From the Laboratoire de Biologie Moléculaire et Cellulaire de l'Ecole Normale Supérieure de Lyon, Unité Mixte de Recherche 49 CNRS, Ecole Normale Supérieure, Institut National de la Recherche agronomique 913, 46, Allée d'Italie, 69364 Lyon cédex 07, France
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ABSTRACT |
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The carbonic anhydrase II gene, whose
transcription is enhanced by 1,25-dihydroxyvitamin D3
(1,25-(OH)2D3), encodes an important enzyme in
bone-resorbing cells derived from the fusion of monocytic progenitors.
We analyzed the 1,25-(OH)2D3-mediated
activation of the avian gene by transient transfection assays with
promoter/reporter constructs into HD11 chicken macrophages and by DNA
mobility shift assays. Deletion and mobility shift analyses indicated
that the 62/
29 region confers 1,25-(OH)2D3
responsiveness and forms DNA-protein complexes. The addition of an
anti-vitamin D receptor (VDR) antibody inhibited binding to this
sequence, whereas anti-retinoid X receptor (RXR) antibody generated a
lower mobility complex. Therefore, we concluded that this element binds
a VDR·RXR heterodimer, but the addition of extra
1,25-(OH)2D3 had no effect on the formation of
this complex. Moreover, the use of nuclear extracts from
1,25-(OH)2D3-treated macrophages led to the
formation of an additional high mobility complex also composed of
VDR·RXR heterodimer. Mutations provided evidence that the
1,25-(OH)2D3-mediated activation of the
carbonic anhydrase II gene is mediated by VDR·RXR heterodimers bound
to a DR3-type vitamin D response element with sequence AGGGCAtggAGTTCG. This vitamin D response element is also functional in the ROS 17/2.8
osteoblasts.
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INTRODUCTION |
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Recent work points to the complexity of the molecular mechanisms
involved in the vitamin D3 signaling pathway. It has been known for some time that in addition to the binding of the vitamin D
receptor (VDR)1 to certain
vitamin D response elements (VDREs) as homodimer (3-7), the in
vitro binding affinity of the VDR is enhanced by dimerization with
accessory factors such as RXRs (8-11). The response elements for these
receptors differ from one another by the number of base pairs (bp)
spacing the hexameric repeats according to the so-called 1 to 5 rule
(1, 2). Following the 1 to 5 rule, optimal VDREs for VDR·RXR
heterodimers should be direct repeats of two hexameric core binding
sites spaced by three nucleotides (DR3) (1, 3). The high specificity of
these DR3-type VDREs was confirmed by identification of some natural
and synthetic VDREs (10). The mouse osteopontin VDRE has been shown to
bind VDR homodimers with low affinity (3, 6, 12, 13) but VDR·RXR
heterodimers with high affinity (3, 9, 14, 15). DR3-type elements have
also been found in numerous promoters such as the rat osteocalcin gene
(16, 17), the rat calbindin D-9k (18), the avian integrin 3 subunit
gene (19), the rat 24-hydroxylase gene (20-22), the avian carbonic
anhydrase II (CAII) gene (23), and mouse p21 (24). However, the binding
sites of the VDREs characterized so far vary considerably in
their sequences, preventing definition of a real VDRE
consensus sequence (7, 25-27).
The differentiating effects of 1,25-(OH)2D3 have been studied extensively in a large number of in vitro systems using cultures of leukemia cells (28, 29), keratinocytes (30-32), or bone cells (33-35). Despite this attention, understanding of the mechanisms that lead to the diverse forms of cell differentiation mediated by 1,25-(OH)2D3 is still fragmentary.
Osteoclasts are large, multinucleated and highly polarized bone-resorbing cells. They belong to the monocytic/macrophage lineage (34, 36), and their differentiation pathway is partly under the control of 1,25-(OH)2D3 (37-40). These cells express some characteristic markers such as tartrate-resistant acid phosphatase, vitronectin receptor, calcitonin receptor, and CAII. CAII, expressed at high levels in osteoclasts (41, 42), plays an important role in the extracellular acidification required for bone resorption and therefore bone remodeling. In particular, CAII deficiency is one of the factors responsible for the osteopetrosis characterized in humans by a renal tubular acidosis and a cerebral calcification (43).
