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J. Biol. Chem., Vol. 277, Issue 21, 18501-18509, May 24, 2002
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From the Department of Biochemistry, University of Kuopio,
FIN-70211 Kuopio, Finland
Received for publication, January 8, 2002, and in revised form, February 11, 2002
Nuclear receptors (NRs) and POU domain factors
form two important transcription factor families for which
several levels of functional interference have been described. In this
study, the adopted orphan receptors constitutive androstane receptor
(CAR) and pregnane X receptor (PXR) were found to perform direct
protein-protein interactions with Pit-1, a representative POU domain
factor. The ligand-dependent interaction profile of Pit-1
with CAR, PXR, and the vitamin D receptor in solution was shown
to be that of a corepressor. In the absence of receptor agonist Pit-1
inhibited the complex formation of NRs with the retinoid X receptor on
DNA. Also in living cells, Pit-1 and Oct-1, another POU domain factor,
behaved like corepressors of NR signaling, and Pit-1-mediated
repression was found to involve histone deacetylases. Conversely
vitamin D receptor, CAR, and PXR were shown to act as repressors of
Pit-1 signaling in different cell lines (MCF-7, HaCaT, and
GH4C1). This repression was found to be independent of histone
deacetylases and seems to be based on a competition of NRs with
coactivator and corepressor proteins for overlaying interaction
interfaces on the surface of Pit-1. Taken together this study suggests
that cross-repression should occur in all tissues in which POU domain factors and non-liganded NRs meet each other.
The adopted orphan receptors constitutive androstane receptor
(CAR,1 NR1I3) and pregnane X
receptor (PXR, NR1I2) (1) are members of the nuclear receptor (NR)
superfamily that regulates target gene transcription in a
ligand-dependent manner. CAR and PXR have a rather broad,
overlapping set of ligands that range from natural steroids to
xenobiotics and also recognize similar DNA binding sites, referred to
as response elements (REs), primarily in promoter regions of the
cytochrome P450 gene family (2). CAR and PXR are closely related to
each other as well as to another NR superfamily member, VDR (NR1I1),
since they share ~40% amino acid identity of their ligand binding
domain. VDR is bound with high affinity (Kd in the
order of 0.1 nM) by its natural ligand
1 Crystal structure analysis of more than 10 presently characterized NR
ligand binding domains has demonstrated a conserved spatial structure
formed by 11 or 12 POU domain factors, such as Pit-1, Oct-1, and
Unc-86, form another large family of transcription factors,
which are of special importance during development (14). Since some
nuclear receptors are also known to have critical roles in development
(15), a possible functional interference between members of the POU
domain factor family and NR superfamily has been investigated quite
extensively. For example, it had been found that pit-1 gene
expression is down-regulated by thyroid hormone (16), that prolactin
gene expression is synergistically activated by Pit-1 and either the
VDR or the estrogen receptor via neighboring binding sites in the
prolactin promoter (17, 18), and that Pit-1 interacts physically with
the thyroid hormone receptor (T3R) and retinoid acid
receptor (RAR) (19).
This study was performed with the aim of attaining a better
understanding of the multitude of protein-protein interactions of NRs.
Therefore, direct protein-protein interactions between VDR, CAR,
and PXR, as representative NRs, and Pit-1, as a representative POU
domain factor, were investigated. It was shown that Pit-1 can act as a
repressor of NR signaling and that conversely NRs can function as
repressors of Pit-1 signaling. This crosswise repressor action,
referred to as cross-repression, was demonstrated only for VDR, CAR,
PXR, Pit-1, and Oct-1, but it possibly applies to all those members of
these two transcription factor families that are coexpressed in the
same tissue and that are able to participate in a direct
protein-protein interaction.
Compounds
Androstanol was from Steraloids (Newport, RI);
1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) was synthesized
and purified according to Honkakoski et al. (20); the HDAC
inhibitor trichostatin A (TSA) was from Biomol (Plymouth Meeting, PA);
sodium butyrate, rifampicin, and valproic acid (2-propylpentanoic acid)
were from Sigma; and 1 DNA Constructs
Protein Expression Vectors--
Full-length cDNAs for human
PXR (21), human VDR (22), human RXR Glutatione S-Transferase (GST) Fusion Protein
Constructs--
The cDNAs for the NR interaction domains of mouse
NCoR (spanning amino acids 1679-2453) and human SRC-1 (spanning amino
acids 596-790) and for full-length rat Pit-1 were subcloned into the GST fusion protein vector pGEX (Amersham Biosciences).
