From the George Whipple Laboratory for Cancer Research, Department of Pathology, Urology, Radiation Oncology, and The Cancer Center, University of Rochester Medical Center, Rochester, New York 14642
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ABSTRACT |
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In situ hybridization analysis
demonstrated that abundant testicular orphan receptor (TR4) transcripts
were detected in kidney, intestine, and bone, which are vitamin
D3 target organs. Cell transfection studies also
demonstrated that the expression of the vitamin D3 target
gene, 25-hydroxyvitamin D3 24-hydroxylase, can be repressed
by TR4 through high affinity binding (Kd = 1.32 nM) to the direct repeat 3 vitamin D3 receptor
response element (DR3VDRE). This TR4-mediated repression of DR3VDRE is in contrast to our earlier report that TR4 could induce thyroid hormone
target genes containing a direct repeat 4 thyroid hormone response
element (DR4T3RE). Electrophoretic mobility shift assay using several TR4 monoclonal antibodies when combined with either TR4-DR3VDRE or TR4-DR4T3RE showed a distinct supershifted
pattern, and proteolytic analysis further demonstrated distinct
digested peptides with either TR4-DR3VDRE or TR4-DR4T3RE.
These results may therefore suggest that TR4 can adapt to different
conformations once bound to DR3VDRE or DR4T3RE. The
consequence of these different conformations of TR4-DR3VDRE and
TR4-DR4T3RE may allow each of them to recruit different
coregulators. The differential repression of TR4-mediated DR3VDRE and
DR4T3RE transactivation by the receptor interacting protein
140, a TR4 coregulator, further strengthens our hypothesis that the
specificity of gene regulation by TR4 can be modulated by protein-DNA
and protein-protein interactions.
Steroid hormones are key physiological mediators of development
and homeostasis (1-4). Understanding the crosstalk between steroid
hormone-dependent and -independent signaling pathways is
critical for gaining further insight into the integration of cellular
regulatory cues that modulate development and tissue-specific gene
expression. The biological effects of steroids and related hormones,
including derivatives of vitamins A and D3, are mediated through their cognate receptors (1-4). These receptors are members of
a large group of ligand-activated proteins that act as transcriptional activators or repressors. However, there is another group of nuclear receptors that shares the same molecular structure as steroid hormone
receptors but has no known ligands. The members of this group have
therefore been named orphan receptors (5). A common characteristic of
many of these orphan receptors is that they exert at least part of
their function as regulators of other receptors. This may occur by
several different mechanisms: competition for the same response
element, heterodimer formation with the regulated receptor, or
heterodimer formation with the retinoid X receptor (RXR),1 thus titrating out
available RXR protein. For instance, chicken ovalbumin upstream
promoter transcription factors have been demonstrated to suppress
ligand-induced gene activation, including that of vitamin A, vitamin
D3, and thyroid hormone target genes (6). This interfering
effect might involve the formation of a transcriptional silencing
complex with RXR or competition with steroid hormone receptors for DNA
binding sites (7).
The human TR4 orphan receptor (TR4) was first identified by using
degenerate polymerase chain reaction cloning. The open reading frames
of TR4 cDNA encode 615 amino acids with a calculated molecular mass
of 67 kDa (8). On the basis of sequence similarities, TR4 is classified
as a member of the steroid hormone receptor superfamily, very close to
the TR2 orphan receptor (9, 10). A comparison of the amino acid
sequence in the p-box of the DNA binding domain groups TR4 in the
estrogen receptor/thyroid hormone receptor (T3R) subfamily,
which recognizes the AGGTCA core consensus motif. From this information
we were able to identify several target genes that are up-regulated by
TR4, including the fifth intron of the ciliary neurotrophic factor
receptor Vitamin D3 is hydroxylated first in the liver at carbon 25 to yield 25-hydroxyvitamin D3 and then in the kidney at the
In this study, we analyzed the regulation by TR4 on the vitamin
D3 signaling pathway and compared the expression pattern of TR4 to that in vitamin D3 target organs. The differential
regulation of target genes containing DR3VDRE and DR4T3RE
by TR4 was further investigated, and the results suggested that
conformational changes because of DNA-protein and protein-protein
interactions might play major roles in this regulation.