We have shown previously that 1,25-(OH)2D3
enhances the expression of CAII in chicken primary blood-derived
macrophages (38). Furthermore, 1,25-(OH)2D3
activates the CAII gene expression at the transcriptional level in the
chicken monocytic BM2 cells induced to differentiate into macrophages
by lipopolysaccharides and phorbol 12-myristate 13-acetate (44) as well
as in the human promyelocytic leukemia cells HL60 after phorbol
12-myristate 13-acetate stimulation (45). CAII gene expression is also
activated transcriptionally by thyroid hormone in normal erythrocytic
cells (46), and several domains in the avian CAII promoter have been
shown to control the thyroid hormone regulation of transcription (47,
48). In previous work, we identified a VDRE between positions 1203 and
1187 of the CAII promoter which mediates
1,25-(OH)2D3 responsiveness to the herpes
simplex virus thymidine kinase (tk) minimal promoter in the
Drosophila SL3 cell line and in human MCF-7 cells (23). This
VDRE, bound by a VDR·RXR heterodimer, is however not functional in an
avian macrophage cell line.
In the present study, we have looked for specific
1,25-(OH)2D3 regulation of the CAII gene
transcription in macrophages. We have studied the
ligand-dependent transactivation of the avian CAII promoter
in the chicken HD11 macrophage cell line in which numerous hormonal
nuclear receptors and CAII gene are expressed endogenously.2 We have looked
for hormone response elements in this promoter and localized an element
conferring 1,25-(OH)2D3-mediated activation (VDRE) to a 34-base pair region located 62/
29 upstream the
transcriptional start site. The VDRE was defined precisely after
methylation interference assays and by using mutated forms of this
sequence. This VDRE, functional in HD11 and in ROS 17/2.8 cell lines,
has a DR3 structure with sequence AGGGCA tgg AGTTCG and is
specifically bound by a heterodimer formed by VDR and the
or
isoform of RXR.
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EXPERIMENTAL PROCEDURES |
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Cells, Medium, and Hormones--
The HD11 avian macrophage cells
(50) were grown in BT88 complete medium described in Ref. 38. The ROS
17/2.8 rat osteoblast-like osteosarcoma cells were grown in Dulbecco's
modified Eagle's medium supplemented with 10% fetal calf serum, 1%
L-glutamine (Life Technologies, Inc.), and antibiotics.
When the medium was supplemented with hormones, we first depleted sera
of small lipophilic compounds by treatment with charcoal.
Charcoal-treated sera were prepared as follows. 250 ml of serum was
added to the activated charcoal/dextran T70 (100:1; Sigma) and
incubated at 55 °C for 30 min with frequent agitation. Serum was
then spun at 2,000 × g for 25 min. After two
incubations, the serum was filtered through 0.45-µm filters and
stored at 20 °C. 1,25-(OH)2D3 was a
generous gift from M. Uskokovic (Hoffman-Laroche).
Reporter Plasmids and Expression Vectors--
Functional
analysis of the CAII promoter was performed using a
CAII-chloramphenicol acetyltransferase (CAT) reporter construct, pHHcaCat (1321+31). The pHHcaCat plasmid and 5'-end deletions of
pHHcaCat were described elsewhere (48). Briefly, constructs were
obtained by restriction enzymes that cut once within the promoter at
positions
932,
619, and
178 to generate pPstCat, pPvuCat, and
pApaCat plasmids, respectively. For functional analysis of the proximal
CAII promoter, three fragments excluding the TATA box were obtained
from pApaCat construct and cloned between HindIII and
XbaI sites of pBLCAT2. These constructs contain the
fragments comprised between residues
177 and
101
(p
177
101tkCat),
120 and
31 (p
120
31tkCat), and
24 and +31
(p
24+31tkCat), respectively. For analysis of the
1,25-(OH)2D3 response domain, three overlapping oligonucleotides of the
120
31 fragment (p
120-83tkCat,
p
90-55tkCat, and p
62
29tkCat) were synthesized and cloned into
the XbaI site of pBLCAT2.