Reporter Gene Constructs--
Three copies of the Pit-1 RE from
the rat growth hormone (GH) promoter (core sequence
5'-CATGAATAAATGTA-3' with core binding
motifs in bold) (35) and four copies of the DR4-type RE from the human
inducible NO synthase promoter (core sequence
5'-GTGGTTCATGCCGGTTCA-3') (36) were fused with
the thymidine kinase (tk) minimal promoter driving the
firefly luciferase (LUC) reporter gene.
In Vitro Translation and Bacterial Overexpression of Proteins
In vitro translated VDR, PXR, CAR, and RXR proteins
were generated by coupled in vitro transcription/translation
using rabbit reticulocyte lysate as recommended by the supplier
(Promega, Mannheim, Germany). By test translation in the presence of
[35S]methionine and taking the individual numbers of
methionine residues per receptor into account, the specific
concentration of the receptor proteins was adjusted to ~4 ng/µl.
Bacterial overexpression of GST-Pit-1FL,
GST-NCoR-(1679-2453), and GST-SRC-1-(596-790) was facilitated
in the Escherichia coli BL21(DE3)pLysS strain (Stratagene). GST-SRC-1-(596-790) and GST-Pit-1FL fusion protein
expression was stimulated with 0.25 mM
isopropyl- GST Pull-down Assays
GST pull-down assays were performed with 50 µl of a 50%
Sepharose bead slurry of GST-Pit-1FL,
GST-NCoR-(1679-2453), or GST-SRC-1-(596-790) (preblocked with 1 µg/µl bovine serum albumin) and 20 ng of in vitro
translated, 35S-labeled NRs in the presence or absence of
their respective ligands. Proteins were incubated in
immunoprecipitation buffer (20 mM Hepes (pH 7.9), 200 mM KCl, 1 mM EDTA, 4 mM
MgCl2, 1 mM dithiothreitol, 0.1% Nonidet P-40,
and 10% glycerol) for 20 min at 30 °C. For competition experiments,
the peptides CoRNR2NCoR (SNLGLEDIIRKAL with core
binding motifs in bold) or CoANR2TIF-2
(KHKILHRLLQDSS) were added before the addition of
ligand. In vitro translated proteins that were not bound to
GST fusion proteins were washed away with immunoprecipitation buffer.
GST fusion protein-bound 35S-labeled NRs were resolved by
electrophoresis through 10% SDS-polyacrylamide gels and quantified on
a Fuji FLA3000 reader (Tokyo, Japan) using Image Gauge software (Fuji).
Gel Shift Assays
Gel shift assays were performed with equal amounts (~10 ng) of
in vitro translated VDR, CAR, PXR, and RXR and bacterially expressed GST-Pit-1FL (~3 µg) and GST (as a control).
Proteins were incubated on ice for 15 min in a total volume of 20 µl
of binding buffer (10 mM Hepes (pH 7.9), 150 mM
KCl, 1 mM dithiothreitol, 0.2 µg/µl poly(dI-dC),
and 5% glycerol). For competition experiments, increasing
concentrations of the peptides CoRNR2NCoR,
CoANR2TIF-2, or AF-2RXR
(TFLMEMLEAPHQMT) were added to the reaction mixture.
Approximately 1 ng of 32P-labeled double-stranded
oligonucleotides (50,000 cpm) corresponding to one copy of the Pit-1 RE
from the rat GH promoter (core sequence 5'-CATGAATAAATGTA-3') (35) or the
DR4-type RE from the rat Pit-1 promoter (core sequence
5'-GAAGTTCATGAGAGTTCA-3') (37), respectively,
were then added, and the incubation was continued for 20 min at room
temperature. Protein-DNA complexes were resolved by electrophoresis
through 8% non-denaturing polyacrylamide gels in 0.5× TBE (45 mM Tris, 45 mM boric acid, and 1 mM
EDTA (pH 8.3)) and quantified on a Fuji FLA3000 reader using Image Gauge software.