Plasmid Construction--
For the transient transfection or
coupled in vitro transcription/translation of the
full-length TR4 protein, the pCMX-TR4 and pET14b-TR4 plasmids were
constructed as described previously (14). The chimeric receptor pCMX4A4
was constructed as described previously (13). The reporter plasmid
P450cc24-CAT, the 5'-flanking region (nucleotide GST Pull-down Assay--
A GST pull-down assay was performed
according to the methods described previously (30). Briefly, the GST
fusion protein and GST control protein were expressed in
Escherichia coli BL21 (DE3) pLys bacterial
culture and recovered on glutathione-Sepharose-4B beads. GST fusion
protein bound to glutathione-Sepharose-4B beads was incubated for
2 h at 4 °C with 5 µl of in vitro translated [35S]methionine-labeled protein in a total volume of 100 µl of incubation buffer (20 mM HEPES, pH 7.9, 150 mM KCl, 5 mM MgCl2, 0.5 mM EDTA, 0.5 mM dithiothreitol, 0.1% Nonidet
P-40, 1 mg/ml bovine serum albumin, 10% glycerol, and protease
inhibitor mixture (1 mM phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, leupeptin, and pepstatin)). The
glutathione-Sepharose-4B beads were then washed three times with wash
buffer (20 mM Tris, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40). The beads were boiled
in 2× SDS sample buffer, loaded onto SDS-polyacrylamide gels, and
visualized by autoradiography.
Other Methods--
In situ hybridization,
electrophoretic mobility shift assay (EMSA), Scatchard analysis, and
transient transfection were performed according to the methods
described previously (11, 14, 31, 32).
TR4 Specifically Binds to DR3VDRE, the AGGTCA Motif with
3-nucleotide Spacing--
EMSA was used to determine the binding
specificity of TR4 to DR3VDRE. In vitro translated TR4
protein was incubated with 32P-labeled DR3VDRE and analyzed
on a 5% polyacrylamide gel. As shown in Fig.
1, free probe was detected at the bottom
of the gel (lane 1), and a specific DNA·protein complex
was identified when 1 µl of in vitro translated TR4
protein was added (indicated by an arrow, lane
2). This complex was competed out by adding 100-fold excess of
unlabeled DR3VDRE (lane 3). This DNA·protein complex was
abolished when anti-TR4 monoclonal antibody 4 was added (lane
4). In contrast, when the anti-TR4 monoclonal antibody 2 was added
to the reaction, a DNA·receptor·antibody complex supershifted band
was visible (indicated by an arrowhead, lane
5).
DR3VDRE Binds to TR4 with Higher Affinity than to the RXR/VDR
Heterodimer--
Vitamin D3 decreases the affinity of the
VDR/VDR homodimer for DNA targets, enhances the formation of RXR/VDR
heterodimers, and potentiates RXR/VDR affinity for VDREs (33, 34).
Comparing the different affinities of these receptors and TR4 for
DR3VDRE might provide more information about how these receptors
regulate their target genes. EMSA was performed to determine receptor
response element Kd. Fixed amounts of in
vitro translated TR4 protein (1 µl) or RXR/VDR (1 µl of each)
were incubated with an increasing amount of 32P-DR3VDRE
(from 0.1 to 12.8 ng) and analyzed by EMSA. As shown in Fig.
2, Scatchard plot analysis of the
DNA·protein complexes in the EMSA demonstrated that the
Kd values of TR4 for DR3VDRE and RXR/VDR for DR3VDRE
were 1.32 nM and 7.31 nM, respectively. Therefore TR4 has an affinity for DR3VDRE that is 5.5-fold higher than
that of the RXR/VDR heterodimer. These data suggest that TR4 was able
to compete with RXR/VDR for the binding to DR3VDRE.
TR4 Represses the Vitamin D3-induced Rat P450cc24 Gene
Expression--
The consequence of high affinity binding between TR4
and DR3VDRE was then investigated by reporter assay. The target gene used here was the 5'-flanking region (nucleotide Localization of TR4 Transcripts in Vitamin D3 Target
Organs during Mouse Embryogenesis--
To determine if TR4 is
expressed in vitamin D target tissues, we applied in situ
hybridization analysis to mouse embryos. As shown in Fig.