Cell Transfection and CAT Assays--
5 × 106
HD11 cells were transfected by electroporation (290 V, 500 microfarads;
Bio-Rad) with 10 µg of CAT reporter constructs and 1 µg of
CMV--galactosidase plasmid as an internal control to normalize for
variations in transfection efficiency. Cells were cultured in BT88
complete medium with 10% charcoal-stripped sera in 60-mm dishes
(Falcon). When appropriate, the medium was supplemented with
10
8 M 1,25-(OH)2D3 or
vehicle (0.01% ethanol) 2 h after transfection. After 24 h
of treatment, the medium was removed, and cells were incubated for an
additional 24 h in 2 ml of fresh medium with the appropriate added
factors. Transient transfections with the ROS 17/2.8 cells were
performed by the calcium phosphate coprecipitation method in 60-mm
dishes with 4.5 µg of reporter construct and 0.5 µg of
CMV-
-galactosidase plasmid. 10
8 M
1,25-(OH)2D3 was added 48 h before
harvesting the cells, and vehicle was added at the same concentration
to the control. After 48 h of treatment, the HD11 or ROS 17/2.8
cells were lysed in Tris-SDS buffer (0.25 M Tris, pH 8, and
0.05% SDS), and extracts were used to measure
-galactosidase and
CAT activities. CAT activity was measured by the CAT assay on TLC
silica plates (Sigma), quantified by PhosphoImager (Molecular
Dynamics), and normalized to
-galactosidase activity.
Electrophoretic Mobility Shift Assays (EMSAs)--
The
macrophage nuclear extracts were prepared from the HD11 cells treated
for 2 days with 108 M
1,25-(OH)2D3 or vehicle (ethanol 0.01%) in
charcoal-treated medium. The nuclear extracts were prepared as
described in Ref. 48. The human recombinant proteins, VDR, RXR
,
RAR
,
, and
, and chicken RXR
were synthesized in TNT
reticulocyte lysate (Promega) from their cDNAs using T7 RNA
polymerase. Unprogrammed lysate and recombinant luciferase were used as
negative controls. The rat anti-chick VDR monoclonal antibody used in
EMSAs was purchased from Chemicon. The mouse anti-RXR (recognizing all
RXR subspecies) and the mouse anti-RAR (recognizing all RAR subtypes)
monoclonal antibodies were generous gifts from P. Chambon (IGBMC,
Illkirch, France). The anti-TR antibody was described elsewhere (48). The anti-Mi antibody was provided by S. Saule (Institut Pasteur, Lille,
France) and the anti-P19, directed against the P19gag
retroviral protein, by J. Samarut (ENS, Lyon, France).
Methylation Interference Experiments--
Dimethyl sulfate
interference assays (51, 52) were performed using the 62/
29
fragment of the p
62
29tkCat construct as a probe. p
62
29tkCat was
linearized by HindIII (or BamHI), dephosphorylated, and the 5'-end was labeled with
[
-32P]ATP. The
62/
29-labeled fragment was obtained
after BamHI digestion for sense-labeled probe to generate an
HindIII-BamHI fragment (or HindIII for
antisense-labeled probe) and purified on an 8% polyacrylamide
nondenaturing gel. Both
62/
29 probes (106 cpm) were
methylated with dimethyl sulfate for 3 min at 18 °C, then used for
EMSAs with 1,25-(OH)2D3-treated nuclear
extracts or recombinant VDR and RXR
proteins. Free and retarded
probes were eluted and sequenced using the Maxam-Gilbert technique. The sequences were analyzed on a 9% polyacrylamide denaturating gel and
compared with G+A sequences obtained with nonmethylated-labeled probes.
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RESULTS |
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The CAII Proximal Promoter Contains a
1,25-(OH)2D3-positive Regulatory
Domain--
We have verified previously that the HD11 cells expressed
the VDR and CAII mRNAs and proteins. We have also checked the
presence of RXR, RAR, and TR mRNAs by using the reverse
transcriptase-polymerase chain reaction technique.2 Then,
to investigate the mechanisms involved in the transcriptional regulation of the CAII gene expression, the 5'-flanking region of the
chicken CAII gene was examined and searched for putative 1,25-(OH)2D3 response domains. The fragment of
the CAII gene, from 1321 bp upstream and +31 bp downstream of the
transcription start site, as well as deleted fragments from positions
932,
619, and
178 were cloned into a CAT reporter plasmid (Fig.