Transfection and Luciferase Reporter Gene Assays
MCF-7 human breast cancer cells, HaCaT human immortalized
keratinocytes, and GH4C1 rat pituitary tumor cells were seeded into six-well plates (105 cells/ml) and grown overnight in
phenol red-free Dulbecco's modified Eagle's medium supplemented with
5% (or 10% in the case of GH4C1 cells) charcoal-treated fetal bovine
serum. Liposomes were formed by incubating 1 µg of the reporter
plasmid and the indicated combinations of expression vectors for Pit-1,
VDR, CAR, PXR, RXR, NCoR, or SRC-1 (each 1 µg if not otherwise
indicated) with 10 µg of
N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate (DOTAP, Roth, Karlsruhe, Germany) for 15 min at room
temperature in a total volume of 100 µl. After dilution with 900 µl
of phenol red-free Dulbecco's modified Eagle's medium, the liposomes
were added to the cells. Phenol red-free Dulbecco's modified Eagle's
medium supplemented with 15% charcoal-treated fetal bovine serum (500 µl, 30% in the case of GH4C1 cells) was added 4 h after
transfection. At this time, NR ligands or histone HDAC inhibitors were
also added. The cells were lysed 16 h after onset of stimulation
using the reporter gene lysis buffer (Roche Diagnostics), and the
constant light signal luciferase reporter gene assay was performed as
recommended by the supplier (Canberra-Packard, Dreieich, Germany). The
luciferase activities were normalized with respect to protein
concentration, and induction factors were calculated as the ratio of
luciferase activity of ligand-stimulated cells to that of solvent controls.
The physical interaction between Pit-1 and a selection of 10 different NRs was assessed by GST pull-down assays using bacterially produced GST-Pit-1FL fusion protein immobilized on
Sepharose beads and in vitro translated,
35S-labeled NR protein (Fig.
1). This qualitative screening suggested a direct protein-protein interaction between Pit-1 and human VDR, chicken T3R The interaction of Pit-1 with the PXR and CAR has not been reported yet
and was investigated in more detail using human PXR and mouse
CAR in reference to human VDR. First, the ligand-dependent interaction of NRs with Pit-1 was compared with their interaction with
the corepressor NCoR and the coactivator SRC-1. GST pull-down assays
were performed with bacterially expressed GST-Pit-1FL, GST-NCoR-(1679-2453), and GST-SRC-1-(596-790) and full-length in vitro translated, 35S-labeled human VDR,
human PXR, and mouse CAR in the absence and presence of their
respective ligands (Fig. 2). The
interaction between Pit-1 and VDR was found to decrease after addition
of the natural VDR agonist 1
Cross-repression, a Functional Consequence of the Physical
Interaction of Non-liganded Nuclear Receptors and POU Domain
Transcription Factors*
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
,25-dihydroxyvitamin D3
(1,25(OH)2D3) (3), whereas CAR and PXR are
bound by an overlapping set of natural and synthetic ligands with
rather low affinity (Kd in the order of 1 µM) (4). Interestingly CAR differs from most other NRs by
displaying in the absence of ligand already a relatively high
constitutive activity, which can be reduced by the binding of the
inverse agonist 5-androstan-3-ol (androstanol) (5). CAR and PXR
each form a heterodimer with RXR on DR4-type REs (6) but also recognize
other RE types. Similarly classical VDR binding sites have only three
spacing nucleotides between the receptor binding motifs, but VDR-RXR
heterodimers show even higher affinity for DR4-type REs (7, 8).
-helices (9). The ligand binding domain has
diverse functions; it is not only involved in ligand binding but also
in interaction with other NRs for the formation of homo- and
heterodimeric complexes, and it is in contact with nuclear mediator
proteins, such as coactivators and corepressors, for modulation of
transcriptional activities. Contact points for coactivators, such as
SRC-1, TIF-2, and RAC3 (10), have been mapped in the activation
function-2 (AF-2) domain of helix 12 and in helix 3 (11), and also
within the less well understood interaction surface for corepressors,
such as NCoR, SMRT (silencing mediator for retinoid and thyroid
hormone receptors), and Alien, the AF-2 domain was found to be of
importance (12). The classical corepressors act as a specific bridge
between transcription factors and histone deacetylases (HDACs), which
are enzymes that locally close chromatin (13).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
,25(OH)2D3 was kindly
provided by L. Binderup (Leo Pharmaceutical Products, Ballerup,
Denmark). 1,25(OH)2D3 was dissolved in
2-propanol, whereas all other compounds were dissolved in dimethyl
sulfoxide (Me2SO); further dilutions were made in Me2SO (for in vitro experiments) or in ethanol
(for cell culture experiments).