5, high levels of TR4 transcripts were
detected in the perichondrium, which contains cells active in bone
formation (Fig. 5B). Distinct TR4 distribution was also
observed in the developing glomeruli and tubule structures of the
kidney as well as the intestinal villi (Fig. 5, C and
D). Fig. 5A shows strong TR4 expression detected
in certain nonclassical vitamin D target tissues, such as lung and hair
follicles (35, 36). Active TR4 expression in these tissues with the
known expression domains of VDR (37) and 24-hydroxylase (38, 39)
suggests TR4 may interact with the in vivo vitamin D
signaling pathway.
The Conformational Differences between TR4·DR3 Complex and
TR4·DR4 Complex--
The above results suggest that TR4 may exert a
repression effect on vitamin D3 responsive target gene
expression by binding to DR3VDRE. In our previous studies, we concluded
that TR4 activates the expression of the genes that contain
DR4T3RE in both HRE sequence- and TR4
dose-dependent manners (13). This contrasting and
differential regulation by TR4 could be because of different
DNA-protein or protein-protein interactions. When compared, we found
the Kd values of TR4 to DR3VDRE and TR4 to
DR4T3RE to be very similar (1.32 versus 2.0 nM). This result eliminates the possibility that a
different binding capacity between TR4 and DR3 or DR4 results in the
distinctive regulation.
The second possibility is that the mechanism of regulation may be
through different protein-protein interactions that are dependent on
the distinct conformations of TR4 once bound to either DR3VDRE or
DR4T3RE. To test this second possibility, EMSA was used to
examine a series of monoclonal antibodies raised against TR4, which are
able to recognize conformational epitopes. Four monoclonal antibodies
(mAbs) were initially characterized by EMSA on the basis of their
ability to supershift or abolish the TR4·DNA complex. As shown in
Fig. 6A, we observed a
specific DR3·TR4 complex (lane 2) that was distinct from
mock-translated protein (lane 1); with the addition of mAbs
1, 2, and 3, supershifted bands were found (lanes 3-5). In
contrast, the specific band formed by DR3·TR4 was abolished when the
mAb 4 was added (lane 6). The same band-shifted pattern was
observed when DR3 was replaced with DR4 (Fig. 6B, lane
1-6). Interestingly, different band-shifted patterns were
observed with various combinations of different antibodies. As shown in
Fig. 6A, an enhanced supershifted band shows migration
positions that are different from the supershifted band (lane
8). This suggests that mAbs 1 and 3 recognize different epitopes
because their simultaneous addition resulted in an increased mobility
shift beyond that of either antibody alone. In contrast, the addition
of mAbs 1 and 2 did not lead to an enhanced supershifted band
(lanes 3 and 4 versus 7). These results suggest
that the mAb 1 recognizes the same epitope of TR4 as the mAb 2 when TR4 is bound to DR3. However, a supershifted band was detected when the mAb
1 was added with the mAb 3 simultaneously to TR4·DR4 complex (Fig.
6B, lane 8). These results indicate that TR4
might fold into different conformations upon binding to diverse HREs
and that different antibodies can recognize these conformations.
This second hypothesis was further proven by proteolytic analysis.
[35S]methionine-incorporated, in vitro
translated TR4 was incubated in the absence or presence of HRE (DR4 or
DR3) at 25 °C for 1 h. As shown in Fig. 4C,
TR4-DR4T3RE has a similar trypsin-resisting fragment
pattern to that of unbound TR4 control. In contrast, the TR4·DR3VDRE
complex was more sensitive to trypsin when 2.5 µg/ml trypsin was
applied. As results showed, the full-length of TR4 was completely
degraded and some trypsin-resisting fragments disappeared compared to
that with TR4·DR4T3RE at the same concentration of
protease treatment. Similar results were also obtained when we replaced
trypsin with RIP140 Interacts with TR4 and Differentially Modulates the
TR4-mediated DR3VDRE-CAT and DR4T3RE-CAT
Activities--
In vitro interaction of TR4 and RIP140 was
performed by GST pull-down assay. As shown in Fig.