1). Each different construct was
transfected together with the CMV-
-galactosidase internal control
into HD11 cells treated with either 10
8 M
1,25-(OH)2D3 or vehicle (0.01% ethanol) and
assayed 48 h later for CAT and
-galactosidase activities. The
transient expression analyses demonstrated that the full-length
promoter and all of the deleted mutants still responded to
1,25-(OH)2D3 but with various efficiencies
(Fig. 1). Indeed, a 5-fold level of induction was seen with pHHcaCat
construct, whereas it was 16-fold with the fragment containing the
178 bp upstream initiation codon (hereafter referred to as the
proximal promoter) (Fig. 1). This proximal promoter was activated by
1,25-(OH)2D3 more efficiently than the full-length promoter and all of the intermediate promoters, indicating that an efficient 1,25-(OH)2D3-positive
regulatory domain must be present between positions
178 and +31 of
the CAII gene and that repressor elements must have been deleted.
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Identification of a Putative 1,25-(OH)2D3
Response Domain--
To characterize further the DNA sequence
necessary for 1,25-(OH)2D3 response, we
constructed different plasmids containing overlapping fragments of the
proximal promoter but excluding the TATA box. Those fragments were
fused to the herpes simplex virus tk minimal promoter and the CAT gene
to generate p177
101tkCat, p
120
31tkCat, p
24+31tkCat,
p
120-83tkCAT, p
90
55ptkCAT, and p
62
29ptkCAT constructs (Fig.
2). These plasmids were transfected into
HD11 cells and tested for 1,25-(OH)2D3
activation. The basal activity of the tk promoter in pBLCAT2 vector was
not modified significantly by the 1,25-(OH)2D3
treatment (Fig. 2, row 1). Under similar conditions, only
p
120
31tkCat and p
62
29ptkCAT construct have retained the ability
to respond to 1,25-(OH)2D3 (Fig. 2, rows
4 and 8 to compare with rows 3,
5, and 8). These analyses showed that the only
proximal regulatory region from position
62 to
29 can confer strong
1,25-(OH)2D3 responsiveness when attached to a
heterologous tk promoter. Then, the regulating domain mediating the
1,25-(OH)2D3-dependent activation
of the CAII promoter is located between positions
62 and
29. This
sequence must contain a potential VDRE conferring
1,25-(OH)2D3 responsiveness to the tk
promoter.
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VDR and RXR Bind Directly to the 62/
29 Fragment as a
Heterodimer--
The 1,25-(OH)2D3DR3-type
activation may result from the binding of nuclear hormonal receptors to
this region. To test this hypothesis, a double-stranded oligonucleotide
with a sequence from positions
62 to
29 was used as a probe for
EMSAs and was incubated with several in vitro translated
nuclear hormonal receptors (Fig.
3A). The synthetic consensus
VDRE DR3, described in Ref. 1 and known to bind VDR·RXR heterodimer,
was used as a positive control and a sequence corresponding to an AP-1
binding site as a negative control. No specific binding was ever
observed when the
62/
29 probe was incubated with unprogrammed
lysate or recombinant luciferase (data not shown). Moreover, VDR, RXR
(
or
isoforms), or RAR (
,
,
isoforms) alone did not
generate any specific complexes with the
62/
29 region (Fig.
3A, lanes 2 and 8-12) or RAR in
combination with VDR (Fig. 3A, lanes 5-7). The
only specific signal was obtained when the
62/
29 probe was
incubated with a mixture of VDR and either RXR
or
isoforms (Fig.
3A, lanes 3 and 4). Mobility shift
experiments clearly demonstrated the RXR requirement for the binding of
the VDR and the cooperative binding of the VDR·RXR heterodimer to
the
62/
29 region of the CAII gene. This signal was not affected by
the addition of 10
6 M
1,25-(OH)2D3 to the binding assay (data not
shown). This VDR·RXR complex, named C1, was competed specifically by
a 100-fold excess of unlabeled DR3 or
62/
29 oligonucleotides (Fig.
