Synergistic Activation of the Prolactin Promoter by Vitamin D Receptor and GHF-1: Role of the Coactivators, CREB-Binding Protein and Steroid Hormone Receptor Coactivator-1 (SRC-1)
Ana I. Castillo,
Ana M. Jimenez-Lara,
Rosa M. Tolon and
Ana Aranda
Instituto de Investigaciones Biomédicas Consejo Superior
de Investigaciones Científicas 28029 Madrid, Spain
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ABSTRACT
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PRL gene expression is dependent on the presence
of the pituitary-specific transcription factor GHF-1/Pit-1,
which is transcribed in a highly restricted manner in cells of the
anterior pituitary. In pituitary GH3 cells, vitamin D increases the
levels of PRL transcripts and stimulates the PRL promoter. We have
analyzed the role of GHF-1 and of the vitamin D receptor (VDR) to
confer vitamin D responsiveness to the PRL promoter. For this purpose
we have used nonpituitary HeLa cells, which do not express GHF-1. We
found that VDR activates the PRL promoter both in a ligand-dependent
and -independent manner through a sequence located between positions
-45/-27 in the proximal 5'-flanking region. This sequence also
confers VDR and vitamin D responsiveness to a heterologous promoter. In
the context of the PRL gene, VDR requires the presence of GHF-1 to
activate the promoter. Truncation of the last 12 C-terminal amino acids
of VDR, which contain the ligand-dependent activation function (AF2),
abolishes regulation by vitamin D, suggesting that binding of
coactivators to this region mediates ligand-dependent stimulation of
the PRL promoter by the receptor. Indeed, expression of the
coactivators, steroid hormone receptor coactivator-1 (SRC-1) and
CREB-binding protein (CBP), significantly enhances the stimulatory
effect of vitamin D mediated by the wild-type VDR but not by the AF2
mutant receptor. Furthermore, CBP also increases the activation of the
PRL promoter by GHF-1 and the ligand-independent activation by both
wild-type and mutant VDR.
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INTRODUCTION
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Tissue-specific and developmental expression of eukaryotic genes
is typically governed by combinations of cell type-specific and
ubiquitous transcription factors (1). GHF-1/Pit-1 is a
pituitary-specific POU-Homeobox transcription factor that regulates
GH, PRL, and TSHß gene expression through binding to specific
promoter sequences (2, 3, 4). The 5'-flanking region of the PRL gene
contains several GHF-1 binding sites clustered in two domains: a distal
enhancer (-1800 to -1500 bp), and a proximal promoter (-422
to +33 bp) both containing four GHF-1 binding sites (5). Multiple
hormones, growth factors, and oncogenes act in conjunction with GHF-1
to regulate the expression of the PRL gene. The DNA elements as well as
the transcription factors responsible for the regulation by some of
these stimuli have been identified. Thus, different ligands of tyrosine
kinase receptors, as well as the src, ras, and
raf oncogenes, activate PRL gene expression, and the
downstream effector for these stimuli appear to be the ubiquitous Ets
factors, which bind to the proximal PRL promoter (6, 7). The proximal
promoter region also contains a basal transcription element (BTE) as
well as sequences conferring cAMP responsiveness [cAMP-response
element (CRE)] to the PRL gene. An estrogen response element
(ERE) located in the distal enhancer binds estrogen receptors and
confers estradiol responsiveness to the PRL gene (8). Other ligands of
nuclear receptors have also been described to regulate PRL gene
transcription both positively and negatively (9).
1,25-Dihydroxyvitamin D3
[1,25-(OH)2D3] (vitamin D), the active form
of vitamin D3, exerts its biological activities through
binding to a specific receptor [vitamin D receptor (VDR)], a member
of the nuclear hormone receptor superfamily, which also includes
retinoid, thyroid hormone, and steroid hormone receptors (10), and it
displays a modular structure with several regions (A/B, C, D, and E).
Transcriptional regulation by nuclear receptors is achieved through
autonomous activation functions (AFs): a constitutive N-terminal AF1
contained in the A/B region, and a ligand-dependent AF2 located in the
C-terminal E region of the ligand-binding domain (LBD). The E region
also contains a dimerization interface. The highly conserved C domain
contains two zinc fingers responsible for DNA binding and
sequence-specific recognition. VDR, as a homodimer or preferentially as
a heterodimer with the retinoid X receptor (RXR), binds to vitamin
D-responsive elements (VDREs) in promoters of vitamin D target genes
and modulates their transcription. In most cases, the VDREs consist of
DR-3 elements (direct repeats of the consensus hexanucleotide motif
(A/G)(A/G)(G/T)N(C/G)A separated by 3 bp) (11).
In the past few years the mechanism of transcriptional activation by
nuclear receptors has been further elucidated by the discovery of a new
class of proteins known as transcriptional coactivators. Some
coactivators, such as the two related proteins CREB (CRE-binding
protein)-binding protein (CBP) and p300, mediate the effects of diverse
groups of transcription factors (12, 13), whereas other coactivators
are more specific for the nuclear receptors. This class of specific
factors include, among others, the steroid hormone receptor
coactivator-1 (SRC-1/NCoA1) (14, 15). Recent crystallographic studies
(15) have shown that ligand binding induces a structural change in the
AF2 region, which allows the recruitment of coactivators and enhances
ligand-induced transactivation.
GH- and PRL-secreting pituitary tumor cells (GH cells), which possess
specific receptor sites for vitamin D (16), have previously been found
to respond to this ligand with a selective increase in PRL synthesis
and secretion, without affecting GH (17). This induction is secondary
to stimulation of PRL gene expression, but the promoter elements
responsible for this effect have not yet been identified.
In this report, we have analyzed the effect of vitamin D on PRL gene
expression in pituitary GH3 cells, as well as the contribution of GHF-1
and VDR to the stimulation of the PRL promoter by vitamin D. To avoid
the problem of endogenous expression of GHF-1 and receptors in
pituitary cell lines, we have performed transient cotransfection assays
with GHF-1 and wild-type and mutant receptors in a heterologous cell
system (HeLa cells). We found that VDR activates the PRL promoter both
in a ligand-dependent and a ligand-independent manner through a VDRE
located in the proximal promoter region, and that this activation
requires the presence of GHF-1. A VDR mutant lacking the AF2 region
exhibits full constitutive activity, but does not confer
ligand-dependent transactivation. Indeed, expression of the coactivator
SRC-1 and CBP dramatically potentiates the vitamin D response mediated
by the wild-type VDR, but not by the mutant receptor. However, both
coactivators have differential effects: CBP (but not SRC-1) increases
the constitutive vitamin D-independent activation of the promoter by
truncated and wild-type VDR. Since CBP also strongly stimulates the
response to GHF-1 in the absence of receptors, these results suggest a
broader role for this cointegrator in PRL gene expression.