7A, TR4 could interact with RIP140 but not RXR (lane 3 versus 4). No
interaction could be detected when GST-TR4 was replaced with GST
(lane 5 and 6). The effects of RIP140 on
TR4-mediated DR3VDRE-CAT and DR4T3TE-CAT activities were
also investigated. As shown in Fig. 7B, RIP140 can further
repress the TR4-mediated DR3VDRE-CAT suppression (lane 8 versus 9). In contrast, RIP140 can repress both TR4 and
T3R-mediated DR4-T3RE-CAT activities (Fig.
7C, lane 5 versus 6 and lane 7 versus 10) significantly, but RIP140 has no
significant effect on the DR4T3RE-CAT induction when TR4
and liganded T3R were co-transfected (Fig. 7C, lane 8 versus 9). It is also worth noting that whereas RIP140 can further
enhance the TR4-mediated DR3VDRE-CAT suppression (lanes 8 versus 9), RIP140 has no significant effect on DR3VDRE-CAT transactivation in the absence of TR4 (Fig. 7B, lane 7 versus 10).
According to the sequence of the TR4 DNA binding domain and our
previous results from EMSA, we conclude that TR4 binds to the AGGTCA
core consensus motif arranged in a direct repeat orientation with
various numbers of nucleotide spacings. In this paper, we demonstrated
that TR4 binds to DR3VDRE with high affinity and thus suppresses
vitamin D3-induced P450cc24 gene activation. However, in
our previous studies, we demonstrated that TR4 activates the genes that
contain DR4-T3RE or nonclassical T3RE by
binding to their response elements (13). The mechanisms of different
binding responses of TR4 to the same sequence in core motifs with
different spacings remain unclear. The responsiveness of genes to
steroid hormones involves both the binding of regulatory proteins to
specific DNA sequences and the formation of critical protein-protein
associations. Throughout the past decade, a number of nuclear receptor
coregulators has been characterized and provides us a more detailed
molecular model of how nuclear receptors regulate their target genes.
Because TR4 binds to DR3VDRE and DR4T3RE with similar
affinities (1.32 versus 2.0 nM), we propose that
whether TR4 works as a transcriptional enhancer or a silencer might be
mediated not only by direct DNA binding but also by protein-protein
interactions. This indicates that in addition to the receptor and the
DNA, other factors may also contribute to the selectivity of receptors
in the recognition of their target genes.
To test this hypothesis, EMSAs and proteolytic analyses were performed.
In Fig. 4, A and B, distinct EMSA patterns were
observed when TR4 was bound 32P-DR3VDRE and
32P-DR4T3RE in the presence of different
combinations of various TR4 monoclonal antibodies. Meanwhile, different
peptide patterns were obtained when DR4- or DR3-bound TR4 was digested
with trypsin. Taken together, these data suggest that TR4 binding to
diverse HREs may result in distinct conformational changes that can
then trigger differential regulation. This finding supports the
existence of a unique spacing of the direct repeat, which serves as a
binding site for an auxiliary protein that modifies receptor activity (39). Similar approaches have also been used to study a thyroid receptor response element, RSVT3RE, which contains an
inverted repeat with a 6-nucleotide space. RSVT3RE allows
strong activation by c-ErbA As previous studies indicated that RIP140 could interact with VDR,
T3R, and TR4 (18, 41), we were interested in determining if
RIP140 could differentially affect TR4-mediated gene induction and
repression. Using the GST pull-down assay, we demonstrated that TR4
could interact in vitro with RIP140. We then proved that RIP140 could also enhance the trans-repressive effect of TR4 on the
vitamin D3-signaling pathway. In contrast, RIP140 has only marginal effects on the vitamin D3-induced VDRE-CAT
activity, although it might interact with VDR (18). On the other hand, RIP140 repressed the transactivation mediated by both TR4 (Fig. 7B, lane 5 versus 6) and liganded
T3R (Fig. 7B, lane 7 versus 10) on DR4T3RE-CAT. However, RIP140 had only
very marginal repressive effects on DR4T3RE-CAT once we
overexpressed TR4 and liganded T3R simultaneously. A
reasonable explanation for the effect of RIP140 on
DR4T3RE-CAT is that TR4 and liganded T3R may
both interact with RIP140 and sequester RIP140 blocking the repressive
effect of RIP140 on DR4-T3RE-CAT (Fig.