3B, lanes 3 and 7 to compare with
lane 2). In contrast, unlabeled oligonucleotides containing
an AP-1 site or corresponding to the
120/
83 and
90/
55 regions
of the CAII promoter failed to compete (Fig. 3B, lanes 4-6). Most importantly, the specific retarded band C1 is
disrupted by the antibody directed against the DNA binding domain of
VDR (Fig. 3B, lane 10) and supershifted by the
anti-RXR antibody (Fig. 3, lane 12), but this complex is not
affected by an unrelated anti-Mi antibody (Fig. 3B,
lane 11). These data confirmed the binding specificity of
the VDR·RXR heterodimer to the
62/
29 region of the CAII promoter.
Therefore VDR and RXR proteins are necessary and sufficient to form a
C1-binding complex on
62/
29 sequence.
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62/
29 Fragment Binds to VDR·RXR Heterodimers Present in HD11
Nuclear Extracts--
Nuclear extracts of HD11 cells treated for 2 days with 1,25-(OH)2D3 were incubated with
labeled probes corresponding to the
62/
29,
120/
83, or
90/
55
DNA fragments of the CAII promoter, and the resulting complexes were
analyzed by EMSA. No specific binding was observed on either
120/
83
or
90/
55 probes (data not shown), but two specific complexes, C1
and C2, were revealed with the
62/
29 sequence (Fig.
4, lanes 2 and 8).
As for the complex obtained with recombinant proteins (Fig.
3B), these two complexes were both specifically competed by
an excess of unlabeled
62/
29 probe (Fig. 4, lane 3) or
DR3 oligonucleotide but not by an excess of AP-1,
120/
83, or
90/
55 oligonucleotides (data not shown). To identify the proteins
implicated in these complexes, antibodies directed against different
transcription factors were used to interfere with the protein-DNA
interactions. The anti-VDR antibody induced a disappearance of the two
binding complexes, and the antibody against RXR further retarded both
C1 and C2 complexes to lower mobility complexes (Fig. 4, lanes
4 and 5). Thus, RXR is present in both C1 and C2
complexes formed with the VDR on this VDRE. No significant binding
interference was observed with the antibodies directed against P19
(anti-gag antibody), RAR, TR
, or Mi (Fig. 4, lanes 6 and
9-11). Thus the C1 and C2 complexes are both composed of
VDR·RXR heterodimers bound to DNA. Taken together, these results
indicated that the nuclear extracts contained the proteins essential
for 1,25-(OH)2D3 activation of the CAII promoter and for the binding to the
62/
29 region. This binding complex is at least composed of RXR and VDR nuclear proteins.
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Role of 1,25-(OH)2D3 on Protein Binding to
DNA--
Mobility shift experiments were also performed with nuclear
extracts from cells treated for 2 days with vehicle. When the 62/
29
probe was incubated with those nuclear extracts only the C1 low
mobility complex was detected (Fig. 5,
lane 6), and this complex was competed by an excess of cold
probe (Fig. 5, lane 9). The high mobility C2 complex, absent
from vehicle-treated nuclear extracts (Fig. 5, lane 6), was
detected only in cells treated with
1,25-(OH)2D3 (Fig. 5, lane 2). Those
different results could be explained by the cell pretreatment with
1,25-(OH)2D3 before the preparation of the
nuclear extracts. Thus, we hypothesized that the C2 complex could be
due to 1,25-(OH)2D3 binding onto its receptor
and inducing a change in complex conformation and in gel mobility.
Therefore we have tested the 1,25-(OH)2D3
impact on DNA binding by the addition of
1,25-(OH)2D3 to the EMSA reaction mixture.
Adding 1,25-(OH)2D3 did not modify the complex
formation observed with 1,25-(OH)2D3 or
vehicle-treated nuclear extracts (Fig. 5, lanes 3 and
4; 7 and 8). These results clearly
show that the C2 complex is not caused by the immediate binding of the
ligand to the VDR. Nevertheless, this C2 complex could be the
consequence of a conformational change of the VDR·RXR heterodimer
bound to the VDRE compared with the C1 complex. The in vivo
bound ligand could induce this conformational change and allow the
recruiting of a third protein. Thus C2 appears to be the physiological
complex since it corresponds to the specific complex formed in
1,25-(OH)2D3-treated cells. These experiments
demonstrated that a VDRE is located in the region between residues
62
and
29 of the CAII promoter which can bind specifically the VDR and
RXR present in the nuclear extracts of the HD11 macrophages.