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RESULTS
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Vitamin D Increases PRL Transcripts and Stimulates PRL Promoter
Activity in Pituitary GH3 Cells
As shown in Fig. 1A
, incubation of
GH3 cells with 100 nM vitamin D for 48 h caused a
significant increase of PRL mRNA levels. Quantification of Northern
blots from three independent experiments showed that this concentration
of vitamin D increased PRL transcripts in GH3 cells by 3.3 ± 0.2
fold. To study whether sequences contained within the 5'-flanking
region of the PRL gene mediate induction by vitamin D in these cells,
the construct -3000PRLCAT was used in transient transfection assays.
Since transcriptional effect of different nuclear receptors can be
interfered by AP-1 factors, this construct lacks the AP-1 binding motif
present in the plasmid backbone (19). As illustrated in Fig. 1B
, vitamin D caused an approximately 3-fold increase in chloramphenicol
acetyltransferase (CAT) activity, a value similar to that found for the
increase in endogenous PRL transcripts. In contrast with the results
obtained in GH3 cells, we were unable to find stimulation of the PRL
gene by vitamin D in GH4C1 cells, another rat pituitary cell
line (data not shown).

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Figure 1. Vitamin D Activates PRL Gene Expression in
Pituitary GH3 Cells
A, Northern blot analysis were carried out with 20 µg of total RNA
from duplicate cultures of control cells and cells treated for 48
h with 100 nM vitamin D. The blot was hybridized with a
labeled cDNA probe for rat PRL. The lower panel shows
the ribosomal 18S RNA. B, GH3 cells were transfected with 5 µg of the
PRL promoter construct -3000 PRLCAT, and CAT activity was determined
after 48 h in untreated cultures and in cultures treated with 100
nM vitamin D. The data show the mean ± SD
values obtained in a representative experiment performed with
triplicate cultures.
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Vitamin D Activates the PRL Promoter in Nonpituitary HeLa Cells
Consistent with the physiological role for vitamin D in the
regulation of PRL gene expression in rat pituitary cells (17), we
decided to analyze the role of VDR on PRL gene activation by vitamin D,
as well as the participation of GHF-1 in this response. For this
purpose we used the nonpituitary HeLa cell line, which is derived from
a human cervical carcinoma and does not express endogenous PRL or the
pituitary-specific factor GHF-1 (18). To characterize the interaction
between GHF-1 and VDR on PRL gene activation by vitamin D, the reporter
plasmid -3000PRLCAT was cotransfected with VDR in the presence or
absence of a GHF-1 expression vector. As shown in Fig. 2
, basal CAT activity was very low in
HeLa cells in the absence of GHF-1 and was not affected by vitamin D.
Furthermore, this activity was not modified by expression of VDR alone,
either in the presence or in the absence of the ligand. However, after
expression of GHF-1, which by itself had little stimulatory effect, a
strong synergistic response was observed and vitamin D caused a marked
promoter stimulation. As can also be observed in Fig. 2
, unliganded VDR
was able to cooperate with GHF-1 to cause a significant increase of CAT
activity, although this constitutive activity was markedly enhanced
upon incubation with the ligand. In contrast, vitamin D did not
activate the PRL construct in the absence of transfected VDR, even when
GHF-1 was present, showing that endogenous VDR levels are not
sufficient to stimulate the PRL promoter in HeLa cells. The same PRL
promoter fragment (-3000 to +74) containing the AP-1 site in the
plasmid backbone showed a stronger response to GHF-1, but the responses
to VDR and vitamin D3 were not affected (data not
shown).

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Figure 2. GHF-1 Cooperates with VDR to Stimulate the PRL
Promoter in HeLa Cells in a Vitamin D-Dependent and -Independent Manner
The plasmid -3000 PRLCAT (5 µg) was cotransfected with expression
vectors for GHF-1 (0.4 µg) and/or VDR (2.5 µg). After 48 h,
CAT activity was determined in untreated cultures and in cultures
treated with 100 nM vitamin D (black bars).
The data show the mean ± SD values obtained in a
representative experiment performed with triplicate cultures.
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5'-Flanking Sequences in the Proximal PRL Promoter Confer Vitamin D
Responsiveness
A series of 5'-deletion constructs were used to determine
the gene elements responsible for PRL promoter stimulation by vitamin
D. All constructs contained the first 74 bp of the coding PRL
region and lacked the AP-1 binding site. In contrast with the results
obtained with the -3000PRLCAT plasmid, CAT levels were essentially
undetectable in GH3 cells transfected with shorter promoter fragments
and, therefore, further mapping could not be carried out in these cells
(Fig. 3B
). In contrast, CAT activity was
detectable in HeLa cells transfected with the different deletions.
Figure 3A
shows that the -1597, -425, and -176 constructs exhibit
similar ligand-dependent activation upon cotransfection with expression
vectors for VDR and GHF-1. As with the -3000PRLCAT plasmid, a weaker
ligand-independent activation was also observed. Deletion to -101 bp
significantly reduced vitamin D response, although constitutive
activation was still observed, and both responses were totally lost in
the construct extending only to -76. This gradual loss of response
suggests either the existence of more than only one VDRE (some of which
could be located between -176 and -101), or that the VDRE cooperates
with other promoter sequences to confer full vitamin D
responsiveness.

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Figure 3. Proximal Promoter Sequences Mediate Stimulation by
Vitamin D
A, HeLa cells were cotransfected with 5 µg of reporter CAT constructs
containing progressive deletions of the PRL promoter and expression
vectors for GHF-1 (0.4 µg) and/or VDR (2.5 µg). The cells
transfected with GHF-1 in addition to VDR were incubated for 48 h
in the presence or absence of 100 nM vitamin D (vit. D).
The data represent the mean CAT activity of four independent assays. B,
GH3 cells were transfected with the constructs indicated and CAT
activity was determined after 48 h treatment in the presence and
absence of vit. D as indicated. The data show the values obtained from
three independent transfections.
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Mapping of the Vitamin D Response Element
To identify the possible vitamin D3 response
element(s) in the proximal promoter of the PRL gene, gel mobility shift
experiments were conducted with a labeled promoter fragment spanning
from -176 to +74 (which confers a full CAT response to vitamin D), a
-101 to +74 fragment (which shows a partial response), and a -70 to
+74 promoter fragment (which is unresponsive to vitamin D in the
transfection assays). The region from -176 to -101 was also used in
the assays. As depicted in Fig. 4A
, the
promoter region between -176 and -101 contains two GHF-1 binding
sites, one of them included in the composite Ets-GHF-1 binding site,
which is required to mediate PRL promoter activation by oncogenic
ras [Ras-responsive element (RRE)] (20). These
elements could participate in the response to vitamin D3.