8). Because RIP140 can repress both
pathways mediated by TR4, it might not be recruited differently by TR4
when it is bound to DR4T3RE or DR3VDRE. However, RIP140 did
show differential effects in the regulation of DR4T3RE-CAT and DR3VDRE-CAT when receptors, TR4 and VDR versus TR4 and
T3R, are co-transfected. These data support the role of the
protein environment surrounding the DNA as a key factor in determining gene regulation. Therefore, the dynamic interaction between the receptors and cofactors in response to their target DNA may determine the specificity of gene regulation.
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(11, 12) and DR4T3RE (13). In contrast, TR4
represses the SV40 major late promoter (14) and retinoic acid
receptor/RXR target genes (15). Similar results were also obtained with
TR2 (16, 17). Molecular mechanisms of the differential regulation by
TR4 remain unclear. Determining whether TR4 is capable of interfering
with gene regulation by binding to other AGGTCA-like motifs and/or interacting with cofactor(s) might provide us more detailed information about this orphan receptor. A vitamin D3 target gene is a
potential candidate of interest for more study, because its receptor
functions through interaction with the vitamin D3 response
element, which contains two AGGTCA repeat motifs with a 3-nucleotide
space between repeats (DR3-VDRE). These response element motifs may
also be recognized by TR4. Despite the similarity of hormone response elements (HRE) recognized by both VDR and TR4, these two receptors have
also been found to interact in vitro with the receptor
interacting protein 140 (RIP140) cofactor (18, 19). Investigating the regulation of TR4 in the vitamin D signaling pathway provides us with a
molecular mechanism to explain the way TR4 regulates the gene
expression in terms of protein-DNA and protein-protein interactions.
-position of carbon 1 to generate
1,25-(OH)2D3, the active form of vitamin D. This has various biological effects, including the maintenance of
calcium homeostasis, regulation of bone remodeling, and modulation of
cell growth and differentiation (20-23). The 25-hydroxyvitamin D3 24-hydroxylase gene (P450cc24) encodes a key enzyme
involved in vitamin D metabolism, which is responsible for the
conversion of 25-(OH)D3 and
1,25-(OH)2D3 to 24,25-dihydroxyvitamin
D3 (24,25-(OH)2D3) and
1,24,25-trihydroxyvitamin D3, respectively (24). These
metabolites are thought to be inactive forms of vitamin D (25).
However, 1,25-(OH)2D3 induces 24-hydroxylase
activity in its target cells, and thus its presence may play a crucial
role in eliminating hormone activity of the vitamin D compound (25).
Two vitamin D-responsive elements (VDRE-1 and VDRE-2) responsible for
1,25-(OH)2D3 stimulation of transcription were
identified at nucleotides
151 to
137 and nucleotides
259 to
245
of the 5'-flanking region of the rat P450cc24 gene (26-28). Both VDREs
contain two AGGTCA-like repeat motifs with a 3-nucleotide space in
sense or antisense orientation and are similar to the VDREs found in
the human P450cc24 gene (29). Examination of regulation via the
24-hydroxylase induction mechanism at the molecular level may
contribute to the understanding of vitamin D in the endocrine system.
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2200 to +188) of the
rat vitamin D3 24-hydroxylase gene, was kindly provided by
Dr. Y. Kato (26). The glutathione S-transferase (GST) fusion
protein was constructed by inserting the full-length TR4 to the
SmaI site of pGEX-3X (Amersham Pharmacia Biotech).
Full-length RIP140 was cloned into the pET-28a(+) vector at the
NotI site for in vitro transcription/translation
reaction. The coupled in vitro transcription and translation
expression plasmids, pET14b-TR4, pCMX-RXR
, and pSG5-VDR (29), were
in vitro transcribed and translated by the TNT system
(Promega) as described previously (14).
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Fig. 1.