(23), human RAR
(24), chicken
T3R (25), mouse PXR (26), human CAR (27),
Xenopus PPAR (28), and human PPAR (29) were subcloned
into the T7/SV40 promoter-driven pSG5 expression vector
(Stratagene, Heidelberg, Germany); full-length cDNAs for mouse CAR
(30), mouse NCoR (31), and human SRC-1 (32) were subcloned into the
T7/cytomegalovirus promoter-driven pCMX expression vector;
full-length cDNAs for rat Pit-1 (33) and human Oct-1 (34) were
subcloned under the control of the Rous sarcoma virus promoter into the
vector pUC18; and the full-length cDNA for rat Pit-1 was also
subcloned into the T7 promoter-driven vector pBluescript
SK(
) (Stratagene). Truncated versions of VDR (VDR
413-27), PXR (PXR421-35), and CAR
(CAR349-58), which were lacking their AF-2 domain, were
generated by introducing via point-directed mutagenesis a stop codon at
positions 413, 349, and 421, respectively, and confirmed by
sequencing. The T7 promoter-driven constructs are suitable
for T7 RNA polymerase-driven in vitro
transcription/translation of the respective cDNAs, and the SV40-,
cytomegalovirus-, and Rous sarcoma virus-driven constructs were used
for overexpression of the respective proteins in mammalian cells.
-D-thio-galactopyranoside for 3 h at
37 °C, and GST-NCoR-(1679-2453) expression was induced with 1.25 mM isopropyl--D-thio-galactopyranoside for
5 h at 25 °C. The fusion proteins were purified and immobilized
by glutathione-Sepharose 4B beads (Amersham Biosciences) according to
the manufacturer's protocol. For gel shift experiments the fusion
proteins were eluted by glutathione.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
, human or mouse PXR, human or mouse CAR,
Xenopus PPAR, or human PPAR but not between Pit-1 and
human RXR or human RAR. GST protein alone was not able to
interact with these receptors (data not shown).
View larger version (25K):
[in a new window]
Fig. 1.
Pit-1 physically interacts with various but
not all NRs. GST pull-down assays were performed with bacterially
expressed GST-Pit-1FL fusion protein and in
vitro translated, 35S-labeled human VDR, human RXR ,
human RAR, chicken T3R, human PXR, mouse PXR, human
CAR, mouse CAR, Xenopus PPAR, and human PPAR. After
precipitation and washing, the proteins were electrophoresed through
10% SDS-polyacrylamide gels. Representative gels are shown. The
upper panel shows the NR input, and the lower
panel shows the specifically bound proteins.
,25(OH)2D3,
which resembles the interaction profile of VDR with NCoR but is in
contrast to that of VDR and SRC-1 (Fig. 2A). A similar
tendency was observed for human PXR and its synthetic agonist
rifampicin: the interaction of PXR with Pit-1 was clearly reduced in
the presence of ligand, and also the interaction of PXR with NCoR was
slightly but significantly reduced, whereas for the interaction of PXR
with SRC-1 no significant ligand-dependent effect was found
(Fig. 2B). The profile of the ligand-dependent
interaction of mouse CAR with Pit-1 was found to be very similar to
that with NCoR and inverse to that of SRC-1 (Fig. 2C). The
synthetic agonist TCPOBOP decreased the interaction between CAR and
Pit-1 or NCoR but increased the contact of CAR with SRC-1. The inverse
agonist androstanol showed the opposite tendency: it slightly increased
the interaction between CAR and Pit-1 or NCoR and decreased the contact
of CAR with SRC-1. The combined treatment with TCPOBOP and androstanol
resulted in effects slightly weaker than that of TCPOBOP alone but
still significantly different from the solvent control.
View larger version (15K):
[in a new window]
Fig. 2.
The direct interaction of Pit-1 with NRs
resembles that of corepressors but not that of coactivators. GST
pull-down assays were performed with bacterially expressed
GST-Pit-1FL, GST-NCoR-(1679-2453), and
GST-SRC-1-(596-790) and full-length in vitro translated,
35S-labeled human VDR (A), human PXR
(B), and mouse CAR (C) in the absence and
presence of their respective ligands. GST alone was used as control.
After precipitation and washing, the samples were electrophoresed
through 10% SDS-polyacrylamide gels, and the percentage of
precipitated NRs in respect to input was quantified using a Fuji
FLA3000 reader. Representative gels are shown. Columns
represent mean values of triplicates, and the bars indicate
S.D. Statistical analysis was performed by two-tailed, paired
Student's t test, and p values were calculated
in reference to respective solvent controls (*, p < 0.05; **, p < 0.01). DMSO,
Me2SO.