Truncation to -101 deletes the RRE (-165/-150). The -76 to +74
fragment, which is unresponsive to vitamin D, has lost an additional
Ets binding site, the CRE (-101/-92), and the BTE (-98/-85). Since
VDR can function as a homodimer or as a VDR/RXR heterodimer (21), we
analyzed the formation of these complexes on the proximal promoter
regions. As shown in Fig. 4B
(left panel), the fragment from
-176 to +74 formed a weak complex with VDR (lane 3). RXR alone also
bound weakly to this fragment (lane 2), but caused the formation of
VDR/RXR heterodimers that bind strongly to the promoter (lane 4),
indicating a preference for heterodimeric binding to the response
element. This heterodimeric complex was removed by antibodies against
both VDR (lane 5) and RXR (lane 6). Identical results were obtained
when the promoter region from -101 to +74 was used in the mobility
shift assays (data not shown), demonstrating that the VDRE was
contained within this fragment. In contrast, as shown in lanes 710,
the region comprised between -176 and -101 did not bind the receptors
although GHF-1 binding to this fragment was readily observed (not
illustrated). These results suggested that the VDRE could be located
between -101 and -76 bp. However, as shown in lanes 11 and 12,
although the -76 to +74 region is not sufficient to confer vitamin D
responsiveness to the PRL promoter in transient transfection studies,
these sequences still contain the VDRE. Again, VDR binding was
significantly enhanced in the presence of its heterodimeric partner
RXR, showing the preference for binding of VDR/RXR heterodimers to this
fragment (lane 12). These results indicate that although the VDRE is
located in this promoter region, other upstream sequences are required
to confer responsiveness to vitamin D3. To further map the
VDRE, the -76 to +74 region was digested with Pst-1 to generate two
new fragments: -76 to -10 and -9 to +74 (Fig. 4A
). Each region was
end labeled with 32P and used in mobility shift experiments
with VDR and RXR. The formation of VDR/RXR complexes was observed with
the -76 to -10 fragment (lane 14), but not with the -9 to +74
fragment (lane 16), demonstrating that the VDRE is located between -76
and -10 bp in the promoter.

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Figure 4. Binding of VDR/RXR Heterodimers to the Region
between Positions -76 and -10 of the PRL Promoter
A, Schematic representation of the proximal PRL promoter from
nucleotides -176 to +74, showing the position of the TATA box, and the
binding sites for GHF-1, Ets factors, the BTE, and the overlapping cAMP
response element (CRE). 32P-labeled promoter fragments
(-176/+74, -176/-101, and -76/+74) were obtained by PCR. The
depicted Pst-1 restriction site was used to generate the labeled
fragments, -76/-10 and -9/+74. B, In the left panels,
gel mobility shift assays were performed with 150 ng of VDR and/or RXR
lacking the N-terminal A/B domain ( A/B-RXR) as fusion proteins with
GST. The receptors were incubated with the 32P-labeled
proximal promoter regions indicated. For supershift assays, 1 µl of
the specific antibodies against VDR or RXR ( VDR and RXR,
respectively) was used in the binding reactions as indicated. The
mobility of the receptor-containing complexes is indicated. ns
represents a nonspecific band present in the labeled -176/+74
fragment. In the right panel the same amount of
receptors was incubated with the -76/+74 promoter fragment in the
presence and absence of recombinant GHF-1 (GHF-1p). As indicated, 1
µM vitamin D (Vit D) or a 50-fold excess of unlabeled
oligonucleotides conforming a consensus VDRE [DR3(oli)], or the GHF-1
binding site [GHF-1(oli)], were included in the reactions.
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Since VDR actions on the PRL promoter depended on the presence of GHF-1
and both transcription factors produced a synergistic effect, we
explored the possibility that binding of GHF-1 and VDR/RXR could be
cooperative. Figure 4B
(right panel, lanes 110) shows
binding of VDR and VDR/RXR alone or in combination with GHF-1 to the
labeled -76 to +74 fragment. This promoter region contains one GHF-1
binding site and the VDRE. The addition of GHF-1 to the VDR or VDR/RXR
binding reactions neither facilitated the binding of the receptors nor
resulted in the formation of new complexes different from those formed
in reactions containing each protein individually. Thus, both factors
can bind independently to the promoter and do not show cooperative
binding. The preference for the formation of VDR/RXR complexes, rather
than VDR homodimers (21), suggests that the VDRE in the PRL promoter
could consist of a direct repeat (DR) motif with a spacer of three
nucleotides. This was confirmed in competition reactions with an
unlabeled consensus DR-3 oligonucleotide. Binding of the VDR/RXR
complex was competed specifically by this oligonucleotide (lanes 6 and
9), but not by a sequence containing a GHF-1 binding site (lane 10).
The latter effectively competed binding of the GHF-1 complex (lanes 3
and 10), which in turn was not affected by the DR-3 oligonucleotide
(lane 9). As also shown in Fig. 4B
, binding of VDR/RXR to the PRL
promoter fragment occurs irrespectively of the presence of vitamin D
(lanes 5 and 8).
Since the data shown in Fig. 4B
indicate that the VDRE must be
contained between the nucleotides -76 and -10 in the PRL promoter, we
screened this promoter region for consensus VDR-binding sites. This
sequence was compared with 18 known natural VDREs (reviewed in Ref.
11). As illustrated in Fig. 5A
, three
overlapping putative VDREs in the -45 to -14 region were found.
Oligonucleotides designed to contain all of them (A), the two most
5'-motifs (B), or the most 3'-motifs (C) were assayed in a mobility
shift experiment. The A and B oligonucleotides bound VDR/RXR
heterodimers, although binding was slightly less intense with the B
sequence (lanes 4 and 8, respectively). Furthermore, the receptors
bound to the promoter fragments and to these oligonucleotides with a
similar strength (compare Figs. 4B
and 5C
). In contrast, the C fragment
bound VDR homodimers with a similar low affinity to the A or B
oligonucleotides (lane 11), but binding of VDR/RXR heterodimers was
almost undetectable (lane 12). These results suggest that the B
sequence, which contains the DR-3 motifs, is the one responsible for
PRL gene response to vitamin D.

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Figure 5. Mapping of the VDRE
A, Schematic representation of the -76 to -10 fragment showing the
most proximal GHF-1 binding site and the TATA Box. B, Synthetic
oligonucleotides corresponding to the region between positions -45 to
-14 designated as A, B, and C, showing the putatives DR3-type VDREs
(black arrows). C, Gel mobility shift assay performed
whit GST-VDR and GST-RXR and the 32P-labeled A, B, and C
sequences as indicated.