Binding of in vitro
expressed TR4 to DR3-VDRE. 32P-labeled DR3VDRE
oligomers were used in EMSA without or with 1 µl of in
vitro translated TR4 (lanes 2-5). A 100-fold excess of
unlabeled DR3VDRE (lane 3) was added as a competitor. One
microliter of anti-TR4 monoclonal antibodies 4 and 2 were added
(lanes 4 and 5). The migration position of the specific
binding formed by the DNA·protein complex and the supershifted band
formed by adding monoclonal antibodies are indicated with an
arrow and an arrowhead, respectively.
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Fig. 2.
Scatchard analysis of RXR/VDR and TR4
affinity for DR3VDRE. One microliter of in vitro
translated TR4, VDR, and RXR was incubated with serially diluted
32P-labeled DR3VDRE (concentrations from 0.1 to 12.8 ng)
and resolved by EMSA. After autoradiography, the respective bands were
excised, placed in scintillation fluid, and quantified directly in a
scintillation counter. The ratio of activity between specific DNA
protein binding (Bound) and free DNA probe with respect to
specific DNA protein binding (bound/free (B/F)) was plotted.
The dissociation constant (Kd) and
Bmax values were generated using the EBDA
program (Biosoft).
2200 to +188) of
the rat P450cc24, containing VDREs that are responsible for the
1,25-dihydroxyvitamin D3 enhancement and are located at
nucleotides
167 to
102 and nucleotides
204 to
129 (22).
Co-transfection of 2.5 µg of pSG5VDR with 3 µg of P450cc24-CAT into
CHO cells enhanced the transactivation of P450cc24-CAT to 32-fold in
the presence of 10
7 M
1,25-(OH)2D3 (Fig.
3B, lane 2 versus 3). However,
there is no CAT activity with the co-transfection of 2.5 µg of
pCMX-TR4 or pCMX-4A4 either in the absence or presence of
1,25-(OH)2D3 (Fig. 3B, lanes 4-7).
The chimeric receptor pCMX-4A4, which replaces the DNA binding domain
of TR4 with that of an androgen receptor (Fig. 3A), was
unable to bind to the AGGTCA DR motif sequence and served here as a
negative control. The transcriptional activity of P450cc24-CAT induced
by vitamin D3 was repressed when co-transfected with
pCMX-TR4, but not when co-transfected with the chimeric receptor, pCMX-4A4 (Fig. 3B, lane 3 versus 8 and 9). These
results suggest that the DNA binding domain of TR4 is essential for the
TR4-mediated repression of the vitamin D3 responsive
enhancement of the P450cc24 gene. To examine the expression levels of
both wild-type and chimeric receptor TR4 proteins after transfection,
polyclonal antibodies against both proteins were produced and examined.
This repression effect was further proven by co-transfection of pSG5VDR
with different amounts of pCMX-TR4 (from 1 to 5 µg) into CHO cells.
As shown in Fig. 4, the repression effect
mediated by TR4 was gradually increased when an increasing amount of
TR4 was co-transfected (lanes 4-8). This result clearly
demonstrated that TR4 could suppress the vitamin D3-induced
P450cc24 gene promoter activity in a TR4 dose-dependent
manner.
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Fig. 3.
Repression of vitamin D3-induced
rat 25-hydroxyvitamin D3 24-hydroxylase gene activity by
TR4. A, the molecular structure of wild-type TR4
(pCMX-TR4) and chimeric receptor 4A4 (pCMX-4A4) plasmids.
CMV, cytomegalovirus promotor. N, DBD, and LBD
indicate the N terminus, DNA-binding domain, and ligand-binding domain
of TR4, respectively. ARDBD indicates the DNA-binding domain of the
androgen receptor. B, P450cc24-CAT reporter plasmids (3 µg
of each) were transfected into CHO cells with or without
co-transfection of a VDR expression vector (pSG5-VDR, 2.5 µg), a TR4
expression vector (pCMX-TR4, 2.5 µg), or a chimeric receptor
(pCMX-4A4, 2.5 µg). Twenty-four hours after transfection,
10 7 M 1,25-(OH)2 vitamin
D3 was added (lanes 3, 5, 7-10), and an equal
volume of ethanol was added in the control experiment. Cells were
harvested 3 days after transfection for CAT analysis. Significant
differences were determined by the Student's t test and are
marked with an asterisk. *, p < 0.005 compared with lane 3. Each value represents the mean ± S.D. of four independent experiments.