In many cases the AF-2 domain of NRs has been shown to mediate their
ligand-dependent protein-protein interaction. Therefore, the agonist-triggered interaction of human VDR and mouse CAR with Pit-1
was compared with that of truncated versions of the receptors, which
lack their C-terminal AF-2 domains. GST pull-down assays were performed
with bacterially expressed GST-Pit-1FL and in
vitro translated, 35S-labeled human
VDRWT, VDR413-27, CARWT,
and CAR349-58 (Fig. 3).
As already shown in Fig. 2, in the presence of the agonists 1,25(OH)2D3 and TCPOBOP the interaction of
full-length VDR and CAR, respectively, with Pit-1 was clearly reduced.
In contrast, in the absence of agonist the interaction of
VDR413-27 and CAR349-58 with Pit-1 was
found to be already at approximately the same low level as the
respective full-length receptor in the presence of agonist but did not
respond further to ligand addition.
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The AF-2 domain has been shown to be critical for the interaction of
NRs both with coactivator proteins as well as with corepressor proteins. Therefore the question of whether Pit-1 competes with corepressors and coactivators for the binding to the AF-2 domain of VDR
and CAR was addressed. GST pull-down assays were performed with
bacterially expressed GST-Pit-1FL and in vitro
translated, 35S-labeled human VDR and mouse CAR in the
presence of increasing concentrations of the peptides
CoRNR2NCoR and CoANR2TIF-2 (Fig. 4). The peptides represent specific NR
interaction domains of the corepressor NCoR and the coactivator TIF-2,
respectively (38, 39). In the absence of ligand, increasing
concentrations of CoRNR2NCoR but not of
CoANR2TIF-2 were shown to be able to reduce the interaction
between Pit-1 and VDR and CAR, i.e. corepressors compete
with Pit-1 for binding to both receptors. In contrast, in the presence
of agonist, CoANR2TIF-2 but not CoRNR2NCoR was found to compete effectively with Pit-1 for binding to VDR and CAR. As
a control, competition experiments were performed with a peptide of the
same length and concentration representing the AF-2 domain of human
RXR (AF-2RXR). This peptide had no effect on the
interaction of Pit-1 with VDR and CAR in either the absence or presence
of receptor agonists (data not shown).
|
The previous experiments demonstrated the interaction of Pit-1 with NRs
in solution. As the next step this interaction was assessed bound to
DNA by performing gel shift experiments with bacterially expressed
GST-Pit-1FL on the Pit-1 RE of the rat GH gene in the
presence of in vitro translated human VDR, mouse CAR, human
PXR, or human RXR (Fig. 5). The RE
showed to be specific for Pit-1 since only the addition of Pit-1 but
not of any of the four NRs resulted in a detectable protein-DNA
complex. Interestingly the preincubation of Pit-1 with VDR, CAR, and
PXR resulted in a slower migrating protein-DNA complex and indicated
that VDR, CAR, and PXR can bind to DNA-bound Pit-1. In contrast, the
addition of RXR was without any effect and confirmed the observation
from Fig. 1 that RXR cannot interact with Pit-1. Competition
experiments with the corepressor peptide CoRNR2NCoR and the
coactivator peptide CoANR2TIF-2 resulted at higher
concentrations in a loss of the "supershifted" protein-DNA complex,
whereas the same amount of the unspecific peptide AF-2RXR
showed no effect. This suggests that corepressor proteins as well as
coactivator proteins are able to compete with DNA-bound Pit-1 for the
binding to NRs.
|
The inverse question to the point addressed in the previous experiment
(Fig. 5) was whether Pit-1 also could interact with DNA-bound NRs.
Therefore, gel shift experiments were performed with in
vitro translated heterodimers between human VDR and human RXR
and between mouse CAR and human RXR and the DR4-type RE of the rat
Pit-1 gene (Fig. 6). As reported
previously (7), VDR-RXR heterodimers demonstrated a clearly
agonist-triggered complex formation on this RE, whereas the DNA binding
of CAR-RXR heterodimers was shown to be ligand-independent.
Interestingly the preincubation of VDR-RXR and CAR-RXR heterodimers
with increasing concentrations of bacterially expressed
GST-Pit-1FL and GST alone (as a control) resulted in a
Pit-1-specific inhibition of DNA complex formation of the heterodimers
only in the absence but not in the presence of receptor agonists.