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The VDRE of the PRL Gene Is Functional
To test the functionality of the VDR-binding sites of the PRL
promoter, the A and B sequences were fused to an heterologous
thymidine kinase (TK) promoter driving the CAT reporter gene
(A-PRLTKCAT and B-PRLTKCAT). These constructs, as well as the
parental TKCAT construct, were transfected into HeLa cells together
with the expression vector for VDR. The effect of vitamin D on a TKCAT
reporter gene containing a consensus DR3 response element as a positive
control was also analyzed. As shown in Fig. 6A
, the plasmid TKCAT, which does not
contain a VDRE, was unresponsive to vitamin D either in the presence or
absence of VDR. On the other hand, when VDR was expressed, vitamin D
induced A-PRLTKCAT and B-PRLTKCAT activity by approximately 6-fold and
5-fold, respectively. A similar induction by vitamin D (4.4-fold) was
found in cells transfected with the TKCAT plasmid containing the
consensus DR3 VDRE. As also shown in Fig. 6A
, unliganded VDR
constitutively activated the A-PRLTKCAT and B-PRLTKCAT construct, but
the unoccupied receptor did not stimulate the DR3 TKCAT plasmid. These
results indicate that the B sequence must be the major element used by
VDR for both the ligand-dependent and -independent activation of the
PRL gene.

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Figure 6. The VDRE of the PRL Promoter Is Functional
A, The A and B sequences of the PRL promoter, as well as a consensus
DR3 element, were fused to a TKCAT construct to give the A-PRLTKCAT,
B-PRLTKCAT, and DR3-TKCAT constructs, respectively. HeLa cells were
transfected with 5 µg of these constructs or the same amount of the
parental TKCAT construct. The cells were cotransfected with 2.5 µg of
VDR expression vector, and CAT activity was determined after 48 h
of incubation in the presence or absence of 100 nM vitamin
D (vit. D). B, HeLa cells were transfected with 5 µg of
-176PRLCAT or the mutated constructs -176 m1PRLCAT and -176
m2PRLCAT. These plasmids were cotransfected with expression vectors for
GHF-1 (0.4 µg) and/or VDR (2.5 µg). After 48 h, CAT activity
was determined in untreated cultures and in cultures treated with 100
nM vitamin D. Schematic representations of the constructs
are shown at the top. The hemisites in the VDRE are
indicated by arrows, and mutations are shown as X.
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To furher test the functionality of the VDRE, this element was mutated
in the context of the -176PRLCAT plasmid. Two different mutations in
the B sequence, one affecting the 5'-hemisite (-176 m1PRLCAT) and the
other affecting both hemisites of the VDRE (-176 m2PRLCAT), were
transfected into HeLa cells. Figure 6B
shows that these mutations
significantly impaired the response of the PRL promoter to vitamin D.
Additionally, ligand-independent stimulation by VDR was reduced by the
mutations. In contrast, the activity of these mutants was strongly
stimulated by incubation with 10 µM forskolin (data not
shown), showing that this element does not contribute to responses
elicited by other signaling pathways.
VDR Activates the PRL Gene through VDR/RXR Heterodimers
Our results indicate that cotransfection of RXR was not needed for
the PRL response to vitamin D. Since HeLa cells contain RXR, the
endogenous receptor levels appear to be sufficient to mediate the
observed response to vitamin D. To analyze whether the activation of
the PRL promoter by vitamin D could be enhanced by overexpression of
RXR, the -176PRLCAT construct was transfected into Hela cells in
combination with the expression vectors for VDR and/or RXR in the
presence of the GHF-1 vector (Fig. 7A
).
The responsiveness of the PRL promoter to vitamin D was only observed
in the presence of VDR. Again, unliganded VDR activated the promoter,
and this effect was increased by vitamin D. Overexpression of RXR did
not affect basal PRL promoter activity, but potentiated both the
constitutive activity of VDR and the promoter activation by vitamin D.
Similar results were obtained in pituitary cells. Figure 7B
shows that
expression of RXR in GH3 cells did not alter basal activity, but
enhanced the response to the vitamin. Thus, it is most likely that VDR
activates the PRL promoter by heterodimerizing with RXR. These results
are in agreement with the stimulation by vitamin D through a VDRE
characterized as a DR-3 motif, since it has been proposed that VDR/RXR
heterodimers mediate transactivation of DR-3-containing promoters (21, 22).

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Figure 7. RXR Cooperates with VDR to Activate the PRL
Promoter
A, HeLa cells were transfected with 5 µg of -176PRLCAT and
expression plasmids for GHF-1 (0.4 µg), VDR (2.5 µg), and/or RXR
(0.5 µg) as indicated. After transfection the cells were incubated
for 48 h in medium alone or with 100 nM vitamin D
(Vit. D). The results shown are the mean ± SD of four
independent transfections. B, GH3 cells were transfected with 5 µg of
-3000PRLCAT alone or in combination with 5 µg of RXR expression
vector. CAT activity was determined after 48 h in triplicate
cultures incubated in the presence and absence of vit. D.
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Transcriptional Activation by Wild-Type and C-Terminally
Truncated VDR Mutants
To evaluate the region of VDR responsible for the ligand-dependent
and ligand-independent activation, we used two truncated mutants of
VDR. In the
ABC mutant, the first 111 amino acids in the N terminus
have been eliminated. This mutant lacks both the constitutive
activation function AF1, and the DNA-binding domain. The
AF2-VDR
mutant lacks the last 12 amino acids in the C terminus. Thus, this
mutant does not contain the transcriptional ligand-dependent activation
domain AF2. AF-2 activity appears to be due to interaction with
coactivators in a ligand-dependent manner. Thus, the coactivator SRC-1
has been shown to interact with the AF-2 region of VDR (23, 24). We
compared in vitro interaction of VDR and
AF2-VDR with
other coactivators [CBP and ACTR (coactivator of thyroid
hormone and retinoic acid receptors)] in gluta-
thione-S-transferase (GST) pull-down assays. As shown in
Fig. 8A
in the presence of vitamin D, a
significant portion of the input of 35S-labeled VDR was
specifically retained by GST-ACTR immobilized in glutathione-agarose
beads, while no significant binding was observed either in the absence
of vitamin D or by GST alone. Deletion of the 12 C-terminal amino acids
abolished binding to the coactivator, and GST-ACTR was not retained
significantly by the 35S-labeled
AF2-VDR mutant in the
presence of vitamin D. Similar results were obtained with GST-CBP.
Incubation with vitamin D increased binding of GST-CBP to the wild-type
receptor, while binding to the
AF2-VDR mutant was unaffected. No
interaction of the GST-CBP with 35S-luciferase used as a
negative control was detected.
Figure 8B
shows the effect of cotransfection of wild-type or mutant VDR
expression plasmids on the induction of -176PRLCAT by vitamin D.