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Fig. 4.
Dose response of P450cc24-CAT repression by
TR4. CHO cells were transfected with P450cc24-CAT reporter plasmid
(3 µg of each) and pSG5VDR (2.5 µg of each) and co-transfected with
increasing amounts of pCMX-TR4 (from 0 to 5 µg). The percentage of
CAT conversion was plotted. All CAT assays were normalized relative to
-galactosidase activity. Significant differences (*,
p < 0.005) of CAT activity reduced by TR4 at different
doses were determined by one-way analysis of variance. Each value
represents the mean ± S.D. of three independent
experiments.
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Fig. 5.
Localization of TR4 transcripts in vitamin
D3 target organs during mouse embryogenesis. Saggital
sections of mouse embryos at gestation day 15 (A) and day 16 (B-D) were analyzed by in situ hybridization with
a 35S-UTP-labeled mouse TR4 antisense riboprobe.
Autoradiograms were photographed under light field illumination
with (B) or without (A, C, and D)
hematoxylin staining. Tissues with strong hybridization signals
(dark areas) are labeled. High magnification
(B-D) shows an intensive TR4 signal in the perichondrium
(PC), kidney (K), and intestine (I).
g, glomerulus; t, renal tubules; v,
villi; HF, hair follicles; L, lung. The
bars represent 1 and 0.1 mm of length in A and
B-D, respectively.
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Fig. 6.
Conformational differences of TR4 induced by
binding to DR3-VDRE and DR4-T3RE. EMSAs were performed
by incubation of 1 µl of in vitro translated TR4 with
various combinations of mAbs prior to adding 32P-DR3VDRE
(A) and 32P-DR4T3RE (B).
The combinations of mAbs are indicated. C, 5 µl of
[35S]methionine-incorporated in vitro
translated TR4 was incubated with 100 ng of DR3-VDRE and
DR4-T3RE at 25 °C for 1 h. 1 µl of different
concentrations of trypsin (25 and 50 µg/µl) and -chymotrypsin
(50 and 100 µg/µl) was incubated with the TR4·DNA complex in 20 µl for another 15 min at 25 °C, and the peptide profiles were
analyzed in 12.5% SDS-polyacrylamide gel electrophoresis.
-chymotrypsin. We concluded that TR4-bound DR4T3RE or DR3V3RE had a different sensitivity to protease
digestion and different protease-resisting fragments could be obtained
with a higher concentration of protease digestion. These results
further confirm our hypothesis that, to exert its proper function, TR4 may adopt distinct conformations when bound to DR3VDRE or
DR4T3RE, leading to different protein environments.
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Fig. 7.
RIP140 interacts with TR4 and exerts
repressive control over DR3VDRE-CAT and DR4T3RE-CAT.
35S-labeled RIP140 and RXR were incubated with GST-TR4
or GST bound to glutathione-Sepharose beads in a pull-down assay. The
input represents 20% of the amount of labeled protein used in the
pull-down assay (A). CHO cells were co-transfected with 1 µg of pCMX-TR4, 2 µg of pSG5VDR (B) or
pCD-T3R
1 (C), 1 µg PEF-RIP140, and 3.5 µg
DR3-VDRE-CAT or DR4-T3RE-CAT in different combination sets.
Twenty-four hours after transfection, the cells were treated with 100 nM vitamin D3 (B) or T3
(C). All CAT assays were normalized relative to
-galactosidase activity. Each value represents the mean ± S.D.
of three independent experiments.
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ABSTRACT
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in the absence of thyroid hormone, and
the results of antibody-induced supershift experiments indicate that
binding to this element may result in a different conformation as
compared with binding to a typical DR4T3RE (40). These
results suggest that different conformational changes may be involved
in determining whether TR4 would function as a positive or negative
regulatory factor.
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Fig. 8.
The molecular model for the action of TR4 and
RIP140 on DR3-VDRE (A) and DR4-T3RE
(B).