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In the previous in vitro experiments (Figs. 2 and 6) the
interaction between NRs and Pit-1 looked more like a crosswise
repression (here referred to as cross-repression) than a synergistic
activation. To study the interaction of Pit-1 with VDR, CAR, and PXR in
living cells, luciferase reporter gene assays were performed in MCF-7 and HaCaT cells, which both represent model cell lines for NR signaling. The cells were transiently transfected with a reporter gene
construct driven by three copies of the rat GH Pit-1 RE and expression
vectors for rat Pit-1, mouse NCoR, human VDR, mouse CAR, and human
PXR (the latter three NRs each in combination with human RXR, see
Fig. 7). The overexpression of Pit-1
resulted for both cell lines in an ~4-fold increase of basal reporter
gene activity (column 4). Both in MCF-7 and in HaCaT cells
Pit-1 signaling was clearly repressed by the overexpression of NCoR
(column 7) and even reduced below basal activity by the
overexpression of VDR, CAR, and PXR (columns 10,
13, and 16). The repression of Pit-1 signaling
was specific to VDR, CAR, and PXR since the overexpression of their
heterodimeric partner RXR alone showed no effect (columns 19 and 21). The NCoR-mediated repression of Pit-1 signaling
could be released by the HDAC inhibitors TSA, sodium butyrate, and
valproic acid (columns 8 and 9). In contrast, the
HDAC inhibitors showed only minor effects on the NR-mediated repression
of Pit-1 signaling (columns 11, 12,
14, 15, 17, and 18).
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GH4C1 rat pituitary tumor cells endogenously express sufficient amounts
of Pit-1 protein so that additional overexpression of the transcription
factor was not able to booster Pit-1 signaling (data not shown).
Reporter gene assays were performed in GH4C1 cells that were
transiently transfected with a reporter gene construct driven by three
copies of the rat GH Pit-1 RE and expression vectors for human SRC-1,
mouse NCoR, human VDR and its AF-2 deletion mutant VDR413-27, mouse CAR and its AF-2 deletion mutant
CAR349-58, and human PXR and its AF-2 deletion mutant
PXR421-35 (Fig. 8). The
overexpression of SRC-1 increased the basal Pit-1 activity
(column 1) by ~40% (column 3), whereas the
overexpression of NCoR reduced it by approximately the same amount
(column 5). All three tested NRs repressed Pit-1 signaling
to a level of 10-30% basal activity (columns 7,
11, and 15). Again this repression was specific
to VDR, CAR, and PXR since the overexpression of RXR showed no effect
(columns 19 and 21). AF-2 domain deletion mutants
of VDR, CAR, and PXR showed less repression of Pit-1 signaling (columns 9, 13, and 17), which
resulted in activities that were approximately double those observed
with the respective full-length receptors (columns
7, 11, and 15). The treatment with the
HDAC inhibitor TSA resulted in an ~50% increase of basal Pit-1
activity (column 2). The same level of Pit-1 signaling was
also reached by TSA-treated, SRC-1- or NCoR-overexpressing cells
(columns 4 and 6) and appears to represent the
maximal Pit-1 activity in GH4C1 cells. TSA treatment also resulted in a
release of NR-mediated repression of Pit-1 signaling (columns
8, 12, and 16). However, this inhibition of
repression was only incomplete since only activities significantly
below the maximal Pit-1 activity were obtained.
|
For the inverse functional experiment, reporter gene assays were
performed in MCF-7 cells that were transiently transfected with a
reporter gene construct driven by four copies of the human inducible NO
synthase DR4-type RE and expression vectors for rat Pit-1, human Oct-1,
mouse NCoR, and mouse CAR (Fig. 9). The
overexpression of CAR increased basal reporter gene activity on average
~10-fold (column 7), whereas the overexpression of Pit-1
(column 3), NCoR (A, column 5), and
Oct-1 (B, column 5) alone showed only minor effects on DR4-type RE-driven gene activity (column 1). The
combined overexpression of CAR and Pit-1 (A, column
9, and B, column 13) or NCoR (A,
column 11) resulted in ~50% reduced gene activity. Interestingly in the presence of agonist TCPOBOP the overexpression of
Pit-1 and NCoR (A, columns 10 and 12)
was of no significant effect on CAR signaling (A,
column 8), i.e. Pit-1 and NCoR act only in the
absence of ligand as corepressors. The repressing effect of Pit-1
overexpression on CAR signaling was found to be dose-dependent (B, columns 9,
11, and 13). Oct-1, another prominent POU domain
family member, showed comparable repressing effects on CAR signaling
(B, columns 15, 17, and
19), and the combined overexpression of Pit-1 and Oct-1 even
resulted in super-repression down to 25% of maximal CAR activity
(B, columns 21, 23, and
25). However, the HDAC inhibitor TSA was found to release
the repressing effect of Pit-1 on CAR signaling completely (compare
column 8 with columns 10, 12, and
14). Moreover, the data appear to suggest that TSA releases
the repression by Oct-1 only partially (compare column 8 with columns 16, 18, 20,
22, 24, and 26), but this effect was
found not to be statistically significant.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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The qualitative screening of 10 different NRs for their ability to perform direct protein-protein interaction with Pit-1 resulted in 80% positive results. Classical endocrine receptors, such as VDR and T3R, as well as adopted orphan receptors, such as CAR, PXR, and PPAR, were found to contact Pit-1 directly. The interaction of Pit-1 with VDR (17), T3R (19), and PPAR (40) has already been shown earlier, whereas the interaction between Pit-1 and CAR or PXR is reported here for the first time. Palomino et al. (19) also have reported a direct contact between Pit-1 and RAR or RXR, which could not been confirmed in this study either in solution (Fig. 1) or bound to DNA (Fig. 5).