Although the
AF2-VDR binds vitamin D with a normal affinity (25),
this mutant exhibited no vitamin D-dependent activation in the presence
of GHF-1. This result indicates that the last 12 amino acids in the C
terminus are absolutely necessary for transcriptional activation in a
ligand-dependent manner and suggests that coactivators that bind to the
AF2 domain play an important role in this response. In contrast, the
AF2-VDR mutant was able to activate the PRL promoter in a
ligand-independent manner with at least the same potency as the
wild-type receptor. In contrast, the
ABC-VDR mutant displayed
neither constitutive transcriptional activity nor vitamin D-dependent
transcriptional activation. This shows that the LBD is not sufficient
for activation and suggests that binding to the VDRE is required.
Role of the Coactivators SRC-1 and CBP on the Activity of the PRL
Promoter
As numerous reports have emphasized the requirement of coactivator
factors to promote full activity of the nuclear receptors in the
presence of their ligands, it was of interest to analyze their role in
the activation of the PRL gene by VDR in the absence and presence of
vitamin D. For this purpose, HeLa cells were transfected with the PRL
promoter construct -3000PRLCAT, and the expression vectors for
VDR, GHF-1, and the nuclear receptor coactivators, SRC-1 and CBP (which
bind to the core AF2 region of nuclear receptors). Figure 9A
shows the functional effects of
these factors on PRL gene stimulation. In the absence of GHF-1, neither
protein activated the PRL promoter (data not shown). In the presence of
GHF-1, SRC-1 neither enhanced the promoter response to this pituitary
factor nor the constitutive activity of VDR, but drastically
potentiated the response to vitamin D. Thus, SRC-1 serves as a good
coactivator for the stimulation of the PRL promoter by VDR in a
ligand-dependent manner. The activating effect of SRC-1 on the PRL
promoter was confirmed in pituitary GH3 cells transfected with
-3000PRLCAT. As shown in Fig. 9B
, overexpression of SRC-1 enhanced
significantly the response to vitamin D without increasing basal
promoter activity. Unlike SRC-1, CBP significantly enhanced the
response to GHF-1 in HeLa cells independently of the presence of VDR.
Furthermore, CBP potentiated not only the ligand-dependent, but also
the ligand-independent, activation mediated by VDR (Fig. 9A
). Finally,
the combination of these coactivators slightly increased the effect
produced by each protein alone. These results suggest that whereas
SRC-1 acts specifically as a nuclear receptor coactivator and in a
vitamin D-dependent manner, CBP exerts a broader role in the regulation
of the PRL gene.

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Figure 9. The Coactivators SRC-1 and CBP Potentiate the
Stimulation of the PRL Promoter by VDR
A, HeLa cells were transfected with 5 µg of -3000PRLCAT, 0.4 µg of
GHF-1, and 2.5 µg of VDR. As indicated, 2 µg of expression vectors
for SRC-1 and CBP were also cotransfected. CAT activity was determined
48 h after transfection in cells treated in the presence or
absence of 100 nM vitamin D. B, GH3 cells were transfected
with 5 µg of -3000PRLCAT alone or in combination with 2 µg of
SRC-1 expression vector. CAT activity was determined in cells treated
for 48 h in the presence and absence of vit. D. The data show the
values obtained from three independent transfections.
|
|
Since coactivators markedly enhanced the vitamin D-dependent activity
of the wild-type VDR, it was also of interest to analyze their role in
the activity of the VDR mutants. For this purpose, the same
transfection assays described above in HeLa cells with wild-type VDR,
SRC-1, and CBP were also performed with the VDR mutants (Fig. 10
). Confirming the results described
above, expression of SRC-1 was unable to activate the promoter
construct in the absence of receptors, whereas CBP increased by
approximately 6-fold the levels found in cells expressing GHF-1. The
ABC-VDR mutant did not activate the PRL promoter either
constitutively or in a ligand-dependent manner in the presence of
coactivators. The
AF2-VDR mutant did not confer vitamin D
responsiveness to the promoter even in the presence of coactivators.
This is not surprising, since as described before (24, 25) and shown in
Fig. 8A
, the AF2 region of VDR was required for interaction with
coactivators. However, the receptor lacking the AF2 region displayed a
normal ligand-independent transcriptional activity and, although this
mutant is not supposed to interact with CBP, expression of this
coactivator substantially increased the constitutive activity of
AF2-VDR. This unexpected finding was specific for CBP, since SRC-1
did not significantly modify ligand-independent induction by the
AF2-VDR mutant.

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Figure 10. CBP, but Not SRC-1, Potentiates the
Ligand-Independent Stimulation of the PRL Promoter by VDR
HeLa cells were transfected with 5 µg of -3000PRLCAT and 0.4 µg of
GHF-1. Additionally, 2.5 µg of expression vector for native VDR, and
the AF2 or the ABC VDR mutants, were cotransfected with 2 µg of
vectors expressing SRC1 and/or CBP. The cells were then incubated with
or without vitamin D for 48 h. The data show CAT activities
obtained in a representative experiment performed with duplicate
cultures with variations of 515%.
|
|
Physical Interaction of VDR with GHF-1
We have previously reported that several nuclear receptors can
interact with GHF-1 (26). Since the stimulation of the PRL promoter by
VDR requires the presence of GHF-1, we asked whether a direct
protein-protein interaction between both factors could be involved in
this functional cooperation. To address this question, a GST-VDR fusion
protein was immobilized on glutathione-sepharose beads and used in
binding assays with in vitro translated GHF-1, labeled with
[35S]methionine. To characterize the role of vitamin D in
the interaction between these proteins, the binding reactions were also
performed in the presence of this ligand. As shown in Fig. 11
, GHF-1 interacted with VDR, and this
association was independent of the presence of vitamin D. To map the
GHF-1 domain responsible for this interaction, different deletion
mutants of the protein were also used in pull-down assays with GST-VDR.
Deletion of the homeodomain, but not of other regions of the protein,
abolished the ability of GHF-1 to interact with VDR. This result
indicates that the DNA-binding domain of GHF-1 is involved in binding
to VDR .

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Figure 11. In Vitro Interaction of VDR with
Wild-Type and Truncated GHF-1 Mutants
Functional domains of GHF-1 and the internal deletion mutants are shown
in a schematic form on the left. The hatched
boxes represent the homeodomains (HB); the striped
boxes, the POU-specific domains (POU); the black
boxes, regions rich in negatively charged residues (OH); and
open boxes, remainder of protein. Thin
lines denote deleted sequences. Mutants are named according to
the deleted sequence. The binding activity of GHF-1 proteins with VDR
was assessed by pull-down experiments. GST-VDR (1 µg) or the same
amount of GST alone (as a negative control) immobilized in
glutathione-sepharose beads were incubated with 5 µl of in
vitro translated GHF-1 or the truncated forms labeled with
[35S]methionine. Incubations were performed in the
absence or presence of 1 µM vitamin D3. After
incubation the beads were washed, and the labeled proteins were
analyzed by SDS-PAGE and visualized by autoradiography.
|
|
 |
DISCUSSION
|
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It has been previously found that somatolactotroph tumor cells
respond to vitamin D with a highly selective increase in PRL synthesis,
raising the level of PRL mRNA without affecting the level of GH mRNA
(17). We have confirmed that vitamin D increases PRL transcripts in GH3
cells and have demonstrated that vitamin D enhances PRL promoter
activity in transient transfection assays. Since pituitary-specific
transcription of the GH and PRL genes involves synergistic interactions
between GHF-1 and other promoter-binding factors, including nuclear
receptors, we decided to analyze the role of GHF-1 in the regulation of
PRL gene expression by the VDR. In this report we show that in HeLa
cells, VDR induces PRL gene activation both constitutively and in a
vitamin D-dependent manner only after the expression of GHF-1.