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Identification of the Putative VDRE by Mutational Analysis--
To
define more precisely the VDR·RXR binding site on the CAII proximal
promoter, the 62/
29 region obtained from the p
62
29tkCat construct was used as a probe for methylation interference assays (Fig.
6A). The guanine residues of
this domain involved in the DNA-protein interactions are shown in Fig.
6B. In this area we could easily point out an
AGGGCA 5'-half site, between positions
53 and
48 (framed sequence in Fig. 6B), quite
homologous to the published consensus VDRE hexamer and including some
of the protected residues (underlined nucleotides). On the other hand, the 3'-putative hexameric half-site could consist of either a GGAGTT between residues
46 and
41 in the case of a DR1
element or an AGTTCG between residues
44 and
39 for a DR3 element.
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DNA Binding Analysis of the 62/
29 Region
Mutants--
Oligonucleotides containing mutations described in Fig. 7
were tested in EMSAs to compete for the formation of DNA-protein complexes on the
62/
29 sequence.
62/
29-labeled probe was
incubated with nuclear extracts from
1,25-(OH)2D3-treated HD11 cells, and mutated
oligonucleotides were used as competitors; the EMSAs are shown in Fig.
8A. The mutants can be
classified within three groups according to their ability to bind
specific protein complexes. The first one, with the m1 and m7 mutants,
localized outside the interfering area, can compete for the DNA-protein
binding (Fig. 8A, lanes 4 and 10)
equally as well as the unlabeled
62/
29 oligonucleotide (Fig.
8A, lane 3). The m3 and m4 mutants, in the second
group, were also able to displace the bound complexes but to a lesser extend than the native sequence (Fig. 8A, lanes 6 and 7). Third, Fig. 8A also shows that the m5
mutant (lane 8) has completely lost the ability to bind,
whereas m2 and m6 mutants seem to have retained a slight binding
(lanes 5 and 9).
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The Identified VDRE Is a DR3 Element--
From the previous
experiments it was clear that the sequence comprised between positions
53 and
39 of the CAII promoter was a VDRE. At this point we could
not completely rule out that it was DR1 element (AGGGCA t GGAGTT cg)
rather than a DR3 element (AGGGCA tgg AGTTCG). It has been shown that
the second base of the 3'-element of a VDRE is the more conserved
residue among all VDREs described so far. We then constructed two other
mutated
62/
29 oligonucleotides, namely m8 and m9 mutants, in which
the G residue in position
45 (for the putative DR1 element) and
43 (for the putative DR3 element) was mutated to a C residue. Transient transfection experiments have revealed that the
1,25-(OH)2D3-mediated response obtained with
m8tkCat reporter plasmid is similar to the p
62
29tkCat construct,
whereas m9tkCat has completely lost the
1,25-(OH)2D3 response capability showing that
the complex bound needs to contact this G residue for efficient
transactivation (Fig. 9A,
lanes 4 and 6, respectively). We then tested
their capability to bind nuclear proteins by using EMSAs with
radiolabeled
62/
29, m8, or m9 probes. As shown in Fig.
9B, the m8 mutant can still compete with the
62/
29
oligonucleotide for the binding of HD11 nuclear extracts (lane
4) and bind VDR·RXR heterodimer (lanes 6-10),
whereas the m9 mutant is unable to do so (lanes 5 and
11-15). These experiments have confirmed that the m8 mutant
is identical in function to the native VDRE and that the m9 mutant is
no more able to bind the VDR·RXR heterodimer specifically. Then, we
concluded that the G residue in position
43 is essential for both
transactivation and binding of macrophage-derived nuclear extracts.
This result clearly confirms that the CAII proximal VDRE is a DR3 with
sequence AGGGCA tgg AGTTCG.
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CAII Proximal VDRE Is Functional in a Heterologous Cellular
System--
To confirm further the validity of the CAII proximal VDRE,
constructs containing this VDRE were tested in a heterologous cellular system. We undertook transient transfection experiments in the rat
osteosarcoma cell line ROS 17/2.8 cells with the different tkCat
reporter constructs described above. These cells were transfected with
the tkCat reporter constructs and the CMV--galactosidase as an
internal control and were then treated for 2 days with
10
8 M 1,25-(OH)2D3.
The results presented in Fig. 10 have
demonstrated that the CAII proximal VDRE is functional in ROS 17/2.8
cells (lane 2) and is also able to confer
1,25-(OH)2D3 responsiveness to the tk promoter
in a heterologous system. Moreover, by using mutated forms of this VDRE
(Fig. 10, lanes 3-9) we have confirmed that, like in HD11
cell line, this VDRE is also of the DR3 type in ROS 17/2.8 cells.