The ligand-dependent interaction profile of Pit-1 with VDR, CAR, or PXR definitely more closely resembles that of the corepressor NCoR than that of the coactivator SRC-1 since the addition of receptor agonist definitely reduces the Pit-1-NR interaction in solution (Fig. 2). Not all regions of the NR ligand binding domains that contribute to the corepressor interaction interface have been mapped yet, but it seems to be clear that helix 12 with its AF-2 domain is involved (12). Therefore, the finding reported herein that the deletion of the AF-2 domain clearly reduces the affinity of NRs for Pit-1 and abolishes the ligand dependence of this interaction (Fig. 3) fits well with the idea that Pit-1 may act as a corepressor to some NRs. Further support of this concept is provided by the observation that the NR interaction domain of NCoR can compete with Pit-1 for binding to VDR and CAR (Fig. 4). This indicates that non-liganded NRs have clearly overlaid interaction interfaces for Pit-1 and NCoR on their surfaces.
The interaction of NRs with Pit-1 was found not to interfere with the DNA binding of Pit-1 since NRs can supershift Pit-1-DNA complexes (Fig. 5). Interestingly peptides that represent the receptor interaction domain of corepressors as well as those that represent interaction domains of coactivators were shown to be able to prevent these supershifts in the absence of NR agonists, but they did not affect the amount Pit-1-DNA complexes. In turn, this suggests that NRs may be able to inhibit the interaction of Pit-1 proteins with corepressor as well as with coactivator proteins, i.e. NRs may block both repression and activation of Pit-1 activity. Conversely in the absence of ligand Pit-1 is able to inhibit (at least partially) the DNA complex formation of VDR-RXR, CAR-RXR, and PXR-RXR heterodimers (Fig. 6). This effect could be explained by a steric hindrance caused by Pit-1 that either blocks heterodimerization of VDR or CAR with RXR (being essential for effective DNA binding) or prevents the contact between the DNA binding domain of the NRs and DNA. However, the observation that Pit-1 is not able to inhibit the DNA binding of NR heterodimers in the presence of their agonist suggests that, at least in complex with DNA, the agonistic receptor conformation does not interact effectively with Pit-1. This is another indication that Pit-1 acts rather as a repressor than as an activator of NR activity.
A repressing effect of NR on Pit-1 signaling as predicted by the in vitro experiments (Figs. 2-5) could be demonstrated in the model cell lines MCF-7 and HaCaT (Fig. 7) as well as in endogenously Pit-1-responding GH4C1 cells (Fig. 8). The repressing function of NCoR, which is known as a repressor of Pit-1 signaling (41), could be released completely by different HDAC inhibitors. In contrast, the same inhibitors caused only a partial release of NR-mediated repression of Pit-1 signaling. In addition, the functional assays confirmed the important role of the AF-2 domains of VDR, CAR, and PXR for the repressing action of the receptors on Pit-1 signaling, but the partial release of repression through deletion of the AF-2 domain also indicated that helix 12 is only a part of the interaction interface with Pit-1. Taken together the functional assays suggest that NRs do not act as classical repressors that recruit HDACs to locally close chromatin, but they most likely inhibit activation of Pit-1 by preventing its interaction with coactivator proteins.