Our data indicate that the response to vitamin D is mediated by a
sequence that binds VDR/RXR heterodimers and is located in the
5'-flanking region of the PRL gene between the nucleotides -45 and
-27. We have identified two putative overlapping VDREs (starting
either in nucleotide -42 or -41). The hexameric core of both elements
shows a high homology with the consensus VDRE sequence RRKNSA
(r = A or G; K = G or T; S = C or G). As in
most VDREs identified in natural vitamin D-responsive gene promoters,
the hemisites in the PRL promoter are arranged as direct repeats spaced
by three nucleotides. As expected for a DR3, the PRL VDRE bound VDR/RXR
heterodimers with high affinity and showed low affinity for VDR
homodimers that have been described to bind preferentially to DR6-type
VDREs (21, 22). It has been reported that vitamin D can increase
binding of VDR/RXR to some VDREs (27) but not to others (22), and our
data show that binding to the PRL VDRE was not affected by vitamin D.
An unusual feature of this VDRE is its close vicinity to the TATA box
of the PRL gene. Our results suggest that binding of receptor
heterodimers to these sequences does not compete with binding of TATA
box-binding protein (TBP) to its recognition sequence, rather a
synergistic action of the receptors with the basal transcriptional
machinery could be facilitated by the close location of the VDRE.
The PRL VDRE fused to a heterologous promoter conferred responsiveness
to vitamin D, and mutation of this element in the PRL promoter
strikingly reduced the response to vitamin D, demonstrating that this
element is functional. Most interestingly, expression of VDR also
causes a constitutive activation of the PRL promoter, and the
heterologous promoter that contains the PRL VDRE was also activated
upon expression of VDR. In contrast, the idealized DR3 sequence used as
a positive control only exhibits ligand-dependent activation. This
result suggests that the activation by the unoccupied receptor is a
specific feature of the PRL VDRE. It has been shown that other nuclear
receptors are able to activate transcription constitutively, and that
this activation is cell and promoter dependent (28, 29). The molecular
mechanisms underlying the transcriptional effects of the unliganded
receptors are not well understood. Either there is an interaction
between VDR and cellular factors that may be necessary for
hormone-independent activity or, alternatively, the receptors may
inhibit the activity of putative inhibitory factors.
The response of successive promoter truncations to liganded or
unliganded VDR shows that a PRL promoter construct extending to -76 bp
does not respond to the actions of this receptor although it contains
the VDRE. The lack of the BTE and the overlapping CRE located between
-101 and -85, which are necessary for the activity of the PRL
promoter (30, 31), could be involved in the unresponsiveness of this
promoter fragment to vitamin D. In fact, this construct which contains
one GHF-1-binding site, does not show activation by GHF-1. However, the
presence of the BTE in the PRL promoter is not sufficient to confer
full responsiveness to vitamin D, since the -101PRLCAT construct
contains this element and only exhibits a partial response to vitamin
D. Thus, other sequences located upstream of -101 bp appear to
contribute to the activation of the PRL promoter by vitamin D. One of
these sequences could be the RRE, a composite element for binding of
GHF-1 and Ets factors (20). A direct protein-to-protein interaction
between both factors has been recently described (18). In most
promoters the hormone response elements are found clustered around
binding sites for other transcription factors, and the requirement for
additional nonreceptor factors is consistent with the finding that VDR
acts with other transcription factors to enhance gene expression
synergistically (32). Sequences located between the nucleotides -176
and -1597 did not further increase activation by vitamin D, suggesting
that factors binding to this region are not involved in this response.
However, the plasmid extending to -3000 bp showed in most experiments
a somewhat stronger regulation by vitamin D. This could be due either
to the presence of additional upstream VDREs or to functional
cooperation of the downstream VDRE with other factors that bind to the
upstream sequences, as, for instance, the GHF-1-binding sites
identified in the distal enhancer. It is interesting that in pituitary
GH3 cells the activity of reporter plasmids that do not contain the
distal enhancer was extremely low, precluding a detailed analysis of
the response element in these cells.
The critical role played by GHF-1 in the stimulatory action of vitamin
D on PRL gene expression is indicated by the finding that in
nonpituitary cells VDR requires the presence of GHF-1 for the
activation of this promoter. A synergistic activation of the PRL
promoter by GHF-1 and other nuclear receptors has been observed
previously. Thus, the estrogen receptor is unable to activate
expression of the PRL gene unless GHF-1 is expressed (33), and as we
have recently observed, peroxisome proliferator-activated
receptor-
activates the PRL promoter, and this activation is
only observed upon expression of GHF-1 (34). There are several models
to explain transcriptional synergism. Synergism could reflect
cooperative binding between adjacently bound factors. For instance,
binding of GHF-1 could facilitate the binding of VDR/RXR to the
promoter, resulting in greater occupancy of the cis-acting
elements, thus promoting transcription. The fact that the most proximal
GHF-1-binding site is very close to the VDRE in the promoter could
suggest the presence of a GHF-1/VDR composite element. This type of
composite element has been described in the distal enhancer of the
GHF-1 gene for GHF-1 and the retinoic acid receptor (RAR) and is
characterized by the cooperative binding of RAR and GHF-1. However, we
do not observe cooperative binding of GHF-1 and VDR/RXR heterodimers
even in the presence of vitamin D, and our gel mobility shift assays
show that the receptors and the GHF-1 bind to the promoter
independently. Therefore, we favor another model for synergism
involving stabilizing interactions of receptors and transcription
factors such as GHF-1 or Ets with multiple target sites (some of them
presumably basal transcription factors as well as coactivator
proteins). The important functional role of coactivators in the
regulation of PRL gene expression by VDR is demonstrated by the
dramatic increase in vitamin D-dependent transactivation of the PRL
promoter by VDR in the presence of SRC-1 and CBP. Both proteins bind
the AF2 domain of the nuclear receptors in a ligand-dependent manner
(12, 24, 25, 35), and our results show that this region is indeed
involved in the recruitment of coactivators and essential for the
response to vitamin D, which was lost in a VDR mutant that lacks the
AF2 region. In contrast, this truncated receptor displayed a
constitutive activation of the PRL promoter as strong as that of the
wild-type receptor, showing that the ligand-independent activation by
VDR does not require binding of coactivators to the AF2 domain. The
finding that the AF2 mutant showed ligand-independent activity also
dismisses the possibility that the constituve action of VDR could be
secondary to activation by residual intracellular levels of vitamin
D.
