP will probably not recruit
corepressors but still antagonize transcriptional activity of its
receptor targets by competition with coactivators for binding to the
AF-2 domain.