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DISCUSSION |
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In this work, we showed that the chicken CAII promoter activity is
induced in response to 1,25-(OH)2D3 in an avian
macrophage cell line. We identified a domain in the CAII promoter
responsible for 1,25-(OH)2D3-mediated
transactivation. Transient transfection assays in HD11 macrophages of
total or deleted fragments of the CAII promoter allowed us to define a
region involved in 1,25-(OH)2D3 responsiveness.
In macrophages, most if not all DNA sequences essential for CAII gene
basal and 1,25-(OH)2D3-induced expression are
within the first 178 bp upstream of the initiation site, referred to as
the proximal promoter. It is noteworthy that the level of 1,25-(OH)2D3 activation increases with the
5'-end progressive deletions of the promoter. A repressor domain
further upstream may be present and would explain the increasing
activation of CAII promoter correlated with deletions. Further
deletions and mutational analyses of the CAII proximal promoter allowed
us to determine a more precise vitamin D-responsive domain, between residues 53 and
39 of the CAII promoter. We showed that this VDRE
is highly functional in macrophages and is bound specifically by a
complex formed by a RXR·VDR heterodimer. Lastly, we showed that
mutations that abolished protein binding to the VDRE inhibit vitamin
D-dependent activation in macrophages.
Many have shown that the VDR has a binding preference for the direct repeat composed of AG(G/T)TCA motifs spaced by 3 bp (2, 3, 53, 54). The CAII proximal VDRE consists of an imperfect tandem of 6 bases spaced by three nucleotides with the sequence AGGGCA for the 5'-motif and AGTTCG for the downstream motif. Mutational analysis revealed the validity of the DR3 structure for the CAII proximal VDRE. Oligonucleotides containing mutations within the spacer motif of this DR3 (m4 and m8 mutants) were still able to bind VDR·RXR heterodimer and have a very mild effect on transactivation efficiency. In contrast, mutations within the two hexameric motifs of the DR3 element inhibited 1,25-(OH)2D3-mediated transcriptional activation and most of the protein-DNA interactions. Furthermore, our results indicate that the 3'-element integrity of this VDRE is more crucial for binding than the 5'-element. This is evidenced by the ability of a mutated 5'-element VDRE (m3 mutant) to compete still for VDR binding with the native sequence, whereas mutations in the 3'-element (m5 and m6 mutants) resulted in a marked decrease in VDR binding to the mutant element. In contrast with others studies showing the important role of the residues in 5'-position outside the VDRE, we have shown here that the nucleotides located outside the VDRE were not essential either for the binding of the protein complex on DNA or for the transactivation activity.
The sequences of numerous known natural positive VDREs, generally of the DR3 type, identified within the promoter regions of different genes, have been aligned. It is of interest to note that the hexameric core binding sites are rather degenerated, although they all can specifically bind VDR complexes and confer transactivation upon 1,25-(OH)2D3 stimulation. We have observed that the upstream motif of the consensus VDRE is more conserved than the downstream one. The difference between the two half-sites may indicate preferential binding of each receptor of the complex bound to DNA. Previous studies have shown that RXRs bind preferentially to the 5'-core binding site in retinoic acid and thyroid hormone response elements as well as in VDREs (1, 7, 25-27, 57, 58).
Haussler et al. (59) have postulated that the guanine in the second position of the 3'-hexamer is absolutely conserved, as we found for the CAII proximal VDRE. Indeed, mutation of this guanine abolishes 1,25-(OH)2D3 responsiveness and the binding of transcription factors to the CAII proximal VDRE. Thus, the 5'-motif of the CAII VDRE could be a high affinity RXR binding motif, whereas the 3'-hexanucleotide sequence could be a VDR binding motif (60).
Consistent with the results obtained with gel retardation experiments, we have demonstrated by transient transfection experiments that the vitamin D stimulation of the CAII gene expression acts, in vivo, through VDR and RXR dimerization.2 Furthermore, we have also shown that, in vivo, addition of RXR ligand does not interfere with the 1,25-(OH)2D3 response and so, does not affect the stability of VDR·RXR heterodimers in the cells nor the transactivation efficiency (49).