Although SRC-1 and CBP had a similar effect on the vitamin D-dependent
activation, a differential effect on the vitamin D-independent action
of VDR was also observed. Thus, whereas VDR constitutive activity was
essentially unaffected by SRC-1, CBP significantly increased the effect
of unoccupied VDR. Furthermore, the vitamin D-independent activation
elicited by the AF2 VDR mutant was also potentiated by CBP. The latter
effect was totally unexpected, since this mutant lacks the
CBP-interacting region (12). This suggests that CBP might exert this
action indirectly by binding to other factor/s that, in turn, associate
with VDR. The best candidate for this interaction is again GHF-1. CBP
significantly potentiates the stimulatory effect of GHF-1 on the
promoter. On the other hand, we have recently observed a direct
protein-to-protein interaction between GHF-1 and CBP (34). This
interaction maps to the POU domain of GHF-1 (36) and to the region of
CBP contained between amino acids 11099 and 16791858, the same
domains of interaction with other transcription factors including Ets
(37).
We have also previously reported a direct association of GHF-1 with
several nuclear receptors (26). This finding, as well as the important
role of GHF-1 in the activation of the PRL gene by VDR, suggested that
this receptor could also interact with GHF-1. Our results demonstrate
that indeed this association exists, and that VDR interacts in a
ligand-independent manner with the homeodomain (the DNA-binding
domain) of GHF-1. Thus, although the interaction might recruit VDR
and GHF-1 to the promoter, binding of VDR/GHF-1 heterodimers to
the promoter was not observed.
As a whole, our data suggest that the simultaneous binding of VDR/RXR
and GHF-1 to their close cognate sites in the promoter, as well as the
direct interactions with other transcription factors, coactivators, and
components of the basal transcription machinery, could
facilitate promoter occupancy and govern transcriptional
activation of the PRL promoter synergistically.
 |
MATERIALS and METHODS
|
---|
RNA Extraction and Hybridization
GH3 cells were cultured in RPMI medium containing 15% horse
serum and 2.5% FCS. For the experiments the cells were incubated for
24 h in a medium containing a hormone-stripped serum by treatment
with resin AG1x8 and activated charcoal. The cells were then treated
for 48 h with 100 nM vitamin D. Total RNA was
extracted from the cell cultures with guanidine thiocyanate. The RNA
was run in 1% formaldehyde-agarose gels and transferred to
nylon-nitrocellulose membranes (Nytran, Schleicher & Schuell, Dassel,
Germany) for Northern blot analysis. The RNA was stained with
0.02% methylene blue. The blots were hybridized with a cDNA probe for
rat PRL labeled by nick translation. Hybridizations were at 42 C with
50% formamide, and the more stringent wash was at 42 C with 1x
SSC-0.1% SDS.
Plasmids
The constructs -3000PRLCAT and -176PRLCAT were generated from
plasmids containing the 5'-flanking region of the rat PRL promoter
(from -3000 to +74, and from -176 to +74 bp, respectively) in a
pBL-CAT2 vector in which the thymidine kinase promoter had been deleted
(38). Since the pBLCAT vector contains an AP-1 like sequence at
+34/+39, which could mask some promoter responses (19), a 301-bp
fragment containing this element was deleted by digestion with
AatII and NarI. The constructs were then
blunt-ended and religated. -1597PRLCAT was obtained by removing a
XbaI/NsiI fragment of 1403 bp from -3000PRLCAT
and then blunt-ended and religated. The -423PRLCAT plasmid was
obtained by digestion of -3000PRLCAT with HindIII and
religation. -101 and -76PRLCAT have been previosly described (7). The
mutated constructs -176 m1PRLCAT and -176 m2PRLCAT were obtained by
PCR. In the first PCR the sense oligonucleotides
5'-CATGAAGCCGTCGAAGTTTA-3' (for M1), and
5'-CATGAAGCCGTCGAAGCCTTATAAAGTC-3'
(for M2) containing the desired nucleotide changes, as well as the
antisense oligonucleotide 5'-GACTCGAGTCGACATCGATGCCATTGGGATATATC-3',
were used to generate mutated fragments. After an elongation phase, a
second PCR was performed using these fragments. The sense
oligonucleotide for this reaction was 5'-cccaagcttTGGCCACTATGTCTTCCT-3'
containing a HindIII site and as antisense
5'-GACTCGAGTCGACAT-3'. The mutated sequences were cut with
HindIII and XhoI and subcloned in pBLCAT3. The
mutations were confirmed by sequencing. The A oligonucleotide,
5'-agctTGAAGGTGTCGAAGGTTTATAAAGTC-AATGTCg-3', which contains the
sequences -45 to -14 of the rat PRL promoter, was cloned into
HindIII/BamHI sites of pBL-CAT2 (without the AP-1
like sequence) upstream of the TK promoter to construct A-PRLTKCAT. The
B oligonucleotide containing the promoter sequences from -45 to -27,
5'-agctTGAAGGTGTCGAAGGTTTA-3', was also cloned into the
HindIII site of pBL-CAT 2 upstream of the TK promoter and
then blunt ended and religated to produce B-PRLTKCAT. The plasmid
DR3-TKCAT, which contains the DR3 consensus oligonucleotide
5'-agctcAGGTCAAGGAGGTCAg-3', has been previously described (39). The
expression vectors for rat GHF-1 (40), human RXR
(41), human VDR
(42), human CBP (43), and human SRC-1 (44) have been described
previously. The expression vector for the
AF2-VDR mutant
was constructed by PCR using the human VDR expression
plasmid as a template and the oligonucleotides
5'-GGAGCAGCAGCGCATCATT-3' and
5'-CGCGGATCCTCACGTTAGCTTCATGCTGC-3' to generate a
872-bp fragment. This fragment was digested with BstXI and
BamHI and cloned into the pSG5 expression vector. This
receptor lacks the last 12 C-terminal amino acids, which
contain the AF2 region. For the
ABC-VDR mutant, the oligonucleotides
5'-GGAATTCCATGGAGGAGGAGGCCTTG-3' and
5'-CGGGATCCTCAGGAGATC TCATTGCC-3' were used to generate
a 972-bp fragment and were then digested with EcoRI and
BamHI and subcloned into pSG5. This construct generates a
truncated receptor lacking 149 N-terminal amino acids that include the
A/B region and the DNA-binding domain (C region). pGST-VDR, which
expresses a fusion protein between glutathione S-transferase (GST) and
VDR, was obtained by PCR using the pSG5-VDR plasmid as a template and
the oligonucleotides 5'-CGGGATCCATGGAGGCAATGGCGG-3' and
5'-GGAATTCTCAGGAGATCTCAT TGC-3' to generate the VDR cDNA (1031 bp).