Although the functional reporter assay presented here was carried out in vitro, the expression patterns of TR4 in the vitamin D target organs support the potential role of TR4 in regulation of the in vivo vitamin D pathway in the endocrine system. Vitamin D and its receptor play essential roles in the regulation of calcium homeostasis and bone formation. Bone, kidney, and intestine are three major targets for such action. In addition, vitamin D is involved in the regulation of cell proliferation, differentiation, and the immune response. At present, the nonclassic targets reported include lung (35) and hair follicles (36). It has been demonstrated that high levels of VDR transcripts are present in bone, kidney, and all identified nonclassic target organs. Throughout the entire process of bone differentiation, VDR transcripts were detected in osteoblasts and osteocytes of both normal and actively remodeling bone tissue (42). In the kidney, VDR and P450cc24 were co-localized in the distal renal tubules, the collecting ducts, the proximal tubes, and the parietal epithelial cells of the glomerulus (37, 43). Our data show TR4 transcripts in perichondrium, kidney, hair follicles, and lung, suggesting TR4 and VDR may be co-localized during the development of these tissues. Among the vitamin D target genes, P450cc24 has been shown to respond to vitamin D treatment in the kidney and intestine. The distribution of this enzyme in the kidney is exclusive to the proximal tubules (38), whereas its location in the intestine was detected in the lower part of the villi and columnar epithelium of the crypt. Our in situ data showed TR4 expression in renal tubules and intestinal villi, indicating TR4 and P450cc24 may be co-expressed in these places, thus providing physiological significance for the interaction between TR4 and VDR on the regulation of the P450cc24 gene. Moreover, immunostaining for VDR revealed that VDR was apparent in rat fetuses as early as embryonic days 9 and 11. In developing limbs at embryonic days 13-15, VDR epitopes were present in the skin, the cytoplasm of condensing mesenchymal cells, and the chondrocytes (44). Whether TR4 could also interact with VDR in regulating other vitamin D target genes, such as osteocalcin and alkaline phosphatase, is an intriguing question to ask.
On the other hand, vitamin D3 decreases VDR/VDR homodimer formation and enhances the formation of RXR/VDR heterodimers. Thus, it potentiates RXR/VDR action by enhancement of the RXR/VDR binding affinity to VDREs and activation of target gene expression (33, 34). We then compared the binding affinity of TR4 and RXR/VDR to DR3VDRE and found that TR4 bound to DR3VDRE with a 5.5-fold higher affinity than the RXR/VDR heterodimer. These binding affinity data suggest that TR4 may be able to compete with RXR/VDR to bind to DR3VDRE and in this way, exert its repressive effect. The ratio of functional TR4·DR3VDRE complex to RXR/VDR·DR3VDRE might also be important in determining target gene action. Additionally, the failure of the repression effect observed when PCMX-TR4 was replaced with the chimeric receptor PCMX-4A4 in a reporter gene assay further supports the idea that DNA binding is essential for TR4 to exert its proper function.
The responsiveness of genes to a steroid hormone receptor is
principally mediated by functional interactions between DNA-bound receptors and components of the transcription initiation machinery. In
this paper we demonstrated that TR4 represses the P450cc24 gene
activation induced by vitamin D3 via in vitro
and in vivo evidence. Both DNA binding and proper
protein-protein interactions may be the key factors in determining the
specific function of TR4. This study may lead to an understanding of
the role of DNA binding in altering the conformation of TR4 and
allowing different protein interactions resulting in a complex that is
capable of mediating differential regulation.
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ACKNOWLEDGEMENTS |
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We thank Dr. Y. Kato for providing the P450cc24-CAT plasmids.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants DK47258, CA68568, and CA71570.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.
Contributed equally to this work.
§ To whom correspondence should be addressed. Fax: 716-756-4133; E-mail: chang{at}pathology.rochester.edu.
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ABBREVIATIONS |
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The abbreviations used are: RXR, retinoid X receptor; TR4, testicular orphan receptor; HRE, hormone response elements; VDRE, vitamin D response element; GST, glutathione S-transferase; EMSA, electrophoretic mobility shift assay; CHO, Chinese hamster ovary; mAb, monoclonal antibody; CAT, chloramphenicol acetyltransferase; DR, direct repeat; VDR, vitamin D3 receptor; T3RE, thyroid response element; RIP140, receptor interacting protein 140; T3R, thyroid receptor.
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REFERENCES |
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