Surprisingly, two specific complexes, C1 and C2, were formed on the
CAII proximal VDRE with nuclear extracts from
1,25-(OH)2D3-treated HD11 cells, whereas only
one complex (C1) was formed with vehicle-treated extracts or
recombinant proteins. However, C1 and C2 complexes bound to this VDRE
are both composed at least of VDR and RXR heterodimers. We have
speculated that in vivo,
1,25-(OH)2D3 induces a specific conformational
change of the VDR·RXR heterodimer leading to the formation of the C2
complex, whereas in vitro, only the C1 complex is observed.
This C2 higher mobility complex could be caused by the binding of
1,25-(OH)2D3 to the VDR, although the addition of extra 1,25-(OH)2D3 to the EMSA reaction
mixture had no effect on the DNA binding affinity of the protein
complex. In such conditions no change in mobility or complex
composition was observed. This suggests that in vitro,
binding of the ligand is not necessary for the binding of proteins to
DNA. In contrast, in vivo, cell exposure to
1,25-(OH)2D3 could change the complex
conformation on DNA and in doing so could either recruit or displace
the binding of either a coactivator or a corepressor. The two
complexes, C1 and C2, obtained by EMSAs using nuclear extracts, may
reflect the possible involvement of known or unknown factors in
addition to the VDR·RXR heterodimer. In fact, the transcription
factor TFIIB, which was shown to interact directly with VDR (61), has been described as forming part of in these complexes and appears to be
required for the VDR to bind the VDREs (62). Other potential unidentified proteins may also be involved in the binding of the VDR to
the VDRE such as positive cofactors, including TRIP1 (63), NCoA62
(64), RIP140, RIP160 (65, 66), and SRC1 (67). These accessory factors
have been shown to contribute to the transcriptional activation
mediated by nuclear hormonal receptors through a direct interaction
with those receptors (49, 64-70). The identified bases of the CAII
proximal VDRE may therefore bind a transcription factor that promotes
1,25-(OH)2D3-mediated transactivation by facilitating interactions between the receptor-occupied VDRE and the
basal transcription apparatus.
Finally, we tested the activity of this CAII proximal VDRE in a heterologous cellular system, the ROS 17/2.8 cell line. These analyses have demonstrated that the CAII proximal VDRE is also functional as a VDRE in the ROS 17/2.8 cells but less efficiently. Thus, the VDRE identified in this study allows 1,25-(OH)2D3 transactivation of the CAII gene after binding of the VDR·RXR heterodimer, is efficient when cloned upstream a heterologous promoter, and is functional in a heterologous cellular system. Although the distal CAII VDRE is not functional in this macrophage cell line, the CAII proximal VDRE is fully active in the cell type (i.e. macrophages) expressing the CAII gene endogenously, and it is indeed the region promoting the 1,25-(OH)2D3 response.
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ACKNOWLEDGEMENTS |
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We thank P. Chambon for the RXR and RAR antibodies, S. Saule for anti-Mi, and M. Uskokovic for 1,25-(OH)2D3. We also thank A. Sergeant and J. Samarut for helpful discussions and P. Herzmark for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by a grant from the Association pour la Recherche sur le Cancer (to I. Q.) and the Ligue contre le Cancer (Nationale and Rhône).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. Fax: 33-472-72-8686;
E-mail: Pierre.Jurdic{at}ens-lyon.fr.
1 The abbreviations used are: VDR, vitamin D receptor; VDRE, vitamin D response element; RXR, retinoid X receptor; bp, base pair(s); DR3, direct repeats of two hexameric core binding sites spaced by three nucleotides; CAII, carbonic anhydrase II; 1,25-(OH)2D3, 1,25-dihydroxyvitamin D3; tk, thymidine kinase; CAT or Cat, chloramphenicol acetyltransferase; CMV, cytomegalovirus; EMSA, electrophoretic mobility shift assay; RAR, retinoic acid receptor; TR, thyroid hormone receptor; Mi, microphalmia transcription factor.
2 I. Quélo, and P. Jurdic, manuscript in preparation.
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REFERENCES |
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