This fragment was then subcloned into BamHI/EcoRI
sites of the pGEX-2T plasmid. pGST-
A/BRXR plasmid (lacking the A/B
domain), was also constructed by PCR but using the pSG5-hRXR and
the oligonucleotides 5'-GGAATTCTGATGGGCCTCAATGGCGTCC-3' and
5'GCTCTA GACTAAGTCATTTGGTGCGG-3' to generate a 1078-bp fragment that
was subcloned into EcoRI/HindIII sites of the
pGEX-2T plasmid. The constructs GST-ACTR and GST-CBP, which express the
cDNAs coding for the amino acids 621821 and 11099 of ACTR and CBP,
respectively, have been previously described (45, 46). These fragments
contain the nuclear receptor-interacting sequences of both
proteins.
Cell Culture and Transfections
HeLa cells were cultured in DMEM containig 10% FCS and
were transfected by calcium phosphate with the reporter CAT
constructs. GH3 cells were shifted to DMEM and transfected with calcium
phosphate with a glycerol shock. In cotransfection experiments the
reporter plasmids were transfected with 2.5 µg of VDR and/or 0.5 µg
of RXR, in the presence or absence of 0.4 µg of GHF-1 expression
vectors. When indicated, 2 µg of vectors encoding SRC-1 or CBP were
also used in cotransfection experiments. In all cases the total amount
of DNA among different transfections was kept constant by addition of
empty noncoding expression vectors. Each transfection also received 0.5
µg of a luciferase vector as a control for transfection efficiency.
CAT activity was determined by incubation of the cell extracts with
[14C]chlor- amphenicol. The unreacted and acetylated
[14C]chloramphenicol was separated by TLC and quantified
with an InstantImager (Packard Instrument Co., Camberra).
The data are expressed as the percentage of acetylated forms after each
treatment. Each experiment was repeated at least three times with
similar results.
Protein Preparations
GHF-1 and its truncated forms cloned in Bluescript SK- (36),
were used for in vitro transcription and translation
following the manufacturers recommendations of the TNTT7 Quick
coupled transcription/translation System (Promega Corp.,
Madison, WI). The reactions were translated in the presence of 40 µCi
of [35S]methionine (Amersham Pharmacia Biotech, Arlington Heights, IL). Five microliters of the
reaction product were resolved in 10% SDS-PAGE. The gel was dried and
autoradiographed overnight. Recombinant purified GHF-1 was a generous
gift from Dr. Castrillo. The GST-fusion proteins, VDR,
ABRXR, CBP,
and ACTR, were expressed in the bacterial strain BL21 (DE3). They were
grown at 37 C in 2x YT [Tryptone, 16 g/liter yeast extract; NaCl, 5
g/liter (pH 7)] until the absorbance reached 0.6. Then the induction
was performed at 30 C for 2 h with 0.4 mM isopropyl
ß-D-thiogalactopyranoside and were purified following the
recommendations of Pharmacia Biotech, (Piscataway,
NJ).
Mobility Shift Assays
Gel retardation assays were performed with the recombinant
GST-fusion proteins. Oligonucleotides corresponding to the A,
B, and C fragments of the PRL promoter were used as probes. A and B
oligonucleotides have been described above, and C
oligonucleotide, 5'-agctCGAAGGTTTATAAAGTCAATGTCg-3', contains
the -36 to -14 promoter sequence. For the binding reaction, the
proteins were incubated on ice for 15 min in a buffer [20
mM Tris HCL (pH 7.5), 75 mM KCl, 1
mM dithiothreitol, 5 µg/ml BSA, 13% glycerol]
containing 3 µg poly (dI-dC) and then for 1520 min at room
temperature with approximately 50,000 cpm of labeled double-stranded
oligonucleotide end labeled with [32P]CTP, using Klenow
fragment as kinase. In addition, the labeled fragment -176 to +74 was
obtained by PCR using the oligonucleotides
5'-cccaagcttTGGCCACTATGTCTTCCT-3' and 5'-AACAGCCAAGTGTCAGCC-3' as
primers. The region -176 to +101 was obtained with the antisense
oligonucleotide 5'-CAATCATCTATTTCCGTCAT-3'. For the PCRs the first
oligonucleotide was previously end labeled with [32P]ATP
using T4-polynucleotide kinase. Similarly, the -101 to + 74 and -76
to +74 fragments were made by PCR with the 5' oligonucleotides
5'-ATGACGGAAATAGATGATTG-3' and 5'-GGAAGAGGATGCCTGAT-3', respectively
(end labeled previously with [32P]ATP). For competition
experiments an excess of unlabeled doubled-stranded oligonucleotides
were added to the binding reactions: as a DR-3 type we used
5'-agctcAGGTCAAGGAGGTCAg-3' and for the GHF-1 binding site we used
5'-CCAGCCATGAATAAATGTATAGGG-3'. For supershift experiments, specific
antibodies against VDR (
VDR) and RXR (
RXR) were added to the
binding reactions before the addition of the labeled fragment. Finally,
DNA-protein complexes were resolved on 6% polyacrylamide gels in 0.5x
TBE buffer. The gels were then dried and autoradiographed at -70
C.
Protein-Protein Interactions
Pull-down assays were performed with 5 µl of in
vitro translated
L-[35S]methionine-labeled GHF-1, VDR, or the
same amount of their truncated forms. These proteins were incubated
with the fusion proteins GST-VDR, GST-ACTR, or GST-CBP or with the same
amount of GST as a control, immobilized in glutathione-sepharose beads
as previously described (34, 35). Where indicated, vitamin D was
included in the binding reaction. The bound proteins were analyzed by
SDS-PAGE and autoradiography.
 |
ACKNOWLEDGMENTS
|
---|
We thank R. Evans, M. Karin, M. Parker, H. Stunnenberg, and H.
Samuels for expression vectors used in this study. We also thank P.
Chambon for anti-RXR antibody. Vitamin D was a gift from
Hoffman-LaRoche, Inc. (Nutley, NJ).
 |
FOOTNOTES
|
---|
Address requests for reprints to: Ana Aranda, Instituto de Investigaciones Biomédicas (CSIC), Arturo Duperier, 4, 28029, Madrid, Spain.
This research was supported by the Comunidad de Madrid, by Grant
PB940094 from the DGICYT, and by the "Fundaci-n Salud
2000" (Serono).
Received for publication June 22, 1998.
Revision received March 4, 1999.
Accepted for publication March 23, 1999.
 |
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