For CAR signaling the repressing potential of Pit-1 on NR activity was demonstrated in the model cell line MCF-7 (Fig. 9). Comparable to NCoR, Pit-1 showed no effect in the presence of receptor agonists and confirmed the results of the in vitro experiments (Fig. 6). Interestingly another POU domain factor, Oct-1, was also found to be able to repress CAR-signaling. This supports the concept of a general interaction potential between members of the POU domain factor family and the NR family. Another interesting aspect is that the repressing effect of Pit-1 on CAR signaling could be released completely by treatment with HDAC inhibitors. This suggests that Pit-1 may be able to recruit HDACs directly (or most likely indirectly). However, this property seems not to be general for POU domain factors since it was found that TSA has only minor effects on Oct-1-mediated repression of CAR signaling.
It is obvious that not all members of the NR superfamily and the POU domain family are expressed in the same tissue or cell line. This makes it well possible that not all interactions that have been detected in vitro are of physiological relevance. However, as demonstrated in this study, Pit-1 is rather promiscuous in its interaction with NRs so that it will certainly find a partner receptor in every Pit-1-expressing tissue. Moreover, since the interaction of Pit-1 with NRs seems to be largely independent from receptor agonists, it may be not very critical which exact member of the NR superfamily Pit-1 finds as a partner. Pit-1 itself has a very restricted expression pattern and is primarily found in the anterior pituitary gland. The gland shows robust expression of VDR (42) but only low levels of CAR and PXR mRNA expression.2 In contrast, Oct-1 is rather ubiquitously expressed and seems to be able to take a similar repressor role as Pit-1 in tissues such as liver and intestine where high expression levels of CAR and PXR are found (26, 27).
For the prolactin gene promoter a synergistic activation by
Pit-1 and VDR has been observed, which appears to be in contrast to the
results reported in this study (17). However, in the case of the
prolactin promoter both transcription factors are able bind to DNA, and
the synergistic activation effects were primarily obtained by treatment
with the agonist 1,25(OH)2D3 and not by VDR
overexpression. In contrast, the effects described here were mainly
observed in the absence of NR agonists. Cross-repression may
apply more likely to those genes that carry a binding site for either a
NR or a member of the POU domain family but not for both together in
their promoter region. In the rare case, such as the prolactin
promoter, where two such binding sites are in close vicinity to each
other, mechanisms as described by Castillo et al. (17) may
overcome the cross-repression described in this study.
In conclusion, this study has demonstrated that Pit-1 can act as a
repressor of NR signaling, and conversely NRs have been shown to act as
repressors of Pit-1 signaling. This cross-repression is mediated by a
direct protein-protein interaction between the NR and Pit-1. Although
this principle has been demonstrated only for the NRs VDR, CAR, and PXR
and the POU domain family members Pit-1 and Oct-1, the cross-repression
described here may apply to all members of the two transcription factor
families for which a direct protein-protein interaction can be demonstrated.
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ACKNOWLEDGEMENTS |
|---|
We thank M. Hiltunen for skilled technical
assistance, L. Binderup for 1,25(OH)2D3, P. Honkakoski for TCPOBOP, and A. Aranda, S. Kliewer, and S. Rhodes for
protein expression vectors.
| |
FOOTNOTES |
|---|
* This work was supported by Academy of Finland Grants 50319 and 50331 (to C. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of
Biochemistry, University of Kuopio, P.O. box 1627, FIN-70211 Kuopio, Finland. Tel.: 358-17-163062; Fax: 358-17-2811510; E-mail:
carlberg@messi. uku.fi.
Published, JBC Papers in Press, March 12, 2002, DOI 10.1074/jbc.M200205200
2 M. Macias Gonzalez and C. Carlberg, unpublished results.
| |
ABBREVIATIONS |
|---|
The abbreviations used are:
CAR, constitutive
androstane receptor;
1, 25(OH)2D3,
1,25-dihydroxyvitamin D3;
AF-2, activation function-2;
androstanol, 5-androstan-3-ol;
DR4, direct repeat spaced by 4 nucleotides;
HDAC, histone deacetylase;
GH, growth hormone;
GST, glutathione S-transferase;
NR, nuclear receptor;
PPAR, peroxisome proliferator-activated receptor;
PXR, pregnane X receptor;
RAR, retinoic acid receptor;
RE, response element;
RXR, retinoid X
receptor;
T3R, thyroid hormone receptor;
TCPOBOP, 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene;
TSA, trichostatin A;
VDR, 1,25(OH)2D3 receptor;
NCoR, nuclear receptor
corepressor;
LUC, luciferase;
tk, thymidine kinase;
FL, full
length;
WT, wild type.
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REFERENCES |
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TOP
ABSTRACT
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MATERIALS AND METHODS
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